Trees are powerful mediators within ecosystem water and nutrient cycles. Through their roots, they take up these essential resources from the soil, distributing them in the system and upward into the canopy to maintain transpiration and photosynthesis.However, understanding and predicting real-world dynamics remains challenging: tree species identity, species mixture, site and soil conditions may shape tree water and nutrient uptake fundamentally, particularly in mature forests. Moreover, in the face of climate change, access to resources in deeper, less drought-prone soil layers is crucial for buffering drought impacts and maintaining forest functioning. Studies targeting tree resource uptake in mature forests are still scarce; but they are emerging, with stable isotopes as a central tool.We investigated root water uptake depth and subsoil water and nitrogen uptake in mature temperate forests of north-western Germany, using 2H, 18O and 15N as tracers. Native European beech, non-native Douglas fir, and native but drought-sensitive Norway spruce were studied, revealing tree species-specific uptake strategies and influences of species mixture. Furthermore, we found consistent site effects: on well-drained, sandy soils, the trees integrated more resources from deeper layers than on loamy soils. Notably, transit times from soil to canopy were slower for nitrogen than for water, highlighting the biotic and abiotic interactions that decouple nitrogen from water.We conclude that species-specific traits in interaction with soil characteristics are crucial for understanding and predicting water and nutrient fluxes in forests. Our findings underscore the importance of belowground processes when assessing forest functioning and resilience.

Consecutive dry periods (e.g., 2014–2016, 2018–2019, 2022) led to persistent long-term impairments in maintaining tree functions such as growth and canopy structure thereby exacerbating drought stress and mortality in temperate forests. Despite growing attention to compound drought impacts on forest ecosystems, the role of deep-water sources at varying positions on hillslopes remains unclear.In this study, we investigated how hillslope position influences growth dynamics and water-use strategies of co-occurring tree species in an unmanaged, structurally diverse forest stand in Lower Saxony, Germany. The stand is composed of the broadleaf deciduous tree species Fagus sylvatica (L.), Carpinus betulus (L.), Fraxinus excelsior (L.), and Quercus robur (L.) which differ in their root structure, stomatal regulation and growth strategies. Over the three years (2023-2025) we employed continuous point-dendrometer, sap flow and soil moisture measurements to monitor growth, soil and stand water use and water potential. Further destructive samples for verifying water potential and stable carbon isotopes of phloem sap were measured.We found that growth patterns were strongly species-specific and closely aligned with contrasting tree water-use strategies. Despite similar climatic conditions in 2023 and 2024, pronounced interannual differences in growth were observed. These differences suggest a delayed recovery from previous long-term drought events (2018-2022), particularly for the shallow-rooted species F. sylvatica, C. betulus and F. excelsior, compared to deep-rooted Q. robur, highlighting long-term effects of compound droughts on productivity. It was notable that the species were able to adapt their strategies according to their position. Additionally, we observed in F. excelsior and Q. robur that high growth rates can be supported by using water storage (e.g., via deep roots and access to deep water sources or via stem water use) or by maintaining high transpiration rates during drought at the risk of cavitation. In conclusion, we postulate that drought mitigation strategies not only depend on species traits, but also on tree positioning and climatic conditions.

Tree rings provide long-term records of tree growth and climate changes, which makes them ideal benchmarks for forest modeling. Tree-ring information has greatly improved the reliability of 3-PG, which is one of the most commonly used process-based forest growth models. Here, we strengthen 3-PG's ability to simulate tree-ring width and stable carbon isotopes (δ13C) by enhancing its descriptions of tree physiology. The major upgrade was adding a carbon storage pool for tree-ring formation using stored carbohydrates. We also incorporated previous modifications (replacing the age modifier with a height modifier) of 3-PG and tested their efficacy in improving tree-ring simulations. We ran the model based on two grand fir (Abies grandis) stands. The updated model greatly improved the simulations for both tree-ring widths and δ13C. The results represent one of the best tree-ring δ13C simulations, which accurately captured the amplitude in annual variations of δ13C. The correlations (R2) between simulations and observations reached 0.50 and 0.73 ​at two stands, respectively. The new model also greatly improved the simulations of raw tree-ring widths and detrended ring-width index (RWI). Because of better descriptions of tree physiology and more accurate simulations of tree rings than the previous model version, the updated 3-PG should provide more reliable simulations than previous 3-PG versions when tree-ring information is used as a benchmark in future studies.

Ongoing changes in climate alter the role of forests in the hydrologic cycle, influencing water transmission to springs and aquifers. Here we compared two forests dominated by either beech or spruce on broadly similar soils (Dystric Cambisols); we monitored the passage of natural-abundance stable isotope signals through the upper meter of soil and onward to springs. The isotopic data were similar between the sites at every time step and at every stage of transit, except at 90-100 cm depth, where the isotopic signal of the beech forest was delayed by approximately 1 month. The data were used in a lumped parameter dispersion model so that physical parameters describing transport could be determined and compared. Modeled residence times were similar between the two forests (123 (sd = 32) vs. 152 (25) days), with high precision to depths of 40 cm. According to the model, rainfall reached 1 meter depth in 200 (8) days under the spruce stand, but required 228 (37) days in the beech. The measurements below the rooting zone (90-100 cm) play a critical role in detecting site/species differences and in prediction of residence times.

