Phenotypic plasticity is defined as the ability of a single plant genotype to produce different phenotypes (morphology, phenology, or physiology) under various biotic and abiotic disturbances, including herbivore grazing, bio-invasion, elevated CO₂, warming, and drought (Nicotra et al., 2010; Dostál et al., 2016; Wang D. et al., 2017). The potential role of phenotypic plasticity on the change of ecosystem function has attracted the interest of grassland ecologists. Furthermore, it provides a new theoretical perspective for understanding how plant functional traits mediate the restoration of grassland ecosystem performance under grazing exclusion (Li et al., 2016a; Wang D. et al., 2017).

In grazing ecosystem, the grazing of large herbivores directly influences the phenotypic characteristics of grassland species by impacting leaf photosynthesis, respiration, and plant development; it can also influence indirectly grassland species by changing the soil micro-environment (Ren et al., 2017; Abdalla et al., 2018). It has long been recognized that plant individuals will become dwarfed in response to overgrazing, whereas plant size will return to normal after exclusion from grazing over a multi-year period (Armitage et al., 2012). Recent results from a long-term grassland trial indicated sheep grazing caused the decline of aboveground biomass of Leymus chinensis, primarily through a ‘bottom-up’ effect due to the asymmetrical response of different plant functional traits with the variations in individual plant size (Li et al., 2015a). During grazing exclusion, however, the patterns of the phenotypic variations of plant species in a long-term study in a degraded pasture are largely unknown.

Over the past few decades, grazing exclusions have become a primary management practice for the restoration of degraded pastures in semi-arid regions worldwide (Han et al., 2008; Jing et al., 2014). Plant–soil interactions play an important role in the maintenance of both plant community composition and soil properties in the transition from degraded grasslands to restored grasslands by grazing exclusion (Laliberté et al., 2012; Odriozola et al., 2017). In general, grazing exclusion can enhance ecosystem stability and productivity in the initial years of the exclusion. (Deng et al., 2017). However, some studies have demonstrated that long-term grazing exclusion could exert a negative influence on species renewal and productivity (Silcock and Fensham, 2013). For example, Jing et al. (2014) reported that the productivity and biodiversity were higher in the mid- and transitional stages of succession, but decreased with increasing restoration time in a semiarid grassland.

Plasticity in plant nutrient traits, including the content, resorption, stoichiometry, and allocation of nutrients, is predicted to play a key role in regulating the morphological plasticity, indirectly affecting the carbon (C) cycle and ecosystem stability (Lü et al., 2012a; Heyburn et al., 2017). The plasticity in the nutrient resorption efficiency and proficiency is an important nutrient strategy for plants subjected to grazing (da Silveira Pontes et al., 2010). In a semi-arid grassland, Lü et al. (2015) found that the dominate grass species displayed decreasing nutrient resorption with the duration of grazing exclusion for nitrogen (N) and phosphorus (P). There is abundant evidence that C:N:P stoichiometry in plant tissues are generally associated with plant growth strategies, which strongly influence the capacity for adaptation to grazing disturbance (Velthuis et al., 2017; Yang et al., 2017). Moreover, nutrient allocation is regulated by grazing primarily through the changes in biomass partitioning and nutrient concentrations (Li et al., 2016b). Grazing-induced shifts in nutrient allocation patterns among different tissues are indirectly influenced by the availability of soil resources (Bai et al., 2012; Zheng et al., 2012). However, there is a lack of long-term investigations into the effects of grazing exclusion on the relationship between plant morphological plasticity and the nutrient strategies of grassland plants.

Native pastures in typical steppe regions cover the largest area in the eastern Eurasian temperate grasslands at the northern boundary of China (Schönbach et al., 2009). In these pastures, the dominant grass species is L. chinensis, which grows rapidly and has a high tolerance to arid conditions. This perennial rhizomatous grass is also very palatable to grazing animals and has a high forage value (Ma et al., 2014; Ren et al., 2014). L. chinensis is considered one of the most promising grass species for grassland rehabilitation and restoration in arid regions of northern China (Liu and Han, 2008; Huang et al., 2015).

