Measurement of stomatal aperture
Main stems of the invasive species were detached between 09:00 and 10:00 h and brought into the lab with the cut end submerged in distilled water. Epidermal layer of the stems of the third internode and mature leaves (including adaxial side and abaxial side) of the invasive species were torn off with fine-tipped tweezers, made wet mount microscope slides and viewed using a Leica DM6000 microscope (Leica, Wetzlar, Germany). Digital micrographs of the epidermal surfaces (20× and 40× magnification) were captured and used to quantify stomatal density and stomatal aperture. The image dimensions were calibrated using the objective micrometer. Stomatal densities were analyzed using the images captured through a the 20x objective and expressed on an area basis (stomata mm−2). The stomatal density of each sample was calculated as the average of four images that were captured from different fields of view. The stomatal lengths and widths were determined using the images captured through the 40× objective, and for each treatment, at least 20 stomata were measured.
Observations of chloroplast ultrastructure
Fresh stems of the third internode and mature leaves were cut into approximately 1 mm×1 mm pieces, vacuum infiltrated and fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0). The samples were then dehydrated in a graded series of ethanol and embedded in Epon 812 epoxy resin. The embedded samples were sliced into ultrathin sections approximately 70 nm in thickness using an ultramicrotome (Leica UC7, Leica), double-stained with uranium lead (a 2% uranyl acetate and lead citrate saturated aqueous solution) and observed under a transmission electron microscope (HT7700, Hitachi Japan). The length and width of the chloroplasts were determined from the images captured at 1200× magnification, and 10 chloroplasts were measured for each treatment.