Abstract
Introduction:
Ecological stoichiometry assessment provides insights into the dynamic interactions between the leaf–litter–humus–soil (LLHS) in different forest types. However, research on ecological stoichiometry in mixed and pure forest ecosystems is relatively weak in the karst region of southwestern China.
Methods:
We investigated how stand types influence the stoichiometric characteristics of carbon (C), nitrogen (N), and phosphorus (P) across the LLHS continuum. Leaf, litter, humus, and soil samples were collected from an evergreen coniferous forest (Cryptomeria japonica var. sinensis Miquel; CJ), a deciduous broadleaf forest (Liquidambar formosana Hance; LF), and a mixed forest composed of both species (C. japonica var. sinensis × L. formosana; CJ × LF) in the fragile karst ecosystem of Southwest China.
Results:
The results showed that stand type had a significant effect on the concentrations of C, N, and P and on the stoichiometric ratios of all LLHS components. Compared with the two pure forests, litter in the mixed forest exhibited significantly higher C/N and C/P ratios, while N and P concentrations were significantly lower. In the broadleaf forest, leaf C/P and N/P ratios were significantly higher than those in the mixed forest and C. japonica monocultures, whereas leaf P concentrations were significantly lower than C. japonica monocultures. Compared with the mixed and broadleaf forests, humus C/N and C/P ratios showed significantly higher in the coniferous forest, while N and P concentrations and the N/P ratio were significantly lower. In the 0–20 cm soil layer, mixed forests had significantly higher C concentration, C/N ratio, and C/P ratio than the two pure forests (p < 0.05). The P nutrient resorption efficiency of plant nutrients in the study area (40.07%–53.69%) was higher than that of N (10.43%–27.04%). Leaf C, N, P, C/N, N/P correlated positively with litter (p < 0.01); leaf N, P, C/N, C/P correlated negatively with humus. Leaf N, C/N, C/P linked positively, leaf P negatively with soil (p < 0.01). The explanatory power of litter, humus, and soil for leaf nutrient concentrations and stoichiometric ratios decreased in the order litter (50.8%) > humus (32.2%) > soil (1.8%).
Discussion:
Overall, these results indicate that the selection of stand type and dominant tree species is central to forest restoration and management in fragile karst ecosystems and plays a crucial role in maintaining the dynamic balance of C, N, and P in forest ecosystems.
1 Introduction
Ecological stoichiometry of carbon (C), nitrogen (N), and phosphorus (P) is a scientific framework that examines the relative balance and dynamics of chemical elements in biogeochemical processes and provides an effective basis for understanding plant growth and nutrient limitation in terrestrial ecosystems (Güsewell, 2004; Lu et al., 2022; Penuelas and Baldocchi, 2019). Nutrient cycling in forest ecosystems is closely linked to ecosystem stability. The dynamic transformation of the leaf–litter–humus–soil (LLHS) continuum represents a primary pathway of nutrient cycling in forests and involves complex interactions among multiple components and nutrients (Arnaud et al., 2020; Muhammad et al., 2023), which directly influence forest productivity (Haghverdi and Kooch, 2019). Within this network, leaves and their decomposition play a critical role in nutrient cycling (Li et al., 2022). Litter is essential for promoting nutrient return, maintaining soil nutrient stability, and contributing to soil formation, thereby acting as a key link between vegetation and soil (Giweta, 2020). Soil serves as both the medium for plant growth and a major nutrient reservoir, exerting a fundamental regulatory influence on plant development (Haghverdi and Kooch, 2019; Liu et al., 2020). Forest humus, located between surface litter and the mineral soil layer, functions as an important interface for nutrient exchange between litter and soil (Wu et al., 2024), while also regulating soil moisture and temperature, enhancing soil structure, and controlling nutrient availability (Aubert and Bureau, 2018). Therefore, elucidating the bidirectional mechanisms of C, N, and P return and supply along the LLHS continuum is crucial for maintaining forest ecosystem stability (Naz et al., 2024).
Changes in forest stand types, particularly the influence of mixed tree species on the ecological stoichiometry of forest components, have become a major focus of ecological research. Significant differences exist in the chemical composition and decomposition rates of litter derived from different tree species (Horodecki et al., 2018). By increasing litter diversity, mixed forests create heterogeneous microenvironments that support more complex soil nutrient conditions and microbial communities. These conditions, in turn, regulate decomposition processes and promote complementary nutrient release (Coll et al., 2018). Numerous studies have shown that mixed forests often exhibit greater stability and sustainability than monoculture forests, partly due to complementary effects of crown and root stratification that enhance nutrient cycling (Khanna et al., 2008). Other studies emphasize that forest ecosystem stability is strongly influenced by species-specific traits, soil nutrient status, and site conditions (He and Dijkstra, 2014; Piaszczyk et al., 2019; Shah et al., 2025), highlighting the importance of tree species composition (Forrester, 2014). Although ecological stoichiometry has been widely examined across different forest stand types and spatial scales, most studies have focused on single components (e.g., leaves or soil) or partial component linkages (e.g., leaf–litter, litter–soil, leaf–litter–soil, or litter–humus–soil) (Bi et al., 2017; Dai et al., 2021; Haghverdi and Kooch, 2019; Li et al., 2022; Liu et al., 2020; Lu et al., 2017; Ren et al., 2012; Wu T. et al., 2010; Yu et al., 2016; Zeng et al., 2016). In contrast, comprehensive investigations of C:N:P stoichiometric differences across the full LLHS continuum among different forest types remain limited. Especially in this process, there is a lack of research on treating humus as a separate layer and paying attention to the flow and coupling or uncoupling of nutrients in LLHS.
