Ecological Archives E087-117-A1

Paul V. A. Fine, Zachariah J. Miller, Italo Mesones, Sebastian Irazuzta, Heidi M. Appel, M. Henry H. Stevens, Ilara Sääksjärvi, Jack C. Schultz, and Phyllis D. Coley. 2006. The growth-defense trade-off and habitat specialization by plants in Amazonian forests. Ecology 87:S150–S162.

Appendix A. Detailed methods for chemical analysis of terpenes, phenolics, and soluble protein.


Approximately 500 mg (fresh weight) leaves from the experimental seedlings were collected at the experimental sites in 2 mL glass vials and filled with dichloromethane (DCM). This leaf-DCM mixture was used for qualitative and quantitative analyses with gas chromatography-mass spectrometry (GCMS) and a gas chromatagraph (GC).

Qualtitative analysis of the plant extracts was performed by GCMS using a capillary column on a ThermoFinnigan TRACE GC coupled with a TRACE Mass Selective Detector at 70 eV with a DB-5 column 30 m long×0.25 mm ID and 0.25 um film with helium carrier gas at a linear velocity of 28 cm/sec. The injector temperature was 250˚ C, detector 280˚ C, and the oven temperature programmed from 60˚ to 240˚ C at 10˚ C/min, then held at 230˚ C for 2 min, flow 1 mL/min. All volatile compounds from plant extracts were identified by matching the Chromatograms obtained to the net-based library maintained by the NIST (National Institute of Standards and Technology) Mass Spectometry Data Center for compound identification, and by comparing the mass spectra and retention times of authentic standards, standard mass spectral libraries and by interpreting the mass spectrum. Reference compounds were alpha-pinene, limonene, caryophyllene, and phytol.

To compare the amount of terpene investment for the 2 species of Oxandra and the 10 species of Protium from the reciprocal transplant experiment, we used a Hewlett-Packard (HP) 5890 gas chromatograph and a 30-m HP-5 capillary column. Operating conditions were splitless injection, and we used the same timing and temperatures as listed above for the GCMS. For each sample, 25 mL of the leaf-DCM extract was diluted in 1 mL of DCM. The dry weight of the leaf material from each vial and volume of DCM from the vial were measured to determine the initial concentration of plant dry weight/ ml solvent of each vial. The monoterpenes alpha-pinene and limonene, the sesquiterpene caryophyllene, and the diterpene phytol were used to calculate standard curves. Standard curves for total terpene quantification (ml terpenes per g of leaf (dry wt.) were prepared by spiking known volumes of DCM with increasing concentrations of analytical grade alpha-pinene and limonene (monoterpenes), caryophyllene (a sesquiterpene), and phytol (a diterpene) together with 250 mg lidocaine/ 1 mL DCM as an internal standard (see Table A1 below). The areas of the known concentrations of terpenes were divided by the internal standard and plotted to fit separate standard curves for monoterpenes, sesquiterpenes and diterpenes. The slopes of the two monoterpenes did not significantly differ from one another and were averaged and used to estimate the concentration of monoterpenes from the samples (y = 0.1 x + 0.00062, R2 =0.99). For sesquiterpenes, the standard curve was y = 0.087 x + 0.00014 (R2 = 0.99), and for diterpenes and other resins it was y = 0.347 x –.0079 (R2 =0.65). Aftter calculating the monoterpene, sesquiterpene, and diterpene concentration for each plant sample, these numbers were then divided by the initial concentration of the plant material/solvent from the field collected vials, and also by the internal standard measurement to standardize comparisons among samples.


Approximately 2 g fresh weight of mature leaves of 16 individuals from each species in the reciprocal transplant experiment were collected and immediately placed in plastic tubes containing silica gel dessicant. Tannin analyses of leaves dried with silica gel gives comparable results with those obtained with fresh leaf analyses (Julkunen-Tiitto and Sorsa 2001). Once samples arrived in the Appel/Schultz laboratory, they were freeze-dried and ground in a UDY cyclone mill to prevent heating, and then stored at -20oC until use.

