DOI: 10.5061/dryad.8cz8w9gsv
title: Data recorded for leaf size, and leaf vein and areole measurements taken from nine Magnoliaceae species

description: Across species, the density of major veins in leaves scales inversely with
 leaf area. Yet, minor vein density manifests no clear relationship with
 respect to leaf are, despite having the potential to provide important
 insights into the trade-off between investments in leaf mechanical
 support, hydraulics, and light interception. To examine this phenomenon,
 between 120−380 mature leaves from each of nine Magnoliaceae species were
 sampled from Nanjing Forestry University campus and Nanjing Botanical
 Garden, Chinese Academy of Sciences, Nanjing, China in 2019 and 2020. The
 leaves were boiled, and the epidermal and mesophyll tissues were removed.
 The remaining leaf skeleton was stained to obtain a clear leaf venation
 network. The software LEAF GUI was used to obtain the data of leaf size,
 leaf vein and areole traits. The large sample size data render it possible
 to explore the scaling relationships between leaf size and each trait of
 leaf veins and areoles. This data set includes the information of leaf
 length, leaf width, leaf area, total vein length, total vein area, mean
 areole area per leaf, and the number of the areole sizes per leaf of the
 nine Magnoliaceae species.
description: <strong>Leaf
 sampling</strong> Leaves of nine species of Magnoliaceae
 (<em>Magnolia amoena, Magnolia denudate, Magnolia soulangeana,
 Magnolia tomentosa, Michelia cavaleriei var. platypetala, Michelia
 chapensis, Michelia compressa, Michelia figo, </em>and
 <em>Michelia maudiae</em>) were collected from Nanjing
 Botanical Garden, Chinese Academy of Sciences and Nanjing Forestry
 University Xinzhuang Campus, Nanjing, China. Fully expanded and fully
 developed leaves, at least three months old were sampled between July to
 September in 2019 and 2020. The sampling of leaves for any one species was
 completed well within one year, in order to avoid phenological changes in
 leaf architecture. However, the sampling of all the leaves from all of the
 species was not performed in the same year owing to the required heavy
 workload this would have necessitated. For each species, 300−500 leaves
 from three to five mature trees were randomly sampled from the middle
 canopy without distinguishing between sun leaves and shade leaves (because
 the objective was to explore general trends rather than focusing on the
 influence of light on the characteristics of leaf veins and areoles, which
 is nevertheless worthy of future research). Only non-damaged healthy
 leaves were sampled. Fresh leaves were wrapped in wet paper to minimize
 desiccation and brought to the laboratory within two hours after
 sampling. <strong>Chemical treatment and image scanning of leaf
 veins</strong> The leaves were labelled by attaching a small
 paper tag to their petioles. Each leaf was subsequently placed into a
 nylon mesh bag and boiled in a 5-10% NaOH solution for 30 minutes in an
 open stainless steel pot (ST24P1, Supor, Wuhan, China; diameter: 28 cm;
 capacity: 1.2 L). The boiled leaves were washed with running water, and a
 soft brush was used to carefully remove epidermal and mesophyll tissues.
 The remaining leaf skeleton was then stained by a 5% safranin solution.
 The chemically cleared leaves were scanned at 600 dpi using an Epson
 scanner (V550, Epson Indonesia, Batam, Indonesia; Figure 1 for
 representative examples of scanned chemically cleared leaves of the nine
 species). We initially sampled and boiled 300−500 leaves. The useable
 samples (with intact leaf veins) were between 120 and 380 leaves. By
 estimate, there was on average less than a 5% loss of leaf veins for each
 of the usable leaves. However, this loss is estimated to not substantially
 influence the results reported here because of the very large sample size
 for each the species examined in this study. 
 <strong>Acquisition of
 leaf vein and areole data</strong>
 The software LEAF GUI
 developed using the Matlab platform (Price et al., 2011) was used to
 obtain leaf area, total leaf vein length, total leaf vein area, the number
 of areoles per leaf, and individual areole area from cleared leaf images.
 The total areole area was calculated by summing the areas of all
 individual areoles. Mean areole area was defined as the mean of all of the
 individual areole areas per leaf. The original RGB images were transformed
 into black and white binary images, and the adaptive threshold and global
 threshold were set in LEAF GUI to obtain ultra-clear leaf vein images. The
 thresholds were determined empirically for each leaf image. LEAF GUI does
 not provide midrib length. Thus, the R script developed by Shi et al.
 (2018) and Su et al. (2019) was used to obtain leaf length, which was
 defined as the distance from the leaf base to leaf apex regardless of
 lamina curvature, and leaf width, which was defined as the maximum
 distance between two orthogonal points of the leaf edge forming a straight
 line perpendicular to the leaf length axis. Leaf length was used as the
 substitute of midrib length, although for curved leaves, the numeric
 values of these traits can moderately differ. Given that most leaves had
 almost perfect bilateral symmetry, the difference between midrib and
 lamina lengths was judged to be negligible (Fig.
 1). <em><strong>The above contents were quoted from Shi et al.
 (2022).</strong></em>
 <strong>References:</strong> Price, C. A., O. Symonova, Y. Mileyko, T. Hilley, and J. S. Weitz. 2011. Leaf extraction and analysis framework graphical user interface: segmenting and analyzing the structure of leaf veins and areoles. P<em>lant Physiology</em> 155: 236–245. Shi, P., Q. Miao, Ü. Niinemets, M. Liu, Y. Li, K. Yu, and K. J. Niklas. 2022. Scaling relationships of leaf vein and areole traits versus leaf size for nine Magnoliaceae species differing in venation density. <em>American Journal of Botany</em> in press. Shi, P., D. A. Ratkowsky, Y. Li, L. F. Zhang, S. Y. Lin, and J. Gielis. 2018. A general leaf-area geometric formula exists for plants–evidence from the simplified Gielis equation. <em>Forests </em>9: 714. Su, J. L., K. J. Niklas, W. Huang, X. Yu, Y. Yang, and P. Shi. 2019. Lamina shape does not correlate with lamina surface area: an analysis based on the simplified Gielis equation. <em>Global Ecology and Conservation</em> 19: e00666.