Nitrogen addition effects on tree growth and soil properties mediated by soil phosphorus availability and tree species identity

Forest Ecology and Management 449 (2019) 117478

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Nitrogen addition effects on tree growth and soil properties mediated by soil phosphorus availability and tree species identity Qiong Zhao, De-Hui Zeng

T

⁎

CAS Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China Daqinggou Ecological Station, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Nitrogen addition Phosphorus addition Pinus sylvestris var. mongolica Populus simonii Soil microbial biomass Nutrient availability

Increasing evidence has suggested that nitrogen (N) deposition has great effects on forest productivity and nutrient cycling, but how these effects are mediated by soil phosphorus (P) availability and tree species identity remains unclear. Here, a two-year factorial experiment was conducted to examine the short-term effects of N addition (100 kg N ha−1 year−1), P addition (50 kg P ha−1 year−1) and their interactions on tree growth and soil nutrient cycling of a Mongolian pine (Pinus sylvestris var. mongolica) plantation and a neighboring Simon poplar (Populus simonii) plantation in Northeast China. Results showed that N addition promoted tree growth (stem increment), reduced soil microbial biomass carbon (C) and N in the Mongolian pine plantation, but did not affect those in the Simon poplar plantation, and these N addition effects were not interacted by P addition. P addition significantly increased microbial biomass C and N in the Simon poplar plantation. N addition elevated soil phosphatase activities and reduced net N mineralization rate, and these N addition effects were alleviated by simultaneous P addition in both plantations. N addition decreased soil labile inorganic P concentration in both plantations and elevated labile organic P in the Mongolian pine plantation, but only the decrease in labile inorganic P with N addition in the Mongolian pine plantation was counteracted by P addition. N addition significantly reduced soil pH, with a greater extent in the Mongolian pine plantation than in the Simon poplar plantation. Moreover, soil pH response explained most of the microbial and nutrient transformation response. Above results indicated that short-term N addition impacts on tree growth and soil microbial biomass were more strongly influenced by tree species identity than by P addition, while N addition effects on soil N and P transformations were strongly influenced by P addition. Drought resistance of trees and acid buffering capacity of soils are key traits influencing the short-term N addition impacts on the growth of trees and microorganisms in this semiarid region.

1. Introduction Human activities have increased the rate of global atmospheric nitrogen (N) deposition from 31.6 Tg N year−1 in 1860 to 103 Tg N year−1 in early 1990s, with this rate still increasing and being predicted to reach 195 Tg N year−1 in 2050 (Galloway et al., 2004). The great increase in N deposition has serious consequences on ecosystem structure and function worldwide, which will have large feedbacks to future climate change by altering ecosystem carbon (C) balance and nitrogen oxide (NOx) emission (Vitousek et al., 1997; Matson et al., 2002; Pregitzer et al., 2008). In forests, increased N deposition can change both tree growth and soil microbial processes via its direct influences on soil N availability and indirect effects on soil pH, availability of phosphorus (P), calcium and potassium, as well as other soil

properties (Matson et al., 2002; Treseder, 2008). Biotic and abiotic factors interact with N deposition to impact aboveground and belowground processes in forest ecosystems, which make the N deposition impacts vary greatly among studies (Matson et al., 2002; Maes et al., 2019). Soil P availability and tree species identity are two important factors that interact with N deposition to influence forest productivity and ecosystem processes. N and P are the most common limiting nutrient elements in terrestrial ecosystems (Elser et al., 2007; Vitousek et al., 2010). Besides the availability, the balance between soil N and P supply is also important to maintain the biological stoichiometry of plant growth, as all organisms require the nutrient elements in determined proportions for incorporation into biomass and metabolism (Elser et al., 2000; Finzi et al., 2011). N deposition can greatly increase soil N availability, and

⁎ Corresponding author at: CAS Key Laboratory of Forest Ecology and Management, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China. E-mail address: [email protected] (D.-H. Zeng).

https://doi.org/10.1016/j.foreco.2019.117478 Received 27 March 2019; Received in revised form 13 June 2019; Accepted 12 July 2019 0378-1127/ © 2019 Published by Elsevier B.V.

