2.3 | Leaf sampling
For each species, we sampled foliage from three juvenile plants in each of six or seven fenced plots and six or seven unfenced plots (six if there were not seven with a sufficient number of plants). In an attempt to limit variation in plant age, we selected individuals that were within the middle 50% of the range of heights for that species in open plots or in fenced plots, based on measurements from fall 2017 in 4 x 1 m belt transects in all plots in the forest. Within the size range, we selected plants with the least amount of visible insect damage. On 26 July 2018, we identified and marked the plants to be sampled and then completed the entire foliage collection the next day, on 27 July. We alternated collection between fenced and unfenced plots, and all collection was completed for one species before proceeding to the next. The youngest fully expanded leaf was collected from the plot’s three plants simultaneously, by having three people work together. The three leaves were combined, formed into a single pellet, wrapped in foil and submerged into liquid nitrogen within 30 sec of collection. They were transferred directly into a -80 C freezer within 2 hr of collection, and 4 days later shipped overnight on dry ice to the Boyce Thompson Institute for metabolite extraction and analysis.
2.4 | Extraction and analysis of metabolites
Leaf samples were ground into fine powder in liquid nitrogen using mortar and pestle. Two hundred milligrams of the fine powder were transferred into pre-chilled microcentrifuge tubes that contain two metal beads and homogenized in 1 mL ice-cold extraction buffer (1: 2: 1 chloroform : methanol : water; v/v) for 2 min. The homogenized samples were vortexed for 20 min at 4 oC and centrifuged for 20 min at 15,000 g. Then, 750 µL of the clear supernatants were transferred into new microcentrifuge tubes and dried under vacuum at room temperature. After adding 100 µL 70% methanol (in water; v/v), the tubes were vortexed for 10 min, centrifuged for 10 min at 15,000 g, and 5 µL of the clean supernatants were analyzed on Q-exactive liquid chromatography-tandem mass spectrometer (LC-MS/MS; Thermo Scientific) in negative ionization mode.