Leaf and canopy temperature have long been recognized as important indicators of plant water status because leaves cool when water is transpired and warm up when leaf stomata close and transpiration is reduced. Unmanned aerial vehicles (UAVs) open up the possibility to capture high resolution thermal images of forest canopies at the leaf scale. However, a careful calibration procedure is required to convert the thermal images to absolute temperatures, in addition, at high spatial resolution, the complexity of forest canopies leads to challenges in stitching overlapping thermal images into an orthomosaic of the forest site. In this study, we present a novel flight planning approach in which the locations of ground temperature references are directly integrated in the flight plan. Six UAV flight campaigns were conducted over a tropical dry forest in Costa Rica. For each flight five different calibration methods were tested. The most accurate calibration was used to analyze the tree canopy temperature distributions of five tree species. From the distribution we correlated its mean, variance, 5th and 95th percentile against individual tree transpiration estimates derived from sapflow measurements. Our results show that the commonly applied calibration provided by the cameras manufacturer (factory calibration) and empirical line calibration were less accurate than the novel repeated empirical line calibration and the factory calibration including drift correction (MAE 3.5°C vs. MAE 1.5°C). We show that the orthomosaic is computable by directly estimating the thermal image orientation from the visible images during the structure from motion step. We found the 5th percentile of the canopy temperature distribution, corresponding to the shaded leaves within the canopy, to be a better predictor of tree transpiration than the mean canopy temperature (R2 0.85 vs. R2 0.60). Although these shaded leaves are not representative of the whole canopy, they may be the main transpiration site in the heat of the day. Spatially high-resolution, validated temperature data of forest canopies at the leaf scale have many applications for ecohydrological questions, e.g., the estimation of transpiration, for comparing plant traits and modeling of carbon and water fluxes by considering the entire canopy temperature distribution in mixed-species forests.

Abstract. Water labeled with stable isotopes provides a conservative tracer, being neither produced nor consumed, for water flowpaths within soils and root systems. We added a strong, evenly distributed 2HHO label to one m2 of soil surface and continuously monitored its passage downward into the soil and upward into the stems of surrounding trees, with the objective of illuminating spatiotemporal lateral root water uptake and overlap. The study was conducted during the historic drought of 2018 in a mature Scots pine (Pinus sylvestris) forest growing on sandy soil in northern Sweden. Continuous in situ isotopic measurements of tree xylem water evidenced root system overlap of six trees within the labeled square meter. This result is consistent with previous estimates from labelled nutrient uptake measurements at this site. However, label uptake differed sharply among trees, even within the same radius; 90 % of the label was taken up by one of the two trees closest to the labelled plot. Horizontal transport rates in tree roots averaged 0.17 ± 0.05 m d-1, meaning that the arrival of label pulse in tree stems was delayed by 6–33 days from first tree to last. Root water uptake by trees appeared restricted to the upper 60 cm of mineral soil, even at the peak of the drought. Label intensity of the mineral soil weakened throughout the drought, consistent with the notion that the label was being dispersed or diluted. Labeled water recovery was low (3–4 %), and we hypothesize that this was due to a significant upward flux of water into the organic surface horizons. Our data provide a daily and three-dimensional description of the passage of a labeled water pulse, highlighting the heterogeneity in horizontal water transport flowpaths and the uneven partitioning of label among individual trees in a boreal forest.

Riparian trees are particularly vulnerable to drought because they are highly dependent on water availability for their survival. However, the response of riparian tree species to water stress varies depending on regional hydroclimatic conditions, making them unevenly vulnerable to changing drought patterns. Understanding this spatial variability in stress responses requires a comprehensive assessment of water stress across broader spatial and temporal scales. Yet, the precise ecophysiological mechanisms underlying these responses remain poorly linked to remotely sensed indices. To address this gap, the implementation of remote sensing methods coupled with in situ validation is essential to obtain consistent results across diverse spatial and temporal contexts. We conducted a multi-tool analysis combining multispectral and thermal remote sensing indices with in situ ecophysiological measurements at different temporal scales to analyze the responses of white poplar (Populus alba) to seasonal changes in drought along a hydroclimatic gradient.Using this approach, we demonstrate that white poplars along the Rhône River (France) exhibit contrasting responses and behaviors during drought depending on the latitudinal context. White poplars in a Mediterranean climate show rapid stomatal closure to reduce water loss and maintain high minimum water potential levels, although this results in a decrease in remotely sensed greenness. Conversely, white poplars located upstream in a temperate climate show high transpiration and stable greenness but lower minimum water potential and water content. A site in the middle of the gradient has intermediate responses. These results demonstrate that white poplars along a climate gradient can have a range of responses to drought along the iso/anisohydricity continuum.These results are important for future climatic conditions because they show that the same species can have different mechanisms of drought resilience, even in the same river valley. This raises questions regarding how these riparian tree populations will respond to future climatic and hydrological conditions.

