How C: N: P stoichiometry in soils and carbon distribution in plants respond to forest age in a Pinus tabuliformis plantation in the mountainous area of eastern Liaoning Province, China

Study site and experimental design

The mountainous area of eastern Liaoning Province, China, belongs to the temperate monsoon climate zone, with a long cold winter and short rainy summer. The average annual precipitation is 700–850 mm, the average annual evaporation is 925–1,284 mm, the average annual temperature is 4-11 °C, and the frost-free period is 120–139 d long. This region belongs to the extension area of the Changbai Mountains, with an altitude of 200–500 m, and a few peaks exceeding 1,000 m. The soil is dominated by dark brown loam, generally acidic or neutral loam.

In the present study, P. tabuliformis plantation sample plots with similar density and different forest ages were established in the Magu Forest Farm of Fushun County (College of Forestry of Shenyang Agricultural University and Magu Forest Farm, Forestry Department of Fushun Mining Group Co. Ltd approval), Liaoning Province, and included five age classes: young forest (YF), middle-aged forest (MAF), near-mature forest (NMF), mature forest (MF), and over-mature forest (OMF). Each age level had three sample plots, for a total of 15 permanent sample plots. The area of each sample plot was 0.06 hm² (20 m × 30 m). Two trees representing the average tree height and diameter at breast height (DBH) in each sample plot were selected as standard trees. The DBH, tree height, height under the branch of P. tabuliformis, stand density, forest age, and other stand indices were measured using a DBH ruler, tape, and altimeter. The basic data of the samples are shown in Table 1.

Aboveground samples

The samples were collected in October 2019. Two standard wood plants were selected from each sample plot and a core was drilled through the standard wood from the DBH along a north-south direction with a growth cone, for a total of 30 cores. The standard wood canopy was divided into three layers: upper, middle, and lower. Two standard branches were selected from each layer, for a total of six standard branches. Each standard branch intercepted one sample branch, and one sample leaf, which were saved in a numbered envelope. A total of 360 samples were collected from 30 standard trees with 180 branch samples and 180 leaf samples. The branch and core samples were transported to the laboratory for drying at 80 °C to a constant weight. The leaves were cured at 105 °C for 30 min, and then dried to a constant weight at 65 °C. The aboveground part totaled 390 samples.

Fine roots and soil samples

Soil and fine root samples were collected in October 2019. The underground part of the standard wood was sampled by using the soil drilling method. A cross section of 1 m × 0.5 m × 0.6 m in length × width × depth was dug at the base of each standard wood, and 180 fine roots samples were taken from three soil layers at different depths (0–20 cm, 20–40 cm, and 40–60 cm), 0.5m and 1 m from the base of the trunk. And 45 soil samples at three different depths (0–20 cm, 20–40 cm, and 40–60 cm) from each plot were collected. All fine roots (<2 mm) were removed from soil and the soil samples and fine roots that had rocks and debris removed were transported to the laboratory. After the fine roots were washed and air-dried naturally, they were placed in an oven, dehydrated at 105 °C for 30 min, and dried to a constant weight at 65 °C. Soil samples were naturally air-dried.

Elements in Pinus tabuliformis and soil samples

Before determining the element content, the samples of leaves, branches, wood cores, fine roots (same distance, same soil layer) from the same sample plot were mixed respectively to form composite samples. The composite samples of wood cores, sample branches, sample leaves, and fine roots were ground and screened with a 60-mesh sieve, whereas the soil composite samples were air-dried and screened with a 100-mesh sieve. All samples were then placed into self-sealing bags, numbered and sealed, and determination.

The TC of plant samples and TC and TN of soil samples were determined using an elemental analyzer (Vario EL III; Elementar. Langenselbold, Germany). Soil TP was determined using the HCLO₄-H₂SO₄-molybdenum-antimony colorimetric method. The soil AP was determined with the molybdenum-antimony colorimetric method.

