Stoichiometric traits (N:P) of understory plants contribute to reductions in plant diversity following long-term nitrogen addition in subtropical forest.

Nitrogen enrichment is pervasive in forest ecosystems, but its influence on understory plant communities and their stoichiometric characteristics is poorly understood. We hypothesize that when forest is enriched with nitrogen (N), the stoichiometric characteristics of plant species explain changes in understory plant diversity. A 13-year field experiment was conducted to explore the effects of N addition on foliar carbon (C): N: phosphorus (P) stoichiometry, understory plant species richness, and intrinsic water use efficiency (iWUE) in a subtropical Chinese fir forest. Four levels of N addition were applied: 0, 6, 12, and 24 g m-2 year-1. Individual plant species were categorized into resistant plants, intermediate resistant plants, and sensitive plants based on their response to nitrogen addition. Results showed that N addition significantly decreased the number of species, genera, and families of herbaceous plants. Foliar N:P ratios were greater in sensitive plants than resistant or intermediate resistant plants, while iWUE showed an opposite trend. However, no relationship was detected between soil available N and foliar N, and soil N:P and foliar N:P ratios. Our results indicated that long-term N addition decreased the diversity of understory plants in a subtropical forest. Through regulating water use efficiency with N addition, sensitive plants change their N:P stoichiometry and have a higher risk of mortality, while resistant plants maintain a stable N:P stoichiometry, which contributes to their survival. These findings suggest that plant N:P stoichiometry plays an important role in understory plant performance in response to environmental change of N.

INTRODUCTION

Over the past century, fossil‐fuel burning and artificial fertilizer application have substantially increased the global class="Chemical">nitrogen (N) depositioclass="Chemical">n (Deciclass="Chemical">na et al. 2020; Galloway et al., 2008; IPCC, 2014). For example, N emissioclass="Chemical">ns have iclass="Chemical">ncreased substaclass="Chemical">ntially iclass="Chemical">n receclass="Chemical">nt years due to rapid iclass="Chemical">ndustrializatioclass="Chemical">n aclass="Chemical">nd urbaclass="Chemical">nizatioclass="Chemical">n (Liu, 2019; Yu et al., 2019), aclass="Chemical">nd aclass="Chemical">n estimated 7.6–20 Tg of reactive N is beiclass="Chemical">ng emitted iclass="Chemical">nto the atmosphere iclass="Chemical">n Chiclass="Chemical">na (Cui et al. 2013). Much research has focused oclass="Chemical">n uclass="Chemical">nderstaclass="Chemical">ndiclass="Chemical">ng how ecoclass="Chemical">n class="CellLine">system processes and plant diversity respond to increasing N deposition (Valliere et al. 2020; Yang et al. 2020; Zak et al. 2019), and many studies show that N deposition poses a significant threat to plant diversity and causes substantial changes in the plant community composition in terrestrial ecosystems (de Vries & Bobbink, 2017; Zhang et al. 2019). Major drivers of such effects of N on plant communities are soil acidification (Lu et al. 2014) and altered plant‐plant interactions (Gilliam et al., 2016; Liu et al. 2017). However, mechanisms explaining reductions in plant diversity in response to long‐term N addition remain unresolved.

Plant elemental allocation, that is, the leaf contents of class="Chemical">carbon (C), N, class="Chemical">n class="Chemical">phosphorus (P), and their C:N:P stoichiometric ratios, is highly species specific (Elser et al., 2007; Reich & Oleksyn, 2004; Tian et al., 2018). N enrichment can directly affect plant N allocation via leaf absorption and root uptake (Bobbink et al., 2010), and thus may change leaf N:P stoichiometry (Mayor et al. 2014) and affect ecosystem properties (Huang et al. 2016; Li et al. 2020). Since plant transpiration or water use efficiency regulates plant nutrient supply in response to N deposition (Lu et al., 2018). Further, due to reduced soil P concentration after N addition (Deng et al. 2017; Marklein & Houlton, 2012), N enrichment can affect leaf P concentrations by altering ecosystem P cycling (Marklein & Houlton, 2012). Recent studies show that both below‐ground community of soil microorganisms (Zheng et al. 2019) and above‐ground plant community (Fan et al., 2015) are strongly related to plant N:P stoichiometry, and that plant C:N:P stoichiometric characteristics regulate ecosystem functions such as plant production and carbon cycling (Li et al., 2020). However, the relationships between the stoichiometric N:P ratios and plant species richness under long‐term anthropogenic N deposition are poorly understood.

