2 Materials and methods
To avoid the heterogeneity of soils and climates, we carried out a
litter-bag experiment in the glasshouse in the Institute of Urban
Environment, Chinese Academy of Science. The leaves were sampled from
Quanzhou Bay Mangrove Reserve (24◦57’24” N -
118◦41’25” E), the south of Fujian, China (Hu et al.,
2011). This site is characterized by an oceanic monsoon climate with a
warm wet winter and hot rainy summer. The annual mean temperature is
20.4 ◦C and the average annual precipitation is 1095.4
mm (Lu et al., 2018). The dominant mangrove species encompassedKandelia obovata , Aegiceras corniculatum , and Avicennia marina. Besides, an alien saltmarsh speciesSpartina alterniflora emerged, which has been invaded into many
mangrove stands in China (Zhang et al., 2012). Three litter mixtures
were set up based on species distributions in the field: A.
corniculatum vs. A. marina , A. corniculatum vs. K.
obovata , and A. corniculatum vs. S. alterniflora .
The
soil was collected from the mangroves in Xiamen city: electronic
conductivity (EC 0.46 ± 0.05 ms cm-1), organic matter
(0.32 ± 0.04 mg g-1), carbon (C 20.09 ± 0.67 mg
g-1), total nitrogen (TN 2.02 ± 0.08 mg
g-1), total phosphorus (TP 0.62 ± 0.03 mg
g-1), sulphate (S 0.72 ± 0.07 mg
g-1), potassium (K 13.73 ± 3.33 mg
g-1), calcium (Ca 8.47 ± 2.39 mg
g-1), magnesium (Mg 9.96 ± 0.22 mg
g-1) and pH (8.11 ± 0.06).
2.1 Experimental design
Healthy green leaves were sampled from the trees of K. obovata, A. corniculatum , A. marina, and S. alterniflora in
the same forest. Thus, all leaves were exposed to similar climatic
conditions, which facilitated interspecies comparison of litter mass
loss. After being taken to the laboratory, the leaves were gently washed
and all dirt particles were then removed by using a soft brush followed
by rinsing in distilled water. Five replicates were set up for each
litter or litter mixture.
All leaf litters were air-dried to constant weight, 5 g of litter for
each single species and 5 g of each litter mixture (2.5 g per species)
was placed in each 75 × 75 mm nylon bag with 1 mm2mesh size. There were 40 bags in total, including 15 bags of litter
mixture (3 pairs) and 25 for single species (5 single species). All the
litter bags were placed on the top of the soil in plastic containers
(500 ml), all the containers consisted of 200 g soil and 200 ml water.
Based on the previous observation that the decomposition of mangrove
leaves generally become slow and steady after 7 weeks (Chanda et al.,
2016), this experiment was then conducted for 90 days. The duration of
90 days is often representing a short-term decomposition experiment
(Keuskamp et al., 2013).
2.2 Element analysis
After 90 days of decomposition, litter and water were collected to the
laboratory for mass and nutrient analyses. The water on the surface of
soils was collected with a syringe and then stored in a polyethylene
bottle. Litters were determined C and N concentrations with an elemental
analyzer (Vario MAX, Vario MACRO, Germany Elementar) after being grounded
through 60 mesh sieves. Phosphorus (P) in litters was quantified with
ICP-OES (Inductively Coupled Plasma Optical Emission Spectrometer,
Optimal 7000DV, PerkinElmer USA) after digested with nitric and
perchloric acid. The ammonium–nitrogen (NH4-N),
nitrate-nitrogen (NO3-N) and nitrate-nitrogen
(NO2-N) were quantified by ultraviolet spectrophotometer
(UV6100, mapada instrument, China) following the standard methods of
water and waste monitoring and analysis method (Wei et al., 2002).
Digestion of the water by alkaline potassium persulfate and potassium
persulfate was carried out after filtration of the water sample, and then
the total N and total P were determined respectively according to the
standard methods (Wei et al., 2002), NH4-N,
NO3-N and NO2-N were then analyzed by
ultraviolet spectrophotometer (UV6100, ma pada instruments, China). Due
to the specific characters of mangrove leaves (Lin et al., 2007), the
condensed tannin of the leaves of the four species were also determined
before decomposition experiment is conducted. The total condensed tannin
was the sum of the extractable, protein-bound and fiber-bound
concentrations. The extraction process was followed by the method of Lin
et al. (2007).
