Daniel Hernández-Cáceres

Alexia Stokes
email: alexia.stokes@cirad.fr

Guillermo Angeles-Alvarez

Josiane Abadie

Fabien Anthelme

Manon Bounous

Grégoire T Freschet

Catherine Roumet

Monique Weemstra

Luis Merino Martin

Luis Merino-Martín

Frédérique Reverchon

Vegetation creates microenvironments that influence soil microbial activity and functional diversity along an elevation gradient

Keywords: alpine, catabolic diversity, Juniperus communis, catabolic activity, Picea abies, multiple substrate induced respiration, root traits, Vaccinium myrtillus Highlights

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Mountainous regions are characterised by abrupt changes in physical, edaphic and biotic environments over short geographic distances because of the presence of elevational gradients. These gradients create a variety of ecological habitats [START_REF] Körner | A definition of mountains and their bioclimatic belts for global comparisons of biodiversity data[END_REF][START_REF] Abbott | Altitudinal gradients, plant hybrid zones and evolutionary novelty[END_REF][START_REF] Hofmann | Distribution of prokaryotic abundance and microbial nutrient cycling across a high-alpine altitudinal gradient in the austrian central alps is affected by vegetation, temperature, and soil nutrients[END_REF], which makes them ideal for studying biodiversity patterns and their drivers. Several studies have emphasised the importance of investigating soil microbial community structure and function along elevation gradients, as soil microorganisms are often more responsive to environmental changes than other organisms [START_REF] Shen | Distinct soil bacterial communities along a small-scale elevational gradient in alpine tundra[END_REF][START_REF] Cui | Diversity patterns of the rhizosphere and bulk soil microbial communities along an altitudinal gradient in an alpine ecosystem of the eastern Tibetan Plateau[END_REF][START_REF] Bardgett | Soil microbial community responses to climate extremes: Resistance, resilience and transitions to alternative states[END_REF]. Soil microorganisms play a key role in a wide range of ecosystem processes, such as decomposition of organic matter, nutrient cycling, erosion control through their action on soil aggregate stability, regulation of water supply, soil development and maintenance of structure [START_REF] Prosser | The role of ecological theory in microbial ecology[END_REF][START_REF] Maron | Soil microbial diversity: Methodological strategy, spatial overview and functional interest[END_REF][START_REF] Aislabie | Soil microbes and their contribution to soil services[END_REF][START_REF] Xu | Microbial functional diversity and carbon use feedback in soils as affected by heavy metals[END_REF][START_REF] Merino-Martín | Interacting effects of land use type, microbes and plant traits on soil aggregate stability[END_REF]. As recently emphasised by several studies, there is a crucial need to consider belowground processes and integrate them with vegetation data in order to better predict soil dynamics and response to climate change [START_REF] Cavicchioli | Scientists' warning to humanity: microorganisms and climate change[END_REF][START_REF] Hagedorn | Above-and belowground linkages shape responses of mountain vegetation to climate change[END_REF]D´Alò et al., 2021). We ask therefore, if it is possible to determine how climatic factors alter microbial activity and functional diversity, depending on soil and vegetation type, in a complex mountainous environment.

In temperate latitudes, (sub)alpine soils are very heterogeneous and are typically rocky, acidic and shallow with poorly developed horizons [START_REF] Price | Mountain Soils[END_REF]. As elevation increases, plant growth and cover are mainly limited by climatic conditions and a shorter growing season, causing a reduction of organic matter content in soils at higher elevations and making soils nutrient-limited for plant growth [START_REF] Zhou | Spatial and resource factors influencing high microbial diversity in soil spatial and resource factors influencing high microbial diversity in soil[END_REF][START_REF] Donhauser | Alpine soil microbial ecology in a changing world[END_REF]Möhl et al., 2018;[START_REF] Stokes | Shifts in soil and plant functional diversity along an altitudinal gradient in the French Alps[END_REF]. Microclimatic conditions strongly influence soil microbial communities [START_REF] Donhauser | Alpine soil microbial ecology in a changing world[END_REF]Frey, 2018, Collins et al., 2020) and therefore microbial activity and functional diversity. Here, we define microbial activity as the global catabolic activity, an indicator of the functional capacity of microbial communities to decompose organic matter. Aditionally, we define microbial functional diversity as the catabolic diversity, i.e. the capacity of a microbial community to use different substrates for respiration, as measured through catabolic response profiles [START_REF] Degens | Development of a physiological approach to measuring the catabolic diversity of soil microbial communities[END_REF][START_REF] Degens | Is the microbial community in a soil with reduced catabolic diversity less resistant to stress or disturbance?[END_REF]. Catabolic activity and diversity are strongly influenced by soil variables, especially pH [START_REF] Moscatelli | Assessment of soil microbial functional diversity: land use and soil properties affect CLPP-MicroResp and enzymes responses[END_REF]) and total and labile organic carbon (C) contents, assessed mainly through studies of increased fertilisation by organic amendments across a wide range of land uses [START_REF] Degens | Decreases in organic C reserves in soils can reduce the catabolic diversity of soil microbial communities[END_REF][START_REF] Margesin | Microbial communities and activities in alpine and subalpine soils[END_REF][START_REF] Tian | Linkages between the soil organic matter fractions and the microbial metabolic functional diversity within a broad-leaved Korean pine forest[END_REF][START_REF] Bongiorno | Soil management intensity shifts microbial catabolic profiles across a range of European long-term field experiments[END_REF]. Moreover, catabolic activity and diversity to increase with warmer temperatures in different grasslands [START_REF] Grayston | Accounting for variability in soil microbial communities of temperate upland grassland ecosystems[END_REF][START_REF] Papatheodorou | The effects of large-and small-scale differences in soil temperature and moisture on bacterial functional diversity and the community of bacterivorous nematodes[END_REF] and alpine climates (D´Alò et al., 2021). As climatic conditions are also determined by topography, they further influence variables such as soil nutrient availability and the quantity of litter input to the soil; altogether, these environmental variables are key to controlling the catabolic activity of soil microorganisms [START_REF] Kang | Variations of soil enzyme activities in a temperate forest soil[END_REF]. However, the linkages between catabolic activity, diversity and soil properties are not yet fully elucidated, particularly in a complex, heterogeneous landscape, where soil type, climate and vegetation all interact to modify microbial community structure and function.

Vegetation can alter global catabolic activity and diversity either at the community level or through specific species effects. Several studies have shown that catabolic diversity increased with higher levels of plant diversity e.g., along a longitudinal gradient in temperate grasslands [START_REF] Liu | Relationship between plant species diversity and soil microbial functional diversity along a longitudinal gradient in temperate grasslands of Hulunbeir, Inner Mongolia, China[END_REF] and along an elevational gradient in temperate forests [START_REF] Klimek | Covariation of soil bacteria functional diversity and vegetation diversity along an altitudinal climatic gradient in the Western Carpathians[END_REF].

These effects were mainly attributed to increases in plant biomass and mediated by soil factors. The influence of increased plant diversity on microbial catabolic activity and diversity is likely through an increase in the variety of C sources, promoting a heterogeneous distribution of soil properties [START_REF] Reverchon | Tree plantation systems influence nitrogen retention and the abundance of nitrogen functional genes in the Solomon islands[END_REF]. However, individual plant species also alter the structure and function of soil microbial communities, through (i) shoot and root litter production [START_REF] Xu | Divergence of dominant factors in soil microbial communities and functions in forest ecosystems along a climatic gradient[END_REF], (ii) root exudation patterns [START_REF] Williams | Root functional traits explain root exudation rate and composition across a range of grassland species[END_REF] and (iii) above-and below-ground functional traits [START_REF] Spitzer | Root trait-microbial relationships across tundra plant species[END_REF]Sweeny et al., 2021).

Microbial activity and functional diversity are enhanced in litter-rich soil because of the large amount of C available as substrate for decomposers [START_REF] Nsabimana | Size, activity and catabolic diversity of the soil microbial biomass as affected by land use[END_REF][START_REF] Nuccio | Niche differentiation is spatially and temporally regulated in the rhizosphere[END_REF], although the quality of litter, especially its content in soluble C, also has a determining effect on microbial catabolic activity [START_REF] Fanin | Litter fingerprint on microbial biomass, activity, and community structure in the underlying soil[END_REF]. In particular, root nitrogen (N) and C content should increase microbial respiration and decomposition of soil organic matter [START_REF] Han | Root functional traits are key determinants of the rhizosphere effect on soil organic matter decomposition across 14 temperate hardwood species[END_REF], whereas roots that are rich in more recalcitrant compounds such as lignin and cellulose could limit microbial activity [START_REF] Poirier | The root of the matter: Linking root traits and soil organic matter stabilization processes[END_REF].