Forest Ecology and Management 449 (2019) 117478

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Simon poplar would respond to N addition more strongly than the Mongolian pine, because of the greater nutrient requirement of trees in the Simon poplar plantation; (2) soil microbial biomass could respond weakly to N and P addition. Soil microbial biomass and activity was limited primarily by C availability in soils with organic C concentration less than 25 g kg−1 (Wardle, 1992; Demoling et al., 2007). Soil organic C in the studied sandy soil was at a very low level (less than 5 g kg−1) (Zhao et al., 2010); (3) N addition would decrease soil available P concentration, because it would elevate the uptake of P by the trees and reduce soil pH in this sandy soil with low acid buffering capacity (Vitousek et al., 2010; Deng et al., 2017). Soil phosphate concentration is at its maximum when pH is 6.5, and the adsorption and precipitation of phosphate with soil minerals increase with both the reduction and raise of soil pH (Parfitt, 1978).

meanwhile alter soil P availability by influencing soil properties such as pH and phosphatase activities, which potentially change the balance between N and P supply (Vitousek et al., 2010; Peñuelas et al., 2013; Deng et al., 2017). Given the coupling between plant N and P nutrition and interactions between soil N and P cycling, impacts of N deposition on tree growth and other ecological processes could largely depend on soil P supply and its balance with N supply. For example, N addition caused an increase in tree growth in many temperate and boreal forests where N is typically a limiting nutrient to net primary productivity, while N addition had a minor effect on tree growth in subtropical and tropical forests whose growth is primarily limited by P (Matson et al., 1999; Tian et al., 2018). Impacts of N deposition on soil microbial biomass and nutrient cycling also varied largely among forests with different N and P limitations (Fanin et al., 2015; Zhou et al., 2017). Despite the increasing recognition of P supply in regulating N deposition effects on forests, the direct test of how soil P addition interact with N deposition to affect tree growth and soil nutrient cycling remains limited. As tree species differ greatly in nutrient requirement and nutrient use strategies, nutrient limitation for tree species grown at the same site can be different, and thus impacts of N deposition on the tree growth and nutrition can be species-specific (Thomas et al., 2010; Vadeboncoeur, 2010). Also, tree species can influence belowground biochemical processes via litter decomposition and rhizosphere effects (Binkley, 1995; Gobran et al., 1998; Cornwell et al., 2010), leading to species-specific belowground responses to N addition, such as soil N transformations (Weand et al., 2010), C storage, enzyme activities and microbial community composition (Christenson et al., 2009; Weand et al., 2010; Lovett et al., 2013). The species-specific responses to N deposition are likely to be important in determining the structure and function of forests, and the feedbacks of N deposition to future climate. Although some plant traits such as mycorrhizal associations and litter chemistry can partly explain the inter-specific variations in the responses of ecosystem processes to N deposition, the N deposition effects on forests at the species level is still unpredictable, and more work is needed (Lovett et al., 2013; Midgley and Phillips, 2016). The main goal of this study was to examine the impacts of shortterm N addition on key plant and soil properties involved in N and P cycling in nutrient-poor forest plantations, and more importantly, how these impacts were mediated by P availability and varied among tree species. To achieve our goal, we conducted a factorial N and P addition experiment in adjacent pure Mongolian pine (Pinus sylvestris var. mongolica) and Simon poplar (Populus simonii) plantations established on a sandy soil deficient in both N and P (Zhao et al., 2009). We compared the responses of stem growth, foliar nutrient concentrations, soil N and P availability and mineralization, as well as microbial biomass to the N and P additions between the two plantations. Mongolian pine and Simon poplar are two of the most widely planted tree species for soil conservation and windbreak in semi-arid regions of Northern China. Both tree species are highly adapted to the nutrient-poor soil conditions, but exhibiting contrasting nutrient cycling rate and characteristics due to their differences in growth rate, leaf habit, nutrient requirement, litter decomposability, and rhizosphere effects on microbial biomass and nutrient transformations (Jiao, 1989; Zhao et al., 2010; Zhao et al., 2013). Mongolian pine is an evergreen conifer and Simon poplar is a broad-leaved deciduous tree species, and nutrient cycling is faster in the poplar plantation than in the pine plantation, because of the great nutrient uptake by trees and easily decomposable leaf litter (Zhao et al., 2010; Zhao et al., 2013). In 15 years old Mongolian pine and Simon poplar plantations adjacent to our study site and with similar stand density to those in the present study, the mean annual N uptake were 16.9 kg ha−1 year−1 and 20.8 kg ha−1 year−1 (Hu et al., 2009). We hypothesized that: (1) N addition would promote the growth of both tree species, and N addition effects on tree growth could be stimulated by simultaneous P addition, because the sandy soil at the study site is deficient in both available N and P (Zhao et al., 2009).

