The experimental site is located in a state-owned forest area in Yiliang County, Kunming City, Yunnan Province (26°11′-26°25′N, 101°27′-101°28′E) at an altitude of 1300-2800 m above sea level. The area has a subtropical monsoon climate, with dry conditions in winter, little precipitation in spring, wet summers, and mild winters. The rainy season lasts from May to October, accounting for about 85% of the annual precipitation, and the dry season is from November to April, accounting for about 15% of the annual precipitation. The average annual precipitation is 912.2 mm, the average annual temperature is 16.3°C, the number of sunshine hours is 2,177.3 hours, and the frost-free period is 260 days.

P. yunnanensis and P. armandii woodlands with uniform vegetation and strong regional representation were selected as sample plots ( Table 1 ). Three standard plots (30 × 30 m) were randomly chosen, and the distance between the plots was greater than 30 m. The plots were located on upper and middle slopes with an average elevation of 2343 m. Six trees (three P. armandii and three P. yunnanensis) were selected in medium-aged stands in each plot, 3 sample plots, a total of nine P. yunnanensis and nine P. armandii were selected from the three sample plots. They had an average diameter at breast height of 20.44 cm and an average height of 12.59 m. The soil type was red loam with 27.1 g·kg^-1 organic carbon, 1.27 g·kg^-1 total nitrogen, and 0.57 g·kg^-1 total phosphorus.

We obtained samples on January 20 (spring), May 29 (summer), August 1 (fall), and November 9 (winter) of 2021. Needles from the middle layer of the canopy were selected and represented the needles of the entire canopy. We collected three first-degree branches (first-degree branches were connected to the trunk, second-degree branches were connected to first-degree branches, and third-degree branches were connected to second-degree branches) from each sample tree to eliminate sampling errors in needle nutrient composition due to differences in branch orientation. Three tertiary branches were randomly cut with high branch shears on selected primary branches of each sample tree in four directions. They were evenly mixed, and the needles of each age group were separated by branch. One mixed needle sample was taken from each of the three sample trees in each plot. The needle samples were bagged and used to determine needle δ¹³C values and the C, N, and P contents. The samples were transported to the laboratory, rinsed, and placed in an oven at 105°C to prevent enzymatic carbohydrate reactions. They were subsequently dried at 80°C to a constant dry weight, crushed in a pulverizer, and stored in a sealed container.

The seasonal variation of the needle δ¹³C values was similar for the two species ( Figure 1 ). There was no significant difference (P > 0.05) in the δ¹³C values of P. armandii needles between the four seasons. They were highest in summer (-27.09‰) and lowest in spring (-27.77‰). In contrast, the δ¹³C values of the P. yunnanensis needles were significantly higher in summer (-27.03‰) than in the other seasons (P< 0.05).

The soluble sugar, starch, and NSC contents of the needles (the sum of soluble sugar and starch concentrations) of both species decreased at different rates seasonally, and the highest values occurred in spring ( Figure 2 ). The season significantly affected (P<0.05) the needles’ soluble sugar, starch, and NSC contents in P. yunnanensis and P. armandii. In P. armandii, the soluble sugar, starch, and NSC contents decreased significantly (P<0.05) from spring to summer and from summer to autumn and maintained a steady decrease from autumn to winter (P>0.05). In P. yunnanensis, the soluble sugar content decreased from spring to winter ( Figure 2A ), the starch content decreased from spring to autumn and increased in winter ( Figure 2B ), and the NSC content decreased from spring to autumn and then remained in winter ( Figure 2C ).

The leaf soluble sugar/starch ratio reflects the distribution of NSC in the leaves and provides insights into the nutrient utilization of plants. The soluble sugar/starch ratio of the P. armandii needles varied seasonally and remained stable ( Figure 2D ), whereas that of the P. yunnanensis needles increased and decreased, reaching a maximum (1.853) in autumn ( Figure 2D ).

