Iris Le Roncé

Jordane Gavinet

Jean-Marc Ourcival

Florent Mouillot

Isabelle Chuine

Jean-Marc Limousin
email: jean-marc.limousin@cefe.cnrs.fr

Holm oak fecundity does not acclimate to a drier world

Keywords: climate change, fecundity, mast seeding, Mediterranean, rainfall exclusion, reproductive cycle, resource allocation

Climate change might impact tree fecundity by altering the relative influences of meteorological and physiological drivers, and by modifying resource investment in reproduction.

(2) Using a 13-year monitoring of Quercus ilex reproduction in a rainfall exclusion experiment, we analysed the interactive effects of long-term increased aridity and other environmental drivers on the inter-annual variation of fecundity (male flower biomass, number of initiated and mature fruits).

(3) Summer-autumn water stress was the main driver of fruit abortion during fruit growth. Rainfall exclusion treatment strongly reduced the number of initiated and mature fruits, even in masting years, and did not increase fruit tolerance to severe drought. Conversely, the relative contribution of the meteorological and physiological drivers, and the inter-annual variability of fruit production were not modified by rainfall exclusion.

(4) Rather than inducing an acclimation of tree fecundity to water limitation, increased aridity impacted it negatively through both lower fruit initiation due to changes in resource allocation, and more severe water and resource limitations during fruit growth. Long-term increased aridity affected tree reproduction beyond what is expected from the current response to inter-annual drought variations, suggesting that natural regeneration of holm oak forest could be jeopardized in the future.

Introduction

Reproduction is a critical step of population demography and population adaptation to climate change through natural selection [START_REF] Lefèvre | Considering evolutionary processes in adaptive forestry[END_REF]. The on-going climate change has been shown to impact the mean seed production of trees either positively [START_REF] Allen | Elevation-dependent responses of tree mast seeding to climate change over 45 years[END_REF][START_REF] Buechling | Climate drivers of seed production in Picea engelmannii and response to warming temperatures in the southern Rocky Mountains[END_REF][START_REF] Caignard | Increasing spring temperatures favor oak seed production in temperate areas[END_REF][START_REF] Shibata | Decadal changes in masting behaviour of oak trees with rising temperature[END_REF]Bogdziewicz et al., 2020c), negatively [START_REF] Mutke | Variability of Mediterranean Stone pine cone production: Yield loss as response to climate change[END_REF], or to have no impact [START_REF] Kelly | Of mast and mean: differential-temperature cue makes mast seeding insensitive to climate change (M Rejmanek[END_REF][START_REF] Bisi | Current and future conifer seed production in the Alps: testing weather factors as cues behind masting[END_REF]. Climate change is also suspected to affect the temporal patterns of tree reproduction [START_REF] Hedhly | Global warming and sexual plant reproduction[END_REF][START_REF] Shibata | Decadal changes in masting behaviour of oak trees with rising temperature[END_REF]Bogdziewicz et al., 2020c). A global synthesis of plant reproductive patterns showed, for example, that seed production has become more variable over the last 115 years [START_REF] Pearse | Inter-annual variation in seed production has increased over time (1900-2014)[END_REF], but other studies reported opposite trends in beech and oak over the last decades [START_REF] Shibata | Decadal changes in masting behaviour of oak trees with rising temperature[END_REF]Bogdziewicz et al., 2020c). Changes in inter-annual variability of seed production are important for the reproductive success because the benefits of increased seed production could be lost if seed production also becomes less variable, thereby increasing predators' population density (Bogdziewicz et al., 2020c). In such an uncertain context, understanding the drivers of tree reproductive success appears essential when predicting how forest natural regeneration may be affected by future climatic conditions.

In the Mediterranean region, it is predicted that temperatures will rise [START_REF] Molina | Future heat waves over the Mediterranean from an Euro-CORDEX regional climate model ensemble[END_REF], rainfall events will become more stochastic into extremes (IPCC, 2013;[START_REF] Lionello | The relation between climate change in the Mediterranean region and global warming[END_REF][START_REF] Vogel | Projected changes in hot, dry and wet extreme events' clusters in CMIP6 multi-model ensemble[END_REF], and summer drought episodes will become more frequent and severe [START_REF] Ruffault | Projecting future drought in Mediterranean forests: bias correction of climate models matters![END_REF][START_REF] Cook | Twenty-First Century Drought Projections in the CMIP6 Forcing Scenarios[END_REF]. Ecosystem rainfall manipulation experiments are powerful tools to anticipate how these changes might impact tree fecundity [START_REF] Pérez-Ramos | Mast seeding under increasing drought: results from a long-term data set and from a rainfall exclusion experiment[END_REF], 2013;Bogdziewicz et al., 2020a). Previous studies using rainfall exclusion experiments in Mediterranean Quercus ilex forests have reported negative effects of increased water deficit on female flowers and total fruit biomass [START_REF] Ogaya | Species-specific drought effects on flower and fruit production in a Mediterranean holm oak forest[END_REF][START_REF] Sánchez-Humanes | Increased drought reduces acorn production in Quercus ilex coppices: thinning mitigates this effect but only in the short term[END_REF][START_REF] Rodríguez-Calcerrada | Is selective thinning an adequate practice for adapting Quercus ilex coppices to climate change?[END_REF][START_REF] Liu | Contrasting impacts of continuous moderate drought and episodic severe droughts on the aboveground-biomass increment and litterfall of three coexisting Mediterranean woody species[END_REF][START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF], although not all of them (Bogdziewicz et al., 2020b). Some studies also reported negative effects on the mean seed biomass during years of high fruit production and an increased number of aborted fruits [START_REF] Pérez-Ramos | Mast seeding under increasing drought: results from a long-term data set and from a rainfall exclusion experiment[END_REF]. Conversely, little effect was observed on male flower biomass [START_REF] Pérez-Ramos | Mast seeding under increasing drought: results from a long-term data set and from a rainfall exclusion experiment[END_REF][START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF] while a reduction of the number of viable pollen grains has been found [START_REF] Bykova | Water deficit disrupts male gametophyte development in Quercus ilex (S Pfautsch, Ed.)[END_REF].

Precipitation reduction experiments, when carried out over a sufficiently long period, can disentangle the effect of the inter-annual drought variability from the long-term effect of (simulated) increased aridity, thereby enabling us to investigate whether trees may acclimate to long-term increased aridity or not [START_REF] Estiarte | Few multiyear precipitation-reduction experiments find a shift in the productivityprecipitation relationship[END_REF] and whether this response could be forecasted from short term responses to episodic droughts. Here we take advantage of a 17-year long rainfall exclusion experiment to examine in detail how long-term increased aridity affects the different mechanisms involved in reproductive success from flower production to fruit maturation, and to determine whether these effects could be predicted from the effects of short term droughts.

