Cryogenic vacuum distillation (CVD) is the most widely used method to extract water from plant tissues aimed at the isotope-based identification of plant water sources. Yet, it can introduce isotopic biases that affect the identification of plant water sources. Alternative methods such as the cavitron (CAV) precisely retrieve the isotopic signal of the plant’s source water by the selective extraction of water from conducting tissues. However, CAV has only been tested for a reduced number of species. We compared CAV and CVD extractions across 17 woody species, analyzing CVD extracts by both cavity ring-down spectroscopy (CRDS) and isotope-ratio mass spectrometry (IRMS). CAV yielded enough water for isotopic analysis in 13 out of 17 species and produced samples without organic compounds that closely resembled local mobile waters. In contrast, CVD-extracted water deviated from local mobile waters, and from CAV values, with a larger discrepancy for δ²H than for δ¹⁸O. The choice of the analyzer was decisive: CVD samples analyzed by CRDS showed the largest δ²H offset (mean Δδ²H = -13.93‰), whereas CVD-IRMS values were closer to CAV although still slightly depleted in δ²H (mean Δδ²H = -4.62‰). The spectral interference was the main driver of isotopic differences in CRDS-based data, whereas extraction efficiency and gravimetric water content had no effect. Overall, CAV provides reliable isotopic signatures of plant water but it is not applicable to all plant species (not even to all large woody plants), while CVD can introduce substantial bias for δ2H, especially when combined with CRDS.

ABSTRACT Isotope‐based Bayesian mixing models (BMM) are widely used in ecohydrology to infer where plants acquire water from the soil, yet clear guidance on their application to root water uptake (RWU) remains limited. This review synthesizes existing BMM applications for RWU estimation and critically examines three fundamental challenges that constrain their robustness and interpretability. First, RWU inference is often severely underdetermined because the number of isotopic tracers is far smaller than the number of potential soil water sources or depth layers, placing fundamental limits on parameter identifiability. Second, RWU estimates are sensitive to model configuration choices, particularly source grouping and prior specification. A key conceptual insight emerging from this review is that so‐called “non‐informative” Dirichlet priors can become strongly informative as the number of sources increases, leading to divergent uptake patterns inferred from the same dataset. Third, inappropriate specification of error structures can misrepresent how isotopic variability is propagated into the likelihood function, inflating posterior uncertainty or biasing inferred RWU proportions. Looking forward, we argue that further progress in BMM‐based RWU inference requires moving beyond discrete, depth‐resolved formulations toward physically grounded and vertically continuous inference frameworks with well‐justified error structures. Such developments, together with explicit consideration of identifiability and model dimensionality, are essential for the robust use of hydrogen and oxygen stable isotopes in quantifying root water uptake patterns. This article is categorized under: Science of Water > Hydrological Processes Science of Water > Methods

Shifts in tree water sources are important for understanding the spatiotemporal dynamics of ecosystem water fluxes. However, our understanding of tree water uptake remains limited, constraining reliable predictions of local and global hydrological processes under ongoing climate change. The isotopic composition of water (δ2H and δ18O) is a powerful tracer of the Earth’s water cycle, as isotopic differences among water reservoirs, together with mixing and fractionation processes, allow water movements to be traced across the hydraulic continuum.This study aims to characterize the tree water sources of Scots pine (Pinus sylvestris L.) in a Mediterranean forest (Pyrenees, NE Spain) during the 2024 growing season. To do so, the isotopic composition of water in several ecohydrological compartments was measured. Precipitation, soil water pools at multiple depths (10, 20, 30, 40, and 60 cm), and xylem water from four individuals were sampled biweekly. Bulk soil water was extracted using cryogenic vacuum distillation, whereas xylem water was obtained using a flow-rotor centrifuge (cavitron). The cavitron enables access to mobile xylem water (e.g., sap) and is not affected by the well-known methodological artifacts associated with cryogenic extraction. Bayesian isotope mixing models were applied to quantify the relative contributions of distinct water pools to xylem water and their temporal evolution. Dynamics of total water uptake were estimated from transpiration data.Our results show that Scots pine predominantly relied on shallow soil water (10 cm) during most of the growing season, with xylem water closely reflecting the isotopic signature of recent precipitation. A decoupling between the isotopic signature of precipitation and xylem water emerged as seasonal drying progressed. Under dry conditions, tree water uptake was low, and tree water sources shifted towards deeper soil layers (40-60 cm). Overall, these patterns indicate a strong coupling between rainfall inputs and tree water use during periods of high transpiration demand, suggesting that the contribution of deeper soil water reserves represent only a very small fraction of tree total water use during a growing season. These findings underscore the ecological importance of shallow soil water and recent precipitation in sustaining forest function and highlight the role of vegetation water use in regulating atmospheric water fluxes.

