Benoît D'angelo

Fabien Leroy
email: fabien.leroy@univ-orleans.fr

Christophe Guimbaud

Adrien Jacotot

Renata Zocatelli

Sébastien Gogo

Fatima Laggoun-Défarge

Carbon balance and spatial variability of CO2 and CH4 fluxes in a Sphagnum-dominated peatland in temperate climate

Keywords: CO2, CH4, DOC, peatland, ecosystem respiration, gross primary production

Introduction

Peatlands are vegetated wetlands that can act as a powerful natural Carbon (C) sink. While they cover only 3% of the emerged lands [START_REF] Lappalainen | Global Peat Resources[END_REF], the C stored in their soils has been estimated to range from 473 to 621 GtC [START_REF] Yu | Global peatland dynamics since the Last Glacial Maximum[END_REF], which represents more than 30% of the C stored in terrestrial soils, estimated between 1500 to 2400 PgC [START_REF] Ciais | The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change[END_REF]. Thus, peatlands are of a great importance in the actual context of global changes and their preservation is essential to prevent the C stored in soil to be released to the atmosphere. However, peatlands are mostly located at high latitudes of the northern hemisphere [START_REF] Strack | Peatlands and climate change[END_REF] where the greatest climate changes are predicted to occur by the IPCC models [START_REF] Christensen | The Physical Science Basis. Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change[END_REF]. Under these modified climatic conditions, the behaviour of peatlands in terms of C cycle is still uncertain.

Actually, the C balance in peatlands covers a wide range of C functioning, from sink to source (Beyer and Höper, 2015;[START_REF] Carroll | Carbon balance of a temperate poor fen[END_REF][START_REF] Koehler | How strong is the current carbon sequestration of an Atlantic blanket bog?[END_REF][START_REF] Vanselow-Algan | High methane emissions dominated annual greenhouse gas balances 30 years after bog rewetting[END_REF]. However, the majority of these estimations were calculated on high latitudes sites, mostly in Northern Europe or in Canada where the winter is cold [START_REF] Peichl | A 12-year record reveals pre-growing season temperature and water table level threshold effects on the net carbon dioxide exchange in a boreal fen[END_REF][START_REF] Strack | Annual carbon balance of a peatland 10 yr following restoration[END_REF][START_REF] Trudeau | Interannual variability in the CO2 balance of a boreal patterned fen, James Bay, Canada[END_REF][START_REF] Waddington | Carbon balance of a boreal patterned peatland[END_REF], even if few studies have been conducted in lower latitudes (below 50°) where warmer climatic conditions prevail (e.g. [START_REF] Bortoluzzi | Carbon balance of a European mountain bog at contrasting stages of regeneration[END_REF].

In addition to climatic pressure, peatlands can be submitted to anthropic disturbances, and temperate peatlands have been widely transformed by the past into agricultural or forestry lands [START_REF] Joosten | Wise Use of Mires and Peatlands[END_REF]. Drainage that leads to major hydrological disruption is usually a prerequisite before peatland exploitation (Beyer et al, 2015). For example, lowering the water table level can change the equilibrium between CO2 and CH4 emissions and thus modify the C balance of the ecosystem [START_REF] Chimner | Multi-decadal changes in water table levels alter peatland carbon cycling[END_REF]. Indeed, increasing the thickness of the oxic layer can lead to (i) higher CO2 fluxes to the atmosphere due to faster decomposition rates of soil organic matter and (ii) lower CH4 fluxes due to the decrease in CH4 production and/or increase in CH4 oxidation during its transport to the surface of the soil [START_REF] Lund | Effects of drought conditions on the carbon dioxide dynamics in a temperate peatland[END_REF][START_REF] Pelletier | Methane fluxes from three peatlands in the La Grande Riviere watershed, James Bay lowland, Canada[END_REF]. As a result of both drainage and climatic change, most temperate peatlands are now invaded by vascular plants [START_REF] Berendse | Raised atmospheric CO2 levels and increased N deposition 16 cause shifts in plant species composition and production in Sphagnum bogs[END_REF][START_REF] Buttler | Experimental warming interacts with soil moisture to discriminate plant responses in an ombrotrophic peatland[END_REF].

Invasive vegetation can play a major role in CO2 and CH4 emissions by (i) producing litter that is more easily degradable than mosses, (ii) altering the growth of subservient species such as Sphagnum mosses, and/or (iii) allowing more CH4 to be transferred to the atmosphere through the plant aerenchyma [START_REF] Bubier | Effects of nutrient addition on vegetation and carbon cycling in an ombrotrophic bog[END_REF][START_REF] Francez | Peat accumulation and peat decomposition after human disturbance in French and Finnish mires[END_REF][START_REF] Gogo | Invasion of a Sphagnum-peatland by Betula spp and Molinia caerulea impacts organic matter biochemistry. Implications for carbon and nutrient cycling[END_REF][START_REF] Armstrong | Biotic and abiotic factors interact to regulate northern peatland carbon cycling[END_REF]. The role of the vegetation on the C fluxes are often related to a comparison between different plant community composition [START_REF] Ward | Warming effects on greenhouse gas fluxes in peatlands are modulated by vegetation composition[END_REF][START_REF] Noyce | Effect of Carex rostrata on seasonal and interannual variability in peatland methane emissions[END_REF]; however only a few of them have attempted to integrate vegetation directly in a C balance model [START_REF] Bortoluzzi | Carbon balance of a European mountain bog at contrasting stages of regeneration[END_REF][START_REF] Kandel | Measurement and modelling of CO2 flux from a drained fen peatland cultivated with reed canary grass and spring barley[END_REF][START_REF] Leroy | CO2 and CH4 budgets and global warming potential modifications in Sphagnumdominated peat mesocosms invaded by Molinia caerulea[END_REF].

In this context, the aim of this study was to investigate what is the C-sink function of a disturbed temperate peatland and the mechanisms controlling it. Here, CO2 fluxes and CH4 emissions were monthly measured on twenty points distributed homogeneously over a temperate peatland recently invaded by a graminoid plants, Molinia caerulea. Then C fluxes measurements were related to biotic and abiotic factors to estimate the annual C budget by using CO2 and CH4 models including, or not, vegetation index. Thus, the hypotheses tested were:

(i)
the drainage and the invasion by vascular plants promoted the C emissions and lead the peatland to act as a C source (ii) the use of vegetation index into models leads to a better representation of fluxes

Materials and methods

Description of the La Guette peatland

The study was performed in La Guette peatland, a Sphagnum peatland located in France (Neuvy-sur-Barangeon, Cher, N 47°1944, E 2°1704; Fig. 1). It is a transitional poor fen (with a pH between 4 and 5 and a conductivity lower than 80 µS m -2 ) with a maximum peat thickness of about 180 cm. Mean annual temperature was 11°C and mean annual rainfall 732 mm for the period 1971-2000 (Gogo et al., 2011). The site is drained in its south part by a road built before 1945 that crosses the peatland. In 2009 the drainage ditch of the road was scraped, lowering the output level and consequently increasing the water losses. This hydrological disturbance in addition to a fire (in 1970's)

Carbon fluxes measurements and calculation

In June 2011, 20 stations were set up on the peatland by dividing the area in twenty squares with a grid and choosing randomly one plot (2 m²) within each square (stratified random sampling). This method allowed a homogeneous spatial covering of the studied site (Fig. 1). Fluxes were monthly measured between March 2013 and February 2015 using a semi-cylindrical transparent static chamber equipped (30 cm of diameter, 30 cm height). The chamber was equipped with a HMP 75 sensor (Vaisala Oyj, Vantaa, Finland) to record the temperature and air humidity variations within the chamber during the incubations. In addition, a small fan allowed the air to be homogenized within the chamber [START_REF] Pumpanen | Comparison of different chamber techniques for measuring soil CO2 efflux[END_REF]. CO2 fluxes were measured in each of the 20 stations during twenty field campaigns thanks to a CO2 sensor (GMP 343 model, Vaisala Oyj, Vantaa, Finland) placed inside the chamber. Fluxes were measured at light for Net Ecosystem Exchange (NEE) and with the chamber covered with a light insulating fabric to simulate night conditions and measure the Ecosystem Respiration (ER). Each measurement lasted between 3 to 5 minutes, and the CO2 concentration inside the chamber was recorded at 5Hz. CH4 fluxes were measured thanks to the SPIRIT instrument, an Infrared Spectrometer developed by the Laboratory of Physics and Chemistry of Environment and Space, (LPC2E, Orléans, France, [START_REF] Guimbaud | A quantum cascade laser infrared spectrometer for CO2 stable isotope analysis: Field implementation at a hydrocarbon contaminated site under bio-remediation[END_REF][START_REF] Guimbaud | A portable infrared laser spectrometer for flux measurements of trace gases at the geosphere-atmosphere interface[END_REF][START_REF] Robert | Simple, stable, and compact multiple-reflection optical cell for very long optical paths[END_REF]. For logistical reasons (weight and long start-up time of SPIRIT), CH4 measurements were only performed during 12 field campaigns and on only 5 stations. These measurements regularly required to access to the plots, so, wooden planks was used as mobile pontoons to limits disturbances CO2 and CH4 fluxes were then calculated as follow:

𝐹 = 𝑑𝑋 𝑑𝑡 * 𝑃 𝑅 * 𝑇 * 𝑉 𝑆 (1)
With F the net gas flux (µmol m -2 s -1 ); dX/dt the gas concentration (µmol mol -1 ) variation during the incubation time (s); P the atmospheric pressure (Pa); R the ideal gas constant (8.3144621 J mol -1 K -1 );

T the average temperature (K) within the chamber during the incubation; V the total volume of the system (m 3 ); and S the chamber surface (0.283 m 2 ). For CO2 fluxes, F corresponds to NEE when measured in light, and to ER when measured in the dark.