There was a significant difference (P<0.05) in the needle C, N, and P contents of the two species in different seasons ( Figure 3 ). The needle C content of P. armandii decreased over the seasons, whereas that of P. yunnanensis decreased and increased, reaching the lowest value (386.100 g·kg^-1) in autumn ( Figure 3A ). The N content of the P. armandii needles decreased, increased, and decreased over the seasons, whereas that of the P. yunnanensis needles decreased and increased, reaching the minimum value (2.559 g·kg^-1) in summer ( Figure 3B ). The P content of the P. armandii needles decreased, increased, and then decreased over the seasons, reaching the maximum value in autumn (1.602 g·kg^-1). In contrast, the N content of the P. yunnanensis needles increased and decreased, reaching the minimum value in autumn (2.019 g·kg^-1) ( Figure 3C ).

The C:N:P ratios of the two species differed significantly (P<0.05) in different seasons ( Figure 3 ). The needle C:N ratios of P. armandii and P. yunnanensis increased, decreased, and increased over the seasons, and both reached the maximum values in summer (223.070 for P. armandii and 199.396 for P. yunnanensis) ( Figure 3D ). The C:P ratio of the P. armandii needles increased and decreased over the seasons, reaching the maximum value in summer (923.774). In contrast, the C:P ratio of the P. yunnanensis needles decreased, reaching the maximum value in spring (1086.716) ( Figure 3E ). The N:P ratios of the needles of P. armandii and P. yunnanensis decreased over the seasons, and both reached the maximum values in spring (9.624 for Pinus sylvestris and 12.535 for P. yunnanensis) ( Figure 3F ).

There was a significant correlation between the C:N:P stoichiometry and the NSC content of the P. yunnanensis and P. armandii needles, whereas the correlation between the δ¹³C values, C:N:P stoichiometry, and NSC was weak ( Figure 4 ). As shown in Figure 4A , the needle δ¹³C values of P. armandii were negatively correlated only with the soluble sugar/starch ratio. The needle C content was negatively correlated with the P content and positively correlated with the C:P and N:P ratios and the contents of soluble sugars, starch, and NSC. The needle N content was negatively correlated with the C:N ratio and positively correlated with the N:P ratio and the soluble sugar, starch, and NSC contents. The needle P content was negatively correlated with the C:N and N:P ratios, and the soluble sugar, starch, and NSC contents.

As shown in Figure 4B , the δ¹³C values of the P. yunnanensis needles were positively correlated only with the C:N ratio. The needle C content was negatively correlated with the P content and the soluble sugar/starch ratio and positively correlated with the C:P and N:P ratios and the soluble sugar, starch, and NSC contents. The needle N content was negatively correlated with the P content, C:N ratio, and soluble sugar/starch ratio and positively correlated with the C:P and N:P ratios and the soluble sugar, starch, and NSC contents. The P content was negatively correlated with the C:P and N:P ratios and the soluble sugar, starch, and NSC contents and positively correlated with the soluble sugar/starch ratio.

Figure 4C shows the correlations for both tree species. The needle δ¹³C values were negatively correlated only with the soluble sugar/starch ratio. The needle C content was negatively correlated with the P content and the soluble sugar/starch ratio and positively correlated with the C:N, C:P, and N:P ratios and the soluble sugar, starch, and NSC contents. The needle N content was negatively correlated with the C:N ratio and positively correlated with the N:P ratio and the soluble sugar, starch, and NSC content. Needle P content was positively correlated with the C:N, C:P, and N:P ratios and the soluble sugar, starch, and NSC content.

The phenotypic plasticity index was calculated for the physiological and biochemical parameters of the needles of P. armandii and P. yunnanensis ( Figure 5 ). The needle δ¹³C had the lowest plasticity indices (0.024 for P. armandii and 0.032 for P. yunnanensis), and the N:P ratio had the largest plasticity indices (0.743 for P. armandii and 0.845 for P. yunnanensis). Among the indicators of P. armandii needles, needle C/P (0.740), N/P (0.743), Soluble sugar (0.741) and NSC (0.722) were the four indicators of plasticity with large variations. Among the indicators of P. yunnanensis needles, needle N/P (0.845), C/P (0.808), P (0.767) and Starch (0.764) were the four indicators with greater d variation in plasticity. The phenotypic plasticity indices of the P. yunnanensis parameters needles were higher than those of the P. armandii parameters.