Statistical analysis

Data processing was performed using SPSS 22.0 software (SPSS, Inc., Chicago, IL, USA). The differences in TC content in the different organs and soil TC, TN, TP, AP and its ratio throughout the entire life cycle (from YF to OMF) were examined using single factor variance analysis. Duncan’s multiple comparison method was used for significance analysis (p < 0.05). Pearson’s correlation analysis was used to analyze the correlation between TC, TN, TP, AP and the ratio of the surface soil (0–20 cm) and the carbon content of different organs over the entire life cycle of the P. tabuliformis plantation.

Carbon distribution of Pinus tabuliformis plantation

Aboveground carbon distribution

The carbon content over the entire life cycle of the P. tabuliformi s plantation was 489.83–509.00 g⋅kg⁻¹ in the leaves (Fig. 1A), 335.00–471.43 g⋅kg⁻¹ in the branches (Fig. 1B), and 489.87–509.73 g⋅kg⁻¹ in the trunks (Fig. 1C). With a change in forest age, the carbon content of the leaves gradually increased and then decreased, reaching a maximum in NMF. There were significant differences between the carbon content in forest ages (p < 0.05), except in MAF and NMF, and MF and OMF (Fig. 1A). The carbon content of the branches decreased from YF to MAF, and decreased after a slight increase in MF. There were significant differences between the carbon content in forest ages (p < 0.05), except in MAF, NMF, and OMF (Fig. 1B). The carbon content of the trunks only in the NMF showed significant differences between MF and OMF (p < 0.05) (Fig. 1C).

Underground carbon distribution

The carbon content of fine roots ranged from 415.20 to 480.37 g⋅kg⁻¹ over the entire life cycle of the P. tabuliformis plantation. There were no significant differences among the carbon content of fine roots (0.5 m) in the soil layers of YF, NMF, and MF. There were significant differences between the 0–20 cm soil layer (p < 0.05) and other soil layers in the MAF and OMF age groups (p <  0.05) (Fig. 2A). In the 0–20 cm soil layer, the carbon content of fine roots (0.5 m) in MAF and YF, NMF and OMF were significantly different (p < 0.05). In the 20–40 cm soil layer, there was significant difference among MF and OMF (p <  0.05). In the 40–60 cm soil layer, YF, MF, and OMF showed significant differences (p < 0.05). However, for the carbon content of fine roots (1 m) in all soil layers of different forest ages, only the 40–60 cm and 0–20 cm layers in NMF showed significant differences (p <  0.05) (Fig. 2B). In the 0–20 cm soil layer, there were significant differences between NMF, YF, and OMF (p < 0.05). In the 20–40 cm soil layer, there was no significant difference among the different forest ages. In the 40–60 cm soil layer, the carbon content of fine roots (1 m) in the YF was significantly different from that in MAF, MF, and OMF (p < 0.05), and between NMF and MF (p <  0.05). The results showed that the carbon content of fine roots decreased with soil layer depth, and there was a trend of first increasing and then decreasing with an increase in forest age.

Table 2:

Carbon distribution over the life cycle of Pinus tabuliformis plantation.

Forest Age	Aboveground Carbon Content			Underground Carbon Content
	Leaf (g⋅kg−1)	Branch (g⋅kg−1)	Trunk (g⋅kg−1)	Fine Roots (0.5m g⋅kg−1)			Fine Roots (1m g.kg−1)
				0–20 cm	20–40 cm	40–60 cm	0–20 cm	20–40 cm	40–60 cm
YF	489.83 ± 1.05 AB	471.43 ± 2.07 BC	503.72 ± 6.43 A	463.83 ± 2.38 CD	461.88 ± 4.07 CD	415.20 ± 5.66 DE	458.20 ± 14.87 CD	448.85 ± 31.74 CDE	422.32 ± 18.50 E
MAF	505.90 ± 2.72 A	335.00 ± 1.59 E	503.17 ± 10.17 A	479.63 ± 6.18 B	458.77 ± 12.65 CD	453.37 ± 9.86 CD	471.27 ± 12.33 BC	445.40 ± 19.89 D	451.20 ± 10.21CD
NMF	509.00 ± 3.72 A	335.47 ± 2.57 F	489.87 ± 2.38 AB	465.13 ± 18.76 CD	456.77 ± 2.21 DE	453.80 ± 12.90 DE	480.37 ± 4.71 BC	451.93 ± 15.85 DE	436.73 ± 19.09 E
MF	497.83 ± 3.62 B	339.67 ± 1.93 D	509.73 ± 10.03 A	463.10 ± 0.76 C	464.43 ± 2.00 C	462.33 ± 3.81 C	471.60 ± 2.42 C	468.67 ± 4.45C	465.97 ± 0.29 C
OMF	494.80 ± 1.11 A	335.17 ± 1.72 F	504.90 ± 6.17 A	471.43 ± 6.26 B	449.07 ± 6.44 DE	439.67 ± 15.88 E	462.57 ± 3.44 BC	456.93 ± 7.80 CD	455.80 ± 4.7CD

