Article Climate Change Effects in a Mediterranean Forest Following 21 Consecutive Years of Experimental Drought Romà Ogaya 1,2, * and Josep Peñuelas 1,2 1 CSIC, Global Ecology Unit CREAF-CSIC-UAB, 08193 Bellaterra, Catalonia, Spain; Josep.Penuelas@uab.cat 2 CREAF, 08193 Cerdanyola del Vallès, Catalonia, Spain * Correspondence: r.ogaya@creaf.uab.cat Abstract: Research Highlights: A small, long-term decrease in the water availability in a Mediterranean holm oak forest elicited strong effects on tree stem growth, mortality, and species composition, which led to changes in the ecosystem function and service provision. Background and Objectives: Many forest ecosystems are increasingly challenged by stress conditions under climate change. These new environmental constraints may drive changes in species distribution and ecosystem function. Materials and Methods: An evergreen Mediterranean holm oak (Quercus ilex L.) forest was subjected to 21 consecutive years of experimental drought (performing 30% of rainfall exclusion resulted in a 15% decrease in soil moisture). The effects of the annual climatic conditions and the experimental drought on a tree and shrub basal area increment were studied, with a focus on the two most dominant species (Q. ilex and the tall shrub Phillyrea latifolia L.). Results: Stem growth decreased and tree mortality increased under the experimental drought conditions and in hot and dry years. These effects differed between the two dominant species: the basal area of Q. ilex (the current, supradominant species) was dependent on water availability and climatic conditions, whereas P. latifolia was more tolerant  to drought and experienced increased growth rates in plots where Q. ilex decay rates were high.  Conclusions: Our findings reveal that small changes in water availability drive changes in species Citation: Ogaya, R.; Peñuelas, J. growth, composition, and distribution, as demonstrated by the continuous and ongoing replacement Climate Change Effects in a of the current supradominant Q. ilex by the subdominant P. latifolia, which is better adapted to tolerate Mediterranean Forest Following 21 hot and dry environments. The consequences of these ecological transformations for ecosystem Consecutive Years of Experimental Drought. Forests 2021, 12, 306. function and service provision to human society are discussed. https://doi.org/10.3390/f12030306 Keywords: basal area increment; climate change; forest dieback; Mediterranean forest; stem growth; Academic Editor: Mariangela Fotelli water availability Received: 14 January 2021 Accepted: 1 March 2021 Published: 6 March 2021 1. Introduction Climate change is increasing the levels of environmental stress conditions for many Publisher’s Note: MDPI stays neutral forest ecosystems around the world due to higher rates of evapotranspiration and increases with regard to jurisdictional claims in in the intensity and duration of extreme climate events [1,2]. Some water-limited forest published maps and institutional affil- ecosystems, such as Mediterranean forests, may be particularly vulnerable to the expected iations. decrease in water availability and may experience associated decreases in stem growth [3,4], greater levels of canopy defoliation [5], and increases in tree mortality [6,7]. While rising concentrations of atmospheric carbon dioxide (CO2 ) are associated with higher growth rates and productivity in forests [8], it is possible that decreases in stem growth may occur Copyright: © 2021 by the authors. in water-limited ecosystems, if the effects of water scarcity exceed those of fertilization Licensee MDPI, Basel, Switzerland. from the increasing levels of CO2 . This article is an open access article Quercus ilex L. is an evergreen tree species and it is the most abundant and representa- distributed under the terms and tive of the Mediterranean forest [9]. Phillyrea latifolia L. is a tall shrub evergreen species, conditions of the Creative Commons and it is also very common in the Mediterranean forest. Both species are well adapted to Attribution (CC BY) license (https:// resist a drought period (usually the summer season) due to their stomatal closure around creativecommons.org/licenses/by/ midday to avoid excessive water loss by transpiration [9]. Despite these two species having 4.0/). Forests 2021, 12, 306. https://doi.org/10.3390/f12030306 https://www.mdpi.com/journal/forests Forests 2021, 12, 306 2 of 11 experienced similar distributions and adaptations for coping with drought conditions, several studies have shown that Q. ilex is more sensitive to dry and hot conditions than other co-occurring tall shrub species, such as P. latifolia [3,4,10,11]. For example: within a shared forest, Q. ilex was found to suffer from high rates of stem mortality during periods of extreme drought, which had little impact on P. latifolia [10,12]; while wood carbon alloca- tion decreased in Q. ilex during dry years and remained stable in P. latifolia [11]; and fruit