Anatomical and physiological adjustments of pubescent oak (Quercus pubescens Willd.) from two adjacent sub-Mediterranean ecosites

Environmental and Experimental Botany 165 (2019) 208–218

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Anatomical and physiological adjustments of pubescent oak (Quercus pubescens Willd.) from two adjacent sub-Mediterranean ecosites

T

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Dominik Vodnika, , Jožica Gričarb, Martina Lavričb, Mitja Ferlanc, Polona Hafnerb, Klemen Elera a

Biotechnical Faculty, Department of Agronomy, University of Ljubljana, Jamnikarjeva 101, SI-1000, Ljubljana, Slovenia Slovenian Forestry Institute, Department of Yield and Silviculture, Vecna pot 2, SI-1000, Ljubljana, Slovenia c Slovenian Forestry Institute, Department of Forest Ecology, Vecna pot 2, SI-1000, Ljubljana, Slovenia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Drought Sap flow Xylogenesis Stomatal conductance Karst

Patterns of drought severity in natural ecosystems depend on ecosite characteristics, particularly on local soil water availability and microclimate (evaporative demand). At the sites with less favourable conditions, the plasticity of fundamental drought adaptive traits can be exceeded under severe drought, even in well adapted species. In our study, anatomical and physiological adjustments to drought were studied in pubescent oak (Quercus pubescens Willd.) growing on adjacent sub-Mediterranean plots (ecosites) differing in bedrock and soil type (limestone with rendzic leptosol, low water retention - L; transition from limestone to flysch with eutric cambisol, high water retention - F). In both years of the study, 2015 and 2016, in the part of the season with sufficient water supply, sap flow density (SFD) was higher in oaks growing on L than those growing on F. With decreasing soil water content (SWC) in July, SFD was reduced on both plots, most in trees growing on limestone. In the period of severe drought, SFD in trees growing on L was decoupled from the changes of VPD, due to increased employment of water conservation strategies, such as decreases in stomatal conductance. This was further translated into stomatal limitation of photosynthesis and contributed to reduced radial stem growth. The seasonal dynamics of traits indicate that Q. pubescens trees use a predisposed anatomical strategy (average size of vessels in different years), adjusted to average ecosite conditions. At the same time, trees respond to local conditions and interanual variability of drought by shortening/prolonging the production and growth of vessels and by adjusting leaf functioning (stomatal conductance, sap flow) in accordance with hydraulic properties.

1. Introduction Forest ecosystems are more and more frequently severely constrained by drought, often accelerated by high temperatures, which affects ecosystem water and carbon fluxes, decreases productivity and increases susceptibility to pests and diseases (Bréda et al., 2006; Allen et al., 2010). The response of forest ecosystems to these constraints varies and may include: (i) persistence of the current forest types, thanks to acclimatization to local conditions and to the phenotypic plasticity of the populations; (ii) local (genetic) adaptation, (iii) migration and substitution of species; and (iv) mortality, extinction of populations with low ecological plasticity (Bussotti et al., 2015). The pattern of drought stress is often quite variable on a finer spatial scale. Ecosite characteristics such as site elevation, aspect, slope, topographic position, bedrock and soil type influence local soil water availability, ⁎

microclimate (evaporative demand) and, consequently, the severity of drought (Allen et al., 2010). On a tree level, low soil water content (SWC) and high evaporative demand impact several steps in water transport along the soil-plantatmosphere continuum (Sperry et al., 2002). Hydraulic resistance increases in the soil, at the soil-root interface and often at the leaf-atmosphere interface, where the closure of the stomata limits water loss and prevents an irreversible drop of plant water potential, but at the cost of reduced CO2 assimilation. At higher drought intensity, the hydraulic conductance of the stem can decrease due to water cohesion break-down and vessel embolism. Stomatal limitations of photosynthesis, drought induced metabolic disorders and restrictions of water flux are translated into reduced growth. They may ultimately lead to tree death due to hydraulic failure, impaired carbon balance or the effects of secondary, frequently biotic, stress factors (McDowell et al., 2008).

Corresponding author. E-mail address: [email protected] (D. Vodnik).

https://doi.org/10.1016/j.envexpbot.2019.06.010 Received 23 April 2019; Received in revised form 31 May 2019; Accepted 17 June 2019 Available online 18 June 2019 0098-8472/ © 2019 Elsevier B.V. All rights reserved.

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typical of karst areas in lowlands where Q. pubescens dominates mid- to late stages of secondary succession of abandoned marginal agricultural lands. On flysch bedrock it prevails on warmer and drier southern slopes; elsewhere on this bedrock it is outcompeted by some faster growing species (Quercus cerris, Ostrya carpinifolia, Robinia pseudoacacia). Despite of its low silvicultural relevance (mostly firewood) the pubescent oak is ecologically important for the region as it prevents degradation of vulnerable, shallow and erosion-prone soil (Brus, 2004). Where it dominates, it defines the ecosystem performance, including carbon sequestration rate as demonstrated by long-term eddy covariance measurements (Ferlan et al., 2011, 2016). The aim of our study was to compare the performance of Q. pubescens trees cooccuring in adjacent stands on two different bedrocks (limestone vs. flysch) each with different soil water retention. The sharp transition between bedrcks (few tens of meters) at our site gives us the possibility to observe the effects of different soil water availability while controlling for the effects of climate. We presumed that at certain parts of the season, the two ecotypes of Q. pubescens experience different levels of drought stress, the later being stronger on limestone. We therefore hypothesise that trees from two ecosites employ different anatomical and functional traits to control and optimize water balance and that trees on limestone are characterised by traits typical for drought acclimation such as narrower xylem conduits, more strict stomatal control etc. To gain an insight into the response of the trees, these traits were integratively studied throughout two seasons (2015, 2016).

There is high diversity among plant species in responses to drought. Their resistance, avoidance or tolerance are based on structural and/or physiological adjustments (Bréda et al., 2006; Kozlowski and Pallardy, 2002), which contribute to functional balance among organs and tissues responsible for water acquisition, transport and transpiration and, at the same time, preserve carbon gain, important for growth and for support to stress response mechanisms (Sterck et al., 2008; Galmés et al., 2012; Flexas et al., 2014). In species from semi-arid ecosystems, these adaptations span from leaf morphology and anatomy (leaf size and shape, pubescense, morphological adaptations of stomata, cuticular conductance to water), foliage aggregation and inclination, plant stature, stem and root morphology and anatomy (hydraulic properties of conducting tissues, stem hydraulic capacitance, root architecture), to physiological adaptations (stomatal regulation, photosynthesis, assimilate allocation) (Galmés et al., 2012). Apart from evolutionary adaptations to limiting water availability and to other associated stresses, plants in semi-arid environments have large phenotypic plasticity, adjusting structure and function in response to environmental conditions (Valladares et al., 2007). Pubescent oak (Quercus pubescens Willd.), a typical Mediterranean deciduous broadleaved tree species, has developed various mechanisms and adaptations to survive in such a drought prone environment (Damesin and Rambal, 1995; Nardini and Pitt, 1999; Nardini et al., 1998; Lavrič et al., 2017; Gričar et al., 2017). It is known for its conservative water use and maintains high transpiration rates despite the incidence of drought. Water potential can consequently decrease substantially (Damesin and Rambal, 1995). Additionally, Q. pubescens is able to maintain high leaf relative water contents under water stress conditions, which is apparently achieved by compensating water loss with an equal amount of water uptake. It has been proposed that such a drought avoidance strategy is made possible by high hydraulic efficiency of stems and roots (Nardini and Pitt, 1999; Nardini et al., 2016), which is largely related to the anatomy of the conducting tissues (Eilmann et al., 2006, 2009; Gričar et al., 2017). When drought is severe, photosynthesis is reduced due to stomatal limitations and to transiently decreased photochemical efficiency, together with metabolic limitation. Diurnal measurements on dry hot days revealed only short-term reversible inhibition of photosystem II, induced by high light and temperature, which indicates efficient photoprotection (Damesin and Rambal, 1995; Haldimann et al., 2008). After summer drought, the species is able to recover leaf carbon gain efficiently (Haldimann et al., 2008). Recent extreme droughts, e.g., in seasons 2003 and 2012, have, however, shown that even higly adapted plants species such as Q. pubescens more and more commonly experience severe drought stress. This has been revealed by studies of carbon and water fluxes at the stand level (Granier et al., 2007; Ferlan et al., 2016), and by observations of tree mortality. In 2012, an extreme summer drought induced species-specific die-back in woody species in northeastern Italy, by which Q. pubescens was heavily impacted (Nardini et al., 2016). During drought, Q. pubescens was characterised by very low predawn water potential (-2.5 MPa), a severe decrease of stomatal conductivity and a substantial (60%) loss of stem hydraulic conductivity (Nardini et al., 2016). Comparison of Q. pubescens with less affected species (e.g., Prunus mahaleb) indicates that the different damage suffered by the different species during extreme drought depends not only on their hydraulic vulnerability but is also profoundly influenced by rooting depth and consequent effects on seasonal changes of plant water status, gas exchange and non-structural carbohydrates content (Nardini et al., 2016). Heterogeneous, patchy patterns of Q. pubescens mortality indicate that ecosite characteristics play an important part in determining the level and the outcome of stress for individual trees. In submediterranean area of Slovenia, Q. pubescens grows on limestone (rendzic leptosol with low water holding capacity), and flysch bedrock (eutric cambisol with higher soil water retention), i.e. on soils typical of the Mediterraenean basin. The first bedrock/soil type is

