This is the accepted version of the article: Ogaya Inurrigarro, Romà; Peñuelas, Josep. «Wood vs. canopy allocation of aboveground net primary productivity in a Mediterranean forest during 21 years of experimental rainfall exclusion». Forests, Vol. 11, Issue 10 (October 2020), art. 1094. DOI 10.3390/f11101094 This version is available at https://ddd.uab.cat/record/232914 under the terms of the license Wood vs. Canopy Allocation of Aboveground Net Primary Productivity in a Mediterranean Forest during 21 years of Experimental Rainfall Exclusion. Romà Ogaya 1,2* and Josep Peñuelas 1,2 1 CSIC, Global Ecology Unit CREAF-CSIC-UAB, 08913 Bellaterra, Catalonia, Spain. r.ogaya@creaf.uab.cat. 2 CREAF, 08913 Cerdanyola del Vallès, Catalonia, Spain. Josep.Penuelas@uab.cat. * Correspondence: r.ogaya@creaf.uab.cat; Tel.: +34-935814036 Received: date; Accepted: date; Published: date Abstract: A Mediterranean holm oak forest was subjected to experimental partial rainfall exclusion during 21 consecutive years, to study the effects of the expected decrease in water availability for the Mediterranean vegetation in the coming decades. Allocation in woody structures and total aboveground allocation were correlated with annual rainfall, whereas canopy allocation and the ratio wood/canopy allocation were not dependent on rainfall. Fruit productivity was also correlated with annual rainfall, but only in Quercus ilex. In the studied site, there were two types of forest structure: high canopy stand clearly dominated by Quercus ilex, and low canopy stand with more abundance of a tall shrub species, Phillyrea latifolia. In the tall canopy stand, the allocation to woody structures decreased in the experimental rainfall exclusion, but not the allocation to canopy. In the low canopy stand, wood allocation in Quercus ilex was very small in both control and plots with rainfall exclusion, but wood allocation in Phillyrea latifolia was even higher than that obtained in tall canopy plots, especially in the plots receiving the experimental rainfall exclusion. These results highlight likely future changes in the structure and functioning of this ecosystem induced by the decrease in water availability. A serious drop of the capacity to mitigate climate change for this Mediterranean forest can be expected, and the ability of Phillyrea latifolia to take advantage of the limited capacity to cope drought conditions detected in Quercus ilex, makes likely a forthcoming change in species dominance especially in the low canopy stands. Keywords: carbon sink; climate change; forest dieback; holm oak; Mediterranean forest; tree growth; wood/canopy allocation 1. Introduction The net primary productivity (NPP) allocation in different tissues is one of the best descriptors of ecosystem functioning [1]. Every woody plant has a trade-off between the resource allocation in canopy structures (leaves, flowers and fruits), or in woody structures (branches and stem). The fraction invested in canopy structures will affect the production (and the consumption) of flowers and fruits, canopy leaf area, their photosynthetic capacity, litter through fall, and its decomposition by soil organisms [1]. The fraction invested in woody structures will be more related to the growth of vegetation and its capacity to ameliorate climate change due to atmospheric CO2 uptake [2]. Variations in wood/canopy allocation could drive important changes in ecosystem functioning and services. Several factors contribute to the variation in resource allocation in trees: tree age [3], stand structure, environment under which the tree develops [4-7], silvicultural treatments [8,9], ontogeny, and many other factors [7,10,11]. Water availability is the main limiting factor for plant productivity in some semiarid ecosystems such as Mediterranean forests, where dry season coincides with the hot summer. For these Mediterranean areas, higher rates of evapotranspiration would be induced by higher air temperatures with no increase of precipitation [12]. Furthermore an increase in the frequency of extreme droughts [13] and heat waves [14] are forecasted for these areas. The Mediterranean forest, seasonally exposed to water stress, is often particularly vulnerable to a decrease in water availability, which can induce reductions in stem growth [15,16], defoliation [17,18], tree mortality and forest dieback [19-24]. The effect of climate change on ecosystem functioning is quite uncertain, but changes in woody and canopy allocation could be a key factor determining some variations of ecosystem structure and functioning, because how carbon is allocated in different tissues determines how long carbon remains in plant biomass and thus remains a central challenge for understanding the global carbon cycle [25]. For