M Voigtlaender

C B Brandani

D R M Caldeira

Florence Tardy

Jean-Pierre Bouillet

J L M Gonçalves

M Z Moreira

F P Leite

Didier Brunet

R R Paula

Nitrogen cycling in monospecific and mixed-species plantations of Acacia mangium and Eucalyptus at 4 sites in Brazil

Keywords: Organic matter, N2 fixation, Eucalypt, litterfall, nutrition, Tree mixtures, tropical 47 plantations. 48

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

Introduction

Mixed-species plantations associating nitrogen-fixing trees (NFT) with other highly productive tree species could be an attractive option to improve both the long-term soil nitrogen (N) status and the overall biomass production [START_REF] Binkley | Twenty years of stand development in pure and mixed stands of Eucalyptus saligna and nitrogenfixing Facaltaria moluccana[END_REF][START_REF] Forrester | Growth dynamics in a mixedspecies plantation of Eucalyptus globulus and Acacia mearnsii[END_REF][START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF]. Although total stem biomass production at the harvest was significantly enhanced in mixed-species stands relative to Eucalyptus monocultures in Australia [START_REF] Forrester | Growth dynamics in a mixedspecies plantation of Eucalyptus globulus and Acacia mearnsii[END_REF], Hawaii [START_REF] Binkley | Twenty years of stand development in pure and mixed stands of Eucalyptus saligna and nitrogenfixing Facaltaria moluccana[END_REF], Congo [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF] and in an experiment in Brazil [START_REF] Santos | Dynamics of aboveground biomass accumulation in monospecific and mixed-species plantations of Eucalyptus and Acacia on a Brazilian sandy soil[END_REF], the mixture was less productive than monospecific Eucalyptus stands in other experiments established in Brazil [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF]. A growing body of evidence suggests an overall trend towards higher biomass production in a mixture than in monocultures of Eucalyptus and NFT in the areas where Eucalyptus growth is limited by N [START_REF] Forrester | The spatial and temporal dynamics of species interactions in mixed-species forests: From pattern to process[END_REF]. A positive effect of diversity on forest production in North America also suggested that facilitation between species could be enhanced relative to competition in stressful environments [START_REF] Paquette | The effect of biodiversity on tree productivity: from temperate to boreal forests[END_REF].

However, light and water use efficiency to produce wood is commonly higher in Eucalyptus plantations growing under favourable conditions than in areas of low productivity (Binkley et al., 2004;[START_REF] Campoe | Stand-level patterns of carbon fluxes and partitioning in a Eucalyptus grandis plantation across a gradient 27 of productivity, in São Paulo State, Brazil[END_REF], which supports an intensive management of highly productive plantations concentrated in small areas to satisfy the growing demand in wood [START_REF] Stape | Eucalyptus production and the supply, use and the efficiency of use of water, light and nitrogen across a geographic gradient in Brazil[END_REF][START_REF] Battie-Laclau | Potassium fertilization increases water-use efficiency for stem biomass production without affecting 26 intrinsic water-use efficiencyintrinsic water-use efficiency in Eucalyptus grandis plantations[END_REF]. While soil N availability is an important factor influencing the interactions between Eucalyptus and NFT, studies dealing with nutrient cycling over entire rotations are scarce in tropical regions [START_REF] Forrester | Mixed plantations of Eucalyptus with nitrogen fixing trees: a review[END_REF].

The gross primary productivity of Brazilian Eucalyptus plantations is higher than 3500 g C m - 2 yr -1 [START_REF] Ryan | Factors controlling Eucalyptus productivity: how resource availability and stand structure alter production and carbon allocation[END_REF][START_REF] Cabral | Fluxes of CO2 above a plantation of Eucalyptus in southeast Brazil[END_REF], which ranks these forests among the most productive in the world [START_REF] Luyssaert | CO2 balance of boreal, temperate, and tropical forests derived from a global database[END_REF]. Input-output budgets show consistently: i) that Eucalyptus plantations can benefit for several decades from an inherited soil fertility from the previous land use, and ii) that N fertilizer additions should increase over successive rotations [START_REF] Laclau | Nutrient cycling in a clonal stand of Eucalyptus and an adjacent savanna ecosystem in Congo. 3. Input-output budgets and consequences for the sustainability of the plantations[END_REF][START_REF] Silva | Fertilizer management of eucalypt plantations on sandy soil in Brazil: Initial growth and nutrient cycling[END_REF]. NFT can be planted in N-deficient soils to enhance soil N status through large inputs of N derived from the atmosphere [START_REF] Binkley | Thinking about efficiency of resource use in forests[END_REF][START_REF] Forrester | The spatial and temporal dynamics of species interactions in mixed-species forests: From pattern to process[END_REF].

The 15 N natural abundance method is commonly used to estimate N2 fixation in forest ecosystems (e.g. [START_REF] Galiana | Nitrogen fixation estimated by the 15N natural abundance method in Acacia mangium Willd. inoculated with Bradyrhizobium sp. and grown in silvicultural conditions[END_REF][START_REF] Forrester | Assessing nitrogen fixation in mixed-and single-species plantations of Eucalyptus globulus and Acacia mearnsii[END_REF][START_REF] Bouillet | Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 2: nitrogen accumulation in the stands and biological N2 fixation[END_REF]) despite strong limitations [START_REF] Shearer | N2-fixation in field settings: estimations based on natural 15 N abundance[END_REF][START_REF] Chalk | Do techniques based on 15 N enrichment and 15 N natural abundance give consistent estimates of the symbiotic dependence of N 2 -fixing plants?[END_REF]. N2 fixation can also be estimated by the accretion method comparing total N stocks in the soil-plant system for NFT and non NFT, and considering that the difference is a result of N2 fixation (similar N inputs and outputs except N2 fixation). A comparison of N2 fixation rates estimated with the accretion method, the 15 N natural abundance method and the 15 N labeling method is recommended to improve the reliability of the estimates [START_REF] Shearer | N2-fixation in field settings: estimations based on natural 15 N abundance[END_REF]Parrota et al., 1996;[START_REF] Forrester | Assessing nitrogen fixation in mixed-and single-species plantations of Eucalyptus globulus and Acacia mearnsii[END_REF][START_REF] Bouillet | Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 2: nitrogen accumulation in the stands and biological N2 fixation[END_REF]. While some studies showed that carbon (C) and N stocks can rapidly increase in mixed-species plantations with NFT relative to eucalypt monocultures in Hawaii [START_REF] Binkley | Thinking about efficiency of resource use in forests[END_REF]), Puerto Rico [START_REF] Resh | Greater soil carbon sequestration under nitrogen-fixing trees compared with Eucalyptus species[END_REF], Brazil [START_REF] Garay | Evaluation of soil conditions in fast-growing plantations of Eucalyptus grandis and Acacia mangium in Brazil: a contribution to the study of sustainable land use[END_REF][START_REF] Santos | Dynamics of aboveground biomass accumulation in monospecific and mixed-species plantations of Eucalyptus and Acacia on a Brazilian sandy soil[END_REF]2017), Australia [START_REF] Forrester | Faster growth of Eucalyptus grandis and Eucalyptus pilularis in mixed-species stands than monocultures[END_REF], and Congo [START_REF] Koutika | Changes in N and C concentrations, soil acidity and P availability in tropical mixed acacia and eucalypt plantations on a nutrient-poor sandy soil[END_REF], no significant changes in C and N stocks were detected after 1 rotation of NFT in Brazil (Voigtlaender et al., 2012;[START_REF] Rachid | Mixed plantations can promote microbial integration and soil nitrate 535 increases with changes in the N cycling genes[END_REF]. The consequences of introducing NFT in eucalypt plantations might be less site-specific for N cycling than for C and N storage in the soil, with a rapid rise in N contents in litterfall and in soil N mineralization rates commonly observed after planting NFT [START_REF] Binkley | Thinking about efficiency of resource use in forests[END_REF][START_REF] Forrester | Faster growth of Eucalyptus grandis and Eucalyptus pilularis in mixed-species stands than monocultures[END_REF]Voigtlaender et al., 2012;Sang et al., 2013;Tchichelle et al., 2017a).

A similar experimental design comparing monospecific and mixed-species plantations of Eucalyptus and Acacia mangium Wild has been replicated at 4 sites in Brazil. [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF] showed that the stemwood biomass at the time of harvest was 14%-57% higher in monospecific Eucalyptus stands (100E) than in A. mangium stands (100A) and an intermediate in mixture at a 1:1 ratio between Eucalyptus and A. mangium (50A50E) at those 4 sites. The biomass of aboveground tree components in 50A50E was strongly influenced by intra-and inter-specific interactions. Mixed-species plantations of Eucalyptus and A. mangium trees might be an interesting option to produce Eucalyptus saw logs with a much higher wood volume from individual Eucalyptus trees than in the commercial monospecific stands managed for pulpwood production [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF].

The objective of our study was to gain insight into the potential of mixed-species plantations of Eucalyptus and NFT to enhance the long-term N status of tropical soils. We hypothesized that introducing A. mangium trees into highly productive eucalypt plantations leads to: 1) large N inputs through biological fixation, estimated consistently by the 15 N natural abundance method and the accretion method, 2) low changes in C and N stocks in the upper soil layers after 1 rotation despite a rise in N cycling through litterfall and soil N mineralization, and 3) an improvement of soil N budgets relative to monospecific eucalypt stands of the same order of magnitude as the amount of N fertilizer commonly applied in commercial plantations. Voigtlaender et al. (2012) showed results for soil C and N stocks, N contents in litterfall as well as soil N mineralization rates at Itatinga (IT). It is one of the 4 sites in the present study. Some results are shown for 3 sites here because the values already published at IT were excluded. Comparisons are made for 4 sites using the results of Voigtlaender et al.'s previously unpublished data for the IT site.