Southwest China, s karst region (540,000 km2) is one of the world’s three largest. Guizhou, at its core, has 73.8% karst terrain (Zhong et al., 2021). Its fragile geology and ecology, plus rocky desertification, severely constrain local socioeconomic development (Sheng et al., 2015). Since the 1990s, Guizhou has implemented national forestry programs to mitigate rocky desertification. By the end of 2023, forest area reached 11.1 million hectares, with 63% coverage. Large-scale afforestation has led to improvements in soil aggregate stability, microbial carbon metabolic activity (Yu and Xie, 2020), soil active carbon fractions (Zhao et al., 2021), and soil enzyme activities, thereby enhancing soil quality (Zhang et al., 2024), water-holding capacity, and physical properties (Hou et al., 2020).
Researchers have increasingly examined the effects of different forest stand types, particularly mixed forests, on ecosystem processes in this region. Overall, soil quality in mixed forests is generally superior to that in pure forests (Cheng et al., 2025). Compared with pure coniferous forests, mixed coniferous–broadleaved forests in rocky desertification areas can significantly increase fungal community diversity by enhancing soil organic carbon and total nitrogen (Liu et al., 2022). Mixed forest establishment also promotes understory plant diversity and aboveground biomass (Ma et al., 2021). Several studies have explored the ecological stoichiometry of leaves, litter, or soil for selected tree species in karst areas of Southwest China, including Pinus armandii (He et al., 2023), Pinus massoniana (He et al., 2019), Sapium rotundifolium Hemsl., Alangium chinense (Lour.) Harms, Vitex negundo Linn. (Zeng et al., 2016), Zenia insignis, Cratoxylum cochinchinense (Liang et al., 2026), and Cryptomeria fortunei (Li et al., 2018). However, in the ecologically fragile karst regions of Southwest China, studies examining LLHS stoichiometric characteristics in pure C. fortunei forests, pure L. formosana forests, and their mixed stands remain scarce.
To address this knowledge gap, we investigated the C, N, and P stoichiometric characteristics of the LLHS continuum in three forest types: pure Cryptomeria japonica var. sinensis Miquel forest, pure Liquidambar formosana Hance forest, and mixed C. japonica var. sinensis × L. formosana forest in the fragile ecological zone of Guizhou Province. We hypothesized the following: (1) the C, N, and P contents and stoichiometry of the LLHS exhibited notably different trends based on forest types. (2) Leaf nutrient resorption rates are affected by forest types, and the P resorption rate was higher than that of N. (3) Leaf stoichiometry are directly regulated by litter, humus, and soil nutrient characteristics, and the contribution of litter C/N is the greatest. The findings of this study will guide species selection for rocky desertification control and is of great significance for forest restoration in fragile karst ecosystems.
2 Materials and methods
2.1 Overview of the study area
The study area is located at the Zhazuo Experimental Forest Farm in Xiuwen County, Guizhou Province (106°21′–106°53′E, 26°45′–27°12′N). The forest farm covers approximately 10,107 ha, with a forest coverage rate of 88.6% (Tan et al., 2021). Elevation ranges from 940 to 1,610 m, and the region has a subtropical humid monsoon climate. The annual mean temperature, annual precipitation, and mean annual sunshine duration are 12.8 °C–14.6 °C, 877–1,121 mm, and approximately 1,300 h, respectively. The frost-free period lasts about 261 days (Chen, 2015). Soils are classified as acidic to slightly acidic yellow loam (Gu, 2017).
Vegetation in the forest farm is dominated by artificial stands, with major tree species including Pinus massoniana Lamb., Cryptomeria fortunei, Pinus armandii Franch., and Betula L. Other associated species include Pinus thunbergii Parl., Sassafras tzumu Hemsl., Cinnamomum bodinieri Nees ex Wall., Paulownia Sieb. et Zucc., Ulmus pumila L., and Liquidambar formosana Hance (Yang, 2019). The main vegetation types are coniferous forest, broadleaved forest, and mixed coniferous–broadleaved forest. As major afforestation species in regional forestry projects, C. fortunei and Liquidambar formosana occupy areas of 143,800 hm2 and 117,200 hm2, respectively, accounting for 2.2% and 1.8% of the total arbor forest area in Guizhou Province (6,679,900 hm2) (Zhang and Ding, 2019; Zhang and Guo, 2020). The litter of coniferous tree species (such as C. fortunei) usually has high C content and C/N ratio, while the litter of broad-leaved tree species (such as Liquidambar formosana) has high N and P content, and decomposes quickly. Its nutrient return rate to the soil is also higher, reflecting different nutrient adaptation strategies (Qin and Shangguan, 2019). A mixed afforestation model combining C. fortunei and L. formosana has been successfully implemented at Zhazuo Forest Farm, where it has improved forest structure (Wu et al., 2025).
2.2 Plot setting and sample collection
Based on the principles of representativeness and consistency, areas with similar site conditions were selected within the forest farm. Sample plots were established for three forest types: Cryptomeria fortunei coniferous forest, Liquidambar formosana broadleaved forest, and C. fortunei–L. formosana mixed coniferous–broadleaved forest. All plots belonged to the same site series and had comparable stand ages and stand densities.
For each forest type, three standard plots were established as replicates. Each plot measured 20 m × 20 m (400 m2). Trees in the arbor layer were surveyed for diameter at breast height, tree height, stand age, and stand density. Detailed plot information has been reported in previously published studies by the research team (Wu et al., 2025).
