Gwenaël Abril
email: gwenael.abril@mnhn.fr

Bruno G Libardoni

Nilva Brandini

Luiz C Cotovicz

Paulo R P Medeiros

Geórgenes H Cavalcante

Bastiaan A Knoppers

Thermodynamic uptake of atmospheric CO2 in the oligotrophic and semi-arid São Francisco estuary (NE Brazil)

Keywords: Carbonate equilibria, estuarine mixing, tidal pumping, heterotrophy, heating, gas exchange

Estuarine carbonate chemistry predicts that thermodynamic equilibration during the mixing of freshwater with seawater will generate a carbon dioxide (CO2) sink for the case of warm and poorly buffered tropical rivers. The São Francisco River estuary has historically become oligotrophic, after the construction on its watershed of a series of hydroelectric dams, where organic matter and nutrients are retained. During two cruises in late winter (Aug. 2014) and early summer (Nov. 2015), dissolved inorganic carbon (DIC) and total alkalinity (TA) increased linearly with salinity in the main estuarine channel, where the mixing time of water was half a day, and showed a close to conservative behaviour. In the main channel, water partial pressure of CO2 (pCO2) recorded at 1min frequency, followed an asymmetric bell-shaped trend versus salinity, close to the curve predicted by thermodynamic conservative mixing of freshwater DIC and TA with seawater DIC and TA. The low (0-3) salinity region was always a source of atmospheric CO2, where, despite low chlorophyll concentrations, a pCO2 diurnal change of about 60 ppmv suggested the occurrence of photosynthesis in summer . At salinities above 3, under-saturated pCO2 values (down to 225 ppmv in winter and neap tides) and invasion of atmospheric CO2 of 0.38-1.70 mmol m -1 h -1 occurred because of predominating thermodynamics during estuarine mixing. In winter and neap tides, higher river discharge, intense estuarine mixing, lower temperatures and limited tidal pumping resulted in observed pCO2 different from theoretical conservative pCO2 by less than 3ppmv at salinities >3. Conversely, in summer and spring tides, recorded pCO2 values were on average +43±35 ppmv above the conservative mixing curve, when tidal pumping, CO2 invasion and surface heating were more significant in the mixing zone, but not sufficient to offset the thermodynamic uptake of atmospheric CO2. By combining carbonate chemistry with estuarine mixing modelling and gas exchange calculations, we estimate that heating contributed to about 15% and gas exchange to about 10% of the positive pCO2 deviation from conservative mixing during summer.

The remaining 75% of the deviation was maximum at ebb tides and within a salinity ranges consistent with the occurrence of tidal pumping from marches and mangrove soils. Indeed, in the mangrove channel, water were supersaturated with pCO2 values of 976±314ppmv, while in the main channel, highest positive pCO2 deviations from conservative mixing (up to +100ppmv during several hours) occurred at ebb tides. An important finding was that, in the São Francisco, the thermodynamic and biological processes compete with each other to CO2 fluxes both at low salinities where evasion and autotrophy occur, and at high salinities where invasion, heterotrophy and tidal pumping occur. Our study suggests that carbonate thermodynamics during mixing is a key process that has been overlooked in estuarine studies, although it can generate important airwater CO2 exchange and contribute significantly to the carbon budget of estuaries and river plumes.

Introduction

The necessity of reducing uncertainties on global estimates of CO2 sources and sinks on Earth has promoted research on the carbon cycle in estuarine and coastal ecosystems particularly during the last three decades [START_REF] Wollast | The coastal organic carbon cycle: fluxes, sources, and sinks[END_REF][START_REF] Smith | Coastal metabolism and the oceanic carbon balance[END_REF][START_REF] Frankignoulle | Carbon dioxide emission from European estuaries[END_REF][START_REF] Cai | Estuarine and coastal ocean carbon paradox: CO2 sinks or sites of terrestrial carbon incineration?[END_REF][START_REF] Borges | Carbon dioxide and methane dynamics in estuaries[END_REF][START_REF] Bauer | The changing carbon cycle of the coastal ocean[END_REF][START_REF] Dinauer | Spatial variability in surface-water pCO2 and gas exchange in the world's largest semi-enclosed estuarine system: St. Lawrence Estuary (Canada)[END_REF]Mucci, 2017, Maher et al., 2018).

Despite their small surface areas, estuaries have been identified as a hotspot of CO2 emissions, able to offset a large part of the CO2 sink on the continental shelves [START_REF] Chen | Reconciling opposing views on carbon cycling in the coastal ocean: continental shelves as sinks and near-shore ecosystems as sources of atmospheric CO2[END_REF].

This conclusion was based on field observations in temperate and boreal regions, where estuaries have been recognized as CO2 sources and as heterotrophic ecosystems, that is, with a negative net ecosystem production (NEP) [START_REF] Smith | Coastal metabolism and the oceanic carbon balance[END_REF][START_REF] Borges | Carbon dioxide and methane dynamics in estuaries[END_REF]. Upper estuarine regions with generally well-mixed channels are almost always net heterotrophic, supersaturated in CO2 and a strong source of CO2 to the atmosphere [START_REF] Frankignoulle | Carbon dioxide emission from European estuaries[END_REF][START_REF] Borges | Carbon dioxide in European Coastal waters[END_REF][START_REF] Sarma | Carbon dioxide emissions from Indian monsoonal estuaries[END_REF]. In contrast, lower estuarine regions with high salinities, including large surfaces of river plumes at sea and coastal embayments, are sites of phytoplankton blooms, and may have a net autotrophic metabolism (positive NEP) at least in the surface layer, and behave as CO2 sinks, at minima seasonally [START_REF] Körtzinger | A significant CO2 sink in the tropical Atlantic Ocean associated with the Amazon river plume[END_REF][START_REF] Borges | Carbon dioxide in European Coastal waters[END_REF][START_REF] Cai | Carbon dioxide dynamics and fluxes in coastal waters influenced by river plumes[END_REF][START_REF] Cotovicz | A strong CO2 sink enhanced by eutrophication in a tropical coastal embayment (Guanabara Bay, Rio de Janeiro, Brazil)[END_REF][START_REF] Lefèvre | Net Heterotrophy in the Amazon Continental Shelf Changes Rapidly to a Sink of CO2 in the Outer Amazon Plume[END_REF].

The global CO2 emissions from estuaries suffer from large uncertainties due to spatial and temporal heterogeneity [START_REF] Chen | Reconciling opposing views on carbon cycling in the coastal ocean: continental shelves as sinks and near-shore ecosystems as sources of atmospheric CO2[END_REF]. In most studies, the relationship between NEP and CO2 fluxes could not be established quantitatively because many processes other than community respiration and primary production can alter the partial pressure of CO2 (pCO2) and the direction and intensity of the air-sea CO2 flux in estuaries [START_REF] Borges | Do we have enough pieces of the jigsaw to integrate CO2 fluxes in the Coastal Ocean?[END_REF]. For instance, in eutrophic estuaries, nitrification can titrate TA to CO2, contrarily to carbonate dissolution that can consume CO2 and generate TA [START_REF] Frankignoulle | Atmospheric CO2 fluxes in a highly polluted estuary (The Scheldt)[END_REF][START_REF] Abril | Nitrogen-Alkalinity interactions in the highly polluted Scheldt basin (Belgium)[END_REF]Abril et al., 2003;[START_REF] Cotovicz | Greenhouse gas emissions (CO2 and CH4) and inorganic carbon behavior in an urban highly polluted tropical coastal lagoon (SE, Brazil)[END_REF]. Some dissolved CO2 transported laterally from freshwaters or tidal marshes and mangroves is ventilated in river-dominated and/or macrotidal estuaries [START_REF] Abril | Excess atmospheric carbon dioxide transported by rivers into the Scheldt Estuary[END_REF][START_REF] Neubauer | Transport of dissolved inorganic carbon from a tidal freshwater marsh to the York River estuary[END_REF][START_REF] Borges | Atmospheric CO2 flux from mangrove surrounding waters[END_REF]2006;[START_REF] Cai | Estuarine and coastal ocean carbon paradox: CO2 sinks or sites of terrestrial carbon incineration?[END_REF]. In general, advection of surface or sub-surface waters enriched in DIC, and/or biodegradable organic carbon (OC), as well as the presence of vegetation at the land-water interface, complicates the calculation of NEP and its relationship with CO2 fluxes [START_REF] Borges | Do we have enough pieces of the jigsaw to integrate CO2 fluxes in the Coastal Ocean?[END_REF][START_REF] Cai | Estuarine and coastal ocean carbon paradox: CO2 sinks or sites of terrestrial carbon incineration?[END_REF][START_REF] Borges | Carbon dioxide and methane dynamics in estuaries[END_REF]Santos et al., 2018;[START_REF] Maher | Beyond burial: lateral exchange is a significant atmospheric carbon sink in mangrove forests[END_REF].