Materials and methods

Study sites

The same experimental design was replicated at Itatinga (IT), Bofete (BO), and Luiz Antônio (LA) in the São Paulo state and at Santana do Paraíso (SdP) in the Minas Gerais state (Figure 1). Large areas of commercial plantations of Eucalyptus grandis Hill ex Maiden and Eucalyptus urophylla S.T. Blake x E. grandis have been established in these regions. A detailed description of the 4 sites can be found in [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF]. In brief, mean annual rainfall was similar at the 4 sites (1240 mm at SdP, 1390 mm at IT and 1420 mm at BO and LA), as well as mean atmospheric humidity (about 70%). The duration of the dry season (monthly rainfall < 30 mm) was 1-2 months at IT and BO, and 3-4 months at LA and SdP (Figure 2). Mean annual temperatures were 20°C at ITA, 21°C at BO, 23°C at LA and 24°C at SdP. The soils were Ferralsols at IT, BO and SdP and Ferralic arenosols at LA (FAO classification). The soil pH was acidic (between 4.5 and 5.8) and the amounts of available phosphorus and base cations were low at all the sites, decreasing sharply with soil depth (Table 1). The soils differed in texture, with clay contents in the 0-5 cm soil layer of 10% at LA, 11% at IT, 12% at BO and 51% at SdP. Our 4 experiments were representative of the range of soil properties and climatic conditions in the regions where the most productive Eucalyptus plantations are managed in Brazil.

Mean annual increment of stemwood at the harvest (6 years after planting) ranged from 18.2 Mg ha -1 yr -1 at IT to 21.8 Mg ha -1 yr -1 at LA in 100E, from 14.7 Mg ha -1 yr -1 at IT to 18.9 Mg ha -1 yr -1 at BO in 50A50E, and from 4.5 Mg ha -1 yr -1 at LA to 11.5 Mg ha -1 yr -1 at BO in 100A [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF]. The biomass production of aboveground tree components at the 4 sites can be found in [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF].

Experimental design

The experiments were set up in former Eucalyptus stands that had been managed for 20-60 years at all the sites. Highly productive Eucalyptus seedlings and cuttings were planted: E. grandis mono-progeny at IT, one E. grandis clone used by the Suzano company at BO, and E. urophylla x E. grandis clones selected by the International Paper company at LA and the Cenibra company at SdP. All the Acacia mangium seedlings were produced from seeds collected in a single highly productive stand in Amazonia (Amapá state). Spacing was 3 m x 3m at all the sites, except at BO where it was 3 m x 2 m.

A complete randomized block design was established at each site with seven treatments and four blocks. Each plot had 10 x 10 trees with two buffer rows (except 8 x 10 trees at BO). Our study was carried out in three of the original seven treatments planted without N fertilization: -100A, monospecific A. mangium stand; -100E, monospecific Eucalyptus stand; -50A50E, mixture in a proportion of 1:1 between Eucalyptus and A. mangium with the same total stocking density as the monospecific stands.

Seedlings were planted between the rows of the previous plantation after soil cultivation with a ripping tine to 40 cm depth. Acacia mangium seedlings were inoculated with Rhizobium strains selected by EMBRAPA for their N2 fixation capacities. In the 50A50E treatment, the two species were planted alternately in the row, and between adjacent rows. The amounts of fertilizer applied (P, K, Ca, Mg and micro-nutrients) were non-limiting to tree growth [START_REF] Laclau | Influence of nitrogen and potassium fertilization on leaf lifespan and allocation of aboveground growth in Eucalyptus plantations[END_REF]) and the lack of N fertilization did not reduce significantly the stemwood biomass of eucalypt trees harvested at 6 years of age [START_REF] Bouillet | Eucalyptus and Acacia tree growth over entire rotation in single-and mixed-species plantations across five sites in Brazil and Congo[END_REF]. Herbicide applications the first two years after planting eliminated grasses and shrubs in these experiments, as in most commercial plantations in Brazil.

Nitrogen contents within tree components

Circumference at breast height (CBH) and tree height (H) were measured in the inner plots at 66, 63, 72 and 73 months after planting at SdP, BO, IT, and LA, respectively. Nitrogen contents in above-ground tree components were estimated by sampling destructively 10 trees of each species in 100A, 100E and 50A50E at each site, distributed over the range of basal area (40 trees per site). The trees were separated into leaves, living branches, dead branches, stemwood and stembark. The stem of each tree was sawn into 3 m sections. The fresh mass of each tree component was measured in the field (± 20 g). The foliage was collected from three different sections of the trees' crown. The biomass of coarse roots, medium-size roots and fine roots were measured for the 2 species in 100A, 100E and 50A50E at the end of the rotation at IT (see [START_REF] Nouvellon | Carbon allocations throughout the development of monospecific and mixed-species plantations of Eucalyptus grandis and Acacia mangium in Brazil[END_REF] for the detailed methodology). Sub-samples of each component were dried at 65°C to constant weight, and the dry biomass of the components in each tree was calculated proportionally. The samples were then ground for chemical analysis.

Quality control procedures were used in the laboratory. Allometric equations were established for each species in each treatment at each site and applied to the corresponding inventory to estimate N contents on a hectare basis from CBH and H measurements.

Litterfall

Litterfall was collected over the last two years of the rotation at BO, LA and SdP. The amounts of C and N returning to soil with litterfall at IT can be found in Voigtlaender et al. (2012). Leaf and fruit litterfall were collected in 4-5 traps (50 cm x 50 cm) per plot installed at various distances from the trees in 100A and 100E, and in 8-10 traps per plot in 50A50E.

Dead branches and bark were collected in an area of 9 m 2 (6 m 2 at BO) delimited between four trees in each plot (replicated in three blocks for the three treatments). The litter traps were set up in three blocks for the three treatments and the litter of the two species was distinguished in 50A50E.

The three sites were far from each other and we could go to each experiment only twice per trimester. Litterfall was collected every 4 weeks (periods of measurement of soil N mineralization) or every 8 weeks (between soil incubation periods). Litter samples were dried at 65°C for 72 h before weighing. Litter dry matter was measured in each plot. The replicates in the three blocks were mixed and composite samples were prepared for each litter component of each species in each treatment at each site every 3 months, then ground for chemical analysis.

Nitrogen contents in the forest floor and the upper soil layers

The methodology described by Voigtlaender et al. (2012) at IT was used at the end of the rotation at BO, LA and SdP. In brief, forest floor and the upper soil layers (0-5 cm, 5-15 cm and 15-30 cm) were sampled in three blocks. Nine positions in each plot were sampled for 100A and 100E and 18 positions were sampled in each plot for 50A50E (9 positions at different distances from each tree species). All the sampling positions were distributed throughout the plots, excluding 2 buffer rows, and replicated in 3 blocks.

The forest floor was sampled with a 15 cm-radius circular frame at each position and divided into three components: Oi (non-fragmented material), Oe (coarse fragments), and Oa (finely fragmented material). The nine samples per component collected in each plot were manually homogenized and one composite sample per plot in mono-specific stands (one sample for each species per plot in 50A50E) was ground for chemical analysis. The ash content was determined by heating sub-samples at 450 °C in an oven for 4 h and used as a correction to determine the ash-free dry mass. Soils were sampled using 5 cm, 10 cm and 15 cm-long metal cylinders (5 cm in diameter) inserted into the upper 0-5 cm, 5-15 cm and 15-30 cm soil layers after collection of the forest floor. All soil samples were air-dried, weighed and the water content was determined from a subsample (dried at 105°C). The roots were removed and the samples were passed through a 2-mm sieve (no gravel in these soils). Bulk density was calculated for all the samples collected as the ratio between oven-dried soil mass and volume of the soil core.

Production of mineral nitrogen

The in-situ coring technique [START_REF] Khanna | In situ core methods for estimating soil mineral-N fluxes: Reevaluation based on 25 years of application and experience[END_REF] was used to estimate N mineralization in the 0-20 cm soil layer in 100A, 100E and 50A50E over the last two years of the rotation at BO, LA and SdP. Soil incubations were conducted for 4-week periods in 12 plots (3 treatments in four blocks) at each site in the middle of each trimester and repeated 8 times over 2 years.

At the onset of each sampling period, three pairs of cores (70 mm in diameter) were driven 20 cm into the soil with a hammer in each plot for 100A and 100E and 6 pairs of cores in each plot for 50A50E (3 pairs close to each tree species). The pairs of soil cores were located 35 cm, 105 cm, and 175 cm from the nearest tree in each plot for a representative sampling of the inter-row. One soil core from each pair was withdrawn immediately and the other core was covered with a plastic cap to prevent mineral N leaching and incubated for 4 weeks. Soil samples were transported in cooled insulated containers and then homogenized manually.

Roots were removed and a subsample was collected for determining the water content at 105°C. Extractions were initiated on the same day for one composite sample in each plot.

Mineral N was extracted by shaking 10 g of soil with 50 ml of 2 M KCl and the concentrations of NH4 + -N and NO3 --N in the extracts were determined by an automated flow injection system (Ruzicka and Hansen 1975). Net ammonification and nitrification were estimated by the difference between post-and pre-incubation concentrations of NH4-N and NO3-N, respectively. Net N mineralization was obtained for each sampling period by summing net ammonification and net nitrification.

Mean annual rates of net ammonification and net nitrification over the study period were estimated multiplying by 13 the mean values across the 8 incubation periods (there are thirteen 4-week periods in 1 year). Voigtlaender et al. (2012) estimated that annual net N mineralization at IT amounted to 123 kg ha -1 yr -1 in 100A and 63 kg ha -1 yr -1 in 100E, from 26 successive incubation periods of 4 weeks (from 4 to 6 yrs after planting). Applying the method used here to their data set (multiplying by 13 the mean mineralization rates estimated over 8 periods of 4 weeks in the middle of each trimester) would estimate mean annual net N mineralization rates at 148 and 65 kg ha -1 yr -1 in 100A and 100E, respectively, i.e. with an overestimation by 20% in 100A and by 3% in 100E. The method used in the present study should therefore be sufficiently accurate to detect the contrasting effects of treatments on the net production of mineral N across the 4 sites.

Assessment of nitrogen fixation Natural abundance of 15 N

The samples collected in 10 trees per species and per site to measure biomass and N contents within aerial tree components (leaf, branch, stemwood and stembark) were used for δ 15 N determinations in 100A, 100E and 50A50E. However, to reduce the number of isotopic analyses, we pooled the samples collected in the 3 dominant, 4 medium-size and 3 smallest trees of each species for each component in each treatment at each site (for a total of 192 samples analyzed). The method described by [START_REF] Bouillet | Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 2: nitrogen accumulation in the stands and biological N2 fixation[END_REF] was used to estimate the percentage of N derived from atmospheric N2 (%Ndfa) in A. mangium trees.

