The carbon isotope composition of leaf tissue is an excellent indicator of a plant's intrinsic water use efficiency, providing crucial insights into vegetation dynamics under global climate change. However, in arid and semiarid grassland ecosystems, the factors driving variations in community foliar δ13C values across different steppe types and the extent to which they can be used to monitor changes in community productivity remain unclear. Based on the community-weighted mean foliar δ13C (foliar δ13CCWM) values of meadow steppe, typical steppe, and desert steppe, which are distributed from east to west as water resources decrease in Inner Mongolia grasslands, our study examines the impacts and regulatory pathways of the community-weighted means of leaf functional traits (LFTCWM) and environmental factors on foliar δ13CCWM values and aboveground productivity. Our results show that the foliar δ13CCWM variations are predominantly influenced by environmental factors in meadow and typical steppe areas but by leaf traits in desert steppe areas. Aboveground productivity in Inner Mongolian grasslands is influenced primarily by temperature, and foliar δ13CCWM values can be used to monitor changes in productivity. Our findings are crucial for understanding how plants drive processes in grassland ecosystems and determining the responses of grassland ecosystems to environmental changes.
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Community-level foliar stable carbon isotope is more influenced by leaf functional traits under drought conditions
https://doi.org/10.21203/rs.3.rs-5228371/v1
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stable carbon isotope
biogeochemical cycles
water use efficiency
leaf functional traits
ecosystem productivity
Plants require sufficient water and nutrients to fix enough carbon for their establishment, persistence and reproduction 1. Trade-offs in the processes of nutrient acquisition and water use have led plants to evolve specific combinations of traits designed to maximize carbon and nutrient uptake (photosynthesis) while minimizing the costs of transpiration, which is achieved mainly by optimizing the opening of stomata 2,3. The stable carbon isotope value of plant foliar (foliar δ13C) is associated primarily with the ratio of the intercellular CO2 concentration (Ci) to the atmospheric CO2 concentration, since Ci is regulated by the balance between stomatal conductance and the photosynthetic rate (A) 4,5; thus, foliar δ13C values reflect the carbon–water trade-offs and can be used as a comprehensive indicator of the intrinsic water use efficiency (iWUE = A/gs) in plants 5,6. Recently, foliar δ13C values have also provided vital insights into vegetation dynamics, climate change, and biogeochemical processes over broad geographical and temporal scales 7–10; thus, understanding the drivers of foliar δ13C values across diverse community types has become a critical challenge for ecologists.
Among the hypothesized fundamental variables driving foliar δ13C values, abiotic factors such as the environment and soil properties have been intensively studied. Researchers have reached relatively consistent conclusions that foliar δ13C values generally decrease with increasing rainfall 11–13. Foliar δ13C values are lower when plants are grown under humid conditions because the relatively high stomatal conductance of the plant increases the intercellular partial pressure of CO2. In contrast, plants generally have lower intercellular CO2 concentrations in arid environments, and plants therefore need to absorb more 13CO2 during photosynthesis, resulting in higher foliar δ13C values 5,14. Past studies on the dependence of δ13C values on the mean annual temperature have been controversial 15,16. Many studies support the existence of positive δ13C–MAT relationships 17,18, whereas other studies report no correlations 11,13, negative correlations 10,19 or even negative to positive correlations 20. This inconsistency may be attributed to the disparate study areas and species used in these previous studies. The main mechanism leading to the positive relationship between δ13C values and MAT may be that lowering the temperature reduces enzyme activity, thus affecting the photosynthesis rate or stomatal conductance and resulting in changes in the Ci and δ13C values of plants 18,21. The negative δ13C‒MAT relationship may be attributed to the increase in mesophyll conductance with increasing temperature, which leads to a decrease in CO2 diffusion resistance in the leaves, resulting in a decrease in foliar δ13C values with increasing temperature 10. In addition to precipitation and temperature, soil properties are also important factors in explaining changes in δ13C values. For example, Yang19 and Niu 22 reported that soil variables such as soil organic carbon and N availability are the best predictors of 13C enrichment in alpine grasslands on the Tibetan Plateau.
