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Article

Responses of Water Use Strategies to Seasonal Drought Stress Differed Among Eucalyptus urophylla S.T.Blake × E. grandis Plantations Along with Stand Ages

by
Zhichao Wang
1,2,3,
Yuxing Xu
2,3,
Wankuan Zhu
2,3,
Runxia Huang
2,3,
Apeng Du
2,3,
Haoyang Cao
2,3 and
Wenhua Xiang
1,*
1
College of Ecology and Environment, Central South University of Forestry and Technology, Changsha 410004, China
2
Research Institute of Fast-Growing Trees (RIFT), Chinese Academy of Forestry (CAF), Zhanjiang 524022, China
3
Guangdong Zhanjiang Eucalyptus Plantation Ecosystem Research Station, Zhanjiang 524022, China
*
Author to whom correspondence should be addressed.
Forests 2025, 16(6), 962; https://doi.org/10.3390/f16060962
Submission received: 28 April 2025 / Revised: 26 May 2025 / Accepted: 5 June 2025 / Published: 6 June 2025
(This article belongs to the Special Issue Advances in Forest Carbon, Water Use and Growth Under Climate Change)
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Abstract

Water use strategies reflect the ability of plants to adapt to drought caused by climate change. However, how these strategies change with stand development and seasonal drought is not fully understood. This study used stable isotope techniques (δD, δ18O, and δ13C) combined with the MixSIAR model to quantify the seasonal changes in water use sources and water use efficiency (WUE) of Eucalyptus urophylla S.T.Blake × E. grandis (E. urophylla × E. grandis) at four stand ages (2-, 4-, 9- and 14-year-old) and to identify their influencing factors. Our results showed that the young (2-year-old) and middle-aged (4-year-old) stands primarily relied on shallow soil water throughout the growing season due to the limitations of a shallow root system. In contrast, the mature (9-year-old) and overmature (14-year-old) stands, influenced by the synergistic effects of larger and deeper root systems and relative extractable water (REW), exhibited more flexibility in water use, mainly relying on shallow soil water in wet months, but shifting to using middle and deep soil layer water in dry months, and quickly returning to mainly using shallow soil water in the episodic wet month of the dry season. The WUE of E. urophylla × E. grandis was affected by the combined effect of air temperature (T), vapor pressure deficit (VPD), and REW. WUE was consistent across the stand ages in the wet season but decreased significantly with stand age in the dry season. This suggests that mature and overmature stands depend more on shifting their water source, while young and middle-aged stands rely more on enhanced WUE to cope with seasonal drought stress, resulting in young and middle-aged stands being more vulnerable to drought stress. These findings offer valuable insights for managing water resources in eucalyptus plantations, particularly as drought frequency and intensity continue to rise.

1. Introduction

Water is an important resource that affects plant growth, development, and distribution [1]. Global climate change has modified precipitation patterns [2,3,4] and increased the frequency, intensity, and duration of droughts in seasonally dry regions [5]. Plants can alter their water use strategies in response to drought and severe soil water deficits [6,7,8,9]. Understanding how a tree species changes its water use strategies in response to drought is important for predicting the future stability of forest ecosystems and precisely designing appropriate water management strategies [9].
Water use strategies of plants involve where water is acquired or absorbed (water sources) and how the acquired water is used (water use efficiency, WUE). The development of stable hydrogen and oxygen isotope tracer techniques has made it possible to identify and quantify the source of water used by plants and has been used extensively [10,11]. Previous studies have found that water sources used by plants are related to plant species [12,13], soil moisture availability [14,15], root distribution, and the interactions between these factors [16,17]. Shallow-rooted plants (such as herbs and some shrubs) usually use unstable surface soil water [18,19], while deep-rooted plants have two water use strategies depending on the soil moisture: one is to continuously use relatively stable deep soil water or/and groundwater regardless of changes in soil moisture [20,21], and the other is to use shallow soil water during the wet season and switch to absorbing stable deep soil water or/and groundwater during the dry season [14,22]. How plants use water also directly determines their ability to withstand drought [12,23]. In general, plants that obtain water primarily from shallow soils are more vulnerable to drought than plants that can utilize deep soil water or groundwater [20,24]. Currently, despite extensive studies of plant water use sources in different ecosystems at regional and global scales, fewer studies have examined how plant water use sources vary with plant development, and how water use source strategies differ among plants at different developmental stages in response to drought stress.
Plants of different stand ages or developmental stages (e.g., young, middle-aged, mature, and overmature stands) may use different water sources because of variations in root distribution and water demand. It was reported that the seedlings of Haloxylon ammodendron (C. A. Mey.) Bunge in the Gurbantunggut Desert used shallow soil water (0–40 cm) but mature trees switched to using deep soil water (>2 m) [6]. The 4- and 9-year-old Caragana intermedia Kuang et H. C. Fu in alpine sandy land mainly use shallow soil water (0–40 cm), 17-year-old trees mainly use middle soil water (40–90 cm), while 31-year-old trees mainly use deep soil water (90–150 cm) [25]. However, there are also some studies that found that stand age has no effect on plant water uptake. For example, Mongolian pines (Pinus sylvestris var. mongolica) of different ages in the Hulunbuir Sandy Land used soil water from the same depth [26]. Plants of different sizes (ages) in a tropical montane cloud forest all used shallow soil water (20–60 cm) throughout the year [27]. These conflicting results suggest that age-related plant water use source strategies may differ between plant species or environmental conditions. Therefore, in order to assess plant viability and community stability in the context of global climate change, it is necessary to elucidate the age-related water use source strategies of different plants in different regions. However, it is still unclear how age-related water use source strategies differ between dry and wet seasons in eucalyptus plantations located in specific areas with abundant annual rainfall but seasonal drought due to the uneven spatial and temporal distribution of rainfall.
Water use efficiency (WUE) is another water use strategy that reflects the strength of a plant’s water use capacity and the degree of a plant’s response to drought stress [28,29,30]. The isotope associated with WUE is 13C. Extensive studies have been conducted to examine the WUE of plants by analyzing the δ13C value in leaves, concluding that more negative δ13C values indicate lower WUE [31,32]. The WUE of plants is influenced by their own biology [33] and varies with environmental factors, particularly soil moisture availability [9,34,35]. However, with decreasing soil moisture availability, the WUE of different plants has shown different trends, increasing [36,37], decreasing [38], or unchanged [39], suggesting that different plant species have different WUE strategies to adapt to drought. In addition, plants undergo changes in physiological conditions and in the uptake and use of external substances during stand development [40], which may result in differences in WUE between plants of different ages, as well as different responses to drought stress. For example, WUE increased with stand age in Pacific Northwest Douglas fir (Pseudotsuga menziesii (Mirb.) Franco) stands [41], but the opposite was true for temperate pine plantation forests in southern Ontario, ON, Canada [42]. Trees of 5-year-old Salix cheilophila C.K.Schneid have a low ability to use stable groundwater, resulting in higher WUE than 9-year-old and 25-year-old trees [43]. WUE in young pine forests in semi-arid regions of the USA is more sensitive to changes in soil moisture than that in mature pine forests due to their shallow root systems [37]. The differences in the WUE of plants at different ages and their different response patterns to drought stress may lead to large differences in their survival and competitiveness under future climate change [44]. However, to date, the understanding of age-related WUE and their pattern and intensity responses to drought stress in eucalyptus plantations in China is still unsystematic and incomplete, which hinders the efficient and sustainable development of China’s eucalyptus industry in the context of increasing drought.
Eucalyptus is a fast-growing and high-yielding forest species introduced to southern China, where the plantation area has reached 5.47 million hectares with typical rotation lengths of 4–7 years for pulpwood and 12–15 years for sawtimber [45]. Its high stand productivity (10~30 m3·ha−1·yr−1) is related to China’s wood supply security. However, there are generally seasonal drought periods every year (from November to April of the following year, a total of six months) in eucalyptus plantation areas. This, coupled with the increasing severity of seasonal drought due to global climate change [5], will make the growth of eucalyptus trees more severely restricted by water stress [46,47], leading to reduced productivity and even tree mortality [48,49]. Therefore, more knowledge on the changes in water sources and WUE and the response patterns to drought stress in eucalyptus plantations at different stages of development is urgently needed to develop management practices for efficient water use and sustainable operations. In the present study, we used Eucalyptus urophylla S.T.Blake × E. grandis (E. urophylla × E. grandis), the most representative eucalyptus species with the largest planted area in China, as the target species to quantify water use sources of E. urophylla × E. grandis at different stand ages using the stable isotopes D and 18O in combination with the MixSIAR model, and investigated the WUE of E. urophylla × E. grandis at different stand ages using the 13C technique, with a focus on examining the changes in water use strategies for adapting to seasonal drought. The objectives of this study were to (i) quantify the sources of water use and differences in their response to drought stress in E. urophylla × E. grandis at different stand ages, (ii) identify the changes in WUE with stand age and differences in the pattern and intensity of WUE responses to drought stress in E. urophylla × E. grandis at different stand ages, (iii) explore the factors influencing the water use strategies of E. urophylla × E. grandis at different stand ages. The results could provide valuable insights into the sustainable management of eucalyptus plantations facing increasing seasonal drought.

