*Article* **Biogeographic Patterns of Leaf Element Stoichiometry of** *Stellera chamaejasme* **L. in Degraded Grasslands on Inner Mongolia Plateau and Qinghai-Tibetan Plateau**

**Lizhu Guo 1,2 , Li Liu <sup>3</sup> , Huizhen Meng <sup>4</sup> , Li Zhang <sup>4</sup> , Valdson José Silva <sup>5</sup> , Huan Zhao <sup>6</sup> , Kun Wang <sup>1</sup> , Wei He <sup>4</sup> and Ding Huang 1,\***


**Abstract:** Plant leaf stoichiometry reflects its adaptation to the environment. Leaf stoichiometry variations across different environments have been extensively studied in grassland plants, but little is known about intraspecific leaf stoichiometry, especially for widely distributed species, such as *Stellera chamaejasme* L. We present the first study on the leaf stoichiometry of *S. chamaejasme* and evaluate its relationships with environmental variables. *S. chamaejasme* leaf and soil samples from 29 invaded sites in the two plateaus of distinct environments [the Inner Mongolian Plateau (IM) and Qinghai-Tibet Plateau (QT)] in Northern China were collected. Leaf C, N, P, and K and their stoichiometric ratios, and soil physicochemical properties were determined and compared with climate information from each sampling site. The results showed that mean leaf C, N, P, and K concentrations were 498.60, 19.95, 2.15, and 6.57 g kg−<sup>1</sup> ; the average C:N, C:P, N:P, N:K and K:P ratios were 25.20, 245.57, 9.81, 3.13, and 3.21, respectively. The N:P:K-ratios in *S. chamaejasme* leaf might imply that its growth is restricted by K- or K+N. Moreover, the soil physicochemical properties in the *S. chamaejasme*-infested areas varied remarkably, and few significant correlations between *S. chamaejasme* leaf ecological stoichiometry and soil physicochemical properties were observed. These indicate the nutrient concentrations and stoichiometry of *S. chamaejasme* tend to be insensitive to variations in the soil nutrient availability, resulting in their broad distributions in China's grasslands. Besides, different homeostasis strength of the C, N, K, and their ratios in *S. chamaejasme* leaves across all sites were observed, which means *S. chamaejasme* could be more conservative in their use of nutrients improving their adaptation to diverse conditions. Moreover, the leaf C and N contents of *S. chamaejasm* were unaffected by any climate factors. However, the correlation between leaf P content and climate factors was significant only in IM, while the leaf K happened to be significant in QT. Besides, MAP or MAT contribution was stronger in the leaf elements than soil by using mixed effects models, which illustrated once more the relatively weak effect of the soil physicochemical properties on the leaf elements. Finally, partial least squares path modeling suggested that leaf P or K contents were affected by different mechanisms in QT and IM regions, suggesting that *S. chamaejasme* can adapt to changing environments by adjusting its relationships with the climate or soil factors to improve its survival opportunities in degraded grasslands.

**Citation:** Guo, L.; Liu, L.; Meng, H.; Zhang, L.; Silva, V.J.; Zhao, H.; Wang, K.; He, W.; Huang, D. Biogeographic Patterns of Leaf Element Stoichiometry of *Stellera chamaejasme* L. in Degraded Grasslands on Inner Mongolia Plateau and Qinghai-Tibetan Plateau. *Plants* **2022**, *11*, 1943. https://doi.org/10.3390/ plants11151943

Academic Editors: Bingcheng Xu, Zhongming Wen and Nikos Fyllas

Received: 5 May 2022 Accepted: 22 July 2022 Published: 26 July 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** biogeographic patterns; leaf stoichiometry; climatic variables; soil physicochemical properties; *Stellera chamaejasme* L.

