2.2. Sample collections and isotope analysis
We collected the samples, including the plant, soil, precipitation and
groundwater. All the samples were frozen at -10°C prior to vacuum
distillation, for consistent handling while avoiding the effects of
fractionation. In addition, our preliminary experimental results showed
that the isotope ratio spectrometer had more stable operating state
after extraction of precipitation and groundwater.
The plant and soil samples were collected from the shrub and grassland
sites during the re-green season, growing season, and withering season
in 2021 (20 May, 15 June, 16 July, 16 August, 7 September, and 12
October). Plant species were randomly chosen on the sampling date in the
two sites. The whole plants of herbs were used for experiments after
removing leaves. Whereas, the stem sections were removing the outer bark
from P. fruticosa were used for isotope analysis (Martín-Gómez et
al., 2017). The soil samples were collected around the selected plants
within 2 m by soil cores, the soil cores were divided into 10 layers
(0–5 cm, 5–10 cm, 10–15 cm, 15–20 cm, 20–30 cm, 30–40 cm, 40–50
cm, 50–60 cm, 60–80 cm, and 80–100 cm), each soil samples were
analyzed as separate samples. Three
replicates for each of the soil
layers and plants samples were collected in the two experimental sites.
A total number of 72 and 360 samples of plants and soils, respectively,
were sealed for isotopic analysis.
Precipitation and groundwater were sampled concurrently from May to
October in 2021. The event-based precipitations were sampled using
bottles at herder’s home, 2 km away from our sampling sites during the
sampling period, the number of replicates (one, two or three) from every
event-based precipitation were determined according to the amount of
precipitation. Groundwater were sampled once a week from a well 2 km
away from our sites, three
replicates were collected. The
well had a depth of 2 m, and was commonly used for groundwater
monitoring. A total of 162 and 72 samples from precipitation and
groundwater, respectively, were all transferred into clean polyethylene
bottles.
The soil water content (SWC) was determined by an automatic soil
moisture monitoring system (CR800; Campbell, USA) with sensors installed
at depths of 5 cm, 10 cm, 15 cm, 20 cm, 30 cm, 40 cm, 50 cm, 60 cm, 80
cm, and 100 cm below the soil surface. Precipitation measurements were
collected using a precipitation gauge (52,203, RM Young, USA) at a
height of 0.5 m. Temperatures were obtained from a meteorological
station (Molis 520; Vaisala, Finland). All data were recorded every 30
min.
2.2.2. Isotopic analysis
Precipitation, groundwater, plant water, and soil water samples were all
extracted by the cryogenic vacuum distillation technique (LI-2100pro,
Lica United Technology Limited, Beijing, China) in order to avoid the
influence of high salinity moisture on the accuracy of the instrument.
After all samples were equilibrated to room temperature, extraction was
started, setting 3 h for plant water and soil water, 2 h for
precipitation and groundwater samples according to West et al. (2006).
All of the extracted water was transferred into 2 ml vials, then
analyzed stable isotopes (δ2H and
δ18O) using an isotope ratio spectrometer
(LGR-TLWIA-912, Los Gatos Research, San Jose, CA, USA). The instrument
was equipped with an autosampler (PAL-LSI) for sample injection, and
post-processing software (LWIA Post Analysis Full Installer v4.4.1) for
test diagnosis, checking, and quantifying problems in the analysis
(e.g., interference from organic pollutants, injection volume error)
through detailed analysis of high-resolution absorption spectra.
The measurement precision was
0.3‰ for δ2H and 0.1‰ for δ18O. The
organic contamination on plant water need correction procedures to
eliminate the influence (Schultz et al., 2011). The isotopic
compositions of δ18O and δ2H were
expressed as an isotope ratio:
\begin{equation}
\delta sam(\%0)=\left(\frac{R\text{sam}}{R\text{std}}-1\right)\times 1000\%0\nonumber \\
\end{equation}where δsam was the
isotopic difference, Rsam was the abundance ratios
(18O/16O and2H/1H) of samples, Rstd was the
abundance ratios of standard.
2.2.3. Statistical analysis
All statistical analyses were conducted using R (software version 4.0.3,
https://www.r-project.org/), and all figures were plotted using Origin
9.1(https://www.originlab.com/). One-way analysis of variance
(ANOVA) followed by the post hoc Turkey’s test at p = 0.05 was used to
assess hydrometeorological parameters of sampling date and sites.
Two-way ANOVA was performed to examine the significant effects of
hydrometeorological parameters and their interactions. Pearson’s
correlations were tested at the p = 0.05 level.
The Bayesian mixing model Mix SIAR (http://conserver.iugo-cafe.org/user/
brice.semmens/Mix SIAR) was used for identifying the proportions of
contributions from each water source according to the
δ2H and δ18O, which were considered
as the mixture data of the potential water sources (Dawson&Ehleringer,
1993; Beyer et al., 2018). The inputs of original data (for example, the
0-20 cm soil layer data was input as 0-10 cm and 10-20 cm isotope data),
the discrimination data (the TDF data in the model), the running time of
Markov Chain Monte Carlo (MCMC), and the diagnosis method of the model
results were according to (Zhou et al., 2021). The average value was
output from the model. Three potential soil water sources were
identified to facilitate subsequent analysis (i.e., shallow soil water
(0–20 cm), middle soil water (20–60 cm), and deep soil water (60-100
cm)), according to the variability in the soil water content and the
impacts of precipitation pulse.
3. Results