Limited inorganic N niche partitioning by nine alpine plant species after long-term nitrogen addition

Journal Pre-proof Limited inorganic N niche partitioning by nine alpine plant species after long-term nitrogen addition

Li Zhang, Tongbin Zhu, Xiang Liu, Ming Nie, Xingliang Xu, Shurong Zhou PII:

S0048-9697(20)30780-4

DOI:

https://doi.org/10.1016/j.scitotenv.2020.137270

Reference:

STOTEN 137270

To appear in:

Science of the Total Environment

Received date:

30 October 2019

Revised date:

15 January 2020

Accepted date:

10 February 2020

Please cite this article as: L. Zhang, T. Zhu, X. Liu, et al., Limited inorganic N niche partitioning by nine alpine plant species after long-term nitrogen addition, Science of the Total Environment (2018), https://doi.org/10.1016/j.scitotenv.2020.137270

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© 2018 Published by Elsevier.

Journal Pre-proof Running head: Plant N acquisition and nitrogen addition

Title: Limited inorganic N niche partitioning by nine alpine plant species after long-term nitrogen addition Li Zhang1, Tongbin Zhu 2, Xiang Liu1, Ming Nie1, Xingliang Xu3, Shurong Zhou1,*

1

Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering,

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Coastal Ecosystems Research Station of the Yangtze River Estuary, Shanghai Institute of

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Eco-Chongming (SIEC), and School of Life Sciences, Fudan University, 2005 Songhu Road,

Karst Dynamics Laboratory, MLR & Guangxi, Institute of Karst Geology, Chinese Academy

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2

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Shanghai 200438, P. R. China

of Geological Sciences, Guilin 541004, P. R. China

Key Laboratory of Ecosystem Network Observation and Modeling, Institute of Geographic

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3

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Sciences and Natural Resources, Chinese Academy of Sciences, 11A Datun Road, Chaoyang

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District, Beijing 100101, P. R. China

Emails of all authors:

Li Zhang: [email protected] Tongbin Zhu: [email protected]

Xiang Liu: [email protected] Ming Nie: [email protected] Xingliang Xu: [email protected] Shurong Zhou: [email protected] *Corresponding author: Shurong Zhou, E-mail: [email protected]

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Journal Pre-proof Abstract 1.

Nitrogen (N) is a major nutrient limiting plant growth in most terrestrial ecosystems. Both niche partitioning and fitness equalizing mechanisms related to N acquisition have been proposed to explain the maintenance of biodiversity and ecosystem functioning. However, their relative importance remains controversial and unclear, especially in worldwide terrestrial ecosystems increasingly threatened by N addition.

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We added NH4NO3 at four levels over 7 years in an alpine meadow on Qinghai-Tibetan

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Plateau. Nine species that all occurred along N addition gradients were selected for

Plants absorbed more ammonium and nitrate with N addition. We found limited

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3.

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in-situ 15N labeling experiment to quantify their uptake of ammonium verse nitrate.

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inorganic N niche partitioning along the N addition gradient. Instead, species tended to prefer the most abundant form of inorganic N in soil, i.e. ammonium. Of all possible

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linear mixed effects models constructed to explain variation in either ammonium or

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nitrate uptake, the most parsimonious one included soil available N. That means plants’ N uptake was influenced by habitat qualities, instead of the amount of added N itself. Our findings suggested that inorganic N niche partitioning may play limited role in the

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4.

maintenance of high diversity in this alpine meadow. Instead, species coexistence might be promoted by minimizing their fitness differences through preferring the most abundant form of inorganic N in the soil. This provides important insights into species coexistence under future N addition in Tibetan alpine meadows. Keywords: Chemical N partitioning, Competitive exclusion, Habitat filtering, N uptake strategy, NH4NO3 addition, Tibetan alpine meadow.

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Journal Pre-proof Introduction Nitrogen (N) is a major nutrient limiting plant growth in most terrestrial ecosystems (Vitousek & Howarth, 1991; LeBauer & Treseder, 2008), and shapes a plant community composition and ecosystem functions (McKane et al., 2002; Silvertown, 2004; Houlton et al., 2007; Andersen et al., 2013). Plants can absorb various forms of N directly from soil solution, e.g. inorganic ammonium (NH4+) and nitrate (NO3−), and organic N with low molecular weight (e.g. free amino acids) to meet their N demands (Jones et al., 2005; Näsholm et al.,

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2009). Although plants can take up organic N, inorganic N has been considered to be a major

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N source for most plants (Chapin, 1980; Marschner, 2011). Alpine meadows are wildly

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distributed over the world with high altitudes, especially on the Qinghai-Tibetan Plateau. N

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limitation is common due to slow mineralization of soil organic matter (SOM) caused by low temperature and water deficiency, although the soil stores a large amount of N (Zhou, 2001;

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Baumann et al., 2009; Zhang et al., 2012). However, such alpine meadows are rich in plant

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species, often with more than 30 species coexisting within a 0.5×0.5 m area (Liu et al., 2017). The conflict between this high diversity and limited N supply has attracted ecologist’s

2015).

