Electronic supplementary material
Deviation from niche optima affects the nature of plant–plant
interactions along a soil acidity gradient
Lei He
Lulu Cheng
Liangliang Hu Jianjun Tang*
Xin Chen*
College of Life Sciences, Zhejiang University, Hangzhou 310058, China
*Corresponding author, chandt@zju.edu.cn or chen-tang@zju.edu.cn
ESM 1-Plants and soil
Three legume species (Lespedeza formosa Koehne, Medicago sativa L., and
Indigofera pseudotinctoria Mats.) commonly grow together in the “red soil area” of
southern China. Lespedeza formosa is a perennial, deciduous shrub that grows to a
height of 1–2 m and produces many branches [1]. It is a pioneer species and is
drought- and acid-tolerant [2]. It grows well in red soil areas with wide range of soil
acidity (pH 4–6). Indigofera pseudotinctoria is also a perennial, deciduous shrub that
grows well in soil with pH 4–5. It flowers from June to September and produces fruit
from November to December. It grows well in degraded soil and helps prevent soil
erosion [3,4]. Medicago sativa is a perennial herb that is grown for forage [5]. It is an
acid-sensitive species and cannot grow well in an acidic soils [6,7]. Seeds of the three
legume species were supplied by the Zhejiang Forestry Academy. The
thousand-kernel weights (mean ± SE) of L. formosa, I. pseudotinctoria, and M. sativa
were 7.49 ± 0.31 g, 2.34 ± 0.10 g, and 6.01 ± 0.22 g, respectively. The soil had a pH
(1:2.5 soil: KCl) of 4.1, an organic matter content of 0.70%, a total nitrogen (N)
content of 0.44 g kg-1, and a NaHCO3-available P content of 3.70 mg kg-1.
ESM2-Preparation of soil acidity gradients
The acid red soil from an abandoned tea garden was air-dried and used as the base soil
for creating an acidity gradient (pH=3.1, 4.1, 5.5, and 6.1). A soil pH of 3.1 was
obtained by adding 40 ml of H2SO4 (pH 2) to 5 L of water, which was added per
mesocosm. A soil pH of 4.1, 5.5, or 6.1 was obtained by adding 0, 100, or 200 g of
CaCO3, respectively, to 5 L of water, which was added per microcosm [8]. The
prepared soils were incubated at 25℃ and at approximately 50% soil moisture content
for 1 month before the experiment.
ESM3-Measurement
At the end of the plant–plant interaction experiment (which started in April and ended
in October), all target plants were harvested from the mesocosms. Shoots were
separated from roots, oven dried at 65 °C for 72, and weighed. The phosphorus (P)
concentration in leaves (0.2 g per sample) was determined with a San++ Continuous
Flow Analyzer (Skalar, Netherlands). Root systems were collected and cleaned for
determination of colonization by arbuscular mycorrhizal fungi (AMF) by the gridline
intersection method [9]. Soil was sampled before harvest. We sampled the top layer
(0-10 cm) in the target plant’s rhizosphere from each mesocosm. The soil samples
were separately air-dried and passed through a screen (<2.0 mm) for further analysis.
For exchangeable aluminium (Al) analysis, soil samples were extracted with a KCl
solution (2 g of soil in 20 ml of 1 M KCl), and the extracts were analysed by
ICP-OES (Optima 8000DV, Perkin Elmer, USA) [10]. Organic matter content was
determined by hydrated heat potassium dichromate oxidation-colorimetry [11]. AMF
spores in soil were extracted by wet sieving and sucrose density gradient
centrifugation, transferred to Petri dishes, and counted with a dissecting microscope
[12].
ESM4-Data analysis
The soil pH that resulted in the greatest biomass of a target species in the absence of a
neighbour was assumed to be the optimum. For each species, we fit the relationship of
ln (target biomass (without neighbour) +10) and soil pH with a binomial equation in
R version 3.1.3. Soil pH at the peak of the polynomial curve was then calculated as
the optimal soil pH. To generate confidence intervals, we conducted bootstrapping for
1000 iterations in R version 3.1.3. For each bootstrap iteration, we randomly chose
five samples under every pH (with replacement) to fit the relationship with a binomial
equation and calculated the optimal soil pH. The lowest and highest 2.5% values were
then chosen to represent the lower and higher 95% confidence limits.
The effect of soil pH on RII and the effects of soil pH and neighbour on the
biomass of target plants, P concentration in target leaves, and soil exchangeable Al
were assessed with the General Linear Model. pH and neighbour were treated as fixed
factors. Homogeneity of variances was checked using Levene's Test before all
statistical analyses. One-way ANOVAs were performed to test for the effects of
neighbour on the number of AMF spores, AMF colonization rate, soil exchangeable
Al, and soil organic matter content. Significance was determined at the 0.05 level.
