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Citation: Yuan, M.; Wang, Y.; Wang,
Y.; Wang, Y.; Wang, S.; Pan, Y.; Zhou,
W.; Xiang, X.; Tong, Y. Ecological
Stoichiometric Characteristics of C, N,
and P in Pinus taiwanensis Hayata
Needles, Leaf Litter, Soil, and
Micro-Organisms at Different Forest
Ages. Forests 2024,15, 1954. https://
doi.org/10.3390/f15111954
Academic Editor: Choonsig Kim
Received: 31 August 2024
Revised: 30 October 2024
Accepted: 4 November 2024
Published: 7 November 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Ecological Stoichiometric Characteristics of C, N, and P in Pinus
taiwanensis Hayata Needles, Leaf Litter, Soil, and
Micro-Organisms at Different Forest Ages
Meng Yuan, Yurong Wang, Yang Wang, Yi Wang, Shiwen Wang, Yang Pan, Wangming Zhou, Xiaoyan Xiang
and Yuewei Tong *
Engineering Technology Research Center for Aquatic Organism Conservation and Water Ecosystem Restoration
of Anhui Province, College of Life Science, Anqing Normal University, 1318, Jixian Road, Yixiu District,
Anqing 246133, China; yuanmeng@stu.aqnu.edu.cn (M.Y.); wangyr@stu.aqnu.edu.cn (Y.W.);
wangyang@stu.aqnu.edu.cn (Y.W.); wangyi24@mails.ucas.ac.cn (Y.W.); wangsw@stu.aqnu.edu.cn (S.W.);
panyang@aqnu.edu.cn (Y.P.); zhouwangming@aqnu.edu.cn (W.Z.); xiaoyanxiang@aqnu.edu.cn (X.X.)
*Correspondence: tongyuewei@aqnu.edu.cn; Tel.: +86-18654064606
Abstract: The ecological stoichiometric characterization of plant and soil elements is essential for
understanding the biogeochemical cycles of ecosystems. Based on three forest ages of Pinus taiwanensis
Hayata (P. taiwanensis) plantations in the Gujingyuan National Nature Reserve (i.e., young (16 years),
middle-aged (32 years), and mature forests (50 years)), we conducted a field experiment to analyzed
C, N, and P stoichiometry and the relationships between needles, litter, soil, and micro-organisms
in P. taiwanensis plantations. We intended to elucidate the nutritional characteristics and stability
mechanisms of the artificial P. taiwanensis forest ecosystem. The results showed that the C contents of
live needles, leaf litter, soil, and micro-organisms in P. taiwanensis plantation forests of the three forest
ages were 504.17–547.05, 527.25–548.84, 23.40–35.85, and 0.33–0.54 g/kg, respectively; the respective
N contents were 11.02–13.35, 10.71–11.76, 1.42–2.56, and 0.08–0.12 g/kg; and the respective P contents
were 0.82–0.91, 0.60–0.74, 0.19–0.36, and 0.03–0.06 g/kg. Forest age significantly influenced both the
C, N, and P contents in live needles, leaf litter, soil, and micro-organisms as well as stoichiometric
characteristics (p< 0.05). Furthermore, although the litter N:P content was comparable to that of
needles, the ratios of C:N and C:P in the litter were notably higher compared to those in needles. Soil
C:P and N:P ratios were the highest in mature forests while microbial C:P and N:P ratios continuously
decreased. Stoichiometric analyses of our findings suggest that forest stand age can influence
divergent changes in element cycling among plants, soil, and micro-organisms. The presented results
can aid in further understanding nutrient utilization strategies and regulatory mechanisms for P.
taiwanensis plantation forest systems.
Keywords: Pinus taiwanensis Hayata; stand age; ecological stoichiometry; plant–soil continuum;
Dabie Mountains
1. Introduction
Ecological stoichiometry is an emerging ecological tool for studying the balance and
cycling of multiple elements, providing an integrated approach for studying the coupling
of C, N, P, and other elements in ecosystem processes [
1
,
2
]. Combining biological studies
from different fields, taxa, and scales has been widely utilized to reveal the functions and
roles of nutrient ratios and the regulatory mechanisms of ecosystem components for the
assessment of nutrient availability in various ecosystems [
3
,
4
]. For the forest ecosystem,
studies on ecological stoichiometry coupling have focused on various study areas, forest
types, successional stages, or the above- or below-ground aspects (e.g., plants, litter, and
soils) of nutrient cycling [
5
–
9
]. For example, the leaf nutrient content status better reflects
the capacity of the soil to provide essential elements to plants and gradually return them
Forests 2024,15, 1954. https://doi.org/10.3390/f15111954 https://www.mdpi.com/journal/forests
Forests 2024,15, 1954 2 of 14
to the soil as litter. In contrast, the soil influences nutrient uptake and leaf stoichiometric
characteristics through nutrient availability, transformation, and release
[5,10–12]
. The eco-
logical stoichiometric characteristics of soil microbial biomass intuitively reflect microbial
retention and the utilization of soil nutrients [
13
]. Meanwhile, the ratios of C, N, and P
in plants and soils are pivotal in determining the composition and structure of microbial
communities [
14
]. The interactions between plant nutrient demand, soil nutrient supply,
plant self-regulation, and nutrient return during litter decomposition increase the com-
plexity of nutrient content studies along the plant–litter–soil continuum [
15
]. Therefore, it
can be observed that the systematic study of plant–litter–soil–microbial stoichiometry is of
paramount importance in elucidating the processes and mechanisms underlying nutrient
cycling within terrestrial ecosystems.
Previous studies have demonstrated that the forest microenvironment is continually
evolving with age [
16
]. As the age of a forest changes, its internal environment, com-
positional structure, and soil properties are altered, thus affecting patterns of nutrient
partitioning [
17
,
18
]. Numerous scholars have examined the associations among forest C,
N, and P contents; their respective stoichiometric characteristics; and stand age, yielding
innumerable findings. The effects of stand age and stand structure on soil micro-organisms,
enzyme activities, and nutrient contents have been reported for Mediterranean black pine
(Pinus nigra Ar. ssp. salzmannii) [
19
]. In a study, the leaf, root, and soil stoichiometric prop-
erties were characterized across different shrub ages of Ammopiptanthus mongolicus, and
it was found that P may act as a restrictive element for the growth of plants and recovery
processes in ecosystems inhabited by Ammopiptanthus mongolicus populations [
20
]. The
dynamics of C, N, and P concerning stand age and spatial distribution contribute to the spa-
tial and temporal patterns of C–N–P stoichiometric characteristics in forest ecosystems [
12
].
However, the precise impact of forest stand age on plant–soil–microbial C:N:P stoichiome-
try changes remains elusive. Therefore, employing the principles and methodologies of
ecological stoichiometry to examine the stoichiometric ratios of C, N, and P in plant and
soil components within forest ecosystems, along with an exploration of the sources of plant
nutrients within the soil nutrient cycle and the equilibrium constraints between them, is
crucial for enhancing the precision of local forestry management practices [21,22].
Pinus taiwanensis Hayata is an essential tree species distributed in the mountainous
regions of the southeast coast and the subtropical eastern regions of China, including
Henan, Jiangxi, Zhejiang, and Anhui. This species possesses significant economic, eco-
logical, and social value [
23
]. As a dominant tree species at high altitudes, P. taiwanensis
fulfills an ecological successional function that is unparalleled among other coniferous
forests in harsh climatic and impoverished soil environments [
24
]. Numerous studies have
been conducted regarding the classification, geographic distribution, genetics, population
ecology, physiology, biochemistry, and wood anatomy of P. taiwanensis [
25
–
27
]. In recent
years, eco-chemometric studies of the plant–soil continuum under artificial management
have emerged as a research hotspot at the regional scale [
28
]. Some scholars have compared
the characteristics of soil nutrient distributions and the primary drivers of soil nutrients in
P. taiwanensis forests located at varying altitudes [
29
,
30
]. However, relatively few studies
have concentrated on the plant–soil–microbial C:N:P nutrient content and stoichiometry of
P. taiwanensis at varying ages. Therefore, we chose the young, middle-aged, and mature
stands of P.taiwanensis in the Gujingyuan National Nature Reserve, in the Dabie Mountains,
and analyzed the C, N, and P contents and their stoichiometric ratios in needles, litter, soil,
and micro-organisms to deepen our insight on the strategies of nutrient utilization of C,
N, and P in P. taiwanensis plantation ecosystems of varying stand ages, thereby providing
a scientific basis for their sustainable management. The research questions we aimed to
address were as follows: (1) How do the C, N, and P nutrient contents vary across forest
ages? (2) Do the biochemical stoichiometric characteristics and nutrient contents of the four
fractions of C, N, and P align with changes in the stand age?