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interests for decades (McKane et al., 2002; Silvertown, 2004; Xu et al., 2011a; Song et al.,

Chemical niche partitioning mechanism has been frequently invoked to explain how plants meet their N demands when competing with a number of plant species (McKane et al., 2002; Xu et al., 2011b; Song et al., 2015). In a given community, species utilizing N resources in a similar way may likely compete more intensively and lead to competitive exclusion. Thus, coexisting species might specify their strategies in using different chemical forms of N to reduce niche overlap and promote stable coexistence (McKane et al., 2002). Usually, the most abundant N form is occupied by the dominant species, leaving the nondominant ones with less dominant N forms (McKane et al., 2002; Ashton et al., 2010). 3

Journal Pre-proof Besides of such stabilizing mechanisms, a coexistence mechanism can also function by minimizing average fitness differences between species according to the modern coexistence theory (Chesson, 2000). In fact, some studies reported that plants meet N demands through preferences for a specific form of N, which largely depend on the most available N forms in their rhizosphere (Houlton et al., 2007; Mayor et al., 2014). The preference for the most abundant N form in the soil could result in reduced inequalities in N uptake and average fitness and thus contribute to coexistence. That particular physiological traits (i.e. preference)

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was ascribed to habitat selection effect, and the preference pattern would be flexible when

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species’ native habitat qualities change accordingly (e.g. ammonium/nitrate ratios) (Wang &

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Macko, 2011; Andersen et al., 2013). However, the relative importance of niche partitioning

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(stabilizing) and fitness equalizing with respect to N acquisition for species coexistence

change.

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remain controversial and unclear, and few studies have addressed this issue in face of global

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Worldwide terrestrial ecosystems are increasingly threatened by N enrichment through anthropogenic activities. Anthropogenic N addition (hereafter, N addition) through N

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deposition, and/or fertilization, can alleviate N limitation and thus lead to concomitant consequences for biodiversity and ecosystem functions (Chen et al., 2016; Niu et al., 2016; Midolo et al., 2018). It has been reported that N addition would stimulate plant N uptake (Lu et al., 2011; Niu et al., 2016), and plasticity in N preference in response to the addition of different forms of N (Song et al., 2015). However, it remains unclear how N addition affects the N acquisition patterns in terrestrial ecosystems mediated by habitat qualities (e.g. available N in soil, microorganisms, and pH) (Niu et al., 2016). Apart from soil N enrichment introduced by N addition, plant-microbial interaction and indirect environmental qualities including pH and Al3+, would also affect plant N uptake of different forms (Niu et al., 2016). Firstly, two processes are mainly responsible for the production of the available N in soil 4

Journal Pre-proof across N addition gradients. At low levels of N addition, plants allocate more carbon and energy to belowground for microorganisms, and activate rhizospheric microorganisms to produce more available N. At high levels, plants would allocate more carbon to aboveground parts and decrease their reliance on microorganisms (Kuzyakov & Xu, 2013; Sun et al., 2014). Thus, microbial biomass would influence soil available N, and then plant N uptake. Secondly, soil acidification can decrease microbial activities. At the same time, soil acidification potentially mobilizes phytotoxic metal ions, such as Al3+, Mn2+ (Tian et al., 2016), which

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suppress a plant’s N uptake through affecting its root physiology. At least three processes can

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affect soil acidification caused by NH4NO3 addition: (i) deposition of H+ with NO3− from the

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oxidation of NH4+, (ii) H+ release when plants or microorganisms absorb NH4+, and (iii)

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leaching of buffering base cations (Chen et al., 2016). However, it remains unclear how habitat qualities caused by N addition affects plant N uptake pattern through mediation by

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soil N enrichment, soil acidification etc.

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To clarify how the N uptake strategy (chemical N partitioning verses preference) of species changes with N addition, we determined the uptake of ammonium and nitrate of 9

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plant species that all appeared along the N addition gradient (0, 5, 10, and 15 g N m-2 year-1) with 15N labeling in a 7-year NH4NO3 addition alpine meadow on the Qinghai-Tibetan Plateau. Specially, we investigated: (i) Whether chemical N partitioning (species specialize in utilizing different forms of N resources) or fitness equalizing mechanism (species prefer the most common form of N) dominated community assembly under conventional land use (i.e., winter grazing) along the N addition gradient; and (ii) How do changes in habitat qualities resulted from N addition would affect plant N uptake.

Materials and methods Study site 5

Journal Pre-proof Our experiment was conducted in an alpine meadow in the eastern part of the Qinghai-Tibetan Plateau (35°58′ N, 101°53′′E), i.e. in Maqu, Gansu, China. The elevation is approximately 3500 m. The mean annual temperature is 1.2°C, ranging from -10.7°C in January to 11.7°C in July. The mean annual precipitation is 620 mm, which mainly falls in summer. This typical alpine meadow is about 54 species, and is dominated by annual and perennial herbaceous species, e.g. Aster diplostephioides, Potentilla potaninii, Saussurea

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stella, Carex aridula, Thalictrum alpinum. The soil is classified as a typical alpine meadow

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kind (Gong, 1999; Liu et al., 2015).

Experiment design

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We fenced a 100×200 m area with a relatively uniform vegetation cover and long-tern

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moderate conventional yak grazing history in June 2009, in which yak grazing was only permitted in winter. Totally forty-eight 5×5 m plots were regularly arranged in June 2011,

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separated by 1 m from adjacent edges. All plots were randomly assigned to one of the four

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concentrations of N addition (NH4NO3): 0, 5, 10, or 15 g N m-2 year-1, with 12 replicates of each treatment. Among the 12 replicates of each N addition treatment, one-half were warmed using reinforced-plastic open-top chambers of 1.5 m2 basal area at the center of the plots, which were not included in this study. The experiment was manipulated annually from 2011, and N was added in the middle of the growing season, twice (June and July) a year. More details about the experiment can be found in Liu et al. (2016).