2. Materials and Methods

2.1. Study Site Description

This study was carried out at the Zhanjiang Eucalyptus Plantation Ecosystem Research Station (21°16′ N, 110°05′ E) on Leizhou Peninsula, Guangdong province, China (Figure 1a). The terrain is mainly flat, with elevations ranging from 80 m to 220.8 m above sea level. The soil is classified as Rhodi-Udic Ferralosols [50], exhibiting a texture ranging from loam to clay loam, with a mean pH of 5.51 at the soil depth of 0–200 cm. The physico-chemical properties within 200 cm soil profiles are shown in Table A1, which demonstrates that as the depth decreases (i.e., closer to the surface), the nutrient content and capillary porosity increase, bulk density decreases, and water holding capacity improves. This area has a maritime monsoon climate. According to the long-term meteorological records (1980–2020) in this area, the mean annual temperature was 23.1 °C, with an extreme minimum of 1.4 °C in January and an extreme maximum of 38.1 °C in July. The mean annual precipitation was 1760.9 mm, of which 77%–85% occurred in the wet season (May–October) [51]. Isotopic enrichment is present in rainfall in the study area and is more pronounced in the dry season than in the wet season (Figure 2a,b). In addition, the groundwater level in the study area exceeded 40 m below the surface, and cannot provide a water source for shallow-rooted plants such as E. urophylla × E. grandis.
During the study period (from January 2021 to April 2022), the daily temperature ranged from 5.8 to 31.3 °C, with an average of 22.4 °C, and the total precipitation was 1531.5 mm. The mean soil water content at 0–100 cm depth at the weather station varied from 21.58% to 33.65% (Figure 1b). During this period, June to August was the wettest months of the wet season, shortened to the “wet months”, with an average monthly rainfall of 173.9 mm. December to January was the driest month of the dry season, shortened to the “dry months”, with an average monthly rainfall of only 30.2 mm. February is usually the driest month of the dry season, but an anomalous rainfall of 111.6 mm was observed in this month (Figure 1b). We refer to this anomalous dry season month as the “wet month of the dry season”. The occurrence of this unexpected period allows us to study water use strategies of eucalyptus in episodic wet periods in the dry season. Therefore, we set the isotope sampling times to these six months (Figure 1b).

2.2. Experimental Design and Stand Characteristics Measurement

E. urophylla × E. grandis plantations at four stand ages (2, 4, 9, and 14 years) were selected to investigate their water use strategy (Figure 1c). The selection of this range of stand ages covers the four growth stages of eucalyptus plantations: young stand, middle-aged stand, mature stand, and overmature stand. The E. urophylla × E. grandis plantations were established on flat land by digging holes and using cloned seedlings of DH32-29. The distance between any two forests was less than 1 km. Differences between the sites before planting was very minor, and the same management regimes were implemented in the four forests. Three plots, each with an area of 20 m × 20 m, were randomly established for each stand age to investigate stand characteristics (Table 1).
The Leaf Area Index (LAI) was measured using a digital plant canopy analysis system (LI-2200C, LI-COR, Lincoln, NE, USA). In the middle of each month, more than 20 points were randomly selected in each experimental plantation for LAI measurements. To avoid the influence of rapid changes in the light environment on the measurement results, early morning or late evening periods with uniform sky scatter was chosen.
In addition, we used a manual excavation method to determine root distribution within a soil depth of 2 m in E. urophylla × E. grandis plantations at four stand ages. Nine sample trees of each stand age were selected for excavation according to average diameter at breast height (DBH), and all fine roots (d < 2 mm) were collected in layers at 10 cm intervals. The collected root samples were washed with water and then were dried to constant weight in an electro-thermostatic blast oven (DHG-9036A, Jinghong, Shanghai, China).

2.3. Sample Collection

To reveal the age-related water use strategy of E. urophylla × E. grandis in response to drought stress, 3 standard trees were selected in each plot (9 trees per stand age), and samples were collected for stable isotope measurements, to minimize the effects of individual plants on the experimental results [52]. Xylem, leaf, and soil samples were collected monthly from June to August in 2021 (wet months), from December 2021 to January 2022 (dry months), and in February 2022 (wet month of the dry season) (Figure 1b). Meanwhile, rainwater samples were collected and separate sampling records kept for all rainfall exceeding 5 mm at any time during the entire monitoring period. The sampling procedures were as follows:
(1)
Plant xylem samples: On each sampling date, to avoid isotopic enrichment due to stomatal transpiration, healthy non-green and corked branches were selected from the canopy of each standard tree [53]. A branch segment of approximately 0.3–0.5 cm in diameter and 3–5 cm in length were collected on a sunny morning (9:00–12:00), immediately stripped of the phloem, and placed in a brown screw top sampling bottle [52]. The bottle was then sealed with parafilm, quickly placed in a bucket filled with dry ice, and transported to the laboratory and stored in a freezer at −20 °C for freezing before water extraction. A total of 216 plant xylem samples were collected (9 replicates per stand × 4 stands × 6 months).
(2)
Leaf samples: On each sampling date (the same as the xylem sampling date), a sample of approximately 20 mature and healthy leaves exposed to full sunlight was collected from the upper canopy of each standard tree on a sunny morning (9:00–12:00) to avoid the canopy effects of leaf δ13C. The leaf samples were placed in a breathable sample bag and returned to the laboratory, where the samples were killed at 105 °C for 20 min, dried at 70 °C for 48 h, then ground at a low temperature in liquid nitrogen, passed through an 80-mesh sieve, sealed, and stored at room temperature. A total of 216 leaf samples were collected (9 replicates per stand × 4 stands × 6 months). The δ13C content of the sampled leaves was measured for long-term WUE analysis.
(3)
Soil samples: Soil samples were collected along with the xylem samples on each sampling date. A hole to expose the soil profile was excavated around three standard trees in each plot. Soil samples were collected at depths of 0.1 m, 0.2 m, 0.3 m, 0.4 m, 0.6 m, 0.8 m, 1.0 m, 1.5 m, and 2 m along the soil profile. To avoid the effects of evaporation on the isotopic composition of the soil, approximately 5.0 cm of soil outside of the profile was removed for each layer sampled. Each collected sample was promptly placed into a 12 mL brown sampling bottle with a screw top, sealed with parafilm, immediately placed into a dry ice bucket, and transported to the laboratory [52]. Soil samples were frozen in a freezer at −20 °C prior to moisture extraction. A total of 648 soil samples were collected (9 depth intervals × 3 replicates per stand × 4 stands × 6 months). Meanwhile, the same number of soil samples were collected using ring knives (inner diameter = 5.1 cm, height = 5.0 cm) and sealed bags from each soil layer to determine soil water content (SWC) and physico-chemical properties.
(4)
Rainwater samples: All precipitation in the study region falls as rain. We collected rainwater samples using the rainwater sampler recommended by the International Atomic Energy Agency’s Global Network of Isotopes in Precipitation. To minimize the effects of evaporation, rainwater samples were collected immediately after rainfall events. About 2 mL of the sample (or whole sample if <2 mL) was removed from the collection bottle and transferred to a brown sample bottle with a screw cap. The bottle was sealed with parafilm and immediately placed in a portable incubator at 4 °C. The sample was stored in a refrigerator at 4 °C until tested. A total of 31 rainfall events were sampled during the monitoring period.

