Methods
This account covers stages we have completed and what we are currently pursuing.
Sampling
The study includes three superlatively tall trees from each species, spanning the North-South axis of the species' ranges. Climbers used rope-based arborist techniques to access and collect samples from the six trees: with a crossbow, they launched a guideline over the lowest branch, then pulled up a weight-bearing rope with the guideline, and climbed the weight-bearing rope using metal tools called ascenders. Ascenders have one-way teeth that grip a rope when they are pulled and release when pushed. The lead climber used a series of ropes to climb to the top of the tree and install a single rope that reached the ground to use during the study. Climbers collected 30 cm long branches at 10-meter height intervals or as available. This study focuses on 3-4 heights per tree that span the canopy. Additional heights were examined in a S. sempervirens here referred to as SESE 001 who grows a very deep canopy.
Samples were completely oven-dried in the lab at 70°C. Twenty-five mature leaves per height were cut from branchlets that did not bear reproductive structures. All of these leaves (N=650) were prepared for whole-leaf analysis. The leaves from one individual of each species (SESE T001 and SEGI T273) were processed as cuticle microscope slides, a subset of which were photographed and further analyzed (N=37) (Figure 1).
Whole leaf analysis
Every cut leaf was weighed in a Mettler Toledo Mx5 microbalance (Columbus, Ohio) and scanned on the abaxial and adaxial—back and front—side in an Epson V30 flatbed image scanner (Seiko Epson, Owa, Suwa, Japan). The digital images were measured in ImageJ (National Institute of Health, Bethesda, Maryland) for leaf length, width, and total area. This data was exported to Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA) and used to calculate the length-to-width ratio and leaf mass area (LMA, noted in g/m²).
Both S. sempervirens and S. giganteum produce stomata in two bands that run parallel to the leaf axis, a band on either side of the midvein. Individual bands contain one to nine rows of stomata. The number of rows in a band is mostly symmetrical across the midvein and varies considerably between leaf faces (abaxial/adaxial) and position (base, middle, or tip) on the leaf (Ma et al. 2002). The number of rows in a stomatal band was counted at the tip, middle, and base of the abaxial and adaxial sides of all leaves using a Leica M165 stereomicroscope at 12x magnification (Leica Microsystems, Wetzlar, Germany). A ratio comparing the number of stomatal rows was calculated for the base, middle, and tip of every leaf.
Cuticle analysis
All of the leaves from SESE T001 and SEGI T273 were scored longitudinally with a razor and macerated in 4% sodium hypochlorite (NaHClO) for 12 to 48 hours, rinsed with demineralized water, and treated with 5% potassium hydroxide (KOH) for 1 min to stop the reaction, followed by another demineralized water rinse. The softened mesophyll was removed and the cuticle stained with 4% Safranin. Cuticles were then gradually dehydrated in 25%, 75%, and 100% glycerin solutions, and mounted in a glycerin-gelatin medium. Per height, 1 to 5 cuticles were—if intact—randomly selected for further analysis. Photos of the cuticles were taken at 50x magnification with a Leica DM2500 microscope, a Nikon DXM1200F camera, and a Nikon DS-L2 control unit (Nikon Corporation, Tokyo, Japan). Even flattened cuticles have depth, so extended depth-of-field images were generated and then stitched into panoramas using Adobe Photoshop CS5 (Adobe Systems Incorporated, San Jose, CA).
For S. sempervirens, three 1mm long counting fields were drawn at the tip, middle, and base of the leaf. For heights added later in the study—70, 80, 90 and 100m—the counting fields were 2mm long. In the smaller leaves of S. giganteum, counting fields encompassed the whole leaf. Every stomatal pit in the field was traced as an ellipse with a longitudinal axis using CorelDraw X4 (Corel Corporation, Ottawa, Ontario, Canada). The traced overlay was exported as a JPEG and analyzed using the particle analysis plugin in ImageJ to find stomatal pit area and orientation to the leaf axis. The data was imported into an Excel spreadsheet.
Specimens are currently housed in the Looy Lab and will be incorporated in the Paleobotanical Collections of the University of California Museum of Palaeontology, Berkeley, CA. Illustrated material will be stored in the Paleobotanical Type and Illustrated Collections. Cuticle descriptions will be logged to the Paleobotanical Database (PBOT).
Proposed Expanded Methods
A new microscope that the Integrative Biology department now shares, one with an automated arm, and Photoshop 24's ability to automatically generate depth-of-field panoramas can help quickly photograph more of the 25 needles per height that have already been prepared as microscope slides. Advances in machine learning have also automated counting stomata (Jayakody et al. 2021, MaskStomata), and hopefully soon measuring stomatal area and angles. Automated stomatal masks currently achieve a .7 intersection-over-union (IoU), a measure of accuracy (Ibid).
Preparing the cut leaves from the other four trees for microscopy study would improve the representation of the two species and may be performed after establishing an efficient workflow with the existing slides.
References
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