Adriane Aupic-Samain
email: adriane.samain-aupic@imbe.fr

Mathieu Santonja

Mathilde Chomel

Susana Pereira

Elodie Quer

Caroline Lecareux

Jean-Marc Limousin

Jean-Marc Ourcival

Guillaume Simioni

Thierry Gauquelin

Catherine Fernandez

Virginie Baldy

Soil biota response to experimental rainfall reduction depends on the dominant tree species in mature Northern Mediterranean forests

Keywords: climate change, Mediterranean forest, mesofauna, microorganism, plant-soil (below-ground) interactions, soil biota

Soil organisms play a major role on litter decomposition process and nutrient cycling in forest ecosystems. These organisms are extremely sensitive to environmental conditions such as soil temperature and moisture conditions which control their demographic parameters and activity.

The ongoing climate change can therefore directly affect soil biota communities and the processes they drive. Besides, climate change can also indirectly affect soil biota by altering tree functional traits (e.g., N, Ca, Mg, water holding capacity) with cascading effects on the litter quality. The aim of this study was to determine the relative effects of increased drought and litter type on microbial biomass (bacteria and fungi) and mesofauna abundance (Collembola and Acari) in three experimental sites representative of the three main forests encountered in the northern part of the Mediterranean Basin (dominated by either Quercus pubescens, Quercus ilex or Pinus halepensis) where rainfall exclusion experiments were taking place. At each site, and in each precipitation treatment (natural and amplified drought plots), we collected and transplanted foliage litters (i.e., species × drought level). After two years, we reported a litter species effect: Q. pubescens litter presented consistently the higher abundance of all soil biota groups compared to Q. ilex and P. halepensis litters in each forest. Surprisingly, despite that the amplified drought treatment induced a modification of the litter quality, we did not reported an indirect reduced precipitation effect on soil biota parameters.

While Oribatid Acari abundance decreased with amplified drought in all three forest types, the direct effects on the other soil biota groups were forest-dependent. In P. halepensis forest, amplified drought resulted in higher bacterial and fungal biomasses but lower Collembola abundance. In Q. ilex forest both Collembola and predatory Acari abundances decreased with amplified drought. In addition, the positive relationships between Collembola and Oribatida abundances and litter mass loss disappeared under amplified drought conditions in both Q. ilex and P. halepensis forests. These results suggest a key role played by Ca, Mg, specific leaf J o u r n a l P r e -p r o o f area (SLA) and water holding capacity (WHC) as drivers of soil biota parameters. Finally, the study highlights that within the same Mediterranean region, climate change could differently alter the soil organisms inhabiting the litter layer and their contributions to the decomposition process depending on the tree species and soil biota group considered.

Introduction

Litter is one of the basal elements of a food web that controls nutrient turnover, carbon sequestration and the overall ecosystem functioning (Wall et al., 2012;Gobat et al., 2013).

Among soil biota, mesofauna (mainly Collembola and Acari) drives many biotic interactions which are fundamental for structuring the soil food web and decomposing leaf litter. Firstly, microbi-detritivore organisms (e.g., Collembola and Oribatid Acari) participate directly to the micro-fragmentation of leaf litter, but also control microbial communities through grazing and dispersing spores and mycelium [START_REF] Berg | Advances in Ecological Research, Litter decomposition: a guide to carbon and nutrient turnover[END_REF][START_REF] Chahartaghi | Feeding guilds in Collembola based on nitrogen stable isotope ratios[END_REF][START_REF] Scheu | Interactions between microorganisms and soil micro-and mesofauna[END_REF][START_REF] Anslan | Temporal changes in fungal communities associated with guts and appendages of Collembola as based on culturing and highthroughput sequencing[END_REF]. Secondly, predators (e.g., Mesostigmatid and some Prostigmatid Acari) regulate microbi-detritivore organisms by feeding on them [START_REF] Koehler | Predatory mites (Gamasina, Mesostigmata)[END_REF][START_REF] Schneider | Top-down control of soil microarthropods -Evidence from a laboratory experiment[END_REF]Thakur et al., 2015) and then indirectly control the leaf litter decomposition.

Chemical and physical characteristics of the leaf litter strongly control soil mesofauna demographic parameters and interactions [START_REF] Hättenschwiler | Drought-induced changes in flavonoids and other low molecular weight antioxidants in Cistus clusii grown under Mediterranean field conditions[END_REF][START_REF] Chomel | Plant secondary metabolites: a key driver of litter decomposition and soil nutrient cycling[END_REF][START_REF] Santonja | Potential shift in plant communities with climate change: outcome on litter decomposition and nutrient release in a Mediterranean oak forest[END_REF][START_REF] Aupic-Samain | Tree litter identity and predator density control prey and predator demographic parameters in a Mediterranean litter-based multi-trophic system[END_REF]. Under the specific Mediterranean climatic conditions (summer drought and episodic drying/rewetting cycles; [START_REF] Larcher | Temperature stress and survival ability of Mediterranean sclerophyllous plants[END_REF][START_REF] Sardans | Plant-soil interactions in Mediterranean forest and shrublands: impacts of climatic change[END_REF][START_REF] Gauquelin | Mediterranean forests, land use and climate change: a socialecological perspective[END_REF], trees generally produce sclerophyllous leaves J o u r n a l P r e -p r o o f [START_REF] Coûteaux | Litter decomposition, climate and litter quality[END_REF] characterized by high lignin concentration (Tian et al., 1992;[START_REF] Gallardo | Leaf decomposition in two Mediterranean ecosystems of southwest spain: influence of substrate quality[END_REF], low specific leaf area (Wright et al., 2005;[START_REF] Pallardy | Physiology of Woody Plants[END_REF] and high diversity and concentration of specialized metabolites (i.e., terpene and phenolic compounds; [START_REF] Macchioni | Chemical composition of essential oils from needles, branches and cones of Pinus pinea, P. halepensis, P. pinaster and P. nigra from central ltaly[END_REF][START_REF] Fernandez | Variations in allelochemical composition of leachates of different organs and maturity stages of Pinus halepensis[END_REF]. These particular characteristics of Mediterranean trees could potentialy lead to distinct litter quality control over soil mesofauna compared to the other temperate forests for which litter nutrient contents (e.g., C, N and P) are frequently reported as key drivers of soil mesofauna demographic parameters [START_REF] Jandl | Forest soil chemistry and mesofauna 20 years after an amelioration fertilization[END_REF][START_REF] Martinson | Detritivory: stoichiometry of a neglected trophic level[END_REF][START_REF] Jacob | Nutrient release from decomposing leaf litter of temperate deciduous forest trees along a gradient of increasing tree species diversity[END_REF][START_REF] Maaroufi | Global change impacts on forest soils: linkage between soil biota and carbon-nitrogen-phosphorus stoichiometry[END_REF]. However, to our knowledge, only few studies investigated this effect of litter quality on soil mesofauna in Mediterranean forests (e.g., [START_REF] Barba | Effects of drought-induced forest die-off on litter decomposition[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][START_REF] Aupic-Samain | Tree litter identity and predator density control prey and predator demographic parameters in a Mediterranean litter-based multi-trophic system[END_REF], necessitating deeper investigation to improve our mechanistic understanding of such relationships.