Bulk tannins were prepared to provide standards for the phenolic assays of individual samples. This is a crude purification, and although non-phenolic materials are unlikely to be present (Hagerman and Klucher 1986; Appel and Schultz unpub. data), the product is merely a more representative sample of extractable polyphenols found in the actual plant than is a commercial standard from some other source (Appel et al. 2001).

In the comparison of species growing on white sand and clay soils, a bulk tannin standard was prepared for each species from a combination of leaf material from both soil types. We were unable to purify bulk standards from several species because the purified samples were too resinous (Mabea subsessilis and Protium calanense) or foamy (Swartzia arborescens and both Parkia species), the latter presumably due to saponins. Two commercial sources of tannins commonly used by ecologists as standards in the Folin Denis assay were also purified with LH20 to remove small molecular weight contaminants: two different sources of the hydrolyzable tannin mixture called “tannic acid” (Sigma T0125 prepared from Rhus sp., Anacardiaceae), and the condensed tannin “quebracho” (Leon Monnier, Inc., from Schinopsis spp., Anacardiaceae). For the genera Oxandra and Protium, we used the self-standards (from the bulks) developed for each species to calculate total phenolics. Examination of the self-standard curves revealed that all of the 20 Protium species had very similar curves, as did the two species of Oxandra. The species Mabea pulcherrima, Swartzia cardiosperma, and Pachira insignis had curves that were very different from the quebracho standard curve. Therefore, we reasoned that for these three genera with only one self-standard curve obtained, we would use that self-standard for both species within a genus rather than using the quebracho standard. For the genus Parkia, since we obtained no self-standard curves in either of the two species due to extreme foam, we used the quebracho standard curve. While not perfect, our multi-species comparisons are still considerably more accurate than those that simply use quebracho standard to compare species differences in total phenolics (Appel et al. 2001).

To make the bulk standards, 3 g of lyophilized leaf powder were washed in 75 mL of ether for 30 minutes to remove pigments and waxes, and then extracted 3X in 125 mL 70% acetone at 4 oC for 1 h under sonication. Ascorbate (10 mM) was added to the acetone to prevent oxidation. Acetone was removed by evaporation under reduced pressure at 35 oC, and distilled water was added to the aqueous extracts to a constant volume of 125 mL. This procedure favors extraction of polymeric tannins (Hagerman and Klucher 1986), and is not exhaustive; substantial amounts of polyphenols may remain covalently bound to cells walls or other cellular components, but many of these are not likely to be defensive because they remain covalently bound in the herbivore gut (Appel and Schultz, unpub.).

Tannins in the polyphenol extracts were separated from non-tannin polyphenols by the method of Hagerman and Klucher (1986). A slurry of 50g of Sephadex LH20 (Pharmacia, Piscataway, NJ) and approximately 1 liter of 95% reagent grade ethanol was equilibrated overnight, and then mixed thoroughly with 125 mL of crude extract. Using a large Buchner funnel and vacuum filtration, non-tannin, monomeric polyphenols were eluted from the slurry by washing it with 95% ethanol until the eluant contained no polyphenols. Polyphenols were detected by the Ferric Chloride Assay (Waterman and Mole 1994), in which a drop of the yellow Ferric Chloride reagent was mixed with a drop of eluant, immediately resulting in a blue color in the presence of polyphenols. Larger polymeric polyphenols (= tannins) were subsequently eluted with 70% acetone until the eluent tested negative with ferric chloride, indicating an absence of polyphenols, irrespective of their composition. Acetone was removed from the filtrate by evaporation under reduced pressure at 35 oC, and the extract was freeze-dried and stored at -20 oC.