Plant foliar δ13C enrichment has been related to leaf biological characteristics and, more specifically, to functional traits 20,23,24. Among trait-based approaches, the global leaf economics spectrum (LES) defines axes of specialization in resource utilization, ranging from "acquisitive" traits to "conservative" traits. Species with "acquisitive" traits usually have fast growth rates and high nitrogen uptake rates, corresponding to high specific leaf areas (SLAs), high leaf nitrogen concentrations (LNCs), high leaf phosphorus concentrations (LPCs), and low leaf dry matter contents (LDMCs), whereas "conservative” traits are associated with the opposite characteristics 25,26. It has been suggested that the physiological mechanisms responsible for carbon isotopic variation (i.e., stomatal conductance and photosynthesis) are associated with shifts in traits associated with fast vs. slow resource-use strategies (i.e., LES traits) 27,28. For example, Reich29 quantified net photosynthesis, SLA, LNC and their interrelationships for 111 species in six biomes and reported that the mass-based net photosynthesis rate increased with the SLA and leaf N content of young mature leaves. Fifteen Mediterranean rangeland species with traits associated with the acquisition or fast use of resources (i.e., high SLA and LNC and low LDMC) presented low δ13C values 24; however, the δ13C values presented a strong positive correlation with LNC in Stipa tenacissima leaves in semiarid Mediterranean steppes 16. The mechanisms driving the correlations between δ13C values and LES traits were related to the CO2 diffusion path length, CO2 supply at carboxylation sites, mesophyll conductivity with respect to CO2, photosynthesis-related enzyme content, etc. 30,31.
Community-weighted mean (CWM) traits have become a key approach not only for explaining variation in ecosystem function but also for elucidating community dynamics 32–34. Collecting information on plant functional traits and species composition simultaneously at the species and ecosystem levels allows ecologists to explicitly link plant and ecosystem functions, since foliar δ13C values are an effective tool for measuring intrinsic water use efficiency at the plant level 5,35; thus, the CWM of foliar δ13C values should be associated with community-level water use efficiency. Aboveground biomass (AGB) represents the major input of carbon into ecosystems and is a key component of carbon and nutrient cycles 36; therefore, the relationship between foliar δ13CCWM value and AGB may reflect trade-offs in the processes of nutrient acquisition and water use at the community level. Here, we assessed the relationship between foliar δ13CCWM values and AGB on the Inner Mongolia Plateau and hypothesized that there should be a negative mutual relationship between foliar δ13CCWM values and AGB. The corresponding sensitivities of foliar δ13CCWM values and AGB to moisture conditions may strongly influence the relationship between them: plant tissue δ13C values are generally relatively high and close to those of the ambient air when WUE is relatively high under dry conditions 5,14,24, whereas aboveground net primary production increases with increasing mean annual precipitation across the Inner Mongolia steppe region in northern China 37.
The Inner Mongolian grasslands are representative of the Eurasian steppe region and are quite different from North American prairies and African savannas 37. In particular, meadow steppe, typical steppe and desert steppe are distributed in sequence from east to west as water resources become less abundant. There are obvious differences in dominant species, soil characteristics, and productivity between different steppe types across the Inner Mongolian grasslands 38. This study was designed to investigate the patterns of foliar δ13CCWM responses to variations in leaf functional traits, environmental factors, and productivity on the basis of a dataset composed of 399 natural arid and semiarid ecosystem sites across the Inner Mongolia region of China. Here, we investigated whether abiotic factors (e.g., temperature, precipitation and soil factors) and biological characteristics (e.g., functional traits) can predict foliar δ13C variation at the community level across three different types of steppes (Fig. 1). We also focused on whether water resource conditions moderate the relationships between foliar δ13CCWM values and leaf functional traits, environmental factors, and AGB.
2.1. Study area
This study focuses on the grasslands of northern China's Inner Mongolia, selecting research sites from three different steppe types (meadow steppe, typical steppe and desert steppe) from east to west (Fig. 2). The meadow steppe is located primarily in the Wulagai Management District and is characterized by a semihumid, semiarid continental climate. The annual mean precipitation is 342 mm, with an average annual temperature of -0.9°C. The dominant plant species in this area are Leymus chinensis, Stipa baicalensis, Cleistogenes squarrosa and Carex pediformis. The typical steppe is located in East Ujimqin Banner and is characterized by a northern temperate continental climate. The area receives an average annual precipitation of 300 mm and has an average annual temperature of 1.6°C. The dominant plant species here are S. grandis, S. krylovii, L. chinensis and C. squarrosa. The desert steppe located in Otog Banner features a mid-temperate semiarid continental climate. This region receives an average annual precipitation of 250 mm and has an average annual temperature of 6.4°C. The grassland community is predominantly composed of S. breviflora, S. glareosa, C. songorica, and Allium polyrrhizum.
2.2. Field sampling
During the peak vegetation growth season (July–August) of 2021, vegetation surveys were conducted across three different types of grasslands, with all selected sites experiencing minimal human disturbance. A total of 399 sites were surveyed. Initially, a 10 × 10 m area was selected at each site, followed by the random placement of three 1 × 1 m quadrats within that area. In each quadrat, we documented the species of plants and collected the aboveground portions. These samples were then dried in an oven at 65°C for 24 hours to achieve a constant weight. Aboveground biomass, a key functional attribute of ecosystems, provides a direct measure of community productivity. Therefore, in this study, the average aboveground biomass from three quadrats was used to represent the community productivity at each site.
