Figure 2.

Ellagitannins, a major class of defense compounds in Tococa quadrialata, increase when symbiotic ants are not present

(A) Base peak chromatogram (BPC) of a methanolic leaf extract run in negative ionization mode on an ion-trap LC-MS -showing that ellagitannins and related compounds are some of the main constituents of the extract (peak numbers correspond to lines in panel B).

(B) Tentative identification of the major peaks of the chromatogram in panel A based on retention time, accurate mass of the most abundant parent ion (usually [M−H]^-), predicted sum formula, and fragmentation pattern (not shown) (for more details, see Table S4, Figure S4).

(C) Structure of the ellagitannin vescalagin, derived from 5 gallic acid moieties (highlighted in green) and a central sugar moiety (black).

(D) Volcano plot of untargeted metabolome analysis of T. quadrialata (with MS in negative ionization mode) comparing features of ant-colonized and ant-deprived plants 2.6 years after ant removal. Several features identified as ellagitannins, precursors, and related compounds were found to be more abundant in ant-deprived plants.

(E) Choice assay with Spodoptera larvae offered leaf discs from ant-colonized (ant) and ant-deprived (no ants) plants (2.7–2.8 years after ant-exclusion) in a Petri dish. Larvae allowed to move freely in the Petri dish for 24 h were found to prefer feeding on leaves of ant-colonized plants (∗∗∗:p < 0.001, paired Wilcoxon rank-sum test, n = 41). Mean and SE are shown. RT, retention time; HR-MS, high-resolution mass spectrometry; HHDP, hexahydroxydiphenoyl.