Within bulk soil, microbial processes can be altered by the proximity of plant roots. Plant roots release organic compounds that modify microbial catabolic activity and functional diversity, since many microorganisms use these root exudates and mucilage as their main energy source [START_REF] Liu | Relationship between plant species diversity and soil microbial functional diversity along a longitudinal gradient in temperate grasslands of Hulunbeir, Inner Mongolia, China[END_REF][START_REF] Hobbie | Microbes in nature are limited by carbon and energy: The starving-survival lifestyle in soil and consequences for estimating microbial rates[END_REF]. These interactions take place with a greater intensity in the rhizosphere, a narrow zone around the root with high concentrations of easily degradable carbon sources, leading to an inflated rate of microbial activity and a greater functional diversity compared to bulk soil [START_REF] Baudoin | Impact of growth stage on the bacterial community structure along maize roots, as determined by metabolic and genetic fingerprinting[END_REF][START_REF] Yang | Comparison of soil microbial community catabolic diversity between Rhizosphere and bulk soil induced by tillage or residue retention[END_REF][START_REF] Kuzyakov | Rhizosphere size and shape: Temporal dynamics and spatial stationarity[END_REF][START_REF] Nuccio | Niche differentiation is spatially and temporally regulated in the rhizosphere[END_REF]. Therefore, plant root traits that promote rhizosphere dimensions, e.g., long, thin roots that have a large surface area, should also enhance microbial catabolic activity and diversity. Woody root traits that have the strongest effect on microbial catabolic activity and biomass include specific root length (SRL: length / mass ratio), root branching intensity and root diameter, whereas root chemical composition (C, phosphorus, calcium and magnesium content) has been found to have less effect [START_REF] Khlifa | Do temperate tree species diversity and identity influence soil microbial community function and composition?[END_REF]Sweeny et al., 2021). Thicker roots increase the volume of cortex available for mycorrhizal fungi colonisation, which would enhance nutrient uptake and soil nutrient cycling [START_REF] Burke | Relationship between soil enzyme activities, nutrient cycling and soil fungal communities in a northern hardwood forest[END_REF][START_REF] Xiao | Effects of re-vegetation type and arbuscular mycorrhizal fungal inoculation on soil enzyme activities and microbial biomass in coal mining subsidence areas of Northern China[END_REF][START_REF] Mccormack | Physical and Functional Constraints on Viable Belowground Acquisition Strategies[END_REF], hence potentially promoting microbial activity in the rhizosphere [START_REF] Chen | Greater variations of rhizosphere effects within mycorrhizal group than between mycorrhizal group in a temperate forest[END_REF]. However, although root exudation is positively correlated to root diameter in grassland species [START_REF] Williams | Root functional traits explain root exudation rate and composition across a range of grassland species[END_REF], it is negatively related to root diameter in trees [START_REF] Han | Root functional traits are key determinants of the rhizosphere effect on soil organic matter decomposition across 14 temperate hardwood species[END_REF]. We therefore expect that rhizospheric microbial communities relying on root exudates as a substrate should be less active around thicker woody roots with relatively small rhizosphere dimensions compared to highly branched roots or roots with a high SRL.

We aimed to unravel the effect that biotic and abiotic factors exert on global catabolic activity and diversity in bulk and rhizospheric soils in a heterogeneous environment. To do this, we examined changes in climatic parameters, soil properties, plant species diversity and root traits along an elevation gradient (without considering microtopography), and their relationships with global catabolic activity and diversity. We investigated the global catabolic activity and diversity in soils beneath three different plant species, in both rhizospheric soil andalso bulk soil where a mixture of roots from other plant species would be present. . We hypothesized that microbial catabolic activity and diversity would : (H1) decrease in colder and nutrient-poor soils from higher elevations, where reduced plant growth limits organic matter supply to soil, compared to soils at lower altitudes; (H2) increase at higher levels of plant diversity, especially in bulk soils, because of the more broad variety of C sources available from litter and root exudates, and (H3) increase around roots possessing traits that enlarge rhizospheric dimensions i.e. longer, thinner roots, that have large exchange surface areas and potentially more available exudates. However, roots that are rich in lignin and cellulose, could limit microbial activity, because these compounds are highly recalcitrant.

Material and Methods

Study site and plant species under study

Fieldwork was conducted along an 8 km elevational gradient located in the Belledonne massif in the French Alps (France, N 45° 7' 1'' E 5° 53' 35', Table 1). The elevation gradient that we used comprised six altimetric bands, each 200 m, and ranged from 1400 to 2400 m a.s.l. The current thermal treeline is situated between 2000 -2100 m (Wang et al., 2018).

Below the treeline, the dominant trees shifted along the gradient from mixed forest of Fagus sylvatica, Pinus sylvestris and Abies alba in the montane belt to mixed forests of Picea abies, Pinus uncinata and Pinus cembra in the subalpine belt. Above the treeline, the vegetation was dominated by (sub)alpine heaths of Ericaceae (Vaccinium spp, Rhododendron ferrugineum, Loiseleuria procumbens (L.) Loisel.) and Juniperus communis subsp. nana and grasslands dominated by graminoid species (Nardus stricta, Carex sempervirens, Festuca spp). Mean annual temperature (MAT), mean annual precipitation (MAP) and mean annual solar radiation (MAR) were calculated using the meteorological AURELHY model [START_REF] Piedallu | Soil water balance performs better than climatic water variables in tree species distribution modelling[END_REF][START_REF] Piedallu | Soil aeration, water deficit, nitrogen availability, acidity and temperature all contribute to shaping tree species distribution in temperate forests[END_REF] during a ten year period (2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011)(2012)(2013)(2014) at each elevation [START_REF] Stokes | Shifts in soil and plant functional diversity along an altitudinal gradient in the French Alps[END_REF] Table 1). MAT decreased from 8.5 ± 0.2 °C to 5.7 ± 0.2 °C up the elevation gradient, while MAP increased from 1024 ± 41 mm to 1187 ± 40 mm. MAR varied from 4204 MJ/m 2 to 4339 MJ/m 2 up along the elevation gradient (Table 1, Fig. S1).

Five plots (20 x 20 m) were selected within each altitudinal band, with a lateral distance of at least 100 m between each plot (Fig. 1, a and b) in June 2018. Plots were selected if they included three species: P. abies, a tall evergreen tree; Juniperus communis L., a prostrate evergreen shrub and Vaccinium myrtillus L., a small deciduous shrub (if these species were present at that elevational band, Table S1). These three plant species were selected because of their different growth forms and occupation of different ecological niches along the elevational gradient. P. abies was the dominant tree species below the tree line; J. communis was abundant locally and V. myrtillus was one of the most frequent species above the treeline, as well as being present in all six elevational bands. P. abies and V. myrtillus are both keystone species [START_REF] Bjune | The development and local stand-scale dynamics of a Picea abies forest in southeastern Norway[END_REF][START_REF] Nybakken | Increased growth and phenolic compounds in bilberry (Vaccinium myrtillus L.) following forest clear-cutting[END_REF]) and although J. communis is not classed as a keystone species, its abundance above the treeline makes it an important species that structures plant communities. Therefore, these three species contribute to shaping the structure of the plant communities in which they are present, and we term them 'structuring species'.

Within each plot, one well-developed individual of each structuring species was selected and at the limit of its canopy, on the downslope side, a 1.0 × 1.0 m quadrat was located for further plant and soil sampling. A botanical survey was first performed in each quadrat (data are provided in [START_REF] Stokes | Shifts in soil and plant functional diversity along an altitudinal gradient in the French Alps[END_REF]. Plants were identified using two floras (Flore Forestière Française, Montagnes, [START_REF] Rameau | Flore forestière française, Montagnes[END_REF]Flora Helvetica, Laufen et al., 2001) and their abundance was estimated making a visual assessment of the relative area covered by different plant species in each quadrat. Simpson diversity index (S) was calculated as following:

𝑆 = 1 - ∑ ( ) ( ) …………………………………………………………………………(1)
where n is the number of individuals of each species and N the total number of individuals of all species.

Bulk and rhizospheric soil sampling

At the center of each 1 x 1 m quadrat, a soil monolith (0.25 × 0.25 × 0.15 m) was excavated using a metal frame (Fig. 1,c). Bulk soil was collected from the monolith´s four lateral faces, sieved at 2 mm, air-dried and stored at room temperature before further analyses. These analyses comprised the assessment of soil physical and chemical properties and catabolic activity and diversity [START_REF] Stokes | Shifts in soil and plant functional diversity along an altitudinal gradient in the French Alps[END_REF]. A total number of 70 bulk soil samples were taken (six elevations × five plots × two to three plant species).

To obtain rhizospheric soil, further soil samples were obtained beneath plants in the same plots and close to the monoliths, using gloves and ethanol sterilized material. Individuals of V. myrtillus were identified and root systems extracted manually. Soil attached to the youngest roots (comprising the root apices) was sampled by gently shaking the roots inside paper bags whilst still in the field. In the case of J. communis and P. abies, whole individuals were not harvested. Instead, a large root was followed from the base of the trunk to a distance of about 0.5 m from the trunk, and fine roots (roots with a diameter ≤2 mm, whose primary function is resource uptake [START_REF] Freschet | Sampling roots to capture plant and soil functions[END_REF]), attached to this large root were collected and then shaken in paper bags. All 70 rhizospheric soil samples were taken to the laboratory and were air-dried for at least one week and stored at room temperature prior to catabolic activity and diversity analyses. We air-dried our soil samples to avoid logistical constraints linked to sampling, transport and storage [START_REF] Gillespie | Tree species mixing affects soil microbial functioning indirectly via root and litter traits and soil parameters in European forests[END_REF].

