This is the submitted version of the article: Preece, Catherine; Farré Armengol, Gerard; Peñuelas, Josep. «Drought is a stronger driver of soil respiration and microbial communities than nitrogen or phosphorus addition in two Mediterranean tree species». Science of the total environment, Vol. 735 (September 2020), art. 139554. DOI 10.1016/j.scitotenv.2020.139554 This version is avaible at https://ddd.uab.cat/record/224354 under the terms of the license Drought is a stronger driver of soil respiration and microbial communities than nitrogen or phosphorus addition in two Mediterranean tree species Catherine Preece1,2*, Gerard Farré-Armengol3, Josep Peñuelas1,2 1 CREAF, Cerdanyola del Vallès, 08193, Catalonia, Spain; 2 CSIC, Global Ecology Unit 3 CREAF-CSIC-UAB, Bellaterra 08193, Catalonia, Spain; Department of Biosciences, University Salzburg, Hellbrunnerstraße 34, 5020, Salzburg, Austria. *Corresponding author Email address: catherine.preece09@gmail.com Highlights • Effects of drought and nutrients on soils may differ under different trees. • Soil respiration and microbial community physiological profiles were measured. • Drought lowered diversity in both species and in Q. ilex also reduced respiration. • Recovery patterns were different for soils of Q. ilex and P. sylvestris. • Drought may be larger driver of changes to soil communities than N or P deposition. 1 Abstract 2 The drivers of global change, such as increasing drought and nutrient deposition, are affecting 3 soils and their microbial communities in many different habitats, but how these factors interact 4 remains unclear. Quercus ilex and Pinus sylvestris are two important tree species in 5 Mediterranean montane areas that respond differently to drought, which may be associated 6 with the soils in which they grow. We measured soil respiration and physiologically profiled 7 microbial communities to test the impact of drought and subsequent recovery on soil function 8 and diversity for these two species. We also tested whether the addition of nitrogen and 9 phosphorus modified these effects. Drought was the stronger driver of changes to the soil 10 communities, decreasing diversity (Shannon index), and evenness for both species and 11 decreasing soil respiration for Q. ilex when N was added. Soil respiration for P. sylvestris 12 during the drought period was positively affected by N addition but was not affected by water 13 stress. P addition during the drought period did not affect soil respiration for either tree species 14 but did interact with soil-water content to affect community evenness. The two species also 15 differed following the recovery from drought. Soil respiration for Q. ilex recovered fully after 16 the drought treatment ended but decreased for P. sylvestris, whereas the soil community was 17 more resilient for P. sylvestris than Q. ilex. Nutrient addition did not affect respiration or 18 community composition or diversity during the recovery period. Soil respiration was generally 19 weakly positively correlated with soil diversity. We demonstrate that short-term water stress 20 and nutrient addition can have variable effects on the soil communities associated with 21 different tree species and that the compositions of the communities can become uncoupled 22 from soil respiration. Overall, we show that drought may be a stronger driver of changes to 23 soil communities than nitrogen or phosphorus deposition. 24 25 Key words: nitrogen; phosphorus; Quercus ilex; Pinus sylvestris; BIOLOG EcoPlate; 26 microbial community diversity 1 27 1 Introduction 28 The interactive effects of drivers of global change on soil communities remain mostly unknown. 29 Drought in the Mediterranean region has long been identified as one of the most important 30 threats to soils and their plant communities. Mediterranean ecosystems are adapted to some 31 amount of water stress, especially during summer, but climatic predictions indicate that more 32 frequent and more severe droughts will become increasingly common (Dai, 2011; Field et al., 33 2014; Touma et al., 2015). Increases in nutrient addition due to greater inputs from 34 industrialisation and farming are an additional disturbance that may interact with drought to 35 affect soils and their biota. Understanding these impacts for predicting the responses of 36 Mediterranean systems and their ecosystem services to the changing future environment is 37 crucial. The combined effects of drought and added nutrients on soil communities, however, 38 remain elusive. 39 Soil respiration comprises respiration by the microbial community (decomposition of 40 soil organic matter and plant tissues in the bulk soil) and respiration by plant roots, associated 41 mycorrhizal fungi, and rhizosphere microbes (using recently fixed carbon [C] from plants; 42 Kuzyakov, 2006). The effects of drought and nutrients on soil respiration can therefore be 43 complex and are likely to be mediated by the species of plants growing in the soil (de Vries et 44 al., 2019). Soil respiration is a large component of the CO2 released into the atmosphere, 45 usually representing ~50-75% of total ecosystem respiration in forest systems (Brændholt et 46 al., 2018; Curiel Yuste et al., 2005; Tang et al., 2008), but it also indicates the activity of the 47 soil. Any changes in soil respiration due to drought or increased nutrient deposition could 48 therefore have large implications for the global C balance (Cascio et al., 2017). Increases in 49 soil respiration can indicate that the soil is increasing its capacity as a C source if the additional 50 efflux of CO2 is derived from organic matter (Kuzyakov, 2006). A lack of soil respiration, 51 however, can suggest that normal soil processes (root and microbial respiration) are not 52 occurring (Allen et al., 2011), which may have future implications for the short-term health of 53 an ecosystem. 2 54 Droughts tend to reduce soil respiration, including in Mediterranean areas (Talmon et 55 al., 2011), and precipitation in a six-year study in a Mediterranean Aleppo pine forest was the 56 dominant factor determining soil respiration in water-limited seasons (Grünzweig et al., 2009). 57 Drought reduces respiration in the short term (days to weeks), because it lowers the biological 58 activities of soil microorganisms, animals, and plants. Water stress leads to long-term (months 59 to years) changes in vegetation patterns, such as reducing plant cover, which has knock-on 60 effects for respiration (Talmon et al., 2011). Many studies, however, have reported that 61 microbial biomass can remain relatively constant during periods of drought, so changes in 62 respiration should not be mistaken for changes in biomass (Schimel, 2018). 63 The availability of soil nutrients can affect soil respiration, but these effects can vary 64 depending on the dose and duration (Cascio et al., 2017). Some ecosystems will be more 65 limited by nitrogen (N), and others will be more limited by phosphorus (P) (Penuelas et al., 66 2020; Peñuelas et al., 2013), and N and P contents can have interactive effects. These 67 differences depend primarily on the soil, especially its age, and can vary greatly between 68 geographical locations and biome types. While effects of N and P addition have long been 69 shown for aboveground communities, soil microbial communities in various systems tend to 70 be limited more by C (Demoling et al., 2007; Ekblad & Nordgren, 2002; Soong et al., 2019). 