Tree rings are an emerging atmospheric mercury (Hg) archive. Questions have arisen, though, regarding their mechanistic controls and reliability. Here, we report contrasting tree-ring Hg records in three collocated conifer species: Norway spruce (Picea abies), Scots pine (Pinus sylvestris), and European larch (Larix decidua), which are from a remote boreal forest. Centennial atmospheric Hg trends at the site, derived from varved lake sediments, peats, and atmospheric monitoring, indicated a steady rise from the 1800s, peaking in the 1970s, and then declining. Prior to ca. 2005, larch and spruce tree rings reproduced the peak in the atmospheric Hg trend, while pine tree rings peaked in the 1930s, likely due to the prolonged sapwood period and ambiguity in the heartwood-sapwood boundary of pine. Since ca. 2005, tree rings from all species showed increasing Hg concentrations in the physiologically active outer rings despite declining atmospheric Hg concentrations. The good agreement between Hg and nitrogen concentrations in active tree-ring cells indicates a similar transport mechanism and cautions against their applicability as atmospheric Hg archives. Our results suggest that tree-ring Hg records are controlled by atmospheric Hg and tree physiology. We provide recommendations for using tree-ring Hg archives that take tree physiology into account.

We compared three methods of estimating gross primary production (GPP) of a boreal forest dominated by spruce and pine with the goals of 1) converging on the best estimate and 2) disaggregating the GPP among the two canopy species and the understory stratum. The three methods were: 1) eddy covariance (EC), 2) a soil-vegetation-atmosphere transfer model, APES, driven by meteorological data, and 3) an ecophysiological approach (Iso/SF) based on sap flux and phloem delta C-13, where sap flux is used to estimate stomatal conductance and delta C-13 is used to estimate intrinsic water-use efficiency (WUEi). The EC and APES methods agreed rather well, which was expected because APES was developed to predict eddy covariance data. The Iso/SF method, which is based on independent data, yielded lower estimates. This was partly because it excluded understory vegetation from the GPP estimate. We also found that the measured sap flux/transpiration estimates for spruce in Iso/SF were much lower than those from APES. In contrast, the absolute values for Scots pines were very similar between the two methods, especially in the summer. In both species, the seasonal dynamics match well among all methods. This multi-method approach allowed us to detect possible problems in the spruce sap-flux measurements, but successfully upscaled pine data from ecophysiological traits to stand and ecosystem functioning.

Large trees in plantations generally produce more wood per unit of resource use than small trees. Two processes may account for this pattern: greater photosynthetic resource use efficiency or greater partitioning of carbon to wood production. We estimated gross primary production (GPP) at the individual scale by combining transpiration with photosynthetic water-use efficiency of Eucalyptus trees. Aboveground production fluxes were estimated using allometric equations and modeled respiration; total belowground carbon fluxes (TBCF) were estimated by subtracting aboveground fluxes from GPP. Partitioning was estimated by dividing component fluxes by GPP. Dominant trees produced almost three times as much wood as suppressed trees. They used 25 ± 10% (mean ± SD) of their photosynthates for wood production, whereas suppressed trees only used 12 ± 2%. By contrast, dominant trees used 27 ± 19% of their photosynthate belowground, whereas suppressed trees used 58 ± 5%. Intermediate trees lay between these extremes. Photosynthetic water-use efficiency of dominant trees was c. 13% greater than the efficiency of suppressed trees. Suppressed trees used more than twice as much of their photosynthate belowground and less than half as much aboveground compared with dominant trees. Differences in carbon partitioning were much greater than differences in GPP or photosynthetic water-use efficiency.

Carbon dioxide sequestration from the atmosphere is commonly assessed using the eddy covariance method. Its net flux signal can be decomposed into gross primary production and ecosystem respiration components, but these have seldom been tested against independent methods. In addition, eddy covariance lacks the ability to partition carbon sequestration among individual trees or species within mixed forests. Therefore, we compared gross primary production from eddy covariance versus an independent method based on sap flow and water-use efficiency, as measured by the tissue heat balance method and δ13C of phloem contents, respectively. The latter measurements were conducted on individual trees throughout a growing season in a mixed broadleaf forest dominated by three tree species, namely English oak, narrow-leaved ash and common hornbeam (Quercus robur L., Fraxinus angustifolia Vahl, and Carpinus betulus L., respectively). In this context, we applied an alternative ecophysiological method aimed at verifying the accuracy of a state-of-the-art eddy covariance system while also offering a solution to the partitioning problem. We observed strong agreement in the ecosystem gross primary production estimates (R2 = 0.56; P < 0.0001), with correlation being especially high and nearly on the 1:1 line in the period before the end of July (R2 = 0.85; P < 0.0001). After this period, the estimates of gross primary production began to diverge. Possible reasons for the divergence are discussed, focusing especially on phenology and the limitation of the isotopic data. English oak showed the highest per-tree daily photosynthetic rates among tree species, but the smaller, more abundant common hornbeam contributed most to the stand-level summation, especially early in the spring. These findings provide a rigorous test of the methods and the species-level photosynthesis offers avenues for enhancing forest management aimed at carbon sequestration.

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.