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N labeling

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N labeling was performed at 9:00 am on August 19th, 2017. Of the 6 plots receiving the

same N addition treatment, one-half were selected for labelling with 15NH4NO3 (60.33 atom% 6

Journal Pre-proof 15

N enrichment) in a 0.5×0.5 m subplot, and the remaining with NH415NO3 labelling (60.21

atom% 15N enrichment). In each subplot, a 40 ml solution providing 6.3 mg 15N was spayed uniformly onto the soil surface, and then additional 20 ml water was sprayed to ensure that no 15

N remained on plant stems and leaves. In order to collect the background values of plant N

content and 15N atom% enrichment, we also sprayed 60 ml water in a 0.5×0.5 m subplot separated by 0.5 m from where 15NH4NO3 was labeled in each plot.

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Plant and soil sampling

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In each plot, four soil cores (5 cm diameter, 0-10 cm depth) were randomly collected, sieved

N labeling to ensure plants absorb 15N directly from labeling solution and to minimize

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15

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(2 mm), and stored at 4 °C to measure soil properties. Plant sampling was performed 4 h after

microbial 15N transformation. The whole subplot in 30 cm depth was pulled out after cutting,

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washed gently with tap water to separate plants (especially mixed roots), and then washed

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with distilled water for three times. Nine species with relative abundance ranged from about 1% to over 10% (Table S1) from 5 families and occurring along all four N addition gradients

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were selected as target species to test how their N uptake strategies change in response to N addition, i.e. Anemone rivularis, Anemone trullifolia, Aster diplostephioides, Carex aridula, Kobresia capillifolia, Ligularia virgaurea, Potentilla potaninii, Saussurea stella, Thalictrum alpinum (Table 1). These nine species on average accounted for over 1/3 of the total species richness in a plot (Table S2). We collected as many plant individuals of the 9 species with intact roots as possible, but still could not maintain the same sample size for every species in subplots with 15N labeling. The root length of the collected individuals ranged from 2.32±0.37cm to 8.39±1.28 cm (Table S3). The aboveground and belowground parts of plants were dried at 65 °C for 48h to measure their aboveground and belowground dry mass respectively and then ground them with the mill (Tissuelyser-48, Jingxin company, Shanghai). 7

Journal Pre-proof Plant N content and 15N atom% were measured using a Sercon SL elemental analyzer with the isotope ratio mass spectrometer (Sercon Ltd., Crewe, UK). Soil pH was measured by a pH analyzer (Metter-S210 SevenCompact™, Switzerland) with a soil water ratio of 1: 2. Soil water content was measured by 100 g fresh soil mass after drying at 105 °C for 24 h. Five grams of fresh soil were extracted with 20 ml 2 M KCl solution, shaken for 1 h (70 rev/s), and then a supernatant fluid was used to measure ammonium and nitrate concentrations, using a continuous flow analyzer (AA3, Bran + Luebbe, Germany). Soil organic carbon content

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(SOC) and total N were measured by an element analyzer (Elementar Vario EL III, Hanau,

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Germany), after removing carbonate with 1 M HCl solution and washing to neutral with

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deionized water. Microbial biomass carbon (MBC) and microbial biomass nitrogen (MBN)

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were measured by chloroform fumigation-direct extraction method (Vance et al., 1987). Extract from soil with ammonium acetate was used to measure the concentration of Al3+, Ca2+,

Calculating N uptake

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Fe2+, K+, and Mg2+, using ICP-AES (Thermo icp6300).

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Natural abundance plant δ15N (‰) value is calculated as:

 15 N 

(R s a m pl eR

)

1000

s t a n d a r d

R s tan dard

(1)

where Rsample is the mol ratio of heavy to light isotope of plant, and Rstandard is the mol ratio of atmospheric N2. 15

NU (μg 15N uptake per capita), the amount of 15N recovered from labeled 15N pool, is

calculated as (McKane et al., 2002; Hood‐Nowotny & Knols, 2007):

15

NU  APE 

N  biomass 15 atom%labeled 15+(100%-atomlabeled ) 14

(2)

where APE  atom%labeled  atom%unlabeled is the atom% excess, i.e. the difference between 8

Journal Pre-proof the 15N atom% in labeled plants and control plants; N is plant N content (%); biomass (g) is the per capita average total biomass (i.e., both aboveground and belowground biomass). NU (μg N uptake per capita) is the amount of plant real N uptake, which is calculated as (McKane et al., 2002):

NU 

m unlabeled 15  NU mlabeled

(3)

where munlabeled and mlabeled are the concentrations of corresponding N form in soil and

N

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tracer, respectively.