2.3. Measurement of leaf traits and foliar δ13C
Leaf traits were measured for the most frequent species in the communities in the three different steppe types (70 species, including both C3 and C4 plants). These species represented, on average, > 80% of the total vegetation composition at each site. Because many species were present at several sites, each species was sampled from a site representing its optimal growth conditions. This approach was based on the assumption that intra-species variability in leaf traits is expected to be less than inter-species variability 39. The selected leaf functional traits closely associated with foliar δ13C variation include leaf thickness (LT), leaf dry matter content (LDMC), specific leaf area, leaf carbon content (LCC), leaf nitrogen content (LNC), and leaf phosphorus content (LPC).
For each species, ten individuals with fully expanded and undamaged leaves were selected. The collected leaf samples were subsequently pooled together. Leaf thickness was measured via a digital calliper, with each species being measured five times; from the pooled samples, 5–10 leaves were randomly selected and placed in a scanner to obtain flat images. The leaf area was then calculated with ImageJ software. After scanning, the fresh weight of the leaves was measured with a precision electronic scale accurate to 0.001 g. The leaves were subsequently dried in an oven at 65°C until a constant weight was achieved to determine the dry weight; the specific leaf area was calculated as the ratio of the leaf dry mass to the leaf area, whereas the leaf dry matter content was determined by the ratio of the leaf dry weight to the leaf fresh weight. The phosphorus content was measured with an inductively coupled plasma optical emission spectrometer (ICP‒OES, Optima 5300 DV, Perkin Elmer, Waltham, USA); the foliar δ13C, leaf carbon content, and leaf nitrogen content were measured via a coupled system consisting of an elemental analyser (Vario Micro cube, Germany) and an isotope ratio mass spectrometer (Delta V Advantage, Thermo Fisher Scientific, Bremen, Germany). The calculation of the foliar δ13C value is expressed as follows:
where \(\:{R}_{sample}\) denotes the 13C/12C molar ratio in the sample and \(\:{R}_{standard}\) denotes the 13C/12C molar ratio in the Pee Dee Belemnite standard.
2.4. Leaf traits and foliar δ13C values at the community level
To scale the leaf traits and foliar δ13C values to the community scale, we used the aboveground biomass of each species at the site to determine the species trait values, which were calculated as follows:
where \(\:s\) represents the number of species within a given site, \(\:{P}_{i}\) represents the proportion of aboveground biomass of the \(\:i\)th plant species within a given site, and \(\:{trait}_{i}\) represents the leaf traits or δ13C value of the \(\:i\)th plant species.
2.5. Environmental variables
The environmental variables were derived from multiple global databases. We collected temperature and precipitation data for 339 sampling sites from WorldClim (https://www.worldclim.org/), on the basis of the availability of location coordinates. The soil bulk density (BD), soil total nitrogen (STN), soil pH (pH), soil organic carbon (SOC) and soil clay content (SC) of topsoil (0–5 cm depth) were collected from SoilGrids (global gridded soil information, https://soilgrids.org/). Soil moisture (SM) data were extracted from the global soil moisture dataset provided by the European Space Agency (http://catalogue.ceda.ac.uk/).
2.6. Statistical analyses
One-way analysis of variance (ANOVA) was performed to compare the differences in foliar δ13CCWM values among the different steppe types. Ordinary least squares regression was conducted to determine the relationships of the foliar δ13CCWM values of the different steppe types with the LFTCWM, climate factors, soil factors and aboveground biomass.
