Florence F Maunoury-Danger
email: florence.maunoury-danger@univ-lorraine.fr

Nicolas Chemidlin Prévost-Bouré

Jérome J Ngao

Daniel D Berveiller

Claude C Brechet

Eric E Dufrene

Daniel D Epron

Jean Christophe

J C Lata

Bernard B Longdoz

Nicolas Chemidlin

Prevost Boure

Jérôme Ngao

Jean-Christophe Lata

Caroline Lelarge-Trouverie

Jean-Yves Pontailler

Kamel Soudani

Claire Damesin

Erwin Dreyer

Jérôme Ngao

Jean-Christophe Lata

Caroline Lelarge Trouverie

Jean-Yves Pontailler

Jérôme Ngao

N Chemidlin Prevost

J.-Y Pontailler

J Ngao

J.-C Lata

C Lelarge

Carbon isotopic signature of CO2 emitted by plant compartments and soil in two temperate deciduous forests

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

& Methods Using manual chambers and isotope ratio mass spectrometry, we recorded simultaneously δ 13 C E and δ 13 C of organic matter in sun leaves, current-year twigs, trunk bases and soil in an oak and a beech forest during 1 year. & Results In the two forests, δ 13 C E displayed a larger variability in the tree components than in the soil. During the leafy period, a pronounced vertical zonation of δ 13 C E was observed between the top (sun leaves and twigs with higher values) and bottom (trunk and soil with lower values) of the ecosystem. No correlation was found between δ 13 C E and δ 13 C of organic matter. Causes for these seasonal variations and the vertical zonation in isotope signature are discussed. & Conclusion Our study shows clear differences in values as well as seasonal dynamics of δ 13 C E among different components in the two ecosystems. The temporal and local variation of δ 13 C E cannot be inferred from organic matter signature or CO 2 emission rates. Carbon isotope composition of CO 2 efflux δ 13 C EECO δ 13 C E of ecosystem δ 13 C EL δ 13 C E of leaves δ 13 C ETG δ 13 C E of twigs δ 13 C ET δ 13 C E of trunk δ 13 C ES δ 13 C E of soil δ 13 C OM δ 13 C of total organic matter δ 13 C OM δ 13 C OM of leaf δ 13 C OMT δ 13 C OM of twig δ 13 C OMT δ 13 C OM of trunk δ 13 C OMS δ 13 C of soil total organic matter Doy Day of year 1 Introduction

The carbon isotope composition (δ 13 C) of CO 2 is now commonly used at the ecosystem level as a natural tracer to investigate carbon processes and their responses to environmental conditions. Approaches coupling isotopic and mass balance are used to partition ecosystem CO 2 efflux (E ECO ) and photosynthetic fluxes [START_REF] Zobitz | Partitioning net ecosystem exchange of CO 2 : a comparison of a Bayesian/isotope approach to environmental regression methods[END_REF]). The δ 13 C of E ECO (δ 13 C EECO ) is used to infer ecosystem or regional C sink strength by inversion modelling. Large uncertainties are remaining about the interpretation of δ 13 C EECO , mainly due to the multi-source nature of E ECO , and the temporal variability of δ 13 C values of E ECO components and their contributions to E ECO [START_REF] Bowling | 13 C content of ecosystem respiration is linked to precipitation and vapor pressure deficit[END_REF][START_REF] Hemming | Pan-European δ 13 C values of air and organic matter from forest ecosystems[END_REF]. Until recently, δ 13 C E values used to interpret δ 13 C EECO were considered to be similar among different ecosystem components (i.e., soil, roots, trunk, twigs and leaves) [START_REF] Kodama | Temporal dynamics of the carbon isotope composition in a Pinus sylvestris stand: from newly assimilated organic carbon to respired carbon dioxide[END_REF]). So far, few studies have examined δ 13 C E of several components concurrently: between leaf and soil [START_REF] Mortazavi | Temporal variability in 13 C of respired CO 2 in a pine and a hardwood forest subject to similar climatic conditions[END_REF], trunk and soil [START_REF] Kodama | Temporal dynamics of the carbon isotope composition in a Pinus sylvestris stand: from newly assimilated organic carbon to respired carbon dioxide[END_REF] or different plant organs [START_REF] Eglin | Leaf and twig δ 13 C during growth in relation to biochemical composition and respired CO 2[END_REF][START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF]; they showed significant differences in δ 13 C E among the targeted components. Moreover, the δ 13 C E for a given component can exhibit a high seasonal or diurnal variability, up to 10‰ for leaves [START_REF] Hymus | Large daily variation in 13 C enrichment of leaf respired CO 2 in two Quercus forest canopies[END_REF][START_REF] Prater | Diurnal variation of the δ 13 C of pine needle respired CO 2 evolved in darkness[END_REF], 4‰ for twigs [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF], 3-5.5 ‰ for trunks [START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF][START_REF] Ubierna | A new method to measure carbon isotope composition of CO 2 respired by trees: stem CO 2 equilibration[END_REF]) and 4.2‰ for soil [START_REF] Ngao | Estimating the contribution of leaf litter decomposition to soil CO 2 efflux in a beech forest using 13 C-depleted litter[END_REF][START_REF] Marron | Seasonal and daily time course of the 13 C composition in soil CO 2 efflux recorded with a tunable diode laser spectrophotometer (TDLS)[END_REF]). However, only a few of these studies included wintertime measurements [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF][START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF][START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF].

