Rafael Costa Pinheiro

Jean-Pierre Bouillet

Bruno Bordron

Lívia Lanzi Aló

Vladimir Eliodoro Costa

Clayton Alcarde Alvares

Karel Van Den Meersche

Jose Luiz Stape

Irae Amaral Guerrini

Jean-Paul Laclau

José Luiz Stape

Iraê Amaral Guerrini

Distance from the trunk and depth of uptake of labelled nitrate for dominant and suppressed trees in Brazilian Eucalyptus plantations: Consequences for fertilization practices

Keywords: nutrient uptake, water uptake, fine roots, 15 N, eucalypt plantations, Brazil

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

Introduction

Climate changes will increase the probability of exceptional droughts, which may dramatically increase tree mortality worldwide [START_REF] Wu | Responses of terrestrial ecosystems to temperature and precipitation change: a meta-analysis of experimental manipulation[END_REF]. Improving our understanding of the structural and physiological mechanisms by which trees maintain tissue hydration and photosynthesis in response to water deficit is therefore essential to predict the effects of climate change on tree survival, carbon sequestration, and use of water in forest ecosystems [START_REF] Mcdowell | Evaluating theories of drought-induced vegetation mortality using a multimodel-experiment framework[END_REF][START_REF] Klein | Towards an advanced assessment of the hydrological vulnerability of forests to climate change-induced drought[END_REF][START_REF] Venegas-González | Drought and climate change incidence on hotspot Cedrela forests from the Mata Atlântica biome in southeastern Brazil[END_REF]. A better understanding of the effects of tree nutrition on the mechanisms involved in tree response to drought is essential to adapt the silviculture to a probable future drier climate (Battie- Laclau et al., 2014).

Both an increase of atmospheric carbon dioxide [START_REF] Iversen | Digging deeper: fine-root responses to rising atmospheric CO2 in forested ecosystems[END_REF][START_REF] Nie | Altered root traits due to elevated CO2: a meta-analysis[END_REF] and prolonged droughts [START_REF] Germon | Consequences of clear-cutting and drought on fine roots dynamics down to a depth of 17 m in eucalypt plantations conducted in coppice[END_REF] should increase the exploration of deep soil layers by plant roots in the future. Deep rooting can enhance the tolerance of some plant species to drought making it possible to reach water stored in the subsoil [START_REF] Chaves | Understanding plant responses to drought -from genes to the whole plant[END_REF][START_REF] Hoekstra | Do belowground vertical niche differences between deep-and shallow-rooted species enhance resource uptake and drought resistance in grassland mixtures?[END_REF]. In a context of climate change, the maximum depth reached by plant roots is therefore an important trait to select genotypes tolerant to prolonged droughts [START_REF] Comas | Root traits contributing to plant productivity under drought[END_REF][START_REF] Pinheiro | A fast exploration of very deep soil layers by Eucalyptus seedlings and clones in Brazil[END_REF].

Roots have been little studied at depths of more than 5 m [START_REF] Jackson | A global budget for fine root biomass, surface area, and nutrient contents[END_REF][START_REF] Pierret | Understanding deep roots and their functions in ecosystems: an advocacy for more unconventional research[END_REF], and the maximum rooting depth has probably been underestimated in many tropical forests [START_REF] Freycon | Tree roots can penetrate deeply in African semideciduous rain forests: evidence from two common soil types[END_REF]. Most of the studies dealt with the description of fine root distributions and fine root dynamics throughout very deep soil profiles [START_REF] Maeght | How to study deep roots -and why it matters[END_REF][START_REF] Pinheiro | A fast exploration of very deep soil layers by Eucalyptus seedlings and clones in Brazil[END_REF][START_REF] Lambais | Contrasting phenology of Eucalyptus grandis fine roots in upper and very deeper soil layers in Brazil[END_REF]. Even though labelling techniques have been used to locate the areas of nutrient uptake in the soil [START_REF] Lehmann | Tracer methods to assess nutrient uptake distribution in multistrata agroforestry systems[END_REF][START_REF] Lehmann | Subsoil root activity in tree-based cropping systems[END_REF][START_REF] Göttlicher | The lateral spread of tree root systems in boreal forests: Estimates based on 15 N uptake and distribution of sporocarps of ectomycorrhizal fungi[END_REF], the contribution of deep roots to supply tree nutrient requirements is still poorly documented in highly weathered tropical soils [START_REF] Poszwa | Do deep tree roots provide nutrients to the tropical rainforest?[END_REF][START_REF] Pradier | Rainfall reduction impacts rhizosphere biogeochemistry in eucalypts grown in a deep Ferralsol in Brazil[END_REF]. A functional specialization of fine roots to take up cations depending on the depth has been shown for tree species [START_REF] Göransson | The vertical distribution of N and K uptake in relation to root distribution and root uptake capacity in mature Quercus robur, Fagus sylvatica and Picea abies stands[END_REF][START_REF] Da Silva | Functional specialization of Eucalyptus fine roots: contrasting potential uptake rates for nitrogen, potassium and calcium tracers at varying soil depths[END_REF] but the uptake of nitrate was little influenced by soil depth for Eucalyptus grandis trees [START_REF] Bordron | Fertilization increases the functional specialization of fine roots in deep soil layers for young Eucalyptus grandis trees[END_REF].