Soil physical and chemical properties

Soil physical and chemical properties were measured in the same monoliths where we sampled bulk soil [START_REF] Stokes | Shifts in soil and plant functional diversity along an altitudinal gradient in the French Alps[END_REF]. Analyses were performed on three replicates of 60- Multiple substrate-induced respiration (MSIR) is a method for characterising and assessing the activity and functional diversity of soil microbiota. This method is used to assess the soil microbial functional capacity in C cycling [START_REF] Creamer | Measuring respiration profiles of soil microbial communities across Europe using MicroResp TM method[END_REF][START_REF] Fromin | Soil microbial activity in a Mediterranean garrigue responds more to changing shrub community than to reduced rainfall[END_REF] and is therefore used as a proxy for global catabolic activity and diversity of soil microbial communities (i.e., the mineralization of organic C into carbon dioxide (CO2), [START_REF] Beare | Measurement of fine root tissue density: A comparison of three methods reveals the potential of root dry matter content[END_REF][START_REF] Nannipieri | Ecological significance of the biological activity in soil[END_REF]. Here, we used the MicroResp™ system (Macaulay Scientific Consulting, Aberdeen, UK) to characterize the community level physiological profiles of the soil microbial communities and assess the capacity to use multiple substrates for respiration (i.e. catabolic diversity). Since the production of CO2 is measured within 6 hours after inducing respiration with the addition of substrates [START_REF] Chapman | Assessing CLPPs using MicroResp TM -A comparison with Biolog and multi-SIR[END_REF], the advantage of this technique is that it gives immediate responses to C substrate decay instead of relying on microbial growth. Briefly, both bulk and rhizospheric soil samples were adjusted to 80 % of their maximum water holding capacity and pre-incubated for one week at 25 °C in deepwell plates, that holds approximately 0.45 g well -1 of soil [START_REF] Baratella | Soil basal respiration and substrate induced respiration (SIR) by MicroResp TM Importance and applications[END_REF], before applying aqueous solutions of the different C substrates and assembling the deepwell-detection plate system. In this study, we used twelve substrates (with four replicates per substrate) of the three main families of organic compounds found in soil and root exudates; sugars (D-glucose, arabinose, N-acetyl-glucosamine), amino acids (Lalanine, L-lysine, L-asparagine, L-glycine) and organic acids (oxalic acid, malic acid, citric acid, vanillic acid) [START_REF] Badri | Regulation and function of root exudates[END_REF]. Sugars and amino acids represent a main source of litterfall-and root-derived C compounds in the soil [START_REF] Jones | Microbial response time to sugar and amino acid additions to soil[END_REF] while organic acids are predominantly liberated in the form of root exudates [START_REF] Farrar | How roots control the flux of carbon to the rhizosphere[END_REF]. All substrates were added at a concentration of 100 g L -1 except for L-asparagine (50 g L -1 ) and vanillic acid (10 g L -1 ) due to their low solubility. Additionally, water was used as a control to measure soil basal respiration that is defined as the steady rate of respiration in soil from the mineralization of organic matter. CO2 emission was estimated using a colorimetric method. Detection plates containing 12.5 µg g -1 cresol red, 150 mM KCl and 2.5 mM NaHCO3 set in 1 % Noble agar were prepared and 150 µl of the solution were distributed per well. After pre-incubation in the dark, each deepwell plate was covered with a detection plate using a silicon seal. The system was secured with a clamp and incubated for six hours at 28 °C. The optical density of each plate was determined using a microplate reader (Victor3 1420 Multilabel Counter, Perkin Elmer, MA, USA) at 570 nm before and after incubation. The optical density was normalized and converted to multiple substrate induced respiration rates (MSIR) expressed in µg CO2 g -1 dry soil h -1 [START_REF] Fromin | Soil microbial activity in a Mediterranean garrigue responds more to changing shrub community than to reduced rainfall[END_REF]). The respiration rates for the different C compounds were summed across all substrates as a proxy of the global catabolic activity. The catabolic diversity index (Shannon index, H'), an indicator of the soil microbial functional diversity [START_REF] Sparling | Resistance to cropping pressure of two New Zealand soils with contrasting mineralogy[END_REF], was calculated for each sample using Equation 2:

𝐻 ´= -∑ 𝑝 * 𝑙𝑛(𝑝 )…………………………………………………… (2)
where 𝑝 was the respiration rate for a given substrate divided by the sum of the total respiration rates of that particular substrate.

2.5 Measurements of community-and species-level root traits

Community level root traits

After the monoliths were extracted, the depth of the litter layer was noted and the litter removed. Half of the monolith was dissected to extract all roots (hereafter community level), that represented a mix of roots from one of the structuring species and from different plant species growing in the monoliths. This community level mixture of roots therefore comprised a large proportion of roots from one of the three structuring species. Within 1 day but sometimes up to 2 days after sampling, roots from the community level were sorted and fine, undamaged roots with a diameter < 2 mm were removed and washed. Absorptive roots are lower root orders, typically the first, second and third root orders (defined as the most distal root orders) [START_REF] Pregitzer | FINE ROOT ARCHITECTURE OF NINE NORTH AMERICAN TREES[END_REF][START_REF] Freschet | Sampling roots to capture plant and soil functions[END_REF]. A subsample of absorptive roots were selected, scanned (Epson Expression 1680, Canada) in a tray of water and analysed (Winrhizo Pro version 2019, Regent Instruments, Canada), following [START_REF] Roumet | Root structure-function relationships in 74 species: Evidence of a root economics spectrum related to carbon economy[END_REF].

At the community level, root length (Lcom), mean root diameter (RDcom), and root volume (Vcom) were estimated in different diameter classes (from 0 to 2 mm diameter with a 0.1 mm bin size). After scanning, root fresh mass (RFMcom) and root dry mass (RDMcom) (60°C for 72h) were determined. Specific root length (SRLcom) as the ratio between Lcom and RDMcom, root tissue density (RTDcom) as the ratio between RDMcom and Vcom, root dry mass content (RDMCcom) as the ratio between RDMcom and RFMcom, root length density (RLDcom) and root mass density (RMDcom) were calculated for roots at the community level (Table S2). Root nitrogen (RNCcom) and carbon (RCCcom) content were measured on absorptive roots using an elemental analyser (CHN model EA 1108; Carlo Erba Instruments, Italy). The content of water-soluble compounds, cellulose, hemicellulose and lignin in absorptive community level roots was determined using the Van Soest method (Van Soest, 1990) in a fiber analyser (Fibersac 24; Ankom, Macedon, NJ, USA).

Species level root traits

Species level root trait data were obtained from a separate study by [START_REF] Weemstra | Patterns in intraspecific variation in root traits are species-specific along an elevation gradient[END_REF] who examined root traits of 11 individual plant species in the same plots and at the same dates, including the three structuring species that we examined, along the same elevational gradient. In their study, roots from the three structuring plant species (P. abies, V. myrtillus and J. communis) were carefully dug out from the top 0.15 m soil horizon and below the litter layer. Approximately 3 to 5 different roots were dug up from 0.5 -1.5 m from the base of the stem and traced back to the stem to verify that they belonged to the chosen plant individuals. Roots were stored in moist plastic bags and kept refrigerated until analysed in the laboratory. Within 1 day but sometimes up to 2 days after sampling, roots were washed and undamaged absorptive roots were scanned and SRLsp, RDMCsp, RTDsp at the species level were calculated as described above for roots harvested at the community level.

Data analysis

We examined the variations in global catabolic activity and catabolic diversity (H´) in bulk and rhizospheric soils with regard to elevation and structuring plant species.

Soil physical and chemical properties, community-and species-level root traits and global catabolic activity and diversity along the elevational gradient, were analyzed as a function of elevational band using one-way analysis of variance (ANOVA) and Tukey's HSD to test significant differences among elevational bands. Unbalanced two-way ANOVA (Type-III sums of squares) was performed to assess the effect of elevation, plant species and their interaction on the different soil physical and chemical properties and community-and species-level root traits.

Spearman's correlation coefficients were assessed tostudy the relationships between catabolic activity and diversity and climatic data, soil properties, plant diversity and community-and species-level root traits, along the elevational gradient for each one of the three structuring species.

Dissimilarity in global catabolic activity between bulk and rhizospheric soils, among elevation bands and structuring plant species was examined using non-metric multidimensional scaling (NMDS) analysis using Bray-Curtis distance. Permutational multivariate analysis of variance (PERMANOVA), implemented with the adonis function from the vegan R package, was used to assess the significance of the observed NMDS differences.

Distance-based redundancy analysis (db-RDA) for constrained ordination based on the Bray-Curtis distance (capscale function of vegan R package) was carried out to determine the extent to which variations in global catabolic activity can be explained by environmental variables along the elevational gradient, followed by a stepwise model selection using Generalized Akaike Information Criterion (AIC, ordistep function of vegan R package with forward and backward direction). Finally, db-RDA analysis was performed only for the variables obtained from the model selection.

Variation partitioning analysis, using the function varpart of the R vegan package, was performed to determine the relative importance of the environmental variables (soil physical and chemical properties, community-and species-level root traits and climatic data), and their contribution to catabolic activity, which was later identified by partial redundancy analysis in Hellinger transformed data.