71 The impacts of N addition on soil respiration are variable and have been reported either as 72 consistently negative (Ramirez et al., 2010), positive only at low levels of addition (Zhu et al., 73 2016), or positive overall, except in forests, where negative impacts have been reported (Zhou 74 et al., 2014). The responses of P addition may largely depend on ecosystem type. A recent 75 meta-analysis found increases in soil respiration for tropical forests and cropland but 76 decreases in wetland and no effect in other ecosystems (Feng & Zhu, 2019). P addition did 77 not significantly affect heterotrophic respiration in any of these ecosystems (Feng & Zhu, 78 2019). 79 Nutrient addition alters soil respiration by affecting both the plants and the soil 80 microbes. N and P can stimulate root growth, leading to an increase in respiration (Ren et al., 3 81 2016; Zhou et al., 2014). A recent meta-analysis, however, found that N addition consistently 82 led to negative effects on microbial growth and respiration across various global terrestrial 83 ecosystems (T. Zhang et al., 2018). Similarly, N addition reduces microbial biomass (Demoling 84 et al., 2008; Treseder, 2008; Wallenstein et al., 2006), perhaps due to toxic osmotic potential 85 that directly inhibits microbial growth or due to the acidification of the soil from N saturation 86 and subsequent limitation of ions such as calcium or magnesium (Treseder, 2008). The effects 87 of P addition on soil microbes are less straightforward, with different studies reporting positive, 88 negative, or no effect on microbial biomass and activity depending on the type of ecosystem 89 (Guo et al., 2017; Illeris et al., 2003; Keith et al., 1997; Liu et al., 2012). 90 The composition and diversity of the soil microbial community also change in response 91 to drought and nutrient addition. Drought can directly affect these communities, e.g. by 92 desiccation and reduction of substrate diffusion, leading to the limitation of resources (Naylor 93 & Coleman-Derr, 2017; Schimel et al., 2007; Schimel, 2018). Though note also that on the 94 other hand, drier soils may increase the availability of gaseous and volatile substrates, which 95 may be beneficial (Schimel, 2018). Low levels of soil moisture can therefore decrease 96 microbial activity, such as nutrient mineralisation, enzymatic activity (Sanaullah et al., 2011; 97 J. Sardans & Peñuelas, 2005) and dormancy, allowing microbes to recover after the drought 98 conditions end (Meisner et al., 2018; Schimel, 2018). The impacts of drought will differentially 99 affect different microbes, with some evidence suggesting that fungi are more tolerant than 100 bacteria (Evans & Wallenstein, 2012; Preece et al., 2019). 101 The impacts of drought on plants can also indirectly affect microbial communities 102 (Bardgett et al., 2008), e.g. by changes in patterns of root exudation, in both quantity and 103 composition (Canarini et al., 2016; Gargallo-Garriga et al., 2018; Preece et al., 2018; Preece 104 & Peñuelas, 2016; Williams & Vries, 2020). Additionally, drought may affect litter inputs from 105 above and below ground, which would likely have knock-on impacts on the microbial 106 community (Bardgett et al., 2008). In terms of microbial diversity, dought has been reported to 4 107 have both positive and negative effects (Bouskill et al., 2013; Acosta-Martínez et al., 2014), 108 which may differ for bacteria and fungi (Preece et al., 2019). 109 Increased N and P in the soil can also affect the composition (Li et al., 2015; Liu et al., 110 2012; Ramirez et al., 2012) and possibly the diversity (Su et al., 2015) of soil microbial 111 communities, tending to favour copiotrophic, fast-growing bacteria (Leff et al., 2015). 112 Responses to added N and P, however, will likely depend strongly on the concurrent effect on 113 the plants in the system. Interactions between nutrient addition and drought that lead to 114 changes in microbial communities are even less well studied than the individual effects. 115 Our aim was to assess the interactive effects of drought and nutrient addition on soil 116 respiration and soil community-level physiological profiles (CLPPs). We also assessed the 117 recovery of soil and its community from a drought and whether nutrients would help or hinder 118 the recovery. Finally, we assessed whether the responses differed between two species that 119 coexist in montane Mediterranean regions, Quercus ilex L. (holm oak) and Pinus sylvestris L. 120 (Scots pine). The two species have very different geographical distributions, with Q. ilex 121 common throughout the Mediterranean Basin, whereas P. sylvestris is more typical in 122 temperate habitats, with the Mediterranean Basin at the southern limit of its range. In fact, Q. 123 ilex is replacing P. sylvestris as the dominant canopy species in Mediterranean montane 124 forests (Aguadé et al., 2015; Galiano et al., 2010; Vilà-Cabrera et al., 2013). We therefore 125 expected that Q. ilex and its associated soil community would be more resistant to the drought 126 treatment. 127 We hypothesised that 1) both drought and nutrient addition would decrease soil 128 respiration and microbial diversity, 2) the effects of drought would be lower in the soils with Q. 129 ilex than P. sylvestris, and 3) the soils with Q. ilex would have more signs of recovery from 130 drought, due to differences in the drought tolerance of the two species. 131 132 5 133 2 Material and methods 134 2.1 Plant and soil material 135 A greenhouse experiment was set up in late April 2016 at the Autonomous University of 136 Barcelona (Spain). The experiment contained 64 three-year-old Q. ilex saplings and 64 three- 137 year-old P. sylvestris saplings (provided by Tres Turons, Barcelona, Spain). The roots of the 138 saplings were gently cleaned using water and small forceps to remove all soil from the 139 previous substrate, and the saplings were then replanted in a new common substrate in 3.5- 140 L pots. This substrate was a natural soil, with stones >1 cm removed. The soil was collected 141 from a natural holm oak forest on a south-facing slope (25% slope) in the Prades Mountains 142 in northeastern Iberian peninsula (41°13′N, 0°55′E; 930 m a.s.l.). All plants were then given 143 adequate water for eight weeks, until late June 2016, to allow them to adjust to the greenhouse 144 environment. 