The drivers of seed production have particularly been investigated in masting species such as oaks, i.e. species showing irregular, but synchronized, fruit production among individuals of a population [START_REF] Kelly | MAST SEEDING IN PERENNIAL PLANTS: Why, How, Where?[END_REF]. The proximate drivers of seed production and of masting are however still debated. Meteorological conditions, trees internal resource dynamics, mineral nutrient uptake, as well as their interactions, are all probably involved in inter-annual variations and synchrony in seed production [START_REF] Tanentzap | Soil nutrient supply modulates temperature-induction cues in mast-seeding grasses[END_REF][START_REF] Crone | Resource depletion, pollen coupling, and the ecology of mast seeding: Mechanisms of mast seeding[END_REF][START_REF] Pearse | Mechanisms of mast seeding: resources, weather, cues, and selection[END_REF]. In oaks, no environmental factor has yet been clearly identified as a reliable predictor of massive fruit production, both across and within species (Koenig & Knops, 2014;[START_REF] Pérez-Ramos | Environmental drivers of mastseeding in Mediterranean oak species: does leaf habit matter?[END_REF]. Moreover, some controversies exist because correlations between environmental factors and fruit production are not necessarily indicative of a causal relationship [START_REF] Kelly | Of mast and mean: differential-temperature cue makes mast seeding insensitive to climate change (M Rejmanek[END_REF][START_REF] Pearse | Cues versus proximate drivers: testing the mechanism behind masting behavior[END_REF][START_REF] Bogdziewicz | Environmental Veto Synchronizes Mast Seeding in Four Contrasting Tree Species[END_REF]. Detrimental weather events, such as drought or frost, can prevent flower production, fruit initiation or fruit maturation and have been called meteorological vetoes [START_REF] Pearse | Mechanisms of mast seeding: resources, weather, cues, and selection[END_REF][START_REF] Bogdziewicz | Correlated seed failure as an environmental veto to synchronize reproduction of masting plants[END_REF][START_REF] Bogdziewicz | Environmental Veto Synchronizes Mast Seeding in Four Contrasting Tree Species[END_REF]. In Mediterranean oaks, the water deficit during spring, summer and autumn might result in seed abortion and is considered as a major meteorological veto (Koenig & Knops, 2014;[START_REF] Pérez-Ramos | Environmental drivers of mastseeding in Mediterranean oak species: does leaf habit matter?[END_REF]Bogdziewicz et al., 2017b[START_REF] Bogdziewicz | Environmental Veto Synchronizes Mast Seeding in Four Contrasting Tree Species[END_REF]. Whether tree acclimation to increasing aridity might modify this seasonal drought effect on tree fecundity remains however to be determined.

The dynamics of resources and their allocation to different tree functions are also commonly identified as drivers of reproduction and are themselves affected by meteorological conditions [START_REF] Obeso | The costs of reproduction in plants[END_REF][START_REF] Barringer | Interrelationships among life-history traits in three California oaks[END_REF]. Schemes of resources allocation to reproduction are indeed central in explaining masting patterns [START_REF] Pearse | Mechanisms of mast seeding: resources, weather, cues, and selection[END_REF]. Besides, the nature of the resources limiting reproduction remains an important issue as phosphorus, nitrogen and nonstructural carbohydrates are all suspected to limit reproduction and drive masting [START_REF] Sala | Masting in whitebark pine (Pinus albicaulis) depletes stored nutrients[END_REF][START_REF] Miyazaki | Nitrogen as a key regulator of flowering in Fagus crenata : understanding the physiological mechanism of masting by gene expression analysis (JMH Knops[END_REF][START_REF] Allen | A Resource Centric View of Climate and Mast Seeding in Trees[END_REF][START_REF] Han | Recent developments in understanding mast seeding in relation to dynamics of carbon and nitrogen resources in temperate trees[END_REF][START_REF] Fernández-Martínez | Nutrient scarcity as a selective pressure for mast seeding[END_REF][START_REF] Satake | Field transcriptome revealed a novel relationship between nitrate transport and flowering in Japanese beech[END_REF]. Reduced water availability might reduce the availability of all these important resources by reducing the tree photosynthesis and productivity [START_REF] Misson | Functional changes in the control of carbon fluxes after 3 years of increased drought in a Mediterranean evergreen forest?[END_REF][START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF], the root soil water uptake [START_REF] Limousin | Long-term transpiration change with rainfall decline in a Mediterranean Quercus ilex forest[END_REF], and the rates of soil litter decomposition and nutrient mineralization [START_REF] Sardans | Drought changes phosphorus and potassium accumulation patterns in an evergreen Mediterranean forest[END_REF][START_REF] Santonja | Plant litter mixture partly mitigates the negative effects of extended drought on soil biota and litter decomposition in a Mediterranean oak forest[END_REF]. As tree acclimation to drought is known to involve changes in biomass allocation among organs [START_REF] Mencuccini | The ecological significance of long-distance water transport: short-term regulation, long-term acclimation and the hydraulic costs of stature across plant life forms[END_REF][START_REF] Poorter | Biomass allocation to leaves, stems and roots: meta-analyses of interspecific variation and environmental control[END_REF][START_REF] Limousin | The temporal response to drought in a Mediterranean evergreen tree: comparing a regional precipitation gradient and a throughfall exclusion experiment[END_REF][START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF], tree fecundity is likely to be modified by drought effects on resource acquisition and allocation [START_REF] Lauder | Fight or flight? Potential tradeoffs between drought defense and reproduction in conifers (A Polle[END_REF]Bogdziewicz et al., 2020b).

Studies investigating the effects of both meteorological conditions and trees' internal resource dynamics on reproduction are scarce, and only a few have tried to disentangle their relative effects [START_REF] Fernández-Martínez | The role of nutrients, productivity and climate in determining tree fruit production in European forests[END_REF]Bogdziewicz et al., 2017b;[START_REF] Nussbaumer | Impact of weather cues and resource dynamics on mast occurrence in the main forest tree species in Europe[END_REF][START_REF] Moreira | Weather cues associated with masting behavior dampen the negative autocorrelation between past and current reproduction in oaks[END_REF]. Three stages of reproduction seem particularly key for determining the number of mature fruits produced at the end of the season in Quercus ilex: female flower initiation and development which determines the number of receptive female flowers at pollination, fertilization which determines fruit set, i.e. the number of fruits initiated, and fruit development until maturity (Sork & Bramble, 1993;[START_REF] Tsuruta | Timing of premature acorn abortion in Quercus serrata Thunb. is related to mating pattern, fruit size, and internal fruit development[END_REF]. Our aim in this study was thus to identify the main drivers of fruit production at the key stages of the reproductive cycle of Q. ilex in a context of increasing aridity, and to disentangle their relative effects.

Using a 13-year monitoring of Q. ilex reproduction in a long-term rainfall exclusion experiment set in a Mediterranean holm oak coppice, we aimed to answer the following questions:

1) How does long-term rainfall reduction affect tree reproduction?

2) Are the drivers of the inter-annual variability of fruit production modified by long-term rainfall reduction?

3) Can tree reproduction acclimate to increasing aridity through changes in resource acquisition and allocation?

Materials and methods

Experimental site and study species

The experiment was conducted in southern France (35 km northwest of Montpellier), on a flat area in the Puéchabon State Forest (43°44'29'' N;3°35'46'' E,270 m a.s.l.). This forest has been coppiced until the last clear cut in 1942. The evergreen Quercus ilex L. forms a dense canopy with a height of c. 5.5 m, a mean basal area of 26.5 m 2 ha -1 , a density of c. 4900 stems ha -1 and a leaf area index (LAI) of 2.2. The evergreen species Buxus sempervirens, Phyllirea latifolia, Pistacia terebinthus and Juniperus oxycedrus compose a sparse understory shrubby layer with c. 25% cover. The bedrock is a hard Jurassic limestone and the soil is extremely rocky with c. 75% of stones and rocks in the top 0-50 cm and 90% below. The stone-free fraction of the soil within the 0-50 cm layer is a homogeneous silty clay loam (38.8% clay, 35.2% silt and 26% sand), leading to a field capacity of 210 mm. The area has a Mediterranean-type climate with a mean annual temperature of 13.5°C (on-site meteorological station, 1990-2019), the coldest month being January (6.0°C mean daily temperature) and the hottest month July (22.4°C mean daily temperature). The mean annual precipitation is 953 mm with a range of 578 -1549 mm (1990-2019). Rainfall mainly occurs during autumn and winter with about 80% between September and April.