2. Material and methods 2.1. Location The study was performed in the sub-Mediterranean phytogeographic region of Slovenia, on the Podgorski kras plateau (SW Slovenia); where carbon and water fluxes have been researched in ecosystems at different stages of succession (pasture, woody plants encroached area) since 2009 (Ferlan et al., 2011, 2016). Two adjacent research plots with Quercus pubescens were selected at the edge of plateau, where the bedrock changes from limestone to flysch. The site with local exchange of sandstone and marlstone is termed F (flysch) for the sake of simplicity (Fig. 1; 45°32′30.48″N, 13°54′44.13″E, 422 m a.s.l.). It is characterised by eutric cambisol, a soil with high water retention potential. The depth of the soil profile was > 0.6 m. The soil on the plot on limestone (L; 45°32′31.82″N, 13°54′46.84″E, 430 m a.s.l.) is rendzic leptosol, shallow soil (depth 0 - 0.5 m) with a rocky ground surface, up to 45% stoniness in the upper 0.5 m of topsoil and, consequently, very poor water retention capacity. Soil depth varies from a few centimetres to several meters in soil pockets. Both soils have a clay texture, with 7.8% of sand, 33.5% of silt and 58.8% clay. The Q. pubescens stands are of similar age (> 50 years) but differ in structure and tree density. Canopy is much more closed and uniform at F plot than on L plot, where oak trees either grow soilatry or in small patches of few ares in size. An overall tree density is much lower at L plot, but can localy, within the patches exceed or is similar to the one at F plot. The site is influenced by the sub-Mediterranenan climate, which is transient between continental and Mediterranean climates. In comparison to the latter, it is characterised by a less pronounced dry summer and colder winter periods. Precipitation is relatively abundant (30-year average (1985–2014): 1313.4 mm; ARSO, 2019) but unevenly distributed throughout the year. Typically, two drought peaks occur annually, one before spring (January-March, 30-year average: 83.9 mm; ARSO, 2019) and another in the summer (July-August, 30-year average: 86.3 mm; ARSO, 2019). 2.2. Weather conditions at the study site in 2015 and 2016 The research was performed in 2015 and 2016. The year 2016 was 209

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Fig. 1. The two research plots, L (limestone bedrock) and F (transition to flysch bedrock), located in the study area of Podgorski kras in the SW part of Slovenia. a) rendzic leptosol on paleogenic limestone bedrock, b) eutric cambisol on a local exchange of sandstone and marlstone.

determined by counting yearly xylem rings using 5 mm diameter increment cores sampled at breast height. These measurements revealed that the selected trees were about the same age (F: 56.3 ± 9.8 years, L: 57.3 ± 7.3 years) but differed in height (F: 15.3 ± 2.3 m, L: 10.6 ± 1.3 m) and trunk circumference at breast height (F: 0.93 ± 0.2 m, L: 0.66 ± 0.1 m).

Table 1 Weather conditions in 2015 and 2016. Precipitation and solar radiation are calculated as daily sum, other variables as daily means. While meteorological parameters reflect the weather for the whole catchment area, SWC differed between the plots and is presented separately for F (flysch bedrock) and L (limestone bedrock); * growing season is the period April–September, from bud break to full leaf senescence (2015: DOY 91–273; 2016: DOY 92–274). Annually

Precipitation [mm] Air temperature [°C] Soil temperature [°C] Vapour pressure deficit [kPa] Solar radiation [MJm−2] Soil water content [m3 m−3]

Growing season*

2015

2016

2015

2016

943.1 14.2 14.2 0.62 4478.7 0.45 (F) 0.33 (L)

1434.1 12.9 11.7 0.51 4682.2 0.45 (F) 0.30 (L)

583.4 18.5 16.3 0.85 3856.7 0.44 (F) 0.32 (L)

571.5 17.7 15.9 0.73 3572.3 0.43 (F) 0.24 (L)

2.4. Collection of micrometeorological parameters Every selected tree was equipped with USB data loggers DL-120TH (VOLTCRAFT, Germany) to measure air temperature (Ta) and air humidity (RH) automatically at 30-minute intervals. They were located in the oaks’ canopies facing north and south. On both plots (F and L), MPS2 Dielectric water potential sensors (Decagon devices Inc., USA) measuring soil matric potential (SMP) and soil temperature (Ts) at 10minute intervals were installed in the soil close to the tree roots at a 0.10-0.15 m soil depth, at three locations on each plot. All sensors were installed in March 2015 and collected data for the two consecutive growing seasons, 2015 and 2016. Precipitation data were collected from Kozina meteorological station (ARSO, 2019) located near the research area. Other meteorological parameters, such as solar radiation (SR), wind speed (WS) and other, were collected at half-hour intervals at a micrometeorological tower in close vicinity to the research plots (Ferlan et al., 2011). Vapour pressure deficit (VPD) was calculated from Ta and RH data following Murray (1967) and Monteith and Unsworth (1990). Soil cores were sampled at the end of the experiment and SWC was obtained from soil retention curves acquired by the Hyprop method (UMS GmbH, Munich, Germany) and SMP data.

generally wet; annual precipitation exceeded the 30-year average by 9%. The year 2015 was much drier, 943.1 mm amounted to 72% of the 30-year average (Table 1). This difference was mainly due to the period January-March, when precipitation in 2016 was higher than 2015 by 65.0%, July and August, on the other hand, were drier in 2016 than in 2015. (Fig. 2). Air temperature was in both cases higher in 2015 than in 2016. Average soil water content (SWC; vol. %) was higher in F than in L. During the growing season, SWC in F did not deviate much from the yearly average, while this was not the case in L, where it was lower, especially in 2016, the year characterised by a dry summer.

2.5. Sap flow measurements 2.3. Tree selection The selected oaks were equipped with SFM1 Sap Flow Meters according to the Heat Ratio Method (SFM1 instrument, ICT International, Australia). Sensors were installed at the beginning of the growing season, i.e., in March 2015, removed in winter and reinstalled in March 2016 to prevent a wounding effect on sap flow readings (Burgess et al., 2001). Sensor installation and data preparation are summarized in detail in Lavrič et al. (2017). Only data from sensors located in sap wood (first thermistor depth (below bark): 8.2 mm) were collected. Sap

At the beginning of the growing season in March 2015, six visually healthy and mature pubescent oaks (Quercus pubescens Willd.) were randomly selected on every research plot. On L plot all trees were selected within woodland patches so that competitive effects of the neighbouring trees were similar between plots. The circumference and diameter at breast height were measured and the height was determined using a Vertex III (Haglöf, Sweden). The age of the trees was 210

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Fig. 2. Micrometeorological variables and Q. pubescens sap flux density in 2015 and 2016 (DOY 131–216) for plots F (transition to flysch bedrock) and L (limestone bedrock). SFD = sap flux density, P = precipitation, VPD = vapour pressure deficit, SWC = soil water content, SR = solar radiation, Ta = air temperature. SFD, Ta, SWC and VPD are presented as daily means and P and SR as daily sums. The dashed line in the SWC panel shows the drought threshold for Q. pubescens dominated forest on limestone (SWCcrit = 0.145 m3 m−3, Ferlan et al., 2016).

flow data are presented as sap flux density (SFD [cm3 cm−2 h-1]; Steppe et al., 2010), which was obtained from Sap Flow Tool software (ICT International, Australia). Zero sapflow was assumed for the leafless period (before and after the growing season) (Do and Rocheteau, 2002) and for night-time data of the growing season with VPD and wind speed close to zero (Plaut et al., 2012; Cleiton et al., 2013). For the period of the assumed zero flow, the mean SF was calculated for each tree for each year; differences between zero and the obtained mean SF were used as the corrections for the whole dataset of each tree-year combination.