Among terrestrial biomes, Mediterranean ecosystems are recognized as being the most sensitive to climatic change [START_REF] Sala | Global biodiversity scenarios for the year 2100[END_REF]Schröter et al., 2005). Regional climate models for the Mediterranean Basin predict a warming of 3.4°C and a decrease of annual precipitations by 30% for the end of the 21 st century, which will result in an intensification of summer drought events [START_REF] Giorgi | Climate change projections for the Mediterranean region[END_REF]IPCC, 2013;[START_REF] Polade | The key role of dry days in changing regional climate and precipitation regimes[END_REF]. Therefore, by decreasing water availability, climate change in Mediterranean ecosystems may have a direct negative impact on soil microorganisms (e.g., [START_REF] Sardans | Soil enzyme activity in a Mediterranean forest after six years of drought[END_REF]Talmon et al., 2011) and mesofauna (e.g., Tsiafouli et al., 2005;[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]. In addition, climate change may indirectly impact microbial and mesofaunal communities by altering litter quality and quantity produced by plants as climatic conditions strongly control plant growth and survival and consequently leaf and litter traits (Wright et al., 2005;[START_REF] Sardans | Drought changes the dynamics of trace element accumulation in a Mediterranean Quercus ilex forest. Environmental Pollution, Air Pollution and Climate Change: A global overview of the effects on forest vegetation[END_REF][START_REF] Rodriguez-Ramirez | Shrub species richness decreases negative impacts of drought in a Mediterranean ecosystem[END_REF]. Indeed, previous studies reported that experimental decrease in water conditions implies lower nutrient content [START_REF] Chen | Leaf nitrogen and phosphorus concentrations of woody plants differ in responses to climate, soil and plant growth form[END_REF] J o u r n a l P r e -p r o o f [START_REF] Allison | Microbial abundance and composition influence litter decomposition response to environmental change[END_REF]García-Palacios et al., 2016a;[START_REF] Santonja | Temporal shifts in plant diversity effects on carbon and nitrogen dynamics during litter decomposition in a Mediterranean shrubland exposed to reduced precipitation[END_REF]. In addition, oaks and pines are dominant tree genera that structure both temperate and Mediterranean forests [START_REF] Ellenberg | Vegetation ecology of central Europe[END_REF][START_REF] Quézel | Ecologie et biogéographie des forêts du bassin méditerranéen[END_REF]. As oak and pine forests exhibit different microclimatic and soil properties (e.g., pH, soil type, humus forms; Table 1; [START_REF] Gauquelin | Mediterranean forests, biocultural heritage and climate change : a social-ecological perspective[END_REF] as well as chemically and structurally different litters (oak leaves vs. pine needles; [START_REF] Aupic-Samain | Tree litter identity and predator density control prey and predator demographic parameters in a Mediterranean litter-based multi-trophic system[END_REF][START_REF] Santonja | Potential shift in plant communities with climate change: outcome on litter decomposition and nutrient release in a Mediterranean oak forest[END_REF], we could expect that climate change may distinctly affect soil biota in these two forest types. However, our current understanding of soil biota responses to climate change drivers in Mediterranean oak and pine forests is still limited by a lack of studies addressing conjointly the relative contributions of environmental conditions and leaf litter quality and both direct and indirect effects of climate change on these organisms.

In this context, we set up a 2-year litter transplant experiment in the three main forests encountered in the northern part of the Mediterranean Basin (Quercus pubescens, Quercus ilex and Pinus halepensis dominated forests) in which we manipulated the amounts of precipitation (natural vs. amplified drought), the litter species identity (leaf / needle litters from the three tree species) and the litter type (litters collected from natural or amplified drought plots) in order to determine their relative effects on soil biota, including both microbial (bacteria and fungi) and mesofaunal (Acari and Collembola) communities. We hypothesized that i) microbial biomass and mesofaunal abundance associated with decomposing oak leaves are higher compared to pine needles due to lower amount of refractory coumpounds (e.g., specialized metabolites); ii) reduced precipitation directly decreases microbial biomass and mesofaunal abundance as water availability is a strong constraining environmental factor; iii) reduced precipitation indirectly decreases microbial J o u r n a l P r e -p r o o f biomass and mesofaunal abundance due to a decrease in leaf/ needle litter quality (e.g., increased specialized metabolite content) and iv) soil biota will be more sensitive to reduced precipitation in oak compared to pine forests where soil biota is already conditioned by more constraining environmental conditions (e.g., litter content).

Materials and Methods

Study site

The study was concurrently set up in three Mediterranean experimental sites (Table 1).

