Impacts of elevated CO 2 levels and temperature on photosynthesis and stomatal closure along an altitudinal gradient are counteracted by the rising atmospheric vapor pressure deficit

vapor-pressure

To assess changes in iWUE and eWUE values over the past 60 years , stable carbon isotope ratios in latewood cellulose (δ 13 C cell ) of annually resolved tree rings were analyzed.Such an approach was sensitive enough to distinguish tree responses to growth environments at different altitudes.Our findings revealed a rising trend in iWUE, particularly in oak trees at low and middle altitudes. Hoever, this increase was negligible at high altitudes.Warmer and drier conditions at lower altitudes likely led to significant stomatal closure and enhanced

Introduction
Forests play a vital role in carbon sequestration, constituting up to 90 % of terrestrial carbon biomass (Pan et al., 2011).Forest ecosystems act as significant carbon sinks, mitigating climate change intensified by fossil fuel burning and rising atmospheric CO 2 concentrations.Predicting how future growth conditions impact tree physiology, especially concerning water and carbon cycles, is paramount.A useful strategy for understanding climate change effects on plants and ecosystems involves comparing populations of the same species across diverse climates along altitudinal and/or latitudinal gradients (De Frenne et al., 2013;Leroy et al., 2020).Altitudinal gradients, characterized by rapid changes in temperature, humidity, precipitation, and sun radiation within small geographic areas (Körner, 2007), offer insights into plant growth responses to varied climate conditions (Jump et al., 2009).These factors not only influence the growing period's onset and duration, but also directly impact plant physiological processes, affecting carbon uptake and plant water status (Bošel'a et al., 2014;Bresson et al., 2011;King et al., 2013;Rajsnerová et al., 2015;Vitasse et al., 2009).
Previous studies have shown decreasing growth rate and tree-ring width (TRW) with increasing altitude (e.g., Hartl et al., 2022;Paulsen et al., 2000;Peñuelas et al., 2008;Ponocná et al., 2016).It is likely due to differences in summer temperatures between low and high altitudes, when TRW is often constrained by low summer temperatures in cold high altitudinal sites (King et al., 2013;Splechtna et al., 2000).Gas exchange studies on the leaf level have further revealed that plants at lower altitudes typically exhibit lower transpiration rates and stomatal conductance compared to their high-altitudinal counterparts (Bresson et al., 2009(Bresson et al., , 2011;;McDowell et al., 2008;Rajsnerová et al., 2015).This reduction in stomatal conductance does not hinder enhanced CO 2 assimilation, likely due to significantly higher RuBisCO (Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase) content in low-altitudinal trees (Mediavilla et al., 2016;Rajsnerová et al., 2015).Such a disparity results in enhanced water use efficiency, calculated as the carbon assimilated per unit of water lost, in low-altitudinal plants, at least in the short term.This parameter, rooted in plant physiology, plays a pivotal role in Earth's interconnected carbon and water cycles and holds great potential in indicating plant adaptability to changing growth conditions (Cheng et al., 2017;El Masri et al., 2019).
Increasing trends in water use efficiency along altitudinal gradients were also confirmed at the level of whole plants using the technique of isotope discrimination (Peñuelas et al., 2008;Wu et al., 2015).In particular, the stable carbon isotope ratio δ 13 C ( 13 C/ 12 C) of tree rings has been found to be a useful indicator of annual changes in intrinsic water use efficiency, iWUE (Adams et al., 2020;Dorado-Liñán et al., 2020;Frank et al., 2015;Oulehle et al., 2023;Waterhouse et al., 2004), defined as the ratio between the rate of CO 2 assimilation and the stomatal conductance to water vapor (Osmond et al., 1980;Ehleringer et al., 1993;Saurer and Voelker, 2022).Higher representation of the 13 C isotope yields less negative δ 13 C values, indicating higher iWUE.This increase may stem from improved CO 2 assimilation (Giammarchi et al., 2017;Guerrieri et al., 2019;Mathias and Thomas, 2021), reduced stomatal conductance (Andreu-Hayles et al., 2011;Lévesque et al., 2014;Saurer et al., 2014;Waterhouse et al., 2004;Yang et al., 2021) or both.While stomata tend to close under conditions of water scarcity or high atmospheric vapor pressure deficit (Grossiord et al., 2020), photosynthetic CO 2 assimilation increases with higher light intensity, nutrient availability, temperature, and CO 2 concentration (Kubiske and Pregitzer, 1996;Moore et al., 2021).Therefore, interpreting annual changes in iWUE over decades requires careful consideration of changes in atmospheric CO 2 concentration and its isotopic composition, along with changes in other abiotic factors such as soil acidification affecting calcium availability, which has a major effect on stomata functioning (Oulehle et al., 2023).Among other nutrients, increased availability of nitrogen and phosphorus has been shown to stimulate iWUE, primarily due to an enhancement in the rate of photosynthetic CO 2 assimilation (Dijkstra et al., 2016).
However, iWUE might not accurately reflect plant water use efficiency because plant water status primarily depends on the total volume of transpired water rather than stomatal conductance (Farquhar et al., 1989a(Farquhar et al., , 1989b;;Ehleringer et al., 1993;Saurer and Voelker, 2022).The total transpiration rate is primarily determined by the evaporative surface size (leaf area) and numerous environmental factors that influence stomatal openness, boundary layer thickness on the leaf surface, and atmospheric conditions.Among these factors, atmospheric vapor pressure deficit (VPD -the difference between the atmospheric water amount at a given temperature and the water amount the atmosphere could hold at saturation) plays a significant role (Cheng et al., 2017;Fletcher et al., 2007;Kolb and Stone, 2000;Treydte et al., 2024).Transpiration rates increase in dry atmospheres but significantly decrease in wet atmospheres.However, iWUE does not capture this VPD-driven phenomenon (Ehleringer et al., 1993).Therefore, the evaporative water use efficiency (eWUE), accounting for atmospheric humidity, has been introduced (Eq.(5) in this study; reviewed in Seibt et al., 2008 or Saurer andVoelker, 2022).
This study evaluates both iWUE and eWUE in oak species growing along their natural altitudinal gradient (130-630 m a.s.l.) in the western regions of Central Europe over the last 60 years .The research is based on the stable carbon isotope composition of tree-ring cellulose from two oak species (Quercus robur L. and Quercus petraea (Matt.)Liebl.)prevalent in Europe (Eaton et al., 2016).The study area, located in the Vihorlat Mountains (eastern part of Slovakia), is unique due to the presence of numerous old trees of the same species, all with the same slope exposure and similar soil conditions (Bosel'a et al., 2021;Hartl-Meier et al., 2015).The primary goal was to test the hypothesis that climate change, characterized by rising atmospheric CO 2 and temperature levels and significant drought periods, leads to a gradual increase in tree water use efficiency over the years.More specifically, we hypothesized that (i) water use efficiency increases differently at different altitudes, assuming (ii) a pronounced effect on iWUE, especially at lower altitudes, but (iii) a minimal effect on eWUE when considering atmospheric humidity.

