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Global Ecology and Conservation - DORA 4RI
Global Ecology and Conservation 26 (2021) e01507

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                                         Global Ecology and Conservation
                                 journal homepage: http://www.elsevier.com/locate/gecco

Original Research Article

Beneficial effects of nitrogen deposition on carbon and
nitrogen accumulation in grasses over other species in Inner
Mongolian grasslands
Xue Wang a, b, *, Meng Wang a, Yimin Tao a, Nina Fang a, Guojiao Yang b,
Jiangping Cai b, Yong Jiang b, Xingguo Han b, Fei-Hai Yu a, Mai-He Li b, c, d
a
  Institute of Wetland Ecology & Clone Ecology, Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conservation,
Taizhou University, Taizhou, 318000, China
b
  Erguna Forest-Steppe Ecotone Research Station, CAS Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology,
Chinese Academy of Sciences, Shenyang, 110016, China
c
  Forest Dynamics, Swiss Federal Research Institute WSL, Zuercherstrasse 111, CH, 8903, Birmensdorf, Switzerland
d
  Key Laboratory of Geographical Processes and Ecological Security in Changbai Mountains, Ministry of Education, School of
Geographical Sciences, Northeast Normal University, Changchun, 130024, China

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

Article history:                                    Globally, chronic nitrogen (N) deposition into terrestrial ecosystems has resulted in
Received 27 October 2020                            changes in community composition, depending on the responses of co-existing species to
Received in revised form 14 February 2021           increasing soil N availability. The levels of plant non-structural carbohydrates (NSCs)
Accepted 15 February 2021
                                                    produced by photosynthesis are associated with leaf N content, and together they repre-
                                                    sent the capital of a plant for its life including competitive ability. However, the specific
Keywords:
                                                    ways that concentrations of plant NSCs, and also N, influence the growth of plants, and
Forb
                                                    thus their productivity, are still abscue. Here, we explored the effect of variations in plant
Grass
Legume
                                                    leaf NSCs (starch and soluble sugars) and N concentrations on the growth of plants with
Nitrogen addition                                   different soil N uptake capacities, i.e. two grasses (Leymus chinensis and Stipa baicalensis),
Non-structural carbohydrate                         two forbs (Potentilla tanacetifolia and Galium verum), and one legume (Thermopsis lance-
Starch                                              olate) in Inner Mongolian grasslands subjected to three N addition rates (i.e., 0, 10, or
                                                    20 g N m2 yr1) and three N forms [i.e., NH4NO3, (NH4)2SO4, or CO(NH2)2] for four years.
                                                    Irrespective of N addition rate and N form, N addition significantly increased aboveground
                                                    biomass of the grasses, but not that of the forbs or the legume. The grasses had higher
                                                    increase in N concentrations than the forbs and the legume. N addition increased the
                                                    starch concentrations of grasses, but decreased that of forbs and the legume. Changes in
                                                    the aboveground biomass of all tested plant species were significantly positively correlated
                                                    with changes in concentrations of both NSCs and N. Our results indicate that, irrespective
                                                    of the N addition rates and N forms, greater increase in NSCs and N concentrations
                                                    contributed to higher aboveground productivity in grasses, but not in forbs and legumes.
                                                    These results imply that future nitrogen deposition will benefit grasses over other plant
                                                    forms, and thus the former may become more dominant in grasslands in northern China.
                                                    © 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC
                                                                       BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 * Corresponding author. Institute of Wetland Ecology & Clone Ecology; Zhejiang Provincial Key Laboratory of Plant Evolutionary Ecology and Conser-
vation, Taizhou University, Taizhou, 318000, China.
    E-mail address: wangxueiae@163.com (X. Wang).

https://doi.org/10.1016/j.gecco.2021.e01507
2351-9894/© 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).
Global Ecology and Conservation - DORA 4RI
X. Wang, M. Wang, Y. Tao et al.                                                             Global Ecology and Conservation 26 (2021) e01507

