Heidy Schimann

Corinne Vacher

Sabrina Coste

Eliane Louisanna

Tania Fort

Lucie Zinger

Assembly processes of bacterial endophytic and epiphytic communities in the canopy of neotropical trees

Keywords: assembly rules, bacterial phyllosphere communities, foliar traits, 16S metabarcoding, endophytes, epiphytes, neotropical tree, within-canopy vertical gradient

Bacterial phyllosphere communities play a key role in the fitness of plants. Understanding the factors and mechanisms shaping them is necessary to predict how trees would respond to a variety of stresses or disturbances. In tropical forests, the steep vertical gradients in light, rainfall or temperature within the canopy are likely to drive the assembly of bacterial communities in leaves. We explored the assembly processes driving epiphytic and endophytic bacterial communities within the canopy of three Neotropical tree species in French Guiana.

We used 16S high-throughput sequencing to characterize leaf bacterial communities and measured foliar traits of host trees to describe variations within the canopy. We found that selection by foliar traits was more important in endophytic communities with a particular effect of traits related to space and available nutrients. Epiphytic communities were also subjected to selection by foliar traits but were more prone to dispersal limitation on top of the canopy. This study provides evidence that the assembly bacterial communities in leaves of tropical trees is jointly driven by determinism and stochasticity. Moreover, the clear differences found between endophytic and epiphytic communities emphasize the major role of the plant host, and provide new perspectives to further investigating the phyllosphere-bacteria interactions.

Plants have a long history of co-evolution with microorganisms and their interactions have proved a key asset to the colonization of land by plants [START_REF] Strullu-Derrien | The origin and evolution of mycorrhizal symbioses: from palaeomycology to phylogenomics[END_REF]. Trees, as all plants, interact with microbes. These interactions do not only take place in tree roots, but also in leaves, an environment called the phyllosphere. A huge diversity of microorganisms colonizes the phyllosphere, either on the surface (epiphytes) or within the internal tissues of leaves (endophytes, [START_REF] Rosado | The importance of phyllosphere on plant functional ecology: a phyllo trait manifesto[END_REF]. These microbial communities encompass most microbial life forms (including filamentous fungi, yeasts and protists), but are largely dominated by bacteria [START_REF] Rosado | The importance of phyllosphere on plant functional ecology: a phyllo trait manifesto[END_REF][START_REF] Stone | The Role of the Phyllosphere Microbiome in Plant Health and Function[END_REF][START_REF] Chaudhry | Shaping the leaf microbiota: plant-microbe-microbe interactions (S Kopriva[END_REF].

Phyllosphere microbial communities (PMCs) have received growing attention in recent years due to their influence on plant fitness [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF][START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF][START_REF] Rosado | The importance of phyllosphere on plant functional ecology: a phyllo trait manifesto[END_REF].

In particular, PMCs can modify photosynthesis by inducing an increase in leaf chlorophyll content, or hydraulics through an increase of the stomatal conductance [START_REF] Rho | Endophyte Effects on Photosynthesis and Water Use of Plant Hosts: A Meta-Analysis[END_REF][START_REF] Rosado | The importance of phyllosphere on plant functional ecology: a phyllo trait manifesto[END_REF]. PMCs produce several beneficial effects on plant/insect interactions by reducing predator insect developmental rate, causing feeding deterrence, or by reducing their survival and oviposition [START_REF] Omacini | Symbiotic fungal endophytes control insect host-parasite interaction webs[END_REF][START_REF] Fernandez-Conradi | Fungi reduce preference and performance of insect herbivores on challenged plants[END_REF][START_REF] Vacher | Trees and Insects Have Microbiomes: Consequences for Forest Health and Management[END_REF]. Last, PMCs upregulate host defensive pathways leading to increased seedling survival and a better plant defense [START_REF] Arnold | Fungal endophytes limit pathogen damage in a tropical tree[END_REF][START_REF] Álvarez-Loayza | Light Converts Endosymbiotic Fungus to Pathogen, Influencing Seedling Survival and Niche-Space Filling of a Common Tropical Tree, Iriartea deltoidea[END_REF][START_REF] Christian | Exposure to the leaf litter microbiome of healthy adults protects seedlings from pathogen damage[END_REF]. Disentangling the factors and mechanisms shaping PMCs should thus improve our understanding of how trees would respond to a variety of stresses or disturbances.

As for communities of macro-organisms, the assembly of PMCS is driven by four fundamental processes: selection (niche-based mechanisms recruiting a particular set of species, i.e. environmental filtering and biotic interactions, [START_REF] Vellend | Conceptual Synthesis in Community Ecology[END_REF], ecological drift (neutral stochastic demographic processes, [START_REF] Chase | Disentangling the importance of ecological niches from stochastic processes across scales[END_REF][START_REF] Hubbell | The Unified Neutral Theory of Biodiversity and Biogeography (MPB-32)[END_REF], dispersal (movement and establishment of species across space preventing local extinction through immigration or mass effect, [START_REF] Chase | Disentangling the importance of ecological niches from stochastic processes across scales[END_REF][START_REF] Hanson | Beyond biogeographic patterns: processes shaping the microbial landscape[END_REF] and evolutionary diversification (processes generating new genetic variation, [START_REF] Nemergut | Patterns and Processes of Microbial Community Assembly[END_REF]. In the case of PMCs, the host may participate in selection and dispersion, e.g. by controlling the quantity and quality of the nutrient pool available, or by producing antimicrobial molecules, thereby selecting particular microbial taxa. The host traits responsible for these selective pressures can be conserved within host phylogenetic clades and leave an imprint on the structure of the microbial community [START_REF] Kohl | Ecological and evolutionary mechanisms underlying patterns of phylosymbiosis in host-associated microbial communities[END_REF]. In the case of a selection by the host, the composition of PMCs should partly result from interactions with the host plant and the host response to its environment, rather than stochastic processes [START_REF] Brooks | Phylosymbiosis: Relationships and Functional Effects of Microbial Communities across Host Evolutionary History (D Relman[END_REF][START_REF] Kohl | Ecological and evolutionary mechanisms underlying patterns of phylosymbiosis in host-associated microbial communities[END_REF]. Hence, the effects of host phylogeny and environmental determinants on the beta-diversity of microbial communities have been disentangle in several studies (for a review see [START_REF] Lim | An introduction to phylosymbiosis[END_REF]. However, host genotype or physiology can also exert strong effects on communities independently of host phylogeny (Amato et al., 2019a,b), or environmental conditions may obscure signals [START_REF] Brooks | Phylosymbiosis: Relationships and Functional Effects of Microbial Communities across Host Evolutionary History (D Relman[END_REF]. Last, priority effects could also play a role in the assembly of plant microbiomes [START_REF] Zheng | Niche Specialization and Functional Overlap of Bamboo Leaf and Root Microbiota[END_REF].

In tropical trees, PMCs remain understudied [START_REF] Harrison | The diversity and distribution of endophytes across biomes, plant phylogeny and host tissues: how far have we come and where do we go from here?[END_REF]. So far, available studies showed variations of PMCs among tree species [START_REF] Redford | The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves[END_REF][START_REF] Kembel | Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest[END_REF], but few have analyzed the processes driving their assembly [START_REF] Donald | A test of community assembly rules using foliar endophytes from a tropical forest canopy[END_REF].