The aim of this study was to investigate the effects of long‐term N addition on the leaf C:N:P stoichiometric characteristics of the understory plant community in a subtropical forest. We wanted to determine if the responses of plant diversity to N addition are regulated by species‐specific stoichiometry. We hypothesized that: (a) long‐term N addition will negatively impact understory plant diversity; (b) class="Species">sensitive, resistaclass="Chemical">nt, aclass="Chemical">nd iclass="Chemical">ntermediate resistaclass="Chemical">nt placlass="Chemical">nt species will have differeclass="Chemical">nt stoichiometric respoclass="Chemical">nses to loclass="Chemical">ng‐term N additioclass="Chemical">n; aclass="Chemical">nd (c) reductioclass="Chemical">ns iclass="Chemical">n placlass="Chemical">nt diversity resulticlass="Chemical">ng from N additioclass="Chemical">n are related to leaf C:N:P stoichiometric characteristics, with class="Chemical">n class="Species">sensitive plant species showing greater changes in their stoichiometric characteristics after N enrichment. These hypotheses reflect previous research showing that N enrichment causes changes in plant diversity, stoichiometric characteristics, and water use efficiency in other forest ecosystems (Maes et al., 2020; Maxwell et al., 2019; Valliere et al. 2020). Our study was investigated in a Chinese fir (Cunninghamia lanceolata) forest, where we explored responses of understory plants to experimental long‐term (13 years) N addition. The understory plant species were subdivided into three N resistant types based on previous studies, that is, resistant, intermediate resistant, and sensitive plants (Wu et al. 2013). Understory plant diversity, foliar C:N:P stoichiometric characteristics, and intrinsic water use efficiency were also measured in the long‐term field experiment.

MATERIALS AND METHODS

Study site

The study was conducted in Chinese fir (C. lanceolata) plantation forest at the Guanzhuang National Forestry Farm (117°43′E, 26°30′N) in Sanming City, Fujian Province, South China. The ecoclass="CellLine">system has a typical subtropical moclass="Chemical">nsooclass="Chemical">n climate with meaclass="Chemical">n aclass="Chemical">nclass="Chemical">nual precipitatioclass="Chemical">n raclass="Chemical">nge of 1,606–1,650 mm aclass="Chemical">nd a meaclass="Chemical">n aclass="Chemical">nclass="Chemical">nual temperature raclass="Chemical">nge of 18.8–19.6°C. Aclass="Chemical">nclass="Chemical">nual average N depositioclass="Chemical">n (iclass="Chemical">ncludiclass="Chemical">ng NO3 − aclass="Chemical">nd NH4 +) from raiclass="Chemical">nfall is 4.2–5.7 g N m−2 year−1 iclass="Chemical">n Saclass="Chemical">nmiclass="Chemical">ng regioclass="Chemical">n (Xiao, 2005). The soil is aclass="Chemical">n class="Chemical">n class="Chemical">acrisol in the U.S. soil taxonomy (soil organic carbon 18.39 g/kg, soil bulk density 1.06 g/cm3, and soil pH 4.68) (Wu et al. 2013). Chinese fir forest was selected for this study because it is widespread in subtropical China and C. lanceolata is of high commercial value (Wei et al. 2012). The forest used in this study was planted in 1992 at a density of 1,660 trees per ha−1 and covered approximately 5,170 ha on land with uniform site characteristics. At the beginning of the experiment, average tree height was 12 m and mean diameter at breast height (DBH, 1.3 m from the ground) was 16.1 cm. The dominant understory species include Ardisia punctate, Smilax china, Arachniodes hasseltii, and Ficus hirta.

Experimental design

The experiment was set up in December 2003 when the C. lanceolata plantations were 12 years old. Briefly, 12 plots with 20 × 20 m were randomly established over a 6‐ha section of the plantation. There were four treatments randomly located in each of three replicated blocks. The treatment names and amount of N added in g N m−2 year−1 are as follows: N0 (control, 0 g N m−2 year−1), N1 (6 g N m−2 year−1), N2 (12 g N m−2 year−1), and N3 (24 g N m−2 year−1). class="Chemical">Urea [class="Chemical">n class="Chemical">CO(NH2)2] is used as N source in our experiment. For each treatment plot, the required amount of urea [CO(NH2)2] was dissolved in 20 L of tap water, and the solution was sprayed onto the forest floor within the plots every month starting in January 2004. The control plots (N0) received an equivalent volume of water without CO(NH2)2.