2. Materials and methods 2.1. Study site This study was conducted at Daqinggou Ecological Station (42°58′N, 122°21′E, 260 m altitude) of the Institute of Applied Ecology, Chinese Academy of Sciences, which is located on the southeastern edge of Keerqin Sandy Land in Northeast China. The climate in this area is temperate semiarid, with annual mean temperature of 6.4 °C and mean annual precipitation of 450 mm. The lowest and highest monthly mean temperature in the study area occurs in January (−12.5 °C) and July (23.8 °C), and more than 60% of precipitation falls from June to August, causing the cold-dry winter and warm-humid summer. The soil is developed from an eolian deposit, with 90.9% sand, 5.0% silt, and 4.1% clay. This poor sandy soil is particularly deficient in N and P, with the organic C, total N and total P concentrations of 3.0 g kg−1, 0.30 g kg−1 and 0.15 g kg−1 at 0–30 cm layer, respectively. The wet N deposition at the study site was up to 18.8 kg ha−1 year−1 in the growing season of 2017 (Song et al., 2019). 2.2. N and P addition experiment A 14-year-old pure Mongolian pine plantation and a 12-year-old Simon poplar plantation were selected for the fertilization experiment. Both plantations were established on degraded grassland on flat topography, sharing the same soil type, and are located within a 1-km radius of each other. The spacing of the plantation is 3 m × 3 m and 2 m × 2 m for Mongolian pine and Simon poplar, respectively. The diameter at breast height at the beginning of the fertilization experiments was 4.96 cm and 10.79 cm for the Mongolian pine and Simon poplar, respectively. The N and P addition experiment was based on a two-factor full factorial design with three replicates, including four treatments: (1) Control, (2) addition of 100 kg N ha−1 year−1 (+N), (3) addition of 50 kg P ha−1 year−1 (+P), and (4) co-addition of N and P in quantities as in +N and +P treatments (+NP). In each stand, 12 plots (20 m × 20 m) separated by at least 10 m from each other were established, and the replicates of all treatments were randomly assigned to the established plots. N was added as ammonium nitrate and P was added as calcium dihydrogen phosphate. Since May 2012, N and P compounds were broadcasted by hand evenly throughout the entire plot monthly from May to September. 2.3. Field sampling Leaf and soil samples were collected in August 2014, after approximately two years of fertilization. Current-year, fully expanded green leaves were taken with a pole pruner on the top third of the canopy of four different directions. Four individual trees were sampled per plot, and leaves in each plot were combined into one composite sample. Leaf materials were ground in a mill to pass through a 0.25-mm 2

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5

Diameter increament (cm)

mesh after being dried at 70 °C to constant weight, and then analyzed for total N and P concentrations. Twelve soil cores (6 cm in diameter) of 0–10 cm layer were collected randomly from each plot and then mixed into one composite sample. Field moist soil samples were sieved to pass a 2-mm mesh and then were thoroughly mixed and divided into two subsamples. One subsample was stored at 4 °C for measuring acid phosphatase activities (APA), NaHCO3 extractable inorganic and organic P, NO−3 –N, NH+4 –N, microbial biomass C, N and P. Another subsample was air-dried for determination of soil pH. Additionally, stem diameter at breast height (1.3 m; DBH) was measured with a DBH tape in November every year for all trees of each plot. A stainless ring with a spring was installed at the breast height of each tree to mark the location of measuring DBH.

Control

+N

+P

N: ** P: ns N P: ns

4

+NP

N: ns P: ns N P: ns

3 2 1 0

2.4. Laboratory analyses

Pine

Poplar Tree speices

Soil pH was measured with a glass electrode (1:2.5 soil:water ratio). For the measurement of NaHCO3 extractable inorganic and organic P (NaHCO3-Pi and NaHCO3-Po), fresh soils were extracted with 0.5 mol L−1 NaHCO3 at pH = 8.5, and the extract was analyzed for inorganic P and total P (digested with H2SO4) colorimetrically using the molybdate blue method. The NaHCO3-Po was calculated as the difference between total P and inorganic P in the NaHCO3 extract (Bowmman and Cole, 1978). Soil APA was assayed by the method described by Tabatabai (1994) using disodium p-nitrophenyl phosphate (pNPP) as the substrate at pH = 6.5, and expressed as µg pNP g−1 h−1. Soil NO−3 –N and NH+4 –N concentrations were analyzed colorimetrically on an autoanalyzer (AutoAnalyzer III, Bran+Luebbe GmbH, Germany) after the soil was extracted with 2 mol L−1 KCl solution. To measure potential net N mineralization, 10 g of field moist soil was incubated in a cup at 25 °C in dark for 30 days, and the moisture was kept constant by adding distilled water every two days. The difference in inorganic N (the sum of NO−3 –N and NH+4 –N) pool before and after the incubation was used to estimate the rate of potential net N mineralization over the incubation period. Soil microbial biomass C, N and P concentrations (Cmic, Nmic and Pmic) were determined by the CHCl3 fumigation-extraction procedure (Brookes et al., 1982, 1985; Vance et al., 1987). The unfumigated and fumigated soil samples were extracted with 0.5 mol L−1 K2SO4 for Cmic and Nmic, and extracted with 0.5 mol L−1 NaHCO3 for Pmic. Organic C in K2SO4 extracts was determined by K2Cr2O7–H2SO4 oxidation method. Total N in K2SO4 extracts was determined colorimetrically on an autoanalyzer after alkaline persulfate oxidation (Cabrera and Beare, 1993). Phosphate in the NaHCO3 extracts was measured colorimetrically using the molybdate blue method. A spike of KH2PO4 equivalent to 25 µg P g−1 soil was used to correct for inorganic P fixation during the NaHCO3 extraction (Brookes et al., 1982). Correction factors of 0.38, 0.45 and 0.40 were applied to estimate the recoveries of Cmic, Nmic and Pmic during fumigation, respectively (Brookes et al., 1982; Jenkinson et al., 2004). All leaf and soil data were expressed on an oven dry weight basis.