Because of the huge diversity in tree species [START_REF] Steege H Ter | Hyperdominance in the Amazonian Tree Flora[END_REF] which develop a wide range of ecological strategies [START_REF] Fortunel | Leaf, stem and root tissue strategies across 758 Neotropical tree species[END_REF]Allié et al., 2015) , partly translated into the chemistry and the morphology of their leaves [START_REF] Hättenschwiler | High variation in foliage and leaf litter chemistry among 45 tree species of a neotropical rainforest community[END_REF][START_REF] Courtois | Differences in volatile terpene composition between the bark and leaves of tropical tree species[END_REF], neotropical trees could thus recruit either generalist or specialist PMCs. Recent studies report, however, contradicting conclusions on the effect of the identity of the host plant species, with some studies reporting a significant effect [START_REF] Laforest-Lapointe | Host species identity, site and time drive temperate tree phyllosphere bacterial community structure[END_REF], 2017;[START_REF] Gomes | Endophytic and Epiphytic Phyllosphere Fungal Communities Are Shaped by Different Environmental Factors in a Mediterranean Ecosystem[END_REF][START_REF] Yao | Phyllosphere epiphytic and endophytic fungal community and network structures differ in a tropical mangrove ecosystem[END_REF][START_REF] Yao | Host identity is more important in structuring bacterial epiphytes than endophytes in a tropical mangrove forest[END_REF], while others reporting no effect [START_REF] Griffin | Plant host identity and soil macronutrients explain little variation in sapling endophyte community composition: Is disturbance an alternative explanation[END_REF]. Part of this inconsistency may lie in that previous studies did not necessarily consider environmental variability. Indeed, PMCs are exposed to strong vertical gradients in light and water availability, which have been previously suggested to influence their composition and diversity [START_REF] Harrison | Vertical stratification of the foliar fungal community in the world's tallest trees[END_REF][START_REF] Izuno | Vertical Structure of Phyllosphere Fungal Communities in a Tropical Forest in Thailand Uncovered by High-Throughput Sequencing (AM Yurkov[END_REF] and could also modify community assembly processes themselves. Such gradients are particularly steep in tropical forests, where the biotic and abiotic conditions in the canopy are very different from those of the understory. The forest top canopy is exposed to strong winds [START_REF] Bittar | Estimation of Throughfall and Stemflow Bacterial Flux in a Subtropical Oak-Cedar Forest[END_REF], sunlight and high temperatures [START_REF] Shaw | Vertical organization of the canopy biota[END_REF], while the understory receives only 3% of the light and 30% of the rainfalls [START_REF] Calder | Canopy processes: implications for transpiration, interception and splash induced erosion, ultimately for forest management and water resources[END_REF].

These environmental heterogeneities are likely to drive PMCs assembly. In addition, PMCs live in two distinct microenvironments: the internal part (endophytic PMCs) and the surface (epiphytic PMCs) of the leaf more exposed to external environmental conditions [START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF]. Consequently, colonization success of the leaf surface may depend on particular microbial functions such as the ability to extract nutrients from the internal tissues while developing on the leaf surface, or live on the few nutrients that leak to the leaf surface, and the ability to withstand dry environments and UVs, through motility or biofilm formation [START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF][START_REF] Chaudhry | Shaping the leaf microbiota: plant-microbe-microbe interactions (S Kopriva[END_REF]. In contrast, internal tissues of the leaf are richer in nutrients, but contain more plant defense metabolites suggesting that endophytic and epiphytic communities could be driven by different factors. Accordingly, in mangrove tree species, epiphytic PMCs are more strongly shaped by the host than endophytic ones [START_REF] Yao | Host identity is more important in structuring bacterial epiphytes than endophytes in a tropical mangrove forest[END_REF].

Additionally, in Olive trees, variations of nutrients and environmental conditions between internal and external plant tissues determined strong differences in the structure of bacterial communities with notably a dominance of phyla resistant to desiccation and radiation at the surface of leaves [START_REF] Mina | Epiphytic and Endophytic Bacteria on Olive Tree Phyllosphere: Exploring Tissue and Cultivar Effect[END_REF].

In this study, we quantified the assembly processes shaping epiphytic and endophytic bacterial communities from the bottom to the top of the canopy of three Neotropical tree species in French Guiana. We used DNA metabarcoding to characterize leaf bacterial communities and measured morphological and chemical foliar traits of host tree to describe variations in the microhabitat of bacteria along the vertical gradient. We specifically tested the hypothesis: (H1) vertical stratification is stronger in epiphytic than in endophytic communities, because epiphytes respond to the within-canopy microclimate gradient ; (H2) the assembly of epiphytic bacterial communities is more deterministic at the top than at the bottom of the canopy, because of harsher abiotic conditions (higher UV radiations and lower humidity) at the top of the canopy ; (H3) foliar traits contribute more to the assembly of endophytic than epiphytic communities, because endophytes are intimately linked to their host tree.

Material and Methods: Study site and sampling design

Leaf sampling was conducted in October 2017 at the Nouragues Experimental Station (4°02 N, 52°414 W) with the help of COPAS (Canopy Operating Permanent Access System) and tree climbers (Figure 1a). Three tree species abundant in the studied area were selected: Eperua falcata Aubl. (Caesalpiniaceae), Macrolobium bifolium Aubl. (Caesalpiniaceae) and Tetrasgastris Gaertn. sp (Burseraceae). E. falcata is a shade hemi-tolerant dominant species growing in relatively dense clusters and on a large spectrum of edaphic conditions [START_REF] Roggy | Leaf natural 15 N abundance and total N concentration as potential indicators of plant N nutrition in legumes and pioneer species in a rain forest of French Guiana[END_REF][START_REF] Baraloto | Seed mass, seedling size and neotropical tree seedling establishment[END_REF]. Macrolobium is a widespread genus throughout the Amazonian basin [START_REF] Murphy | On the Monophyly of Macrolobium Schreb., an Ecologically Diverse Neotropical Tree Genus (Fabaceae-Detarioideae)[END_REF]. Tetragastris sp. belongs to the family Burseraceae, an important Neotropical tree lineage known for their chemical defenses including terpenes and phenolics [START_REF] Fine | Phylogenetic community structure and phylogenetic turnover across space and edaphic gradients in western Amazonian tree communities[END_REF]. The sampling area was approximately 2 ha of homogeneous forest habitat. Five individuals of each tree species were selected, tall enough to have leaves on top of the canopy in full light, and geolocalized (Figure 1b). On each individual, we sampled leaves at 3 heights (Figure 1c): on top of the crown (Top Canopy, TC), in the middle of the crown (Middle Canopy, MC) and on the lower branch of the crown (Bottom Canopy, BC). Three leaves were collected at each height for DNA analysis. Each leaf was collected in a separate plastic bag to avoid cross contaminations, resulting in 135 leaf samples (3 tree species x 5 individuals x 3 heights x 3 leaves). In addition, we collected 2 leaves for each height and each individual, that were further used for leaf traits measurements. Leaves were sealed immediately after sampling in pre-weighted zip-lock plastic bags and placed in a cooler until return to the lab.

Microclimate measurements

Irradiance at each height (TC, MC, BC) was recorded with a photosynthetically active radiation linear sensor (Accepter model LP-80 PAR/LAI Ceptometer, Decagon Devices). Instantaneous measurements were realized at the level of sampled leaves for 4 trees and values were then compared to a reference sensor located at the top of a nearby flux tower (NOURAFLUX, nouragues.cnrs.fr), which provide an estimate of the incident irradiance above the canopy (i.e. the full irradiance). For each height, relative irradiance (%) was computed as the ratio of the local and above the canopy irradiance.