In addition with gas fluxes, dissolved organic carbon (DOC) flux was estimated monthly during the studied period. Water was sampled at the peatland outlet, filtered at 0.45 µm and in-situ acidified with 2 drops of H3PO4. The DOC concentration was then determined in the laboratory thank to a TOC-LCPH analyser (Shimadzu, Kyoto, Japan).

Monitoring of environmental variables and vegetation cover

Soil-meteorological variables were automatically monitored at a 30-min frequency during the entire studied period thanks to an automatic station installed on the site in November 2010 (Fig. 1).

Parameters recorded were the total rainfall, net solar radiation, atmospheric pressure, wind direction and speed, air temperature, relative humidity and soil temperature at -5,-10, -20, and -40 cm depths. In addition, four automatic piezometer spread into the peatland allowed to record the Water Table Level (WTL) variations at the ecosystem scale [START_REF] Binet | A water-table dependent reservoir model to investigate the effect of drought and vascular plant invasion on peatland hydrology[END_REF]. In addition to automatic measurements, manual recording of WTL, Photosynthetically Active Radiation (PAR), soil temperatures (-5, -10, -15, -20, -25, -30, -40, -50, -60, -70, -80, -90, -100 cm depth) and atmospheric pressure were realized at each measurement campaign.

A vegetation Index (VI) was calculated (Eq. 2) by summing the percentage of vegetation cover in each soil collar following three distinct plant strata: the muscular (Sphagnum spp.), herbaceous (Molinia caerulea and Eriophorum augustifolium), and shrub (Erica tetralix and Calluna vulgaris) strata divided by the total potential cover TC (TC = n x 100, n being the number of vegetation strata recorded):

𝐼𝑉 = 𝑀𝑆+𝐻𝑆+𝑆𝑆 𝑇𝐶 (2)
With MS, HS and SS the percentages of cover of the muscular, herbaceous and shrub strata, and TC the sum of percentages of the three strata.

Modelling of high-frequency ER, GPP, FCH4 and DOC

The C balance estimation was conducted in 3 successive steps: i) calibration to establish the relationship between gas fluxes and environmental variables; ii) evaluation to determine the relevance of these relationships in a broader context; and iii) interpolation to integrate these relationships over time and to calculate a Net Ecosystem Carbon Balance (Chapin et al, 2006):

NECB=GPP-ER-FCH4-FDOC-FDIC-FPC-FVOC-FCO (3)
with GPP (Gross Primary Production), FCH4, FDOC, FDIC, FPC, FCOV, FCO, the fluxes of CH4, Dissolved Organic Carbon (DOC), Dissolved Inorganic Carbon (DIC), Particulate Carbon (PC), Volatile Organic Carbon (VOC) and Carbon Monoxide (CO), respectively. In terms of quantity, the first 4 terms are the most abundant and were estimated in the present study to calculate the C budget. By convention the negative sign refers to carbon lost by the ecosystem.

The available data were averaged by field campaign to reduce the spatial variability of the fluxes and then used to produce a high-frequency (1-hour) dataset. For this, the relationship between gas fluxes and environmental variables was fitted using non-linear regression curves. Robustness of adding a variable to the models was evaluated following three comparative criteria. First, the adjusted R² (R 2 a;

Eq. 4) were used to estimate the proportion of explained variance:

𝑅 𝑎 2 = 1 -(1 -(1 - ∑(𝑦-𝑦 ^)2 ∑(𝑦-𝑦 ¯)2 )) * 𝑛-1 𝑛-𝑝-1 (4)
with R 2 the traditional R 2 , y the measured data, ŷ the modelled data, n the number of observations and p the number of predictors.

Then, the Normalized Root Mean Square Error (NRMSE; Eq. 5) was applied to evaluate the differences between measured and modelled data points respectively:

𝑁𝑅𝑀𝑆𝐸 = 100 * √( ∑(𝑦-𝑦 ^) 𝑛 ) 𝑦 ¯
(5) with y the average of the measured data.

Finally, the relevance of adding a new variable in the model was estimated by the Akaike Information Criterion (AIC; Eq. 6; [START_REF] Akaike | A new look at the statistical model identification[END_REF][START_REF] Burnham | Model selection[END_REF].

𝐴𝐼𝐶 = -2 × 𝑙𝑜𝑔(𝐿) + 2 × 𝑝 (6)
with L the maximum likelihood.

The NEE, ER, and CH 4 fluxes were modelled separately and the annual carbon balance was calculated as the sum of the model interpolations at a 1h time step. This interpolation was done made using (i) the high frequency measurement values for temperature and (ii) a linear interpolation between the punctual measurements for vegetation. For all gaseous fluxes two models are presented: one using temperature as this model is widely used [START_REF] Ballantyne | Long-term water table manipulations alter peatland gaseous carbon fluxes in Northern Michigan[END_REF] and, the other being the best model estimation using vegetation [START_REF] Bortoluzzi | Carbon balance of a European mountain bog at contrasting stages of regeneration[END_REF][START_REF] Kandel | Measurement and modelling of CO2 flux from a drained fen peatland cultivated with reed canary grass and spring barley[END_REF]. Evaluation of models 1 and 2 for GPP, ER and FCH4 was realized using an independent dataset that includes CO2 and CH4 fluxes conducted in the same peatland in 2014 and using similar techniques as in the present study (unpublished data).

For GPP, the saturated GPP (GPPsat) was first calculated following [START_REF] June | A simple new equation for the reversible temperature dependence of photosynthetic electron transport: a study on soybean leaf[END_REF] (Eq. 7), and secondly by a modified version of the equation that incorporates the vegetation index (Eq. 8):

𝐺𝑃𝑃 𝑠𝑎𝑡 -1= a*𝑒 ( 𝑇-𝑏 𝑐 ) 2 (7) 𝐺𝑃𝑃 𝑠𝑎𝑡 -2= (a*VI+d)*𝑒 ( 𝑇-𝑏 𝑐 ) 2 (8)
Then, GPP-1 and GPP-2 were calculated using Eq. 9 proposed by [START_REF] Bubier | Ecological controls on methane emissions from a Northern Peatland Complex in the zone of discontinuous permafrost, Manitoba, Canada[END_REF] and reused in many studies (e.g. [START_REF] Bortoluzzi | Carbon balance of a European mountain bog at contrasting stages of regeneration[END_REF][START_REF] Worrall | The multi-annual carbon budget of a peat-covered catchment[END_REF]:

𝐺𝑃𝑃 = 𝐺𝑃𝑃 𝑠𝑎𝑡 * 𝑖 * 𝑃𝐴𝑅 𝐺𝑃𝑃 𝑠𝑎𝑡 +𝑖 * 𝑃𝐴𝑅 (9)
with a the rate of electron transport at light saturation (µmol m -2 s -1 ), b the optimal temperature for a The two models of CH4 fluxes were estimated following:

𝐹 𝐶𝐻4 -1= a*𝑒 (𝑏 * 𝑇) (12) 𝐹 𝐶𝐻4 -2= a*𝑒 (𝑏 * 𝑉𝐼) (13)
with T the air temperature (°C); a and b the fitted parameters and IV the vegetation index.

High frequency of DOC fluxes (FDOC) have been extrapolated using the monthly measured concentrations of DOC and an estimation of the water quantities leaving the ecosystem (D) using a hydrological model specifically calibrated for the La Guette peatland [START_REF] Binet | A water-table dependent reservoir model to investigate the effect of drought and vascular plant invasion on peatland hydrology[END_REF]:

FDOC=D*[DOC] (14)

Spatial variability of CO2 fluxes

To estimate the spatial variability of the CO2 fluxes, the best models were also calibrated for each of the 20 plots individually. Hence the model parameters R 2 a and NRMSE were estimated for each measurement point as well as the annual CO2 flux.