and 2016, mean vessel tangential diameter, mean vessel area, vessel density (i.e., number of cells mm−2) and percentage water conductive area were assessed in earlywood, also separately for the first ring of earlywood vessels (i.e., initial earlywood vessels). Explanations and definitions of the determined phenological phases of cambial activity, xylem development and wood-anatomical analyses are given in detail in Gričar et al.(2017). 2.7. Leaf gas exchange, leaf water potential Leaf measurements were conducted on fully developed sun exposed leaves from the southern part of the crown by using two LI-6400 XT portable photosynthesis systems (LI−COR, Lincoln, USA). Measurements were taken simultaneously on both plots, from 9 to 11 a.m., at a constant reference CO2 concentration (400 μmol mol−1) and photon flux density (1500 μm m-2 s−1), controlling temperature and water vapour deficit at the ambient level (targeting to average for measuring time respecting daily weather conditions). Six leaves from three trees were measured per plot. Net photosynthesis (A), transpiration (E), stomatal conductance (gs), intercellular leaf CO2 concentration (Ci) and photochemical efficiency (Fv’/Fm’; yield) were recorded when steady state conditions were reached. Intrinsic water use efficiency (WUE = A/E; μmol CO2 mmol H2O−1) was derived from gas exchange measurements. The measured leaves were collected, the chlorophyll content was determined by SPAD (Konica-Minolta Sensing Inc., Osaka, Japan). Homogeneity in the light exposure of the measured leaves was tested by assessing specific leaf area, after the leaves were scanned (area measurements), dried and weighed in the lab. Midday water potential (Ψmd) was determined in the leaves, similar to ones measured by Li6400, using the pressure chamber technique (Scholander et al., 1964; chamber 3005-1223, Soil Moisture Equipment Corp., Goleta, USA). Three leaves from three trees were sampled for

2.6. Observations of leaf phenology and leaf area and the radial tree growth Leaf phenological observations were performed from budbreak until leaf fall, in the period from March to November in both investigated years. Phenological stages were followed according to a 10-stage scale described in Gričar et al. (2017). Leaf Area Index (LAI) was determined on the plot level using LAI-2200 Plant Canopy Analyzer (LI−COR Inc., USA). LAI measurements were performed in several transects in the small woodland patches in which the selected trees were growing. Mean LAI per plot was calculated for each measurement term (7-10-day interval). Micro-core collection was performed from the stem 1.0–1.3 m above the ground using a Trephor tool (Rossi et al., 2006) at 7-10-day intervals in the period March-October in 2015 and 2016. Sample collection, preparation and measurements were performed as described in Lavrič et al. (2017) and Gričar et al. (2017). Stained and embedded sections were observed under a Leica DM4000 B light microscope (Leica, Microsystems CMS GmbH, Wetzlar, Germany) using transmission and polarized light. Histometrical analyses were performed with a Leica Application Suite, version 4.9.0 (Leica Microsystems Ltd., Switzerland). Samples with callus or tension wood were excluded from further analysis. In addition, in completely developed xylem increments of 2015 211

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Table 2 Sap flux density (SFD) for different periods on plots F and L in 2015 and 2016 presented as average daily values. (1) 2015: days of the year 133–200, 2016: 133–190; (2) 2015: 201–211, 2016: 191–216; (3) 2015: 212-273. * data from DOY220 on are missing due to a wildfire that occurred on August 7, 2016. SFD [cm3 h−1 cm-2] / Period

2015

2016

F Full leaf unfolding-summer drought (1) Summer drought (SWC < 0.145 m3 m−3) (2) End of summer drought-end of September (3) Total summer (1+2)

5.42 5.56 5.92 5.44

L ± ± ± ±

1.71 0.86 1.49 1.61

7.13 5.05 7.91 6.84

± ± ± ±

1.75 0.58 2.36 1.79

F

L

6.07 ± 1.35 6.88 ± 0.99 * 6.32 ± 1.30

6.66 ± 2.26 6.08 ± 0.64 * 6.48 ± 1.92

and there was also a consistent difference in SWC between the plots. SWC was constantly lower at L plot than at F plot. SWC was relatively stable until July and August, with one exception - a significant decrease in SWC at L plot in response to a short drought period at the beginning of June 2015. The most prominent, however, are differences in SWC for August, when rain in 2015 contributed to the recovery of SWC to values close to the seasonal average. In contrast, low SWC persisted in 2016, characterised by a dry second half of July and beginning of August, until the rain period in the second half of August. In both years, SWC at L plot decreased below SWCcrit (0.145 m3 m−3) in July and August. On L plot, SWCcrit was reached round DOY 200 in 2015 and around DOY 190 in 2016.

each plot. 2.8. Data processing and analysis Analyzes were performed in statistical software R version 3.4.3 (R Core Team, 2018). First, data of the different parameters were displayed as time-series and analyzed with descriptive statistics. Sapflow data was analysed on both diurnal and seasonal time scales. Daytime mean SFD (7 a.m. - 6 p.m.) was calculated to examine the seasonal course of sap-flow. SFD dependence on VPD and SR was tested using nonlinear (negative exponential) and linear models, respectively. We also tested whether including a separate categorical variable that indicated soil water availability, significantly improves the model. For this, we defined two periods of the season, one with sufficient water availability and the dry period. Based on eddy-covariance measurements with limestone, we have previously defined 0.145 m3 m−3 as the critical SWC value (SWCcrit) below which evapotranspiration of ecosystem and Q. pubescens as its dominant species starts to be strongly influenced by water shortage (Ferlan et al., 2016). On this basis, a time of the season when SWCL plot is smaller than SWCcrit can be clearly defined as a dry period for the area, while SWC > SWC crit indicates periods of sufficient soil water availability. This, however, does not imply the actual water availability at different ecosites within the area, nor the level of drought stress experienced by trees growing at these sites. Comparison of a joint model (without SWC class) and separate models (SWC classes included) was tested using a partial F-test. Half‐hourly values of SFD were used to investigate the diurnal sensitivity of SFD to VPD and SR. This analysis was restricted to two periods within season 2016, a period with sufficient soil water availability (3-day window around DOY 174) and drought period (3-day window around DOY 200). For stem anatomy, gas exchange and leaf water parameters, the significance of the difference between plots and years was determined using linear mixed-effects models with plots and years and fixed factors and the tree within a plot as a random factor. Assumption of normality was assessed graphically (Q-Q plot) and homogeneity of variance was tested using the Levene test.

3.2. Sap flow The phenological development of oaks was similar on F and L in both years. LAI, however, reached much higher values on F plot than on L plot (4.2 ± 1.1 vs. 3.9 ± 1.1 in 2015 and 2.8 ± 0.8 vs. 2.4 ± 0.8 in 2016) at full leaf unfolding. Prior to the summer drought, the average SFD was 23.9% (2015) and 8.9% (2016) higher on L plot than on F plot (Fig. 2, Table 2). In both years, the course of SFD corresponded well to the changes in VPD. The seasonal, plot specific maximum of SFD (SFDmax) was about the same in both years. The two-year average of SFDmax was 8.4 cm3 cm−2 h-1 for F plot and 10.7 cm3 cm−2 h-1 for L plot. With decreasing SWC in July, SFD was reduced on both plots, most in trees growing on limestone (Fig. 2). In the period of extreme drought, the average SFD was 9.2% (2015) and 11.6% (2016) lower on L plot than on F plot. These differences diminished at the end of August with decreasing evaporative demand. In the post-drought period in September 2015, SFD was again higher on L plot than on F plot (Table 2). Analysis of non-aggregated, half‐hourly data for the period with sufficient water supply (DOY 173–175) revealed that SFD tended to preceed VPD (Fig. 3), resulting in a clockwise hysteresis loop (Fig. 4). During severe drought (DOY 199–201), however, SFD and VPD were more coupled in trees on L. In this period, their SFD reached half of the maximum value recorded on DOY 173-175.

3. Results

3.3. Relation between sap flow and weather characteristics (VPD, SR)

3.1. Water availability

SFD was shown to be related to both VPD and SR, with SFD vs. VPD showing an asymptotic relationship and SFD vs. SR showing a positive linear relationship (Fig. 5). Data measured on L plot were grouped according to SWC into those measured during drought (SWCL plot < 0.145 m3 m−3) and the remainder of the data. For F plot SFD-VPD and SFD-RS dependencies, the effect of these SWC groups was not significant, whereas for L plot the responsiveness of SFD to RS and VPD decreased significantly under dry conditions (p-value of partial F-test is < 0.001). On the other hand, high evaporative demand (high VPD) stimulated SFD in the period of favourable soil water availability. In this case, the VPD-related increase of SFD was higher in trees growing on L plot than those growing on F plot.

