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).