Plant investigation and sampling analyses

Understory plant communities were investigated in September 2011, 7 years after the start of the N addition treatment (Wu et al. 2013). One 5 × 5 m subplot was established in each 20 × 20 m plot, and all plants taller than 5 cm were recorded. Plant richness (family, genera, and species) and percentage cover were evaluated within each subplot. Understory plant species were divided into two functional groups: woody plants and herbaceous plants. In September 2016 (13 years after N addition started), the understory plants were reassessed using the same methods as in 2011 (Wu et al. 2013). We divided the understory species into three resistance types: resistant plants (RP), intermediate resistant plants (IRP), and n class="Species">sensitive plants (SP) based oclass="Chemical">n the meaclass="Chemical">n preseclass="Chemical">nce aclass="Chemical">nd abseclass="Chemical">nce of the placlass="Chemical">nt species recorded iclass="Chemical">n 2011. Briefly, placlass="Chemical">nt species fouclass="Chemical">nd iclass="Chemical">n plots with aclass="Chemical">ny of the three N additioclass="Chemical">n treatmeclass="Chemical">nts aclass="Chemical">nd iclass="Chemical">n the coclass="Chemical">ntrol (N0) plots, were classified as RP. Placlass="Chemical">nt species fouclass="Chemical">nd iclass="Chemical">n the N0, N1, aclass="Chemical">nd N2 treatmeclass="Chemical">nt plots, but class="Chemical">not iclass="Chemical">n the N3 treatmeclass="Chemical">nt plots, were categorized as IRP. Ficlass="Chemical">nally, placlass="Chemical">nt species fouclass="Chemical">nd oclass="Chemical">nly iclass="Chemical">n N0 treatmeclass="Chemical">nt plots were categorized as SP.

After the second plant community assessment (species presence and abundance in each treatment plot) in 2016, plant leaves were immediately collected in each plot to measure leaf C, N, P, and their C:N:P stoichiometric traits. Leaves from 22 plant species were collected from the twelve plots of four treatments for foliar chemical analysis. For each individual plant, 5 mature leaves were sampled and bulked to make a composite sample. The fresh plant leaves were oven‐dried at 60°C then ball milled prior to analysis natural abundance of class="Chemical">13C, aclass="Chemical">nd total C, N, aclass="Chemical">nd P. Foliar P was aclass="Chemical">nalyzed by class="Chemical">n class="Chemical">persulfate oxidation followed by colorimetric analysis (Bao, 2000), whereas foliar C and N were analyzed by Elemental Analyzer (Flash 200 EA‐HT, Thermo Fisher Scientific, Inc.). Foliar δ13C was analyzed by Isotope Ratio Mass Spectrometer (Deata V Advantage, Thermo Fisher Scientific, Inc.). The standards for foliar 13C were Pee Dee Belemnite. The calculation as the following:where R represents the isotope ratio (13C/12C) from samples or standards. The analytical precision for δ13C was better than 0.1‰. Based on the value of δ13C, we calculated the intrinsic water use efficiency (iWUE) according to the descriptions from Lu et al. (2018). Briefly, 13C isotopic discrimination (Δ13C) was calculated by (δ13Ca − δ13Cplant)/(1 + δ13Cplant), where δ13Ca and δ13Cplant are the δ13C values of atmospheric CO2 and plant leaves. The δ13C values of atmospheric CO2 and the CO2 concentrations were cited from the Global Monitoring Division of the Earth System Resrearch Laboratory at Mauna Loa Observatory (www.esrl.noaa.gov/gmd/index.html). According the reference of Farquhar et al. (1982), Δ13C = a + (b − a)C/C, where a and b refer to the fraction from diffusion through stomata (4.4‰) and the fraction by ribulose‐1,5 bisphosphate carboxylase/oxygenase enzyme during carboxylation (27‰), respectively. And C/C means the ratio of intercellular to atmospheric CO2 concentration. Based on the above equations, iWUE = (C − C)/1.6.

Statistical analyses

The effect of treatment on foliar stoichiometric characteristics was analyzed using a one‐way analysis of variance (ANOVAs). Repeated measures were used to determine the effects of N addition and sampling year (2011 and 2016) on plant communities. The effects of N addition, plant resistant type, and functional group on foliar C:N:P stoichiometric characteristics were assessed with three‐way ANOVAs. Statistical analyses were performed with R version R 3.3.2 (R Core Team, 2016). Differences were considered significant at the 0.05 level. The relationship between foliar N concentration and P concentration was fitted as: y = ax + b, and correlation coefficients were calculated. All correlations were assessed using regression function in Sigmaplot 12.0 (n class="CellLine">Systat Software Iclass="Chemical">nc.).

RESULTS

Understory plant species diversity

A total of 68 plant species, 54 genera, and 37 families were found across all plots in 2016. There were 51 species found in N0, 32 species in N1, 28 species in N2 and N3. Long‐term N addition decreased significantly the number of families of understory plants compared with N0 treatment (Figure 1a). The number of genera, species richness, and coverage also showed a declining trend with increasing N addition (Figure 1b–d), although the differences between N treatments were not statistically significant. The combined results for 2011 and 2016 analyzed with a repeated measures ANOVA showed N addition significantly reduced the number of families, genera, and species of understory plants (Table 1). Further, the cover of plant families, genera, and species was decreased by N treatment, although this trend was not significant. There was no interaction for species, genera, or family richness between sampling years (2011 and 2016) and N addition treatments (Table 1).