145 146 2.2 Experimental design 147 The experimental design comprised a drought period and a recovery period, with 128 pots in 148 total, divided equally between the two species. During the drought period, a drought treatment 149 (control or drought) and a nutrient treatment (control, N, P and both N and P), were applied to 150 all pots in a factorial design, with equal numbers of replicates for each treatment combination 151 (n = 8). Pots were divided into four blocks (for two replicates of each drought-nutrient 152 combination in each block). The nutrient treatment began on June 10th 2016 with the addition 153 of N, the addition of P, and the addition of both N and P. N was added as ammonium nitrate 154 (NH4NO3) at a rate of 50 kg N ha-1, and P was added as calcium phosphate (Ca3(PO4)2) at a 155 rate of 25 kg P ha-1. The diameter of the pots required application rates of 0.22 g of Ca3(PO4)2 156 and 0.25 g of NH4NO3 as aqueous solutions per pot. A second round of nutrient addition was 157 performed in mid-September. The drought treatment was applied from June 27th 2016 by 158 reducing the addition of water by approximately 50% compared to the controls. Soil-water 159 content (SWC) was measured using an ML3 Theta Probe connected to an HH2 Moisture 6 160 Meter (Delta-T Devices, Cambridge, UK) in each pot once a week to maintain the content at 161 approximately 20% for the control plants and 10% for the drought-treated plants. These SWC 162 values were chosen based on previous experiments by the group in similar conditions, that 163 were known to provide sufficient water (20%) and moderate drought (10%) (Preece et al., 164 2018; Zhang et al., 2017). Soil respiration was measured and soil samples were collected 165 (described in detail below) for all pots after 14-17 weeks (September 30th to October 21st 166 2016), which signified the end of the drought treatment. Measurements were temporally 167 staggered at one week per block for logistical reasons due to the large number of replicates. 168 The period of drought was considered long enough for the SWC to have stabilised long before 169 the measurements were taken and to avoid differences in soil respiration or CLPPs due to the 170 timing of the measurement. The pots were re-watered at optimal amounts for eight weeks to 171 represent a recovery period, after which respiration was again measured and soil samples 172 were collected (November 25th to December 16th 2016). 173 Mean air temperature during the experiment, monitored using five EL-USB-2 data 174 loggers (Lascar Electronics, Wiltshire, UK), was 21.3°C. Soil temperature was monitored at a 175 fine scale in four pots across the different soils using a Decagon Em50 data logger with 5TM 176 soil probes (Decagon Devices, Pullman, USA) and averaged 22.3°C throughout the 177 experiment. SWC in each pot was measured at the start of the experiment and at the end of 178 the drought period, and at the end of the recovery period if relevant, using the ML3 Theta 179 Probe connected to the HH2 Moisture Meter. Mean SWC during the drought treatment in the 180 Q. ilex pots was 18.6% in the control water-treatment pots and 10.6% in the drought-treated 181 pots. Mean SWC in the P. sylvestris pots was 21.3% in the control water-treatment pots and 182 12.5% in the drought-treated pots. Nutrient addition did not affect SWC. 183 184 2.3 Measurements 185 Soil respiration was measured in each pot using an EGM-4 portable gas analyser connected 186 to an SRC-1 soil-respiration chamber (PP Systems, Amesbury, MA, USA) at the end of the 187 drought and recovery periods. Care was taken when inserting the respiration chamber into the 7 188 pots to avoid damaging the plants and to ensure a tight seal with the soil. Measurements were 189 taken at least 24 h after watering to limit the effect of water pulses on soil respiration. 190 Respiration was expressed as g CO2 m-2 h-1. 191 CLPPs were assessed using BIOLOG (Hayward, CA, USA) EcoPlates, which measure 192 the metabolism of 31 C sources, to identify changes between microbial communities in the 193 treatments. Soil samples of approximately 10 g were collected from each pot soon after soil 194 respiration was measured, at the ends of the drought and recovery periods. The two replicate 195 samples for each of the eight drought-nutrient combinations per block were pooled and mixed 196 well. A 5 g subsample of soil for each treatment per block was placed in a 100 mL sterile 197 plastic flask, and 50 mL of a sodium pyrophosphate (Na4P2O7·10(H2O)) solution (pH 7) were 198 added. This solution was shaken for 20 min, left to settle for 1 min, and then 2 mL were added 199 to 18 mL of Ringer solution and agitated to mix. One hundred and forty microlitres of this new 200 solution were pipetted into each well of an EcoPlate. A microplate reader (Tecan Sunrise, 201 Männedorf, Switzerland) was used to read the absorbance at 590 nm in each well. The plates 202 were incubated in the dark at 25 °C for 4 d, with absorbance readings taken at 0, 24, 48, 60, 203 and 72 h. This process was repeated for each sample. 204 Absorbance readings after 72 h were corrected for initial absorbance for each plate by 205 subtracting the corresponding readings taken at 0 h, with any negative values removed. The 206 mean absorbance for the three wells designated as blanks in each plate was calculated, which 207 was then subtracted from the absorbances for each of the C sources. The mean of these 208 blanked absorbances was calculated to give the average well colour development (AWCD) 209 per plate. All absorbances were then standardised by dividing by the AWCD, and the three 210 replicate samples for each C source were then used to calculate the mean standardised 211 absorbance for each source. Each of these values was divided by the sum of the mean 212 standardised absorbance of all 31 C sources in each sample, to give the relative abundance. 213 This procedure allowed the calculation of the Shannon H index, as a measure of diversity, and 214 evenness per plate (with a plate for each drought-nutrient treatment per block). 215 8 216 2.4 Statistical analyses 217 The data were grouped into two subsets (named as ‘experiment’ in the statistical 218 models), the drought period and the recovery period. There was no statistical difference 219 between the two species in terms of soil respiration or CLPP diversity (Shannon H and 220 evenness). There was a small difference between species in terms of CLPP composition 221 (PERMANOVA), however the r2 of this model was very low (<0.04) and the species effect 222 disappeared when other variables (SWC and nutrients) were included in the model. Therefore, 223 all data were analysed separately for each species. The data for soil respiration were log- 224 transformed before analysis, and a constant was first added for the data for Q. ilex to improve 225 the normality of the data. All statistical analyses were performed using R (R Core Team, 2018). 226 Linear mixed-effect models were used to test the effect of drought and nutrient (N and P) 227 addition on soil respiration using the lmer function in the lme4 package. Soil respiration per 228 pot was thus the response variable, and the explanatory variables were SWC (as a continuous 229 variable) and N and P addition as two categorical variables. Block was added as a random 230 effect. Interactions between variables were included if they were significant in the model (P < 231 0.05). We also used mixed-effect models for analysing the CLPP data, with the Shannon H 232 index or evenness as the response variable and with SWC, N addition, and P addition as the 233 explanatory variables, with any interactions where significant. 