Forests 2024,15, 1954 3 of 14
2. Materials and Methods
2.1. Overview of the Study Area
The research site was positioned in the Gujingyuan National Nature Reserve, Yuexi
County, Anqing City, Anhui Province (Figure 1), in the southeast of the Dabie Mountains,
with an altitude of 210 to 1465 m. The geographic coordinates are 116
◦
25
′
02
′′
–116
◦
33
′
23
′′
E and 30
◦
56
′
43
′′
–31
◦
07
′
37
′′
N, spanning a north–south width of 20.02 km and an east–
west length of 13.25 km, covering a total area of 7904.3 hectares. The study region is
typified by a northern subtropical mesic monsoon regime, exhibiting a mean annual
thermometric mean of 14.4
◦
C, a median annual precipitation of 1445.82 mm, an average
annual evaporation of 1444 mm, a normative annual frequency of precipitation events at
147 days, and a vernalization-free period ranging from 180 to 220 days. The soil matrix is
primarily composed of hemp gravel, hemp sand, and hemp clay. Pinus taiwanensis is the
most prevalent species in the region, which is often mixed with broad-leaved forests to
form mixed conifer and broad-leaved forests [31].
Forests 2024, 15, x FOR PEER REVIEW 3 of 14
2. Materials and Methods
2.1. Overview of the Study Area
The research site was positioned in the Gujingyuan National Nature Reserve, Yuexi
County, Anqing City, Anhui Province (Figure 1), in the southeast of the Dabie Mountains,
with an altitude of 210 to 1465 m. The geographic coordinates are 116°25′02″–116°33′23″ E
and 30°56′43″–31°07′37″ N, spanning a north–south width of 20.02 km and an east–west
length of 13.25 km, covering a total area of 7904.3 hectares. The study region is typified by
a northern subtropical mesic monsoon regime, exhibiting a mean annual thermometric
mean of 14.4 °C, a median annual precipitation of 1445.82 mm, an average annual evapo-
ration of 1444 mm, a normative annual frequency of precipitation events at 147 days, and
a vernalization-free period ranging from 180 to 220 days. The soil matrix is primarily com-
posed of hemp gravel, hemp sand, and hemp clay. Pinus taiwanensis is the most prevalent
species in the region, which is often mixed with broad-leaved forests to form mixed coni-
fer and broad-leaved forests [31].
Figure 1. Geographic location of the study area. The study region is located in Gujingyuan National
Nature Reserve, Dabie Mountainous Region, China. Different shapes represent different P. taiwan-
ensis stand age groups.
2.2. Sample Site Seings
In early July 2023, based on a field survey, three forest age classes of P. taiwanensis
forests were selected in the Gujingyuan National Nature Reserve, namely, young (16
years), middle-aged (32 years), and mature (50 years) forests, and three 20 m × 20 m sam-
ple plots were established corresponding to each forest age class. The distance between
the sample plots was not less than 1 km. The per-tree checking was conducted on the P.
taiwanensis trees in the sample plots with the average tree height, DBH, and stand density
measured, and also, the vegetation status under three forests was investigated (Figure 2).
As P. taiwanensis is typically encountered in arid and barren mountainous regions, thriv-
ing in diverse environments across high-elevation gradients, the understory vegetation is
seldom herbaceous. Coniferous forests possess smaller canopies, allowing sunlight to
penetrate the understory more directly, fostering a conducive environment for shrub
growth. The survey revealed that the young forest sample maintained an open structure,
abundant with shrub plants and ample light. Conversely, mature forests exhibited a lower
richness of understory vegetation. The main tree species in the understory include Lindera
glauca, Lindera reflexa, and Quercus serrata, among others. Basic information on the three
forest age classes is shown in Table 1.
Figure 1. Geographic location of the study area. The study region is located in Gujingyuan National
Nature Reserve, Dabie Mountainous Region, China. Different shapes represent different P. taiwanensis
stand age groups.
2.2. Sample Site Settings
In early July 2023, based on a field survey, three forest age classes of P. taiwanensis
forests were selected in the Gujingyuan National Nature Reserve, namely, young (16 years),
middle-aged (32 years), and mature (50 years) forests, and three 20 m
×
20 m sample plots
were established corresponding to each forest age class. The distance between the sample
plots was not less than 1 km. The per-tree checking was conducted on the P. taiwanensis
trees in the sample plots with the average tree height, DBH, and stand density measured,
and also, the vegetation status under three forests was investigated (Figure 2). As P.
taiwanensis is typically encountered in arid and barren mountainous regions, thriving in
diverse environments across high-elevation gradients, the understory vegetation is seldom
herbaceous. Coniferous forests possess smaller canopies, allowing sunlight to penetrate
the understory more directly, fostering a conducive environment for shrub growth. The
survey revealed that the young forest sample maintained an open structure, abundant
with shrub plants and ample light. Conversely, mature forests exhibited a lower richness
of understory vegetation. The main tree species in the understory include Lindera glauca,
Lindera reflexa, and Quercus serrata, among others. Basic information on the three forest age
classes is shown in Table 1.
Forests 2024,15, 1954 4 of 14
Forests 2024, 15, x FOR PEER REVIEW 4 of 14
Figure 2. Conditions of three experimental sites. (a–c) show the three stand profiles of young, mid-
dle-aged, and mature-age forests, respectively.
Table 1. Basic information and variables of standard plots in different age groups.
Forest Type Soil Layer()(0–10 cm)
Forest Type Age Alti-
tude()(m)
Mean
DBH()(cm)
Mean
Height()(m)
Mean
Stand()Den-
sity()(trees⋅ha
−1)
Mean Can-
opy Density pH Mois-
ture()(%)
Young Forest 16 1208.8 11.12 ± 0.33 9.44 ± 0.40 533 0.83 ± 0.03 5.43 ± 0.17 25.72 ± 1.53
Middle-aged
Forest 32 1234.5 18.06 ± 0.17 13.39 ± 0.48 500 0.64 ± 0.04 5.67 ± 0.16 25.39 ± 0.99
Mature Forest 50 1241.5 28.22 ± 0.55 15.31 ± 0.62 470 0.59 ± 0.03 5.60 ± 0.18 26.29 ± 1.12
Mean values ± standard errors are shown.
2.3. Sample Collection and Determination
Five average trees were chosen as the standard trees in each mature-age plot. Healthy
and mature live needles were collected from the middle and upper crowns of each tree,
evenly distributed across the east, west, south, and north directions, using high-branching
scissors. In three plots of each forest age class, five 2 × 2 m lier traps were set up at the
four corners and centers to collect lier. The collected samples were thoroughly mixed
and placed in a paper bag. Firstly, live needle samples were killed at 105 °C for 30 min to
inactivate the enzymes in the needles for a short period of time, then the temperature was
reduced to 70 °C to remove the water from the needles until a stable weight was aained.
Subsequently, the needles and lier samples were ground into a powder with a particle
size of 0.15 mm to ascertain the C, N, and P contents. Concurrently, soil samples were
collected beneath the same trees where coniferous lier was gathered; in particular, 5 soil
specimens were gathered from the uppermost 0 to 10 cm of the topsoil stratum using a
soil drill in each square. These soil samples were thoroughly mixed. One part was air-
dried and screened with a 0.25 mm sieve to determine soil C, N, and P contents while the
other part was sieved and stored at 4 °C to assess soil microbial C, N, and P.
The total plant (needles and lier) and soil organic C contents were assessed with a
revised Walkley–Black acid dichromate (FeSO4) exothermic titration method [32]. Plant
total N and soil total N contents were determined according to the Kjeldahl method after
digestion with H2SO4 and H2O2. The P content was also ascertained through the molyb-
denum anti-colorimetric method [33]. Moreover, the chloroform fumigation extraction
method was employed to evaluate the soil microbial biomass C, N, and P contents. The
Figure 2. Conditions of three experimental sites. (a–c) show the three stand profiles of young,
middle-aged, and mature-age forests, respectively.
Table 1. Basic information and variables of standard plots in different age groups.