The first is the Oak Observatory at the "Observatoire de Haute Provence" (O 3 HP) located in the Luberon Natural Regional Park (43°45′34.26″N; 5°17′57.84″E), in Provence, SE France [START_REF] Gauquelin | L'O3HP (Oak Observatory at OHP): un site expérimental pour l'étude J o u r n a l P r e -p r o o f du fonctionnement et de la biodiversité de la chênaie pubescente face aux changement climatiques[END_REF]. This oak forest is dominated by deciduous downy oak (Quercus pubescens Willd.). The second site is located in the Puéchabon State Forest (43°44'30" N; 3°35'40" E) in Occitanie, SE France [START_REF] Misson | Functional changes in the control of carbon fluxes after 3 years of increased drought in a Mediterranean evergreen forest?[END_REF]. This oak forest is dominated by the evergreen holm oak (Quercus ilex L.). The third site is located in the departmental forest of Font-Blanche (43°14'25''N; 5°40'40''E) in Provence, SE France (Simioni, 2011). This is a mixed forest, but Aleppo pine (Pinus halepensis Mill.) is the most abundant species, contributing around 70% of the basal area. During the two years of field experiment, the mean annual precipitation ranged from 635.6 mm in the P. halepensis forest to 1020.9 mm in the Q. ilex forest, while the mean annual temperature ranged from 12.3°C in the Q. pubescens forest to 14.1°C in the P. halepensis forest (Supplementary Fig. S2).

In order to simulate the intensification of the summer drought period, each site is equipped with a rain exclusion device reducing approximatively 30% of annual precipitation (similar to climatic models projection -A2 scenario; [START_REF] Giorgi | Climate change projections for the Mediterranean region[END_REF]IPCC, 2013).

In the Q. pubescens forest the rain exclusion device consists of a 15 m × 20 m rainout-shelter J o u r n a l P r e -p r o o f above the canopy which dynamically excluded precipitations by deploying automated shutters during rainfall events of the vegetation growing season (i.e., from spring to autumn) (Supplementary Fig. S2a). In the Q. ilex and P. halepensis forests the rain exclusion is performed by using fixed PVC gutters installed below the forest canopy, excluding about 30% at each rainfall event (Supplementary Fig. S2b andc). In each site, we compared control plots (natural drought -ND) and rain exclusion plots (amplified drought -AD) (Table 1 and Supplementary Fig. S1).

Litter collection

Freshly abscised leaves and needles of Q. pubescens, Q. ilex and P. halepensis were collected in ND and AD plots over the litterfall period in 2014. For that, litter traps were used during the abscission period that occurred from June to September for the needles (P. halepensis) and from October to November for the leaves (Q. ilex and Q. pubescens).

Immediately after collection, the leaves/needles were air dried at room temperature and stored until the beginning of the experiment. Several aliquots of senescent leaves or needles were also frozen at -20 °C, freeze-dried for 72 h and ground prior to chemical analyses.

Experimental design

Plant litter decomposition was studied over 730 days using the litterbags method (Swift et al., 1979). In December 2014, 10 g (in equivalent dry weight) of senescent leaves or needles of either Q. pubescens, Q. ilex and P. halepensis collected from trees either in ND or AD plots were placed in a 4-mm mesh litterbag (20 × 20 cm) designed to allow colonization by microbes and mesofauna. Litter transplants were made between each site for the three species considered, i.e., a litter bag containing the litter of each species placed on each forest site, under the two precipitation conditions, i.e., ND and AD (see Fig. 1).

J o u r n a l P r e -p r o o f

Thus, the experiment consisted in 36 treatment combinations corresponding to 3 forest sites (Q. pubescens, Q. ilex and P. halepensis forests) × 3 litter species (Q. pubescens, Q. ilex and P. halepensis) × 2 litter types (litters collected from ND or AD plots) × 2 precipitation treatments (ND and AD) (Fig. 1). Each modality had 5 replicates for a total of 180 litterbags.

Litterbags were placed perpendiculary to the gutters system in Q. ilex and P. halepensis forests and under the rain exclusion device in the Q. pubescens forest by using 5 transects (i.e., 5 replicates of the 6 litterbag modalitites) equidistant from each other (1 m distance between the 5 transects and 0.6 m between the 6 litterbags). Transects were oriented E-W.

They were placed on the ground after the removal of the litter layer and fixed to the soil with galvanized nails to prevent movement by animals or wind. The litter layer was then replaced over the litterbags. In December 2016, i.e., after 730 days of decomposition, all the litterbags were harvested and sealed in plastic bags to prevent the further loss of biological material.

Initial litter characteristics

Initial litter quality of the three litter species (Q. pubescens, Q. ilex and P. halepensis) collected from the two precipitation treatments (ND and AD) was determined from five samples.

Carbon (C) and nitrogen (N) concentrations were determined by thermal combustion on a Flash EA 1112 series C/N elemental analyzer (Thermo Scientific®, Waltham, MA, USA). Phosphorus (P) and cations, i.e., calcium (Ca), sodium (Na), potassium (K) and magnesium (Mg), were extracted from 80 mg of grounded litter with 8 ml of HN0 3 and 2 ml of H 2 O 2 . Then, samples were heated at 175 °C for 40 min using a microwave digestion system (Ethos One, Milestone SRL, Sorisole, Italy). After this mineralization step, every sample was adjusted to 50 ml with demineralized water. P concentration was measured colorimetrically using the molybdenum blue method [START_REF] Grimshaw | Nutrient elements[END_REF]. 100 µl of sample, 100 µl of

J o u r n a l P r e -p r o o f

NaOH, 50 µl of mixed reagent (emetic tartrate and ammonium molybdate solution) and 50 µl of ascorbic acid were mixed directly in a 96 well microplate. After 45 min at 40 °C, the reaction was completed, and P concentration was measured at 720 nm using a microplate reader (Victor, Perkin Elmer, Waltham, MA, USA). Cations concentrations were determined by atomic absorption spectrophotometer. Total phenolic compounds were measured colorimetrically by the adapted method of [START_REF] Peñuelas | Litter traits and rainfall reduction alter microbial litter decomposers: the evidence from three Mediterranean forests[END_REF] using gallic acid as standard (expressed as equivalent acid gallic). 250 mg of litter sample were extracted in 20 ml of a 70 % aqueous methanol solution, shaken for 1 h, and then filtered (0.45 µm filter); 50 µl of filtered extract were mixed with 200 µl of saturated aqueous Na 2 CO 3 (to stabilize the color reaction), 1650 µl of distilled water and 100 µl Folin-Ciocalteu reagent [START_REF] Folin | A colorimetric method for the determination of phenols (and phenol derivatives) in urine[END_REF]. After 30 min, the reaction was completed, and the concentration of phenolics was measured at 765 nm on a UV/Vis spectrophotometer (Thermo Scientific®, Waltham, MA, USA). Lignin, cellulose and hemicellulose as well as water soluble compounds (WSC) concentrations of initial litter materials were determined according to the Van Soest extraction protocol (Van Soest, 1963) using a fiber analyzer (Fibersac 24, Ankom, Macedon, NJ, USA).