Results

Changes in soil physical and chemical properties along the elevation gradient

Beneath V. myrtillus and P. abies, soil nutrient content (SOC, TN and P), CEC and C/N ratio varied little along the elevational gradient except at 1600 m where they were significantly higher than at all other elevations (Table S3). Beneath J. communis, changes in these properties were negligible along the gradient (Table S3). In bulk soil beneath P. abies, NH4 + content was significantly higher at 1600 m than at all other elevations, but under V. myrtillus and J. communis, there were no significant differences among elevational bands (Table S3).

With regard to NO3 -content, there were no significant changes along the gradient beneath any structuring species (Table S3). Litter depth beneath P. abies decreased significantly from 1400 m to 2000 m while under V. myrtillus the main change was between 1400 and 1600 m.

No trends beneath J. communis occurred along the gradient (Table S3). Soils beneath all three plant species along the gradient were acidic with a pH ranging from 4.40 to 5.60, and the percentage of soil water content was significantly lower at 1400 -1600 m than at 1800 -2400 m (Table S3). Both elevation and structuring plant species were not significantly related to changes in the majority of soil physical and chemical properties (ANOVA analysis, Tables S4). However, elevation had a significant influence on silt and sand content (p<0.001)

and structuring species had a significant impact on MWDtop and soil water content (p<0.001; Table S4).

Global catabolic activity and diversity (H') in bulk and rhizosphere soil along the elevation gradient

No significant differences were found between basal respiration and global catabolic activity, therefore, to simplify results, we present values for global catabolic activity only (Table S5).

In bulk soil beneath V. myrtillus and P. abies, global catabolic activity fluctuated along the gradient with a significant peak (pairwise comparisons between elevational bands, p < 0.05;

Table S6) at 1600 m (Fig. 2 a,b). No changes in bulk soil catabolic activity under J. communis occurred between elevation bands (Fig. 2 c; Table S6). Global catabolic activity in rhizospheric soil beneath V. myrtillus and P. abies gradually decreased with increasing elevation and had significantly greater values at 1400 -1600 m compared to those at higher elevations (Fig 2 a,b; Table S6). However, global catabolic activity in rhizospheric soil of J.

communis did not show any significant differences amongelevation bands (Fig. 2 c; Table S6).

Global catabolic activity in rhizospheric soil from V. myrtillus and P. abies at 1400, 1600

and 1800 m was higher compared to bulk soil (Fig 2 a,b). Above 1800 m, bulk and rhizospheric catabolic activity values from V. myrtillus and P. abies converged and were not significantly different (Fig. 2 a,b; Table S6). In samples from beneath J. communis, although catabolic activity in rhizospheric soil was generally higher than in bulk soil, differences were not significant (Fig. 2 c; Table S6). The mean catabolic diversity index (H´) of both bulk and rhizospheric soils was 2.4 and no significant differences were found (data not shown) either in bulk or rhizospheric soil, among plant species or along the elevation gradient.

No clear trend between global microbial catabolic activity and elevational bands in bulk and rhizospheric soil (NMDS plot, -Fig. S3), however the PERMANOVA test showed that elevation explained the majority of variation in catabolic activity in both rhizospheric soil (p<0.001, R 2 =0.36; Table 2) and bulk soil (p=0.01, R 2 =0.10; Table 2). In contrast, the variation in catabolic activity not explained by elevation was greater in bulk soil (residuals=0.87; Table 2) than in rhizosphere soil (residuals=0.52; Table 2). In addition, there was a significant influence of the interaction between plant species and elevation (p=0.006, R 2 =0.08) and of plant species (p=0.03, R 2 =0.05) on catabolic activity in rhizospheric soil.

Finally, there was no significant influence of plant species (p=0.43, R 2 =0.02) nor of the interaction between plant species and elevation (p=0.87, R 2 =0.01; Table 2) on catabolic activity in bulk soil.

Relationships between abiotic factors and global catabolic activity and diversity (H') in bulk and rhizospheric soil

Climatic data did not explain global catabolic activity in either bulk (p=0.40, adjusted R 2 =0.01; Table 3) and rhizospheric soil (p=0.11, adjusted R 2 =0.04; Table 3).. Nor were any significant relationships were found between either MAP, MAT or MAR and catabolic activity or H' beneath P. abies or J. communis in bulk soil (Spearman correlation coefficients, Table 4) However, MAT was positively correlated with catabolic activity beneath V.

myrtillus, whereas both MAP and MAR were negatively correlated (Table 4; Fig. S3). Bulk soil H' under V. myrtillus was negatively correlated with MAP (Table 4). In the rhizosphere of V. myrtillus, MAP and MAR were negatively and significantly correlated while MAT was significantly and positively correlated with catabolic activity (Table 4; Fig. S3). In the rhizosphere of P. abies, MAR was negatively and significantly correlated with catabolic activity (Table 4; Fig. S4). No climatic parameters were significantly correlated to global catabolic activity in the rhizospheric soil of J. communis.

The only significant relationships between H' and climatic variables were with MAP and MAT in the rhizosphere of V. myrtillus. MAP was positively correlated while MAT was negatively correlated with rhizospheric catabolic diversity (Table 4).

Along the elevation gradient, variations in soil physical and chemical properties were the main drivers explaining global catabolic activity in bulk soil even though the proportion of the variance was still limited (p=0.01, adjusted R 2 =0.09; Table 3). Soil texture, C, P and N contents, cation exchange capacity and pH were particularly important since catabolic activity was strongly and positively related to bulk soil SOC, soil TN, P, CEC and clay content but decreased with higher pH and sand content (Fig. 3, Table S7). Regarding catabolic diversity, it was significantly and positively correlated with silt content in bulk soil beneath V. myrtillus and P. abies (Table S7) but no relationships were found beneath J.

communis (Table S7).

According to the partition of variance analysis, global catabolic activity in rhizospheric soil was not significantly explained by soil physical and chemical properties (p=0.71, adjusted R 2 =0.02; Table 3). However, when examining plant species individually, rhizospheric soil global catabolic activity was, to a lesser extent than bulk soil, positively related to SOC, TN, P, CEC and clay content, but decreased at higher pH and sand content, mostly in the rhizosphere of V. myrtillus (Table S7). Underneath P. abies, pH was the only soil property to be negatively and significantly correlated to rhizospheric soil catabolic activity (Table S7).

Changes in H' were few, but occurred mostly in the rhizosphere of V. myrtillus, where they were strongly related to litter depth, soil water content, topsoil aggregate stability and ammonium content (Table S7).

Relationships between plant diversity and global catabolic activity and diversity (H') in bulk and rhizospheric soil

Vegetation cover varied along the elevational gradient (Table S8). Vegetation cover and trees decreased with elevation while shrub cover increased [START_REF] Stokes | Shifts in soil and plant functional diversity along an altitudinal gradient in the French Alps[END_REF][START_REF] Weemstra | Patterns in intraspecific variation in root traits are species-specific along an elevation gradient[END_REF]. Graminoid and forb covers were greater at 1800 m -2000 m. Simpson diversity in the quadrats containing V. myrtillus and P. abies changed significantly with elevation. At mid-elevations (1800 m -2000 m), plant diversity peaked and slowly decreased at 2200 m -2400 m (Table S8). However, plant diversity in the quadrats containing J. communis did not vary along the elevation gradient (Table S8).

Catabolic activity was negatively correlated with plant diversity in rhizospheric soil beneath V. myrtillus and bulk soil beneath P. abies, with no other significant relationships found among structuring plant species (Table S9). No significant relationships were found between H' and plant diversity in either bulk or rhizospheric soil (Table S9).

Relationships between plant root traits (community-and species-) and global catabolic activity and diversity (H´) in bulk and rhizospheric soil

Overall, global catabolic activity was best explained by RDcom, SOC, MAT, MWDtop and RNCcom (db-RDA, Fig 4)

In bulk soil, catabolic activity was best explained by community level root traits (partition of variance analysis: p=0.02, adjusted R 2 =0.11; Table 3) and to a lesser extent by species level root traits (p=0.06, adjusted R 2 =0.04; Table 3) although in both cases the proportion of variance was low.

Spearman correlation coefficients using community level root traits showed that RD and lignin content were always significantly and positively correlated with catabolic activity in bulk soil beneath all three plant species, whereas cellulose content was always negatively correlated (Fig. 5; Table S9). Hemicellulose was significantly and negatively correlated with bulk soil catabolic activity beneath J. communis only. SRLcom and RLDcom were both significantly and negatively correlated with bulk soil catabolic activity beneath V. myrtillus only (Table S9). With regard to H' in bulk soil, the only relationships between community level root traits were beneath V. myrtillus, whereby RNCcom was positively correlated and RC:Ncom was negatively correlated (Table S9).

At the species level, the only root trait that was significantly related to global catabolic activity in bulk soil was RDMCsp that was negatively correlated beneath P. abies only (Table S9). There were no other significant relationships between H' and root traits at the species level in bulk soil beneath all three plant species (Table S9).

Global catabolic activity in rhizospheric soil was not significantly explained by either community-(partition of variance analysis: p=0.64, adjusted R 2 = -0.07; Table 3) or species-(p=0.24, adjusted R 2 = -0.03; Table 3) level root traits.