example, if carbon allocation in tissues with long turnover time (such as stem and branches) decreases, and carbon allocation in tissues with short turnover time (leaves, flowers and fruits) proportionally increases, the function of atmospheric CO2 sink and their capacity to ameliorate the climate change will strongly decrease. Climate manipulating experiments have the potential to verify the ecosystem responses when it is subjected to a continuous climate change [26-28]. Furthermore, long-term experiments could highlight the usual dampening effect size of treatments reported in global-change experiments [29]. As a result, the use of experiments simulating the environmental conditions projected by models of global change, as long as possible, is the best tool to study the long-term climate change effects on natural ecosystems. The Mediterranean forest studied here is subjected to a rainfall exclusion since 1999, and is one of the longest ongoing climate manipulation experiments conducted in natural ecosystems in the world [28]. Plant development in Mediterranean forests is mainly limited by summer drought, and it is projected a future decrease in water availability in these ecosystems (about 15%) [15,16,23]. For these reasons, we aim to study the effects on carbon allocation in woody or canopy structures, derived from an experimental 15% decrease of water soil moisture. We also aimed to discuss the possible variations in the structure and functioning of the Mediterranean forest as a consequence of these effects on carbon wood/canopy allocation. 2. Materials and Methods 2.1. Study site. The study was carried out in a south-face slope (25%) in Prades Holm oak forest, in Catalonia, NE Spain (41°21′N, 1°2′E; 930 m a.s.l.). This forest was not perturbed during the last 70 years, but as a result of ancient coppicing, the vegetation is a dense multi-stem forest (15 433 stems ha-1) dominated by Quercus ilex L., with a high presence of evergreen tall shrub species such as Phillyrea latifolia L (Figure A1). Along forest distribution, there are two types of forest structure: a tall canopy forest (8-10m tall) clearly dominated by Q. ilex, and a low canopy forest (4-6m tall) with more abundance of P. latifolia. Different soil deep determined different water and nutrient availability, and finally this different forest structure [30]. Eight 15 x 10m plots were delimited at the same altitude along the slope (four in the tall canopy area, and other four in the low canopy area). In four of these plots (two in the tall canopy area, and other two in the low canopy area) a partial rainfall exclusion system was installed, whereas the other four plots were control. The partial rainfall exclusion treatment consisted in transparent PVC strips installed 1m above ground level, and covering ca. 30% of plot surface, as shown in (Figure A1); these PVC strips covered also a buffer of 2m above and below plot area. Soil moisture in these drought plots was on average 15% smaller than in control plots during the overall studied period [23]. 2.2. Carbon allocation measurements. Before the start of the experiment and outside the plots, different size 10 Q. ilex, and 10 P. latifolia trees were cut (covering the range of stem sizes of trees from the experimental plots). The circumference of each stem at 50 cm height was measured with a metric tape, and after that, each stem was cut from the base and all the aboveground biomass was dried in an oven at 105ºC during enough time to reach constant weight. For each species, allometric relationships between the stem circumference and the weight of dry leaves and total aboveground dry biomass were determined, and these allometric relationships were used to estimate total aboveground and leaf biomass of all stems from the plots (Table A1). Every winter, the circumference at 50 cm height of all stems from the experimental plots were measured, and the aboveground and leaf biomass of all species per plot was estimated using the allometric relationships. We estimated annual wood Net Primary Productivity (Wood NPP) from the difference of the total aboveground biomass increment and the leaf biomass increment, during the overall studied period (1999-2019). In each plot, 20 baskets (27 cm diameter, and with a 1.5 mm mesh inside them) were randomly placed over the ground (Figure A1). The litter fallen in the baskets, was collected every two months, and separated per species and litter fraction (leaves, flowers, and fruits), finally, litter was weighed after drying in an oven at 70ºC. Litter collected in the baskets during one year was used to estimate total flower, fruit, and leaves production per species and plot; it was assumed that all fallen leaves were replaced by new ones. The canopy Net Primary