Results

Environmental monitoring

Variations of air temperature, rainfall, and water table level are presented in Fig. 2. Between 2011 and 2015 the mean annual air temperature measured by the meteorological station varied between 9.5 and 11.5°C (Fig. 2A), with a high seasonal variability. In addition, average air temperature measured in the 20 stations during the field campaigns ranged from 6 to 32°C. Total rainfall recorded on the site for 2014 and 2015 were quite similar with 935 and 940 mm, respectively (Fig. 2B). The WTL measured by the automatic piezometers ranged between -17.5 and -0.1 cm during the studied period, with clear seasonal variability (Fig. 2C). The average WTL manually measured in the 20 stations during the field campaigns showed a similar cyclicity (Fig. 2C) with deeper level during summer (maximum depth of -18 and -10.5 cm in 2013 and 2014, respectively) than during winter. The seasonal variability of vegetation was controlled by the graminoids which started to grow earlier in 2014 (April) than in 2013 (May) (Fig. 3). Also, in 2014 the vegetation index (VI) was slightly higher than in 2013, at 0.45 and 0.51 respectively.

CO2, CH4 and DOC fluxes

Results of CO2 fluxes (NEE, ER and calculated GPP) are presented in Fig. 2. CO2 fluxes (GPP and ER) showed a clear seasonal trend with a maximum in summer. In 2013 the GPP averaged maximum occurred at the end of July reaching 12.80±4.91 µmol m -2 s -1 and ER 9.43±3.48 µmol m -2 s -1 , with the maximum of both variables reached at the end of July (Fig. 2A andB). In 2014 the GPP and ER averaged maxima were reached earlier, in June with 13.16±4.70 µmol m -2 s -1 and 7.83±2.55 µmol m -2 s -1 respectively (Fig. 2A andB). Averages values of GPP for 2013 and 2014 were 7.12±5.19 and 6.56±4.72 µmol m -2 s -1 respectively. For ER, the average values were, for 2013 and 2014, 4.27±3.16 and 3.63±2.56 µmol m -2 s -1 , respectively (Fig. 4 A andB). Mean values of NEE for 2013 and 2014 were 2.85±3.05 and 2.93±2.77 µmol m -2 s -1 , respectively (Fig. 4 C). In comparison to CO2, CH4 emissions showed a lower magnitude with fluxes below 0.3 µmol m -2 s -1 . A clear difference was also visible between 2013 and 2014 respectively (Fig. 5). In 2013 CH4 average fluxes were 0.04±0.03 µmol m -2 s -1 , whereas they reached 0.10±0.08 µmol m -2 s -1 in 2014. For both years, the average DOC concentrations measured at the outlet of the peatland were 18.5±7.7 mg L -1 . Thus, the average cumulated flux of DOC leaving the peatland for 2013 and 2014 was 12±1 gC m -2 yr -1 (8±1 and 16±1 gC m -2 yr -1 in 2013 and 2014, respectively).

Model selection

Comparisons between modelled and measured values of ER, GPP and CH4, using models 1 and 2 during calibration and evaluation are presented in Fig. 6. During models' calibration, incorporation of vegetation in the ER model (leading to ER-2) improved the representation of ER, yielded to a higher R 2 and a lower NRMSE than without vegetation (ER-1, Fig. 6A and Table 1). However, the difference between ER-1 and ER-2 is low but the AIC, which decreased from 47 to 35, confirmed this observation (Table 1). ER was estimated to 1286±231 and 1261±164 gC m -2 yr -1 for ER-1 and ER-2, respectively.

Concerning GPP, GPP-2 realized a higher R²a score, a lower NRMSE, and a lower AIC compared to GPP-1 (Fig. 6C and Table 1). Resulting cumulated fluxes were 1290±400 and 1070±203 gC m -2 yr -1

for GPP-1 and GPP-2. For CH4, only CH4-2 (including vegetation) that showed a satisfying result (Fig. 6E and Table 1) was used to estimate the CH4 cumulated fluxes. Evaluation of the GPP models showed high NRMSE values with 47 and 58 % for GPP-1 and GPP-2 respectively (Fig. 6D and Table 1). CH4 evaluation led to even higher NRMSE values with 68 % for FCH4-2. R²a negative values due to the use of non-linear regression were here irrelevant (Fig. 6F and Table 1). On the contrary, the ER models led to high R²a and low NRMSE values with ER-2 performing better than ER-1: R²a values increased from 0.16 to 0.59 and NRMSE values decreased from 35 to 23 % (Fig. 6E and Table 1).

Calculation of Net Ecosystem Carbon Balance (NECB)

NECB (estimated here as GPP-ER-FCH4-FDOC) was calculated first without inclusion of vegetation in the model (GPP-1, ER-1) and then with vegetation (GPP-2, ER-2), at the exception of the calculation of CH4 for which only the model with vegetation was used (CH4-2). Whether model 1 or 2 was used, the NECB showed that the peatland was a source of carbon to the atmosphere in 2013 and 2014 with -26 gC m -2 yr -1 and -220 gC m -2 yr -1 for model 1 or 2, respectively (Table 2). The high difference in the estimation of NECB was principally due to the GPP estimations (Table 1). In addition, NECB was higher in 2013 than in 2014 (Table 2). NECB was mainly driven by CO2 fluxes as CH4 and DOC fluxes are two orders of magnitude lower. However, in terms of the Global Warming Potential (GWP), CH 4 fluxes are not negligible with CH4 having a GWP 34 times higher than CO2 for the 100-year time horizon (Myhre et al, 2013).

Spatial variability

The calibration of ER-2 and GPP-2 on each measurement plot enabled the spatial variability of the CO2 fluxes on the site to be estimated. For ER-2, the R²a of the individual plots were above 0.5 (with the exception of measurement plot n°10) and the NRMSE values were below 50% (Fig. 7). The model parameter variations ranged from 0 to 0.015 for a, from 0.035 to 0.11 for b, and from 0.18 to 1.03 for c. For GPP-2 the R²a were above 0.5 and the NRMSE values between 20 and 60% (with the exception of point n°10). Model parameters for GPP-2 showed higher variations: between -6.1 and 66 for a, 23.9 and 90.4 for b, 6.2 and 60.0 for c and -10.7 and 27.1 for d (Fig. 8). For both measurement years, cumulated GPP fluxes ranged from 511 to 1420 gC m -2 yr -1 , with most values around 1100 gC m -2 yr - 1 and an average of 1052±238 gC m -2 yr -1 . For ER, the cumulated fluxes ranged from 842 to 2363 gC m -2 yr -1 with an average of 1215±362 gC m -2 yr -1 (Fig. 7 and8). approximately for sites located in Quebec, Canada and in Northern Sweden respectively. However, these large fluxes were close to those found for sites with a high Mean Annual Temperature (MAT).

For instance, Beyer and Höper (2015) found for a site with similar vegetation (Molinia caerulea, Eriophorum augustifolium, Sphagnum spp.) and with a MAT of 8.6°C, GPP fluxes between 534 and 1058 gC m -2 yr -1 and ER fluxes between 420 and 1052 gC m -2 yr -1 . Thus, the large fluxes measured in La Guette peatland can be related to the relatively warmer climate compared to peatlands at higher latitudes. However, a MAT alone cannot explain why the system functioned as a source of C as other studies have shown that peatlands with a MAT above 8°C can still be a C sink. For instance, [START_REF] Koehler | How strong is the current carbon sequestration of an Atlantic blanket bog?[END_REF] estimated an uptake of -29.7 gC m -2 yr -1 in an Atlantic blanket bog with a MAT of 10.6°C for a 6-year average (2003)(2004)(2005)(2006)(2007)(2008). The water table drawdown favours oxic reactions in a thicker soil layer, thus it is often adduced to explain the sources of C in peatlands. During the two years of measurements, La Guette peatland experienced favourable hydrological conditions due to heavy rainfall leading to high water table levels [START_REF] Bernard-Jannin | Hydrological control of dissolved organic carbon dynamics in a rehabilitated Sphagnum-dominated peatland: a water-table based modelling approach[END_REF]. In spite of this, the system functioned as a source to the atmosphere. Moreover, these high water table levels was spatially and temporally similar during the time course of the experiment (2013-2014, two exceptionally wet years), which did not allow to include this factor during the modelling processes. Higher variation of the water table depth could have brought to use equations with this parameters as Luan et al. (2015) to explain the GPP or [START_REF] Leroy | CO2 and CH4 budgets and global warming potential modifications in Sphagnumdominated peat mesocosms invaded by Molinia caerulea[END_REF] to model the CO2 and CH4 emissions. Nevertheless, inclusion of environmental parameters have to be considered up to their contribution to the models and to the models parsimony [START_REF] Baird | Modelling time-integrated fluxes of CO2 and CH4 in peatlands: A review[END_REF].