The years 2015 and 2016 significantly differed in distribution of precipitation during the growing season (Fig. 2). A very wet first third of 2016 was followed by a relatively dry spring with a few minor rain events, while spring was considerably wetter in 2015, when there was a longer rain period in the last decade of May (DOY 140–150). In both years, June was relatively wet, except the dry and warm first week of 2015. In the same year, there was an extreme rain peak in the last week of June. July and August were, as usual, dry in both years. An important difference affecting the development of drought, however, is that there was some rain at the end of August in 2015 but not in 2016. Seasonal precipitation patterns were reflected in soil water content 212

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Fig. 3. Q. pubescens sap flux density (SFD) and vapour pressure deficit (VPD) for plots F (transition to flysch bedrock) and L (limestone bedrock). Data are shown for 3-day windows during the period with sufficient water supply (SWC > SWCcrit; left panels) and during summer drought (SWC < SWCcrit, right panels). SFD represents the average of non-aggregated half-hourly data for four trees.

3.4. Leaf gas exchange and leaf water potential

3.5. Xylem-ring and earlywood vessel properties

On June 22 (DOY 174), in the period with sufficient water availability, trees from the two locations had similar photosynthetic rates but clearly differed in stomatal conductivity and transpiration, which were both smaller in trees from L plot (Table 3). These trees were also characterised by lower midday water potential. In the drought period (DOY 200), the midday leaf water potential on both plots was below −2 MPa, but it was still significantly lower in L trees, which substantially reduced their stomatal conductivity (gs), limiting both transpiration and photosynthesis. The gs in trees from F plot was in a similar range to that on DOY 174, as was the net photosynthesis. In conditions of normally wetted soil (DOY 174, 2016), oaks from the drier L plot exhibited 65% higher intrinsic water use efficiency (WUEi = 8.16 ± 1.18 μmol CO2 mmol H2O−1) than oaks from the wetter F plot (2.83 ± 0.43 μmol CO2 mmol H2O−1). At that time, SWC was relatively high on both plots (F: 0.46 m3 m-3, L: 0.36 m3 m-3). The opposite trend was observed in July (DOY 200, 2016) during summer drought, when a higher WUEi was maintained by oaks from the wetter plot F (SWC = 0.44 m3 m-3, WUEi = 6.68 ± 1.95 μmol CO2 mmol H2O−1) than those from the drier plot L (SWC < 0.05 m3 m-3, WUEi = 3.79 ± 0.45 μmol CO2 mmol H2O−1).

The measured selected wood variables showed statistically significant anatomical differences between trees growing on different bedrock/soil (Table 4). In general, trees from F plot were characterised by wider annual xylem increments, wider earlywood vessels and bigger conducting area than L trees. The mean earlywood vessel diameter was smaller on L plot by 15.7% in 2015 and 11.0% in 2016, resulting in a 22.7% smaller mean earlywood vessel area (18.2% of total earlywood vessel area) in 2015 and 19.3% smaller (34.2% of total earlywood vessel area) in 2016 than on F plot. Corresponding to the wider earlywood vessels, their density was lower on plot F by about 22.9%. The percentage of water conductive area in earlywood, on the other hand, was comparable on the two plots and in both years (from 35.7% to 37.3%). The same pattern was observed in initial earlywood vessels (i.e., 1st ring), in which mean vessel diameter, mean vessel area and total vessel area were in all cases smaller in trees from plot L in both years than those from plot F. Interannual comparison of wood-anatomy parameters revealed no statistically significant differences. However, the width of latewood tended to be wider in 2016 on both plots than in 2015 (p = 0.058). A significant interaction of plot x year was revealed for total vessel area in the first ring. On F plot, these values were higher in 2016 than in 2015, while the opposite was found for L plot (0.207 mm2 ± 0.064 in 2015, Fig. 4. Hysteresis loops for vapour pressure deficit (VPD) and sap flux density (SFD) for pubescent oak (Q. pubescens) growing on transition to flysch (F; upper panels) and limestone (L; lower panels) bedrock. Hysteresis is shown for 3-day windows during a period with sufficient water supply (SWC > SWCcrit; DOY 173–175; left panels) and a period of drought (SWC < SWCcrit; DOY 199–201; right panels).

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Fig. 5. Relation between daily average sap flux density (SFD) and mean daily vapour pressure deficit (VPD; upper panels) and SFD and daily sum of solar radiation (SR; lower panels). For all variables, daytime data (7 a.m. - 6 p.m.) from both years (2015 and 2016) were used in the analysis. Open symbols are used for the period of sufficient soil water availability, closed symbols are for the drought period (SWCL plot < 0.145 m3 m−3). For F (transition to flysch bedrock) a joint model for all data was used; for L (limestone bedrock) models were significantly different for wet and dry soil conditions. Coefficients of determination (R2) and mean absolute error (MAE) of different models are given.

0.179 mm2 ± 0.046 in 2016).

wind in the area. It can be concluded that, at certain periods, oak stands on L are more exposed to air mixing and thus to higher evaporative demand. As far as the light regime within the canopy is concerned, it can be presumed that it is more homogeneous in the sparse canopy on L plot than in the dense canopy on F, where more light is attenuated by foliage in the outer part of the crowns. Tree density and the level of canopy closure do not only influence microclimate but also affect soil water content In dense stands water and other resources can be limited due to competition which is reflected in canopy processess and has a consequence in higher vurnelability to drought (McDowell et al., 2006; Bottero et al., 2017). However, when comparing two plots with large difference in water holding capacity, such as F and L, different tree density can be regarded as secondary factor influencing SWC. As revealed from results, it does not prevail defining water availability. In both years, a significant decrease of SWC was detected in the midsummer period, which is a typical drought period for the area (Ferlan et al., 2016). As expected, a much larger decrease of SWC was observed in rendcic leptosol, due to the shallow soil profile and very low water retention. Data for 2015, however, revealed fast recovery of SWC when there was sufficient input from precipitation.

4. Discussion The integrative structural-functional approach presented in this work, i.e. linking ecophysiological and anatomical measurements of individual tree levels with an ecological evaluation (Steppe et al., 2015), revealed that ecosite properties substantially influenced drought acclimation of Quercus pubescens. Adjacent pubescent oak stands with different soil characteristics did not differ much in meteorological conditions. At both plots, air temperature, solar radiation and vapour pressure deficit exhibited similar seasonal patterns, which, however, slightly differed in magnitude. These differences can be partly related to the density of the canopy, which significantly affects microclimatic conditions by influencing light penetration and air circulation. It contributes to variation in temperature and relative humidity regimes (Chen et al., 1999; Heithecker and Halpern, 2007). Throughout the 2015 season, we observed a slightly higher VPD on L plot where the stand has sparser canopy (as indicated by slightly lower LAI values). However, this does not match observations by Rambo and North (2009), who reported lower VPD in a less dense (understorey thinned) forest stand. It is likely that in our case the observed differences are more a result of local topography. Due to a small (8 m) but quite sharp difference in altitude (Fig. 1), F plot is fairly well protected from the NE, which is the direction of the prevailing

4.1. Tree functioning in the part of the season with sufficient SWC In the period of sufficient water availability, evaporative demand

Table 3 Net photosynthesis (A), stomatal conductance (gs), transpiration (E), intrinsic water use efficiency (WUEi = A/E) and water potential (Ψ) of Q. pubescens growing on flysch (F) and limestone bedrock (L). Measurements were taken on June 22 (DOY 174) and July 18 (DOY = 200). Means ± standard errors are shown, N = 9. Siginificant factor effects (p < 0.05) are indicated in bold and marked by *. DOY 174

−2

s ] A [μmol CO2 m gs [mol H20 m−2 s-1] E [mmol H2O m−2 s-1] WUEi [μmol mmol−1] Ψ [MPa] -1

DOY 200

ANOVA (p value)

F

L

F

L

Plot

Doy

Plot x Doy

14.29 ± 3.09 0.25 ± 0.06 5.14 ± 1.26 2.83 ± 0.43 −1.63 ± 0.38

14.79 ± 3.20 0.10 ± 0.03 1.84 ± 0.46 8.16 ± 1.18 −2.08 ± 0.26

13.74 ± 4.19 0.23 ± 0.10 2.32 ± 1.08 6.68 ± 1.95 −2.23 ± 0.20

3.99 ± 1.33 0.03 ± 0.01 1.05 ± 0.31 3.79 ± 0.45 −2.40 ± 0.14

0.000* 0.000* 0.000* 0.000* 0.005*

0.000* 0.000* 0.000* 0.312 0.000*

0.000* 0.000* 0.069 0.000* 0.144

214

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Table 4 Anatomical characteristics of Q. pubescens wood. Means ± standard deviations are shown for F (flysch bedrock) and L (limestone bedrock) for 2015 and 2016. Siginificant factor effects (p < 0.05) are indicated in bold, N = 6. F