234 The effect of the recovery period on respiration, Shannon H, and evenness for each 235 species was also tested using linear models, combining the drought and recovery data, with 236 ‘experiment’ as a fixed factor interacting with the drought treatment. The C sources were 237 grouped into five guilds (Supplementary Material, Table S1), carbohydrates, amino acids, 238 carboxylic and acetic acids, amines/amides, and polymers (following Weber & Legge, 2009), 239 to identify the effect of the drought or nutrient addition on the potential activity (as a percentage 240 of all C sources) of each C guild. The simplest model was always selected using the Akaike 241 information criterion. A permutational multivariate analysis of variance (PERMANOVA) was 242 also performed, using adonis2 in the vegan package, to test the effects of SWC and N and P 243 addition on the composition of the carbohydrate sources that could be metabolised. The 9 244 relative abundance of the mean standardised absorbance was the response variable, and as 245 before, block was included as a random effect. A principal component analysis (PCA) was 246 performed to visualise the relationships between the potential metabolisms of the 31 C 247 sources and SWC during the drought treatment and recovery period. The nutrient treatments 248 were not included, because the PERMANOVA indicated that they were not relevant to the 249 changes in potential community composition. Significant differences between the control and 250 drought groups were identified using an analysis of variance (ANOVA). The relationship 251 between soil CLPP diversity (Shannon H) and respiration for all data was tested using a linear 252 mixed-effect model, with species as a random effect. Finally, to analyse the correlations 253 between SWC, nitrogen and phosphorus and the individual carbon sources, a partial least 254 squares regression was performed (pls function in MixOmics R package), with results shown 255 in clustered image maps (cim function in MixOmics R package, Rohart et al., 2017). 256 257 3 Results 258 3.1 Effects of drought and nutrient addition on soil respiration and CLPPs 259 Soil respiration ranged from 0.01 to 0.54 µmol C m-2 s-1. Respiration in the drought period was 260 lower in the soils with Q. ilex, but only when N was added (Table 1, Fig. 1a; interaction between 261 SWC and N, P < 0.05). Although respiration in P. sylvestris soil was unaffected by drought, it 262 was positively affected by N addition (Table 1, Fig. 1b; effect of N, P < 0.05). P addition during 263 the drought treatment did not affect soil respiration for either Q. ilex or P. sylvestris. 264 PERMANOVA indicated that the CLPPs did not differ significantly between the soils 265 with Q. ilex and P. sylvestris under control conditions. SWC strongly affected the CLPPs for 266 both species (Table 1; effect of SWC, P < 0.001 for both). Similarly, the PCA clearly separated 267 the control and water-stressed groups for both species during the drought period, with an 268 ANOVA indicating that PC1 differed significantly between the two groups, and PC2 also for P. 269 sylvestris (Fig. 2a, PC1, P < 0.001; PC2, P = 0.07 for Q. ilex; Fig. 2b, PC1, P < 0.01; PC2, P 270 < 0.05 for P. sylvestris). 10 271 The drought treatment negatively affected CLPP diversity for both species (Table 1, 272 Fig. 3a, b; effect of SWC, P < 0.05 for both). CLPP evenness for Q. ilex during the drought 273 period was positively correlated with SWC (Table 1, Fig. 3c; effect of soil-moisture content, P 274 < 0.01). CLPP evenness in pots with P. sylvestris during the drought period was affected by 275 an interaction between SWC and P addition (Table 1, Fig. 3d; P < 0.05), such that SWC was 276 strongly positively correlated with evenness when no P was added but was not correlated 277 when P was added. 278 In soils with Q. ilex, drought led to increases in the potential metabolism of 279 carbohydrates (Supplementary Material, Table S2, negative effect of SWC, P < 0.001), in 280 particular D-cellobiose, pyruvic acid methyl ester and D-mannitol (Supplementary Material, 281 Fig S1). Drought led to decreases in the amine/amide group (positive effect of SWC, P < 0.01) 282 and polymers (P < 0.05), with no significant nutrient effects. In pots with P. sylvestris, drought 283 increased the potential metabolism of amino acids (P < 0.001), in particular L-asparagine 284 (Supplementary Material, Fig S2), and there was an interaction between soil moisture and 285 phosphorus addition; when there was no additional P, there was a positive effect of the drought 286 (negative effect of increasing SWC) and when there was additional P, there was a negative 287 drought effect (interaction between SWC and P, P < 0.05). 288 289 3.2 Recovery of soil respiration and CLPPs 290 Soil respiration did not differ significantly between the drought period and the recovery period. 291 Respiration in the soils with Q. ilex fully recovered when watering recommenced at normal 292 levels (Table 2, Fig. 4a). Respiration in the soils with P. sylvestris had a delayed effect of 293 drought, because it was affected by SWC during the period of drought (Table 2, Fig. 4b; effect 294 of SWC, P < 0.001). The addition of the nutrients had no effect on soil respiration for either 295 species during the recovery period. 296 The CLPP patterns during the recovery from drought were more complex. The 297 PERMANOVA indicated that Q. ilex CLPP was affected by SWC during recovery and the 11 298 previous drought period (Table 2; P < 0.01). P. sylvestris CLPP was also affected by SWC, 299 but only from the drought period (P < 0.05). Nutrient addition did not affect the CLPPs 300 (diversity, evenness or composition) for either species in the recovery period. The PCA 301 indicated that the communities overlapped more than after the drought treatment, and the 302 ANOVA for Q. ilex found no difference between the control and drought groups, although the 303 first principal component (PC1) for P. sylvestris differed significantly between the control and 304 drought groups (Fig. 5b, P < 0.05). 305 The diversity indices did not differ significantly between the drought period and the 306 recovery period for either species. The drought, however, had a continued effect for Q. ilex 307 during the recovery period; the drought treatment had a negative impact on Shannon H when 308 measured at the end of the recovery period (Table 2, Fig. 6a; P < 0.01). Diversity, however, 309 tended to recover for P. sylvestris, as SWC did not affect H (Table 2, Fig. 6b). Similarly, with 310 evenness, soil under Q. ilex showed a continued negative effect of SWC during the drought 311 period (Table 2, Fig. 6c; P < 0.05), but not for P. sylvestris (Table 2, Fig. 6d). Nutrient addition 312 was not important for determining the Shannon diversity or evenness during the recovery 313 period. 