Hydrolysis, derivatization and analyses of samples for CSIA –  For Exps. 3-4, samples (1-2 mg dry weight) for CSIA-AA were added to 1 ml 6 N HPLC-grade HCl, then flushed with N2, capped with a Teflon-lined cap, and hydrolyzed at 150°C for 70 min.  The resulting hydrolysate was evaporated to dryness under N2 at 55°C, redissolved in 1 ml 0.01 N HCl, purified by filtration (0.45-μm hydrophilic filter), washed with 1 ml of 0.01 N HCl, and further purified using cation-exchange chromatography with a 5-cm resin column (Dowex 50WX8-400) in a glass Pasteur pipette.  AAs were eluted with 4 ml of 2 N NH4OH and evaporated to dryness under a stream of N2 at 80°C, then reacidified with 0.5 ml of 0.2 N HCl, flushed with N2, heated to 110°C for 5 min and evaporated to dryness under N2 at 55°C.  Hydrolyzed samples were esterified with 2 ml of 4:1 isopropanol:acetyl chloride, flushed with N2 and heated to 110°C for 60 min.  After drying at 60°C under N2, the samples were acylated by adding 1 ml of 3:1 methylene chloride:trifluoracetic anhydride (TFAA) and heated to 100°C for 15 min.  The derivatized AAs were further purified by solvent extraction following (Ueda et al. 1989).  The acylated AA esters were evaporated at room temperature under N2 and redissolved in 3 ml of 1:2 chloroform:P-buffer (KH2PO4 + Na2HPO4 in Milli-Q water, pH 7).  Vigorous shaking ensured that the derivitized AAs were partitioned into chloroform while contaminants remained in the P-buffer.  The solvents were separated by centrifugation (10 min at 600 g), the chloroform was transferred to a clean vial, and the solvent extraction process repeated.  Finally, to ensure derivatization, the acylation step was repeated.  Samples were stored at –20°C in 3:1 methylene chloride:TFAA for up to 2 weeks until analysis.  AA derivatives were then analyzed by isotope monitoring gas chromatography-mass spectrometry.  We used a Delta V Plus mass spectrometer interfaced with a Trace GC gas chromatograph through a GC-C III combustion furnace (980°C), reduction furnace (650°C), and liquid nitrogen cold trap.  Internal reference compounds (aminoadipic acid and norleucine) of known nitrogen isotopic composition were co-injected with samples and used to normalize the measured δ15N values of unknown AAs, and a suite of eight AAs with known isotopic composition was analyzed every 3 injections for additional quality control.  At least three injections per sample were analyzed.  For Exps. 5 and 6, CSIA-AA samples were hydrolyzed in 6N HCl at 110°C for 20 h.  The hydrolysate was evaporated to dryness at temperature under a continuous stream of N2 gas, then esterified with 4:1 isotopropanol:acetyl chloride at 110°C for 60 min, acylated in a 1:1 solution of methylene chloride:trifluoracetic anhydride (DCM:TFAA) for 10 min at 110°C.  Samples were stored at -20°C in 1:1 DCM:TFAA for up to 3 months before isotope analysis using a Delta V Plus mass spectrometer (Thermo Scientific) interfaced through a Conflo IV to a GC 1310 gas chromatograph coupled to a GC Isolink combustion-reduction furnace (1000°C) and liquid nitrogen cold trap.  All samples were injected (splitless injector) onto a forte BPx5 capillary column (60 m x 0.32 mm x 1.0-μm film thickness) at an injector temperature of 250°C with a constant helium flow rate of 1.4 ml min–1.  The column was initially held at 50°C for 2 min and then increased to 125 °C at a rate of 15°C min-1.  Once at 125°C, the temperature was increased at a rate of 3°C min-1 to 160°C and then to 190°C at a rate of 4°C min-1.  The final temperature of 300°C was reached by ramping to 275°C at 6°C min-1 and then 15°C min-1 afterward.  Samples were analyzed in triplicate and normalized to the known δ15N values of a suite of 14 AAs analyzed before and after each set of 3 samples.