Nitrogen accretion method

Nitrogen fixation at the 4 sites was also estimated using the N accretion method [START_REF] Forrester | Assessing nitrogen fixation in mixed-and single-species plantations of Eucalyptus globulus and Acacia mearnsii[END_REF] by comparing the N contents within soil, forest floor and biomass in 100A, 100E and 50A50E. All the N stocks were measured at each site, and for IT we used the values in the mineral soil and in the forest floor shown by Voigtlaender et al. (2012). The amount of N in the belowground biomass (NBG) was only measured at IT (unpublished data), and we considered that the proportions of N in aboveground (NAG) and belowground tree components were similar at the other sites for each treatment. NBG was therefore estimated in each plot at BO, LA and SdP, multiplying the aboveground N content by the NBG/NAG ratio measured at IT.

Rates of N2 fixation over six years after planting were estimated by calculating the difference in total N stock (summing N contents in tree biomass, forest floor and 0-15 cm soil layer) between 100E and the plots containing A. mangium (100A and 50A50E). We hypothesized that differences among treatments resulted only from N2 fixation and that N losses via leaching or denitrification were negligible in our plots, which did not receive N fertilization. It was shown at IT that N leaching at a depth of 3 m was lower than atmospheric inputs over a Eucalyptus rotation [START_REF] Binkley | Connecting ecological science and management in forests for scientists, managers and pocket scientists[END_REF], and the same feature was observed at IT the first four years after planting in 100A and 50A50E (unpublished data). In addition, some measurements during the rainy season at IT also showed very low denitrification fluxes (unpublished data), as commonly reported in forest plantations and savanna woodland [START_REF] Livesley | Seasonal variation and fire effects on CH4, N2O and CO2exchange in savanna soils of northern Australia[END_REF]).

Nitrogen budgets relative to monospecific Eucalyptus stands

A simple N budget was calculated in the soil to estimate the enrichment in soil N over the first rotation after introducing N fixing trees, relative to monospecific Eucalyptus plantations. In each experiment, we used the equation:

B = (F -E ) -(0 -E ) = F -E + E (1)
where BN is the soil N budget in 100A (resp. in 50A50E) relative to 100E, FN is the biological fixation of N2 in 100A estimated from the N accretion method (resp. in 50A50E), EA is the exportation of N within stemwood at the harvest in 100A (resp. in 50A50E), and EE is the amount of N exported with stemwood at the harvest in 100E.

Laboratory analyses

Total N in plant samples was determined by acid-base titration (TE036/01-Tecnal, Piracicaba, Brazil) after Kjeldahl mineralization. Carbon contents in the forest floor were estimated from dry matter values, assuming that the C concentration in each litter layer was similar to that measured at IT by Voigtlaender et al. (2012) for the same treatments. Carbon and N contents in the mineral soil at SdP, LA and BO were determined using NIR Spectrometry (Foss NIRSystems 5000, Silver Spring, MD, USA) for the 810 samples collected [START_REF] Brunet | Determination of carbon and nitrogen contents in Alfisols, Oxisols and Ultisols from Africa and Brazil using NIRS analysis: Effects of sample grinding and set heterogeneity[END_REF]. A CHN analyzer (Fisons/Carlo Erba NA 2000, Milan, Italy)

was used to determine C and N concentrations for 150 samples selected to cover the range of spectra (110 samples for a specific calibration and 40 for cross-validation). The validation data set showed that NIRS predictions were accurate (R 2 = 0.94 for C and 0.95 for N).

Statistical analyses

We analyzed the main effects and interactions between treatments in each experimental area using the GLM procedure in SAS. When significant differences (P < 0.05) between treatment levels were detected, the Tukey test was used to compare treatment means. Then we used the average results for each treatment at each site to test the effects of the treatments and the sites as well as their interaction, as a global response study. Nitrogen content models were adjusted for each tree component by NPL procedure of SAS 9.2 (SAS Institute, Cary, NC, USA).

Global and local models were established by treatment with up to three parameters (y = a + bx C ) and the Akaike's information criterion was used to select the best models. Homogeneity of variance was tested by Levene's test and original values were transformed when the variances were unequal. The data were analyzed with SAS statistical software (SAS Institute Inc., 2000), where linear regressions were established to assess the relationships between annual values of net N mineralization and N content in litterfall for each tree species across the plots studied.

Results

Nitrogen accumulation in the trees

Mean annual N accumulation in the aboveground (ABG) tree components over the rotation of 6 years ranged from 40 to 110 kg ha -1 yr -1 depending on the treatment and the site, and was approx. 40% higher in 100A than in 100E at IT, SdP, and BO (Figure 3). At LA by contrast, the ABG N content in 100A was only half of the amount in 100E. The ABG N content in the trees was intermediate in 50A50E relative to 100A and 100E, regardless of the site. About half of the N content in the ABG biomass in 100A was accumulated in the leaves and the stembark. In 100E and 50A50E, the stemwood contained from 40 to 60% of the ABG N content. Only 5-20% of the total amount of N in ABG tree components was accumulated in the branches whatever the site and the treatment.

Dry matter and N content in litterfall

Litterfall dry matter differed between treatments (Table 2). It was 50% lower in 100A than in 100E at LA, and 25% lower in 100A than in 100E at BO, and amounted to about 9 Mg ha -1 yr - 1 in all the treatments at SdP (Figure 4a). In 50A50E, the proportion of A. mangium dry matter in the total litterfall was highly dependent on the site, ranging from 15% at LA to 40% at SdP.

Nitrogen contents in litterfall were on average 1.7-fold as high in 100A as in 100E across the three sites (Figure 4b). The amounts of N in litterfall ranged from 75 to 103 kg ha -1 yr -1 in 100A depending on the site and from 49 to 62 kg N ha -1 yr -1 in 100E. N contents in litterfall were intermediate in 50A50E and A. mangium components represented between 30% (at LA) and 60% (at SdP) of the total amount of N in litterfall.

Carbon and nitrogen stocks in the forest floor and in the upper soil layers

The C stocks in the forest floor and in the 0-15 cm soil layer were not significantly influenced by the treatments but they differed between the sites (Table 2 and Appendix 1). While the total amounts of C in the forest floor ranged from 7.2 to 9.5 Mg ha -1 at BO and LA, they were close to 5 Mg ha -1 at SdP (Appendix 1). The pattern was different in the 0-15 cm soil layer, with higher C stocks at SdP (35 Mg ha -1 ) than at BO (28 Mg ha -1 ) and LA (23 Mg ha -1 ).

The N content in the forest floor (FN) was 30-90% higher in 100A than in 100E and intermediate in 50A50E at the three sites (Table 2 andAppendix 2). The effect of the site was much more marked in 100A (FN ranging from 136 kg N ha -1 at SdP to 231 kg N ha -1 at LA) than in 100E (from 107 kg N ha -1 at SdP to 141 kg N ha -1 at BO) and in 50A50E (from 133 kg N ha -1 at SdP to 151 kg N ha -1 at BO). In the 0-15 cm soil layer, the N stocks were not significantly different between treatments but were higher at SdP (about 2500 kg N ha -1 ) than at BO (about 1500 kg N ha -1 ) and LA (about 1200 kg N ha -1 ) (Table 3). The total N stocks in the upper soil layers (summing the amounts in the forest floor and in the 0-15 cm layer) were not significantly influenced by the treatments 6 years after planting, whatever the site.

Nitrogen mineralization rates

The pools of mineral N in the 0-20 cm soil layer (non-incubated) and the N mineralization rates were significantly different between treatments and between sites (Table 2). On average over the study period, mineral N pools in the 0-20 cm layer were 16.6 kg ha -1 in 100A, 12.8 kg ha -1 in 50A50E and 12.4kg ha -1 in 100E with minimum values during the cold and dry season (data not shown). Mean N mineralization rates across the three sites amounted to 158.5 kg N ha -1 yr -1 in 100A, 131.2 kg N ha -1 yr -1 in 50A50E and 99.8 kg N ha -1 yr -1 in 100E (Figure 5 and Table 2). Net N mineralization rates were significantly higher at BO (199 kg N ha -1 yr -1 on average) than at LA (100 kg N ha -1 yr -1 ) and at SdP (95 kg N ha -1 yr -1 ). Net ammonification represented about 20% of the total net N mineralization in the three treatments at BO and was negligible at the two other sites (Figure 5).

Relationships between N content in litterfall and soil N mineralization

The amount of N returning to soil in litterfall was positively correlated with net soil N mineralization across the studied plots at the end of the rotation (Figure 6). However, the relationship was very different for eucalypt and acacia monocultures (not significant in A. mangium stands). Litterfall N return to soil could be used as a proxy for net soil N mineralization at the end of the rotation in eucalypt plantations. Tree plasticity to recycle N in litterfall when the soil N availability increased was much higher for A. mangium than for E.

grandis. For soil N mineralization rates ranging from 50 to 300 kg ha -1 yr -1 in E. grandis monocultures, N content in litterfall ranged from 50 to 70 kg ha -1 yr -1 . N content in litterfall ranged from 70 to 130 kg ha -1 yr -1 for soil N mineralization rates between 100 and 250 kg ha -1 yr -1 .

N2 fixation estimates

The accretion method estimated N2 fixation in 100A over the rotation of 6 years at 413, 348 and 367 kg N ha -1 at IT, BO and SdP, respectively (Table 3). A high mortality of A.

mangium trees at the end of the rotation prevented us from using this method at LA for the 100A treatment. In 50A50E, N2 fixation over the rotation ranged from 84 kg N ha -1 at LA to 277 kg N ha -1 at SdP. Nitrogen concentrations in the aboveground tree components of the 50A50E treatment were significantly higher in A. mangium trees than in Eucalyptus trees (Table 4). N concentrations differed depending on the site and a species x site interaction was significant in living branches and stembark but not in leaves and stemwood.

Unexpectedly, δ 15 N values in natural abundance within leaves and living branches were significantly lower for Eucalyptus than for A. mangium trees (Table 4). In stemwood and stembark, the differences between species were not significant. δ 15 N values differed significantly depending on the site with higher values at SdP than at the other sites in all the aboveground tree components.