314 The five guilds of C sources responded differently to SWC in the pots with Q. ilex at 315 the end of the recovery period, with interactive effects between SWC during drought and 316 recovery for the carboxylic and acetic acids group and for polymers (Supplementary Material, 317 Table S2, P < 0.01 and P < 0.05, respectively). SWC in the two periods was positively 318 correlated with the potential metabolism of carboxylic and acetic acids and negatively 319 correlated with the potential metabolism of polymers. Regarding individual C sources, L-serine 320 (amino acid) was strongly positively correlated with SWC in drought, and D,L-alpha-glycerol 321 phosphate (a carbohydrate) was strongly positively correlated with SWC in recovery, while 322 there was a strong positive effect of drought on the polymer alpha-cyclodextrin 323 (Supplementary Material, Fig S3). Nutrient addition did not affect the metabolism of the five C 324 guilds in the pots with Q. ilex. The results for the pots with P. sylvestris were slightly more 12 325 complicated. Carbohydrate metabolism was affected by an interaction between the SWC in 326 the drought treatment and the recovery period (P < 0.05). Amino acid metabolism was 327 positively affected by N addition, particularly L-arginine (Supplementary Material, Fig S4), and 328 carboxylic and acetic acid metabolism was affected by an interaction between P addition and 329 SWC during drought (P < 0.05) and by an interaction between the SWC in the drought period 330 and the recovery period (P < 0.01). 331 332 3.3 Are diversity and respiration linked? 333 The combined data for both periods of the experiment (drought period and recovery period) 334 indicated a positive correlation between CLPP diversity (Shannon H) and soil respiration (Fig. 335 7, P < 0.05). 336 337 4 Discussion 338 4.1 Effects of drought on soil respiration and CLPPs 339 Soil respiration tended to be resistant to drought, at least in the short term, with no change for 340 P. sylvestris and a negative effect for Q. ilex only when extra N was added. Previous studies 341 have consistently found that drought tended to decrease soil respiration, including in 342 Mediterranean regions (Curiel Yuste et al., 2007; Misson et al., 2010). The drought treatment 343 in our study may not have been severe enough (in duration or in water reduction) to induce 344 significant changes in soil respiration, particularly autotrophic respiration by the sapling roots. 345 In comparison with our drought treatment, studies which found reductions in soil respiration 346 tended to have lower soil moisture than was achieved in our study (e.g. less than 5% in Curiel 347 Yuste, 2009 and less than 10% in Mission et al., 2010 compared with 10.6 and 12.5% mean 348 moisture in Q. ilex and P. sylvestris pots respectively in our study). This result was supported 349 by indications that foliar greenness (chlorophyll fluorescence measurements, data not shown) 13 350 was not greatly affected by the drought, and this resistance may have extended to the other 351 organs of the plant, such as the roots. The decrease in respiration in the droughted soils with 352 Q. ilex in the presence of high levels of N could indicate a decrease in the activity of the soil 353 microbial community when the N was added, which is a common response to N addition 354 (Ramirez et al., 2010). The respiration of the system then decreased at low levels of soil 355 moisture with the additional stress of the drought. 356 The use of EcoPlates allowed us to monitor the potential microbial communities by 357 measuring the metabolic responses to different C sources. Reducing SWC had strong 358 negative effects on the CLPPs for both species, reducing diversity and evenness and affecting 359 the composition. Some evidence suggests that Mediterranean soils may be resistant to mild 360 long-term water stress (Curiel Yuste et al., 2014), but microbial communities may still be 361 responsive to shorter-term changes in water availability (Preece et al., 2019) before a new 362 steady state is reached, especially because bacteria and fungi tend to have fast turnover times 363 of days to weeks (Blazewicz et al., 2014; Rousk & Bååth, 2011). The drought treatment was 364 a 50% reduction in water for approximately four months, similar to other studies using 365 EcoPlates to study the effects of drought and which have detected changes in CLPPs (e.g. 366 Kassem et al., 2008; Hueso et al., 2012), supporting our use of this method. Drought had a 367 negative, positive, or no impact on the C guilds, and these effects were not consistent between 368 the two species. The drought treatment thus affected the potential composition of microbial 369 communities, but not consistently. The role of plant species in shaping the rhizosphere has 370 been confirmed in previous studies that have showed that distinct microbial communities occur 371 in the root-zones of different species (Burns et al., 2015), mediated, at least in part, by root 372 exudates (Bais et al., 2006; Sasse et al., 2018). Thus, differences in the effects on CLPPs 373 between the soils of Q. ilex and P. sylvestris in our study may well be due to differences in 374 their root exudation patterns. 375 14 376 4.2 Recovery after drought 377 Soil respiration is known to show a large pulse of CO2 efflux when drought conditions end, 378 known as the Birch effect, which is thought to be due to the microbially mediated 379 decomposition of soil substrates or dead microbes that accumulate during dry periods, or to 380 the mobilisation of stable soil C that can be respired by the surviving microorganisms (Birch, 381 1958; Göransson et al., 2013; Schimel, 2018). Respiration fully recovered in the soils with Q. 382 ilex but not P. sylvestris, where the effect of drought was delayed. We do not know how long 383 a period of recovery, within the constraints of our study, would be needed for soil respiration 384 with P. sylvestris to return to pre-drought levels, but soil respiration was less resilient for P. 385 sylvestris than Q. ilex. 386 The CLPPs had opposite patterns between the species, with diversity and evenness 387 for Q. ilex negatively affected by drought with no signs of recovery at the end of the recovery 388 period. SWC did not affect diversity or evenness during the recovery period for P. sylvestris, 389 indicating that the microbial community had either recovered or had adjusted to the new 390 conditions. Our experiment could not determine which of these options was correct, but the 391 fact that there was also a change in recovery CLPP composition due to the prior drought 392 treatment suggests a change to a new stable condition. The uncoupling of the responses of 393 the CLPPs and soil respiration to drought suggests that autotrophic respiration may have been 394 more important in our experiment, because the respiration data was often not explained by 395 the CLPP data. 396 Previous studies have demonstrated the high capability of respiration to recover in soil. 397 For example, soil respiration in a field study