This C source function if the La Guette peatland is now also shown thanks to an Eddy-covariance tower and this with C fluxes similar as reported here (Jacotot et al., in prep). The main hypothesis concerning the mechanisms behind the C source function of the La Guette is linked to recent invasion of the site by Molinia caerulea, which is suspected to modify the C dynamic of the ecosystem. The observed C flux intensities and losses are often related to the plant community of the peatland. Vascular plants have higher gaseous C fluxes than bryophytes in poor fens [START_REF] Leroy | CO2 and CH4 budgets and global warming potential modifications in Sphagnumdominated peat mesocosms invaded by Molinia caerulea[END_REF][START_REF] Rydin | The biology of peatlands, 2e[END_REF] and a shift in peatland plant communities, especially those reducing Sphagnum dominance in favor of vascular vegetation, is expected to have significant effects on carbon storage [START_REF] Dieleman | Climate change drives a shift in peatland ecosystem plant community: implications for ecosystem function and stability[END_REF]. Now, the site is almost entirely invaded by vascular plants, especially Molinia caerulea in the graminoid strata [START_REF] Gogo | Invasion of a Sphagnum-peatland by Betula spp and Molinia caerulea impacts organic matter biochemistry. Implications for carbon and nutrient cycling[END_REF]. The occurrence of this plant can stimulate the C uptake compared to Sphagnum peatland [START_REF] Leroy | CO2 and CH4 budgets and global warming potential modifications in Sphagnumdominated peat mesocosms invaded by Molinia caerulea[END_REF]. However it also significantly increase the ER respiration and CH4 emissions through different physiological, phenological, and ecological traits [START_REF] Gogo | Invasion of a Sphagnum-peatland by Betula spp and Molinia caerulea impacts organic matter biochemistry. Implications for carbon and nutrient cycling[END_REF][START_REF] Leroy | CO2 and CH4 budgets and global warming potential modifications in Sphagnumdominated peat mesocosms invaded by Molinia caerulea[END_REF]. The ratio GPP/ER should be further assessed, especially in the context of interaction between different functional types (D'Angelo, 2015)

Implication of taking vegetation into account in the C budget estimation

The plants functional type often explained large of variation of the CO2 and CH4 fluxes [START_REF] Armstrong | Biotic and abiotic factors interact to regulate northern peatland carbon cycling[END_REF]. In this way, it was thus important to include the vegetation in our models, especially with the occurrence of Molina caerulea. Indeed, this species shows a high growth variability during the growing season (e.g. number of leaves, leave length) that could impact the C fluxes (more leaves, more GPP). However, its integration into models is seldom carried out [START_REF] Bortoluzzi | Carbon balance of a European mountain bog at contrasting stages of regeneration[END_REF][START_REF] Görres | Comparative modeling of annual CO2 flux of temperate peat soils under permanent grassland management[END_REF], probably because of the difficulty in measuring the pertinent variables (leaf area index, leaves number). Including vegetation in models has led to a better representation of CO2 fluxes. These results were in accordance with [START_REF] Bortoluzzi | Carbon balance of a European mountain bog at contrasting stages of regeneration[END_REF] who also found a lower NRMSE when vegetation was included in the model. Another way to take the vegetation into account is to use the Ratio Vegetation Index (RVI), which is a measurement of incoming and reflected radiation. This method was used by [START_REF] Görres | Comparative modeling of annual CO2 flux of temperate peat soils under permanent grassland management[END_REF] and led to an improvement for GPP modelling but not for ER.

However, such an improvement using the RVI for ER modelling was found by [START_REF] Kandel | Measurement and modelling of CO2 flux from a drained fen peatland cultivated with reed canary grass and spring barley[END_REF] on a cultivated fen peatland in Denmark. Although the calibration of the model was as good for ER as for GPP, evaluation of the models presented more contrasted results since only ER-2 showed a high R 2 a and a low NRMSE. For FCH4-2 the evaluation revealed that the interpretation of the model outputs should be limited to the current study and could not be used to extrapolate fluxes for other times and spaces. Nevertheless, as hypothesis, model evaluations indicated that the inclusion of vegetation increased the model's capability to represent CO2 fluxes.

The DOC exports

The quantities of DOC exported by La Guette peatland are in the same order of magnitude of those present in the literature (e.g. [START_REF] Waddington | Carbon balance of a boreal patterned peatland[END_REF][START_REF] Worrall | The multi-annual carbon budget of a peat-covered catchment[END_REF] but have a low impact on C balance compared to CO2 fluxes. The doubling of the DOC flux observed in 2014 compared to 2013 is linked to a greater quantity of water leaving the bog with similar DOC contents. At the same time, the average water table measured in 2014 is slightly higher than that measured in 2013 and precipitation is of the same order of magnitude (Fig. 2).

ER and GPP spatial variability

Calibration of the individual CO2 fluxes at the 20 measurement points showed a large range of cumulated flux estimation (Fig. 7 and8). Nonetheless, the average of these fluxes was coherent with those estimated at the ecosystem scale with the average of the 20 points: 1215 vs. 1261 gC m -2 yr -1 and 1052 vs. 1070 for ER and GPP respectively (Table 1). The ER estimates are very close for the two years, which is consistent with the relatively similar water table also observed. The difference in air temperature between 2013 and 2014 (9.1 and 10.1 •C respectively) is not sufficient to observe a significant difference. The order of magnitude of the spatial variability was the same between ER and GPP estimated within one site (this study) and ER and GPP estimated in different sites by [START_REF] Jacobs | Variability of annual CO2 exchange from Dutch grasslands[END_REF]. For ER, the spatial variability found within La Guette peatland was also larger than the inter-annual variability measured by Beyer et al. (2015), in which the difference between the maximum and minimum annual fluxes was 429 gC m -2 yr -1 on a temperate fen used as grassland. With the acquired data, no patterns have been found between the location of the points and the model parameters or with the measured environmental variables that would enable this spatial variability to be linked to specific local environmental conditions. Indeed, this variability does not seem geographically distributed or related to gradient of environmental parameters with spatial and temporal similarity of the air temperature and water table levels. However, a first approach by grouping points per vegetation classes (with dominance of the muscular, herbaceous or shrubs strata) shows promising results in order to explain this spatial variability [START_REF] D'angelo | Variabilité spatio-temporelle des émissions de GES dans une tourbière à Sphaignes: effets sur le bilan carbone[END_REF].

Conclusions

In this study we have shown that despite high water table levels, La Guette peatland acted as a C source to the atmosphere for the 2 years of measurements, in 2013 and 2014. This was partly explained by the high mean annual temperatures that led to large fluxes. However, it is probable that the site history also played a significant role: after several dry years, the peatland was just starting to refill during the 2 measurement years, probably leading to air trapped in the porosity that favoured oxic organic matter degradation. Longer observations are needed to catch the long-term dynamics in terms of hydrological cycle and site history leading to hysteresis effects. The vegetation had a significant effect when included in ER models despite the difficulty in monitoring the vegetation profile without disturbances.

However, such an effect was not as clear for the GPP models. As a result, the ER models had a narrower range of estimations (more precise when vegetation is included) than the GPP ones, indicating that ER models seem more reliable. As a result, this study advocates the inclusion of vegetation in models, even in a simplistic way. These results also emphasize the importance of model evaluation as it outcomes may be quite different from the calibration and this even for models with very well-fitted parameters. Accessing these differences might help to estimate the interpretative power of such models in a broader context. Finally, the estimations of the CO2 fluxes on the 20 measurement points showed a large spatial variability. The order of magnitude of this variability estimated on one site was the same as those reported between different sites. More studies are needed to better quantify the spatial variability within a site, and to allow a better estimation of the uncertainty of the greenhouse gas budget estimated at larger scales.