All concentrations were expressed in mg g -1 of litter dry weigh.

To determine the Water Holding Capacity (WHC), intact leaf or needle were soaked in distilled water for 24 h, drained and weighed. The dry weight was determined after drying the samples at 60 °C for 48 h. WHC was calculated as (moist weight / dry weight) × 100 and expressed in % [START_REF] Santonja | Potential shift in plant communities with climate change: outcome on litter decomposition and nutrient release in a Mediterranean oak forest[END_REF]. Specific Leaf Area (SLA) was calculated as the ratio between leaf area (determined by using the Image J software; https://imagej.nih.gov/ij/, MA, USA) and dry weight and was expressed in cm 2 g -1 of dry weigh.

Mesofauna extraction and identification

J o u r n a l P r e -p r o o f

Mesofauna was extracted from one litterbag using the Tullgren funnel method for 10 days [START_REF] Berlese | Apparecchio per raccogliere presto ed in gran numero piccoli arthropodi[END_REF]. Collected arthropods were stored in 70 % ethanol, counted by using a binocular microscope and separated between Collembola and Acari, with different suborders for the latter: Oribatid, Mesostigmatid and Prostigmatid Acari [START_REF] Gisin | Collembolenfauna Europas. Museum d'Histoire Naturelle[END_REF][START_REF] Hopkin | Biology of the Springtails: (Insecta: Collembola)[END_REF].

Collembola and Oribatid Acari were assigned as microbi-detritivores and Mesostigmatid and Prostigmatid Acari as predators [START_REF] Coleman | Fundamentals of soil ecology[END_REF][START_REF] Donoso | Trees as templates for trophic structure of tropical litter arthropod fauna[END_REF][START_REF] Crotty | Divergence of feeding channels within the soil food web determined by ecosystem type[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].

Litter mass loss estimation

After mesofauna extraction, the litter samples were freeze-dried (Lyovac GT2) for 72 h and the remaining dry mass (%) after 730 days of decomposition was calculated.

PLFA analyses

Since, the phospholipid fatty acids (PLFA) are essential components of all living cells (Tollefson and McKercher, 1983;Zelles, 1999) with a wide structural diversity (Zelles, 1997;Tornberg et al., 2003), we used PLFA as biomarkers of litter microbial communities. The PLFA were extracted from freeze-dried ground litter according to the method from [START_REF] Buyer | High throughput phospholipid fatty acid analysis of soils[END_REF] ). The total PLFA concentration was used as measure of the total microbial biomass, while fungal and bacterial biomasses were estimates through PLFA markers summed [START_REF] Frostegård | The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil[END_REF]. Biomasses were expressed in µg g -1 dw of litter. Among the 24 identified PLFAs in the samples, 12 microbial specific PLFAs were analysed. The fatty acids i15:0, a15:0, i16:0 and i17:0 were used as biomarkers for Gram-positive bacteria; 16:1ω7, 18:1ω7 and cy19:0 were used as biomarkers of Gram-negative bacteria; 15:0, 17:0 were used as general bacterial markers and 19:1ω8 were used as biomarkers of methane oxidizing bacteria [START_REF] Frostegård | Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis[END_REF]. The total bacterial PLFA biomass was calculated by adding Gram-positive, Gram-negative and general bacterial biomarkers while 18:2ω6,9 was used as a biomarker of fungi (Bååth and [START_REF] Aupic-Samain | Tree litter identity and predator density control prey and predator demographic parameters in a Mediterranean litter-based multi-trophic system[END_REF][START_REF] Klamer | Estimation of conversion factors for fungal biomass determination in compost using ergosterol and PLFA 18:2ω6,9[END_REF].

Statistical analysis

Statistical analyses were performed using a combination of univariate and multivariate techniques with R software (version 3.3.1). Statistical significance was evaluated in all cases at P < 0.05. Normality and homoscedasticity of the residuals were checked using Shapiro-Wilk and Levene tests, respectively.

To analyse the differences of the initial characteristics of the three litter species (Q. pubescens, Q. ilex and P. halepensis) and the two litter types (leaves or needles collected from trees in ND or AD plots) we performed a Principle Component Analysis (PCA) followed by pairwise tests with permutational multivariate analyses of variance (PERMANOVA; [START_REF] Anderson | Permutational multivariate analysis of variance[END_REF] using the adonis function of the vegan package [START_REF] Oksanen | The vegan package[END_REF][START_REF] Oksanen | Package 'vegan[END_REF].

Four-way analyses of variance (ANOVA), followed by Tukey tests for post-hoc pairwise comparisons, were used to test the effects of forest site, litter species identity, precipitation treatment, litter type and their interactions on the 5 soil biota parameters J o u r n a l P r e -p r o o f previously log-transformed: abundances of Collembola, Oribatida, and predator and biomasses of bacteria and fungi. The full models were then simplified to determine the most parsimonious models using the dredge function of MuMIn package [START_REF] Barton | Package "MuMIn": Multi-Model Inference[END_REF], an established model selection procedure with both forward and backward selection algorithms, which ranks all candidate models (all possible combinations of the initial explanatory variables included in the full model) based on the lowest Akaike Information Criterion (AIC).

Thus, litter type treatment initially included in the full models, was never retained in the most parsimonious models.

Finally, Spearman correlations were performed to link litter mass loss after 24 months of decomposition to the 4 soil biota directly involved in the decomposition process (bacteria, fungi, Collembola and Oribatida).