Within each plot, representative standard trees were selected through individual tree measurements. Healthy, mature leaves of the dominant tree species were collected from sun-exposed branches and placed in envelopes for subsequent analysis. In addition, within each plot, all litter and surface humus were collected from five 1 m × 1 m quadrats located at the southwest, northwest, northeast, southeast, and center positions, and were stored separately for laboratory analysis. The humus we collected is mainly the humification layer, located below the fermentation layer and above the mineral soil layer A. Its main characteristic is that organic matter has been completely decomposed, losing its original plant tissue form and presenting a dark, finely fragmented humus state.
Soil samples were collected along an “S-shaped” sampling pattern within each plot. At five sampling points, soil profiles were excavated and samples were collected from three depth intervals (0–20, 20–40, and 40–60 cm) using a 100 cm3 cutting ring. Soil samples from the same depth within each plot were thoroughly mixed and transported to the laboratory for further analysis.
2.3 Sample processing and nutrient content determination
Leaf, litter, and humus samples were oven-dried at 80 °C to constant weight, ground, and passed through a 0.25 mm sieve prior to analysis. Soil samples were air-dried naturally, sieved through a 2 mm mesh to remove roots and stones, and then further ground and sieved through a 0.25 mm mesh for chemical analyses.
Organic carbon in the LLHS components was determined using the potassium dichromate oxidation–external heating method. Total nitrogen was measured using the semi-micro Kjeldahl method, and total phosphorus was determined using the molybdenum–antimony anti-spectrophotometric colorimetric method with ultraviolet spectrophotometry (Chen et al., 2025). All measurements were conducted in quadruplicate.
2.4 Data analysis
The plant nutrient resorption efficiency was calculated as (Pang et al., 2021):
Data were compiled using Excel 2019, and statistical analyses were performed using SPSS 22.0. One-way analysis of variance (ANOVA) was used to test differences in C, N, and P contents, stoichiometric ratios, and leaf N and P nutrient resorption efficiency among the three forest stand types. Differences were assessed using the least significant difference (LSD) test at a significance level of p < 0.05. Pearson correlation analysis was applied to examine relationships between leaf C, N, and P contents and stoichiometric ratios and the corresponding indicators in litter, humus, and soil. Redundancy analysis (RDA) was conducted using CANOCO 5.0, with litter, humus, and soil nutrient contents and stoichiometric ratios as explanatory variables and leaf nutrient contents and stoichiometric ratios as response variables.
3 Results and analysis
3.1 C, N, P contents and their stoichiometric characteristics in leaves, litter, and humus of different forest stand types
Overall, the contents of C, N, and P followed the pattern leaves > litter > humus, and significant differences were observed among forest stand types for all three components (Figures 1A–C). For leaf nutrients, C, N, and P contents were highest in coniferous forests and differences with broadleaved and mixed forests ranging from 3.79% to 40.61% (Figures 1A–C). For litter, C content in coniferous and mixed forests was significantly higher than that in broadleaved forests by 6.36% and 6.09%, respectively (Figure 1A). In contrast, N content in coniferous and broadleaved forests was 1.15 and 1.21 times higher, respectively, than that in mixed forests (p < 0.05). Similarly, P content in coniferous and broadleaved forests was 1.07 and 1.05 times higher, respectively, than that in mixed forests (p < 0.05) (Figures 1B,C). For humus, N and P contents followed the order broadleaved forest > mixed forest > coniferous forest. Specifically, N and P contents in broadleaved forests were significantly higher than those in coniferous forests by 51.13% and 16.0%, respectively (p < 0.05) (Figures 1B,C). No significant differences in humus C content were detected among the three forest types.
Figure 1
The C/N and C/P ratios of leaves, litter, and humus followed the order humus > litter > leaves, whereas the N/P ratio followed the order litter > humus > leaves. Forest stand type significantly influenced the C/N, C/P, and N/P ratios of all three components (Figures 1D–F). In leaves, the C/N ratio in mixed forests was significantly higher than that in coniferous and broadleaved forests by 32.99% and 35.17%, respectively (p < 0.05) (Figure 1D). Leaf C/P and N/P ratios in broadleaved forests were significantly higher than in coniferous and mixed forests, with differences ranging from 7.12% to 48.56% (p < 0.05) (Figures 1E,F). In litter, the C/N ratio in mixed forests was significantly higher than that in coniferous and broadleaved forests by 13.15% and 24.75%, respectively (p < 0.05), while the C/P ratio was higher by 5.81 and 11.79%, respectively (p < 0.05) (Figures 1D,E). The N/P ratio in broadleaved forests was 1.05 and 1.13 times higher than that in coniferous and mixed forests, respectively (Figure 1F). In humus, the C/N ratio in coniferous forests was significantly higher than that in broadleaved and mixed forests by 66.88% and 48.68%, respectively (p < 0.05), and the C/P ratio was higher by 20.78 and 8.69%, respectively (p < 0.05) (Figures 1D,E). In contrast, the N/P ratio in broadleaved and mixed forests was 1.27 and 1.25 times higher, respectively, than that in coniferous forests (Figure 1F).