Xylem ring width [μm] Earlywood width [μm] Latewood width [μm] Vessel density [no mm−2] Mean diameter of initial earlywood vessels [μm] Mean area of initial earlywood vessels [mm2] Total area of initial earlywood vessels [mm2] Mean earlywood vessel diameter [μm] Mean earlywood vessel area [mm2] Total earlywood vessel area [mm2] Percentage of water conductive area in earlywood [%]

L

ANOVA (p value)

2015

2016

2015

2016

Plot

Year

Plot x Year

1556.4 ± 753.2 635.5 ± 120.3 920.9 ± 669.1 5.7 ± 1.6 312.7 ± 35.7 0.079 ± 0.014 0.214 ± 0.055 270.6 ± 38.9 0.066 ± 0.013 0.363 ± 0.113 36.3 ± 5.1

1881.8 ± 741.3 637.0 ± 250.8 1244.8 ± 639.3 6.5 ± 1.4 298.8 ± 38.9 0.081 ± 0.018 0.259 ± 0.081 243.6 ± 43.5 0.057 ± 0.016 0.439 ± 0.175 35.7 ± 6.9

716.1 ± 305.5 401.6 ± 124,5 314.5 ± 201.6 7.6 ± 1.5 274.2 ± 35.0 0.068 ± 0.016 0.207 ± 0.064 228.1 ± 30.6 0.051 ± 0.013 0.297 ± 0.851 37.3 ± 7.6

1100.6 ± 636.0 464.5 ± 189.8 636.1 ± 460.8 8.2 ± 1.6 253.8 ± 25.8 0.059 ± 0.008 0.179 ± 0.046 216.7 ± 29.5 0.046 ± 0.009 0.289 ± 0.079 37.0 ± 5.8

0.036 0.020 0.058 0.004 0.000 0.001 0.018 0.007 0.004 0.008 0.607

0.105 0.690 0.063 0.095 0.157 0.484 0.546 0.134 0.116 0.366 0.716

0.885 0.704 0.994 0.747 0.778 0.342 0.025 0.522 0.526 0.285 0.907

and solar radiation were the main factors determining sap flow. In agreement with other studies (Chen et al., 2014; Shen et al., 2015), we found a strong linear relation between SFD and SR. The responsiveness of SFD was similar on both plots. SFD vs. VPD exhibited a commonly reported negative exponential curve; increases in VPD led to a relatively linear response in SFD, until a critical threshold in VPD, at which SFD rates plateaud (Monteith, 1995; Du et al., 2011; Wu et al., 2018). Here the responsiveness of SFD on VPD was higher on L plot than on F plot, which means that acceleration of SFD for a certain VPD rise was greater in trees growing on limestone. In agreement with this observation, the daily averaged SFD values in this period were substantially higher on L plot than on F plot, as were the seasonal SFDmax. These differences can be only partly attributed to microclimate, since the difference in VPD between the plots was only minor. Irradiance, on the other hand, could promote higher canopy conductance in the sparser crowns of trees on limestone. Furthermore, SFD could be influenced by the total leaf area (AL) generating a pulling force for the water column in the tree. It is generally suggested that AL is reduced under drought, which contributes to higher leaf specific conductivity (LSC = Kp / AL), assuming that stem conductivity (Kp) remains unchanged or is less decreased (Martínez-Vilalta et al., 2002). However, comparison of two populations of Q. pubescens trees, growing in persistently wet or dry soil in the alpine valley of Valais, Switzerland, revealed the opposite (Sterck et al., 2008). In this Swiss study, modelling morphological and anatomical data showed low LSC in trees on the dry site, which was associated with a smaller functional sapwood area and narrower conduits, resulting in a stronger reduction in Kp than AL. It was suggested that relatively high investment in leaves compared with sapwood contributes to carbon gain over an entire season, by enabling rapid whole-plant photosynthesis during periods of high water availability (e.g., in spring, after rain events and during morning hours, when the leaf to- air vapour pressure deficit is small) (Sterck et al., 2008). In our study, we lack insight into AL and LSC, since we made no destructive leaf area determinations and an assessment of AL of individual trees by LAI measurements was not possible since the trees were not solitary. However, a possible explanation suggested by Sterck et al. (2008) would explain the fairly high SFD values in L trees in the period of sufficient SWC. This presumption cannot be fully supported by our leaf gas-exchange measurements, since they were performed on south exposed sunny leaves at the time of the day when stomata could reduce conductivity in response to increased VPD, despite relatively high SWC. Knowing that light availability within the forest canopy is heterogeneous in spatio-temporal terms (Kitao et al., 2009), the response of measured leaves may not neccessarily reflect the response at the crown level. Measurements revealed that sampled leaves of L trees operated under lower stomatal conductivity (gs) and a higher tension of the water column in xylem (lower Ψ) than F trees. They still reach similar

photosynthetic rates to those on F plot and comparable to rates reported for non-stressed Q. pubescens trees (Damesin and Rambal, 1995; Haldimann et al., 2008; Gallé et al., 2007). They surpassed F trees in WUE. This indicates that trees on limestone employ conservative water use even in the period of sufficcient SWC, in order to optimize hydraulic function (and carbon gain). This is where the response of gs to environmental factors accords with the anatomy of the conducting tissues. Analysis of diurnal courses showed that SFD preceeds VPD when trees were well supplied with water. On both plots, SFD rapidly increased in the morning and reached a diurnal maximum earlier than VPD (ca. 2.5–3 hours on F plot). Similarly, a fairly unusual lagging of VPD behind SFD was recently reported by Looker et al. (2018) for subalpine fir (Abies lasiocarpa) and Engelmann spruce (Picea engelmannii). The most plausible explanation is that this diurnal pattern is driven by solar radiation. Stomata respond to light by opening, increasing gs and E, which generates tension enabling a faster mass flow of water in xylem conduits. Not only gs but also leaf hydraulic conductance is known to be promoted by light (Tyree et al., 2005; Voicu et al., 2008). 4.2. Tree functioning in the drought period The trees from the two ecosites differed in functional response to environmental conditions during summer drought. Due to low soil water availability on limestone, the water upatke to roots was insufficient and leaf water potential dropped to −2.4 MPa. According to previous studies (Damesin and Rambal, 1995; Nardini et al., 2016), this value of Ψ indicates moderate to strong stress of Q. pubescens. In our case, the severitiy of stress was clearly reflected in leaf physiology, with a strong reduction of stomatal conductivity and photosynthesis. Furthermore, the sap flow density of L trees decreased and was fairly unresponsive to short rain events bringing insufficient water input (Fig. 2, 2016, DOY 196). In F trees, there was no severe stomatal limitation of photosynthesis and the SWC related fluctuations of SFD were less prominent. These trees were more buffered against drier hydroclimatic conditions due to higher availability of soil water. This is also reflected in changes in the sensitivity of SFD to VPD and SR (daily averages, Fig. 5), which decreased significantly in L trees but not in F trees. Decoupling of SFD from the changes of VPD and SR indicates that plant water potential (which is actually a result of the water uptake: water loss ratio) dominates the regulation of stomatal conductivity, preventing irreversible turgor loss and preserving hydraulic function. Nardini et al. (2016) reported a 60% loss of hydraulic conductivity of Q. pubescens stem when midday leaf Ψ decreased from −1.5 (June 2012) to −3.5 MPa (August 2012), which is in accordance with previous reports on Q. pubescens hydraulic vulnerability (Tyree and Cochard, 1996). Despite the drought in 2016 not being as severe as in 2012, the response of trees on limestone shows that they were 215

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might indirectly influence plant performance during and after drought events via effects on xylem hydraulic functioning, with special reference to processes of postdrought embolism recovery (Trifilo et al., 2017). However, our analysis of non-structural carbohydrates revealed no clear difference in bark and wood NSC content of trees from the two ecosites (Gričar et al., 2018). In general, the role of NSC storage and allocation in drought stress and mortality in trees has yet to be fully investigated (Hartmann and Trumbore, 2016).

approaching limits at which they cannot easily compensate for water loss. Diurnal sap flow measurements provided a deeper insight into water conservation strategies of L trees during summer drought (Fig. 4). SFD patterns indicate a midday depression in stomatal conductivity that has often been measured on Mediterranean plants and has been demonstrated on different oak species, including Q. pubescens (Damesin and Rambal, 1995; Lange et al., 1982). In the morning and late afternoon, however, leaf gas exchange promoted SFD and supplied photosynthesis, contributing to carbon gain. This indicates that the level of stress was not extreme. Haldimann et al. (2008), for example, reported a whole day stomatal closure and complete reduction of Q. pubescens photosynthesis during an exceptionally dry and hot summer period in 2003. On the diurnal scale, SFD was decoupled from VPD and SR only in the middle of the day, while it was very well synhronized (no lag) with both environmental factors during the remainder of the light period. The lack of hysteresis reflects fine tuning of hydraulic conductance, neccessary when a tree is functioning close to the safety limits.