To understand the responses of forest carbon (C) cycling to environmental change, it is important to attribute the observed C release to the correct respiratory sources. One area where considerable uncertainty still exists is the role of the xylem in transporting CO2 from the roots and/or soil to be released from, or assimilated by, the stems or the foliage (e.g. Hölttä & Kolari, 2009; Bloemen et al., 2013; Stutz et al., 2017; Salomón et al., 2021; Tarvainen et al., 2021). While several studies have quantified the magnitude of CO2 transport by the xylem sap of different tree species, they have yielded contradictory results regarding the importance of this process for tree-scale C cycling. It has been consistently reported that xylem CO2 transport rates can be considerable in ring-porous and diffuse-porous species with high sap flow rates and small conducting areas (McGuire & Teskey, 2004; Teskey et al., 2008; Bloemen et al., 2013, 2014; Salomón et al., 2019; Mincke et al., 2020). However, xylem transport has been found to be of limited magnitude in large conifers with slow sap flow rates (e.g. Ford et al., 2007; Ubierna et al., 2009; Tarvainen et al., 2021). While differences relating to the ease of water movement through the xylem, such as wood anatomy and sap flow rate, undoubtedly explain some of the among-species variation in xylem CO2 transport, the chemistry of the sap also matters. The CO2 partial pressure (pCO2) of the sap has been reported to vary over a very broad range from near zero up to 26.3% among species and studies (e.g. Teskey et al., 2008; Tarvainen et al., 2014; Salomón et al., 2021), and directly affects the amount of CO2 dissolved in the xylem sap ([CO2*], McGuire & Teskey, 2002) that is available for transport as described by Henry's law (Eqn 1). However, previous observations of pCO2 show that both high and low pCO2 can be found in species with generally higher xylem CO2 transport rates, such as diffuse- and ring-porous species, as well as in those with generally lower transport rates, such as conifers (Teskey et al., 2008). The pH of the xylem sap is another factor that can affect the potential for xylem CO2 transport through its effects on the solubility of CO2 (Eqn 1; Levy et al., 1999; Erda et al., 2014) and is also known to vary among species (e.g. Teskey et al., 2008). Previous studies on trees have reported diel (e.g. Aubrey et al., 2011; Erda et al., 2014; Salomón et al., 2016) and seasonal (e.g. Aubrey et al., 2011; Salomón et al., 2016; Losso et al., 2018; Pramsohler et al., 2022) variation in xylem pH. Such patterns may be linked to several interacting factors known to affect xylem pH including within-tree variation in sap chemistry (Schill et al., 1996), phenological stage (Pramsohler et al., 2022), water and nutrient availabilities (e.g. Gollan et al., 1992; Jackson et al., 2003; Sharp & Davies, 2009; Wang et al., 2012), site elevation (Pramsohler et al., 2022), solar radiation, vapor pressure deficit, and air and soil temperatures (Wilkinson & Davies, 2002, 2008; Wan et al., 2004; Pramsohler et al., 2022). Although the magnitude of, and causes for, within-species variation in xylem pH has received considerable attention, few studies to date have compared xylem pH in tree species from different functional types growing under similar climatic and soil conditions. Thomas & Eamus (2002) studied six Australian savanna species, including two deciduous species, two semi-deciduous species and two evergreens. They found a wet to dry season difference in xylem pH that correlated with changes in leaf water potential in the semi-deciduous and the evergreen species, but not in the deciduous species, suggesting that among-species differences in sap pH are seasonally variable. However, they did not specifically analyze the among-species variation in pH. For nonwoody species, Sharp & Davies (2009) measured the xylem pH of 22 perennial species growing under similar conditions in pots and found a pH range of more than three units. Thus, the previous observations suggest that xylem pH varies over a broad enough range to matter for the CO2 transport rates but do not directly address the question of whether different tree species operate at different xylem pH when exposed to similar conditions. Many tree species have been found to have xylem pH values that can be near the point where solubility of CO2 increases strongly, pH c. 6–6.5 (e.g. Levy et al., 1999; Erda et al., 2014; Fig. 1). Thus, the accuracy of the pH estimates is of key importance for studies analyzing xylem CO2 transport with the commonly-used mass balance approach (e.g. McGuire & Teskey, 2004; Bowman et al., 2005; Salomón et al., 2018; Tarvainen et al., 2021), which relies on Henry's law to calculate [CO2*]. A potential issue with the accuracy of the pH data is the method used for sap extraction. If the sap is forced out of the xylem sample in a way that damages the parenchyma cells, such as crushing the tissue using a vice, the sample may be contaminated by leakage from the damaged cells. If such contamination occurs it will cause an overestimation of sap pH, given that the cytoplasmic pH is in the 7.0–7.4 range (Madshus, 1988). As an alternative, it has been suggested that the displacement method (e.g. Bennett et al., 1927; Glavac et al., 1990), where a water column is used to create pressure to force out the sap from, for example, a cut branch, leads to minimal sample contamination due to the large sap volume collected (Dambrine et al., 1995). However, this method also requires the cutting of cells and is likely to result in some contamination of the first sap collected. To our knowledge, it has not been previously determined whether, and by how much, parenchyma damage during sampling may affect the pH estimates in different tree species, and how this in turn affects the estimates of [CO2*] and xylem CO2 transport. The study was carried out at the Svartberget research station located in Vindeln, northern Sweden (64°42′N, 19°77′E, 260 m asl). The 30-yr mean annual temperature and precipitation in at the site are 1.8°C and 614 mm, respectively (Laudon et al., 2013). The sampling was conducted at an experimental site set up as a blocked design (three blocks) with several tree species. Each sub-block was c. 0.12 ha in size and included 289 trees. Six trees from four species growing at the site were included in this study: Pinus sylvestris (Scots pine), Larix sukaczewii (Siberian larch), Sorbus aucuparia (rowan), and Alnus incana (grey alder). The trees chosen for this study were also used as a part of a nitrogen fertilization experiment where KNO3 fertilizer (corresponding to a single dose of 70 kg N ha−1) was applied around three individuals of each of the four studied species. Thus, the overall design consisted of a total of six individuals of each species, divided into three control and three fertilized trees. The experiment was initiated on 26 August 2015 when the fertilizer was applied. The sampling was carried out 6 d later in the morning of 1 September 2015. We note that nitrogen (especially nitrate) content has been shown to correlate with sap pH in several plant species (e.g. Gollan et al., 1992; Wang et al., 2012). Because of this, the data are presented here separately for the control and fertilized trees. During the sampling, the Scots pine and larch branches were collected from mature trees growing at an experimental site. Since alder and rowan were not included in the original blocked design, the alder branches were collected from mature trees growing adjacent to the blocked experiment and the rowan samples consisted of main stems of younger trees interspersed within the blocked experiment. The selected branches were cut near the trunk and had similar diameters for all species (c. 25 mm at the proximal end). The site is located on a gentle westward facing slope with all the sampled trees thus exposed to similar soil and climatic conditions. After collection, the samples were placed in large opaque plastic bags and transported by car, for c. 1 h, before being stored in cold room (8°C) in darkness until sap extraction. The sap was extracted by the displacement method (e.g. Glavac et al., 1990) as described by Tarvainen et al. (2021). Briefly, the sample branches were recut under water at both ends and the bark removed with a knife before the xylem sap extraction. The length of the branch sections used for the analysis varied among the samples but was generally > 30 cm to allow for sufficient amount of sap to be collected. The branch was fastened to one end of a 25-mm-diameter PVC tube that was mounted vertically above the branch and c. 600 ml deionized water including food colouring (Dr Oetker Sverige AB, Gothenburg, Sweden; blue, pH 3.37) was poured into the tube. The xylem sap was pushed out of the branch by the resulting pressure and collected in a series of successive 1.5-ml Eppendorf vials until colored water arrived. Despite the use of similar external pressures, the sap extraction rates and the total amount of sap extracted per branch were variable, likely in response to the among-species differences in wood anatomy. The pH of each sample was measured immediately following sap extraction with a PHM202 pH meter (Radiometer, Copenhagen, Denmark). The room temperature during the sap extraction and pH measurement was c. 23°C. Analysis of the pH in each vial allowed us to determine the potential biasing effect of the initial parenchyma cell damage during sap extraction on the pH estimates. When analyzing the change in pH with sample number, each pH observation was normalized relative to the average pH of the last five samples collected before dye breakthrough, representing asymptotic steady-state pH. For some branches (one rowan and one larch branch), the dye broke through so early that such averaging was not possible. For three pine branches, some pH observations were likely affected by pitch (which was visible at the cut surface and appeared to cause the pH to increase) and were removed from further analyses. In these cases, we used the pH observed in the single last sample before dye breakthrough or appearance of pitch for all analyses. The steady-state data, showing no evidence of dye breakthrough, were used for the among-species comparison of xylem pH. The effect of species and soil nitrogen availability on sap pH was analysed by two-way independent ANOVA with species and treatment (i.e. control or fertilizer addition) as fixed factors. The data were checked for normality and homogeneity of variance, and a Bonferroni correction was used in the post hoc analyses. All statistical analyses were performed with IBM SPSS Statistics 20 (IBM Corp., Armonk, NY, USA). Destructive sampling of xylem sap can cause cell damage, leading to cytoplasmic contamination of the sample, potentially biasing the pH measurement (e.g. Glavac et al., 1990). Accordingly, in the current study, the pH estimates based on the first extractions from the larches and pines were up to 8% higher than for latter samples (Fig. 2), corresponding to c. 0.45 pH units. Such an error would have had two major consequences for the continued data analysis and the interpretation of the results had we not continued sampling until a pH asymptote was reached. First, the error is large enough that it would have led us to incorrectly conclude that the xylem pH did not vary significantly among the four studied species. Second, because the studied trees all varied around pH = 6, where bicarbonate solubility begins to increase exponentially (Fig. 1), even small increases in pH could have considerable effects on the estimated amount of dissolved CO2 present in the sap (Table 1; Levy et al., 1999; Erda et al., 2014). For example, the observed +0.45 pH-unit error would have led us to overestimate the [CO2*] of the two conifer species by up to 50% under the Ts and pCO2 (which is linearly related to [CO2*] under constant Ts according to Eqn 1) used in our calculations. This would, in turn, have considerably biased any further estimates of C cycling in the studied trees. Furthermore, given that the pH estimates for pine and larch did not stabilize until between 5 and 10 ml of sap were collected (Fig. 2), our results suggest that sample contamination issues likely will be exacerbated for small sample sizes, such as sap extracted from twigs using a Scholander type pressure bomb. Notably, the contamination effect was much smaller, < 2% deviation from the pH asymptote, in the two broadleaf species included in the study (Fig. 2). This raises the question of whether the pH effect from damage during sampling is especially large in species, such as Scots pine and larch, which may excrete large quantities of pitch in response to wounding. In addition, we note that the accuracy of the sap pH measurement is particularly important in studies where it is used for inferring xylem CO2 transport rates, such as when using the mass balance approach (e.g. McGuire & Teskey, 2004; Bowman et al., 2005; Salomón et al., 2018; Tarvainen et al., 2021). On the contrary, studies that utilize isotopic tracers (e.g. Ubierna et al., 2009; Bloemen et al., 2013; Salomón et al., 2019; Tarvainen et al., 2021) avoid these problems by directly tracking the CO2 movement inside the stems. There was no overall short-term xylem sap pH response to the fertilizer application detected on the samples collected once the pH had reached an asymptote, F(1, 16) = 0.35, P = 0.56 (Fig. 3). However, a significant main effect of species on the xylem sap pH, F(3, 16) = 35.37, P < 0.001, was found. The post hoc tests indicated that the xylem sap pH was similar for alder and rowan (P = 1.00), and for larch and pine (P = 1.00), but significantly higher for alder and rowan compared with larch and pine (P < 0.001; Fig. 3). Furthermore, the calculations with Henry's law showed that, given the observed pH differences among the tree species, the amount of CO2 dissolved in the xylem could differ by nearly 80% over a reasonable range of temperatures and CO2 partial pressures (Table 1). A significant Fertilization × Species interaction was also detected (F(3, 16) = 7.080, P = 0.003), reflecting the lower pH in the fertilized larches compared to the controls. This may simply present a statistical artifact given the small sample sizes and the short time between the fertilizer application and the sap collection in the current study. However, it is clear that sap chemical composition influences the xylem pH in general (e.g. Raven, 1985; Wilkinson & Davies, 2002) and previous studies have shown correlations between sap pH and nitrogen concentrations and forms in several plant species (e.g. Gollan et al., 1992; Dodd et al., 2003; Wang et al., 2012). Based on these previous findings and the observation in this study of a significant among-species variation in sap pH in trees growing under the same conditions, it would seem useful to study further whether among-species differences in the forms in which nitrogen is present in the sap could explain the observed pH differences and the solubility of CO2 in the xylem sap. Given the limited scope of this study, two species per taxonomic group, it is important that the generality of the finding of among-species variation in xylem sap pH is further evaluated by other studies. The simplest way for accomplishing this would be to use a similar study design but increase the number of species and include greater within-species replication, for example, by utilizing arboretums or species trials established within forestry research. Such studies would greatly benefit from additional measurements to allow for full mass balance calculations over extended periods of time, including continuous observations of sap flow rates, sap CO2 concentrations, and stem temperatures, as well as from repeated measurements of sap pH to account for its temporal variability and from continuous monitoring of environmental factors to link the changes in sap pH and CO2 transport to their drivers. This would both allow for better evaluations of the among-species variation in xylem sap pH and provide information about the other potential physiological causes behind among-species differences in xylem CO2 transport rates. The isotopic tracer approach (e.g. Ubierna et al., 2009; Bloemen et al., 2013; Salomón et al., 2019; Tarvainen et al., 2021) has great potential for studies of among-species variation in xylem CO2 transport rate but needs to be supported by accurate measurements of other factors, including sap flow rate and sap pH, to provide improved mechanistic understanding. Furthermore, a meta-analysis approach would allow for utilizing large data sets to study the causes behind the observed among-species variation in the magnitude of xylem CO2 transport. Notably, based on the findings presented here, such an analysis would need to be done with careful consideration of the methods used for sap pH determination in mass balance-based studies to account for the possible bias caused by cell damage during sampling. The findings of this study suggest among-species variation in xylem pH to be large enough to significantly affect a species' capacity for stem CO2 transport and hence be important for the tree-scale C cycling. However, we note that these results should be seen as preliminary given the limited number of species and trees investigated, and thus need to be corroborated by other studies. Detection of the among-species pH differences required careful elimination of cell contents from the analysed samples with continuous sap extraction. The two needle-leaf species, Scots pine and Siberian larch, had lower pH and, thus, weaker capacity for holding CO2 dissolved in the xylem sap compared with the two broadleaves, rowan and alder, growing under similar soil and environmental conditions. Previous work has shown that gymnosperms exhibit lower sap flow rates than angiosperms (e.g. Flo et al., 2021). Thus, the combination of low sap pH and low xylem transport rates may help to explain the limited magnitude of internal CO2 transport observed for conifers. This work was supported by The Kempe foundations, The Knut and Alice Wallenberg Foundation (2015.0047 and 2018.0259), The Swedish University of Agricultural Sciences (TC4F and Bio4E) and the research councils: The Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning, The Swedish Research Council and The Swedish Governmental Agency for Innovation Systems. We would like to thank Jonas Lundholm at the analytical lab at the Department of Forest Ecology and Management, the Swedish University of Agricultural Sciences in Umeå, Sweden for his help with preparing the pH analyses. None declared. LT, NH, TN and JDM contributed to the design of the research; LT and NH performed the measurements; LT analysed the data and wrote the manuscript with input from the other authors. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Microdialysis is a useful tool for measuring in situ fluxes of soil compounds with minimal disturbance of soil structure and function. Fluxes of sampled compounds are commonly calculated per unit of membrane surface area, assuming that the entire membrane surface is capable of exchange – which is unlikely given varying soil moisture and the occlusion of membrane pores by the soil solid phase. We present a method to quantify the degree of connectivity of the microdialysis probe membrane to the surrounding soil by means of water exchange between a microdialysis perfusate and soil solution using deuterium (2H2O; equilibrated to DHO) as an internal standard. We applied the method to a range of probe membrane surface areas and soil moisture conditions to generate empirical models that estimate membrane surface area active in exchange. Our results suggest that even in a saturated sandy soil, active membrane surface areas reach only 40.3% of the probe surface area, perhaps due to occlusion by soil particles. However, when accounting for volumetric water content of the soil, active surface areas approached 80–90% of the area likely in contact with water, indicating that sampling efficiency of water-filled pores may still be high, particularly at slow flow rates. Furthermore, our method enables assessment of local soil water content around the probe. Models estimating soil water content were applied to field measurements of DHO exchange in three soil horizons (Organic, B1, B2) at two boreal sites, and in situ estimates were similar to those from conventional soil moisture methods when models were calibrated with the same soil type. We present DHO exchange as a powerful method for improving microdialysis flux interpretations in future studies, and for exploring small-scale water variability in relatively undisturbed soils.