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Statistical analysis

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Tukey's HSD test followed by one-way ANOVA was used for multiple comparisons (at P < 0.05 level) to detect the differences in plant N content and δ15N values among the 9 species,

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and differences in plant N content, δ15N values, topsoil characteristics (including pH, water

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content, soil organic matter, total nitrogen, carbon to nitrogen ratio (C: N), ammonium, nitrate,

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Al3+, Ca2+, Fe2+, K+, and Mg2+), MBC, and MBN along the N addition gradient. Plant N uptake was lg-transformed to meet the assumption of normality. Preference for ammonium

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and nitrate of the 9 species was examined using Wilcoxon rank-sum test because of the non-normality and unequal sample sizes. Linear models were performed to test the effect of N addition on ammonium and nitrate uptake for each plant species, aboveground biomass, soil characteristics, MBC, and MBN. Liner mixed models were applied to test whether ammonium and nitrate uptake varied significantly across the four N addition levels, or the preference for two chemical N forms varied among the 9 species. To do so, we set the four N addition levels or N labeling forms as the fixed effect, and ~(1|species+1|plot) as random effect, using lmer function in lme4 package. Then, the glht function in the multcomp package was used to conduct ANOVA (Tukey’s HSD test) for multiple comparisons, by evaluating the pair-wise differences at P < 0.05 level. 9

Journal Pre-proof In order to search for the most parsimonious model in explaining ammonium and nitrate uptake, we generated a series of possible linear mixed models based on the information theoretic method, with ~(1|species+1|plot) as a random effect (dredge function in MuMIn package). Factors included the amount of N added, soil available N (ammonium, nitrate, AN), SOC, total N, C: N, pH, Al3+, soil base cation (Ca2+, Mg2+, Fe2+, and K2+), microbial biomass (MBC, and MBN). PCA was performed to facilitate our analyses (rda function in vegan package). The PCA result showed that there were significant correlations between variables

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and the first principal component (PC1) (Table S6). The Pearson correlation test was used to

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avoid setting high correlation predictors in one model (p > 0.9). All factors were put into a

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full model except SOC based on the Pearson correlation test (Table S7). Next, model

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averaging was used to get estimates and relative importance of that parameter when considering models with changes in ΔAIC < 2 (model.avg function in MuMIn package).

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Information-theoretic Akaike’s information criterion corrected for small samples sizes (AICc),

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ΔAIC (difference between AICc of one model and lowest AICc), and AICc weight (wAICc) were calculated for model ranking (Burnham et al., 2011). In all linear models, the percentage

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of deviance explained (De) was used to represent a model’s goodness-of-fit, and the evidence ratio (ER, wAICc [slope model]: wAICc [null model]) was calculated as a support for the slope model if ER>1.5. In all the linear mixed models, marginal and conditional R2 (R2m and R2c) were calculated as the variance explained by only the fixed, and by the both fixed and random effects using function r.squaredGLMM (Nakagawa & Schielzeth, 2013). All the statistical analyses were conducted using R version 3.5.2 (R Development Core Team 2019).

Results Plant N content, δ15N and aboveground biomass N content (%) varied among the 9 plant species. Without N addition (i.e., 0 g m-2 year-1), 10

Journal Pre-proof plant N content ranged from 1.09% (L. virgaurea) to 1.81% (Anemone trullifolia) (Table 1). Under N addition, Anemone trullifolia had the highest N content (2.01%, 4.08%, and 4.75% under N addition of 5, 10, 15 g m-2 year-1, respectively, whereas C. aridula had the lowest (1.05%, 1.45%, and 1.5%, respectively). The 9 species also varied in δ15N (‰) except under N addition of 5 g m-2 year-1. Of all the 9 species, L. virgaurea had the lowest δ15N values. Both N content and δ15N (‰) increased with the amount of N added in 6 out of the 9 species, whereas N content and δ15N (‰) of L. virgaurea, Anemone rivularis, and C. aridula did not

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respond to N addition (Table 1). Plant aboveground biomass exhibited no significant

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difference across four N addition levels among eight species, and only T. alpinum showed a

Soil characteristics, MBC, and MBN

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negative correlation with N addition (ER = 9.032, De = 0.288, Table S4).

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Concentration of ammonium was much higher than that of nitrate in the topsoil across four N

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addition levels (Table 2). Without N addition, the average concentration of ammonium and nitrate was 2.38±0.20 (mg kg-1) and 1.18±0.40 (mg kg-1), respectively. The concentration of

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soil ammonium was 4.8, 9.8, and 18.1 times higher, and soil nitrate was 1.7, 6.2, and 13.9 times higher under N addition of 5, 10, 15 g m-2 year-1 than that in the control plots (0 g m-2 year-1), respectively. The average concentration of ammonium was generally higher than nitrate, although this trend was not statistically significant under N addition of 5 and 10 g m-2 year-1. Nitrogen addition significantly decreased soil pH, water content, C: N, MBC and MBN (Table 2). For instance, the mean soil pH was 6.2 in the control plots, but this value was reduced to 5.8 under N addition of 15 g m-2 year-1. MBN was 235±17.4 mg kg-1 in the control plots, whereas the lowest value of 142±12.1 mg kg-1 was found under the highest N addition of 15 g m-2 year-1. There was no significant change in SOC, total N as well as acid ion (Al3+), 11

Journal Pre-proof and base ions (Ca2+, Fe2+, K+, and Mg2+) with N addition (Table 2).

Effects of N addition on inorganic N acquisition There was no significant difference between ammonium and nitrate uptakes for any of the 9 species and the N addition gradient (Wilcoxon rank-sum test, P > 0.05) (Fig. 1). Nonetheless, the per capita uptake of ammonium was generally higher than that of NO3−-N (Fig. 1). Along the N addition gradient, 7 out of 9 species’ per capita uptake of ammonium increased, while

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the remaining ones decreased with N addition. Of them, 3 species’ (Anemone rivularis, C.

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aridula, and T. alpinum) uptake of ammonium significantly increased with N addition (Fig. 1,

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Table S5). All the 9 species’ per capita uptake of nitrate increased with N addition, with 4

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species (Aster diplostephioides, L. virgaurea, P. potaninii, and T. alpinum) significantly having increased.