In estuaries, water pCO2 is also strongly modified by the displacement of the acid-base equilibria of the carbonate system during the mixing of freshwater with seawater. As a consequence, the concentrations of bicarbonate (HCO3 -) and carbonate (CO3 2-) ions, as well as pCO2 in a mixture of two waters with different salinities and temperatures, are very different from the arithmetic mean of these parameters in the two waters. In their pioneer theoretical work, [START_REF] Whitfield | The carbon dioxide system in estuaries -An inorganic perspective[END_REF] applied newly published carbonate dissociation and gas solubility constants to calculate the theoretical distribution of pCO2 along the salinity gradient induced by the conservative mixing of DIC and TA from the freshwater and marine end-members. These authors showed that "the mixing of river water and sea water […] tends to produce significant disequilibrium between the estuary and the atmosphere which may manifest itself as a deficit or as an excess of carbon dioxide, depending on the pH and alkalinity of the river water." However, in recent years, studies rarely consider in detail carbonate thermodynamic during mixing as a potential driver of estuarine CO2 fluxes, although it can generate some atmospheric CO2 release or uptake additional to NEP (Cai et al., 2013;[START_REF] Lefèvre | Net Heterotrophy in the Amazon Continental Shelf Changes Rapidly to a Sink of CO2 in the Outer Amazon Plume[END_REF][START_REF] Delaigue | Spatial variations in CO2 fluxes in the Saguenay Fjord (Quebec, Canada) and results of a water mixing model[END_REF][START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. Most studies have focused on the correlation between biological components of NEP and CO2 fluxes [START_REF] Borges | Carbon dioxide and methane dynamics in estuaries[END_REF], and lateral C fluxes induced by tidal inundation and pumping [START_REF] Cai | Estuarine and coastal ocean carbon paradox: CO2 sinks or sites of terrestrial carbon incineration?[END_REF][START_REF] Maher | Groundwater-derived dissolved inorganic and organic carbon exports from a mangrove tidal creek: The missing mangrove carbon sink?[END_REF]2018). In the Amazon plume, a large part of the CO2 sink at salinities higher than 17 could be attributed to thermodynamics, but unfortunately no pCO2 field data are available at salinities lower than 17 [START_REF] Körtzinger | A significant CO2 sink in the tropical Atlantic Ocean associated with the Amazon river plume[END_REF][START_REF] Cai | Carbon dioxide dynamics and fluxes in coastal waters influenced by river plumes[END_REF][START_REF] Lefèvre | Net Heterotrophy in the Amazon Continental Shelf Changes Rapidly to a Sink of CO2 in the Outer Amazon Plume[END_REF]. Recently, [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF] reported a CO2 sink in the estuarine delta of the Paraiba do Sul River induced by a combination of carbonate thermodynamics, ecosystem metabolism and temperature effects.

Although it is essential in the open ocean, the role of surface water heating or cooling on CO2 fluxes is poorly documented in estuarine waters. In tropical and sub-tropical coastal systems, surface heating and evaporation can have a large impact on CO2 water-air fluxes [START_REF] Yao | Responses of carbonate system and CO2 flux to extended drought and intense flooding in a semiarid subtropical estuary[END_REF][START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF][START_REF] Yao | Hydrologic controls on CO2 chemistry and flux in subtropical lagoonal estuaries of the northwestern Gulf of Mexico[END_REF]).

In the present study, we report high resolution data of the carbonate system in a tropical, oligotrophic estuary with short mixing time, where thermodynamics alone explains a large part of the well-defined pCO2 distribution. Owing to warm temperatures and low TA at the river endmember, the São Francisco estuary presents conditions that theoretically generate CO2 invasion during estuarine mixing, even when supersaturation prevails in the river, a case similar to large tropical rivers such as the Amazon and the Congo [START_REF] Cai | Carbon dioxide dynamics and fluxes in coastal waters influenced by river plumes[END_REF]. The oligotrophic character of the estuary and the short mixing time make chemical and physical processes predominate over biological processes. The estuary is also connected to a small mangrove forest that may export dissolved carbon to waters [START_REF] Maher | Groundwater-derived dissolved inorganic and organic carbon exports from a mangrove tidal creek: The missing mangrove carbon sink?[END_REF], and is subjected to important heating owing to the semi-arid evaporative conditions in summer.

Material and methods

Study area

The São Francisco River (Fig. 1) drains an area of 634 000 km 2 in Brazil that includes Pleistocene-Holocene plains [START_REF] Dominguez | The São Francisco strandplain: a paradigm for wave-dominated deltas?[END_REF]. Precambrian rocks of the Brazilian shield dominate the geology of the basin, and the climate varies from tropical humid in the upper and lower regions, to semiarid in the middle region [START_REF] Bernardes | Notas sobre o clima da bacia do Rio são Francisco[END_REF]. The River course has been progressively intensively modified between the 50's and the 90's by the construction of five hydroelectric reservoirs, which had strong impacts on the estuary. Decreased precipitations combined to an increased evaporation due to the presence of dams on the watershed have reduced the average annual freshwater discharge from about 2,895 m 3 s -1 to about 1,800 m 3 s -1 (after Xingó Dam) in 1995 and to less than 1000m 3 s -1 during the last decade [START_REF] Bernardes | Landsea interface features of four estuaries on the South America Atlantic coast[END_REF][START_REF] Cavalcante | Circulation and salt balance in the São Francisco river Estuary (NE/Brazil)[END_REF][START_REF] De Jong | Hydroelectric production from Brazil's São Francisco River could cease due to climate change and inter-annual variability[END_REF]. Floods that strongly influenced the estuarine morphology were suppressed, and most of the riverine suspended sediments, nitrogen, phosphorus and silica were retained in reservoirs, turning the estuary oligotrophic, with chlorophyll concentrations lower than 3 µg L -1 in more than 95% of the samples from recent years [START_REF] Medeiros | Changes in Nutrient Loads (N, P and Si) in the São Francisco Estuary after the Construction of Dams[END_REF]2018;[START_REF] Melo | Nutrients Load Estimation in a Regulated Streamflow Estuary: The São Francisco Estuary (NE/Brazil)[END_REF][START_REF] Melo-Magalhães | Microphytoplankton biomass and trophic state of the estuarine region of the São Francisco River (Northeastern Brazil) Braz[END_REF]. Today, the estuarine morphology consists in a stratified, salt-wedge type, main channel, separated from the sea by a shallow area of submerged dynamic sandy banks, and a secondary channel in the South, surrounded by mangroves that have been partly deforested for shrimp farming. Water depth in the main channel in on average 3m, with a maximum of 12m and very shallow depth at the mouth, where sandy banks separate the estuary from the shelf. Strong saline stratification occurs in the main channel and tidal movements generate large variations in surface water salinity and extension of the plume on the shelf (Cavalcantes et al. 2020). Residence time of freshwater in the inner estuary with the surface area of 19km 2 is about half a day [START_REF] Cavalcante | Circulation and salt balance in the São Francisco river Estuary (NE/Brazil)[END_REF]. About 5% of the freshwater transits through the secondary channel and ends-up in a shallow coastal lagoon parallel to the coast and connected to the sea at high tide through several narrow canals (Fig. 1).