Site description
The forest under investigation is situated on the southwestern slope of the Vihorlat Mountains, a volcanic massif spanning eastern Slovakia and western Ukraine in Central Europe.Oak, beech, hornbeam, and pine are the most common naturally growing tree species in this area.The region has a continental climate with hot summers, cold winters, and N. Pernicová et al. moderate amounts of precipitation.The growing season, occurring from April to August, is characterized by mean temperatures and precipitation amounts ranging between 11.2 and 19.8 • C and between 168 and 721 mm, respectively.However, in the past two decades, there has been an increase in the frequency of dry seasons (total seasonal precipitation below 345 mm; first quartile for the investigated period 1961-2020) and hot seasons (mean seasonal temperatures above 15.5 • C; third quartile), accompanied by increased interannual variability in climate parameters (Supplementary Tables S1 and S2).
In this study, primarily sessile oak trees (Quercus petraea) were sampled at low altitude (LA; 280 m a.s.l.), middle altitude (MA; 400 m a. s.l.), and high altitude (HA; 630 m a.s.l.).Based on the available professional forestry maps, these altitudes represent the natural occurrence range for sessile oak trees in the given region.Additionally, we sampled English oak trees (Q.robur) at the altitude of 130 m a.s.l. as a supplementary material.Sampling sites are located approximately 90 km NE of Košice (SK), 240 km SW of Lviv (UA), 330 km SE of Krakow (PL), and 350 km NE of Budapest (HU) (see the map in Graphical Abstract and Supplementary Fig. S1).The specific coordinates for each site are provided in Table 1.
Daily minimum, mean and maximum temperatures and air humidity from ~300 climatological stations and daily precipitation totals from 1100 rain-gauge stations of the Czech Hydrometeorological Institute (CHMI) and Slovak Hydrometeorological Institute (SHMI) were used as source of climate data.The vapor pressure deficit (VPD) for each site was estimated using a variation of Tetens' (1930) formula following Allen et al. (1998).The location of the nearest SHMI stations is shown in Supplementary Fig. S1.The entire dataset of CHMI and SHMI data was then used to estimate daily values for 0.5-km × 0.5-km grids for the entire 1961-2020 period.Daily data are interpolated by regression through kriging, which uses geographical coordinates, altitude, and other terrain characteristics as predictors.Within the CHMI and SHMI domain, the average minimum distance between two neighboring stations is approximately 22 km for elements measured at climatological stations and <10 km for elements measured at precipitation stations.For each sampling site, the most corresponding 0.5-km grid was used in terms of distance and altitude.
Climate data for the vegetation period (April-August) from 1961 to 2020 are shown in Fig. 1.While the mean air temperatures during this period (15.5, 14.7, and 13.5 • C) and the vapor pressure deficit (0.57, 0.52, and 0.46 kPa) decrease with increasing altitude, the mean relative air humidity (67.8, 69.1, and 70.8 %) and the precipitation (393, 418, and 458 mm) increase with altitude.These trends were confirmed at the altitude of 130 m a.s.l., where temperature, VPD, relative air humidity, and precipitation amount to 16.6 • C, 0.65 kPa, 65.8 %, and 345 mm, respectively (Supplementary Fig. S2 and Supplementary Table S1).