In the rhizosphere, there were few significant relationships between global catabolic activity and community level root traits across the structuring species. Root N content was positively and significantly correlated with catabolic activity beneath V. myrtillus (Table S9) and RC:Ncom was negatively correlated beneath P. abies and V. myrtillus (Table S9). As in the bulk soil, root cellulose content was negatively correlated with catabolic activity, but in the rhizosphere of V. myrtillus only (Table S9). RLDcom was also negatively and significantly correlated with catabolic activity in the rhizosphere of V. myrtillus (Table S9). The only significant relationship between H' in the rhizosphere and community level root traits was beneath V. myrtillus and in contrast to that found in the bulk soil, RNCcom was negatively correlated with H' in the rhizosphere (Table S9).

With regard to species-level root traits, as in bulk soil, RDMCsp was negatively and significantly correlated with catabolic activity beneath P. abies only (Table S9). RTDsp was negatively correlated with catabolic activity in the rhizosphere of V. myrtillus only (Table S9). However, no significant relationships were found between H' and species level root traits in the rhizosphere of any plant species.

Discussion

In agreement with our first hypothesis (H1), catabolic activity decreased at higher elevations (above 1800 m in the case of rhizospheric soil from P. abies and V. myrtillus). Along the elevation gradient, variations in soil physical and chemical properties were the main drivers of catabolic activity, especially texture,pH, cation exchange capacity, carbon and nitrogen content Changes in catabolic diversity were few, but occurred mostly in the rhizosphere of V. myrtillus, where they were strongly related to litter depth and topsoil aggregate stability.

Contrary to our H2, catabolic activity was negatively correlated with plant diversity in rhizospheric soil beneath V. myrtillus and bulk soil beneath P. abies, with no other significant relationships found among plant species. Also refuting our H2, we found no relationships between plant diversity and catabolic diversity in either bulk or rhizospheric soil. With regard to root traits, our H3 was not corroborated in that root traits that enhanced rhizospheric dimensions had little effect on global catabolic activity or diversity in the rhizosphere. Also contrary to our H3, we found that at the community level, root diameter (for V. myrtillus and J. communis) and lignin content (for V. myrtillus ) were positively correlated with catabolic activity in bulk soil only, and had no effect on diversity. Also, root nitrogen and carbon contents had a negligible effect on catabolic activity and diversity, except for roots from V.

myrtillus. Root cellulose content was negatively related to catabolic activity in bulk soil beneath all plant species and rhizospheric soil from V. myrtillus only, and had no effect on catabolic diversity. Trends in catabolic activity with root traits measured at the species level were only found for rhizospheric soil beneath P. abies and V. myrtillus, where more dense root tissue decreased catabolic activity, but had no effect on functional diversity.

Changes in catabolic activity and diversity along the elevation gradient and relationship with climatic factors

At elevations below 1800 mglobal catabolic activity in the rhizosphere was 2-3 times greater than in bulk soil for P. abies and V. myrtillus, converging beyond 1800 m. Juniperus communis was not present at lower elevations, therefore biasing results for this species. As sampling was performed in early June, vegetation at lower elevations would have already commenced growth and photosynthesis several weeks previously (Wang et al., 2018), thus stimulating the production of exudates during root elongation. At higher elevations, the growing season was only just beginning, and plants were commencing physiological processes after winter dormancy. Therefore, root exudate production and microbial activity in the rhizosphere would be less than at lower elevations. In line with this, changes in climate also partially explained trends in catabolic activity in the rhizosphere: increased MAP and MAR decreased catabolic activity in the rhizosphere of V. myrtillus, but activity increased with higher MAT. Similar relationships were found in bulk soil beneath this structuring species, but were less distinct. The only relationship found between a climatic variable and catabolic activity for another structuring species, was in the rhizosphere beneath P. abies, where, as for V. myrtillus, high MAR decreased catabolic activity. Incident solar radiation can reach very high levels with increasing altitude, and causes photodegradation of plant litter (through the production of volatile compounds via photochemical mineralization) and photofacilitation (stimulation of biotic activity due to changes in litter chemistry) [START_REF] Méndez | Solar radiation exposure accelerates decomposition and biotic activity in surface litter but not soil in a semiarid woodland ecosystem in Patagonia, Argentina[END_REF]. Therefore, at the treeline, where tree density is very sparse (stand basal area was only 18 m² ha -1 at our treeline site, [START_REF] Mao | Characterizing Above-and Belowground Carbon Partitioning in Forest Trees along an Altitudinal Gradient using Area-Based Indicators[END_REF]), aboveground C input to mineral soil would be reduced via the biotic acceleration of C turnover in the litter layer [START_REF] Méndez | Solar radiation exposure accelerates decomposition and biotic activity in surface litter but not soil in a semiarid woodland ecosystem in Patagonia, Argentina[END_REF].

Unlike the study by [START_REF] Esch | Direct and indirect effects of shifting rainfall on soil microbial respiration and enzyme activity in a semi-arid system[END_REF], where manipulated rainfall increased soil microbial activity, we found that high MAP decreased catabolic activity beneath V. myrtillus along the elevation gradient. As this species is an understory herb, with minimal buffering of precipitation by a large canopy, the effects of rainfall will be accentuated as precipitation reaches the soil surface directly. Similar results were found by [START_REF] Shi | An increase in precipitation exacerbates negative effects of nitrogen deposition on soil cations and soil microbial communities in a temperate forest[END_REF], who performed a rainfall manipulation experiment in a moist, temperate deciduous forest. In their study, increased precipitation rapidly decreased fungal biomass but with no effect on bacterial biomass, and it was suggested that high rainfall reduced sodium concentrations in the soil with a detrimental effect on fungal activity. However, it seems more likely that high MAP will impact photosynthesis via stomatal closure [START_REF] Zhang | Benefits of flooding-induced aquatic adventitious roots depend on the duration of submergence: Linking plant performance to root functioning[END_REF][START_REF] Li | Ecological responses to heavy rainfall depend on seasonal timing and multi-year recurrence[END_REF], decreasing the flow of photoassimilates and the subsequent production of C-rich root exudates into the rhizosphere. Also, heterotrophic respiration can be suppressed in extremely moist conditions [START_REF] Horz | Ammonia-oxidizing bacteria respond to multifactorial global change[END_REF][START_REF] Zhao | Soil microbial community composition and respiration along an experimental precipitation gradient in a semiarid steppe[END_REF].

We also found a positive relationship between MAT and global catabolic activity in the rhizosphere of V. myrtillus, as several studies have shown that soil warming increases microbial activity [START_REF] Grayston | Accounting for variability in soil microbial communities of temperate upland grassland ecosystems[END_REF][START_REF] Papatheodorou | The effects of large-and small-scale differences in soil temperature and moisture on bacterial functional diversity and the community of bacterivorous nematodes[END_REF]. [START_REF] Adamczyk | Strong shifts in microbial community structure are associated with increased litter input rather than temperature in High Arctic soils[END_REF] showed that increased microbial respiration rates at higher temperatures may be explained by additional C input to cold, nutrient-poor soils that are becoming warmer as a result of climate change. Additionally, increases in temperature stimulate microbial metabolism through changes in physiology or enzyme functioning involved in organic matter decomposition [START_REF] Tang | Effects of temperature, soil substrate, and microbial community on carbon mineralization across three climatically contrasting forest sites[END_REF][START_REF] Nottingham | Adaptation of soil microbial growth to temperature: Using a tropical elevation gradient to predict future changes[END_REF]. Warmer temperatures also increase plant growth and biomass, stimulating photosynthesis and hence the production of C rich root exudates [START_REF] Rossi | Pathways to persistence: plant root traits alter carbon accumulation in different soil carbon pools[END_REF]. Enhanced root exudation with warmer temperatures has also been shown to increase microbial catabolic diversity [START_REF] Papatheodorou | The effects of large-and small-scale differences in soil temperature and moisture on bacterial functional diversity and the community of bacterivorous nematodes[END_REF].

Nevertheless, [START_REF] Klimek | Soil physicochemical and microbial drivers of temperature sensitivity of soil organic matter decomposition under boreal forests[END_REF] found a decrease in catabolic diversity at higher temperatures because less functionally diverse microbial communities were unable to effectively degrade various organic substrates. Our results show that catabolic diversity decreased with higher MAT in rhizospheric soil, possibly indicating that colder temperatures lead to specialised microorganisms in these alpine soils [START_REF] Donhauser | Alpine soil microbial ecology in a changing world[END_REF][START_REF] Collins | Belowground impacts of alpine woody encroachment are determined by plant traits, local climate and soil conditions[END_REF].