Fig. 1. Stem diameter increment of the Mongolian pine and Simon poplar after 2 years of N and P additions (mean ± SE, n = 3). ** denotes significant level at p < 0.01 and ns denotes non-significant (p > 0.05) effect within each tree species (following the general linear model of two-way ANOVA).

significant level was set at 0.05. ANOVAs were conducted separately for the pine plantation and poplar plantation, and were performed using the SPSS statistical software package version 11.5 (SPSS Inc., 2002). To analyze the relationships among soil variables, we employed principal component analysis (PCA) of all soil variables using CANOCO 4.5 software. Data were ln (x + 1) transformed prior to PCA analysis. 3. Results 3.1. Tree growth and foliar nutrient concentrations There were no N × P interactions on the stem growth of both pine and poplar, and the main effects of N and P additions differed between the two species. Stem diameter increment (SDI) of Mongolian pine increased significantly by 41% with N addition compared to the control, but was not influenced by P addition. In contrast, SDI of poplar was neither affected by N addition nor by P addition (Fig. 1). Foliar N and P concentrations and N/P ratio responded similarly to N and P additions in the pine and poplar plantations. P addition significantly increased foliar N concentration in both plantations, while there were no significant treatment effects on foliar P concentration and N/P ratio (Table 1). 3.2. Soil P and N transformations N addition significantly reduced soil NaHCO3-Pi concentration in both plantations, but the magnitude of the effects and the N × P interactions depended on tree species (Fig. 2A). There were significant N × P interactions on NaHCO3-Pi concentration in the pine plantation, because NaHCO3-Pi concentration was 52% lower in the +N plots than that in the control plots (P < 0.001, LSD post-hoc test, one-way ANOVA), but did not differ between +P and +NP plots (P = 0.015, Fig. 2A). In the poplar plantation, no N × P interaction on NaHCO3-Pi concentration was observed, and NaHCO3-Pi concentration was 20% lower in the plots with N addition (+N and +NP) than in the plots without N addition (control and +P) (Fig. 2A). There were no N × P interactions on soil NaHCO3-Po concentration in both plantations, and N addition significantly increased soil NaHCO3-Po concentration by 61% in the pine plantation but not in the poplar plantation (Fig. 2B). Soil NaHCO3-Po concentration also significantly increased with P addition in both plantations, by 24% increase in the pine plantation and 52% increase in the poplar plantation (Fig. 2B). N and P additions interacted to influence soil APA in both plantations, and the effects differed between the two plantations (Fig. 2C). In the pine plantation, soil

2.5. Statistical analyses Firstly, we conducted two-way analysis of variance (ANOVA) using General Linear Model procedures to test for the main effects of N and P addition and their interactions on tree and soil variables. One-way ANOVA with LSD post-hoc tests was then conducted to compare the differences in all variables among four treatments, when there were significant N × P interactions. Our study focused on the N addition effects and how they were interacted by simultaneous P addition, so we described the N × P interactions by comparing the N addition effects when N was added alone and when N was added together with P. Data were tested for normality and homogeneity of error variances prior to ANOVAs, and were ln (x + 1) or square-root transformed to achieve normality and homogeneity of variances when necessary. Statistically 3

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Table 1 Foliar N and P concentrations and N/P ratio of Mongolian pine and Simon poplar after 2 years of N and P additions. Mongolian pine N (g kg Control +N +P +NP

12.4 12.5 12.9 13.5

Simon poplar

−1

)

P (g kg

(0.11) (0.08) (0.25) (0.04)

1.17 1.06 1.24 1.16

P values of two-way ANOVA N 0.075 P 0.001 N×P 0.176

−1

)

(0.08) (0.10) (0.13) (0.12)

0.403 0.473 0.880

N/P

N (g kg−1)

P (g kg−1)

N/P

10.7 (0.64) 12.0 (1.14) 9.7 (1.28) 11.9 (1.29)

13.2 13.4 13.6 13.6

1.04 0.99 1.14 1.20

12.8 13.5 12.0 12.1

0.164 0.654 0.742

0.477 0.026 0.648

(0.02) (0.20) (0.06) (0.03)