FIGURE 1

Effects of long‐term N addition on plant richness (species‐1c, genera‐1b, and families‐1a) and percentage plant coverage (1d) of understory plants, under the tree canopy of Cunninghamia lanceolata plantation in 2016. Values are means ± SE, n = 3. N0, N1, N2, and N3 refer to addition of 0, 6, 12, and 24 g of N m−2 year−1 in each plot, respectively. The statistical effects (F and p values) of N addition are indicated in figures based on ANOVA and Tukey's honest significant difference test. Different lowercase letters indicate significant differences among N treatments

TABLE 1

The effect of long‐term N addition on understory plant species diversity for woody and herbaceous plants in a Cunninghamia lanceolate plantation

Factors	F (p) values
	Family	Genera	Species	%Coverage	Woody	Herbaceous
SY	0.03 (0.87)	3.82 (0.09)	2,084 (0.13)	3.96 (0.08)	2.17 (0.18)	2.97 (0.12)
N	9.42 (0.005)	9.27 (0.006)	9.50 (0.005)	1.73 (0.24)	1.91 (0.21)	4.18 (0.047)
SY × T	0.45 (0.73)	0.14 (0.94)	0.13 (0.94)	1.19 (0.37)	0.51 (0.69)	0.03 (0.99)

F and (p) values are given for the effects of repeated measures with sampling year (SY: 2011 and 2016), nitrogen treatments (N), and their interactions, on the plant richness and percentage coverage after long‐term N addition

Effects of long‐term N addition on plant richness (species‐1c, genera‐1b, and families‐1a) and percentage plant coverage (1d) of understory plants, under the tree canopy of n class="Species">Cunninghamia lanceolata placlass="Chemical">ntatioclass="Chemical">n iclass="Chemical">n 2016. Values are meaclass="Chemical">ns ± SE, class="Chemical">n = 3. N0, N1, N2, aclass="Chemical">nd N3 refer to additioclass="Chemical">n of 0, 6, 12, aclass="Chemical">nd 24 g of N m−2 year−1 iclass="Chemical">n each plot, respectively. The statistical effects (F aclass="Chemical">nd p values) of N additioclass="Chemical">n are iclass="Chemical">ndicated iclass="Chemical">n figures based oclass="Chemical">n ANOVA aclass="Chemical">nd Tukey's hoclass="Chemical">nest sigclass="Chemical">nificaclass="Chemical">nt differeclass="Chemical">nce test. Differeclass="Chemical">nt lowercase letters iclass="Chemical">ndicate sigclass="Chemical">nificaclass="Chemical">nt differeclass="Chemical">nces amoclass="Chemical">ng N treatmeclass="Chemical">nts

F and (p) values are given for the effects of repeated measures with sampling year (class="CellLine">SY: 2011 aclass="Chemical">nd 2016), class="Chemical">n class="Chemical">nitrogen treatments (N), and their interactions, on the plant richness and percentage coverage after long‐term N addition

Foliar C:N:P stoichiometric characteristics

Foliar C did not respond to the N addition treatments (Figure 2a; Table 2), but foliar N was greater for herbaceous plants of the SP than IRP and RP categories (p =0.009). Foliar N of herbaceous plants was higher than woody plants within RP group (Figure 2b). Foliar P of SP was slightly higher than for IRP and RP within the herbaceous plant group (p =0.066, Figure 2c). The species S. china was selected as representative of RP as it was found and sampled in all four (N0, N1, N2, and N3) treatments. Stoichiometric characteristics of foliar C, N, and P for S. china did not respond to long‐term N addition (Figure S1).

FIGURE 2

Concentrations (foliar C‐2a, foliar N‐2b, and foliar P‐2c) and stoichiometric characteristics (C:N‐2d, C:P‐2e, and N:P‐2f) of foliar C, N, and P for understory species with different functional groups (woody and herbaceous plants) after long‐term N addition. Plants were also divided into three resistant types, that is, sensitive species (SP), intermediate resistant species (IRP), and resistant species (RP). Values are means ± SE, n = 3. Within each functional group, means with different letters are significantly different among resistant types based on ANOVA and Tukey's honest significant difference test (p <.05). Different lowercase letters indicate significant differences among resistant types for woody plants, different uppercase letters indicate significant differences among resistant types for herbaceous plants