Results

Initial litter characteristics

The PCA (Fig. 2) and PERMANOVA (Pseudo-F litter species = 42.5, P = 0.001) revealed a considerable variation of initial litter quality between the three plant species. The first axis of the PCA (43 % of the variance explained) discriminated leaves of Q. pubescens from leaves of Q. ilex with higher SLA, Mg and lower Na and lignin contents in the former. The second axis of the PCA (34 % of the variance explained) distinguished P. halepensis from the two Quercus species, with a higher WSC concentration and, on the opposite, lower WHC, Ca and hemicellulose concentrations in the former. In addition, PERMANOVA revealed an effect of litter type (i.e., leaves or needles collected from trees in ND or AD plots; Pseudo-F litter type = 3.3 ; P = 0.033) indicating a modification of litter quality under amplified drought conditions, as well as an interaction between litter species and litter type (Pseudo-F litter species × litter type = J o u r n a l P r e -p r o o f 2.4; P = 0.046) indicating a modification of litter traits acconding to the tree species considered. AD induced an increase of Mg and Na for both oak species, a decrease of Ca content the three tree species (Supplementary Table S1). SLA and WHC decreased with AD for the two Quercus species but did not change for P. halepensis (Supplementary Table S1).

Finally, AD induced an increase of leaf phenolic content for Q. ilex whereas no change was observed for Q. pubescens leaves and P. halepensis needles (Supplementary Table S1).

Microbial community

Bacterial and fungal biomasses were respectively 16% and 15% higher in Q. pubescens litter compared to the two other litters (Table 2; Fig. 3a andb) and were not affected by the litter type (i.e., leaves or needles collected from trees in ND or AD plots, Supplementary Fig. S3). The effect of AD on microbial biomass was dependent on the forest considered (significant forest × precipitation interaction, Table 2). AD treatment had no effect on the fungal or bacterial biomasses in the Q. pubescens or Q. ilex forests, but increased them by 29 % in the P. halepensis forest (Fig. 4a andb).

Soil mesofauna

We collected a total of 27 292 individuals of microarthropods from all the litterbags.

Collembola were the most abundant microbi-detritivore arthropods (49 %), compared to Oribatida (39 %), while predatory Acari represented 12 % of the microarthropods community.

As reported for microbial biomass, mesofaunal abundance varied according to litter species identity but was not affected by litter type (Table 2, Supplementary Fig. S3). The abundance of all mesofauna groups associated to Q. pubescens litter was always two times higher compared to Q. ilex and P. halepensis litters (Fig. 3c-e).

J o u r n a l P r e -p r o o f

Except for Oribatid Acari, forest type and precipitation treatment interactively affected Collembola and predator abundances (significant forest × precipitation interaction, Table 2). AD treatment did not affect Collembola abundance in Q. pubescens forest but decreased Collembola abundance by 46 % and 48 % in Q. ilex and P. halepensis forests, respectively.

Consequently, the Collembola abundance was similar level across the three Mediterranean forests under AD treatment (Fig. 4c). The AD treatment had a significant effect only in the Q. ilex forest, with a 50 % decrease of the predator abundance (Fig. 4d). Finally, Oribatid Acari abundance was respectively 87 % and 62 % higher in Q. ilex forest compared to Q. pubescens and P. halepensis forests (Fig. 5a) and was reduced by 33 % under AD conditions in the three forest types (Fig. 5b).

Relationships between soil biota parameters and litter mass loss

While Collembola and Oribatida abundances were positively correlated with litter mass loss under ND condition in both Q. ilex and P. halepensis forests (Table 3), these relationships disappeared under AD conditions. Collembola abundance was only marginally correlated with litter mass loss under ND treatment in Q. pubescens forest (Table 3), and this trend also disappeared under drier conditions. Finally, fungal biomass was positely related to litter mass only in Q. ilex forest under AD conditions (Table 3).

Discussion

Our study highlighted that litter identity strongly controls soil biota in Mediterranean forests after a 24-month field litterbag experiment. In each of the three forest, Q. pubescens litter had consistently more abundance of all soil biota groups compared to Q. ilex and P. halepensis litters. Our results suggest a key role played by Ca, Mg, specific leaf area (SLA) and water holding capacity (WHC) as drivers of soil biota parameters. In addition, amplified J o u r n a l P r e -p r o o f drought differently affects soil biota with an increase in microbial biomass and a decrease in soil fauna abundance. However, except for Oribatida Acari, negative effect of amplified drought were dependent on the forest considered. Surprisingly, amplified drought did not indirectly affect the soil biota by altering the tree litter quality.

Effects of litter traits on soil biota

We provided clear evidence that soil biota communities are strongly controlled by litter species identity, but we only partly confirm our first hypothesis of higher abundance of soil biota in oak compared to pine forests. More precisely, the abundance of all soil biota associated with Q. pubescens litter was higher compared to Q. ilex and P. halepensis litters, whatever the forest in which the litter decomposed. Q. pubescens leaves exhibited higher specific leaf area (SLA) and water holding capacity (WHC) compared to Q. ilex leaves and P. halepensis needles. Higher litter SLA has been reported to induce an increase in water availability [START_REF] Castro-Díez | Leaf morphology and leaf chemical composition in three Quercus (Fagaceae) species along a rainfall gradient in NE Spain[END_REF][START_REF] Makkonen | Highly consistent effects of plant litter identity and functional traits on decomposition across a latitudinal gradient[END_REF][START_REF] Makkonen | Do physical plant litter traits explain non-additivity in litter mixtures? A test of the improved microenvironmental conditions theory[END_REF] or in habitat structure for Collembola [START_REF] Kalinkat | Habitat structure alters top-down control in litter communities[END_REF][START_REF] Santonja | Potential shift in plant communities with climate change: outcome on litter decomposition and nutrient release in a Mediterranean oak forest[END_REF][START_REF] Aupic-Samain | Tree litter identity and predator density control prey and predator demographic parameters in a Mediterranean litter-based multi-trophic system[END_REF]. WHC is a physical trait corresponding to the litter ability to hold the water, which is necessary for soil biota development and activity [START_REF] Pflug | Influence of drought and litter age on Collembola communities[END_REF][START_REF] Makkonen | Highly consistent effects of plant litter identity and functional traits on decomposition across a latitudinal gradient[END_REF][START_REF] Makkonen | Do physical plant litter traits explain non-additivity in litter mixtures? A test of the improved microenvironmental conditions theory[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]. In addition, Q. pubescens leaves also exhibited higher Ca and Mg concentrations than the two other litters. Ca positively affects fungal growth and activity [START_REF] Eriksson | Microbial and enzymatic degradation of wood and wood components[END_REF][START_REF] Jenkins | The influence of water chemistry on the enzymatic degradation of leaves in streams[END_REF] and is a key constitutive of invertebrate cuticles [START_REF] Cairns | A review of the influence of low ambient calcium concentrations on freshwater daphniids, gammarids, and crayfish[END_REF]. Mg plays an important role in the growth and metabolic functions of microbial cells (Walker, 1994) and is an essential element for invertebrates required for enzymatic reactions, nerve connections or muscle function (National Research Council, 2005). Some previous studies identified these physical (SLA and WHC) and chemical (Ca and Mg) litter traits as important drivers of the litter decomposition process in