DOI: 10.7717/peerj.11873/table-2

Notes:

Different uppercase letters indicate significant differences between the carbon content of different organs in the same forest age (p < 0.05).

YF

Young forest

MAF

Middle-aged forest

NMF

Near-mature forest

MF

Mature forest

OMF

Over-mature forest

Soil ecological stoichiometric characteristics

Soil TC, TN, TP, and AP content

In the surface soil (0–20 cm), the value of soil TC content was varied between 11.25–37.82 g⋅kg⁻¹, the value of soil TN content was varied between 0.69–2.95 g⋅kg⁻¹, the value of soil TP content was varied between 0.28–1.32 g⋅kg⁻¹, and the value of soil AP content was varied between 13.57–18.57 mg⋅kg⁻¹. And in all soil layers, over the entire life cycle of the P. tabuliformis plantation, the value of soil TC content was varied between 3.29 and 37.82 g⋅kg⁻¹ (Fig. 3A), the value of soil TN content was varied between 0.22 and 2.95 g⋅kg⁻¹ (Fig. 3B), the value of soil TP content was varied between 0.23 to 1.61 g⋅kg⁻¹ (Fig. 3C), and the value of soil AP content was varied between 13.52–20.99 mg⋅kg⁻¹ (Fig. 3D). In some forest ages, the TC and TN content in the 0–20 cm soil layer was significantly different from those in the other soil layers; however, the TC and TN content in the 20–40 cm and 40–60 cm soil layers were not significantly different over the entire life cycle. The TP and AP content in the soil did not change significantly with soil layer and forest age, except for the AP content in NMF. Therefore, the TC and TN content in the soil decreased with soil layer depth, and that of TC, TN, TP, and AP was the highest in the OMF in each soil layer, except for AP in the 20–40 cm soil layer.

Stoichiometric ratio of soil TC, TN, and TP

In the surface soil (0–20 cm), soil TC: TN ratio ranged from 12.82 to 18.00, soil TC: TP ratio ranged from 11.01 to 67.84, and soil TN: TP ratio ranged from 0.73 to 3.97. And in all soil layers, over the entire life cycle of the P. tabuliformis plantation, soil TC: TN ratio ranged from 10.64 to 18.00 (Fig. 4A), soil TC: TP ratio ranged from 4.01 to 67.84 (Fig. 4B), and soil TN: TP ratio ranged from 0.29 to 3.97 (Fig. 4C). There was no significant difference in soil TC: TN ratio among the different soil layers in the same forest age. In YF, MAF, and NMF, soil TC: TP and soil TN: TP ratio showed significant differences among the soil layers. The soil TC: TN, TC: TP and TN: TP ratio varied with the content of soil TC, TN and TP. So they show different significant differences at different ages. In the same forest age, soil TC: TN, soil TC: TP, and soil TN: TP ratio showed a downward trend with soil layer depth, and soil TC: TP and soil TN: TP ratio first increased and then decreased with an increase in forest age.

Correlation of soil ecological stoichiometry and its ratio with the carbon content in different organs

There was a significant positive correlation between the carbon content of leaves and soil TN: TP ratio in the YF (Fig. 5A). The carbon content of leaves had a significant positive correlation with soil AP content (Fig. 5B), and the carbon content of trunks had a significant positive correlation with soil TN: TP ratio in MF (Fig. 5C). The results showed that soil stoichiometry and its ratio had a weak correlation with the aboveground carbon content.