(0.04) (0.04) (0.09) (0.23)

0.113 0.545 0.321

(0.44) (0.36) (0.94) (1.97)

0.065 0.921 0.324

Notes: Values are means with SE in the parentheses (n = 3). Significant P values (P < 0.05) are highlighted in bold.

were much stronger in the pine plantation than in the poplar plantation. The concentration of NH4+-N was 16 times higher in the +N and +NP plots than in the control and +P plots in the pine plantation, while it was only 4 times higher in the poplar plantation (Fig. 2D). Similarly, NO3−-N concentration was 6.9 and 1.8 times higher in the +N and +NP plots than in the control and +P plots in the pine and poplar plantation, respectively (Fig. 2E). N and P additions interacted to affect

APA did not differ between +N and control plots, but was significantly lower in the +NP plots than in the +P plots. In the poplar plantation, soil APA was 75% higher in the +N plots than in the control plots, but did not differ between +NP and +P plots (Fig. 2C). Soil NH4+-N and NO3−-N concentrations were enhanced greatly by N addition, but not affected by P addition and N × P interactions in both plantations (Fig. 2D and E). In addition, the N addition effects

Control

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N: ns P: ** N P: ns

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NaHCO3-Po (mg kg )

+N

20

Poplar

b

15 10

a a

5 0

Pine

c

Pine

Poplar Tree species

Tree species

Fig. 2. Soil available P and N concentrations, acid phosphatase activities (APA) and potential net N mineralization rate (Nmin) in the Mongolian pine and Simon poplar plantations after 2 years of N and P additions (mean ± SE, n = 3). ** and * denote significant levels at p < 0.01 and p < 0.05 respectively, ns denotes nonsignificant (p > 0.05) effects, within each tree species (following the general linear model of two-way ANOVA). Treatments with different letters are significantly different within each tree species (p < 0.05, LSD post-hoc test following a one-way ANOVA). 4

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-1

Cmic (mg kg )

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250 200

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+N

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c

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30 20

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N:* P:* N P: ns

D

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Pmic (mg kg )

40

0 15

Pine

Tree species

Poplar

Tree species

Fig. 3. Soil microbial biomass C, N, P and the ratio of microbial biomass C to N in the Mongolian pine and Simon poplar plantations after 2 years of N and P additions (mean ± SE, n = 3). ** and * denote significant levels at p < 0.01 and p < 0.05 respectively, ns denotes non-significant (p > 0.05) effects, within each tree species (following the general linear model of two-way ANOVA). Treatments with different letter are significantly different within each tree species (p < 0.05, LSD post-hoc test following a one-way ANOVA).

10

potential net N mineralization rates in both plantations, also with stronger effects in the pine plantation than in the poplar plantation (Fig. 2F). Specifically, in the pine plantation, potential net N mineralization rate was 74% lower in the +N plots than in the control plots, but was only 40% lower in the +NP plots than +P plots. In the poplar plantation, N mineralization rate did not differ between the +N and control plots, but was significantly higher in the +NP plots than in the +P plots (Fig. 2F).

Soil pH

8

Control

N:** P: ns N P: ns

+N

N: ns P: * N P: **

+P

a

+NP

b

a

a

6 3.3. Soil microbial biomass and pH Soil Cmic and Nmic responded similarly to fertilization, but the responses differed obviously between the two plantations (Fig. 3). In the pine plantation, soil Cmic and Nmic were significantly reduced by N addition, but not affected by P addition and N × P interactions. In the poplar plantation, Cmic and Nmic were not affected by N addition but increased significantly with P addition, with significant N × P interactions for Cmic (Fig. 3A and C). Soil Pmic responded to N and P additions similarly in both plantations. It increased sharply with P addition by 4.6–10.8 times and decreased slightly with N addition, with no N × P interactions (Fig. 3B). The microbial biomass C/N ratio increased significantly with both N and P additions in the pine plantation with no N × P interactions, but was not affected by any treatment in the poplar plantation (Fig. 3D). N addition reduced soil pH in both plantations, with significant N × P interactions in the poplar plantation (Fig. 4). In the pine plantation, soil pH decreased by one unit with N addition; in the poplar plantation pH decreased by 0.5 unit in the +N plots relative to the control plots, but did not differ between the +NP and +P plots (Fig. 4). P addition alone did not influence soil pH in both plantations.