The ample diversity in grapevine genotypes and their contrasting physiological responses to drought can be used by winegrowers to adapt to the increasingly drier conditions imposed by climate change. Still, the exposure to drought is more strongly associated with the rootstock genotype onto which grape scions are grafted. Indeed, certain rootstocks help maintain higher yields in drought conditions. Although some morphological, developmental, and physiological rootstock traits have been associated with greater drought resistance, information on how grapevine rootstocks access and use belowground water sources in field conditions is scarce. Besides, the rootstock-scion interaction could modulate the root growth that may dictate the root depth and root water uptake. Deciphering the grapevine belowground activity is critical to decide which rootstock is more suitable for each agronomic situation. Isotopic tracers (δ18O and δ2H) are a promising tool to study grapevine water sources, but they have been seldom used for phenotyping. Here we aimed to test whether different rootstocks influence the grapevine’s access to water, impacting whole-plant water status, plant growth, and yield, and if the interaction rootstock-scion has a role in the magnitude of this impact. For that, we combined isotopic-based determination of grapevine water sources with the monitoring of physiological and agronomical performance in an experimental vineyard with 9 rootstock-scion combinations (3 rootstocks x 3 scions). We found that the above-ground plant water use was mainly governed by the scion (e.g. midday water potential), whereas the rootstock has significant effects on the exposure to drought (e.g. predawn water potential). Surprisingly, isotopic tracers revealed that all rootstocks were accessing a similar belowground water pool, despite different predawn water potentials. This suggests that the lower exposure to drought offered by one of the studied rootstocks (110-R) is not caused by a deeper root system, but by a more efficient water uptake provided by non-explored morphological, anatomical, or physiological traits. In addition, we observed that plants rapidly took water up after the first substantial rain events following the summer drought, around harvest. Overall, we show that isotopic techniques can provide information on grapevine’s access to water that can guide vineyard management and rootstock phenotyping.

Temperate forests on their warm and dry distribution limits are expected to be most vulnerable to reductions in water availability. This prediction is mostly based on studies assessing single forest functions, mainly growth. Water and nutrient cycling are functions that rely on tree roots and their symbiotic association with ectomycorrhizal (ECM) fungi. Trees can compensate for seasonal reductions in water availability by shifting root water-uptake (RWU) towards deeper soil layers, but ECM fungi mostly dwell in the topsoil, thus suffering from desiccation and compromising nutrient uptake. We hypothesised that drier sites should depict larger seasonal shifts in RWU, but at the expense of lower ECM fungal diversity and colonization of fine roots by ECM fungi. We selected three beech Fagus sylvatica forests in their warm distribution limit with contrasting geographic locations and mean annual precipitation: northern Atlantic (2500 mm), intermediate transitional (1150 mm) and southern Mediterranean (780 mm). We collected soil, stem and root samples in spring (wet) and summer (dry) to: 1) quantify fine-root density and colonization by ECM fungi, 2) infer RWU from isotopic composition of plant and soil water and 3) characterize ECM communities through DNA-metabarcoding. Generalized and linear mixed models revealed that high topsoil moisture benefited ECM diversity, but higher diversity and ECM colonization did not imply larger contributions of the topsoil to RWU. The prevailing climate and abiotic conditions determined how ECM communities were structured, more than seasonal climatic variability. Across sites, communities differed in their functional diversity: ECM fungi with long hyphae, more vulnerable to water scarcity, dominated at the southernmost site, where water availability was the highest. Our results suggest that, in a climate change scenario, increasing drought might not compromise RWU, but it would still be detrimental for ECM communities, compromising key ecosystem services such as nutrient cycling and productivity.