The dominant species, Quercus ilex L. or holm oak, is an evergreen wind-pollinated monoecious tree that usually flowers in May in the study area. The male inflorescences, called catkins, bear around 20-25 staminate flowers [START_REF] Yacine | Self-and cross-pollination effects on pollen tube growth and seed set in holm oak Quercus ilex L (Fagaceae)[END_REF][START_REF] Gómez-Casero | Pollen biology in four Mediterranean Quercus species[END_REF]. Female pistillate flowers mature a few days after male flowers. Fertilization occurs in late June to early July, leading to the initiation of fruit (acorn) growth. Fruits achieve their maturation in November-December [START_REF] Yacine | Self-and cross-pollination effects on pollen tube growth and seed set in holm oak Quercus ilex L (Fagaceae)[END_REF]. In Q. ilex, two to three cohorts of leaves produced in different years usually coexist on the branches [START_REF] Limousin | Morphological and phenological shoot plasticity in a Mediterranean evergreen oak facing long-term increased drought[END_REF]. Floral initiation period remains unknown in Q. ilex, but sporadic observations of second flowering during summer and autumn lead us to suspect that staminate flowers might be initiated in early summer as observed in Q. alba [START_REF] Merkle | Development of Floral Primordia in White Oak[END_REF].

Experimental design of the rainfall exclusion

In March 2003, a throughfall exclusion experiment was set up on three replicated blocks located 50-100 m apart one from the other and situated on a flat forested area with no lateral flow. Each block comprised two contiguous 10 × 10 m plots subjected to either control precipitation or throughfall exclusion (further named "dry treatment"). For the throughfall exclusion treatment, the experimental plot was equipped with parallel 14 m long and 0.19 m wide PVC gutters hung below the canopy with a slope, between 1 m and 2 m height, so as to cover 33% of the ground area under the tree canopy within the 10 x 10 m plot and a 2 m buffer zone. Taking into account rainfall interception and stemflow, the net input of precipitation was reduced by 27% compared with the control plots [START_REF] Limousin | Modelling rainfall interception in a mediterranean Quercus ilex ecosystem: Lesson from a throughfall exclusion experiment[END_REF]. The experimental design reduces significantly the surface soil water content (García de [START_REF] De Jalón | Microhabitat and ectomycorrhizal effects on the establishment, growth and survival of Quercus ilex L. seedlings under drought[END_REF], and induced significantly more negative tree water potentials in most summers since treatment installation in 2003 [START_REF] Bykova | Water deficit disrupts male gametophyte development in Quercus ilex (S Pfautsch, Ed.)[END_REF]. In the control plots, identical gutters were installed upside down to homogenize albedo and understorey microclimate without reducing precipitation inputs. Stand density and mean DBH did not differ significantly between throughfall exclusion and control plots at the start of the experiment, with respectively 5930 and 6430 stems ha -1 , and 6.5 and 7.6 cm DBH [START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF]. In total, 178 and 193 Q. ilex trees, were respectively included in the three replicated blocks with control and dry treatments.

Reproduction, growth and phenology data

Stem DBH was inventoried every year in winter from 2003 to 2019 for all the stems with a DBH > 2 cm and converted into aboveground wood biomass (stem and living branches) using an allometric equation calibrated on the study site [START_REF] Rambal | The growth respiration component in eddy CO2 flux from a Quercus ilex mediterranean forest[END_REF]:

Biomass (g) = 191.6 × DBH 2.171
Eq (1)

Wood biomass increment at the plot scale was calculated by summing annual biomass increment of the trees. Relative plot biomass increment of the year was calculated as the plot aboveground biomass increment divided by the plot biomass.

Litterfall was collected monthly from 2003 to 2019 in 12 litter traps per plot placed regularly on a grid over the gutters at a height of 2 m and representing a total collecting area of 1.1 m 2 per plot. Litterfall was oven-dried at 60°C for 3 days, separated into leaves, wood, flowers and fruits and weighted. Fallen fruits were further categorized into three different categories and counted from 2007 to 2019: (1) aborted (not completely or mal-developed seeds, with length < 13 mm and/or diameter < 7 mm), (2) insect infested (having signs of larvae predation, such as gnaw marks or holes), and (3) mature (attaining mature seed size). The number of initiated fruits was calculated as the sum of the three categories. Annual litterfall was calculated as the sum of monthly litterfall from the 1 st of May of the year until the 30 th of April the following year in order to consider all the flowers and fruits produced during one reproductive season. Fruit number (initiated and mature), fruit biomass and male flower biomass produced per year and per plot were divided by the plot estimated aboveground biomass so that investment in reproduction was comparable among blocks. These variables are thus expressed per kg of aboveground biomass in the following.

The phenology of leaves and catkins was monitored weekly during the season on six trees and three ramets per tree in each treatment of one of the blocks where scaffolds allow access to branches from the top of the canopy. A median date of catkin anthesis was calculated for each year and the mean length of the period between anthesis and flower senescence was 9 ± 4 days. We therefore considered a 25-day pollination period comprising 8 days before median anthesis (2 SD), and 17 days after median anthesis (mean flowering period + 2 SD).

Meteorological variables, water stress modelling and carbon fluxes data

Continuous meteorological data were collected by a weather station located in a clearing less than 100 m away from the experimental plots. For each year, precipitation during pollination was calculated as the sum of daily precipitation during the 25 days of the pollination period and was considered similar for the two treatments because it impacts airborne pollen above the gutters. The number of days of torrential rain, previously identified as a potential determinant of mature acorn production in Q. ilex [START_REF] Pérez-Ramos | Mast seeding under increasing drought: results from a long-term data set and from a rainfall exclusion experiment[END_REF], was determined as the number of days between

July and November (period between fruit initiation and fruit maturity) during which daily rainfall exceeded 76 mm (threshold determined as the 0.99 percentile value of daily precipitations between July and November from 1984 to 2019).

Tree water stress, assessed by the predawn leaf water potential (Ψlpd), was simulated at a daily time scale using the water balance module of the SIERRA vegetation model [START_REF] Mouillot | A generic process-based SImulator for meditERRanean landscApes (SIERRA): design and validation exercises[END_REF] calibrated and validated on our experimental site by [START_REF] Cabon | Thinning increases tree growth by delaying drought-induced growth cessation in a Mediterranean evergreen oak coppice[END_REF]. Soil parameters were kept constant for the two treatments, the LAI was reduced by 18% in the dry treatment relatively to the 2.2 value in control to reflect the lower leaf production in this treatment [START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF], and the net precipitation input to the dry treatment was reduced by 27% compared to the control [START_REF] Limousin | Modelling rainfall interception in a mediterranean Quercus ilex ecosystem: Lesson from a throughfall exclusion experiment[END_REF]. Model performance was evaluated against 11 years of discrete predawn leaf water potential measurements (described in [START_REF] Bykova | Water deficit disrupts male gametophyte development in Quercus ilex (S Pfautsch, Ed.)[END_REF]) and was similarly good in the two treatments (control treatment: R² = 0.85, RMSE = 0.58 MPa; dry treatment: R² = 0.89, RMSE = 0.57 MPa). Daily absolute values of simulated Ψlpd were summed over the period from April to June to calculate the water stress index (WSI, defined by [START_REF] Myers | Water stress integral-a link between short-term stress and long-term growth[END_REF]) during the period of flower development and fertilization, and from July to November to calculate the WSI during the period of fruit development and maturation. WSI was also calculated for the summer (July-September) of the previous year, which corresponds to the suspected period of initiation of male flowers.

