The study was conducted in Saihanba Mechanized Forest Farm, located in Hebei Province, northern China (42°02′ - 42°36′N, 116°51′ - 117°39′E), at an altitude ranging from 1010.0 to 1939.9 m. The typical soils of the area are aeolian sandy soil, meadow soil, brown soil and gray forest soil. The climate is a typical temperate continental monsoon climate. It has an average annual temperature of -1.3°C, with extreme minimum and maximum temperatures of -43.3°C and 33.4°C, respectively. The area experiences an average snow cover for seven months, with an annual precipitation of 460 mm. The frost-free period lasts an average of 64 days. The total operating area of the forest farm is 94,000 hm², with 73,000 hm² designated as forest land, including 57,000 hm² of planted forests and 16,000 hm² of natural forests. The forest coverage rate is 80%, the total forest volume is 5.025 million m³, and the average annual growth rate is 9.7% (Xu et al., 2022). The main tree species include L. principis-rupprechtii, Picea asperata, Betula platyphylla, and P. sylvestris (Wu et al., 2023). The soil organic carbon content of the near-mature forests of L. principis-rupprechtii in this region is about 43.7 g/kg, total nitrogen 2.4 g/kg, total phosphorus 0.3 g/kg and total potassium 19.4 g/kg.

In 1970, an initial density of 5000 trees/hm² of L. principis-rupprechtii was planted in the Saihanba Mechanized Forest Farm. To enhance the microenvironment of the forest and facilitate the establishment and growth of the replanted saplings, thinning was conducted on the upper layer of larch and retaining different tree densities. The retention densities for the upper layer of L. principis-rupprechtii consisted of four levels: control (clear-cut, providing a full-light environment without upper larch trees, CK), 150 trees/hm² (Treatment 1, T1), 225 trees/hm² (Treatment 2, T2), and 300 trees/hm² (Treatment 3, T3).

In 2012, 4-year-old P. sylvestris saplings were planted under the L. principis-rupprechtii plantation (42 years old). The P. sylvestris saplings in the understory were spaced at 3 × 3 m, resulting in approximately 70-75 P. sylvestris per acre. All saplings were container-grown and planted using the “pit planting” method, ensuring consistent afforestation practices. In 2022, standard plots measuring 30 m × 20 m were established for each UFD treatment of L. principis-rupprechtii. The average height of the upper larch trees was 20.8 m, and the mean diameter at breast height (DBH) was 28.6 cm. Three replicates were set up for each treatment and control, resulting in 12 sample plots (i.e., 4 treatments × 3 replicates).

To demonstrate that there were differences in understory environments due to different UFD treatments between experimental gradients, canopy images were used to calculate canopy openness to better account for the effects of canopy structure on understory environments. Canopy images were captured using a wide-angle lens at a height of 2 m in different UFD treatments. The photographs were taken under overcast conditions to minimize glare from direct sunlight, following the method described by Beaudet and Messier (Beaudet and Messier, 2002). The canopy light environment data were obtained by analyzing the images using Hemiview software (Li et al., 2014). The results revealed canopy openness values of 51.9%, 43.2%, 28.4%, and 100% for T1, T2, T3, and CK, respectively.

One-way ANOVA and Duncan’s multiple comparisons were employed to assess the differences in survival, growth, and biomass of young P. sylvestris trees, leaf morphology, nutrient content, and anatomical characteristics across different UFD treatments. Principal component analysis (PCA) was utilized to visualize the overall coordination and correlation between tree growth and leaf traits in the various canopy treatments. The experimental data were analyzed using ANOVA in SPSS 25.0 statistical software, followed by Duncan’s test (p< 0.05) for post-hoc comparisons. PCA and all figures were generated using R statistical software. The FactoMineR and factoextra packages were employed for the PCA analysis. All data are presented as mean ± standard error.