AimClimate change challenges temperate forest trees by increasingly irregular precipitation and rising temperatures. Due to long generation cycles, trees cannot quickly adapt genetically. Hence, the persistence of tree populations in the face of ongoing climate change depends largely on phenotypic variation, that is the capability of a genotype to express variable phenotypes under different environmental conditions, known as plasticity. We aimed to quantify phenotypic variation of central Europe's naturally dominant forest tree across various intraspecific scales (individuals, mother trees (families), populations) to evaluate its potential to respond to changing climatic conditions.LocationEurope.Time Period2016-2019.Major Taxa StudiedEuropean beech (Fagus sylvatica L.).MethodsWe conducted a fully reciprocal transplantation experiment with more than 9000 beech seeds from seven populations across a Europe-wide gradient. We compared morphological (Specific Leaf Area), phenological (leaf unfolding) and fitness-related (growth, survival) traits across various biological scales: within single mother trees, within populations and across different populations under the contrasting climates of the translocation sites.ResultsThe experiment revealed significant phenotypic variation within the offspring of each mother tree, regardless of geographic origin. Initially, seedling height growth varied among mother trees and populations, likely due to maternal effects. However, the growth performance successively aligned after the first year. In summary, we observed a consistent growth response in different beech populations to diverse environments after initial maternal effects.Main ConclusionsThe study strikingly demonstrates the importance of considering intraspecific variation. Given the surprisingly broad spectrum of phenotypes each mother tree holds within its juvenile offspring, we conclude that Fagus sylvatica might have the potential for medium-term population persistence in face of climate change, provided that this pattern persists into later life stages. Hence, we also suggest further investigating the inclusion of passive adaptation and natural dynamics in the adaptive management of forests.

Riparian corridors often act as low-land climate refugia for temperate tree species in their southern distribution range. A plausible mechanism is the buffering of regional climate extremes by local physiographic and biotic factors. We tested this idea using a 3-year-long microclimate dataset collected along the Ciron river, a refugia for European beech ( Fagus sylvatica ) in southwestern France. Across the whole network, canopy gap fraction was the main predictor for spatial microclimatic variations, together with two other landscape features (elevation above the river and woodland fraction within a 300m radius). However, within the riparian forest only (canopy gap fraction < 25%, distance to the river < 150m), variations of up to -4°C and + 15% in summertime daily maximum air temperature and minimum relative humidity, respectively, were still found from the plateau to the cooler, moister river banks, only ~ 5-10m below. Elevation above the river was then identified as the main predictor, and explained the marked variations from the plateau to the banks much better than canopy gap fraction. The microclimate measured near the river is as cool but moister than the macroclimate encountered at 700-1000m asl further east in F. sylvatic a's main distribution range. Indeed, at all locations, we found that air relative humidity was higher than expected from a temperature-only effect, suggesting that extra moisture is brought by the river. Our results explain well why beech trees in this climate refugium are restricted to the river gorges where microtopographic variations are the strongest and canopy gaps are rare.