Eddy covariance fluxes of CO2 between the ecosystem and the atmosphere have been measured continuously during the experiment using a three-dimensional sonic anemometer and a closed path infrared gas analyser set up at the top of a 12-m-high tower located near the experimental plots (see [START_REF] Allard | Seasonal and annual variation of carbon exchange in an evergreen Mediterranean forest in southern France[END_REF] for details). Processing schemes of FLUXNET have been used for filling data gaps and partitioning net ecosystem productivity (NEP) into gross primary productivity (GPP) and ecosystem respiration Reco [START_REF] Reichstein | On the separation of net ecosystem exchange into assimilation and ecosystem respiration: review and improved algorithm[END_REF][START_REF] Papale | Towards a standardized processing of Net Ecosystem Exchange measured with eddy covariance technique: algorithms and uncertainty estimation[END_REF]. Half-hourly values of GPP were summed over the period from July to November as an estimate of ecosystem-scale carbon assimilation during fruit development and maturation.

Leaf chemical analyses

From 2007 to 2018 (except 2013), leaves from the top canopy of the 6 to 8 trees per treatment in the scaffold-equipped block, where we also monitored phenology and fruit growth, were collected in winter (mid-November to mid-February, depending on years) oven-dried at 60°C for 3 days and ground for chemical analyses. Leaves were previously sorted between leaves produced during the current year (less than 1 year old) and leaves produced the year before. Nitrogen (N) mass-based concentration was determined by thermal combustion, using a Flash Smart NC Elemental Analyzer (ThermoFisher Scientific, USA) and phosphorus (P) mass-based concentration was measured colorimetrically using the molybdenum blue method. A mean N and P winter leaf concentration for each leaf cohort was calculated from 2007 to 2018 (except for missing P data in 2013 and 2017, and N data in 2013 and 2018).

Variables selection and statistical analyses

The four main reproduction variables that we studied are the biomass of male flowers, the number of initiated fruits, the fruit abortion rate and the number of mature fruits.

First, we tested for a treatment effect on each reproductive variable, by running generalized linear mixed models (GLMMs) using the R package lme4 [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF] with treatment as a fixed effect, and block and year as random effects. We used negative binomial distribution with a log link for count variables to account for over-dispersion issues.

Second, we did an extensive review of the literature to identify all the factors potentially driving the inter-annual variability of Q. ilex fruit production (summarised in Table 1). Due to the large number of potential explanatory variables and of time periods on which they can be considered, and to the high collinearity among some weather variables, we narrowed our analyses based on existing knowledge about the main drivers of fruit production in Q. ilex. We only considered the factors that we could associate to a physiological mechanism, and therefore excluded variables such as the difference of annual temperature between current and previous year [START_REF] Koenig | Is the relationship between mast-seeding and weather in oaks related to their life-history or phylogeny?[END_REF] or January minimum temperature [START_REF] García-Mozo | Influence of pollen emission and weather-related factors on variations in holm-oak (Quercus ilex subsp. ballota) acorn production[END_REF]. We excluded the factors which had a very large temporal coverage, such as annual rainfall [START_REF] Alejano | Influence of pruning and the climatic conditions on acorn production in holm oak (Quercus ilex L.) dehesas in SW Spain[END_REF], evapotranspiration between January and August (Bogdziewicz et al., 2017b) or precipitations during the 10 months before fruit maturation [START_REF] Ogaya | Species-specific drought effects on flower and fruit production in a Mediterranean holm oak forest[END_REF], or a very large spatial coverage, such as the remote sensing enhanced vegetation index [START_REF] Camarero | An increase in canopy cover leads to masting in Quercus ilex[END_REF][START_REF] Fernández-Martínez | Temporal trends in the enhanced vegetation index and spring weather predict seed production in Mediterranean oaks[END_REF]. We then attributed the explanatory variables to each period of the reproductive cycle according to the time of the year they cover. When some variables were too strongly correlated to one another (such as temperature, rainfall and water stress in autumn), we chose to include in the model selection only the water stress index (WSI) which is the variable that best integrates the physiological state of the tree.

Third, in order to determine the set of variables that best explain the inter-annual variability of the four reproductive response variables, we applied a model selection procedure on the full models including all the variables that we had previously selected, and in which variance inflation factors never exceeded 3 as recommended by [START_REF] Zuur | A protocol for data exploration to avoid common statistical problems: Data exploration[END_REF]. The four models are described in Table 2, indicating both the variables retained after model selection and the variables excluded by model selection. The model with the best empirical support was selected by minimizing the corrected AIC for small sample size (AICc) using the R package MuMIn [START_REF] Bartoń | MuMIn: Multi-Model Inference[END_REF]. The best models exhibited a ΔAICc > 2 from others for all the reproduction variables, except for the number of initiated fruits for which we chose the most parsimonious model (ΔAICc = 0.6 with the one including male flower biomass). We proceeded in two steps. First, we run the full models (GLMMs with block as random factor) on the control treatment only to identify the main drivers of interannual variability in tree fecundity. Second, we run the selected model, with the same characteristics (response variables, model distribution and random effect), on both the control and dry treatments in order to determine the impact of long term increased aridity on the trees' response to seasonal drivers (Table 2). As fixed effects, we included the variables retained by model selection for each response variable (except for GPP, which was not measured in the dry treatment), treatment and the interactions between treatment and all the other variables. We then simplified the model by sequentially removing the insignificant terms, starting with the weakest and least significant interaction.

In order to determine whether the production of initiated fruits and of male flowers was impacted by nitrogen and phosphorus concentration in leaves during the winter before bud break, we used data from the two cohorts of leaves and the two treatments. We fitted GLMMs with a negative binomial distribution for the number of initiated fruits produced per kg of aboveground biomass and with a Gaussian distribution for male flower biomass per kg of aboveground biomass.

Treatment, the standardised nutrient concentration and their interaction were included as fixed effects.

All statistical analyses and visual representations were conducted using the software R version 3.6.1 (2019) and the ggplot2 package [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF]. Marginal (R²m) and conditional (R²c) were calculated with the package MuMIn according to Nakagawa's method [START_REF] Nakagawa | A general and simple method for obtaining R2 from generalized linear mixed-effects models[END_REF] to estimate the variance explained by fixed effects and fixed plus random effects, respectively.

Results

Treatment effect on tree water stress and reproductive variables

Water stress (measured by WSI) of spring (April-June), summer-autumn (July-November) and summer (July-September) of the previous year were significantly increased by 20.6%, 16.8% and 13.4%, respectively, in the rainfall exclusion treatment compared to the control (Supporting Information Fig. S1). Rainfall exclusion significantly reduced the male flower biomass, the number of initiated fruits and the number of mature fruits, while it significantly increased the fruit abortion rate (Fig. 1a,b,c,d). Rainfall exclusion had, however, no impact on leaf nutrient concentrations in winter (Supporting Information Table S1).

Inter-annual variability of reproduction

The number of initiated fruits and the biomass of male flowers were less variable among years (mean CVp 42% and 57%, respectively, in control treatment), than the number of mature fruits (CVp 126 % in control treatment) and the rate of fruit abortion between initiation and maturity (Fig. 2a,b,c,d, Supporting Information Table S2). Rainfall exclusion did not affect any of these coefficients of variation (Fig. 3).

The mean mass of mature fruits did not differ between treatments (Supporting Information Table S2, Fig. S2a). There was no trade-off between mean mature fruit mass and mature fruit number.

On the contrary, there was a positive relationship between the two variables: years of high fruit production were also years during which mature fruits were heavier (Supporting Information Fig. S2b).