Seasonal variations in δ 13 C E have been related to several factors such as environmental conditions several days before measurements [START_REF] Bowling | 13 C content of ecosystem respiration is linked to precipitation and vapor pressure deficit[END_REF][START_REF] Ekblad | Forest soil respiration rate and δ 13 C is regulated by recent above ground weather conditions[END_REF], canopy stomatal conductance [START_REF] Dowell | Response of the carbon isotopic content of ecosystem, leaf, and soil respiration to meteorological and physiological driving factors in a Pinus ponderosa ecosystem[END_REF]), the nature and/or δ 13 C of respiratory substrates [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF][START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF] or to other metabolic processes, such as variations in respiratory pathways [START_REF] Tcherkez | Metabolic origin of carbon isotope composition of leaf dark-respired co 2 in french bean[END_REF][START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF]. Recently, a comprehensive study concluded that seasonal δ 13 C E patterns in one ecosystem were similar for the different components of beech and spruce and between both species [START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF], also showing maximal differences for the trunk between summer and winter. The question now arising is how recurrent are seasonal patterns (1) for a given species at different environmental conditions and (2) between closely related species depending on leaf phenology.

In this context, we focused on the spatio-temporal δ 13 C E dynamics of the main tree components (leaves, twigs and trunks) and soil in two temperate broadleaved forest ecosystems. The main objectives of this study were (1) to quantify differences in δ 13 C E among components and (2) to characterize their seasonal variation, in particular between the two main phenological periods (i.e. leafy and winter period), and their potential link with environmental parameters (air and soil temperature, relative humidity, vapour pressure deficit), CO 2 efflux rates and the respective total organic matter δ 13 C. Synchronous in situ measurements of CO 2 efflux rates and δ 13 C E were performed throughout 1 year in an oak (Quercus petraea L.) and a beech (Fagus sylvatica L.) forest using isotope mass spectrometry measurements. In order to validate CO 2 field-sampling methods adapted to leaf and twig components [START_REF] Prater | Diurnal variation of the δ 13 C of pine needle respired CO 2 evolved in darkness[END_REF][START_REF] Werner | Evaluating high time-resolved changes in carbon isotope ratio of respired CO 2 by a rapid in-tube incubation technique[END_REF], we compared two methods of tissue incubation, with CO 2 -free air or N 2 flushes usable under field conditions.

Materials and methods

Study sites and experimental setup

The study was conducted in two French forest sites belonging to the CARBOEUROPE IP network (http:// www.carboeurope.org/). The Barbeau forest (cluster_FR1, FR-Fon site, 48°29′ N, 02°47′ E, Table 1) is a managed mature oak-dominated (Q. petraea) stand with an understory of Carpinus betulus L. Soil is a gleyic luvisol [World Reference Base (WRB) classification] of 80 cm depth, on millstone bedrock and covered with an oligomull humus type, named "Barbeau" in the following. The Hesse forest (cluster_FR1, FR-He site, 48°40′ N, 7°05′ E, Table 1), is a young beech-dominated (F. sylvatica) stand with a dystric cambisol (WRB classification) of 120 cm depth and an oligomull humus type. This site is subsequently named "Hesse".

The study was conducted from March 2005 (before budburst) to January 2006 during nine field campaigns in Barbeau (06/04, 18/04, 02/05, 01/06, 21/06, 11/07, 09/09, 23/11, and 12/01) and four field campaigns in Hesse (16/03, 18/05, 05/07, and 14/09). Gas exchange measurements, CO 2 , and organic matter sampling were always performed between 10:00 and 13:00 UT to avoid diurnal variations in δ 13 C E as previously observed [START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF]. Each four dominant oak and beech trees were randomly selected for the whole campaign. Soil measurements were performed around the sampled trees, within about 20 m 2 .

Environmental and phenological parameters

At both sites and for each campaign, soil temperature was measured at 10 cm depth using a temperature probe (LM35CZ) in Barbeau and five copper-constantan thermocouples (Faculty of Agronomy of Gembloux, Belgium) in Hesse. Soil surface moisture (in the 0-6 cm layer) was measured using a capacitive ML2x Thetaprobe (Delta-T Device, Cambridge, UK) in Barbeau. Mean air temperature at 2 m height (sensor LM35CZ), rainfall and air relative humidity (to calculate water vapour pressure deficit, VPD) were determined every 30 min by meteorological stations installed on-site.

Budburst dates (Table 1) were determined by field observations as either 50 % of trees showing 50 % of bursted buds in Barbeau, or as the beginning increase in the leaf area index (LAI), measured at regular intervals before and during the leafy period (LI-COR LAI 2000) in Hesse. The leaf fall period was also recorded at both sites. The trunk growth period was determined by tape measurements of the radius at 1.30 m height every week in Barbeau and during each field campaign in Hesse (Table 1). The growing period is defined as the time of trunk growth, while the leafy period includes the time span between bud burst and leaf fall.