Air temperature and relative humidity were measured with environmental HOBO sensors (model U23-001, HOBO Pro V2 Temp/RH Data logger, Amanvillers, France) placed at each canopy height (TC, MC, BC) on two trees over 24 hours.

Morphological and chemical leaf traits measurements

We measured 10 leaf traits that may influence the diversity, composition and function of leaf bacterial communities, because they are indicative of (1) water availability, (2) space available for bacterial colonization, (3) sugars availability, or (4) defense metabolism.

To approximate (1) water availability for leaf bacteria, we measured leaf water content (LWC) and stomatal density (SD). The aperture and density of stomata control the exchange of gases (water and carbon dioxide) between the interior of the leaf and the atmosphere, participating in the control of water by the whole plant [START_REF] Hetherington | The role of stomata in sensing and driving environmental change[END_REF]. Stomata are also a major route of bacterial colonization inside the leaf [START_REF] Chaudhry | Shaping the leaf microbiota: plant-microbe-microbe interactions (S Kopriva[END_REF]. To approximate (2) space available for bacterial colonization, we measured 4 morphological traits: leaf dry matter content (LDMC), leaf mass per area (LMA), leaf thickness (LT) and leaf area (LA). All four are informative of the production of biomass by the plant and the density of leaf [START_REF] Garnier | Consistency of species ranking based on functional leaf traits[END_REF]. To approximate (3) sugars availability for leaf bacteria, we measured Chlorophyll and Nitrogen contents, which reflect the photosynthesis and the metabolism activities of the plant [START_REF] Field | photosynthesis--nitrogen relationship in wild plants. On the economy of plant form and function[END_REF][START_REF] Evans | The allocation of protein nitrogen in the photosynthetic apparatus: Costs, consequences, and control[END_REF]. Finally, leaf carbon content and leaf construction cost (CCM) were used as proxies of (4) plant defense, given that the defense response involves a wide array of mechanisms which demand a massive redistribution of energy [START_REF] Chaudhry | Shaping the leaf microbiota: plant-microbe-microbe interactions (S Kopriva[END_REF]. One sampled leaf was used to measure SD while the other one was used to measure LWC and all other traits. To measure SD, a band of lamina was fixed with a FAA buffer (5% formalin, 5% acetic acid, 50% ethanol). Stomatal density of the adaxial surfaces of the lamina (SD, number of stomata per cm 2 ) was then determined in the lab with imprints made using transparent nail varnish. The number of stomata was recorded using an optical microscope for each lamina as the average of five randomly selected areas of 1cm 2 each [START_REF] Leroy | Ants mediate foliar structure and nitrogen acquisition in a tank-bromeliad[END_REF]. To measure the other traits, fresh weight (fw, g) was determined by weighing the sealed pre-weighted bags containing the fresh leaves. Leaf area (LA, cm2) of fresh leaves was measured with a scanner and the ImageJ software [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]. Leaf thickness (LT, µm) was estimated as the mean of three to eight measurements (four per simple leaf and one per leaflet for compound leaf) with a digital micrometer (Digimatic micrometer, Mitutoyo, Japan). We took care to avoid the main veins for these measurements. Chlorophyll contents (Chl, µg cm -2 ) were estimated with a portable chlorophyll meter (SPAD-502 Konica-Minolta, Osaka, Japan). Four to eight SPAD measurements per leaf (one per leaflet for compound leaf) were averaged to obtain the SPAD estimate of the leaf. We then used the calibration equation proposed by [START_REF] Coste | Assessing foliar chlorophyll contents with the SPAD-502 chlorophyll meter: a calibration test with thirteen tree species of tropical rainforest in French Guiana[END_REF]. Leaves were then dried at 60°C for 72h and weighted (dry weight, g). Leaf water content (LWC=fw-dw/fw, %), leaf mass per area (LMA=dw/LA, g m -2 ) and leaf dry matter content (LDMC=dw/fw, mg g -1 ) were calculated.

Leaves were then ground to determine C, N and ash contents (%) at the USRAVE platform of Bordeaux (France). Leaf specific construction cost (CCM, g glucose g -1 dw) was estimated from C, N and ash content, according to [START_REF] Poorter | Construction costs and payback time of biomass: a whole plant perspective[END_REF]. As in [START_REF] Coste | A cost-benefit analysis of acclimation to low irradiance in tropical rainforest tree seedlings: leaf life span and payback time for leaf deployment[END_REF], this computation assumes all nitrogen is absorbed as nitrate hence an additional cost for nitrate reduction in non-photosynthetic tissues was taken into account.

DNA sampling

To collect epiphytic DNA, the whole upper and lower surfaces of each leaf were carefully wiped with a piece (2x2 cm) of Whatman paper sterilized by autoclaving (120°C, 20 min) and soaked in sterile CTAB buffer (2% cetyl trimethylammonium bromide, 1% polyvinyl pyrrolidone, 100 mM Tris-HCl, 1.4 M NaCl, 20 mM EDTA), within 4 hours after leaves harvesting. The Whatman paper was stored in a 2ml Eppendorf tube filled with CTAB for downstream DNA extraction. Surface-sterilization protocol was performed as recommended by [START_REF] Compant | The plant endosphere world -bacterial life within plants[END_REF] to collect endophytic communities. Each leaf was surface-sterilized in 0.525% sodium hypochlorite (2 min) and 70% ethanol (2 min) and two segments of 2 cm 2 area from the lamina were cut and stored in a 2ml Eppendorf tube filled with CTAB for downstream DNA extractions close to a field Bunsen burner to create a sterilized environment. This resulted in 270 DNA samples (3 tree species x 5 individuals 3 heights x 3 leaves x 2 DNA types (epiphytic or endophytic)).

DNA extraction, amplification and sequencing

DNA was extracted using a CTAB extraction method [START_REF] Carrell | Pinus flexilis and Picea engelmannii share a simple and consistent needle endophyte microbiota with a potential role in nitrogen fixation[END_REF]. We added 800 µl of CTAB solution to 0.6 g of tissue, incubated for 2 h at 60°C, and homogenized with glass beads for 3 min. Proteins were removed with the addition of an equal volume of chloroform, centrifuged for 10 min at 16 000 g, and placed the top aqueous phase in a sterile tube. Nucleic acids were precipitated with the addition of 1/10 volume of cold 3 M sodium acetate and 1/2 volume cold isopropanol, froze them at -20°C for 12 h, and centrifuged for 30 min at 16 000 g.

The supernatant was discarded, 700 µl of 70% ethanol was added to the solution, and centrifuged for 10 min. The air-dried pellet was resuspended with 30 µl of DNA resuspension fluid (1.0 M Tris-HCL and 0.1 M EDTA) and stored it at -20°C. The V5-V6 region of the bacterial 16S rRNA gene was amplified using the chloroplast-excluding forward primer 799f [START_REF] Chelius | The Diversity of Archaea and Bacteria in Association with the Roots of Zea mays L[END_REF] and the reverse primer 1115R [START_REF] Reysenbach | Reliable amplification of hyperthermophilic archaeal 16S rRNA genes by the polymerase chain reaction. Archaea: a laboratory manual[END_REF]. Forward and reverse primers were tagged in 5' with a combination of two different 8-nucleotide labels.