4

Poplar

Pine Tree species

Fig. 4. Soil pH in the 0–10 cm layer of Mongolian pine and Simon poplar plantations after 2 years of N and P additions (mean ± SE, n = 3). ** and * denote significant levels at p < 0.01 and p < 0.05 respectively, ns denotes non-significant (p > 0.05) effects, within each tree species (following the general linear model of two-way ANOVA). Treatments with different letter are significantly different within each tree species (p < 0.05, LSD post-hoc test following a one-way ANOVA).

among soil variables (Fig. 5). The first principal component (PC1) explained 55.8% of the total variance, and mainly represented the variation related to NaHCO3-Pi. The second principal component (PC2) explained 29.2% of total variance, and represented the variation associated with inorganic N (Fig. 5). The PC1 was significantly correlated with NaHCO3-Pi concentration and Pmic (P < 0.001), and marginally correlated with NaHCO3-Po concentration (P = 0.088). The PC2 was highly correlated with pH, NaHCO3-Po concentration, inorganic N concentration, N mineralization rate, Cmic, Nmic and the ratio of Cmic/ Nmic (P ≤ 0.01). The ordination diagram also confirmed that N addition

3.4. Correlations among soil variables The ordination diagram from PCA clearly showed the relationships 5

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important plant trait influencing N deposition effects on tree growth in the semiarid region. Soil microbial biomass responded to N addition differently from the tree growth, and also differently between the Mongolian pine and Simon poplar plantations. Inconsistent with our hypothesis that soil microorganisms could respond weakly to N and P addition, N addition decreased microbial biomass C in the Mongolian plantation, but not in the Simon poplar plantation (Fig. 3). These results suggest that high dose of N addition suppressed microbial biomass at the study site. Reduction in soil microbial biomass with N addition was observed in many ecosystems around the world, and the reduction become stronger with increasing duration and rate of N addition (Treseder, 2008). Excess N addition can inhibit microbial growth and activity directly by the toxic effects of too much ions on osmotic potentials, and indirectly via decreasing soil pH (Treseder, 2008). Although we did not investigate the mechanisms underlying the reduction in microbial biomass, high correlations between soil microbial biomass C, soil pH (r = 0.79, P < 0.01) and inorganic N (r = −0.59, P < 0.01) suggest reduction in soil pH could be largely responsible for the depressed soil microbial biomass. Soil acidification with N inputs was reported in numerous studies, in particular with high N addition rates and on soils with low cation exchange capacity (Vitousek et al., 1997; Tian and Niu, 2015). N addition can result in reduction of soil pH via ammonia volatilization, nitrification, nitrate leaching, unbalanced plant uptake and immobilization of NH4+ and NO3− (Matson et al., 2002; Gundersen et al., 2006; Lucas et al., 2011). Elevated nitrification and nitrate leaching were considered as the main reason for N addition induced soil acidification (Bergkvist and Folkeson, 1992). However, nitrate leaching is very low (about 60 mg m−2 year−1) and even lower than the ammonia volatilization at our study site, so ammonia volatilization may play a nonnegligible role in the soil acidification in semi-arid regions (Zhang et al., 2011). In the present study, P addition significantly improved soil microbial biomass C and N in the Simon poplar plantation, suggesting the Plimitation of microbial biomass. Non-significant negative effects of N addition on soil microbial biomass C in the poplar plantation could be attributed to the much less response of available N and soil pH to N addition (Figs. 2 and 4). Also, the root activities could regulate the N deposition effects on soil microbial biomass. Simon poplar exhibits stronger positive rhizosphere effects on microbial biomass C than Mongolian pine (Zhao et al., 2010), which may partly counteract the negative effects of N addition on soil microbial biomass. Above results suggest that tree species can mediate the responses of soil microbial biomass to N addition mainly by their differences in buffering soil acidification. The decrease in microbial biomass under N addition may have consequences for C fluxes, given that soil CO2 emissions declined in concert with decreases in microbial biomass. Soil microbial biomass N and P were controlled by different soil factors and thus responded differently to N and P additions at our study site. Microbial biomass N was highly correlated with microbial biomass C (r = 0.91, P < 0.01), and varied with N and P additions in the similar pattern to the microbial biomass C (Figs. 3 and 5), reflecting the close coupling of microbial C and N demands. The close coupling of microbial C and N has been indicated by the relatively consistent microbial biomass C:N ratios across biomes (typically varying between 8:1 and 12:1 on a mass basis) (Paul and Clark, 1996; Cleveland and Liptzin, 2007). Besides, soil organic C and N are stabilized together and C and N mineralization are coupled processes (McGill and Cole, 1981). In contrast, microbial biomass P was highly correlated with soil available P (r = 0.96, P < 0.01) but not with microbial C and N, and it responded to N and P additions in a quite different pattern from microbial biomass C and N (Figs. 3 and 5). Greatly increased microbial biomass P with P addition can be ascribed to the luxury absorption of P by microorganisms, a phenomenon that has been found in some inorganic P addition experiments (Enwezor, 1966; Malik et al., 2012). Variations in soil microbial biomass C/N ratio are commonly related

Fig. 5. Ordination diagram with variables and samples from principal component analysis (PCA) of soil variables across the Mongolian pine and Simon poplar plantations. Each symbol represents a sample. Lines with arrows represent soil variables. The smaller angle between two variable arrows indicates stronger correlation, as the cosine of the angle between variable arrows equals their correlation coefficients.

effects on the measured variables were stronger in the pine plantation than in the poplar plantation, as indicated by the longer distance between sample points of the treatments with N addition (+N and +NP) and treatments without N addition (control and +P treatments) in the pine plantation (Fig. 5).

