Statistical analysis
The relationship between plant height and DAP was fitted using a
three-parameter sigmoidal model, and when DAP for individual height
approached 50% of its maximum height, (H50) was
calculated and designated as a proxy of growth rate. Leaf AT data were
fitted to a quadratic function and the optimum temperature for
photosynthesis (Topt, oC) and
Asat at Topt were estimated (Gunderson,
O’hara, Campion, Walker, & Edwards, 2010). We were limited to four
glasshouse bays for our experiment, so we did not have replicate bays
for the growth temperature treatments. Therefore, acknowledging
pseudo-replication as a function of our complex experimental design, we
were limited to considering individual plants within each bay as
independent experimental units (i.e. replicate), and subsequently tested
the treatment effects with a general linear model, as has been done in
our previous studies (Lewis et al. 2013; Duan et al. 2014), as well as
in other studies (Apgaua et al. 2019). We used a two-way ANOVA to test
the main and interactive effects of temperature and water deficit stress
on gas exchange variables, biomass and AT response parameters. We also
used a two-way ANOVA to assess the effects of water deficit stress and
nocturnal warming within each growth temperature regime (i.e. cool and
warm). A three-way ANOVA was used to test the instantaneous effects of
growth temperature, water deficit stress and heatwave on leaf gas
exchange variables during the heatwave and recovery stage, as well as on
harvested biomass at the end of the experiment. The differences in
physiological and growth variables across treatment combinations were
assessed using Tukey’s HSD post hoc . Data were tested for
normality and homogeneity of variance before ANOVA was performed.
Statistically significant differences were considered if P ≤0.05. All
data and statistical analysis were performed in R 3.5.3 statistical
computing environment (R Development Core Team 2014). ANOVA was
performed using the lm() in combination with the Anova() function in thecar package, and multiple comparisons were conducted with the
HSD.test() function in the agricolae package.