Nitrogen budgets

The amounts of N exported with stemwood at the harvest ranged from 148 to 238 kg ha -1 and were little influenced by the treatments at each site, except in 100A at LA where tree mortality was high (Table 3). Relative to a rotation of monospecific eucalypt trees, a monoculture of A. mangium increased the N budget by about 400 kg ha -1 over 6 years at IT, BO and SdP. The N budget over the rotation was about 200 kg ha -1 higher in 50A50E relative to 100E at the same sites, but lower at LA where the climate was not suitable for A. mangium trees.

Discussion

Biological N2 fixation

While the accretion method showed large N inputs through biological N2 fixation in 100A and 50A50E at the 4 sites, in agreement with our first hypothesis, the 15 N natural abundance method was unable to estimate biological N2 fixation. Indeed, the δ 15 N values in the leaves at the four sites were lower in non-NFT (both in 100E and 50A50E) than in NFT, which suggests that A. mangium trees did not fix N2. However, the accretion method at the 4 sites in our study and 15 N labeling at IT [START_REF] Bouillet | Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 2: nitrogen accumulation in the stands and biological N2 fixation[END_REF][START_REF] Paula | Nitrogen fixation rate of Acacia mangium Wild at mid rotation in Brazil is higher in mixed plantations with Eucalyptus grandis Hill ex Maiden than in monocultures[END_REF] show that large amounts of N were actually fixed. Lower δ 15 N values in E. grandis leaves than in A. mangium leaves consistently at the four sites confirm the risk of misuse of the 15 N natural abundance method pointed out in previous studies [START_REF] Shearer | N2-fixation in field settings: estimations based on natural 15 N abundance[END_REF][START_REF] Binkley | Ecology and Management of Forest Soils[END_REF][START_REF] Chalk | Do techniques based on 15 N enrichment and 15 N natural abundance give consistent estimates of the symbiotic dependence of N 2 -fixing plants?[END_REF]. If the δ 15 N values had been lower for A. mangium trees than for E. grandis trees, those values might have been used to estimate the N2 fixation rates whereas the method would not be suitable. Lower δ 15 N values in E. grandis trees than in A. mangium leaves might reflect different fractionation patterns of the 15 N taken up in the soil, which might be a consequence of the uptake of N by different mycorrhizal strains. [START_REF] Forrester | Assessing nitrogen fixation in mixed-and single-species plantations of Eucalyptus globulus and Acacia mearnsii[END_REF] showed a strong relationship between the mycorrhizal status and δ 15 N values within eucalypt and acacia trees in Australia and stressed the difficulties in using the 15 N natural abundance method for plants with different mycorrhizae strains. Further studies are needed to improve our understanding of the role of mycorrhiza in the nutrition of eucalypt and acacia trees in tropical plantations, and their effect on the isotopic fractionation of N.

Although the accretion method led to high standard errors of N2 fixation in our study, the estimates were consistent across the four sites. This method requires an accurate quantification of N content in the trees, in the forest floor and in the soil. Despite an intensive sampling (160 trees harvested and N concentrations determined in 640, 144, and 972 samples of plant, forest floor and soil, respectively), the variability between blocks at each site was high. However, the global picture is consistent across the four sites and a large amounts of N input to the ecosystem through the N2 fixation of acacia trees is consistent with more active N cycling in 100A and 50A50E than in 100E. The accretion method assumes that N inputs (except biological fixation) and N outputs in the soil are similar in the stands including NFT and in the stands with only non-NFT. The conditions for using the accretion method were propitious in our study. Nitrogen atmospheric deposition was probably of the same order of magnitude in eucalypt and acacia stands (low differences between eucalypt and acacia leaf areas probably led to similar filter effects on dry deposition) and very low losses of N by deep drainage have been shown in eucalypt plantations growing on deep tropical soils [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 seriesP[END_REF][START_REF] Binkley | Connecting ecological science and management in forests for scientists, managers and pocket scientists[END_REF]. Monitoring soil solution chemistry in A. mangium stands and mixed-species stands with eucalypt and acacia over the first four years after planting at IT showed that N leaching at a depth of 3 m was of the same order of magnitude as atmospheric inputs (unpublished data). The depth of the soil was > 3 m at the four sites and whether the losses of N by deep drainage were higher under acacia than under eucalypt at some sites (as a result of higher soil N mineralization rates than in eucalypt plantations), this bias would lead to an underestimation of the N2 fixation rates through the accretion method. The estimates of N2 fixation at the end of one rotation of A. mangium (about 400 kg ha -1 from planting to age 6 years, except at LA) and in mixed-species plantations including 50% of A. mangium trees (about 250 kg ha -1 over one rotation, except at LA) are therefore conservative values that might be slightly underestimated at some sites. High N2 fixation rates were also estimated, from 15 N labeling methods, in Congolese Acacia mangium plantations where the amount of N derived from the atmosphere in the mixture one year after labelling (220 kg ha -1 ) was 60% higher than expected on the basis of the amount found in 100A (276 kg ha -1 ), taking into account a 50% lower density of acacia trees in mixture (Tchichelle et al., 2017b). The low N2 fixation rates at LA compared with the other sites can be explained by a high mortality of A.

mangium trees at the end of the rotation resulting from unsuitable pedo-climatic conditions for this species.

Nitrogen cycling

Planting A. mangium trees in soils cultivated over several decades with eucalypt plantations dramatically changed nitrogen cycling in only 6 years. Even though a strong effect of NFTs on N cycling in planted forests is well documented [START_REF] Binkley | Nitrogen fixation in tropical forest plantations[END_REF][START_REF] Forrester | Mixed plantations of Eucalyptus with nitrogen fixing trees: a review[END_REF], the originality of our results relies on the speed of the changes in highly productive plantations (from 4 to 6 years after planting NFTs) and the consistency of the results at 4 sites.

The main N fluxes of the biological cycle strongly increased in the stands including acacia trees in comparison with monospecific eucalypt plantations: N accumulation in the trees, N content in litterfall and soil N mineralization. A strong effect of NFTs on the production of mineral N in the soil has been shown in tropical plantations with eucalypts and acacias in the Congo (Tchichelle et al., 2017a), in Brazil [START_REF] Rachid | Mixed plantations can promote microbial integration and soil nitrate 535 increases with changes in the N cycling genes[END_REF][START_REF] Santos | Dynamics of aboveground biomass accumulation in monospecific and mixed-species plantations of Eucalyptus and Acacia on a Brazilian sandy soil[END_REF]2017), in Australia [START_REF] Adams | Role of Acacia Spp. in Nutrient Balance and Cycling in Regenerating Eucalyptus regnans F. Muell. Forests. Temporal Changes in Biomass and Nutrient Content[END_REF][START_REF] Forrester | Nutrient cycling in a mixed-species plantation of Eucalyptus globulus and Acacia mearnsii[END_REF], and in Hawaii [START_REF] Binkley | Twenty years of stand development in pure and mixed stands of Eucalyptus saligna and nitrogenfixing Facaltaria moluccana[END_REF].

N accumulation in the aboveground biomass was higher in 50A50E and 100A than in 100E at all the sites (except LA), which probably reflects the increase in N availability for the trees resulting from the high N2 fixation rates of A. mangium trees (Wang et al., 2010;[START_REF] Inagaki | Nutrient accumulation in aboveground biomass of planted tropical trees: a meta-analysis[END_REF]. About half of the N content in the aboveground components of A. mangium trees was accumulated in leaves and stembark at 6 years of age whereas the stemwood contained half of the N stock in eucalypt trees. High accumulation rates of N in leaves and bark of acacia trees relative to eucalypt trees have already been reported [START_REF] Bouillet | Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil: 2: nitrogen accumulation in the stands and biological N2 fixation[END_REF][START_REF] Koutika | Changes in N and C concentrations, soil acidity and P availability in tropical mixed acacia and eucalypt plantations on a nutrient-poor sandy soil[END_REF]. The contrasting proportions of N accumulated in leaf, branch, stemwood and stembark for eucalypt and acacia trees at harvesting age suggest that the management of harvest residues could strongly influence the N bioavailability for the next rotation. 15 N-labeling of eucalypt harvest residues (leaf, branch, bark) showed that the amount of N released throughout the decomposition of the forest floor is highly dependent on the mixture between leaves and ligneous residues, which can strongly influence tree growth after replanting [START_REF] Versini | The role of harvest residues to sustain tree growth and soil nitrogen stocks in a tropical Eucalyptus plantation[END_REF]2016). Even though the amounts of N are higher in A.

mangium residues than in eucalypt residues, the dynamics of N release is also influenced by the C quality of the litter [START_REF] Freschet | A plant economics spectrum of litter decomposability[END_REF]. The faster decomposition of E. grandis leaves than A. mangium leaves at IT might be a consequence of higher concentrations of lignin and condensed tannins as well as lower concentrations of water soluble compounds in acacia leaves [START_REF] Bachega | Decomposition of Eucalyptus grandis and Acacia mangium leaves and fine roots in tropical conditions did not meet the Home Field Advantage hypothesis[END_REF].

Soil N mineralization rates in the topsoil are generally higher under NFTs than under non-NFTs (e.g. [START_REF] Forrester | Nutrient cycling in a mixed-species plantation of Eucalyptus globulus and Acacia mearnsii[END_REF]Wang et al., 2010;[START_REF] Koutika | Changes in N and C concentrations, soil acidity and P availability in tropical mixed acacia and eucalypt plantations on a nutrient-poor sandy soil[END_REF]. This feature can be explained by larger amounts of N returning to soil with litterfall as well as rapid changes in soil biological activity and microbial community composition [START_REF] Bini | Microbial biomass and activity in litter during the initial development of pure and mixed plantations of Eucalyptus grandis and Acacia mangium[END_REF][START_REF] Rachid | Mixed plantations can promote microbial integration and soil nitrate 535 increases with changes in the N cycling genes[END_REF][START_REF] Rachid | Intercropped silviculture systems, a key to achieving soil fungal community management in eucalyptus plantations[END_REF]. The most noticeable difference in net N mineralization among the sites in our study is the very high production of soil mineral N in eucalypt monocultures at BO, which could be a consequence of silvicultural practices over the rotations before trial establishment.

