DOI: 10.5061/dryad.95x69p8vc
title: Mixing oak and pine trees in Mediterranean forests increases aboveground hydraulic dysfunctions

description: Increasing tree species diversity in Mediterranean forests could reduce
 drought‐induced hydraulic impairments through improved microclimate and
 reduced competition for water. However, it remains unclear if and how
 species diversity modulates tree hydraulic functions and how impacts may
 shift during the growing season.  Using unmanaged Mediterranean
 forest stands composed of one (i.e., monospecific) or four (i.e.,
 multispecific) tree species, we examined the seasonal dynamics of in‐situ
 hydraulic traits (predawn and midday leaf water potential – Ψpd and Ψmd,
 xylem‐ and leaf‐specific hydraulic conductivity – KS and KL, percentage
 loss of conductivity – PLC, specific leaf area – SLA, and Huber value –
 HV) in four co‐existing Pinus and Quercus species over two years. We
 mainly observed adverse impacts of species diversity with lower Ψpd, Ψmd,
 KS, KL, and higher PLC in multispecific compared to monospecific stands,
 especially for the two pines. These impacts were observed all along the
 growing season but were stronger during the driest periods of the summer.
 Beneficial impacts of diversity were rare and only occured for oaks (Q.
 faginea) after prolonged and intense water stress. Our findings reveal
 that mixing oaks and pines could mainly enhance hydraulic impairments for
 all species during the dry season, suggesting a potential decline in mixed
 Mediterranean forests under future climate.
description: <em>Study site</em>            
 The study was conducted in Mediterranean forests in the Alto Tajo natural
 park (Guadalajara, Castilla La Mancha, 40.66°N, 02.27°W) in central Spain,
 where 15 stands (30m x 30m) within a 20 km<sup>2</sup> area
 were selected from the FunDivEUROPE Exploratory Platform (Fig.1, Baeten et
 al., 2013). To assess the effect of tree species diversity, the stands
 were established in 2011 in non-managed, mature, and even-aged (i.e., more
 than 50 years old) forests with limited variation in altitude (i.e., from
 980 to 1300 m a.s.l.), topography, soil type, and density (see Table S1
 and Baeten et al (2013) for more details on plot selection). However,
 while all stands have not been managed for more than 50 years, their
 original species composition resulted from past land management where
 monospecific stands naturally occurred, and mixed stands were encouraged
 for wood production. The soils in all stands were shallow (from 20 to 40
 cm) calcic cambisol soils (FAO/UNESCO soil classification) on a cracked
 limestone bedrock but with plant roots that may extend down to several
 meters through the fractured bedrock (Peñuelas & Filella,
 2003). The study area has a continental Mediterranean
 climate with hot and dry summers and cold and snowy winters. The long-term
 mean annual temperature at the site was 11°C (1990-2022), with 12°C and
 11°C in 2021 and 2022 (during the measurements), respectively. Maximum
 daily temperatures were 31°C and 33°C for 2021 and 2022, respectively
 (Fig. 2a-b). The long-term annual precipitation sum was 436 mm
 (1990-2022), with 516 and 367 mm in 2021 and 2022, respectively (Fig.
 2c-d). We used the monthly Standardized Precipitation Evaporation Index
 (SPEI) extracted from a database (https://spei.csic.es/; Vicente-Serrano
 et al., 2010). Based on SPEI drought classifications (Agnew, 2000), the
 summer 2021 (i.e., from June to August) was not particularly dry, while
 the summer of 2022 was extremely dry with a mean SPEI of -1.9, reaching
 its minimum over the past 30 years (Fig. 2e-f).      
 The area is characterized by the natural dominance of four tree
 species, i.e., two coniferous species (<em>Pinus nigra subsp.