The depth of water uptake by tree roots is commonly estimated monitoring soil water contents throughout soil profiles [START_REF] Guderle | Using measured soil water contents to estimate evapotranspiration and root water uptake profiles -a comparative study[END_REF], through modelling approaches (e.g. [START_REF] Kumar | Evaluation of root water uptake models -a review[END_REF][START_REF] Christina | Importance of deep water uptake in tropical eucalypt forest[END_REF], or using stable isotopes of hydrogen (deuterium, 2 H) and oxygen ( 18 O) [START_REF] Beyer | A deuterium-based labelling technique for the investigation of rooting depths, water uptake dynamics and unsaturated zone water transport in semiarid environments[END_REF][START_REF] Beyer | Examination of deep root water uptake using anomalies of soil water stable isotopes, depthcontrolled isotopic labeling and mixing models[END_REF]Koeniger et al., 2016;[START_REF] Trogisch | Spatiotemporal water uptake patterns of tree saplings are not altered by interspecific interaction in the early stage of a subtropical forest[END_REF]. Deuterium and 18 O techniques can be used to estimate the depth of water uptake when their natural abundances in soil solutions follow a clear gradient from the soil surface to deep soil layers [START_REF] Roupsard | Reverse phenology and dry-season water uptake by Faidherbia albida (Del.) A. Chev. in an agroforestry parkland of Sudanese West Africa[END_REF][START_REF] Brum | Hydrological niche segregation defines forest structure and drought tolerance strategies in a seasonal Amazon forest[END_REF]. Injecting 2 H and 18 O at different depths makes it possible to detect the areas of water uptake in the soil at the date of sampling. For example, a dualisotope labelling technique in a tropical rainforest ( 2 H applied at soil surface and 18 O injected at 120-cm depth) showed that some tree species had a high plasticity in their depth of water uptake, exhibiting an efficient strategy for water resource acquisition below 100-cm depth during dry periods [START_REF] Stahl | Depth of soil water uptake by tropical rainforest trees during dry periods: does tree dimension matter?[END_REF]. However, the depth of water uptake in tropical forests can be highly variable from one week to another depending on the occurrence of rainfall events [START_REF] Christina | Importance of deep water uptake in tropical eucalypt forest[END_REF]. It would be therefore necessary to repeat deuterium and 18 O sampling along the year to estimate the maximum distance of water uptake from trees. Moreover, isotopic techniques based on deuterium and 18 O measurements are expensive and time-consuming. Repeated measurements of 2 H and 18 O uptake at different depths in the soil throughout the year are therefore rare in tropical forests.

Da [START_REF] Da Silva | Functional specialization of Eucalyptus fine roots: contrasting potential uptake rates for nitrogen, potassium and calcium tracers at varying soil depths[END_REF] showed that 15 N from labelled nitrates injected at a depth of 3 m were only detected in young leaves of 6-year-old Eucalyptus trees when the gravitational soil solutions reached 3 m depth, which suggests that NO3 --15 N uptake could be a tracer of water uptake in these plantations. Nitrate is highly mobile in soil solutions and the uptake of large amounts of nitrates is dependent on mass flow transport up to fine roots [START_REF] Hinsinger | Acquisition of phosphorus and other poorly mobile nutrients by roots. Where do plant nutrition models fail?[END_REF][START_REF] White | Matching roots to their environment[END_REF][START_REF] Mcmurtrie | Quantifying the contribution of mass flow to nitrogen acquisition by an individual plant root[END_REF]. Nitrate labelling could therefore be a proxy of the cumulative uptake of water between the date of NO3 --15 N injection in the soil and leaf sampling a few months later, since a clear enrichment in foliar 15 N content can only occur if large amounts of NO3 --15 N are transported through mass flow up to fine roots at the vicinity of the injection area.

Eucalyptus planted forests cover more than 20 million hectares worldwide and are expanding rapidly in tropical and subtropical regions [START_REF] Booth | Eucalypt plantations and climate change[END_REF][START_REF] Gonçalves | Integrating genetic and silvicultural strategies to minimize abiotic and biotic constraints in Brazilian eucalypt plantations[END_REF]. In Brazil, these fast-growing plantations are intensively managed over 7.4 million hectares and account for 75 % of the total area of planted forests (IBGE, 2017). However, highly productive Eucalyptus plantations are sensitive to prolonged drought periods. The cumulative area affected by tree mortality as a result of drought over the last decade in the state of Minas Gerais (the largest producer of Eucalyptus in Brazil) was about 200,000 hectares [START_REF] Gonçalves | Eucalypt plantation management in regions with water stress[END_REF]. The variability among trees within the same stand to reach water and nutrients far from the trunk (horizontally and in depth) might be an important factor likely to help explain why only some trees survive during drought periods. While the influence of deep roots on drought-adaptative mechanisms of tree species is well documented [START_REF] Mcdowell | Mechanisms of plant survival and mortality during drought: why do some plants survive while others succumb to drought?[END_REF][START_REF] Fang | General mechanisms of drought response and their application in drought resistance improvement in plants[END_REF], the relationship between the social status of trees and their ability to withdraw water in very deep soil layers has been little investigated in monospecific forests.

Our study aimed to assess the maximum distance from the trunk where Eucalyptus fine roots take up water and mobile nutrients in soil solutions in very deep sandy soils. We hypothesized that mobile nutrients are taken up farther from the trunk and more deeply for dominant trees than for suppressed trees.

Material and Methods

Study site

Our study was carried out in one Eucalyptus stand at Avaré (23º00'41''S, 48º55'10''W) and in two neighboring Eucalyptus stands at Itatinga (23º14'54''S,48º35'28''W),in southeast Brazil (São Paulo State). The distance between the stands sampled at Itatinga and Avaré was 40 km. The same clone of the Eucalyptus urophylla (S.T. Blake) x Eucalyptus grandis (Hill ex Maid) hybrid was managed by the Suzano SA Company at both sites with the same silvicultural practices. The annual rainfall and the mean temperature over the study period were 1578 mm and 20.2 ºC at Itatinga, and 1980 mm and 20.6 ºC at Avaré, respectively (Figure 1). The soils at both sites were very deep Ferralic Arenosols (FAO classification) developed on Cretaceous sandstone of the Marilia formation, Bauru group. This soil type is common for commercial Eucalyptus plantations in Brazil (Table 1). The topography was a plateau at an elevation of 700-900 m at both sites and the water table was very deep (more than 15 m, which was the deepest hole made to inject the tracer in our study).