4. Discussion 4.1. Species-specific responses of tree and soil properties to N addition We hypothesized that N addition would promote the growth of both species and with stronger impacts on the Simon poplar, considering the greater nutrient requirement of trees in the Simon poplar plantation than in the Mongolian pine plantation. Inconsistent with this hypothesis, N addition only promoted the stem growth of Mongolian pine in the present study. No effect of N and P additions on the stem growth of Simon poplar is likely not because N and P are not the limiting factors, but is the result of other stronger limiting resource. In this semi-arid region, the primarily limiting factor to the growth of Simon poplar is probably soil water availability rather than N availability. Although both tree species are highly adapted to the nutrient-poor soil conditions and semi-arid climate, the slow-growing Mongolian pine is more tolerant to the drought stress than the fast-growing Simon poplar (Li, 1994). The foliar transpiration rate of Simon poplar (2.02 mg g−1 min−1) is more than twice of that of Mongolian pine (0.92 mg g−1 min−1) in our study region (Jiao, 1989). Furthermore, the irrigation and N addition experiments on Mongolian pine and poplar seedlings showed that N addition alone had stronger effects on the seedling growth than irrigation alone for Mongolian pine, while irrigation alone exerted great influence on the growth and N addition alone did not affect the growth of poplar seedling (Wang et al., 2004; Deng, 2006). These results suggest that drought resistance ability is an 6

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rhizosphere effects on phosphatase activities (27–60%) (Zhao et al., 2010). At the same time, the P use efficiency is high for both species as indicated by the high P resorption efficiency (44–60%) (Chen et al., 2004). In addition, N addition significantly elevated labile organic P concentration while reducing labile inorganic P concentration (Fig. 2), which may alleviate the P deficiency to some extent. The strong ability of Mongolian pine and Simon poplar to mobilize soil recalcitrant organic P could be related to their ectomycorrhizal associations. Mongolian pine is an ectomycorrhizal fungi-dependent species, and the colonization rate was about 75% at our study site. Simon poplar is still mainly associated with ectomycorrhizal fungi at our study site, and the colonization rate was about 50% (Data not shown). Ectomycorrhizal fungi can not only produce hydrolytic extracellular enzymes to mineralize P-bearing inositol phosphates, but also can weather minerals by releasing low-molecular-weight organic chelators and hydrogen ions to increase P availability (van Breemen et al., 2000; Taylor et al., 2009). Similar to the tree growth, N addition effects on soil microbial biomass was also not influenced by P addition, reflecting that the suppressed microbial growth by high doses of N addition was not caused by the N addition induced reduction in available inorganic P in this short-term experiment. Although short-term N addition impacts on tree growth and microbial biomass were not interacted by P addition, simultaneous P addition significantly alleviated both the positive N addition effects on soil P mineralization (indicated as phosphatase) and negative N addition effects on net N mineralization in the present study, suggesting that N and P transformations interact through soil biochemical processes. P addition was found to increase net N mineralization in many Australian P-limited or N and P co-limited forests (Falkiner et al., 1993). However, non-significant effects of P addition on net N mineralization were observed in P-limited tropical forests in China (Wang et al., 2014). Clear mechanisms underlying the effects of P addition on soil N transformation remain poorly understood, though P addition effects on protease enzyme production, microbial community, and organic matter solubility were thought to be the possible mechanisms (Falkiner et al., 1993; Wang et al., 2014). In the present study, P addition decreased net N mineralization rate, but alleviated the suppression of net N mineralization by N addition, which could be related to the concomitant variation in soil pH and microbial biomass. As discussed above, soil pH and microbial properties were key factors influencing N mineralization, which were positively correlated with soil net N mineralization (Fig. 5). As an inducible enzyme, soil phosphatase activities are driven by the deficiency of inorganic P and repressed by the excess of inorganic P (Nannipieri, 2011). Great elevation in soil phosphatase activity with N addition was alleviated by P addition in the poplar plantation, suggesting that aggravation of microbial P limitation with N addition was alleviated by P addition.

