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Effects of P addition on plant C:N:P stoichiometry in an N-limited temperate wetland of Northeast China
Science of the Total Environment 559 (2016) 1–6
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Effects of P addition on plant C:N:P stoichiometry in an N-limited temperate wetland of Northeast China Rong Mao a,b,⁎, Hui-Min Chen a,c, Xin-Hou Zhang a, Fu-Xi Shi a, Chang-Chun Song a,⁎ a b c
Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China The Three Gorges Institute of Ecological Environment, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China University of Chinese Academy of Sciences, Beijing 100049, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Effects of P enrichment on plant C:N:P stoichiometry at different scales are unclear. • Species- and community-level plant stoichiometry responded similarly to P addition. • Six years of P addition increased plant N and P concentrations. • Six years of P addition decreased plant C:N, C:P, and N:P ratios. • Altered C:N:P stoichiometry following P enrichment would accelerate nutrient cycles.
a r t i c l e
i n f o
Article history: Received 23 December 2015 Received in revised form 22 March 2016 Accepted 22 March 2016 Available online xxxx Editor: D. Barcelo Keywords: C:N:P stoichiometric ratios Deyeuxia angustifolia Eutrophication Freshwater marsh Glyceria spiculosa Species dominance
a b s t r a c t . Phosphorus (P) enrichment induced by anthropogenic activities results in modified plant nutrient status, which potentially alters the stoichiometry of carbon (C), nitrogen (N), and P in plants. However, how increased P availability changes plant C:N:P stoichiometry at different hierarchical scales is unclear in N-limited ecosystems. In this study, we conducted a four-level P addition experiment (0, 1.2, 4.8, and 9.6 g P m−2 year−1) to elucidate the effect of P enrichment on plant C:N:P stoichiometric ratios at both the species and community levels in a freshwater wetland in the Sanjiang Plain, Northeast China. We found that species- and community-level plant C:N:P stoichiometry responded consistently to six years of P addition, although there was a shift in species dominance. Phosphorus addition increased plant N and P concentrations and thus decreased C:N, C:P, and N:P ratios irrespective of the P addition levels. These similar change trends at different scales resulted from the identical responses of plant N and P concentrations in different species to P addition. Moreover, plant N concentration exhibited an increasing trend with increasing P addition levels, whereas plant C:N ratio showed a declining trend. At the community level, P addition at the rates of 1.2, 4.8, and 9.6 g P m−2 year−1 decreased the C:N ratio by 24%, 27%, and 34%; decreased the C:P ratio by 33%, 35%, and 38%; and decreased the N:P ratio by 12%, 10%, and 6%, respectively. Our results indicate that the stoichiometric responses to P addition are scaleindependent, and suggest that altered plant C:N:P stoichiometry induced by P enrichment would stimulate organic matter decomposition and accelerate nutrient cycles in N-limited temperate freshwater wetlands. © 2016 Elsevier B.V. All rights reserved.
⁎ Corresponding authors at: Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 4888 Shengbei Road, Changchun 130102, China. E-mail addresses: [email protected] (R. Mao), [email protected] (C.-C. Song).
http://dx.doi.org/10.1016/j.scitotenv.2016.03.158 0048-9697/© 2016 Elsevier B.V. All rights reserved.
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R. Mao et al. / Science of the Total Environment 559 (2016) 1–6
1. Introduction
2. Materials and methods
Nitrogen (N) and phosphorus (P) are the essential nutrients for plant growth in terrestrial and aquatic ecosystems (Elser et al., 2007). Thus, the stoichiometry or relative abundances of carbon (C), N, and P in plants can substantially influence plant-mediated ecological processes and thus modulate the structure and function of the ecosystem (Elser et al., 2010; Bian et al., 2013; Zechmeister-Boltenstern et al., 2015). Plant C:N:P stoichiometry is strongly influenced by nutrient availability, and can effectively indicate the changes in the C, N, and P cycles (Sardans et al., 2012; Hessen et al., 2013). In recent decades, anthropogenic activities have altered global P cycle mainly through excessive applications of fertilizer and the discharge of sewage, leading to increased P input to terrestrial and aquatic ecosystems worldwide (Bennett et al., 2001; Peñuelas et al., 2013). Increased P loading can disrupt the balance between C, N, and P in plants, and thus alter the plant C:N:P stoichiometric ratios (Peñuelas et al., 2013). Previous studies have found that P enrichment increases the P concentration and lowers the C:P ratio in plant tissues (Rejmánková and Snyder, 2008; Ostertag, 2010; Bracken et al., 2014; Yuan and Chen, 2015). However, the effects of P enrichment on plant N concentration, C:N ratio, and N:P ratio may vary with the type of nutrient limitation. In P-limited ecosystems, increased P availability generally causes a decline in plant N concentration due to the dilution effect of plant growth enhancement, and thus an increase in C:N ratio and a decrease in N:P ratio (Feller et al., 2007; Yuan and Chen, 2015). In N-limited ecosystems, the responses of plant N concentration, C:N ratio, and N:P ratio to P enrichment are elusive because of the different nutrient use strategies among species (Yuan and Chen, 2015). Hence, large uncertainties remain regarding the plant stoichiometric responses to P enrichment. Phosphorus enrichment has caused substantial changes in plant community composition and structure through the effects on plant growth rates and on the competitive abilities of different species (Chiang et al., 2000; Rejmánková, 2001; Rejmánková et al., 2008; Cusell et al., 2014). Moreover, previous studies have found that the response of plant nutrient concentration to P enrichment is speciesspecific due to the inherent nutrient use strategies (Shaver and Chapin, 1980; Ostertag, 2010; Mayor et al., 2014). Therefore, the effect of P enrichment on plant-mediated ecological processes depends not only on the changes in the C:N:P stoichiometry at the species level, but also at the community level. Given that community-level plant C:N:P stoichiometry is influenced by both element ratios at the species level and species composition and biomass effects (Han et al., 2014), the effect of P enrichment on plant stoichiometric ratios may be scaledependent. Unfortunately, the information on the effect of P enrichment on plant C:N:P stoichiometry at the community level is extremely scarce, especially in N-limited ecosystems. To verify the effects of P enrichment on plant C:N:P stoichiometry in N-limited ecosystems, we established a long-term multi-level P addition experiment in a freshwater wetland in the Sanjiang Plain, Northeast China. The Sanjiang Plain is composed of the largest freshwater wetland areas in China, and the total area of the wetlands was approximately 9600 km2 in 2005 (Guo et al., 2014). In these wetland ecosystems, plant growth is generally limited by N availability (Mao et al., 2014). In recent decades, freshwater wetlands have been widely reclaimed to meet the increasing food needs of China (Guo et al., 2014). Consequently, the freshwater wetlands in this region have received increasing P inputs mainly due to the application of mineral P fertilizer during farming (Mao et al., 2015). Here, we report the changes in organic C, N, and P concentrations and their ratios in aboveground plant biomass at both the species and community levels following six years of P addition. The main aims of this study are to (1) investigate whether P addition alters plant C:N:P stoichiometry and (2) examine whether species- and community-level plant stoichiometric ratios respond similarly to P addition.
2.1. Study site and experimental design This study was performed in a Deyeuxia angustifolia-dominated freshwater marsh at the Sanjiang Mire Wetland Experimental Station, which is located in the centre of the Sanjiang Plain, Heilongjiang Province, Northeast China (47°35′N, 133°31′E; 56 m above sea level). The D. angustifolia-dominated freshwater marsh is the main wetland type in this region (Guo et al., 2014). The mean annual precipitation of the study site is 566 mm, and the mean annual temperature is 2.5 °C. The soil is a typical meadow mire soil according to the Soil Classification of China, and the soil organic C, N, and P concentrations in the 0–10 cm depth are 167.0, 6.8, and 1.5 mg g−1, respectively. The annual P input to the marshes is approximately 0.4 g P m−2 during the growing season (early May to late August) (Mao et al., 2015). In September 2006, 12 plots (1 m × 1 m) were established in a D. angustifolia-dominated freshwater marsh. The dominant species in this freshwater marsh were D. angustifolia and Glyceria spiculosa. Each plot was separated by a 1-m buffer zone and was fenced with stainless steel frames (1 m × 1 m, 0.5 m in depth) to prevent the loss of the added P. Meanwhile, boardwalks were set up across the plots to avoid soil disturbance during sampling. To mimic future P enrichment in this freshwater marsh, P was applied at four levels (CK, 0 g P m − 2 year − 1 ; P1, 1.2 g P m − 2 year − 1 ; P2, 4.8 g P m − 2year− 1 ; and P3, 9.6 g P m − 2 year− 1 ) during the growing season. The study was arranged in a complete randomized block design, and each P addition level was replicated three times. Phosphorus was added as NaH 2 PO 4 . For each P addition level, P fertilizer has been equally applied 10 times over the growing season each year since 2007.