Figure 6 shows the results of the principal component analysis. Differences were observed in the seasonal variation of the physiological indicators of P. armandii and P. yunnanensis. The cumulative variance contributions of the first two principal components for P. armandii and P. yunnanensis were 70.9% ( Figure 6A ; axis 1 = 45.4% and axis 2 = 25.5%) and 78.3% ( Figure 6B ; axis 1 = 58.7% and axis 2 = 19.6%), respectively. The ranking of the physiological indicators of the P. armandii needles was the starch content, NSC content, N:P ratio (positive axis), and P content (negative axis) on the first axis, and the N content (positive axis) and C:N ratio (negative axis) on the second axis. The ranking of the physiological indicators of the P. yunnanensis needles was the starch content, NSC content, soluble sugar content, N:P ratio (positive axis), and the P content on the first axis (negative axis), and in the C:N ratio (positive axis) and N content (negative axis) on the second axis. The cumulative variance contributions of the first two principal components of the two species were 67.9% ( Figure 6C ; axis 1 = 46.2% and axis 2 = 21.7%). The ranking of the physiological indicators of the two species was the starch content, NSC content, N:P ratio (positive axis), and P content on the first axis (negative axis) and the N content (positive axis) and C:N ratio on the second axis (negative axis).

Leaf δ¹³C is a reliable indicator of the long-term WUE of plants (Farquhar et al., 1989; Shen et al., 2017). Temperature and water availability are the primary factors affecting plant WUE, and the latter is the most significant factor (Liu, 1998). Plants adapt their physiological functions to different water conditions. When plant growth is constrained by water conditions, plants reduce water transpiration and stomatal conductance to maintain growth (Wu et al., 2017). The needle δ¹³C values of P. armandii and P. yunnanensis reached the maximum in summer ( Figure 1 ). This season is the maximum growth period of needles, and the plants have the highest demand. Drought stress requires adaptation, resulting in the high WUE of both species ( Table 1 ). This finding indicates that precipitation substantially affected the WUE of the two tree species experiencing drought stress. Our results are consistent with those of Garten et al. (1992) for Quercus prinus and Nie et al. (2014) for Radermachera sinica, Sapium rotundifolium, Sterculia euosma, Schefflera octophylla, Alchorena trewioides, and Vitex negundo. The plants responded to a dry environment by increasing the leaf δ¹³C values (higher WUE). The seasonal differences can be attributed to the plants’ water use strategy, shifting from regular water use in the rainy season to conservative water use in the dry season, depending on seasonal precipitation. However, the phenotypic plasticity index analysis showed that the δ¹³C values of the needles of both two tree species ( Figure 5 ) were the smallest. And the principal component analysis ( Figure 6 ) also showed low loading values. These two results again explain the low sensitivity of the two species’ plant needle δ13C values to seasonal changes, and one of the main strategies by which the two species have a strong fitness for use as pioneer tree species for the restoration and improvement of fragile karst habitats in subtropical China.

NSC represent an energy source for the growth and metabolism of plants (O’Brien et al., 2014; Hartmann and Trumbore, 2016; Liu et al., 2020). Soluble sugars are photosynthetic products required for plant growth, development, and osmoregulation (Dietze et al., 2014). Starch is stored to meet the plant’s energy needs (Hartmann and Trumbore, 2016). The NSC content reflects the relationship between stored C (photosynthesis) and plant respiration and growth (Richardson et al., 2015; Yang et al., 2016).