Cryogenic vacuum distillation (CVD) is a widely used technique for extracting plant water from stems for isotopic analysis, but concerns about potential isotopic biases have emerged. Here, we leverage the Cavitron centrifugation technique to extract xylem water and compare its isotopic signature to that of CVD-extracted bulk stem water as well as source water. Conducted under field conditions in tropical northern Australia, our study spans seven tree species naturally experiencing a range of water stress levels. Our findings reveal a significant deuterium bias in CVD-extracted bulk stem water when compared to xylem water (median bias -14.9 parts per thousand), whereas xylem water closely aligned with source water (median offset -1.9 parts per thousand). We find substantial variations in deuterium bias among the seven tree species (bias ranging from -19.3 parts per thousand to -9.1 parts per thousand), but intriguingly, CVD-induced biases were unrelated to environmental factors such as relative stem water content and predawn leaf water potential. These results imply that inter-specific differences may be driven by anatomical traits rather than tree hydraulic functioning. Additionally, our data highlight the potential to use a site-specific deuterium offset, based on the isotopic signature of local source water, for correcting CVD-induced biases. This paper addresses the drivers of the deuterium bias associated with cryogenic vacuum distillation (CVD) of stem water. Using the Cavitron centrifugation technique to extract xylem water and compare its isotopic signature to that of CVD-extracted bulk stem water, we find substantial variations in deuterium bias among seven tree species under field conditions. Interestingly, the deuterium bias exhibits no correlation with environmental factors such as relative stem water content and drought stress (as estimated by pre-dawn leaf water potentials). image

Journal Article Origin and fate of carbon and nitrogen reserves in trees Get access Adrià Barbeta, Adrià Barbeta Department of Evolutionary Biology, Ecology and Environmental Sciences, University of Barcelona, Edifici Margalef, Facultat de Biologia, Diagonal, 643, E-08028 Barcelona, Catalonia, Spain Corresponding authors (adria.barbeta.margarit@gmail.com; eli.martinez@ub.edu) Search for other works by this author on: Oxford Academic PubMed Google Scholar Elisabet Martínez-Sancho Elisabet Martínez-Sancho Department of Evolutionary Biology, Ecology and Environmental Sciences, University of Barcelona, Edifici Margalef, Facultat de Biologia, Diagonal, 643, E-08028 Barcelona, Catalonia, Spain Corresponding authors (adria.barbeta.margarit@gmail.com; eli.martinez@ub.edu) Search for other works by this author on: Oxford Academic PubMed Google Scholar Tree Physiology, Volume 44, Issue 3, March 2024, tpae021, https://doi.org/10.1093/treephys/tpae021 Published: 13 February 2024 Article history Received: 27 November 2023 Accepted: 07 February 2024 Published: 13 February 2024 Corrected and typeset: 14 March 2024

The analysis of the stable isotopic composition of hydrogen and oxygen in water samples from soils and plants can help to identify sources of vegetation water uptake. This approach requires that the heterogeneous nature of plant and soil matrices is carefully accounted for during experimental design, sample collection, water extraction and analyses. The comparability and shortcomings of the different methods for extracting water and analyzing isotopic composition have been discussed in specialized literature. Yet, despite insightful comparisons of extraction methods and benchmarking methodologies of laboratories worldwide, the community still lacks a roadmap to guide sample collection, extraction, and isotopic analyses, and many practical issues for potential users remain unresolved: for example, which (soil or plant) water pool(s) does the extracted water represent? These constitute a hurdle for the implementation of the approach by newcomers. Here, we summarize discussions led in the framework of the COST Action WATSON ("WATer isotopeS in the critical zONe: from groundwater recharge to plant transpiration"-CA19120). We provide guidelines for (1) sampling soil and plant material for isotopic analysis, (2) methods for laboratory or in situ water extraction, and (3) measurements of isotopic composition. We highlight the importance of considering the process chain as a whole, from experimental design to isotopic analysis to minimize biased estimates of the relative contribution of different water sources to plant water uptake. We conclude by acknowledging some of the limitations of this methodology and advice on the collection of key environmental parameters prior to sample collection for isotopic analyses. This article is categorized under: Science of Water > Hydrological Processes Science of Water > Water and Environmental Change Science of Water > Water Extremes