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The linear mixed effects model analysis, setting species and plots as random effects,

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showed that nitrate uptake significantly increased with N addition across the 9 species, whereas there was only an increasing trend in ammonium uptake (Fig. 2). With a given

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amount of N added, species generally preferred taking up more ammonium than nitrate, and such a pattern was of significant meaning under N addition of 0 and 15 g m-2 year-1 (Fig. 2). When averaging over all the 9 species and across the N addition gradient, the mean ammonium uptake was significantly higher than nitrate uptake (Fig. 3).

The effects of soil characteristics and MB on plant ammonium and nitrate uptake The result of the model averaging revealed that soil available N (AN) was the only factor that positively contributed to plant ammonium uptake (parameter value = 0.35, Table 3a). Soil C: N and pH possibly influenced ammonium uptake (parameter value = -0.41, and 0.27). Water content (WC), C: N, and AN were three most important factors affecting nitrate uptake. Soil 12

Journal Pre-proof WC and AN positively (parameter values = 0.08 and 0.63), while C: N negatively (parameter values = -0.81), affected nitrate uptake (Table 3a). Of all possible linear mixed models (i.e. setting species and plot as random factors) for explaining ammonium uptake variation, the most parsimonious model consisted of AN (wAICc = 0.292), with pH and C: N ratio supplying little extra explanatory power for the variation in ammonium uptake (ΔAICc < 2) (Table 3b). The model including AN, C: N, and WC was the most parsimonious one for

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nitrate uptake (wAICc = 0.582).

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Discussion

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As N is one major nutrient limiting plant growth in most terrestrial ecosystems, N uptake

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strategy may play a critical role in species coexistence (McKane et al., 2002; Silvertown, 2004; Houlton et al., 2007; Andersen et al., 2013). Here we examined whether species N

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uptake strategy changes after 7 year’s N (NH4NO3) addition using the 15N labeling approach

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in an alpine meadow. We found that instead of specialized on different inorganic N forms, diverse species could track changes in N cycle and exploit the most available form of N in the

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soil with or without N addition. Our findings may have important implications on species diversity maintenance associated with N acquisition.

Plant N uptake strategy along the N addition gradient Depending on species, ammonium and nitrate uptake were positively or neutrally, but never negatively, related to the amount of NH4NO3 added. This produces limited inorganic N niche partitioning along the N addition gradient, which is consistent with previous studies conducted in this region (Song et al., 2015; Zhang et al., 2019). For example, Zhang et al. (2019) also found limited N niche partitioning with or without neighbor competition in 3 species. In another study, although partitioning in N form uptake was observed, only 13

Journal Pre-proof aboveground part under one N addition level was considered (Song et al. 2015). Such plasticity could also be found in a few species at a specific N addition level in our study, however it was rather limited when all the species and all the N addition treatments were considered. Instead of N partitioning along the N addition gradient, all the 9 plant species tended to prefer the most abundant form of N in the soil, i.e., ammonium, which is consistent with some previous studies (Houlton et al., 2007; Andersen et al., 2013; Mayor et al., 2014). A

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species’ ability to adjust to different N resources will determine whether it can successfully

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adapt to environmental change. If species specialize on a particular form of N in the soil with

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little plasticity, any change in N cycle may lead to species extinction, turnover, and thus

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changes in community composition. In contrast, species that can dominate along an environment gradient might be those that capable of adjusting their N acquisition to the most

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abundant N form in the soil. For example, Houlton et al. (2007) showed that a functionally

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diverse group of dominant species in Hawaiian tropical forest are able to switch their N acquisition to mirror changes in soil N forms caused by mean annual precipitation. Here in

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this study, all the 9 species are functionally different species that present along the N addition gradient. We found that these species almost consistently accessed the most abundant form of inorganic N in the soil. Plant growth is costly in the energy use associated with the uptake and assimilation of N. Hence, the strategy that tracking the abundant N forms in their environment is consistent with minimizing the energy cost in resource acquisition from both the physiological and evolutionary perspective (Marie et al., 2009; Wang & Macko, 2011; Boudsocq et al., 2012). Further studies on the fate and transfer of inorganic N in Tibetan alpine meadow under nitrogen addition would provide dynamic changes of N cycling (e.g. Zhang et al., 2017). In this study, a group of functionally diverse species tend to take the strategy for 14

Journal Pre-proof addressing the abundant N forms in their environment rather than diverge in their inorganic N uptake strategy. These findings do not support the chemical niche partitioning mechanism for the maintenance of the high plant diversity in this alpine meadow (Hutchinson, 1961). In contrast, species might

minimize their fitness inequalities by utilizing the abundant N form

in the soil and coexist in the alternative and dynamical way (Chesson, 2000). In this case, the relative abundance of N forms in the soil acts like a sieve to select those species possessed a plastic ability to switch their N acquisition strategy in pace of changes in N cycle in the soil,

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i.e., habitat filtering. Previous studies taking habitat filtering as a mechanism of plant

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community assembly were heavily biased on aboveground traits (e.g. plant height, seed mass,

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leaf N, Lyu et al., 2017). However, belowground traits are vital for nutrient acquisition and

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competition (Ma et al., 2018). This study provided unique evidence in the alpine meadow

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from this perspective.