Productivity (canopy NPP) was estimated as the sum of all fallen leaves, fruits and flowers, and the Aboveground Net Primary Productivity (ANPP) was estimated as the sum of annual wood NPP and canopy NPP. 2.3. Statistics. General Linear Models were performed to test the effect of climatic conditions, and rainfall exclusion treatment on ANPP, wood NPP, canopy NPP (flower and fruit production, and leaf replacement), and the proportion of wood allocation in function of canopy allocation (wood/canopy allocation). For each type of canopy plot, several analyses of variance (ANOVAs) were conducted with the percentage of ANPP, wood NPP, canopy NPP (related to the initial biomass per species and plot, at the start of the experiment), and wood/canopy allocation as dependent variables, and species (only the two dominant species, Q. ilex and P. latifolia were considered separately) and rainfall exclusion treatment as independent factors. Other ANOVAs were conducted with each fraction of canopy NPP as dependent variable, and species, rainfall exclusion, and type of canopy as independent factors. Analyses of covariance (ANCOVAs) were performed with ANPP, wood NPP, canopy NPP, and wood/canopy allocation as dependent variables, species, canopy type, and rainfall exclusion treatment as independent factors, and mean annual temperature and total annual rainfall as covariates. Finally, linear regressions with the same variables were conducted to determine the effect of annual climatic conditions on each dependent variable. The proportions of ANPP, wood NPP, and canopy NPP (p) were arc sin p 0.5 transformed to reach the assumptions of a normal distribution. All statistical analyses were performed with Statistica 12 software package (StatSoft Inc., Tulsa, OK, USA). 3. Results 3.1.1. Climatic data. Mean annual temperature was on average 12.38 ºC, and mean annual precipitation 619 mm during these 21 years of experiment in the studied site (Figure A1). But both temperature and rainfall fluctuated a lot depending on the year, for example in 2015, it was recorded the highest mean temperature (13.25 ºC) and also the lowest precipitation (355 mm), on the contrary, the coldest year 2010 (10.98 ºC) coincided with the second wettest year (902 mm, just below the 1021 mm corresponding to year 2018) (Figure A2). 3.1.2. Relationships between productivity and climatic conditions. Forest wood NPP and total ANPP were positively correlated with annual rainfall (P<0.01 and P=0.05 for wood NPP and ANPP, respectively), but there were no any significant relationship between canopy NPP or wood/canopy allocation and annual rainfall (Figure 1), and none of wood NPP, canopy NPP, wood/canopy, and ANPP variables were significantly correlated with mean annual temperature, at least there is a trend towards wood NPP inversely correlated with temperature (P=0.1) (Figure 1). When both dominant species were analysed separately, the wood NPP of both species were correlated with annual rainfall (P<0.05), and Q. ilex ANPP was positively correlated with annual rainfall (P<0.05) (Figure 2), but not with mean annual temperature. However, neither canopy NPP or wood/canopy allocation ratio were correlated with meteorological data. Finally, ANPP was correlated with annual rainfall (P<0.05) in Q. ilex, but not in P. latifolia (Figure 2). The meteorological data determined also the fruit allocation, which was correlated with annual rainfall only in Q. ilex (P<0.05) (Figure 3). The effect of annual rainfall on the studied variables was similar in plots located in tall and low canopy areas, and also in control and plots with the partial rainfall exclusion system. 3.2.1. Net Primary Productivity allocation. The average ANPP of the studied forest during all the studied period was 3.6 Mg ha -1 y-1, about one third of this ANPP corresponded to wood NPP (1.2 Mg ha -1 y-1), and about two thirds of total ANPP corresponded to canopy NPP (2.4 Mg ha-1 y-1), so wood/canopy allocation was on average 0.56. ANPP was higher in tall canopy plots than in low canopy plots (4.0 and 3.2 Mg ha - 1 y-1, respectively) (P<0.01), but the wood/canopy allocation ratio was similar in both canopy-type plots. When ANPP was examined in function of the previous aboveground biomass in each plot, Q. ilex experienced similar % of ANPP in both tall and low canopy plots, but ANPP in P. latifolia was relatively higher in low canopy plots (P<0.05). 3.2.2. Net Primary Productivity allocation in tall canopy forest stand. In tall canopy plots, the % of wood NPP was similar in both species, but the partial rainfall exclusion reduced wood NPP in both species (P<0.05) (Figure 4), the % of canopy NPP was higher in P. latifolia than in Q. ilex (P<0.01), as a result of this, the % of ANPP was also higher in P. latifolia (P<0.05) (Figure 4). In these tall canopy plots, wood/canopy allocation was higher in Q. ilex than in P. latifolia (P<0.01), and this wood/canopy allocation ratio decreased in both species when submitted to rainfall exclusion treatment (P=0.01) (Figure 4). 