Euparal mounting medium

Wings were removed from adult flies, dehydrated in 100% ethanol for 5 min and placed on a microscope slide to allow ethanol to evaporate. A small drop of Euparal Mounting Medium (Roth, Karlsruhe, Germany) was dropped onto the wing and a glass coverslip placed on top. Images were captured with a Spot digital camera and a Nikon E1000 microscope (Nikon Instruments Europe, Tokyo, Japan).

The rightwing of each male and female mosquito was detached from the thorax and processed following the protocol described by Lorenz and Suesdek [31 (link)], with some modifications. Each wing was bathed using 3% sodium hypochlorite (NaClO) for 20 min. The scales were removed from the wings using cotton swabs, followed by a 99.5% ethanol wash. Afterward, the wings were bathed in an acid fuchsin solution for 1 h and then washed with 70% ethanol twice. Finally, the wings were mounted between a slide and a cover slip with a drop of Euparal© mounting medium (Carl Roth, Karlsruhe, Germany). The mounted wings were photographed using a camera(SI 3000 version I8, Warpsgrove Ln, Chalgrove OX44 7XZ, UK) with a 10× magnification on a stereoscopic microscope (CETI Steddy Stereo Trinocular Microscope, Warpsgrove Ln, Chalgrove OX44 7XZ, UK). In total, 19 landmarks (LMs) [32 (link)] were identified and digitized using Tps Dig2 (V2.31) software [33 ] to generate the Cartesian coordinates in two dimensions for each individual mosquito (Supplemental Table S2, Supplemental Figure S1).

For microscopy, the main stem of field-grown WT trees was harvested 1 m above soil level in August 2017 of the second growth season. At that time, the WT tetraploids already showed the brittle apex phenotype. Harvested parts were fixed in 70% (v/v) ethanol. Multiple transversal stem sections of 20 μm thickness were made per plant using a Reichert-Jung 2040 Autocut Microtome (Leica) and stained with 0.5% (w/v) astra blue, 0.5% (w/v) chrysiodine and 0.5% (w/v) acridine red for 10 min. To prepare the triple staining solution, 0.5% (w/v) astra blue (Santa Cruz Biotechnology) in 2% tartaric acid was mixed with 0.5% (w/v) chrysoidine (Sigma Aldrich) in 5% (w/v) ammonium aluminum sulfate and 0.5% (v/v) glacial acetic acid and 0.5% (w/v) acridine red (Santa Cruz Biotechnology) in 5% ammonium aluminum sulfate and 0.5% (v/v) glacial acetic acid in a 4:1:1 ratio. After dehydration in isopropyl alcohol, the stained sections were mounted in Euparal mounting medium (Carl Roth). Images were acquired using a Zeiss Axioskop 2 microscope and processed using automated software described by Andriankaja et al. (2012 (link)) to quantify the average number and average area of vessels and fibers per selected area. The number of vessels was divided by the number of fiber cells to provide a ratio. The proportion of vessel lumen was defined as the total vessel area per selected area.