About 200 kg N ha -1 rotation -1 was applied at BO whereas the doses were commonly <100 kg N ha -1 rotation -1 by other forest companies (unpublished data). A very fast root development in deep soil layers combined with high water and nutrient demand to produce leaves and fine roots after planting prevent the loss of large amounts of N in eucalypt plantations growing on deep tropical soils [START_REF] Versini | The role of harvest residues to sustain tree growth and soil nitrogen stocks in a tropical Eucalyptus plantation[END_REF][START_REF] Laclau | Biogeochemical cycles of nutrients in tropical Eucalyptus plantations. Main features shown by intensive monitoring in Congo and Brazil[END_REF]. The large amounts of N applied at BO over the rotations before the establishment of our trial were therefore probably not lost, and might account for the very high N mineralization rates measured in the topsoil whatever the treatment (100A, 100E and 50A50E).

Influence of A. mangium trees on soil C and N stocks

While soil C and N stocks commonly increase in NFTs relative to monospecific eucalypt stands [START_REF] Resh | Greater soil carbon sequestration under nitrogen-fixing trees compared with Eucalyptus species[END_REF]Wang et al., 2010;[START_REF] Forrester | Soil Organic Carbon is Increased in Mixed-Species Plantations of Eucalyptus and Nitrogen-Fixing Acacia[END_REF][START_REF] Koutika | Changes in N and C concentrations, soil acidity and P availability in tropical mixed acacia and eucalypt plantations on a nutrient-poor sandy soil[END_REF], the changes were not significant after one rotation of A. mangium trees at the study sites, in agreement with our second hypothesis and the results already published at IT (Voigtlaender et al., 2012). Larger C and N stocks in the topsoil at SdP than at the other sites reflect the differences in clay contents and the well-documented relationship between soil texture and soil organic matter accumulation [START_REF] Feller | Physical control of soil organic matter dynamics in the tropics[END_REF]. While other studies showing that NFTs can greatly enhance soil N stocks were carried out in degraded tropical lands [START_REF] Resh | Greater soil carbon sequestration under nitrogen-fixing trees compared with Eucalyptus species[END_REF][START_REF] Koutika | Changes in N and C concentrations, soil acidity and P availability in tropical mixed acacia and eucalypt plantations on a nutrient-poor sandy soil[END_REF], the field trials in our study were set up after decades of cultivation of highly productive eucalypt plantations. Soil C increased after 60 years of eucalypt monocultures relative to the native savanna at IT but, in agreement with the results in the present study, soil N stocks were not modified [START_REF] Maquère | Influence of land use (savanna, pastures, Eucalyptus plantations) on soil organic matter stocks in Brazil[END_REF].

Soil N budgets and consequences for fertilization regimes

In agreement with our third hypothesis, introducing A. mangium trees in commercial eucalypt plantations strongly enhanced soil N budgets in 100A and 50A50E. Atmospheric N2 fixation led to an input of approx. 250 kg N ha -1 in mixed-species stands and 400 kg N ha -1 in monospecific A. mangium stands, which was much higher than the amounts of N fertilizer commonly applied (about 100 kg N ha -1 ) in Brazilian commercial plantations (Gonçalves et al., 2008). However, the budgets have been computed here relative to eucalypt monocultures and comprehensive input-output budgets including all the major fluxes (in particular atmospheric deposition, run off and deep leaching) would be necessary to assess the longterm changes in soil N stocks. Moreover, fine roots of E. grandis trees were found 4 years after planting down to the water table at a depth of 17 m at Itatinga while the A. mangium roots reached a depth of 12 m [START_REF] Germon | Consequences of mixing Acacia mangium and Eucalyptus grandis trees on soil exploration by fine-roots down to a depth of 17 m[END_REF], which might provide access to deep soil profile sources of soil N [START_REF] Houlton | Convergent evidence for widespread rock nitrogen sources in Earth's surface environment[END_REF]. The larger amounts of N accumulated in A.

mangium trees than in eucalypt trees at our study sites, consistent with the results of a metaanalysis [START_REF] Inagaki | Nutrient accumulation in aboveground biomass of planted tropical trees: a meta-analysis[END_REF], lead to much larger N inputs at the soil surface with harvest residues in A. mangium stands than in eucalypt plantations. A strong relationship between the N content in harvest residues, heterotrophic respiration, and early tree growth after replanting was shown in Congolese eucalypt plantations [START_REF] Versini | The manipulation of organic residues affects tree growth and heterotrophic CO2 efflux inatropical Eucalyptus plantation[END_REF]. Further studies are needed to improve our understanding of the biogeochemical processes driving the mineralization of harvest residues in short-rotation forests in order to optimize the fertilization regimes over successive rotations [START_REF] Versini | The role of harvest residues to sustain tree growth and soil nitrogen stocks in a tropical Eucalyptus plantation[END_REF][START_REF] Rocha | Decomposition rates of forest residues and soil fertility after clear-cutting of Eucalyptus grandis stands in response to site management and fertilizer application[END_REF].

In conclusion, the 15 N natural abundance method was unsuitable to estimate N2 fixation despite high N fixation rates in A. mangium stands, consistently shown by the accretion method at 4 sites in Brazil. Planting A. mangium trees strongly increased the availability of mineral N in the topsoil as well as N cycling through litterfall. Nitrogen budgets suggest that introducing NFTs in eucalypt plantations can contribute to increasing their sustainability through a reduction of the need for N fertilizer addition. Table 2. Effects of treatments (T), sites (S) and interaction between treatment and site (T x S) on forest floor dry matter (MF, kg ha -1 yr -1 ), N stock in the forest floor (NF, kg ha -1 ), C stock in the 0-15 cm soil layer (SC, kg ha -1 ), N stock in the 0-15 cm soil layer (SN, kg ha -1 ), mineral N stock in the 0-20 cm soil layer (NM, kg ha -1 ), net N mineralization (MN, kg ha -1 yr -1 ), litterfall dry matter (LDM, kg ha -1 yr -1 ), N content in the litterfall (LN, kg ha -1 yr -1 ), and total N stock in trees + forest floor + 0-15 cm soil layer (NT, kg ha -1 ). Different letters in the same row are indicated when the differences between treatments or between sites are significant (P < 0.05).

P values

Mean

Figure 1 .Figure 2 .

 12 Figure 1. Localization of the experimental sites in Brazil.

Figure 3 .

 3 Figure 3. Mean annual N accumulation in the aboveground tree components over a rotation of 6 years (harvesting age) in 100A, 50A50E and 100E at IT (a), BO (b), LA (c) and SdP (d). Vertical bars show standard errors between blocks for each treatment (n = 3 at IT and n = 4 at BO, LA and SdP). Different letters indicate significant differences between treatments in total N content aboveground at the same site (P < 0.05).

Figure 4 .

 4 Figure 4. Annual litterfall dry matter (Mg ha -1 yr -1 ) from 4 to 6 years after planting a) and annual N content (kg ha -1 yr -1 ) in litterfall b) in 100A, 50A50E and 100E at BO, LA and SdP. Vertical bars show standard errors between blocks for each treatment (n = 3). Different letters indicate significant differences between treatments at the same site (P < 0.05).

Figure 5 .

 5 Figure 5. Net ammonification and nitrification rates in 100A, 50A50E and 100E at BO a), LA b), and SdP c). Vertical bars show standard errors between blocks (n = 3).

Figure 6 .

 6 Figure 6. Relationship between net nitrogen mineralization in the top soil and nitrogen content in litterfall from 4 to 6 years after planting in 100A, 50A50E and 100E. The dotted and the full lines show linear regressions for 100A and 100E, respectively, across all the studied plots.

  Mean and standard deviation between blocks (n = 3) of selected soil properties in the 4 experiments. The fertilization regimes in each experiment are indicated.

  The new frontiers of afforestation in Brazil are close to the Amazonia region where high temperatures are more suitable for A. mangium trees than for eucalypt trees. Biomass production and N cycling in mixed-species plantations with eucalypt and A. mangium trees should be studied in this context where facilitation processes might lead to transgressive overyielding as shown recently under a similar climate in the Congo[START_REF] Pretzsch | Transgressive overyielding in mixed compared with pure stands of Norway spruce and European beech in Central Europe: evidence on stand level and explanation on individual tree level[END_REF]).Voigtlaender M., Laclau J.-P., Gonçalves J.L.M., de Cássia Piccolo M., Moreira M.Z., Nouvellon Y., Ranger J., Bouillet J.-P., 2012. Introducing Acacia mangium trees in Eucalyptus grandis plantations: consequences on soil organic matter stocks and nitrogen mineralization. Plant Soil, 352, 99-111. Wang F, Li Z, Xia H, Zou B, Li N, Liu J, Zhu W., 2010. Effects of nitrogen-fixing and nonnitrogen-fixing tree species on soil properties and nitrogen transformation during forest restoration in southernChina. Soil Sci Plant Nutr 56,[297][298][299][300][301][302][303][304][305][306]

  N content in boiler ash was < 0.1%; N.D.: not determined.

							0.40 (0.21) 0.11 (0.00) 0.11 (0.00)	5.87 (0.19) 1.90 (0.43) 0.85 (0.22)	P:37; K: 186 Boiler ash: 3000* B, S
	Luiz Antônio (LA)	0-5 50-100	1.38 (0.11) N.D.	4.8 (0.1) 4.8 (0.2)	1.7 (0.3) 0.4 (0.1)	14.0 (4.6) 1.7 (0.6)	0.84 (0.12) 0.13 (0.04)	4.32 (0.08) 1.98 (0.27)	N:4; P:29; K:147 Lime: 1200
		200-300	N.D.	4.8 (0.2)	0.2 (0.0)	1.0 (0.0)	0.11 (0.00)	1.02 (0.05)	Cu, Zn, B
	Santana do Paraíso (SdP)	0-5 50-100 200-300	1.04 (0.14) N.D. N.D.	5.5 (0.5) 4.7 (0.3) 5.0 (0.3)	3.8 (0.8) 1.2 (0.2) 0.1 (0.0)	6.4 (2.5) 1.3 (0.6) 1.0 (0.0)	5.78 (4.64) 0.14 (0.02) 0.16 (0.01)	10.75 (4.96) 3.79 (0.43) 2.15 (0.43)	N: 6; P: 45; K: 162; dolomite: 1500; Cu, B, Zn

*

Acknowledgements

We are grateful for the support of Cenibra, International Paper, Suzano Papel e Celulose. The Itatinga experiment is included in the SOERE F-ORE-T network, which is supported annually by Ecofor, Allenvi and the French National Research Infrastructure ANAEE-F (http://www.anaee-france.fr/fr/). We are grateful to the staff in the forest companies and at the Itatinga Experimental Station, in particular Rildo Moreira e Moreira (Esalq, USP) and Eder Araujo da Silva (http://www.floragroapoio.com.br), for their technical support.

