2.4 | Phenotyping
We measured eight morphological traits of leaves and shoots, which were thought to be distinctive between Qc and Qd and associated with the tolerance to coastal stress (Nagamitsu et al. 2019). Measurements for three leaves and three shoots of branches collected from each tree were averaged and regarded as a phenotypic value of each tree.
To describe leaf shape, three traits were recorded: relative leaf width, lateral vein interval, and tooth angle (Figure S4). Among the three traits, the two latter traits are important to identify Qc andQd taxa (Ohba 2006; Aizawa et al. 2021). The length between the apex and base of each leaf and the width at the widest part of the leaf (mm) were measured, and the number of lateral veins were counted. Relative leaf width was calculated from (leaf width)/(leaf length), and lateral vein interval (mm) was calculated from (leaf length)/(number of lateral veins + 1). Tooth angle (˚) was measured on a serration at the central part of each leaf.
The density and size of stellate hairs on leaves vary between Qcand Qd (Ishida et al. 2003) and may have ecological and physiological functions (Bickford 2016). Stellate hairs on the lower surface of each leaf were observed in a circular leaf area with a 3.1 mm diameter between lateral veins using a stereo microscope (Figure S4). The number of stellate hairs in each area were counted, and the density (mm–2) was obtained. As the size of stellate hairs, the length of a radial filament (mm) of a typical stellate hair was measured (Figure S4).
Leaf mass per area (LMA) and shoot diameter may be related with adaptation to intense light, severe drought, and strong wind in coastal habitats (Sancho-Knapik et al. 2021). A leaf disc with a 9 mm diameter was collected from the central part of each leaf (Figure S4), and the dry weight (mg) of each disc was measured after dehydration at 60˚C for 48 h to calculate LMA (mg mm–2). Shoot diameter (mm) was measured at the upper part of shoots. Salt spray and harsh wind in winter cause the mortality of buds, particularly those in the upper part of shoots (Asai et al. 1986). Bud production in the lower part of shoots is likely to convey the tolerance to coastal stress, because this bud production can compensate for high bud mortality in the upper part of shoots. Thus, the number of axillary buds at bud-scale scars located in the lowest part of shoots was recorded (Figure S4).