SPSS 16.0 was used for the analyses.
ESM 5-acid-tolerance of neighbour plant L. formosa
Before the plant-plant experiment, we conducted a one-factor experiment to text how
the pH-levels of the soil affected the growth of L. formosa. The mesocosms and soil
pH levels (3.1, 4.1, 5.5, 6.1) were the same as the experiment of plant-plant
interaction. The experiment was set up in a common garden located at the University
of Zhejiang in Hangzhou, China. The mesocosms were arranged in a fully randomized
manner with five replicate mesocosms, giving a total of 20 mesocosms. Seeds were
sown in soil in a plant nursery. After 1 month, one seedling was transplanted into each
mesocosm. After 3 months, all plants were harvested from the mesocosms, oven dried
at 65 °C for 72 h, and weighed. One-way ANOVA was performed to test for the
effects of pH on biomass of L. formosa. Homogeneity of variances was checked using
Levene's Test before all statistical analyses. Significance was determined at the 0.05
level. SPSS 16.0 was used for the analyses.
Biomass of L. formosa significantly reduced at the treatment of pH 3.1, while
there was no significant difference of biomass among the treatments of pH 4.1, 5.5
and 6.1 (Fig. S2).
ESM 6- The P concentration in the leaves of target plants
The P concentration in the leaves of I. pseudotinctoria was significantly affected by
pH (F2, 24=14.709, P<0.0001), neighbour (F1, 24=12.203, P=0.002), and the interaction
of pH and neighbour (F2, 24=5.71, P=0.009). The P concentration in the leaves of M.
sativa was significantly affected by pH (F2, 24=9.403, P=0.001) and the interaction of
pH and neighbour (F2,
24=10.57,
P=0.001). The P concentration in leaves of I.
pseudotinctoria without the neighbour decreased as pH increased from 4.1 to 6.1 (Fig.
S3). The P concentration in M. sativa leaves was highest at pH 5.5 without the
neighbour and at pH 4.1 with the neighbour (Fig. S3). The P concentration in I.
pseudotinctoria leaves was affected by the neighbour treatment (0.93±0.13 mg/g
without neighbour vs. 0.56±0.05 mg/g with neighbour) at pH 4.1 (Fig. S3). At pH 5.5,
the P concentration in M. sativa leaves was higher without than with neighbour
(0.94±0.09 mg/g without neighbour vs. 0.53±0.07 mg/g with neighbour) (Fig. S3).
ESM 7-Biomass of target plants
The biomass of the target plant I. pseudotinctoria was significantly affected by the pH
(F2,
24=4.4,
P=0.024) and the interaction of pH and neighbour (F2,
24=21.931,
P<0.0001). The biomass of the target plant M. sativa was significantly affected by the
pH (F2, 24=214.609, P<0.0001), neighbour (F1, 24=6.058, P=0.021), and the interaction
of pH and neighbour (F2, 24=37.149, P<0.0001). Biomass of I. pseudotinctoria without
the neighbour decreased as pH increased from 4.1 to 6.1, but the biomass with the
neighbour was largest at pH 5.5 (Fig. S3). Biomass of M. sativa was highest at pH 5.5
both with and without the neighbour (Fig. S3). At pH 3.1, all plants grew slowly and
then died after 3 months whether the neighbour was present or absent. The neighbour
treatment significantly affected the biomass of M. sativa only at pH 4.1 and 5.5 (Fig.
S3). At pH 4.1, M. sativa biomass was higher with than without the neighbour. The
biomass of I. pseudotinctoria was significantly lower without than with the neighbour
at pH 6.1 (Fig. S4).
Fig. S1. Diagram of the experiment concerning plant-plant interactions as affected by
a soil acidity gradient (the figure represents one soil acidity level). (a) represents the
“with neighbour” treatment; (b) represents the “without neighbour” treatment.
Fig.S2 The biomass of the neighbour plant L. formosa along the soil pH levels.
Means with different lowercase letters are significantly different (P< 0.05).
Fig. S3. Phosphorus concentration in leaves of target plants with and without the
neighbour along a soil acidity gradient. All plants had died after 3 months at pH
3.1. Values are means ± SE (n=5). For each species of target plant, means with
different lowercase letters are significantly different (P< 0.05).
Fig. S4. Biomass of target plants with and without the neighbour along a soil
acidity gradient. All plants had died after 3 months at pH 3.1. Values are means ± SE
(n=5). For each target plant, means with different lowercase letters are significantly
different (P < 0.05).
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