Experimental design

Experiments comparing plots with all trees planted in a single day (uniform treatment) with plots where planting was spread over 80 days (heterogeneous treatment) showed that the social status of the trees is established very early in monoclonal Eucalyptus plantations [START_REF] Binkley | Explaining growth of individual trees: Light interception and efficiency of light use by Eucalyptus at four sites in Brazil[END_REF]. Suppressed trees the first months after planting in these experiments remain suppressed throughout the entire rotation.

In the youngest stand at Avaré, 15 circular plots (20 m in radius) were randomly located with a minimum distance of 50 meters between the centre of each plot: 5 depths of tracer injection (0.1, 1.5, 3.0, 4.5 or 6.0 m) and 3 replications per depth. At 3 months after planting, 50% of the trees were totally defoliated manually in each plot to induce a variability of growth between trees making it possible to study dominant and suppressed trees. This manual defoliation mimicked the activity of ants, which can remove all the leaves from young Eucalyptus trees. The NO3 --15 N tracer was applied in the middle of the inter-row at the centre of each plot, at age 0.6 years (7 months after planting) (Figure 2). At Itatinga, 27 circular plots (20 m in radius) were randomly located in a 9-month-old stand, with a minimum distance of 50 meters between the centre of each plot: (i) 12 plots to apply the NO3 --15 N tracer at 4 depths (0.1, 3.0, 6.0 or 9.0 m) and 3 replications per depth at 1.2 years after planting, and (ii) 15 plots to apply the NO3 --years after planting. In order to induce a growth heterogeneity among trees in this stand, 50% of the trees were defoliated manually (about 80% of leaf area removed) at age 9 months. The NO3 --15 N tracer was applied in the middle of the inter-row at the centre of each plot.

In a 6-year-old stand at Itatinga (near the other sampled stand), 18 circular plots (20 m in radius) were randomly located with a minimum distance of 50 meters between the centre of each plot. The NO3 --15 N tracer was applied at age 6.4 years at the centre of each plot. The 18 plots in this stand corresponded to 6 depths of tracer injection (0.1, 3.0, 6.0, 9.0, 12.0 or 15.0 m) and 3 replications per depth. The dominant and suppressed trees sampled in this stand were selected based on the natural variability of tree growth during development (50% of the young trees were not defoliated as in the other sampled stands).

An inventory in each stand at the date of NO3 --15 N tracer injection showed large differences in basal area between dominant and suppressed trees (Appendix 1). In each plot, 4 dominant and 4 suppressed trees were selected at different distances from the injection site to sample recently expanded leaves in the upper part of the crown. The distances between the trunk of the sampled trees and the injection site were approx. 2 m, 4 m, 6 m and 10 m in the youngest stand, and approx. 2 m, 6 m, 10 m and 14 m in the other stands (Figure 2). For each depth of tracer injection at each stand age, a total of 24 trees were sampled (4 dominant and 4 suppressed trees in 3 plots).

Tracer applications

Our study (aiming to assess the maximum depth where labelled nitrates can be taken up) was carried out during the dry season because a recent study showed that Eucalyptus trees withdraw preferentially water in the topsoil after rainfall events [START_REF] Christina | Importance of deep water uptake in tropical eucalypt forest[END_REF]. One day before application in the field, a labelled solution was prepared in the laboratory and maintained at 4 ºC. 16.6 g of NH4 15 NO3 commercial compound (10 atom% or 29,221.98 ‰ NO3 --15 N) dissolved in 20 ml of distilled water were injected at a single depth at the centre of each plot. The holes were drilled down to the target application depth using a cylindrical auger with an inner diameter of 9 cm and the soil water content was measured every meter in all samples collected during drilling (Appendix 2). Soil blocks from the inner part of the auger (to avoid any contamination with fine roots from upper soil layers) were collected for the deepest meter in each hole to assess fine root densities close to the area of NO3 --15 N injection.

A PVC tube (2.5 cm in diameter) was inserted into each hole to avoid any contamination of the soil with NO3 --15 N during tracer application. A 0.4-cm polyethylene tube, attached to an iron rod, was inserted into the PVC tube, with a length depending on the application depth. A plastic sheet was placed around each hole to avoid any contamination with NO3 --15 N at the soil surface. 20 ml of the labelled solution was applied at the selected depth using a syringe. Thereafter, 280 ml of distilled water were injected to rinse the polyethylene tube and to promote nitrate uptake in the soil area where the tracer was applied. Lastly, the polyethylene and PVC tubes were carefully withdrawn from the holes, which were filled with the soil removed during drilling, respecting the original order of the soil layers. Similar methodologies were used by da Silva et al. ( 2011) and [START_REF] Bordron | Fertilization increases the functional specialization of fine roots in deep soil layers for young Eucalyptus grandis trees[END_REF] to study fine root specialization to take up nutrients in Eucalyptus plantations.

Leaf sampling

Thirty young leaves (from 2 weeks to 3 months after bud emergence) were collected in the upper half of the crown of each sampled tree, about 5 months after tracer injection. da Silva et al. (2011) showed that the enrichment with 15 N of leaves sampled in the upper crown of Eucalyptus grandis trees was roughly constant between 2 and 7 months after NO3 --15 N injection in the soil.