Trunk and soil CO 2 efflux rates

Trunk CO 2 efflux rate (E T ) was measured using a closed chamber system (see [START_REF] Damesin | The carbon isotope composition of CO 2 respired by trunks: comparison of four sampling methods[END_REF] for a detailed description). Briefly, a cylindrical polymethyl methacrylate (PMMA) chamber was temporarily fixed on the trunk, cleaned of mosses and lichens, with a rubber sealant (Terosta-7, Teroson, Ludwigsburg, Germany) and connected to an infrared gas analyser (IRGA, EGM4, PPSystems, Hitchin, UK). The installation was considered to be leakfree if blowing air along the seals caused no increase in the CO 2 level inside the chamber. A fan provided air mixing within the chamber. Once tightly fixed to the trunk, the chamber was purged from accumulated CO 2 by opening a 5-cm diameter lid. Once back to ambient CO 2 concentrations, the lid was closed to allow CO 2 accumulation. Three to four E T measurements of 2 min during linear CO 2 increase were performed each time. The E T values were determined from the slope of CO 2 concentration increase and expressed per unit volume of living tissue (i.e., phloem and sapwood; in μmolm -3 living tissues -1 ). For beech, the whole trunk volume was considered because living cells occur all along the trunk radius [START_REF] Ceschia | Spatial and seasonal variations in stem respiration of beech trees (Fagus sylvatica)[END_REF]. For each oak tree, the living tissue cross-section was determined from a trunk core collected near the chamber at the end of the campaign. The chambers were reinstalled at the same place during each campaign.

Soil CO 2 efflux (E S ) was measured using two different closed dynamic systems [START_REF] Ngao | Crosscalibration functions for soil CO 2 efflux measurment systems[END_REF][START_REF] Prévost-Bouré | Root exclusion through trenching does not affect the isotopic composition of soil CO 2 efflux[END_REF]): In Barbeau, an EGM4 was connected to a homemade PMMA chamber (25.4 L, 12 cm height), while in Hesse, a Li-6200 (LI-COR Inc., Lincoln, NE, USA) IRGA was used with the Li-6000-9 chamber. In both cases, the soil chamber was put on collars previously inserted into the soil under the canopy (500 and 110 mm diameter in Barbeau and Hesse respectively, inserted 2-3 cm deep) at the beginning of the year, allowing measurements at the same place during field sessions. Two collars were installed at Barbeau and three at Hesse. E S values were calculated from the slope of CO 2 increase and expressed per surface area (in μmolm -2 soils -1 ).

CO 2 sampling for isotopic analysis

Incubation tests

The gaseous medium (N 2 vs. CO 2 -free air) of an incubation setup may have an immediate impact on the CO 2 efflux rate by shortening the oxygen supply to living tissues and potentially influencing the isotope composition of emitted CO 2 . We tested this effect using a system close to that described in [START_REF] Werner | Evaluating high time-resolved changes in carbon isotope ratio of respired CO 2 by a rapid in-tube incubation technique[END_REF]. Entire mature sun leaves and twigs were sampled from the top of the canopy of three oaks and three beeches at the end of summer. Each sample was immediately inserted into a 50-ml flask previously purged with either pure N 2 or CO 2 -free air. Leaves (n044) and twigs (n020) were incubated in the dark under ambient temperature. In preliminary CO 2 efflux rate measurements (data not shown), we determined that an incubation time between 10 and 25 min was required to collect enough emitted CO 2 (800-900 ppm). The δ 13 C in air from the incubations was analysed by isotope ratio mass spectrometry (IRMS) as described below. These tests revealed no significant effect of the gaseous medium used, neither for beech or oak leaves nor for twigs (p00.72 for leaves and p00.53 for twigs, Fig. 1).

Sampling of CO 2 emitted by ecosystem components

For CO 2 emissions at the leaf and twig levels, these components were incubated as described above. Sun leaves and twigs from the tree canopy were sampled using a rifle. Leaves and twigs were immediately inserted into 50-ml airtight syringes with valves (SGE, Australia) prepurged with pure N 2 . Air from the incubation syringe was then transferred into an empty syringe in Barbeau or into a 12ml Exetainer vial (Labco Ltd, High Wycombe, UK) in Hesse, according to the equipment available (see IRMS analyses below).

For trunk CO 2 emissions, the trunk chamber was purged after each E T measurement with N 2 for 15 min until the CO 2 concentration dropped to near 0 ppm. Then, outlet and inlet tubes of the chamber were connected to allow the accumulation of CO 2 emitted by the trunk. After an increase of 700-800 ppm (during approximately 10 min in summer and 90 min in winter), the air in the chamber headspace, containing CO 2 originating only from E T , was sampled using a 50-ml syringe and analysed by IRMS. Again, this N 2 flushing approach gave the same results as that using a CO 2 free-air flush or estimating the δ 13 C of emitted CO 2 with the Keeling plot method [START_REF] Damesin | The carbon isotope composition of CO 2 respired by trunks: comparison of four sampling methods[END_REF].

For CO 2 emitted by soil, the Keeling plot method [START_REF] Keeling | The concentration and isotopic abundances of atmospheric carbon dioxide in rural areas[END_REF]) was applied to determine the δ 13 C E of E S (Chemidlin [START_REF] Prévost-Bouré | Root exclusion through trenching does not affect the isotopic composition of soil CO 2 efflux[END_REF]). The sampling setup consisted of 50-ml airtight syringes in Barbeau, or a homemade sampling device by-passing the Li-6200 air circuit (see [START_REF] Ngao | Estimating the contribution of leaf litter decomposition to soil CO 2 efflux in a beech forest using 13 C-depleted litter[END_REF] for details) in Hesse. After each E S measurement, CO 2 concentration was allowed to increase again within the closed system. During this increase, five air samples were taken at steps of 50-100 ppm within a 400-1000 ppm range inside Exetainer vials or 50-ml syringe and analysed by IRMS. From the Keeling plots, the δ 13 C ES for each collar was estimated using the ordinary least square regression model [START_REF] Zobitz | Partitioning net ecosystem exchange of CO 2 : a comparison of a Bayesian/isotope approach to environmental regression methods[END_REF]). δ 13 C ES was determined as the intercept of the linear regression between the inverse of the CO 2 concentration and the δ 13 C of the air samples. δ 13 C ES values having a standard error >5 % of the estimated value were discarded.