Therefore, in this study, L. chinensis (the dominant grassland species) was chosen as a model plant for exploring ecological processes. We examined the effects of grazing exclusion on morphological plasticity, nutrient concentration, accumulation, allocation, and ecological stoichiometry of L. chinensis along a time series in a degraded pasture located in Xilinhot, Inner Mongolia, China. Specifically, we addressed the following four questions: (i) How do morphological plasticity and biomass allocation change across a grazing exclusion chronosequence? (ii) How does grazing exclusion influence the relationships between the size of L. chinensis and the concentrations of carbon (C), nitrogen (N), and phosphorous (P) in leaves and stems? (iii) How does grazing exclusion affect C, N, and P allocation strategies in L. chinensis in plants of varying sizes? (iv) How does grazing exclusion affect the relationship between L. chinensis C:N:P stoichiometry and morphological plasticity?

Morphological plasticity, which exists in various terrestrial plant species, is a comprehensive strategy to adapt to heterogeneous habitats. This strategy allows plants to effectively gain access to more resources (de Vries et al., 2016). Grazing affects natural pastures in the macro-scale performance of ecosystems and the plant function at the micro-scale in semi-arid regions (Sanjuán et al., 2017). Our results demonstrated that L. chinensis leaves and stems exponentially tended to be larger after grazing exclusion over time compared with plants in free-grazing conditions. This is consistent with the findings described in several previous studies (Deléglise et al., 2011; Wu et al., 2013). We detected a dramatic change (0.05–6.8) in the plasticity of different functional traits with the increase of grazing exclusion time. The sensitivity of stems was higher than that of leaves with the increase of grazing exclusion time, indicating that L. chinensis can exhibit rapid pant growth by promoting stem elongation. A previous study on leaf traits indicated that the strategies for stability and plant protection under grazing disturbance provides some indirect evidence for our findings (Jaurena et al., 2013).

In our study, individual L. chinensis leaf and stem mass accumulation exponentially increased during the peak-growth season with the increase of grazing exclusion time. This is consistent with results from previous studies conducted in a variety of grassland habitats (Courtois et al., 2004; Shrestha and Stahl, 2008). However, there was a slight decrease in biomass accumulation after long-term grazing exclusion (plot from 1979) compared to the medium-term plot (established in 1999). This may be related to the tradeoff between leaf and stem growth after long-term grazing exclusion (35-years in this study). Specifically, intraspecific and interspecific competition for light resources will be intensified in grazing exclusion habitats (Borer et al., 2014); therefore, plant stem elongation will be stimulated as a shade avoidance response to light limitations (Baldissera et al., 2016). Photoassimilates will be preferentially allocated to the stem to rapidly increase the plant individual height during the stem elongation process (Williamson et al., 2012), which may be the underlying mechanism for our finding that the leaf to stem mass ratio significantly decreases in the long-term grazing exclusion habitats.

We initially hypothesized that increased nutrient concentrations in plant tissues would support plant morphological plasticity in response to grazing exclusion, but this was not supported by our results that grazing exclusion significantly decreased N and P concentrations. Theoretically, this may be explained by three previously discussed hypotheses. First, the growth dilution hypothesis: if increased accumulation of shoot mass is more than the increase in nutrient acquisition under grazing exclusion, nutrient concentrations will decrease (Li et al., 2016b). Second, the decreasing metabolism hypothesis: decreased N and P concentrations might weaken the number and activity of photosynthetic enzymes and improve the photosynthetic rate when plants are grown under grazing exclusion conditions (Scogings et al., 2013). Third, the nutrient competition hypothesis: competition for soil nutrients under grazing exclusions became significantly more intense compared to grazed habitats, contributing to the decrease of nutrient concentrations (Lü et al., 2015). In general, our finding that grazing exclusion significantly decreased N and P concentrations in L. chinensis is similar with previous studies on experimental grazing, mowing, elevated CO₂, and drought, supported by the mechanisms discussed above (Fernando et al., 2014; Li et al., 2016b).

We observed a sharp decrease of N and P concentrations after grazing exclusion in the degraded pasture; however, only minor changes in N concentration and increases in P were detected across grazing exclusions established in 2005, 1999, and 1979. Therefore, the growth dilution hypothesis provides only a partial explanation for these findings. It is also likely associated with the drivers of the metabolism regulation hypothesis. In contrast, we found that C concentrations in the three sampling positions rapidly increased after grazing exclusions. This is most likely related to greater photosynthetic function and an adequate supply of carbon assimilates in the grazing excluded pastures. This suggests that there was a tradeoff of C concentration with N and P concentrations, and it may provide new insight into plant nutrient strategies in response to grazing exclusion.