2.4. Soil Physico-Chemical Properties Analysis

Soil pH was determined by an electronic pH meter (PHS-3C, Shanghai INESA Scientiffc Instrument Co., Shanghai, China) from a solution prepared at a soil–water ratio of 1:2.5. The bulk density (B), soil total porosity (Tpo), and soil capillary porosity (Cpo) were measured using the cutting ring method, and the saturated water holding capacity (SWHC), capillary water holding capacity (CWHC), and field water holding capacity (SFHC) were determined using the drying method [54]. Soil organic matter (SOM) was quantified using the potassium dichromate-sulfate colorimetric method [55]. Total nitrogen (TN) was determined by the Kjeldahl method [56]. Available nitrogen (AN) was calculated as the sum of ammonium nitrogen (NH4-N) and nitrate nitrogen (NO3-N). NH4-N and NO3-N were determined by 2 mol⋅L−1 KCl leaching–indophenol blue colorimetry and UV spectrophotometry, respectively [57]. Total phosphorus (TP) and available phosphorus (AP) were measured with the sodium hydroxide fusion–molybdenum antimony colorimetric [58] and Mehlich 3 methods, respectively [59]. Total potassium (TK) and available potassium (AK) were obtained from the alkali fusion–flame spectrophotometry and alkali diffusion methods, respectively [57].

2.5. Meteorological Factors and Soil Water Content Measurement

Meteorological factors were continuously monitored using an automatic meteorological observation system set up in an open area near the studied forest stands. Precipitation (P; mm) was measured with a tilting rain gauge (TE525MM; Campbell, Logan, UT, USA). Air temperature (T; °C) and relative humidity (RH; %) were measured with a thermo recorder (HMP155A, Vaisala, Helsinki, Finland). All meteorological data were collected at 1 min intervals, averaged every 30 min, and recorded using a datalogger (CR3000; Campbell, Logan, UT, USA). The VPD (kPa) was calculated from Ta and RH according to the following Equation:
VPD = 0.611   ×   e 17.502   ×   Ta Ta + 240.97   ×   ( 1 − RH )
Soil samples from each soil layer collected in ring knives were weighed (M1) and then placed in an oven and dried at 105 °C ± 2 °C until a constant weight was reached (M2). Soil water content (SWC, cm3 cm−3) was calculated as follows [52]:
SWC = M 1 − M 2 M 2   ×   B i
where M1 and M2 are wet soil weight (g) and dry soil weight (g) in each soil layer, respectively, and Bi is soil bulk density (g cm−3) in i soil layer. To determine the state of soil water deficit and to ensure that the results are comparable with other regions with different soil hydrological characteristics, SWC was converted into relative extractable water (REW), which was calculated as follows [60]:
REW = SWC − SWC m SWC FC − SWC m
where SWCm is the value of SWC at the wilting point (defined as SWC held at −1.5 MPa) measured using a HYPROP system (HYPROP2; METER, Pullman, Germany), and SWCFC is the soil field capacity. Related studies have shown that REW < 0.4 can be defined as the presence of drought stress [61,62]. In addition, based on the soil depth and coefficient of variation (CV, %) of REW between dry and wet seasons, we divided the soil into three potential water source layers [52]: shallow soil layer (0–0.6 m, CV > 20%), middle soil layer (0.6–1.0 m, 10% < CV < 20%), and deep soil layer (1.0–2.0 m, CV < 10%).

2.6. Measurement of Stable Isotopic Compositions of Water Samples and Leaf Samples

Plant xylem and soil samples were firstly extracted using an LI-2100 automated vacuum distillation system (LICA, Beijing, China), which subjected the samples to a maximum allowable vacuum pressure of 1.5 kPa and a temperature difference of 225 °C (heating temperature 130 °C; cooling trap −95 °C) for 3 h to ensure that more than 99% of the water was extracted from the samples [63]. After the extraction, the δD and δ18O values of soil water, rainwater, and xylem water were measured using an Isotope Ratio Mass Spectrometer (IRMS) (Delta V Advantage, Thermo Fisher Scientific, Inc., Waltham, MA, USA), which has been shown to be effective in eliminating interference from organic distraction [16,63,64]. The precision of this instrument was ±<1‰ for δD and ±<0.2‰ for δ18O. The leaf sample was first completely combusted through an elemental analyzer (EA-HT, Thermo Fisher Scientific, Inc., Bremen, Germany) to generate CO2, then δ13C was determined by IRMS. The precision of this instrument was ±<0.1‰ for δ13C. Additionally, Vienna Standard Mean Ocean Water (VSMOW) and international standard object (Pee Dee Belemnite or PDB) were used as standard reference materials for liquid water δD and δ18O and leaf δ13C, respectively, to correct for instrumental drift and mass bias (i.e., standard–sample–standard bracketing). The stable isotope value of the sample was calculated using the following formula:
δ X ‰ = ( R sam R std −   1 )   ×   1000
where δX is the measured stable isotope value of the corresponding sample, Rsam is the ratio of heavy to light isotopes of elements in the sample, and Rstd is the ratio of heavy to light isotopes of elements in international standards (such as D/H, 18O/16O, and 13C/12C).

2.7. Plant Water Source and WUE Identification

Water source identification: The isotopic composition of plant xylem water is a mixture of isotopes from different layers of soil water. Since the δ18O and δD values of soil water in the four stands differed significantly (p < 0.05) between soil layers (Table A2), the observed differences in the isotopic composition of xylem water among stand ages in different seasons (Figure A1) represent the differences in the proportion of soil water absorbed by the stands from different soil layers. The MixSIAR model was used to calculate the relative use ratio of E. urophylla × E. grandis from various potential water sources by analyzing and comparing the isotopic composition of xylem water with soil water at each depth [65]. This model has been shown to fully account for uncertainty in source values, categorical and continuous covariates, and a priori information factors [16,66]. Its rationale for quantifying the proportion of plant uptake from different water sources is as follows:
δ X s = ∑ 1 n f n ×   δ X sn + r n ,   ( ∑ 1 n f n   = 1 ,   0   ≤   f n   ≤   1 )
where δXs is the δD or δ18O value of xylem water, n is the different soil layers, δXsn is the δD or δ18O value of soil water from different soil layers, fn is the proportion of different water sources used by plants, and rn is the fractional parameter for δD or δ18O.
However, recent studies have shown a limitation in discriminating plant water sources using the MixSIAR model due to the occurrence of isotopic mismatch between xylem water and soil water resulting from water extraction techniques and/or isotopic fractionation during plant water uptake [16,63]. In this study, both soil and xylem water extraction efficiencies exceeded 99%. Meanwhile, the δD-δ18O data points for xylem water closely aligned with those for soil water in the dual isotope space (Figure 2c,d), proving that there was no significant isotopic fractionation during root water uptake and water transport in E. urophylla × E. grandis. In addition, non-fractionation of hydrogen and oxygen isotopes during water uptake and transport by E. urophylla × E. grandis has been reported in other studies [67]. Therefore, it is reasonable to use the stable isotopes D and 18O in combination with the MixSIAR model to study water uptake by E. urophylla × E. grandis, and the results can provide an important basis for understanding the proportional water use of different sources. In this study, we entered the δD and δ18O values of soil water from different soil layers (source data) and xylem water of four aged E. urophylla × E. grandis (mixture data) into the MixSIAR model. Fractionation parameters for δD and δ18O were set to 0 because isotopic fractionation does not occur during water uptake. To ensure the accuracy of the model’s computational results, the running step size of the Markov Chain Monte Carlo was set to “long” and “process + residual” was selected for the model error. Gelman–Rubin and Geweke were used to determine whether the results of the runs converged or not. If they did not, the run length was increased until the results converged [68].
WUE identification: The WUE of E. urophylla × E. grandis was indirectly reflected by the measured leaf δ13C content. Previous studies have confirmed that leaf δ13C values are closely related to plant WUE, with more negative δ13C values indicating lower WUE [31,32].