2.2. Field sampling and measurement In early August 2012, aboveground plants were clipped above the soil surface in two randomly selected subplots (0.4 m × 0.4 m) in each plot, and a total of 24 subplots were harvested. In each subplot, the plant biomass was separated by species, over-dried at 65 °C, weighed, and then ground. In all of the plots, D. angustifolia and G. spiculosa occupied N95% of the total aboveground biomass. Therefore, we only measured the C, N, and P concentrations of the two dominant species in this community. Plant C concentration was measured by the dry combustion method using a multi N/C 2100 analyzer (Analytik Jena, Germany). For each plant sample, a 0.1-g subsample was digested with concentrated H2SO4, and then total N and P concentrations in the digests were determined by the sodium salicylate-sodium hypochlorite method and the molybdenum blue method on a continuous-flow autoanalyzer (AA3, Seal Analytical, Germany), respectively. At the community level, plant C, N, and P concentrations were calculated using the biomass-weighted means of the two selected species. Plant C, N, and P concentrations were expressed on a mass basis. Plant C, N, and P pools were calculated by multiplying the dry mass with the corresponding concentrations. In each plot, soil pore water at 0–15 cm depth was sampled according to the procedures proposed by Höll et al. (2009) when the plant samples were collected. Approximately 200 mL of water was sampled, transferred to a bottle, transported to the laboratory in a cool box, and then filtered through a 0.45-μm membrane filter prior to chemical \ \ analysis. For the soil pore water, NH+ 4 -N, NO3 N, and inorganic P concentrations were analyzed by the sodium salicylate-sodium hypochlorite method, the hydrazine reduction method, and the molybdenum blue method using a continuous-flow autoanalyzer, respectively. Inorganic N concentration in the soil pore water was the sum of NH+ 4 -N and \ NO\ 3 N concentrations. Total N and P concentrations in the soil pore water were assessed by the hydrazine reduction method and the
R. Mao et al. / Science of the Total Environment 559 (2016) 1–6
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Fig. 1. Effect of P addition on aboveground plant biomass and species dominance. Different lowercase letters indicate significant differences among the four treatments.
molybdenum blue method described above following peroxodisulfate oxidation (Ebina et al., 1983).
2.3. Statistical analyses All of the statistical analyses were performed using the SPSS statistical package (v. 13.0) for Windows, and the accepted significance level was α = 0.05. Before the statistical analysis, the normality of the data was tested by Levene's test. When necessary, the data were natural log transformed to meet the assumptions of normal distribution. Twoway analysis of variance (ANOVA) was used to examine the effects of P addition levels, species, and their interactions on plant C, N, and P concentrations, and their stoichiometric ratios at the species level. One-way ANOVA was used to test the effect of P addition levels on the nutrient concentration in the soil pore water and on the communitylevel plant C:N:P stoichiometry. Tukey's honest significant difference test was used to assess the significant difference in the selected parameters among all of the treatments.
3. Results The aboveground biomass and dominance of D. angustifolia exhibited a decline with increasing P addition levels, whereas the aboveground biomass and dominance of G. spiculosa showed an increase (Fig. 1). In the soil pore water, six years of P addition did not affect NH+ 4 -N, \ NO\ 3 N, and inorganic N concentrations, but significantly increased the concentrations of inorganic P, total N, and total P (Table 1). Moreover, inorganic P, total N, and total P concentrations in the soil pore water generally increased with increasing P addition levels. Plant species and P addition levels independently affected plant N concentration, but produced an interactive effect on plant P concentration (Table 2). Moreover, plant C concentration significantly varied with plant species (Table 2). Across all of the P addition treatments, D. angustifolia had greater C concentration and lower N and P concentrations than G. spiculosa. For both D. angustifolia and G. spiculosa, the P addition treatments had greater N and P concentrations than the CK treatment. Moreover, plant N concentration significantly increased with increasing P addition levels for the two selected species.
Table 1 Effect of P addition on nutrient concentrations in the soil pore water. Treatments
NO− 3 N
Inorganic N
Inorganic P
Total N
Total P
μg L
μg L−1
μg L−1
μg L−1
μg L−1
μg L−1
7.86 (0.23) 7.64 (0.29) 7.48 (0.42) 7.57 (0.24) 0.3
12.9 (0.6) 12.6 (0.6) 12.2 (0.4) 12.3 (0.2) 0.4
20.7 (0.7) 20.2 (0.5) 19.7 (0.1) 19.8 (0.3) 1.0
4.20 (0.18)c 6.98 (0.18)b 7.07 (0.09)b 8.71 (0.10)a 163.0⁎⁎⁎
555 (14)d 659 (8)c 809 (30)b 943 (23)a 69.1⁎⁎⁎
38.6 (1.0)c 68.5 (1.1)b 85.3 (2.3)a 90.1 (0.9)a 257.3⁎⁎⁎
NH+ 4 -N −1
CK P1 P2 P3 F-value
Data in parentheses are standard errors of the means (n = 3). Different lowercase letters in the same column indicate significant differences among the four treatments. ⁎⁎⁎ p b 0.001.
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Table 2 Effect of P addition on plant C, N, and P concentrations and their stoichiometric ratios at the species level. Treatments
C concentration
N concentration
P concentration
mg g-1
mg g-1
mg g-1
C:N ratio
C:P ratio
N:P ratio
D. angustifolia CK P1 P2 P3
461 (2) 456 (1) 458 (6) 462 (4)
7.39 (0.26)c 9.97 (0.20)ab 9.96 (0.42)b 11.18 (0.07)a
1.34 (0.07)c 1.93 (0.11)b 1.90 (0.03)b 2.35 (0.04)a
62.5 (2.4)a 45.8 (1.0)b 46.1 (1.4)b 41.3 (0.6)b
345 (18)a 237 (14)b 241 (2)b 197 (5)b
5.51 (0.11)a 5.18 (0.23)ab 5.23 (0.18)ab 4.76 (0.08)b
G. spiculosa CK P1 P2 P3
440 (2) 437 (4) 444 (6) 444 (3)
9.99 (0.08)c 11.29 (0.13)b 11.98 (0.24)ab 12.61 (0.31)a
1.64 (0.09)b 2.28 (0.07)a 2.33 (0.06)a 2.33 (0.04)a
44.0 (0.5)a 38.7 (0.8)b 37.1 (0.6)bc 35.2 (0.9)c
269 (15)a 192 (8)b 191 (3)b 190 (3)b
6.13 (0.40)a 4.95 (0.10)b 5.15 (0.03)b 5.41 (0.05)ab
F-value of two-way ANOVAs Species P addition level Species × P addition level
43.1⁎⁎⁎ 0.9 0.3
116.9⁎⁎⁎ 62.8⁎⁎⁎
30.4⁎⁎⁎ 57.6⁎⁎⁎ 4.1⁎
153.2⁎⁎⁎ 63.2⁎⁎⁎ 12.0⁎⁎⁎
37.6⁎⁎⁎ 49.1⁎⁎⁎ 3.9⁎
3.2 7.4⁎⁎ 3.0
3.0
Data in parentheses are standard errors of the means (n = 3). Different lowercase letters in the same column indicate significant differences among the four treatments. ⁎ p b 0.05 ⁎⁎ p b 0.01 ⁎⁎⁎ p b 0.001.