3.2.3. Net Primary Productivity allocation in low canopy forest stand. In low canopy plots, the % of wood NPP and the % of canopy NPP were higher in P. latifolia than in Q. ilex (P<0.05 and P<0.01 for wood NPP and canopy NPP, respectively), and as a result of this, the % of ANPP was also higher in P. latifolia than in Q. ilex (P<0.01) (Figure 5). In low canopy plots, there was no significant difference between both species and plots with different treatment for the wood/canopy allocation (Figure 5). Surprisingly, the only significant effect of rainfall exclusion was an increase of ANPP in P. latifolia (Figure 5). Figures. Figure 1. Linear relationships between average NPP values (wood NPP, canopy NPP, ANPP, and wood/canopy allocation) and meteorological data (annual rainfall and mean annual temperature) of all species analyzed together. Figure 2. Linear relationships between wood NPP and ANPP values, and annual rainfall in the two dominant species of the studied forest. Figure 3. Linear relationships between fruit allocation and annual rainfall in the two dominant species of the studied forest. Figure 4. Average % of wood NPP, canopy NPP, ANPP, and wood/canopy allocation of the two dominant species, in control and plots with partial rainfall exclusion (drought plots), located in the tall canopy stand. Depicted data correspond to the NPP during the overall studied period (1999-2019). Figure 5. Average % of wood NPP, canopy NPP, ANPP, and wood/canopy allocation of the two dominant species, in control and plots with partial rainfall exclusion (drought plots), located in the low canopy stand. Depicted data correspond to the NPP during the overall studied period (1999-2019). 4. Discussion A decrease in water availability usually drives to lower carbon allocation in stem and branches compared to the allocation in roots and canopy [31]. In our experiment, wood allocation decreased during the drier years, especially in Q. ilex. In plots located in the tall forest stand, where water availability is not so limited as in low canopy stand, rainfall exclusion decreased wood allocation in both studied species, as expected, while no effect was observed in canopy allocation; as a result, wood/canopy allocation decreased in both species. In the low forest stand, wood and canopy allocation were very low in Q. ilex, in both control and rainfall exclusion plots, showing that growth in this species is seriously constrained by the low water availability of this low canopy stand. In contrast, P. latifolia seems to be more able to obtain advantage of the decay of Q. ilex, because P. latifolia experienced larger wood and canopy allocation in the low canopy stand, even larger allocation in plots with rainfall exclusion, as a result of the decrease in stem density induced by the tree mortality observed in Q. ilex [22,23]. As observed in previous studies conducted in the same experimental site, P. latifolia was more able to cope better with drought conditions than Q. ilex [15,16,23]. Analyzing the fruit production, once again Q. ilex was also more dependent than P. latifolia to water availability, because fruit allocation decreased when annual rainfall decreased in Q. ilex, but not in P. latifolia. Despite a slight decrease of Q. ilex fruit production in rainfall exclusion plots, detected during the first years of study [32], this effect disappeared with time, and globally there was no effect of rainfall exclusion on fruit allocation during the overall studied period [33], in contrast with what was observed in another rainfall exclusion experiment, where Q. ilex acorn production decreased in plots with rainfall exclusion [34]. However, rainfall exclusion exerted a decrease in wood growth of Q. ilex, but not in P. latifolia, so this more drought sensitive species, Q. ilex, was able to maintain their fecundity by shifting their allocation of resources away from growth [33]. Our results highlight a strong decrease in the capacity of Mediterranean forest to mitigate climate change induced by future limitations in water availability, because of the high decrease in wood growth. But this forest seems to be able to maintain leaf turnover and the production of flowers and fruits Moreover, our estimation of wood allocation was provided from stem radial growth, but sometimes water scarcity could decrease wood density in tree rings instead of radial growth [35], as observed in Q. ilex [36]. If water scarcity drives also to a lower wood density, the decrease in resource wood allocation could be