The results of the ANOVA indicated a significant effect (P<0.05) of the UFD treatments on the survival, growth, leaf morphology, leaf anatomy, and leaf nutrients of young P. sylvestris trees ( Table 1 ). The survival rate of P. sylvestris in the T3 was only 67.5%, 30.2%, 18.3%, and 19.5% lower than the CK, T1, and T2, respectively ( Figure 1A ). As the UFD increased, the growth of under-canopy P. sylvestris initially increased and then decreased. The maximum values of tree height, DBH, and crown width were observed in the T1, significantly higher than in the other treatments ( Figures 1B–D ).

The total and above-ground biomass followed a similar trend with different UFD treatments. There were no significant differences between the CK and T1, but both were significantly higher than the T2 and T3 ( Figures 2A, C ). The proportion of trunk biomass to total biomass in the CK was 10.4%, 15.3%, and 20.4% lower than T1, T2, and T3, respectively ( Figure 2B ). However, the leaf biomass ratio in the CK was significantly higher than in the other treatments. There were no significant differences in the above-ground and below-ground biomass ratios among the treatments (p>0.05, Figures 2E, F ).

Mean leaf length, leaf width, and leaf area all decreased with increasing UFD ( Figure 3A, B, D ). The leaf area of CK was 13.2%, 32.8%, and 48.9% higher than that of T1, T2, and T3, respectively. However, LMA showed the opposite trend, increasing with increasing UFD ( Figure 3E ). Leaf length-to-width ratio did not differ significantly among treatments (p>0.05, Figure 3C ).

Anatomical profiles showed that P. sylvestris leaves were semicircular in cross-section, with undifferentiated mesophyll tissue consisting of irregularly arranged but tightly packed cells. Multiple resin ducts were between the leaf tissue cells ( Figure 4A ). As the UFD increased, the thickness of the leaf, mesophyll tissue, and epidermis showed an overall decreasing trend ( Figures 4B–D ).

The pattern of variation for leaf nutrient indicators with UFD treatments was inconsistent. Leaf C content decreased with increasing UFD ( Figure 5C ), whereas leaf P content of the T3 was significantly higher than that of the other treatments ( Figure 5B ). Leaf nutrient stoichiometric ratios indicated that N/P was not significantly different between CK and T1, but both were significantly higher than T2 and T3 ( Figure 5D ). The C/N was significantly higher in CK, T1, and T2 than in T3 ( Figure 5E ).

The principal component analysis based on 18 variables of P. sylvestris under different UFD treatments showed that the first two principal components explained 51.8% of the total phenotypic variance ( Figure 6 ). The first dimension (Dim 1) explained 32.2% of the total variance. Leaf thickness, mesophyll tissue thickness, leaf width, and leaf area had significant positive correlations with the first dimension, all of which are leaf morphology and anatomy indicators, suggesting that the first principal component mainly reflects the variation of leaf structure. The second dimension (Dim 2) explained 19.6% of the total variance. LMA, leaf length-to-width ratio, and leaf C/N ratio had significant correlations with the second dimension, all of which are leaf functional traits ( Figure 6A ). The P. sylvestris samples of the CK differed significantly from those of the T3, of which CK had positive coordinates, and T3 had negative coordinates on the first dimension ( Figure 6B ). However, there was no significant separation from the T1 and T2, and there was a degree of clustering.

P. sylvestris young trees exhibited the highest survival rate under full light conditions. However, under the T3, characterized by extreme low light conditions, there was a significant decrease in the survival of P. sylvestris. This finding aligns with the results of Zhang et al. (2013) on Pinus koraiensis and Quercus mongolica, where reduced light intensity resulted in decreased survival of light-demanding species. However, the growth of P. sylvestris young trees in the understory did not follow the same pattern as survival. In our study, tree height, DBH, and crown width were significantly higher in the T1 compared to the other treatments. This suggests that the mild shading resulting from a lower UFD was conducive to promoting the growth of P. sylvestris young trees in the understory. This phenomenon, known as the shade-avoidance response (Fiorucci and Fankhauser, 2017), indicates that the species adapts to a low-light environment by altering its morphology. However, when the UFD exceeded 150 plants/hm², the growth of young trees in the understory began to exhibit a decreasing trend. It has been confirmed that weak light resulted in low photosynthesis, reducing the plant’s carbon assimilation potential (Sharkey et al., 2007; Dai et al., 2009). Consequently, the photosynthetic carbon assimilation products could not meet the plant’s normal growth requirements, leading to growth suppression. This finding is consistent with previous studies on Torreya grandis seedlings (Tang et al., 2015).

