This study was conducted at the Sierra Morena mountain range in Southwestern Spain (38° 22′ 50.64″ N, 4° 45′ 27.69″ W; Pozoblanco, Córdoba). This area has a continental-Mediterranean climate characterised by cold winters and hot, dry summers. Annual precipitation is 439 mm year−1 and mean annual temperature is 15.2 °C. The studied ecosystem is a “Mediterranean dehesa”, a savannah-type forest managed as a traditional sylvo-pastoral system, with an herbaceous stratum of native pasture and a tree layer of scattered oak trees (Quercus ilex L.). Tree density is 14.5 ± 1.3 trees ha−1, and the herbaceous layer is dominated by a diverse community of annual native species such as: Hordeum murinum (L.), Senecio vulgaris (L.), Bromus madritensis (L.), and Sinapis alba (L.), with a mean species richness of 9.6 ± 0.3 species m−2.
In September 2016, we selected three sites separated by 4.3 ± 1.9 km with similar tree density, topography, slope, orientation, and soil type. At each site, we sampled two areas: a partially shaded site under Q. ilex canopy (under tree canopy) and a nearby open grassland site (open grassland). These two habitat types are characteristic of the dehesa ecosystem in the region. Then, within each area (i.e. habitat type) at each site we established six permanent 4 × 6 m (1.2 m high) fenced plots (N = 36 plots). Each plot was divided into two subplots (N = 72 subplots), one subplot was subjected to a rainfall reduction treatment whereas the other was exposed to ambient rainfall. Rainfall was reduced by placing a 2.5 × 2.5 m rain-exclusion shelter over one of the subplots (Fig. 2), resulting in a 30% reduction in annual rainfall22 as predicted by the IPCC for Mediterranean regions23. In addition, within each subplot we sampled two ca. 1-m2 quadrats (144 quadrats in total, i.e. 2 quadrats × 72 subplots): one was subjected to a warming treatment and the other was not manipulated (i.e. ambient temperature). Warming was achieved by placing a 40 × 50 × 32 cm (ca. 0.65 m2) hexagonal open top chamber of methacrylate material without UV filter (Faberplast, Madrid) at the centre of the quadrat (Fig. 2), increasing air temperature by ca. 2 °C compared to ambient temperature (17.5 ± 0.1 vs. 15.7 ± 0.1 °C, respectively). This matches the temperature increase for the study region by climatic forecasting models (SRES A-2 model by the Intergovernmental Panel on Climate Change23). Ambient air temperature was slightly lower (approx. 0.5 °C) in the shaded habitat than in the open habitat, but the experimental temperature increase was of similar magnitude in both cases. Temperature in each quadrat was measured with data loggers. Based on the above set-up, the experiment followed a randomized split–split plot design replicated across three sites, with habitat type as the whole factor (plots as replicates), rainfall manipulation as the split factor (subplots), and temperature manipulation as the split–split factor (quadrats).
Sampling, measurements and chemical analyses
In April 2017 and April 2018, i.e. 1 and 2 years after establishment of climatic treatments, we identified all plant species and estimated their frequency within each quadrat. Then, in April 2018, after the second survey of species frequencies, we collected 3–4 fully expanded leaves from each plant for chemical analyses which were pooled to obtain a single sample per species and quadrat. Plant sampling was restricted to a central portion of 0.65 m2 in each quadrat (within chamber in the case of warming quadrats) as a function of the surface covered by and sampled within the hexagonal chamber placed in quadrats subjected to warming (see above). Due to lack of plant material, we only sampled 127 out of the 144 quadrats. In total, we collected 436 samples from 28 annual plant species (Table S1). Immediately after collection, we oven-dried leaves for 48 h at 40 °C, ground them with liquid nitrogen, and stored them at room temperature before conducting chemical analyses.
We determined total phenolic content colorimetrically by the Folin-Ciocalteu method24,25. Briefly, we extracted phenolics from 20 mg of plant tissue with 70% methanol in an ultrasonic bath for 15 min, followed by centrifugation. We determined total phenolic content colorimetrically by the Folin–Ciocalteu method in a Biorad 650 microplate reader (Bio-Rad Laboratories, PA, USA) at 740 nm, using tannic acid as standard. We performed three technical replicates of each sample in order to estimate variations due to the experimental procedure, and expressed concentrations based on dry weights (d.w.). We decided to use total phenolics as these could be measured across all species in order to address the goal of testing for community-level effects of climatic stressors on plant secondary chemistry.
For each quadrat, we calculated the CWMphenolics using the weighted.mean function in R software26. For this, we used phenolic values at the species level weighed by the abundance (i.e. frequency) of each species. We then performed linear mixed models using PROC MIXED in SAS 9.4 (SAS Institute, Cary, NC)27 to test for the effects of habitat type, rainfall manipulation, temperature manipulation, and their two-way and three-way interactions (all fixed factors) on CWMphenolics. We included site, the site × habitat type interaction, and the site × habitat type × rainfall manipulation interaction as random factors. The two- and three-way interaction interactions tested the main effect of habitat type and rainfall manipulation (respectively) with the appropriate error terms according to a split-split plot design. We log-transformed original values to achieve normality of residuals.
We also assessed whether climatic manipulations influenced the community-level expression of phenolic compounds through changes in plant composition with a PERMANOVA (‘vegan’ package in R) that included the effects of climatic treatment, year, and their interaction.