3.2 Soil C, N, and P contents and stoichiometric characteristics under different forest stand types
Significant differences in soil C, N, and P contents were observed among forest stand types, with patterns varying across soil layers (Figures 2A–C). In the 0–20 cm layer, soil C content in mixed forests was 1.31 times higher than that in broadleaved forests (p < 0.05). In the 20–40 cm and 40–60 cm layers, soil C content in mixed and broadleaved forests was significantly higher than that in coniferous forests (Figure 2A). Differences in soil N content among forest types were mainly observed in the 20–40 cm and 40–60 cm layers. In both layers, soil N content was highest in broadleaved forests and lowest in coniferous forests, with differences of 1.38- and 1.80-fold, respectively (p < 0.05) (Figure 2B). Soil P content also differed significantly among forest types, but the patterns varied by depth. In the 0–20 cm layer, soil P content differed significantly between coniferous forests (0.44 g·kg−1) and broadleaved forests (0.39 g·kg−1) (p < 0.05). In the 40–60 cm layer, soil P content in mixed forests was significantly higher than that in coniferous and broadleaved forests by 11.5 and 20.8%, respectively (p < 0.05) (Figure 2C).
Figure 2
Soil C/N, C/P, and N/P ratios also differed significantly among forest stand types and soil layers (Figures 2D–F). The soil C/N ratio was consistently highest in mixed forests across all three layers and was significantly higher in the 0–20 cm layer than in coniferous and broadleaved forests (p < 0.05) (Figure 2D). Differences in soil C/P ratios varied with depth. In the 0–20 cm layer, the C/P ratio in mixed forests was significantly higher than that in coniferous and broadleaved forests by 27.6% and 25.8%, respectively. In the 20–40 cm layer, the C/P ratio in mixed and broadleaved forests was significantly higher than that in coniferous forests by 55.24% and 44.1%, respectively. In contrast, in the 40–60 cm layer, the C/P ratio in broadleaved forests was significantly higher than that in mixed and coniferous forests by 24.5 and 79.3%, respectively (p < 0.05) (Figure 2E). In the 20–40 cm and 40–60 cm layers, soil N/P ratios were highest in broadleaved forests and lowest in coniferous forests, with increases of 45.4% and 109.6%, respectively (p < 0.05) (Figure 2F).
With increasing soil depth, C, N, and P contents generally declined across all forest types, as did the C/P and N/P ratios in coniferous and mixed forests (p < 0.05). In contrast, in broadleaved forests, C/P and N/P ratios in the 40–60 cm layer were significantly higher than those in the 0–20 cm and 20–40 cm layers (p < 0.05). No significant differences in soil C/N ratios were detected among forest types across soil layers (p > 0.05).
3.3 Characteristics of N and P nutrient resorption efficiency in leaves and litter under different forest types
As shown in Figures 3, N nutrient resorption efficiency ranged from 10.43% to 27.04% among forest types, whereas P nutrient resorption efficiency ranged from 40.07% to 53.69%. Overall, the mean P nutrient resorption efficiency (47.07%) was substantially higher than the mean N nutrient resorption efficiency (19.76%) in the karst fragile ecological zone of Guizhou. The N nutrient resorption efficiency followed the order coniferous forest > broadleaved forest > mixed forest, with values in coniferous and broadleaved forests being 2.45 and 1.99 times higher, respectively, than those in mixed forests (p < 0.05). The P nutrient resorption efficiency followed the order coniferous forest > mixed forest > broadleaved forest, with significant differences among all three forest types (p < 0.05). The P nutrient resorption efficiency in coniferous forests was 1.35 times higher than that in broadleaved forests.
Figure 3
3.4 Relationships between leaf and litter–humus–soil C, N, and P contents and stoichiometric ratios
Significant positive correlations were observed between leaf and litter C, N, and P contents and the C/N and N/P ratios (R2 = 0.09–0.77, p < 0.01; Figure 4). In contrast, significant to highly significant negative correlations were detected between leaf N and P contents and the C/N and C/P ratios and the corresponding humus indicators (Figure 5). Leaf N content and the C/N and C/P ratios increased with increasing soil N content and soil C/N and C/P ratios (R2 = 0.09–0.14, p < 0.001), whereas leaf P content decreased with increasing soil P content (p < 0.01; Figure 6).
Figure 4
Figure 5
Figure 6
Redundancy analysis was performed using leaf C, N, and P contents and C/N, C/P, and N/P ratios as response variables, and litter, humus, and soil nutrient contents and stoichiometric ratios as explanatory variables (Table 1). The first and second RDA axes explained 44% and 29% of the variance, respectively, with a cumulative explanation of 73.17%. Significant explanatory variables included litter C/N (F = 76.3, p = 0.002), C/P (F = 13.4, p = 0.002), N/P (F = 18.4, p = 0.002), and N (F = 8.2, p = 0.002), as well as humus N (F = 31.1, p = 0.002), P (F = 26.3, p = 0.002), N/P (F = 17.1, p = 0.002), C/N (F = 21.9, p = 0.002), and soil P (F = 6.3, p = 0.004). The explained proportions of these variables were 41.9, 4.1, 3.5, 1.3, 13.3, 9.0, 4.5, 4.8, and 1.1%, respectively. Overall, the cumulative explanatory power followed the order litter (50.8%) > humus (32.2%) > soil (1.8%), indicating that litter nutrient status and stoichiometric ratios exerted the strongest influence on leaf nutrient characteristics.