Fieldwork, chemical analyses and calculations

Sampling occurred in late winter the 5-8 th of August 2014 and in early summer the 11-14 th of November 2015, when river discharges were, respectively, 1220 and 950m 3 s -1 , and the tidal ranges were 1.2 m (end of neap tides) and 1.8 m (spring tides). Detailed descriptions of all methods for in-situ measurements, discrete water sampling and preservation can be found in [START_REF] Cotovicz | A strong CO2 sink enhanced by eutrophication in a tropical coastal embayment (Guanabara Bay, Rio de Janeiro, Brazil)[END_REF] and [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. Field work consisted in measurement every minute of pCO2 salinity and temperature, and sampling of surface water from freshwater to coastal waters (Salinity >30) in both channels. Discrete samples were taken along the salinity gradient in the two channels at approximately two-salinity intervals, except in the salinity range 10-25, where changes were too abrupt to allow sampling at regular salinity intervals. Three end-members could be sampled (Fig. 1): the centre of the river main channel, the mouth of the main channel (salinity ~ 30), and a shallow coastal lagoon at the outlet of the secondary mangrove channel (Salinity 30-34). The 11-12 th of Nov. 2015, measurements were also performed during 25h at an anchored station in the main channel, were salinity varied from 1.2 at low tide to 32.2 at high tide.

Continuous measurements were performed during the vessel's travels or at the anchored station, by pumping surface water at 8L min -1 to a 1L vial containing the sondes and a marble-type equilibrator [START_REF] Frankignoulle | A new design of equilibrator to monitor carbon dioxide in highly dynamic and turbid environments[END_REF]. The marble equilibrator consisted in an acrylic tube filled with glass marbles where water flows from the top, and a closed air circuit with a flux of about (1 L min -1 ), flowing in the opposite direction from the bottom to the top. The air is dried with drierite and pumped to a non-dispersive infrared (NDIR) gas analyser (LICOR© LI-820). Before and after each sampling campaigns, the NDIR was calibrated using gas standards with air pCO2 values of 410, 1007 and 5035 ppmv (White Martins Certified Material, RJ, Brazil). Fresh soda lime was used to set the zero and the standard at 1007 ppmv to set the span. The air standards of 410 and 5035 ppmv were used to verify the linearity of the response. The accuracy of the pCO2 measurements is estimated at ±5 ppmv. Water was continuously overflowing from the 1L vial that hosted a YSI© Professional Plus multiparameter sonde that measured Temperature, salinity and dissolved oxygen (DO) , and a WTW© 3310 pH meter equipped with a Sentix 41 electrode. The pH meter was calibrated daily with a three-point standard (pH 4.01, pH 7.00 and pH 10.01) according to the National Institute of Standards and Technology (NIST). The precision of the pH measurements estimated after seven verifications against standards was about 0.01 NBS units. The salinity probe was calibrated before the cruise with IAPSO standard seawater and deionized water. DO was measured with a polarographic electrode calibrated every morning at 100% saturation in vaporsaturated air. Temperature values measured with the YSI© salinity probe differed from those measured with the WTW© pH-meter by less than 0.1°C. For continuous measurement performed during sailing, the ship speed was adapted according to the salinity changes and varied between Total alkalinity (TA) was titrated on the discrete samples with certified 0.1N HCl on duplicate 50 mL 0.7µm-filtered samples, using the Gran function to determine the equivalent point.

The concentration of DIC carbonate system was calculated in the discrete samples from the measured pCO2 and TA, with the CO2sys program [START_REF] Pierrot | MS Excel Program Developed for CO2 System Calculations[END_REF]) using the CO2 solubility from [START_REF] Weiss | Carbon dioxide in water and seawater: the solubility of a non-ideal gas[END_REF] and the dissociation constants of [START_REF] Mehrbach | Measurements of the apparent dissociation constants of carbonic acid in seawater at atmospheric pressure[END_REF], refitted by [START_REF] Dickson | A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media[END_REF]. One important precaution for the use of this set of constants is to check for the absence of organic alkalinity at significant concentrations compared to carbonate alkalinity, particularly in the freshwaters (Cai et al., 1998;[START_REF] Hunt | Contribution of non-carbonate anions to total alkalinity and overestimation of pCO2 in New England and New Brunswick rivers[END_REF][START_REF] Cotovicz | A strong CO2 sink enhanced by eutrophication in a tropical coastal embayment (Guanabara Bay, Rio de Janeiro, Brazil)[END_REF]. In order to identify an eventual bias induced by the interference of organic alkalinity in the calculations of DIC, we used our pH measurements concomitant to TA measurements in order to calculate pCO2 (pH/TA) in our discrete samples. The pCO2 values calculated from pH and TA and those measured directly with the equilibrator were fairly consistent (< ±10%), except in a freshwater small tributary on the left edge of the São Francisco River (North of Fig. 1 and Fig. S1) that drains a small wetland, and where the calculated pCO2 (pH/TA) was more than 4 times higher than the measured pCO2 [START_REF] Cotovicz | Comparações entre medições em tempo real da pCO2 aquática com estimativas indiretas em dois estuárioss tropicais contrastantes: o estuário eutrofizado da Baia de Guanabara (RJ) e o estuário oligotrofico do Rio São Francisco (AL)[END_REF]). However, this tributary does not contribute significantly to the river discharge, as confirmed by our conductivity monitoring at the confluence, and organic alkalinity does not appear to significantly interfere in the calculation of the carbonate system in the São Francisco main channel and mangrove channel.

In this work, we analyse and compare the distribution along the salinity gradient of four types of pCO2: the pCO2 observed in situ, noted O-pCO2, the pCO2 resulting from conservative mixing of river water with seawater, noted C-pCO2, the pCO2 normalized to temperature, noted T-pCO2, and the pCO2 resulting from conservative after being corrected for gas exchange, noted Ex-pCO2. For each campaign we established the equations of the conservative mixing lines for TA, DIC and temperature as a function of salinity, using the values at the two end-members: the reference freshwater station at the centre of the main channel upstream and the most saline sample at the mouth downstream (Fig. 1). Then the C-pCO2 thermodynamic conservative mixing curves were calculated for each salinity value where continuous measurements were performed, using the calculated conservative (linear) values of temperature, TA and DIC between the two end-members in the main channel. We evaluated the impact of water heating on pCO2 in the main channel and in the secondary channel passing through the mangrove, by normalizing the O-pCO2 observed at a given salinity at the temperature corresponding to the linear interpolation of the temperatures in the freshwater and marine end-members. To that end, we calculated for each salinity point measurement , a temperature-normalized T-pCO2 from the measured pCO2 the conservative TA mixing line, and the lowest temperatures observed at each end-member, generally at night and early morning. In winter and neap tides (Aug. 2014), two distinct temperature marine end-members should be considered: one at the mouth of the main channel and another one in the coastal lagoon at the mouth of the mangrove channel (Fig. 1). In contrast, a single set of freshwater and seawater temperature end-members could be used during summer and spring tides (Nov. 2015) when consistent cooler night-time waters were observed in both marine end-members (see results

section).

The evasion or invasion of CO2 at the water-air interface was calculated according to: FCO2 = kα∆pCO2

Eq.1

where FCO2 is the water-air CO2 flux (mol m -2 h -1 ), k is the gas transfer velocity (m h -1 ), α is the solubility coefficient (mol m -3 atm -1 ) and ∆pCO2 is the difference between water and air pCO2 (in atm). Atmospheric pCO2 was measured every morning and evening by pumping with the NDIR gas analyser and daily average values were used for the flux calculation. To calculate k, we apply three equations as a function of the concomitant wind speed at 10 meters (U10) recorded at a station located 4km landward (Fig. 1). The equations of Wanninkhof 1992 (noted W92) and [START_REF] Raymond | Gas exchange in rivers and estuaries: choosing a gas transfer velocity[END_REF] (noted RC01) provide the gas transfer velocity as a function of U10, and the equation of [START_REF] Abril | Turbidity limits gas exchange in a large macrotidal estuary[END_REF] (noted A09) as a function of U10, estuarine surface area and water current velocity. In the A09 model, we used a surface area of 19 km 2 , and an average water current of 0.4 m s -1 as representative for the São Francisco main channel [START_REF] Cavalcante | Circulation and salt balance in the São Francisco river Estuary (NE/Brazil)[END_REF]. For all calculations, pCO2 and wind speed data were selected in order to calculate FCO2 in winter and summer at daytime and in summer at night-time.