Changes in catabolic activity and diversity along the elevation gradient and relationship with soil physical and chemical properties

Along the elevation gradient, bulk soil physical and chemical properties were major drivers of catabolic activity and to a lesser extent, catabolic diversity., SOC, TN, P, CEC, NH4 + , sand and clay content were all strongly related to each other and generally decreased at higher altitudes (except for sand content that increased). Several authors report similar findings regardless of climate type (e.g., [START_REF] Xu | Soil microbial community structure and activity along a montane elevational gradient on the Tibetan Plateau[END_REF][START_REF] Kotas | Soil microbial biomass, activity and community composition along altitudinal gradients in the High Arctic (Billefjorden, Svalbard)[END_REF][START_REF] Hofmann | Distribution of prokaryotic abundance and microbial nutrient cycling across a high-alpine altitudinal gradient in the austrian central alps is affected by vegetation, temperature, and soil nutrients[END_REF][START_REF] Praeg | Microbial diversity in bulk and rhizosphere soil of ranunculus glacialis along a high-alpine altitudinal gradient[END_REF]. Therefore, we show that along an elevation gradient, nutrient content and texture had a greater influence on microbial activity in bulk soil than climate and vegetation.

The influence of soil properties on catabolic activity in the rhizosphere was less evident. Soil water content and pH had the strongest influence on catabolic activity in rhizospheric soil of V. myrtillus, with most activity in drier, acidic soils. Catabolic activity in the rhizosphere of P. abies was also greatest in the most acidic soils, suggesting that they were dominated by acid-tolerant microbial species [START_REF] Shen | Soil pH drives the spatial distribution of bacterial communities along elevation on Changbai Mountain[END_REF]. The respiratory activity of fungi increases under acidic conditions while that of bacteria decreases [START_REF] Blagodatskaya | Interactive effects of pH and substrate quality on the fungal-to-bacterial ratio and qCO2 of microbial communities in forest soils[END_REF].

Therefore, if soil pH increases, the fungal contribution to respiration and decreasing overall catabolic activity could decrease, as observed in our results. A strong negative relationship did exist between catabolic diversity, litter depth and NH4 + in the rhizosphere of V. myrtillus.

NH4 + is the nitrogen source on which V. myrtillus typically relies [START_REF] Roth | Nitrogen deposition is positively correlated to foliar nitrogen content in Vaccinium myrtillus and other understory species in temperate forests on acidic soil[END_REF] but [START_REF] Morvan | Into the wild blueberry (Vaccinium angustifolium) rhizosphere microbiota[END_REF] found a high proportion of di-nitrogen (N2) fixers (Rhizobiales) in the rhizosphere of Vaccinium angustifolium. Therefore, the availability of NH4 + in soil beneath Vaccinium sp. could reduce catabolic diversity if N2-fixing bacteria dominate the rhizosphere, reflected in the negative relationship that we found between root nitrogen content and catabolic diversity in the rhizosphere of V. myrtillus. Alternatively, nutrient-poor soils could moderate competition among microbial species. [START_REF] Eldridge | Competition drives the response of soil microbial diversity to increased grazing by vertebrate herbivores[END_REF] found that altered levels of soil carbon changed the competitive abilities of different microbial phyla, with reduced carbon promoting bacterial diversity, but decreasing fungal diversity. Carbon input to soil and litter depth are strongly associated, therefore, although litter depth increases catabolic activity in the rhizosphere of V. myrtillus, competition between microbial phyla may be increased, leading to less catabolic diversity with increasing litter depth.

An increase in aggregate stability in topsoil was negatively related to catabolic activity in the rhizosphere of V. myrtillus, but catabolic diversity increased. However, catabolic activity increased in the more stable subsoil aggregates. The decrease of catabolic activity with soil aggregate stability is likely due to physical protection of carbon in more stable aggregates (Chevalier, 2011), that may induce a reduction of catabolic activity. Many studies have highlighted the prominent role of microbial communities promoting aggregate stability (e.g. [START_REF] Bossuyt | Influence of microbial populations and residue quality on aggregate stability[END_REF][START_REF] Cosentino | Aggregate stability and microbial community dynamics under drying-wetting cycles in a silt loam soil[END_REF]. Therefore, although [START_REF] Aspiras | The role of microorganisms in the stabilization of soil aggregates[END_REF] postulated that the status of soil aggregation is determined by the cumulated effects of synthesis and degradation of binding materials by microbial populations, rather than the activity of specific microorganisms, our results suggest that species-specific microbial activity can have diverse effects on aggregate stability .

Relationships between plant diversity and catabolic activity and diversity

Generally,the more diverse a plant species assemblage, the more diverse the chemistry of the root exudates and litter produced [START_REF] Klimek | Covariation of soil bacteria functional diversity and vegetation diversity along an altitudinal climatic gradient in the Western Carpathians[END_REF]. Therefore, when plant diversity indices are high, more ecological niches should be available for soil microorganisms, as the spatial heterogeneity of soil resources and increased availability of labile carbon sources should promote catabolic activity [START_REF] Creamer | Measuring respiration profiles of soil microbial communities across Europe using MicroResp TM method[END_REF][START_REF] Bongiorno | Soil management intensity shifts microbial catabolic profiles across a range of European long-term field experiments[END_REF]. However, the relationship between species assemblage and the microbial communities of any given plant species is not necessarily direct. Individual plant species may be positively or negatively affected by increased plant diversity (Losapio et al.,2021) ), also affecting the production of root exudates and microbial communities closely related to them. Here, we found that contrary to our second hypothesis, catabolic activity was negatively correlated with plant diversity in rhizospheric soil of V. myrtillus and bulk soil beneath P. abies, with no other significant relationships found among structuring species or sampling location.

Unlike [START_REF] Klimek | Covariation of soil bacteria functional diversity and vegetation diversity along an altitudinal climatic gradient in the Western Carpathians[END_REF], we did not discriminate between bacterial and fungal catabolic diversity in our study. It is therefore possible that the relative contribution of fungi and bacteria to global catabolic activity varied depending on the soil under study. The presence of various exudates may also impact catabolic activity differently, for example, [START_REF] Yuan | Exudate components exert different influences on microbially mediated C losses in simulated rhizosphere soils of a spruce plantation[END_REF] found that the addition of three common components of root exudates (oxalic acid, glucose and glycine) altered considerably microbial communities, activities and related processes. Hence, this disparity could explain the observed lower catabolic diversity in the rhizosphere of V. myrtillus and in the bulk soil under P. abies.

Influence of plant root traits on catabolic activity and diversity

Along this complex environmental gradient, where multiple properties simultaneously change, root traits of different species varied in diverse ways, leading to species-specific patterns in intraspecific root trait variation (described in detail by [START_REF] Weemstra | Patterns in intraspecific variation in root traits are species-specific along an elevation gradient[END_REF] and associated microbial responses. We expected that in bulk soil, where litter is a major carbon source and microbial decomposers dominate, an increase in root nitrogen and carbon content should enhance global catabolic activity and diversity, but this hypothesis was only true for V. myrtillus, where catabolic diversity was positively related to root nitrogen content.

Additionally, our hypothesis that plant root traits that increase rhizosphere dimensions or fungal colonisation (e.g., longer, thinner roots with large exchange surface areas), would increase global catabolic activity and diversity was not corroborated in either bulk or rhizospheric soil. However, at the community level, thicker roots did increase catabolic activity in bulk soil beneath V. myrtillus and J. communis. Root thickness is partially linked to root age, and so reflects chemical composition. Thin, woody roots are usually young and possess a greater quantity of cellulose [START_REF] Genet | The influence of cellulose content on tensile strength in tree roots[END_REF][START_REF] Hales | Topographic and ecologic controls on root reinforcement[END_REF], that is negatively related to lignin content [START_REF] Thomas | Lignin and cellulose concentrations in roots of Douglas fir and European beech of different diameter classes and soil depths[END_REF]. Our results showed that at the community level, root diameter and cellulose content were negatively related in P. abies and V. myrtillus, and lignin content was positively correlated with root diameter beneath all three plant species ( Figure S4). Therefore, relationships between catabolic activity and root diameter may not be causal, but rather due to root age and chemical composition. We found a strong positive relationship between catabolic activity and root lignin content (at the community level) in all bulk soils. Lignin is considered difficult to degrade [START_REF] Roumet | Root structure-function relationships in 74 species: Evidence of a root economics spectrum related to carbon economy[END_REF][START_REF] Poirier | The root of the matter: Linking root traits and soil organic matter stabilization processes[END_REF], but within microbial fungal and bacterial phyla, diverse strategies exist to facilitate the decomposition of lignin, allowing microbial specialists to thrive in different ecological niches [START_REF] Janusz | Lignin degradation: Microorganisms, enzymes involved, genomes analysis and evolution[END_REF]. Therefore, lignin-degrading fungal and bacterial specialists are likely to be dominant in these root-and leaf-litter rich soils.

Trends in catabolic activity with root traits measured at the species level were only found for rhizospheric soil beneath P. abies and V. myrtillus, where more dense root tissue decreased catabolic activity. The changes in root chemical composition that we observed will be reflected in tissue density: cellulose increases wood density and lignin decreases density (Nuopponen et al., 2006), thus explaining why dense tissue decreases catabolic activity.