1. Introduction

    The amount of nitrogen (N) deposited into terrestrial ecosystems has significantly increased due to an upsurge in
anthropogenic activities (Erisman et al., 2013; Liu et al., 2013). This N increment can differentially affect the growth and
competitive capacity of different plant species and functional groups in plant communities, resulting in a shift in the
dominance of different species and functional groups (Cleland and Harpole, 2010). For instance, simulated N deposition has
generally been shown to increase the abundance of grasses and decrease that of forbs in grassland communities (Xia and
Wan, 2008; Xu et al., 2015; Fu and Shen, 2016; Mitchell et al., 2017; Tang et al., 2017).
    The growth-related bio-physicochemical processes of a plant, such as carbon balance and N uptake, could significantly
respond to N addition (Pornon et al., 2019; Li et al., 2020). For example, the level of non-structural carbohydrates (NSCs, i.e.
soluble sugars and starch) in plant tissues is the result of carbon gain (photosynthesis) minus consumption (growth and
maintenance respiration) (Chapin et al., 1990; Li et al., 2013), representing a plant’s capital for growth and defence (Li et al.,
2002, 2018). The photosynthetic capacity and thus the amount of NSCs produced by photosynthesis are positively correlated
with leaf N (Evans, 1989) and soil N availability (Zhao et al., 2005). Therefore, N supply influences plant photosynthesis, NSCs
status and growth, and further affects their productivity and ultimately their survival in a plant community. However, the
specific ways that concentrations of plant NSCs and N influence the growth of plant species with different competitive N
uptake capacities, such as grasses vs. forbs/legumes, are still obscure.
    Plant species vary greatly in their N uptake capacities, depending on their physiological and biochemical characteristics
and different soil N forms (Song et al., 2012; Sheppard et al., 2014; Yan et al., 2019), and thus show different magnitudes and
directions of plant N responses to N addition with various soil N forms (Liu et al., 2016; Du et al., 2020). Consequently, plants
exhibit differential responses of NSCs that are closely associated with N addition rates, N application duration, and even the
specific N compounds applied (Humbert et al., 2016; van den Berg et al., 2016). For instance, some plants have higher NO        3 but
lower organic N absorption capacity than others, even under optimal N environments (Nordin et al., 2006). Plants that
dominate acidic soils absorb more NHþ   4 relative to other plants (van den Berg et al., 2008). Thus, the bioavailability of soil N in
the various forms affects plant leaf N levels, which in turn influences photosynthesis and NSC concentrations, and finally plant
growth. However, we still lack information on how different soil N forms affect the relationship between carbon balance and
the growth of different species in grassland ecosystems.
    Increased aboveground biomass and cover of grasses, accompanied with decreased biomass and cover of forbs, were
widely reported in N addition experiments in Inner Mongolian grasslands (Song et al., 2011, 2012; Xu et al., 2012, 2015; Zhang
et al., 2015). To our knowledge, however, the plant carbon- and nitrogen-physiological mechanisms underlying those changes
have rarely been studied. Thus, we examined the relationships between aboveground biomass and the concentrations of leaf
N and NSCs (i.e. soluble sugars and starch) in two C3 grasses (Leymus chinensis and Stipa baicalensis), two forbs (Potentilla
tanacetifolia and Galium verum), and one legume (Thermopsis lanceolate) in an Inner Mongolian grassland ecosystem sub-
jected to various N addition rates and N forms for four years. We tested the following hypotheses: (1) N addition leads to a
higher accumulation of leaf N and NSCs in grasses than in forbs and legumes, and thus a higher productivity; and (2) the
magnitude of the stimulating effects varies with N forms and N addition rates.