To complete our study, we corrected the theoretical C-pCO2 conservative mixing curves for the airwater CO2 exchange, building Ex-pCO2 curves. We applied an iterative calculation every salinity unit from 0 to 34, of the loss or gain of DIC as CO2 evasion or invasion during the mixing of water at salinity s with seawater to reach the value of salinity s+1.

DICgain(s) = -FCO2 (s).RT.H -1

Eq.2 where FCO2(s) is the flux (mol m -2 h -1 ), RT is the residence time (h) and H is the water depth (m).

FCO2 (s) is calculated with Eq.1 and the C-pCO2 value at salinity s, deduced from the mixing of DIC(s) with DIC(34) and TA(s) with TA(34). Then, the new corrected DIC concentration at s+1, DIC(s+1), is obtained by adding the DICgain(s) to the DIC(s):

Corrected DIC(s+1) = DIC(s+1) + DICgain(s) Eq.3

At that step, we recalculate a corrected C-pCO2(s+1) from new mixing between corrected DIC(s+1)

and DIC(34), and TA(s+1) and TA(34). The next iteration consisted in calculating the FCO2(s+1) from the corrected C-pCO2(s+1) and the DICgain(s+2) using Eq.1. The model was set-up in the CO2sys spreadsheet [START_REF] Pierrot | MS Excel Program Developed for CO2 System Calculations[END_REF], each line representing a salinity increment of one unit, from zero to 34, with the possibility to choose the gas transfer equation, the U10, RT and H.

Incrementation of the model allowed the building of thermodynamic Ex-pCO2 mixing curves corrected from gas exchange according to various combinations of these parameters. We used an RT/H ratio of 0.17 day m -1 which corresponds to a short RT value in the São Francisco estuary of 0.5 day as estimated from the discharge during the cruise, a mean H of 3m and an estuarine surface of 19km 2 . In order to simulate conditions closer to the ones occurring in large river plumes, we also tested the influence of increasing RT/H to 0.33 and 0.5 day m -1 , and we applied three different wind speeds of 0.5, 3 and 7 m s -1 , as representative of estuarine and shelf meteorological conditions. In addition, we assumed that the residence time was uniformly distributed along the salinity gradient, although there might be some exceptions as for instance longer residence times (several tidal cycles) in the low salinity range.

Results

DIC and TA increased linearly with salinity in the main channel, respectively from 553±40 and 539±27 µmol kg -1 in the river end-member to 1719±127 and 1975±146 µmol kg -1 at the mouth with salinity>30 (Fig. 2). Some significant positive anomalies of both parameters were observed at high salinity stations in the mangrove channel (Fig. 2). During both cruises, O-pCO2 was highly variable in the freshwater with some lateral heterogeneity between the different channels, and was above the atmospheric pCO2 at the centre of the main channel, with a value of ~700ppmv (Fig. 3A&B).

Downstream in the main channel, O-pCO2 sharply decreased below 400ppmv at salinity 3, and followed an asymmetric bell-shaped curve along the salinity gradient. In winter, O-pCO2 distribution in the main channel was almost perfectly predicted by thermodynamics during mixing with average difference between O-pCO2 and C-pCO2 in the 3-30 salinity range being 3±13 ppmv (Table 1), that is, within the error of pCO2 measurements and mixing model calculations. In summer, however, O-pCO2 in the main channel was on average 43±35 ppmv above the theoretical mixing curve of C-pCO2 along the 3-30 salinity range at night-time and daytime. Measured O-pCO2 included undersaturated values and invasion of atmospheric CO2 at salinities above 3 during both cruises (Fig. 3A&B). The most marine waters with salinities >30 were under-saturated in Aug. 2014 (O-pCO2: 332 ppmv) and almost at equilibrium (O-pCO2: 403±18 ppmv) in Nov. 2015 (Fig. 3E&F). In the mangrove channel, O-pCO2 was higher and more variable than in the main channel: between 298 and 975 ppmv (mean 512 ppmv) in winter and between 307 and 2007 ppmv (mean 976 ppmv) in summer (Fig. 3A&B). Several O-pCO2 peaks occurred in the mangrove channel and the adjacent coastal lagoon, at intermediate and high salinities and variations also occurred according to the tidal phase, with higher values in Nov. 2015 corresponding to low tide at spring tide period. A maximum of 2000 ppmv was recorded in the coastal lagoon at the outlet of the mangrove channel during the afternoon at low tide (Fig. 3B). During the two cruises DO remained between 99% and 101% saturation throughout most of the salinity gradient in the main channel (Fig. S2). At both seasons, significant DO undersaturation occurred in the river at low salinities and in the mangrove channel, with lower minimum values in Nov. 2015 compared to Aug. 2014. In Nov. 2015, slight DO oversaturation (max 103%) was observed in the main channel at salinities around 3 and above 31 (Fig. S2).

Water temperatures were lower in Aug. 2014 than in Nov. 2015 (Fig. 3C.D). During winter and neap tide (Aug. 2014), temperature remained constant in the main channel, and about 1°C above the conservative mixing line in the mangrove channel, showing that water heating occurred (Fig. 3C). In Aug. 2014, O-pCO2 was higher than the T-pCO2 by 5-20ppmv in the mangrove channel, but less than 2ppmv in the main channel (Fig. 3E). In Nov. 2015, when measurements were also performed at night, surface heating was evident from night-time to daytime, as well as, within the mixing zone in comparison with the two end-members. The entire salinity gradient in both channels was more than 1°C cooler at night and early morning (before 9h) than at daytime and dusk (Fig. 3D), which resulted in O-pCO2 higher than the T-pCO2 by 10-15ppmv (Fig. 3F). A peak temperature of 29.8°C occurred in Nov. 2015 during the afternoon in the shallow lagoon at the outlet of the mangrove channel at low tide, which corresponded to a O-pCO2 120ppmv higher than T-pCO2, when the O-pCO2 reached 2000 ppmv (Fig. 3F).

During the 25h cycle at the anchored station in the main channel the 11 th and 12 th of Nov. 2015, salinity varied between 1.2 and 32.2, and water pCO2 varied between 238 ppmv at intermediate salinities and 476ppmv at low salinity. The record at one minute frequency revealed a remarkable parallelism between O-pCO2 and C-pCO2 calculated from the mixing of the two end-members and the salinity value. However, O-pCO2 was on average +43±35ppmv above the conservative mixing curve o C-pCO2 (Fig. 4). Maximum deviations of O-pCO2 from C-pCO2 occurred during the ebb tides, both at sunset and night (hours 1.5 to 4.2: 52±19ppmv) and at sunrise and early morning (hours 13.5 to 17.5: 92±30ppmv). In addition, at low tide when salinities were below 3, O-pCO2 values were close to the C-pCO2 at daytime (hours 19-21.5), but were ~60ppmv higher than C-pCO2 at night-time (hours 7-10) indicating diurnal variations in this low salinity range (Fig. 3).

Finally, throughout the 25h cycle, some heating effect on pCO2 was detected during the afternoon at high tide and high salinity (hours 1 and 25) and at low tide and low salinity (hour 20), when O-pCO2 was at maximum +25ppmv higher than T-pCO2 (Fig. 4B).

Wind speed was low during the cruises, always below 3.5 m s -1 , on average between 1.1 m s -1 in Nov. 2015 and 2.2 m s -1 in April at day time, and 0.5 m s -1 at night-time (Table 1). In the main channel, FCO2 fluxes varied between 0.01 and 0.79 mmol m -2 h -1 (positive fluxes for CO2 evasion) in the 0-3 salinity range and between -0.04 and -1.68 mmol m -2 h -2 in the 3-30 salinity range. In the mangrove channel, calculated FCO2 varied between 1.23 and 10.72 mmol m -2 h -1 . For the observed wind speed range, calculated FCO2 strongly depended on the choice of the k600 parameterization, the W92 equation giving lower fluxes than the RC01 and A09 equations (Table 1).