The PCR amplification was done in 25 μl with 1x buffer, 0.22 mM dNTP each, 0.45 μM each tagged primer, 2.84 mM MgCl2, 0.11 mg/ml bovine serum albumin and 0.04 U/μl Taq polymerase (Solis Biodyne). The thermocycling conditions were as follows: 5 min at 94°C, 30 cycles of 30 sec at 94°C, 30 sec at 58°C and 40 sec at 78°C. The PCR reactions were done for each sample separately and amplicons were quantified with a fluorescence-based method (Qubit 3.0, Invitrogen Life Tech) and pooled in equimolar conditions. The library was built using the Fasteris MetaFast protocol (FASTERIS SA, Plan-les-Ouates, Switzerland) and sequenced on one run of a MiSeq Illumina platform (FASTERIS SA,) using the paired-end sequencing technology. To control for potential contaminants [START_REF] Salter | Reagent and laboratory contamination can critically impact sequence-based microbiome analyses[END_REF] and false positive sequences caused by tag-switching events [START_REF] Esling | Accurate multiplexing and filtering for highthroughput amplicon-sequencing[END_REF] the sequenced multiplexes comprised extractions/PCR negative controls and unused tag combinations.

Bioinformatics

A total of 3.181.610 sequencing reads were obtained and curated using the OBITools3 package [START_REF] Boyer | obitools: a unix-inspired software package for DNA metabarcoding[END_REF] and the R software version 3.5.3 (R Core Team 2019) following the procedure described in [START_REF] Zinger | Body size determines soil community assembly in a tropical forest[END_REF]. Paired-end reads were assembled and assigned to their respective samples. After dereplication, low quality sequences (i.e. containing Ns, being shorter than 80 pb or singletons) were excluded. PCR/sequencing errors were removed from the dataset and remaining reads were clustered into operational taxonomic units (OTUs) at 97% similarity using the Sumaclust algorithm [START_REF] Kopylova | Open-Source Sequence Clustering Methods Improve the State Of the Art[END_REF]. The most abundant read was considered representative of the OTU and taxonomically assigned using the SILVAngs r138.1 [START_REF] Quast | The SILVA ribosomal RNA gene database project: improved data processing and web-based tools[END_REF]. Finally, we checked the taxonomic assignment and kept only sequences assigned to the Bacteria kingdom. Curation procedure yielded to 649,212 reads corresponding to 10152 bacterial OTUs in 203 samples. Finally, because the sequencing depths were uneven across samples (Figure S1), the sequencing depth of each sample was standardized by randomly resampling a number of reads equal to the first quartile of read number across samples (n=1864) which removed 51 samples and kept 152 samples for subsequent analyses.

Statistical analyses

All analyzes were conducted using the R software (R Development Core Team) and with packages vegan 2.5-7 [START_REF] Oksanen | vegan: Community Ecology Package[END_REF], adespatial 1.7-17 [START_REF] Dray | adespatial: Multivariate Multiscale Spatial Analysis[END_REF] with 999 permutations [START_REF] Anderson | Permutation tests for univariate or multivariate analysis of variance and regression[END_REF] and dissimilarities among samples were visualized using a Principal Coordinate Analyses (PCA). The model included geographical position of trees (3 significant PCNMS), host species (E. falcata, M. bifolium, Tetragastris sp.), vertical position in the canopy (low, middle and top of the canopy), position on the leaf (epiphytes and endophytes) and their interactions as fixed effects. The homogeneity of variance among groups (host species, position in the canopy and on the leaf) groups were tested as PERMANOVA analysis are sensitive to heterogeneity in within-group variance [START_REF] Oksanen | vegan: Community Ecology Package[END_REF].

The relationships between foliar traits and the alpha-(Shannon index) and beta-diversity (Bray-Curtis distance) of endophytic and epiphytic communities were assessed using a linear mixed model and a permutational multivariate analysis of variance, respectively. Both models included leaf traits related to water resources (LWC, SD), available space (LDMC, LMA, LT, LA), available sugars (Chlorophyll and N contents) and defense metabolism (CCM and C content) implemented as fixed effects, and host species implemented as random effect.

Homogeneity of variance among host species was also tested after performing the PERMANOVA model.

Significant variations of the leaf traits mentioned above were beforehand tested individually by performing an analysis of variance (ANOVA, p<0.01). The model included the vertical positions in the crown, the host species and their interactions. Normality of each variable was tested with a Shapiro test and variables were transformed with a box-cox when necessary.

To disentangle the relative contribution of niche-based and neutral processes in the assembly of epiphytic and endophytic communities in leaves separately, we implemented a null modeling approach as proposed by [START_REF] Stegen | Quantifying community assembly processes and identifying features that impose them[END_REF] and [START_REF] Vass | Using null models to compare bacterial and microeukaryotic metacommunity assembly under shifting environmental conditions[END_REF]. Pairwise phylogenetic turnover between communities were calculated with the mean-nearest-taxon-distance (βMNTD) metric [START_REF] Fine | Phylogenetic community structure and phylogenetic turnover across space and edaphic gradients in western Amazonian tree communities[END_REF] to infer the strength of selection. MUSCLE (Edgar 2004) with default parameters was used to align sequences found in communities of each vertical position (TC, MC, BC). Pairwise distances were computed with a Juke-Cantor model (JC69) and built using a phylogenetic tree with the UPGMA method for each community. The pairwise phylogenetic turnover between communities was calculated as the mean nearest taxon distance metric (βMNTD) while accounting for taxa relative abundance. According to [START_REF] Stegen | Quantifying community assembly processes and identifying features that impose them[END_REF], the degree to which βMNTD deviates from a null model expectation should reflect the degree to which community composition is driven by selection, which is expected to recruit phylogenetically related OTUs due to niche conservatism. To quantify the deviation, we built a null model by shuffling randomly taxa names and abundances across tips of the phylogeny, and then calculated βMNTD. This allowed us to obtain the null distribution of βMNTD, after 999 randomizations. Then, β-nearest taxon index (βNTI) was computed as the difference between observed βMNTD and the mean of the null distribution in units of s.d. of the null distribution. For this procedure we used the R codes provided by [START_REF] Tripathi | Soil pH mediates the balance between stochastic and deterministic assembly of bacteria[END_REF].

According to [START_REF] Stegen | Quantifying community assembly processes and identifying features that impose them[END_REF], when |βNTI|>2, the observed difference in phylogenetic turnover between communities is the result of selection. When |βNTI|<2, the observed difference in phylogenetic community composition should result from dispersal limitation, ecological drift or homogenizing dispersal. We quantified these processes by computing Bray-Curtis based Raup-Crick metric (RCbray) on pairwise comparisons when |βNTI|<2. Following the method proposed by [START_REF] Dini-Andreote | Disentangling mechanisms that mediate the balance between stochastic and deterministic processes in microbial succession[END_REF], the relative contribution of each process to variations in community composition were estimated as follow: dispersal limitation was estimated as the percentage of pairwise comparisons with |βNTI| <2 and RCbray> +0.95; homogenizing dispersal was estimated as the percentage of pairwise comparisons with |βNTI| <2 and RCbray< -0.95 ; and ecological drift was estimated as the percentage of pairwise comparisons with |βNTI| <2 and |RCbray|< 0.95. We then computed the contribution of each process (selection, dispersal limitation, homogenizing dispersal or ecological drift) for each position in the canopy in epiphytic and endophytic communities.

Results

Variations in leaf microclimate, physiology and phenology within the canopy and across tropical tree species

Temperature and relative humidity showed no significant differences between the top and the bottom of the canopy (86% +-12 % of relative humidity and 26 +-3°C), but the relative irradiance varied from 77 % + 34% of the PAR at the top of the canopy to 1.55% +-1.5% on the soil (Figure 1c). All leaf traits exhibited strong and significant variations among host species and positions within the canopy (Figure 2). Interactions between both factors were only significant for C, N contents and CCM (Figure 2). Most leaf trait variations followed the same trends across the different tree species. Leaf SD, LDMC, LMA, LT and chlorophyll content decreased from the top to the bottom of the canopy while LWC and LA increased.