2. Materials and methods

2.1. Study site and experimental design

    The experiment was conducted at the Erguna Forest-Steppe Ecotone Research Station (N50100 46.100, E119 220 56.400 ),
northeastern China. The field experiment was carried out in a natural steppe that has remained fenced since 2013 to prevent
livestock grazing. Before then, the grassland was annually mown for hay-harvesting. The site has a long-term mean annual
precipitation of 363 mm and a mean annual temperature of 2.45  C (1957e2016). According to the US soil taxonomy
classification, the site has chernozem soil. The pH of the topsoil (0e10 cm) is 6.8e7.0, and the dominant species in the
ecosystem are Leymus chinensis, Stipa baicalensis, Cleistogenes squarrosa, Thermopsis lanceolata, Cymbaria dahurica, and Carex
duriuscula.
    The study was part of a long-term, large experiment (Yang et al., 2019) with 60 treatments and eight replications. Our
sampling involved only seven of the treatments, i.e. two rates of N addition (i.e., 10, and 20 g N m2 yr1) crossed with three
forms of N compounds [i.e., NH4NO3, (NH4)2SO4, and CO(NH2)2], and a control (no N addition). The N addition experiment
began in late May of 2014 and the treatments were applied in a completely randomized block design (eight blocks) in plots
measuring 10 m  10 m. In the plots with N addition, a certain amount of the N compounds were broadcasted by hand once a
year. To avoid potentially confounding effects, similar quantities of sand (0.5 kg per plot) were applied in the control plots.

2.2. Sampling

   In mid-August 2017, after four years of continuous N addition, leaf samples were collected from six dominant or abundant
species in each plot, including two grasses (Leymus chinensis and Stipa baicalensis), two forbs (Potentilla tanacetifolia and
Galium verum), and two legumes (Thermopsis lanceolate and Vicia sepium). The climatic condition during the sampling was
shown in Fig. S1. For each species, two to three healthy, fully expanded leaves were collected from each of 10 randomly

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X. Wang, M. Wang, Y. Tao et al.                                                           Global Ecology and Conservation 26 (2021) e01507

selected plant individuals, if they were present, at the center of each plot to avoid edge effects. The samples were pooled to
obtain a mixed sample for each species in each plot. We stored the samples in boxes with ice until they were oven-dried at
65  C for 48 h and then finely ground to allow passage through a 0.15 mm mesh size. Since we could not find all the six species
in all eight replicate plots, we used the four replicate plots where all the six species were found. However, the quantities of the
samples of V. sepium obtained still did not meet the minimum requirement for analysis. Therefore we excluded this species
from the analysis.
    To assess aboveground biomass, we clipped all vascular plants at the soil surface in 1 m  1 m quadrats (in mid-August
2017). The quadrats were randomly placed in each plot without a spatial overlap of the quadrats and at a minimum of 50 cm
from the border of each plot to avoid edge effects. All living vascular plants were sorted according to species. The plant
materials were then oven-dried at 65  C for 48 h and then weighed.
    A five mixed soil sample (0e15 cm soil depth) was taken from each plot, using soil cores with an inner diameter of 3.0 cm.
Fresh soil samples were sieved through a 2 mm mesh size, air-dried and ground into fine powder before measuring total soil
N.
    Analysis of the concentrations of non-structural carbohydrates, nitrogen and chlorophyll content.
    The powdered leaf material (0.1 g) was put into a 10 ml centrifuge tube, and 5 ml of 80% ethanol was added. The mixture
was incubated in a water bath-shaker maintained at 80  C for 30 min, then centrifuged at 3500 rpm for 10 min. The pellets
were extracted at least twice with 80% ethanol. Supernatants were retained, pooled, and stored at 4  C until further use for
assessment of the total soluble sugars.
    The ethanol-insoluble pellet was air-dried to evaporate the ethanol, and then the residue was used for starch extraction. To
obtain the starch, the residue was dispersed in 2 ml distilled water and heated for 15 min in a boiling water-bath. After cooling
to room temperature, 2 ml of 9.2 M perchloric acid (HClO4) was added, and the mixture was maintained at room temperature
for 15 min to allow for starch hydrolysis. Subsequently, 4 ml of distilled water was added to each sample, and then centrifuged
at 4000 rpm for 10 min. The pellets were used for starch extraction one more time with 2 ml of 4.6 M HClO4. Supernatants
were retained, combined, and topped up to 25 ml, and then used to determine starch concentrations. The concentrations of
soluble sugars and starch were evaluated spectrophotometrically (ultravioletevisible spectrophotometer 752S, Cany Preci-
sion Instruments Co., Ltd., Shanghai, China) at 620 nm by the anthrone method. Starch quantities were calculated by
multiplying the glucose concentrations by a conversion factor of 0.9. Glucose was used as an internal standard (Li et al., 2008).
    The chlorophyll content was determined colorimetrically by chloroplast pigment extract from 95% ethanol (Lichtenthaler
1987) (Fig. S2). A C/N analyzer (Vario Micro cube, Germany) was used to evaluate the concentration of leaf N and total soil N
(Fig. S3).