Table 3: Nitrogen stocks in tree biomass, in the forest floor and in the 0-15 cm soil layer (kg ha -1 ) approx. 6 years after planting in 100A, 100E, and 50A50E at 4 sites in Brazil. Nitrogen fixation rates in 100A and 50A50E were estimated by difference between total N stocks relative to 100E (accretion method). Standard errors between blocks are indicated (n = 3). Significant effects are indicated in bold (P < 0.05). Different letters in the same row are indicated when the differences between treatments are significant.


J B Foulquier

P Berneau

A Frérou

M C Verdier

F Saint-Marcoux

A Petitcollin

C Tron

E Bellissant

Dr Florian Lemaitre
email: florian.lemaitre@chu-rennes.fr

Liposomal amphotericin B pharmacokinetics in a patient treated with extracorporeal membrane oxygenation

Keywords: amphotéricine B, oxygénation par membrane extracorporelle, pharmacocinétique amphotericin B, extracorporeal membrane oxygenation, pharmacokinetics

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

1 Introduction Amphotericin B is a macrocyclic polyene with a broad-spectrum antifungal activity indicated in the management of invasive fungal infections and leishmaniasis. Lipid formulations were developed to improve drug tolerability. The recommended regimen for liposomal amphotericin B in invasive pulmonary aspergillosis is 3-5 mg/kg daily infused over 30-60 minutes.

Extracorporeal membrane oxygenation (ECMO) is a complex life-support technique which aims to compensate cardiac and/or respiratory function in case of organ failure. Its potential for interacting with the pharmacokinetics of several drugs used in the intensive care unit (ICU) is well documented.

Drug sequestrations within ECMO components, as well as release from binding sites and increase in drug clearance or volume of distribution have been reported [START_REF] Shekar | Pharmacokinetic changes in patients receiving extracorporeal membrane oxygenation[END_REF]. Liposomal amphotericin B may interact with ECMO device due to its physicochemical properties, but limited data is available on this subject.

Patient and methods

We report the case of a 33-year-old woman admitted to our ICU for acute respiratory distress syndrome in April 2017 (day 0). The patient had a history of Hodgkin lymphoma diagnosed in 2008, which required hematopoietic stem cell transplantation and intensive chemotherapy in April 2017.

Two weeks before ICU admission, the patient presented with cough associated with chest pain and dyspnea initially treated with amoxicillin. Despite antibacterial treatment, her status worsened, and she was admitted to the ICU. On day 1 Aspergillus fumigatus, Streptococcus pneumonia, and Moraxella catarrhalis were isolated from bronchoalveolar lavage (BAL) culture. Voriconazole and piperacillintazobactam were initiated. Invasive pulmonary aspergillosis diagnosis was confirmed by chest imaging and elevated galactomannan antigen in BAL initially at 1.956 (threshold <0.5). Minimum inhibitory concentrations for voriconazole, micafungin, and amphotericin B were 0.19, 0.012, and 0.38 µg/mL. On day 16 hemiplegia associated with microbiological failure (galactomannan antigen >6 in BAL and positive culture) led to the addition of micafungin. Respiratory symptom worsening on day 34 led to switching to liposomal amphotericin B (4 mg/kg/day, 6-hour infusion). Veno-venous ECMO was required while culture and antigen for Aspergillus fumigatus were still positive. Due to the complexity of the situation, a complete pharmacokinetic profile of amphotericin B was assessed at steady state on day 47 to guide therapy. Despite antifungal therapy, the patient's clinical condition continued to worsen until death on day 55. Blood samples were collected before infusion and 2, 4, 6, 8, 16, and 20 hours after drug infusion. Drug levels were determined using liquid chromatography paired with a photodiode array detector.

Pharmacokinetic parameters were estimated using a non-compartmental analysis with the PKSolver software [START_REF] Zhang | PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in microsoft excel[END_REF].

Results and discussion

Despite the non-linear kinetic of liposomal amphotericin B, the pharmacokinetic parameters obtained in our case patient were similar to those reported in critically ill non-ECMO patients [START_REF] Heinemann | Pharmacokinetics of liposomal amphotericin B (Ambisome) in critically ill patients[END_REF] (Table 1). As previously published, the use of ECMO may significantly alter pharmacokinetic profiles of commonly used drugs. Physiological changes such as a larger volume of distribution or a prolonged elimination have been reported [START_REF] Touchard | Predictors of insufficient peak amikacin concentration in critically ill patients on extracorporeal membrane oxygenation[END_REF][START_REF] Lemaitre | Impact of extracorporeal membrane oxygenation and continuous venovenous hemodiafiltration on the pharmacokinetics of oseltamivir carboxylate in critically ill patients with pandemic (H1N1) influenza[END_REF]. The increase in volume of distribution is usually related to a dilutional effect associated with a loss of drug in the membrane oxygenator or polyvinylchloride tubing. Due to its molecular size and lipophilicity, liposomal amphotericin B is expected to be absorbed or sequestrated by circuit component. However, the volume of distribution calculated for our patient was similar to values observed in critically ill patients without ECMO [START_REF] Heinemann | Pharmacokinetics of liposomal amphotericin B (Ambisome) in critically ill patients[END_REF]. This result also depends on the severity of the patient's underlying illness which could explain the difference with neutropenic patients and healthy volunteers [START_REF] Coukell | Liposomal amphotericin B[END_REF][START_REF] Bekersky | Pharmacokinetics, excretion, and mass balance of liposomal amphotericin B (AmBisome) and amphotericin B deoxycholate in humans[END_REF]. The elimination of the drug can also be prolonged in case of organ failure and increase in volume of distribution. In the present case, the drug elimination was comparable to critically ill patients and longer than in the two other groups of patients (i.e., neutropenic patients and healthy volunteers) [START_REF] Heinemann | Pharmacokinetics of liposomal amphotericin B (Ambisome) in critically ill patients[END_REF][START_REF] Coukell | Liposomal amphotericin B[END_REF][START_REF] Bekersky | Pharmacokinetics, excretion, and mass balance of liposomal amphotericin B (AmBisome) and amphotericin B deoxycholate in humans[END_REF]. Hence, the ECMO device did not seem to alter the clearance of liposomal amphotericin B. Furthermore, the exposure (i.e., the area under the curve [AUC]) was, in the present case, closer to the value reported in ICU patients and healthy volunteers than in neutropenic patients (133, 171, 171 versus 555 µg.h/mL, respectively). Moreover, the trough concentration measured 20 hours after the onset of infusion was 4.9 µg/mL, a result close to a very similar case where liposomal amphotericin B measured at H13 and H18 was respectively 5.8 and 6.2 µg/mL [START_REF] Ruiz | Potential voriconazole and caspofungin sequestration during extracorporeal membrane oxygenation[END_REF]. On the contrary, the C max measured in our patient was lower than that described in critically ill non-ECMO patients (8.7 versus 14.4 µg/mL). This may result from the extended infusion duration performed in our patient.

Extending infusion duration, which aimed to decrease renal toxicity, could have decreased treatment efficacy due to the concentration-dependent antifungal activity of amphotericin B. These findings might be in favor of a minor impact of ECMO on liposomal amphotericin B pharmacokinetics.

Conclusion

We presented the first case of a complete pharmacokinetic profile of amphotericin B in a patient treated with ECMO. ECMO did not seem to have modified the pharmacokinetics of amphotericin B suggesting that no dose adjustment should be necessary in similar patients. However, further studies are warranted to confirm this observation.

Declaration of interest

The authors declare no competing interest.

Contribution of authors

JBF, MCV, and CT collected the data and wrote the article.

Tableau 1 .

 1 PB and AF ensured the clinical management of the patient. AP performed the pharmacokinetic analysis.EB and FL supervised the work, wrote and approved the article. Paramètres pharmacocinétiques de l'amphotéricine B liposomale chez des patients sous ECMO versus sans ECMO.

Table 1 .

 1 Pharmacokinetic parameters of liposomal amphotericin B in patients with ECMO vs non-ECMO patients. AUC 0-24 = area under the plasma concentration-time curve from 0 to 24 hours; C max = peak plasma drug concentration; CL = total plasma clearance; t 1/2 = mean elimination half-life; V d = volume of distribution.

		Present case Heinemann et	Coukell&Brogden [4]	Bekersky et al. [5]
			al. [3]
	Patients	ECMO	Critically ill	Neutropenic patients	Healthy volunteers
			patients
	Number of	1	16	10	5
	patients
	Dose (mg/kg/day)	4	1.2 to 4.2	5	2
	Infusion duration	6	1	1-2	2
	(hours)
	C max (µg/mL)	8.7	14.4	31.4	22.9
	AUC 0-24h (µg h/mL)	133	171	555	171
	V d (L/kg)	0.40	0.42	0.10	0.77
	t 1/2 (h)	18.3	13.1	6.8	6.0
	CL (mL/h/kg)	16.2	22.0	11.0	9.7


Y Yang

J B Zhan

J B Sui

C Q Li

K Yang

P Castany

T Gloriant

Functionally graded NiTi alloy with exceptional strain-hardening effect fabricated by SLM method

Keywords: SLM, NiTi alloys, functionally graded, phase transformation

In this study, we have employed a repetitive laser scanning strategy to fabricate a three-dimensional functionally graded NiTi alloy by SLM method. The microstructure shows gradient characterized by the increase of volume fraction for B19' phase. An exceptional strain-hardening associated with continuous increase of mechanical recoverable strain is obtained in such material. The gradient of functionality is attributed to the superimposition of multi-deformation mechanisms originating from the microstructural gradient. The deformation mechanisms are dominated by reorientation of B19' variants at early stage of deformation and then by stress-induced martensitic transformation at higher level of stress.

The NiTi alloy is the most widely used metallic shape memory alloy due to its superior shape memory effect and superelasticity. The maximum recoverable strain is proved to be up to 8% [START_REF] Otsuka | Recent developments in the research of shape memory alloys[END_REF].