with beech (Fagus sylvatica) trees recovered 398 within three days of the end of a drought treatment, even exceeding control values as the 399 recovery period progressed, compensating for the decrease in respiration during drought 400 (Hagedorn et al., 2016). In that study it was reported that the majority of soil respiration was 401 plant derived as opposed to microbe derived, and the quick recovery was thought to be due 15 402 to the accumulation of carbon compounds in the roots, such as sugars, starch, and amino 403 acids, which enabled the fast re-activation of trees when re-watering began (Hagedorn et al., 404 2016). This finding was supported by another mesocosm experiment in a montane 405 grassland, which also found that C from root exudates accumulated in the rhizosphere during 406 drought, accompanied by a low activity of soil microbes, and in re-wetting this C disappeared 407 and microbial activity returned (Karlowsky et al., 2018). Root exudates from droughted plants 408 can also stimulate soil respiration compared to controls, presumably due to changes in 409 exudate composition (Gargallo-Garriga et al., 2018). Experimental work has shown that the 410 effect of these exudates on soil respiration can be much greater than the effect of a soil 411 having a legacy of drought, illustrating that plant mediated signals during recovery might be 412 critical for sustained and successful soil-ecosystem function after drought (de Vries et al., 413 2019). Additionally, it has been shown, in Q. ilex, that exudate amount and composition 414 varies between drought and recovery periods, changing from higher exudation rates, 415 dominated by secondary metabolites in drought to lower exudation rates of mainly primary 416 metabolites in recovery (Gargallo-Garriga et al., 2018; C. Preece et al., 2018). This has 417 important implications for the composition of the rhizosphere microbiome, which can be 418 directly affected by exudation patterns (Williams & Vries, 2020), suggesting that differences 419 in CLPP profiles in the current study may be largely driven by changes in root exudation. 420 421 4.3 Does nutrient addition affect soil respiration and CLPPs during drought and recovery? 422 Although the Mediterranean region is thought to be primarily limited by water, in terms of plant 423 growth and microbial activity, it is also often limited by N and P (Ochoa-Hueso et al., 2011; 424 Rutigliano et al., 2009; Jordi Sardans et al., 2004). This may explain the main effect of nutrient 425 addition on soil respiration in our study, which was the positive impact that N addition had in 426 the soil with P. sylvestris during the drought period. This finding was inconsistent with previous 427 studies where N and P addition have had widespread negative effects on soil respiration 16 428 (Bowden et al., 2004; Guo et al., 2017; Ramirez et al., 2010; T. Zhang et al., 2018), although 429 responses varied with site and treatment. These effects included both heterotrophic and root 430 (autotrophic) respiration and were larger at less productive sites, where N was more likely to 431 limit plant growth (Janssens et al., 2010). This finding is associated with long-term (chronic) N 432 fertilisation, so results in short-term experiments such as ours may be less likely to have any 433 impacts. Moreover, a previous study in a Mediterranean system showed that an increase in 434 nitrogen supply can lead to an increase in water use efficiency and reduced water loss of 435 Pinus pinaster (Fernández et al., 2006). Thus, in our study, positive effects of nitrogen addition 436 on P. sylvestris could lead to feedbacks in the soil, such as maintenance of root exudation, 437 which could have a follow-on benefit on soil respiration. 438 In addition to soil respiration, the effects of N and P addition on the soil community 439 were also identified by analysing CLPP composition, diversity and evenness. The effects of 440 nutrient addition on CLPP were generally small and varied depending on the species and the 441 SWC, and overall, the potential soil community composition was mostly unaffected by the 442 addition of both N and P. A previous large-scale experiment found that adding N to a variety 443 of bare soils directly and consistently affected bacterial communities and decreased the 444 activities of extracellular enzymes, indicating a decrease in soil microbial activity (Ramirez et 445 al., 2012). Our experiment, however, found no evidence of an effect of nutrient addition on the 446 structure of the soil communities, perhaps due to the larger impact of the drought. 447 Drought in our study, as in many similar experiments, was a treatment whose effects 448 increased over time, concurrent with the length of time that the plants had less than adequate 449 water, whereas the effects of N and P addition decreased in intensity over time, due to the 450 uptake by plants and soil microbes and to leaching during watering. This may help to explain 451 why drought effects seemed to be more important in their impacts on soil respiration and the 452 CLPPs compared with nutrient addition. We found no evidence that nutrient addition improved 453 respiration or the diversity indices during the recovery from drought for either species. 17 454 455 4.4 Are responses the same for Q. ilex and P. sylvestris? 456 The Mediterranean region is characterised by dry summers to which the vegetation is adapted, 457 but this adaptation does not prevent the plants and soils from responding to environmental 458 changes. Although the two study species can coexist in montane Mediterranean regions, Q. 459 ilex has been shown to be replacing P. sylvestris as the dominant species (Aguadé et al., 460 2015; Galiano et al., 2010; Vilà-Cabrera et al., 2013). It was therefore expected that Q. ilex, 461 and its associated soil community would be the more resistant to the drought treatment. In 462 contrast to this hypothesis, the drought treatment negatively affected soil respiration faster for 463 Q. ilex than P. sylvestris, but only when N was added. However, the drought treatment did not 464 affect respiration at the end of the drought period for P. sylvestris. The CLPP analysis indicated 465 that the potential compositions of the soil communities did not differ significantly between the 466 species under control conditions, excluding the presence of measurably distinct soil 467 communities (at the CLPP level) prior to the drought treatment, which led to the divergent 468 responses to drought. In terms of CLPP, the species responded similarly to drought, which 469 negatively affected diversity and strongly affected community functional composition. 