even greater than the detected in our experiment. The allocation to fine roots is another fraction of resource allocation competing with wood and canopy allocation, and it is described that fine root allocation could be more variable depending on water availability than wood and canopy allocation [1]. We reported the effects of rainfall exclusion in aboveground biomass allocation, but there is a lack of any estimation about the allocation to fine roots. Usually, water scarcity enhanced the growth of fine roots [1], also, another experiment conducted nearby the study site, reported shorter fine-root longevity in Q. ilex when water availability decreased [37], so more resource allocation is needed to restore dead roots. Also, we did not measure how much is lost in the form of volatiles, root exudates, through mycorrhizal associations, consumption by animals, and as dissociated dead parts [38]. Previous measurements conducted in the same experimental site highlighted a decrease of volatile organic compounds (VOCs) emission under heavy drought in Q. ilex, whereas no VOCs emission decrease was observed in P. latifolia [39], and it was also observed that rainfall exclusion increased 21% of Q. ilex root exudates [40]. Despite of these changes in resource allocation described before, other authors reported higher plant ability to alter organ morphology, such as specific leaf area (SLA), to cope climatic variations than to adjust allocation [7]. The small values of wood allocation described in Q. ilex trees from low canopy plots, and the high wood and total aboveground allocation in P. latifolia in the same plots, are in agreement with the defoliation and tree mortality observed in Q. ilex in the same study area when is subjected to severe drought conditions [22,23], and highlight the ability of P. latifolia to take a competitive advantage of Q. ilex decay. In the low canopy stand, Q. ilex cannot actually adjust more the allocation of resources to wood structures to cope the drier conditions posed by climate change. The dominant tree species of this Mediterranean forest, Q. ilex, is starting to be currently replaced especially in the low canopy stand by P. latifolia, which is more adapted to the current and forthcoming drier and hotter conditions [15,16,23]. 5. Conclusions Increasing water deficits characterized by lower soil moisture and higher atmospheric aridity are leading to several changes in the ecosystem functioning of the Mediterranean forest, such as a dramatic decrease in the capacity to mitigate climate change; but it seems that this forest will be able to maintain leaf turnover and the production of reproductive structures. However, if current climate evolution leads to forest dieback, as occurring in Q. ilex in the low canopy stand, the maintenance of the production and dispersion of seeds will be not enough to compensate Q. ilex decay. A progressive substitution of the most sensitive species to water scarcity (Q. ilex) by other species more adapted to drought (such as P. latifolia) is expected, and it may transform the current Mediterranean forest to a tall shrubland, with different species composition and different ecosystem behavior and services [41]. Author Contributions: Conceptualization, design of the experiment, statistical analyses, and discussion of the results R.O. and J.P.; writing R.O.; and supervision J.P. Funding: This research was financially supported by the European Research Council Synergy grant ERC- 2013-SyG-2013-610028 IMBALANCE-P, the Spanish government projects PID2019-110521GB-I00, and the Catalan government grant SGR-2017-1005. Acknowledgments: This study was undertaken with the approval of the staff of the PNIN de Poblet Natural Park. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Figure 1. View of the study site and its localization, and view of the partial rainfall exclusion treatment installation and litter collectors. The PVC strips were only placed in plots subjected to partial rainfall exclusion, whereas litter collector baskets were placed in all plots. Figure 2. Mean annual temperature and annual rainfall values of the study site during the overall duration of the experiment. Table 1. Allometric relationships between total aboveground biomass (AB) and total biomass of leaves (LB) from stem diameter at 50cm height (D50), in Q. ilex and P. latifolia. Species Total aboveground biomass Total biomass of leaves Quercus ilex ln AB=4.900+2.277 ln D50 ln LB=3.481+1.695 ln D50 Phillyrea latifolia ln AB=4.251+2.463 ln D50 ln LB=1.433+2.426 ln D50 References 1. Malhi, Y.; Doughty, C.; Galbraith, D. The allocation of ecosystem net primary productivity in tropical forests. Phil. Trans. R. Soc. B. 2011, 366, 3225-3245. 2. 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