In each Eucalyptus stand, young leaves were also sampled in 12 control trees (6 dominant and 6 suppressed trees) located far from the injection site (> 50 m) in order to estimate the prediction interval of foliar δ 15 N values in natural abundance. Control trees were sampled away from each other throughout the stand to cover the variability of the 15 N natural abundance. In the young stands (1 year and 1.6 years after planting), the leaves were collected from the ground using a long pruner. In the 2.7-and in the 6.8-year-old stands, a professional team of tree climbers used a 5-m pruner to cut the branches from the selected trees. In total, 30 young leaves (from 2-4 branches per tree) were collected on 132, 108, 132 and 156 trees at 1, 1.6, 2.7 and 6.8 years after planting, respectively.

Isotopic analyses of 15 N

In the laboratory, the leaves were washed in deionized water and oven-dried at 65 ºC for 72 hours. The dried samples were ground (< 60 μm) in a cryogenic mill ( 2010Geno Grinder, SPEX Sample Prep, Metuchen, USA) at -196 °C for homogenization and stored in sealed acrylic pots until isotopic analysis. An aliquot of 4500-5000 μg of each dry and milled sample was weighed into a 6 mm height, 4 mm diameter cylindrical tin capsule (D1106 -Elemental Microanalysis, Okehampton, UK) with a 1 μg resolution scale (XP6 -Mettler Toledo, Greifensee, Switzerland). The 15 N analyses were performed at the Centre for Stable Isotopes at São Paulo State University using a continuous flow system in a CF-IRMS isotope ratio mass spectrometer (Delta V Advantage -Thermo Scientific, Bremen, Germany) coupled to an elemental analyzer (Flash 2000 OEA -Thermo Scientific, Bremen, Germany) with an interface (ConFlo IV Universal -Thermo Scientific, Bremen, Germany) that determines the isotope ratio of the sample (Rsample= 15 N/ 14 N).

The 15 N values in isotopic delta (δ 15 N, expressed in ‰) were calculated according to the following equation [START_REF] Coplen | Guidelines and recommended terms for the expression of stable-isotope-ratio and gas-ratio measurement results[END_REF]):

= -1 • 1000‰
where Rsample is the 15 N/ 14 N ratio in the sample and Rair is the 15 N/ 14 N ratio of atmospheric air (Rair=0.0036765) as international standard. The standard uncertainty in δ 15 N was ± 0.3 ‰ for samples slightly enriched with 15 N.

Data analysis

For each Eucalyptus stand, a prediction interval with a threshold of 99% for δ 15 N natural abundance was calculated from the δ 15 N values of the 12 control trees using the equation ± 1 + 1/ , where and are the average and standard deviation of the control values, the number of observations, and the 99.5 percentile of a Student's t-distribution with (n-1) degrees of freedom [START_REF] Geisser | Predictive Inference: An Introduction[END_REF].

The statistical software R was used (R Core Team, 2019). Foliar δ 15 N values higher than the upper boundary of the prediction interval were considered significantly different from the control population (p<0.01), which indicated that the sampled trees took up the NO3 --15 N tracer injected in the same plot. The δ 15 N values of all the sampled tree (dominant and suppressed) were plotted relative to the horizontal distance between the trunk and the tracer injection site. one dominant tree took up the tracer at a distance of 3.7 m from the injection site (Figure 3D). Soil coring to inject NO3 --15 N showed that fine roots were present in all the areas of tracer injection down to a depth of 4.5 m in the 0.6-year-old stand (Appendix 3). However, it cannot be excluded that fine roots belonging to the previous Eucalyptus stand (before clearcutting and replanting) were not distinguishable from the fine roots of young trees. While soil texture was roughly constant throughout the soil profile (Table 1), soil water contents sharply increased below the depth of 4 m at age 0.6 years (Appendix 2), which suggests that water was mainly withdrawn by tree roots in the upper 4 m before the first dry season.

Results

3

3.2. NO3 --15 N uptake from 1.2 to 1.6 years after planting Dominant and suppressed trees took up large amounts of the NO3 --15 N tracer applied at a depth of 0.1 m, within 2 m from the injection site with a peak of foliar δ 15 N at 420 ‰ for a suppressed tree. The foliar δ 15 N value in one suppressed tree at a distance of 7.5 m from the injection site was slightly above the upper boundary of the prediction interval of natural abundance (Figure 4A). When the NO3 --15 N tracer was applied at a depth of 3 m, foliar δ 15 N values were higher than the upper boundary of the prediction interval of natural abundance for all the dominant trees located within 2 m from the injection site and only for one suppressed tree. No tracer uptake was detected at greater distances from the injection site (Figure 4B). Only dominant trees within a distance of 2 m from the injection site took up large amounts of NO3 --15 N applied at a depth of 6 m. Foliar δ 15 N values were slightly above the upper boundary of the prediction interval of natural abundance for two trees at a distance of 6 m from the injection site (Figure 4C). Although some fine roots were sampled at a depth of 9 m when the NO3 --15 N tracer was injected (Appendix 3), the foliar δ 15 N values for all the sampled trees were within the interval of prediction of natural abundance, which showed that large amounts of the tracer were not taken up by the sampled trees at this depth.

3.3. NO3 --15 N uptake from 2.2 to 2.7 years after planting Large amounts of the NO3 --15 N tracer injected at a depth of 0.1 m were taken up both by dominant and suppressed trees within 2 m from the injection site (Figure 5A). The same pattern was observed when the NO3 --15 N tracer was applied at a depth of 3 m (Figure 5B). Only one dominant tree took up a detectable amount of NO3 --15 N at a depth of 6 m and it was located within 2 m from the injection site (Figure 5C). Foliar δ 15 N values for all the sampled trees were within the prediction interval of natural abundance in the plots where the NO3 --15 N tracer was injected at depths of 9 m and 12 m (data not shown). Soil coring to inject NO3 --15 N showed that fine roots were present in all the areas of tracer injection down to a depth of 12 m in the 2.2-year-old stand (Appendix 3). Changes in soil water contents between 1.2 and 2.2 years after planting, in the same stand (in June 2016 and June 2017), suggest that tree roots withdrew water between the depths of 4 m and 9 m (Appendix 2).