Table 1
| Name | Explains % | pseudo-F | p | RDA ordination figure |
|---|---|---|---|---|
| Littter C:N | 41.9 | 76.3 | 0.002 |  |
| Humus N | 13.3 | 31.1 | 0.002 | |
| Humus P | 9 | 26.3 | 0.002 | |
| Humus C:N | 4.8 | 21.9 | 0.002 | |
| Humus N:P | 4.5 | 17.1 | 0.002 | |
| Litter C:P | 4.1 | 13.4 | 0.002 | |
| Litter N:P | 3.5 | 18.4 | 0.002 | |
| Litter N | 1.3 | 8.2 | 0.002 | |
| Soil P | 1.1 | 6.3 | 0.004 | |
| Soil C:P | 0.7 | 3.9 | 0.022 | |
| Humus C:P | 0.6 | 3.5 | 0.024 |
RDA Analysis of litter-humus-soil C, N, P and stoichiometric ratio with leave.
4 Discussion
4.1 Impact of stand type on C, N, P content and stoichiometric ratios in leaves and litter
4.1.1 The impact of stand type on leaf C, N, P content and stoichiometric ratios
As a fundamental component of terrestrial ecosystems, plants play a critical role in maintaining ecosystem stability, and nutrient elements in leaves are essential for plant growth and development (John et al., 2007). Leaf C content reflects the carbon assimilation capacity of plants (Elser et al., 2000); higher C content generally indicates thicker and larger leaves and stronger defensive capabilities. In contrast, higher leaf N and P contents are associated with enhanced resource acquisition and competitive ability (Wang et al., 2011). In this study, the C and P contents of leaves in the Cryptomeria japonica coniferous forest were significantly higher than those in the broadleaf forest (Figure 1), suggesting that among the three forest types, the coniferous forest exhibits the strongest defensive capacity and competitive ability in response to environmental change. However, some previous studies have reported lower N and P contents in the leaves of coniferous species (e.g., Cunninghamia lanceolata and Taxus chinensis) compared with broadleaf species (Chen et al., 2024), which contrasts with our findings. These discrepancies likely reflect differences in leaf morphology, photosynthetic metabolism, and nutrient requirements among tree species (Chen et al., 2024; Rauf et al., 2023; Sardans and PEñUELAS, 2013). In addition, variations in sampling season, climatic conditions, and soil properties may also influence leaf nutrient contents.
Leaf stoichiometric characteristics not only affect plant growth but also reflect internal physiological status and adaptive strategies in response to environmental conditions (Koerselman and Meuleman, 1996; McIntyre et al., 1999). Leaf C/N and C/P ratios indicate the balance between organic matter accumulation and mineral nutrient uptake and can be used to evaluate nutrient-use efficiency (Elser et al., 2000). A higher C/P ratio reflects greater phosphorus-use efficiency. In this study, the leaf C/N ratio of the mixed forest (43.78) was significantly higher than that of the broadleaf forest (32.39) and coniferous forest (32.92) (Figure 1), indicating that mixed forests have a stronger capacity to accumulate carbon per unit nutrient (Liu et al., 2020). Leaf C/P ratios in all three forest types exceeded the global average value (232) (Zeng et al., 2016), following the order broadleaf forest (371.76) > mixed forest (347.06) > coniferous forest (297.65) (Figure 1). This indicates that plants in karst forests exhibit high phosphorus-use efficiency, with the highest efficiency in broadleaf forests and the lowest in coniferous forests. These results are consistent with those reported by Qiu et al. (2017) and may be related to soil nutrient availability and inherent genetic characteristics of tree species (Liu et al., 2020).
The N/P ratio is commonly used to diagnose nutrient limitation (Tang et al., 2013): N/p > 16 indicates P limitation, 14 < N/p < 16 suggests co-limitation by N and P, and N/p < 14 indicates N limitation (Koerselman and Meuleman, 1996). The leaf N/P ratio in this study was consistently below 14 (Figure 1), indicating nitrogen limitation of plant growth, in agreement with the findings of Pi et al. (2016). In the karst areas of southwestern China, the fundamental reason for nitrogen limitation is that the high content of CaCO3 in karst soil enhances the stability of soil organic matter. Calcium ions “lock” organic nitrogen in the soil organic matter complex through bridging, resulting in a low rate of nitrogen mineralization and severe shortage of available nitrogen for plants (Pan et al., 2018). Moreover, the P resorption rate (40.07%–53.69%) was significantly higher than that of N (10.43%–27.04%) (Figure 3), which aligns with the findings of Shah et al. (2024) on the reabsorption efficiency of N and P. This likely due to the shallow soils and severe erosion in the study area, which result in substantial nutrient losses and limited nutrient availability (Zeng et al., 2016). Consequently, forest plants enhance P resorption to cope with phosphorus deficiency (Li et al., 2018). Nutrient reabsorption in plant leaves is not solely driven by “limiting nutrients.” Research has shown that multiple reabsorption strategies can coexist simultaneously in the same ecosystem–stoichiometric control strategy, nutrient limitation control strategy, and nutrient concentration control strategy (Chen et al., 2021). Based on our research findings, it is speculated that the high reabsorption rate of phosphorus in karst systems may be driven by stoichiometric control mechanisms. The available phosphorus in karst soil is easily affected by high calcium environments, leading to the formation of insoluble calcium phosphate precipitation (He et al., 2026). Therefore, plants have evolved efficient internal preservation strategies for phosphoru (Peng et al., 2023).
