2.2 Leaf anatomy
Five mature leaves were used to determine the area occupied by the vascular elements of the leaf midrib: consecutive hand transverse sections were cut with a micro scalpel at three different positions in the leaves: the petiole, the mid part of mid vein, and about 2cm back from leaf tip. Each section was stained with 0.1% w/v aniline blue in PO4K3 (pH 10) (Linskens & Esser, 1957), observed with a Zeiss Axiophot epifluorescent microscope using the DAPI narrow filter band (excitation 365 nm, bandpass 12 nm; dichroic mirror FT 395 nm; barrier filter LP397 nm), and photographed with an AxioCam 512 Color linked to the AxioVision software (Zeiss, Oberkoche, Germany). The cross-sectional area of xylem and phloem was measured (totaling 45 measures for each tissue) and, for the 27 cross sections in which all vascular elements were visible, the number of vascular elements in each tissue were counted
Five additional leaves were selected for measurements of individual sieve tubes and xylem vessels. Longitudinal sections of the petiole, midrib, second, third, and fourth order veins were obtained with a scalpel, placed in a solution containing acetic acid: hydrogen peroxide 1:1 v/v, left incubating at 60ÂșC for two days, and then mounted onto glass slides for microscopy observation and image acquisition (n>250 total vessel elements measured). Similar areas were used to obtain fresh longitudinal sections (less than 1mm thick), mounted onto slides, stained with 0.1% aniline blue, counterstained with calcofluor white for cellulose (Hughes & McCully, 1975), photographed and measured (at least 35 sieve tubes per vein order, n=250).