#### **1. Introduction**

Ecological stoichiometry plays an important role in analyzing the composition, structure, and function of a concerned community and ecological system [1–3]. Over the last a few decades, one particular focus of ecological stoichiometry has been to document largescale patterns of and the driving factors for plant carbon: nitrogen: phosphorus (C:N:P) stoichiometry [4–8]. The relationship between leaf stoichiometry, geographic patterns, and climate factors have been studied on both global and regional scales. Geographical variation in foliar ecological stoichiometry is a challenging issue to plant ecologists [5,9–11]. Meanwhile, the homeostasis (*H*) of element composition is one of the central concepts of ecological stoichiometry, and its strength is related to the ecological strategy and adaptability of species [2,12]. Stoichiometric homeostasis can help predict the strategies that are used by different plant species to cope with limited resources [2,13]. The nutrient conservatism of high *H*-species could be important mechanism contributing to their success, particularly in natural (unmodified) terrestrial ecosystems, where nutrient supply is often limited and highly variable [14,15]. Indeed, the stoichiometric homeostasis of plants varied with species, growth stages, and element types [16–19].

*Stellera chamaejasme* L. is a native perennial weed that has distributed abundantly in the alpine meadow on the eastern Tibetan Plateau and typical steppe on eastern Inner Mongolia Plateau of China [20,21]. It competes with forage-grass species for water, nutrition, and space, thereby decreasing the quality of the forage grass and shortening the use of grasslands [22]. The whole plant of *S. chamaejasme* is poisonous and its roots and pollens are most toxic, therefore, livestock may be poisoned by inadvertently inhaling the pollen while grazing [23]. It has become one of the most serious weeds threatening a wide range of grasslands, which were grazed heavily, posing potential hazards to the grassland ecological safety and its impact on animal husbandry sustainability [21]. Previous studies of *S. chamaejasme* focused on its nutrient uptake efficiency and water use efficiency compared to co-existing species [21], its allelochemicals and allelopathic effects on forages [24], and weed control techniques and use [25], but no similar phylogeographical study had ever been conducted on *S. chamaejasme*. Plant nutrient and stoichiometry are key foliar traits with great ecological importance, but previous publications provide limited insight into the biogeographic leaf nutrient and stoichiometry patterns for *S. chamaejasme* [21]. As habitat heterogeneity tends to increase with geographical scale, wide-ranging species can usually use a wide array of resources and tolerate broad environmental conditions or physiological stresses and flourish over a larger area [26,27]. Recent studies have assumed that wide-ranging species always have stronger homeostasis or a weak relationship with nutrient concentrations than narrow-ranging species in response to environmental factors (e.g., soil fertility) [15,26]. The widespread nature of *S. chamaejasme* may be associated with its stoichiometric homeostasis.

Several studies on a regional and global scale reported that changes of the leaf N and P stoichiometry are associated with many biotic and abiotic factors, including climate variables, soil properties, species type, and plant functional groups [4–7,10,28–30]. However, sample collection is commonly limited to a few individuals, a few populations, and averaged at the population or species level, disregarding the intraspecific variability [31]. Investigating the geographic variation within species can help uncover the mechanisms of relationships between plant tissue nutrients and environments [32] by excluding the confounding effects of taxonomic and phylogenetic structure such as those that have been found to influence the geographic patterns in leaf nutrients, and their linkages to climate and soil. Since relationships between environment and plant traits along environmental gradients could be presented as evidence of environmental control over species distribution,

examining plant-environment (e.g., climate and soil nutrient availability) interactions may provide some insights into the underlying mechanisms of *S. chamaejasme* distribution in degraded grasslands. However, no studies have yet incorporated information on the geographic patterns in leaf stoichiometry of *S. chamaejasme* in relation to environmental factors.

This study aimed to assess the element stoichiometry of *S. chamaejasme* leaves in degraded grasslands across northern China. The distinct regions of the Qinghai-Tibetan Plateau (QT) and Inner Mongolia Plateau (IM) provide a unique opportunity to test whether there are significant differences in leaf stoichiometry under different environmental conditions and to examine how and to what extent soil and climate modify leaf stoichiometry of *S. chamaejasme* across degraded grasslands. In general, most researchers focused on the roles of C, N, and P stoichiometry in the ecological process from individuals to ecosystems, but potassium (K) is an essential macronutrient that has been partly overshadowed by C, N, and P [5,8,33,34]. Our study also focuses on leaf K concentrations of *S. chamaejasme*, which broadens the contents of ecological stoichiometry. We hypothesized that: (1) *S. chamaejasme*, a wide-spread weed, would exhibit small variation in leaf stoichiometry and tolerate broad environmental conditions; in other words, *S. chamaejasme* may have stoichiometric homeostasis and, (2) due to the differences in limiting factors to vegetation in QT and IM, the relationship between *S. chamaejasme* and environmental factors may be related to different factors in the two regions. To test our hypotheses, we first explored the overall biogeographic patterns of C, N, P, and K stoichiometry of *S. chamaejasme* leaves from 29 sampling sites in the two grassland ecosystems in northern China. We then disentangled the effects of soil and climate on the overall plant stoichiometry pattern and compared the difference between the two regions.