Forest Type Soil Layer
(0–10 cm)
Forest Type Age Altitude
(m)
Mean DBH
(cm)
Mean
Height
(m)
Mean Stand
Density
(trees·ha−1)
Mean
Canopy
Density
pH Moisture
(%)
Young Forest 16 1208.8 11.12 ±0.33 9.44 ±0.40 533 0.83 ±0.03 5.43 ±0.17 25.72 ±1.53
Middle-aged Forest
32 1234.5 18.06 ±0.17 13.39 ±0.48 500 0.64 ±0.04 5.67 ±0.16 25.39 ±0.99
Mature Forest 50 1241.5 28.22 ±0.55 15.31 ±0.62 470 0.59 ±0.03 5.60 ±0.18 26.29 ±1.12
Mean values ±standard errors are shown.
2.3. Sample Collection and Determination
Five average trees were chosen as the standard trees in each mature-age plot. Healthy
and mature live needles were collected from the middle and upper crowns of each tree,
evenly distributed across the east, west, south, and north directions, using high-branching
scissors. In three plots of each forest age class, five 2
×
2 m litter traps were set up at the
four corners and centers to collect litter. The collected samples were thoroughly mixed
and placed in a paper bag. Firstly, live needle samples were killed at 105
◦
C for 30 min to
inactivate the enzymes in the needles for a short period of time, then the temperature was
reduced to 70
◦
C to remove the water from the needles until a stable weight was attained.
Subsequently, the needles and litter samples were ground into a powder with a particle
size of 0.15 mm to ascertain the C, N, and P contents. Concurrently, soil samples were
collected beneath the same trees where coniferous litter was gathered; in particular, 5 soil
specimens were gathered from the uppermost 0 to 10 cm of the topsoil stratum using a soil
drill in each square. These soil samples were thoroughly mixed. One part was air-dried
and screened with a 0.25 mm sieve to determine soil C, N, and P contents while the other
part was sieved and stored at 4 ◦C to assess soil microbial C, N, and P.
The total plant (needles and litter) and soil organic C contents were assessed with a
revised Walkley–Black acid dichromate (FeSO
4
) exothermic titration method [
32
]. Plant
total N and soil total N contents were determined according to the Kjeldahl method after
digestion with H
2
SO
4
and H
2
O
2
. The P content was also ascertained through the molyb-
denum anti-colorimetric method [
33
]. Moreover, the chloroform fumigation extraction
method was employed to evaluate the soil microbial biomass C, N, and P contents. The
microbial C and N contents were derived through comparing the unfumigated and fumi-
gated soil samples. A colorimetric determination of microbial P was carried out using UV
spectrophotometry [34].
Forests 2024,15, 1954 5 of 14
2.4. Data Analysis
Excel 2019 was utilized to organize the experimental data. One-way ANOVAs were
conducted for the live needle, litter, soil, and soil microbial C, N, and P contents, along
with the stoichiometric ratios of P. taiwanensis across different ages using SPSS 27.0 (IBM
Corp., Armonk, NY, USA). The least significant difference (LSD) test was employed at a
95% confidence interval (p< 0.05) to assess whether the sample means differed significantly
from a normal distribution, and the mean
±
standard deviation values have been utilized
in the graphical representations. Pearson correlation coefficient assessment was conducted
to ascertain the interrelationships among C, N, and P in plant and soil samples and their
respective ecological stoichiometric proportions. The results were depicted graphically
utilizing Origin 2021 software (OriginLab Corp., Northampton, MA, USA).
3. Results
3.1. C, N, and P Contents in Live Needle, Leaf Litter, Soil, and Microbial Communities Across
Different Forest Ages
The C, N, and P contents of P. taiwanensis forests exhibited a pattern of live
needle > leaf
litter > soil > micro-organisms, except for the leaf litter C content being greater than that
of needle C (Table 2). Firstly, the live needle, leaf litter, soil, and microbial C contents in
different P. taiwanensis plantations ranged from 504.17 to 547.05, 527.25 to 548.84, 23.40
to 35.85, and 0.33 to 0.54 g/kg, respectively. An analysis of variance (ANOVA) revealed
that the C contents of all the four fractions varied significantly across different stand ages
(
p< 0.05
). With the advancement of forest age, the needle and litter C contents reduced first
and then increased and exhibited an inverted U-shaped trend. However, the soil C content
continued to decline, and the microbial C content was also highest in the young forests.
Table 2. Chemical properties of C, N, and P contents in live needles, leaf litter, soil, and micro-
organisms of P. taiwanensis plantations across different age groups.
Chemical
Properties
(g/kg)
Different
Fractions
Young
Forest
Middle-Aged
Forest
Mature
Forest
C
Live needle 504.17 ±5.72 b547.05 ±6.01 a543.84 ±5.76 a
Leaf litter 535.66 ±4.94 b548.84 ±8.00 a527.25 ±5.98 b
Soil 35.85 ±1.45 a25.16 ±2.02 b23.40 ±0.58 b
Micro-organisms 0.54 ±0.018 a0.33 ±0.012 c0.36 ±0.007 b
N
Live needle 11.02 ±0.46 b12.26 ±0.29 a13.34 ±0.59 a
Leaf litter 10.71 ±0.45 b11.03 ±0.35 b11.76 ±0.35 a
Soil 2.56 ±0.12 a1.42 ±0.22 b1.53 ±0.11 b
Micro-organisms 0.12 ±0.004 a0.09 ±0.014 b0.08 ±0.019 b
P
Live needle 0.85 ±0.02 b0.92 ±0.15 a0.82 ±0.07 b
Leaf litter 0.74 ±0.04 a0.60 ±0.01 b0.70 ±0.01 a
Soil 0.36 ±0.022 a0.23 ±0.025 b0.19 ±0.014 b
Micro-organisms 0.03 ±0.002 b0.05 ±0.009 a0.06 ±0.002 a
Different superscript lowercase characters denote statistically significant (p< 0.05) variations in the C, N, and
P contents within needles, litter, soil, and micro-organisms across various stand age categories. Live needles,
leaf litter, and soil were collected from each sample plot for each of the three stand ages, with five replicates per
sample plot.
Then, the N contents of P. taiwanensis plantations were in the ranges of 11.02 to
13.34, 10.71 to 11.76, 1.42 to 2.56, and 0.08 to 0.12 g/kg for needles, litter, soil, and micro-
organisms, respectively. The N contents of needles, leaf litter, and micro-organisms differed
significantly among forest ages (p< 0.05). The age of the forest exerted a comparable
influence on the N content in the needles and litter of P. taiwanensis; mature forests showed
the highest level of N in their needles and litter. However, soil N content exhibited a
U-shaped trend with an increasing stand age, and the highest levels of soil and microbial N
contents were found in the young forests.
Forests 2024,15, 1954 6 of 14
Moreover, the needle, litter, soil, and microbial P contents of three P. taiwanensis
plantations in the study area were 0.82 to 0.92, 0.60 to 0.74, 0.19 to 0.36, and 0.03 to
0.06 g/kg, respectively. The P contents of needles, litter, soil, and micro-organisms differed
significantly among forest ages (p< 0.05). In particular, the needle P content was also
showed an inverted U-shaped trend, with the needle P content being higher in middle-aged
forests compared to young and mature ones. Leaf litter and soil P contents were highest in
young forests while microbial P content was highest in mature forests.
3.2. Ecological Stoichiometric Ratios Among Needles, Leaf Litter, Soil, and Micro-Organisms in
Different Stand Ages
The C:N, C:P, and N:P ratios across the four distinct fractions revealed the order to
be leaf litter > live needles > soil > micro-organisms, and the differences between the four
pools were significant (p< 0.05). The effects of forest age on the four fractions of C:N, C:P,
and N:P were not uniform. The values of C:N for live needles, litter, soil, and microbes
in P. taiwanensis plantations were 40.81 to 45.79, 44.86 to 50.08, 14.01 to 17.91, and 3.53 to
4.58, respectively (Figure 3). The C:N ratios of needles and litter in mature forests differed
significantly from those in young and middle-aged forests (p< 0.05); both litter and needle
C:N decreased with an increasing stand age. Meanwhile, variations in soil C:N ratios were
observed across the three forest age classes, and soil C:N reached its highest value and
microbial C:N reached its lowest value in the middle-aged forest.
Forests 2024, 15, x FOR PEER REVIEW 7 of 14
(A) (B)
(C)
Figure 3. Stoichiometry of live needle, leaf lier, soil, and microbial C:N (A), C:P (B), and N:P (C) in
P. taiwanensis plantations of different ages. Capital leers (A, B, C, D) denote substantial variations
(p < 0.05) among needles, leaf lier, soil, and micro-organisms at the same stand age; lowercase
leers (a, b, c) signify significant differences (p < 0.05) across various stand ages within the same
research component.