TABLE 2

The effect of long‐term N addition, plant resistant type, and functional group on plant C, N, P, C:N:P stoichiometric characteristics and δ13C in a Cunninghamia lanceolate plantation

Factors	F (p) values
	C	N	P	C:N	C:P	N:P	δ13C
N	0.61 (0.66)	0.15 (0.96)	0.04 (0.99)	0.23 (0.92)	0.06 (0.99)	0.15 (0.96)	0.80 (0.53)
RT	0.79 (0.46)	3.83 (0.03)	4.24 (0.02)	2.55 (0.09)	6.93 (<0.01)	2.34 (0.11)	8.74 (<0.01)
FG	0.06 (0.81)	8.07 (0.01)	3.84 (0.06)	11.79 (<0.01)	3.40 (0.07)	3.87 (0.06)	0.55 (0.46)
N × RT	1.43 (0.25)	0.80 (0.46)	0.63 (0.54)	1.07 (0.35)	1.65 (0.21)	2.07 (0.14)	2.38 (0.11)
N × FG	0.61 (0.61)	0.99 (0.41)	1.15 (0.34)	1.14 (0.35)	0.91 (0.45)	1.32 (0.28)	0.90 (0.44)
RT × FG	0.29 (0.75)	0.80 (0.46)	0.25 (0.78)	1.51 (0.24)	0.28 (0.76)	1.57 (0.22)	0.38 (0.69)
N × RT×FG	0.02 (0.90)	1.89 (0.18)	1.10 (0.30)	3.02 (0.09)	1.863 (0.18)	0.83 (0.37)	1.23 (0.27)

F and p values for the effects of three nitrogen treatments (N), plant resistant type (RT), functional group woody or herbaceous (FG), and their interactions on foliar C, N, P, C:N:P stoichiometric characteristics and δ13C after long‐term N addition.

Concentrations (foliar C‐2a, foliar N‐2b, and foliar P‐2c) and stoichiometric characteristics (C:N‐2d, C:P‐2e, and N:P‐2f) of foliar C, N, and P for understory species with different functional groups (woody and herbaceous plants) after long‐term N addition. Plants were also divided into three resistant types, that is, n class="Species">sensitive species (SP), iclass="Chemical">ntermediate resistaclass="Chemical">nt species (IRP), aclass="Chemical">nd resistaclass="Chemical">nt species (RP). Values are meaclass="Chemical">ns ± SE, class="Chemical">n = 3. Withiclass="Chemical">n each fuclass="Chemical">nctioclass="Chemical">nal group, meaclass="Chemical">ns with differeclass="Chemical">nt letters are sigclass="Chemical">nificaclass="Chemical">ntly differeclass="Chemical">nt amoclass="Chemical">ng resistaclass="Chemical">nt types based oclass="Chemical">n ANOVA aclass="Chemical">nd Tukey's hoclass="Chemical">nest sigclass="Chemical">nificaclass="Chemical">nt differeclass="Chemical">nce test (p <.05). Differeclass="Chemical">nt lowercase letters iclass="Chemical">ndicate sigclass="Chemical">nificaclass="Chemical">nt differeclass="Chemical">nces amoclass="Chemical">ng resistaclass="Chemical">nt types for woody placlass="Chemical">nts, differeclass="Chemical">nt uppercase letters iclass="Chemical">ndicate sigclass="Chemical">nificaclass="Chemical">nt differeclass="Chemical">nces amoclass="Chemical">ng resistaclass="Chemical">nt types for herbaceous placlass="Chemical">nts

The effect of long‐term N addition, plant resistant type, and functional group on plant C, N, P, C:N:P stoichiometric characteristics and δn class="Chemical">13C iclass="Chemical">n a Cuclass="Chemical">nclass="Chemical">niclass="Chemical">nghamia laclass="Chemical">nceolate placlass="Chemical">ntatioclass="Chemical">n

F and p values for the effects of three n class="Chemical">nitrogen treatmeclass="Chemical">nts (N), placlass="Chemical">nt resistaclass="Chemical">nt type (RT), fuclass="Chemical">nctioclass="Chemical">nal group woody or herbaceous (FG), aclass="Chemical">nd their iclass="Chemical">nteractioclass="Chemical">ns oclass="Chemical">n foliar C, N, P, C:N:P stoichiometric characteristics aclass="Chemical">nd δclass="Chemical">n class="Chemical">13C after long‐term N addition.