### **2. Results**

## *2.1. Pattern of Leaf Ecological Stoichiometry and Soil Physicochemical Properties of S. chamaejasme*

Leaf C, N, P, K, and C:N, C:P, N:P, N:K, K:P of *S. chamaejasme* varied little across all the study sites (Table 1 and Table S1). The mean leaf C, N, P, and K across all sites were 498.60 g kg−<sup>1</sup> , 19.95 g kg−<sup>1</sup> , 2.15 g kg−<sup>1</sup> , and 6.57 g kg−<sup>1</sup> , respectively, and the CV% of leaf P was the largest. Moreover, the mean leaf C:N ratio was 25.20, C:P ratio 245.57, N:P ratio 9.81, N:K ratio 3.13, and K:P ratio 3.21. Inconsistent with the pattern of leaf results, the soil physicochemical properties of *S. chamaejasme*-infested areas varied remarkably (Tables 2 and S2). The soil C, N, P, and K exhibited large variations, primarily ranging c. 5.87–84.74 g kg−<sup>1</sup> for C; 0.24–7.43 g kg−<sup>1</sup> for N, 0.20–0.82 g kg−<sup>1</sup> for P, and 0.95–30.55 g kg−<sup>1</sup> for K. The variation in the soil K content across all the study sites was about 32 times (maximum/minimum), which was the most variable element among the four total elements. The soil mean C:N, N:P, C:P, N:K, and K:P ratios were 13.54, 77.72, 6.34, 0.20, and 38.73, respectively. For the available soil nutrients, soil NN variation was considerably larger than that for the AP, AK, and AN content, as evidenced by coefficients of variation (CVs). Similarly, soil WC, pH, and Ec showed a greater variation throughout the sampling areas.

When comparing the leaf element contents and stoichiometry of *S. chamaejasme* in QT and IM, we found that only the leaf K concentrations, N:K and K:P ratio were significantly different between the two regions (Table 1). Moreover, most soil physicochemical properties were higher in QT than those in IM, except soil AN, NN, and Ec. Specifically, soil P, K, AP, WC, and pH were significantly higher in QT than IM, but soil Ec was significantly lower in QT. Similarly, soil C, N, P, and K stoichiometry showed no significant difference between QT and IM (Table 2).


**Table 1.** Regional *S. chamaejasme* leaf ecological stoichiometry. SD is the standard deviation and CV is the coefficient of variation. Differences between QT and IM were tested using independent *t*-test; significant differences at *p* < 0.05 are indicated by different letters.

**Table 2.** Regional *S. chamaejasme* soil physicochemical properties. SD is the standard deviation and CV is the coefficient of variation. Differences between QT and IM were tested using independent *t*-test; significant differences at *p* < 0.05 are indicated by different letters.


#### *2.2. Ecological Stoichiometry Homeostasis of S. chamaejasme in Degraded Grassland*

Pearson correlations analysis indicates that there are only weak or no correlations between leaf ecological stoichiometry and soil physicochemical properties (Table S3). Furthermore, the relationships between leaf elements and stoichiometry of *S. chamaejasme* and soil by using the homeostasis model were analyzed (Table 3). For C, N (vs. soil N and nitrate N), and K (vs soil K and available K) content, and C:N, N:K, K:P ratios of *S. chamaejasme* leaves were categorized as 'strictly homeostatic' (*p* > 0.1). The leaf P content and C:P were 'weakly plastic', and the leaf N (vs. soil ammonium N) and N:P ratio were classified as 'weakly homeostatic'.