(

  °C), and c the difference in temperature from b at which GPPsat equals to e -1 of its value at b[START_REF] June | A simple new equation for the reversible temperature dependence of photosynthetic electron transport: a study on soybean leaf[END_REF], T the air temperature (°C), and VI the vegetation index.Concerning ER, the equation proposed byLuo et Zhou (2006) was used for ER-1 and modified with the integration of the herbaceous strata (HS) for ER-2: ER-1= a*𝑒(𝑏 * 𝑇) (10)ER-2= (a*HS+c)*𝑒 (𝑏 * 𝑇) (11)with T the air temperature (°C); a, b and c the fitted parameters and HS the cover of the herbaceous strata (%).Finally, modelled NEE have been calculated from GPP and ER as NEE = GPP -RE. Negative values of NEE correspond to an emission of carbon from the ecosystem to the atmosphere and positive values to a fixation of carbon.

  behind the high C fluxes recorded and the C source functioning CO2 fluxes measured at La Guette peatland were above 1000 gC m -2 yr -1 for the GPP and the ER, whatever the model used. These fluxes were high compared to those of boreal peatlands.[START_REF] Trudeau | Interannual variability in the CO2 balance of a boreal patterned fen, James Bay, Canada[END_REF] and[START_REF] Peichl | A 12-year record reveals pre-growing season temperature and water table level threshold effects on the net carbon dioxide exchange in a boreal fen[END_REF] found GPP and ER fluxes between 100 and 500 gC m -2 yr -1

  has probably contributed to the invasion of the site by vascular plants, mainly Pinus sylvestris, Betula spp. (Betula verrucosa and pubescens) and Molinia caerulea; This Poaceae is now invading numerous peatlands in Europe mainly, due to an increase of the nitrogen deposition and drainage(Chambers et al., 1999) and thus at the detriment of the specific peatland species composed of Sphagnum cuspidatum and Sphagnum rubellum, Eriophorum augustifolium, Erica tetralix and Calluna vulgaris. .

Acknowledgements This work was supported by the Labex VOLTAIRE (ANR-10-LABX-100-01) and CARBIODIV project (2012-00073566). The authors gratefully acknowledge A. Menneguerre for his contribution to gas measurements and P. Jacquet and C. Robert for their assistance in SPIRIT maintenance. We also thank E. Rowley-Jolivet for revision of the English version.

Author contribution.

BD, SG and FLD designed the experiment BD, SG, CG, FL and FLM collected data BD, SG, CG, FL and FLM performed model simulations and data analysis BD and FL prepared the manuscript with contributions from all co-authors AJ and RZ reviewed and corrected the manuscript


Ambre Salis
email: ambre.salis@univ-lyon1.fr

Jean-Paul Léna

Thierry Lengagne

Jean-Paul Lena

How subtle protocol choices can affect biological conclusions? An example with how great tits respond to allopatric

Keywords: Animal communication, Heterospecific communication, Mobbing, Replication crisis, Syntax

How subtle protocol choices can affect biological conclusions? An example with how great tits respond to allopatric mobbing calls.

During the last decade, failure to reproduce published results in various fields or research (e.g., in psychology [START_REF] Bohannon | Many psychology papers fail replication test[END_REF], or in epidemiology Lash et al., 2018) alerted the scientific community about a potential low reliability of published results. This replication crisis, complex and heavily debated, can be explained in different ways [START_REF] Fanelli | Opinion : Is science really facing a reproducibility crisis, and do we need it to[END_REF][START_REF] Maxwell | Is psychology suffering from a replication crisis? What does "failure to replicate" really mean?[END_REF]. Indeed, while models have shown that the global "publish or perish" problem probably increases misconduct [START_REF] Grimes | Modelling science trustworthiness under publish or perish pressure[END_REF][START_REF] Higginson | Current Incentives for Scientists Lead to Underpowered Studies with Erroneous Conclusions[END_REF], direct frauds (i.e., fabrication or falsification of data) remain scarce according to empirical evidence [START_REF] Fanelli | Opinion : Is science really facing a reproducibility crisis, and do we need it to[END_REF]. More probable is the combined effect of several inconspicuous and ordinary factors [START_REF] Ioannidis | Why Most Published Research Findings Are False[END_REF] leading to the publication of false results and/or interpretations, with one major example being conscious or unconscious phacking (i.e., to collect data, select data or use statistical analyses until nonsignificant results become significant, [START_REF] Head | The Extent and Consequences of P-Hacking in Science[END_REF]. In the field of animal behaviour and especially in animal communication, three specific factors are specifically of importance. Firstly, behaviour is by nature an external proxy of internal state of animals, needing researchers to interpret each variable under study. Such behaviours are prone to flexibility between researchers regarding the type of recording, the definition of behaviour, and their relative relevance to the question asked. Indeed, each scientist is the sum of their past experience, knowledge, and personal background, which can affect their biological conclusions at the creation of the protocol (Tang-Martínez, 2020), when analysing results (Silberzahn et al., 2018) or when interpreting those results [START_REF] Tang-Martínez | The history and impact of women in animal behaviour and the ABS : A North American perspective[END_REF]. Greater flexibility increases the opportunity to transform negative results into positive ones [START_REF] Ioannidis | Why Most Published Research Findings Are False[END_REF], and in a general manner creates disparities between papers investigating the same question. Secondly, the difficulty to obtain large numbers of wild subjects as much as the ethics considerations often lead behaviour studies to obtain restricted sample sizes (Schwagmeyer & Mock, 1997).

Small sample sizes are less likely to detect small differences between treatments (type I error, [START_REF] Button | Power failure : Why small sample size undermines the reliability of neuroscience[END_REF] but are also prone to stochastic variation so that the probability of a positive result is inflated despite no biological difference (type II error, [START_REF] Button | Power failure : Why small sample size undermines the reliability of neuroscience[END_REF].

Thirdly, fields of research such as language evolution in animal communication are quite new, with several teams working simultaneously on similar questions. This increases the risk of more spectacular positive results being published in priority, as each team aims at showing their most influential work [START_REF] Ioannidis | Why Most Published Research Findings Are False[END_REF]. Replicating behavioural studies should consequently be of great interest, with however one caveat: while the original author may have been prone to p-hacking, the replicating author may in opposition (probably unwillingly) possess a "null hacking bias" (i.e., the motivated pursuit of null results by replicating investigators, [START_REF] Bryan | Replicator degrees of freedom allow publication of misleading failures to replicate[END_REF]. As a result, replication studies are often as questionable as the study they wish to replicate (Schmidt & Oh, 2016). To circumvent this problem, one would need two researchers having the exact same question, at the same time, testing the same population, without being influenced by each other. Such a situation occurred in our laboratory: two independent researchers, one leaving and one arriving in the laboratory, had by chance the same idea, and communicated too late about it. This led to two datasets answering the same question, obtained with quite similar yet not exactly equal protocols. Since the disparities between protocols are relatively low and all justified by authors, this article is therefore one great opportunity to compare how slight changes of protocol between two researchers can affect, or not, the resulting biological conclusions.

The biological question at stake concerns the currently hotly debated question of compositional syntax in birds. Compositional syntax is defined as when the meaning of a sequence is related to its different parts and in the way they are combined (Suzuki et al., 2019;[START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. Recent studies have proposed that some species, when mobbing a predator (i.e., actively harass it instead of flying away, [START_REF] Carlson | Mobbing[END_REF], use a combinatorial call in a fixed order: the first part (hereafter called FME: Frequency Modulated Elements, [START_REF] Dutour | Mobbing calls : A signal transcending species boundaries[END_REF] elicits vigilance, while the second part (called the D notes, following [START_REF] Hailman | The 'chick-a-dee' calls of Parus atricapillus : A recombinant system of animal communication compared with written English[END_REF] elicits approach from the receiver [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF]Suzuki et al., 2016, Figure 1). Combined, the resulting sequence engender behaviours such as scanning, approaching and calling in receivers, typical behaviours linked to mobbing [START_REF] Carlson | Sparrowhawk movement, calling, and presence of dead conspecifics differentially impact blue tit (Cyanistes caeruleus) vocal and behavioral mobbing responses[END_REF]Salis et al., 2020;Suzuki et al., 2016). Furthermore, the reversed order (i.e., D notes then FME) results in lower responses from the birds [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF]Suzuki et al., 2016). Debates on whether such coding strategy can be designated as compositional syntax in the human linguistics sense have been profuse (Bolhuis et al., 2018a(Bolhuis et al., , 2018b;;[START_REF] Griesser | From bird calls to human language : Exploring the evolutionary drivers of compositional syntax[END_REF]. The reasonable response to such critics is that this young subject deserves more studies on the same species to conclude on such potentially high cognitive abilities in birds. One way to dig into that question can be to test whether a species known to use a FME-D combinatoriality also respond to mobbing calls of an allopatric species exhibiting a similar ordering sequence in mobbing call but made up of acoustically different notes. Two adequate species for such an experiment are the great tit (Parus major), living in Europe and for which the use of compositional syntax have already been investigated [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF], and the North American black-capped chickadee (Parus atricapillus), for which the mobbing calls are also made up of a FME-D notes combination with fixed syntactic rules (although the idea that FME notes are related to vigilance behaviour and D notes to approach has not been tested yet in the black-capped chickadee, [START_REF] Baker | Mobbing calls of black-capped chickadees: effects of urgency on call production[END_REF]Otter, 2007, Figure 1). Great tits respond to the mobbing calls of the blackcapped chickadee in the same way they do for conspecific calls: they mob to the complete sequence, are vigilant when hearing FME notes, and approach to D notes [START_REF] Randler | A possible phylogenetically conserved urgency response of great tits (Parus major) towards allopatric mobbing calls[END_REF], Salis et al. 2020). One could therefore expect great tits to respond with mobbing behaviour when presented to an unknown call sequence when it has the same composition, while failing to do so when the ordering of the call sequence is reversed, as they did for conspecific calls [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF]. Such questions were addressed by two studies in a few months of interval [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] and the present study). These two experiments are one great opportunity to test whether replication without prior knowledge of the previous studies reaches the same conclusion.