Within the herbaceous plant group, foliar C:N ratios were lower for SP than IRP and RP plant types (p =0.019), but within the woody plant group there was no effect of plant resistant type on foliar C:N (Figure 2d). For herbaceous plants, foliar C:N ratios were lower than woody plants within RP and SP groups (p =0.003 and p =0.072, respectively, Figure 2d). Within both herbaceous (p =0.018) and woody plant groups (p =0.041, Figure 2e), foliar C:P ratios were significantly lower for SP than IRP and RP. Within the herbaceous plant group foliar, N:P ratios were greatest in SP and were higher for herbaceous plants than woody plants (Figure 2f). For the species resistant types, foliar N (p =0.03), P (p =0.02) and C:P ratio (p =0.002) were differed significantly (Table 2). Foliar N and P concentrations were related significantly (Figure 3), but the regression slope of relationship was greater for SP than IRP and RP plant types (Figure 3a), and for herbaceous than woody plants (Figure 3b). No relationship was detected between soil available N and foliar N, or soil N:P and foliar N:P (Figure S2).

FIGURE 3

Relationships between foliar N and P concentrations of understory plants with different resistant types (a) and functional groups (b) after long‐term N addition. In the upper (a), open circles with long dash line mean resistant species, solid squares with solid line mean intermediate resistant species, and solid diamonds with short dash line mean sensitive species. In the lower (b), solid circles with solid line mean woody plants and solid squares with long dash line mean herbaceous plants

Relationships between foliar N and P concentrations of understory plants with different resistant types (a) and functional groups (b) after long‐term N addition. In the upper (a), open circles with long dash line mean resistant species, solid squares with solid line mean intermediate resistant species, and solid diamonds with short dash line mean n class="Species">sensitive species. Iclass="Chemical">n the lower (b), solid circles with solid liclass="Chemical">ne meaclass="Chemical">n woody placlass="Chemical">nts aclass="Chemical">nd solid squares with loclass="Chemical">ng dash liclass="Chemical">ne meaclass="Chemical">n herbaceous placlass="Chemical">nts

Foliar δ13C and iWUE

Foliar δclass="Chemical">13C was highest for the RP placlass="Chemical">nt type aclass="Chemical">nd was differed sigclass="Chemical">nificaclass="Chemical">ntly for both woody aclass="Chemical">nd herbaceous placlass="Chemical">nt groups (p =0.05 aclass="Chemical">nd p =0.017, respectively). There was class="Chemical">no effect of fuclass="Chemical">nctioclass="Chemical">nal group oclass="Chemical">n foliar δclass="Chemical">n class="Chemical">13C within the same plant resistant type (Figure 4a). The two‐way ANOVA showed that plant resistant types, but not N treatment, functional group, or their interactions, significantly affected foliar δ13C (Table 2). iWUE showed the similar trend with δ13C values (Figure 4b). For S. china, foliar δ13C were from −32.2‰ to −33.8‰ without significant variation among N addition treatments (Figure S3).

FIGURE 4

Foliar δ13C (a) and intrinsic water use efficiency (iWUE, b) for understory species with different functional groups (woody and herbaceous plants) after long‐term N addition. Values are means ± SE, n = 3. See Figure 2 for abbreviations

Foliar δclass="Chemical">13C (a) aclass="Chemical">nd iclass="Chemical">ntriclass="Chemical">nsic class="Chemical">n class="Chemical">water use efficiency (iWUE, b) for understory species with different functional groups (woody and herbaceous plants) after long‐term N addition. Values are means ± SE, n = 3. See Figure 2 for abbreviations

DISCUSSION

Tropical and subtropical forests are at risk from increasing levels of N deposition (de Vries & Bobbink, 2017; Yu et al., 2019), and previous studies have suggested that increasing N deposition reduces plant diversity (Lu et al., 2018; Wu et al. 2013). We conducted a field experiment in a subtropical Chinese fir (C. lanceolata) forest to examine long‐term N addition on understory plant diversity and investigate if species class="Chemical">water use efficieclass="Chemical">ncy iclass="Chemical">nflueclass="Chemical">nces their respoclass="Chemical">nses to N depositioclass="Chemical">n. Coclass="Chemical">nsisteclass="Chemical">nt with the results of Wu et al. (2013) usiclass="Chemical">ng data from these plots collected iclass="Chemical">n 2011, aclass="Chemical">nd our first hypothesis, N additioclass="Chemical">n class="Chemical">negatively affected placlass="Chemical">nt diversity, causiclass="Chemical">ng a loss of 33.3%–49.3% of placlass="Chemical">nt species richclass="Chemical">ness duriclass="Chemical">ng 13 years of N additioclass="Chemical">n treatmeclass="Chemical">nt. Siclass="Chemical">nce the light regime was similar iclass="Chemical">n each plot (Wu et al. 2013), we do class="Chemical">not believe the effect of caclass="Chemical">nopy structure oclass="Chemical">n uclass="Chemical">nderstory shade was a maiclass="Chemical">n driver of species distributioclass="Chemical">n. A reductioclass="Chemical">n iclass="Chemical">n placlass="Chemical">nt richclass="Chemical">ness after N additioclass="Chemical">n has beeclass="Chemical">n reported for other ecoclass="Chemical">n class="CellLine">systems such as grassland (Liu et al. 2017), shrubland (Valliere et al. 2020), and forest (Gilliam, 2019). The potential mechanisms for the decline of plant diversity with N enrichment are widely reported to include factors such as soil acidification, increased aluminium mobility, and increased plant competition from fast‐growing, exploitive species (Bobbink et al., 2010; Gilliam, 2019; Lu et al. 2010). However, our results for this subtropical plantation forest suggest a potential mechanism for the survival of understory plant diversity under N enrichment, namely differences in plant species N:P stoichiometric characteristics. Plants N:P stoichiometry in understory plants showed significant differences among species categorized by presence and abundance in the plots into N resistance types.

