Supplementary Methods 2: Chemical analysis of total
phenolics and total triterpenoid
Leaves were flash frozen in liquid nitrogen and stored at -80°C for
analysis. Total phenolic concentration was calculated with the modified
Folin-Ciocalteau method (Singleton & Rossi, 1964). Briefly, an aliquot
of 1 mL of free phenolic acid extract was added to 5 mL of water, 1.0 mL
Folin-Ciocalteau reagent, and 3 ml of saturated sodium carbonate
solution (7.5%) and mixed in a screw-top test tube. The absorbance was
measured at 765 nm with a spectrophotometer, after incubation for 2 h at
room temperature. Quantification was based on the standard curve,
established with 0, 50, 100, 150, 200 and 250 mg/L of gallic acid, and
the results were expressed as gallic acid equivalent in milligrams per
gram dry weight (mg GAE/g DW). Total triterpenoid concentration was
determined via vanillin-glacial acetic acid-perchloric acid
spectrophotometry using oleanolic acid as the standard (Fan & He,
2006). Briefly, total triterpenoids were extracted by ultrasonic
extraction using anhydrous ethanol as a solvent. Oleanolic acid was used
as the reference, 5% vanillin-acetic acid glacial solution and
perchlorate acid were used as the chromogenic agent, followed by water
bath at 70°C for 15min, and the absorbance was measured at 560 nm.
Because the amount of one leaf was not sufficient for each chemical
analysis, we gathered leaves from six plants as a biological
replication. There were 12 replicates for each combination of health
plant and herbivory treatment for each non-native species or their
native congeners.