Our results demonstrate that relationships between microbial functioning and abiotic and biotic variables are complex in a highly heterogeneous environment. Nevertheless, we show that the main drivers of microbial activity and functioning are mean annual temperature and soil physicochemical properties but the presence of vegetation enhances these relationships through belowground characteristics such as root exudation, root chemical composition and changes in litter production that affect soil carbon. Therefore, in this environment, shifts in functional diversity of soil microbes under future climate scenarios could be mediated primarily by plant species, because soil properties will be slower to change. To better understand these relationships, future studies should examine temporal fungal and bacterial community taxonomic diversity and functioning, and how they relate to plant growth and root demography in a wide range of plant species. SOC is soil organic carbon; TN is total nitrogen; C/N is soil carbon-nitrogen ratio; CEC is cation exchange capacity; Kfs is soil hydraulic conductivity; MWDsub is mean weight diameter of subsoil aggregates; MWDtop is mean weight diameter of topsoil aggregates Table S4. Effect of structuring plant species and elevation on soil physical and chemical properties along the elevation gradient tested with unbalanced two-way ANOVA (Type-III sums of squares). Bold values show a significant effect (p<0.05).

Tables

Two

Figure 1 . 21 Figure 4 .Figure 5 .

 12145 Figure 1. Sampling design along the elevational gradient. (a) Six elevational bands, situated at 200 3 m from each other, were located along the gradient, ranging from 1400 m to 2400 m. Five plots (20 4 m x 20 m) containing two or three selected structuring plant species were located at each altitude. (b) 5At the canopy limit of the structuring plant species, a 1 m x 1 m botanical survey was performed. (c) 6In the center of this quadrat, a soil monolith (0.25 m x 0.25 m x 0.15 m) was extracted. 7

  34 myrtillus (orange), P. abies (green) and J. communis (blue) along the elevational gradient. 35 Correlation coefficients (⍴), p-values, regression equations and R 2 are shown. Lines are 36 linear regressions through data.

Table 1 .

 1 Main characteristics of the elevation gradient. Mean values and standard deviation in brackets for climatic data, elevation and GPS coordinates measured in each of the five plots (further detail on the data description available in the data paper[START_REF] Stokes | Shifts in soil and plant functional diversity along an altitudinal gradient in the French Alps[END_REF] and raw data available at https://data.inrae.fr/dataverse/ecopics).

	Elevational band (m)	Elevation (m.asl.)	GPS coordinates	MAT (°C)	MAP (mm)	MAR (MJ/m 2 )	Life zone
	1400	1366 (15)	N 45°08ʹ15ʺ E 5°85ʹ79ʺ	8.5 (0.2) 1024 (41)	4204 (0)	Montane
	1600	1601 (12)	N 45°09ʹ25ʺ E 5°86ʹ91ʺ	7.3 (0.7) 1066 (20)	4181 (0)	Subalpine
	1800	1799 (20)	N 45°10ʹ83ʺ E 5°89 ʹ 27 ʺ	8.1 (0.5)	1110 (7) 4575 (102) Subalpine
	2000	1971 (11)	N 45°11ʹ73ʺ E 5°90ʹ28ʺ	5.7 (0.7) 1155 (12)	4463 (0)	Subalpine
	2200	2208 (19)	N 45°12ʹ73 ʺ E 5°92ʹ29ʺ	3.8 (0.1) 1205 (20)	4339 (0)	Alpine
	2400	2405 (17)	N 45°12ʹ95ʺ E 5°93ʹ03ʺ	5.7 (0.2) 1187 (40)	4339 (0)	Alpine

MAT: Mean annual temperature, MAP: Mean annual precipitation, MAR: Mean annual solar radiation

Table 2 .

 2 Effect of structuring plant species and elevation on microbial global catabolic activity in bulk and rhizospheric soil assessed with PERMANOVA.

				Bulk soil					Rhizospheric soil
	Factors	Df Sums	Means	F	R 2	P(>F)	Df Sums	Means	F	R 2	P(>F)
			of Sqs	sqs					of Sqs	sqs
	Plant species	2	0.05	0.02	0.84	0.02	0.43	2	0.19	0.09	3.20	0.05	0.03
	Elevation	1 0.210	0.21	7.35	0.1	0.01	1	1.29	1.29	44.01	0.36 0.001
	Plant	2	0.01	0.006	0.02	0.005	0.87	2	0.28	0.14	4.72	0.08 0.006
	species*Elevation
	Residuals	64	1.83	0.03		0.87		64	1.87	0.03		0.52
	Total	69	2.11			1.0		69	3.63			1.0

Table 3 .

 3 Partition of variance in constrained ordination distance-based redundancy analysis (db-RDA) for four environmental variables (soil physical and chemical properties, community-and species-level root traits and climatic data) influencing microbial catabolic activity and diversity for bulk and rhizospheric soil samples.

				Bulk soil			Rhizospheric soil
		Df	R 2	R 2 adjusted	P	Df	R 2	R 2 adjusted	P
	Climate	3	0.05	0.01	0.40	3	0.08	0.04	0.11
	Soil properties	15	0.29	0.09	0.01	15	0.23	0.02	0.71
	Community level	12	0.26	0.11	0.02	12	0.11	-0.07	0.64
	root traits
	Species level root	3	0.08	0.04	0.06	3	0.02	-0.03	0.24
	traits
	Values in bold are significant at p<0.05; values in italics are significant at p<0.1

Table 4 .

 4 Spearman correlations between global microbial catabolic activity and diversity (H') and climatic data of the three structuring plant species along the elevational gradient (significance levels p < 0.0001 ****; p < 0.001 ***, p < 0.01 **, p < 0.05 *).

	Bulk soil

MAT is mean annual temperature; MAP is mean annual precipitation; MAR is mean annual solar radiation.

Table S3 .

 S3 Mean values of soil physical and chemical properties along the elevation gradient beneath Vaccinium myrtillus, Juniperus communis and Picea abies. Standard deviation is shown in brackets. Different lowercase letters indicate significant differences (Tukey´s HSD test; p<0.05) between elevational bands following a one-way ANOVA.

	Clay (g/kg)	295 (48.0) ab	431.8 (160.7) a	342.4 (25.6) ab	346.2 (54.0) ab	373.4 (58.2) ab	238.4 (54.1) b	359 (63.4) a	319.8 (75.0) a	352.2 (104.4) a	254.2 (89.0) a	323.6 (31.1) b	488 (151.9) a	368.6 (21.7) ab	366.2 (56.5) ab
	Plant species Silt (g/kg)	215.9 (15.2) a	211.4 (71.3) ab	Vaccinium myrtillus 225.9 200.1 (18.1) a (30.3) ab	195.5 (28.4) ab	161.2 (14.2) b	232 (29.2) b	Juniperus communis 198.9 207.1 (41.0) ab (60.6) ab	159.7 (28.4) a	224.6 (23.9) a	Picea abies 192.1 218.3 (43.3) a (16.2) a	191.1 (37.9) a
	Elevation (m)	1400	1600	1800	2000	2200	2400	1800	2000	2200	2400	1400	1600	1800	2000
	SOC (g/kg) Sand (g/kg)	127.5 (66.5) b 136.6 (29.6) ab	313.7 (126.1) a 72.7 (89.4) a	140.9 (55.36) b 102.9 (25.4) a	107.3 (39.1) b 126.8 (60.8) ab	183.8 (65.2) ab 117.8 (44.2) a	93.1 (26.5) b 219.6 (84.0) b	178.1 (111.1) a 88.5 (33.5) b	86.4 (31.9) a 141.2 (75.6) ab	209 (86.9) a 116.8 (45.3) ab	104.3 (39.2) a 213.2 (74.2) a	169.0 (71.9) b 113.6 (24.0) a	336.4 (136.9) a 63.9 (80.4) a	136.6 (24.2) b 97.4 (15.9) a	145.4 (62.0) b 125.8 (69.3) a
	Litter Depth TN (g/kg) (cm)	6.3 8.30 (2.0) b (3.46) a	13.8 3.10 (5.6) a (1.95) b	7.4 2.80 (1.3) b (0.27) b	7.2 1.40 (2.5) b (1.04) b	9.8 3.40 (3.0) ab (0.55) b	6.1 1.20 (1.2) b (0.84) b	9.1 4.0 (4.5) a (1.46) b	6.2 1.30 (2.1) a (0.76) a	11.9 3.80 (5.9) a (1.68) b	6.6 1.10 (2.2) a (0.22) a	7.6 8.20 (1.7) b (3.49) b	13.3 5.80 (4.2) a (2.80) ab	7.7 5.30 (1.0) b (1.86) ab	8.8 1.80 (1.1) b (0.91) a
	P (g/kg) Kfs (cm/h)	0.021 (0.011) b 9.57 (5.69) a	0.061 (0.032) a 9.97 (2.88) a	0.028 (0.012) ab 8.02 (6.73) a	0.031 (0.018) ab 6.11 (6.94) a	0.028 (0.011) ab 4.22 (3.18) a	0.016 (0.005) b 4.18 (4.52) a	0.028 (0.012) a 6.38 (5.17 )a	0.023 (0.017) a 11.43 (10.62) a	0.027 (0.011) a 7.02 (7.89) a	0.017 (0.006) a 6.21 (5.56) a	0.024 (0.008) b 10.09 (4.76) a	0.079 (0.05) a 19.04 (11.32) a	0.020 (0.004) b 8.85 (8.65) a	0.039 (0.014) ab 12.73 (9.95) a
	MWDsub	3.11	2.95	3.14	2.55	2.92	2.33	3.06	2.49	2.91	2.69	3.16	2.94	3.12	2.95
	NH4 (mg/kg) (mm) MWDtop (mm) NO3 (mg/kg) Water	36.05 (0.08) a (34.27) a 0.58 0 (0) b (0.70) a 10.16	45.84 (0.32) a (24.02) a 0.31 2.98 (0.44) a (0.23) a 10.18	33.98 (0.14) a (11.21) a 0.34 3.18 (0.09) a (0.48) a 43.59	29.34 (0.81) a (11.82) a 1.04 2.87 (0.41) a (1.47) a 40.43	13.30 (0.20) a (4.36) a 0.15 3.05 (0.24) a (0.004) a 56.37	10.62 (0.63) a (7.40) a 0.15 2.53 (0.69) a (0.00) a 42.38	37.83 (0.31) a (15.57) a 1.04 3.01 (0.10) a (1.43) a 44.68	23.80 (0.87) a (12.70) a 0.27 2.89 (0.33) a (0.14) a 38.34	25.93 (0.35) a (25.42) a 0.80 2.50 (1.41) a (1.43) a 58.66	10.80 (0.39) a (2.54) a 0.28 2.68 (0.29) a (0.28) a 39.69	31.70 (0.12) a (8.56) b 0.36 0 (0) b (0.26) a 9.98	77.34 (0.27) a (52.54) a 0.28 2.92 (0.23) a (0.13) a 10.17	48.59 (0.22) a (10.56) b 1.43 2.99 (0.24) a (1.37) a 45.31	27.15 (0.21) a (18.29) b 2.90 (0.43) a 1.18 (2.23) a 39.13
	C/N content (%)	(5.0) ab 19.2 (0.18) a	(2.4) a 22.5 (0.15) a	(4.2) ab 18.4 (19.46) b	(0.7) b 14.7 (12.02) b	(1.2) ab 18.4 (12.52) b	(1.8) b 15.04 (5.12) b	(3.3) b 18.7 (20.66) a	(0.8) a 13.7 (6.69) a	(2.4) b 18.3 (11.37) a	(1.6) ab 15.6 (9.36) a	(4.7) a 21.2 (0.19) a	(4.1) a 24.4 (0.38) a	(3.5) a 17.8 (20.87) b	(6.4) a 16.4 (23.27) b
	CEC	12.95	31.80	16.96	14.83	13.92	8.83	21.78	14.47	16.25	9.08	17.84	36.62	18.74	19.86
	(cmol + /kg)	(6.67) b	(15.62) a	(6.27) ab	(3.54) b	(4.08) b	(3.13) b	(13.32) a	(3.54) a	(6.97) a	(3.0) a	(5.72) a	(17.59) a	(4.97) a	(8.19) a
	pH	4.50 (0.24) ac	4.37 (0.43) a	4.9 (0.51)abc	5.35 (0.39) b	4.58 (0.10) ac	5.17 (0.19) bc	4.98 (0.60) ab	5.58 (0.42) a	4.75 (0.39) b	5.13 (0.35) ab	4.51 (0.30) a	4.63 (0.28) a	5.06 (0.51) a	5.08 (0.29) a