 salzmannii (Dunal) Franco</em> and <em>Pinus sylvestris
 L.</em>) and two broadleaved ones (<em>Quercus faginea
 Lam.</em> and <em>Quercus ilex L.</em>), which were
 selected for this study. We selected stands where the target species
 represents more than 90% of the total basal area (i.e., for monospecific
 stands; Fig. 1b-e) and where the four tree species were mixed in similar
 abundances (i.e., multispecific stands; Fig. 1f). Each species richness
 level (monospecific <em>vs</em>. multispecific) was replicated
 three times for all species (Table S1; Baeten et al., 2013), leading to a
 total of 15 stands (three monospecific stands per species and three
 multispecific ones). The understory vegetation (representing less than 10%
 of the total basal area) was mainly composed of shrub species
 (<em>Arctostaphylos uvaursi, Buxus sempervirens,
 </em>and<em> Genista scorpius</em>) and juveniles of the
 dominant tree species.             We randomly selected
 five dominant or co-dominant trees per species in each plot, leading to
 120 trees. To assess the seasonal dynamics of aboveground hydraulic
 traits, we conducted <em>in-situ</em> measurements (detailed
 below) on each tree in 2021 and 2022 at the beginning (i.e., in May when
 soil moisture was high), middle (i.e., in July, corresponding to the
 driest and hottest period), and the end of the growing season (i.e., in
 September, representing the recovery transition from dry to wet
 soils). <em>In-situ hydraulic
 traits:</em> <em>            </em>One
 twig of about 0.5 to 1 cm diameter per tree was collected in the higher
 part of the canopy with a pool pruner before sunrise
 (Ψ<sub>pd</sub>) and at midday (Ψ<sub>md</sub>) to
 measure the leaf water potential (MPa) with a Scholander-type pressure
 chamber (M1505D, PMS Instruments, USA). On the same day
 as the leaf water potential measurements, one sun-exposed branch of 1.5 m
 for broadleaves and 50 cm-long for conifers (to cut branches that were
 longer than the longest vessels or tracheids), was cut on four from the
 five selected trees (n=4) and placed in sealed black plastic bags with wet
 tissues to keep a humid atmosphere. Within the next two days, the samples
 were processed using a commercial conductivity meter (XYL’EM, Bronkhorst,
 France), according to Cochard et al., (2005). Three 3- to 5-cm-long stem
 segments were cut underwater from the terminal part of each branch,
 corresponding to the current and previous year. Due to the low growth rate
 of Mediterranean tree species, short segments are commonly used to
 determine the yearly hydraulic conductivity at the risk of open-vessel
 artifacts (e.g., Torres-Ruiz et al., 2014; Vilagrosa et al.,
 2003). The proximal end of each segment was connected
 to the tubing system of the XYL’EM, which was filled with deionized
 filtered water with 10mM KCl and 1mM CaCl<sub>2</sub> that had
 been previously degassed. A low-pressure (70kPa) flow was applied to
 measure the initial hydraulic conductivity of each shoot
 (K<sub>ini</sub>, kg m MPa<sup>-1</sup>
 s<sup>-1</sup>). Due to the high resin content in pines, only
 the segments of oak species were flushed at 0.15MPa for 1.30 min to
 measure their maximum hydraulic conductivity (K<sub>max</sub>,
 kg m MPa<sup>-1</sup> s<sup>-1</sup>). A second
 flush at 0.15MPa for 30s was performed to confirm the maximal conductivity
 value. The K<sub>ini</sub>/K<sub>max</sub> ratio
 was used to compute the percentage loss conductivity for oaks (PLC, %).
 Stem xylem-specific conductivity (K<sub>s</sub>, kg
 m<sup>-1</sup> MPa<sup>-1</sup>
 s<sup>-1</sup>) was calculated for every shoot by dividing
 K<sub>ini</sub> by the sapwood area, estimated as the
 cross-section area (pith included) without bark. Due to the limited
 intra-specific variability in pith area (Larios Mendieta et al., 2021),
 its inclusion in the cross-section calculation should have negligeable
 impacts on our results assessing the seasonal variability in Ks within
 species.   In addition, all distal leaves and needles
 of each segment were scanned using a flatbed scanner (CanoScan LiDE 300,
 CANON, UK), followed by analysis with ImageJ (Schindelin et al., 2019) to
 extract the total leaf area per segment (LA, m²). The leaf-specific
 hydraulic conductivity (K<sub>L</sub>, kg
 m<sup>-1</sup> MPa<sup>-1</sup>
 s<sup>-1</sup>) was calculated by dividing
 K<sub>ini</sub> by LA, and the Huber value (HV,
 m<sup>2</sup> m<sup>-2</sup>) was estimated as the
 sapwood area:LA ratio. The mature leaves and needles collected in July
 2021 and 2022 were dried at 60°C for 24h and weighed using a high
 precision scale (MS104, Mettler Toledo, CH) to calculate the specific leaf
 area (i.e., the ratio dry mass:LA, SLA, m²
 g<sup>-1</sup>). <em>Statistical
 analyses</em>             The effects of tree
 species diversity on Ψ<sub>pd</sub>,
 Ψ<sub>md</sub>, K<sub>s</sub>,
 K<sub>L</sub>, PLC, HV, and SLA were determined through linear
 mixed-effects models for each species using the function
 <em>lmer</em> in the package<em> lme4 </em>(Bates
 et al., 2015). The effect of the season (i.e., spring, summer, fall), year
 (i.e., 2021, 2022), species diversity (i.e., monospecific/multispecific),
 and stand density (i.e., stem ha<sup>-1</sup>) were used as
 fixed effects, and the individual plot was treated as a random effect.