3.4. NO3 --15 N uptake from 6.4 to 6.8 years after planting

A foliar enrichment with NO3 --15 N was only detected when the NO3 --15 N tracer was injected in the upper soil layer at a depth of 0.1 m. Both dominant and suppressed trees took up NO3 --15 N within 3 m from the injection site. The suppressed trees had higher δ 15 N values within leaves than the dominant trees, with a peak at 349 ‰ (Figure 6A). At all the other depths of injection of the NO3 --15 N tracer (i.e. 3, 6, 9, 12 and 15 m), foliar δ 15 N values were within the prediction interval of natural abundance for all the sampled trees (Figure 6B and 6C -data not shown below 6 m of depth).

Soil coring to inject NO3 --15 N in the 6.4-year-old stand showed that fine roots were present in all the areas of tracer injection down to a depth of 15 m (Appendix 3).

Discussion

Reliability of our method to detect the maximum distance of water uptake from trees

The depths of NO3 --15 N uptake throughout the rotation in our study are consistent with other studies estimating the depth of water withdrawal in tropical Eucalyptus plantations, which suggests that the uptake of labelled nitrate could be a simple proxy of cumulative water uptake in specific soil areas. In particular, a modelling approach based on soil moisture and eddy-covariance measurements over 4.5 years after planting in a commercial Eucalyptus stand at about 20 km from our study sites (in a similar soil) showed that water was withdrawn from the soil surface to a depth of 6-7 m the first year after planting, from the soil surface to a depth of 10 m the second dry season, then only in the upper soil layers and close to the water table at a depth of more than 12 m until harvesting [START_REF] Christina | Importance of deep water uptake in tropical eucalypt forest[END_REF]. The depth of uptake of labelled nitrate by Eucalyptus trees in our study is consistent with the dynamics of water uptake throughout deep soil profiles over long periods in other Eucalyptus plantations in the same region [START_REF] Laclau | Dynamics of soil exploration by fine roots down to a depth of 10 m throughout the entire rotation in Eucalyptus grandis plantations[END_REF][START_REF] Marsden | Modifying the G'DAY process-based model to simulate the spatial variability of Eucalyptus plantation growth on deep tropical soils[END_REF][START_REF] Christina | Simulating the effects of different potassium and water supply regimes on soil water content and water table depth over a rotation of a tropical Eucalyptus grandis plantation[END_REF]. However, a strong demonstration of the validity of our method (enrichment in foliar 15 N after injection of NO3 --15 N in the soil) as a proxy of water uptake by tree roots would require to monitor foliar 15 N as well as 2 H and 18 O in the trees after the injection in the same soil areas of NO3 --15 N, 2 H and 18 O tracers.

Although we injected NO3 --15 N down to a depth of 15 m, we did not detect any significant uptake of labelled nitrate at depths of more than 9 m. This pattern does not demonstrate that water was not taken up at these depths but suggests that large amounts of labelled nitrate were not taken up by neighboring trees. Fine root densities are very low at depths of more than 6 m in Eucalyptus plantations [START_REF] Christina | Almost symmetrical vertical growth rates above and below ground in one of the world's most productive forests[END_REF][START_REF] Laclau | Dynamics of soil exploration by fine roots down to a depth of 10 m throughout the entire rotation in Eucalyptus grandis plantations[END_REF], and it is possible that the trees sampled to determine foliar 15 N contents in our study did not explore the small soil areas where the tracer was injected. The simple method used here to detect water and nitrate uptake is also probably less sensitive than isotopic methods based on 2 H and 18 O measurements to detect small amounts of water taken up throughout the study period.

Influence of the social status of the tree on the distance of uptake of the labelled nitrate

Our study clearly shows in monoclonal stands that dominant trees have a higher This pattern is consistent with a recent study in a seasonal Amazon forest using the stable isotope ratios (δ 18 O and δ 2 H) of water collected from tree xylem and soils down to a depth of 10 m during an extreme dry event. Rooting depth differences were strongly related to tree size and water uptake by understory species was limited to shallow soil layers. Coexisting trees were mainly segregated along a single hydrological niche axis defined by root depth, access to light and tolerance of low water potential [START_REF] Brum | Hydrological niche segregation defines forest structure and drought tolerance strategies in a seasonal Amazon forest[END_REF]. Our results in monoclonal Eucalyptus stands suggest that the strong relationship between tree size and rooting depth showed for co-existing species in this tropical forest could be valid between big and small trees of the same species. However, another isotopic study in a tropical rainforest showed that tall trees preferentially extracted water from layers below a depth of 1 m, while smaller trees exhibited large variations in the mean depth of water uptake, which prevented the use of tree dimensions to parameterize functional models in this multispecific forest [START_REF] Stahl | Depth of soil water uptake by tropical rainforest trees during dry periods: does tree dimension matter?[END_REF]. Further studies on the relationship between tree size and water uptake down to a depth of 10 m (or more) are needed to get an insight into the sharing of limited water resources between plants during drought events in tropical forests.