Ex-pCO2, the theoretical thermodynamic pCO2 mixing curves corrected for gas exchange, were below the standard conservative curve between salinities 0 and 3-5, where CO2 evasion occurs, and above the curve between salinities 3-5 and 34, where invasion of atmospheric CO2 occurs (Fig. 5). Computed deviation from conservative mixing, i.e. Ex-pCO2 minus C-pCO2 strongly depended on the selected parameters, the gas transfer parametrization, the U10 and the residence time / depth ratio. These deviations varied from -0.3 to -167 ppmv (average -29 ppmv) in the 0-3 salinity range and from -8 to +76 ppmv (average +16 ppmv) in the 3-30 salinity range.

Applying a residence time in the São Francisco estuary of 0.5 days during the cruises, an average water depth of 3m and the U10 values of 0.5 and 3 m s -1 as representative for the conditions during the cruise, gives Ex-pCO2 -C-pCO2 values of -12±9ppmv in the 0-3 salinity range and 4±3ppmv in the 3-30 salinity range (Table 1).

Discussion

Predominance of thermodynamics

In the main channel, the pCO2 measurements (O-pCO2) and the thermodynamic estuarine conservative mixing model (C-pCO2) follow the same asymmetric bell-shape curve, with a steep decrease from salinity zero to 3, and undersaturation of surface waters all along the 3-30 salinity gradient (Fig. 3). To date, such well-defined pCO2 curve, with high resolution measured values very consistent with the theoretical conservative mixing curve, have been reported in the Amazon plume only for salinities higher than 17, and in the tropical Paraiba do Sul along the entire salinity gradient, with continuous pCO2 measurements [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. In the mesotrophic Paraiba do Sul River Estuary, autotrophic and heterotrophic activities had strong influences on the pCO2 distribution along the salinity gradient at low river discharge, but not at high discharge and shorter residence time, when the pCO2 versus salinity plot followed the theoretical curve [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. Because of the oligotrophic character of the São Francisco estuary [START_REF] Medeiros | Changes in Nutrient Loads (N, P and Si) in the São Francisco Estuary after the Construction of Dams[END_REF]2018) and the short residence time of water in the mixing zone (half a day), thermodynamics predominates over biological processes in controlling estuarine pCO2 distribution along the estuarine gradient. Some theoretical works have described how thermodynamics during estuarine mixing can generate positive or negative air-water CO2 fluxes depending on the DIC and TA concentrations in the river end-member, the concentrations in the marine end-member being considered as constant in comparison [START_REF] Whitfield | The carbon dioxide system in estuaries -An inorganic perspective[END_REF][START_REF] Cai | Carbon dioxide dynamics and fluxes in coastal waters influenced by river plumes[END_REF][START_REF] Delaigue | Spatial variations in CO2 fluxes in the Saguenay Fjord (Quebec, Canada) and results of a water mixing model[END_REF]). In the case of the São Francisco with a low TA river end-member (around 500 µmol kg -1 ), and warm waters (above 25°C), thermodynamics during mixing turns the estuary into a sink of atmospheric CO2 [START_REF] Whitfield | The carbon dioxide system in estuaries -An inorganic perspective[END_REF]; this is also the same for the Amazon (TA of 450 µmol kg -1 ) and the Congo (300 µmol kg -1 ) but not the Mississippi (>2000 µmol kg -1 ), where carbonate thermodynamics generates a CO2 source and where the observed CO2 invasion is biological as revealed by nitrate uptake [START_REF] Cai | Carbon dioxide dynamics and fluxes in coastal waters influenced by river plumes[END_REF]. Although the conditions found in the largest world rivers potentially generate a CO2 sink in the coastal zone, very few in-situ pCO2 data cover a complete estuarine salinity gradient consistent with the pCO2 theoretical mixing curve [START_REF] Cai | Carbon dioxide dynamics and fluxes in coastal waters influenced by river plumes[END_REF][START_REF] Lefèvre | Net Heterotrophy in the Amazon Continental Shelf Changes Rapidly to a Sink of CO2 in the Outer Amazon Plume[END_REF][START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. In winter in the São Francisco estuary, the average difference between O-pCO2 and C-pCO2 was only 3±13 ppmv along the 3-30 salinity range in the main channel and thermodynamics was apparently the unique driver of the carbonate system, creating a permanent CO2 influx in this salinity range. In summer, the average difference between O-pCO2 and C-pCO2 at salinities > 3 was +49±39 ppmv at daytime and +36±22 at nighttime (Table 1), but O-pCO2 always remained below the atmospheric value of 400 ppmv at salinities above 3, and thermodynamic uptake of atmospheric CO2 was predominant.

Our continuous measurements in the São Francisco also allow an interpretation of these O-pCO2 deviations from C-pCO2 as the result of other internal CO2 sources and sinks. First, estuarine NEP and lateral transport from river and intertidal areas can be a source of CO2 [START_REF] Borges | Carbon dioxide and methane dynamics in estuaries[END_REF][START_REF] Bauer | The changing carbon cycle of the coastal ocean[END_REF]. Second, C-pCO2 simulations were based on the conservative mixing of temperature at daytime at the two end-members, although heating and cooling of water is a common feature in this semi-arid region, and can potentially change pCO2 at daily and tidal time scales, particularly in shallow regions of the estuary such as the lagoon. Third, while air-water exchange of CO2 is much slower than acid-base equilibration of DIC species, evasion and invasion of CO2 occur during estuarine mixing, potentially altering the thermodynamic pCO2 curve. All these processes can generate pCO2 changes of few tens of ppmv that can add up to the values of pCO2 deviations from the mixing curves in summer [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. However, when interpreting these deviations, we must also keep in mind that the shape of the modelled C-pCO2 curve depends on the choice of the freshwater end-member, which was difficult to define because of lateral heterogeneity. For example, we calculated that using a freshwater end-member with 5µmol kg -1 higher TA (or DIC) (a change within the range of analytical precision for these parameters),

generates mixing pCO2 values 10 ppmv lower (or 9 ppmv higher) at salinity 10. In the case of our study, we choose to use the TA and pCO2 values at the central and deepest station upstream all the small islands (Fig. 1), where most freshwater flows and with little tidal influence, and not an average of all measurements at the lowest salinity (<0.1) region. Owing to the significant heterogeneity in the different canals, we cannot totally exclude lateral input of freshwater from small tributaries in this region that alter the DIC concentrations in the end-member and the modelled mixing pCO2 curve.