4.4. Ecological implications In the context of changing climate, intra-annual variability in physiological and growth responses of pubescent oak to micro-environmental conditions contribute to a better understanding of its future survival changes and growth performance. As shown in the case of pubescent oak, bedrock type acting via soil water availability can substantially affect several tree processes. It modulates weather effects and in this respect, it is the factor to be included in predicting tree and forest ecosystem functioning, productivity and stability within the climate change scenarios. According to these scenarios the subMediterranean ecosystems are predicted to be highy affected by lower precipitation, particularly during the vegetation period (Keenan et al., 2011). More importantly, anticipated increase in frequency and severity of extreme weather events, such as heat waves, drought and fires, may jeopardize tree health and raise the possibility of amplified forest mortality (Bréda et al., 2006; Allen et al., 2010), which would eventually lead to community composition changes. In contrast to evergreen Mediterranean trees which can supplement growth and carbon storage during favourable winter periods (Sanz-Perez et al., 2009), the functioning of deciduous trees, such as pubescent oak, in the sub-Mediterranean will likely be more constrained in the future climate by both, winter temperatures and summer drought. Finally, woodlands in drought-prone environments on shallow (e.g. limestone) soils will be replaced by shrublands of lower economic value, lower carbon sequestration potential and increased fire risk, which will also have significant implications on global carbon cycling (Bodner and Robles, 2017).

4.3. Ecosite specific adjustment of stem anatomy The hydraulic function of trees relies significantly on the anatomical properties of conducting tissues (Pfautsch, 2016). As expected, oaks on the L plot had considerably narrower conduits and higher conduit densities in earlywood, which are typical anatomical traits indicating low water availability in the ecosystem. Similar anatomical characteristics have been reported for dry site populations of Q. pubescens in Valais, Switzerland in dry years compared to mesic years (Sterck et al., 2008; Eilmann et al., 2006, 2009). This accords with the explanation that water availability during vessel differentiation is a key factor determining the size of conduits (Cherubini et al., 2003). The cell expansion rate and ultimate cell size decrease with decreasing water availability (Sass and Eckstein, 1995; Villar-Salvador et al., 1997; Zweifel et al., 2006), which is related to the fact that the cell expansion rate and cell size are controlled by turgor pressure (Hsiao and Acevedo, 1974; Steppe et al., 2006). Furthermore, a smaller diameter of vessels reduces vulnerability to cavitation (Sperry et al., 1994), which is important since Q. pubescens avoids drought by a water spending strategy (Levitt, 1980; Nardini and Pitt, 1999). The dynamic equlibrium between water loss and uptake is reached here by increasing the driving force for water uptake (tension generated by transpiration), and the tree can undergo a marked drop in xylem water potential, which might reach the threshold for xylem cavitation (Nardini and Pitt, 1999; Nardini et al., 2016). However, conduit diameters are predominantly linked to susceptibility to freeze/thaw–induced embolism, while pit architecture is mainly related to vulnerability to drought-induced embolism (Pfautsch, 2016). Due to the relatively harsh winter conditions at our study site, trees may be at risk of cold stress. This is supported by increased raffinose amounts during the winter (Gričar et al., 2018), which are known to contribute to cell protection against environmental stress (Lintunen et al., 2016). Whether the structure of intervessel pits in L-trees and F-trees is also adapted to different micro-environmental conditions remains an open question for further investigation. Cambial activity, cell growth and differentiation are costly processes and therefore influenced by the carbon budget of the tree. In our accompanying study of seasonal dynamics of radial growth (Lavrič et al., 2017; Gričar et al., 2018), we showed that earlier cessation of cell production (cca 1-week difference between L and F plots) and a considerably lower rate of cell production are the main causes of reduced xylem stem growth in L-trees (over 40% in 2016 compared to F-trees). Interestingly, growth was not considerably prolonged at F plot, which might be an adaptation of Q. pubescens to stop radial growth before stressful conditions appear (Zweifel et al., 2006). Investment in wood formation depends on the yearly carbon gain of a tree, which was significantly lower on L plot, as indicated by growth parameters and the limitation of photosynthesis during drought periods. Plant carbon status

5. Conclusions The persistence of forests trees under changing climate is based on adjustments occurring in traits at morphological and physiological levels. For two adjacent populations of Q. pubescens, we showed large differences in growth, anatomy of stem conducting tissues and shortterm stomatal regulation. In the population occupying limestone, smaller stem radial increments and narrower vessels were found throughout the years, which indicates that insufficient water availability is a persisting environmental constraint. Trees achieve homeostasis in water transport by a combination of short-term stomatal regulation and optimal sapwood conductivity. The seasonal dynamics of traits indicates that trees use a predisposed anatomical strategy (average size of vessels in different years), adjusted to average ecosite conditions and, at the same time, respond to local conditions and interanual variability of drought by shortening/prolonging the production and growth of vessels. Under given environmental conditions, trees adjust leaf functioning in accordance with hydraulic properties. There are important other traits, not studied here, such as tree rooting to access different water sources, root hydraulic conductivity, carbon allocation to roots and leaves etc. These traits should be addressed in further research. Depending on species and population, some traits can have higher plasticity than others, with consequences on adaptive processes. Severe droughts in the past, however, have shown that plasticity of fundamental adaptive traits of Q. pubescens can be exceeded, which could affect its local geographical distribution. 216

