I measured 14 functional traits, including leaf area (LA), specific leaf area (SLA), leaf
dry-matter content (LDMC), leaf thickness (Lth), succulence, chlorophyll content (Chl),
leaf water repellency (Dropupper and Dropbelow), venation density (VD), wood density
(WD), stable isotope ratio of nitrogen and carbon of leaf (δ15N and δ13C) and content of
nitrogen and carbon per mass in leaf (Nmass and Cmass). If not stated otherwise, the
measurement methods followed the handbookof Pérez-Harguindeguy et al. (2013).
For the measurement of functional traits, branches with leaves enclosed in plastic
bags were taken out from the refrigerator. Six entire leaves, from the branch sampled from
the same individual tree without damaged by the insect or covered by mosses were cut
and their petioles removed. These leaves were separated into two groups, each group has
three leaves. I then put the leaf into two transparent folders as soon as possible to avoid
the leaves losing water. After I finished collecting three to five individuals, I put the
remaining samples and one other folder which contains the other three leaves back to the
refrigerator. Samples in a prepared folder were waiting for the follow-up measurements.
For each measurement I used three leaves from each individual.
2.6.1 Leaf morphology measurements
Leaf fresh weight was measured by an electric balance (OHAUS Adventurer AR2140,
USA) with precision of 0.0001 g. Leaf thickness (Lth) was measured by digital display
thickness gauge (DML digital thickness gauge, UK) with precision of 0.001 mm. Primary,
secondary or obvious tertiary veins were avoided during the measurement. Leaf thickness
at upper right, upper left, lower right and lower left of the lamina were measured and
averaged. Leaf area (LA) was estimated by a scanner (Perfection V370 Photo, EPSON).
I put three leaves on the screen of scanner, upper lamina facing down, avoiding individual
leaves to overlap. Petioles of the leaves were positioned to the same direction. A ruler 5
centimeters long was placed on the corner of the screen as a scale. The scan resolution
was set to 300 dpi. After the image was scanned, leaf area was estimated by ImageJ
program (Fiji), which is a freeware software. After leaf scanning, each lamina was put
into an envelope folded from newspapers. All leaf samples in envelopes were dried in the
oven at 70°C for three or more days, to make sure the leaf was dry. I used again the
four-digit scales weight to measure the dry weight (LDW) of the leaf.
Specific leaf area (SLA) was calculated as one-side leaf area divided by leaf dry
weight (LA/LDW). Leaf dry-matter content (LDMC) was calculated from leaf dry weight
divided by the fresh weight (LDW/LFW). Leaf succulence was calculated from leaf dry
weigh, leaf fresh weight and leaf area (Mantovani 1999). The equation is following:
Succulence = (Leaf fresh weight- Leaf dry weight) *1000 / Leaf area (eq. 2)
2.6.2 Chlorophyll content (Chlmass)
A chlorophyll meter (SPAD-502, KONICA MINOLTA, Japan) was used for the
measurement of leaf chlorophyll content. I divided lamina into two parts, left and right,
and for each part I took the measurement at three random points. The average of these six
measurements represents the chlorophyll content for each leaf. In the previous project,
the chlorophyll content was measured by a different instrument (CCM-200, APOGEE,
USA). Because I wanted to use values measured both by SPAD and CCM in the same
analysis, I calibrate values measured by these two instruments, I did a small test to convert
the value of CCM-200 to SPAD-502 (Appendix 1). According to the result, I used the
regression line as calibration equation. The equation of calibration is following:
SPAD = -12.85+log(CCM)х18.26 (eq. 3)
I also transfer the chlorophyll content measured by SPED to chlorophyll content per
mass (Chlmass). The equation is Chlmass= ((117.1*SPAD)/(148.84-SPAD))*SLA (Coste et
al. 2010).
