All stem samples were collected before dawn. For each tree, a large
branch (\($\approx$\)0.8 to \($\approx$\)2 m length;
\($\approx$\)10 to \($\approx$\)25 mm base diameter) was removed
from the canopy using a telescopic pruner. The cut edge of each branch
was immediately sealed with parafilm and electric tape, and sealed
branches were transported to our base (an air-conditioned, darkened
room) within 30 minutes of cutting. Upon arrival, leaves were cut with a
sharp razor blade for pre-dawn leaf water potential (LWP) measurements
(see description below). Two separate stems from the same branch were
then cut, sealed at both ends with parafilm, wrapped in cling wrap, and
double Ziploc bagged. Stems were subsequently stored at 4°C for later
water extraction through CVD and Cavitron centrifugation methods. The
sampled stems typically ranged from 210 to 260 mm in length and 6 to 13
mm in diameter.
We used a Model-1000 pressure chamber (PMS Instrument Company, United
States) with nitrogen gas and a mounted eye lens for the LWP
measurements. Two replicate measurements were made for each branch
sample, plus a third measurement when the difference between the first
two measurements was > 10%. All LWP measurements were
finalised within two hours of sampling. For each sample we report the
average of the two to three measurements.
Source water sampling
To characterise the spectrum of isotopic compositions of potential
source waters, we sampled both groundwater and soil water at various
locations and depths within and around Elsey National Park. We obtained
groundwater from five observation bores and soil water from six soil
cores. For groundwater, we used a submersible pump (Tornado, Proactive,
USA) except at one site where we used a pre-installed solar-powered
pump, and collected samples once three bore volumes had been purged
and/or once pH and conductivity measurements had stabilised. For soil
samples, we used a hand auger at five sites to extract shallow soil
(maximum depth of 2.0 m), while at one site, we used a small drill rig
to extract deeper soil horizons (maximum depth of 5.4 m). We collected a
total of 36 soil samples at depths ranging from 0.1 to 5.4 m. Each
sample comprised approximately 100 to 300g of soil material, was sealed
in double Ziplock bags with minimised headspace, and kept at 4°C until
further analysis. Additionally, a local meteoric water line was obtained
from 18 rainfall samples collected on site between 2019 and 2021
(Lamontagne et al., in prep.).
Cavitron extractions
To extract xylem water from stem samples, we used a standard 270-mm
diameter Cavitron manufactured by DG-Meca (France) and fitted to an
ultracentrifuge (Avanti J-E, Beckman Coulter, United States) at Charles
Darwin University. We broadly followed the method outlined in Barbeta et
al. (2022). Briefly, small plastic containers were inserted into each
end of the stem and sealed with parafilm to collect xylem water. Samples
were spun for two minutes at speeds ranging from 3,000 to 9,000 rpm,
corresponding to xylem pressures of –0.57 to –6.40 MPa based on stem
length (Alder et al., 1997; Cochard, 2002). Extracted water was then
collected from the containers using a micropipette, filtered through
0.45 μm and stored in 2 mL glass vials fitted with 0.1 mL micro-inserts.
Samples were then preserved at 4°C until analysis. Stem samples were
weighed before and after centrifugation, oven-dried at 105°C for 24h
after centrifugation and reweighed to determine the relative stem water
content (RSWC). A more detailed description of our operating procedure
is provided in the Supplementary Information.
Cryogenic extractions
Bulk stem water and soil water were extracted at the West Australian
Biogeochemistry Centre (WABC), University of Western Australia,
following the CVD procedure outlined in West et al. (2006). Samples were
fully frozen using liquid nitrogen, after which they were subjected to a
vacuum with pressure < 10 Pa. Frozen samples were then heated
under vacuum conditions, causing water vapour to be collected in a
liquid nitrogen cold trap. Extraction times were set at 60 and 90 min
for soil and stem samples, respectively, aligning with the
recommendations of West et al. (2006). To ensure the quality and
accuracy of the extraction process, water was also extracted from four
different standards using the same procedure.
Isotopic analyses
All extracted water samples were analysed for oxygen and hydrogen
isotopic ratios (δ18O and δD) at the WABC using a
cavity ring-down spectrometer (Picarro Inc., model L2130-I) fitted with
a micro-combustion module to remove organic compounds that may be
present in extracted water. The raw isotopic values are expressed
relative to VSMOW and are reported in per mil (\($\textperthousand$\)). According to analyses
on replicate stem samples, overall precision for the CVD extraction and
measurement procedure was ±0.5\($\textperthousand$\) and ±3.0\($\textperthousand$\) for δ18O and
δD, respectively. Cavitron-extracted xylem water and groundwater samples
had a precision of ±0.1\($\textperthousand$\) and ±0.5\($\textperthousand$\) for δ18O and δD,
respectively.
Data analyses
Statistical analyses and plotting were conducted using MATLAB R2022a. We
define deuterium bias as the difference between δD of CVD-derived
bulk stem water and that of Cavitron-derived xylem water of the same
branch:
δDbias = δDbulk –
δDxylem (1)
We define deuterium offset as the difference between δD of xylem
or bulk stem water and their expected δD based on the source water line:
δDoffset = δDxylem or bulk –
δDsource (2)
with δDsource = αswl *
δ18Oxylem or bulk +
βswl (3)
where αswl and βswl are the slope and
intercept of the source water line, respectively.
To test whether the means of the δD offsets of xylem and bulk stem water
were significantly different from zero, we used a one-sample t-test (MATLAB function ttest). To test whether the
difference between the δD offsets of xylem and bulk stem water was
significant, we used the non-parametric two-sample Kolmogorov-Smirnov
test (MATLAB function kstest2). Unlike other tests (e.g. paired t-test) that only compare the means of each group, this test
evaluates the entire distribution of each group. To test cross-species
differences in mean δD bias, we used a Kruskal-Wallis test (MATLAB
function kruskalwallis) followed by a Dunn-Sidák post-hoc test
for pairwise comparison (MATLAB function multcompare).
To test the potential effect of tree species, RSWC, pre-dawn LWP and
xylem water isotopic composition on the CVD-induced δD bias, we used
linear mixed-effects models (MATLAB function fitlme). We used
species, pre-dawn LWP, δD of xylem water and RSWC as predictor variables
and δD bias as the response variable. In addition, we included sampling
site as a random effect in the mixed-effects model.