Temperate forests on their warm and dry distribution limit are expected to be most vulnerable to reductions in water availability. This prediction is mostly based on studies assessing single forest functions, mainly growth. Water and nutrient cycling are functions that rely on tree roots and their symbiotic association with ectomycorrhizal (ECM) fungi. Trees can compensate for seasonal reductions in water availability by shifting root water-uptake (RWU) towards deeper soil layers, but ECM fungi dwell in the upper soil, thus suffering from desiccation and compromising nutrient uptake. We hypothesised that drier sites should depict larger seasonal shifts in RWU, but at the expense of lower diversity and colonization of fine roots by ECM fungi. We selected three beech ( Fagus sylvatica ) forests in their warm distribution limit with contrasting geographic locations and mean annual precipitation: northern Atlantic (2500mm), intermediate transitional (1150mm) and southern Mediterranean (780mm). We collected soil, stem and root samples in spring (wet) and summer (dry) to quantify fine-root density and colonization by ECM fungi, to infer RWU from isotopic composition of plant and soil water, and to characterize ECM fungal diversity through DNA-metabarcoding. High moisture in the upper soil benefited the ECM community, but higher diversity and fine-root colonization by ECM fungi in the upper soil did not imply larger contributions of this soil layer to RWU. The prevailing climate and local abiotic conditions determined how ECM communities structured, more than seasonal variability. Across sites, ECM communities differed in their functional diversity: ECM fungi with long hyphae, more vulnerable to water scarcity, dominated at the site with the highest water availability. Our results suggest that transient reductions in soil water availability might not compromise RWU but could be detrimental for maintaining ECM-mediated nutrient uptake in beech forests experiencing longer and more severe drought periods under current climate change. ### Competing Interest Statement The authors have declared no competing interest.

Summary A fundamental assumption when using hydrogen and oxygen stable isotopes to understand ecohydrological processes is that no isotope fractionation occurs during plant water uptake/transport/redistribution. A growing body of evidence has indicated that hydrogen isotope fractionation occurs in certain environments or for certain plant species. However, whether the plant water source hydrogen isotope offset (δ 2 H offset) is a common phenomenon and how it varies among different climates and plant functional types remains unclear. Here, we demonstrated the presence of positive, negative, and zero offsets based on extensive observations of 12 plant species of 635 paired stable isotopic compositions along a strong climate gradient within an inland river basin. Both temperature and relative humidity affected δ 2 H offsets. In cool and moist environments, temperature mainly affected δ 2 H offsets negatively due to its role in physiological activity. In warm and dry environments, relative humidity mainly affected δ 2 H offsets, likely by impacting plant leaf stomatal conductance. These δ 2 H offsets also showed substantial linkages with leaf water 18 O enrichment, an indicator of transpiration and evaporative demand. Further studies focusing on the ecophysiological and biochemical understanding of plant δ 2 H dynamics under specific environments are essential for understanding regional ecohydrological processes and for conducting paleoclimate reconstructions.

Present and future climatic trends are expected to markedly alter water fluxes and stores in the hydrologic cycle. In addition, water demand continues to grow due to increased human use and a growing population. Sustainably managing water resources requires a thorough understanding of water storage and flow in natural, agricultural, and urban ecosystems. Measurements of stable isotopes of water (hydrogen and oxygen) in the water cycle (atmosphere, soils, plants, surface water, and groundwater) can provide information on the transport pathways, sourcing, dynamics, ages, and storage pools of water that is difficult to obtain with other techniques. However, the potential of these techniques for practical questions has not been fully exploited yet. Here, we outline the benefits and limitations of potential applications of stable isotope methods useful to water managers, farmers, and other stakeholders. We also describe several case studies demonstrating how stable isotopes of water can support water management decision-making. Finally, we propose a workflow that guides users through a sequence of decisions required to apply stable isotope methods to examples of water management issues. We call for ongoing dialogue and a stronger connection between water management stakeholders and water stable isotope practitioners to identify the most pressing issues and develop best-practice guidelines to apply these techniques.