2.3. Data analysis

   For each of the five species, we calculated the change (x) in each variable (aboveground biomass, N, soluble sugars, starch,
and total NSCs) between each of the six N rate and N form treatments (T) and the control (C) as:

             variable in T  variable in C
        x¼                                  100%
                     variable in C

    A three-way analysis of variance (ANOVAs) was performed to determine the effects of plant species, soil N rate, soil N form,
and their interactions on the change in aboveground biomass, N, soluble sugars, starch, and total NSCs in leaves (Table S1). We
further used two-way ANOVAs to test these effects for each species separately. We also employed one-way ANOVAs to test the
effect of species on aboveground biomass, N, sugars, starch, and NSCs in the control, and one sample t-tests to determine
whether the change in each variable for each N rate and N form treatment (i.e. the difference between each of the six N rate
and N form from treatments and the control) differed significantly from zero. Tukey’s HSD was used for multiple comparisons.
Linear regressions were used to examine the relationships between the change in aboveground biomass and the change in N,
soluble sugars, starch and total NSCs. All data were checked for normality with the Kolmogorov-Smirnov test and for ho-
mogeneity of variance with Levene’ s test. Data that did not meet the condition were transformed to log or square-root
transformed prior to analysis. All statistical analyses were performed using the SPSS software version 22.0 (IBM Corp.,
Armonk, NY, USA).

3. Results

3.1. Changes in aboveground biomass

   The net changes in aboveground biomass varied among the different species (P < 0.001; Table S1). Compared to the
control, N addition significantly increased aboveground biomass of L. chinensis (Fig. 1A, P < 0.001) but decreased that of P.
tanacetifolia (Fig. 1D, P ¼ 0.017) and did not affect that of G. verum or T. lanceolate (Fig. 1C, E, P ¼ 0.235 and P ¼ 0.611,
respectively). Neither two high N addition rates nor N forms affected the change in aboveground biomass of any of the five
species, except for a significant effect of N rate for S. baicalensis, which had higher values with 10 g N m2 yr1 (N10) than with
20 g N m2 yr1(N20) (Fig. 1B).

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X. Wang, M. Wang, Y. Tao et al.                                                                                Global Ecology and Conservation 26 (2021) e01507

3.2. Changes in leaf N and NSCs

    In the control plots, the concentrations of N and NSCs in leaves varied among species (Fig. 2). The legume T. lanceolate had
the highest leaf N concentration, and forbs had higher leaf N concentration than grasses (Fig. 2A). The concentrations of
soluble sugars, starch, and total NSCs were in general higher in the forbs than in the grasses and the legume (Fig. 2BeD).
    In the plots with N addition, the changes in the concentrations of N, soluble sugars, starch, and total NSCs in leaves varied
significantly among species (P < 0.001; Table S1). For all the five species, we detected significant increases in leaf N con-
centration after N addition (Fig. 3AeE). However, the extent of the increase in the N concentration varied among species, and
across the N rates and N forms, the grasses (þ44.4%) showed larger increases than the forbs (þ24.3%) and the legume (þ3.7%)
(Fig. 3AeE, P < 0.001). Neither two high N rates nor N forms affected the leaf N concentrations of any of the five species, except
for P. tanacetifolia where the N concentration was greater with the higher N addition rate (Fig. 3D). For L. chinensis, urea
addition resulted in larger increases in the concentrations of soluble sugars and total NSCs than with the addition of
ammonium nitrate (NH4NO3) or ammonium sulfate (NH4)2SO4) (Fig. 3F, P, P ¼ 0.037 for soluble sugars and P ¼ 0.025 for total
NSCs). Compared with the control, N addition led to an increase in the concentration of starch in the grasses (Fig. 3K and L),
but a decrease in the forbs (Fig. 3M, N). Compared with the control, N addition significantly increased the sugar: starch ratio of
the forb P. tanacetifolia (Fig. 3X).

