2. Materials and Methods
2.1 Site description
The experiment was conducted at the Dongzhaigang National Nature Reserve
(110°32´–110°37´E and 19°51´–20°01´N) in northeastern Hainan Province,
China. The reserve is the earliest established mangrove reserve in
China, covering 3337.6 ha. This area is characterized by a semi-enclosed
estuary with a muddy bottom, nourished by four small rivers. It
experiences semidiurnal tidal cycles, averaging a tidal range of 1.6 to
1.8 meters. The climate is characterized as a tropical maritime monsoon
with an average annual rainfall of 1676.4 mm and a mean annual
temperature ranging from 23.3 to 23.8°C (Li et al. , 2016). A
total of thirty-five species of mangrove plants have been documented
across 25 genera and 18 families. This included 24 species of true
mangroves, which belong to 14 genera and 10 families, as well as 11
species of minor mangroves, categorized under 11 genera and 8 families
(Jiang et al. , 2023). Deforestation ceased in 1986 when the bay
was declared a national nature reserve. The dominant mangrove species
are Avicennia marina , Aegiceras corniculatum ,Bruguiera sexangula , Ceriops tagal , and Rhizophora
stylosa .
2.2 Field survey
The field survey was conducted during the peak of the rainy season. We
selected five tree species and five shrub species for this study based
on previous field investigations and literature research (Bai et
al. , 2021; Yu et al., 2023). Among our sampled
species, five were located in the low intertidal zone, and five were in
the high intertidal zone (Table 1). Four plots (10 m×10 m, >1
km apart) were established for each species. The height and diameter at
breast height (DBH) or basal diameter of each individual tree and shrub
were recorded. For each species, we collected 30 current-season, fully
expanded, light-exposed mature and healthy green leaves from three adult
individuals per plot and mixed them as a composite sample. All leaves
were placed in plastic bags and immediately stored in a cooler with ice.
Subsequently, we transported the samples to the laboratory for the
measurements of leaf structural traits.
2.3 Leaf traits
The fresh leaf chlorophyll content (LCC) was estimated with a portable
optical chlorophyll meter (SPAD-502, Konika-Minolta Inc., Tokyo, Japan).
The leaf area (LA) was determined with a leaf area meter (LI-3000c,
Lincoln, Nebraska, USA). Additionally, leaf thickness (LT) was measured
using a digital micrometer (Digimatic micrometer, Mitutoyo, Japan). This
measurement was derived from the average of three randomly selected
positions on each leaf, deliberately avoiding the prominent veins to
ensure accuracy on flat leaf surfaces. Leaf fresh mass (LFM) was
weighted using a balance (0.0001 g, Meilen, Meifu Electronics Co. Ltd.,
Shenzhen, China). Following the rehydration procedure, the leaves were
carefully dabbed with tissue paper to eliminate any residual surface
moisture prior to measuring the leaf saturated mass (LSM). Samples were
subsequently oven-dried to a constant mass at 65°C for at least 48 h and
then weighed to obtain the leaf dry mass (LDM).
Leaf volume (LV) was estimated using LA multiplied by LT. The leaf mass
per area (LMA), the reciprocal of the specific leaf area (SLA), was
calculated using the LDM divided by the LA. Leaf density (LD) was
calculated by dividing LMA by LT. The leaf dry matter content (LDMC) was
calculated as the ratio of LDM to LSM. Finally, water saturation deficit
(WSD), a critical parameter widely utilized for assessing plant
tolerance to temporary water shortages, was calculated as follows (Lalet al. , 2009):
\begin{equation}
\text{WSD}\left(\%\right)=\frac{\left(LSM-LFM\right)}{\left(LSM-LDM\right)}\times 100\%\nonumber \\
\end{equation}2.4 Statistical analyses
All the statistical analyses were conducted using R (version 4.3.0, R
Core Team 2023). Normality, homoscedasticity, and model fit were
assessed using residual plots, Shapiro-Wilk test, and Levene’s test.
First, we conducted two-way analysis of variance (ANOVA) using general
linear model procedures to test for the main effects of intertidal
gradients and growth forms and their interactions on leaf traits. When
the effects of treatments were significant, mean comparisons were
performed using the ‘emmeans ’ package. Second, phylogenetic
signals of all traits were calculated with Blomberg’s K statistic
(Blomberg et al. , 2003) using the ‘picante ’ package. This
test compares the variance of the phylogenetically independent contrast
of the study trait against those obtained with data randomly reshuffled
in the phylogeny. A K value close to 1 indicates a significant
phylogenetic effect, while a value close to 0 suggests no phylogenetic
signal. In this study, the K values were less than 1, and the
corresponding p values were greater than 0.05 for all traits,
suggesting a lack of phylogenetic conservatism (Appendix Table S1). To
investigate multivariate trait relationships, we performed principal
component analysis (PCA) on all 11 leaf traits and plant sizes using the
‘vegan ’ package. Finally, we used simple regression analyses to
examine the effects of plant height and diameter on LCC, LD, LDMC, WSD,
and LMA and used general linear models to test the difference in
regression slopes between intertidal zones and growth forms.