Biological CO2 sources and sinks

The diurnal O-pCO2 change at low salinity (Fig. 4) reveals some photosynthetic activity in this region, despite the chlorophyll concentration around 3 µg L -1 (unpublished data). During our measurements (Fig. 4), the respiration of this plankton apparently generated a deviation of +60 waters would be about 2%sat. In fact, in Nov. 2015, the DO concentration at salinity 3 was 100% at night time and 103% at daytime (Fig. S2), but these differences remained close to the error in DO measurements. Consequently, pCO2 monitoring was much more powerful than O2 monitoring to detect photosynthetic activity in the oligotrophic São Francisco estuary. It is well known that freshwater phytoplankton can still be active in estuaries at low salinities, but generally not at higher salinities in the mixing zone, where they die due to saline stress [START_REF] Lancelot | Trends in Estuarine Phytoplankton Ecology[END_REF]. This is probably the case in the São Francisco estuary, where, despite the general low biomass, freshwater phytoplankton potentially fuels heterotrophy downstream in the main channel. However, the residence time of half a day is probably too short to allow the well establishment of an active heterotrophic community [START_REF] Crump | Microbial biogeography along an estuarine salinity gradient: combined influences of bacterial growth and residence time[END_REF]. Benthic heterotrophy is probably limited in the sandy sediments of the São Francisco estuary where strong hydrodynamics limits the deposition of fine organic biodegradable material [START_REF] Medeiros | Aporte fluvial e dispersão de matéria particulada em suspensão na zona costeira do Rio São Francisco (SE/AL)[END_REF]). Along the salinity gradient, the deviation of O-pCO2 from C-pCO2 was spatially and temporally linked to tidal advection from mangroves and other brackish marshes. Many authors have reported high CO2 degassing fluxes from mangrove surrounding waters [START_REF] Borges | Atmospheric CO2 flux from mangrove surrounding waters[END_REF][START_REF] Maher | Groundwater-derived dissolved inorganic and organic carbon exports from a mangrove tidal creek: The missing mangrove carbon sink?[END_REF][START_REF] Santos | Carbon outwelling and outgassing vs. burial in an estuarine tidal creek surrounded by mangrove and saltmarsh wetlands[END_REF][START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. High water pCO2 come from the tidal pumping of mangrove-saturated soil CO2 [START_REF] Maher | Groundwater-derived dissolved inorganic and organic carbon exports from a mangrove tidal creek: The missing mangrove carbon sink?[END_REF][START_REF] Santos | Carbon outwelling and outgassing vs. burial in an estuarine tidal creek surrounded by mangrove and saltmarsh wetlands[END_REF], and from the respiration of mangrove-derived organic matter in creek waters and sediments [START_REF] Bouillon | Inorganic and organic carbon biogeochemistry in the Gautami Godavari estuary (Andhra Pradesh, India) during pre-monsoon: the local impact of extensive mangrove forests[END_REF][START_REF] Borges | Carbon dioxide and methane dynamics in estuaries[END_REF]. In the mangrove's soils, a large part of the metabolism is sulphate reduction that stoichiometrically produces the same amount of TA than CO2 [START_REF] Borges | Atmospheric CO2 flux from mangrove surrounding waters[END_REF]. This explains the positive TA (and associated DIC) anomalies observed at high salinities in the mangrove channel of the São Francisco Estuary (Fig. 2). Although water O-pCO2 was similar during both campaigns in the main channel, in the secondary channel, pCO2 values were much higher, particularly in Nov. 2015 compared to Aug. 2014. This can be attributed to the spring tide conditions during sampling in 2015, enhancing the tidal pumping [START_REF] Maher | Groundwater-derived dissolved inorganic and organic carbon exports from a mangrove tidal creek: The missing mangrove carbon sink?[END_REF][START_REF] Santos | Carbon outwelling and outgassing vs. burial in an estuarine tidal creek surrounded by mangrove and saltmarsh wetlands[END_REF]. During the 25h cycle in the main channel at spring tide in 2015 (Fig. 4), the highest (>100 ppmv) and longest positive deviations of O-pCO2 from C-pCO2 occurred during the ebb tides, particularly at late night and early morning (hours 14-17) which reveals an export from the mangrove waters to the main channel that affects the 5-8 salinity range (Fig. 3B). The monitoring point during 25h was located at less than two km from confluence between the main channel and the mangrove channel, and also close to brackish tidal marshes (Fig. 1) that could contribute, together with mangroves to the pCO2 peaks at high and ebb tide. In addition, the short residence time of waters in the estuary suggests that heterotrophs would spend little time in the main channel and would be exposed to strong salinity stress to generate significant respiratory CO2, which is apparently coming in majority from tidal pumping.

Impact of heating

Semi-arid coastal ecosystems are environments where heating and evaporation can have a strong influence on the water chemistry and where very few studies document the behaviour of the carbonate system [START_REF] Marchand | Water Biogeochemistry of a Mangrove-Dominated Estuary Under a Semi-Arid Climate (New Caledonia)[END_REF]Yao and Hu, 2018;[START_REF] Yao | Hydrologic controls on CO2 chemistry and flux in subtropical lagoonal estuaries of the northwestern Gulf of Mexico[END_REF]. Heating of water will first increase the pCO2 and favour outgassing [START_REF] Takahashi | Global sea-air CO2 flux based on climatological surface ocean pCO2, and seasonal biological and temperature effects[END_REF]. If evaporation is intense and leads to hypersaline conditions, consumption of TA and release of CO2 due to carbonate precipitation can occur [START_REF] Murgulet | Temporal and spatial fluctuations of groundwater-derived alkalinity fluxes to a semiarid coastal embayment[END_REF][START_REF] Yao | Hydrologic controls on CO2 chemistry and flux in subtropical lagoonal estuaries of the northwestern Gulf of Mexico[END_REF]. In the São Francisco estuary, higher temperature relative to the mixing of the two end-members was most pronounced during summer and in the mangrove secondary channel (Fig. 3C.D). In summer, despite the short residence time of waters, the temperature increased in both channels by more than 1°C during daytime compared to night-time (Fig. 3D), which corresponds to a theoretical pCO2 increase (difference between O-pCO2 and T-pCO2) of ~15ppmv (Fig. 3F). In the main channel, the 25h cycle (Fig. 4) reveals that heating at daytime has a significant impact on pCO2 at both low tide (hour 21) and high tide (hours 1 and 25). In Nov. 2015 at salinities > 3, averaged observed pCO2 in the main channel in summer were 33ppmv higher during daytime than during night-time, and the thermal effect contributed to 25% of the deviation of pCO2 above the conservative curve at daytime and 10% at night, when estuarine waters still remained warmer than predicted from the mixing of the end-members (Table 1). At salinities < 3, the thermal effect was partially offset by biological productivity and the O-pCO2 were similar at night-time and daytime. However, the daytime data included salinities below 1.4 with higher O-pCO2, whereas night-time data did not (Table 1). In the secondary channel, the maximal thermal effect occurred at salinity ~28 in the coastal lagoon in the afternoon at low tide, with an increase in temperature of ~3°C concomitant with a maximum pCO2 of ~2000ppmv (Fig. 3C,F). The thermal effect at this site (Fig. 3F) contributed to 15% of the deviation of pCO2 above the conservative curve at maximum (Fig. 3B), and this pCO2 peak is attributed to the maximum pumping of mangrove-derived CO2 at low tide. Although water depth was less than half a meter and temperature reached 29.8°C, we found no evidence of carbonate precipitation during this event as those reported in other semi-arid coastal lagoons [START_REF] Yao | Hydrologic controls on CO2 chemistry and flux in subtropical lagoonal estuaries of the northwestern Gulf of Mexico[END_REF]. Indeed, the salinity was only 28 during the pCO2 peak and increased only when the tide started to enter the system through the small canals (Fig. 1); no significant TA consumption could confirm carbonate precipitation, TA was above the mixing line in this region, as a result of tidal pumping of DIC from predominant sulphate reduction and other anaerobic processes in the mangrove soils [START_REF] Borges | Atmospheric CO2 flux from mangrove surrounding waters[END_REF][START_REF] Santos | Carbon outwelling and outgassing vs. burial in an estuarine tidal creek surrounded by mangrove and saltmarsh wetlands[END_REF].

Impact of gas exchange

The fact that thermodynamic mixing in the main channel generates undersaturated pCO2 conditions with respect to the atmosphere implies that uptake of atmospheric CO2 will occur at salinities > 3. Consequently, the hypothesis of conservative DIC mixing on which the thermodynamic pCO2 curve relies will become invalid, whereas that of conservative TA will remain valid. If we consider the average and standard deviation air-water CO2 fluxes calculated with the conservative pCO2 mixing curve and the three gas exchange equations (Table 1), then thermodynamic during mixing in the main channel emits +2.5±1.7 mmol m -2 h -1 of CO2 at salinity lower than 3 and absorbs -1.3±0.6 mmol m -2 h -1 of CO2 between salinities 3 and 34. A precise integration of these fluxes would require an estimate of the relative surface area of different salinity ranges according to the shape of the pCO2 bell curve. In particular, it is necessary to separate the 0-3 salinity range that behaves as a source (as does the river) from the estuarine waters with salinities above 3 that behave as a sink (Table 1). During our study, we could sample the marine end-member in the shallow waters of the mouth of the estuary at high tide (Fig. S1). However, at low tide as well as on average over the tidal cycle, salinity at the mouth is generally lower, and a significant part of the salinity gradient occurs in a plume offshore. Based on numerical modelling, [START_REF] Cavalcante | Temporal streamflow reduction and impact on the salt dynamics of the São Francisco River Estuary and adjacent coastal zone (NE/Brazil)[END_REF] showed that the tide-integrated salinity at the mouth of the São Francisco

Estuary can be as low as 22 during concomitant neap tides and high river flow. In addition, surface waters with salinities lower than 34 extends 5-20km offshore, with a surface area up to 50km 2 , that is, an area much larger than the 19km 2 of the inner estuary [START_REF] Cavalcante | Temporal streamflow reduction and impact on the salt dynamics of the São Francisco River Estuary and adjacent coastal zone (NE/Brazil)[END_REF]. Considering the low river discharge during the study period, an area of 6km 2 for the 0-3 salinity range, which corresponds to one third of the inner estuary, emits 4.3tC per day. An area 30 km 2 (13km 2 in the inner estuary and 17km 2 in the plume offshore) for the 3-34 salinity range will absorb -11.2tC per day. With these hypotheses on the surface areas, the São Francisco Estuary absorbs 7tC per day of CO2. In comparison, the input of DIC by the river was 95 and 70 tC per day, respectively in Aug.