Sample collection
First, 15 representative trees were selected at each site and one core per tree was taken at breast height (1.3 m) using an increment borer (Haglöf Company Group, Långsele, Sweden) with an inner diameter of 5 mm (Gut et al., 2019).Tree-ring width (TRW) series were measured on all samples using a VIAS TimeTable (SCIEM, Brunn, Austria) with an accuracy of 0.01 mm.The obtained TRW series were cross-dated using PAST4 software (SCIEM, Brunn, Austria), corrected for missing and false rings, and visually compared (Rybníček et al., 2010).The site TRW chronologies were calculated using bi-weight robust means.The most recent version of the Slovakia oak TRW composite chronology (Prokop et al., 2016) was used for verification and absolute dating of the site TRW chronologies.Mean values of inter-site correlations of TRW series (Rbar) ranged between 0.32 and 0.52 over the period 1961-2020 (p < 0.01).
Based on the TRW analysis, six trees from each site were subsequently selected for the analysis of stable carbon isotopes in non-pooled tree rings.Thus, TRW series was used for absolute dating of the samples and selection of representative trees for isotopic analysis.The set of six trees represents a good compromise between the loss of explained variance and the cost of isotopic analysis (Rybníček et al., 2021).

Cellulose extraction and δ 13 C cell determination
The absolutely dated annual increments of individual trees from the period 1961-2020 were cut into small pieces with a scalpel under a stereomicroscope and packed into F57 Teflon bags (Ankom Technology, Macedon, NY, USA) (Rybníček et al., 2021).Samples included particularly latewood, considered a robust archive of stable carbon isotopes reflecting the climate of the current year, but samples from the most narrow tree rings could contain a greater proportion of earlywood.A previous study on oaks showed that the relative proportion of latewood in a sample does not have a statistically significant effect on the ratio of stable carbon isotopes (Torbenson et al., 2023), although the carbon isotope composition of the earlywood could be affected by the remobilization of carbohydrate reserves from the previous year (Hill et al., 1995).
The modified Jayme-Wise isolation method was used to extract the α-cellulose from each tree ring (Boettger et al., 2007).Teflon filter bags were washed twice in a 5 % NaOH solution at 60 • C for 2 h, followed by another wash in a 7 % NaClO 2 solution at 60 • C for an additional 30 h.To keep the pH at 4-5, acetic acid (99.8 %) was added to the solution.
Teflon bags containing extracted α-cellulose were washed and then rinsed three times in hot distilled water (90 • C).Such an extraction procedure has been tested and proven sufficient for extracting cellulose from the wood of living oaks that have not been contaminated or chemically treated (Urban et al., 2021).The samples were subsequently dried at 50 • C for 24 h, locked in Eppendorf microtubes, and stored in the dark at 21 • C until analysis.
For the determination of δ 13 C cell , homogenized α-cellulose (approximately 1.0 mg) was weighed into tin capsules (Elementar Analysensysteme, Langenselbold, Germany) and combusted at 960 • C to CO 2 using an elemental analyzer varioPYRO cube (Elementar Analysensysteme, Germany).The abundance of 12 C and 13 C stable isotopes in CO 2 was determined by a continuous flow isotope ratio mass spectrometer ISOPRIME100 (Isoprime, Manchester, UK).The system was internally calibrated using certified reference materials of known isotopic ratios from the International Atomic Energy Agency (IAEA, Vienna, Austria) and the US Geological Survey (USGS, USA): caffeine (IAEA-600) and graphite (USGS24).The 13 C/ 12 C ratio in tree-ring cellulose (δ 13 C cell in ‰) was related to the Vienna Pee Dee Belemnite (VPDB) standard according to the formula: where R is the ratio of the heavy to light isotope ( 13 C/ 12 C) in cellulose (cell) and VPDB.
In our previous study on an unexceptionally large oak dataset, it was demonstrated that there are no age trends in tree-ring stable isotopes (Büntgen et al., 2020).The raw δ 13 C cell data, obtained for individual trees with absolute dating and annual resolution, were directly used to calculate water use efficiency without the need for any additional standardization and/or correction.