3.3. Correlations between aboveground biomass and N, soluble sugars, starch, and total NSCs

   Changes in aboveground biomass were positively correlated with the changes in the concentrations of leaf N, starch, and
total NSCs (Fig. 4A, B, and D). However, there was no significant correlation between mean changes in aboveground biomass
and soluble sugars (Fig. 4C).

4. Discussion

4.1. Effects of N addition

   The concentration of non-structural carbohydrates (NSCs) and starch in the grasses increased, whereas it decreased or
unchanged in the forbs and the legume under N addition (Fig. 3KeO). Meanwhile, the grasses had a greater increase in leaf N
concentrations than the forbs and the legume in response to N addition (Fig. 3AeE). Further, the changes in aboveground

Fig. 1. Changes in aboveground biomass of each plant species under six nitrogen (N) rate and N form treatments relative to the control. U, AN and AS stand for the
addition of urea, ammonium nitrate, and ammonium sulfate, respectively; Nf and Nr represent for N form and N rate treatments, respectively. P values of two-
way ANOVAs are given. An asterisk above a bar indicates a significant difference between the N addition treatment and the control (*P < 0.05).

                                                                                4
X. Wang, M. Wang, Y. Tao et al.                                                                                  Global Ecology and Conservation 26 (2021) e01507

Fig. 2. Concentrations of nitrogen and mobile carbohydrates (soluble sugars, starch, and NSCs) in each of the five species in the control plots (without N addition).
Different lowercase letters indicate significant differences among the different species (P < 0.05).

biomass were positively correlated with the changes in leaf NSCs and N (Fig. 4). These results are consistent with our first
hypothesis that higher competitiveness regarding NSCs accumulation and N uptake contribute to higher productivity in
grasses under N addition.
    Exogenous N addition significantly increased aboveground biomass of the grasses and decreased that of forbs. This finding
is in agreement with previous studies (Bobbink et al., 2010; Tian et al., 2016; Mitchell et al., 2017; You et al., 2017). In our study,
in response to N addition, the grasses exhibited a larger increase in leaf N concentrations than the forbs, as well as a greater
increase in aboveground biomass. This increase in leaf N concentrations of grasses, according to You et al. (2017), can be
attributed to the branched fibrous root systems in grasses which have the advantage of absorbing more nutrients from the soil
than other species. Indeed, we found that rhizomatous grass (L. chinensis) and bunchgrass (S. baicalensis) had lower leaf N
concentrations than the forbs and the legume in the control plots, but a larger increase in leaf N concentrations under N
addition (Figs. 2A, 3AeE).
    Exogenous N addition can enhance the photosynthetic rate of plants (Zhang et al., 2018; Liang et al., 2020) as well as leaf
area development (Be  langer et al., 1992), thereby increasing the concentrations of non-structural carbohydrates and sub-
sequently increasing aboveground biomass (Niu et al., 2010). N addition resulted in an increase in both aboveground biomass
and starch concentration in the grasses (Fig. 1A and B; Fig. 3K and L), probably because grasses have higher photosynthetic
rates than forbs (Tjoelker et al. (2005), as we found a higher chlorophyll content in grasses than in forbs (Fig. S2). In general,
the mobile carbohydates can be stored as starch in case that the carbon supply is greater than the carbon need. Thus, the
strong competitive advantage in photosynthesis (starch accumulation) and the greater increase in leaf N concentrations in
response to N addition contributed to the increase in aboveground biomass of grasses, especially for L. chinensis.