Carbon dioxide [CO2] has reached almost 420 ppm in 2022 (Friedlingstein et al. 2022) and may increase to 600 ppm by the year 2100. Understanding plant responses to increasing CO2 is essential for predictions of plant productivity and of future climate (Ehlers et al. 2015). The hydrogen isotopes protium (1H) and deuterium (2H or D) exhibit the largest isotope effects, and D is fractionated by both physical and biochemical processes. Thus, hydrogen isotope compositions of plant compounds have a remarkable potential to further our knowledge about plant physiological and environmental processes. However, whole-molecule δD depends on the δD of the plant’s water source, fractionation by transpiration, and enzyme isotope effects. To disentangle these influences, isotopomer analysis is required since enzyme isotope effects influence stable isotope abundance in specific intramolecular positions (Ehlers et al. 2015), called isotopomers. As CO2 increases over decades, plant responses to T and CO2 over decades are important. For forests, opposing effects of CO2 and T determine if forests will in the future be a sink or source of CO2 (Van der Sleen et al. 2015; Sperry et al. 2019). Furthermore, a mechanistic understanding of physiological responses is essential to be able to estimate future C assimilation using ecosystem models. Photorespiration is a side reaction of photosynthesis that reduces C assimilation in most vegetation, and photorespiration is reduced by increasing CO2 yet exacerbated by rising T (Van der Sleen et al. 2015; Sperry et al. 2019). Therefore, we aim to unravel how photorespiration will develop under scenarios of rising CO2 and climate change.Tree rings help us understand interactions of plants and environmental drivers over decades-millennia. Variables that can be measured on tree rings fall into two groups. Variables like ring width are valuable for integrating effects of several environmental drivers on tree growth. In contrast, isotopomers depend on individual biochemical events and are therefore better for mechanistic studies.We use an NMR (nuclear magnetic resonance) method to analyze isotopomers of the glucose units of tree-ring cellulose, to elucidate physiological changes in trees during past decades of increasing CO2. In this contribution, we will report results of two kinds of experiments to investigate long-term tree responses.First, in manipulation experiments we calibrate isotopomer responses to environmental drivers, in particular CO2 and T. Second, we analyse tree-ring series over previous decades of rising CO2, and use the calibrations from the manipulation experiments to deduce shifts in photosynthetic metabolism over decades. For selected tree species, we will present combined results from both kind of experiments, conclusions on physiological changes of these trees over past decades, and implications for future C assimilation by broadleaved trees. ReferencesEhlers et al., 2015. https://doi.org/10.1073/pnas.1504493112.Friedlingstein et al., 2022. https://doi.org/10.5194/essd-14-1917-2022.Sleen et al., 2015. https://doi.org/10.1038/ngeo2313.Sperry et al., 2019. https://doi.org/10.1073/pnas.1913072116.