Plant species and P addition levels significantly interacted to affect plant C:N and C:P ratios, whereas only P addition levels significantly influenced plant N:P ratio at the species level (Table 2). Across all of the treatments, D. angustifolia had higher C:N and C:P ratios than G. spiculosa. For the two selected species, the P addition treatments had lower C:N, C:P, and N:P ratios than the CK treatment. In addition, only G. spiculosa in the P3 treatment had a lower C:N ratio than that in the P1 and P2 treatments, respectively. At the community level, P addition did not significantly affect plant C concentration, but increased plant C pool (Table 3). In addition, P addition increased the concentrations and pools of N and P in the aboveground plant biomass (Table 3). Consequently, the P addition treatments had lower plant C:N, C:P, and N:P ratios than the CK treatment (Table 3). Moreover, the P3 treatment generally had greater plant N concentration, and C, N, and P pools, but a lower C:N ratio than the P1 and P2 treatments, respectively. 4. Discussion In this study, we found that six years of P addition markedly changed plant nutrient concentrations, and hence the C:N:P stoichiometry in a temperate wetland in the Sanjiang Plain, Northeast China, and the change trends were similar at both the species and community scales. Increased P loading increased plant N and P concentrations, and thus decreased plant C:N, C:P, and N:P ratios, irrespective of the P addition levels. These similar change trends at different scales could be caused by the consistent responses of the C:N:P stoichiometric ratios in different species to P addition, considering the altered plant community
structure following P addition and the large interspecific variations in nutrient concentrations between the two selected plant species. Surprisingly, P addition increased plant N concentration and decreased plant C:N ratio, although there was no change in inorganic N availability in the soil pore water. Moreover, the extents of the increase in plant N concentration and the decrease in plant C:N ratio increased with increasing P addition levels. Previous studies have observed negative (Feller et al., 2007; Ostertag, 2010), neutral (Verhoeven and Schmitz, 1991; Mayor et al., 2014), and even positive (Rejmánková and Snyder, 2008; Iversen et al., 2010) effects on plant N concentration. These inconsistent results were probably caused by the differences in the nutrient use strategies of plant species and substrate nutrient availability among ecosystems (Danger et al., 2008; Townsend and Asner, 2013). Generally, temperate wetlands store a large amount of dissolved organic N in soils, although soil inorganic N availability is relatively low in these ecosystems (Neff et al., 2003). In addition, plants in cold biomes could directly absorb organic N from soils (Chapin et al., 1993). These results implied that P enrichment appeared to enhance the abilities of plant N acquisition from the soils in temperate wetlands, and thus increased the N concentration and pool in aboveground plant biomass (Iversen et al., 2010). Increased plant N concentration following P addition could enhance litter quality through altered N resorption processes (Mao et al., 2015), and thus stimulate litter decomposition and N release (Aerts et al., 1999). Indeed, we observed an increase in the total N concentration in soil pore water following six years of P addition, although the inorganic N concentration in the soil pore water exhibited no response to increased P availability (Table 1). Therefore, increased N return to soils via litter decomposition
Table 3 Effect of P addition on plant C, N, and P concentrations and their stoichiometric ratios at the community level. Treatments
CK P1 P2 P3 F-value
C concentration
N concentration
P concentration
C pool
N pool
P pool
mg g−1
mg g−1
mg g−1
g m−2
g m−2
g m−2
453 (2) 445 (2) 449 (5) 445 (3) 1.5
8.32 (0.23)c 10.72 (0.14)b 11.29 (0.30)b 12.44 (0.25)a 53.3⁎⁎⁎
1.46 (0.01)b 2.15 (0.08)a 2.21 (0.05)a 2.33 (0.04)a 56.9⁎⁎⁎
115 (12)b 130 (4)ab 139 (12)ab 165 (4)a 5.2⁎
2.09 (0.23)c 3.08 (0.07)bc 3.45 (0.35)b 4.55 (0.20)a 19.3⁎⁎
0.36 (0.04)c 0.61 (0.02)b 0.67 (0.07)ab 0.85 (0.03)a 21.0⁎⁎⁎
C:N ratio
C:P ratio
N:P ratio
54.5 (1.7)a 41.6 (0.7)b 39.8 (0.9)bc 35.8 (0.8)c 56.2⁎⁎⁎
310 (3)a 208 (9)b 203 (4)b 191 (3)b 105.2⁎⁎⁎
5.69 (0.13)a 5.00 (0.14)b 5.11 (0.04)b 5.33 (0.04)ab 9.5⁎⁎
Data in parentheses are standard errors of the means (n = 3). Different lowercase letters in the same column indicate significant differences among the four treatments. ⁎ p b 0.05 ⁎⁎ p b 0.01 ⁎⁎⁎ p b 0.001.
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would enhance the N supply to plants and provide a positive feedback to plant N concentration and ecosystem productivity. Phosphorus addition increased plant P concentration and hence caused a decline in the plant C:P ratio, which was also observed in the previous studies (e.g., Rejmánková and Snyder, 2008; Ostertag, 2010; Mayor et al., 2014; Chen et al., 2015). Moreover, the magnitude of the increase in the plant P concentration did not vary with P addition levels. In temperate and boreal wetlands, soil nutrient availability is generally low because of the slow rates of decomposition induced by the cool temperatures, anoxic conditions, and the recalcitrant substrates (Aerts et al., 1999). Thus, plants in these ecosystems often could absorb nutrients in excess of their growth needs (i.e., through luxury consumption) during nutrient flushes, and these reserved nutrients may be used to support plant growth when external nutrient availability was low (Chapin, 1980). In this study, P addition drastically increased inorganic P and total P concentrations in the soil pore water (Table 1). Given that plant growth in this wetland was limited by N availability (Mao et al., 2014), the positive effect of P addition on the plant P concentration would be attributed to the luxury consumption. Despite the simultaneous increases in N and P concentrations in plants, P addition caused a decline in the plant N:P ratio, which was consistent with the previous studies conducted in N-limited forests (Ostertag, 2010), grassland (Li et al., 2011), and subarctic tundra (De Long et al., 2016). Because P addition levels were much N1.0 g P m−2 year− 1 in these fertilization experiments, the magnitude of the increase in plant P concentration exceeded that in plant N concentration, which resulted in an imbalance between N and P in plants (Sistla et al., 2015). Considering that the biomass N:P ratio can indicate nutrient limitation for plant growth (Güsewell et al., 2003), our result implies that increased P availability has the potential to aggravate the N limitation of the plant community in this wetland ecosystem. P addition markedly altered plant community structure in this wetland, which was consistent with the previous studies conducted in temperate and boreal wetlands (Thormann and Bayley, 1997; Bedford et al., 1999; Liu et al., 2015). In the present study, the dominance of D. angustifolia decreased with increasing P addition levels, whereas the dominance of G. spiculosa exhibited an opposite trend. Generally, increased nutrient availability could shift belowground competition for nutrients to aboveground competition for light resources (Grace, 1999). Compared with D. angustifolia, G. spiculosa had greater leaf area and leaf:stem biomass ratio (Mao et al., 2014), and hence may capture more light to enhance the growth rate (Grace, 1999). Therefore, G. spiculosa gained an advantage over D. angustifolia following six years of P addition. Because of the substantial differences in plant functional traits between D. angustifolia and G. spiculosa (Mao et al., 2014, 2015), the altered vegetation community structure induced by P enrichment would further exert marked influences on plant-mediated biogeochemical cycles in this freshwater wetland. 5. Conclusions In summary, species- and community-level plant C:N:P stoichiometry exhibited similar responses to six years of P addition, although there was a shift in dominance from D. angustifolia to G. spiculosa in a temperate wetland, Northeast China. At both the species- and communitylevel, P addition increased plant N and P concentrations, and thus decreased C:N, C:P, and N:P ratios. These results indicate that the effects of P enrichment on plant C:N:P stoichiometry are scale-independent, and imply that the stoichiometric responses to increased P availability at the community level would be mirrored by those at the species level. Moreover, altered plant C:N:P stoichiometry and vegetation community structure induced by P enrichment would enhance organic matter decomposition and nutrient release, and thus further intensify the eutrophication process and cause C loss from soils in N-limited temperate freshwater wetlands.