**Table 3.** Standardized major axis regression analysis and stoichiometric homeostasis coefficients (*H*) for leaf C, N, P, and K contents and leaf C:N:P:K ratio in *S. chamaejasme* (*n* = 29). All data have been log10-transformed before analysis. If the regression was non-significant (*p* > 0.1), 1/H was set to zero, and the organism was considered to be 'strictly homeostatic'. Species with 1/H = 1 were considered not homeostatic. All datasets with significant regressions and 0 < H < 1 were categorized as: 0 < 1/H < 0.25: 'homeostatic'; 0.25 < 1/H < 0.5: 'weakly homeostatic'; 0.5 < 1/H < 0.75: 'weakly plastic'; 1/H > 0.75 'plastic'. For 1/H > 1, 1/H close to 1 indicates weak or no stoichiometric homeostasis, and 1/H much larger than 1 indicates 'homeostatic'.


### *2.3. Spatial Variation of Leaf Elements of S. chamaejasme in Relation to Climatic Factors*

No significant relationships among the leaf C and N content and two climatic factors (MAT and MAP) were found using data for all the sample sites or regions (Figure 1). For all the study sites, only the leaf K content was correlated with MAT (*p* < 0.001, Figure 1d). For the regions, it should be noted that in IM, the relationship between the leaf P and climatic factors was significant, but K was not; on the contrary, the K content of *S. chamaejasme* leaves was related to climatic factors but P was not in QT. To be specific, the leaf P concentration increased with increasing MAT and MAP in IM. Moreover, with increasing MAT, leaf K had an increasing trend, but increasing MAP showed an opposite trend in QT.

#### *2.4. Relative Roles of Soil and Climatic Factors in Leaf Elements of S. chamaejasme*

Variation in the leaf C and P heterogeneity across all the sites was mainly explained by the MAP (C: 46.46%; P: 36.49%; Table S4). MAT explained a relatively larger percentage of variation in the leaf N heterogeneity (72.62%) and leaf K heterogeneity (51.46%). The interaction of soil, MAP, and MAT explained the different percentage of the variation in four elements (C: 22.16%; N: 6.99%; P: 22.03%; K: 11.85%).

What is more, both the leaf P and K contents of *S. chamaejasme* were affected by soil and climatic factors. Thus, a more in-depth analysis using partial least squares path modeling revealed direct and indirect effects of the environmental drivers on leaf P and K content of *S. chamaejasme* in different regions (Figure 2, Table S5). Firstly, the influence of climatic factors on soil were bigger in IM than that in QT, and the effect of climatic factors on soil was significant on LP in IM. Secondly, we found that soil factors had a significant effect only on LP in QT only. Thirdly, the effect of climate factors on LP was significant in IM, but the direct effect of climate factors on LP or LK in IM and QT were greater than the indirect. These results suggest that LP or LK were affected by different mechanisms in QT and IM regions. Moreover, the goodness of fit (GOF) was 0.3205 and 0.3556 for LP and LK in QT, respectively, and 0.5490 and 0.4431 in IM. The relatively low predictive power of the model of QT suggested that most variation remained unexplained.

**Figure 1.** Relationships between the leaf C, N, P, and K content of *S. chamaejasme* with MAT & MAP in the Qinghai−Tibet Plateau (green circles, *n* = 19) and Inner Mongolia Plateau (red triangles, *n* = 10). Linear regression model analyses were utilized. Colored dotted lines represented significant relationships (*p* < 0.05) in different region (red, IM; green, QT; grey, all sampling sites). (**a**) MAT vs. leaf C; (**b**) MAT vs. leaf N; (**c**) MAT vs. leaf P; (**d**) MAT vs. leaf K; (**e**) MAP vs. leaf C; (**f**) MAP vs. leaf N; (**g**) MAP vs. leaf P; (**h**) MAP vs. leaf K. **Figure 1.** Relationships between the leaf C, N, P, and K content of *S. chamaejasme* with MAT & MAP in the Qinghai−Tibet Plateau (green circles, *n* = 19) and Inner Mongolia Plateau (red triangles, *n* = 10). Linear regression model analyses were utilized. Colored dotted lines represented significant relationships (*p* < 0.05) in different region (red, IM; green, QT; grey, all sampling sites). (**a**) MAT vs. leaf C; (**b**) MAT vs. leaf N; (**c**) MAT vs. leaf P; (**d**) MAT vs. leaf K; (**e**) MAP vs. leaf C; (**f**) MAP vs. leaf N; (**g**) MAP vs. leaf P; (**h**) MAP vs. leaf K.