2.8. Statistical Analyses

Statistical analyses were conducted using SPSS v19.0 (IBM, Armonk, New York, NY, USA). Data met assumptions of normality (Shapiro–Wilk’s test) and homogeneity of variance (Levene’s test) prior to ANOVA. One-way analysis of variance (ANOVA) was used to test the differences in fine root biomass between stand ages within each soil layer, as well as the differences in δD and δ18O of soil water across different soil layers within each stand age. Additionally, factorial ANOVA was used to test the differences in REW among stand ages, soil layers, and seasons, and to test the differences in δD and δ18O of xylem water and δ13C of leaves among stand ages and seasons. Tukey’s HSD post hoc tests identified specific group differences when ANOVAs were significant (p < 0.05). Linear regression was used to investigate the relationships between the δD and δ18O of atmospheric precipitation, xylem water, and soil water for each stand age and the relationship between leaf δ13C content and REW in the four stands. Pearson’s correlation analyses were used to analyze the correlations between δD and δ18O values of xylem water, δ13C values of leaves, and environmental factors for the four stands, and the correlations between the water contribution rate from different soil layers and fine root biomass, REW, and their interaction at corresponding depths. We used backward elimination multiple regression analysis to identify an optimal predictive model for water contribution rate from different soil layers, based on the lowest Akaike Information Criterion (AIC) as the criterion for variable selection and rejection [69,70]. This procedure was performed with the Step AIC function in the MASS package of the R statistical program 4.2.2 (R Core Team 2022 [71]). Figures and tables were produced using Origin 2019b (Origin Software Inc., Fairview, TX, USA) and Excel 2021, respectively.

3. Results

3.1. Relative Extractable Water (REW) and Fine Root Distribution

Relative extractable water (REW) in four E. urophylla × E. grandis plantations increased and then stabilized with soil depth (Figure 3a). The mean REW in the four stands differed significantly among the three periods, with the lowest in the dry months and no significant differences between the wet months and the wet month of dry season. The difference among the three periods gradually narrowed with increasing depth (Figure 3a). The REW did not vary significantly between the stand ages within a given period. The REW in different soil layers across the four stands ranged from 0.47 to 0.75 during the wet months and the wet month of the dry season, indicating that the soil in all layers of the four stands was not under drought stress. However, during the dry months, the REW in the shallow, middle, and deep soil layers ranged from 0.12 to 0.44, 0.41 to 0.56, and 0.49 to 0.66, respectively. This suggests that drought stress only occurred in the shallow soil layers during the dry months, whereas drought stress was absent in the middle and deep soil layers.
The fine roots of the E. urophylla × E. grandis plantations at four stand ages initially increased and then decreased with soil depth (Figure 3b), with most of them distributed in the shallow soil layer (0–60 cm), ranging from 62.5% (14-year-old) to 87.4% (2-year-old). At a given soil layer, fine root biomass gradually increased with stand age, with total biomass increasing from 43.9 g/m2 in the 2-year-old stand to 144.4 g/m2 in the 14-year-old stand. The fine root biomass at 60–200 cm soil depth (middle and deep soil layer) was significantly (p < 0.05) lower in the 2- (5.52 g/m2) and 4-year-old stands (11.98 g/m2) than in the 9- (33.23g/m2) and 14-year-old stands (54.18g/m2) (Figure 3b).

3.2. Shifts in Water Use Sources

Seasonal variations in the potential water source uptake ratios were observed in the E. urophylla × E. grandis plantations at four stand ages (Figure 4). During the wet months, all stands primarily relied on shallow soil water (0–60 cm), with uptake rates ranging from 70.2% to 74.3%. However, in the dry months, the uptake rates of shallow soil water decreased to 35.8% and 32.0% for 9- and 14-year-old plants, respectively, and shifted to predominantly accessed middle and deep soil water (60–200 cm), where no drought stress was present, with uptake rates of 64.3% and 68.0%, respectively. In contrast, the 2- and 4-year-old stands continued to rely on shallow soil water in the dry months, although the uptake rates of middle and deep soil water (60–200 cm) increased by 5.8% and 8.5%, respectively. During the wet month of dry season, the 9- and 14-year-old stands shifted back to mainly using shallow soil water, with uptake rates of 83.4% and 65.0%, respectively. The 2- and 4-year-old stands also increased their uptake rates of shallow soil water to 83.1% and 79.1%, respectively.

3.3. Leaf δ13C and WUE

The δ13C values of leaves in E. urophylla × E. grandis plantations decreased with stand age, with significant differences (p < 0.05) observed only in the dry months and the wet month of the dry season (Figure 5). In addition, leaf δ13C varied significantly among the three periods, with the magnitude of variations decreasing as stand age increased (Figure 5). The δ13C values of leaves in the 2-year-old stands in the wet months (range: −31.90 to −28.51‰; mean: −30.31‰) were significantly lower than those in the wet month of the dry season (range: −28.53 to −27.88‰; mean: −28.23‰), which were also significantly lower than those in the dry months (range: −27.81 to −26.82‰; mean: −27.53‰). The δ13C values of leaves in the 4- and 9-year-old stands in the wet months (ranging from −31.89 to −28.97‰, with a mean value of −30.20‰ and ranging from −32.02 to −28.51‰, with a mean value of −30.52‰ for 4- and 9-year-old stands, respectively) were significantly lower than those in the dry months (ranging from −28.88 to −27.30‰, with a mean value of −28.26‰ and ranging from −31.25 to −27.76‰, with a mean value of −29.50‰ for 4-, and 9-year-old stands, respectively) and the wet month of the dry season (ranging from −29.69 to −28.11‰, with a mean value of −28.60‰ and ranging from −30.94 to −28.91‰, with a mean value of −29.96‰ for 4- and 9-year-old stands, respectively). In contrast, the δ13C values of leaves in the 14-year-old stand were significantly lower only in the wet months (range: −31.38 to −29.53‰; mean: −30.46‰) than that in the dry months (range: −31.38 to −28.21‰; mean: −29.73‰).

3.4. Factors Influencing Water Use Sources and WUE

The correlation analysis showed that the δ13C values of leaves in the four stands showed significant negative correlations with T, VPD, and REW (Figure 6). The slope of the linear regression line between δ13C and REW gradually decreased with increasing stand age, from 9.98 in 2-year-old to 3.08 in 14-year-old stands (Figure A2). This suggests that the WUE of E. urophylla × E. grandis is jointly affected by T, VPD, and REW and that the younger the stand, the more negatively affected it is by changes in REW and the greater the increase in WUE during the dry months. The δ18O and δD values of xylem water in 9- and 14-year-old stands were significantly positively correlated with REW (p < 0.01), whereas no significant correlation was observed in 2- and 4-year-old stands (Figure 6). This indicates that the 9- and 14-year-old stands can quickly shift their primary water sources in response to changes in soil moisture. In addition, the multiple linear regression showed that the water contribution rate from different soil layers was mainly controlled by the corresponding fine root biomass in 2- and 4-year-old stands, whereas it was mainly controlled by the interaction between the corresponding fine root biomass and REW in 9- and 14-year-old stands (Figure 7).