Other features including low Young's modulus, high damping capacity, good biocompatibility and corrosion resistance all enable the NiTi alloy an excellent candidate material for actuators in both aerospace and bio-engineering areas [START_REF] Huang | On the selection of shape memory alloys actuators[END_REF][START_REF] Elahinia | Manufacturing and processing of NiTi implants[END_REF]. As the origin of these unique properties, the reversible thermo-elastic martensitic transformation between the B2 phase and the B19' phase has been investigated [START_REF] Carroll | Multiple-step martensitic transformation in Ni-rich NiTi shape memory alloys[END_REF]. However, the intrinsic narrow martensitic transformation temperature (MTT) range (sometimes called intervals) brings difficulties to well control the actuating process [START_REF] Meng | Transformation intervals and elastic strain energies of B2-B19' martensitic transformation of NiTi[END_REF]. The lack of strain-hardening on the strain-stress curve also hinders the application of NiTi alloys. In recent years, functionally graded NiTi alloys characterized by progressive transformation on loading or on cooling have been regarded as an efficient solution to resolve the problems mentioned above [START_REF] Meng | Compositionally graded NiTi plate prepared by diffusion annealing[END_REF][START_REF] Naebe | Functionally graded materials: A review of fabrication and properties[END_REF].

Functionally graded properties can be achieved by introducing microstructural, compositional or geometrical gradients in NiTi alloys. Relevant technologies for formation of the gradients mentioned above actually include physical vapor deposition (PVD) [START_REF] Rodrigues | Thermo-mechanical characterization of NiTi orthodontic archwires with graded actuating forces[END_REF], diffusion annealing treatment [START_REF] Meng | Compositionally graded NiTi plate prepared by diffusion annealing[END_REF][START_REF] Shariat | Functionally graded shape memory alloys: Design, fabrication and experimental evaluation[END_REF][START_REF] Meng | Functionally graded NiTi strips prepared by laser surface anneal[END_REF], chemical treatment [START_REF] Chu | Graded surface structure in chemically polished NITi shape memory alloy after NaOH treatment[END_REF] and laser annealing [START_REF] Meng | Laser annealing of functionally graded NiTi thin plate[END_REF][START_REF] Mahmud | Gradient anneal of functionally graded NiTi[END_REF][START_REF] Wang | Laser annealing of amorphous NiTi shape memory alloy thin films to locally induce shape memory properties[END_REF]. The selective laser melting (SLM) method as a kind of additive manufacturing technology can be another novel way to fabricate a functionally graded NiTi alloy [START_REF] Naebe | Functionally graded materials: A review of fabrication and properties[END_REF][START_REF] Wang | Selective laser melting produced layer-structured NiTi shape memory alloys with high damping properties and elinvar effect[END_REF]. In our previous studies, the laser beam energy dependence of martensitic transformation in SLM fabricated NiTi alloy was observed [START_REF] Yang | Laser beam energy dependence of martensitic transformation in SLM fabricated NiTi shape memory alloy[END_REF]. The microstructure, crystallographic texture, functional performance vary with the SLM processing parameters [START_REF] Yang | Evolution of functional properties realized by increasing laser scanning speed for the selective laser melting fabricated NiTi alloy[END_REF][START_REF] Khoo | Effect of heat treatment on repetitively scanned SLM NiTi shape memory alloy[END_REF]. Based on the obtained results, the three-dimensional functionally graded NiTi alloy is designed, fabricated and investigated in the present study.

The near equiatomic NiTi alloy powder was produced through electrode induction-melting gas atomization (EIGA) from a Ni59.68Ti40.32 (wt. %) ingot. The chemical composition of the powder was analyzed to be Ti: 44.48%, Ni: 55.98%, O: 0.046%, Fe: 0.004% and C: 0.0025% with methods of inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and infrared method after fusion under inert gas. The phase constitution determined by XRD analysis shows solely the B2 phase for the alloying powder. A commercial SLM machine of Concept Laser (Mlab-R) was used to fabricate samples under argon atmosphere (99.99%) with the oxygen content stabilized at the level of 300 ppm. A meander pattern with the scanning angle alternated by 67° upon the precedent layer was employed to minimize the thermal stress between layers. A repetitive laser scanning strategy with the schematic illustration shown in Fig. 1 has been explored to fabricate the functionally graded NiTi alloy.

As shown in Fig. 1, the gradient direction (GD) referring to the changing direction of microstructures and functionality is set to be perpendicular to the building direction (BD). In each single powder layer, a first laser scan with constant power of 60 W is initially proceeded as shown in Fig. 1a, followed by another subsequent laser scan with varied laser power from 0 W to 95 W amongst different gradient areas as shown in Fig. 1b. The power increment was set to be 5 W, hence a gradient of twenty parts with the second repetitive laser power of 0, [START_REF] Meng | Transformation intervals and elastic strain energies of B2-B19' martensitic transformation of NiTi[END_REF][START_REF] Meng | Functionally graded NiTi strips prepared by laser surface anneal[END_REF][START_REF] Wang | Selective laser melting produced layer-structured NiTi shape memory alloys with high damping properties and elinvar effect[END_REF][START_REF] Zeng | Wire and arc additive manufacturing of a Ni-rich NiTi shape memory alloy: Microstructure and mechanical properties[END_REF][START_REF] Otsuka | Physical metallurgy of Ti-Ni-based shape memory alloys[END_REF]30,35,40,45,50,55,60,65,70,75,80,85,90, 95 W was obtained. Different gradient areas were labeled as 60 W-X W, where the "X" refers to the second repetitive laser power. Except the laser power, other scanning parameters all keep constant: layer thickness (t) is 25 µm, scanning speed (v) is 440 mm/s and hatch space (h) is 110 µm. It is worth noting that the gradient width marked in Fig. 1b can also be purposely adjusted. The Fig. 1c shows a cubic specimen with the size of 10 mm×10 mm×10mm and gradient width of 0.5 mm, as well as another rectangular specimen with size of 5mm×8 mm×40mm and gradient width of 2 mm.

The samples for SEM observations were prepared by mechanical polishing followed by chemical etching with the reagent composed of 3.2% HF, 14.1% HNO 3 and 82.7% H 2 O in volume fraction. The XRD detection on the single gradient area was performed on samples which were cut individually from the graded NiTi alloy sample (gradient width of 2 mm). The XRD tests were performed at ambient temperature on Rigaku D-Max 2400 with a scanning step of 0.01 o and a wavelength of 0.15406 nm (Cu-Kα1). The EBSD analysis was performed on the central part of the graded tensile specimen in the FEI NanoSEM450 field-emission SEM equipped with a NordlysMax2 EBSD detector. The specimen for DSC related to the graded NiTi alloy was prepared by several steps. Firstly, a piece of thin sheet on the GD-TD plane was cut, then a needle with axial direction parallel to the GD was cut from the thin sheet. Finally, the needle was cut into small pieces with scissor and collected for DSC experiment. A Netzsch DSC 200 F3 with a heating/cooling rate of 10 o C/min in nitrogen atmosphere was used to estimate the MTTs. The normalized flat tensile specimens with 5 mm × 20 mm × 2 mm gage dimensions and gradient width of 1 mm was directly deposited on the NiTi alloy substrate. Tensile test was conducted at room temperature with approximately 25 o C. The tensile direction and transverse direction are parallel to the GD and the BD, respectively. The cyclic tensile tests were conducted with strain increments of 0.3 % and the strain rate of 10 -3 s -1 on the INSTRON 3369 machine. Fig. 2 displays the primary microstructural characterization on the graded NiTi alloy. It is observed from the SEM images in Fig. 2a that microstructural gradients exist on the BD-GD plane amongst the six single gradient areas (60W-15W, 60W-30W, 60W-45W, 60W-60W, 60W-75W and 60W-90W). The trace of molten pools can be clearly seen in all images. More B19' phase can be observed and its grain size seems coarsened with the increase of the second repetitive laser power. Further, small number of spherical defects was noticed, especially for the single gradient samples of 60W-75W and 60W-90W. The molten pool was probably over-heated when the second repetitive scanning laser power is higher enough than 75 W.

The EBSD maps conducted on the central part of the tensile specimen are displayed in Fig. 2b and 2c in terms of inverse pole figures (IPFs-BD). Some black areas corresponding to high internal residual stress or defects were revealed. As the gradient width was set to be 1 mm, the EBSD maps contain at least two gradient boundaries. Accordingly, the microstructure around the gradient boundaries is metallurgically continuous. Concerning on the texture shown by the IPFs-BD, there is no big difference between the gradient areas contained in the maps. That is because the adjacent gradient areas were fabricated with similar SLM fabrication processing parameters. Indeed, the incremental of repetitive laser power with 5 W was purposely designed in order to avoid sharp changes of microstructure and further guarantee the continuous change of microstructures.

Fig. 2d reveals the XRD profiles corresponding to the SEM images shown in Fig. 2a. Peak separation and fitting processes were performed. Final indexations were displayed above the profiles. An example of the separated and fitted peaks for the single gradient area of 60W-90W was enlarged in Fig. 2d. It is clearly seen the martensitic B19' phase is prone to form in the sample fabricated with higher repetitive laser scanning powers. In detail, the volume fraction of B19' phase was measured to increase from 28.3 % for the single gradient area of 60W-15W to 85.6% for the single gradient area of 60W-90W. Such increasing volume fraction of B19' phase with higher repetitive laser power is in accordance with microstructure gradient. This is also consistent with the conclusion obtained in previous investigations that higher energy density leads to more volume fraction of B19' phase [START_REF] Wang | Selective laser melting produced layer-structured NiTi shape memory alloys with high damping properties and elinvar effect[END_REF][START_REF] Yang | Laser beam energy dependence of martensitic transformation in SLM fabricated NiTi shape memory alloy[END_REF][START_REF] Yang | Evolution of functional properties realized by increasing laser scanning speed for the selective laser melting fabricated NiTi alloy[END_REF][START_REF] Khoo | Effect of heat treatment on repetitively scanned SLM NiTi shape memory alloy[END_REF]. Reasons can be attributed to the evaporation of Ni element under higher energy density that eventually leads to the increase of MTTs [START_REF] Saedi | On the effects of selective laser melting process parameters on microstructure and thermomechanical response of Ni-rich NiTi[END_REF][START_REF] Zeng | Wire and arc additive manufacturing of a Ni-rich NiTi shape memory alloy: Microstructure and mechanical properties[END_REF].