2014 and Nov. 2015. Consequently, the invasion of CO2 from the atmosphere due to thermodynamics during mixing will increase the DIC export to the sea by 7-10%. This modest increase in the DIC discharge of the São Francisco Estuary to the ocean might be also partly explained by its morphology and hydrological feature induced by very shallow depths at the mouth and a very steep longitudinal salinity gradient between 10 and 25 inside the lagoon (Fig. 4).

By calculating for each salinity increment the quantity of CO2 absorbed from the atmosphere during the residence time of waters in the estuary, our mixing Ex-pCO2 model provides a precise evaluation of the impact of gas exchange on the C-pCO2 curves (Fig. 5). Evasion occurs first at low salinity, and the correction is negative during a restricted amount of time; invasion occurs at salinity above 3-5 with a positive correction and presumably during a longer amount of time. In the case of the São Francisco Estuary, with a water depth of 3m, a residence time of half a day and relatively low wind speed values (Table 1), the model results in a difference between C-pCO2 and Ex-pCO2 of only 4ppmv in the 3-30 salinity range in Nov. 2015, corresponding to ~10% of the O-pCO2 deviation above the C-pCO2 mixing curve. However, strong stratification of the water column at intermediate salinities may alter this conclusion in two opposite ways: on one hand, if we consider a mixed surface layer of only 1m height as representative in-situ conditions [START_REF] Cavalcante | Circulation and salt balance in the São Francisco river Estuary (NE/Brazil)[END_REF]), CO2 invasion will generate a larger difference between Ex-pCO2 and C-pCO2, on average ~12ppmv, which corresponds to ~25% of the O-pCO2 deviation above the C-pCO2 mixing curve at salinity > 3 in Nov. 2015; on the other hand, stratification of the water column implies that part of the mixing of estuarine water occurs below the water surface, that is, without contact with the atmosphere and without gas exchange. Wind speed, fetch length, and water current all contribute to surface turbulence in estuaries, where gas exchange is generally faster than in the ocean [START_REF] Borges | Gas transfer velocity of CO2 in three European estuaries (Randers Fjord, Scheldt and Thames)[END_REF][START_REF] Orton | Tidal and atmospheric influences on nearsurface turbulence in an estuary[END_REF]. Most of the proposed k600 parameterization in estuaries are based on data in relatively well-mixed estuaries [START_REF] Borges | Gas transfer velocity of CO2 in three European estuaries (Randers Fjord, Scheldt and Thames)[END_REF][START_REF] Abril | Turbidity limits gas exchange in a large macrotidal estuary[END_REF][START_REF] Orton | Tidal and atmospheric influences on nearsurface turbulence in an estuary[END_REF]. To our best knowledge, no gas exchange study has been performed in highly stratified systems, where buoyancy fluxes dissipate kinetic energy as turbulence (Etemad-Shahidi and Imberger, 2002) potentially enhancing gas exchange, whereas stratification limits the vertical diffusion of atmospheric CO2 to the whole water column. In large tropical estuaries and plumes such as the Amazon and the Congo, most of the salinity gradient occurs offshore where wind speed is higher. Gas exchange in these plumes can significantly alter the C-pCO2 mixing curves and the impact of stronger wind and longer residence time in the mixing zone might be partially offset by a deeper mixed layer in the range of 5-30 m [START_REF] Grodsky | Year-to-year salinity changes in the Amazon plume: Contrasting 2011 and 2012 Aquarius/SACD and SMOS satellite data Remote Sens[END_REF][START_REF] Phillipson | Assimilation of Satellite Salinity for Modelling the Congo River Plume[END_REF].

Relative contributions of pCO2 deviations from conservative mixing

In August 2014 in the main channel of the São Francisco estuary, heating and tidal pumping were almost negligible and we found little deviation of O-pCO2 from C-pCO2, except in the salinity range 0-3, where the O-pCO2 were on average 45 ppmv lower than the modelled conservative pCO2 (Fig. 3A), suggesting the occurrence of autotrophic activity by phytoplankton and/or CO2 evasion in the area, consistent with observations at low tides in Nov. 2015 during the 25h cycle (Fig. 4). In contrast, the deviation from conservative mixing was more pronounced in Nov. 2015, during summer and spring tides, and we could calculate the distribution of the three major active processes that make pCO2 deviate from the conservative curve along the salinity gradient, that is, (i) surface water heating or cooling based on temperature field data (T-pCO2), (ii) evasion or invasion of CO2 based on modelling (Ex-pCO2), and (iii) autotrophy or the sum of heterotrophy and tidal pumping from mangrove creeks as the residual part of the deviation between modelled and observed data, calculated as O-pCO2 -T-pCO2 -Ex-pCO2 [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. We have binned our Nov. 2015 data, calculating the means and standard deviations of O-pCO2 and T-pCO2 for each salinity increment. In Aug. 2014, the deviation of pCO2 from conservative mixing was too low (Table 1). The difference between O-pCO2, C-pCO2, T-pCO2 and Ex-pCO2 were within the uncertainties of measurements and calculations, meaning that thermodynamics alone drives almost all the O-pCO2 distribution. The 2015 binned O-pCO2 and T-pCO2 could be compared with the modelled C-pCO2 and Ex-pCO2 (Fig. 6). We also separated the day and night periods for the regions with salinities above and below 3 and we calculated the CO2 fluxes corresponding to these three processes (heating, gas exchange and the residual as biological activity) using wind speed data and three gas transfer parameterizations (Table 2). The calculations indicate that in Nov.

2015, heating was consistently stronger at daytime compared to night-time, and contributed to a peak at 20 ppmv at salinity 28 (Fig. 6), and a maximum average deviation from conservativity of +12 ppmv at daytime and salinity above 3, corresponding to a heating CO2 flux of 0.2 mmol m -2 h -1 (Table 2). Gas exchange had a slightly lower impact on pCO2 values than heating, with rapid evasion of river-borne CO2 occurring at low salinity and CO2 invasion at salinity between 3 and 34.