In this study, the soluble sugar, starch, and NSC contents of P. armandii and P. yunnanensis needles decreased at different rates over the seasons ( Figure 2 ). Moreover, the soluble sugar, starch, and NSC contents of P. armandii and P. yunnanensis needles were important indicators of phenotypic plasticity, and the degree of change in the content reflected the degree of the species’ response to seasonal variation. The plants had to use soluble sugars and NSC for growth and development under drought conditions, resulting in an increase in the soluble sugar and NSC contents in the needles (Ayub et al., 2011; Ramírez-Briones et al., 2017). The starch in the needles had accumulated in the previous year due to the sprouting of new leaves, causing the highest needle starch content in P. yunnanensis in January. As the growing season progressed, the stored carbohydrates were utilized (soluble sugars in needles and starch decomposition into soluble sugars) during protoplast extension. The needles synthesized small amounts of soluble sugars for their growth. Thus, the carbon assimilation rate was lower than the carbon consumption rate, and the NSC content decreased. This result agrees with those of Mei et al. (2015) for Xing’an larch (Larix gmelinii) and ash (Fraxinus chinensis). However, in our study, the NSC content (including the soluble sugar and starch contents) did not increase during the late growing season, this may be an internal regulatory mechanism in plants.

The soluble sugar/starch ratio in plants reflects the mutual conversion of soluble sugar and starch in response to environmental changes (Li et al., 2008; Han et al., 2020). In this study, the soluble sugar/starch ratio of P. armandii needles varied seasonally and remained stable, whereas that of P. yunnanensis needles increased and decreased, reaching a maximum in autumn ( Figure 2 ). The phenotypic plasticity index of the soluble sugar/starch ratio was higher for P. yunnanensis than for P. armandii ( Figure 5 ). The principal component loading value of the soluble sugar/starch ratio was higher for P. yunnanensis than for P. armandii ( Figure 6 ). These findings indicated that P. yunnanensis responded to seasonal variation by regulating the conversion of needle soluble sugar and starch, whereas P. armandii did not. The results of the phenotypic plasticity analysis ( Figure 5 ) and principal component analysis ( Figure 6 ) demonstrated that the needle starch content (storage of starch and conversion to soluble sugars) was an indicator of the physiological strategies of both tree species to cope with seasonal variation.

Ecological stoichiometry is used to analyze plant energy use efficiency and their ability to maintain nutrient levels and produce photosynthetic products (Zhang et al., 2018). Plant leaves absorb carbon dioxide through photosynthesis, which requires enzyme (N) catalysis. Enzyme synthesis requires the replication of RNA (P), reflecting the coupling between the three elements C, N, and P (Domingues et al., 2010; He et al., 2019). The mean C content of the needles of P. yunnanensis in young and medium-aged stands in this study ( Figure 3 ) was close to the global leaf C content of terrestrial plants (464 g-kg-1) (Elser et al., 2000). The needle N and P contents ( Figure 3 ) were lower than the global-scale N and P contents (20.60 g-kg-1, 1.99 g-kg-1) (Elser et al., 2000) and the national average of 753 terrestrial plant species (18.6 g-kg-1, 1.21 g-kg-1) (Han et al., 2005). The C:N:P stoichiometric ratios of P. armandii and Pinus yunnanensis needles in this study showed seasonal dynamics. The C content of P. armandii needles decreased over the seasons, and that of P. yunnanensis needles was lowest in autumn ( Figure 3A ). During the rapid growth phase of the tree needles, the biomass increases rapidly, reducing the C content (Sardans and Peñuelas, 2008). The ranking of the N contents of P. armandii and P. yunnanensis was spring > autumn > winter > summer ( Figure 1B ). Thus, more N is stored in the plants, and when nutrient uptake occurs during drought, a higher nutrient level increases the N content. The nutrients are used for the growth of non-photosynthetic tissues or organs, increasing cellular osmotic pressure and conserving water (Gessler et al., 2017). The P content of P. armandii and P. yunnanensis needles was higher in autumn and winter ( Figure 3C ) and reached the minimum value in summer, in agreement with Ågren (2008), who observed a higher P concentration in plant tissues during rapid growth periods.