Root exploration limited to shallow soil layers for small trees seems contradictory with the observation that suppressed trees can survive better to drought events than dominant trees [START_REF] Bennett | Larger trees suffer most during droughts in forests worldwide[END_REF][START_REF] Mcdowell | Darcy's law predicts widespread forest mortality under climate warming[END_REF]. However, many factors other than the maximum depth reached by fine roots can account for tree survival during drought periods. Low water demand of suppressed trees relative to dominant trees might help avoid hydraulic failure during drought [START_REF] Grote | Importance of tree height and social position for drought-related stress on tree growth and mortality[END_REF].

While drought periods impacted less the growth of suppressed trees relative to dominant trees in some forests [START_REF] Martín-Benito | Growth response to climate and drought in Pinus nigra Arn. trees of different crown classes[END_REF]Martínes-Vilalta et al., 2012;[START_REF] Zang | Size-dependent responses to summer drought in Scots pine, Norway spruce and common oak[END_REF], the pattern was less clear in other forests and a more pronounced impact of drought in the understory than for dominant trees has also been reported [START_REF] Castagneri | Temporal variability of sizegrowth relationships in a Norway spruce forest: the influences of stand structure, logging, and climate[END_REF][START_REF] Trouvé | Growth partitioning in forest stands is affected by stand density and summer drought in sessile oak and Douglas-fir[END_REF][START_REF] Merlin | Effects of stand composition and tree size on resistance and resilience to drought in sessile oak and Scots pine[END_REF].

The horizontal distance of NO3 --15 N uptake from the trunk was not clearly influenced by the social status of the trees in our study. Only a small proportion of the sampled trees took up the NO3 --15 N tracer, which is consistent with an uneven distribution of tree roots shown in other studies. In monospecific boreal forests, the average lateral root spread of trees was 4-5 m but not all trees took up the 15 N tracer close to the injection area, which suggested that the root system was highly asymmetric [START_REF] Göttlicher | The lateral spread of tree root systems in boreal forests: Estimates based on 15 N uptake and distribution of sporocarps of ectomycorrhizal fungi[END_REF]. Throughout deuterium pulses, small trees in an Amazonian tropical forest showed different rooting patterns, while most species took up the deuterium tracer only close to the trunk, a few species had high concentrations of deuterium up to 10 m of horizontal distance from the injection area [START_REF] Sternberg | Uptake of water by lateral roots of small trees in an Amazonian Tropical Forest[END_REF].

Consequences for the management of Eucalyptus stands in deep sandy soils

Our study was also designed to answer a question from forest managers in Brazil:

which proportion of the inter-rows in Eucalyptus plantations must be fertilized (after weeding) to make it possible for all the trees to take up the applied nutrients?

Fertilization and weeding are seasonal activities with a peak at the mid-rainy season, and an extension of these activities throughout the year could be useful to reduce the cost of the silviculture. The staff and the equipment needed to cope with the peak of activity over the rainy season could indeed be reduced if the intervention could be postponed of a few months in a significant proportion of the inter-rows within each stand.

Whatever stand age and tree social status, most of the labelled nitrate injected at different soil depths was taken up by trees located within 2-3 m from the injection site. Our results show that N fertilizers must be applied close to the trunks to be taken up by all the trees, whatever stand age. Spacing in commercial Eucalyptus plantations managed in short rotations is commonly 2 m x 3 m or 3 m x 3 m. Our results suggest that fertilization (and weeding) in half of the inter-rows during the rainy season could be sufficient to make it possible for all the trees to take up the applied nutrients. However, the proportion of fertilized inter-rows cannot be lower than 50%. Applying fertilizers in 1/3 of the inter-rows would prevent most trees that are not adjacent to the fertilized inter-row from taking up the nutrients and would probably lead to a high heterogeneity of growth within the stand.

The risk of nutrient losses by deep drainage will be higher if only 50% of the interrows are fertilized because only about half of the root system would have access to the applied nutrients. [START_REF] Laclau | Nutrient dynamics throughout the rotation of Eucalyptus clonal stands in Congo[END_REF][START_REF] Iversen | Digging deeper: fine-root responses to rising atmospheric CO2 in forested ecosystems[END_REF] showed that nutrient requirements are high in the first two years after planting Eucalyptus trees to produce leaves and fine roots. A combination of high nutrient requirements and high transpiration rates of Eucalyptus trees in highly weathered tropical soils account for very low nutrient losses by deep drainage [START_REF] Mareschal | Nutrient leaching and deep drainage under Eucalyptus plantations managed in short rotations after afforestation of an African savanna: Two 7-year time series[END_REF][START_REF] Versini | Effects of litter manipulation in a tropical Eucalyptus plantation on leaching of mineral nutrients, dissolved organic nitrogen and dissolved organic carbon[END_REF][START_REF] Binkley | Connecting ecological science and management in forests for scientists, managers and pocket scientists[END_REF]. We show that nitrates leached in deep sandy soils can be taken up by tree roots at a depth of 6 m between 0.6 and 2.5 years after planting in commercial Eucalyptus plantations. However, the velocity of exploration of deep soil layers depends on the social status of the trees. When N fertilization is concentrated in the first months after planting (and even more if fertilizers are only applied in 50% of the inter-rows), nitrate leaching in very deep soil layers might increase the heterogeneity of stands since deep nitrates could only be available for dominant trees. Field trials are needed to assess if weeding and fertilizing only 50% of the inter-rows during the rainy season (and the other 50% a few months later) would affect stand productivity.

In drought-prone areas, an increase in stand evapotranspiration due to weeds during the rainy season might reduce the availability of water for trees during the next dry season and therefore might increase the risk of tree mortality during prolonged drought periods. The suitability of an extension of weeding and fertilization throughout the year is therefore probably dependent on climate and soil properties.

Further studies dealing with the relationship between the social status of the trees and the distance of water and nutrient uptake are also needed to improve tree-based modeling approaches that intend to predict the response of tropical forests to climate change. The lower and the upper boundaries of the prediction interval calculated at this age were -0.9 ‰ and 5.0 ‰, respectively.