3.3. Correlation of Factors in Different Fractions of P. taiwanensis Plantation Forests
The ecological stoichiometry of C, N, and P in the needles, lier, soil, and microbiota
of P. taiwanensis plantations exhibited strong correlations (Figure 4). As the results
showed, the needle C and N contents exhibited a strong, significant correlation with soil
and microbial C, N, and P values. The leaf lier N showed a significant correlation with
microbial and soil P, and leaf lier P was significantly correlated with microbial C content
and soil N. Meanwhile, there was a highly significant correlation between soil C, N, and
P contents with microbial C, N, and P contents (p < 0.01). For the ecological stoichiometric
ratios, the needle C:N was significantly and positively correlated with leaf lier C:N (p <
0.05), and the needle C:P and N:P ratios displayed highly significant correlations (p < 0.01)
with the lier C:N ratio. Additionally, there was a notably inverse relationship between
lier N:P and microbial C:P and N:P (p < 0.01), and the lier C:N ratio was significantly
associated with soil N:P, while the lier C:P ratio showed a significant correlation with
both microbial and soil C:N ratios (p < 0.05).
Figure 3. Stoichiometry of live needle, leaf litter, soil, and microbial C:N (A), C:P (B), and N:P (C) in
P. taiwanensis plantations of different ages. Capital letters (A, B, C, D) denote substantial variations
(p< 0.05) among needles, leaf litter, soil, and micro-organisms at the same stand age; lowercase
letters (a, b, c) signify significant differences (
p< 0.05
) across various stand ages within the same
research component.
Forests 2024,15, 1954 7 of 14
The needle, litter, soil, and microbial C:P ratios of P. taiwanensis plantations within the
study region were in the ranges of 593.97 to 667.24, 724.15 to 911.71, 94.70 to 125.56, and
5.99 to 19.26 g/kg, respectively. The effects of forest age on needle, litter, soil, and microbial
C:P ratios were all significant (p< 0.05); leaf litter C:P was significantly higher than needle
C:P; microbial C:P decreased with an increasing forest age and had a highest level in young
forests compared to middle and mature forests.
The N:P ratios of the needle, litter, soil, and micro-organisms of P. taiwanensis plan-
tations were 12.98 to 16.36, 14.45 to 18.32, 6.27 to 7.14, and 1.35 to 4.36 g/kg, respectively.
The effects of different forest ages on needle, litter, soil, and microbial N:P ratios were also
significant (p< 0.05). The needle N:P ratio continued to increase while stand age advanced,
and the microbial N:P diminished. The N:P ratio in needles was highest in the mature
forest stage while it was highest in the middle-aged forest for the leaf litter.
3.3. Correlation of Factors in Different Fractions of P. taiwanensis Plantation Forests
The ecological stoichiometry of C, N, and P in the needles, litter, soil, and microbiota of
P. taiwanensis plantations exhibited strong correlations (Figure 4). As the results showed, the
needle C and N contents exhibited a strong, significant correlation with soil and microbial
C, N, and P values. The leaf litter N showed a significant correlation with microbial and
soil P, and leaf litter P was significantly correlated with microbial C content and soil N.
Meanwhile, there was a highly significant correlation between soil C, N, and P contents
with microbial C, N, and P contents (p< 0.01). For the ecological stoichiometric ratios, the
needle C:N was significantly and positively correlated with leaf litter C:N (p< 0.05), and
the needle C:P and N:P ratios displayed highly significant correlations (p< 0.01) with the
litter C:N ratio. Additionally, there was a notably inverse relationship between litter N:P
and microbial C:P and N:P (p< 0.01), and the litter C:N ratio was significantly associated
with soil N:P, while the litter C:P ratio showed a significant correlation with both microbial
and soil C:N ratios (p< 0.05).
Forests 2024, 15, x FOR PEER REVIEW 8 of 14
Figure 4. Stoichiometric relationships in the plant-soil continuum of four components: needles, leaf
lier, soil, and micro-organisms. Correlations between live needle, leaf lier, soil, and microbial C,
N, and P contents and ecological stoichiometric ratios in P. taiwanensis plantations are shown. Sym-
bols * and ** indicate correlations of significance at the 0.05 and 0.01 probability levels, respectively.
4. Discussion
4.1. Changes in Live Needle, Leaf Lier, Soil, and Microbial CNP Contents with Forest Age
The circulation of elements C, N, and P is a crucial element influencing the function-
ality of forest ecosystems [3]. Most previous studies have shown that the C, N, and P con-
tents of plants and their ecological stoichiometric ratios vary with forest age, with different
paerns of change being influenced by forest age [18]. In this research, needle C, N, and P
contents were significantly influenced by the stand age, suggesting variations in the eco-
logical adaptations of the plants across different developmental stages [35]. The mean
value of C content in P. taiwanensis needles in this study area exceeded the global mean C
content for terrestrial plant leaves [36], indicating that the content of organic compounds
synthesized by P. taiwanensis needles was high. Meanwhile, the mean value of N content
in P. taiwanensis needles was significantly lower than the mean value in leaves of terrestrial
plants in China [37] and the mean value in leaves of plants globally [36]. Furthermore, the
mean value of P content in live needles was significantly lower than the mean value in
land plant leaves [37]. All these results indicated that the distribution paern in the live
needle of P. taiwanensis was “high C, low N and P”, consistent with the findings of Ashfaq
[38]. This paern may be related to the characteristics of the tree species as conifers typi-
cally have a high C content and low N and P contents [39]. The growth rate of P. taiwan-
ensis gradually increases in the middle-aged stage, thus increasing dry maer synthesis,
and more rRNA is required for protein synthesis [40]. In stands ranging from young to
mature, leaf C contents in middle-aged forests were higher, indicating that middle-aged
stands have higher leaf organic maer content and greater C storage, compared to other
stand ages. The reason for this phenomenon may have been the influence of plant age on
photosynthesis [41]. On the other hand, lier acts as the hub connecting vegetation and
soil and is the primary way in which plants return nutrients to the ecosystem [42]. The
lier P content was lower than the global averages for senescent leaves while the C and N
Figure 4. Stoichiometric relationships in the plant-soil continuum of four components: needles, leaf
litter, soil, and micro-organisms. Correlations between live needle, leaf litter, soil, and microbial C, N,
and P contents and ecological stoichiometric ratios in P. taiwanensis plantations are shown. Symbols *
and ** indicate correlations of significance at the 0.05 and 0.01 probability levels, respectively.
Forests 2024,15, 1954 8 of 14
4. Discussion
4.1. Changes in Live Needle, Leaf Litter, Soil, and Microbial CNP Contents with Forest Age
The circulation of elements C, N, and P is a crucial element influencing the functionality
of forest ecosystems [
3
]. Most previous studies have shown that the C, N, and P contents
of plants and their ecological stoichiometric ratios vary with forest age, with different
patterns of change being influenced by forest age [
18
]. In this research, needle C, N, and
P contents were significantly influenced by the stand age, suggesting variations in the
ecological adaptations of the plants across different developmental stages [
35
]. The mean
value of C content in P. taiwanensis needles in this study area exceeded the global mean C
content for terrestrial plant leaves [
36
], indicating that the content of organic compounds
synthesized by P. taiwanensis needles was high. Meanwhile, the mean value of N content in
P. taiwanensis needles was significantly lower than the mean value in leaves of terrestrial
plants in China [
37
] and the mean value in leaves of plants globally [
36
]. Furthermore, the
mean value of P content in live needles was significantly lower than the mean value in land
plant leaves [
37
]. All these results indicated that the distribution pattern in the live needle of
P. taiwanensis was “high C, low N and P”, consistent with the findings of Ashfaq [
38
]. This
pattern may be related to the characteristics of the tree species as conifers typically have a
high C content and low N and P contents [39]. The growth rate of P. taiwanensis gradually
increases in the middle-aged stage, thus increasing dry matter synthesis, and more rRNA is
required for protein synthesis [
40
]. In stands ranging from young to mature, leaf C contents
in middle-aged forests were higher, indicating that middle-aged stands have higher leaf
organic matter content and greater C storage, compared to other stand ages. The reason for
this phenomenon may have been the influence of plant age on photosynthesis [
41
]. On the
other hand, litter acts as the hub connecting vegetation and soil and is the primary way in
which plants return nutrients to the ecosystem [
42
]. The litter P content was lower than
the global averages for senescent leaves while the C and N contents were higher [
43
]. The
trends in leaf litter content and live needle C and N contents with an increasing stand age
were consistent, indicating a close relationship between the P. taiwanensis litter and needles.