Variation in the leaf C and P heterogeneity across all the sites was mainly explained by the MAP (C: 46.46%; P: 36.49%; Table S4). MAT explained a relatively larger percentage of variation in the leaf N heterogeneity (72.62%) and leaf K heterogeneity (51.46%). The interaction of soil, MAP, and MAT explained the different percentage of the variation in

What is more, both the leaf P and K contents of *S. chamaejasme* were affected by soil and climatic factors. Thus, a more in-depth analysis using partial least squares path modeling revealed direct and indirect effects of the environmental drivers on leaf P and K

*2.4. Relative Roles of Soil and Climatic Factors in Leaf Elements of S. chamaejasme*

four elements (C: 22.16%; N: 6.99%; P: 22.03%; K: 11.85%).

climatic factors on soil were bigger in IM than that in QT, and the effect of climatic factors on soil was significant on LP in IM. Secondly, we found that soil factors had a significant effect only on LP in QT only. Thirdly, the effect of climate factors on LP was significant in IM, but the direct effect of climate factors on LP or LK in IM and QT were greater than the indirect. These results suggest that LP or LK were affected by different mechanisms in QT and IM regions. Moreover, the goodness of fit (GOF) was 0.3205 and 0.3556 for LP and LK in QT, respectively, and 0.5490 and 0.4431 in IM. The relatively low predictive power of

the model of QT suggested that most variation remained unexplained.

**Figure 2.** Effects of different soil and climatic variables on the leaf P and K of *S. chamaejasme* in the Qinghai−Tibet Plateau (QT) and Inner Mongolia Plateau (IM) based on partial least squares path modeling. The blue arrows represent positive pathways, the red arrows indicate negative pathways, both are direct effects. The grey arrows show the indirect effects. The standard path coefficients are shown on the arrow. A significant effect is indicated by an \* (*p* < 0.05). GOF, goodness of fit of the statistical model. (**a**,**b**) PLS−PM describing the relationships in QT; (**c**,**d**) PLS−PM describing the relationships in IM. **Figure 2.** Effects of different soil and climatic variables on the leaf P and K of *S. chamaejasme* in the Qinghai−Tibet Plateau (QT) and Inner Mongolia Plateau (IM) based on partial least squares path modeling. The blue arrows represent positive pathways, the red arrows indicate negative pathways, both are direct effects. The grey arrows show the indirect effects. The standard path coefficients are shown on the arrow. A significant effect is indicated by an \* (*p* < 0.05). GOF, goodness of fit of the statistical model. (**a**,**b**) PLS−PM describing the relationships in QT; (**c**,**d**) PLS−PM describing the relationships in IM.

#### **3. Discussion 3. Discussion**

#### *3.1. Leaf Ecological Stoichiometry and Soil Physicochemical Properties of S. chamaejasme 3.1. Leaf Ecological Stoichiometry and Soil Physicochemical Properties of S. chamaejasme*

