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.

Quercus 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:

1. Control: No removal of tissues (Fig. 2a).
2. 25% apical removal: Removal of the dominant apical meristem and 25% apical shoot (Fig. 2b).
3. 75% apical removal: Removal of the dominant apical meristem and 75% apical shoot (Fig. 2c).
4. 25% auxiliary removal: Removal of all apical meristems (except for dominant meristem) and 25 % of auxiliary shoots (Fig. 2d).
5. 75% auxiliary removal: Removal of all apical meristems (except for dominant meristem) and 75 % of auxiliary shoots (Fig. 2e).

Measurements of Quercus defensive traits

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 Quercus 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 Quercus 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.

Quercus growth and leaf morphology

After treatments were applied, individual Quercus tree growth was measured. We measured the height of the apical shoot (height), and the lengths of all auxiliary shoots (auxiliary growth). The Quercus 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 RGR in the equation:

RGR=ln⁡(W₂ )-ln⁡(W₁)t₂-t₁

where W₁ and W₂ are a measurement of the plant’s height or auxiliary growth at times t₁ and t₂. RGR calculations minimize bias caused by variance in initial measurements of plant size (Hoffmann and Poorter, 2002; Rees et al., 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 et al. (2012).

Quercus nutrient allocation responses

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 et al., 2014). Non-structural carbohydrate analyses were done in a single lab to avoid differences from varying labs and techniques (Landhäusser et al., 2018). Nitrogen was analyzed using a rapid N exceed^® nitrogen analyzer.

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.