Table S5 .

 S5 Mean comparison of bulk and rhizospheric soil basal respiration and global catabolic activity for the three structuring species (V. myrtillus, P. abies and J. communis) along the elevational gradient using a two tailed t-test (p <0.05).

				-way ANOVA	Bulk soil					Rhizospheric soil
		Factor	Elevation Plant Species Elevation * Plant Species Basal Respiration Global catabolic activity		Basal Respiration	Global catabolic activity
	SOC (g/kg) Elevational band Structuring Species	0.620 Mean	0.787 SD	Mean	SD	0.825 p-value	Mean	SD	Mean	SD	p-value
	TN (g/kg) P (g/kg) 1400 Vaccinum myrtillus NH4 (mg/kg) 1600 Vaccinum myrtillus 1800 Vaccinum myrtillus	0.796 0.557 3.14 0.090 6.45 3.63	0.986 0.965 0.44 0.918 2.52 0.40	3.31 6.08 3.62	0.972 0.62 0.931 2.82 0.846 0.40	0.21 0.25 0.95	9.35 8.47 4.74	3.08 1.99 1.55	10.77 8.96 4.96	3.38 1.84 1.58	0.51 0.70 0.15
	NO3 (mg/kg) 2000 Vaccinum myrtillus	0.379 2.44	0.165 0.31	2.42	0.118 0.33	0.66	2.89	0.92	2.97	1.02	0.26
	C/N 2200	Vaccinum myrtillus	0.513 3.23	0.227 0.81	3.20	0.323 0.72	0.70	3.76	0.85	3.93	0.71	0.74
	CEC (cmol + /kg) 2400 Vaccinum myrtillus	0.078 2.68	0.745 0.93	2.95	0.691 0.87	0.21	3.24	1.03	3.15	0.86	0.56
	pH			0.668	0.132			0.150
	Clay (g/kg) 1400 Picea abies		0.137 2.96	0.646 0.69	3.06	0.551 0.74	0.14	6.87	2.20	7.56	2.10	0.17
	Silt (g/kg) 1600 Picea abies	<0.001 5.49	0.139 1.25	5.83	0.190 1.49	0.14	6.22	1.69	7.21	2.67	0.14
	Sand (g/kg) 1800 Picea abies		0.005 3.60	0.251 0.39	3.67	0.245 0.32	0.47	4.98	2.00	5.16	2.38	0.58
	Litter Depth (cm) 2000 Picea abies		0.157 2.78	0.060 0.47	2.86	0.077 0.68	0.48	2.79	0.33	2.86	0.66	0.68
	Water Content (%)		0.863	0.021			0.025
	Kfs (cm/h) 1800 Juniperus communis 0.734 4.88	0.895 2.21	5.17	0.748 2.75	0.32	3.57	1.30	3.83	1.53	0.11
	0.437 Juniperus communis 2.48 MWDsub (mm) 2000 MWDtop (mm) 0.443 2200 Juniperus communis 3.64 2400 Juniperus communis 2.53	0.701 0.44 <0.001 1.57 0.96	2.46 3.62 2.87	0.622 0.45 1.49 <0.001 1.19	0.43 0.77 0.71	4.05 4.74 3.31	1.14 1.09 0.58	4.40 5.12 3.27	1.86 2.02 0.68	0.63 0.59 0.81

SOC is soil organic carbon; TN is total nitrogen; C/N is soil carbon-nitrogen ratio; CEC is cation exchange capacity; Kfs is soil hydraulic conductivity; MWDsub is mean weight diameter of subsoil aggregates; MWDtop is mean weight diameter of topsoil aggregates

Acknowledgements

We thank our very helpful students and colleagues for their huge contribution in the field and in the laboratory: Nereyda Cruz-Maldonado, Mathieu Fontaine, Stephane Fourtier, Leonor Jiménez, Beatriz Marín-Castro, Zhun Mao, Awaz Mohamed, Hans Reijnen, Hervé Rey and Katrin Sieron. Funding was provided by the French and Mexican governments (ECOPICS project, ANR-16-CE03-0009 and CONACYT-2 73659). DHC was funded by a doctoral bursary from CONACYT (471700). Thanks are due to the Mairie de Chamrousse for access to field sites. Partial data were presented in the session "Forest Management Practices and the Responses of Soil Carbon, Water, Nutrients and Their Interactions" organised at the XXV IUFRO World Congress 2019, "Forest Research and Cooperation for Sustainable Development," held at Curitiba, Brazil; 29 September -5 October 2019.

1.1 Supplementary materials (tables) Estimation of soil volume explored by a root system [START_REF] Barber | Effect of Tillage Practice on Corn (Zea mays L.) Root Distribution and Morphology[END_REF] Root length density (RLD) 𝑅𝐿𝐷 = 𝑅𝑀𝐷 𝑥 𝑆𝑅𝐿 Estimation of soil volume explored by a root system [START_REF] Barber | Effect of Tillage Practice on Corn (Zea mays L.) Root Distribution and Morphology[END_REF] L is root length (mm); V is root volume (cm 3 ); RDM is root dry mass (g); RFM is root fresh mass (g); SRL is specific root length (m/g -1 ), RTD is root tissue density (g/cm -3 ); RDMC is root dry mass content (mg/g -1 ); RMD is root mass density (g/cm -3 ); RLD is root length density (m/cm -3 ); Soil volume (cm -3 ) where roots were sorted.

Plant diversity

Simpson index -0.19 -0.21 -0.53* 0.14 0.14 0.22 -0.82**** 0.27 -0.26 -0.12 0.11 0.17 is root diameter; RDMC is root dry mass content; SRL is specific root length; RTD is root tissue density; RMD is root mass density; RLD is root length density; RNC is root nitrogen content; RCC is root carbon content, RC:N is root carbon to nitrogen ratio; subscriptcom denotes community level traits and subscriptsp denote species level root traits. [START_REF] Piedallu | Soil water balance performs better than climatic water variables in tree species distribution modelling[END_REF][START_REF] Piedallu | Soil aeration, water deficit, nitrogen availability, acidity and temperature all contribute to shaping tree species distribution in temperate forests[END_REF], Stokes et al., 2021).