In this article, we will therefore not discuss the importance of the resulting biological conclusions regarding compositional syntax in birds, since Dutour and her colleagues (2020) already did so. We will focus on whether slight differences in protocol choices engendered contrasting results, discuss which parameters may be of importance in such potential disparities and conclude on how this affect our field of research.

Methods

General organisation

Our experiment is aimed at answering two specific questions: (i) do great tits respond to allopatric mobbing sequences never eared before in the same way they do for conspecific calls (question 1, hereafter designated as the "species comparison"), and (ii) whether they would do so for allopatric calls for which order is reversed (i.e., D-FME, question 2, hereafter designated as "order comparison").

To do so, a in a field study we presented great tits with mobbing recordings of great tits, natural calls of black-capped chickadees, reversed calls of black-capped chickadees, or background noise (control). We measured their vigilance with the number of scans they produced, and whether they approached the loudspeaker. If they respond to allopatric mobbing sequences, they should scan and approach as much as when hearing conspecific calls. Secondly, if order is important in the decoding process, they should not respond anymore when the allopatric mobbing sequence is reversed (less scanning and less approaching).

We here describe our protocol and for each point, describe the similarities or difference with [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. Our protocols are similar on most points but, while [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] created two separate experiments with somewhat different protocols (see Table 1), all our different treatments were tested in the same global experiment. We will consequently separate the two questions (species comparisons and order comparison) only in the statistical analysis and results section. For clarity sake, Table 1 summarize the common ground and differences with [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF].

Preparation of soundtracks

In both our experiment and in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], 4 different types of soundtracks were built: first, soundtracks with the complete (FME-D) mobbing call sequence of the great tit (GT) or black-capped chickadee (BC), to check whether great tits responded in a similar way to allopatric calls and conspecific ones. Secondly, artificially reversed black-capped chickadees sequences (D-FME) to test the importance of order. At last, we both used a control, background noise (BN).

Our soundtracks of great tits and black-capped chickadees were built using recordings obtained from the Xeno-canto online database (www.xeno-canto.org) and the Macaulay Library (www.macaulaylibrary.org). [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] used the same websites (recordings previously used in other studies) in addition to three recordings of their own of great tits. For both species, we only conserved good quality recording files (A or B grades) under the denomination "Alarm call"/"Call". Then, to ensure that the selected recordings truly represented mobbing call sequences, several features were controlled: first, in both studied species a mobbing call correspond to an association of FME and D notes (Fig. 1). Hence, selected recordings were all made of the same FME and D notes reported in [START_REF] Baker | Mobbing calls of black-capped chickadees: effects of urgency on call production[END_REF] and [START_REF] Templeton | Allometry of Alarm Calls : Black-Capped Chickadees Encode Information About Predator Size[END_REF] for the black-capped chickadee and in Randler (2012) and [START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF] for the great tit. In addition, for both species, the D notes length is known to vary with the context [START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF][START_REF] Templeton | Allometry of Alarm Calls : Black-Capped Chickadees Encode Information About Predator Size[END_REF]; we therefore checked that our soundtracks had the same length as the D notes used in mobbing calls (X = 0.05 s ± 0.01 for the great tit, X = 0,18 s ± 0,02 for the black-capped chickadee, mean ± standard deviation). Finally, for the great tit, we verified that no FME used in food or flight related contexts were the most preponderant in any of our recordings (i.e., G, H, I and M notes associated with food for the great tit, Kalb et al., 2019a).

From these recording files, we built 40 soundtracks of 1 min mobbing sequences of great tits and black-capped chickadees (20 for each species, each provided from a different emitter) using Avisoft-SASLab software (files were converted into a Wav format). To allow comparison between black-capped chickadees and great tits responses, we constructed every mobbing sequence with a similar duty cycle [START_REF] Landsborough | Variation in chick-adee call sequences, not in the fine structure of chick-a-dee calls, influences mobbing behaviour in mixed-species flocks[END_REF] and mobbing calls repetition (30 calls/minute, natural range of repetition rate, Suzuki et al., 2016).

Consequently, each mobbing call emitted by both species had a total similar FME duration (0.31 ± 0.06 sec/call, mean ± SD) and D duration (0.50 ± 0.07 sec/call), but not the same number of notes, since BC D notes are longer (Figure 1). [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] created the same number of playbacks but chose to control for the number of D notes per call (8 D notes/call) and global duty cycle instead of the number of calls/min. Therefore, the number of calls per playback was lower in the black-capped chickadees playbacks (14 calls/min) compared to the playbacks of great tits (26 calls/min). In both our experiment and [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], reversed playbacks were constructed by putting the FME notes after the D notes.

We made sure that the space between the FME and D notes was the same before and after the manipulation. The reversed playbacks therefore possessed the exact same duty cycle and rhythm that the natural order playbacks. We also constructed 20 background noise soundtracks extracted from the original recordings (control soundtrack hereafter referred as BN). Each of these 80 soundtracks were cleared of any other bird calls, background noise was reduced, and amplitude homogenized.

Field tests

In our experiment, data were collected in the east of France at during the breeding season (March/April 2019), in a radius of 25 km around Lyon. Data of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] were collected in the same territory, also during the reproductive season, but the year before (February/March for question 1, May for question 2). For each type of soundtrack, 20 fully independent tests were performed (each bird tested was tested only once, and each bird received a different playback).

In both [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] and our experiment, each test was performed by two field assistants. One of them was assigned to the soundtrack operation, while the other was kept unaware of the selected soundtrack (using headphones with music) and assigned to the behavioural recording of the focal bird. For each test, after detecting an individual using binoculars, the focal bird was observed for at least 1 min, and the pre-test behaviour (singing or foraging) was noted. If the animal was displaying an alarm behaviour, no test was performed. A loudspeaker was placed 16 m from the bird (16.79 ± 6.27 m), and at less than 3 m to a potential roost (bushes/trees) to allow the approach of the focal bird. The two field assistants were then placed in retreat (minimum of 15 m to both the bird and the loudspeaker) before launching the soundtrack with a remote control. All soundtracks were broadcast using a Shopinnov 20 W loudspeaker with an intensity of 79.8 ± 1.9 dB(C) (measured at 1 m from the loudspeaker using Lutron SL-4001, C weighting, slow settings, re: 20 µPa). The field procedure for question 1 of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] and in our experiment were both based on a complete randomized design and very similar, excepted for three details: 1/ the main observer in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] was aware of the playback launched, as she did not wear any sound protection, 2/ the loudspeaker was placed at ~ 30 m from the bird in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], and 3/ the amplitude of the sound was of 83 dB in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. In experiment 2 of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], the tests were carried out at the nest, the loudspeaker was placed at 20 m, and birds were tested several times using a crossover design.

Tests were carried out between 06:00 and 13:00 h during calm and dry weather days. Each of the 4 soundtracks were tested each day in a different order to avoid any temporal effect. To avoid pseudoreplication, each selected focal bird was separated from each other by at least 100 m [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF]. Although birds were not individually ringed, great tits are known to be strongly territorial during the breeding period [START_REF] Krebs | Territory and Breeding Density in the Great Tit, Parus major L[END_REF][START_REF] Wilkin | Density effects on life-history traits in a wild population of the great tit Parus major : Analyses of long-term data with GIS techniques: Great tit breeding density[END_REF]) so that spacing between neighbouring individuals is often used to ensure sampling of different specimens in field tests. As several other studies (e.g. [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF], we used a distance that roughly correspond to the highest average distance expected according to territorial sizes reported in this species (c.a. 1.5 ha, [START_REF] Wilkin | Density effects on life-history traits in a wild population of the great tit Parus major : Analyses of long-term data with GIS techniques: Great tit breeding density[END_REF]. Moreover, in the present study, two or three singing birds were often concurrently detected within 100 m, suggesting territorial size to be substantially inferior to 1.5 ha in the study area. As all sampled birds were at least distant by 100 m from each other, we are thus well confident that the risk of testing the same individual twice remained quite low.