To analyse the regulatory effects of LFTCWM, climate factors, and soil factors on foliar δ13CCWM values and aboveground productivity across different steppe types, we fitted a structural equation model (SEM). This model includes the following variables: (1) LFTCWM, including LT, LDMC, SLA, LCC, LNC and LPC; (2) climate factors, including MAT and MAP; and (3) soil factors, including BD, pH, SOC, STN, SC and SM. Before constructing the SEM, to simplify the analysis and avoid overfitting, a principal component analysis (PCA) was performed on all the factors (soil factors and LFTCWM) containing more than three variables. We applied the Kaiser rule to retain PC axes with eigenvalues greater than 1, where the cumulative variance explained by the variables reached over 80%, satisfying the default variance capture threshold (≥ 70%)40. In meadow steppe, the first two PC axes for the community-weighted means of LT, LDMC, SLA, LCC, LNC, and LPC explained 40.2% (LFTCWMPC1) and 21.7% (LFTCWMPC2) of the variation in these traits (Figure S1a). In the typical steppe, the first two PC axes of the community-weighted means of traits explained 49.7% (LFTCWMPC1) and 28.8% (LFTCWMPC2) of the variation (Supplementary Fig. 2a). In the desert steppe, the first two PC axes explained 41.7% (LFTCWMPC1) and 30.1% (LFTCWMPC2) of the variation in the community-weighted means of the traits (Supplementary Fig. 3a). In Inner Mongolia grasslands, the first two PC axes of the community-weighted means of traits explained 45.8% (LFTCWMPC1) and 22.7% (LFTCWMPC2) of the variation (Supplementary Fig. 4a). In the meadow steppe, the first two PC axes of the soil variables (BD, pH, SOC, STN, SC and SM.) explained 47.8% (SoilPC1) and 21.0% (SoilPC2) of the variation (Figure S1b). In the typical steppe, the first two PC axes explained 46.3% (SoilPC1) and 18.6% (SoilPC2) of the variation in the soil variables (Supplementary Fig. 2b). In the desert steppe, the first two PC axes explained 39.9% (SoilPC1) and 19.5% (SoilPC2) of the variation in the soil variables (Supplementary Fig. 3b). In the Inner Mongolia grasslands, the first PC axis of the soil variables explained 68.7% (SoilPC1) of the variation (Supplementary Fig. 4b). Initially, we constructed an initial structural equation model that incorporated all potential biotic and abiotic interactions. We then used the stepwise AIC procedure to select the best model on the basis of the initial structural equation model. This process selects the most important pathways and eliminates most of the nonsignificant pathways. Standardized path coefficients are used to measure the direct and indirect effects of the predictors. The fit of the SEM was evaluated via Fisher’s C statistic and associated p value 41.
The one-way ANOVA, PCA (via the "FactoMineR" package), ordinary least squares regression, and structural equation modelling (via the "piecewise SEM" package) were performed in R (version 4.3.1).
3.1. Variations in the foliar δ13CCWM values of the different steppe types
The foliar δ13CCWM values of the Inner Mongolia steppe (n = 399) ranged from − 28.80‰ to − 13.72‰, with an average value of − 25.43‰ ± 0.14‰ (Supplementary Table 1). Overall, we observed that foliar δ13CCWM values varied significantly across different steppe types (p < 0.05). Foliar δ13CCWM values were high under dry conditions but substantially reduced under wet conditions, with the foliar δ13CCWM values being highest in the desert steppe (average value of − 23.81‰ ± 0.31‰, n = 130) and lowest in the meadow steppe (average value of − 26.73‰ ± 0.12‰, n = 144) (Fig. 3; Supplementary Table 1).
3.2. Relationships between foliar δ13CCWM values and environmental factors
Our results confirmed the general lack of foliar δ13CCWM–MAP relationships in the three types of steppe, but at the scale of the entire Inner Mongolia grassland, the foliar δ13CCWM values were negatively correlated with MAP (Fig. 4a). Furthermore, the foliar δ13CCWM values in the meadow steppe, typical steppe and entire Inner Mongolia grassland region significantly increased with increasing MAT, whereas the values in the desert steppe did not show this trend (Fig. 4b).
We tested whether soil factors could account for the foliar δ13CCWM values in Inner Mongolian grasslands. Our results highlighted the trend that foliar δ13CCWM values increased with increasing soil pH and soil bulk density but decreased with increasing STN and SOC; however, these trends were not observed in the desert steppe, where foliar δ13CCWM values were not affected by the four soil factors (Figs. 5a-d). In the meadow steppe and typical steppe, foliar δ13CCWM values were negatively correlated with clay (Fig. 5e), and a similar trend was found for the foliar δ13CCWM–soil moisture relationship in the entire Inner Mongolia grassland (Fig. 5f). However, in the desert steppe, foliar δ13CCWM values showed the opposite pattern (positive correlation) with soil clay content and soil moisture (Figs. 5e-f).
3.3. Relationships between foliar δ13CCWM and LFTCWM
The foliar δ13CCWM values were positively associated with acquisitive plant functional traits and negatively associated with conservative plant functional traits (Fig. 6). Specifically, foliar δ13CCWM values increase with increasing LNCCWM and LPCCWM; however, in meadow steppe, the CWM of foliar δ13C values is not influenced by these two traits (Figs. 6b-c). In meadow, typical, and desert steppes, foliar δ13CCWM values are positively correlated with SLACWM (Fig. 6a); a similar trend is observed in the foliar δ13CCWM–LTCWM relationship throughout the entire Inner Mongolian grassland (Fig. 6d). However, in the typical steppe, the foliar δ13CCWM values shows the opposite pattern (i.e., a negative correlation with the LTCWM) (Fig. 6d). Furthermore, the results indicate that the foliar δ13CCWM value is negatively correlated with LDMCCWM and LCCCWM (Figs. 6e-f); however, in meadow and typical steppes, LCCCWM does not exhibit this relationship with the foliar δ13CCWM value. In the typical steppe, LCCCWM is positively correlated with the foliar δ13CCWM value, whereas in the meadow steppe, there is no significant relationship between LCCCWM and foliar δ13CCWM values (p > 0.05; Fig. 6f).