Sampling of plant and soil material for isotopic analysis

At both sites and for each campaign, trunk phloem samples of the four trees were taken using a core borer (0.5 cm diameter) at the chamber level or up to 30 cm above. Leaves and twigs used for the incubations and phloem samples were lyophilised and powdered using a ball mill (Type MM200, Retsch, Haan, Germany). Four soil cores (0-15 cm depth and 1.2 cm diameter) were sampled about 15 cm away from each collar. Soil samples did not include litter or roots.

IRMS analyses

To maintain airtight conditions, gas-filled syringes were processed within 12 h by laboratories close to the sites. Gas samples from the Barbeau were analysed with a NA-1500 elemental analyser (Carlo Erba, Milan, Italy) coupled to a VG Optima IRMS (Fison, Villeurbanne, France), as described by [START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF]. Those from Hesse were injected into a gas purification device (Gas-Bench II, ThermoFinnigan, Bremen, Germany) coupled to a Delta S IRMS (ThermoFinnigan, Bremen, Germany). All solid samples were analysed with the NA-1500/IRMS setup.

All δ 13 C values were expressed relative to the Vienna Pee Dee Belemnite international standard. Different laboratory working standards (glutamic acid, -28.06‰ for organic matter samples; air with 500 μmolmol -1 of CO 2 , -53.10‰ for air samples) were measured after each group of 12 samples to correct for any offset of the IRMS. The precision for isotopic measurements was ± 0.2 ‰, based on repeated measurements of the laboratory working standards. Both IRMS systems were inter-calibrated for gas analyses using the same reference gas as above, revealing a discrepancy of 0.7‰ that was removed to the values measured at Hesse to have comparable values between both sites.

Statistical analysis

Pearson's correlation coefficients were calculated between δ 13 C E of each component and climatic data or CO 2 efflux rates solely at Barbeau where measurements were more frequent. All climatic variables from the measurement day and the day before were tested.

Pearson correlations were also established between δ 13 C E of different components. Non-parametric Kruskal-Wallis rank sum tests should reveal differences among components at each site, and during the two main phenological periods, followed by Mann-Whitney tests to compare one component to another.

A one-way ANOVA was applied to compare δ 13 C E measurements in the incubation tests with N 2 or CO 2 -free air flushings.

All analyses were performed using Statistica (version 7, Statsoft Inc., USA) and R 2.11.1 (R development core team 2010).

Results

CO 2 efflux rates

At both sites, the trunk CO 2 efflux E T showed a pronounced seasonal evolution and ranged from 10 (April) to 130 (June) μmol CO 2 m -3 of living tissue s -1 in Barbeau (Fig. 2a), and from 10 (March) to 88 (July)μmol CO 2 m -3 of living tissues -1 in Hesse (Fig. 2b). Variations of soil CO 2 efflux were also marked, especially in Barbeau where E S ranged from 0.7 (November) to 5.1 (July)μmol CO 2 m -2 s -1 . In Hesse, it ranged from 0.7 (March) to 1.8 (September)μmol CO 2 m -2 s -1 but summer efflux data, which are assumed to be highest, are missing. The maximum observed values of trunk and soil CO 2 efflux at Barbeau (Fig. 2a) occurred during the trunk growth period when air and soil temperatures were high (Fig. 3). The lowest CO 2 efflux rates occurred during winter.

Carbon isotope composition of emitted CO 2

In oak forest, the carbon isotope composition of leaves (δ 13 C EL ) showed the largest seasonal variations (4.6‰, Fig. 2c). It was always higher than δ 13 C of CO 2 emitted by buds in spring, i.e. the first measurement before budburst (-22.3‰). It increased during the whole growing season, with a small decrease in June and then decreased from September onwards. δ 13 C EL showed a similar pattern in Hesse albeit at a smaller observed range (2.3‰) and lower values (Fig. 2d).

At both sites, δ 13 C E of twigs (δ 13 C ETG ) clearly increased between budburst and May (Fig. 2c,d). In September, the values were near those before budburst for oak or those in May for beech. During the trunk growth period, the δ 13 C ET was lower than the δ 13 C E of the two canopy components at both sites (Fig. 2c,d). In Barbeau, δ 13 C ET increased during winter and reached a maximum of -18.9‰ in January 2006 (Fig. 2c).

Soil δ 13 C ES was rather similar at both sites, and seasonal variations were low (Fig. 2c,d). E S and δ 13 C ES were not correlated (Table 2).

In Barbeau, δ 13 C ETG was not related to any climatic variable, whereas δ 13 C EL was related to soil moisture 2) and δ 13 C ET was related to air and soil temperature (p00.011 and p00.012, Table 2) and vapour pressure deficit (p00.033). δ 13 C ES values were only related to soil moisture (p 00.006). Moreover, δ 13 C ET was negatively related to E T and to E S (r0-0.715; p00.030 and r0-0.667; p00.050, respectively, Table 2). If only the values from the leafy period were kept, correlations between δ 13 C EL and δ 13 C ETG (r00.955; p00.011), δ 13 C ES (r00.917; p 00.029) and soil moisture (r 00.960; p 00.041), and between δ 13 C ES and soil moisture (r00.966; p00.034) were remaining.