470 Therefore, overall, we do not have support for our second hypothesis, that the effects of 471 drought would be lower in the soils with Q. ilex. 472 It was also hypothesised that soils with Q. ilex would recover more quickly from the 473 drought compared with soils with P. sylvestris, which is supported by the soil respiration data. 474 Respiration in soils with Q. ilex returned to control levels during the recovery period, whereas 475 in soils with P. sylvestris, respiration was negatively affected during the recovery period, 476 presumably a delayed effect of the previous water stress. Both species continued to show the 477 effects of the drought (SWC during the drought period) on community composition into the 478 recovery period, although these effects for Q. ilex were also linked to SWC at the time of 479 measurement (recovery). However, the Shannon index and evenness recovered completely 18 480 for P. sylvestris but not for Q. ilex, contradicting our prediction that soils with Q. ilex would be 481 able to recover better than the soils with P. sylvestris. 482 4.5 Are soil diversity and respiration linked? 483 The relationship between the composition and function of soil microbial communities is still 484 not completely understood. Some evidence suggests a positive relationship between microbial 485 diversity and soil respiration (Ren et al., 2018) and that information about the composition of 486 microbial communities can improve predictions of soil respiration (Cleveland et al., 2007; Liu 487 et al., 2018), but other studies report a lack of effect of diversity on respiration (Griffiths et al., 488 2001; Nannipieri et al., 2003). CLPP diversity in our study was positively correlated with soil 489 respiration, but the correlation was weak, likely due to both the method for assessing the 490 communities (CLPPs with the EcoPlates) and the influence of other experimental factors, 491 particularly SWC, that had a large impact on both respiration and diversity. Our results 492 generally reinforced the potential for using CLPP techniques to identify links between soil 493 diversity and soil functioning, but our relatively coarse assessment of the communities 494 indicates that data must be carefully interpreted. 495 496 5 Conclusions 497 This study has demonstrated that responses to drought in Mediterranean forest soils may vary 498 strongly depending on the dominant tree species and the timescale of the drought. Respiration 499 in the soils with Q. ilex decreased during water stress but recovered quickly, whereas 500 respiration in the soils with P. sylvestris had a delayed effect and a subsequent slower 501 recovery. Interestingly, the opposite pattern was found, for the two species, regarding the 502 diversity of the microbial communities, indicating that changes in soil respiration may be more 503 closely linked to autotrophic (root) than heterotrophic (microbial) respiration. The effects of 504 nutrient addition on the soils were generally weaker than the effects of drought, reaffirming the 19 505 importance of water availability for soil function and community diversity in Mediterranean 506 ecosystems. 507 20 508 Figure Captions 509 Fig. 1. Effect of SWC and N and P addition on soil respiration at the end of the drought period 510 for pots with (a) Q. ilex and (b) P. sylvestris. Each datapoint represents a different pot. 511 512 Fig. 2. PCA of the CLPP data for (a) Q. ilex and (b) P. sylvestris at the end of the drought 513 period. Control pots are blue points, and drought-treated pots are yellow points. Carbon 514 sources are colour-coded as follows: red = carboxylic & acetic acids, orange = carbohydrates, 515 green= amines/amides, blue = amino acids, purple = polymers. 516 517 Fig. 3. Effect of SWC and N and P addition on diversity (Shannon H index) for pots with (a) 518 Q. ilex and (b) P. sylvestris and on evenness for pots with (c) Q. ilex and (d) P. sylvestris at 519 the end of the drought period. Each datapoint represents a different pot. 520 521 Fig. 4. Effects of SWC and N and P addition on soil respiration at the end of the recovery 522 period for pots with (a) Q. ilex and (b) P. sylvestris. Each datapoint represents a different pot. 523 NS, not significant. 524 525 Fig. 5. PCA of the CLPP data for (a) Q. ilex and (b) P. sylvestris at the end of the recovery 526 period. Control pots are blue points, and drought-treated pots are yellow points. Carbon 527 sources are colour-coded as follows: red = carboxylic & acetic acids, orange = carbohydrates, 528 green= amines/amides, blue = amino acids, purple = polymers. 529 21 530 Fig. 6. Effects of SWC and N and P addition on diversity (Shannon H index) for pots with (a) 531 Q. ilex and (b) P. sylvestris and evenness for pots with (c) Q. ilex and (d) P. sylvestris at the 532 end of the recovery period. Each datapoint represents a different pot. NS, not significant. 533 Fig. 7. Relationship between soil respiration and CLPP diversity (Shannon H) (P < 0.05) for 534 all pots in the experiment (drought period and recovery period). 22 535 References 536 Acosta-Martínez, V., Cotton, J., Gardner, T., Moore-Kucera, J., Zak, J., Wester, D., & Cox, 537 S. (2014). Predominant bacterial and fungal assemblages in agricultural soils during a 538 record drought/heat wave and linkages to enzyme activities of biogeochemical cycling. 539 Applied Soil Ecology, 84, 69–82. doi: 10.1016/j.apsoil.2014.06.005 540 Aguadé, D., Poyatos, R., Rosas, T., & Martínez-Vilalta, J. (2015). 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Positive effect of SWC with added N Composition Effect of SWC, P < 0.001 Effect of SWC, P < 0.001 (PERMANOVA) Shannon H Positive effect of SWC, P < Positive effect of SWC, 0.05 P < 0.05 Evenness Positive effect of SWC, P < SWC content × P addition interaction. 0.01 Positive effect of SWC with no added P, P < 0.05 Table 2. Effects of SWC on soil respiration and CLPP diversity (Shannon H) and evenness at the end of the recovery period. NS, not significant. Q. ilex P. sylvestris Soil respiration NS Positive effect of SWC during drought, P < 0.001 Composition SWC during drought × SWC Effect of SWC during drought, (PERMANOVA) during recovery, P < 0.01 P < 0.05 Shannon H Positive effect of SWC during NS drought, P < 0.01 Evenness Positive effect of SWC during NS drought, P < 0.05 (a) (b) Q. ilex at the end of drought P. sylvestris at the end of drought 0.0 ln(soil respiration + 0.1) g CO 2m-2 h-1 SWC × N, P < 0.05 N, P < 0.05 ln(soil respiration) g CO2 m-2h-1 1 0.5 Nutrients 1.0 Control N 2 P 1.5 NP 2.0 3 2.5 10 15 20 25 10 15 20 25 Soil water content (%) during drought Soil water content (%) during drought (a) (b) Q. ilex at the end of drought P. sylvestris at the end of drought 0.75 0.4 L-asparagine Tween 80 Phenylethylamine 0.50 Putrescine Drought treatment 0.2 Tween 40 Gamma- 4-Hydroxy benzoic acid Glycyl-L-glutamic acid L-phenylalanine PC2 (13.37%) PC2 (16.15%) D-galacturonic acid hydroxybutyric acid Control D-glucosaminic acid Glycogen Drought i-erythritol Alpha-cyclodextrin 0.25 Pyruvic acid methyl ester Itaconic acid L-arginine L-threonine 0.0 Pyruvic acid methyl ester D-malic acid Alpha-ketobutyric acid D-cellobiose Alpha-D-lactose D-mannitol Tween 80 Alpha-ketobutyric acid 2-Hydroxy benzoic acid D-galactonic acid- L-arginine 2-Hydroxy benzoic acid N-acetyl-D-glucosamine gamma-lactone D-galacturonic acid L-phenylalanine Beta-methyl-D-glucoside Glucose-1- D-galactonic acid-gamma-lactone 0.00 Itaconic acid L-asparagine i-erythritol phosphate Glycyl-L-glutamic acid