3.4 Dominant determinants of foliar δ13CCWM values and AGB variations
We analysed the relationship between foliar δ13CCWM values and AGB because AGB represents the major input of carbon into ecosystems and is a key component of the carbon and nutrient cycles. We found negative relationships between the AGB and the CWM of foliar δ13C values in the Inner Mongolia grasslands except for the desert steppe (Fig. 7).
Structural equation models (SEMs) were used to disentangle the various pathways through which LFTCWM, climate factors and soil factors influenced the foliar δ13CCWM values and AGB within different steppe types in Inner Mongolia (Fig. 8). The results revealed that LFTCWM is the dominant determinant of foliar δ13CCWM variation, but remarkably, for the meadow steppe, foliar δ13CCWM values are mainly directly influenced by MAT (Fig. 8a). Environmental factors affect δ13CCWM variations directly or indirectly via leaf functional traits. Additionally, the results of the SEMs revealed that climate factors have a greater influence on foliar δ13CCWM variations than do soil factors (Fig. 8). The SEMs suggested that climate factors have a direct positive effect on the AGB in the meadow steppe, desert steppe and Inner Mongolia grassland (Figs. 8a, c and d). In addition to climate factors, the AGB of the Inner Mongolia grassland is also directly affected by soil factors (Figs. 8a and d). Moreover, in the typical steppe, the AGB is also influenced by LFTCWM PC1 (Fig. 8b). Notably, in the typical steppe ecosystem and the whole Inner Mongolian grassland ecosystem, the AGB is regulated by the foliar δ13CCWM value (Figs. 8a, b and d).
Inner Mongolian grasslands are among the driest regions in the world and face greater drought risks than other biomes do42. In an era of rapidly intensifying droughts in grasslands due to global climate change 43, understanding the contributors to the water use efficiency (WUE) of plant communities is of ecological importance. The leaf stable carbon isotope value of plants can provide insights into how WUE responds to climate change 44,45. For both an individual steppe and the entire Inner Mongolian grassland, our study provides, to our knowledge, the first evidence that community-weighted mean foliar δ13C values vary across environmental gradients.
4.1. Responses of foliar δ13CCWM variations in different steppe types to environmental factors
In this study, we analysed the δ13CCWM values in three steppe types under different moisture conditions and found that as precipitation decreases, the foliar δ13CCWM value gradually increases, increasing from − 26.73‰ in the meadow steppe to -23.81‰ in the desert steppe (Fig. 3; Supplementary Table 1), indicating that plants have greater water use efficiency in arid environments 46. This may be because, compared with C3 plants, the additional C4 carbon fixation in C4 plants enables them to assimilate more 13C; therefore, C4 plants have higher water use efficiency than C3 plants 5,47. As water availability decreases, the proportion of C4 plants increases; hence, there are more in desert steppes than in meadow steppes 48. This pattern may lead to increased foliar δ13CCWM values in arid regions, particularly in the desert steppe 49. Under conditions of limited water resources, an insufficient CO2 supply resulting from reduced stomatal conductance leads to higher δ13C values in forbs 49. This process may further explain the differences in foliar δ13CCWM values among different steppe types.
At the steppe level, our results confirmed the general prevalence of nonsignificant relationships between foliar δ13CCWM values and MAP in the three steppes (Fig. 4a). Following the biomass ratio hypothesis 50, the community-weighted mean foliar δ13C values were determined to a large extent by the δ13C values of the dominant species 32,51. The results at the steppe level may be attributed to the fact that the dominant species in different communities in the same steppe have comparable levels of WUE under long-term similar environmental conditions. Moreover, the responses of plants to precipitation are influenced not only by changes in rainfall amounts but also by the plants’ physiological characteristics and other factors 52. The nonsignificant relationship between foliar δ13CCWM values and MAP may further reveal that plant community-level WUE can maintain a certain degree of stability under mild precipitation fluctuations or drought stress. In contrast to the results at the steppe level, the foliar δ13CCWM values were negatively and significantly associated with MAP across the Inner Mongolia grassland (Fig. 4a), which is in line with our hypothesis and previous empirical evidence 9,13,18,20. The inconsistent results between the two scales may be attributed mainly to the difference in MAP gradient length and the large differences in the WUE of the dominant species among the different steppes (Fig. 4a).