Leaf bacterial community composition associated to tropical tree species

Leaf bacterial communities were dominated by 6 classes including Alphaproteobacteria (49.06%), Actinobacteria (23.64%), Sphingobacteria (5.79%), Gammaproteobacteria (4.50%), Chloroflexia (5.43%) and Cytophagia (2.44%) (Figure S2). 5 orders represented 76% of the OTUs: Rhizobiales, Actinomycetales, Sphingomonadales, Sphingobacteriales and Rhodospirillales (Figure S3). We were able to assign 200 families and 630 genera with mainly Methylobacterium (12.60%), and Sphingomonas (10.58%).

Factors driving leaf bacterial communities at the leaf, the canopy and tree scales

Bacterial community composition varied significantly across host species, geographical position and within the leaf (Table 1). Host species was the main driver of the bacterial assemblage in leaves, explaining 10.7% (Table 1) of bacterial composition and influencing significantly its diversity (Table S1). However, tree species showed no deep differences regarding their bacterial communities at the Class or Family taxonomic rank (Figure S2) indicating that variations of bacterial communities among their host species occur at lower taxonomic ranks. Surprisingly, the variation in community composition within the canopy was less important than differences between leaf inside tissues and its surface suggesting that community assemblage of Bacteria in tropical tree species occur at the leaf spatial scale.

Assembly processes in endophytic and epiphytic communities

In endophytic communities, deterministic assembly processes (bNTI < |2|) represented 50.3%, 52.1% and 43.7% at the top, middle and bottom of the canopy respectively (Figure 3a). Overall, no significant difference of pairwise comparisons of bNTI was observed suggesting that deterministic and stochastic factors are equally important in endophytic communities within the canopy. However, when estimating the effect of each process within the canopy we found that endophytic communities were mainly driven by selection (52.10% at TC, 43.7% at MC and 50.3% at BC) and ecological drift (41% at TC, 39.5% at MC and 47 % at BC) whereas dispersal limitation had limited effects at the top (6.8%) and bottom of the canopy (2.6%) (Figure 4a). Interestingly, the latest had more effect in the middle of the canopy (16.8%).

In epiphytic communities, deterministic assembly processes (bNTI < |2|) represented 34.1%, 49.6% and 43.1% at the top, middle and bottom of the canopy respectively (Figure 3b). We found significant differences of pairwise comparisons of βNTI values overall among the three vertical positions (Figure 3b). As for endophytic communities, epiphytic communities were also mainly driven by selection (34% at TC, 49.6% at MC and 43.1% at BC) and ecological drift (46.5% at TC, 44% at MC and 50.6% % at BC). Dispersal limitation had increasing effect on epiphytic communities from the top to the bottom of the canopy (19.4% at TC, 6.55% at MC and 6.25% at BC, Figure 4b).

Effects of leaf traits in bacterial epiphytic and endophytic communities:

Leaf traits accounted for 21.6 % and 18.4% of the variation in endophytic and epiphytic community composition, respectively (Table 2). In endophytic communities, 9.17% of the variation were explained by LDMC (3.6%), Chlorophyll (2.97%) and C contents (2.60%). In epiphytic communities, 9.17% of the variation were significantly explained by LT (2.57%), Chlorophyll (1.88%), N (2.07%) and C (2.48%) contents. Leaf traits had no significant effects on endophytic and epiphytic alpha diversity (Table S2).

Discussion

Host species is a strong driver of bacterial leaf communities

Bacterial communities in the phyllosphere were largely dominated by Alphaproteobacteria, Actinobacteria and Bacteroidetes which is in line with previous observations in tropical trees [START_REF] Kembel | Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest[END_REF][START_REF] Griffin | Tree Endophytes: Cryptic Drivers of Tropical Forest Diversity[END_REF][START_REF] Donald | A test of community assembly rules using foliar endophytes from a tropical forest canopy[END_REF]. Proteobacteria are known to carry methylotrophy, nitrification or nitrogen fixation, Bacteroidetes phyllosphere bacteria are often aerobic and pigmented suggesting a good adaptation to this environment and Actinobacteria are plant pathogens, nitrogen-fixers, fungal antagonists or decomposers [START_REF] Stone | The Role of the Phyllosphere Microbiome in Plant Health and Function[END_REF]. Sphingomonas (Sphingomonales) and Methylobacterium (Rhizobiales), the two most abundant genera, are common in phyllosphere communities [START_REF] Bulgarelli | Structure and Functions of the Bacterial Microbiota of Plants[END_REF].

Methylobacterium uses the methanol produced by the leaf metabolism as source of carbon and energy and Sphingomonas produces multiple transport proteins suggesting an ability to scavenge diverse plant metabolites [START_REF] Delmotte | Community proteogenomics reveals insights into the physiology of phyllosphere bacteria[END_REF]. In line with previous studies on tropical tree species [START_REF] Kembel | Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest[END_REF][START_REF] Laforest-Lapointe | Host species identity, site and time drive temperate tree phyllosphere bacterial community structure[END_REF][START_REF] Harrison | The diversity and distribution of endophytes across biomes, plant phylogeny and host tissues: how far have we come and where do we go from here?[END_REF], host species were the most important drivers of PMC, together with the geographical factors. Simultaneously, the position on the leaf was also significant to a lesser extent, contrary to what was observed in [START_REF] Laforest-Lapointe | Host species identity, site and time drive temperate tree phyllosphere bacterial community structure[END_REF]Herrmann et al. (2021). Contrary to our first hypothesis, no prominent impact of the vertical stratification on diversity or composition was observed, neither in endophytic or in epiphytic communities.

Deterministic and stochastic processes equally govern the assembly of endophytic and epiphytic communities across the vertical gradient