Behavioural observations

In both our experiment and [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], during 1 min of playback, two types of behavioural states were assessed, respectively (1) Vigilance effort as indicated by the number of horizontal scans displayed (the number of movements that birds made with their heads from left to right or right to left, approximately a 180° turn [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF]Suzuki et al., 2016); (2) Approach inferred using a dichotomic variable (approaching at least halfway from their starting point) measured with a Leica DISTO D210 telemeter. In the field, we reported for each test the distance of the bird from the loudspeaker at the beginning of the test, and the closest distance of the bird during the test. We then divided the closest distance by the distance at the beginning and the bird was considered as approaching when the ratio was < 0.5. This way of defining approach allowed us to take into account the initial distance of the bird (even if we tried to be at 15 m from the bird, we sometimes were at 13 m or 17 m). All observations were done using binoculars and recorded on a voice recorder (Sony ICD-PX370) by the same pair of observers in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] while two trained pairs of observers ensured the field observations in the present study. We limited birds' disturbance with two decisions: tests were of short duration, and birds were tested only once. Moreover, after our tests, we checked that all birds returned to their pre-test behaviour in less than 5min.

Statistical Analysis

We followed the same methodology as [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] to analyse our results. We therefore split our tests into two questions: first, we compared the response of great tits to natural conspecific and natural allopatric calls (species comparison). Then, we compared responses to the control (Background noise), the natural allopatric call, and the reversed allopatric call (order comparison). We used GLMM (glmer, package lme4) for both the scanning and approach behaviour, with the original soundtrack as a random effect. Posthoc comparisons were achieved with functions emmeans and multcomp::cld (packages emmeans and multcomp) with a Tukey adjustment for multiple comparisons. The number of scan produced was analysed with a Poisson distribution and log link function, since no overdispersion was detected (checked with glmm.overdisp, package RVAideMemoire). Note that Dutour et al. 2020 used a quasi-Poisson distribution because of overdispersion of their data; and that the analysis of experiment 2 took into account the identity of the bird tested, as they were tested multiple times. We also corrected the analyses for the actual observation time using the time the bird was actually seen as an offset. For the approach behaviour, we set a logistic regression (binomial distribution and logit link function). All fixed effects introduced in the models were tested using Wald tests (Anova, package car).

Since the raw data available in the supplementary material of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] is incomplete regarding the cross over design used for the second experiment, it was not possible to embed both our dataset and the one of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] in a same analysis in order to compare both studies. Nevertheless, the available information published in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] was sufficient to calculate the effect sizes of each relevant comparison, and we therefore used these metrics to compare our results from those of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. We computed odds ratio (hereafter OR, odds.ratio, package questionr) for the approach behaviour, and Cliff's d for the scanning behaviour as this variable does not follow a normal distribution (cliff.delta, package effsize). One should nevertheless note that the computed effect size does not take into account the non-independence of the observations done in the second experiment of Dutour et al. (2020, i.e., the cross-over design where different acoustic tests were performed on the same subjects).

Results

Question 1: Response to natural mobbing calls from a conspecific or an allopatric species

In our experiment, great tits scanned an average of 7.30 ± 3.16 scans (mean ± standard deviation) when presented with conspecific calls, and 6.80 ± 3.12 scans when presented with black-capped chickadees calls (Figure 2). No statistical difference was detected in our model (X² = 0.93, df = 1, p = 0.33), and the calculated effect size of the difference was 0.18 (Cliff's d, 95% CI [0.42; 5.24]). In [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], great tits produced 10 ± 5.33 scans in response to conspecific calls and 9.05 ± 5.62 scans in response to black-capped chickadees calls. The resulting effect size is 0.12 (Cliff's d, 95% CI [ -0.22; 0.44]), hence very similar to the one we detected (Figure 3). 60% of the great tits tested (n = 20 for each treatment) approached the loudspeaker when hearing conspecific calls, but only 30% when hearing black-capped chickadees calls (Figure 2), and the difference between both treatments approached statistical significance (n = 40, X² = 3.51, df = 1, p = 0.06), with an odds ratio of 3.5 (95% CI [0.94;12.97]). This difference was stronger than in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], who found an odds ratio of 1.5 (95% CI [0.42; 5.24]) between the two treatments. Nonetheless, the confidence intervals of the effect sizes being large [START_REF] Cumming | Error bars in experimental biology[END_REF], the difference between our two studies cannot be considered as statistically significant (Figure 3).

Question 2: Response to reversed allopatric calls

Great tits scanned differently background noise, natural allopatric calls, and reversed allopatric calls (n = 60, X² = 56.04, df = 2, p < 0.001, Figure 2). Indeed, they scanned less to the background noise than to either of the two allopatric soundtracks (BN vs BC Natural: z = 7.41, p < 0.001; BN vs BC Reversed: z = 6.59, p < 0.001, Figure 2). They produced on average 6.8 ± 3.12 scans toward the natural calls, and 5.9 ± 3.54 scans toward the reversed, leading to an effect size of 0.16 (95% CI [-0.20; 0.48]), which is a non-statistically significant difference as indicated by post-hoc tests (z = 1.15, p = 0.48). Birds only produced 1.55 ± 2.06 scans when hearing control tests. In contrast, in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], great tits scanned on average 14.3 ± 6.80 scans to the natural calls, 11 ± 6.55 scans to the reversed calls, and 8.85 ± 6.33 the control tests, leading to a substantial difference between natural and reversed calls (0.27, 95% CI [0.07; 1.52]) and no significant difference between reversed calls and background noise (0.21, 95% CI [-0.15; 0.52]). Nonetheless, the effect sizes associated to these differences remain comparable between the two studies (Figure 3). Only 5% of great tits approached the loudspeaker when hearing background noise, but 30% when hearing naturally ordered allopatric calls and 10% when hearing reversed allopatric calls (Figure 2). Even though the odds ratio of the difference between our treatments was superior to 1 (Figure 3), it was not statistically significant (X² = 4.29, df = 2, p = 0.12). Our effect sizes parallel the ones from [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] who also did not detect statistically significant difference between natural and reversed calls (Figure 3). Nonetheless, the percentage of approach in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] were overall higher, with 55% of birds approaching in response to natural allopatric calls, 35% for the reversed calls, and 15% for BN.

Discussion

Two researchers with the same idea, very similar protocol and statistical analysis have obtained similar effect sizes for the differences of interest, nonetheless differed about the great tit's ability to respond to allopatric calls when considering results based on the p-value. Indeed, we detected a lower response to black-capped chickadees calls compared to conspecific ones, while the responses to both calls were similar in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. We detected no difference between responses to natural and reversed allopatric calls while [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] detected one for scanning. While the difference between great tit and black-capped chickadee natural calls can easily be explained by a subtle protocol choice; the difference regarding the second question (i.e., effect of reversion on great tits' response) could be explained both by a protocol choice and/or by the p-value fluctuating especially with low sample sizes (n = 20 for each treatment in both studies). These two disparities are therefore of different kind and will be discussed below.

Allopatric versus conspecific mobbing calls

In our experiment, great tits approached less to allopatric calls than to conspecific ones, a result different from [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] who did not detect any difference. A lower response from GT to BC has previously been detected in [START_REF] Randler | A possible phylogenetically conserved urgency response of great tits (Parus major) towards allopatric mobbing calls[END_REF], while a similar level of response was found in [START_REF] Dutour | Mobbing calls : A signal transcending species boundaries[END_REF]. One could hypothesize that such difference is explained by the distance of the loudspeaker from the focal bird (30 m for [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] versus 16 m for us). Indeed, amplitude of the sound is probably a proxy for urgency in birds [START_REF] Hingee | Flights of fear : A mechanical wing whistle sounds the alarm in a flocking bird[END_REF] and calls uttered at larger distance could consequently engender lower approach. In addition, increased distance implies both the attenuation of the sound (lower sound to noise ratio) and the degradation of some sound characteristics (e.g., high frequencies are degraded more easily, [START_REF] Kroodsma | Acoustic communication in birds[END_REF].

Sound attenuation and degradation have been repeatedly shown to modify birds' response, especially in studies investigating anthropogenic noise [START_REF] Jung | Traffic noise and responses to a simulated approaching avian predator in mixed-species flocks of chickadees, titmice, and nuthatches[END_REF]Shannon et al., 2016). In our situation, the differences between the mobbing calls of the allopatric blackcapped chickadee and the sympatric marsh tit (who possess a similar mobbing call) could therefore be less salient at longer distances. However, two points should be raised: firstly, sound attenuation and degradation of a sound at 30 m [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] versus 15 m (our experiment) in a semi-open environment is probably extremely similar. Secondly, the discriminative skills of Parids are known to be particularly precise. For example, blackcapped chickadees and mountain chickadees (Poecile gambeli) can distinguish each other calls' based on features of their D notes [START_REF] Bloomfield | All "chick-a-dee" calls are not created equally Part II. Mechanisms for discrimination by sympatric and allopatric chickadees[END_REF].