Intrinsic and evaporative water use efficiency
To determine annual values of intrinsic water use efficiency (iWUE) based on δ 13 C cell data, the standardized approach described in Mathias and Hudiburg (2022) was followed.Firstly, we calculate the carbon isotope discrimination (Δ 13 C in ‰) as: where δ 13 C atm and δ 13 C cell are the stable carbon isotope ratios in the atmosphere and tree-ring cellulose of a given year, respectively, and d represents correction for the post-photosynthetic fractionations (2.2 ‰).Values of δ 13 C atm reported by McCarroll and Loader (2004) and values from direct measurements on Mauna Loa (https://www.esrl.noaa.gov/gmd/dv/data/), which are implicitly assumed to be global (Belmecheri and Lavergne, 2020).Consequently, an intercellular CO concentration (Ci in μmol CO 2 mol − 1 ) was calculated as: where a, b, and f ´are correction factors of carbon isotope fractionations due to CO 2 diffusion across the stomata (4.4 ‰), carboxylation by Rubisco (28 ‰), and photorespiration (12 ‰), respectively, Ca is mean annual atmospheric CO 2 concentration (μmol CO 2 mol − 1 ), while pCa is the equivalent of Ca expressed as the partial pressure in Pa, and Г* is CO compensation point.Because carbon fractionation through photorespiration is temperature dependent, Г* values were adjusted to mean temperatures of a given vegetation period and altitude (Fig. 1A, Supplementary Fig. S2A, and Supplementary Table S1) following the equations described in Bernacchi et al. (2001).
Finally, iWUE in μmol mol − 1 was calculated according to Farquhar et al. (1989b) as: where the coefficient of 1.6 represents the ratio of stomatal conductance to CO 2 and water vapor diffusions.Values of iWUE thus represent the ratio between photosynthetic CO 2 assimilation rate (A) and stomatal  S1 for details.
conductance for water vapor (g s ).
In contrast, the calculation of evaporative water use efficiency (eWUE) was designed to reflect the ratio between the rates of photosynthesis and transpiration (Farquhar et al., 1989b): where VD is the water vapor density in the atmosphere in kg m − 3 converted from VPD (vapor pressure deficit) in kPa according to Strange et al. (2023) and studies cited there.Such conversion allows an accurate representation of photosynthesis (A) to transpiration (E) ratio and an expression of eWUE in mmol CO 2 mol − 1 H 2 O.

Statistical analysis
Linear regression, based on the least squares method, was used to assess changes in iWUE and eWUE over the period 1961-2020.Consequently, statistically significant differences between regression slopes for individual altitudes were evaluated using the t-test.Student's t-test was also employed, after testing for normality (Shapiro-Wilk test) and equality of variance (Brown-Forsythe test), to compare the 30-year periods 1961-1990 and 1991-2020 for the investigated ecophysiological and climatic parameters.SigmaPlot ver.15 software (Systat Software, San Jose, CA, US) was used for all statistical tests and graph preparation.