Few studies have considered plant N:P stoichiometry as a potential mechanism for the survival of plants in response to N addition, which is surprising given the important role that foliar N:P ratios play in regulating plant production and community dynamics (Tian et al., 2018). Consistent with our second hypothesis, foliar N:P stoichiometry of the different N resistant types responded differently to 13 years of N addition. The category of plant resistance type was the main distinguishing feature reflecting foliar N, P, and N:P stoichiometric characteristics, while the N addition treatment was not statistically significant. In particular, we found that resistant plant type showed negligible response of foliar N and P and their N:P stoichiometric characteristics to long‐term N addition, which we suggest these understory plants to persist under high N inputs. In contrast, the class="Species">sensitive plant type (SP) had greater leaf coclass="Chemical">nceclass="Chemical">ntratioclass="Chemical">ns of N thaclass="Chemical">n resistaclass="Chemical">nt (i.e., IRP aclass="Chemical">nd RP placlass="Chemical">nt types) species, which may have iclass="Chemical">nduced sigclass="Chemical">nificaclass="Chemical">nt chaclass="Chemical">nges iclass="Chemical">n N:P values aclass="Chemical">nd iclass="Chemical">ncreased their vulclass="Chemical">nerability to loss uclass="Chemical">nder elevated N. This is coclass="Chemical">nsisteclass="Chemical">nt with the class="Chemical">notioclass="Chemical">n that placlass="Chemical">nts with plastic stoichiometry caclass="Chemical">n chaclass="Chemical">nge their iclass="Chemical">ntriclass="Chemical">nsic elemeclass="Chemical">ntal balaclass="Chemical">nce, which iclass="Chemical">ncreases their risk of loss wheclass="Chemical">n experieclass="Chemical">nciclass="Chemical">ng eclass="Chemical">nviroclass="Chemical">nmeclass="Chemical">ntal chaclass="Chemical">nge (e.g., N depositioclass="Chemical">n) (Sterclass="Chemical">ner & Elser, 2002). Further support for the class="Chemical">notioclass="Chemical">n that placlass="Chemical">nt stoichiometry uclass="Chemical">nderlies placlass="Chemical">nt respoclass="Chemical">nses to N additioclass="Chemical">n comes from a receclass="Chemical">nt report that respoclass="Chemical">nses of foliar N of domiclass="Chemical">naclass="Chemical">nt uclass="Chemical">nderstory species were margiclass="Chemical">nal after 8 years of N additioclass="Chemical">n, which caclass="Chemical">n be categorized as resistaclass="Chemical">nt placlass="Chemical">nt type (Zou et al. 2019). Iclass="Chemical">n additioclass="Chemical">n, a study across class="Chemical">niclass="Chemical">ne terrestrial ecoclass="Chemical">n class="CellLine">systems in North America demonstrated that both the abundance‐ and functional trait‐based mechanisms explained plant diversity losses to N fertilization (Suding et al., 2005). In our study, insignificant relationships between soil available N and foliar N, or soil N:P and foliar N:P supported the idea that plant stoichiometric traits may play important roles in the response of plants to N addition. Since plant N:P values do not always reflect soil nutrient values, and this was also found in other studies (Deyn, 2017).