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of xylem sap flow with thermal dissipation probes. 2. Advantages and calibration of a noncontinuous heating system. Tree Physiol. 22 (9), 649–654. https://doi.org/10. 1093/treephys/22.9.649. Du, S., Wang, Y.-L., Kume, T., Zhang, J.-G., Otsuki, K., Yamanaka, N., Liu, G.-B., 2011. Sapflow characteristics and climatic responses in three forest species in thesemiarid Loess Plateau region of China. Agric. For. Meteorol. 151 (1), 1–10. https://doi.org/ 10.1016/j.agrformet.2010.08.011. Eilmann, B., Weber, P., Rigling, A., Eckstein, D., 2006. Growth reactions of Pinus sylvestris L. And Quercus pubescens Willd. To drought years at a xeric site in Valais, Switzerland. Dendrochronologia 23 (3), 121–132. https://doi.org/10.1016/j.dendro.2005.10.002. Eilmann, B., Zweifel, R., Buchmann, N., Fonti, P., Rigling, A., 2009. Drought-induced adaptation of the xylem in Scots pine and pubescent oak. Tree Physiol. 29 (8), 1011–1020. https://doi.org/10.1093/treephys/tpp035. Ferlan, M., Alberti, G., Eler, K., Batič, F., Peressotti, A., Miglietta, F., et al., 2011. Comparing carbon fluxes between different stages of secondary succession of a karst grassland. Agric. Ecosyst. Environ. 140 (1-2). https://doi.org/10.1016/j.agee.2010. 12.003. Ferlan, M., Eler, K., Simončič, P., Batič, F., Vodnik, D., 2016. Carbon and water flux patterns of a drought-prone mid-succession ecosystem developed on abandoned karst grassland. Agric. Ecosyst. Environ. 220, 152–163. https://doi.org/10.1016/j.agee. 2016.01.020. Flexas, J., Diaz-Espejo, A., Gago, J., Gallé, A., Galmés, J., Gulías, J., Medrano, H., 2014. Photosynthetic limitations in Mediterranean plants: a review. Environ. Exp. Bot. https://doi.org/10.1016/j.envexpbot.2013.09.002. Gallé, A., Haldimann, P., Feller, U., 2007. Photosynthetic performance and water relations in young pubescent oak (Quercus pubescens) trees during drought stress and recovery. New Phytol. 174, 799–810. https://doi.org/10.1111/j.1469-8137.2007. 02047.x. Galmés, J., Flexas, J., Medrano, H., Niinemets, Ü, Valladares, F., 2012. Ecophysiology of photosynthesis in semi-arid environments. In: Flexas, J., Loreto, F., Medrano, H. (Eds.), Terrestrial Photosynthesis in a Changing Environment: A Molecular, Physiological, and Ecological Approach. Cambridge University Press, Cambridge, pp. 448–464. https://doi.org/10.1017/CBO9781139051477.035. Granier, A., Reichstein, M., Bréda, N., Janssens, I.A., Falge, E., Ciais, P., et al., 2007. Evidence for soil water control on carbon and water dynamics in European forests during the extremely dry year : 2003. Agric. For. Meteorol. 143, 123–145. https:// doi.org/10.1016/j.agrformet.2006.12.004. Gričar, J., Lavrič, M., Ferlan, M., Vodnik, D., Eler, K., 2017. Intra-annual leaf phenology, radial growth and structure of xylem and phloem in different tree parts of Quercus pubescens. Eur. J. For. Res. 136 (4). https://doi.org/10.1007/s10342-017-1060-5. Gričar, J., Zavadlav, S., Jyske, T., Lavrič, M., Laakso, T., Hafner, P., et al., 2018. Effect of soil water availability on intra-annual xylem and phloem formation and non-structural carbohydrate pools in stem of Quercus pubescens. Tree Physiol. https://doi.org/ 10.1093/treephys/tpy101. tpy101. Haldimann, P., Gallé, A., Feller, U., 2008. Impact of an exceptionally hot dry summer on photosynthetic traits in oak (Quercus pubescens) leaves. Tree Physiol. 785–795. Hartmann, H., Trumbore, S., 2016. Understanding the roles of non-structural carbohydrates in forest trees – from what we can measure to what we want to know. New Phytol. 211, 386–403. https://doi.org/10.1111/nph.13955. Heithecker, T.D., Halpern, C.B., 2007. Edge-related gradients in microclimate in forest aggregates following structural retention harvests in western Washington. For. Ecol. Manage. 248 (3), 163–173. https://doi.org/10.1016/j.foreco.2007.05.003. Hsiao, T.C., Acevedo, E., 1974. Plant responses to water deficits, water-use efficiency, and drought resistance. Agric. Meteorol. 14 (1-2), 59–84. https://doi.org/10.1016/00021571(74)90011-9. Keenan, T., Serra, J.M., Lloret, F., Ninyerola, M., Sabate, S., 2011. Predicting the future of forests in the Mediterranean under climate change, with niche- and process-based models: CO2 matters!. Glob. Chang. Biol. 17, 565–579. https://doi.org/10.1111/j. 1365-2486.2010.02254.x2011. Kitao, M., Löw, M., Heerdt, C., Grams, T.E.E., Häberle, K.-H., Matyssek, R., 2009. Effects of chronic elevated ozone exposure on gas exchange responses of adult beech trees (Fagus sylvatica) as related to the within-canopy light gradient. Environ. Pollut. 157, 537–544. https://doi.org/10.1016/j.envpol.2008.09.016. Kozlowski, A.T.T., Pallardy, S.G., 2002. Acclimation and adaptive responses of woody plants to environmental stresses acclimation and adaptive responses of woody plants to environmental stresses. BioOne 68 (2), 270–334. https://doi.org/10.1663/00068101(2002)068[0270:AAAROW]2.0.CO;2. Lange, O.L., Tenhunen, J.D., Braun, M., 1982. Midday stomatal closure in Mediterranean type sclerophylis under simulated habitat conditions in an environmental chamber. I. Comparison of the behaviour of various European Mediterranean species. Flora 172 (6), 563–579. Lavrič, M., Eler, K., Ferlan, M., Vodnik, D., Gričar, J., 2017. Chronological sequence of leaf phenology, xylem and phloem formation and sap flow of Quercus pubescens from abandoned karst grasslands. Front. Plant Sci. 8, 1–11. https://doi.org/10.3389/fpls. 2017.00314. Levitt, J., 1980. Responses of Plants to Environmental Stresses. Volume II. Water, Radiation, Salt, and Other Stresses. Academic Press, London 607 pp. Lintunen, A., Paljakka, T., Jyske, T., Peltoniemi, M., Sterck, F., von Arx, G., et al., 2016. Osmolality and non-structural carbohydrate composition in the secondary phloem of trees across a latitudinal gradient in Europe. Front. Plant Sci. 7, 726. https://doi.org/ 10.3389/fpls.2016.00726. Looker, N., Martin, J., Hoylman, Z., Jencso, K., Hu, J., 2018. Diurnal and seasonal coupling of conifer sap flow and vapour pressure deficit across topoclimatic gradients in a subalpine catchment. Ecohydrology 11 (7), 1–16. https://doi.org/10.1002/eco. 1994. Martínez-Vilalta, J., Prat, E., Oliveras, I., Piñol, J., 2002. Hydraulic properties of roots and

Authors’ contributions All authors conceived and designed the work and participated in data collection. ML carried out anatomical and sap flow analyses; ML and KE performed statistical analyses and figures; DV, ML and JG led the writing of the manuscript; all authors critically revised the manuscript and approved its final to be published. Agreement/Declaration There's no financial/personal interest or belief that could affect the objectivity of the study. Funding This work was supported by the Slovenian Research Agency, Young Researchers Program (ML), research core funding nos. P4-0085, P40107, Project J4-7203 (Short and long term responses of oak in the submediterranean region to extreme weather events using tree anatomical analysis and ecophysiological measurements), and by a scholarships (ML) from the National Scholarship Fund of the World Federation of Scientists, Erasmus + programme and COST Action MaPFGR (FP1202). Acknowledgement The authors gratefully acknowledge the help of Gregor Skoberne, Urša Pečan, Jan Eržen, Boštjan Županc, Gabrijel Leskovec and Robert Krajnc in the field and laboratory. We thank Kathy Steppe for consultations on sap-flow measurements, Nives Ogrinc and Bor Krajnc for analyses and Martin Cregeen for language editing. References Allen, C.D., Macalady, A.K., Chenchouni, H., Bachelet, D., McDowell, N., Vennetier, M., et al., 2010. Forest Ecology and Management A Global overview of Drought and HeatInduced Tree Mortality Reveals Emerging Climate Change Risks for Forests, vol. 259. pp. 660–684. https://doi.org/10.1016/j.foreco.2009.09.001. ARSO. 2019. http://www.arso.gov.si/o%20agenciji/knji%C5%BEnica/mese%C4%8Dni %20bilten/ (accessed, 15.4.2019). Bodner, G.S., Robles, M.D., 2017. Enduring a decade of drought: patterns and drivers of vegetation change in a semi-arid grassland. Journa of Arid Environments 136, 1–14. https://doi.org/10.1016/j.jaridenv.2016.09.002. Bottero, A., D’Amato, A.W., Palik, B.J., Bradford, J.B., Fraver, S., Battaglia, M.A., Asherin, L.A., 2017. Density-dependent vulnerability of forest ecosystems to drought. J. Appl. Ecol. 54, 1605–1614. https://doi.org/10.1111/1365-2664.12847. Bréda, N., Huc, R., Granier, A., Dreyer, E., 2006. Temperate forest trees and stands under severe drought : a review of ecophysiological responses, adaptation processes and long-term consequences. Ann. For. Sci. 63 (6), 625–644. https://doi.org/10.1051/ forest:2006042. Brus, R., 2004. Drevesne vrste na Slovenskem. 1. izd. Ljubljana, Založba Mladinska knjiga. d.d.: 399 p.. . Burgess, S.S., Adams, M.A., Turner, N.C., Beverly, C.R., Ong, C.K., Khan, A.A., Bleby, T.M., 2001. An improved heat pulse method to measure low and reverse rates of sap flow in woody plants. Tree Physiol. 21 (9), 589–598. https://doi.org/10.1093/ treephys/21.9.589. Bussotti, F., Pollastrini, M., Holland, V., Brüggemann, W., 2015. Functional traits and adaptive capacity of European forests to climate change. Environ. Exp. Bot. 111, 91–113. https://doi.org/10.1016/j.envexpbot.2014.11.006. Chen, J., Saunders, S.C., Crow, T.R., Naiman, R.J., Brosofske, K.D., Mroz, G.D., Brookshire, B.L., Franklin, J.F., 1999. Microclimate in forest ecosystem and landscape ecology. BioScience 49 (4), 288–297. https://doi.org/10.2307/1313612. Chen, D.Y., Wang, Y.K., Liu, S.Y., Wei, X.G., Wang, X., 2014. Response of relative sap flow to meteorological factors under different soil moisture conditions in rainfed jujube (Ziziphus jujuba Mill.) plantations in semiarid Northwest China. Agric. Water Manag. 136, 23–33. https://doi.org/10.1016/j.agwat.2014.01.001. Cherubini, P., Gartner, B.L., Tognetti, R., Braker, O.U., Schoch, W., Innes, J.L., 2003. Identification, measurement and interpretation of tree rings in woody species from mediterranean climates. Biol. Rev. 78 (1), 119–148. https://doi.org/10.1017/ S1464793102006000. Damesin, C., Rambal, S., 1995. Field study of leaf photosynthetic performance by a Mediterranean deciduous oak tree (Quercus pubescens) during a severe summer drought. New Phytol. 131 (2), 159–167. https://doi.org/10.1111/j.1469-8137.1995. tb05717.x. Do, F., Rocheteau, A., 2002. Influence of natural temperature gradients on measurements