From May to September (leafy period), δ 13 C E exhibited a vertical zonation in both ecosystems, revealing significant differences between components (Kruskal-Wallis ANOVA, p<0.001 for each site, Fig. 4).δ 13 C EL values were globally the highest, and δ 13 C ETG values were lower in Barbeau (p0 0.003) but not in Hesse (p00.380). δ 13 C ETG values were always higher than those of trunk (p00.001, both systems) and soil (p00.001 in Barbeau and p00.002 in Hesse). At both sites, δ 13 C ET and δ 13 C ES values were not significantly different (p00.07 in Barbeau and p00.16 in Hesse) and represented the lowest δ 13 C E values.

During leaf fall (October) and winter (January), the vertical zonation was not maintained (Fig. 4). This period was characterised by an increase in δ 13 C ET .

δ 13 C of total organic matter

In contrast to δ 13 C E , δ 13 C of total organic matter (δ 13 C OM ) showed weak temporal variations. At both sites, the most Only significant r values are presented (p<0.05)

Instant T air and Instant T soil air and soil instantaneous temperature during measurements (°C); VPD daily mean vapour pressure deficit (mBar) during the day of measurement; H v soil (%) soil volumetric moisture pronounced variations occurred in leaves and twigs with a slight decrease in July and a slight increase towards September (Fig. 2e,f). Variations in δ 13 C OMT during the year were very narrow, with the same annual average of -26.5‰ for the two forests (Fig. 2e,f). δ 13 C OMS values were stable and averaged -27.01‰±0.06 in Barbeau and -26.21‰±0.08 in Hesse.

When comparing organic matter to CO 2 efflux δ 13 C, differences between δ 13 C EL and δ 13 C OML were up to 9.3‰ (Barbeau) and 8.5‰ (Hesse). Differences between δ 13 C ETG and δ 13 C OMTG for oak and beech were lower, with maxima of 5.6 and 6.1‰, respectively. During spring and summer, the difference between δ 13 C ET and δ 13 C OMT was maximum 1.7 ‰ (Barbeau) and ranged between -2.4 and 1.3 ‰ (Hesse). After leaf fall in Barbeau, this difference reached values as high as 7.6‰ in January. Differences between δ 13 C ES and δ 13 C OMS were always positive in Barbeau (1.0-2.5‰) but could be negative in Hesse (-1.6-1.0‰.).

Finally, the differences in δ 13 C OM between organs were lower compared to those for δ 13 C E . Even if δ 13 C OMTG was generally less negative than the δ 13 C OM of other components, no stable vertical zonation was apparent (Fig. 4).

Discussion

Incubation methods for leaf and twig in the field

Field protocols to collect CO 2 efflux from tissue at the top of the canopy showed that δ 13 C E of attached leaves was similar to that of detached ones [START_REF] Prater | Diurnal variation of the δ 13 C of pine needle respired CO 2 evolved in darkness[END_REF]. Furthermore, an in-tube incubation method using CO 2 -free air flushes had been validated for δ 13 C E measurements and showed no difference to online gas exchange measurement [START_REF] Werner | Evaluating high time-resolved changes in carbon isotope ratio of respired CO 2 by a rapid in-tube incubation technique[END_REF]). Here, we complete the methods debate on which approaches are compatible to field conditions, by testing two variants of tissue incubation with CO 2 -free air or N 2 flushes. Our results clearly demonstrate that the different gases used do not change the measured δ 13 C E of leaves or twigs. Tissue incubation in vials previously flushed with N 2 or CO 2 -free air can thus be used in the field to sample the emitted CO 2 from current-year branches.

4.2 Vertical zonation of δ 13 C E from canopy to soil during the leafy period In both forests, our measured δ 13 C E values were comparable to those obtained for leaves in deciduous [START_REF] Hymus | Large daily variation in 13 C enrichment of leaf respired CO 2 in two Quercus forest canopies[END_REF] or coniferous forests [START_REF] Prater | Diurnal variation of the δ 13 C of pine needle respired CO 2 evolved in darkness[END_REF], twigs [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF], trunks [START_REF] Damesin | The carbon isotope composition of CO 2 respired by trunks: comparison of four sampling methods[END_REF][START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF][START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF]) and soil of deciduous forests [START_REF] Ngao | Estimating the contribution of leaf litter decomposition to soil CO 2 efflux in a beech forest using 13 C-depleted litter[END_REF], coniferous forests [START_REF] Ekblad | Forest soil respiration rate and δ 13 C is regulated by recent above ground weather conditions[END_REF] or rainforests [START_REF] Buchmann | Interseasonal comparison of CO2 concentrations, isotopic composition and carbon dynamics in an Amazonian rainforest (French Guiana)[END_REF].

We revealed that during the leafy period, δ 13 C E values significantly differed among the ecosystem components, overall decreasing from the top of the canopy to the soil (Fig. 4). We observed this zonation at both sites, two different deciduous tree forests in distinct climatic conditions. Interestingly, no such zonation was found by [START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF] in spruce and beech forests during the leafy period.