According to the β-nearest taxon index (βNTI), both deterministic and stochastic processes are jointly responsible for the assembly of leaf PMCs [START_REF] Stegen | Quantifying community assembly processes and identifying features that impose them[END_REF][START_REF] Vass | Using null models to compare bacterial and microeukaryotic metacommunity assembly under shifting environmental conditions[END_REF], Zhou & Ning 2017[START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF]. Niche-based factors included ecological filters imposed by the environment, here we showed a wide variation in microclimate (mainly irradiance) from the top of the canopy to the base of the crown, but also by the host, namely the physiological and morphological variations of leaves across tree species and induced by the irradiance vertical gradient. Overall, and contrary to our expectations, separate analyses of endophytic and epiphytic bacterial communities showed that endophytic communities were more prone to determinism (46%) than epiphytic ones (37%), but with variations according to the position in the canopy. The assembly of the endophytic and epiphytic was also significantly driven by stochastic processes such as dispersal limitation, ecological drift or diversification. The importance of these effects differed between epiphytes and endophytes communities and across the vertical gradient. Dispersal limitations had more effects on endophytic PMCs assembly in the middle of the crown of the tree whereas it had higher effects at the top of the canopy for epiphytic PMCs. Beside the seed and germination environment, bioaerosols are one of the major sources of the colonization of the leaf by bacteria [START_REF] Bulgarelli | Structure and Functions of the Bacterial Microbiota of Plants[END_REF]; together with rainfall and subsequent splashing of raindrops [START_REF] Morris | Phyllosphere[END_REF] or transmission by herbivorous insects or animals [START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF][START_REF] Vacher | Trees and Insects Have Microbiomes: Consequences for Forest Health and Management[END_REF]. While it is easy to imagine that the top of the canopy is more subject to wind and rainfalls [START_REF] Bittar | Estimation of Throughfall and Stemflow Bacterial Flux in a Subtropical Oak-Cedar Forest[END_REF], their effects inside the crown are more difficult to understand. Moreover, the contribution of ecological drift (or evolutionary diversification) on the turnovers was similar on endophytic or epiphytic communities and whatever the positions. Here, we cannot distinguish between ecological drift and evolutionary diversification. It is assumed that ecological drift should occur in communities where the strength of deterministic processes is low [START_REF] Chase | Disentangling the importance of ecological niches from stochastic processes across scales[END_REF], which is rather not the case in this study, and interactions between ecological drift and other processes may be important in host-associated microbial communities [START_REF] Kohl | Ecological and evolutionary mechanisms underlying patterns of phylosymbiosis in host-associated microbial communities[END_REF]. On the other hand, host-associated communities should evolve in time in a manner that is congruent with host phylogeny [START_REF] Brooks | Phylosymbiosis: Relationships and Functional Effects of Microbial Communities across Host Evolutionary History (D Relman[END_REF][START_REF] Kohl | Ecological and evolutionary mechanisms underlying patterns of phylosymbiosis in host-associated microbial communities[END_REF][START_REF] Lim | An introduction to phylosymbiosis[END_REF].

Epiphytic communities are selected by similar traits as endophytic communities in leaves

The identity of host species and leaf traits related to the plant economics spectrum strongly shaped the composition (beta-diversity) of both endophytic and epiphytic communities. We expect this to be mainly due to the physiology of the leaf, as suggested by our results, as already shown by [START_REF] Kembel | Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest[END_REF]. The impact of leaf resource uptake strategies on bacterial communities can be explained through their impact on local leaf nutrients and water availability for bacteria [START_REF] Kembel | Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest[END_REF][START_REF] Lajoie | Plant-bacteria associations are phylogenetically structured in the phyllosphere[END_REF]. As expected, foliar traits explained up to 21.6% and 18.4 % of the variation of beta-diversity between samples in endophytic and epiphytic bacterial communities respectively, which suggests a more important role of leaf attributes on endophytic than on epiphytic communities. Leaf dry matter content for endophytic communities and leaf thickness for epiphytic ones, i.e. traits related to space for colonization, explained similar proportions of variation. The environment at the surface of a leaf and its chemistry (cuticle layer, stomata, hydathodes) is known to create more stressful and nutrientpoor conditions for bacteria. To cope with them, bacteria develop growth strategies such as forming large aggregates, key to successful colonization [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF][START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF][START_REF] Chaudhry | Shaping the leaf microbiota: plant-microbe-microbe interactions (S Kopriva[END_REF]. On another hand, stomatal chambers or apoplastic spaces inside leaves are large intercellular spaces known to host microbial communities [START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF] which can explain the effect of leaf thickness we showed. These host-adapted colonizers are also more tolerant to harmful UV radiation oxidative stress and desiccation, particularly important for topcanopy leaves [START_REF] Delmotte | Community proteogenomics reveals insights into the physiology of phyllosphere bacteria[END_REF][START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]. Epiphytic communities were more strongly shaped by traits related to available sugars (3.95% of the variation), namely chlorophyll and nitrogen contents than epiphytic communities (2.97 %), and similarly by traits linked to defense such as carbon content (2.48% and 2.6% of the variation in epiphytic and endophytic communities respectively). These results suggest a selection by nutrients resulting from photosynthesis. Indeed, leaf nitrogen content is linked to the maximum photosynthetic rate (Cornelissen 2003) and correlates with composition of bacterial communities [START_REF] Kembel | Relationships between phyllosphere bacterial communities and plant functional traits in a neotropical forest[END_REF][START_REF] Laforest-Lapointe | Host species identity, site and time drive temperate tree phyllosphere bacterial community structure[END_REF][START_REF] Lajoie | Adaptive matching between phyllosphere bacteria and their tree hosts in a neotropical forest[END_REF]. Leaf nutrients include also volatile organic compounds such as methanol exported to the surface of leaf [START_REF] Vacher | The Phyllosphere: Microbial Jungle at the Plant-Climate Interface[END_REF][START_REF] Lemanceau | Plant communication with associated microbiota in the spermosphere, rhizosphere and phyllosphere[END_REF], which may explain the slightly higher abundance of Methylobacterium we found in epiphytic communities. Interestingly, leaf traits related to water content (leaf water content and stomatal density) showed no impact on communities contrary to what is expected in the literature. Leaf surface microtopographic features is known to regulate the assembly of the bacterial communities [START_REF] Vorholt | Microbial life in the phyllosphere[END_REF]; in particular stomatal density of leaf vein density because they improve the supply of available water to the surrounding microorganisms [START_REF] Yan | Leaf surface microtopography shaping the bacterial community in the phyllosphere: evidence from 11 tree species[END_REF]. Last, most of the leaf traits investigated decreased or increased significantly with the vertical light gradient within the canopy, which was expected [START_REF] Markesteijn | Light-dependent leaf trait variation in 43 tropical dry forest tree species[END_REF][START_REF] Poorter | A meta-analysis of plant responses to light intensity for 70 traits ranging from molecules to whole plant performance[END_REF], and suggest an indirect effect of the irradiance on the composition of endophytic and epiphytic communities in leaves.

To conclude, our study provides evidence that the assembly of bacterial communities in tropical leaves was governed by selection imposed on endophytic communities; and with epiphytic communities more exposed to dispersal limitation on top of the canopy. We found that internal features of leaf were selective factors for epiphytic communities in the same way as for endophytic ones. This emphasizes the global selective pressure (i.e. the phyllosphere effect) of the plant on microbial communities, but with marked differences. However, stochastic process is also important and ecological drift (or evolutionary diversification) was similar in both communities within the canopy, which raises questions about how strongly the phylogeny of the host might also drives the composition of bacterial communities.

Tables:

Table 1: Bacterial community structure explained by host species, vertical position in the canopy, position on the leaf (epiphyte vs endophyte), their interactions and geographical

  , ape 5.5[START_REF] Paradis | ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R[END_REF], phyloseq 1.36.0[START_REF] Mcmurdie | phyloseq: An R Package for Reproducible Interactive Analysis and Graphics of Microbiome Census Data (M Watson[END_REF], picante 1.8.2[START_REF] Kembel | Picante: R tools for integrating phylogenies and ecology[END_REF], lme4 1.1-27.1[START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF], FactoMineR 2.4[START_REF] Lê | FactoMineR: A Package for Multivariate Analysis[END_REF]) and ggplot2 3.3.5(Wickham, 2016, p. 2).The importance of deterministic processes in the assembly of PMCs was assessed by estimating their variation in composition (i.e. beta-diversity) and diversity (i.e. species richness) among host species, within the canopy (TC, MC, BC), within the leaf (epiphytes or endophytes), and across spatial location of the host trees. The diversity and composition of bacterial communities were assessed using exponential of the Shannon index (see Supplementary) and Bray-Curtis distance (Lou Jost & Chazdon, 2010). Because spatial autocorrelation may arise from both spatially structured environmental factors and dispersal, spatial variation was account for by including Principal Components of Neighbours Matrices (PCNM) spatial eigenvectors based on the geographical coordinates of tree individuals in the model. PCNMs significance were tested by computed Moran's I with 1000 permutations and retained only three PCNMs that showed a significant and positive value of Moran's I. Variations in PMCs composition were estimated by performing a Permutational Multivariate Analyses of Variance (PERMANOVA)