Male flower biomass

According to model selection, male flower biomass was best explained by (in order of importance)

the male flower biomass of the previous year and the summer WSI of the previous year which both had a negative effect. The two variables explained collectively 22% of the variance, which was less than the variance explained by the random effect (Table 2, Fig. 4a). The effect of the rainfall exclusion on male flower biomass remained negative when combined with other variables (Fig. 4b).

Number of initiated fruits

The number of initiated fruits in the control plots was best explained by (in order of importance) cumulative rainfall during pollination, which had a negative effect, wood biomass increment and mean June temperature, which both had positive effects (Table 2, Fig. 5a). The variance explained by the fixed effects was around 39% which is larger than for the male flowers. Besides, contrary to the male flowers, the number of fruits was very little explained by random effects. There was no trade-off between wood biomass increment and reproduction standardised by the plot biomass, on the contrary, years during which large amounts of wood biomass were produced were also years during which a large number of fruits were initiated. Interestingly, neither the fruit crop of the previous year, the male flower biomass nor the spring WSI were retained by model selection as strong predictors of the number of initiated fruits (Table 2). The long-term rainfall exclusion treatment had an additional strong negative effect on fruit initiation (Fig. 5b). There was no interaction between wood biomass increment and the rainfall exclusion effect.

Abortion rate between fruit initiation and maturity

Fruit abortion rate between initiation and maturity was best explained by (in order of importance)

July-November WSI, which had a positive effect, and by July-November GPP, the number of initiated fruits and wood biomass increment which had a negative effect, with all the fixed effects explaining 21% of the variance (Table 2, Fig. 6a). Years during which trees initiated a large number of fruits tended to be years during which fruits survived better. As WSI and the number of initiated fruits were already strongly affected by the dry treatment (Fig. 1b and Supporting Information Fig. S1f), the latter did not have a significant effect on abortion when tested in addition to these two variables (Fig. 6b). However, treatment and July-November WSI interacted significantly (Fig. 6b), indicating that trees from the dry treatment experienced less fruit abortion than control trees during years with low water stress in summer and autumn (Supporting Information Fig. S3a,b).

Number of mature fruits

The number of mature fruits was best explained by (in order of importance) the number of initiated fruits which had a positive effect, July-November WSI which had a negative effect, and July-November GPP which had a positive effect (Table 2, Fig. 7a). Our model explained as much as 65% of the variance in mature fruits, probably as a result of the inclusion of the number of initiated fruits as a predictor of the number of mature fruits. However, the final number of mature fruits was driven more by the rate of abortion than by the number of initiated fruits (Supporting Information Fig. S4a,b). The rainfall exclusion treatment did not affect the number of mature fruits when tested in addition to the number of initiated fruits and summer-autumn WSI (Fig. 7b).

Effect of leaf nutrients content

Male flower production and the number of initiated fruits were both positively related to winter nitrogen concentration in old leaves (cohort produced two years before) (Table 3). They were not correlated to nitrogen concentration in young leaves, nor with phosphorus concentrations. There were no interactions between treatment and nutrient concentrations. Conversely, the total biomass of fruits produced during summer and autumn (i.e. aborted and mature fruits) and the treatment had no effect on the nitrogen and phosphorus concentration of the leaves the following winter in either treatment (Supporting Information Table S1).

Discussion

Increased aridity has detrimental effects on flower production, fruit set and fruit growth

The long-term experimental reduction of precipitation by 27% increased the water stress during flower development and fruit initiation in spring, fruit growth in summer and autumn, and male flower initiation in summer. Increased aridity significantly decreased the production of male flowers, the number of initiated and mature fruits, and increased fruit abortion during summer and autumn. Consequently, the final fruit crop under rainfall exclusion was reduced due to two cumulative effects, i.e. a lower number of initiated fruits in spring and a higher rate of fruit abortion in summer and autumn. Our results confirm those from other rainfall exclusion experiments in Q.

ilex forests, in which a 15 % rainfall exclusion reduced both the number of initiated fruits and of mature acorns (Sánchez-Humanes & Espelta, 2011), as well as the biomass of male flowers and fruits [START_REF] Ogaya | Species-specific drought effects on flower and fruit production in a Mediterranean holm oak forest[END_REF][START_REF] Liu | Contrasting impacts of continuous moderate drought and episodic severe droughts on the aboveground-biomass increment and litterfall of three coexisting Mediterranean woody species[END_REF].

Conversely, the long-term precipitation reduction did not modify the inter-annual variability in seed production, which can result from variation in either flower production, fruit initiation rate or fruit abortion rate [START_REF] Pearse | Mechanisms of mast seeding: resources, weather, cues, and selection[END_REF]. In our study, the number of mature fruits was driven more by fruit abortion than by fruit initiation and our results confirm that Q. ilex is a 'fruit maturation masting' species (Sork & Bramble, 1993;[START_REF] Espelta | Masting mediated by summer drought reduces acorn predation in Mediterranean oak forests[END_REF][START_REF] Bogdziewicz | Environmental Veto Synchronizes Mast Seeding in Four Contrasting Tree Species[END_REF], i.e. that fruit crop is determined more by what happens after fruit set than before it. With a 3-fold higher

CVp for the number of mature fruits than for initiated fruits, the difference of inter-annual variability among reproduction stages was larger here than in previous studies on Mediterranean oaks [START_REF] Espelta | Masting mediated by summer drought reduces acorn predation in Mediterranean oak forests[END_REF][START_REF] Pérez-Ramos | Why Is Seed Production So Variable among Individuals? A Ten-Year Study with Oaks Reveals the Importance of Soil Environment (S Delzon[END_REF]. In any case, our experimental reduction of precipitation did not modify the relative variability of either flower production or fruit abortion, and it did not increase the inter-annual variability of seed production. This result thus does not support the hypothesis proposed by some authors that inter-annual variability of fruit production should increase in more stressful long-term conditions [START_REF] Espelta | Masting mediated by summer drought reduces acorn predation in Mediterranean oak forests[END_REF][START_REF] Bogdziewicz | Correlated seed failure as an environmental veto to synchronize reproduction of masting plants[END_REF][START_REF] Wion | Aridity drives spatiotemporal patterns of masting across the latitudinal range of a dryland conifer[END_REF].

Several studies have shown that fruit abortion rate was strongly influenced by water stress during summer and autumn [START_REF] Espelta | Masting mediated by summer drought reduces acorn predation in Mediterranean oak forests[END_REF][START_REF] Alejano | Influence of pruning and the climatic conditions on acorn production in holm oak (Quercus ilex L.) dehesas in SW Spain[END_REF][START_REF] Carevic | Plant water relations and edaphoclimatic conditions affecting acorn production in a holm oak (Quercus ilex L. ssp. ballota) open woodland[END_REF][START_REF] Pérez-Ramos | Mast seeding under increasing drought: results from a long-term data set and from a rainfall exclusion experiment[END_REF][START_REF] Liu | Contrasting impacts of continuous moderate drought and episodic severe droughts on the aboveground-biomass increment and litterfall of three coexisting Mediterranean woody species[END_REF]. [START_REF] Alejano | Influence of pruning and the climatic conditions on acorn production in holm oak (Quercus ilex L.) dehesas in SW Spain[END_REF] suggested that water stress may act as a climatic veto when leaf water potential is lower than -3.5 MPa, which is close to the turgor loss point of Q. ilex [START_REF] Tognetti | Water relations of oak species growing in the natural CO 2 spring of Rapolano (central Italy)[END_REF]. In our study, the two years during which fruit production was the highest (2007 and 2015) were years during which the simulated water potential never reached this threshold in any treatment (in 2015) or only for a few days (in 2007). However, the same condition was also met during other years and did not result in a high fruit crop. A low summer water stress therefore appears as a necessary but not sufficient condition for reproductive success.