Results
All sampling sites (Table 1) are characterized by significantly different microclimatic conditions with a typical distribution of temperature, VPD and precipitations along the altitudinal gradient (Fig. 1, Supplementary Fig. S2, Supplementary Table S1).Moreover, the mean air temperature during the vegetation season significantly (p < 0.001) increased by 4.3-11.1 % (i.e. between 0.72 • C at the lowest altitude and 1.42 • C at the highest altitude) in the period 1991-2020 when compared with 1961-1990.Similarly, VPD values in the period 1991-2020 increased by 10.8 % at the lowest altitudes while it was by 22.0 % at high altitudes when compared to 1961-1990.Conversely, no changes in total precipitation during the growing season between these two periods were observed (p = 0.390-0.917).Such variability in growth conditions led to the changes in δ 13 C cell values of the tree-ring cellulose and δ 13 Cderived ecophysiological parameters (Figs. 2 and 3).
Inter-site correlations of the δ 13 C cell series revealed significant (p < 0.05) coherence across individual trees at the same altitude (Rbar ranged between 0.32 and 0.38), but it was only 0.17 for Q. petraea trees at an altitude of 280 m a.s.l.(p > 0.05) over the period 1961-2020.Gradual decreases in raw δ 13 C cell values were observed during the investigated period in Q. petraea at all altitudes (280-630 m a.s.l.; Fig. 2A-C) and are consistent with δ 13 C cell decline in Q. robur (130 m a.s.l.; Supplementary Fig. S3).Such declines, being more pronounced at high than low altitudes, reflect not only the physiological adjustments of trees, but also the changes in the isotopic composition of atmospheric CO 2 (δ 13 C atm ) during the last decades (black lines in Fig. 2A-C).
The Ci time series (calculated according to Eqs. ( 2) and ( 3)) have an increasing tendency at all altitudes (Fig. 2D-F).These increases in Ci correspond to an increase in the atmospheric CO 2 concentration (Ca) from 317.4 to 412.4 μmol CO 2 mol − 1 over the past 60 years (black line in Fig. 2D-F).While the mean Ci values ranged between 196 and 205 μmol CO 2 mol − 1 over the period 1961-1990 at all altitudes (statistically nonsignificant differences; p > 0.05), Ci significantly increased (p < 0.01) in Q. petraea by 16.3, 14.2, and 24.1 % at the LA, MA, and HA sites, respectively, over the period 1991-2020 (Supplementary Fig. S4).However, these increases in Ci were not followed by a change in the Ci/ Ca ratio, which remained constant (0.59-0.61) at the LA and MA sites.Noticeably, similar changes in the Ci and Ci/Ca ratio were observed in Q. robur (Supplementary Fig. S3) when compared to Q. petraea at the LA site; however, the absolute values for Q. robur are slightly higher (Supplementary Fig. S4).On the contrary, a significant (p < 0.01) increase in the Ci/Ca ratio by 9.8 % was found in the HA trees when comparing the N. Pernicová et al. 1961Pernicová et al. -1990 (mean Ci/Ca value 0.61) and 1991-2020 periods (mean Ci/ Ca value 0.67).Generally, the highest Ci/Ca values (0.726) were found in cold years (1997), while the lowest Ci/Ca values (0.553) were observed in dry years (1964).
During the investigated period, iWUE linearly increased in Q. robur (Supplementary Fig. S3) as well as Q. petraea trees from the LA and MA sites, but not in those from the HA (Fig. 3A-C).The slope of linear regression is significantly (p < 0.01) lower at the HA site (increase by 0.43 μmol mol − 1 per decade) when compared to LA and MA ones (increase by 3.04-3.58μmol mol − 1 per decade).Noticeably, the highest increase in iWUE (4.52 μmol mol − 1 per decade) was found in Q. robur trees at the altitude of 130 m a.s.l.(Supplementary Fig. S3).At low altitude (Fig. 3A), the iWUE values of Q. petraea varied between 79.2 μmol mol − 1 in 1966 (wet year) and 104.7 μmol mol − 1 in 2016 (hot and dry year; mean seasonal temperature > 15.5 • C and total seasonal precipitation <345 mm).The same pattern was observed for Q. robur.Also at middle and high altitudes, the minimal iWUE values (72.7 and 62.3 μmol mol − 1 for MA and HA sites, respectively) were associated with wet (total seasonal precipitation >475 mm) and/or cold years (mean seasonal temperature < 13.8 • C), while maximum iWUE values (103.6 and 94.9 μmol mol − 1 for MA and HA sites, respectively) have been achieved in hot and dry years (Fig. 3B, C; Supplementary Table S2).In line with these results, a comparison of mean iWUE values over the 1961-1990 and 1991-2020 periods revealed significant (p < 0.01) increases by 17.3 % in Q. robur and by 11.8 and 14.9 % in Q. petraea trees grown at the LA and MA sites, respectively, but no change in the HA trees was observed (Supplementary Fig. S5).
The mean eWUE values for the investigated period were 38.9 mmol mol − 1 for LA trees, 40.5 for MA trees, and 42.6 for HA trees (Q.petraea).However, the lowest eWUE value of 32.3 mmol mol − 1 was observed for Q. robur grown at the lowest altitude.These differences among sites are statistically significant (p < 0.01).Compared to iWUE, different trends in eWUE development during the period 1961-2020 were observed (Fig. 3D-F).Although weak or no trends were observed in the LA and MA trees, a significant (p < 0.01; r = 0.499) decrease in eWUE was observed in the HA trees.The mean eWUE value amounted 47.1 mmol mol − 1 during the 1961-1990 period while it was only 38.1 mmol mol − 1 during 1991-2020, i.e. a significant (p < 0.01) decrease by 19.1 % (Supplementary Fig. S5).
At all altitudes, the minimal eWUE values (29.7, 31.1, and 29.6 mmol mol − 1 at LA, MA, and HA, respectively) were associated with hot and dry years, while maximum values (50.4,55.7, and 62.39 mmol mol − 1 at LA, MA, and HA, respectively) were found in cold and wet years.