When there was a difference in δ 13 C E between twigs and leaves, it systematically consisted of a 13 C impoverishment of the CO 2 emitted by twigs relative to leaves. This difference cannot be explained by differences in substrate δ 13 C because leaves have generally a significantly lower δ 13 C for starch and soluble sugars than twigs [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF]; [START_REF] Eglin | Leaf and twig δ 13 C during growth in relation to biochemical composition and respired CO 2[END_REF]). The gap between δ 13 C EL and δ 13 C ETG might be related to a difference in the balance of CO 2 released by either pyruvate decarboxylation (resulting in 13 C-enriched CO 2 ) or by the Krebs cycle (resulting in 13 Cdepleted CO 2 , [START_REF] Tcherkez | Metabolic origin of carbon isotope composition of leaf dark-respired co 2 in french bean[END_REF][START_REF] Gessler | On the metabolic origin of the carbon isotope composition of CO 2 evolved from darkened light-adapted leaves in Ricinus communis[END_REF]).

The main hypotheses explaining differences in δ 13 C E between twigs and trunks are (1) an isotope discrimination during the assimilate transport in the phloem along twigs and trunk [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF][START_REF] Gessler | δ 13 C of organic matter transported from the leaves to the roots in Eucalyptus delegatensis: short-term variations and relation to respired CO 2[END_REF]), (2) changes in PEPc activity [START_REF] Gessler | On the metabolic origin of the carbon isotope composition of CO 2 evolved from darkened light-adapted leaves in Ricinus communis[END_REF][START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF], known to discriminate against 12 C during carbon fixation [START_REF] Cernusak | Why are non-photosynthetic tissues generally 13 C enriched compared with leaves in C 3 plants? Review and synthesis of current hypotheses[END_REF]), (3) or a substantial contribution of belowground-evolved CO 2 brought by the xylem sap stream to the upper part of the tree, i.e., in the trunk [START_REF] Aubrey | Root-derived CO 2 efflux via xylem stream rivals soil CO 2 efflux[END_REF][START_REF] Grossiord | Transpiration alters the contribution of autotrophic and heterotrophic components of soil CO2 efflux[END_REF]. Other processes may contribute to the 13 C impoverishment in trunk compared to twigs, especially the progressive mixing along metabolite translocation from sun and shade leaves via twigs to trunk [START_REF] Eglin | Linking intra-seasonal variations in climate and tree-ring δ13C: A functional modelling approach[END_REF]). It will be interesting to address in future studies in more detail the reasons for the differential isotope discrimination in twigs and trunk found in our study.

Comparable values of isotope composition of CO 2 efflux in trunk and soil may be explained by the coupling of both components via C assimilates. Carbon substrates are rapidly transferred in broadleaved species from trunk to roots and via root exudates also to soil microorganisms (Dannoura et al. 2011;[START_REF] Epron | Seasonal variations of belowground carbon transfer assessed by in situ 13 CO 2 pulse labelling of trees[END_REF]. The comparable values for trunk and soil also suggest that contrary to substrate translocations from leaves to twigs, there is no apparent isotope discrimination during carbon translocation from trunk to soil. 4.3 Seasonal variations in δ 13 C E Seasonal ranges of emitted CO 2 δ 13 C were in agreement with ranges previously observed for beech leaves [START_REF] Eglin | Leaf and twig δ 13 C during growth in relation to biochemical composition and respired CO 2[END_REF], beech twigs [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF], oak trunks [START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF]) and hardwood forest soil [START_REF] Mortazavi | Temporal variability in 13 C of respired CO 2 in a pine and a hardwood forest subject to similar climatic conditions[END_REF][START_REF] Marron | Seasonal and daily time course of the 13 C composition in soil CO 2 efflux recorded with a tunable diode laser spectrophotometer (TDLS)[END_REF]. Our maximum δ 13 C E values of leaves and twigs have been obtained during summer, in agreement with δ 13 C E of a coniferous forest ecosystem [START_REF] Bowling | 13 C content of ecosystem respiration is linked to precipitation and vapor pressure deficit[END_REF][START_REF] Mortazavi | Temporal variability in 13 C of respired CO 2 in a pine and a hardwood forest subject to similar climatic conditions[END_REF] but oppositely to δ 13 C E of a deciduous forest ecosystem [START_REF] Mortazavi | Temporal variability in 13 C of respired CO 2 in a pine and a hardwood forest subject to similar climatic conditions[END_REF].

δ 13 C E seasonal variations in leaves might be related to changes in respiratory substrate δ 13 C due to variable 13 C discrimination during C assimilation. The latter is itself linked to environmental conditions like soil moisture [START_REF] Mortazavi | Temporal variability in 13 C of respired CO 2 in a pine and a hardwood forest subject to similar climatic conditions[END_REF]. The correlation between δ 13 C EL and δ 13 C ETG (Table 2) suggests that δ 13 C ETG variability is linked to the same mechanisms as that of δ 13 C EL . The increase in δ 13 C ET in winter might be explained by a switch of respiratory substrates from photosynthesis-derived sugars (lower δ 13 C) during the leafy period to stored carbohydrates, i.e. starch with higher δ 13 C, in the dormancy period as suggested before [START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF][START_REF] Kuptz | Seasonal dynamics in the stable carbon isotope composition (δ 13 C) from non-leafy branch, trunk and coarse root CO 2 efflux of adult deciduous (Fagus sylvatica) and evergreen (Picea abies) trees[END_REF]). Another explanation for these winter values is that, during winter time, transpiration is null and δ 13 CE T was no more influenced by a possible contribution of belowgroundevolved CO 2 . Surprisingly, we did not observe any increase in δ 13 C E for twigs, which, like trunks, probably use starch as main respiratory substrate. This unexpected difference between trunk and twig δ 13 C E may be explained by a metabolic discrepancy such as, e.g. differences in PEPc activity, during winter. Correlations between δ 13 C ET and E T or air temperature have already been observed by [START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF] in the same oak forest, with comparable correlation coefficients, suggesting that the respiration rate, which is influenced by air temperature, affects δ 13 C ET .