It is essential to maintain nutrient elements in sufficient amounts and relatively stable ratios for plants to survive and grow [1,2,35–38]. This study presents, to our knowledge, the first analysis of leaf element concentrations (C, N, P, K) and ratios (C:N, C:P, N:P, N:K, K:P) of *S. chamaejasme* across degraded grasslands in northern China. Our results show that the leaf C (498.60 g kg−1), N (19.95 g kg−1), and P (2.15 g kg−1) of *S. chamaejasme* were higher than the mean value of all species average in the the China Grassland Transect [38], and that there was no obvious difference between two regions of *S. chamaejasme.* N and P are the most important limiting nutrients for primary productivity in terrestrial ecosystems [39], and a high concentration of N and P in *S. camazepams* leaves suggests its high nutrient uptake efficiency in degraded grasslands, which could facilitate its competitive advantage over other species in nutrient-poor environments. Moreover, K is one of the essential macronutrients that plays a critical role in various metabolic processes, but it has been partly overshadowed in ecological stoichiometry by nitrogen and phosphorus [40,41]. It is worth noting that K concentrations of *S. chamaejasme* were greater in QT than that in IM. The reason may be that the content of nutrients in plants are constrained by nutrient supply in the soil, and the content of soil K is significantly higher in QT, therefore It is essential to maintain nutrient elements in sufficient amounts and relatively stable ratios for plants to survive and grow [1,2,35–38]. This study presents, to our knowledge, the first analysis of leaf element concentrations (C, N, P, K) and ratios (C:N, C:P, N:P, N:K, K:P) of *S. chamaejasme* across degraded grasslands in northern China. Our results show that the leaf C (498.60 g kg−<sup>1</sup> ), N (19.95 g kg−<sup>1</sup> ), and P (2.15 g kg−<sup>1</sup> ) of *S. chamaejasme* were higher than the mean value of all species average in the the China Grassland Transect [38], and that there was no obvious difference between two regions of *S. chamaejasme.* N and P are the most important limiting nutrients for primary productivity in terrestrial ecosystems [39], and a high concentration of N and P in *S. camazepams* leaves suggests its high nutrient uptake efficiency in degraded grasslands, which could facilitate its competitive advantage over other species in nutrient-poor environments. Moreover, K is one of the essential macronutrients that plays a critical role in various metabolic processes, but it has been partly overshadowed in ecological stoichiometry by nitrogen and phosphorus [40,41]. It is worth noting that K concentrations of *S. chamaejasme* were greater in QT than that in IM. The reason may be that the content of nutrients in plants are constrained by nutrient supply in the soil, and the content of soil K is significantly higher in QT, therefore generating this difference. Generally, C:N:P can be used as an effective tool to analyze coupled relationships and differences between each element in the plant-soil system [1,2]. The average leaf C:N and C:P ratio of *S. chamaejasme* were 25.20 and 245.57, respectively, which were lower than the national grassland average leaf C:N (26.86) and C:P (439.84) [38]. The results indicated that *S. chamaejasme* have higher P utilization rates and N utilization efficiency. Previous studies found that nutrient ratios in aboveground vascular plants can be used to distinguish (1) N-limited sites, (2) P- or P+N-limited sites, and (3) K- or K+N-limited sites from each [7,29,42]. The N:P < 14.5, N:K > 2.1, and K:P < 3.4 in *S. chamaejasme* leaf might imply that its growth is restricted by K- or K+N-limited. Both the leaf and soil K content

were significantly different between two sampling regions and fertilizer experiments should be conducted to test the validity of this idea in the future.

We found that *S. chamaejasme* could survive in a soil environment with considerable variation, which is consistent with the fact that *S. chamaejasme* is a wide-ranging species in the grasslands of China [22]. The soil conditions for *S. chamaejasme* growth varies considerably from site to site. Soil physicochemical properties varied with a difference of more than 10 times between the maximum and the minimum included C (14.43 times), N (30.94 times), K (32.27 times), NN (26.66 times), and WC (10.60 times), Ec (21.86 times). This may provide a competitive advantage for *S. chamaejasme* against other plant species and help explain its rapid expansion in various environments, even in heavily degraded grasslands. Generally, Tibetan alpine grasslands and Inner Mongolian temperate grasslands, which have different limiting factors, are both zonal grassland types in China [43]. Alpine grasslands are mainly limited by low temperatures in the growing season, while temperate grasslands are affected by drought [38]. Accordingly, our analysis indicated that some soil physicochemical properties of *S. chamaejasme*for the regions were significantly different. Soil WC and pH for Qinghai-Tibet were significantly higher, and the Ec was lower than those for Inner Mongolia. However, apart from SP, SK and SAP, soil C and N concentrations, and other soil available nutrients (AN, NN, AK) for the regions were insignificantly different. These findings suggest that climate imposes important controls on some soil nutrients.