Changes in climate conditions along the altitudinal gradient
In Europe, ongoing climate change, besides an increase in average temperature, is often marked by prolonged periods of drought and heatwaves (Brázdil et al., 2021;Seneviratne et al., 2021).However, these trends vary with altitude (Kotlarski et al., 2012(Kotlarski et al., , 2015)).Generally, warming intensifies with altitude, while the influence of altitude on precipitation changes is relatively minor.These patterns align with the observed trends in air temperature and precipitation during the vegetation period (April-August) in our study area (Fig. 1 and Supplementary Fig. S2).Considering the anticipated ongoing rise in temperature and VPD in Central Europe (Treydte et al., 2024), the observed reactions of lowland trees to current climate conditions can serve as a valuable indicator of future behavior and the long-term acclimation capacity of highland trees (Leroy et al., 2020).Additionally, it can be utilized as a predictor for potential alterations in highland forest biodiversity, structure, or treeline shifts (Körner, 2007).Notably, a more significant increase in atmospheric VPD occurred at higher altitudes (an increase in atmospheric VPD by 10.8 % (130 m a.s.l.) and 22.0 % (630 m a.s.l.) comparing the periods 1991-2020 to those of .This suggests a potentially stronger impact of climate change on eWUE at higher altitudes.

Environmental changes improve iWUE but not eWUE values
The range of observed δ 13 C cell values in our study (Fig. 2A-C) is consistent with values reported for oak species at different European sites (ISONET Project Members et al., 2023).The isotope-ratio mass spectrometry was shown to be sensitive enough to distinguish between trees growing along a relatively small altitudinal gradient.Our 60-year (1961-2020) dataset based on δ 13 C cell values supports the hypothesis that ongoing environmental changes (associated with increasing CO 2 concentration, temperature, and drought) lead to increased water use efficiency in oak trees.This effect is particularly noticeable for oak trees at low and middle altitudes, specifically concerning intrinsic iWUE, reflecting changes in the stomatal aperture openness (Fig. 3A, B).In addition to climatic factors, it is likely that the natural hybridization of Q. robur and Q. petraea contributes to a gradual improvement of water status and greater resistance of oaks to drought (Petit et al., 2003).
Similar findings documenting an increase in iWUE during recent decades have been reported for various tree species including oaks, pine, beech, and spruce (Dorado-Liñán et al., 2020;Kannenberg et al., 2021;Peñuelas et al., 2008;Waterhouse et al., 2004;Wu et al., 2015).For example, Dorado-Liñán et al. (2020) reported an increase of mean iWUE values from 64.8 (period 1880-1915) to 81.1 μmol mol − 1 (period 1980-2015) in high-altitude (1500 m a.s.l.) Q. petraea tress.Such iWUE values are comparable to those reported in this study for HA trees in the period 1961-2020 (62.3-95.0μmol mol − 1 ; Fig. 3C).Similarly, the iWUE values observed for LA trees (71-127 μmol mol − 1 for Q. robur and 72-122 μmol mol − 1 for Q. petraea) are in line with those previously presented for Q. robur growing at altitudes of 50-150 m a.s.l.(75-115 μmol mol − 1 ; Waterhouse et al., 2004).A cross-European study by Frank et al. (2015) also noted that iWUE values increase more rapidly in coniferous than broadleaved tree species, with the highest increases in temperate forests of Central Europe.However, a decline in iWUE has been noted in Silver fir during the last 30 years, which was probably related to the deposition of sulfur and nitrogen in Central Europe and consequent changes in the availability of micronutrients including calcium (Oulehle et al., 2023).Similarly, Bauters et al. (2020) reported a decline in iWUE for tropical trees in central Africa over the past century, likely due to significant nutritional constraints on forest growth and productivity.
While δ 13 C cell values in oak tree rings are generally considered independent of cambial age (Büntgen et al., 2020), an increase in iWUE may occur with tree age, especially when comparing small juvenile trees with tall middle-aged and mature trees (Wu et al., 2018).Such an increase is often attributed to rising hydraulic stress associated with increasing tree size, leading to reduced stomatal conductance (reviewed in Bond, 2000).However, it is anticipated that the age trends of iWUE in our study will be relatively low because the average tree age at the beginning of the study (1961) was >60 years (Table 1).The metaanalysis by Mathias and Thomas (2021) revealed that the increase in iWUE is primarily modulated by increasing the CO 2 assimilation rate (observed in 83 % of the examined studies), while the reduced stomatal conductance played the dominant role in only 17 % of the examined studies.The stimulatory effect is mainly attributed to elevated CO 2 concentration in the atmosphere leading to an increase in the CO 2 assimilation rate and a reduction of stomatal conductance (Poorter et al., 2022;Urban, 2003;Weiwei et al., 2018).However, the stimulating effect of CO 2 on photosynthesis can be negligible in cold, dry, and/or nutrient-poor environments (Albert et al., 2011;Delgado et al., 1994;Hättenschwiler and Körner, 1997;Ofori-Amanfo et al., 2023;Poorter et al., 2022;Šigut et al., 2015).Unlike iWUE, the eWUE time series (Fig. 3D-F) did not show any increasing trends in evaporation-based water use efficiency over the last 60 years.This result indicates that the positive effects of elevated CO 2 concentration and temperature on photosynthesis and stomatal closure, leading to increased iWUE, might be suppressed by the increasing deficit of water vapor in the atmosphere (Fig. 1).This deficit accelerates transpiration rates through plants, potentially resulting in higher transpiration even when stomata are partially or fully closed (Marchin et al., 2016;Leonardi et al., 2000).
The different trends between iWUE and eWUE suggest the necessity of considering changes in atmospheric VPD in WUE calculations, especially at ecosystem and global scales.Furthermore, it emphasizes the importance of accurate interpretation of the WUE based on stomatal conductance or transpiration rate.iWUE appears to be a robust indicator of long-term ecophysiological responses to climate change and CO concentration, while eWUE is closely linked with environmental impacts on plant water use and directly related to water and carbon cycles (Strange et al., 2023).