In recent years, the widespread use of laser-based analyzers of the isotopic composition of water (δ18O and δ2H) resulted in an increase in the temporal and spatial resolution of measurements of plant water and their sources. Such datasets revealed previously undetected mismatches between the isotopic composition of subsurface water pools and bulk xylem water usually extracted by cryogenic distillation. To understand the underlying cause of these isotopic mismatches, plant ecophysiologists and ecohydrologists have conducted numerous experiments to address a range of hypotheses. Measurement artifacts produced by water extraction techniques in both bulk xylem water and soil water were claimed to be behind the observed mismatches. However, there is not yet a consensus on a sole mechanism to explain all cases. On the other hand, our research demonstrated the existence of isotopic heterogeneities between the water in different xylem compartments, which also have contrasting degrees of hydraulic connectivity with the transpiration stream. Analogous isotopic patterns were observed in soil water pools and attributed to physicochemical interactions with soil particles. Altogether, it seems that the water pools that are measured matter, and that not all isotopic mismatches can be attributed to methodological artifacts. Given the widespread occurrence of these isotopic mismatches, it is urgent to identify the cause, either natural, artificial, or both. This will allow us to make informed choices of the extraction techniques in each situation and eventually, we could be able to correct potentially biased old datasets. In this regard, we will summarize the most recent findings and suggest research strategies to unravel the underlying mechanisms of isotopic mismatches. In addition, we will outline how such strategies can also provide important insights for closely related disciplines such as plant hydraulics or isotopic analyses of tree-ring archives.

The thermal balance of forests is the result of complex land–atmosphere interactions. Different climate regimes and plant functional types can have contrasting energy budgets, but little is known about the influence of forest structure and functional traits. Here, we combined spaceborne measurements of surface temperature from ECOSTRESS with ground‐based meteorological data to estimate the thermal balance at the surface (∆ T can−air ) during four summers (2018–2021), at the Mediterranean–temperate ecotone in the NE Iberian Peninsula. We analyzed the spatiotemporal drivers of ∆ T can−air by quantifying the effects of meteorology, forest structure (stand density, tree height) and ecophysiology (hydraulic traits), during normal days and hot spells. Canopy temperatures ( T can ) fluctuated according to changes in air temperature ( T air ) but were on average 4.2 K warmer. During hot spells, ∆ T can−air was smaller than during normal periods. We attribute this decrease to the advection of hot and dry air masses from the Saharan region resulting in a sudden increase in T air relative to T can . Vapor pressure deficit (VPD) was negatively correlated with ∆ T can−air , since the highest VPD values coincided with peaks in heat advection. Nonetheless, T can increased with VPD due to decreased transpiration (following stomatal closure), even though sufficient soil water availability enabled some degree of evaporative cooling. Our findings demonstrate that plot‐scale forest structural and hydraulic traits are key determinants for the forest thermal balance. The integration of functional traits and forest structure over relevant spatial scales would improve our ability to understand and model land–atmosphere feedbacks in forested regions.

SummaryWe compiled hydrogen and oxygen stable isotope compositions (δ2H and δ18O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ2H was more closely correlated with δ2H of xylem water or atmospheric vapour, whereas leaf water δ18O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ2H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ18O. We next expressed leaf water as isotopic enrichment above xylem water (Δ2H and Δ18O) to remove the impact of xylem water isotopic variation. For Δ2H, leaf water still correlated with atmospheric vapour, whereas Δ18O showed no such correlation. This was explained by covariance between air relative humidity and the Δ18O of atmospheric vapour. This is consistent with a previously observed diurnal correlation between air relative humidity and the deuterium excess of atmospheric vapour across a range of ecosystems. We conclude that 2H and 18O in leaf water do indeed reflect the balance of environmental drivers differently; our results have implications for understanding isotopic effects associated with water cycling in terrestrial ecosystems and for inferring environmental change from isotopic biomarkers that act as proxies for leaf water.