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The effects of habitat qualities on the uptake of ammonium and nitrate by plants Our study also provides evidence that plant N uptake can be mediated by soil characteristics.

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Of all possible linear mixed effects models constructed to explain variation in either ammonium or nitrate uptake, the most parsimonious one included soil available N. Continuous increase in ammonium and nitrate uptake across the 9 species with N addition suggested that thresholds in the effect of soil available inorganic N supply on plant N absorption did not occur (Niu et al., 2016), even after 7 years of N addition. The increase in plant N uptake may lead to increase in biomass production (Niu et al., 2016). It has been reported that plant N uptake increased on an average by 48%, and aboveground biomass production increased by 29% in response to N addition (LeBauer & Treseder, 2008; Lu et al., 2011). However, in this study, none of the 9 target species’ biomass was positively related to N addition, suggesting that N addition did not necessarily optimize plant aboveground 15

Journal Pre-proof biomass. This may be ascribed to: i) biomass was not only limited by N alone, or may shift to P, or by K limitation (Fujita et al., 2010; Tian et al., 2016; Li et al., 2016); ii)Mechanisms regulating aboveground biomass may shift from belowground nutrients competition (when N is limited) to aboveground competition for light under N addition (Hautier et al., 2009); iii) Changes in community composition (i.e. species turnover) driven by N addition would affect the 9 target species’ biomass (Lepš et al., 2011). One extreme example was Legumes failed to adapt to eutrophication, lost their competitive advantage, and went extinct as a consequence

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(Liu et al., 2017); and iv) soil acidification caused by N addition might suppress plant

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biomass (Fujita et al., 2010; Tian et al., 2016), i.e., species are more resistant to soil

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acidification would have higher biomass; and v) Winter grazing may partially remove the

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reproductive parts of specific plants and thus reduce their biomass production. In addition to the supply of soil enriched available N for plants under N addition, changes

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in other aspects of habitat qualities can also affect plant N uptake. Our study showed that

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high water content and low soil C: N can increase plant nitrate uptake across the 9 species. Plant nitrate uptake increased with water content because of more rapid diffusion of nitrate to

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roots under high water content (Hodge et al., 2000; Boudsocq et al., 2012). The negative correlation between soil C: N and nitrate uptake may be ascribed to a high SOM mineralization rate with low C: N (Niu et al., 2016). Contrary to previous studies (e.g. a long-term N addition in the Inner Mongolian grassland), soil Al3+, Ca2+, Mg2+ and Na+ content did not change with N addition, and pH, Al3+ did not suppress plant N uptake. Their result showed a decreasing trend of soil acidification buffering ions (e.g. Ca2+, Mg2+, Na+), and an increasing trend of phytotoxic metal ions (e.g. Al3+) with N addition (Tian & Niu, 2015; Chen et al., 2016; Ye et al., 2018). That can be ascribed to a higher SOM content and cation exchange capacity in the Qinghai-Tibetan alpine meadow, compared with temperate grasslands, which act to suppress soil acidification (Tian & Niu, 2015). We also found that 16

Journal Pre-proof microbial biomass did not significantly affect plant N uptake. This may because although the demand for the same nutrients can lead to competition between plants and microorganisms, it can be weakened by temporal niche differentiation between them (Kuzyakov & Xu, 2013). Furthermore, soil microorganisms could regulate their N uptake according to C acquisition to maintain stable C:N stoichiometry (Table 1), thus switching N limitation to C limitation under N addition.

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Conclusions

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In summary, we conclude that there is an apparent community-wide trend of tracking the

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most abundant form of inorganic N in the soil after N addition in the alpine meadow. Our

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findings suggest that the fitness equalizing associated to N resource, rather than niche partitioning mechanism, might contribute to the community assembly in the alpine meadow.

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However, investigations with considerable sampling of species and on systems without

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anthropogenic disturbance are needed to assess the generality of our conclusion. Furthermore, we find that plants’ N uptake capability under different levels of N addition is species specific.

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Species differed in their increasing, decreasing, or neutral trend in ammonium and nitrate uptake along the N addition gradient. Plant community composition also changed in response of N addition (Liu et al. 2017). Hence, further studies should assess the relative contributions of these two processes (species turnover and species-specific response) to changes in the community level N uptake under N addition.

Acknowledgements: This work was supported by the National Natural Science Foundation of China (31830009, 31770518 and 41877089). The work was done in the Research Station of Alpine Meadow and Wetland Ecosystems of Lanzhou University. We thank Dexin Sun, Shengman Lyu for helping us perform the experiment. 17

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Authors’ Contribution: SZ designed the experiment. LZ, TBZ conducted experiments, and collected data. LZ, XLX, MN and SZ developed the hypothesis. LZ and XL analyzed the data. LZ, XLX, and SZ wrote the manuscript. All authors approved the final manuscript.

Data accessibility: All data supporting the manuscript are presented, and additional

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ro

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information related is available from the corresponding author if reasonable request.

18

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Journal Pre-proof Supporting Information Table S1: Species richness and relative abundance of the nine species accounted for in one plot. Table S2. Species richness, number of coexisting species out of the nine species within a subplot, and total relative abundance of the nine species under different N addition treatments.

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Table S3. Root length of the 9 species selected for 15N labeling across four N addition level. Table S4. The relationship between the aboveground biomass and N addition (NH4NO3): 0, 5,

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10, and 15 g m-2 year-1.

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Table S5. The relationship between ammonium and nitrate uptake and N addition.