4.1.2 Effects of stand type on litter C, N, and P contents and stoichiometric ratios
Approximately 70%–90% of the nutrients required for subtropical forest growth are derived from litter decomposition (Zhao et al., 1991). The decomposition rate of litter determines nutrient release and is influenced by litter quality, internal N and P concentrations, and environmental conditions (Freschet et al., 2013). In this study, The litter N and P contens in mixed forests are significantly lower than that in coniferous and broadleaf pure forests, while the C/N and C/P ratios are significantly higher than that in pure forests (Figure 1), indicating that stand type strongly influences nutrient dynamics during litter decomposition, consistent with the findings of Chen et al. (2017). Litter decomposition is also constrained by nutrient availability (Güsewell and Freeman, 2005), which can be assessed using the N/P ratio; values below 25 indicate nitrogen limitation (Liu et al., 2020). Accordingly, litter decomposition in this area was constrained by nitrogen. Research in karst areas further confirms that plants are generally limited by both nitrogen and phosphorus, which drives more efficient nutrient recovery mechanisms (Gong et al., 2024). In mixed forests, interspecific competition (especially for underground resources) is more intense than in pure forests. The soil in karst areas is shallow and nutrient poor, and this competitive pressure forces plants to undergo stronger N and P reabsorption before leaf litter to maintain their own growth needs. The result is that the content of N and P in litter is significantly reduced (Pan et al., 2018), thereby increasing C/N and C/P in mixed forests (Andrés, 2019).
Litter serves as a critical link in nutrient cycling between plant leaves and soil. Lower C/N and C/P ratios favor microbial activity, thereby accelerating litter decomposition and nutrient release into the soil (He et al., 2019; Manzoni et al., 2010). Previous studies suggest threshold values for nutrient release (Huang et al., 2021): when C/N < 40 and C/p < 600, microbial nutrient requirements are met and N and P are released into the soil (Huang et al., 2021; Li et al., 2018). In the study area, both C/N and C/P ratios of broadleaf forest litter (38.91 ± 1.72 and 584.94 ± 12.69, respectively) were below these thresholds, whereas litter C/N ratios in coniferous forests (42.90 ± 1.37) and mixed forests (48.54 ± 0.65) exceeded 40, and C/P ratios exceeded 600. This suggests that litter from broadleaf forests provides more favorable conditions for microbial growth and nutrient release (Huang et al., 2021). Of course, these values treated as universal breakpoints may vary with climate, microbial community composition, and substrate chemistry. The nutrient release experiments of the three types of forest litter should be carried out in subsequent work and further discussed.
4.2 Effects of stand type on humus and soil C, N, and P contents and stoichiometric ratios
4.2.1 Effects of stand type on humus C, N, and P contents and stoichiometric ratios
Forest humus is an important indicator of environmental conditions and soil fertility (Ponge, 2013). Organic matter accumulates on the forest floor in the form of humus, and its morphological characteristics, stratification, and structure reflect decomposition dynamics and nutrient availability (Jabiol et al., 2013; Prescott et al., 2000). Previous studies have shown that lower forest floor quality (high C content and high C/N ratio) is associated with thicker humus layers and slower decomposition rates (Bayranvand et al., 2017).
In this study, the humus C/N and C/P ratio in coniferous forests was significantly higher than that in broadleaf and mixed forests (Figure 1), indicating thicker humus layers and slower decomposition rates in coniferous forests. Because lower C/N ratios correspond to faster decomposition and nutrient cycling (Bayranvand et al., 2021), humus decomposition rates were highest in broadleaf forests, followed by mixed forests, and lowest in coniferous forests. Higher humus N content may also promote microbial biomass and activity, further enhancing decomposition (Guo et al., 2019). Consistently, humus N and P contents in broadleaf and mixed forests were significantly higher than those in coniferous forests (Figure 1), suggesting stronger microbial activity and faster nutrient cycling (Chen et al., 2017). The differential transformation during the decomposition process of litter from different tree species is the main factor affecting humus nutrients and stoichiometry. Due to the high C/N ratio and lignin content, the microbial decomposition rate of coniferous forest litter is slower (Gong et al., 2024). However, due to the lower C/N ratio and fewer anti decomposition substances in broad-leaved tree litter, microorganisms can decompose and release N and P more quickly, leading to the accumulation of more N and P in humus (Liu et al., 2016). Although high C/N ratio of humus in coniferous forests means slow nutrient cycling, it also represents slow organic carbon loss and high carbon storage potential. Due to protective mechanisms such as calcium and organic matter complexation, the carbon sequestration rate in karst areas is about 31% higher than that in non karst areas (Zhu et al., 2022). Therefore, the high C/N ratio of humus in coniferous forests has a positive impact on carbon sequestration function, but it needs to be balanced with nutrient availability.
4.2.2 Effects of stand type on soil C, N, and P contents and stoichiometric ratios
Soil is the primary source of nutrients for plant growth, and its nutrient status directly affects vegetation composition and productivity (Wu W. et al., 2010). Soil nutrient contents are largely determined by litter and humus decomposition and nutrient return (Li et al., 2004). In this study, the average C, N, and P contents of soils (0–60 cm) in the karst fragile ecological zone of Guizhou were 19.91, 0.81, and 0.34 g·kg−1, respectively, while surface soil (0–20 cm) values were 26.51, 1.18, and 0.44 g·kg−1. These values are comparable to those reported by Li et al. (2018) for surface soils in Guizhou Province. Compared with reported forest soil values (0–20 cm) for C (18.2–19.4 g·kg−1), N (1.20–6.35 g·kg−1), and P (0.41–1.50 g·kg−1) (Bi et al., 2017; Lu et al., 2022; Ren et al., 2012; Wang et al., 2011; Wu T. et al., 2010), soil C content in the study area was relatively high, whereas N and P contents were near the lower limit of the average range. This is likely due to severe soil erosion in karst landscapes, which results in substantial nutrient losses (Liu et al., 2021). Soil N content in broadleaf forests (0.88 ± 0.03 g·kg−1) was significantly higher than that in coniferous forests (0.73 ± 0.06 g·kg−1), consistent with the findings of Bi et al. (2017). Broadleaf litter contains more easily decomposable organic compounds, which enhances soil N input, whereas acidic residues from coniferous litter decomposition may reduce soil N availability (Li et al., 2018; Li et al., 2021).