The positive correlation of foliar δ13CCWM values with MAT in the meadow steppe and typical steppe (Fig. 4b) is consistent with previous observations that higher temperatures lead to increased foliar δ13C values 53. Compared with desert steppes, meadow steppes and typical steppes receive more precipitation and have lower temperatures, making plants more susceptible to temperature stress. Low temperatures reduce enzyme activity; subsequently, low levels of photon flux or enzyme production result in reduced photosynthetic rates 18,21. WUE = (Ca-Ci)/1.6ΔW 5,54. In this equation, Ca is the CO2 concentration in the atmosphere, Ci is the CO2 concentration in the plant tissue, and ΔW is the water pressure difference inside and outside the leaves. Generally, the entry and exit channels for CO2 and water vapour are stomata, and the higher the Ci is, the more CO2 diffuses from the atmosphere into the interior of the leaf, which means that water vapour can also escape from the stomata and that the WUE will naturally be lower. The photosynthetic rate is the main factor controlling Ci, so a low photosynthetic rate caused by low temperatures leads to an increase in Ci and a corresponding decrease in the δ13C value. In addition, in high-temperature environments (e.g., the desert steppe in this study), plant leaves may close some stomata to reduce water loss, whereas in low-temperature environments (e.g., the meadow steppe in this study), they are more likely to open stomata to increase the photosynthetic rate 55. The increase in the stomatal opening rate caused by low temperatures reduces the air-to-leaf water vapour pressure deficit, resulting in an increase in the leaf internal to ambient partial CO2 pressure (Ci/Ca) ratio and a decrease in the δ13C value 5,18.
The relationships between foliar δ13C values and soil factors vary among the different steppe types and are influenced by the interactions among soil, vegetation, and climate factors. We found that the foliar δ13CCWM values were positively related to the soil pH in the meadow steppe, typical steppe and Inner Mongolian grassland (Figs. 5a-b). Similar to our results, 56 reported a significant positive correlation between foliar δ13C values and soil pH. This is because, in soils with higher pH values, roots are generally healthier and have greater absorption capabilities, which aids plants in more effectively absorbing water and nutrients. Consequently, this leads to higher δ13C values in the leaves 57. The correlation between STN and foliar δ13CCWM values in our study is consistent with the significant negative correlation found by a previous study on these two variables of major species in the Xilin River Basin 58. When the soil nitrogen supply is sufficient, plants can maintain relatively high photosynthetic efficiency and growth rates without significantly closing their stomata to conserve water. Open stomata facilitate the uptake of CO2 but also increase transpiration losses, reducing the WUE. Consequently, a lower WUE is associated with lower foliar δ13C values 59. The foliar δ13CCWM values in the meadow steppe and Inner Mongolian grassland decrease with increasing SOC content (Fig. 5d). The negative correlation pattern observed could be explained by the following two factors. First, in soils with a relatively high SOC content, there is a relatively high proportion of fresh carbon, which has relatively little isotopic discrimination within plants. Consequently, this results in lower δ13C values in plant leaves 60. Second, under barren soil conditions, the proportion of microbially derived organic matter increases with the SOC content, whereas in fertile soils, the proportion of plant-derived organic matter increases with the SOC content. Compared with plant-sourced organic matter, microbially sourced organic matter typically has a higher δ13C content 61. In the fertile soils of the meadow steppe, an increase in the proportion of plant-derived organic matter may lead to a decrease in foliar δ13C values as the SOC content increases 19. We observed that in the desert steppe, the foliar δ13CCWM values were positively correlated with soil moisture, whereas across the whole Inner Mongolian steppe ecosystem, the foliar δ13CCWM values were negatively correlated with soil moisture (Fig. 5f). This contrasting pattern may be attributed to differences in species composition across the different types of grasslands. C4 plants are often considered dominant competitors in arid regions 62, with their foliar δ13C values increasing as soil moisture availability increases 63. However, in the whole Inner Mongolian grassland, the species composition is dominated by C3 plants, whose foliar δ13C values decrease with increasing soil moisture availability 63. These findings indicate that C3 and C4 plants respond differently to soil moisture conditions and that the relationship between foliar δ13CCWM values and soil moisture varies among different steppe types. We also found that the foliar δ13CCWM values are influenced by the soil bulk density and soil clay content (Figs. 5b and e). The clay content and bulk density affect the ability of soil to retain carbon (C), water, and nutrients, thus affecting the δ13C values in plant leaves 64,65.