The fruit abortion rate was the only reproductive variable to exhibit an interactive effect between the dry treatment and summer-autumn water stress (Fig. 6). Fruit abortion had a greater sensitivity to water stress in the dry treatment than in the control, suggesting that increased aridity worsens the effect of summer drought on fruit growth. Although this greater sensitivity to water stress in the dry treatment translated into a lower abortion than in the control in favourable years, it did not allow for a better fruit development in dry years and fruit production remained lower in the dry treatment irrespectively of the drought conditions.

Fruit set has also been shown to be limited by pollination success, which depends on meteorological conditions during pollination because rainfall during flowering washes pollen out of the atmosphere and alters pollination success [START_REF] García-Mozo | Influence of pollen emission and weather-related factors on variations in holm-oak (Quercus ilex subsp. ballota) acorn production[END_REF]. As our experiment was not designed to manipulate rainfall over the tree canopy, we cannot estimate how less precipitation in spring may affect fruit initiation due to pollen washing. Nevertheless, in a previous study, we found that rainfall exclusion decreased viable pollen production by the trees [START_REF] Bykova | Water deficit disrupts male gametophyte development in Quercus ilex (S Pfautsch, Ed.)[END_REF], which may reduce pollen availability in the future. Previous studies on oaks have observed a pollen limitation effect on fruit production, but only in low tree density savannah-like landscapes [START_REF] García-Mozo | Influence of pollen emission and weather-related factors on variations in holm-oak (Quercus ilex subsp. ballota) acorn production[END_REF], 2012;[START_REF] Pesendorfer | Individual resource limitation combined with population-wide pollen availability drives masting in the valley oak (Quercus lobata) (K Whitney, Ed.)[END_REF], and not in dense forests [START_REF] Fernández-Martínez | Masting in oaks: Disentangling the effect of flowering phenology, airborne pollen load and drought[END_REF]Bogdziewicz et al., 2017b). Finally, pollination may be positively affected by warmer spring temperatures in Mediterranean oaks because they favour flowering synchrony and thereby the efficiency of pollination [START_REF] Koenig | What drives masting? The phenological synchrony hypothesis[END_REF]Bogdziewicz et al., 2017b), and fertilization (Sork et al., 1993;[START_REF] Cecich | Influence of weather at time of pollination on acorn production of Quercus alba and Quercus velutina[END_REF] possibly through a positive influence on pollen tube growth [START_REF] Hedhly | Global warming and sexual plant reproduction[END_REF]. Our results validate the positive effect of June temperature, during the fertilization period, on the number of initiated fruits, as previously observed by [START_REF] García-Mozo | Influence of pollen emission and weather-related factors on variations in holm-oak (Quercus ilex subsp. ballota) acorn production[END_REF], but not an effect of the temperatures during flowering (April-May).

Increased aridity has additive carry-over effects on male flower production and fruit set

Experimentally increased aridity had long-term effects on tree allocation to reproductive organs in our experiment. This was revealed by the strong treatment effect on the initial fruit set which is highly correlated to female flower biomass and on male flower biomass, that could not be explained merely by an increased water stress during critical periods of the reproductive cycle.

Indeed, the treatment effect on male flower biomass remained significant in addition to the previous year WSI effect, and seasonal water stress had no significant effect on fruit initiation (Fig. 4, Fig. 5). This means that the effect of experimentally increased aridity was additive with the other drivers, so that trees in the dry treatment always produced less male flowers and initiated less fruits than the control trees, independently of the meteorological conditions. Conversely, the long-term increased aridity did not interact with the meteorological drivers of fruit production and thus did not modify their hierarchy of importance in explaining the inter-annual variation in fecundity.

Increased aridity thus lowered the intercept of the relationship between reproduction and climatic conditions but without affecting the slope (no interaction). Therefore, the sensitivity of the reproductive organs to temperature and seasonal drought remained similar across treatments while the overall capacity to allocate to reproduction was diminished by increased aridity. In other words, the dry treatment reduced fruit production more strongly than what we would predict from a 27% decrease in precipitation with the model adjusted on the control treatment data. Consequently, we argue that Q. ilex acorn production may be more affected by increasing aridity in the future than what current inter-annual variations would lead us to forecast.

We suggest that this additive effect of increased aridity might be induced partly by the carry-over effects of previous summer water stress on bud development. In oak species, primary growth is largely determined by preformed organs inside the buds [START_REF] Fontaine | Preformation and neoformation of growth units on 3-year-old seedlings of Quercus petraea[END_REF][START_REF] Alla | Seasonal and inter-annual variability of bud development as related to climate in two coexisting Mediterranean Quercus species[END_REF].

Bud enlargement [START_REF] Montserrat-Martí | Summer-drought constrains the phenology and growth of two coexisting Mediterranean oaks with contrasting leaf habit: implications for their persistence and reproduction[END_REF], leaf primordia initiation [START_REF] Alla | Seasonal and inter-annual variability of bud development as related to climate in two coexisting Mediterranean Quercus species[END_REF] and male floral primordia initiation [START_REF] Merkle | Development of Floral Primordia in White Oak[END_REF] have all been shown to occur during the previous growing season and to be potentially affected by summer drought. Our results indeed show a significant effect of previous year WSI on male flower production, and previous studies of the same experiment found a 1-year lagged effect of drought on leaf production (decreased leaf number per growth unit) [START_REF] Limousin | Morphological and phenological shoot plasticity in a Mediterranean evergreen oak facing long-term increased drought[END_REF][START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF], and a lower number of viable pollen grains [START_REF] Bykova | Water deficit disrupts male gametophyte development in Quercus ilex (S Pfautsch, Ed.)[END_REF]. Female flowers, contrary to other organs, were not affected by water stress of the previous summer. However, because they are generally initiated on the larger shoots bearing numerous leaves [START_REF] Alla | Acorn production is linked to secondary growth but not to declining carbohydrate concentrations in current-year shoots of two oak species[END_REF], their lower number in the dry treatment might be caused indirectly by the reduced number of ramifications and leaves per shoot in this treatment [START_REF] Limousin | Morphological and phenological shoot plasticity in a Mediterranean evergreen oak facing long-term increased drought[END_REF].

Fewer resources are allocated to reproduction under increased aridity

In our study, years during which conditions were favourable for growth were also favourable for fruit production. Fruit crop and fruit abortion rate in the control treatment were, respectively, positively and negatively correlated to cumulated GPP from July to November. This result is consistent with previous studies showing that the carbon used for fruit development is provided mainly by recent uptake of photo-assimilates [START_REF] Hoch | Fruit production in three masting tree species does not rely on stored carbon reserves[END_REF]Ichie et al., 2013;[START_REF] Han | Reproduction-related variation in carbon allocation to woody tissues in Fagus crenata using a natural 13C approach[END_REF], and it suggests that Q. ilex acorn production might be limited by the availability of carbon resources. Other studies conducted in temperate forest ecosystems on Q. petraea or F. sylvatica have, however, failed to observe such a relationship between GPP and seed production [START_REF] Delpierre | Wood phenology, not carbon input, controls the interannual variability of wood growth in a temperate oak forest[END_REF][START_REF] Mund | It is not just a 'trade-off': indications for sink-and source-limitation to vegetative and regenerative growth in an old-growth beech forest[END_REF]. In the Mediterranean context of our study, where the ecosystem carbon uptake is driven by water availability in summer [START_REF] Rambal | How drought severity constrains gross primary production (GPP) and its partitioning among carbon pools in a Quercus ilex coppice?[END_REF], GPP and acorn production are both largely influenced by water stress. In spite of this, the WSI and GPP were both retained by model selection as significant drivers of the annual fruit crop and fruit abortion rate (Fig. 6, Fig. 7), thereby suggesting an effect of carbon resources on acorn production, in addition to the effect of water availability. The greater sensitivity of the fruit abortion rate to summerautumn WSI in the dry treatment could be partly explained by a reduced availability of recently assimilated carbon given that leaf carbon assimilation and ecosystem GPP are reduced in this treatment compared to the control [START_REF] Misson | Functional changes in the control of carbon fluxes after 3 years of increased drought in a Mediterranean evergreen forest?[END_REF][START_REF] Limousin | Do photosynthetic limitations of evergreen Quercus ilex leaves change with long-term increased drought severity? Plant[END_REF].