L-serine 4-Hydroxy benzoic acid D-mannitol L-serine D-xylose Glycogen L-threonine D-malic acid -0.2 Alpha-cyclodextrin Gamma-hydroxybutyric acid Tween 40 D-glucosaminic acid Alpha-D-lactose Beta-methyl-D-glucoside D,L-alpha-glycerol D-xylose D,L-alpha-glycerol Glucose-1- D-cellobiose Putrescine phosphate phosphate -0.25 phosphate N-acetyl-D-glucosamine Phenylethylamine -0.4 -0.2 0.0 0.2 -0.2 0.0 0.2 0.4 PC1 (19.47%) PC1 (27.5%) (a) (b) Q. ilex at the end of drought P. sylvestris at the end of drought 3.4 3.4 SWC, P < 0.05 SWC, P < 0.05 3.2 3.2 Shannon H Shannon H 3.0 3.0 2.8 2.8 10 15 20 25 10 15 20 25 Soil water content (%) during drought Soil water content (%) during drought (c) (d) Q. ilex at the end of drought P. sylvestris at the end of drought 1.00 1.00 SWC, P < 0.01 SWC × P, P < 0.05 0.95 0.95 Evenness Evenness 0.90 0.90 0.85 0.85 10 15 20 25 10 15 20 25 Soil water content (%) during drought Soil water content (%) during drought (a) (b) Q. ilex at the end of recovery P. sylvestris at the end of recovery 0.0 0 ln (Soil respiration + 0.1) g CO 2 m-2 h-1 ln (Soil respiration) g CO 2 m-2 h-1 NS SWC in drought, P < 0.001 −0.5 Nutrients −1 Control −1.0 N P −2 NP −1.5 −2.0 −3 −2.5 10 15 20 25 10 15 20 25 Soil water content (%) during drought Soil water content (%) during drought (a) (b) Q. ilex at the end of recovery P. sylvestris at the end of recovery 0.4 D-galactonic acid-gamma-lactone L-serine Putrescine D-mannitol L-arginine Gamma-hydroxybutyric acid D-mannitol 0.2 Itaconic acid D-glucosaminic acid D-galactonic acid- 0.2 D-galacturonic acid L-asparagine gamma-lactone L-arginine Drought treatment L-serine D,L-alpha-glycerol phosphate Tween 80 D-cellobiose i-erythritol D-malic acid Control Drought PC2 (14.32%) D-glucosaminic acid PC2 (18.19%) D-malic acid D,L-alpha-glycerol 2-Hydroxy benzoic acid i-erythritol phosphate 0.0 Tween 40 4-Hydroxy benzoic acid D-xylose N-acetyl-D-glucosamine 0.0 L-threonine D-galacturonic acid N-acetyl-D-glucosamine Glycyl-L-glutamic acid Glucose-1-phosphate Itaconic acid Alpha-D-lactose L-threonine Gamma-hydroxybutyric acid Beta-methyl-D-glucoside Tween 40 2-Hydroxy benzoic acid Glucose-1- Putrescine Glycogen Alpha-cyclodextrin 4-Hydroxy phosphate L-asparagine Alpha-cyclodextrin Phenylethylamine Alpha-ketobutyric benzoic acid D-cellobiose -0.2 acid -0.2 Beta-methyl-D-glucoside Glycyl-L- D-xylose Pyruvic acid methyl ester L-phenylalanine glutamic acid L-phenylalanine Alpha-ketobutyric acid Tween 80 Pyruvic acid methyl ester Glycogen Alpha-D-lactose Phenylethylamine -0.4 -0.4 -0.4 -0.2 0.0 0.2 -0.2 0.0 0.2 0.4 PC1 (24.34%) PC1 (24.15%) (a) (b) Q. ilex in recovery Q. ilex at the end of recovery P. sylvestris at the end of recovery 0.0 3.4 SWC in drought, P < 0.01 3.4 NS −0.5 (Soil respiration) 3.2 −1.0 3.2 Nutrients H Shannon H Control lnShannon N −1.5 P NP 3.0 3.0 −2.0 −2.5 2.8 2.8 10 15 20 25 10 15 20 25 10 Soil 15 moisture (%) in20drought 25 Soil water content (%) during drought Soil water content (%) during drought (c) (d) Q. ilex at the end of recovery in recovery P. sylvestris at the end of recovery 0.0 1.00 1.00 SWC in drought, P < 0.05 −0.5 NS respiration) 0.95 0.95 −1.0 Nutrients Evenness Evenness Control N −1.5 P ln (Soil NP 0.90 0.90 −2.0 0.85 −2.5 0.85 10 15 20 25 10 10 15 15 20 20 25 25 Soil water content (%) during drought Soil water content (%) during drought −1.0 ln (Soil respiration) g CO 2 m-2 h-1 −1.5 P. sylvestris −2.0 Q. ilex −2.5 −3.0 2.8 2.9 3.0 3.1 3.2 3.3 Shannon H 1 Drought is a stronger driver of soil respiration and microbial communities than nitrogen 2 or phosphorus addition in two Mediterranean tree species – Supplementary Material 3 4 5 Table S1 Guild groupings of the BIOLOG EcoPlate carbon sources (following Weber and 6 Legge, 2009). Carbon source Guild Alpha-D-lactose Carbohydrates Beta-methyl-D-glucoside Carbohydrates D-cellobiose Carbohydrates D-mannitol Carbohydrates D-xylose Carbohydrates D,L-alpha-glycerol phosphate Carbohydrates Glucose-1-phosphate Carbohydrates i-erythritol Carbohydrates N-acetyl-D-glucosamine Carbohydrates Pyruvic acid methyl ester Carbohydrates Alpha-ketobutyric acid Carboxylic & acetic acids 2-Hydroxy benzoic acid Carboxylic & acetic acids 4-Hydroxy benzoic acid Carboxylic & acetic acids D-galactonic acid-gamma-lactone Carboxylic & acetic acids D-glucosaminic acid Carboxylic & acetic acids D-galacturonic acid Carboxylic & acetic acids Gamma-hydroxybutyric acid Carboxylic & acetic acids Itaconic acid Carboxylic & acetic acids 1 D-malic acid Carboxylic & acetic acids L-arginine Amino acids L-asparagine Amino acids L-phenylalanine Amino acids L-serine Amino acids L-threonine Amino acids Glycyl-L-glutamic acid Amino acids Phenylethylamine Amines/amides Putrescine Amines/amides Alpha-cyclodextrin Polymers Glycogen Polymers Tween 40 Polymers Tween 80 Polymers 7 2 Table S2. Effects of drought and nutrient addition on the potential metabolisms of the five C guilds identified by linear models, with species (Q. ilex and P. sylvestris) and experiment (drought and recovery) separated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant. Close to significant P-values are also shown. Positive and negative refers to the type of correlation between the relative abundance of the mean standardised absorbance for each guild with the explanatory variable. Note that when there is a significant interaction, the direction (positive or negative) of the individual treatment effect is not described. Drought period Carbohydrates Amino acids Carboxylic and Amines/amides Polymers acetic acids Quercus ilex SWC Negative *** NS NS Positive ** Positive * N addition NS NS NS NS NS P addition NS NS NS NS NS 3 Pinus sylvestris SWC NS Negative *** NS * NS N addition NS NS NS NS NS P addition NS NS NS * NS Moisture content × P addition NS NS NS Moisture content NS has positive effect with P addition but negative effect without P addition * 4 Recovery period Carbohydrates Amino acids Carboxylic and Amines/amides Polymers acetic acids Quercus ilex SWC during drought NS NS Positive ** NS Negative * SWC during recovery NS NS Positive *** NS Negative ** N addition NS NS NS P addition NS NS NS SWC (drought) × SWC (recovery) NS NS ** NS * SWC (recovery) × P addition NS NS NS SWC (drought) × N addition NS NS NS SWC (drought) × P addition NS NS NS 5 Pinus sylvestris SWC during drought * P = 0.07 Positive ** NS NS SWC during recovery * P = 0.05 Positive ** NS NS N addition NS Positive * NS NS P addition P = 0.08 Positive * NS NS SWC (drought) × SWC (recovery) * P = 0.06 ** NS NS SWC (drought) × N addition P = 0.09 NS NS SWC (drought) × P addition * NS NS SWC (recovery) × P addition P = 0.08 NS NS 6 Figure S1. Clustered image map showing the results of the PLS analysis for Quercus ilex in the drought period. 7 Figure S2. Clustered image map showing the results of the PLS analysis for Pinus sylvestris in the drought period. 8 Figure S3. Clustered image map showing the results of the PLS analysis for Quercus ilex in the recovery period. 9 Figure S4. Clustered image map showing the results of the PLS analysis for Pinus sylvestris in the recovery period. 10