The leaf C/N and C/P ratios are critical physiological indicators of the plant growth rate and carbon assimilation capacity (Wang et al., 2018). The leaf C/N and C/P ratios are inversely correlated with the N and P use efficiencies, i.e., the amount of total organic matter lost or stored per unit of nutrients (Vitousek, 1982). Leaf C/N and C/P ratios are important physiological indicators related to plant growth rate and plant carbon assimilation capacity (Wang et al., 2018); they can also reflect the efficiency of plant use of N and P nutrients. Leaf C/N and C/P are inversely proportional to the efficiency of N and P use by the plant, i.e. the amount of total organic matter lost or stored per unit of nutrients (Vitousek, 1982). In this study, the C:N ratios of P. armandii and P. yunnanensis needles were highest in summer ( Figure 3D ), whereas the C:P ratio was higher in spring and summer than in the other seasons ( Figure 3E ). This result indicates that both species had the highest N use efficiency during summer and the highest P use efficiency and assimilation ability during spring and summer. The leaf N/P ratio is an indicator of N saturation and nutrient limitations (Wang et al., 2018). Rong et al. (2015) found that plant growth was limited by P when the N/P ratio exceeded 16 and limited by N when the N/P ratio was less than 14. In this study, the N:P ratios of the needles of the two tree species decreased over the seasons and were less than 14 ( Figure 3F ), indicating that growth was limited by N. Both tree species had larger phenotypic plasticity indices ( Figure 5 ) and larger principal component loadings ( Figure 6 ), indicating this indicator of needle N:P ratio is important for seasonal variation in both tree species. An analysis of the seasonal dynamics of the ratios is critical to understanding nutrient storage and the long-term productivity of the two tree species.

The nutrient status (N and P) of trees affects WUE by influencing the photosynthetic rate or stomatal conductance. The leaf N concentration is positively correlated with the photosynthetic CO₂ assimilation rate as N deposition increases (Evans, 1989). Jacob and Lawlor (1991) showed that leaf conductance affected the photosynthetic rate more than stomatal conductance when phosphorus shortage occurred. Talbi-Zribi et al. (2011) found a positive correlation between leaf phosphorus concentration and photosynthetic rate. Garrish et al. (2010) observed that plant WUE was related to soil N availability but not soil phosphorus availability. In contrast, Huang et al. (2015) showed that plant WUE in subtropical regions was not significantly correlated with leaf N concentrations under phosphorus limitations but was significantly and positively correlated with leaf phosphorus concentration.

In this study, the δ¹³C values of P. armandii needles were negatively correlated with the soluble sugar/starch ratio, whereas those of P. yunnanensis needles were positively correlated with the C:N ratio. This finding indicates that the WUE of P. armandii is related to NSC transformation during growth. P. armandii responds to seasonal variation by regulating the C sources and sinks. The WUE of P. yunnanensis was positively correlated with N utilization efficiency. N and P are the limiting factors of plant growth and development. Their levels are closely related to photosynthetic processes and govern the production and distribution of NSC (Xie et al., 2018). The leaf N content is positively correlated with NSC fixation capacity, and P is critical for plant metabolism (McGroddy et al., 2004). Therefore, the leaf N and P contents influence the photosynthetic capacity and NSC synthesis. We analyzed the individual and interactive effects of needle N and P contents, NSC storage and conversion, and the δ¹³C values of the two tree species to determine the key factors affecting their survival and growth in different seasons. The results showed that needle C:N:P stoichiometry was significantly correlated with the NSC content, indicating that the needle N and P concentrations and the C:N and C:P ratios were related to NSC storage in the two tree species. Our results are consistent with studies of other plants (Xie et al., 2018). In this study, the N content was positively correlated with the NSC content (including soluble sugar and starch contents) and increased as NSC were stored. In contrast, the NSC content (including soluble sugar and starch contents) was negatively correlated with the P content and positively correlated with the N:P ratio ( Figure 4 ). These results demonstrated that in this region limited by N, the needle P content affected the NSC storage capacity and content. There are two reasons. First, P is involved in plant metabolism and energy and protein synthesis, and the P content is closely related to the leaf N content. Second, limitations of the soil conditions may have affected the results.