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Acknowledgments We would like to thank Dr. De-Yan Liu, Gui-Sheng Yang, Yan-Yu Song, and Gui-Yan Ai for the field and laboratory works and two anonymous reviewers and the editor for their helpful comments and suggestions for improving the manuscript. This study was funded by the “Strategic Priority Research Program — Climate Change: Carbon Budget and Related Issue” of the Chinese Academy of Sciences (No. XDA05050508), the National Natural Science Foundation of China (Nos. 31570479 and 41125001), and the Youth Innovation Promotion Association of CAS (No. 2013152). References Aerts, R., Verhoeven, J.T.A., Whigham, D.F., 1999. Plant-mediated controls on nutrient cycling in temperate fens and bogs. Ecology 80, 2170–2181. Bedford, B.L., Walbridge, M.R., Aldous, A., 1999. Patterns in nutrient availability and plant diversit of temperate North American wetlands. Ecology 80, 2151–2169. Bennett, E.M., Carpenter, S.R., Caraco, N.F., 2001. Human impacts on erodible phosphorus and eutrophication: a global perspective. Bioscience 51, 227–234. Bian, J., Berninger, J.P., Fulton, B.A., Brooks, B.W., 2013. Nutrient stoichiometry and concentrations influence silver toxicity in the aquatic macrophyte Lemna gibba. Sci. Total Environ. 449, 229–236. Bracken, M.E.S., Hillebrand, H., Borer, E.T., Seabloom, E.W., Cebrian, J., Cleland, E.E., Elser, J.J., Gruber, D.S., Harpole, W.S., Ngai, J.T., Smith, J.E., 2014. Signature of nutrient limitation and co-limitation: response of autotroph internal nutrient concentrations to nitrogen and phosphorus additions. Oikos 124, 113–121. Chapin, F.S., 1980. The mineral nutrition of wild plants. Annu. Rev. Ecol. Syst. 11, 233–260. Chapin, F.S., Moilanen, L., Kielland, K., 1993. Preferential use of organic nitrogen for growth by a non-mycorrhizal arctic sedge. Nature 361, 150–153. Chen, F.S., Niklas, K.J., Liu, Y., Fang, X.M., Wan, S.Z., Wang, H., 2015. Nitrogen and phosphorus additions alter nutrient dynamics but not resorption efficiencies of Chinese fir leaves and twigs differing in age. Tree Physiol. 35, 1106–1117. Chiang, C., Craft, C.B., Rogers, D.W., Richardson, C.J., 2000. Effects of 4 years of nitrogen and phosphorus additions on Everglades plant communities. Aquat. Bot. 68, 61–78. Cusell, C., Kooijman, A., Lamers, L.P.M., 2014. Nitrogen or phosphorus limitation in rich fens? Edaphic differences explain contrasting results in vegetation development after fertilization. Plant Soil 384, 153–168. Danger, M., Daufresne, T., Lucas, F., Pissard, S., Lacroix, G., 2008. Does Liebig's law of the minimum scale up from species to communities? Oikos 117, 1741–1751. De Long, J.R., Sundqvist, M.K., Gundale, M.J., Giesler, R., Wardle, D.A., 2016. Effects of elevation and nitrogen and phosphorus fertilization on plant defence compounds in subarctic tundra heath vegetation. Funct. Ecol. 30, 314–325. Ebina, J., Tsutsui, T., Shirai, T., 1983. Simultaneous determination of total nitrogen and total phosphorus in water using peroxodisulfate oxidation. Water Res. 17, 1721–1726. Elser, J.J., Bracken, M.E.S., Cleland, E.E., Gruner, D.S., Harpole, W.S., Hillebrand, H., Ngai, J.T., Seabloom, E.W., Shurin, J.B., Smith, J.E., 2007. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol. Lett. 10, 1135–1142. Elser, J.J., Fagan, W.F., Kerkhoff, A.J., Swenson, N.G., Enquist, B.J., 2010. Biological stoichiometry of plant production: metabolism, scaling and ecological response to global change. New Phytol. 186, 593–608. Feller, I.C., Lovelock, C.E., McKee, K.L., 2007. Nutrient addition differentially affects ecological processes of Avicennia germinans in nitrogen versus phosphorus limited mangrove ecosystems. Ecosystems 10, 347–359. Grace, J.B., 1999. The factors controlling species density in herbaceous plant communities: an assessment. Perspect. Plant Ecol. 2, 1–28. Guo, Y.D., Lu, Y.Z., Song, Y.Y., Wan, Z.M., Hou, A.X., 2014. Concentration and characteristics of dissolved carbon in the Sanjiang Plain influenced by long-term land reclamation from marsh. Sci. Total Environ. 466-467, 777–787. Güsewell, S., Koerselman, W., Verhoeven, J.T.A., 2003. Biomass N:P ratios as indicators of nutrient limitation for plant populations in wetlands. Ecol. Appl. 13, 372–384. Han, X., Sistla, S.A., Zhang, Y.H., Lü, X.T., Han, X.G., 2014. Hierarchical responses of plant stoichiometry to nitrogen deposition and mowing in a temperate steppe. Plant Soil 382, 175–187. Hessen, D.O., Elser, J.J., Sterner, R.W., Urabe, J., 2013. Ecological stoichiometry: an elementary approach using basic principles. Limnol. Oceanogr. 58, 2219–2236. Höll, B.S., Fiedler, S., Jungkunst, H.F., Kalbitz, K., Freibauer, A., Drösler, M., Stahr, K., 2009. Characteristics of dissolved organic matter following 20 years of peatland restoration. Sci. Total Environ. 408, 78–83. Iversen, C.M., Bridgham, S.D., Kellogg, L.E., 2010. Scaling plant nitrogen use and uptake efficiencies in response to nutrient addition in peatlands. Ecology 91, 693–707. Li, L.J., Zeng, D.H., Yu, Z.Y., Fan, Z.P., Mao, R., Peri, P.L., 2011. Foliar N/P ratio and nutrient limitation to vegetation growth on Keerqin sandy grassland of North-East China. Grass Forage Sci. 66, 237–242. Liu, C., Bu, J., Ma, J., Yuan, M., Feng, L., Liu, S., 2015. Comparative study on the response of deciduous and evergreen shrubs to nitrogen and phosphorus input in Hani Peatland of Changbai Mountains. Chin. J. Ecol. 34, 2711–2719. Mao, R., Zhang, X.H., Song, C.C., 2014. Effects of nitrogen addition on plant functional traits in freshwater wetland of Sanjiang Plain, Northeast China. Chin. Geogr. Sci. 24, 674–681.
6
R. Mao et al. / Science of the Total Environment 559 (2016) 1–6
Mao, R., Zeng, D.H., Zhang, X.H., Song, C.C., 2015. Responses of plant nutrient resorption to phosphorus addition in freshwater marsh of Northeast China. Sci. Rep. 5, 8097. Mayor, J.R., Wright, S.J., Turner, B.L., 2014. Species-specific responses of foliar nutrients to long-term nitrogen and phosphorus additions in a lowland tropical forest. J. Ecol. 102, 36–44. Neff, J.C., Chapin, F.S., Vitousek, P.M., 2003. Breaks in the cycle: dissolved organic nitrogen in terrestrial ecosystems. Front. Ecol. Environ. 1, 205–211. Ostertag, R., 2010. Foliar nitrogen and phosphorus accumulation responses after fertilization: an example from nutrient-limited Hawaiian forests. Plant Soil 334, 85–98. Peñuelas, J., Poulter, B., Sardans, J., Ciais, P., van der Velde, M., Bopp, L., Boucher, O., Godderis, Y., Hinsinger, P., Llusia, J., Nardin, E., Vicca, S., Obersteiner, M., Janssens, I.A., 2013. Human-induced nitrogen-phosphorus imbalances alter natural and managed ecosystems across the globe. Nat. Commun. 4, 2934. Rejmánková, E., 2001. Effect of experimental phosphorus enrichment on oligotrophic tropical marshes in Belize, Central America. Plant Soil 236, 33–53. Rejmánková, E., Snyder, J.M., 2008. Emergent macrophytes in phosphorus limited marshes: do phosphorus usage strategies change after nutrient addition? Plant Soil 313, 141–153. Rejmánková, E., Macek, P., Epps, K., 2008. Wetland ecosystem changes after three years of phosphorus addition. Wetlands 28, 914–927.
Sardans, J., Rivas-Ubach, A., Peñuelas, J., 2012. The C:N:P stoichiometry of organisms and ecosystems in a changing world: a review and perspectives. Perspect. Plant Ecol. 14, 33–47. Shaver, G.R., Chapin, F.S., 1980. Response to fertilization by various plant growth forms in an Alaskan tundra: nutrient accumulation and growth. Ecology 61, 662–675. Sistla, S.A., Appling, A.P., Lewandowska, A.M., Taylor, B.N., Wolf, A.A., 2015. Stoichiometric flexibility in response to fertilization along gradients of environmental and organismal nutrient richness. Oikos 124, 949–959. Thormann, M.N., Bayley, S., 1997. Response of aboveground net primary plant production to nitrogen and phosphorus fertilization in peatlands in southern boreal Alberta, Canada. Wetlands 17, 502–512. Townsend, A.R., Asner, G.P., 2013. Multiple dimensions of resource limitation in tropical forests. Proc. Natl. Acad. Sci. U. S. A. 110, 4864–4865. Verhoeven, J.T.A., Schmitz, M.B., 1991. Control of plant growth by nitrogen and phosphorus in mesotrophic fens. Biogeochemistry 12, 135–148. Yuan, Z.Y., Chen, H.Y.H., 2015. Negative effects of fertilization on plant nutrient resorption. Ecology 96, 373–380. Zechmeister-Boltenstern, S., Keiblinger, K.M., Mooshammer, M., Peñuelas, J., Richter, A., Sardans, J., Wanek, W., 2015. The application of ecological stoichiometry to plant–microbial–soil organic matter transformations. Ecol. Monogr. 85, 133–155.
