Leaf temperature
To explore the question I in Introduction, we conducted leaf-temperature
measurements in the field conditions. To determine the effect of leaf
trichomes on leaf temperature across the elevational gradient, we
measured leaf temperature of both intact and trichome‐shaved leaves at
the four elevational sites (700, 1280, 1800, 2400 m). We excluded the
100 m site for this measurement because the studied individuals in this
site were tall and the sun‐exposed leaves were out of reach
(>4 m). We selected three individuals with typical leaf
traits in each site, i.e., glabrous individuals in the 700 m site and
pubescent individuals in the other sites. The mean trichome thickness of
the three individuals was 0, 0.278, 0.433, and 0.716 mm at 700, 1280,
1800, and 2400 m, respectively. We selected two sun‐exposed leaves per
individual and carefully shaved the leaf trichomes on either one of
these two leaves by using a rubber thimble. These two leaves per each
individual were close to each other and their microenvironmental
conditions can be assumed to be almost same. In August 2017, we measured
diurnal variations in leaf temperature of these leaves with 0.2‐mm
diameter copper/constantan thermocouples connected with a data logger
(0.1 °C precision; ADL12, AsOne, Japan). The thermocouple was attached
to the lower surface of the leaf. For intact leaves with leaf trichomes,
the thermocouple was inserted beneath the trichome layer and in contact
with the lamina. During this measurement, we also measured the air
temperature and humidity (TR‐72WF‐H, T&D Co. Ltd., Nagano, Japan) every
ten minutes at each site.
To determine the association between trichome thickness and leaf
temperature, we measured leaf temperature of both intact and
trichome‐shaved leaves collected at the 1800‐m site where the variation
in the amount of trichomes was largest (Table 3; Tsujii et al., 2016).
We studied ten randomly selected individuals at the 1800 m site. One
pair of opposite leaves that were fully exposed to sun was selected per
individual, and the leaf trichomes on either one of the paired leaves
were carefully shaved by using a
rubber thimble. We confirmed that this trichome‐shaved treatment itself
did not directly affect the physiological activities such as stomatal
conductance and transpiration rate through damaging stomata and cuticle
layer (Figure S2). At least two days later, we measured leaf temperature
on the upper surfaces of intact and trichome‐shaved leaves using a
radiation thermometer (0.1 °C precision; 62 MAX+, Fluke Corp., USA)
between 11:30 and 12:30 for three times each on three sunny days in July
2016 (total nine times for each leaf; air temperature 22.0 ± 1.0 °C,
PPFD > 2000 μmol m⁻2s⁻1) when the solar angle was nearly vertical which
minimized leaf direction effect between the paired leaves. We used the
averages of nine data for each pair of leaves to analyze the association
between leaf temperature and trichome thickness. After the measurements,
the measured leaves were collected and used for the measurements of
trichome thickness, as mentioned above.