production correlated with annual rainfall in Q. ilex, but not in P. latifolia [11]. Mediterranean vegetation is characterized by the physiological adaptations that allow its tolerance to, and recovery from, episodes of dry environmental conditions; however, if extreme drought events exceed the tipping points of climate tolerance, some sensitive species may fail to survive, whereby species diversity, composition, and associated ecosys- tem function become permanently altered, leading to perturbations in the ecosystem service provision [13]. Q. ilex is the current dominant species of the Mediterranean forest, and is widely distributed across the whole Mediterranean Basin in South Europe and North Africa, and from Portugal to Syria. Under hotter and drier environmental conditions, a decrease in the abundance of Q. ilex in Mediterranean forests may be expected, along with a reduction in forest capacity to mitigate the effects of climate change through the sequestration of atmospheric CO2 ; there may also be a progressive replacement of Q. ilex by other tall shrub species, such as P. latifolia, that transforms the structure and ecosystem function of the forest. In water-limited ecosystems, the principal effects of hydrological processes on vegeta- tion are mediated through soil water content [14]; thus, experimental methods that affect soil moisture effectively simulate the likely impacts of decreases in water availability on plants. Furthermore, long-term climate manipulation experiments in natural ecosystems allow the prediction of the effects of environmental change on ecosystem function and development [15,16] and may highlight the dampening effect on the size of treatments that has been widely reported in global-change experiments [17]. Here, our main objective was to determine the effects of climate change-mediated drought on the development of the Mediterranean forest, paying special attention to the development of the different species and their possible divergent evolution. This study was conducted as a long-term climate manipulation experiment that simulates a 15% decrease in soil moisture, as projected by general circulation models for the Mediterranean region [18]. 2. Materials and Methods 2.1. Study Site The study was conducted in a Mediterranean holm oak forest located on a south facing slope (25%) of the Prades Mountains, Catalonia, in north-eastern Spain (41◦ 200 38” N, 1◦ 20 0” E, 950 m a.s.l.), in which the vegetation comprises dense multi-stem, coppiced woody species (16.616 stems ha−1 ), created for charcoal production; no coppicing activity has taken place in the forest during the preceding 70 years. The dominant species are Q. ilex and P. latifolia (both species represent more than 80% of the total forest basal area). There are also other evergreen shrub species such as Arbutus unedo L., Erica arborea L., and Juniperus oxycedrus L.; deciduous tree species include the occasional Sorbus torminalis L. and Acer monspessulanum L. The soils comprise a Dystric Cambisol, and the range in soil depth (35 to 100 cm) results in contrasting levels of water and nutrient availability, reflected by two forest-type structures [19]: a tall canopy forest (8–10 m high) dominated by Q. ilex, and a low canopy forest (4–6 m high), with a greater abundance of P. latifolia than in tall canopy (Table A1). 2.2. Experimental Design Eight 15 × 10 m plots were delimited at the same altitude along the slope (four in the tall canopy stand and another four in the low canopy stand) in 1999. Four of these plots (two each in the tall and low canopy stands) were randomly selected as drought Forests 2021, 12, 306 3 of 11 plots, and received an experimental partial rainfall exclusion; the four remaining plots were left untreated as a control. In the drought plots, six plastic strips, comprising four rigid PVC tubes connected with transparent PVC, were installed under the canopy along the longitudinal slope, at about 1 m above the soil surface and extending to 2 m outside the plot margin to reduce edge-effects; the strips covered 30% of the plot surface area for the interception of rain throughfall and its removal downslope. Soil moisture was measured monthly using a time domain reflectometer (Delta-T Devices, Cambridge, UK) placed in the upper 15 cm of the soil profile of four randomly selected loci per plot, along with the leaf water potential (measured seasonally, in two leaves from two different trees per species, collected in the upper layer of the canopy and fully exposed to solar radiation) at the same loci using a Scholander-type chamber pressure (PMS Instrument Company, Albany, OR, USA). An automatic meteorological station installed at the study site monitored temperature and rainfall during the 1999–2019 study period. 