4. Discussion

4.1. Water Source Used by E. urophylla × E. grandis Plantations at Four Stand Ages

The water source strategies used by trees to cope with drought stress can also vary considerably among different stand ages due to the differences in tree structure and the surface and subsurface environments [6,8]. In this study, regardless of the presence or absence of drought stress in the shallow soil layers (both dry and wet seasons), the young (2-year-old) and middle-aged (4-year-old) E. urophylla × E. grandis mainly absorbed water from shallow soil layers (0–60cm) (Figure 4). This suggests that the main source of water used in the two stands does not change with changing soil moisture, as evidenced by the lack of correlation between δD and δ18O in their xylem water and REW (Figure 6). Similar results were also observed in young stands of Pinus sylvestris L. [52], Ziziphus jujube Mill. [72], and Salix psammophila C. Wang et Ch. Y. Yang [16]. This may be closely related to their shallow root systems [65,73]. Multiple regression analyses showed that the contribution rate of different soil water sources to 2- and 4-year-old stands was mainly limited by the distribution of fine root biomass (Figure 7). The fine root system of 2- and 4-year-old E. urophylla × E. grandis was mainly distributed in the 0–60 cm soil layers, with lower root biomass in the middle and deep soil layers (Figure 3b), resulting in a lack of physiological and structural capacity to absorb water from deeper soils. This is further supported by the fact that the proportion of middle and deep soil water used by the 2- and 4-year-old stands during the dry months, although all increased, was minimal (Figure 4).
In contrast, the mature (9-year-old) and overmature (14-year-old) E. urophylla × E. grandis had more flexible water use strategies, mainly absorbing water from shallow soil layers in the wet months, shifting to mainly using water from middle and deep soil layers in the dry months when the shallow soil layers were under drought stress (Figure 3a), as evidenced by the significant positive correlation between REW and δD and δ18O in their xylem water (Figure 6). Multiple regression analyses showed that this dynamic plasticity in the water use strategy of mature and overmature E. urophylla × E. grandis was mainly controlled by the synergistic effects of fine root biomass and REW (Figure 7). The following are possible drive mechanisms: Deeper and higher root biomass, combined with dimorphic roots, give mature and overmature E. urophylla × E. grandis the ability to change the water absorption layer and to absorb water from deeper soil layers [52,65,74]. The REW directly mediated shifts in water use strategies. When the REW in shallow soil was high, the larger distribution of fine roots in shallow layers drove the mature and overmature E. urophylla × E. grandis to preferentially use mainly shallow soil water, a water use strategy consistent with both the “least-cost hypothesis” [75,76] and the “two-pool hypothesis” of plant water use [65,77]. The closer the soil is to the surface, not only is the nutrient content higher, but the looser the soil is (Table A1), the shorter the water transport distances, and the less energy is required for plant uptake and water transport [52,65]. This results in the preferential use of shallow soil water during primary acquisition and growth [27,65,78,79]. Only when drought stress occurs in shallow soils (dry months) did mature and overmature stands expend more energy to absorb more middle and deeper soil water, relying on their considerable distribution of deep fine roots to meet the needs of tree transpiration and carbon fixation [80,81], reflecting a trade-off between energy efficiency and survival.
Notably, our study also uniquely documented that the mature and overmature E. urophylla × E. grandis rapidly returned to mainly using shallow soil water during occasional wet periods within the dry season (the wet month of the dry season) (Figure 4). This contrasts with the findings of Huo et al. [72] on seasonal water use patterns of rainfed jujube (Ziziphus jujube Mill.) trees in stands of different ages under semiarid plantations in China, who found that the heavy rainfall did not increase the contribution of shallow soil layer water to jujube trees of all ages, reflecting a more conservative water use strategy. This may be related to the deep root system and its long-term adaptation to water-unstable environments [52,73]. However, our results are consistent with the findings of Schwinning et al. [82,83], who found that both shallow- and deep-rooted plants in the Colorado Plateau region increased their uptake of shallow soil water during rainfall ‘pulses’. This ability highlights the opportunistic water use strategy of mature and overmature E. urophylla × E. grandis for maximizing water resources and increasing growth under fluctuating conditions while maintaining drought tolerance.

4.2. Seasonal Changes in WUE in E. urophylla × E. grandis Plantations at Four Stand Ages

Previous studies have verified that lower δ13C values in tree leaves reflect lower long-term water use efficiency (WUE) [31,32]. In this study, the δ13C values in the leaves of E. urophylla × E. grandis at four stand ages were generally lower in the wet season (ranging from −32.02 to −28.51‰, with a mean value of −30.37‰) than in the dry season (ranging from −31.38 to −26.82‰, with a mean value of −28.90‰) (Figure 5), implying that E. urophylla × E. grandis usually adopts a profligate water use strategy (low WUE) in the wet season, but switches to a frugal water use strategy (high WUE) in the dry season. This shift in water use strategy is very favorable for its adaptation to seasonal drought. Correlation analyses showed that the increase in WUE during the dry season was attributed to the combination of lower air temperature, VPD, and REW in the dry season (Figure 6), all of which could directly reduce the stomatal conductance of the plant [84,85]. Due to the difference in the diffusion coefficients between CO2 and water, the decrease in stomatal conductance has a stronger effect on the transpiration rate than the photosynthetic rate [63,86], leading to an increase in WUE.
The WUE in the E. urophylla × E. grandis plantations decreased with stand age (Figure 5), with significant differences observed only in the dry season. Furthermore, the variation in the amplitude of the δ13C in leaves between the dry and wet seasons increased with decreasing age (Figure 5). Supported by the lack of significant differences in WUE among the four stands during the wet season, we assume that the leaf stomatal conductance of the same species responds equally to changes in meteorological factors (T and VPD). Therefore, we believe that the above differences are mainly due to the fact that the degree of drought stress that E. urophylla × E. grandis is subjected to under the same REW conditions in the dry season gradually decreases with increasing stand age, as evidenced by the decreasing magnitude of the negative effect of REW on WUE of E. urophylla × E. grandis gradually decreasing with stand age (Figure A2). This could be explained by the deeper root distribution and larger root biomass in older stands (Figure 3b) and their greater ability to obtain a stable water supply from deeper soils (Figure 4) to mitigate the effects of drought stress [52,84]. For example, 5-year-old Salix cheilophila C.K.Schneid cannot use stabilized deep soil water or groundwater during drought stress periods due to their shallow root systems, resulting in their significantly higher WUE than 9- and 25-year-old stands [43]. Additionally, the younger the stand, the lower the hydraulic resistance of the trees and the greater the likelihood that drought stress will lead to xylem cavitation and, thus, an imbalance in the hydraulic system [87,88]. As a result, stomatal conductance may be more constrained under drought stress in plantations of a younger stand age. For example, as soil water deficit increased in the late summer in the Pacific Northwest, the stomatal conductance recorded for young pine was only 3/7 that of mature pine, resulting in the WUE of young pine being 2.8 times that of mature pine trees [84]. This is also a side effect of the fact that trees become more resistant to drought stress with increasing stand age.
The WUE of the four stands remained higher in the episodic wet months of the dry season than in the wet months (Figure 5), suggesting that E. urophylla × E. grandis maintains a conservative WUE strategy in the face of episodic wet periods after experiencing prolonged drought stress in the dry season. This may be due to a combination of hydraulic and non-hydraulic factors in the tree [89], in addition to the continued influence of low T and VPD. Previous studies have shown that there is a temporal delay in the hydraulic recovery of trees after an improvement in soil moisture conditions [90,91], and about 10%–30% of hydraulic conductivity is unrecoverable [89,92,93]. In addition, a study on E. pauciflora found that stomatal conductance did not fully recover, even after 10 days of favorable water status after stem hydraulics were recovered, suggesting that non-hydraulic factors have a control over stomatal behavior [89]. This result indicates that E. urophylla × E. grandis has a conservative WUE strategy to protect against rainfall anomalies in the dry season and ensure their long-term stable survival.