Key message The physiological responses expressed by variation in carbon and oxygen stable isotopes and iWUE in five provenances of maritime pine grown in four common gardens were primarily determined by genotype differences in phenotypic plasticity and secondarily by genotype. Abstract Given the impacts of climate change on forest resources and considering the slowness of evolutionary processes in trees, a need arises to understand the interplay between tree species adaptation to climate, genetic variation, and their impact on tree growth and productivity. Broadening knowledge of the capacity of tree populations to respond to climate-related disturbances is a prerequisite for the development of resilience strategies, including assisted migration and climate-smart forestry. This study tests the physiological ability of different maritime pine provenances, comparing Mediterranean (Corsica, Sardinia, and Tuscany) and Atlantic (Portugal) provenances, to adapt to progressively drier conditions that have occurred in the last thirty years. Four provenance trials with randomized blocks of the five maritime pine provenances were used as test sites in Sardinia (Italy). Wood cores were collected from the 40-year-old plants. Cores were split into five-year segments to determine provenance-related variations in carbon and oxygen stable isotopes and provide information on long-term patterns in intrinsic water use efficiency (iWUE). The provenance × site interaction was the most important source of variation, meaning that the genotypes responded differently to the planting sites. Considering the main effects, both genotype and environmental conditions at the planting sites influenced stable isotope composition in tree rings. This suggests that iWUE was determined by phenotypic plasticity that differed among genotypes. In contrast, provenance responses were stable with time, and the provenance × site interaction was stable across time periods. These findings suggest that provenance selection to improve iWUE in maritime pine may need to consider site conditions but point more to soil conditions than to climate. In any case, they limit our ability to recommend maritime pine provenances based on iWUE until the missing site factors can be identified.