Soil stoichiometric ratios are important indicators of soil organic matter quality and nutrient mineralization capacity (Wang and Yu, 2008). Lower C/N and C/P ratios indicate higher net mineralization of N and P (Marty et al., 2017). When soil C/N exceeds 25–30, carbon mineralization is relatively rapid, but nitrogen mineralization is slow, leading to nitrogen limitation of plant growth (He et al., 2019; Huang et al., 2024). In this study, surface soils (0–20 cm) in broadleaf forests exhibited the lowest C/N and C/P ratios (Figure 2), indicating higher net N and P mineralization and reduced nutrient limitation compared with coniferous and mixed forests. Soil N/P ratios provide a useful indicator of nutrient limitation: N/p < 10 indicates N limitation, 10–20 indicates co-limitation, and >20 indicates P limitation (Lu et al., 2023). In the study area, soil N/P ratios were below 10 and lower than the national average (5.2) (Guo, 2013), confirming that forest growth in karst ecosystems is primarily nitrogen-limited (He et al., 2019; Li et al., 2018; Liu et al., 2020). Although surface soil N/P did not differ significantly among forest types, mixed forests exhibited the highest ratio, suggesting that mixing Cryptomeria with Liquidambar formosana may help alleviate nitrogen limitation.
4.3 Transmission patterns and coupling/decoupling mechanisms of C, N, and P in the continuum of LLHS in karst forests
In the three forest stands studied, the content of C, N, and P showed a decreasing sequence of leaves>litter>humus>soil. The core driving force of this pattern is the gradual release and dilution of nutrients during the decomposition process, presenting a continuous coupled state. Before the aging and shedding of living leaves, plants will recover some N and P through nutrient reabsorption, but still retain a high initial nutrient concentration (Zhang et al., 2022). During the stage from litter to humus, microorganisms continuously decompose organic matter and release nutrients, gradually reducing the nutrient content at different decomposition stages. Humus eventually mixes with mineral soil, and nutrients are diluted by a large number of mineral particles. Some nutrients leave the system through leaching and biological absorption, resulting in the lowest concentrations of C, N, and P in the soil (Chen et al., 2022). In the LLHS continuum, decoupling phenomena have also occurred. The N/P ratio of litter reaches its highest point, which is a critical location for decoupling between N and P. The reabsorption efficiency of P by plants before leaf senescence is usually higher than that of N, resulting in a greater loss of P than N in litter and a sharp increase in N/P. In addition, in the early stages of decomposition, P is released first due to leaching and rapid mineralization by microorganisms (Liu et al., 2016). The development of rock fissures and underground drainage systems in karst areas leads to the rapid flow of leached P into the ground, exacerbating the loss of P in the litter layer (Shen and Chen, 2019). N is retained due to microbial fixation and chemical binding with organic matter. An increase in N/P in the litter layer indicates that microbial activity in the later stages of decomposition is limited by P. In karst areas, this P restriction can drive oligotrophic bacteria to dominate the microbial community, altering the overall structure of the decomposer community (Yang et al., 2023).
4.4 Relationships among C, N, and P contents and stoichiometric ratios in leaves, litter, humus, and soil
Carbon absorbed by leaves through photosynthesis is transferred to humus and soil primarily via litter input, while soil provides the nutrients required for plant growth. The coupling of C, N, and P contents and their stoichiometric ratios across the leaf–litter–humus–soil (LLHS) continuum represents the intrinsic regulatory mechanism of nutrient cycling in forest ecosystems (Chen et al., 2024; Johnson and Turner, 2019).
This study revealed highly significant positive correlations between C, N, P contents and C/N and N/P ratios in leaves and litter (Figure 4). Compared with litter, leaves contained higher N and P but lower C/N, C/P, and N/P ratios (Figure 1), indicating that litter nutrients originate primarily from leaf senescence (Shah et al., 2024; Wang and Zheng, 2021). Redundancy analysis (RDA) showed that litter C/N was the primary factor controlling variation in leaf C, N, and P contents and stoichiometric ratios, with an explanatory power of 41.9% (Table 1). This suggests that in karst ecosystems, leaf nutrient status and adaptive strategies are largely regulated by litter quality (Parton et al., 2007), highlighting the critical role of litter “self-fertilization” in maintaining ecosystem productivity and stability. However, the relatively high litter C/N ratios (mean value of 43.45; Figure 1) across all forest types indicate slow decomposition and nitrogen deficiency, which limits nutrient release into the soil. Consequently, plants adjust nutrient resorption strategies to reduce dependence on soil nutrients (Killingbeck, 1996)—an important adaptation to nutrient-poor karst environments (He et al., 2019; Li et al., 2018).
