Carbon isotope composition (δ13C)
Six shoots with leaves were collected for carbon isotope analysis from both sides of the sun-exposed canopy for three trees per replication at three different times from June to August in 2018 and 2019. Means were calculated for all sampling times during the season. Leaves and stems were separated and placed in a chamber with constant air flow to dry at 25 °C for at least 30 days. Dry leaves and stems were well-mixed, and 2 g subsamples were ground to a fine powder using a VWR Homogenizer (VWR, Radnor, PA). 3 mg samples for leaves and 4 mg samples for stems were weighed using a high-precision analytical balance (XSE105 DualRange, Metler Toledo, Greifensee, Switzerland) into 5mm x 9mm tin capsules (Costech Analystycal Technologies, Inc., Valencia, CA, USA). Prepared capsules were shipped for analysis to the Stable Isotope Core Laboratory at Washington State University. Samples were analyzed using an elemental analyzer (ECS 4010, Costech Analytical, Valencia, CA, USA) coupled with a continuous flow isotope ratio mass spectrometer (Delta PlusXP Thermofinnigan, Bremen). Carbon isotope ratios were determined using the standard Vienna PeeDee belemnite (VPDB) and the values reported in “delta” notations as δ values in permil (‰) (Sharp, 2005) as described in Equation 2 and 3:
Equation 2
\(\delta=\left(\left(\frac{\text{R\ sample\ \ }}{\text{R\ standard\ }}\right)\ -1\right)\ X\ 1000\)
Equation 3
\begin{equation} \delta^{13}C=\left(\left(\frac{\left(\frac{{}^{13}C}{{}^{12}C}\right)\text{\ sample}}{\left(\frac{{}^{13}C}{{}^{12}C}\right)\text{standard}}\right)\ -1\right)\ X\ 1000\nonumber \\ \end{equation}
where, the ratio of the heavy (13C) over the lighter (12C) isotope was calculated for the sample and then was divided by the 13C/12C ratio of the standard (PeeDee Belemnite).