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.

Study site

            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 km² 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 (Pinus nigra subsp. salzmannii (Dunal) Franco and Pinus sylvestris L.) and two broadleaved ones (Quercus faginea Lam. and Quercus ilex L.), 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 vs. 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 (Arctostaphylos uvaursi, Buxus sempervirens, and Genista scorpius) 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 in-situ 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).

In-situ hydraulic traits:

            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_pd) and at midday (Ψ_md) 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 CaCl₂ that had been previously degassed. A low-pressure (70kPa) flow was applied to measure the initial hydraulic conductivity of each shoot (K_ini, kg m MPa^-1 s^-1). 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_max, kg m MPa^-1 s^-1). A second flush at 0.15MPa for 30s was performed to confirm the maximal conductivity value. The K_ini/K_max ratio was used to compute the percentage loss conductivity for oaks (PLC, %). Stem xylem-specific conductivity (K_s, kg m^-1 MPa^-1 s^-1) was calculated for every shoot by dividing K_ini 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_L, kg m^-1 MPa^-1 s^-1) was calculated by dividing K_ini by LA, and the Huber value (HV, m² m^-2) 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^-1).

Statistical analyses

            The effects of tree species diversity on Ψ_pd, Ψ_md, K_s, K_L, PLC, HV, and SLA were determined through linear mixed-effects models for each species using the function lmer in the package lme4 (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^-1) 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 in-situ 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 lmerTest (Kuznetsova et al., 2017). Non-linear and linear regressions were used to test the relationships between Ψ_pd and K_L. 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.