Photosynthetic heat tolerance
Photosynthetic heat tolerance was measured using a chlorophyll
fluorometer (PAM-2000, Walz) following a protocol (Leon-Garcia & Lasso,
2019; Slot et al., 2021) modified from Krause et al (2010). We sampled
leaf material early in the morning, from north facing branches within
one metre of the top of the canopy. For a subset of individuals in the
lowland provenance of C. australe and H. novo-guineensis ,
we also sampled leaf material from lower in the canopy. We randomised
the species and individuals that were measured each day, making sure to
always include at least one replicate from each provenance of each
species.
To ensure assessment at steady state with respect to the photosynthetic
system, fresh leaf material was dark adapted for 30 minutes, and then
the ratio of Fv/Fm was recorded.
Subsequently a cork corer was used to cut 4 to 6 discs per leaf, which
were pooled per tree. From this pool, six leaf discs were randomly
assigned to each temperature treatment. Leaf discs were heated in a
water bath for 15 min at temperatures of either 34, 38, 40, 42, 44, 46,
48, 50, 52, 54, or 58 °C. Control discs were kept in a water bath at
ambient lab temperature of 25 °C. Treatment duration times of 15 min
were selected as is common for other studies of thermal tolerance (Perez
& Feeley, 2020; Slot et al., 2021; Tiwari et al., 2020). After the heat
treatment, leaf discs were stored in the dark in petri dishes with their
abaxial side face down on a moist paper towel. After 24 hours,
Fv/Fm was measured on dark-adapted leaf
discs to determine irreversible damage to photosystem II. The
temperature response of Fv/Fm was fitted
using the following equation:
\begin{equation}
F_{v}/F_{m}=\ \frac{F_{v}/F_{\text{m.max}}}{1+\ e^{b(Tleaf-T50)}}\nonumber \\
\end{equation}Where F v/F m.max is the
upper horizontal asymptote that represents theF v/F m of non-stressed,
healthy leaves and Tleaf is the temperature treatment of the water bath.
The ‘nls_multstart’ function in the ‘nls_multstart’ package was used
to fit curves and obtain estimates of T50 for each
plant, whereas Tcrit was defined as the temperature
where the horizontal line indicatingF v/F m.max intersects with
the line of the slope of theF v/F m decline at
T50.