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Mediterraean forests (García-Palacios et al., 2016a), wich support our study demonstrating for the first time that these litter traits directly control soil biota in Mediterranen forests and then ecosystem processes. However, we acknowledge that soil biota parameters were analysed only at one sampling time in the present study (after 730 days of decomposition in litterbags), preventing an extrapolation to soil biota dynamics throughout litter decomposition time and necessiting additional experimentations to confort our findings.

Direct and indirect effect of precipitation treatment on soil biota

We confirmed our second hypothesis that decreasing precipitation directly affects of soil biota biomass and abundance. Conversely, we did not evidence an indirect effect of amplified drought on soil biota mediated by a shift in litter quality at intraspecific level (Fig. 2), in contrast to our third hypothesis. In a climate change context, these findings clearly highlight that reduced water availability prevails the intraspecific shift in litter quality due to plant water stress as a major driver of soil biota communities in our Mediterranean forests.

In temperate forest ecosystems, bacteria and fungi are often positively correlated with soil water availability (e.g., [START_REF] Pflug | Influence of drought and litter age on Collembola communities[END_REF]Taylor and Wolters, 2005;[START_REF] Lensing | Impact of changes in rainfall amounts predicted by climatechange models on decomposition in a deciduous forest[END_REF]. In Mediterranean ecosystems, in contrast, the regular summer droughts could have select adapted phenotypes among microbial species [START_REF] Criquet | Annual variations of phenoloxidase activities in an evergreen oak litter: influence of certain biotic and abiotic factors[END_REF]Curiel Yuste et al., 2014;[START_REF] Peñuelas | Litter traits and rainfall reduction alter microbial litter decomposers: the evidence from three Mediterranean forests[END_REF], leading to only weak or an absence of drought effect on soil bacterial and fungal communities (Wilkinson et al., 2002. Sherman et al., 2012;Curiel Yuste et al., 2014). In addition, higher mortality and lower fecundity rates were reported for both Collembola and Acari under drier condition, due to altered physiological processes [START_REF] Holmstrup | Dehydration tolerance and water vapour absorption in two species of soil-dwelling Collembola by accumulation of sugars and polyols[END_REF][START_REF] Houck | Mites: ecological and evolutionary analyses of life-history patterns[END_REF][START_REF] Poinsot-Balaguer | L'anhydrobiose: un problème biologique nouveau chez les Collemboles (Insecta)[END_REF], species behavior (Verhoef and van Selm, 1983) or predator-prey interaction [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].

Consistently with these previous studies, microbial biomass was not negatively affected, J o u r n a l P r e -p r o o f while soil fauna abundance decreased under amplified drought in the three studied Mediterranean forests.

Except for Oribatida Acari, the drought responses of the soil biota varied according to the forest type considered. In P. halepensis forest, we observed lower Collembola abundance but higher microbial biomass with the amplified drought treatment. Since Collembola are known to be microbial feeders [START_REF] Makkonen | Do physical plant litter traits explain non-additivity in litter mixtures? A test of the improved microenvironmental conditions theory[END_REF] and soil microbial communities to be drought-tolerant in Mediterranean ecosystems (Sherman et al., 2012;Curiel Yuste et al., 2014), higher microbial biomass under reduced precipitation could be the result of lower Collembola feeding activity on them. Moreover, specialized microorganisms species could also benefit on specific conditions from P. halepensis forest (higher specialized metabolites from P. halepensis litter) to proliferate at the expense of generalist species. In contrast, in Q. ilex forest we reported lower Collembola and predatory Acari abundances with amplified drought, but no differences in microbial biomass. Since Acari Mesostigmata and Prostigmata are active predators of Collembola [START_REF] Koehler | Predatory mites (Gamasina, Mesostigmata)[END_REF][START_REF] Schneider | Top-down control of soil microarthropods -Evidence from a laboratory experiment[END_REF] and are more drought-tolerant than Collembola [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], this finding could suggest that amplified drought in Q. ilex forest led to a negative cascading effect from Collembola to their predators. However, in Q. pubescens forest we observed no effect of reduced precipitation on Collembola, nor on their basale resource (microorganisms) and nor on their predators (Acari).

An explanation may lie in the differences among the precipitation reduction set-ups used in three Mediterranean forests: precipitation reduction (~30%) in the Q. pubescens forest is concentrated in the summer months, as expected by the climatic models (IPCC, 2013;[START_REF] Polade | The key role of dry days in changing regional climate and precipitation regimes[END_REF], while in Q. ilex and P. halepensis forests, reduced precipitation is effective throughout the year. As sampling was done in winter, soil biota was likely able to recover from the amplified summer drought in Q. pubescens forest, given that precipitation patterns, and not only the amounts, could impact the soil biota. A previous assessment of the soil J o u r n a l P r e -p r o o f microbial community associated to litter performed in the same Q. ilex forest in 2013 reported a significant decrease of their biomass under amplified drought (García-Palacios et al., 2016b), which was not significantly confirmed in December 2016 by the present study. In addition, [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] reported a negative effect of amplified drought on both Collembola and Oribatid Acari in the same studied Q. pubescens forest between 2012 and 2013, while this negative effect was maintained only for Oribatid Acari for the present study.