4.2 Effects of LFTCWM on foliar δ13CCWM variations in different steppe types
The foliar δ13C value, as a physiological trait that reflects a plant's intrinsic WUE, is related to and has trade-offs with other leaf functional traits 66. At the community level, we found a generally positive relationship between foliar δ13CCWM values and acquisitive plant functional traits and a negative relationship between foliar δ13CCWM values and conservative plant functional traits (Fig. 6), which contrasts with theoretical predictions and previous findings 20,24,67. According to the global LES, species at the quick return end of the LES should theoretically have high leaf nutrient concentrations and high photosynthesis rates 26,28; therefore, the CO2 in the plant tissue should be quickly consumed, which would result in a decrease in the leaf internal to ambient partial CO2 pressure (Ci/Ca) ratio and a decrease in the δ13C value 5,18. Therefore, theoretically, species with high SLA values, high LNC values and low LDMC values should have low foliar δ13C values. At the species level, 24 tested the relationships between LES leaf traits and δ13C values across 15 Mediterranean rangeland species in southern France and reported that these species exhibit resource-acquisitive strategies (high SLA and leaf N, P and K) and low leaf δ13C values. At the plant functional type (PFT) level, 20 investigated the foliar δ13C values of different PFTs on the eastern Qinghai–Tibetan Plateau and reported that the foliar δ13C values of C3 plants increased with increasing LDMC and Narea and decreasing Nmass, whereas the foliar δ13C values of C4 plants were not significantly correlated with these leaf functional traits. Since the δ13C value is a proxy for intrinsic WUE (iWUE), communities with high foliar δ13CCWM values presented more nutrient and carbon resource-acquisitive strategies, revealing carbon‒nitrogen‒water coupling in dominant species in Inner Mongolian grasslands 68.
The discrepancies between the results of our study and those of previous studies may be attributed to the unique species composition of the Inner Mongolian grasslands. This region is characterized by a mix of C3 and C4 plants, predominantly composed of tall and dense Stipa spp. and tussock grasses. These plants possess high LDMC values and small SLA values, with generally lower foliar δ13C values (compared with those of C4 plants) 69. Therefore, at the community level, owing to the main influence of these dominant species, the foliar δ13C value is negatively correlated with LDMC and positively correlated with SLA (Figs. 6a and e), which is consistent with the results reported for Stipa tenacissima leaves in semiarid Mediterranean steppes 16. The plant leaf nitrogen content can influence the transport of CO2 by altering the stomatal density in leaves, thereby leading to variations in foliar δ13C values 70. Our results revealed that the foliar δ13CCWM value was positively correlated with LNCCWM (Fig. 6b). The relationship we obtained between these two traits is essentially consistent with the results of previous studies 71. However, these findings are inconsistent with findings from studies in high-elevation areas, where low atmospheric pressure and temperature modify how nitrogen relates to photosynthetic capacity, thereby altering the δ13C–N relationship 66. The primary reason for the positive correlation is that a relatively high leaf nitrogen content enhances the carboxylation capacity of plants, which reduces the CO2 concentration at the site of carbon fixation, thereby lowering the Ci/Ca ratio and subsequently increasing the δ13C value 72. The foliar δ13C value has been reported to be positively correlated 73, negatively correlated 66, and not significantly correlated 74 with LPC. Our findings revealed a positive relationship between foliar δ13CCWM values and LPCCWM (Fig. 6c). This relationship is attributed to the impact of leaf phosphorus on photosynthetic capacity through its influence on rubisco 75. Compared with studies focusing on LNC and LPC, research exploring the relationship between the foliar δ13C value and leaf carbon content is limited. However, studies have shown that the foliar δ13C value is negatively correlated with ash content; generally, a relatively high ash content corresponds to a relatively low carbon content 66. Our findings from the typical steppe are consistent with this observation (Fig. 6f). We also observed that, across the Inner Mongolian grasslands, thicker leaves presented higher δ13C values and that these leaves are associated with increased leaf mesophyll thickness and nitrogen content 76.
4.3 Direct and Indirect Effects of Abiotic and Biotic Attributes on δ13CCWM values and AGB
In the meadow steppe, our SEM confirmed that the most important determinant of foliar δ13CCWM values was the direct positive effect of MAT, which was further reinforced via its indirect positive effects on soil factors (Supplementary Fig. 5). Analogous results revealed that variations in foliar δ13C values are influenced primarily by climatic factors 12,13. In addition, MAT is also the most important determinant of AGB; MAT directly negatively affects AGB, and this effect is further strengthened through indirect negative effects on soil factors. Strangely, MAP only weakly affects foliar δ13CCWM values and AGB indirectly through soil factors; thus, our study highlights the dependence of foliar δ13CCWM values and AGB on temperature rather than precipitation in Inner Mongolian meadow steppes. Typical steppe is in the transition zone between meadow steppe and desert steppe and features intermediate temperature and precipitation values 37. Compared with the values observed in the meadow steppe, the δ13CCWM values in the typical steppe were less affected by climate factors and gradually increased with soil factors and leaf traits (Fig. 7). In addition, δ13CCWM values and AGB were negatively correlated in both the linear regression model and SEM (Figs. 7–8), which may indicate a trade-off between WUE and productivity in typical grasslands. Our results are in line with the trade-off between long-term WUE and nitrogen use efficiency (NUE) found in the typical steppe zone of the Inner Mongolia Plateau 77. This result may be because the leaf intercellular CO2 concentration is generally unsaturated for carbon assimilation, and any increase in Ci will increase the NUE of photosynthesis but will also increase transpiration. Therefore, plants cannot simultaneously maximize the NUE and the WUE. Our results further emphasize the need to consider the relationship between AGB and plant WUE when evaluating the carbon balance and water cycles within grassland ecosystems. Using an SEM, we examined the relative contributions of LFTCWM and environmental factors to foliar δ13CCWM variations in the desert steppe (Fig. 8). The results revealed that the LFTCWM PC1 was the most important determinant of foliar δ13CCWM values. This may be because plant leaf traits are more sensitive to environmental changes in the desert steppe than in the meadow and typical steppe ecosystems 78,79. Additionally, in desert steppe ecosystems, plants employ a range of strategies to adapt to the arid conditions 80–83, thereby increasing their WUE. Thus, variations in foliar δ13CCWM values are more significantly regulated by LFTCWM.