4.3. Age-Related Water Use Strategies and Implications for Plantation Management

Drought is a major environmental stress that limits plant growth and survival [94]. This study revealed significant differences in the water use strategies of E. urophylla × E. grandis of different ages under seasonal drought conditions. The 2- and 4-year-old stands, limited by their shallow root distribution, relied mainly on shallow soil water throughout the year. Such a water use strategy has obvious ecological risks. First, the narrow ecological niche may exacerbate competition for water with surface plants [95]. Second, the high variability and susceptibility to the depletion of shallow soil water makes it more vulnerable and severe to drought stress [6,73], leading it to increase its WUE in response to drought stress through greater stomatal control during the dry season [10,65]. However, the cost of this drought-adapted water use strategy may be a reduction in productivity and an increased risk of mortality [49,96]. In contrast, the 9- and 14-year-old stands adopted a dynamic plasticity water use strategy that could regulate its depth of water use according to soil moisture conditions through a larger and deeper root system, and also responded quickly to episodic wet periods (Figure 4), demonstrating a sensitive opportunistic water use strategy. The ecological advantage of this flexible water use strategy is that it allows for efficient access to water resources, minimizes exposure to drought stress [97] and its stomatal limitation (lower WUE) during the dry season [86,98,99], and, thus, ensures greater productivity and carbon sequestration. The result that the mean annual increment (MAI) of E. urophylla × E. grandis monitored by us increases with the increase in stand age also indirectly supports the age-related water use strategy in this study (Table 1). In conclusion, as E. urophylla × E. grandis develops, its water use strategy to adapt to drought stress gradually shifts from physiological regulation dominated (stomatal control to increase WUE) to structural adaptation dominated (use of a large root system to regulate the water use layer) (Figure 8).
Based on the differences in water use strategies for drought adaptation in different ages of E. urophylla × E. grandis and their respective characteristics, we propose the following water retention and water augmentation management measures for eucalyptus plantations, especially young and middle-aged forests, to ensure the stable development of China’s eucalyptus industry in the context of global climate change: (i) Increasing ground cover by retaining logging residues not only increases nutrient return [100,101] but also reduces surface evaporation while decreasing the incidence of surface runoff and increasing soil infiltration [102]. (ii) Appropriate pruning or thinning of young and middle-aged trees (typically 2–3 years old), which improves wood quality [103] while reducing transpiration [104,105] and canopy retention of evapotranspiration, increases the amount of precipitation that penetrates the forest [106]. (iii) Appropriate water replenishment measures (e.g., drip irrigation) for young and middle-aged forests during seasonal dry periods can improve shallow soil water deficits, which can significantly increase the productivity of eucalyptus plantations [47,107,108]. These water management practices can also serve as a reference for plants with similar age-related water use strategies in other seasonally dry regions of the world.

4.4. Limitations and Perspectives for Future Exploration

This study reveals the differences in water use strategies among E. urophylla × E. grandis plantations of different ages and emphasizes that fine root distribution and REW are the key driving factors of water use strategies. However, some limitations should be noted in this study. Firstly, our analysis relied on isotopic and root distribution data but did not directly measure plant hormones (e.g., abscisic acid, cytokinin, etc.) and physiological traits (e.g., stomatal conductance, xylem hydraulic conductivity), which could further reveal how physiological regulation mediates age-related water use strategies for drought adaptation [109,110]. Secondly, phenological variations (e.g., seasonal leaf growth, senescence) were not monitored, though they may interact with water uptake patterns under drought. Future studies could integrate high-resolution phenological observations with stable isotope techniques to better disentangle age-related growth water use trade-offs. Finally, this study only focused on a single hybrid genotype (DH32-29) in one specific region. Replicating these methods across genetically diverse eucalyptus plantations and different regions would help validate the universality of our conclusions.

5. Conclusions

This study used stable isotope technology to investigate the water use strategies of E. urophylla × E. grandis across four stand ages (2-, 4-, 9-, and 14-year-old) in response to seasonal drought and to analyze the factors influencing them on the Leizhou Peninsula, China. Young (2-year-old) and middle-aged (4-year-old) stands primarily relied on shallow soil water throughout the growing season. In contrast, mature (9-year-old) and overmature (14-year-old) stands showed higher plasticity in their water use, mainly using shallow soil water during the wet months. During the dry months, they shifted to middle and deep soil water, and returned to shallow soil water sources during the wet months of the dry season. A fine root biomass distribution was the main limiting factor for the water uptake rates of young and middle-aged E. urophylla × E. grandis to different soil layers, whereas soil water uptake rates of mature and overmature stands to different soil layers were mainly influenced by the interaction between fine root biomass distribution and soil moisture. The WUE of E. urophylla × E. grandis was affected by the combined effect of T, VPD, and REW. The water use efficiency (WUE) of E. urophylla × E. grandis did not differ significantly in the wet season, but decreased significantly with increasing stand age in the dry season. These results indicate that young and middle-aged stands mainly relied on an enhanced WUE to cope with drought stress, whereas mature and overmature stands adapted by shifting their water source. Consequently, young and middle-aged E. urophylla × E. grandis stands are more vulnerable to drought stress. To mitigate these effects under future climate change, water management practices should be considered for eucalyptus plantations, especially young and middle-aged stands, such as retaining logging residues (to reduce evaporation), pruning/thinning (to reduce transpiration), and targeted drip irrigation during dry periods. Moreover, monitoring the δ13C values can further guide resource allocation to optimize the effectiveness of these management practices. These findings offer valuable insights for the sustainable management of eucalyptus plantations.

Author Contributions

Conceptualization, A.D. and Z.W.; methodology, W.X.; software, H.C.; validation, R.H., H.C. and W.X.; formal analysis, Z.W.; investigation, Z.W., W.Z. and Y.X.; resources, A.D.; data curation, Z.W.; writing—original draft preparation, Z.W.; writing—review and editing, W.X.; visualization, H.C. and Z.W.; supervision, W.X.; project administration, A.D. and Y.X.; funding acquisition, Z.W. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by the Natural Science Foundation of Guangdong Province (2025A1515011402); the special funds for the basic research and development program in the central non-profit research institutes of China (CAFYBB2024MA018); the National Key R&D Program of China (NO. 2023YFD2201005).

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We thank Zhong Wang and Yajun Cheng for the help with field data collection. We thank the assistance of the staff from the South China Experiment Nursery for support during the selection of suitable plots for this study.

Conflicts of Interest

The authors declare no conflicts of interest.