However, the influences of forest age on the needle and litter C, N, and P contents were
not uniform, suggesting that forest age had a non-uniform influence on their variability.
Nevertheless, both are significantly influenced by the age of the forest.
The C, N, and P contents of the soil are affected by changes in soil physicochemical
properties and the return of apoplastic nutrients [
44
]. According to the stoichiometric values
in this study, C and N contents in 0–10 cm soils were high, compared to national levels, while
the P content was low [
45
]. This imbalance further led to soil C:N:P stoichiometry alterations
as the C and N accumulation rate is usually faster than that of P [
46
]. Additionally, there
was a general decline in soil C, N, and P contents as the forest age progressed. This suggests
a specific degree of soil degradation by P. taiwanensis in this area, consistent with the study
by Zeng et al. on Larix gmelinii (Rupr.) [
47
]. The reason may be that as a plant grows,
the roots draw nutrients from the soil and store them in the trunk and leaves [
48
]. The
vegetation canopy, plant biodiversity, topographical features, soil textural attributes, and
substrate lithology are pivotal in modulating the pools of soil organic carbon and essential
nutrients [
49
,
50
]. Soil N is primarily derived from litter decomposition and atmospheric
deposition [
51
]. Young forest stands are distinguished by their low canopy density, rich
shrubs and herbaceous plants, abundant light, and high air temperatures. Consequently,
soil microbial activity is heightened, facilitating the decomposition of organic matter and
the conversion of N into soil nutrients, thus leading to higher soil C and N contents in young
forests [
52
]. Soil P is derived from rock weathering and the decay of organic matter such as
decomposing plant material [
53
]. The pattern of change in P levels of soil is analogous to
that of N, and similar findings have been observed across diverse mixed forests located in
the Qinling Mountains region of China [54].
Soil micro-organisms are the main agents responsible for the decomposition of soil
organic matter and the turnover of nutrients [
4
]. The microbial C content can indicate the
abundance of living soil micro-organisms [
55
]. The microbial N content comprehensively
Forests 2024,15, 1954 9 of 14
reflects the nitrogen mineralization and fixation process by microbial bodies while microbial
P is a crucial source of available P for plants, with a fast turnover rate and susceptibility
to environmental influences [
56
]. The responses of microbial C and N to stand age in P.
taiwanensis plantations in this study were consistent with the soil C and N. This response
reflects the characteristics of the soil microbial biomass, affected by soil nutrients [
57
].
The depletion of soil C and N in middle-aged and mature forests affected the activities of
soil micro-organisms, leading to a reduction in soil microbial C and N levels. Microbial
P gradually increased, favoring the conversion and release of soil-available P, similar to
the results obtained by Fang et al. when considering Quercus serrata forests of different
ages [58].
4.2. Changes in Live Needle, Litter, Soil, and Microbial CNP Stoichiometric Ratios with Stand Age
The C:N, C:P, and N:P ratios of live needles, leaf litter, soil, and micro-organisms
represent the competition between different components to maintain ecological balance
and their efforts to adapt to the environment in order to meet their own needs, thus
characterizing the changes in total productivity at the elemental level [
59
]. Needle N:P
serves as an indicator for assessing plant limitations in terms of N and P. Notably, the
N:P ratio in needles varied significantly across different stand ages. P. taiwanensis was
found to be limited by different nutrients at various stand ages. The observed increase in
needle N:P ratios with age suggests that P. taiwanensis forests progressively become more
P-limited [
60
]. As important physiological indices of plants, C:N and C:P can indicate both
a plant’s capacity for carbon assimilation as well as the effectiveness of nutrient acquisition
and utilization [
61
]. The mean values of C:N and C:P in P. taiwanensis needles at different
forest ages were higher than the global mean values for plant leaves [
36
], suggesting that P.
taiwanensis exhibits an efficient use of N and P along with a significant capacity for carbon
sequestration. The mature forest stand exhibited elevated leaf C:P and N:P ratios compared
to the other stands, suggesting a greater capacity for C assimilation at this stage.
The conditions of soil nutrients and the absorption and utilization of nutrients by
plants significantly influence the regulation of the ecological stoichiometric properties
of C, N, and P in litter. When the soil nutrient supply is inadequate, plants may resort
to the re-uptake of nutrients from the litter, thereby altering its ecological stoichiometric
characteristics [
62
]. The N and P contents of leaf litter were lower than those in live
needles while the C:N and C:P ratios significantly exceeded those found in live needles,
which, as in the study by Wang et al., indicated that P. taiwanensis is characterized by
nutrient reabsorption [
63
]. Studies have shown that the litter N:P ratio is a crucial factor
influencing litter decomposition and nutrient cycling and negatively correlates with the
litter decomposition rate [
64
]. The litter N:P ratio of P. taiwanensis initially rose before
declining as the forest age advanced, indicating that the rate of litter decomposition initially
slowed down before accelerating as the forest age increased, similar to the results of the
study by Jiang et al. on Pinus tabulaeformis forests of different ages [65].
Previous studies have indicated that the soil C:N ratio is indicative of the rate at which
organic matter undergoes mineralization, the C:P ratio demonstrates the magnitude of P
potential released through the microbial degradation of soil organic matter into inorganic
forms, and the N:P ratio reflects soil P availability [
66
]. The mean values of the C:N,
C:P, and N:P ratios in P. taiwanensis soils under different forest ages were higher than the
respective national mean values for terrestrial soils [
45
]. This indicates that the conversion
of soil organic matter into minerals in P. taiwanensis forests in this region is slow, which is
conducive to the accumulation of soil organic C and enhancement of the function of soil C
pools in P. taiwanensis forests. The slow decomposition of N and P is not conducive to the
release of N and P, easily restricting the growth of P. taiwanensis [
67
]. The C:P ratio in the soil
is vital for the development of plants and development, indicating the potential of plants
to release P from the environment and to absorb and utilize it [
68
]. A higher soil C:P ratio
reflects low soil P availability [
69
]. In this study, soil C:P ratios changed minimally with
an increasing forest age, suggesting that the trend of soil P availability remained relatively
Forests 2024,15, 1954 10 of 14
stable during the subsequent growth of P. taiwanensis forests. Furthermore, numerous
studies have proven that a high microbial C:N ratio is associated with a high proportion
of soil fungi in the microbial community [
70
]. The differences in microbial C:N ratios
among the three stands in this study were not significant, indicating that the abundance
of soil microbial fungi in P. taiwanensis plantations did not vary significantly among the
three stands. The microbial C:P ratio continuously decreased with an increasing forest
age, suggesting an enhancement in the ability of micro-organisms to fix P, which was the
strongest in mature forests [
71
]. The demand for and utilization of resources by micro-
organisms are motivated by the biochemical ratios in their biomass and their capacity
for growth; however, they may also be influenced by the relative supply of available
resources [72].
4.3. Coupling of Needle, Litter, Soil, and Microbial CNP and Stoichiometric Ratios
Correlation analyses revealed significant correlations among the C, N, and P stoichio-
metric characteristics of P. taiwanensis live needles, leaf litter, soil, and micro-organisms.
These results indicated that C, N, and P elements were transported and transformed among
the four components (i.e., plants, litter, soil, and micro-organisms) in forest ecosystems [
73
].
The soil microbial biomass is pivotal in regulating soil nutrient cycling and energy flow [
74
].
Furthermore, the soil environment is a crucial factor influencing the growth of micro-
organisms as soil is intimately linked to micro-organisms [
75
]. This study also revealed
a substantial association among soil-based metrics and microbial stoichiometric ratios.
The highly significant correlation between soil C, N, and P contents and needle C and N
contents indicates that the stoichiometric balance of soil C, N, and P significantly affects
leaf nutrient uptake. Therefore, it is crucial to explore the profound connections between C,
N, and P within plants, soils, and micro-organisms [
76
]. In this study, there was a notable
correlation between the needle C:P and N:P ratios and the litter C:N ratio. Additionally,
leaf litter N:P ratio exhibited a significant negative correlation with the microbial C:P and
N:P ratios. The litter C:P and N:P ratios showed a strong, positive correlation with the
soil C:N ratio, indicating that live needles are (directly or indirectly) correlated with each
indicator of litter, soil, and micro-organisms [
77
,
78
]. This result demonstrates the signif-
icant coupling relationships between the ecological stoichiometric characteristics of the
four components, namely, needles, leaf litter, soil, and micro-organisms. This suggests the
mutual transportation and transfer of C, N, and P among these four components during
the growth of P. taiwanensis.