During the leafy period, respiratory substrates are partially derived from C recently assimilated by leaves and transported by the phloem towards the trunk base and roots [START_REF] Dannoura | In situ assessment of the velocity of carbon transfer by tracing 13 C in trunk CO 2 efflux after pulse labelling: variations among tree species and seasons[END_REF][START_REF] Epron | Seasonal variations of belowground carbon transfer assessed by in situ 13 CO 2 pulse labelling of trees[END_REF]) and may finally end up via root exudates as organic matter in the soil. Such a substrate similarity between trunk and soil may result in δ 13 C ES values close to those measured for trunk during the leafy period. In contrast, during winter, a lack C assimilation lowers dramatically this recent C source for soil respiration, while trunk stored compounds with higher δ 13 C values supply substrates for E T . Furthermore, carbon supplies to the soil via root exudates are much lower or nil during winter. In addition, the heterotrophic component, i.e. microbial respiration, contributes more to the soil CO 2 efflux and to δ 13 C ES than during the leafy period [START_REF] Epron | Seasonal dynamics of soil carbon dioxide efflux and stimulated rhizosphere respiration in a beech forest[END_REF]. This decoupling between trunk and soil respiratory substrate pools therefore may explain the significant differences in δ 13 C E of both components in winter.

At the soil and ecosystem level, temporal variations of δ 13 C E have been explained by environmental conditions with or without time lag (from 1 to 10 days) before measurement (e.g. [START_REF] Bowling | 13 C content of ecosystem respiration is linked to precipitation and vapor pressure deficit[END_REF][START_REF] Ekblad | Forest soil respiration rate and δ 13 C is regulated by recent above ground weather conditions[END_REF][START_REF] Marron | Seasonal and daily time course of the 13 C composition in soil CO 2 efflux recorded with a tunable diode laser spectrophotometer (TDLS)[END_REF]). The present study showed a differential impact of environmental conditions on the δ 13 C E of the different components studied., Specifically, in contrast to other studies, we found no correlation between δ 13 C EL or δ 13 C ETG and climate [START_REF] Dowell | Response of the carbon isotopic content of ecosystem, leaf, and soil respiration to meteorological and physiological driving factors in a Pinus ponderosa ecosystem[END_REF]Mortazavi et al. 2005), which might be explained by the lower measurement frequency in our study. Yet, we found several correlations of environmental factors with δ 13 C ET , suggesting that the trunk data integrate the overall climate effect on the tree. This argument can be supported by the facts that (1) a time lag exists for substrate transport in phloem from leaves to soil [START_REF] Barnard | Evaporative enrichment and time lags between δ 18 O of leaf water and organic pools in a pine stand[END_REF]) and (2) a progressive mixing of several different substrates (e.g. recent vs. stored compounds, top vs. bottom of tree canopy) along this C translocation.

Lastly, the general enrichment of δ 13 C E in comparison with δ 13 C OM has been classically highlighted in previous studies [START_REF] Damesin | Carbon isotope composition of currentyear shoots from Fagus sylvatica in relation to growth, respiration and use of reserves[END_REF][START_REF] Klumpp | C-isotope composition of CO 2 respired by shoots and roots: fractionation during dark respiration?[END_REF][START_REF] Maunoury | Seasonal, daily and diurnal variations in the stable carbon isotope composition of carbon dioxide respired by tree trunks in a deciduous oak forest[END_REF]. Our results show a mismatch between the δ 13 C E of each component and δ 13 C OM of the mature leaf, but these discrepancies were not stable throughout the year. Thus, our results invalidate the hypothesis of [START_REF] Bowling | Carbon isotopes in terrestrial ecosystem pools and CO 2 fluxes[END_REF] that bulk leaf δ 13 C can be used as a reference value to predict differences between δ 13 C E and δ 13 C OM of plant components and ecosystem.

Conclusion

Our study highlights isotopic differences of CO 2 emitted by the top (sun leaves and twigs) and the bottom of the forest (trunk base and soil) both with regard to higher δ 13 C values during the leafy period (higher at the top) and of seasonal dynamics (higher at the top). Variations in substrate δ 13 C (via the use of stored compounds or a mixing effect) might be the major-but not the only-explanation for these differences. Our study confirmed that δ 13 C of CO 2 emitted by the forest components cannot be deduced from the δ 13 C of the total organic matter of the component, or from 2 efflux intensity. Nowadays, high frequency measurements by tunable diode laser spectroscopy allow the analysis of temporal dynamics in δ 13 C E [START_REF] Marron | Seasonal and daily time course of the 13 C composition in soil CO 2 efflux recorded with a tunable diode laser spectrophotometer (TDLS)[END_REF], especially during winter time, and offer thus better possibilities to understand inherent variability its link to metabolic processes.