Soil TC: TN ratio was significantly positively correlated with the fine roots (0.5 m) in the 20–40 cm soil layer. Soil TN and TP content was significantly negatively correlated with the fine roots (1 m) in the 0–20 cm soil layer, and TC: TP ratio was significantly positively correlated with the fine roots in the YF (Figs. 6A–6D). The soil AP content was significantly negatively correlated with the fine roots (0.5 m) in the 20–40 cm soil layer in the MAF (Fig. 6E). Soil TC content was significantly negatively correlated with the fine roots (0.5 m) in the 40–60 cm soil layer (Fig. 6F). The soil TN content soil was significantly negatively correlated with the fine roots (0.5 m) in the 20–40 cm soil layer in the NMF (Fig. 6G). Soil TC: TP ratio was significantly positively correlated with the fine roots (0.5 m) in the 40–60 cm soil layer in the MF (Fig. 6H), and the soil TN content was significantly negatively correlated with fine roots (1 m) in the 20–40 cm soil layer in the OMF (Fig. 6I). Therefore, the soil stoichiometry and its ratio strongly correlated with the carbon content of fine roots.

Carbon distributions over the life cycle of Pinus tabuliformis plantation

The result of this part of the study is consistent with our first hypothesis. The carbon content in the leaves of P. tabuliformis was obviously higher than that (464 g⋅kg⁻¹) of the 492 terrestrial plants worldwide studied by Elser et al. (2000), indicating a high content of organic compounds in P. tabuliformis leaves ((Wang & Zheng, 2018). The carbon content of leaves, branches, and trunks was obviously different from that reported by Liu et al. (2015a); Liu et al. (2015b) and which may be due to the comprehensive influence of different factors, including sampling time, forest age, site conditions, climate, and environment of the sampling site (Wang & Zheng, 2018). The carbon content of the branches decreased with an increase in forest age, whereas the carbon content of the trunks did not change significantly with an increase in forest age, which was consistent with that reported by Zhang et al. (2018). This may be due to the difference in the carbon synthesis rate and distribution strategy in the leaves and branches at different ages (Lei, 2019). The carbon content in the leaves and branches of P. tabuliformis with an increase in forest age, indicating that the fixed carbon content decreased with the growth of plant organs, and the change in content was easily affected by a variety of factors, including sampling time, forest age, and soil physical and chemical properties (Zhang et al., 2013).

During the early growth stage of trees, with an increase in growth rate, the carbon distribution of fine roots closer to the trunk of P. tabuliformis increased, and to meet the needs of tree growth, the fine roots absorbed water and nutrients continuously. However, during the later growth stage of trees, when the growth rate reached the maximum, the growth rate slowed down or stopped, and the roots mainly grew and spread around to expand the absorption area of nutrients. Therefore, during the later growth stage of trees, the fine roots farther away from the trunk had higher carbon content.

Soil ecological stoichiometry characteristics over the life cycle of Pinus tabuliformis plantation

The accumulations of soil TC, TN, and AP are long-term process, and the soil TP content had the most obvious accumulation trend with an increase in forest age. This is consistent with our second hypothesis. The present study was consistent with the previous research have also found that the TC, TN, and TP content of soil in OMF was the highest (Zhao et al., 2012).The results of the present study also showed that the surface layer (0–20 cm) had the highest TC and TN content, showing the phenomenon of “surface accumulation,” which is consistent with the results of previous studies and might be related to the high activity of soil surface soil microorganisms (Wang & Zheng, 2018). The surface soil TN content (2.95 g⋅kg⁻¹) was higher than the national average total nitrogen content (1.88 g⋅kg⁻¹), indicating that the P. tabuliformis plantation in this area had a certain effect on increasing the soil nitrogen content.