Trees acclimate to environmental changes differently at different altitudes
Our data support the hypothesis that changes in WUE over the past 60 years vary along the altitudinal gradient.We observed a significant increase in iWUE in low-altitude oak trees (by 26.9 % in Q. robur and by 20.3 % in Q. petraea), whereas this increase was marginal in highaltitude Q. petraea trees (by 4.4 %; Fig. 3A, C and Supplementary Fig. S3).Similar trends in iWUE time series were observed for European beech in the Montseny Mountains, Spain (Peñuelas et al., 2008), and Schrenk spruce in the Tienshan Mountains, China (Wu et al., 2015).For the period 1978-2003, Peñuelas et al. (2008) ) reported an increase in δ 13 C-based iWUE values by 10 % and 6 % at altitudes of 990 and 1130 m a.s.l., respectively, while no increasing trend in iWUE was observed at high altitudes (1640 m a.s.l.).The more significant increase in iWUE at lower altitudes is primarily attributed to warmer and drier conditions, which regulate stomatal aperture and maintain high photosynthetic CO uptake efficiency stimulated by increasing CO 2 concentration (Rajsnerová et al., 2015;Zhang et al., 2005).On the contrary, the stimulatory effect of elevated CO 2 on photosynthesis is minimized at suboptimally low temperatures typical for high altitudes (Bernacchi et al., 2001;Šigut et al., 2015;Zhang et al., 2005), thus contributing to a smaller increase in iWUE.This reduction in photosynthetic efficiency may lead to a progressive rise in intercellular CO 2 concentration (Farquhar et al., 1982;Zhang et al., 2005), supported by a significant increase in Ci/Ca ratio at high altitudes over the past 30 years (average increase of 9.8 %).On the contrary, the ratio remained relatively stable at low and middle altitudes (average increase of up to 1.7 %) compared to the 1961-1990 period (Supplementary Fig. S4).Similar trends in Ci/ Ca ratio along the altitudinal gradient were reported for both broadleaved (Peñuelas et al., 2008) and coniferous tree species (Wu et al., 2015).
However, contrasting trends were observed in eWUE (Fig. 3), especially in high-altitude trees, where a nearly 20 % decrease in eWUE corresponded to a 22 % increase in atmospheric VPD (Fig. 1).This finding implies a lower ability of trees to cope with changing conditions, higher water loss at the ecosystem level due to stimulated transpiration, and thus a growing risk of drought stress for forest stands at higher altitudes, a risk previously considered low.Opposing trends between iWUE and eWUE were also observed in forest populations during the megadrought in the southwest of the US (Strange et al., 2023).Trees accessing underground water levels operated with lower iWUE and higher eWUE values, indicating less physiological stress compared to trees at dry sites.
To our knowledge, this is the first study demonstrating the development of eWUE along an altitudinal gradient based on δ 13 C cell values in tree rings.Several studies combining CO 2 and H 2 O flux measurements with modeling approaches have shown an increase in WUE at the ecosystem level with altitude.However, at high altitudes, where adverse conditions for growth, photosynthesis, and transpiration prevail, ecosystem WUE may decrease, as reported for forest (Sun et al., 2020) and grassland ecosystems (Han et al., 2013).Parametrization of a coupled model for evapotranspiration and gross primary production using satellite MODIS products revealed a slight increase in WUE up to N. Pernicová et al. altitudes of 1100 m a.s.l. in China, irrespective of vegetation cover type (Nandy et al., 2022;Zhang et al., 2023).However, WUE dramatically decreased at altitudes above 5000 m a.s.l.(Xue et al., 2015).