5. Conclusions
Consequently, the current study examined P. taiwanensis plantations of young, middle-
aged, and mature forests in the Gujingyuan National Nature Reserve, a representative
region in northern subtropical China. By analyzing C, N, and P contents in lived needles,
litter, soil, and micro-organisms, we investigated the patterns of changes in the stoichiomet-
ric ratios of C, N, and P across different forest ages. The results showed that the nutrient
requirements of the plantations varied with the stand age. Specifically, significant differ-
ences in the stoichiometric characteristics of C, N, and P in needles, leaf litter, soil, and
micro-organisms were observed between different stand ages of P. taiwanensis plantation
forests. Litter can serve as a nutrient hub between leaf and soil systems. According to the
needle N:P reabsorption theory, the N:P ratio increased with forest age, suggesting a transi-
tion from N to P limitation as the forest matured. At the same time, soil C and P contents
decreased under all three stand ages, suggesting a certain degree of soil degradation in the
area. Microbial C:P values decreased as stand age advanced, implying that the microbial
capacity to fix P increased. The understory soil of P. taiwanensis in the study area is sandy
and low in nutrients, as well as in microbial C, N, and P contents. P. taiwanensis fulfills an
ecological successional function unparalleled among coniferous forests in harsh climatic
and impoverished soil environments. Overall, the C, N, and P contents of P. taiwanensis
needles, litter, soil, and micro-organisms were closely related to their ecological stoichio-
Forests 2024,15, 1954 11 of 14
metric ratios. Forest age is an important factor influencing elemental cycling among plants,
litter, soil, and micro-organisms.
Author Contributions: Formal analyses, surveys, and writing—original manuscript, M.Y.; data
compilation, analysis, and investigation, Y.W. (Yi Wang) and Y.W. (Yurong Wang); conceptualization
and methodology, X.X. and W.Z.; investigation, Y.W. (Yang Wang), S.W. and Y.P.; writing—reviewing
and editing, Y.T. All authors have read and agreed to the published version of the manuscript.
Funding: This research was financially supported by the Project of the Natural Science Foundation of
Anhui Province (2208085QC72 and 1908085MC58) and the Scientific Research Projects of Universities
in Anhui Province (2022AH051052).
Data Availability Statement: Data are available on request from the authors.
Acknowledgments: The authors sincerely thank the Gujingyuan National Nature Reserve in Anhui
Province for supporting and permitting this study. The authors are thankful to the staff of the reserve
for their help.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1.
Elser, J.J.; Dobberfuhl, D.R.; MacKay, N.A.; Schampel, J.H. Organism size, life history, and N:P stoichiometry: Toward a unified
view of cellular and ecosystem processes. BioScience 1996,46, 674–684. [CrossRef]
2.
Sistla, S.A.; Schimel, J.P. Stoichiometric flexibility as a regulator of carbon and nutrient cycling in terrestrial ecosystems under
change. New Phytol. 2012,196, 68–78. [CrossRef] [PubMed]
3.
Leroux, S.J.; Wal, E.V.; Wiersma, Y.F.; Charron, L.; Ebel, J.D.; Ellis, N.M.; Hart, C.; Kissler, E.; Saunders, P.W.; Moudrá, L.; et al.
Stoichiometric distribution models: Ecological stoichiometry at the landscape extent. Ecol. Lett. 2017,20, 1495–1506. [CrossRef]
[PubMed]
4.
Van de Waal, D.B.; Elser, J.J.; Martiny, A.C.; Sterner, R.W.; Cotner, J.B. Editorial: Progress in ecological stoichiometry. Front.
Microbiol. 2018,9, 1957. [CrossRef] [PubMed]
5.
Wang, Z.; Zheng, F. Ecological stoichiometry of plant leaves, litter and soils in a secondary forest on China’s Loess Plateau. PeerJ
2020,8, e10084. [CrossRef]
6.
Wang, W.; Peng, Y.; Chen, Y.; Lei, S.; Wang, X.; Farooq, T.H.; Liang, X.; Zhang, C.; Yan, W.; Chen, X. Ecological stoichiometry and
stock distribution of C, N, and P in three forest types in a Karst region of China. Plants 2023,12, 2503. [CrossRef]
7.
Yu, M.F.; Tao, Y.; Liu, W.; Xing, W.; Liu, G.; Wang, L.; Ma, L. C, N, and P stoichiometry and their interaction with different plant
communities and soils in subtropical riparian wetlands. Environ. Sci. Pollut. Res. Int. 2020,27, 1024–1034. [CrossRef]
8.
Asada, K.; Kanda, T.; Yamashita, N.; Asano, M.; Eguchi, S. Interpreting stoichiometric homeostasis and flexibility of soil microbial
biomass carbon, nitrogen, and phosphorus. Ecol. Model. 2022,470, 110018. [CrossRef]
9.
Jiang, J.; Lu, Y.; Chen, B.; Ming, A.; Pang, L. Nutrient resorption and C:N:P stoichiometry responses of a Pinus massoniana
plantation to various thinning intensities in Southern China. Forests 2022,13, 1699. [CrossRef]
10.
Zhang, G.; Zhang, P.; Peng, S.; Chen, Y.; Cao, Y. The coupling of leaf, litter, and soil nutrients in warm temperate forests in
northwestern China. Sci. Rep. 2017,7, 11754. [CrossRef]
11.
Manivannan, S.; Chadha, K.L. Standardization of time of sampling for leaf nutrient diagnosis on Kinnow mandarin in North-West
India. J. Plant Nutr. 2011,34, 1820–1827. [CrossRef]
12.
Bui, E.N.; Henderson, B.L.C. N:P stoichiometry in Australian soils concerning vegetation and environmental factors. Plant Soil
2013,373, 553–568. [CrossRef]
13.
He, X.; Hou, E.; Veen, G.F.; Ellwood, M.D.F.; Dijkstra, P.; Sui, X.; Zhang, S.; Wen, D.; Chu, C. Soil microbial biomass increases
along elevational gradients in the tropics and subtropics but not elsewhere. Glob. Ecol. Biogeogr. 2020,29, 345–354. [CrossRef]
14.
Yang, Y.; Liu, H.; Yang, X.; Yao, H.; Deng, X.; Wang, Y.; An, S.; Kuzyakov, Y.; Chang, S.X. Plant and soil elemental C:N:P ratios are
linked to soil microbial diversity during grassland restoration on the Loess Plateau, China. Sci. Total Environ. 2022,806, 150557.
[CrossRef] [PubMed]
15. Agren, G.I.; Bosatta, E. Theoretical ecosystem ecology: Understanding element cycles. J. Ecol. 1998,18, 1025–1026.
16.
Ren, Y.; Gao, G.L.; Ding, G.D.; Zhang, Y.; Zhao, P.S. Patterns and environmental drivers of C, N, and P stoichiometry in the
leaf–litter–soil system associated with Mongolian pine forests. Ecol. Evol. 2024,14, e11172. [CrossRef]
17.
Inagaki, Y.; Miura, S.; Kohzu, A. Effects of forest type and stand age on litterfall quality and soil N dynamics in Shikoku District,
southern Japan. For. Ecol. Manag. 2004,202, 107–117. [CrossRef]
18.
Shen, J.; Fan, S.; Zhang, J.; Liu, G. Stoichiometric homeostasis of N and P in the leaves of different–aged Phyllostachys edulis after
bamboo forest expansion in Subtropical China. Forests 2024,15, 1181. [CrossRef]
19.
Lucas-Borja, M.E.; Hedo, J.; Cerdá, A.; Candel-Pérez, D.; Viñegla, B. Unravelling the importance of forest age stand and forest
structure driving microbiological soil properties, enzymatic activities and soil nutrients content in Mediterranean Spanish black
pine (Pinus Nigra Ar. Ssp. Salzmannii) Forest. Sci. Total Environ. 2016,562, 145–154. [CrossRef]
Forests 2024,15, 1954 12 of 14
20.