217

Environmental and Experimental Botany 165 (2019) 208–218

D. Vodnik, et al.

Acad. Sci. U.S.A. 52, 119–125. https://doi.org/10.1073/pnas.52.1.119. Shen, Q., Gao, G.Y., Fu, B.J., Lu, Y.H., 2015. Sap flow and water use sources of shelter-belt trees in an arid inland river basin of Northwest China. Ecohydrology 8 (8), 1446–1458. https://doi.org/10.1002/eco.1593. Sperry, J.S., Hacke, U.G., Oren, R., Comstock, J.P., 2002. Water Deficits and Hydraulic Limits to Leaf Water Supply. pp. 251–263. Sperry, J., Nichols, K., Sullivan, J., Eastlack, S., 1994. Xylem embolism in ring-porous, diffuse-porous, and coniferous trees of northern Utah and interior Alaska. Ecology 75, 1736–1752. Steppe, K., De Pauw, D.J.W., Lemeur, R., Vanrolleghem, P.A., 2006. A mathematical model linking tree sap flow dynamics to daily stem diameter fluctuations and radial stem growth. Tree Physiol. 26 (3), 257–273. https://doi.org/10.1093/treephys/26.3. 257. Steppe, K., Vandegehuchte, M.W., Tognetti, R., Mencuccini, M., 2015. Sap flow as a key trait in the understanding of plant hydraulic functioning. Tree Physiol. 35 (4), 341–345. https://doi.org/10.1093/treephys/tpv033. Sterck, F.J., Zweifel, R., Sass-Klaassen, U., Chowdhury, Q., 2008. Persisting soil drought reduces leaf specific conductivity in Scots pine (Pinus sylvestris) and pubescent oak (Quercus pubescens). Tree Physiol. 28 (4), 529–536. https://doi.org/10.1093/ treephys/28.4.529. Trifilo, P., Casolo, V., Raimondo, F., Petrussa, E., Boscutti, F.-, et al., 2017. Effects of prolonged drought on stem non-structural carbohydrates content and post-drought hydraulic recovery in Laurus nobilis L.: the possible link between carbon starvation and hydraulic failure. Plant Physiol. Biochem. 120 (2017), 232–241. Tyree, M.T., Cochard, H., 1996. Summer and winter embolism in oak: impact on water relations. Annales des Sciences Forestieres 53 (2-3), 173–180. https://doi.org/10. 1051/forest:19960201. Tyree, M.T., Nardini, A., Salleo, S., Sack, L., El Omari, B., 2005. The dependence of leaf hydraulic conductance on irradiance during HPFM measurements: any role for stomatal response? J. Exp. Bot. 412, 737–744. Valladares, F., Gianoli, E., Gómez, J.M., 2007. Ecological limits to plant phenotypic plasticity. New Phytol. 176 (4), 749–763. https://doi.org/10.1111/j.1469-8137. 2007.02275.x. Villar-Salvador, P., Castro-Diez, P., Perez-Rontome, C., Montserrat-Marti, G., 1997. Stem xylem features in three Quercus (Fagaceae) species along a climatic gradient in NE Spain. Trees - Structure and function 12 (2), 90–96. https://doi.org/10.1007/ PL00009701. Voicu, M.C., Zwiazek, J.J., Tyree, M.T., 2008. Light response of hydraulic conductance in bur oak (Quercus macrocarpa) leaves. Tree Physiol. 28 (7), 1007–1015. https://doi. org/10.1093/treephys/28.7.1007. Wu, Y.Z., Zhang, Y.K., An, J., Liu, Q.J., Lang, Y., 2018. Sap flow of black locust in response to environmental factors in two soils developed from different parent materials in the lithoid mountainous area of North China. Trees – Structure and function 32 (3), 675–688. https://doi.org/10.1007/s00468-018-1663-6. Zweifel, R., Zimmermann, L., Zeugin, F., Newbery, D.M., 2006. Intra-annual radial growth and water relations of trees: implications towards a growth mechanism. J. Exp. Bot. 57 (6), 1445–1459. https://doi.org/10.1093/jxb/erj125.

stems of nine woody species from a holm oak forest in NE Spain. Oecologia 133 (1), 19–29. https://doi.org/10.1007/s00442-002-1009-2. McDowell, N.G., Adams, H.D., Bailey, J.D., Hess, M., Kolb, T.E., 2006. Homeostatic maintenance of ponderosa pine gas exchange in response to stand density changes. Ecol. Appl. 16, 1164–1182. McDowell, N., Pockman, W.T., Allen, C.D., Breshears, D.D., Cobb, N., Kolb, T., et al., 2008. Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought? New Phytol. 178 (4), 719–739. https://doi.org/10.1111/j.1469-8137.2008.02436.x. Monteith, J.L., 1995. A reinterpretation of stomatal responses to humidity. Plant Cell Environ. 18 (4), 357–364. https://doi.org/10.1111/j.1365-3040.1995.tb00371.x. Monteith, J.L., Unsworth, M.H., 1990. Priciples of Environmental Physiscs, 4th ed. Elsevier, Amsterdam 401 pp. Murray, F.W., 1967. On the computation of saturation vapor pressure. J. Appl. Meteorol. 6, 203–204. https://doi.org/10.1175/1520-0450(1967)006<0203:OTCOSV>2.0. CO;2. Nardini, A., Pitt, F., 1999. Drought resistance of Quercus pubescens as a function of root hydraulic conductance, xylem embolism and hydraulic architecture. New Phytol. 143 (3), 485–493. https://doi.org/10.1046/j.1469-8137.1999.00476.x. Nardini, A., Casolo, V., Dal Borgo, A., Savi, T., Stenni, B., Bertoncin, P., et al., 2016. Rooting depth, water relations and non-structural carbohydrate dynamics in three woody angiosperms differentially affected by an extreme summer drought. Plant Cell Environ. 39 (3), 618–627. https://doi.org/10.1111/pce.12646. Nardini, A., Lo Gullo, M.A., Salleo, S., 1998. Seasonal changes of root hydraulic conductance (KRL) in four forest trees: an ecolological interpretation. Plant Ecol. 139 (1), 81–90. Pfautsch, S., 2016. Hydraulic anatomy and function of trees - basics and critical developments. Curr. For. Rep. 2 (4), 236–248. https://doi.org/10.1007/s40725-0160046-8. Plaut, J.A., Yepez, E.A., Hill, J., Pangle, R., Sperry, J.S., Pockman, W.T., McDowell, N.G., 2012. Hydraulic limits preceding mortality in a piñon-juniper woodland under experimental drought. Plant Cell Environ. 35 (9), 1601–1617. https://doi.org/10.1111/ j.1365-3040.2012.02512.x. R Core Team, 2018. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing., Vienna. Rambo, T.R., North, M.P., 2009. Canopy microclimate response to pattern and density of thinning in a Sierra Nevada forest. For. Ecol. Manage. 257 (2), 435–442. https://doi. org/10.1016/j.foreco.2008.09.029. Rossi, S., Anfodillo, T., Menardi, R., 2006. Trephor: a new tool for sampling microcores from tree stems. IAWA J. 27, 89–97. https://doi.org/10.1163/22941932-90000139. Sanz-Perez, V., Castro-Diez, P., Joffre, R., 2009. Seasonal carbon storage and growth in Mediterranean tree seedlings under different water conditions. Tree Physiol. 29 (9), 1105–1116. https://doi.org/10.1093/treephys/tpp045. Sass, U., Eckstein, D., 1995. The variability of vessel size in beech (Fagus sylvatica L.) and its ecophysiological interpretation. Trees - Structure and function 9 (5), 247–252. Scholander, P.F., Hemmingsen, E.A., Hammel, H.T., Bradstreet, E.D.P., 1964. Hydrostatic pressure + osmotic potential in leaves of mangroves + some other plants. Proc. Natl.

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