Our results also show that biomass increment was positively related to the number of initiated fruits per kilogram of aboveground biomass and negatively related to the fruit abortion rate, thereby indicating an absence of causal trade-off between growth and reproduction. In masting Fagaceae, massive fruit crops have been either associated to reduced growth [START_REF] Pearse | Mechanisms of mast seeding: resources, weather, cues, and selection[END_REF] or increased growth [START_REF] Pérez-Ramos | Mast seeding under increasing drought: results from a long-term data set and from a rainfall exclusion experiment[END_REF][START_REF] Fernández-Martínez | Temporal trends in the enhanced vegetation index and spring weather predict seed production in Mediterranean oaks[END_REF]Bogdziewicz et al., 2020b), although these relationships might not be causal [START_REF] Knops | Negative correlation does not imply a tradeoff between growth and reproduction in California oaks[END_REF][START_REF] Knops | Sex Allocation in California Oaks: Trade-Offs or Resource Tracking?[END_REF], or not associated to growth at all [START_REF] Yasumura | Resource allocation to vegetative and reproductive growth in relation to mast seeding in Fagus crenata[END_REF][START_REF] Pulido | Resource manipulation reveals flexible allocation rules to growth and reproduction in a Mediterranean evergreen oak[END_REF]. The absence of trade-off between growth and reproduction in Q. ilex might be explained by a temporal lag between stem and fruit growth. Q. ilex stem growth takes place mostly in spring and early summer, before the drought and concomitantly to flower development and fruit initiation, but also in autumn when water availability is restored after the drought (Supporting Information Fig. S5, Methods S1). Stems and fruits are thus in competition for resources mainly in autumn, consistently with [START_REF] Martín | Trade-off between stem growth and acorn production in holm oak[END_REF] who observed a negative correlation between acorn production during mast years and autumn stem growth, but not spring growth. Here, we observed no interactive effect between stem growth and the treatment, but the biomass increment and treatment were both retained by model selection as significant drivers of fruit initiation and abortion (Fig. 5, Fig. 6).

This means that for a given allocation to wood growth, the allocation to fruits was lower in the rainfall exclusion treatment. Our results thus confirm an earlier conclusion by Bogdziewicz et al.

(2020b) that increased aridity modifies the relative allocation to growth versus reproduction in Q.

ilex, although in our case the allocation to fruits was more impacted by aridity than the allocation to wood. In our experiment, stand aboveground net primary productivity was reduced by 11% in the dry treatment compared to the control, but while wood production was not significantly impacted by the dry treatment, acorn mean biomass production was reduced by as much as 34% over the course of our experiment [START_REF] Gavinet | Rainfall exclusion and thinning can alter the relationships between forest functioning and drought[END_REF]. This suggests that short-term maintenance of tree functioning is prioritized over reproduction under increased aridity, raising concerns about long-term persistence of Q. ilex populations under climate change as well as on its ability to colonize newly favorable areas.

Male flower biomass and the number of initiated fruits were both positively correlated to mean nitrogen concentration in 2 yr-old leaves sampled during the winter before. These old leaves usually fall in early summer, shortly after new leaves and flowers have been produced. Our result thus suggests that nitrogen used for flower production might be remobilized from old leaves before they fall [START_REF] Cherbuy | Internal remobilization of carbohydrates, lipids, nitrogen and phosphorus in the Mediterranean evergreen oak Quercus ilex[END_REF] and that male flowers development might be limited in part by nitrogen availability. There is increasing evidence that stored nitrogen or phosphorus contribute to masting events [START_REF] Sala | Masting in whitebark pine (Pinus albicaulis) depletes stored nutrients[END_REF][START_REF] Ichie | Dynamics of mineral nutrient storage for mast reproduction in the tropical emergent tree Dryobalanops aromatica[END_REF][START_REF] Miyazaki | Nitrogen as a key regulator of flowering in Fagus crenata : understanding the physiological mechanism of masting by gene expression analysis (JMH Knops[END_REF][START_REF] Han | Recent developments in understanding mast seeding in relation to dynamics of carbon and nitrogen resources in temperate trees[END_REF][START_REF] Satake | Field transcriptome revealed a novel relationship between nitrate transport and flowering in Japanese beech[END_REF], and fertilisation experiments in oak forests have sometimes increased acorn production (Bogdziewicz et al., 2017a), although not always [START_REF] Brooke | Effects of fertilization and crown release on white oak (Quercus alba) masting and acorn quality[END_REF].

Conversely, the absence of an interactive effect between rainfall exclusion and leaf nutrient content on flower and fruit production suggests that the long-term increased drought effect was not mediated by a nutrient limitation.

Conclusion

Taken together our results have important implications for forecasting the response of tree reproduction to increasing aridity because they suggest that it may affect tree reproduction beyond what is expected from the current response to seasonal drought. Under the drier conditions simulated by our experiment, trees initiate fewer flowers and fruits, and these fruits are less likely to withstand summer drought and reach maturity. We interpret the strong negative effect of increased aridity on fruit production as caused by more severe water stress during fruit development, stronger limitations by resources and changes in resources allocation, all suggesting that holm oak forest natural regeneration could be jeopardized in a drier future.

Tables Table 1: treatment and all covariables were first included, and non-significant interactions were sequentially removed from the model starting with the least significant ones. The positive interaction between July-November WSI and Dry treatment reflects the fact that trees from the dry treatment experienced less fruit abortion than control trees during years with lower water stress in summer and autumn. Note that in the driest years both treatments showed 100% fruit abortion (see Fig. S3b). We report marginal (R²m) and conditional (R²c) R² for the reduced, final model. WSI = water stress index, GPP = gross primary production. Interactions between treatment and all covariables were first included, and non-significant interactions were sequentially removed from the model starting with the least significant ones.

We report marginal (R²m) and conditional (R²c) R² for the reduced, final model. WSI = water stress index, GPP = gross primary production. The following Supporting Information is available for this article: Table S1 Effect of fruit biomass production and treatment on leaf nutrient concentration the following winter.

Table S2 Population Coefficient of Variation (CVp) for male flower biomass, numbers of initiated and mature fruits and mean fruit mass.

Fig. S2

Mean mature fruit mass according to year, treatment and the number of mature fruits produced.

Fig. S3

Relationship between summer-autumn water stress index and fruit abortion rate according to treatment.

Fig. S4

Effects of the number of initiated fruits and the fruit abortion rate on the number of mature fruits.

Fig. S5

Intra-annual variations of phenological phases, fruit and stem growth, water potential, precipitations, gross primary production and temperature.

