Appendix A. Detailed methodology of leaf trait measurements.
Following each of the six censuses, we collected three leaves from each experimental tree; we collected leaves haphazardly but chose average-sized or large leaves with minimal damage. Leaves were kept chilled on ice after collection and weighed within three hours. We measured toughness using a penetrometer (average of three punches per leaf, nine total punches per tree, Force Dial FDK 32, Wagner Instruments, Greenwich, CT). In 2008, we punched one 6 mm diameter leaf disk from each leaf. Leaves and disks were lyophilized for 72–96 hours and reweighed to obtain dry weight. We calculated water content as (wet weight – dry weight) / wet weight, and weighed leaf disks to calculate specific leaf area (SLA, dry weight / 0.283 cm2).
We assayed condensed and hydrolyzable tannins for each tree and compared them to standards purified from bulk leaf tissue pooled from all trees. Bulk tissue was washed with 95% ethanol and extracted with 70% acetone on Sephadex LH-20 in a Büchner funnel. Samples from each tree were rinsed with diethyl ether, and tannins were extracted in 70% acetone followed by rotary evaporation; the resulting aqueous samples were brought to common volume. We assayed condensed tannins using the acid-butanol technique (Rossiter et al. 1988, Waterman and Mole 1994) and hydrolysable tannins using the potassium iodate technique (Bate-Smith 1977, Schultz and Baldwin 1982). Absorbances of each tree were compared to a curve constructed from bulk standard samples of known concentration and expressed as percentages of starting leaf tissue mass. An error in the May 2007 condensed tannin assays resulted in questionable values, so these data were discarded and excluded from analyses. We determined carbon and nitrogen content by microcombustion on a Perkin-Elmer Series II CHNS/O Analyzer 2400.
Bate-Smith, E. C. 1977. Astringent tannins of Acer species. Phytochemistry 16:1421–1426.
Rossiter, M. C., J. C. Schultz, and I. T. Baldwin. 1988. Relationships among defoliation, Q. rubra phenolics, and gypsy moth growth and reproduction. Ecology 69:267–277.
Schultz, J. C. and I. T. Baldwin. 1982. Oak leaf quality declines in response to defoliation by gypsy moth larvae. Science 217:149–151.
Waterman, P. G. and S. Mole. 1994. Analysis of phenolic plant metabolites. Blackwell Scientific Publications, Oxford.