Overall patterns of leaf ecological stoichiometry and soil physicochemical properties of S. chamaejasme across northern China
The patterns of N and P status in plant biomass, and especially in leaves, have been intensely studied [4-6, 35-38], but few studies have attempted to document intraspecific leaf stoichiometry, especially for poisonous weeds in grasslands. 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) of S. chamaejasme across degraded grassland in northern China. Our results show that 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 in the China Grassland Transect , and there was no obvious difference between two habitats of S. chamaejasme. N and P are the most important limiting nutrients for primary productivity in terrestrial ecosystems , and a high concentration of N and P in S. chamaejasme leaves means high nutrient uptake efficiency of S. chamaejasme in degraded grasslands, which could facilitate S. chamaejasme outcompete other species in nutrient-poor environments. Moreover, K is one of the essential macronutrients that play critical roles in various metabolic processes, but it has been partly overshadowed in ecological stoichiometry by nitrogen and phosphorus [40, 41]. Although most studies did not involve the K content in plant leaves, it is worth noting that K concentrations of S. chamaejasme were significantly greater in QT than that in IM. The reason may be that the content of nutrients in plants can be constrained by nutrient supply in the soil, and the content of soil K is significantly higher in QT, therefore generating this difference. The leaf C:N ratio of S. chamaejasme was 25.46, C:P ratio 246.22, N:P ratio 9.84. Generally, it is not uncommon that using N:P ratios of plant biomass as indicators of N or P limitation in various studies [35, 42]. The low N/P ratio in S. chamaejasme might imply that its growth is restricted by N, which was consistent with the results reported by Guo et al .
We found that S. chamaejasme could survive in a soil environment with considerable variation, which is consistent with the fact that S. chamaejasme is wide-ranging species with a wide geographic range in China grassland . The soil condition 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), WC (10.60 times), Ec (21.86 times). Moreover, one sampling site (Haiyan in QT) showed the greatest soil physicochemical properties. For example, the content of soil P was lowest (0.20 g· kg-1), and soil WC was the minimum (0.03), but pH reached a maximum (8.81), indicating S. chamaejasme could tolerate an extreme environment. This may provide a competitive advantage for S. chamaejasme against other plant species and help explain its rapidly expansion in degraded grasslands. Additionally, we also found that the CV of NN was 100.57%, greater than for AN (41.16 %), indicating that S. chamaejasme was less sensitive to the NN variation.
Generally, Tibetan alpine grasslands and Inner Mongolian temperate grasslands, which have different limiting factors, are both zonal grassland types in China . Alpine grasslands are mainly limited by low temperatures in the growing season, while temperate grasslands are affected by drought . 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 Ec 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 soil nutrients.
Relationships between leaf ecological stoichiometry and environmental variables
Plant nutrient concentrations and their correlations with soil nutrient conditions are considered effective tools for exploring plant adaptation and resource utilization strategies in a severe environment [28, 45]. In our study, few significant correlations between S. chamaejasme leaf ecological stoichiometry and soil physicochemical properties were observed, implying insensitive response to the changes in soil nutrient supply of S. chamaejasme. This supports the finding of Geng et al. , and provides confirmation that wide-ranging species are usually able to use a wide array of resources and to 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 grasslands. Further, stoichiometric homeostasis is the ability of plants to maintain their element composition relatively stable regardless of changes in nutrient availability via various physiological mechanisms [2, 11], and the degree of stoichiometric homeostasis can be indicated by the homeostatic coefficient (H) [12, 46, 47]. It is well known that stoichiometric homeostasis had been reported in many dominant palatable species [15, 18, 48] in grasslands. However, this has not been established in unpalatable species. Since poisonous plants represent the majority of the plant species detected after grasslands have been degraded, reveal the eco-physiology characteristics of poisonous weeds will help us better understand how the communities dominated by poisonous weeds form. We found HC and HN of S. chamaejasme were 0, indicating that S. chamaejasme could not maintain carbon and nitrogen internally. Compared with other grassland species, S. chamaejasme had no HN, which was different from previously reported results for other species [17, 49]. Our data clearly show that the stoichiometric homeostasis coefficient of P (HP) of S. chamaejasme was higher than HN, indicating S. chamaejasme was relatively less sensitive responses to soil P than soil N. However, HN was consistently higher than HP at the levels of community, family, and species in China reported by previous studies [15, 18]. Like many other terrestrial ecosystems, grassland ecosystems face an ongoing increase of atmospheric nitrogen deposition in recent decades . The increase of N availability in the soil leads to ecosystems limited by N have gradually transformed to P-limitation or other elements [51, 52]. Thus S. chamaejasme with HP could survive outstandingly than species without HP in the future P-limited surroundings. Moreover, although globally N and P are considered of paramount importance to plant function, it is widely known that many other elements are also important in specific contexts or regions . We also found that HK was greater than HP, implying K could be used as another important element that indicates the degree to which an organism maintains homeostasis.
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 conducted in the grassland biomes of China . The weak relationships 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 leaf P and K and climatic factors (Fig. 4). The relationship between 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 (R2 = 0.5523) was greater than the relationship between P and MAT (R2 = 0.4886) in IM, and the relationship between K and MAT (R2 = 0.3338) was greater than that with MAP (R2 = 0.2920) in QT. These again reflect the different limiting factors of plant growth in different regions . 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 like Inner Mongolian Plateau temperate grasslands. In contrast, the temperature is more likely to have a greater effect on leaf element concentrations than precipitation in Qinghai Tibet Plateau alpine grasslands with high-altitude and low temperature. We also found that only leaf K was negative related with MAP in QT. In fact, K leaches more easily from leaves than N and P ; hence it is easy to ascertain the increasement of MAP in the studies leading more leaf leaching of K in S. chamaejasme.
To explore complex relationships between soil and climatic factors on leaf P and K contents of S. chamaejasme, we conducted a PLS-PM analysis. We found soil exerted a significant effect on leaf P and climate affected leaf K directly in QT, while leaf P appeared to be limited mainly by climatic factors and leaf K was not affected significantly by any environmental factors in IM. This was contradicting our knowledge that climate factors often affect leaf elements through their influence on soil nutrient status . The arid conditions of the Inner Mongolia Plateau (Arid and Semi-Arid Areas) have no doubt restricted grassland plants growth by insufficient water, but the results of our previous study  have proved that high water use efficiency plus high nutrient uptake efficiency of S. chamaejasme ensure its competitive advantage on degraded grasslands in Inner Mongolia Plateau, which makes the relationship between leaf P or K of S. chamaejasme and the soil factors was insignificant in IM region. However, the leaf P content was significantly positively correlated with soil factors (soil P, available P, nitrate N and pH), which was not entirely consistent with the result obtained in IM. The negative influence of climatic variables on leaf K was significant in QT 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, 54]. Our model suggests that underlying mechanisms behind the leaf P or leaf K content of S. chamaejasme were different in two habitats studied, which means S. chamaejasme developed adjustable relationships with environmental factors to adapt to different growth conditions, thus facilitating the spread of S. chamaejasme in degraded grasslands.
In addition, species natural habitats will be subject to more disturbances in the future due to climate change and habitat degradation caused by intensive anthropogenic activities [55, 56]. In future works, continuing wide-scale sampling and considering the influence of human activities are required to further develop a deeper understanding of the geographic patterns in S. chamaejasme.