Methods S1

Fruit growth and stem growth measurements. relationship between mean mature fruit mass and the number of mature fruits depending on 3 treatment. Mature fruit mean mass was significantly and positively correlated to the number of 4 mature fruits (Wald χ² = 11.4, p < 0.001), whereas treatment and the interaction between treatment 5 and fruit number were not significant using a linear mixed model with block as a random effect, 6 mean fruit mass as response variable, number of fruits, treatment and their interaction as fixed 7

Figure 1 :

 1 Figure legends

Figure 2 :

 2 Figure 2: Mean inter-annual variability (± SD) of the production of (a) male flower biomass, (b) number of initiated fruits, (c) fruit abortion rate and (d) number of mature fruits in the control (blue) and dry (orange) treatments in Q. ilex. Points indicate the mean value of the 12 traps for each plot and each year.

Figure 3 :

 3 Figure 3: Treatment effect on the inter-annual variability (population coefficient of variation) of Q. ilex mean male flower biomass, mean number of initiated fruits and mean number of mature fruits among the three plots per treatment. CI = Confidence interval, AB = aboveground biomass.

Figure 4 :

 4 Figure 4: Generalized linear mixed models standardised estimates ± SE and associated p-values (*: p < 0.05, **: p < 0.01) of the variables explaining Q. ilex male flower biomass per kg of aboveground biomass (a) after model selection in the control plots for 2007-2019 and (b) with the variables retained by model selection in (a) plus the dry treatment effect in control and dry plots. Interactions between treatment and all covariables were first included, and non-significant interactions were sequentially removed from the model starting with the least significant ones. We report marginal (R²m) and conditional (R²c) R² for the reduced final model. WSI = water stress index.

Figure 5 :

 5 Figure 5: Generalized linear mixed models standardised estimates ± SE and associated p-values (*: p < 0.05, **: p < 0.01, ***: p < 0.001) of the variables explaining the number of initiated

Figure 6 :

 6 Figure 6: Generalized linear mixed models standardised estimates ± SE and associated p-values (***: p < 0.001) of the variables explaining fruit abortion rate between initiation and maturity in Q. ilex (a) after model selection in control plots for 2007-2019 and (b) with the variables retained by model selection in (a) plus the dry treatment effect in control and dry plots. GPP was not included in this last model, as it was not measured in the dry treatment. Interactions between

Figure 7 :

 7 Figure 7: Generalized linear mixed models standardised estimates ± SE and associated p-values (**: p < 0.01, ***: p < 0.001) of the variables explaining the number of mature fruits per kg of aboveground biomass in Q. ilex (a) after model selection in control plots for 2007-2019 and (b)with the variables retained by model selection in (a) plus the dry treatment effect in control and dry plots. GPP was not included in this model as it was not measured in the dry treatment.

  Figure 1

Fig. S1

 S1 Fig.S1Treatment effect on the water stress index of spring, summer of the previous year and summer-

Fig. S1

 S1 Fig. S1 Mean inter-annual variability of the water stress index (WSI) of (a) spring, (c) summer of the previous year and (e) summer-autumn of the current year in the control and dry treatments, and effect of the treatment on (b) spring WSI, (d) summer WSI of the previous year and (f) summer-autumn WSI of the current year. P-values correspond to the treatment effect in the linear mixed model (LMM) (***: p < 0.001). Treatment effect on WSI was tested with a LMM with treatment as fixed effect and year as a random effect. The boxplots represent the three quartiles with the median depicted as a solid thick horizontal line. The whiskers extend towards 1.5 times the inter-quartile range. In (b), (d) and (f), each dot represents the value for one plot in one year (points have been jittered to avoid over-plotting).

1Fig.

  Fig. S2 (a) Inter-annual variability of mean mature fruit mass (± SD) depending on treatment, and (b) 2

  Fig. S3 Relationship between absolute values of summer-autumn water stress index (WSI) and fruit 13 abortion rate depending on treatment observed in data from 2007 to 2019 (a) and predicted by the 14 generalized linear mixed model described in Fig. 6b (b). 15

Table S1

 S1 Standardised estimates ± SE of the linear model testing the effect of fruit biomass, rainfall exclusion treatment and their interaction on leaf nutrient concentration the following winter between 2007 and 2018. None of the associated p-values are significant.

	Response variables		Fixed effects
		Total fruit biomass	Treatment	Fruit biomass x
		produced during year n		treatment
	[N] in leaves produced during	0.02 ± 0.02	0.10 ± 0.10	-0.007 ± 0.05
	year n
	[N] in leaves produced during	-0.008 ± 0.02	0.02 ± 0.11	-0.01 ± 0.06
	year n-1
	[P] in leaves produced during	-0.0003 ± 0.002	0.006 ± 0.008	-0.002 ± 0.004
	year n
	[P] in leaves produced during	-0.0004 ± 0.001	-0.004 ± 0.007	0.005 ± 0.004
	year n-1

Table S2

 S2 Population coefficient of variation (CVp) in % for male flower biomass, number of initiated fruits, number of mature fruits and mean fruit mass at the plot level between 2007 and 2019.

	Treatment	Control		Dry
	Block	R1 R2 S	Mean	R1 R2 S	Mean
	Male flower biomass per kg of	49 52 71	57	52 52 46	50
	aboveground biomass
	Number of initiated fruits per kg of	50 51 25	42	46 65 37	49
	aboveground biomass
	Number of mature fruits per kg of	108 162 107	126	135 157 119	137
	aboveground biomass
	Mean fruit mass	36 23 31	30	35 39 32	35

Acknowledgments

We thank Manon Vaudois and Matthias Grenié for field help for their very useful and constructive comments. This work was supported by LabEx CeMEB, an Agence Nationale de la Recherche (ANR) "Investissements d'avenir" program (ANR-10-LABX-04-01); by the ANR FOREPRO (ANR-19-CE32-0008), and by a grant to ILR from Agroparistech and the French ministry of environment. The Puéchabon experimental site is annually supported by the OSU OREME (UMS 3282) and the French national research infrastructure ANAEE-France (ANR-11-INBS-0001).

Authors Contribution

ILR, IC and JML conceived and designed the study; JMO and JML collected data; FM developed the water stress model; ILR and JG analysed data; ILR led the writing. All authors contributed critically to the drafts and gave final approval for publication.

Supplementary

Fig. S1 Treatment effect on the water stress index of spring, summer of the previous year and summer-autumn of the current year.

Table S1 Effect of fruit biomass production and treatment on leaf nutrient concentration the following winter.

Table S2 Population Coefficient of Variation (CVp) for male flower biomass, numbers of initiated and mature fruits and mean fruit mass. Methods S1 Fruit growth and stem growth measurements.

Methods S1 Fruit growth and stem growth measurements. 38

We tagged 20 to 30 initiated fruits per tree, located in the top upper canopy of 4 to 6 trees in the 39 control treatment of the scaffold-equipped block. From July to November, fruit abortion and fruit size 40 were monitored monthly. Fruit size was measured with an electronic calliper. During the early stage 41 of fruit development, we measured the diameter of the fruit because the seed is invisible inside the 42 acorn cup and the fruit has a spherical shape. As soon as the seed protrudes from the cup, we 43 measured fruit length from the basis of the cup to the top of the seed excluding the remains of the 44 style. 45 Stem circumference changes were continuously recorded using automatic band-46 dendrometers installed approximately 1.3 m above the ground (ELPA-98, University of Oulu, Finland). 47

The daily maximum circumference value was used to calculate the tree daily basal area, and basal area 48 increment was calculated by subtracting the previous day value. Analyses and graphs were performed 49 with averaged individual values expressed in relative to the maximal individual yearly basal area. 50