2.3. Basal Area Determination In 1999, all of the living tree and shrub stems in the plots were marked at 50 cm above ground level using permanent paint, and their circumference (diameter > 2 cm) was measured at the painted mark annually in winter. The basal area of each stem was determined from the stem circumference measurements, assuming each stem section to be a circle; the annual basal area increment was calculated from the difference in the basal area measured in two consecutive years. For each plot, species, and year, we determined the basal area increment (BAI), basal area lost by stem mortality (BAL), and total basal area increase (TBAI) which was determined as the basal area increment minus basal area lost by stem mortality. 2.4. Data Analysis Seasonal differences in soil moisture and leaf water potential were tested using re- peated measures analysis of variance (ANOVA). January, February and March were com- bined in “winter”, April, May and June were combined in “spring”, July, August and September were combined in “summer”, and October, November and December were combined in “autumn”. The relationships between the BAI, BAL, and TBAI per species and canopy stand (dependent variables) and the annual mean temperature, rainfall, and SPEI climatic indices [3,4,20] (independent variables) were tested using linear and polynomial regression analyses. We used general linear modeling to test the determinant factors of the annual BAI, BAL, and TBAI of Q. ilex and P. latifolia. The species, treatment and canopy stand were considered independent factors, and the annual mean temperature and rainfall were covariates. Finally, we conducted the same models without the species factor, and including all tree and shrub species present in the plots named “overall species”. All analyses were performed using Statistica 10.0 (StatSoft Inc., 2010, Tulsa, OK, USA). 3. Results 3.1. Soil Moisture and Leaf Water Potential The meteorological conditions of the study site were typically Mediterranean: sum- mers were hot and dry, winters were relatively cold, and rainfall events tended to be concentrated in spring and autumn (Figure 1). Accordingly, both soil moisture and leaf water potential levels showed a clear seasonality; their values decreased in summer, recov- ered in autumn, and were greatest in spring and winter when evapotranspiration rates were lower. Soil moisture levels were 15% lower in the drought plots than in the control plots (p < 0.05) (Figure 2). The decrease in leaf water potential values in summer was stronger in P. latifolia than in Q. ilex (p < 0.05), whereas in autumn, Q. ilex maintained a higher water potential than P. latifolia; in winter and spring, the levels were similar in both species (Figure 2). The drought treatment did not exert any significant additional effect on the leaf water potential levels. Forests 2020, 11, x FOR PEER REVIEW Forests 2021, 12, 306 4 of 11 Figure 1. Average meteorological conditions at the study site during the experimental period Figure 1. Average meteorological conditions at the study site during the experimental period (1999–2019). Red dots indicate mean monthly air temperature and grey columns indicate mean 148 (1999–2019). Red dots indicate mean monthly air temperature and grey columns indicate mean monthly rainfall. Vertical bars in the grey columns and red dots indicate the standard error of the 149 monthlyFigure mean rainfall. (n = 21 1. Vertical years). Average bars in the grey columns meteorological and red conditions dotsstudy at the indicate sitethe standard during the error of the experimental perio 150 mean (n(1999–2019). = 21 years). Red dots indicate mean monthly air temperature and grey columns indicate m 151 monthly rainfall. Vertical bars in the grey columns and red dots indicate the standard error 152 mean (n = 21 years). Figure 2. Average seasonal leaf water potential (upper panel) and soil moisture (lower panel) during the study period Figure 2. Average seasonal leaf water potential (upper panel) and soil moisture (lower panel) during the study period (1999–2019). Vertical bars indicate the standard error of the mean (n = 21 years). (1999–2019). Vertical bars indicate the standard error of the mean (n = 21 years). 153 154 Figure 2. Average seasonal leaf water potential (upper panel) and soil moisture (lower panel) during the study peri 155 (1999–2019). Vertical bars indicate the standard error of the mean (n = 21 years). 6 3.2. Climate Effects on Stem Growth 7 Forests 2020, 11, x FOR PEER REVIEW BAI was positively related to the annual rainfall in the two species separately (Q.5 ilex: of 12 8 Forests 2021, 12, 306 p < 0.05; P. latifolia: p < 0.01) and for all of the tree and shrub species (p <0.01) (Figure 5 of 11 3); 96 there was a Effects 3.2. Climate trend towards a positive relationship between the BAL and the mean annual on Stem Growth 0 temperature in Q. ilex (p = 0.06) and all of the tree and shrub species (p = 0.06), but not in 7 BAI was positively related to the annual rainfall in the two species separately (Q. ilex: 1 P. latifolia (Figure 4). As a consequence of lower stem growth in dry years and greater stem 8 p 3.2. < 0.05; P. latifolia: Climate Effects p