4.2. Effects of N addition rates

    Although the higher N addition (20 g N m2 yr1) corresponded to higher total soil N concentrations (Fig. S3), it did not
result in a significantly greater increase in leaf concentration (except for P. tanacetifolia), aboveground biomass or NSC
concentrations in comparison to lower N addition (10 g N m2 yr1). According to Aber et al. (1998), nitrogen levels exceeding
a specific range could have an inhibitory effect on plants. Our results indicated that the biomass of the selected species could
be saturated at a rate of >10 g N m2 yr1. This is in agreement with Bai et al. (2010), who found that the aboveground biomass
in Inner Mongolian grassland saturated at an N addition rate of approximately 10.5 g N m2 yr1. The legume T. lanceolate was
less sensitive to N addition, which indicates that a surge in the amount of available N in the environment causes legumes to
lose their advantage of fixing N (Suding et al., 2005). Increased leaf N concentration can facilitate photosynthesis and promote
the accumulation of mobile carbohydrates. On the other hand, N addition can accelerate growth, respiration, and other
physiological processes that consume mobile carbohydrates (Hoch et al., 2003; Wang et al., 2017; Li et al., 2020). Thus, these
processes could increase, reduce, or not affect the levels of mobile carbohydrates, depending on the dominant processes. In
our study, For all the species in our study, no difference in mobile carbohydrate concentrations was detected between N

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X. Wang, M. Wang, Y. Tao et al.                                                                                Global Ecology and Conservation 26 (2021) e01507

Fig. 3. Changes in nitrogen (N), soluble sugars, starch, and total non-structural carbohydrate concentrations and in the sugar: starch ratio of each plant species
between each of the six N rate and N form treatments and the control. U, AN and AS stand for the addition of urea, ammonium nitrate and ammonium sulfate,
respectively; Nf and Nr represent N form and N rate treatments, respectively. P values of two-way ANOVAs are given. An asterisk above a bar indicates the
significant a difference between the N addition treatment and the control (P < 0.05).

addition rates of 10 g N m2 yr1and 20 g N m2 yr1 (Fig. 3). This result suggests that increased leaf N concentrations
promote other processes, but not mobile carbohydrate accumulation.

4.3. Effects of N forms

    Different soil N forms applied significantly affected the concentration of mobile carbohydrates in grasses, although this
effect did not induce any changes in aboveground biomass. This result is inconsistent with our second hypothesis.

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X. Wang, M. Wang, Y. Tao et al.                                                                            Global Ecology and Conservation 26 (2021) e01507

Fig. 4. Relationships between the changes in aboveground biomass of the five plant species and the changes in concentrations of nitrogen (N), soluble sugars,
starch and total non-structural carbohydrates (NSCs).

    Across the species, aboveground biomass and leaf N concentrations did not vary among the different N forms, suggesting
that the five species did not exhibit differences in N uptake according to the form of N applied. The ratio of N to P is regarded as
an important index to explain nutrient limitation pattern (He et al., 2006). According to Koerselman and Meuleman (1996)
and Güsewell (2004), an N: P ratio
X. Wang, M. Wang, Y. Tao et al.                                                                                    Global Ecology and Conservation 26 (2021) e01507

Funding

   This work was supported by the Natural Science Foundation of Zhejiang Province of China (LQ20C160008), Research and
Innovation Initiatives of Taizhou University (2017PY033), and the Ten Thousand Talent Program of Zhejiang Province
(2018R52016).

Declaration of competing interest

    The authors declare that they no conflicts of interest.

Acknowledgments

    The authors thank the staff of Erguna Forest-Steppe Ecotone Research Station for facilitating this study, and Melissa Dawes
for English language corrections.

Appendix A. Supplementary data

    Supplementary data to this article can be found online at https://doi.org/10.1016/j.gecco.2021.e01507.

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X. Wang, M. Wang, Y. Tao et al.                                                                                    Global Ecology and Conservation 26 (2021) e01507

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