Water uptake patterns
On each sampling date, in the monospecific and four-species mixtures
(n=15 plots), we collected three 10 cm-long twig samples across the
canopy from each tree between 9 am and 3 pm (local time). After removing
the bark, the samples were immediately sealed in airtight vials
(Exetainer, Labco Limited, UK). The vial lid was wrapped with parafilm
and placed in cool conditions to avoid evaporation. On the same day as
the twig sampling, soil samples were collected every 10 cm at four
depths (0–10, 10–20, 20–30, and 30-40 cm) and three random positions
in each plot, using a manual soil corer or/and a pickaxe when the soil
was too rocky. The soil samples were immediately placed in vials and
stored like twigs. As the depth of the limestone bedrock varied within
and between plots (between 20 and 70 cm; Table S1), the maximum depth of
soil sample collection varied by date, plot, and position. Nevertheless,
we could extract all the soil layers from 0 to 40 cm in every plot at
each campaign, except for the Q.ilex monocultures in spring 2021,
due to technical limitations. Precipitation water (used as a proxy of
the groundwater isotopic values, see below) was collected by two
Tube-dip-in-water collector types with pressure equilibration (RS1,
Palmex, HR), spread into the study area (Fig. S1). Due to the unique
design of the rain samplers avoiding evaporation for up to one year
(Gröning et al., 2012), we collected the rainwater every two months
during the growing season and once during the winter each year. The
water was placed in vials and sealed with a lid and parafilm.
Water from xylem and soil samples was extracted using a custom-made
cryogenic vacuum distillation system at the Swiss Federal Institute for
Forest, Snow, and Landscape Research (WSL, Birmensdorf, CH) (Diao et
al., 2022). The extraction system consisted of 20 tubes connected to 20
U-shaped collection tubes specifically designed for this system. A
frozen sample was placed in the extraction tube and submerged in water
at 80°C, while the associated collection tube was submerged in liquid
nitrogen. The system was then evacuated to 5.10-2mBar. The extraction was maintained for 2 h for both xylem and soil
samples to achieve a complete extraction following the recommendations
of West et al. (2006) (i.e., a minimum of 60 min extraction time for a
broad range of plant and soil materials). This process led to an
extraction of 99.96% of the water in the samples, with more than 1 mL
extracted for each sample, limiting the uncertainties in plant water
isotopic composition due to cryogenic vacuum distillation (Diao et al.,
2022). After the extraction, water samples were transferred into
cap-crimp 2-ml vials and stored at −20 ◦C until the
isotopic analysis. Uncertainties associated with bulk water extractions
using cryogenic distillation systems could occur that would
underestimate the contributions of soil water and overestimate the ones
from groundwater (Barbeta et al., 2021). Yet, as all samples were
treated similarly, the errors would only affect the actual values, not
the comparison between mixtures and seasons.
The δ2H and δ18O of all water
samples (i.e., twig, soil, and precipitation) were measured with a
high-temperature conversion elemental analyzer coupled to a DeltaPlus XP
isotope ratio mass spectrometer (TC/EA-IRMS; Thermo, DE). Isotope ratios
were reported in per mil (‰) relative to Vienna Standard Mean Ocean
Water (VSMOW). Calibration versus the international standards was
achieved by analysis of a range of certified water of different isotope
ratios, resulting in a precision of 2‰ for δ2H and
0.3‰ for δ18O.