2.3 Calculation
The litter mass loss was calculated by comparing the initial mass and
the mass after decomposition. The initial mass remaining after
decomposition was calculated based on the following formula (Wu et al.,
2013):
\begin{equation}
X_{r}=\frac{X_{i}}{X_{t}}\times 100\nonumber \\
\end{equation}
Where Xr is the percentage (%) of mass remaining after
decomposition, Xi is the initial litter mass,
Xt is the mass of the remained litter in the litter bags
after a given time period (t) of decomposition.
The decomposition rate (k) was calculated by exponential decay model,
the litter mass remaining after decomposition and initial litter mass:
\begin{equation}
\frac{X_{t}}{X_{i}}=\mathbb{e}^{-kt}\nonumber \\
\end{equation}
Where, k is the decomposition rate coefficient; t is the time duration
of decomposition.
The percentage of the initial litter nutrient remaining
(Nr) during decomposition was calculated by the
following equation (Zhang et al., 2014):
\begin{equation}
N_{r}=\left(X_{t}\times{[N}_{t}]+X_{i}\times[X_{i}]\right)\times 100\nonumber \\
\end{equation}
We also calculated the expected rate of the litter mixture after
decomposition using the following formula:
\begin{equation}
X_{\exp}=\left(\frac{X_{i1}}{X_{i1}+X_{i2}}\times X_{r_{1}}+\frac{X_{i2}}{X_{i1}+X_{i2}}\times X_{r_{2}}\right)\times 100\nonumber \\
\end{equation}
Where Xexp is the percentage of the expected mass
remaining (MR) after decomposition, X1 and
X2 are the initial dry masses in single species which
was 2.5 g, and Xr1 and Xr2 are the mass
remaining from the single decomposition species. The effect strength was
calculated by the difference between the observed mass remaining (%)
and the expected mass remaining (%) (O-E): additive (no significant
difference between observed and expected values), synergistic
non-additive (negative value, meaning an acceleration of litter
decomposition), antagonistic non-additive (positive value, meaning a
deceleration of litter decomposition) (Lecerf et al., 2011).
The community-weighted mean value of traits (CWM) was calculated as the
mean value of each species in the mixture, because the two litters mixed
as 1: 1 of mass in this study (Roscher et al., 2018).
2.4 Statistical analysis
Statistical analysis was performed with IBM SPSS Statistics 23. All data
were removed outlier and checked for the normal distribution and
homogeneity of variances before the statistical analysis was carried
out. One-way ANOVA was used to detect the differences in decomposition
rate and nutrients in water among litters, as well as in trait
dissimilarity and CWM among each trait. The differences in mass
remaining and nutrients in water between observed and expected values
were analysed by independent t-test. The variation of the difference in
mass remaining between observed and expected values from zero (i.e the
observed equals to expected value) was detected by one-sample t-test
analysis. The linear correlations of decomposition rate or mass
remaining with leaf trait dissimilarity or CWM were assessed through the
correlation process. This process
was also used to identify the correlation between nutrients in water and
in leaves.
3 Result
Non-additive effect on litter decomposition were found in the litter
mixtures from three mangrove species.
Nutrients in water, the initial
trait dissimilarity and CWM of litter mixtures varied with species
composition.
3.1 Decomposition rate (k) and mass
remaining
Non-additive effect on litter decomposition was detected in the litter
mixtures from mangrove species, but not in the mixture of A.
corniculatum vs. S. alterniflora (Fig. 1a, b). The observed mass
remaining of the mixture of A. corniculatum vs. K obovatawas substantially higher than the expected value, whereas the observed
mass remaining of the mixture of A. corniculatum vs. A.
marina was lower than the expected one (p < 0.05),
only the mixture of A. corniculatum vs. S. alterniflora did not show significant difference between the observed and the
expected values (p > 0.05; Fig. 1b).