*3.2. Relationships between Leaf Ecological Stoichiometry and Environmental Variables*

Plant nutrient concentrations and their correlations with soil nutrients are considered effective tools for exploring plant adaptation and resource utilization strategies in a severe environment [28,44]. In our study, few significant correlations between leaf ecological stoichiometry of *S. chamaejasme* and grassland soil physicochemical properties were observed, implying an insensitive response to the changes in the soil nutrient supply of *S. chamaejasme*. This supports the finding of Geng et al. [26] and provides confirmation that wide-ranging species are usually able to use a wide array of resources and tolerate broad environmental conditions or physiological stresses, and hence flourish over a larger area. The poor synchronization with local edaphic conditions demonstrates a capacity of *S. chamaejasme* to maintain a high level of function at both high and low resource levels, resulting in their broad distributions in China's grasslands. Further, stoichiometric homeostasis can help the plants to maintain their element composition at a relatively stable level regardless of changes in nutrient availability via various physiological mechanisms [2,12], and the degree of stoichiometric homeostasis can be indicated by the homeostatic coefficient (*H*) [12,45,46]. Stoichiometric homeostasis had been reported in many dominant palatable species [15,18,47] in grasslands. However, this has not been established in poisonous species. Since unpalatable plants represent the majority of the plant species that were detected after grasslands have been degraded globally [48–50], revealing the ecophysiology characteristics of poisonous weeds will help us better understand how the communities that are dominated by poisonous weeds form. Generally, species-level stoichiometric homeostasis was positively correlated with the stability of vegetation [15,18,51]. Meanwhile, the species with the highest degree of N homeostasis consistently had the relatively highest growth rates [19] and well-developed storage systems [15,52]. Therefore, resource utilization and storage functions of these species mitigates environmental variations [53], resulting in spatiotemporal stability in abundance [54]. Our results showed that *S. chamaejasme* leaves contain different homeostasis strength of C, N, and K contents and its ratios, which means *S. chamaejasme* could be more conservative in their use of nutrients improving their adaptation to diverse conditions.

Besides, growing plants induce changes in the composition of soil communities and the physicochemical soil environment [55,56]. A previous study in an alpine meadow ecosystem has shown that *S. chamaejasme* produced more aboveground litter with higher tissue N and lower lignin:N than each of the co-occurring species, and significantly increased the surface soil organic matter [19]. Another study found that *S. chamaejasme* had

different ammonia oxidizing bacterial (AOB) that were present with low ammonia oxidation rates, which means greater N availability for *S. chamaejasme* growth due to losses of N reduction [57]. *S. chamaejasme* have positive feedback on soil processes, especially soil C and N, which could be the reason that why leaf C and N contents of *S. chamaejasme* were not different among the two sites of distinct environments, and both were unaffected by local soil factors. Besides, the soil biota plays a pivotal role in modulating primary production by controlling decomposition and nutrient availability, as well as affecting root grazing and plant nutrient uptake [58,59]. Generally, roots of invasive plants enhance or reduce their mutualistic associations with different mycorrhizal fungi or N fixing bacteria [60], which potentially feedback to plant invasion by enhancing N uptake of invaders [61] or by lowering the dependence of plant invaders on arbuscular mycorrhizal fungi (AMF) compared to native species [62]. A recent study suggested that *S. chamaejasme* possessed high ratios of plant growth-promoting proteobacterial endophytes such as *Pseudomonas*, *Acinetobacter,* and *Brevundimonas* [63], and its invaded soils had a lower relative abundance of AMF, but greater pathogenic fungi [64], which in consistent with many invasive plants. Past studies have revealed that *S. chamaejasme* can cultivate the soil environment differently, facilitating it spread rapidly in degraded grassland although it is a native species.