To extract tannins from the individual leaf samples, 0.02 g freeze-dried and ground plant tissue was washed 3X with 1 mL ether to remove pigments and other interfering substances. Tannins were then extracted from the tissue 3X with 0.275 mL 70:30 acetone (containing 1mM ascorbate to prevent oxidation) by sonication on ice for 10 min. The acetone was removed from the combined supernatants by evaporation under reduced pressure at 35 oC and the extracts were brought to 1 mL volume with DD water and refrigerated.

Extracts were analyzed for “total phenolics” by the Folin Assay which measures the ability of phenolics to reduce a mixture of phosphomolybdic and phosphotungstic acids. The data from all assays are reported as average and standard deviations of triplicate measurements of single extracts of each sample, relative to the standard used (Waterman and Mole 1994).

In a microtitre plate adaptation of the Folin Assay (Appel et al 2001), serial dilutions were made of each bulk standard to provide final concentrations of dry wt per mL of DD water of 2, 8, 14, and 20 mg/mL, and extracts were diluted as necessary to reside on the standard curve. Reactions were carried out in a microtitre plate with the following volumes: 0.067 mL sample or standard, 0.067 mL Folin Assay Reagent (1.76 g lithium sulfate + 10mL concentrated Folin reagent + 10 mL DD water) + 0.067 mL 2N sodium carbonate. After a 1-hour incubation at room temperature, absorbance at 725nm was determined with a microplate reader (SpectraMax, Molecular Devices).

Soluble Protein Assays

The amount of available foliar protein was measured at the Appel/Schultz laboratory using the same dried leaf samples collected for the tannin analyses. Soluble protein was extracted from 0.003 – 0.004 g freeze-dried ground leaf material by incubation at 104oC in 1.5 mL 0.1N NaOH for 2 hr in 2mL microfuge tubes with a perforated lid. After a 10 min cooling period, extracts are analyzed for soluble protein using the BioRad assay (micromethod). Serial dilutions were made of bovine serum albumin to provide final concentrations of 0.00025, 0.0005, 0.0010, 0.005, 0.010 and 0.025 mg/mL. The reaction contains 0.2 mL of standards or 0.01 mL of extract in 0.190 mL DD water and 0.05 mL Bio-Rad dye reagent in a microtitre plate incubated for a minimum of 5 min and a maximum of 1 hr before absorbance at 595 nm is determined with a microplate reader.

TABLE A1: Retention times and list of identified compounds from a survey of the species in the experiment using the GCMS. Chemicals in bold were confirmed with industrial standards.

Retention Time (GCMS)

NIST-identified Compounds

6–9 minutes

Monoterpenes (C10H16): alpha-pinene, limonene, bicyclohexane, alpha-phellandrene, beta-myrcene, bicyclohexene

10–18 minutes

Sesquiterpenes (C15H24): germacrene D, alpha-cubebene, ylangene, caryophyllene, alpha-caryophyllene, germacrene B, copaene, cyclomenthene

19–22 minutes

Diterpenes and Alkanes (C20H38, C20H40O, C14H28O): 9-eicosyne, 3,7,11 tetramethanol, hexadecanol, pentadecanol, oxirane



Appel, H. M., H. Govenor, D’Ascenzo, E. Siska, and J. C. Schultz. 2001. Limitations of folin assays of foliar phenolics in ecological studies. Journal of Chemical Ecology 27:761–778.

Hagerman, A. E., and K. M. Klucher. 1986. Tannin-protein interactions. Pages 67–76 in V. Cody, E. Middleton, and J. Harborne, editors. Plant flavonoids in biology and medicine: biochemical, pharmacological and structure activity relationships. Alan R. Liss, New York, New York, USA.

Julkunen-Tiitto, R., and S. Sorsa. 2001. Testing the effects of drying methods on willow flavonoids, tannins and salicylates. Journal of Chemical Ecology 27:779–789.

Waterman, P. G., and S. Mole. 1994. Analysis of phenolic plant metabolites. Blackwell Scientific, London, UK.

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