Significant negative correlations were observed between leaf nutrients and humus N and P, as well as soil P (Figure 5), suggesting that humus and soil nutrients largely originate from leaf nutrient return. Although phosphorus is often considered a geogenic element controlled by parent material, the observed correlations indicate active biological regulation (Wang et al., 2016). Significant positive correlations were also found between leaves and soil for N, C/N, and C/P (Figure 6), consistent with studies in temperate rainforests of Australia and New Zealand showing strong coupling between soil and leaf nutrient stoichiometry (Bui and Henderson, 2013; Lambers et al., 2010; Parfitt et al., 2005). Nonetheless, some studies have reported weak or absent correlations (Chen et al., 2024; Ladanai et al., 2010), likely due to the influence of additional factors such as soil temperature, moisture, microbial activity, and species-specific nutrient-use strategies (Reich and Oleksyn, 2004). The low explanatory power of soil nutrients for leaf nutrient variation in this study (Table 1) further supports the conclusion that species traits and litter-mediated processes play a dominant role in regulating nutrient dynamics in karst forest ecosystems (Wang and Zheng, 2020).
5 Conclusion
This study investigated the nutrient contents and stoichiometric characteristics of the leaf–litter–humus–soil (LLHS) system in Cryptomeria-dominated coniferous forests, Liquidambar-dominated broadleaved forests, and mixed Cryptomeria–Liquidambar forests in the fragile karst ecological zone of Guizhou Province, China. The main conclusions are as follows: Stand type had a significant effect on the concentrations of C, N, and P and the stoichiometric ratios of all LLHS components. The growth of forest vegetation in the research area is mainly limited by nitrogen, and compared to coniferous forests and mixed forests, broad-leaved forests have relatively smaller nitrogen limitations. The coniferous forest has good carbon sequestration function, stronger defense ability, and resource competitiveness. Plant phosphorus nutrient resorption efficiency were significantly higher than nitrogen nutrient resorption efficiency across all forest types, indicating that forest plants in the karst region enhance phosphorus resorption as an adaptive strategy to cope with soil phosphorus deficiency. Significant correlations were observed between leaf nutrient contents and stoichiometric ratios and those of litter, humus, and soil. Among these components, litter C/N was identified as the primary controlling factor influencing leaf nutrient status and stoichiometric balance, highlighting the dominant role of litter quality in regulating plant nutrient strategies in nutrient-poor karst ecosystems. Overall, this study enhances understanding of the stoichiometric coupling of carbon, nitrogen, and phosphorus within the LLHS system and underscores the critical role of stand type and litter-mediated processes in nutrient cycling. The findings provide a scientific reference for tree species selection in fragile karst ecological regions, e.g., broadleaf forests for rapid nutrient cycling, coniferous forests for long-term carbon storage.mixed forests, and introducing nitrogen fixing tree species into mixed forests.
6 Limitations
Although this study systematically reveals the stoichiometric characteristics of C, N, and P in the LLHS continuum across different forest types in the fragile karst ecosystems of Southwest China, providing valuable foundational data for understanding forest nutrient cycling, it also has limitations. This study collected samples at a single point scale, but there is significant heterogeneity in karst ecosystems across different regions. Therefore, whether the conclusions drawn apply to other karst areas or non-karst forest ecosystems requires cross-regional multi-site comparative studies for validation. Additionally, this study did not measure litter decomposition rates and nutrient release dynamics, limiting the in-depth analysis of the driving mechanisms behind stoichiometric ratio variations. Microorganisms, as the core drivers of litter decomposition and soil nutrient transformation, exert profound regulatory effects on stoichiometric characteristics through their community structure, functional gene expression, and enzyme activity. However, the study did not include microbial community composition and diversity data, making it impossible to explain the intrinsic reasons for stoichiometric ratio differences among different forest stands from a biological mechanism perspective. Since the nutrient resorption efficiency in this study was not corrected for mass loss during litter decomposition, this method may underestimate reuptake rates. In summary, future research should expand to multi-site networks, combining decomposition experiments and high-throughput sequencing to deeply integrate stoichiometric characteristics with decomposition dynamics and microbial ecological functions, aiming to establish a more systematic nutrient cycling theoretical framework and provide a stronger scientific basis for the sustainable management of karst forests.
Statements
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.
Author contributions
YW: Writing – original draft, Writing – review & editing. JC: Conceptualization, Data curation, Writing – original draft. XT: Writing – review & editing. LS: Data curation, Software, Writing – review & editing. XL: Data curation, Funding acquisition, Writing – review & editing. BD: Funding acquisition, Writing – original draft, Writing – review & editing. GD: Investigation, Writing – original draft, Writing – review & editing.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This work was supported by Guizhou Province General Program of Basic Research (Qiankehe Foundation MS [2026] No. 482); and Scientific research project of characteristic forestry industry in Guizhou Province (2020–23).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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The author(s) declared that Generative AI was not used in the creation of this manuscript.
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Summary
Keywords
fragile ecological zone, karst, leaf-litter-humus-soil, stand type, stoichiometry
Citation
Wu Y, Cen J, Tan X, Shi L, Liu X, Ding B and Di G (2026) Eco-stoichiometric characteristics of carbon, nitrogen, and phosphorus in leaves, litter, humus, and soil across different forest types in the karst region of Southwest China. Front. For. Glob. Change 9:1882910. doi: 10.3389/ffgc.2026.1882910
Received
16 May 2026
Revised
14 August 2026
Accepted
14 August 2026
Published
09 September 2026
Volume
9 - 2026
Edited by
Xiting Zhang, Leshan Normal University, China
Updates
Copyright
© 2026 Wu, Cen, Tan, Shi, Liu, Ding and Di.
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*Correspondence: Bo Ding, 754181531@qq.com; Guili Di, diguili59@163.com
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