Leaf Morphometric Analysis of Madera Plants

Between 12 and 14 leaves were collected from six shoots per sample from plants in Madera, California, USA in June 2022. The sampled shoots grew from retained nodes. Leaves were pressed in an herbarium press at Madera, California, USA and shipped in the press to East Lansing, Michigan, USA for scanning and analysis. The leaves were scanned using a CanoScan 9000 F Mark II (Canon U.S.A., Inc) at 600 DPI. The leaves were landmarked manually by placing 21 landmarks from Bryson et al. [11 (link)] on leaf scans using ImageJ v1.53k [12 ]. Scans were saved as x- and y-coordinates in centimeters. The shoelace algorithm, originally described by Meister [13 ], was used to calculate leaf, vein, and blade areas using the landmarks. The landmarks were used as the vertices of polygons and the following formula, as described in Chitwood et al. [14 (link)], was used to then calculate the areas (where n represents the number of polygon vertices defined by the landmarked x and y coordinates):
To investigate changes in leaf shape between WT and WB leaves, a generalized Procrustes analysis and a principal components analysis (PCA) was performed using the shapes package v1.2.7 [15 ] in R v4.2.2 [16 ] and RStudio v2022.12.0.353 [17 ], with scaling and rotation. The shapes package v1.2.7 [15 ] in R and RStudio was also used to test for mean shape differences using a Hotelling’s T² test.