The total biomass was significantly higher in the CK and T1 (strong light environment) compared to T2 and T3 (weak light environment). This finding confirms previous studies showing a positive correlation between strong light conditions and dry matter accumulation (Toledo-Aceves and Swaine, 2008; Lu et al., 2018). The proportion of trunk biomass to total biomass gradually increased with increasing UFD. This suggests that P. sylvestris young trees preferentially allocate biomass to the trunk to enhance tree height growth and improve their ability to compete for light resources in the presence of shading from the upper canopy. This finding aligns with our initial hypothesis and supports the acclimation strategy, which suggests that plants preferentially allocate biomass to organs that most efficiently acquire light, water, and nutrients. It also suggests that biomass allocation within a plant may involve an optimization process in response to stress (Garnier, 1991). However, there were no significant differences in above- and below-ground biomass ratios among the UFD treatments. This indicates that the response of biomass allocation in young P. sylvestris trees to the light environment is limited. Additionally, biomass allocation is not fixed and varies with time, environment, and species (Poorter et al., 2012). Therefore, long-term monitoring may be necessary to further understand the treatment effects.

Considering the observed differences in morphology among young P. sylvestris trees under different light conditions, it is evident that the variation in biomass allocation is relatively smaller than in plant morphology, but tree morphology exhibits greater plasticity than biomass allocation in response to the light environment. This finding is consistent with previous studies on species such as Fagus sylvatica seedlings (Curt et al., 2005) and Camellia oleifera (Zhang et al., 2022b).

In this study, mean leaf length, leaf width, and leaf area decreased, but LMA increased with increasing UFD. However, previous studies on Acer saccharum (Coble and Cavaleri, 2015), Camellia oleifera (Zhang et al., 2022b), and Medicago sativa (Tang et al., 2022) found that plants typically increase leaf area to capture more light resources by expanding the light-receiving surface, decreasing LMA in shaded environments. This may be a difference caused by different life types of trees. Structural diversification in response to different light availability was smaller in leaves of evergreen conifers, as noted in previous studies on conifers (Youngblood and Ferguson, 2003; Wyka et al., 2007, 2012) and other evergreen species (Valladares et al., 2000). In contrast, deciduous broadleaf species, with their shorter leaf lifespan, typically invest more dry matter into plant growth and leaf area expansion. Therefore, inferring the light-trapping capacity of conifers based solely on their morphological size is limited. Furthermore, there were no significant differences in the leaf length and width ratio among the different upper stand density treatments. This indicates that the leaves of P. sylvestris young trees adapt to low-light environments by altering their size rather than their shape.

Previous studies on leaf structure have mainly focused on broadleaf species, revealing that plants tend to reduce leaf thickness, particularly the thickness of palisade tissues, in response to decreased light intensity as an adaptation to low-light environments (Kong et al., 2016; Zhang et al., 2022a, b). In our study, which focused on a coniferous species, the mesophyll tissue did not differentiate into palisade and spongy tissue. However, our findings also demonstrated that P. sylvestris, similar to other broadleaf species, exhibited a gradual decrease in leaf thickness, mesophyll tissue thickness, and epidermis thickness with decreasing light intensity. The thickening of the epidermis under high light intensity may reduce stomatal conductance, enhancing water use efficiency. These changes can protect photosynthetic tissues under conditions of high light intensity and drought (Hanba et al., 2002; Ivancich et al., 2012). Conversely, leaf thickness and mesophyll tissue thickness are reduced under low light conditions. Previous studies have indicated that thinner leaves have a higher likelihood of light interception, but this structural adaptation can be detrimental to CO₂ fixation and transport (Terashima et al., 2011).