 However, stand density had no significant effects on the studied hydraulic
 traits (Table S2), suggesting that other parameters, including species
 interactions, prevailed over stand density in driving the
 <em>in-situ</em> hydraulic traits. The two-way interactions
 between year, season, and species diversity were then tested as described
 in Table S2. To reveal significant differences between species diversity
 levels for each measurement at each sampling date and each species, post
 hoc analyses were performed with Tukey's HSD test, with FDR
 correction for multiple testing using the package
 <em>lmerTest</em> (Kuznetsova et al., 2017). Non-linear and
 linear regressions were used to test the relationships between
 Ψ<sub>pd</sub> and K<sub>L</sub>. All analyses
 were performed using the R v.4.2.2 statistical software (R Development
 Core Team, Austria, 2022). Before performing each model, the homogeneity
 of variances and the normality of residuals were assessed, and data were
 log-transformed if necessary.
description: # Mixing oak and pine trees in Mediterranean forests increases aboveground
 hydraulic dysfunctions
 [https://doi.org/10.5061/dryad.95x69p8vc](https://doi.org/10.5061/dryad.95x69p8vc) ## Description of the data and file structure Using unmanaged Mediterranean forest stands composed of one (i.e., monospecific) or four (i.e., multispecific) tree species, we examined the seasonal dynamics of *in-situ* hydraulic traits (predawn and midday leaf water potential – Ψpd and Ψmd, xylem- and leaf-specific hydraulic conductivity - KS and KL, percentage loss of conductivity – PLC, specific leaf area- SLA, and Huber value - HV) in four co-existing *Pinus* and *Quercus* species over two years. ### Files and variables #### File: data.csv **Description:** Raw data belonging to: "Mas, E., Vilagrosa, A., Morcillo, L., Valladares, F., Grossiord, C. (2024), Mixing oak and pine trees in Mediterranean forests increases aboveground hydraulic dysfunctions." ##### Variables | Column | Entry | Value | Unit | Explaination | Remark | | :----- | :------------ | :---- | :--------------- | :--------------------------------------------------------------------------------------------------------------- | :----------------------------------------- | | A | Year | 2 | | Year (2021; 2022) | | | B | Plot | 30 | | Plot number | | | C | Tree | 117 | | Tree number | | | D | Species | 4 | | Species (PS:Pinus sylvestris, PN: Pinus nigra, QF: Quercus faginea, QI: Quercus ilex) | | | E | Mixture | 2 | | Species richness level (1, 4) | | | F | Group | 2 | | Type of mixture (monospecific, multispecific) | | | G | Date | 702 | | Date of the measuring campaign (format: Year-Month) | | | H | Campaign | 6 | | Campaign number | | | I | Season | 3 | | Season (Spring, Summer, Fall) | | | J | Predawn | 697 | MPa | Predawn leaf water potential | | | K | Midday | 686 | MPa | Midday leaf water potential | | | L | PLC | 282 | % | Pourcentage loss of conductivity (difference between Kh and Khmax) | measured only for oaks | | M | Khmax | 283 | kg.m-1.Mpa-1.s-1 | Maximal Hydraulic conductivity (hydraulic conductivity after flushing) | measured only for oaks | | N | Kh | 549 | kg.m-1.Mpa-1.s-1 | Native hydraulic conductivity (the volume flow rate divided by the pressure gradient (i.e. length of the sample) | | | O | Ks | 550 | kg.m-1.Mpa-1.s-1 | Specific hydraulic conductivity (Kh divided by the segment's cross-sectional xylem area) | | | P | Ksmax | 282 | kg.m-1.Mpa-1.s-1 | Maximal Specific Hydraulic conductivity (Khmax divided by the segment's cross-sectional xylem area) | measured only for oaks | | Q | KL | 550 | kg.m−1.MPa−1.s−1 | Leaf specific hydraulic conductivity (Kh divided by the leaf area distal to the measured segement) | | | R | KLmax | 273 | kg.m−1.MPa−1.s−1 | Maximal Leaf specific hydraulic conductivity (Khmax divided by the leaf area distal to the measured segement) | measured only for oaks | | S | SLA | 184 | m².g-1 | Specific leaf area (Leaf area / Dry mass) | measured only in Summer (Campaign 2 and 5) | | T | HV | 188 | m2.m-2 | Huber value for July (Sapwood area / LA) | measured only in Summer (Campaign 2 and 5) | | U | Stand-density | 117 | stem.ha-1 | Number of stem per hectar | | ## Code/software To view the data, use Excel or R statistical software (v.4.2.2, R Development Core Team, Austria, 2022).