. 1 .

 1 NO3 --15 N uptake from 0.6 to 1 year after planting Foliar δ 15 N values showed that the trees within 2 m from the injection site (both dominant and suppressed) took up the largest amounts of the NO3 --15 N tracer injected at a depth of 0.1 m (Figure3A). The highest δ 15 N value was measured in a suppressed tree (273 ‰). The foliar δ 15 N value was slightly above the upper boundary of the prediction interval of natural abundance for one dominant tree at a distance of 4 m from the injection site. The behavior was similar for the injection depth of 1.5 m, but with lower foliar δ 15 N peaks in dominant and suppressed trees than for the injection depth of 0.1 m (data not shown).When NO3 --15 N was injected at a depth of 3 m, both dominant and suppressed trees took up the tracer within 2 m from the injection site (Figure3B). Only dominant trees took up the NO3 --15 N tracer applied at a depth of 4.5 m and only within 2 m from the injection site. High foliar δ 15 N values (> 100 ‰) showed that large amounts of tracer were taken up by those trees (Figure3C). At an injection depth of 6 m, only dominant trees took up the NO3 --15 N tracer (foliar δ 15 N values reached 94.4 ‰ in one tree) and

  capacity to use soil resources in very deep soil layers than suppressed trees. From 0.6 to 1 year after planting, most of the trees close to the injection sites took up NO3 --15 N in the upper 3 m of the soil profile and only dominant trees took up the tracer at depths of 4.5 m and 6 m. While some dominant trees took up large amounts of 15 N at a depth of 6 m the second and third years after planting, the lack of clear enrichment in foliar 15 N for all the suppressed trees suggests that their ability to access to soil resources in very deep soil layers was low relative to dominant trees.

Fig. 1 .Fig. 2 .

 12 Fig. 1. Time course of monthly rainfall and mean temperature over the study period at Avaré (A) and at Itatinga (B). Continuous arrows show the 15 N tracer injection periods in 1.2-and 6.4-yearold stands in June 2016, and in 0.6-and 2.2-year-old stands in June 2017. Dashed arrows indicate the leaf sampling periods, five months after tracer injection.

Fig. 3 .

 3 Fig. 3. Foliar δ 15 N values in 1-year-old E. urophylla x E. grandis trees, 5 months after application of NO3 --15 N tracer A) at a depth of 0.1 m, B) at a depth of 3 m, C) at a depth of 4.5 m, and D) at a depth of 6 m. Dominant trees (empty circle) and suppressed trees (solid triangle) were sampled.The prediction interval (P < 0.01) was calculated from δ 15 N values in natural abundance measured for 12 trees (6 dominant and 6 suppressed trees). The lower and the upper boundaries of the prediction interval calculated at this age were 0.8 ‰ and 5.5 ‰, respectively.

Fig. 4 .

 4 Fig. 4. Foliar δ 15 N values in 1.6-year-old E. urophylla x E. grandis trees, 5 months after application of the NO3 --15 N tracer A) at a depth of 0.1 m, B) at a depth of 3 m, and C) at a depth of 6 m. Dominant trees (empty circle) and suppressed trees (solid triangle) were sampled. The prediction interval (P < 0.01) was calculated from δ 15 N values in natural abundance measured for 12 trees (6 dominant and 6 suppressed trees). The lower and the upper boundaries of the prediction interval calculated at this age were -1.2 ‰ and 3.6 ‰, respectively.

Fig. 5 .

 5 Fig.5. Foliar δ 15 N values in 2.7-year-old E. urophylla x E. grandis trees, 5 months after application of NO3 --15 N tracer A) at a depth of 0.1 m, B) at a depth of 3 m, and C) at a depth of 6 m. Dominant trees (empty circle) and suppressed trees (solid triangle) were sampled. The prediction interval (P < 0.01) was calculated from δ 15 N values in natural abundance measured for 12 trees (6 dominant and 6 suppressed trees). The lower and the upper boundaries of the prediction interval calculated at this age were -0.6 ‰ and 2.2 ‰, respectively.

Fig. 6 .

 6 Fig.6. Foliar δ 15 N values in 6.8-year-old E. urophylla x E. grandis trees, 5 months after application of NO3 --15 N tracer A) at a depth of 0.1 m, B) at a depth of 3 m, and C) at a depth of 6 m. Dominant and suppressed trees were sampled. The prediction interval was calculated from δ 15 N values in natural abundance measured for 12 trees sampled at more than 50 m from all the injection points. The lower and the upper boundaries of the prediction interval calculated at this age were -0.9 ‰ and 5.0 ‰, respectively.

Table 1 .

 1 Main physical and chemical soil properties at Itatinga and Avaré. Mean values and standard errors are indicated (n = 3).