Appendix A

Table A1. Basic physico-chemical properties of soils in the four studied forest stands.
Table A1. Basic physico-chemical properties of soils in the four studied forest stands.
IndicatorSoil Depth (cm)
0–1010–2020–3030–4040–6060–8080–100100–150150–200
pH5.33 (0.28)5.41 (0.27)5.45 (0.3)5.48 (0.32)5.51 (0.29)5.56 (0.32)5.51 (0.29)5.67 (0.27)5.71 (0.35)
BD (g·m−3)0.99 (0.04)1.11 (0.03)1.13 (0.05)1.19 (0.05)1.27 (0.03)1.26 (0.07)1.28 (0.05)1.28 (0.08)1.27 (0.05)
Tpo (%)56.94 (2.7)56.86 (3.03)57.5 (2.11)57.9 (1.56)55.4 (1.48)55.2 (1.75)53.2 (3.59)52.47 (2.27)50.44 (2.93)
Cpo (%)56.16 (2.31)53.44 (2.97)55.6 (2.12)52 (1.52)50.4 (1.30)53.1 (1.87)50.7 (2.66)51.42 (1.94)49.26 (2.3)
SWHC (%)57.46 (5.72)59.62 (6.59)58.69 (5.04)57.87 (3.51)55.73 (2.80)52.52 (5.01)48.36 (7.78)43.36 (5.2)42.85 (6.49)
CWHC (%)56.68 (5.00)56.08 (6.44)55.91 (4.99)52.77 (3.52)50.7 (2.42)50.52 (5.01)46.09 (6.15)42.49 (4.53)41.85 (5.34)
FWHC (%)56.27 (5.01)53.41 (6.60)53.42 (5.02)52.44 (3.47)49.6 (2.37)47.57 (4.99)45.18 (6.06)42.05 (4.47)41.48 (5.27)
SOM (%)2.88 (0.43)2.76 (0.66)2.13 (0.3)1.83 (0.10)1.66 (0.32)1.09 (0.15)1.66 (0.32)1.63 (0.26)1.65 (0.29)
TN (g·kg−1)2.51 (0.1)2.39 (0.2)2.29 (0.88)1.95 (0.39)1.96 (0.05)1.80 (2.05)1.96 (0.05)1.94 (0.72)1.88 (0.38)
TP (g·kg−1)1.23 (0.24)1.21 (0.29)1.18 (0.16)0.72 (0.1)1.03 (0.22)0.84 (0.10)1.03 (0.22)0.95 (0.18)0.89 (0.2)
TK (g·kg−1)2.95 (0.7)2.85 (0.93)2.92 (0.92)3.02 (0.73)3.26 (0.59)3.24 (1.10)3.26 (0.59)3.22 (0.76)3.25 (0.68)
AN (mg·kg−1)351.22 (48.13)347.74 (41.72)292.3 (44.05)267.78 (47.92)267.31 (51.34)267.62 (42.50)267.31 (51.33)267.43 (48.39)266.98 (49.86)
AP (mg·kg−1)3.4 (2.02)3.34 (1.29)3.29 (1.32)2.79 (1.9)3.47 (2.39)2.75 (0.76)3.47 (2.39)2.89 (1.85)3.21 (2.12)
AK (mg·kg−1)31.54 (10.1)30.94 (8.57)26.81 (12.02)24.21 (9.93)18.69 (5.86)18.44 (6.56)18.69 (5.86)19.26 (6.09)18.77 (5.98)
Note: BD, bulk density; Tpo, total porosity; Cpo, capillary porosity; SWHC, saturated water holding capacity; CWHC, capillary water holding capacity; FWHC, field water holding capacity; SOM, soil organic matter; TN, total nitrogen; TP, total phosphorus; TK, total potassium; AN, available nitrogen; AP, available phosphorus; AK, available potassium. Values shown as means of four E. urophylla × E. grandis plantations. Values in parentheses represent standard deviations.
Table A2. Isotope distribution values (δD and δ18O) of soil water in different soil layers in 2-, 4-, 9- and 14-year-old E. urophylla × E. grandis stands in wet months, dry months, and wet month of the dry season.
Table A2. Isotope distribution values (δD and δ18O) of soil water in different soil layers in 2-, 4-, 9- and 14-year-old E. urophylla × E. grandis stands in wet months, dry months, and wet month of the dry season.
AgesLayersWet MonthsDry MonthsWet Month of the Dry Season
δ18O (‰)δD (‰)δ18O (‰)δD (‰)δ18O (‰)δD (‰)
2 yrShallow−8.22 (1.38) a−57.32 (7.38) a−7.40 (2.13) a−55.73 (14.77) a−6.35 (0.61) a−50.80 (2.84) a
Middle−10.03 (0.54) b−67.05 (7.11) b−10.08 (0.81) b−70.35 (2.92) b−8.86 (0.81) b−63.59 (2.27) b
Deep−10.58 (0.81) b−70.20 (4.20) b−9.62 (1.20) b−68.82 (7.64) b−9.34 (0.05) b−67.54 (0.22) b
4 yrShallow−9.39 (2.34) a−64.38 (15.99) a−6.89 (1.24) a−51.49 (11.93) a−7.32 (0.71) a−50.11 (4.71) a
Middle−10.31 (1.62) ab−68.10 (7.89) ab−9.22 (1.34) b−62.32 (5.04) ab−9.89 (0.57) b−64.90 (1.76) b
Deep−10.75 (0.30) b−73.72 (2.22) b−10.8 (0.44) b−72.42 (1.49) b−10.43 (0.21) b−69.20 (3.60) b
9 yrShallow−7.27 (0.09) a−56.47 (4.5) a−9.31 (2.13) a−63.23 (14.33) a−7.15 (1.09) a−55.84 (1.83) a
Middle−9.47 (0.3) b−76.87 (13.35) b−11.11 (0.47) b−73.78 (2.25) b−10.15 (0.90) b−68.26 (2.91) b
Deep−10.96 (0.21) c−81.79 (0.63) c−10.09 (0.17) ab−67.38 (5.09) ab−10.48 (0.74) b−67.22 (2.09) b
14 yrShallow−8.55 (0.75) a−58.92 (3.24) a−8.96 (1.10) a−64.53 (7.74) a−6.56 (0.17) a−51.47 (1.97) a
Middle−11.68 (0.66) b−78.51 (5.58) b−10.42 (1.00) ab−69.99 (5.43) ab−9.20 (1.16) b−65.94 (1.00) b
Deep−12.07 (0.36) b−80.58 (6.03) b−11.03 (0.74) b−73.91 (3.28) b−9.91 (0.69) b−67.50 (2.51) b
Note: Different lowercase letters indicate significant (p < 0.05) differences in hydroxide isotope values between different soil layers for the same stand age groups at a given period. Values in parentheses represent standard deviations.

Appendix B

Forests 16 00962 g0a1
Figure A1. Isotopic composition (δD and δ18O) of stem xylem water in 2-, 4-, 9-, and 14-year-old E. urophylla × E. grandis stands in wet months, dry months, and wet month of the dry season. Box plots show the median values (black lines in the boxes), the average values (red dots in the boxes), interquartile range (extent of the boxes), and range of data (whiskers). Different lowercase letters indicate differences at the significance level p < 0.05.
Figure A1. Isotopic composition (δD and δ18O) of stem xylem water in 2-, 4-, 9-, and 14-year-old E. urophylla × E. grandis stands in wet months, dry months, and wet month of the dry season. Box plots show the median values (black lines in the boxes), the average values (red dots in the boxes), interquartile range (extent of the boxes), and range of data (whiskers). Different lowercase letters indicate differences at the significance level p < 0.05.
Forests 16 00962 g0a1
Forests 16 00962 g0a2
Figure A2. Linear regression between leaf δ13C content and relative extractable water (REW) in E. urophylla × E. grandis plantations of four stand ages (2-, 4-, 9- and 14-year-old). ** Represents p < 0.01, and *** represents p < 0.001.
Figure A2. Linear regression between leaf δ13C content and relative extractable water (REW) in E. urophylla × E. grandis plantations of four stand ages (2-, 4-, 9- and 14-year-old). ** Represents p < 0.01, and *** represents p < 0.001.
Forests 16 00962 g0a2