  Figure 1: a) Localization of the sampling site in French Guiana, b) localization of each tree in

Figure 2 :

 2 Figure 2: Variations of each individual foliar leaf trait in the canopy and between host species.

Figure 3 :

 3 Figure 3: Patterns of between-community nearest taxon intext (ßNTI) in bacterial endophytic

Figure 4 :

 4 Figure 4: Relative contribution of deterministic (selection by environment, biotic interactions

Table 2 :

 2 Results of a PERMANOVA testing the effects of 10 leaf traits categorized in 4 groups on dissimilarity matrices of endophytic and epiphytic communities, host species was implemented as random factor and 1000 permutations were done. LWC: Leaf water content, SD: Stomatal density, LDMC: Leaf Dry Matter Content, LMA: Leaf Mass per Area, LA: Leaf Area, LT: Leaf Thickness, CCM: Leaf construction cost.Significant p-values (<0.05) and associated Rsquare are indicated in bold. The direction of variation of values of foliar traits from the top (TC) to the bottom (BC) of the canopy is also indicated (but see also Figure2)

	position. PERMANOVA analysis of Bray-Curtis dissimilarities. Statistical significance was
	assessed from 999 permutations
		Df	SumSq	R 2	F	Pr(>F)
	Host species (H)	2	5.167	0.107	9.676	0.001
	Position in the canopy (C) 2	0.484	0.010	0.907	0.605

Acknowledgments: This work received financial support from an 'Investissement d'Avenir' grant managed by the Agence Nationale de la Recherche (CEBA, ref. ANR-10-LABX-25-01). In accordance with Article 17, paragraph 2 of the Nagoya Protocol, this work benefits of the Access and Benefit Sharing Agreement ABSCH-IRCC-FR-245926-1. Authors contribution: HS planned and designed the research, HS, CV, SC, EL, TF, LZ conducted filed and laboratory work. HS, CV, SC and LZ analyzed the data. HS and LZ wrote the manuscript with inputs from all authors. All authors gave approval for publication.


domain: scco shs

text:
Laurent Cordonier
email: laurent.cordonier@unil.ch

Peer imitation in children: imitators show communicative attitude only when they copy "effect-less" actions

This field study assesses children's relational attitude when they imitate other children. We observe that 3.7-to 5.4-years-old children show communicative attitude toward the model when they reproduce "effect-less" actions, but not when they copy target actions.

INTRODUCTION

As pointed out by [START_REF] Elsner | Infants' imitation of goal-directed actions: The role of movements and action effects[END_REF], imitation studies in developmental psychology have predominantly focused on the reproduction of target actions (or "effective" actions), that is, sequences of movements performed in order to initiate determined and desired changes in the environment (e.g., [START_REF] Brugger | Doing the right thing: Infants' selection of actions to imitate from observed event sequences[END_REF][START_REF] Flynn | Imitation of hierarchical structure versus component details of complex actions by 3-and 5-year-olds[END_REF][START_REF] Lyons | The hidden structure of overimitation[END_REF]McGuigan et al., 2007;[START_REF] Nielsen | Young children overimitate in third-party contexts[END_REF]. In contrast, imitation of "effect-less" actions (such as the production of a mere succession of body movements) has been less studied. Some studies show that preverbal children, from at least 12 months of age, are able to encode and reproduce "effect-less" actions, but that they are much more likely to reproduce actions oriented toward determined external targets, and more effective in their reproduction than in the reproduction of "effect-less" actions [START_REF] Bellagamba | Re-enacting intended acts: comparing 12-and 18-month-olds[END_REF][START_REF] Carpenter | Twelve-and 18-month-olds copy actions in terms of goals[END_REF][START_REF] Elsner | Infants' imitation of goal-directed actions: The role of movements and action effects[END_REF][START_REF] Huang | Infants' behavioral reenactment of "failed attempts": exploring the roles of emulation learning, stimulus enhancement, and understanding of intentions[END_REF][START_REF] Meltzoff | Understanding the Intentions of Others: Re-Enactment of Intended Acts by 18-Month-Old Children[END_REF]. Yet, studies carried out by [START_REF] Nadel | Imitation et communication entre jeunes enfants[END_REF][START_REF] Nadel | Imitation and imitation recognition: Functional use in preverbal infants and nonverbal children with autism. The imitative mind: Development, evolution, and brain bases[END_REF] clearly show that children between 18 and 30 months frequently and spontaneously engage in imitations of actions that are devoid of such external targets -as, for example, carrying a small chair above the head while freely moving in a room.

What could explain these seemingly paradoxical results is that, unlike the other studies mentioned, [START_REF] Nadel | Imitation et communication entre jeunes enfants[END_REF][START_REF] Nadel | Imitation and imitation recognition: Functional use in preverbal infants and nonverbal children with autism. The imitative mind: Development, evolution, and brain bases[END_REF] research focuses on peer imitation in children: the model is not an adult who performs a specific action, but a child whose way of playing with an object is imitated by another child. In such a context, imitation fulfills a function of social communication: by reproducing the way the model plays with the object, the imitator communicates to him his similarity, his proximity (on the communicative function of imitation, see: [START_REF] Chartrand | The chameleon effect: the perception-behavior link and social interaction[END_REF][START_REF] Lakin | I am too just like you. Nonconscious mimicry as an automatic behavioral response to social exclusion[END_REF][START_REF] Meltzoff | Imitation and other minds: The "Like Me" hypothesis[END_REF][START_REF] Nadel | The evolving nature of imitation as a format for communication[END_REF]. It may well be that preverbal children spontaneously use this mode of communication mainly between them. This would explain the fact that adults' actions imitated by children are preferentially those that allow them to learn something, that is to say, "effective" actions (on the learning function of imitation, see [START_REF] Gergely | The social construction of the cultural mind: Imitative learning as a mechanism of human pedagogy[END_REF][START_REF] Nielsen | The imitative behaviour of children and chimpanzees: A window on the transmission of cultural traditions[END_REF][START_REF] Tomasello | The cultural origins of human cognition[END_REF][START_REF] Want | How do children ape? Applying concepts from the study of non-human primates to the developmental study of "imitation" in children[END_REF].

In a natural setting, do older children spontaneously imitate "effect-less" actions performed by other children? And do they display a similar communicative attitude when they reproduce "effective" and "effect-less" actions? To answer these questions, we observed spontaneous immediate peer imitations on 3.7-to 5.4-years-old children in a daycare center (Study 1). However, we had the opportunity to observe very few imitations of "effective" actions in this context (unlike imitations of "effect-less" actions). To supplement the data from our observations, we then encouraged the same children to produce imitations of "effective" actions by making them take part in a learning task requiring them to imitate a peer (Study 2). In both studies, we recorded for each imitation sequence whether the children involved exchanged glances, whether they synchronized their movements, and whether the imitator smiled (or laughed) to the model -these behaviors being indicative of communicative attitude [START_REF] Nadel | Imitation et communication entre jeunes enfants[END_REF][START_REF] Nadel | Imitation and imitation recognition: Functional use in preverbal infants and nonverbal children with autism. The imitative mind: Development, evolution, and brain bases[END_REF].