Contents lists available at ScienceDirect
Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Effects of P addition on plant C:N:P stoichiometry in an N-limited temperate wetland of Northeast China Rong Mao a,b,⁎, Hui-Min Chen a,c, Xin-Hou Zhang a, Fu-Xi Shi a, Chang-Chun Song a,⁎ a b c
Key Laboratory of Wetland Ecology and Environment, Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Changchun 130102, China The Three Gorges Institute of Ecological Environment, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China University of Chinese Academy of Sciences, Beijing 100049, China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Effects of P enrichment on plant C:N:P stoichiometry at different scales are unclear. • Species- and community-level plant stoichiometry responded similarly to P addition. • Six years of P addition increased plant N and P concentrations. • Six years of P addition decreased plant C:N, C:P, and N:P ratios. • Altered C:N:P stoichiometry following P enrichment would accelerate nutrient cycles.
a r t i c l e
i n f o
Article history: Received 23 December 2015 Received in revised form 22 March 2016 Accepted 22 March 2016 Available online xxxx Editor: D. Barcelo Keywords: C:N:P stoichiometric ratios Deyeuxia angustifolia Eutrophication Freshwater marsh Glyceria spiculosa Species dominance
a b s t r a c t . Phosphorus (P) enrichment induced by anthropogenic activities results in modified plant nutrient status, which potentially alters the stoichiometry of carbon (C), nitrogen (N), and P in plants. However, how increased P availability changes plant C:N:P stoichiometry at different hierarchical scales is unclear in N-limited ecosystems. In this study, we conducted a four-level P addition experiment (0, 1.2, 4.8, and 9.6 g P m−2 year−1) to elucidate the effect of P enrichment on plant C:N:P stoichiometric ratios at both the species and community levels in a freshwater wetland in the Sanjiang Plain, Northeast China. We found that species- and community-level plant C:N:P stoichiometry responded consistently to six years of P addition, although there was a shift in species dominance. Phosphorus addition increased plant N and P concentrations and thus decreased C:N, C:P, and N:P ratios irrespective of the P addition levels. These similar change trends at different scales resulted from the identical responses of plant N and P concentrations in different species to P addition. Moreover, plant N concentration exhibited an increasing trend with increasing P addition levels, whereas plant C:N ratio showed a declining trend. At the community level, P addition at the rates of 1.2, 4.8, and 9.6 g P m−2 year−1 decreased the C:N ratio by 24%, 27%, and 34%; decreased the C:P ratio by 33%, 35%, and 38%; and decreased the N:P ratio by 12%, 10%, and 6%, respectively. Our results indicate that the stoichiometric responses to P addition are scaleindependent, and suggest that altered plant C:N:P stoichiometry induced by P enrichment would stimulate organic matter decomposition and accelerate nutrient cycles in N-limited temperate freshwater wetlands. © 2016 Elsevier B.V. All rights reserved.
⁎ Corresponding authors at: Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, 4888 Shengbei Road, Changchun 130102, China. E-mail addresses: [email protected] (R. Mao), [email protected] (C.-C. Song).
http://dx.doi.org/10.1016/j.scitotenv.2016.03.158 0048-9697/© 2016 Elsevier B.V. All rights reserved.
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1. Introduction
2. Materials and methods
Nitrogen (N) and phosphorus (P) are the essential nutrients for plant growth in terrestrial and aquatic ecosystems (Elser et al., 2007). Thus, the stoichiometry or relative abundances of carbon (C), N, and P in plants can substantially influence plant-mediated ecological processes and thus modulate the structure and function of the ecosystem (Elser et al., 2010; Bian et al., 2013; Zechmeister-Boltenstern et al., 2015). Plant C:N:P stoichiometry is strongly influenced by nutrient availability, and can effectively indicate the changes in the C, N, and P cycles (Sardans et al., 2012; Hessen et al., 2013). In recent decades, anthropogenic activities have altered global P cycle mainly through excessive applications of fertilizer and the discharge of sewage, leading to increased P input to terrestrial and aquatic ecosystems worldwide (Bennett et al., 2001; Peñuelas et al., 2013). Increased P loading can disrupt the balance between C, N, and P in plants, and thus alter the plant C:N:P stoichiometric ratios (Peñuelas et al., 2013). Previous studies have found that P enrichment increases the P concentration and lowers the C:P ratio in plant tissues (Rejmánková and Snyder, 2008; Ostertag, 2010; Bracken et al., 2014; Yuan and Chen, 2015). However, the effects of P enrichment on plant N concentration, C:N ratio, and N:P ratio may vary with the type of nutrient limitation. In P-limited ecosystems, increased P availability generally causes a decline in plant N concentration due to the dilution effect of plant growth enhancement, and thus an increase in C:N ratio and a decrease in N:P ratio (Feller et al., 2007; Yuan and Chen, 2015). In N-limited ecosystems, the responses of plant N concentration, C:N ratio, and N:P ratio to P enrichment are elusive because of the different nutrient use strategies among species (Yuan and Chen, 2015). Hence, large uncertainties remain regarding the plant stoichiometric responses to P enrichment. Phosphorus enrichment has caused substantial changes in plant community composition and structure through the effects on plant growth rates and on the competitive abilities of different species (Chiang et al., 2000; Rejmánková, 2001; Rejmánková et al., 2008; Cusell et al., 2014). Moreover, previous studies have found that the response of plant nutrient concentration to P enrichment is speciesspecific due to the inherent nutrient use strategies (Shaver and Chapin, 1980; Ostertag, 2010; Mayor et al., 2014). Therefore, the effect of P enrichment on plant-mediated ecological processes depends not only on the changes in the C:N:P stoichiometry at the species level, but also at the community level. Given that community-level plant C:N:P stoichiometry is influenced by both element ratios at the species level and species composition and biomass effects (Han et al., 2014), the effect of P enrichment on plant stoichiometric ratios may be scaledependent. Unfortunately, the information on the effect of P enrichment on plant C:N:P stoichiometry at the community level is extremely scarce, especially in N-limited ecosystems. To verify the effects of P enrichment on plant C:N:P stoichiometry in N-limited ecosystems, we established a long-term multi-level P addition experiment in a freshwater wetland in the Sanjiang Plain, Northeast China. The Sanjiang Plain is composed of the largest freshwater wetland areas in China, and the total area of the wetlands was approximately 9600 km2 in 2005 (Guo et al., 2014). In these wetland ecosystems, plant growth is generally limited by N availability (Mao et al., 2014). In recent decades, freshwater wetlands have been widely reclaimed to meet the increasing food needs of China (Guo et al., 2014). Consequently, the freshwater wetlands in this region have received increasing P inputs mainly due to the application of mineral P fertilizer during farming (Mao et al., 2015). Here, we report the changes in organic C, N, and P concentrations and their ratios in aboveground plant biomass at both the species and community levels following six years of P addition. The main aims of this study are to (1) investigate whether P addition alters plant C:N:P stoichiometry and (2) examine whether species- and community-level plant stoichiometric ratios respond similarly to P addition.
2.1. Study site and experimental design This study was performed in a Deyeuxia angustifolia-dominated freshwater marsh at the Sanjiang Mire Wetland Experimental Station, which is located in the centre of the Sanjiang Plain, Heilongjiang Province, Northeast China (47°35′N, 133°31′E; 56 m above sea level). The D. angustifolia-dominated freshwater marsh is the main wetland type in this region (Guo et al., 2014). The mean annual precipitation of the study site is 566 mm, and the mean annual temperature is 2.5 °C. The soil is a typical meadow mire soil according to the Soil Classification of China, and the soil organic C, N, and P concentrations in the 0–10 cm depth are 167.0, 6.8, and 1.5 mg g−1, respectively. The annual P input to the marshes is approximately 0.4 g P m−2 during the growing season (early May to late August) (Mao et al., 2015). In September 2006, 12 plots (1 m × 1 m) were established in a D. angustifolia-dominated freshwater marsh. The dominant species in this freshwater marsh were D. angustifolia and Glyceria spiculosa. Each plot was separated by a 1-m buffer zone and was fenced with stainless steel frames (1 m × 1 m, 0.5 m in depth) to prevent the loss of the added P. Meanwhile, boardwalks were set up across the plots to avoid soil disturbance during sampling. To mimic future P enrichment in this freshwater marsh, P was applied at four levels (CK, 0 g P m − 2 year − 1 ; P1, 1.2 g P m − 2 year − 1 ; P2, 4.8 g P m − 2year− 1 ; and P3, 9.6 g P m − 2 year− 1 ) during the growing season. The study was arranged in a complete randomized block design, and each P addition level was replicated three times. Phosphorus was added as NaH 2 PO 4 . For each P addition level, P fertilizer has been equally applied 10 times over the growing season each year since 2007.