Differences in foliar δclass="Chemical">13C values have beeclass="Chemical">n related to class="Chemical">n class="Chemical">water use efficiency and associated photosynthetic capacity (Palmroth et al. 2014). For understory species of N sensitive (SP) plants, foliar δ13C was lower than resistant plant (RP) type, which may reflect the lower water use efficiency (Townsend et al. 2007). Foliar δ13C in our study of a representative N resistant species (S. china) did not significantly respond to long‐term N addition. The resistant species may have higher water use efficiency as indicated by the values of intrinsic water use efficiency in this study, which can reduce water uptake during growth. Consequently, resistant species can reduce N nutrient uptake from soil to plant and facilitate them maintaining stable N:P stoichiometry after N enrichment as predicted by our third hypothesis. In support of this, past study has reported that tropical forests with high N background would acclimate N deposition during their evolutionary history (Lu et al., 2018). Our results were also partially supported the Stability of Limiting Elements Hypothesis (Han et al. 2011), where limiting elements in plants have low variability and environmental sensitivity. Since the resistant plants have more stable N:P stoichiometric characteristics and may be less sensitive to environmental gradients, which helps them survival in N loading sites. In tropical and subtropical forests, N enrichment affects N and P concentrations in plants (Chen et al. 2015; Homeier et al., 2012) and shows species‐specific traits (Huang et al. 2016; Lu et al., 2018). Our findings suggest that decreased understory plant diversity is related to plant N:P stoichiometric characteristics.

CONCLUSIONS

Our long‐term experimental results have several implications. First, we confirm that long‐term N deposition can decrease plant diversity in subtropical plantation forests. Understory plants are important components of forest ecoclass="CellLine">systems aclass="Chemical">nd the coclass="Chemical">nsequeclass="Chemical">nce of losiclass="Chemical">ng placlass="Chemical">nt diversity class="Chemical">needs to be coclass="Chemical">nsidered uclass="Chemical">nder global chaclass="Chemical">nge pheclass="Chemical">nomeclass="Chemical">noclass="Chemical">n (e.g. iclass="Chemical">ncreasiclass="Chemical">ng N depositioclass="Chemical">n). Secoclass="Chemical">nd, our results iclass="Chemical">ndicate that the chaclass="Chemical">ngeable stoichiometry of class="Chemical">n class="Species">sensitive plants, that also contain higher N concentration, may result in an elemental imbalance and consequentially put sensitive plants at a higher risk of loss. In contrast, resistant plant types that do not respond to N addition display stable N:P stoichiometric characteristics that enable them to thrive in an environment that may be affected by N deposition. Overall, greater biodiversity is important for ecosystem functions (Landuyt et al., 2019; Mori, 2017), the effects of decreased plant diversity after N deposition on the performance of ecosystem functions need more investigations in the forest ecosystems.

CONFLICT OF INTEREST

We declare no conflict of interest.

AUTHOR CONTRIBUTIONS

Jianping Wu: Conceptualization (equal); data curation (equal); formal analysis (equal); funding acquisition (equal); investigation (equal); methodology (equal); project administration (equal); resources (equal); software (equal); validation (equal); visualization (equal); writing – original draft (equal); writing – review and editing (equal). Fangfang Shen: Data curation (equal); formal analysis (equal); investigation (equal); methodology (equal); writing – original draft (equal). Jill Thompson: Conceptualization (equal); formal analysis (equal); methodology (equal); software (equal); supervision (equal); writing – original draft (equal); writing – review and editing (equal). Wenfei Liu: Data curation (equal); formal analysis (equal); investigation (equal); project administration (equal); resources (equal); writing – original draft (equal). Honglang Duan: Data curation (equal); formal analysis (equal); software (equal); writing – original draft (equal). Richard D. Bardgett: Conceptualization (equal); data curation (equal); formal analysis (equal); supervision (equal); writing – original draft (equal); writing – review and editing (equal).

Supplementary Figure 1. Stoichiometric characteristics of foliar C, N and P for n class="Species">Smilax china after loclass="Chemical">ng‐term N additioclass="Chemical">n. Values are the meaclass="Chemical">ns ± SE of three plots. N0, N1, N2, aclass="Chemical">nd N3 refer to additioclass="Chemical">n of 0, 6, 12, aclass="Chemical">nd 24 g of N m‐2 yr‐1 iclass="Chemical">n each plot, respectively. The statistical effects (F aclass="Chemical">nd P values) of N additioclass="Chemical">n were iclass="Chemical">ndicated iclass="Chemical">n figures based oclass="Chemical">n ANOVA aclass="Chemical">nd Tukey’s hoclass="Chemical">nest sigclass="Chemical">nificaclass="Chemical">nt differeclass="Chemical">nce test.

Supplementary Figure 2. The regressions between soil available N and foliar N (upper), and soil N:P and foliar N:P (lower) after long‐term N addition.

Supplementary Figure 3. Foliar δclass="Chemical">13C for class="Chemical">n class="Species">Smilax china after long‐term N addition. Values are the means ± SE of three plots. N0, N1, N2, and N3 refer to addition of 0, 6, 12, and 24 g of N m‐2 yr‐1 in each plot, respectively. The statistical effects (F and P values) of N addition were indicated in figures based on ANOVA and Tukey’s honest significant difference test.

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