The average leaf C content increased with increasing light intensity, which supports the findings of Coble and Cavaleri (2015) that higher light levels lead to increased carbon investment in leaf structures during adaptation. The leaf N/P ratio can indicate the nutrient limitation status of plants in their environment (Güsewell, 2004). Generally, plant growth is limited by P when the N/P ratio is greater than 16, N when it is less than 14, and both elements when it falls in between (Koerselman and Meuleman, 1996). In our study, under the CK and T1 treatments, which experienced higher light intensity, the N/P ratio of young P. sylvestris leaves was significantly higher than that of P. sylvestris under low light conditions. On the other hand, the average N/P ratio of the four treatments was about 14. It indicated that the growth of P. sylvestris was more limited by N in our study environment. Further investigation reveals that the variation in N and P contents under the canopy is influenced by both the plant’s inherent supply-demand dynamics and the ability of the mixed forest to modify soil conditions (Wang et al., 2013; Chen et al., 2022). A previous study reported a total soil nitrogen content of 0.60 ± 0.02 g/kg in this area (Niu et al., 2019), indicating a nutrient-poor state according to the nutrient classification standard of the second national soil census. Therefore, the observed nitrogen limitation in this region may be attributed to the depletion of nitrogen nutrients in the soil. In this study, although the C/N of CK, T1, and T2 under better light conditions was significantly higher than that of T3 under low light conditions, the values of C/N did not have a significant trend with decreasing light intensity. Furthermore, it has been demonstrated that the variation in leaf C/N was primarily influenced by soil water content (Lu et al., 2023). Therefore, the changes in leaf nutrient and stoichiometric ratios may be more closely associated with soil physicochemical properties.

Different UFD treatments were distributed in different regions of the PCA score plot, with significant differences observed between CK and T3 along the first principal component axis. Leaf anatomy and morphology exhibited higher loading coefficients in the first principal component. This finding emphasizes the importance of leaf structure, which includes both external morphological features and internal anatomical structures, as a key indicator for the adaptation of young P. sylvestris trees to diverse understory environments and resource utilization. This observation aligns with our initial hypothesis. However, T1 and T2 showed considerable overlap in the PCA score plot. This finding suggests that understory P. sylvestris young trees are well adapted to moderate shade environments without much variability.

Previous studies on Tetrastigma hemsleyanum (Dai et al., 2009) have found that photosynthetic activity and growth are depressed under light intensities greater than 50% shade. Carbon assimilation is limited and plant growth decreases under irradiance levels lower than 75% shade. As can be seen, optimal light intensity varies from species to species. In this study, when UFD is less than 225 trees/hm², the inhibitory effect of upper canopy shading on the growth and development of understory P. sylvestris young trees decreases. Therefore, it is not advisable to maintain a high UFD when planting P. sylvestris young trees under the forest canopy. Furthermore, the light requirements of plants vary at different developmental stages. Thus, conducting long-term monitoring of the growth status of understory seedlings is necessary to optimize forest stand structure. These results provide a reference for regulating interspecific relationships and promoting the growth and development of P. sylvestris young trees in mixed forests under the forest canopy.<5. Conclusion and management implication.

This experiment investigated the effects of UFD treatments on the individual development, biomass allocation, leaf morphology, nutrients, and anatomical structure of young P. sylvestris trees. We found that P. sylvestris young trees exhibited better growth under the T1, with higher tree height, DBH, crown width, and total biomass, which supported that it would be practicable for under-canopy afforestation using P. sylvestris as long as sufficient light transmittance could be ensured. Under shading conditions, P. sylvestris young trees allocated more photosynthetic products to the main trunk area, improving their light competition and survival ability. Light availability had greater impacts on tree morphology than biomass allocation. However, long-term monitoring is necessary during the young tree stage to identify potential trends in response changes. The introduction of P. sylvestris into pure larch forests can optimize stand structure, including species composition and vertical layering, and promote near-natural succession in these monoculture forests.