Our results indicate that in the macro scale, leaf C and N do not directly correlate with meteorological factors (MAT and MAP), which is in agreement with previous studies that were conducted in the grassland biomes of China [7]. The weak relationships that was observed between leaf C, N, and climatic variables may result from plant growth, development, metabolism, phenological, and life-history traits rather than from the specific geographic environment. On the contrary, there were close relationships between the leaf P and K and climatic factors (Figure 1). The relationship between the leaf P and climate factors was significant only in IM, and the K content of *S. chamaejasme* leaves was significantly related to climate factors only in QT. We noticed that the correlation of leaf P and MAP (R<sup>2</sup> = 0.5523) was greater than the relationship between P and MAT (R<sup>2</sup> = 0.4886) in IM, and the relationship between K and MAT (R<sup>2</sup> = 0.3338) was greater than that with MAP (R<sup>2</sup> = 0.2920) in QT. These again reflect the different limiting factors of plant growth in different regions [38]. It is a reasonable assumption that precipitation is a more important limiting factor than the temperature for vegetation growth in arid and semi-arid regions such as Inner Mongolian Plateau temperate grasslands. However, the variation in MAP seems very small and the positive relationships among MAP, soil water content, soil P, and AP were weak (Figure S1). We suggested that altitude changes may the reason behind a positive association between MAP and leaf P in IM considering the ranges of geographical distribution and altitude (N 41.34◦~44.77◦ , E 115.30◦~118.16◦ , 1060 m to 1535 m). Our results for the strong relationships between leaf P, soil P and AP, and soil water content and altitude in IM supported our suggestion (Figure S1). In contrast, the temperature is more likely to have a greater effect on the leaf element concentrations than precipitation in QT alpine grasslands with high-altitude and low temperature. We also found that only leaf K was negatively correlated with MAP in QT. One possible explanation is that K leaches more easily from leaves than N and P, hence it is easy to ascertain the increase of MAP in the studies area leading to more leaf K leaching of *S. chamaejasme*. Another possible explanation may be that K plays many fundamental physiological and metabolic roles in terrestrial plants in relation to water-use efficiency [34]. Some recent studies have observed that K concentrations are related to drought resistance [65,66], therefore, the leaf K content tends to decrease with MAP increase.

Moreover, the MAP or MAT contribution was stronger in leaf elements than soil, which illustrated once more the relatively weak effect of soil physicochemical properties on leaf elements. To explore complex relationships between soil and climatic factors on the leaf P and K contents of *S. chamaejasme*, we conducted a PLS-PM analysis. We found that soil exerted a significant effect on leaf P content and climate affected leaf K directly in QT, while the leaf P content appeared to be limited mainly by climate but the leaf K content was not affected significantly in IM. This does not fit with the fact that climate factors which often

affect leaf elements through their influence on soil nutrient status [67]. The arid conditions of the IM may have restricted grassland plants growth by insufficient water supply, but the results of our previous study [21] have proven that high water use efficiency plus high nutrient uptake efficiency of *S. chamaejasme* ensures its competitive advantage on degraded grasslands in IM, which makes the relationship between leaf P or K of *S. chamaejasme* and the soil factors weak in the IM region. However, the leaf P content was positively correlated with soil factors (soil P, available P, nitrate N, and pH), which was not entirely consistent with the result that was obtained in IM. The negative influence of climatic variables on leaf K was significant in QT. This may be the result of the negative relationship between MAP and leaf K, because K shows a greater loss from the plant canopy by foliar leaching than other nutrients such as N and P [34,68]. Our model suggests that the underlying mechanisms behind the leaf P or leaf K content in *S. chamaejasme* were different in the two regions that were studied, which means *S. chamaejasme* developed adjustable relationships with environmental factors to adapt to different growth conditions, thus facilitating its spread in degraded grasslands.

In addition, species' natural habitats will be subjected to more disturbances in the future due to climate change and habitat degradation that is caused by intensive anthropogenic activities [69,70]. Thus, continuing wide-scale sampling and considering the influence of human activities are required to further develop a deeper understanding of the geographic patterns of leaf stoichiometry in *S. chamaejasme*.