STUDY 1

Materials and Methods

Study 1 focuses on observations of spontaneous immediate peer imitations on 3.7-to 5.4-years-old children (mean: 4.44 y/o) in a daycare center. The selected group of children consisted of 11 girls and 14 boys. They all regularly attended the daycare center and were thus familiar with each other. The observations were made at intervals of two hours twice a week for 2 months (about 36 hours of observation in total). These observations were made during free playtime and collation time in the morning. We used the method of "behavioral sampling", which systematically records the occurrence and the characteristics of a specific behavior -in our case, immediate peer imitation -in a group of individuals and within a defined time [START_REF] Altmann | Observational study of behavior: sampling methods[END_REF][START_REF] Lehner | Handbook of ethological methods[END_REF]. Data collection was performed by "direct written recording" (McGrew, 1972). The two authors were present simultaneously in the field, and they took their written records separately.

The observed imitations were coded according to the following three indicator behaviors considered separately:

1. Presence of exchange(s) of glances between the imitator(s) and the model. 2. Production of laughter and/or smile(s) directed at the model. 3. Presence of behavioral synchrony between the imitator(s) and the model (at least two successive movements simultaneously performed by the children).

A sequence of imitation involving several imitators is coded as meeting one of these indicator behaviors only if all imitators have presented at least once the behavior in question during the sequence. In a sequence of imitation, the "model" is defined as the child who first began to perform the action that is reproduced by other children (the "imitators").

We then separated the observed imitations of "effective" actions from those of "effect-less" actions.

An "effective" action is defined as a sequence of movements performed to initiate a determined and desired change in the environment [START_REF] Elsner | Infants' imitation of goal-directed actions: The role of movements and action effects[END_REF].

The two authors coded the imitations separately, on the basis of their respective field notes.

Results

We observed 39 cases of immediate peer imitations in total:

-37 are imitations of "effect-less" actions: in 32 of them we notice the presence of exchange(s) of glances between the imitator(s) and the model; in 32 of them we notice the production of laughter and/or smile(s) directed at the model; in 35 of them we notice the presence of behavioral synchrony between the imitator(s) and the model. (See Table 1) -Only 2 are imitations of "effective" actions: in those 2 imitations, there is no exchange of glances between the imitator and the model, nor production of laughter and/or smile directed at the model, nor presence of behavioral synchrony between the imitator and the model.

The observed imitations (N=39) involve 1 to 7 imitators (mean: 1.74), and last 10 to 208 seconds (mean: 59 sec.). Nineteen children (12 boys, 7 girls; min.: 3.7 y/o, max.: 5.2 y/o, mean: 4.53 y/o.) of the 25 constituting the group took part as imitators in at least one (max.: 10, mean: 3.58) of the 39 imitations. Twelve of the imitations of "effect-less" actions include the use of an object (e.g., playing with a doll in the same and unconventional way as another child). The other 25 imitations of "effectless" actions consist in the simultaneous reproduction of sequences of repeated, fragmented, and exaggerated body movements. The 2 imitations of "effective" actions consist instances where a child copies the way another child is assembling two objects in order to obtain a new one, and uses it as the model does.

STUDY 2

Materials and Methods

Study 2 focuses on imitations of "effective" actions in children. The group of children is the same as in Study 1. We encouraged them to copy a sequence of manipulations produced by another child on a specific device, namely an opaque box which releases a "bouncing ball" (the reward) after three ordered separate manipulations are performed.

The procedure is as follows: a child is randomly chosen as a model, and he is brought into a separate room. One researcher explains and demonstrates the correct use of the box to the child, who can then practice on it as needed, and he tells him that he can keep the ball if he repeats the task several times in the presence of one of his mates. Another child, the imitator, is then invited to sit next to his friend. A second researcher tells him that the box is a new toy, which allows him to win a ball, and that his mate (the model) already knows how to use it. After the model carries out two demonstrations in the presence of the imitator, a second box, identical to the first, is given to him. The researcher informs him that he can now also play with his own box, and that if he succeeds in getting the ball 3 times, he can keep it. A ball is then introduced into each of the boxes at the same time, and the two children are observed while they manipulate their boxes. The sequence ends after the imitator gets the ball three times. Until this is the case, the model continuously manipulates his own box to get the ball (the ball is put back into the box every time he gets it). A child who has been the imitator in a sequence can take on the role of the model in a subsequent sequence.

The sequences were all recorded in their entirety, with two digital cameras, one filming children's faces, the other filming their manipulations of the box. Both authors did the coding, from recordings viewed separately, and according to the same three indicator behaviors as in Study 1.

Results

We conducted a total of 13 sequences of imitation following this procedure. Three of them were excluded because of the child's uncooperativeness. The remaining 10 sequences of imitation last 79 to 365 seconds (mean: 214 sec.). During these 10 sequences, as they were trying to get the ball from their own box, 7 on the 10 imitators have observed many times the manipulations of the model. Yet, in the 10 observed imitations, there is no exchange of glances between the imitator and the model, nor production of laughter and/or smile directed at the model (the imitators focused their attention on their own manipulations and on those of the model, and they sometimes looked and smiled at the researchers). We notice the presence of behavioral synchrony in only one case. (See Table 1)

GLOBAL RESULTS

Table 1 -Comparison of the attitude of the imitator(s) toward the model in imitations of "effect-less" actions (Study 1) and in imitations of "effective" actions (Study 2). The two imitations of "effective" actions observed in Study 1 are not included in the table.

DISCUSSION

Results of studies 1 and 2 show that the attitude of 3.7-to 5.4-years-old children dramatically differs depending on whether they reproduce other children's "effective" or "effect-less" actions. Contrary to what happens when they imitate "effective" actions, children adopt a clear communicative attitude when they copy "effect-less" actions: they exchange glances with the model, they smile at him, and they synchronize their movements with him.

This difference in attitude may certainly be explained by the fact that, when children reproduce "effective" actions, they are learning what those actions allow them to do. Their attention is therefore focused on the actions they are observing in order to reproduce them, and not on the model. In this case, the model is an informant for the imitator, and it is the information contained in the sequence of movements (produced with a well-defined purpose) that attracts the attention of the imitator. Conversely, when children copy "effect-less" actions, it is the interaction with the model that interests them, more than the specific movement he produces. In this case, the model is a playmate for the imitator. This interpretation of our results is in line with Over andCarpenter's (2012, 2013) hypothesis that, when imitation is performed to fulfill a learning goal, "[…] children are more focused on the characteristics of the task […] than in their relationship, or interaction with the model", whereas when "[…] social goals predominate, the child seeks to be like the model without any attempt to learn a new skill. In these cases, the match between the child's behavior and the model's behavior is of paramount importance" (Over & Carpenter, 2012: 186-187). For Over and Carpenter, the matching between the partners' behaviors is achieved through the faithfulness of the imitation (faithful imitation, versus selective imitation). But, as shown by [START_REF] Nadel | Imitation et communication entre jeunes enfants[END_REF][START_REF] Nadel | Imitation and imitation recognition: Functional use in preverbal infants and nonverbal children with autism. The imitative mind: Development, evolution, and brain bases[END_REF] and in this study, this matching can also be carried out by the temporal synchronism of the children's movements during imitation.

Acknowledgments:

We would like to thank Prof. Laurence Kaufmann and Prof. Fabrice Clément for the supervision of this study, Dr. Stéphane Bernard and Danny Dukes for their valuable comments on a previous version of this paper, and Ibanda daycare center's team, the children, and their parents for their cooperation.

This research was partially supported by the Swiss National Science Foundation (Doc.Mobility Grant n° 148524).