Such discrepancies among studies could be explained by a number of factors, among which are the year to year climatic differences, and the different sampling periods and methods. The experiment of [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] in the Q. pubescens forest was performed just after the installation of the rain exclusion device in 2011 while the present study was performed 5 years later. As Oribatid Acari exhibit lower dispersal ability [START_REF] Hopkin | Biology of the Springtails: (Insecta: Collembola)[END_REF], lower fecundity rate [START_REF] Houck | Mites: ecological and evolutionary analyses of life-history patterns[END_REF] or higher habitat specialization (Wehner et al., 2016) than Collembola, we could also speculate why these Oribatid Acari are still negatively affected by amplified drought treatment after several years, while Collembola were able to adapt to the drier conditions. Finally, while increasing Collembola and Oribatida abundances were positively related to litter mass loss in Q. ilex and P. halepensis forests, amplified drought conditions suppressed these relationships. This last finding highlights that, in addition to the impact on soil biota demographic paramaters, drier environmental conditions alter the contributions of soil organisms to the processes they drive in Mediterranean forests.

Conclusion

The focus of this study was to assess how litter quality and reduced precipitation drive soil biota in three Mediterranean forests. We provide clear evidence that soil biota communities are strongly controlled by litter traits with a common pattern for all taxonomic groups studied. Q. pubescens litter exhibited the highest microbial biomasses and mesofaunal J o u r n a l P r e -p r o o f abundances in comparison to Q. ilex and P. halepensis litters whatever the forest in which the litter decomposed, likely due to a better microhabitat (SLA and WHC) as well as nutritive resource (Ca and Mg) conditions. Surprisingly, despite the amplified drought treatment inducing a modification of litter quality, we did not observe an indirect climate change effect on soil biota due to this intraspecific shift in litter quality. However, we observed a direct effect of amplified drought on soil biota and their contributions to the litter decomposition process with different response patterns depending on both the taxononomic group and the Mediterranean forest considered. Sabaté, S., Sitch, S., Smith, B., Smith, J., Smith, P., Sykes, M.T., Thonicke, K., Thuiller, W., Tuck, G., Zaehle, S., Zierl, B., 2005. Ecosystem Table 1. Main characteristics of the three studied forests. MAT and MAP correspond respectively to the annual mean values of temperature and precipitation between 2008 and 2019 in natural precipitation (ND) and amplified drought (AD) plots (Supplementary Fig. S2).

These values did not significantly differ between the forest sites (One-way ANOVAs, F site = 0.6; P > 0.05, F site = 1.7; P > 0.05 and F site = 1.8; P > 0.05 for MAT, MAP in ND plots and MAP in AD plots, respectively).

  with modifications. Four ml of Bligh-Dyer extractant containing 4 µl of 1,2dinonadecanoyl-sn-glycero-3-phodphocholine (C19:0; Avanti® Polar lipids, Inc.) as internal standard were added to 0.5 g of samples. Lipids separation was performed by solid-phase extraction (SPE) on Phenomenex® (Strata SI-1 with 50 mg of silica, 55 µm, 70 Å). The resulting fatty acid methyl ester (FAMEs) were analysed by gas-chromatography/massspectrometry (GC-MS) on an Agilent 7890 system equipped with an MSD5977A Network mass detector, an ALS7693 automatic injector and an HP5-MS apolar column (30 m × 0.25 mm × 0.25 μm; J&W Agilent Technologies) and MassHunter software. Qualitative analysis J o u r n a l P r e -p r o o f of FAMEs resulted by retention time comparison via FAMEs mixture (range between C4 to C24

  service supply and vulnerability to global change in europe. Science 310, 1333-1337 Sherman, C., Sternberg, M., Steinberger, Y., 2012. Effects of climate change on soil respiration and carbon processing in Mediterranean and semi-arid regions: an experimental approach. European Journal of Soil Biology 52, 48-58 Simioni, G., 2011. Le site d'étude à long terme de Fontblanche. Forêt Méditerranéenne. Swift, M.J., Heal, O.W., Anderson, Jonathan Michael, Anderson, J. M., 1979. Decomposition in terrestrial ecosystems. University of California Press. Talmon, Y., Sternberg, M., Grünzweig, J.M., 2011. Impact of rainfall manipulations and biotic controls on soil respiration in Mediterranean and desert ecosystems along an aridity gradient. Global Change Biology 17, 1108-1118 Taylor, A.R., Wolters, V., 2005. Responses of oribatid mite communities to summer drought: The influence of litter type and quality. Soil Biology and Biochemistry 37, 2117-2130 Thakur, M.P., Herrmann, M., Steinauer, K., Rennoch, S., Cesarz, S., Eisenhauer, N., 2015. Cascading effects of belowground predators on plant communities are densitydependent. Ecology and Evolution 5, 4300-4314 Tian, G., Kang, B.T., Brussaard, L., 1992. Biological effects of plant residues with contrasting chemical compositions under humid tropical conditions-Decomposition and nutrient release. Soil Biology and Biochemistry 24, 1051-1060 Tollefson, T.S., McKercher, R.B., 1983. The degradation of 14C-labelled phosphatidyl choline in soil. Soil Biology and Biochemistry 15, 145-148 J o u r n a l P r e -p r o o f Tornberg, K., Bååth, E., Olsson, S., 2003. Fungal growth and effects of different wood decomposing fungi on the indigenous bacterial community of polluted and unpolluted soils. Biology and Fertility of Soils 37, 190-197 Tsiafouli, M.A., Kallimanis, A.S., Katana, E., Stamou, G.P., Sgardelis, S.P., 2005. Responses of soil microarthropods to experimental short-term manipulations of soil moisture. Applied Soil Ecology 29, 17-26 Van Soest, P.J., 1963. Use of detergents in the analysis of fibrous feeds. A rapid method for the determination of fiber and lignin. Journal of the Association of Official Agricultural Chemists 46, 829-835. Verhoef, H.A., van Selm, A.J., 1983. Distribution and population dynamics of Collembola in relation to soil moisture. Ecography 6, 387-388 Walker, G.M., 1994. The roles of magnesium in biotechnology. Critical Reviews in Biotechnology 14, 311-354 Wall, D.H., Ritz, K., Six, J., Strong, D.R., Putten, W.H. van der, 2012. Soil Ecology and Ecosystem Services. OUP Oxford Wehner, K., Norton, R.A., Blüthgen, N., Heethoff, M., 2016. Specialization of oribatid mites to forest microhabitats-the enigmatic role of litter. Ecosphere 7, e01336. Wilkinson, S.C., Anderson, J.M., Scardelis, S.P., Tisiafouli, M., Taylor, A., Wolters, V., 2002. PLFA profiles of microbial communities in decomposing conifer litters subject to moisture stress. Soil Biology and Biochemistry 34, 189-200 Wright, I.J., Reich, P.B., Cornelissen, J.H.C., Falster, D.S., Groom, P.K., Hikosaka, K., Lee, W., Lusk, C.H., Niinemets, Ü., Oleksyn, J., Osada, N., Poorter, H., Warton, D.I., Westoby, M., 2005. Modulation of leaf economic traits and trait relationships by climate. Global Ecology and Biogeography 14, 411-421 J o u r n a l P r e -p r o o f Zelles, L., 1999. Fatty acid patterns of phospholipids and lipopolysaccharides in the characterisation of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111-129 Zelles, L., 1997. Phospholipid fatty acid profiles in selected members of soil microbial communities. Chemosphere, Experimental and Theoretical Approaches in Environmental Chemistry 35, 275-294 J o u r n a l P r e -p r o o f Tables