2.6.3 Leaf water repellency (Dropupper and Dropbelow)
Remaining leaves which were not used for other trait measurement were used for
measuring leaf water repellency and venation density (VD). For leaf water repellency
measurement, leaves were placed on flat custom-made platform (Figure 4) consisting of
one box (17.5 cm × 10 cm × 8 cm), one clipboard (10.6 cm × 18.5 cm), two rulers (around
12 cm) and four binder clips (two 32 mm and two 19 mm). I bounded the clipboard on
the box, using two bigger binder clips, and fixed the ruler on the clipboard from beside
and other two clip at the front side of the platform. Distance between two rulers was
around 0.7 cm. The following measurements were done for both adaxial (upper) and
abaxial (lower) surface of each leaf sample. Three leaves from each individual were
measured. The leaf was mounted horizontally between clipboard and rulers by binder
clips. One 5-μl droplet of distilled water was placed on the surface of lamina by a
micropipette (HIRSCHMANN labopette 2–20 μl single channel, Germany). Because
Figure 4: Platform for measuring droplet. (a) Front view (b) Top view
(a) (b)
droplet will spread and influence the angle of droplet, picture were taken as soon as
possible when water droplet was dropped on the leaf surface. Photos were taken by a
camera (NIKON D5500, Japan) equipped with lens (TAMRON SP AF 17-50 mm F2.8
XR Di II VC, Japan). Two or more photos for one treatment were taken. ImageJ (ImageJ
1.51u) with DropSnake plugin (Stalder et al. 2006) was used to analyze the photos. The contact angle (θ) was calculated following Aryal & Neuner (2010). Leaf surface was set
as the baseline, and the contact angle (θ) between a line at a tangent of the droplet running
through the point of contact between
the droplet and the leaf surface and
baseline was measured (Figure 5).
According to Crisp (1963), the leaves
with droplet angle higher than 110°
are considered as water repellent, and leaves with lower angle as not repellent.
2.6.4 Venation density (VD)
Leaves were cut into sections of 0.25–1 cm2, with the section size depending on the size
of the original lamina. This section is located at the middle of the leaf and avoids primary
and secondary veins. Three sections from one individual (one section per leaf, three leaves
per individual) were measured. In order to make vein more visible, I used clearing method Figure 5: Determination of the contact angle (θ). Figure is modified from Fig.2 of Aryal
& Neuner (2010).
to make the section transparent. Sections from the same individual were put into a 20 ml
of Vials Scintillation Glass, containing 5% NaOH–H2O. The sections were soaked for
24–72 hours. Soaking time depends on the species. Leaves of some species need longer
time to become transparent. The transparent leaf sections were rinsed the leaf in distilled
water for several times. 1% safranin O in distilled water was transferred inside the Vials
staining the sections for 15 minutes. After staining, sections were rinsed again with
distilled water until no visible red color in the rinsed water. The sections were temporarily
mounted by water on the slide. I then used microscope (OLYMPUS CX31, Japan) to
observe the sections and used a CCD camera (MICROTECH HDC200, USA) to take
pictures. Several pictures were taken to complete one section. When all photographs were
finished, I used Image Composite Editor (2015 Microsoft Corporation, Version 2.0.3.0)
to assemble all pictures together. After these procedures, image for the analysis of
venation density was completed. The transect method applied by Blonder & Enquist
(2014) was then used to estimate the venation density. I randomly draw line segments on
vein image to get the parameter d (in equation 4), then counted the number of veins the
line crossed. Parameter d is calculated as the total length of a line (70 mm) divided by the
number of veins the line crossed. I calculated the vein density using the following
equation:
VD = 0.629 × (1/d) + 1.073 (eq. 4)
2.6.5 Wood density (WD)
Two approaches were used to estimate wood density. For wood core collected by the borer,
I directly took out the core from straw. If core broke into several segments, I removed the
segments shorter than 1 centimeter. For measuring the volume of the core, I used Archimedes’ principle with a box filled of 80% of water inside. The core segment was
inserted into water, and sink into water without touch the bottom of the box. The
increment of the weight represents the volume of this segment. After the volume being
measured, the segments were wrapped into aluminum foil and then dried in oven on
105°C for four to five days. Dry weight of the segments were measured on dried samples,
and wood density was calculated as volume divided by dry weight. For branch sample,
most of the steps were the same as those for the wood core. The bark of the branch was
removed before measuring its density.
2.6.6 Stable isotopes of leaf nitrogen and carbon content
Leafs from 160 individuals were analyzed for stable isotopes ratio. For each species, at
least one individual was analyzed. If this species occurs in more than one elevation zone
or is present in plots of all three of topography, I took more duplicates for analysis. Leaves
were ground into fine powder by mortar and pestle. Around two microgram of the powder
samples was weighted and wrapped into a tin capsule. The elemental analyzer (FlashEA
1112 series element analyzer, Thermo Fisher Scientific, Italy) was used to analyze carbon
(Cmass) and nitrogen (Nmass) contents. The ratio of stable carbon (δ13C) and stable nitrogen (δ15N) isotopes was measured with an isotope ratio mass spectrometer (Delta V
Advantage, Finnigan Mat, Germany). The stable isotopes ratio δ13C (or δ15N) is expressed
as: δ13C (or δ15N) = [(R sample/R standard) − 1]1000, where R sample is 13C/12C (or
15N/14N). The global standard for δ13C is PeeDee belemnite (PDB) and for δ15N is the
nitrogen in atmosphere.