In the present study, the soil TP content (1.61 g⋅kg⁻¹) was lower than the global average (2.8 g⋅kg⁻¹) (Ren et al., 2007), which is consistent with the fact that the soil TP content in China is generally lower than the global level (Yang et al., 2014). It may be related to environmental conditions, weathering, and soil erosion in the mountainous areas of eastern Liaoning Province. The results was consistent with the previous research have also found that there was no significant difference in the TP content among all soil layers (Wei & Shao, 2007). This was mainly due to the different carbon, nitrogen, and phosphorus sources (Wang & Yu, 2008). Carbon and nitrogen elements accumulate on the soil surface, migrate downward by leaching, and are affected by plant absorption and utilization. Phosphorus is mainly affected by the weathering of the soil parent material. Moreover, phosphorus is a sedimentary mineral that migrates relatively little in the soil, and is evenly distributed throughout the entire soil layer (Cui, Cao & Chen, 2015). The weathering of rocks and minerals is a stable and lengthy process, which is a major source of soil AP. Therefore, their spatial variability in the soil is small and changes in the soil layer depths are not significant (Liu et al., 2010).

In general, the soil TC: TN ratio is inversely proportional to the decomposition rate of soil organic matter (Majdi & Ohrvik, 2004). The soil TC: TN ratio (18.00) in the present study area was higher than the Chinese soil TC: TN ratio (10.1–12.1) and the global average (13.33). Therefore, the decomposition rate of organic matter and mineralization in the study area were relatively slow. However, except for NMF in this study, soil TC: TN ratio in other aged P. tabuliformis plantations decreased with forest age, indicating that with the increase of forest age, the mineralization rate of organic matter increased, and the demand for soil nutrients gradually increased. If the soil TC: TP ratio is relatively low, it is conducive to the release of nutrients from microorganisms in the process of organic matter decomposition and to promote the increase of available phosphorus in the soil. On the contrary, when soil TC: TP ratio is relatively high, there will be limited phosphorus in the process of decomposition of organic matter by microorganisms. Therefore, there will be competition with plants for soil inorganic phosphorus, which is not conducive to the growth of plants and the increase of NPP (Wang et al., 2014). In the present study, soil TC: TP ratio (67.84) was higher than the average value of 61 in China, indicating that microorganisms and plants in the surface soil layer of the study area compete for phosphorus, which is not conducive to growth (Ning, 2020). In the present study, soil TN: TP ratio (3.97) was lower than the mean value of soil TN: TP ratio in China (5.2), and the mean value increasing at the beginning and then decreasing with the change in forest age, but the difference was not statistically significant. The reason may be that with the growth of P. tabuliformis, the contents of nutrient elements in soil decreased to different degrees, and the demand for phosphorus was stronger than that for carbon and nitrogen. With the increase in forest age, the uptake of nutrients slows down, and the return of nutrients from the litter supplements the soil. In the present study, soil TN content, TC: TN and TC: TP ratios were higher than the national average, whereas TN: TP ratio was lower, indicating that carbon, nitrogen, and phosphorus content in the soil was adequate.

Correlation among components over the life cycle of Pinus tabuliformis plantation

According to Garnier (1998) theory, there is a positive correlation between the content of an element in the soil and the content of an organ in the plant, and this element is the limiting nutrient. In the present study, the results showed that the carbon content of MF leaves was significantly positively correlated with the soil AP content, whereas the soil TN, TP, and AP content were significantly negatively correlated with the fine roots carbon content. Therefore, the soil AP content in MF was the limiting nutrient for the leaves, and soil TN, TP, and AP were the limiting nutrients for fine roots. Given that soil nutrients have significant effects on fine root biomass and nutrients (Chen et al., 2016), it is not surprising that the fine root carbon and nitrogen contents were correlated with the soil carbon and nitrogen contents. There were similar findings, which was consistent with our third hypothesis (Chen et al., 2018; Yuan, Chen & Reich, 2011). According to the third hypothesis proposed by Nadelhoffer (2000) (which is the most well-supported hypothesis), with the improvement of soil nutrient availability, the fine root productivity of trees increases, the life span is shortened, turnover is accelerated, and the fine root biomass decreases. The reduction in the biomass of fine roots of trees can be explained by the optimal allocation and cost-effectiveness theory, that is, the improvement of nutrient availability might reduce the carbon allocation of fine roots. In the P. tabuliformis plantation soil in this area, the TN, TP, and AP content in the soil was assumed to be too high, which might reduce the carbon distribution of the fine roots. Among the different organs of P. tabuliformis in this study area, only the leaves were significantly correlated with the soil AP content. There was no correlation between branches, trunks, and soil nutrients. These results were consistent with those of Jiang et al. (2016).