to shifts in microbial community composition (bacteria vs. fungi), since the fungi have higher C:N ratios than the bacteria (Cleveland and Liptzin, 2007). Different responses of microbial biomass C/N ratio to N and P additions in the two studied plantations reflect the different resistance of soil microbial community composition to N and P additions (Fig. 3). The increase in microbial C/N ratio with the N and P addition in the Mongolian pine plantation was likely caused by a shift towards a more fungal-dominated microbial community. In contrast, soil microbial community composition kept unchanged with N and P additions in the Simon poplar plantation. Although decrease in microbial biomass C/N ratio and fungi/bacteria was found in many N addition experiments (Zhou et al., 2017), increase in C/N ratio or fungi/bacteria ratio in forests were also reported (Bejarano et al., 2014; Fanin et al., 2015; Treseder, 2008). Soil pH is one of the most powerful determinants of the microbial community composition. Increased fungi/bacteria ratio in the present study could be related to the reduced soil pH with N addition because fungi are more acid tolerant than bacteria (Rousk et al., 2011). Impacts of N addition on soil N and P mineralization and availability were generally stronger in the pine plantation than in the poplar plantation. N addition can affect soil net N mineralization via altering soil N availability, pH, microbial properties and enzyme activities. Thus, soil net N mineralization can increase with N addition as a result of increased quality and quantity of substrates, microbial activity or the priming effect. Also, N mineralization can be inhibited by decreased soil pH with N addition (Nave et al., 2009; Cheng et al., 2019). Much greater reduction in potential net N mineralization rate in the Mongolian pine plantation than in the poplar plantations was probably due to the greater decreases in soil pH and microbial biomass with N addition in the Mongolian pine plantation, considering the close correlations between net N mineralization and pH, microbial biomass C (Fig. 4). N addition can influence soil phosphatase activity and P availability both positively and negatively, via several interactive pathways, such as by altering N supply, microbial activity, pH, and P supply, generating widely varied net N addition impacts in different cases (Marklein and Houlton, 2012; Deng et al., 2017). In the present study, greater reduction in soil bicarbonate extractable inorganic P with N addition in the Mongolian pine plantation than in the Simon poplar plantation could be largely related to the greater decrease in soil pH in the pine plantation. Soil pH is the primary abiotic factor determining soil P availability, and soil inorganic phosphate reaches its maximum at pH 6.5 and decreases with either increase or decrease of soil pH (Parfitt, 1978). Variation in soil phosphatase activities is widely used to indicate the P limitation because the phosphatase production is stimulated by the biotic demand for P (McGill and Cole, 1981; Olander and Vitousek, 2000). Greatly elevated soil acid phosphatase with N addition in the Simon poplar could be related to the aggravated P limitation of microbial biomass with N addition in this plantation.

5. Conclusions 4.2. Interactive effects of N and P additions In this short-term experiment with high doses of N and P additions, both tree growth and soil microbial biomass responded differently to N and P additions between the Mongolian pine and Simon poplar plantations, reflecting differential resource limitation of the trees and soil microorganisms in these plantations. Non-significant effect of N and P additions on the stem growth of Simon poplar is likely because of the stronger water limitation. Therefore, drought resistance ability of tree species and interaction with variation in precipitation should be taken into account when studying N deposition impacts on forest productivity in semi-arid regions. Although the soil at the study site is deficient in both available N and P, P addition did not affect the N addition impacts on the tree growth and soil microorganisms, reflecting the strong ability of the tree species and microorganisms to acquire and utilize P. However. P addition exerted great influences on the N addition effects on soil N and P mineralization and P availability, suggesting the close coupling of soil N and P transformations through soil biochemical

One of the aims of the present study was to examine how the impacts of N addition were mediated by P supply. Inconsistent with our hypothesis that the N addition effects on tree growth would be stimulated by simultaneous P addition, P addition did not affect the responses of stem growth to N addition for both tree species. These results could be because P is not the primary limiting or co-limiting nutrient element for both species. As discussed above, growth of Mongolian pine was primarily limited by N and Simon poplar was primarily limited by water availability. Even N was added at the rate of about five times of ambient N deposition and decreased soil available P greatly in the present study, two-years of N addition still did not intensify the P limitation, indicating the strong ability of these two species to acquire and utilize P on this nutrient-poor sandy soil with available inorganic P less than 3 mg kg−1. The both tree species have strong ability to increase mineralization of soil organic P as indicated by their positive 7

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processes. Soil acidification caused by N addition and the different acidification degrees between the two plantations largely explained the soil microbial and nutrient transformation responses to N addition. Despite the N addition rate was about 5 times of the ambient N deposition and the fertilization only last for two years in the present study, which is impossible to obtain the same results as the real N deposition effects, our results highlight the importance of P supply and tree species in regulating the N deposition effects on forest productivity and nutrient transformation, and reveal the role of soil acidification and the type of nutrient limitation (N vs. P) in determining the influence of P supply on the N addition effects. Measures to elevate soil buffering capacity can be taken to alleviate the negative impacts of the N deposition on soil microorganisms and P availability.

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