Fig. 1

 1 Fig. 1 Mean values of δ 13 C E measured for leaves and twigs of oak and beech incubated after flushing with CO 2 -free air (white) or N 2 (black). Error bars represent standard errors of the mean. n (leaf)027 for oak and 17 for beech, and n (twig)010 for oak and 10 for beech

Fig. 3

 3 Fig. 3 Seasonal changes in rainfall (gray histogram), soil (open triangles) and air (filled triangles) temperature, soil moisture (open diamonds) and air relative humidity (filled diamonds), in Barbeau (oak forest) (a) and Hesse (beech forest) (b). Error bars represent standard errors of the mean

Fig. 4

 4 Fig. 4 Mean values (±standard errors of the mean) of δ 13 C E (bold) and δ 13 C OM (italic) observed for each component (leaf, twig, trunk and soil) during the leafy period and winter, in Barbeau (oak forest) (left) and Hesse (beech forest) (right)

  Keywords Carbon isotopic composition . CO 2 efflux . Oak forest . Beech forest

	Abbreviations
	E T	Trunk CO 2 efflux
	E S	Soil CO 2 efflux
	E ECO	Ecosystem CO 2 efflux
	δ 13 C	Carbon isotope composition
	δ 13 C E

Table 1

 1 Stand characteristics for Barbeau and Hesse in 2005

		Barbeau (oak forest)	Hesse (beech forest)
	Location	48°29′ N, 02°47′ E	48°40′ N, 7°05′ E
	Elevation (m above sea level)	90	300
	Climate	Modified temperate maritime	Continental climate
		climate
	Mean annual temperature (°C)	10.5	9.2
	Annual rainfall (mm)	690	820
	Soil type (WRB classification)	Gleyic luvisol	Stagnic luvisol
	Humus type	Oligomull	Oligomull
	Stand age in 2005 (years)	100-150	40
	Ground area (m 2 ha -1 )	20.7	25.3
	Tree density (stems ha -1 )	3480	1134
	Max tree height (m)	30	20
	Dominant species	Quercus petraea L.	Fagus sylvatica L.
	Understorey	Dense	Sparse
	Represented tree species	Carpinus betulus L.
	a Deduced from the four
	dominant trees selected at both
	sites

Quercus petraea L. Betula pendula L. Carpinus betulus L. Budburst (dominant species) Doy 110 Doy 119 Leaf fall From doy 287 to 337 From doy 297 to 310 Trunk growth a Between doy 115 and 239 Between doy 115 to 214 Trunk diameter at 1.30 m a (cm) 25-39 24-27

  trunk (filled squares) and total soil (open circles). Vertical dashed lines delimitate the budburst date and the leaf fall period. Before budburst, δ 13 C E and δ 13 C OM of leaves and twigs were measured on buds and previous year-twig. The trunk growth period is indicated in gray. Error bars represent standard errors of the mean

			Barbeau forest					Hesse forest
	Budburst			Leaffall			Budburst			Leaffall
		Trunk growth					Trunk growth			14
												12
												10
												8
												6
												4
												2
												0
	(e)
	Mar	May	July	Sept	Nov	Jan	Mar	May	July	Sept	Nov	Jan
	Fig. 2 Seasonal changes in trunk and soil respiration rate in Barbeau
	(oak forest) (a) and Hesse (beech forest) (b), in δ 13 C of emitted CO 2 in Barbeau (c) and in Hesse (d), and in δ 13 C of total organic matter in
	Barbeau (e) and in Hesse (f), of leaf (filled circles), twig (filled
	triangles),

(p00.041, Table

Table 2

 2 Pearson's correlation coefficient (r) matrix between carbon isotope signatures of emitted CO 2 in leaves, twigs, trunk, soil and soil and climatic conditions in oak forest or δ 13 C E of leaves, twigs, trunk and soil

		Instant T air	VPD	Instant T soil	H v soil	E T	E S	δ 13 C EL	δ 13 C ETG	δ 13 C ET	δ 13 C ES
	δ 13 C EL	ns	ns	ns	0.960	ns	ns		0.955	ns	0.917
		p00.853	p00.611	p00.316	p00.041	p00.334	p00.220		p00.011	p00.953	p00.029
	δ 13 C ETG	ns	ns	ns	ns	ns	ns	0.955		ns	ns
		p00.438	p00.373	p00.370	p00.953	p00.228	p00.156	p00.011		p00.301	p00.625
	δ 13 C ET	-0.789	-0.708	-0.909	ns	-0.715	-0.667	ns	ns		ns
		p00.011	p00.033	p00.012	p00.527	p00.030	p00.050	p00.953	p00.301		p00.174
	δ 13 C ES	ns	ns	ns	0.934	ns	ns	0.917	ns	ns
		p00.817	p00.940	p00.702	p00.006	p00.755	p00.759	p00.029	p00.625	p00.174

F.Maunoury-Danger et al.

δ 13 C of CO 2 emitted by tree compartments and soil

Acknowledgements The authors are grateful to the Office National des Forêts, especially M. Bénard, for facilitating experimental work at Barbeau. We acknowledge N. Bréda (INRA Nancy, France) for trunk growth measurements at Hesse. The platform Métabolisme-Métabolome of the IFR87 is acknowledged for the isotope measurements. We are grateful to M. Danger and X. Raynaud for valuable discussions on the manuscript and to E. M. Gross for revising the manuscript. We thank two anonymous reviewers and the editor for their helpful comments and improvements to the manuscript.

Funding This research was funded by the French projects 'Ministère délégué à la recherche-ACI Jeunes Chercheurs' (no. JC10009) and 'Programme National ACI/FNS ECCO, PNBC' (convention no. 0429 FNS) and by the ESE laboratory thanks to funds from Paris-Sud University and CNRS.

designing the experiment and coordinating the research projects 'Ministère délégué à la recherche-ACI Jeunes Chercheurs' (no. JC10009) and 'Programme National ACI/FNS ECCO PNBC' (convention no. 0429 FNS)