Conclusions
In this study, we have confirmed significant differences in climate factors along the natural altitudinal gradient (130-630 m a.s.l.) where oak species grow in Central Europe.Amplified atmosphere warming and desiccation increase significantly with altitude, particularly in the last three decades.Changes in total precipitation during the vegetation season remain relatively stable.These climatic shifts, combined with increasing CO 2 concentration, have influenced intrinsic (stomata-based) and evaporative (transpiration-based) WUE, determined through a highly sensitive detection of stable carbon isotope ratios in the latewood cellulose (δ 13 C cell ) of annually resolved tree rings.While iWUE significantly increased in the last 60 years in oaks grown at low (irrespective of Quercus species) and middle altitudes, this trend was negligible at high altitudes.The marked increase in iWUE at lower altitudes suggests more efficient photosynthesis and stomatal closure in response to warmer and drier conditions.On the contrary, the stimulatory effects of elevated CO 2 and temperature on iWUE appear to be limited by the rising deficit of water vapor in the atmosphere at higher altitudes.
In contrast to iWUE, eWUE did not exhibit any increasing trends over the past 60 years.A slight decrease in eWUE was observed in highaltitude trees, corresponding to a 22 % increase in atmospheric VPD.These divergent trends between iWUE and eWUE emphasize the importance of accurate interpretation of these two measures of plant physiological status.Furthermore, they underscore the necessity of correcting for atmospheric VPD changes, especially when evaluating changes in WUE on ecosystem and global scales.

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Carbon isotopes assess water use efficiency (WUE) in oaks along altitudinal gradient.• High sensitivity allows to distinguish even among trees of small mountain ranges.• Trees at low altitudes show increased stomata-based WUE over the past 60 years.• Increasing vapor-pressure deficit (VPD) offsets CO 2 and temperature benefits on WUE.• Atmospheric VPD correction is necessary when calculating WUE at large scales.water use in plants, a critical ecophysiological parameter closely related to water and carbon cycles, is essential for understanding the interactions between plants and their environment.This study investigates the effects of ongoing climate change and increasing atmospheric CO 2 concentration on intrinsic (stomata-based; iWUE) and evaporative (transpiration-based; eWUE) water use efficiency in oak trees along a naturally small altitudinal gradient (130-630 m a.s.l.) of Vihorlat Mountains (eastern Slovakia, Central Europe).

Fig. 2 .
Fig. 2. Changes in stable carbon isotope ratio (δ 13 C cell ; A-C) and intercellular CO 2 concentration (Ci; D-F) over the past 60 years (1961-2020) derived from the annually resolved tree-ring cellulose of sessile oak (Q.petraea) trees growing at low (LA; 280 m a.s.l.), middle (MA; 400 m a.s.l.), and high altitude (HA; 630 m a.s.l.).Coloured lines represent the mean values (n = 6), while coloured areas indicate the range between the first and the third quartiles.Black lines show (A-C) stable carbon isotope ratio in the atmosphere (δ 13 C atm ) and (D-F) mean annual atmospheric CO 2 concentration (Ca).Statistical differences in Ci/Ca ratio between the periods 1961-1990 and 1991-2020 are shown in figs.D-F: n.s.= statistically non-significant difference (p > 0.05), ** = statistically highly significant difference (p < 0.01).

Table 1
Summary information for sites and trees used for the isotope measurement.Abbreviations: HA (high altitude), MA (middle altitude), LA (low altitude), and DBH (diameter at breast height of all analyzed trees).