2. Materials and methods
2.1 Study area
The study was conducted at the Longyandong Forest Farm (113°21’
E–113°27’ E and 23°10’ N–23°18’ N) in southern China. The study area
has a tropical monsoon climate that is characteristic of the area.
Throughout the year, the average temperature is 21 °C and the relative
humidity is 80%. There are approximately 1, 900 mm of precipitation per
year in the area, during the wet season (April–September), the region
receives approximately 80% of rainfall; while, in the dry season
(October–March), it receives approximately 20% (Liu et al., 2013). The
region has experienced considerable N deposition (34 kg N
ha−1 yr−1) due to rapid urbanization
since 1978 (Huang et al., 2015). The soil type is lateritic red soil and
is mainly composed of granite and sand shale.
C. hystrix plantations were planted in 1986, 1990, 1995, 2005,
2010, and 2014 after clear cutting. All plantations created in different
years were subjected to the same artificial cultivation measures,
including fertilization and the removal of understory vegetation, in the
third and fifth years, followed by the cessation of artificial
disturbance. All lands had similar geological and land use history
before planting; all lands were planted with Acacia mangium using
the same methods beginning in the 1970s, before which all lands were
covered with evergreen broad-leaved forest, dominated by C.
hystrix , Castanopsis chinensis , Syzygium rehderianum , andSchima superba (Zhou et al., 2013). The main shrub species wereFicus hirta , Psychotria asiatica , and Melicope
pteleifolia , and the main herb species were Lophatherum gracile ,Woodwardia japonica , and Blechnum orientale .
2.2 Plot design
After carefully selecting and replicating plots, a chronosequence
approach was used to survey forest stands (Walker et al., 2010). In
August 2020, a sample survey was conducted in six stands of different
ages (6, 10, 15, 25, 30, and 34 years old). Three plots (20 m × 20 m)
were randomly set up in each stand (a total of 18 plots; three plots ×
six stand ages). A distance of at least 20 m was maintained between each
plot and the forest edge, and the slope positions and aspects of all
selected plots were similar. Plots between different stand ages were
spaced at least 1 km apart to minimize spatial autocorrelation. The data
of all stand ages were obtained from the records of the Longyandong
Forest Farm (Table 1).
2.3 Field measurements
In August 2020, mature leaves and litter (1 m × 1 m on the ground) were
collected at each stand age of the C. hystrix plantation (three
replicates × six stand ages = 18 samples). All live trees were measured
simultaneously to determine the diameter at breast height (DBH, cm; ≥ 5
cm) and tree height (m). The species richness and stand density (tree
ha−1) were calculated by summing the stand basal area
(m2 ha−1) for each species at the
plot level (Feng et al., 2017; Yang et al., 2021).
Seven subsamples were collected from each of the three soil layers
(0–10, 10–20, and 20–40 cm) using
a 4-cm diameter auger, and the three subsamples from each soil layer
were combined into a single composite sample. The soil samples were
quickly refrigerated using ice packs and handheld storage boxes so they
could be tested in the laboratory within 72 hours.
2.4 Aboveground productivity of C.
hystrix
plantations
The tree and understory biomass were measured in each plot of theC. hystrix plantation in August 2020. The tree biomass was
estimated using allometric equations that correlated biomass with the
tree height and DBH (Zhou et al., 2018). All understory vegetation
(shrubs and herbs) was harvested from three sub-quadrats (2 m × 2 m)
randomly located in each plot. Litter samples were collected from three
sub-quadrats (1 m × 1 m) located within each plot. Then, all plant
samples were dried at 65°C to obtain a constant weight for determining
the biomass. The annual aboveground productivity of the C.
hystrix plantation was calculated as the aboveground (tree + understory
+ litter) biomass/stand age.
2.5 Laboratory assessments
A minimum of 72 hours were required for the oven drying of the leaf and
litter samples. The Walkley–Black wet digestion method as described by
Nelson and Sommers (1982) was used to determine the concentrations of C
in leaf and litter samples (g kg−1). The Kjeldahl
method (Bremner and Mulvaney, 1982) was used to determine the N
concentrations (g kg−1) in leaf and litter
samples. The leaf and litter P
concentrations (g kg−1) were measured using a
photometer after digesting the samples with
H2SO4–H2O2.
An ultraviolet spectrophotometer was used to determine the
concentrations (mg kg−1) of
NO3−–N and
NH4+–N in soil that had been
extracted with KCl solution at 1 M. A solution containing 0.03 M
NH4F and 0.025 M HCl was used to extract the soil
available P concentration (mg kg−1) (the ratio between
soil and extractant was 1:7). Then, an ultraviolet spectrophotometer was
used to measure the results. Dichromate oxidation and titration with
ferrous ammonium sulfate as described by Nelson and Sommers (1982) were
used to determine the soil organic C (SOC, g kg−1).
The activities of soil enzymes involved in the cycling of C, N, and P
were measured, as well as four hydrolytic enzymes, namely
cellobiohydrolase (CBH), β -glucosidase (BG), AP, and NAG, and two
oxidases, namely peroxidase and
PhOx. The methodology described by Lie et al. (2019) was used to measure
the BG, CBH, NAG, and AP activities. Hydrolytic enzymes were measured
colorimetrically using a Multiskan EX (Thermo Scientific, Waltham, MA,
USA) at 405 nm, and oxidase enzymes were measured at 450 nm (Tabatabai,
1994). To calculate the specific enzyme activities, this study followed
the methodology described by Trasar-Cepeda et al. (2007) and divided the
enzyme activities by the soil microbial biomass carbon (MBC).
The fumigation–extraction method was used to analyze the soil MBC, MBN,
and MBP (Vance et al., 1987). The organic C and N of moist soil were
extracted using a solution containing 0.5 M
K2SO4, while P was extracted with
0.03 M NH4F and
0.025 M HCl. Following fumigation with chloroform for 24 hours at 25°C,
the same extraction methods were conducted. A Total Organic Carbon
Analyzer (TOC-VCSH, Shimadzu, Japan) was used to determine the extracted
C and N in K2SO4, while the extracted P
was determined using inductively coupled plasma optical emission
spectroscopy. Based on the differences between the fumigated and
unfumigated subsamples multiplied by their conversion factors, the
microbial biomass values were calculated (Jenkinson et al., 2004).
2.6 Statistical analysis
To understand the mechanisms associated with the indirect and direct
effects of altered leaf, litter, and soil nutrients triggered by stand
ages, linear regression was used to examine the associations
between stand ages and
leaf, litter,
soil characteristics, and microbial
parameters. The relationships between variables (leaf, litter, and soil
C:N:P concentrations and ratios) were represented by Pearson correlation
coefficients. To determine the effects of stand ages, soil depth, and
their interactions on soil characteristics and microbial parameters,
two-way analyses of variance (ANOVAs) were used. All analyses were
performed in R 4.0.4. and SPSS 20.0. The level of statistical
significance was set at 0.05.