2.2. Field sampling and measurement In early August 2012, aboveground plants were clipped above the soil surface in two randomly selected subplots (0.4 m × 0.4 m) in each plot, and a total of 24 subplots were harvested. In each subplot, the plant biomass was separated by species, over-dried at 65 °C, weighed, and then ground. In all of the plots, D. angustifolia and G. spiculosa occupied N95% of the total aboveground biomass. Therefore, we only measured the C, N, and P concentrations of the two dominant species in this community. Plant C concentration was measured by the dry combustion method using a multi N/C 2100 analyzer (Analytik Jena, Germany). For each plant sample, a 0.1-g subsample was digested with concentrated H2SO4, and then total N and P concentrations in the digests were determined by the sodium salicylate-sodium hypochlorite method and the molybdenum blue method on a continuous-flow autoanalyzer (AA3, Seal Analytical, Germany), respectively. At the community level, plant C, N, and P concentrations were calculated using the biomass-weighted means of the two selected species. Plant C, N, and P concentrations were expressed on a mass basis. Plant C, N, and P pools were calculated by multiplying the dry mass with the corresponding concentrations. In each plot, soil pore water at 0–15 cm depth was sampled according to the procedures proposed by Höll et al. (2009) when the plant samples were collected. Approximately 200 mL of water was sampled, transferred to a bottle, transported to the laboratory in a cool box, and then filtered through a 0.45-μm membrane filter prior to chemical \ \ analysis. For the soil pore water, NH+ 4 -N, NO3 N, and inorganic P concentrations were analyzed by the sodium salicylate-sodium hypochlorite method, the hydrazine reduction method, and the molybdenum blue method using a continuous-flow autoanalyzer, respectively. Inorganic N concentration in the soil pore water was the sum of NH+ 4 -N and \ NO\ 3 N concentrations. Total N and P concentrations in the soil pore water were assessed by the hydrazine reduction method and the
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Fig. 1. Effect of P addition on aboveground plant biomass and species dominance. Different lowercase letters indicate significant differences among the four treatments.
molybdenum blue method described above following peroxodisulfate oxidation (Ebina et al., 1983).
2.3. Statistical analyses All of the statistical analyses were performed using the SPSS statistical package (v. 13.0) for Windows, and the accepted significance level was α = 0.05. Before the statistical analysis, the normality of the data was tested by Levene's test. When necessary, the data were natural log transformed to meet the assumptions of normal distribution. Twoway analysis of variance (ANOVA) was used to examine the effects of P addition levels, species, and their interactions on plant C, N, and P concentrations, and their stoichiometric ratios at the species level. One-way ANOVA was used to test the effect of P addition levels on the nutrient concentration in the soil pore water and on the communitylevel plant C:N:P stoichiometry. Tukey's honest significant difference test was used to assess the significant difference in the selected parameters among all of the treatments.
3. Results The aboveground biomass and dominance of D. angustifolia exhibited a decline with increasing P addition levels, whereas the aboveground biomass and dominance of G. spiculosa showed an increase (Fig. 1). In the soil pore water, six years of P addition did not affect NH+ 4 -N, \ NO\ 3 N, and inorganic N concentrations, but significantly increased the concentrations of inorganic P, total N, and total P (Table 1). Moreover, inorganic P, total N, and total P concentrations in the soil pore water generally increased with increasing P addition levels. Plant species and P addition levels independently affected plant N concentration, but produced an interactive effect on plant P concentration (Table 2). Moreover, plant C concentration significantly varied with plant species (Table 2). Across all of the P addition treatments, D. angustifolia had greater C concentration and lower N and P concentrations than G. spiculosa. For both D. angustifolia and G. spiculosa, the P addition treatments had greater N and P concentrations than the CK treatment. Moreover, plant N concentration significantly increased with increasing P addition levels for the two selected species.
Table 1 Effect of P addition on nutrient concentrations in the soil pore water. Treatments
NO− 3 N
Inorganic N
Inorganic P
Total N
Total P
μg L
μg L−1
μg L−1
μg L−1
μg L−1
μg L−1
7.86 (0.23) 7.64 (0.29) 7.48 (0.42) 7.57 (0.24) 0.3
12.9 (0.6) 12.6 (0.6) 12.2 (0.4) 12.3 (0.2) 0.4
20.7 (0.7) 20.2 (0.5) 19.7 (0.1) 19.8 (0.3) 1.0
4.20 (0.18)c 6.98 (0.18)b 7.07 (0.09)b 8.71 (0.10)a 163.0⁎⁎⁎
555 (14)d 659 (8)c 809 (30)b 943 (23)a 69.1⁎⁎⁎
38.6 (1.0)c 68.5 (1.1)b 85.3 (2.3)a 90.1 (0.9)a 257.3⁎⁎⁎
NH+ 4 -N −1
CK P1 P2 P3 F-value
Data in parentheses are standard errors of the means (n = 3). Different lowercase letters in the same column indicate significant differences among the four treatments. ⁎⁎⁎ p b 0.001.
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Table 2 Effect of P addition on plant C, N, and P concentrations and their stoichiometric ratios at the species level. Treatments
C concentration
N concentration
P concentration
mg g-1
mg g-1
mg g-1
C:N ratio
C:P ratio
N:P ratio
D. angustifolia CK P1 P2 P3
461 (2) 456 (1) 458 (6) 462 (4)
7.39 (0.26)c 9.97 (0.20)ab 9.96 (0.42)b 11.18 (0.07)a
1.34 (0.07)c 1.93 (0.11)b 1.90 (0.03)b 2.35 (0.04)a
62.5 (2.4)a 45.8 (1.0)b 46.1 (1.4)b 41.3 (0.6)b
345 (18)a 237 (14)b 241 (2)b 197 (5)b
5.51 (0.11)a 5.18 (0.23)ab 5.23 (0.18)ab 4.76 (0.08)b
G. spiculosa CK P1 P2 P3
440 (2) 437 (4) 444 (6) 444 (3)
9.99 (0.08)c 11.29 (0.13)b 11.98 (0.24)ab 12.61 (0.31)a
1.64 (0.09)b 2.28 (0.07)a 2.33 (0.06)a 2.33 (0.04)a
44.0 (0.5)a 38.7 (0.8)b 37.1 (0.6)bc 35.2 (0.9)c
269 (15)a 192 (8)b 191 (3)b 190 (3)b
6.13 (0.40)a 4.95 (0.10)b 5.15 (0.03)b 5.41 (0.05)ab
F-value of two-way ANOVAs Species P addition level Species × P addition level
43.1⁎⁎⁎ 0.9 0.3
116.9⁎⁎⁎ 62.8⁎⁎⁎
30.4⁎⁎⁎ 57.6⁎⁎⁎ 4.1⁎
153.2⁎⁎⁎ 63.2⁎⁎⁎ 12.0⁎⁎⁎
37.6⁎⁎⁎ 49.1⁎⁎⁎ 3.9⁎
3.2 7.4⁎⁎ 3.0
3.0
Data in parentheses are standard errors of the means (n = 3). Different lowercase letters in the same column indicate significant differences among the four treatments. ⁎ p b 0.05 ⁎⁎ p b 0.01 ⁎⁎⁎ p b 0.001.