An interesting result was that leaf to stem ratios of N, P, and C accumulation were negatively correlated with plant size induced by grazing exclusion. We also found that the degrees of plasticity of L. chinensis individuals in stem traits were markedly higher than that in leaf traits under grazing exclusion. We speculate that there may be some theoretical correlation between nutrient allocation and leaf-stem allometry (Li et al., 2015a). Our SEM results demonstrated that the changes in N, P, and C allocation induced by grazing exclusion were derived from leaf to stem ratios of biomass and nutrient concentration. A previous study has shown that light competition affects plant growth in restored grassland communities (Loydi et al., 2015). For example, shade avoidance can significantly promote the nutrient allocation and growth of plant stems (Heger, 2016). Hence, with the increase of grazing exclusion time, the increased stem nutrient allocation in response to light competition may explain the observed stimulation of stem elongation and biomass accumulation.

The C:P and C:N ratios of plant illustrate the C assimilation efficiency when absorbing one unit N and P. In general, these ratios can be a predictive indicator of nitrogen- and phosphorus-use efficiency in many ecosystems (Zhou et al., 2013). Increased nutrient use efficiency in plants is known to be an important strategy for adaptation to nutrient-poor habitats. Our finding that grazing exclusion increased C:N and C:P ratios and decreased N and P concentrations is not consistent with previous studies on soil nutrient enrichments (Feyisa et al., 2017; Wang T. et al., 2017), thus suggesting that soil nutrient changes induced by grazing exclusion are not the primary reason for changes in plant nutrient strategies. In addition, we found that grazing exclusion significantly increased the C:N and C:P ratios in leaves, stems, and the aboveground parts of L. chinensis individuals. This implies that changes in C:N and C:P ratios of plant tissues may be a temporary response to livestock grazing, which differs from the lag effect of plant morphology, community productivity, and soil features.

Nutrient limitation for grassland plants in a plant-herbivore interaction system has long been a controversial topic. The N:P ratio in ecological stoichiometry is often adopted to estimate nutrient limiting factors in plants across many ecosystems (Zhan et al., 2017). A previous study revealed that a N:P ratio > 16 indicates P limitation at a community level, while a N:P ratio < 14 is indicative of N limitation (Bai et al., 2012). We found that N:P in L. chinensis individuals was lower than the threshold of 14:1 for N limitation, and that N:P gradually decreased with the increase of grazing exclusion time, especially in L. chinensis stems. This may be related to the increasing rapid growth and N requirements of stems with the increase of the duration of grazing exclusion.

Our findings have important implications for understanding the potential effects of grazing on plant traits and nutrient strategies in grassland regions, and for developing useful management practices to improve pasture productivity (Wu et al., 2014). Theoretically, we provide new evidence for understanding the relationship between plant size and nutrients in grazing ecosystems. Practically, grazing-induced changes in individual plant size under increased human activity will dramatically affect pasture productivity, and the effects will be especially profound in the pastures in Inner Mongolia, China (Bai et al., 2012). Based on our findings that there were potentially P and N limitations of the dominated L. chinensis in free-grazing and grazing exclusion grasslands respectively, we suggest promoting pasture utilization reasonably and P-optimized management should be preferred in degraded grasslands, whereas N-optimized management should be the primary measure to increase pasture productivity in grazing exclusion pastures.

We concluded that the increase of grazing exclusion time exponentially increased plant morphological traits, and nutrient concentrations and stoichiometry in the degraded pasture were also strongly impacted. There was a significant negative correlation between the degrees of plasticity and stability of various morphological traits. The patterns of biomass allocation of L. chinensis were significantly correlated with the increased duration of grazing exclusion, primarily determined by changes in stem traits. Phenotypic traits were negatively correlated with N and P concentrations, but they were positively correlated with C concentration. Further, we detected that there was a tradeoff between concentrations and the accumulation of N and P, but coordinated variation of C. We discovered that changes in N, P, and C allocation induced by grazing exclusion were derived from biomass partitioning and leaf to stem ratios of nutrient concentrations. In general, our findings suggest that there is a significant negative effect between plant sizes and nutrient traits under grazing exclusion.

ZL: wrote the paper and the data analysis. TB: designed the study. JD: carried out the experiments. GY, JS, and XL collected the data and data analysis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.