2.3 Xylem vessel structural traits
We measured hydraulically weighted diameters (D h)
of xylem vessels in the branches and roots. Three transverse sections
(20-30 μm thick) per tree (total n = 90) were cut from the branch
and root segments using a sliding microtome (SM2010R; Leica, Wetzlar,
Germany). The sections were stained with safranine, rinsed in deionized
water, and mounted in glycerol on a glass slide. For each section, six
to ten images per cross-section were taken at 100× magnification using a
light microscope (DM2500; Leica, Wetzlar, Germany) equipped with a
high-resolution digital camera (ICC50W; Leica, Wetzlar, Germany). These
images were combined to produce an integrated image covering the whole
cross-section (or at least a radial transect based on the sectional
quality), using graphics software (Photoshop CS6; Adobe Systems
Incorporated, USA). A minimum of 300 vessels per section were analyzed.
After the images were downloaded into a computer, we measured the lumen
area of all intact vessels using image analysis software (ImageJ;
National Institutes of Health, USA). Vessel diameter (D , in μm)
was determined as the diameter of a circle with an area equal to the
lumen area. We calculated the hydraulically weighted mean diameter
(D h, in μm) for each segment as
(∑D 4/N )1/4 (Tyreeet al ., 1994), where N is the total number of vessels in
an integrated image.
For pairs of vessels, we measured maximum span across a vessel
(b ) and double wall thickness (t ) on digital images using
ImageJ, so that t 2/b 2was proportional to vessel wall reinforcement to resist cavitation
(Hacke et al ., 2001). Thet 2/b 2 value was
calculated from the mean of a minimum of 10 vessel pairs per branch or
root. We measured the above traits on the same segment used to measure
vulnerability curves.