Plant species and P addition levels significantly interacted to affect plant C:N and C:P ratios, whereas only P addition levels significantly influenced plant N:P ratio at the species level (Table 2). Across all of the treatments, D. angustifolia had higher C:N and C:P ratios than G. spiculosa. For the two selected species, the P addition treatments had lower C:N, C:P, and N:P ratios than the CK treatment. In addition, only G. spiculosa in the P3 treatment had a lower C:N ratio than that in the P1 and P2 treatments, respectively. At the community level, P addition did not significantly affect plant C concentration, but increased plant C pool (Table 3). In addition, P addition increased the concentrations and pools of N and P in the aboveground plant biomass (Table 3). Consequently, the P addition treatments had lower plant C:N, C:P, and N:P ratios than the CK treatment (Table 3). Moreover, the P3 treatment generally had greater plant N concentration, and C, N, and P pools, but a lower C:N ratio than the P1 and P2 treatments, respectively. 4. Discussion In this study, we found that six years of P addition markedly changed plant nutrient concentrations, and hence the C:N:P stoichiometry in a temperate wetland in the Sanjiang Plain, Northeast China, and the change trends were similar at both the species and community scales. Increased P loading increased plant N and P concentrations, and thus decreased plant C:N, C:P, and N:P ratios, irrespective of the P addition levels. These similar change trends at different scales could be caused by the consistent responses of the C:N:P stoichiometric ratios in different species to P addition, considering the altered plant community
structure following P addition and the large interspecific variations in nutrient concentrations between the two selected plant species. Surprisingly, P addition increased plant N concentration and decreased plant C:N ratio, although there was no change in inorganic N availability in the soil pore water. Moreover, the extents of the increase in plant N concentration and the decrease in plant C:N ratio increased with increasing P addition levels. Previous studies have observed negative (Feller et al., 2007; Ostertag, 2010), neutral (Verhoeven and Schmitz, 1991; Mayor et al., 2014), and even positive (Rejmánková and Snyder, 2008; Iversen et al., 2010) effects on plant N concentration. These inconsistent results were probably caused by the differences in the nutrient use strategies of plant species and substrate nutrient availability among ecosystems (Danger et al., 2008; Townsend and Asner, 2013). Generally, temperate wetlands store a large amount of dissolved organic N in soils, although soil inorganic N availability is relatively low in these ecosystems (Neff et al., 2003). In addition, plants in cold biomes could directly absorb organic N from soils (Chapin et al., 1993). These results implied that P enrichment appeared to enhance the abilities of plant N acquisition from the soils in temperate wetlands, and thus increased the N concentration and pool in aboveground plant biomass (Iversen et al., 2010). Increased plant N concentration following P addition could enhance litter quality through altered N resorption processes (Mao et al., 2015), and thus stimulate litter decomposition and N release (Aerts et al., 1999). Indeed, we observed an increase in the total N concentration in soil pore water following six years of P addition, although the inorganic N concentration in the soil pore water exhibited no response to increased P availability (Table 1). Therefore, increased N return to soils via litter decomposition
Table 3 Effect of P addition on plant C, N, and P concentrations and their stoichiometric ratios at the community level. Treatments
CK P1 P2 P3 F-value
C concentration
N concentration
P concentration
C pool
N pool
P pool
mg g−1
mg g−1
mg g−1
g m−2
g m−2
g m−2
453 (2) 445 (2) 449 (5) 445 (3) 1.5
8.32 (0.23)c 10.72 (0.14)b 11.29 (0.30)b 12.44 (0.25)a 53.3⁎⁎⁎
1.46 (0.01)b 2.15 (0.08)a 2.21 (0.05)a 2.33 (0.04)a 56.9⁎⁎⁎
115 (12)b 130 (4)ab 139 (12)ab 165 (4)a 5.2⁎
2.09 (0.23)c 3.08 (0.07)bc 3.45 (0.35)b 4.55 (0.20)a 19.3⁎⁎
0.36 (0.04)c 0.61 (0.02)b 0.67 (0.07)ab 0.85 (0.03)a 21.0⁎⁎⁎
C:N ratio
C:P ratio
N:P ratio
54.5 (1.7)a 41.6 (0.7)b 39.8 (0.9)bc 35.8 (0.8)c 56.2⁎⁎⁎
310 (3)a 208 (9)b 203 (4)b 191 (3)b 105.2⁎⁎⁎
5.69 (0.13)a 5.00 (0.14)b 5.11 (0.04)b 5.33 (0.04)ab 9.5⁎⁎
Data in parentheses are standard errors of the means (n = 3). Different lowercase letters in the same column indicate significant differences among the four treatments. ⁎ p b 0.05 ⁎⁎ p b 0.01 ⁎⁎⁎ p b 0.001.
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would enhance the N supply to plants and provide a positive feedback to plant N concentration and ecosystem productivity. Phosphorus addition increased plant P concentration and hence caused a decline in the plant C:P ratio, which was also observed in the previous studies (e.g., Rejmánková and Snyder, 2008; Ostertag, 2010; Mayor et al., 2014; Chen et al., 2015). Moreover, the magnitude of the increase in the plant P concentration did not vary with P addition levels. In temperate and boreal wetlands, soil nutrient availability is generally low because of the slow rates of decomposition induced by the cool temperatures, anoxic conditions, and the recalcitrant substrates (Aerts et al., 1999). Thus, plants in these ecosystems often could absorb nutrients in excess of their growth needs (i.e., through luxury consumption) during nutrient flushes, and these reserved nutrients may be used to support plant growth when external nutrient availability was low (Chapin, 1980). In this study, P addition drastically increased inorganic P and total P concentrations in the soil pore water (Table 1). Given that plant growth in this wetland was limited by N availability (Mao et al., 2014), the positive effect of P addition on the plant P concentration would be attributed to the luxury consumption. Despite the simultaneous increases in N and P concentrations in plants, P addition caused a decline in the plant N:P ratio, which was consistent with the previous studies conducted in N-limited forests (Ostertag, 2010), grassland (Li et al., 2011), and subarctic tundra (De Long et al., 2016). Because P addition levels were much N1.0 g P m−2 year− 1 in these fertilization experiments, the magnitude of the increase in plant P concentration exceeded that in plant N concentration, which resulted in an imbalance between N and P in plants (Sistla et al., 2015). Considering that the biomass N:P ratio can indicate nutrient limitation for plant growth (Güsewell et al., 2003), our result implies that increased P availability has the potential to aggravate the N limitation of the plant community in this wetland ecosystem. P addition markedly altered plant community structure in this wetland, which was consistent with the previous studies conducted in temperate and boreal wetlands (Thormann and Bayley, 1997; Bedford et al., 1999; Liu et al., 2015). In the present study, the dominance of D. angustifolia decreased with increasing P addition levels, whereas the dominance of G. spiculosa exhibited an opposite trend. Generally, increased nutrient availability could shift belowground competition for nutrients to aboveground competition for light resources (Grace, 1999). Compared with D. angustifolia, G. spiculosa had greater leaf area and leaf:stem biomass ratio (Mao et al., 2014), and hence may capture more light to enhance the growth rate (Grace, 1999). Therefore, G. spiculosa gained an advantage over D. angustifolia following six years of P addition. Because of the substantial differences in plant functional traits between D. angustifolia and G. spiculosa (Mao et al., 2014, 2015), the altered vegetation community structure induced by P enrichment would further exert marked influences on plant-mediated biogeochemical cycles in this freshwater wetland. 5. Conclusions In summary, species- and community-level plant C:N:P stoichiometry exhibited similar responses to six years of P addition, although there was a shift in dominance from D. angustifolia to G. spiculosa in a temperate wetland, Northeast China. At both the species- and communitylevel, P addition increased plant N and P concentrations, and thus decreased C:N, C:P, and N:P ratios. These results indicate that the effects of P enrichment on plant C:N:P stoichiometry are scale-independent, and imply that the stoichiometric responses to increased P availability at the community level would be mirrored by those at the species level. Moreover, altered plant C:N:P stoichiometry and vegetation community structure induced by P enrichment would enhance organic matter decomposition and nutrient release, and thus further intensify the eutrophication process and cause C loss from soils in N-limited temperate freshwater wetlands.