As a consequence of global change, forests worldwide are undergoing a restructuring process. The expectations for forests of the future are ambitious: Providing resilient forest ecosystems that capture large amounts of carbon but also provide stable groundwater recharge rates. To balance the interests of both forestry and water management authorities, scientists and practitioners need to be able to investigate and predict which forest types and combinations of tree species are most likely to fulfill these needs.Methods based on the analysis water stable isotopes have been used extensively for studying water uptake depths of vegetation, groundwater recharge, transit times, and water sources in general. Despite being arguably the superior tool when not only amounts but also knowledge of the sources of an (eco-)hydrological flux are needed, the highly dynamic nature of water transport processes within the soil-plant-atmosphere continuum (SPAC) could hardly be captured in the past. With the advent of laser spectroscopy in the last decade, we are now able to measure water stable isotopes continuously and in all compartments of the SPAC.In this keynote, we present and review the most recent advances (2016-now) of combined soil and plant in-situ water isotope measurements carried out in different ecosystems worldwide. We then critically discuss the gain in process-understanding of in-situ monitoring approaches and demonstrate how in-situ methods could be integrated with traditional and novel methods to advance forest hydrology.In-situ and semi-in-situ (i.e., sampling of water vapor) water stable isotope methods have been greatly improved within the last five years. Initial disadvantages (e.g., comparability to traditional methods, complicated & laborious setup & maintenance, expensive) have been carefully addressed, and improvements have been implemented. Recent research has proven that i.) highly dynamic and heterogenous processes (e.g., stem flow, groundwater recharge through preferential pathways, change of uptake depths in response to rainfall/drought, disentangling water use of different tree species in mixed forests) can be captured exceptionally well using in-situ isotope methods; ii.) water-vapor equilibration methods represent the isotope composition of mobile water better compared to destructive methods, and iii.) using continuous water isotope data reduces parameter uncertainties in SPAC modeling.In summary, we state that the benefits of using in-situ or semi in-situ techniques outweigh the disadvantages by far and strongly encourage the water stable isotope community to integrate them regularly into studies of dynamic soil-plant-atmosphere feedbacks.