We rather suggest that such difference may lie in the soundtrack preparation, and particularly in the number of D notes. Indeed, D notes possess a general recruitment function in some species of Parids [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF]Suzuki et al., 2016) and the number of D notes per call is thought to code for urgency in Parids [START_REF] Kalb | Subtle variations in mobbing calls are predator-specific in great tits (Parus major)[END_REF][START_REF] Templeton | Allometry of Alarm Calls : Black-Capped Chickadees Encode Information About Predator Size[END_REF].

One recent study demonstrated that the increase of D notes may not be as important as the resulting increase of duty cycle (i.e., the amount of time a signal is present over a specified time, [START_REF] Landsborough | Variation in chick-adee call sequences, not in the fine structure of chick-a-dee calls, influences mobbing behaviour in mixed-species flocks[END_REF]. In our case, since black-capped chickadees' notes are longer than great tits' notes, each researcher chose to either control for the duty cycle of each type of note per call or for the number of D notes per call. [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] chose to control the number of D notes, with 8 notes per call, while we chose to control the duty cycle resulting in only 2 or 3 D notes per call (Figure 1). Future experiment disentangling the effect of D note number and duty cycle with a crossed design may be of interest. Importantly however, even if the response to BC calls was lower, the effect sizes of the differences between natural and reversed order in our second question were similar in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] and our own experiment: different choices in protocol did not hamper subsequent differences of interest.

Difference in scan number

Our second disparity lies in the difference in scanning behaviour for the second question. The absolute number of scans was extremely different, with rarely more than 10 scans counted in our study, while most observations from Dutour et al. counted more than 10 scans. In addition, the difference between control and reversed BC playbacks was strong in our study, but not significant in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. The scanning variable could be criticized: counting 180° head turn in real time may be difficult and is probably impacted by the observer's personal definition of scanning. However, the two observers in our study only varied in their scan number for 1 scan on average. Such a result is in accordance with [START_REF] Dutour | Syntax manipulation changes perception of mobbing call sequences across passerine species[END_REF] who tested the differences in scan count between two experienced ornithologists and detected a high concordance between observers. The difference in absolute scores between our two studies may consequently rather be explained by the context in which the birds were tested. Indeed, while we tested free ranging birds while foraging, Dutour et al. tested birds when arriving at their nest box. In particular, birds are probably more vigilant (hence increasing the number of scan) in the vicinity of their nest, and the perceived risk associated to conspecific and allopatric calls could also differ according to the distance of the caller from the nest. This subtle variation of context between both studies could thus well explain both the stronger difference between BN and Reversed playback in our study compared to [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], and the overall disparities of the absolutes scores between the two studies. A question that remains is whether, in addition to difference in absolute scores of scanning, such difference in context may also affect the differences between treatments. Since the effect sizes of the differences between treatments were similar between our experiment and the one from [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF], we think that the context overall increased the scanning behaviour but did not affect the differences between treatments.

Similar effect sizes, but dissimilar p-values

Obtaining similar effect sizes of the difference between natural and reversed calls indicates two important things. Firstly, this indicates that even if [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] were not fully blinded when doing their playback tests, they were not affected by an expectancy effect (i.e., unknowingly distorting the observations to make them fit with your hypothesis, [START_REF] Holman | Evidence of Experimental Bias in the Life Sciences : Why We Need Blind Data Recording[END_REF]Rosenthal & Fode, 1963). Secondly, obtaining similar effect sizes but dissimilar p-values between the two studies indicates a discrepancy between effect sizes and analyses based on p-values. The use of p-value is increasingly criticized [START_REF] Anderson | Null Hypothesis Testing : Problems, Prevalence, and an Alternative[END_REF]. Indeed, p-values are known to flicker even with great sample sizes [START_REF] Halsey | The fickle P value generates irreproducible results[END_REF]. In our case, natural variability combined with the difference of experimental design between both studies (i.e., completely random versus partly cross over design) could well have contributed to this phenomenon. Indeed, the slightly lower p-value reported by [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] may have arisen from a higher statistical power of the cross over design permitted by the subtraction of the predicted individual variability from the residual variance (i.e., through the inclusion of a random individual effect). Unfortunately, the estimate of the subject effect was not reported in [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF] precluding the possibility to examine this point more formally. This emphasizes the need to rely more on effect sizes and on the biological relevance of them [START_REF] Nakagawa | Effect size, confidence interval and statistical significance : A practical guide for biologists[END_REF], especially since they seem more stable with low sample sizes [START_REF] Halsey | The fickle P value generates irreproducible results[END_REF]. Clustering several tiny, repeated experiments may be another solution to control natural and protocol variability [START_REF] Von Kortzfleisch | Improving reproducibility in animal research by splitting the study population into several 'mini-experiments[END_REF]. More generally, the various sources of variability between the two experiments (protocol choices and natural between-year variability) show how much replicated studies and meta-analysis approaches are needed.

Conclusion

In conclusion, we found that the context in which the birds are tested (here, different distances from the nest) as much as the playback preparation can modify the behavioural cues assessed in language related studies. These different protocol choices seem to have mainly affected the absolute scores rather than the differences between treatments, as we found similar effect sizes between the two experiments. However, relying only on the p-value would here have led to different biological conclusions regarding complex syntax use in great tits. We believe this work provides a clear demonstration that the confrontation between two similar experiments is not a matter of who did wrong and who did right, but rather that both experiments grabbed one aspect of reality, at one precise moment. In our field of research, the flexibility present in behavioural measures and the limited sample sizes are probably the major explanations for disparities between similar experiments. Repeated experiments are therefore one great opportunity to understand variability in natural experiments and to approach, at best, biological reality.

TABLES

Table 1. Protocol comparison between the experiment of M. Dutour & et al. and A. Salis et al. Experiments consisted in recording behaviour of birds when hearing specific soundtracks.

We listed the factors that could potentially influence different results in the two studies. Bold text emphasizes the differences between protocols. CRD = Completely randomized design, GLM = generalized linear model. turn of the head to explore the environment) made by great tits during the one minute test.

For both figures, the 95% confidence intervals are given. Q1 represents the comparison of interest for the first question (emitter species comparison), comparable to the experiment 1 of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. Q2 represents the comparison of interest for the second question (order comparison), comparable to the experiment 2 of [START_REF] Dutour | Great tit responses to the calls of an unfamiliar species suggest conserved perception of call ordering[END_REF]. Statistical inference can be made using the overlap of such CI: if they overlap at less than halfway, the difference can be considered as statistically significant for an alpha = 5% [START_REF] Cumming | Error bars in experimental biology[END_REF]. BN = Background noise, GT = great tit, BC = black-capped chickadee. the comparisons for the approach variable, with odds ratio. For each effect size given, the associated 95% confidence intervals are given. For both (a) and (b), left part concerns question 1 (species comparison: GT-Natural vs BC-Natural) and right part concerns question 2 (order comparison: BC-Natural vs BC-Reversed). Statistical inference can be made using the overlap of such CI: if they overlap at less than halfway, the difference can be considered as statistically significant for an alpha = 5% [START_REF] Cumming | Error bars in experimental biology[END_REF]. Associated p-value found in respective models are indicated below each comparison. A table summarizing the results and effect sizes of the two studies can be found in Sup.Mat.

FIGURESFigure 1 .

 1 FIGURES

Figure 2 .

 2 Figure 2. Results of our experiment, with (a) the proportion of individuals that approached

Figure 3 .

 3 Figure 3. Comparison of effect sizes between our own experiment (white dots) and results

Acknowledgments

We thank Mylène Dutour, Toshitaka Suzuki and David Wheatcroft for their transparency, allowing us this comparison study. Collaborations with M. Dutour on other projects are currently being reviewed; but the current study was done independently from her work. We thank the Fondation Vérots for access on their property. Finally, we thank Charlotte Bourbon, Jean Capelle and Julie Ruffion for useful help in the field.

The authors comply with the ASAB guidelines for the use of animals in research. The fieldwork did not require any special permit but followed the laws of the Rhône county and the rules of the ethics committee of the University Lyon 1.

SUPPLEMENTARY MATERIAL

Sup.Mat 1. Comparisons of p-value and effect sizes of both studies (Salis et al. or Dutour et al.) regarding the differences between treatments (Experiment 1: between species comparison, Experiment 2: order comparison). Effect sizes from the scanning behaviour are Cliff's d given with their 95% confidence intervals (CI). If such CI encompass 0, the difference can be considered as non-statistically significant. Effect sizes from the approach behaviour are Odds Ratio given with their 95% CI. If such CI encompass 1, the difference can be considered as non-statistically significant.