Dong, X.; Xu, D.; Wang, D.; Han, C.; Huang, Y.; Zhang, J. Leaf-root-soil stoichiometric characteristics in different shrub ages of
Ammopiptanthus mongolicus.Plants 2023,12, 3103. [CrossRef]
21.
Wang, Y.; Zhang, Y.; Wang, L.; Jing, X.; Yu, L.; Liu, P. Response of leaf biomass, leaf and soil C:N:P stoichiometry characteristics to
different site conditions and forest ages: A case of Pinus Tabuliformis plantations in the temperate mountainous area of China.
Front. Plant Sci. 2022,13, 1060406. [CrossRef] [PubMed]
22.
Gong, Z.; Sheng, M.; Zheng, X.; Zhang, Y.; Wang, L. Ecological stoichiometry of C, N, P, and Si of Karst Masson Pine Forests:
Insights for the forest management in southern China. Sci. Total Environ. 2024,912, 169490. [CrossRef] [PubMed]
23.
Li, Q.Z. Population Structure and Dynamics of Pinus taiwanensis Hayata at Songyang County, Zhejiang Province, China. Plant
Ecol. 1990,86, 119–129.
24.
Su, S.J.; Liu, J.F.; Lan, S.R.; Hong, W.; Li, W.Z. A review of Pinus taiwanensis studies (1960–2014) and the knowledge domain
analysis. Fujian J. Agric. For. Univ. 2015,44, 478–486. (In Chinese)
25.
Chu, M.H.; Hsiao, S.W.; Kao, Y.C.; Yin, H.W.; Kuo, Y.H.; Lee, C.K. Cytotoxicity effect of constituents of Pinus taiwanensis Hayata
Twigs on B16–F10 Melanoma Cells. Molecules 2022,27, 2731. [CrossRef]
26.
Lyu, M.; Sun, M.; Peñuelas, J.; Sardans, J.; Sun, J.; Chen, X.; Zhong, Q.; Cheng, D. Thermal acclimation of foliar CarbonMetabolism
in Pinus taiwanensis along an elevational gradient. Front. Front. Plant Sci. 2021,12, 778045.
27.
Fang, M.F.; Wang, Y.J.; Zu, Y.M.; Dong, W.L.; Wang, R.N.; Deng, T.T.; Li, Z.H. The complete Chloroplast Genome of the Taiwan
Red Pine Pinus taiwanensis (Pinaceae). Mitochondrial DNA Part A 2016,27, 2732–2733. [CrossRef]
28.
Xiang, Y.; Pan, P.; Ouyang, X.; Zang, H.; Rao, J. The chemical stoichiometry characteristics of plant-soil carbon and nitrogen in
subtropical Pinus massoniana natural forests. Sci. Rep. 2024,14, 5031. [CrossRef]
29.
Jiang, M.; He, Y.; Liu, J.; Xing, H.; Gu, Y.J.; Wei, X.; Zhu, Z. Elevation gradient altered soil C, N, and P stoichiometry of Pinus
taiwanensis forest on Daiyun Mountain. Forests 2019,10, 1089. [CrossRef]
30.
Chen, B.; Chen, L.; Jiang, L.; Zhu, J.; Chen, J.; Huang, Q.; Liu, J.; Xu, D.; He, Z.C. C:N:P stoichiometry of plant, litter and soil along
an elevational gradient in subtropical forests of China. Forests 2022,13, 372. [CrossRef]
31.
Wang, X.J. Study on the distribution and biodiversity of the plant communities in Gujingyuan national nature reserve. Anhui For.
Sci. Technol. 2019,45, 10–14. (In Chinese)
32. Bao, S.D. Soil and Agricultural Chemistry Analysis; China Agriculture Press: Beijing, China, 2000. (In Chinese)
33. Kuo, S. Phosphorus. In Methods of Soil Analysis; John Wiley & Sons, Ltd.: Hoboken, NJ, USA, 1996; pp. 869–919.
34.
Wu, J.; Joergensen, R.G.; Pommerening, B.; Chaussod, R.; Brookes, P.C. Measurement of soil microbial biomass C by fumigation-
extraction-an automated procedure. Soil Biol. Biochem. 1990,22, 1167–1169. [CrossRef]
35.
Li, H.; Crabbe, M.J.C.; Xu, F.; Wang, W.; Ma, L.; Niu, R.; Gao, X.; Li, X.; Zhang, P.; Ma, X.; et al. Seasonal variations in carbon,
nitrogen and phosphorus concentrations and C:N:P stoichiometry in different organs of a Larix Principis-Rupprechtii Mayr.
plantation in the Qinling Mountains, China. PLoS ONE 2017,12, e0185163. [CrossRef] [PubMed]
36.
Elser, J.J.; Fagan, W.F.; Denno, R.F.; Dobberfuhl, D.R.; Folarin, A.; Huberty, A.; Interlandi, S.; Kilham, S.S.; Mccauley, E.; Schulz,
K.L. Nutritional constraints in terrestrial and freshwater food webs. Nature 2000,408, 578–580. [CrossRef]
37.
Han, W.; Fang, J.Y.; Reich, P.B.; Woodward, F.I.; Wang, Z.H. Biogeography and variability of eleven mineral elements in plant
leaves across gradients of climate, soil and plant functional type in China. Ecol. Lett. 2011,14, 788–796. [CrossRef]
38.
Ashfaq, A.; Hussain, M.; Ali, S.; Akhtar, K.; Mamoona, W.M.; Zamir, A. Ecological stoichiometry in Pinus massoniana L. plantation:
Increasing nutrient limitation in a 48–Year chronosequence. Forests 2022,13, 469. [CrossRef]
39.
Stierle, A.A.; Stierle, D.B. Bioactive secondary metabolites produced by the fungal endophytes of conifers. Nat. Prod. Commun.
2015,10, 1671–1682. [CrossRef]
40.
Matamouros, S.; Gensch, T.; Cerff, M.; Sachs, C.C.; Abdollahzadeh, I.; Hendriks, J.; Horst, L.; Tenhaef, N.; Tenhaef, J.; Noack, S.;
et al. Growth-rate dependency of ribosome abundance and translation elongation rate in Corynebacterium glutamicum differs from
that in Escherichia coli.Nat. Commun. 2023,14, 5611. [CrossRef]
41. Bielczynski, L.W.; Ł ˛acki, M.K.; Hoefnagels, I.; Gambin, A.; Croce, R. Leaf and plant age affects photosynthetic performance and
photoprotective capacity. Plant Physiol. 2017,175, 1634–1648. [CrossRef]
42.
Hu, T.; Xiong, K.; Yu, Y.; Wang, J.; Wu, Y. Ecological stoichiometry and homeostasis characteristics of plant-litter-soil system with
vegetation restoration of the karst desertification control. Front. Plant Sci. 2023,14, 122469131. [CrossRef]
43.
Yuan, Z.; Chen, H.Y.H. Global trends in senesced-leaf nitrogen and phosphorus. Glob. Ecol. Biogeogr. 2009,18, 532–542. [CrossRef]
44.
Shaver, G.R.; Chapin, E.S., III. Long–term responses to factorial, NPK fertilizer treatment by Alaskan wet and moist tundra sedge
species. Ecography 1995,18, 259–275. [CrossRef]
45.
Tian, H.; Chen, G.; Zhang, C.; Melillo, J.M.; Hall, C.A.S. Pattern and variation of C:N:P ratios in China’s soils: A synthesis of
observational data. Biogeochemistry 2010,98, 139–151. [CrossRef]
46.
Zhang, W.; Qiao, W.; Gao, D.; Dai, Y.; Deng, J.; Yang, G.; Han, X.; Ren, G. Relationship between soil nutrient properties and
biological activities along a restoration chronosequence of Pinus tabulaeformis plantation forests in the Ziwuling Mountains, China.
Catena 2017,161, 85–95. [CrossRef]
47.
Zeng, F.P.; Chi, G.Y.; Chen, X.; Shi, Y. The stoichiometric characteristics of C, N and P in soil and root of larch (Larix spp.)
plantation at different stand ages in mountainous region of eastern Liaoning Province, China. Chin. J. Ecol. 2016,35, 1819–1825.
(In Chinese)
Forests 2024,15, 1954 13 of 14
48.
Chang, Y.; Zhong, Q.; Yang, H.; Xu, C.; Hua, W.; Li, B. Patterns and driving factors of leaf C, N, and P stoichiometry in two forest
types with different stand ages in a mid–subtropical Zone. For. Ecosyst. 2022,9, 100005. [CrossRef]
49.
