DOI: 10.5061/dryad.fttdz08s1
title: Growth, defense, and storage responses of 12 oak (Quercus) species to varying locations and intensities of simulated herbivory

description: The evolution of plant defenses is often constrained by phylogeny. Many of
 the differences between competing plant-defense theories hinge upon the
 differences in the location of meristem damage (apical vs. auxiliary) and
 the amount of tissue removed. We analyzed the growth and defense responses
 of 12 Quercus (oak) species from a well-resolved molecular phylogeny using
 phylogenetically independent contrasts. Access to light is paramount for
 forest-dwelling tree species, such as many members of the genus Quercus.
 We therefore predicted a greater investment in defense when apical
 meristem tissue was removed. We also predicted a greater investment in
 defense when large amounts of tissue were removed and a greater investment
 in growth when less tissues were removed. We conducted five
 simulated-herbivory treatments including a control with no damage and
 alterations of the location of meristem damage (apical vs. auxiliary
 shoots) and intensity (25% vs. 75% tissue removal). We measured growth,
 defense, and nutrient re-allocation traits in response to simulated
 herbivory. Phylomorphospace models were used to demonstrate the
 phylogenetic nature of trade-offs between characteristics of growth,
 chemical defenses, and nutrient re-allocation. We found that
 growth-defense trade-offs in control treatments were under phylogenetic
 constraints, but phylogenetic constraints and growth-defense trade-offs
 were not common in the simulated-herbivory treatments. Growth-defense
 constraints exist within the Quercus genus, although there are adaptations
 to herbivory that vary among species.
description: <i>Quercus</i> saplings were purchased from Mossy Oak
 Nativ Nursery in West Point, MS, United States. We used saplings that were
 the same age to avoid adaptive responses to damage caused by ontogenetic
 differences (Gruntman & Novoplansky, 2011). We applied five
 treatments to mimic variations in location and intensity of simulated
 herbivory. Each species received all five treatments, which were
 replicated five times for a total of 25 individuals per species. The five
 treatments were as follows: 
 Control: No removal of tissues (Fig.
 2a). 25% apical removal:
 Removal of the dominant apical meristem and 25% apical shoot (Fig.
 2b). 75% apical removal:
 Removal of the dominant apical meristem and 75% apical shoot (Fig.
 2c). 25% auxiliary removal:
 Removal of all apical meristems (except for dominant meristem) and 25 % of
 auxiliary shoots (Fig. 2d).
 75% auxiliary removal: Removal of all apical
 meristems (except for dominant meristem) and 75 % of auxiliary shoots
 (Fig. 2e). 
 <b>Measurements of Quercus defensive
 traits</b> Trees were harvested one year after
 treatment application. For chemical defense traits, leaf and root samples
 were dried in an oven at 65 ℃ for 48 h until plant tissues were completely
 dry. To assess possible differential investments in different types of
 tannins, we measured total polyphenols and two types of tannins (tannins
 constitute a type of polyphenol). Polyphenols and tannins were extracted
 from the oven-dried plant tissues (Hagerman, 1988) using a 70% acetone
 solvent (Graca & Barlocher, 2005; Hagerman, 2011). Once extracted,
 total polyphenol concentrations in the <i>Quercus</i> tissue
 were analyzed using the Prussian Blue Assay (Price & Butler, 1977)
 with modifications for use on a microplate reader (Hagerman, 2011). We
 used gallic acid as a standard (gallic acid equivalents “G.A.E.”). Total
 tannin concentrations in the <i>Quercus</i> tissue were
 analyzed using the Radial Diffusion Assay and standardized against tannic
 acid (tannic acid equivalents “T.A.E.”) (Hagerman, 1987). Condensed tannin
 concentrations were analyzed using the Acid Butanol Assay for
 proanthocyanidins (Gessner & Steiner, 2005; Hagerman, 2011), and
 standardized against quebracho tannin (quebracho equivalents “Q.E.”). Note
 that there are no unique concentrations for polyphenols or tannins, so
 they are expressed as equivalents of a specific polyphenol or tannin
 (Hagerman, 2011). To calculate trichome density, a hole
 punch was used to punch discs of 7 mm diameter from each leaf. The discs
 were placed on a microscope, and trichome density was calculated as the
 number of trichomes/dry mass (g) of the 7 mm disc. The average number of
 trichomes/dry mass (g) of the three discs from each sapling was recorded
 as the trichome density. <b>Quercus growth and
 leaf morphology</b> After treatments were
 applied, individual <i>Quercus</i> tree growth was measured.
 We measured the height of the apical shoot (<i>height</i>),
 and the lengths of all auxiliary shoots (<i>auxiliary
 growth</i>). The <i>Quercus</i> saplings were kept in a
 greenhouse under optimal conditions for one year. Growth measurements for
 each individual tree were measured biweekly for analysis of relative
 growth rates. Relative growth rates were calculated for each growth
 variable (height and auxiliary growth) defined as <i>RGR</i>
 in the equation: <i>RGR=</i>ln⁡<i>(W<sub>2 </sub>)-</i>ln⁡<i>(W<sub>1</sub>)t<sub>2</sub>-t<sub>1</sub></i> where <i>W</i><sub>1 </sub>and <i>W</i><sub>2</sub><i> </i>are a measurement of the plant’s height or auxiliary growth at times <i>t</i><sub>1 </sub>and <i>t</i><sub>2</sub>. RGR calculations minimize bias caused by variance in initial measurements of plant size (Hoffmann and Poorter, 2002; Rees <i>et al</i>., 2010;). All growth measurements were taken biweekly throughout the year following treatment application. The final growth measurements were taken once trees were harvested, one year after treatments were applied. Leaf morphological samples were taken during harvesting, one year after treatment application. Leaves were scanned on a CI-202 leaf area meter from CID BioScience. After scanning, leaves were dried and weighed. We measured specific leaf area (leaf area divided by the leaf’s dry weight), leaf aspect ratio (maximum leaf breadth/ maximum leaf length), and leaf shape factor (leaf area/ perimeter) by removing three leaves from each sapling and following leaf measurement protocols, as described by Lu <i>et al</i>. (2012). <b>Quercus nutrient allocation responses</b> The samples were tested for the concentration of total non-structural carbohydrates using the method by Fournier (2001) that uses a phenol-sulfuric acid solvent for a colorimetric reaction of sugars and starches extracted from leaf tissues (see Tomlinson <i>et al., </i>2014). Non-structural carbohydrate analyses were done in a single lab to avoid differences from varying labs and techniques (Landhäusser <i>et al</i>., 2018). Nitrogen was analyzed using a rapid N exceed<sup>®</sup> nitrogen analyzer.
description: These responses are for manipulated treatments. The treatments were (1)
 control- no simulated herbivory; (2) removal of apical meristem and 25% of
 apical growth; (3) removal of apical meristem and 75% of apical growth;
 (4) removal of 25% of lateral growth; (5) removal of 75 percent of lateral
 growth.