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Table S6. Results of the correlations between the variables and PC1 of three groups (soil available N, soil microorganisms, and soil base cations).

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nitrate uptake, respectively.

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Table S7. Pearson’s correlation matrix for raw input variables in explaining ammonium and

25

Journal Pre-proof Table 1. Basic characteristics of the 9 species selected for 15N labeling. Nitrogen content (%) and δ15N (‰) was of the whole plant (including aboveground part and intact root), and the values were mean ± SE of 3 replicates. Relative abundance of focal species was mean ± SE across 24 plots. Different superscript characters indicate significant differences among all the species under a given N addition, and different subscript characters indicate significant differences along all the levels of N addition of one species.

f o

N content (%)

Species

Family

Life

Dispersal

history

mode

0 g m-2

Asteraceae

n r u

Aster Asteraceae diplostephioides

Saussurea stella

Kobresia capillifolia

Asteraceae

Cyperaceae

l a

Perennial Wind

Perennial Wind

o J

Annual

-p

(δ15N (‰))

e r P

5 g m-2

10 g m-2

15 g m-2

1.21±0.14ab

2.37±0.49ab

1.66±0.65a

(-0.96±0.79a)

(0.1±1.3)

(2.01±0.43a)

(1.27±1.13a)

1.12±0.13aa

1.61±0.06bcda

1.99±0.06aba

3.83±0.38bcb

(2.17±0.69bca)

(2.68±0.51a)

(2.94±0.45aba)

(6.39±0.72bb)

1.43±0.02aba

1.27±0.07aba

3.01±0.26bcb

2.6±0.53abab

(0.39±0.38abcab)

(-0.31±0.25a)

(3.83±0.55abc)

(2.49±0.66abbc)

1.13±0.03aa

1.48±0.04abca

2.68±0.17abcb

2.43±0.35abb

(0.69±0.24abca)

(0.37±0.43a)

(4.02±0.07abb)

(3.97±1.09abb)

1.09±0.08a

Ligularia virgaurea

ro

Wind

Perennial Wind

26

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Potentilla potaninii

Carex aridula

Anemone rivularis

Rosaceae

Poaceae

Ranunculaceae

1.23±0.07aba

1.55±0.17abcda

2.36±0.16abb

2.42±0.22abb

(0.12±0.44aba)

(1.68±0.25b)

(3.87±0.24abc)

(3.52±0.35abc)

1.57±0.11bc

1.05±0.05a

1.45±0.34a

1.4±0.11a

(2.71±0.37c)

(0.31±0.58)

(1.4±0.79a)

(1.64±0.94a)

1.3±0.06ab

1.36±0.14abc

2.01±0.07ab

2.15±0.48ab

(2.57±0.19)

(3.85±0.68ab)

(3.49±0.65ab)

1.88±0.06cdab

2.69±0.32abcbc

2.85±0.21abcc

a

(-0.92±0.67 a)

(-0.1±0.82a)

(3.05±0.36abb)

(3.58±0.39abb)

1.81±0.02ca

2.01±0.12da

4.08±0.49cb

4.75±0.36cb

(0.33±0.09abca)

(0.41±0.54a)

(5.19±1.09bb)

(4.49±0.84abb)

Perennial Unassisted

Perennial Wind

o r p

Perennial Adhensive (2.35±0.49bc)

e

1.58±0.07bca Thalictrum alpinum

Anemone trullifolia

Ranunculaceae

Ranunculaceae

Perennial Adhensive

l a

Perennial Unassisted

n r u

r P

o J

27

f o

Journal Pre-proof Table 2. Soil characteristics, microbial biomass carbon (MBC), and microbial biomass nitrogen (MBN) of topsoil (0-10cm) at different levels of N addition. Soil characteristics include pH, water content (WC), soil organic carbon (SOC), total N, C: N, ammonium, nitrate, Al3+, Ca2+, Fe2+, K+, and Mg2+. Table a: The values are mean ± SE of 6 replicates. Different subscript characters indicate significant differences across the four levels of N addition. Different superscript characters indicate significant differences between ammonium and nitrate at a given level of N addition. Table b: Significant linear relationships (ER>1.5)

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between different variables and N addition. Shown are the intercept, slope,

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information-theoretic Akaike’s information criterion corrected for small samples sizes (AICc),

-p

changes in AICc relative to the null model (ΔAICc), AICc weight (wAICc), evidence ratio

a

5 g N m-2 year-1

10 g N m-2 year-1

15 g N m-2 year-1

6.11±0.03bc

5.98±0.04ab

5.84±0.06a

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0 g N m-2 year-1

re

(ER), and model’s goodness-of-fit (De).

6.16±0.04c

WC (%)

39.20±2.72

39.22±0.53

37.56±0.90

34.50±2.16

SOC (g kg-1)

84.42±6.64

91.12±3.29

78.72±2.12

81.42±5.40

7.16±0.53

7.86±0.27

6.84±0.15

7.11±0.44

11.78±0.11

11.58±0.04

11.50±0.13

11.43±0.12

Ammonium (mg kg-1)

2.38±0.20ab

13.89±4.57ab

25.61±8.73bc

45.35±6.22cb

Nitrate (mg kg-1)

1.18±0.40aa

3.20±0.79ab

8.46±2.23b

17.63±2.71ca

MBC (mg kg-1)

1744.97±130.17c

1720.31±78.85bc 1287.45±130.24ab

1078.26±95.81a

MBN (mg kg-1)