Aponte, C.; Marañón, T.; García, L.V. Microbial C, N and P in soils of Mediterranean oak forests: Influence of season, canopy
cover and soil depth. Biogeochemistry 2010,101, 77–92. [CrossRef]
50.
Deng, W.; Wang, X.; Hu, H.; Zhu, M.; Chen, J.; Zhang, S.; Cheng, C.; Zhu, Z.; Wu, C.; Zhu, L. Variation characteristics of soil
organic carbon storage and fractions with stand age in north subtropical Quercus acutissima Carruth. Forest in China. Forests 2022,
13, 1649. [CrossRef]
51.
Chen, Y.; Song, Q.; Pan, L.; Jia, M.; Li, C.; Hu, B.; Wu, G. Trace metals, organic carbon and nutrients in the Beidagang Wetland
Nature Reserve, northern China. PLoS ONE 2018,13, e0204812. [CrossRef]
52.
Chen, X.; Chen, H.Y.H. Plant mixture balances terrestrial ecosystem C:N:P stoichiometry. Nat. Commun. 2021,12, 4562. [CrossRef]
53.
Gong, Y.; Lv, G.; Guo, Z.; Chen, Y.; Cao, J. Influence of aridity and salinity on plant nutrients scales up from species to community
level in a desert ecosystem. Sci. Rep. 2017,7, 6811. [CrossRef] [PubMed]
54.
Pang, Y.; Tian, J.; Zhao, X.; Chao, Z.; Wang, Y.; Zhang, X.; Wang, D. The linkages of plant, litter and soil C:N:P stoichiometry
and nutrient stock in different secondary mixed forest types in the Qinling Mountains, China. PeerJ 2020,8, e9274. [CrossRef]
[PubMed]
55.
Liu, S.; Plaza, C.; Ochoa-Hueso, R.; Trivedi, C.; Wang, J.; Trivedi, P.; Zhou, G.; Piñeiro, J.; Martins, C.S.C.; Singh, B.K.; et al. Litter
and soil biodiversity jointly drive ecosystem functions. Glob. Chang. Biol. 2023,29, 6276–6285. [CrossRef] [PubMed]
56.
Gavazov, K.; Ingrisch, J.; Hasibeder, R.; Mills, R.T.E.; Buttler, A.; Gleixner, G.; Pumpanen, J.; Bahn, M. Winter ecology of a
subalpine grassland: Effects of snow removal on soil respiration, microbial structure and function. Sci. Total Environ. 2017,590,
316–324. [CrossRef] [PubMed]
57.
Xu, X.; Hui, D.; King, A.W.; Song, X.; Thornton, P.E.; Zhang, L. Convergence of microbial assimilations of soil carbon, nitrogen,
phosphorus, and sulfur in terrestrial ecosystems. Sci. Rep. 2015,5, 17445. [CrossRef]
58.
Fang, P.; Hu, H.; Wang, X. Soil ecological stoichiometry characteristics of different aged Quercus acutissima plantations. J. Southwest
For. Univ. 2022,42, 39–47. (In Chinese)
59.
Ghaley, B.B.; Sandhu, H.S.; Porter, J.R. Relationship between C:N/C:O stoichiometry and ecosystem services in managed
production systems. PLoS ONE 2015,10, e0123869. [CrossRef]
60.
Koerselman, W.; Meuleman, A.F.M. The vegetation N:P ratio: A new tool to detect the nature of nutrient limitation. J. Appl. Ecol.
UK 1996,33, 1441–1450. [CrossRef]
61.
Zhang, G.; Han, X.; Elser, J.J. Rapid top-down regulation of plant C:N:P stoichiometry by grasshoppers in an Inner Mongolia
grassland ecosystem. Oecologia 2011,166, 253–264. [CrossRef]
62.
Franklin, O.; Ågren, G.I. Leaf senescence and resorption as mechanisms of maximizing photosynthetic production during canopy
development at N limitation. Funct. Ecol. 2002,16, 727–733. [CrossRef]
63.
Weiqi, W.; Linglin, X.U.; Congsheng, Z.; Chuan, T.; Linhai, Z. Carbon, nitrogen and phosphorus ecological stoichiometric ratios
among live plant-litter-soil systems in estuarine wetland. Acta Ecol. Sin. 2011,31, 7119–7124.
64.
Barantal, S.; Schimann, H.; Fromin, N.; Hättenschwiler, S. C, N and P fertilization in an Amazonian rainforest supports
stoichiometric dissimilarity as a driver of litter diversity effects on decomposition. Proc. Biol. Sci. 2014,281, 20141682. [CrossRef]
[PubMed]
65.
Jiang, P.; Cao, Y.; Chen, Y.; Wang, F. Variation of C, N, and P stoichiometry in plant tissue, litter, and soil during stand development
in Pinus tabulaeformis plantation. ACTA Ecol. Sin. 2016,36, 6188–6197. (In Chinese)
66.
Quan, Q.; Wang, C.; He, N.; Zhang, Z.; Wen, X.; Su, H.; Wang, Q.; Xue, J. Forest type affects the coupled relationships of soil C and
N mineralization in the temperate forests of northern China. Sci. Rep. 2014,4, 6584. [CrossRef] [PubMed]
67.
Liu, A.N.; Zhang, Y.; Hou, Z.F.; Hui Lü, G. Allometric scaling of biomass with nitrogen and phosphorus above- and below-ground
in herbaceous plants varies along Water-Salinity gradients. AoB Plants 2021,13, plab030. [CrossRef]
68.
Jiang, W.; Gong, L.; Yang, L.; He, S.; Liu, X. Dynamics in C, N, and P stoichiometry and microbial biomass following soil depth
and vegetation types in low mountain and hill region of China. Sci. Rep. 2021,11, 19631. [CrossRef]
69.
Awoonor, J.K.; Dogbey, B.F.; Salis, I. Human-induced land use changes and phosphorus limitation affect soil microbial biomass
and ecosystem stoichiometry. PLoS ONE 2023,18, e0290687. [CrossRef]
70.
Fanin, N.; Fromin, N.; Buatois, B.; Hättenschwiler, S.; Cleland, E. An experimental test of the hypothesis of non-homeostatic
consumer stoichiometry in a plant litter-microbe system. Ecol. Lett. 2013,16, 764–772. [CrossRef]
71. Pokharel, P.; Chang, S.X. Biochar decreases and nitrification inhibitor increases phosphorus limitation for microbial growth in a
Wheat-Canola rotation. Sci. Total Environ. 2023,858, 159773. [CrossRef]
72.
Elser, J.J.; Acharya, K.; Kyle, M.; Cotner, J.B.; Sterner, R.W. Growth rate-stoichiometry couplings in diverse biota. Ecol. Lett. 2003,
6, 936–943. [CrossRef]
73.
Mcgroddy, M.E.; Daufresne, T.; Hedin, L.O. Scaling of C:N:P stoichiometry in forests worldwide: Implications of terrestrial
Redfield-type ratios. Ecology 2004,85, 2390–2401. [CrossRef]
74.
Yang, G.; Ma, Y.; Ma, X.; Wang, X.; Lu, C.; Xu, W.; Luo, J.; Guo, D. Changes in soil organic carbon components and microbial
community following spent mushroom substrate application. Front. Microbiol. 2024,15, 1351921. [CrossRef] [PubMed]
75.
Woli ´nska, A.; Podlewski, J.; Słomczewski, A.; Grz ˛adziel, J.; Gał ˛azka, A.; Ku´zniar, A. Fungal indicators of sensitivity and resistance
to long-term Maize Monoculture: A culture-independent approach. Front. Microbiol. 2022,12, 799378. [CrossRef] [PubMed]
Forests 2024,15, 1954 14 of 14
76.
Chang, Y.; Liu, W.; Mao, Y.; Yang, T.; Chen, Y. Biochar addition alters C: N: P stoichiometry in Moss Crust-Soil continuum in
Gurbantünggüt Desert. Plants 2022,11, 814. [CrossRef] [PubMed]
77.
Kück, P.; Hita Garcia, F.; Misof, B.; Meusemann, K. Improved phylogenetic analyses corroborate a plausible position of Martialis
heureka in the ant tree of life. PLoS ONE 2011,6, e21031. [CrossRef]
78.
Ahemad, M. Phosphate-solubilizing bacteria-assisted phytoremediation of metalliferous soils: A review. 3 Biotech 2015,5, 111–121.
[CrossRef]
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