The DSC curves for single gradient areas and the whole graded NiTi alloy sample are displayed in Fig. 3a and Fig. 3b, respectively. The MTTs including M s , M f , A s , and A f were all measured and listed in Table 1. In fact, each MTT increases with the second repetitive laser power.

That is most probably due to evaporation of Ni element at higher laser power as shown by chemical composition analysis via inductively coupled plasma (ICP) method [START_REF] Speirs | On the transformation behavior of the NiTi shape-memory alloy produced by SLM[END_REF]. The MTT interval (ΔT A→M =M s -M f ) shown in the fourth columns of Table 1 What is also interesting to mention is some additional peaks highlighted by red arrows in Fig. 3. In contrast to the origin of the additional peaks as microstructural non-uniform or R phase [START_REF] Wang | Selective laser melting produced layer-structured NiTi shape memory alloys with high damping properties and elinvar effect[END_REF][START_REF] Yang | Laser beam energy dependence of martensitic transformation in SLM fabricated NiTi shape memory alloy[END_REF][START_REF] Saedi | On the effects of selective laser melting process parameters on microstructure and thermomechanical response of Ni-rich NiTi[END_REF] in Fig. 3a for single gradient area, a more reasonable explanation for additional peaks in Fig. 3b is the overlapping of MTT peaks resulting from multi-gradients. Therefore, the phase transformation gradient on cooling and heating was realized on the graded NiTi alloy.

The cyclic tensile curves consisting of strain increment of 0.3% followed by stress release until fracture are displayed in Fig. 4a. The Young's modulus, initial yielding stress, elongation and ultimate tensile stress were estimated to be approximately 32 GPa, 130 MPa, 6.88 % and 449 MPa, respectively. It can be observed that hysteresis loops between loading and unloading paths become widened once the applied strain exceeds the first yielding point of 0.35 %. Additionally, the tensile curves for the functionally graded NiTi alloy reveal an exceptional strain-hardening effect of more than 300 MPa, which is unusual for NiTi alloys. These two facts on the functionality gradient are due to involvement of multiple deformation mechanisms.

In order to characterize the functionality gradient, the evolution of mechanical recoverable strain with the underlying deformation mechanisms was analyzed. As shown in Fig. 4b, the obtained mechanical recoverable strain calculated to be the difference value between the applied strain and the residual strain was plotted as the function of applied strain and highlighted by the solid black circle lines. It is surprising to find that the mechanical recoverable strain increases continuously with the applied strain. Compared to this continuous increasing relationship, the SLM fabricated NiTi alloys fabricated with non-functional gradients do behave differently as shown in the Fig. 4b with open grey circle lines. Similar evolution of mechanical recoverable strain shown in other shape memory alloys with non-functional gradient can also be evidenced [START_REF] Yang | Stress release-induced interfacial twin boundary ω phase formation in a β type Ti-based single crystal displaying stress-induced α" martensitic transformation[END_REF][START_REF] Besse | Mechanisms of deformation in gum metal TNTZ-O and TNTZ titanium alloys: a comparative study on the oxygen influence[END_REF][START_REF] Héraud | In Situ synchrotron X-ray diffraction of the martensitic transformation in superelastic Ti-27Nb and NiTi Alloys: a comparative study[END_REF][START_REF] Otsuka | Physical metallurgy of Ti-Ni-based shape memory alloys[END_REF].

The underlying deformation mechanisms are explored hereafter to explain the continuously linear increase of mechanical recoverable strains. As observed from SEM images in Fig. 2a and confirmed by XRD profiles in Fig. 2d, more B19' martensite but less B2 phase were formed in the gradient areas fabricated with higher repetitive laser power. So, when the external stress imposed on the graded NiTi alloy exceeds the elastic limit, the gradient areas containing more B19' take a priority to deform via the reorientation of the pre-existing martensitic B19' twinning variants. As the reorientation process cannot be reversed after stress releasing in the present case, the loading and unloading paths are nearly overlapping at this stage (0.5%<applied strain< 1.5%). With the increase of the applied stress, the gradient areas which contain more B2 phase with lower repetitive laser power deform subsequently via stress-induced martensitic B19' transformation.

Larger hysteresis loops were hence left on the cyclic tensile curves as clearly shown in Fig. 4b.

The activation of the latter deformations enables the mechanical recoverable strain to increase linearly.

The functionally graded NiTi alloys have already been successfully fabricated with various methods, like diffusion annealing [START_REF] Rodrigues | Thermo-mechanical characterization of NiTi orthodontic archwires with graded actuating forces[END_REF] and laser annealing [START_REF] Meng | Functionally graded NiTi strips prepared by laser surface anneal[END_REF][START_REF] Meng | Laser annealing of functionally graded NiTi thin plate[END_REF][START_REF] Mahmud | Gradient anneal of functionally graded NiTi[END_REF][START_REF] Wang | Laser annealing of amorphous NiTi shape memory alloy thin films to locally induce shape memory properties[END_REF]. The excellent strain-hardening highlighted by Δσ in Fig. 4a was also found to be 300 MPa by other reference [START_REF] Shariat | Functionally graded shape memory alloys: Design, fabrication and experimental evaluation[END_REF].

The importance of the present study lies on two points. Firstly, the graded NiTi alloy can be fabricated with one-way SLM process without additional treatment. Further, the intrinsic feature of the additive manufacturing ensures a potentially high precision of gradient control via adjustment of the gradient width, density and fabrication strategy. Although the transformation gradient was characterized previously [START_REF] Meng | Compositionally graded NiTi plate prepared by diffusion annealing[END_REF][START_REF] Naebe | Functionally graded materials: A review of fabrication and properties[END_REF][START_REF] Rodrigues | Thermo-mechanical characterization of NiTi orthodontic archwires with graded actuating forces[END_REF][START_REF] Shariat | Functionally graded shape memory alloys: Design, fabrication and experimental evaluation[END_REF][START_REF] Meng | Functionally graded NiTi strips prepared by laser surface anneal[END_REF][START_REF] Chu | Graded surface structure in chemically polished NITi shape memory alloy after NaOH treatment[END_REF][START_REF] Meng | Laser annealing of functionally graded NiTi thin plate[END_REF][START_REF] Mahmud | Gradient anneal of functionally graded NiTi[END_REF][START_REF] Wang | Laser annealing of amorphous NiTi shape memory alloy thin films to locally induce shape memory properties[END_REF][START_REF] Wang | Selective laser melting produced layer-structured NiTi shape memory alloys with high damping properties and elinvar effect[END_REF][START_REF] Yang | Laser beam energy dependence of martensitic transformation in SLM fabricated NiTi shape memory alloy[END_REF], the value of MTT intervals (ΔT A→M and ΔT M→A ) which can be measured to be approx. 80K [START_REF] Meng | Laser annealing of functionally graded NiTi thin plate[END_REF] and 100 K [START_REF] Meng | Functionally graded NiTi strips prepared by laser surface anneal[END_REF] were emphasized rarely. This value reached approximately 120 K in the present case. As a matter of fact, the mechanical recoverable strain shown in Fig. 4b would further increase and finally stabilize at quite higher stress level where the plastic deformation would comprehensively occur. Therefore, processing parameters for the SLM needs to be further optimized.

In summary, a functionally graded NiTi alloy has been fabricated with selective laser melting method based on the design of a repetitive scanning strategy. The microstructural observations reveal a continuous microstructure evolution amongst different gradient areas and a well metallurgical fusion zone on the gradient boundaries. The microstructure graident is explicated by the increasing volume fraction of martensitic B19' phase along the gradient direction. The martensitic transformation as the origin of the functional properties is characterized by MTT intervals. The forward and backward MTT intervals of ΔT A→M and ΔT M→A are higher than 118 o C and 125 o C, respectively. The functionality of the graded NiTi alloy were characterized via analysis of mechanical recoverable strain and deformation mechanisms. The gradient area containing more B19' phase deforms initially via the reorientation of martensitic B19' variants.

Other gradient area containing more B2 phase deforms subsequently via stress-induced martensitic transformation at higher applied stress level. The superimposed multi-deformation mechanisms lead to the continuous increase of mechanical recoverable strain and exceptional strain-hardening effect.

More works concerning on the in situ microstructural observation and in situ synchrotron X-ray radiation diffraction in order to characterize more precisely the deformation details is now ongoing. A more accurate evaluation to unveil the gradient change of shape for the functionally graded NiTi is also planned. The prospects on the functionally graded NiTi alloy can be explored for biomedical and smart engineering devices as controllable self-driving actuator.

Fig. 1

 1 Fig. 1 Schematic illustration for the functionally graded NiTi alloy: (a) the first laser scan with a constant laser power of 60 W, (b) the repetitive laser scan with the varied laser power from 5 W to 95 W, (c) two typical graded NiTi specimens.

Fig. 2

 2 Fig. 2 Primary characterization for the functionally graded NiTi alloy: (a) the SEM microstructures of several selected gradient areas, (b, c) the IPFs mapping observed from different planes of SLMed sample, (d) the XRD profiles of several selected gradient areas.

Fig. 3 (

 3 Fig. 3 (a) DSC curves for (a) different single gradient samples and (b) the whole functionally graded NiTi alloy

  actually locates between 66.6 o C and 74.1 o C for single gradient areas. But this value is as higher than 118.1 o C for the graded NiTi alloy. The ΔT M→A values (ΔT M→A =A s /R s -A f ) listed in the seventh columns shows similarity with the ΔT A→M . Apparently, a nearly doubled MTT interval was obtained in the graded NiTi alloy.

Table 1 :

 1 Characteristic martensitic transformation temperatures (MTTs) measured from several selected gradient areas and from the whole graded NiTi alloys.

	Specimens	M s	M f	ΔT A→M ,	A s or R s	A f	ΔT M→A ,
		o C	o C	o C	o C	o C	o C
	60W-15W	14.5	-52.1	66.6	-19.6	28.3	47.9

The authors acknowledge Dr. J.X. Lin in Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences for providing facilities of additive manufacturing. This work was financially supported by the project supported by the National Science Foundation for Young Scientists of China (Grant No. 51801199).