FiguresFig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

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Table 2 .

 2 Effects of the forest type (Quercus pubescens, Quercus ilex and Pinus halepensis forests), litter species identity, precipitation treatment (natural vs. amplified drought), and their interactions on microbial (bacterial and fungal biomasses) and mesofaunal (Collembola, Oribatida, and predator abundances) parameters. d.f. = degrees of freedom. F-values and associated P-values (with the respective symbols * for P < 0.05, ** for P < 0.01, and *** for P < 0.001) are indicated. Litter type treatment was initially included in the full models but it was never retained in the most parsimonious models.

	Forest (F) Species (S) Precipitation (P) F × S F × P	Bacteria F 2.9 7.6 *** P 0.9 2.5 3.4 * J o u r n a l P r e -p r o o f Fungi Collembola Oribatida df F P F P F P 2 3.3 * 16.9 *** 10.8 *** 2 7.5 *** 12.0 *** 20.2 *** 1 1.5 4.6 * 11.9 *** 4 2.4 2 3.4 * 6.1 ** 2.1	Predator F 29.0 *** P 6.9 ** 0.2 4.1 *

Table 3 .

 3 Matrix of Spearman correlations between litter mass loss after 24 months of decomposition and the 4 soil biota directly involved in the decomposition process (bacteria, fungi, Collembola and Oribatida) according to forest site and precipitation treatment. Values

	in this matrix can range from -1.0 to 1.0, with 1.0 indicating perfectly correlated variables and
	-1.0 indicating perfectly negative correlations. Significant correlations are indicated with the
	respective symbols *** for P < 0.001, ** for P < 0.01, * for P < 0.05 and ms for P < 0.07. ND
	= natural drought and AD = amplified drought.
		Q. pubescens forest	Q. ilex forest	P. halepensis forest
		ND	AD	ND	AD	ND	AD
	Bacteria	-0.10	-0.13	-0.14	0.29	-0.17	0.10
	Fungi	-0.10	-0.18	-0.14	0.42*	-0.18	0.07
	Collembola	0.34 ms	0.11	0.53**	0.19	0.36*	0.09
	Oribatida	0.10	-0.08	0.61***	0.29	0.45**	0.34 ms

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Acknowledgement

We specially thank to Sylvie Dupouyet, Jean-Philippe Orts and Adoudé Adotevi for their technical assistance during the laboratory experiment and Amélie Saunier for his reviewing of the English. Chemical analyses were performed at the Plateforme d'Analyses Chimiques en Ecologie (PACE, LabEx Centre Méditerranéen de l'Environnement et de la Biodiversité, Montpellier, France), as well as at the Mediterranean Institute of Biodiversity and Ecology (IMBE, Marseille, France). We thank Raphaëlle Leclerc, Bruno Buatois and J o u r n a l P r e -p r o o f Nicolas Barthes for their assistance during chemical analyses. Funding was provided by the French National Research Agency (ANR) through the project SecPriMe2 (no. ANR-12-BSV7-0016-01) and the program BioDivMeX (BioDiversity of the Mediterranean expriment) of the meta-program MISTRALS (Mediterranean Integrated STudies at Regional And Local Scales). This research is also a contribution to the Labex OT-Med (no ANR-11-LABX-0061) funded by the "Investissements d'Avenir" program of the French National Research Agency through the A*MIDEX project (no ANR-11-IDEX-0001-02). The three experimental sites were annually supported by the research infrastructure AnaEE-France (no ANR-11-INBS-0001) and by Allenvi through the SOERE F-ORE-T. We acknowledge the supporters of the COOPERATE database (Reiter IM, Castagnoli G, Rotereau A, 2015, "COOPERATE database", https://cooperate.obs-hp.fr/db): CNRS FR3098 ECCOREV, LABEX OT-Med and OSU-Pythéas. Finally, the authors declare no conflict of interest.

Data accessibility

Upon acceptance of this manuscript, the data supporting this article will be available from the Dryad Digital Repository.

Author contributions

VB and CF designed the experiment. AAS, SP, CL, MS , CF and VB performed the experiment. AAS and MS analyzed the data and led the writing of the manuscript. All authors contributed critically to the drafts and discussion and gave final approval for publication.

Conflict of interest:

The authors declare that they have no conflict of interest.

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