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Acknowledgments We would like to thank Dr. De-Yan Liu, Gui-Sheng Yang, Yan-Yu Song, and Gui-Yan Ai for the field and laboratory works and two anonymous reviewers and the editor for their helpful comments and suggestions for improving the manuscript. This study was funded by the “Strategic Priority Research Program — Climate Change: Carbon Budget and Related Issue” of the Chinese Academy of Sciences (No. XDA05050508), the National Natural Science Foundation of China (Nos. 31570479 and 41125001), and the Youth Innovation Promotion Association of CAS (No. 2013152). References Aerts, R., Verhoeven, J.T.A., Whigham, D.F., 1999. Plant-mediated controls on nutrient cycling in temperate fens and bogs. Ecology 80, 2170–2181. Bedford, B.L., Walbridge, M.R., Aldous, A., 1999. Patterns in nutrient availability and plant diversit of temperate North American wetlands. Ecology 80, 2151–2169. Bennett, E.M., Carpenter, S.R., Caraco, N.F., 2001. Human impacts on erodible phosphorus and eutrophication: a global perspective. Bioscience 51, 227–234. Bian, J., Berninger, J.P., Fulton, B.A., Brooks, B.W., 2013. Nutrient stoichiometry and concentrations influence silver toxicity in the aquatic macrophyte Lemna gibba. Sci. Total Environ. 449, 229–236. Bracken, M.E.S., Hillebrand, H., Borer, E.T., Seabloom, E.W., Cebrian, J., Cleland, E.E., Elser, J.J., Gruber, D.S., Harpole, W.S., Ngai, J.T., Smith, J.E., 2014. Signature of nutrient limitation and co-limitation: response of autotroph internal nutrient concentrations to nitrogen and phosphorus additions. Oikos 124, 113–121. Chapin, F.S., 1980. The mineral nutrition of wild plants. Annu. Rev. Ecol. Syst. 11, 233–260. Chapin, F.S., Moilanen, L., Kielland, K., 1993. Preferential use of organic nitrogen for growth by a non-mycorrhizal arctic sedge. Nature 361, 150–153. Chen, F.S., Niklas, K.J., Liu, Y., Fang, X.M., Wan, S.Z., Wang, H., 2015. Nitrogen and phosphorus additions alter nutrient dynamics but not resorption efficiencies of Chinese fir leaves and twigs differing in age. Tree Physiol. 35, 1106–1117. Chiang, C., Craft, C.B., Rogers, D.W., Richardson, C.J., 2000. Effects of 4 years of nitrogen and phosphorus additions on Everglades plant communities. Aquat. Bot. 68, 61–78. Cusell, C., Kooijman, A., Lamers, L.P.M., 2014. Nitrogen or phosphorus limitation in rich fens? Edaphic differences explain contrasting results in vegetation development after fertilization. Plant Soil 384, 153–168. Danger, M., Daufresne, T., Lucas, F., Pissard, S., Lacroix, G., 2008. Does Liebig's law of the minimum scale up from species to communities? Oikos 117, 1741–1751. De Long, J.R., Sundqvist, M.K., Gundale, M.J., Giesler, R., Wardle, D.A., 2016. Effects of elevation and nitrogen and phosphorus fertilization on plant defence compounds in subarctic tundra heath vegetation. Funct. Ecol. 30, 314–325. Ebina, J., Tsutsui, T., Shirai, T., 1983. Simultaneous determination of total nitrogen and total phosphorus in water using peroxodisulfate oxidation. Water Res. 17, 1721–1726. Elser, J.J., Bracken, M.E.S., Cleland, E.E., Gruner, D.S., Harpole, W.S., Hillebrand, H., Ngai, J.T., Seabloom, E.W., Shurin, J.B., Smith, J.E., 2007. Global analysis of nitrogen and phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol. Lett. 10, 1135–1142. Elser, J.J., Fagan, W.F., Kerkhoff, A.J., Swenson, N.G., Enquist, B.J., 2010. Biological stoichiometry of plant production: metabolism, scaling and ecological response to global change. New Phytol. 186, 593–608. Feller, I.C., Lovelock, C.E., McKee, K.L., 2007. Nutrient addition differentially affects ecological processes of Avicennia germinans in nitrogen versus phosphorus limited mangrove ecosystems. Ecosystems 10, 347–359. Grace, J.B., 1999. The factors controlling species density in herbaceous plant communities: an assessment. Perspect. Plant Ecol. 2, 1–28. Guo, Y.D., Lu, Y.Z., Song, Y.Y., Wan, Z.M., Hou, A.X., 2014. Concentration and characteristics of dissolved carbon in the Sanjiang Plain influenced by long-term land reclamation from marsh. Sci. Total Environ. 466-467, 777–787. Güsewell, S., Koerselman, W., Verhoeven, J.T.A., 2003. Biomass N:P ratios as indicators of nutrient limitation for plant populations in wetlands. Ecol. Appl. 13, 372–384. Han, X., Sistla, S.A., Zhang, Y.H., Lü, X.T., Han, X.G., 2014. Hierarchical responses of plant stoichiometry to nitrogen deposition and mowing in a temperate steppe. Plant Soil 382, 175–187. Hessen, D.O., Elser, J.J., Sterner, R.W., Urabe, J., 2013. Ecological stoichiometry: an elementary approach using basic principles. Limnol. Oceanogr. 58, 2219–2236. Höll, B.S., Fiedler, S., Jungkunst, H.F., Kalbitz, K., Freibauer, A., Drösler, M., Stahr, K., 2009. Characteristics of dissolved organic matter following 20 years of peatland restoration. Sci. Total Environ. 408, 78–83. Iversen, C.M., Bridgham, S.D., Kellogg, L.E., 2010. Scaling plant nitrogen use and uptake efficiencies in response to nutrient addition in peatlands. Ecology 91, 693–707. Li, L.J., Zeng, D.H., Yu, Z.Y., Fan, Z.P., Mao, R., Peri, P.L., 2011. Foliar N/P ratio and nutrient limitation to vegetation growth on Keerqin sandy grassland of North-East China. Grass Forage Sci. 66, 237–242. Liu, C., Bu, J., Ma, J., Yuan, M., Feng, L., Liu, S., 2015. Comparative study on the response of deciduous and evergreen shrubs to nitrogen and phosphorus input in Hani Peatland of Changbai Mountains. Chin. J. Ecol. 34, 2711–2719. Mao, R., Zhang, X.H., Song, C.C., 2014. Effects of nitrogen addition on plant functional traits in freshwater wetland of Sanjiang Plain, Northeast China. Chin. Geogr. Sci. 24, 674–681.
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Mao, R., Zeng, D.H., Zhang, X.H., Song, C.C., 2015. Responses of plant nutrient resorption to phosphorus addition in freshwater marsh of Northeast China. Sci. Rep. 5, 8097. Mayor, J.R., Wright, S.J., Turner, B.L., 2014. Species-specific responses of foliar nutrients to long-term nitrogen and phosphorus additions in a lowland tropical forest. J. Ecol. 102, 36–44. Neff, J.C., Chapin, F.S., Vitousek, P.M., 2003. Breaks in the cycle: dissolved organic nitrogen in terrestrial ecosystems. Front. Ecol. Environ. 1, 205–211. Ostertag, R., 2010. Foliar nitrogen and phosphorus accumulation responses after fertilization: an example from nutrient-limited Hawaiian forests. Plant Soil 334, 85–98. Peñuelas, J., Poulter, B., Sardans, J., Ciais, P., van der Velde, M., Bopp, L., Boucher, O., Godderis, Y., Hinsinger, P., Llusia, J., Nardin, E., Vicca, S., Obersteiner, M., Janssens, I.A., 2013. Human-induced nitrogen-phosphorus imbalances alter natural and managed ecosystems across the globe. Nat. Commun. 4, 2934. Rejmánková, E., 2001. Effect of experimental phosphorus enrichment on oligotrophic tropical marshes in Belize, Central America. Plant Soil 236, 33–53. Rejmánková, E., Snyder, J.M., 2008. Emergent macrophytes in phosphorus limited marshes: do phosphorus usage strategies change after nutrient addition? Plant Soil 313, 141–153. Rejmánková, E., Macek, P., Epps, K., 2008. Wetland ecosystem changes after three years of phosphorus addition. Wetlands 28, 914–927.
Sardans, J., Rivas-Ubach, A., Peñuelas, J., 2012. The C:N:P stoichiometry of organisms and ecosystems in a changing world: a review and perspectives. Perspect. Plant Ecol. 14, 33–47. Shaver, G.R., Chapin, F.S., 1980. Response to fertilization by various plant growth forms in an Alaskan tundra: nutrient accumulation and growth. Ecology 61, 662–675. Sistla, S.A., Appling, A.P., Lewandowska, A.M., Taylor, B.N., Wolf, A.A., 2015. Stoichiometric flexibility in response to fertilization along gradients of environmental and organismal nutrient richness. Oikos 124, 949–959. Thormann, M.N., Bayley, S., 1997. Response of aboveground net primary plant production to nitrogen and phosphorus fertilization in peatlands in southern boreal Alberta, Canada. Wetlands 17, 502–512. Townsend, A.R., Asner, G.P., 2013. Multiple dimensions of resource limitation in tropical forests. Proc. Natl. Acad. Sci. U. S. A. 110, 4864–4865. Verhoeven, J.T.A., Schmitz, M.B., 1991. Control of plant growth by nitrogen and phosphorus in mesotrophic fens. Biogeochemistry 12, 135–148. Yuan, Z.Y., Chen, H.Y.H., 2015. Negative effects of fertilization on plant nutrient resorption. Ecology 96, 373–380. Zechmeister-Boltenstern, S., Keiblinger, K.M., Mooshammer, M., Peñuelas, J., Richter, A., Sardans, J., Wanek, W., 2015. The application of ecological stoichiometry to plant–microbial–soil organic matter transformations. Ecol. Monogr. 85, 133–155.