235.34±17.41c

235.20±10.76c

182.31±17.71ab

141.83±12.13a

Al3+ (mg kg-1)

39.03±3.25

33.67±4.38

41.82±2.53

41.21±3.01

Ca2+ (mg kg-1)

4470.72±111.21

4543.72±227.91

4340.22±193.19

4302.22±133.33

C: N

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Total N (g kg-1)

na

pH

28

Journal Pre-proof Fe2+ (mg kg-1)

129.72±9.76

119.06±3.06

131.19±4.31

145.07±14.53

K+ (mg kg-1)

349.83±29.18

360.27±29.21

353.23±14.94

365.23±22.04

Mg2+ (mg kg-1)

325.64±9.13

316.71±22.57

309.72±23.38

302.66±16.17

b Slope

AICc

ΔAICc

wAICc

ER

De

~pH

6.183

-0.022

-34.341

-18.528

1.000

10550.264

0.586

~WC

39.984

-0.315

143.334

-1.393

0.667

2.007

0.154

~C: N

11.743

-0.022

7.677

-2.998

0.817

4.477

0.209

~Ammonium

0.716

2 .812

199.240

-18.521

1.000

10515.710

0.586

~Nitrate

-0.572

1.092

146.687

-22.959

1.000

96698.482

0.656

~MBC

1822.696

-48.660

342.626

-14.905

0.999

1724.520

0.518

~MBN

248.638

-6.663

246.573

1.000

2023.017

0.525

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Intercept

29

-15.225

Journal Pre-proof Table 3. Table a is the summary of the relative importance and parameter values of soil characteristics in determining mean ammonium and nitrate uptake of the 9 plant species. Shown are pH, water content (WC), soil available N (PC1 from ammonium, nitrate, AN), carbon to nitrogen ratio (C: N), total N. Values were derived through a model averaging approach (model.avg function in MuMIn package). Shown are the relative importance with the estimates in the brackets. Factors with parameter values highlighted in bold show a significant difference (P < 0.05). Table b shows the results of linear mixed-effects models

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(setting ~(1|species+1|plot) as random effect) to explain ammonium and nitrate uptake with

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environment qualities respectively. Shown are the number of model parameters (k),

-p

maximum log-likelihood (logLik), information-theoretic Akaike’s information criterion

re

corrected for small samples sizes (AICc), changes in AICc relative to the best model (ΔAICc), AICc weight (wAICc), and the variance explained by only fixed, and both fixed and random

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effects (R2m and R2c).

pH

na

a

0.34 (-0.41)

Nitrate uptake

0.30 (-0.38)

b

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Ammonium uptake

WC

1 (0.08)

AN

C: N

1 (0.35)

0.18 (0.27)

1 (0.63)

1 (-0.81)

k

logLik

AICc

ΔAICc

wAICc

R2m

R2c

~ AN

1

-70.897

152.627

0.000

0.292

0.456

0.688

~ AN+pH

2

-70.077

153.338

0.711

0.205

0.454

0.693

~ AN+C: N

2

-70.712

154.608

1.981

0.108

0.457

0.691

~ AN+C: N+pH

3

-69.887

155.374

2.748

0.074

0.450

0.697

Model Ammonium uptake

30

Journal Pre-proof Nitrate uptake 3

-55.932

127.378

0.000

0.582

0.518

0.692

~ AN+C: N+WC+pH

4

-55.564

129.101

1.723

0.246

0.516

0.689

~ AN+C: N+WC+Total N

4

-57.754

133.481

6.103

0.028

0.516

0.689

Jo ur

na

lP

re

-p

ro

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~ AN+C: N+WC

31

Journal Pre-proof Figure Legends Fig. 1. Mean per capita ammonium, and nitrate uptake (lg-transformed) of the 9 species at different levels of N addition. The lines on the upper right corner of each frame represent significant correlations between ammonium (red line) or nitrate uptake (blue line) and N addition.

Fig. 2. Mean per capita ammonium, and nitrate uptake (lg-transformed) of the 9 species at

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different N addition levels. Different lowercase letters (a/b/c) indicate a significant difference

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between ammonium, and nitrate uptake at a specific level of N addition (linear mixed-effects

-p

models (setting ~(1|species+1|plot) as random effect), at P < 0.05 level). Uppercase letters

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(A/B) denote variations in nitrate uptake with N addition (Tukey’s HSD test for multiple

na

at P < 0.05 level).

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comparisons after linear mixed-effects models (setting ~(1|species+1|plot) as random effect),

Fig. 3. Mean per capita ammonium, and nitrate uptake (lg-transformed) across all the 9

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species along the N addition gradient. Different lowercase letters indicate a significant difference between ammonium and nitrate uptake by the 9 species (linear mixed-effects models (setting ~(1|species+1|plot) as random effect), at P < 0.05 level).

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Fig. 1

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Fig. 2

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Fig. 3

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All authors has no conflict of interest to declare.

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Graphical abstract

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Journal Pre-proof Highlights 

Nine species that all occurred along N addition gradients were selected for in-situ 15N labeling to quantify ammonium and nitrate uptake.

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Plants absorbed more ammonium and nitrate with N addition.

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Plants’ N uptake was influenced by habitat qualities, instead of the amount of N added.

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Inorganic N niche partitioning played limited role in the maintenance of high diversity. Instead, species coexistence may be promoted by minimizing their fitness differences

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through preferring the most abundant form of inorganic N in the soil.

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