Trees’ annual rings provide a remarkable record of the past, dated precisely by the annual cycle of xylem cell formation. Although dendrochronology often focuses on ring widths, there have been many studies exploring the chemistry of the material comprising the rings as well. Particularly interesting in this respect is the isotopic composition of the cell-wall material, which provides detailed information about the weather conditions and the physiological state of the tree during the period of growth (McCarroll and Loader 2004, Siegwolf et al. 2022). These quality data do not come easily or cheaply, however. Isotope analysis can be tricky, and the manual splitting of rings is tedious. Therefore, any improvement in precision or ease of sampling would be most welcome. Nearly 20 years ago, laser ablation seemed to offer such an opportunity (Schulze et al. 2004), however its initial promise has been slow to mature. Its challenges have been addressed in the recent paper of Saurer et al. (2022), which describes two systems in regular use at WSL in Switzerland and LUKE in Finland. The paper includes detailed assessments of system performance and a comparison of alternatives for sample preparation. Considered alongside other recent advances (e.g., Loader et al. 2017), the time may finally be ripe for efficient adoption and standardization of this technique. ‘Ablation’ refers to the removal of material from the surface, often implying gradual loss of particles or fragments, but the products may also include escaping gases and liquids. In the systems described here, ablation breaks molecules free from a point on the surface of a tree-ring sample and delivers the ablated material to a ratioing mass spectrometer for isotope analysis. It is a simple way to feed the instrument with material from a known location on the wood sample. The ablated material is gathered into a helium stream and transferred to an oven with an abundance of oxygen, where carbon compounds are converted quantitatively to CO2. The CO2 is then collected, concentrated and cleaned. The clean CO2 pulse is then transferred to a ratioing mass spectrometer, where natural abundance of the stable isotopes can be precisely determined. One advantage of this technique is that the tight focus of a laser provides precise control of the sampled position on the wood surface. This would be advantageous in any discipline, but it is especially so in dendrochronology, where space implies time. Because a tree ring is produced from the inside outward over a growing season (Cuny et al. 2013), position in the ring can be used to estimate the date of deposition of the material being analysed. This capacity has distinct advantages when seeking short-term events, e.g., a short drought period in the middle of a growing season (Schiestl-Aalto et al. 2021). It is also possible to detect these events using a microtome to slice an annual ring into thin sections (Loader et al. 1995, Helle and Schleser 2004), but the microtome technique is difficult to apply. More common is to split out whole rings from a core without attempting to subsample them, or perhaps to divide the rings into earlywood and latewood sections (e.g., Marshall and Monserud 1996). But even this becomes difficult when rings are narrow, as frequently observed in harsh climates and on large-diameter trees. A second advantage of the laser is that it is relatively noninvasive (Loader et al. 2017), charring points on the surface of a wood core without influencing the remainder of