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Figure 1. Location of the study area (a), seasonal variation in environmental factors during the study period (b), photographs of the forests investigated (2-, 4-, 9- and 14-year-old E. urophylla × E. grandis) (c). Vertical dashed lines, red line, red shaded area, blue line, and blue shaded area in (b) indicate sampling times, monthly mean air temperature (T), monthly T range, monthly mean vapor pressure deficit (VPD), and monthly mean soil water content (SWC) at 0–100 cm depth, respectively.
Figure 1. Location of the study area (a), seasonal variation in environmental factors during the study period (b), photographs of the forests investigated (2-, 4-, 9- and 14-year-old E. urophylla × E. grandis) (c). Vertical dashed lines, red line, red shaded area, blue line, and blue shaded area in (b) indicate sampling times, monthly mean air temperature (T), monthly T range, monthly mean vapor pressure deficit (VPD), and monthly mean soil water content (SWC) at 0–100 cm depth, respectively.
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Figure 2. Variations in precipitation (P, mm) and isotopic composition (δD and δ18O) of rainwater during the monitored period (a). Comparison of local meteoric water line (LMWL) and global meteoric water line (GMWL) during dry and wet seasons (b). Isotopic composition of soil water (c) and xylem water (d) for 2-, 4-, 9-, and 14-year-old E. urophylla × E. grandis plantations during the study period. The fitted curves represent the plant water line (PWL) and soil water line (SWL) for E. urophylla × E. grandis plantations of each stand age, respectively. The black dashed lines represent the LMWL. *** Represents p < 0.001.
Figure 2. Variations in precipitation (P, mm) and isotopic composition (δD and δ18O) of rainwater during the monitored period (a). Comparison of local meteoric water line (LMWL) and global meteoric water line (GMWL) during dry and wet seasons (b). Isotopic composition of soil water (c) and xylem water (d) for 2-, 4-, 9-, and 14-year-old E. urophylla × E. grandis plantations during the study period. The fitted curves represent the plant water line (PWL) and soil water line (SWL) for E. urophylla × E. grandis plantations of each stand age, respectively. The black dashed lines represent the LMWL. *** Represents p < 0.001.
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Figure 3. Mean relative extractable water (REW) of each soil layer in 2-, 4-, 9-, and 14-year-old E. urophylla × E. grandis plantations in wet months, dry months, and wet month of the dry season (a), and the vertical distribution of fine root biomass (d < 2 mm) for the four stands (b). Gray shaded areas in (a) represent the presence of water stress. Different lowercase letters in (b) indicate differences in fine root biomass between stand ages in the same soil layer at the significance level p < 0.05.
Figure 3. Mean relative extractable water (REW) of each soil layer in 2-, 4-, 9-, and 14-year-old E. urophylla × E. grandis plantations in wet months, dry months, and wet month of the dry season (a), and the vertical distribution of fine root biomass (d < 2 mm) for the four stands (b). Gray shaded areas in (a) represent the presence of water stress. Different lowercase letters in (b) indicate differences in fine root biomass between stand ages in the same soil layer at the significance level p < 0.05.
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Figure 4. Contribution of potential water sources to plant water uptake in E. urophylla × E. grandis plantations of four stand ages (2-, 4-, 9- and 14-year-old) in wet months, dry months, and wet month of the dry season.
Figure 4. Contribution of potential water sources to plant water uptake in E. urophylla × E. grandis plantations of four stand ages (2-, 4-, 9- and 14-year-old) in wet months, dry months, and wet month of the dry season.
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Figure 5. Differences in leaf δ13C content among four stand ages (2-, 4-, 9- and 14-year-old) and three periods (wet months, dry months, and wet month of the dry season). Different lowercase letters in figure a indicate significant (p < 0.05) differences between different periods for the same stand age groups and different capital letters indicate significant (p < 0.05) differences between different stand ages in the same periods.
Figure 5. Differences in leaf δ13C content among four stand ages (2-, 4-, 9- and 14-year-old) and three periods (wet months, dry months, and wet month of the dry season). Different lowercase letters in figure a indicate significant (p < 0.05) differences between different periods for the same stand age groups and different capital letters indicate significant (p < 0.05) differences between different stand ages in the same periods.
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Figure 6. The correlations between the xylem water hydrogen oxygen isotopic composition (δ18O and δD) and leaf δ13C content of E. urophylla × E. grandis at four ages and under various environmental factors. REW, relative extractable water; T, air temperature; VPD, vapor pressure deficit; RH, relative air humidity. * Represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.
Figure 6. The correlations between the xylem water hydrogen oxygen isotopic composition (δ18O and δD) and leaf δ13C content of E. urophylla × E. grandis at four ages and under various environmental factors. REW, relative extractable water; T, air temperature; VPD, vapor pressure deficit; RH, relative air humidity. * Represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.
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Figure 7. Correlations and multiple linear regression equations between water contribution rate (RATE) from different soil layers and fine root biomass (ROOT), relative extractable water (REW), and their interaction (ROOT × REW) at corresponding depths. * Represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.
Figure 7. Correlations and multiple linear regression equations between water contribution rate (RATE) from different soil layers and fine root biomass (ROOT), relative extractable water (REW), and their interaction (ROOT × REW) at corresponding depths. * Represents p < 0.05, ** represents p < 0.01, and *** represents p < 0.001.
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Figure 8. Water use strategies of different stand ages of E. urophylla × E. grandis in response to seasonal drought. The numbers in the boxes indicate the absorption ratio of shallow, middle, and deep soils in the wet months (orange box), dry months (light-blue box), and wet month of the dry season (green box). The numbers in the aboveground part are the δ13C values, and the size of the blue arrows indicates the difference in water use efficiency between dry and wet seasons.
Figure 8. Water use strategies of different stand ages of E. urophylla × E. grandis in response to seasonal drought. The numbers in the boxes indicate the absorption ratio of shallow, middle, and deep soils in the wet months (orange box), dry months (light-blue box), and wet month of the dry season (green box). The numbers in the aboveground part are the δ13C values, and the size of the blue arrows indicates the difference in water use efficiency between dry and wet seasons.
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Table 1. Stand characteristics of E. urophylla × E. grandis plantations at four stand ages.
Table 1. Stand characteristics of E. urophylla × E. grandis plantations at four stand ages.
Stand Age (Years)Stand Density (Trees·ha−1)Mean DBH (cm)Mean Height (m)Leaf Area Index (LAI)Crown Breadth (m)Mean Annual Increment (m3 cha−1·yr)
216669.1 (1.44)11.5 (0.64)1.24 (0.14)2.3 (0.81)22.50 (10.51)
4166612.8 (1.02)16.4 (0.72)1.79 (0.21)3.1 (1.15)24.78 (9.13)
9150019.4 (1.61)19.7 (1.02)2.08 (0.14)4.8 (1.36)26.68 (6.45)
14130024.7 (2.04)27.1 (1.36)2.48 (0.31)5.6 (1.74)27.05 (4.63)
Note: Values in parentheses represent standard deviations.
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Wang, Z.; Xu, Y.; Zhu, W.; Huang, R.; Du, A.; Cao, H.; Xiang, W. Responses of Water Use Strategies to Seasonal Drought Stress Differed Among Eucalyptus urophylla S.T.Blake × E. grandis Plantations Along with Stand Ages. Forests 2025, 16, 962. https://doi.org/10.3390/f16060962

AMA Style

Wang Z, Xu Y, Zhu W, Huang R, Du A, Cao H, Xiang W. Responses of Water Use Strategies to Seasonal Drought Stress Differed Among Eucalyptus urophylla S.T.Blake × E. grandis Plantations Along with Stand Ages. Forests. 2025; 16(6):962. https://doi.org/10.3390/f16060962

Chicago/Turabian Style

Wang, Zhichao, Yuxing Xu, Wankuan Zhu, Runxia Huang, Apeng Du, Haoyang Cao, and Wenhua Xiang. 2025. "Responses of Water Use Strategies to Seasonal Drought Stress Differed Among Eucalyptus urophylla S.T.Blake × E. grandis Plantations Along with Stand Ages" Forests 16, no. 6: 962. https://doi.org/10.3390/f16060962

APA Style

Wang, Z., Xu, Y., Zhu, W., Huang, R., Du, A., Cao, H., & Xiang, W. (2025). Responses of Water Use Strategies to Seasonal Drought Stress Differed Among Eucalyptus urophylla S.T.Blake × E. grandis Plantations Along with Stand Ages. Forests, 16(6), 962. https://doi.org/10.3390/f16060962

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