	Soil layer	Sand	Silt	Clay	pH	O. M.	Presin	H+Al	K	Ca	Mg	SB	CEC
	(m)	Particle size distribution (% )	CaCl2	g.kg	-1	mg.kg	-1			mmolc.kg-1
	Itatinga
	0-0.25 92.8 ± 0.3 1.4 ± 0.5 5.8 ± 0.4 4.20 ± 0.18 14.13 ± 3.50 12.59 ± 3.47 27.94 ± 4.96 0.36 ± 0.04	22.16 ± 4.95	4.54 ± 0.33	27.05 ± 4.79	55.00 ± 6.20
	0.25-0.5 91.0 ± 0.4 2.1 ± 0.4 6.9 ± 0.5 4.04 ± 0.06 6.48 ± 0.35 3.87 ± 0.91 24.26 ± 2.59 0.26 ± 0.11	15.48 ± 0.55	2.68 ± 0.58	18.42 ± 0.68	42.68 ± 3.22
	0.5 -1 89.3 ± 0.4 1.9 ± 0.4 8.8 ± 0.1 4.03 ± 0.06 5.52 ± 0.36 2.48 ± 0.41 18.87 ± 2.41 0.22 ± 0.01	14.36 ± 1.01	2.05 ± 0.35	16.63 ± 0.76	35.49 ± 2.94
	1 -2	88.7 ± 0.4 2.0 ± 0.9 9.3 ± 0.6 4.07 ± 0.11 5.63 ± 0.25 2.28 ± 0.27 17.92 ± 2.71 0.35 ± 0.22	12.58 ± 3.20	2.29 ± 0.33	15.21 ± 3.35	33.14 ± 4.01
	2 -4	88.8 ± 0.7 1.8 ± 0.8 9.4 ± 0.2 4.23 ± 0.20 3.28 ± 0.88 1.87 ± 0.37 11.59 ± 1.74 0.35 ± 0.24	12.54 ± 1.52	2.57 ± 0.34	15.46 ± 1.86	27.04 ± 1.87
	4 -6	86.9 ± 1.3 2.6 ± 0.5 10.5 ± 0.8 4.17 ± 0.10 2.66 ± 0.67 1.59 ± 0.21 10.38 ± 0.80 0.16 ± 0.04	12.27 ± 2.05	2.47 ± 0.20	14.89 ± 2.15	25.27 ± 2.53
	6 -8	87.1 ± 0.5 2.3 ± 1.0 10.7 ± 0.6 4.20 ± 0.03 1.99 ± 0.23 2.08 ± 0.61 10.29 ± 0.52 0.14 ± 0.00	12.44 ± 0.49	2.12 ± 0.13	14.70 ± 0.44	24.99 ± 0.14
	8 -10 86.3 ± 0.2 3.8 ± 1.5 9.9 ± 1.5 4.19 ± 0.07 1.93 ± 0.14 1.73 ± 0.09 9.81 ± 0.42 0.14 ± 0.00	15.32 ± 3.39	1.87 ± 0.24	17.34 ± 3.16	27.15 ± 3.42
	10 -12 87.0 ± 0.4 2.4 ± 0.3 10.6 ± 0.3 4.12 ± 0.08 2.27 ± 0.21 1.77 ± 0.18 10.17 ± 0.44 0.14 ± 0.00	13.63 ± 3.48	2.11 ± 0.27	15.88 ± 3.66	26.05 ± 3.96
	Avaré
	0-0.25 84.7 ± 2.4 3.4 ± 1.6 11.9 ± 1.6 4.13 ± 0.39 22.5 ± 4.84 8.24 ± 2.51 43.36 ± 10.73 0.81 ± 0.47	8.82 ± 6.27	3.16 ± 2.13	12.79 ± 8.38	56.15 ± 5.92
	0.25-0.5 81.8 ± 2.3 4.8 ± 1.9 13.4 ± 1.1 3.94 ± 0.10 9.82 ± 0.46 4.27 ± 1.13 37.78 ± 4.66 0.38 ± 0.17	6.59 ± 8.96	1.41 ± 1.52	8.38 ± 10.64 46.16 ± 8.05
	0.5 -1 81.5 ± 1.6 4.7 ± 0.9 13.8 ± 1.2 3.87 ± 0.06 8.81 ± 1.54 2.49 ± 0.61 30.34 ± 2.07 0.26 ± 0.18	4.88 ± 6.85	1.11 ± 1.03	6.25 ± 8.07	36.59 ± 7.65
	1 -2	77.3 ± 3.7 6.3 ± 3.8 16.4 ± 0.6 3.97 ± 0.05 6.43 ± 1.24 1.89 ± 0.13 23.83 ± 3.64 0.24 ± 0.08	7.63 ± 11.58 1.51 ± 1.70	9.39 ± 13.35 33.23 ± 9.98
	2 -4	77.6 ± 2.8 5.6 ± 2.8 16.7 ± 0.3 4.21 ± 0.05 4.12 ± 0.79 1.93 ± 0.14 14.75 ± 0.72 0.13 ± 0.05	6.38 ± 8.63	1.34 ± 1.41	7.86 ± 10.07 22.61 ± 10.03
	4 -6	75.9 ± 1.5 5.6 ± 1.0 18.5 ± 0.9 4.33 ± 0.04 2.55 ± 0.58 1.96 ± 0.12 11.80 ± 0.40 0.16 ± 0.00	4.96 ± 6.17	1.22 ± 1.20	6.33 ± 7.37	18.13 ± 7.59

N tracer at 5 depths (0.1, 3.0, 6.0, 9.0 or 12.0 m) and 3 replications per depth at 2.2

Acknowledgements

We thank Raul Chaves and forest engineers in the Eucflux project (http://www.ipef.br/eucflux/en/) for interesting questions relative to the necessity or not to apply fertilizers in 100% of the inter-rows within Eucalyptus stands. This work was supported by the São Paulo Research Foundation (FAPESP, project 2015/25946-0). Additional and essential funding came from Suzano SA Company (http://www.suzano.com.br/en/), which also provides the study areas and assistance in the field activities supported by forest technician Dileto Benedito Bau. We are grateful to the staff at the Itatinga Experimental Station (in particular Rildo Moreira e Moreira -ESALQ/USP), Eder Araujo da Silva (http://www.floragroapoio.com.br) and tree climbers of the Forestry Science and Research Institute

(http://www.ipef.br/english/seeds/) for their technical support. We also acknowledge the Stable Isotopes Centre from the Institute of Biosciences (UNESP).