Alternative water uptake pathways through leaves and bark complement water supply with interception, fog or dew. Bark water-uptake contributes to embolism-repair, as demonstrated in cut branches. We tested whether bark water-uptake could also contribute to supplement xylem-water for transpiration. We applied bandages injected with 2 H-enriched water on intact upper-canopy branches of Pinus sylvestris and Fagus sylvatica in a boreal and in a temperate forest, in summer and winter, and monitored transpiration and online isotopic composition (δ2 H and δ18 O) of water vapour, before sampling for analyses of δ2 H and δ18 O in tissue waters. Xylem, bark and leaf waters from segments downstream from the bandages were 2 H-enriched whereas δ18 O was similar to controls. Transpiration was positively correlated with 2 H-enrichment. Isotopic compositions of transpiration and xylem water allowed us to calculate isotopic exchange through the bark via vapour exchange, which was negligible in comparison to estimated bark water-uptake, suggesting that water-uptake occurred via liquid phase. Results were consistent across species, forests and seasons, indicating that bark water-uptake may be more ubiquitous than previously considered. We suggest that water taken up through the bark could be incorporated into the transpiration stream, which could imply that sap-flow measurements underestimate transpiration when bark is wet.

We compiled hydrogen and oxygen stable isotope compositions (δ2 H and δ18 O) of leaf water from multiple biomes to examine variations with environmental drivers. Leaf water δ2 H was more closely correlated with δ2 H of xylem water or atmospheric vapour, whereas leaf water δ18 O was more closely correlated with air relative humidity. This resulted from the larger proportional range for δ2 H of meteoric waters relative to the extent of leaf water evaporative enrichment compared with δ18 O. We next expressed leaf water as isotopic enrichment above xylem water (Δ2 H and Δ18 O) to remove the impact of xylem water isotopic variation. For Δ2 H, leaf water still correlated with atmospheric vapour, whereas Δ18 O showed no such correlation. This was explained by covariance between air relative humidity and the Δ18 O of atmospheric vapour. This is consistent with a previously observed diurnal correlation between air relative humidity and the deuterium excess of atmospheric vapour across a range of ecosystems. We conclude that 2 H and 18 O in leaf water do indeed reflect the balance of environmental drivers differently; our results have implications for understanding isotopic effects associated with water cycling in terrestrial ecosystems and for inferring environmental change from isotopic biomarkers that act as proxies for leaf water.

Summary The long‐standing hypothesis that the isotopic composition of plant stem water reflects that of source water is being challenged by studies reporting bulk water from woody stems with an isotopic composition that cannot be attributed to any potential water source. The mechanism behind such source–stem water isotopic offsets is still poorly understood. Using a novel technique to extract selectively sap water from xylem conduits, we show that, in cut stems and potted plants, the isotopic composition of sap water reflects that of irrigation water, demonstrating unambiguously that no isotopic fractionation occurs during root water uptake or sap water extraction. By contrast, water in nonconductive xylem tissues is always depleted in deuterium compared with sap water, irrespective of wood anatomy. Previous studies have shown that isotopic heterogeneity also exists in soils at the pore scale in which water adsorbed onto soil particles is more depleted in deuterium than unbound water. Data collected at a riparian forest indicated that sap water matches best unbound soil water from depth below −70 cm, while bulk stem and soil water differ markedly. We conclude that source–stem isotopic offsets can be explained by micrometre‐scale heterogeneity in the isotope ratios of water within woody stems and soil micro‐pores.

Thermal balance of forests in the Mediterranean-temperate ecotone. This dataset comprises the data used in the manuscript "Disentangling the role of Forest structure and functional traits for the thermal balance in the Mediterranean–Temperate Ecotone", to be submitted to a scientific journal shortly after the publication date of this dataset. Data comprise 54 variables including case categorization, meteorological, climatic and forest structural variables and the thermal balance of the forest estimated from ECOSTRESS remote sensing measurements.