In addition, we noted that the regulation of foliar δ13CCWM values changes with environmental factors and that leaf traits vary among different steppe types. The foliar δ13CCWM variations within meadow and typical steppes are predominantly influenced by climate and soil factors. This is mainly because climate factors can influence elements such as soil nutrients and moisture levels. Under conditions of ample soil moisture, the stomatal openness of plants increases, facilitating greater exchange of CO2 with the atmosphere, influencing the proportion of 12C, which subsequently affects the foliar δ13C value. Furthermore, soil nutrients, particularly nitrogen, can influence plant photosynthesis and thus modulate changes in foliar δ13C values 84,85. Climate change is changing the spatial distribution and characteristics of vegetation (Higgins et al, 2023). Our results may provide certain predictions for changes in meadow and typical steppes against the background of climate change; that is, if drought continues in the grasslands of Inner Mongolia in the future, plants with high WUE (as indicated by high foliar δ13C values) may replace plants with low WUE. However, in the Inner Mongolian desert steppe, foliar δ13CCWM values appear to be insensitive to both climate and soil factors (Figs. 4–5), whereas leaf traits have a much greater direct impact on foliar δ13C values. Under long-term drought stress, desert steppe plants may have adapted to the drought environment through the adjustment of traits, reducing their sensitivity to environmental factors such as climate and soil. Variations in foliar δ13C values are not only influenced by physiological differences among leaves 86 but also controlled phylogenetically 10. Therefore, the influence of leaf traits on foliar δ13CCWM changes may exceed that of external environmental factors.
On the basis of measurements of the community-weighted mean foliar δ13C values in three steppe types with different moisture conditions, this study elucidates the impacts and regulatory pathways of leaf traits and environmental factors on foliar δ13CCWM values and aboveground productivity. The foliar δ13CCWM gradually increases as precipitation decreases, with the foliar δ13CCWM being highest in the desert steppe and lowest in the meadow steppe. At the steppe level, our results confirmed the general prevalence of nonsignificant relationships between foliar δ13CCWM values and MAP in the three steppes and the positive correlation of foliar δ13CCWM values with MAT in the meadow steppe and typical steppe. Our results highlighted the trend that foliar δ13CCWM values increase with increasing soil pH and soil bulk density but decrease with increasing STN and SOC in the meadow steppe and typical steppe, whereas foliar δ13CCWM values are not affected by the four soil factors in the desert steppe. Foliar δ13CCWM values are positively associated with acquisitive plant functional traits and negatively associated with conservative plant functional traits. We found negative relationships between the AGB and foliar δ13CCWM values in the Inner Mongolian grasslands except for the desert steppe. The variations in foliar δ13CCWM values in the meadow and typical steppe types are predominantly influenced by climate and soil factors, whereas in the desert steppe, leaf functional traits make greater contributions than abiotic factors to the variations in foliar δ13CCWM values.
Competing interests
The authors declare no competing interests.
Author contributions
X.W. conceived and designed the main conceptual ideas of this work, performed data analysis, and wrote the manuscript. Y.B., S.J. and K.L. contributed to field investigation. H.L., L.W., Y.W., L.D., and C.L. contributed to the revising of the manuscript. J.Z. supervised the study, contributed to the revising of the manuscript, and contributed to the funding resources.
Acknowledgments
The work was supported by the National Key Research and Development Program of China (grant number 2022YFF1300601), the Natural Science Foundation of Inner Mongolia (grant number 2022MS03027 and 2022QN03010), the Science and Technology Program of Inner Mongolia Autonomous (grant number 2023YFHH0065). We would like to thank Ziqing Gong, Xiaoyang Gong, Yiwei Tang, Qiang Liu, Qingqing Wang, Tao Liu and Xinlei Guo for their contributions to the data collection.
Data availability
The data that support the findings of this study are openly available in figshare: https://doi.org/10.6084/m9.figshare.27129891.
Code availability
The code that supports the findings of this study is openly available at: https://doi.org/10.6084/m9.figshare.27129891.
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There is NO Competing Interest.