According to our quantitative analysis, concomitant CO2 evasion and net autotrophy occur in the 0-3 salinity range, whereas concomitant CO2 invasion and net heterotrophy associated to tidal pumping from mangroves occur at salinity above 3 (Fig. 6). This trend in the São Francisco Estuary is similar to the one recently reported in the mesotrophic Paraiba do Sul Estuary during high river discharge [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]). These two river-dominated tropical estuaries have low TA concentrations in the river end-member. At low salinities, inputs of river-borne CO2 combined with low buffering capacity favour outgassing, whereas little salinity stress allows some production of freshwater phytoplankton [START_REF] Cloern | Phytoplankton bloom dynamics in coastal ecosystem: a review with some general lessons from sustained investigation of San Francisco Bay, California[END_REF][START_REF] Lancelot | Trends in Estuarine Phytoplankton Ecology[END_REF][START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF]. At salinities below 3, the O-pCO2 values were 109 ppmv below the modelled C-pCO2 values at daytime, but 34 ppmv above at night-time (Table 2), consistent with phytoplanktonic photosynthetic activity. The different salinity ranges sampled at daytime (0-3) and night-time (1.4-3) do not allow to accurately estimate a NEP from diurnal variations in the upper estuary. At increasing salinities, the mixing with buffered seawater creates thermodynamics conditions for CO2 invasion and offsets some apparent heterotrophy combined with tidal advection of mangrove-derived CO2. The latter generates a deviation (calculated as O-pCO2 -T-pCO2 -Ex-pCO2 ) of about 40 ppmv of pCO2 along the 4-25 salinity range (Fig. 6) and +33 ppmv (daytime) and +27 ppmv (night time) on average over the 3-34 salinity gradient (Table 2). Interestingly, our approach also suggests some autotrophic activity in the 30-33 salinity range (Fig. 6B), which is also a classical pattern of phytoplankton distribution in estuaries; indeed, blooms of marine phytoplanktonic species are a common feature in high salinities regions of river plumes, where they can take benefit from the available light and nutrients and from the stratification of the water column [START_REF] Cloern | Phytoplankton bloom dynamics in coastal ecosystem: a review with some general lessons from sustained investigation of San Francisco Bay, California[END_REF][START_REF] Lancelot | Trends in Estuarine Phytoplankton Ecology[END_REF]. Heating of water made O-pCO2 higher than T-pCO2 all along the salinity gradient with a maximum difference between O-pCO2 and T-pCO2 of +22 ppmv at salinity 32, precisely where the bloom of marine phytoplankton apparently occurred, possibly favoured by warmer surface waters and a temporarily more stratified water column.

After a conversion to CO2 fluxes by applying a gas transfer velocity, the deviation between the T-pCO2 and the modelled Ex-pCO2 corrected for gas exchange, provide an indirect and integrative estimate of NEP (Table 2). Quantitatively, the computed NEP (including tidal pumping) with this original method in the São Francisco (Table 2), as well as in the Paraiba do Sul [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF] are around 1 or less mmol m -2 h -1 , when NEP reported in estuaries of the world varies between +5 (autotrophy) and -12 mmol m -2 h -1 (heterotrophy) [START_REF] Borges | Carbon dioxide and methane dynamics in estuaries[END_REF]. Despite these low values, our method is quite sensitive and the sign and spatial distribution of the calculated NEP are consistent with classical concepts of estuarine plankton ecology [START_REF] Crump | Microbial biogeography along an estuarine salinity gradient: combined influences of bacterial growth and residence time[END_REF][START_REF] Lancelot | Trends in Estuarine Phytoplankton Ecology[END_REF], tidal advection and the short residence-time in the mixing zone. This confirms the robustness of our approach that could be applied to many estuarine environments, whether they act as CO2 sink or source.

Conclusions

The São Francisco Estuary displays specific conditions in terms of carbonate chemistry and airwater CO2 exchange: because of warm and poorly buffered freshwaters, oligotrophic conditions and short residence time, carbonate thermodynamic equilibration during mixing predominate over biological processes and determine water pCO2 and air-water CO2 fluxes. This results in an abiotic uptake of atmospheric CO2 by the estuary at salinities above 3 in the main channel and the DIC export to the sea is increased by 7-10%. During winter and neap tide period, tidal pumping and heating were weak and the observed pCO2 almost matched the theoretical conservative mixing curve, within the error of measurements and computations. During summer and spring tide period, the observed pCO2 was still below the atmospheric value, but on average +49ppmv above the conservative mixing curve. At that season and salinities > 3, heating of water relative to the mixing of the two end-members contributed to ~15%, and gas exchange contributed to ~10% of the deviation of observed pCO2 from conservativity, the remaining ~75% being attributed in majority to tidal pumping from mangroves and brackish marshes, maybe with some respiratory activity in estuarine waters despite the short residence time. Following the approach of [START_REF] Cotovicz | Carbon dioxide sources and sinks in the delta of the Paraíba do Sul River (Southeastern Brazil) modulated by carbonate thermodynamics, gas exchange and ecosystem metabolism during estuarine mixing[END_REF] in the mesotrophic and tropical Paraiba do Sul River delta that also behaves as a CO2 sink in the mixing zone, our study is a second detailed report of CO2 air-water exchange, based on the combination of high-resolution pCO2 field data and theoretical computation that cover the complete salinity gradient. We highlight a singular situation, not only occurring in the oligotrophic São

Francisco, but also the mesotrophic Paraiba do Sul Estuary, of concomitant CO2 uptake and heterotrophy at salinities higher than 3. Similar thermodynamics conditions, i.e., warm and poorly buffered freshwaters that potentially generates CO2 invasion in the mixing zone, occur in large tropical river plumes like the Amazon, Congo and Orinoco. High resolution pCO2 measurements along the entire salinity gradient of these large estuaries and plumes are thus necessary. In the Amazon plume, the available equilibrator pCO2 data covers only the >17 salinities range with undersaturated values with respect to the atmosphere, well below those predicted by the conservative mixing of Amazon river water with Atlantic equatorial water [START_REF] Körtzinger | A significant CO2 sink in the tropical Atlantic Ocean associated with the Amazon river plume[END_REF][START_REF] Lefèvre | Net Heterotrophy in the Amazon Continental Shelf Changes Rapidly to a Sink of CO2 in the Outer Amazon Plume[END_REF]. This indicates that in the Amazon, thermodynamic and biological processes contribute together to CO2 invasion because the plume is autotrophic, whereas in the Paraiba do Sul and São Francisco Estuaries, the two processes are competing with each other both at low salinities (evasion and autotrophy) and high salinities (invasion and heterotrophy). High-resolution surface pCO2, temperature and salinity measurements coupled with modelling of estuarine mixing provided some detailed and precise information that could be applied in many plumes of large tropical rivers that contribute more significantly to the riverine carbon load to the world ocean.

Table 1. Synthesis of measured and calculated parameters during the two cruises. O-pCO2 is the observed pCO2 as measured in situ, C-pCO2 is the conservative pCO2 modelled from mixing of river water with seawater, T-pCO2 is the pCO2 normalized to temperature resulting from the mixing of the two end-members and Ex-pCO2 is the pCO2 resulting from conservative after being corrected for gas exchange. *Calculated using the mixing/gas exchange model described in Fig. 5, for the measured U10, a value of RT of 0.5 days and H of 3m. # During night, measured and modelled conservative pCO2 values are lower than expected when compared to the daytime, because night-time measurements did not include samples with salinities lower than 1.2 and highest pCO2. $ In order to account for the protection of mangrove trees to creek waters, we divided by 2 the wind speed values to calculate CO2 fluxes from the mangrove secondary channel, as a first approximation. Standard deviation of the differences between two numbers were calculated as the square root of the sum of the squares of the two errors. 1.1±0.5 $ 0.5-1.6 $ k600 (cm h -1 ) W92 k600 (cm h -1 ) RC01 k600 (cm h -1 ) A09 Calculations were made using Temperature, Salinity, DIC and TA observed in the freshwater and marine end-members in Nov. 2015 and the gas exchange was computed every salinity unit increment (see section 2.2). RT/H is the ratio between residence time and water depth, and the selected value of 0,17 is representative for estuarine systems such as the São Francisco, whereas the value may apply to larger tropical rivers such as the amazon or congo.

  photosynthetic/respiratory quotient of 1, the equivalent diurnal change in DO in these oligotrophic

Figure 1 .

 1 Figure 1. Map of the São Francisco Estuary. Arrows show the regions of freshwater-estuarine

Figure 2 :

 2 Figure 2: Distribution of Total Alkalinity (TA) (A. and B.) and Dissolved Inorganic Carbon (DIC) (C.

Figure 3 .

 3 Figure 3. Distribution of O-pCO2 (A. and B.), temperature (C. and D.), and the difference between

Figure 4 .

 4 Figure 4. Time course of physico-chemical parameters in surface water of the main channel of the

Figure 5 .

 5 Figure 5. Impact of air-water CO2 exchange on thermodynamic conservative pCO2 mixing curves.

Figure S2 Figure S2 .

 S2S2 Figure S2

Table 2 .

 2 Comparison of binned data with modelled data for the Nov. 2005 campaign. *Calculated as O-pCO2 minus T-pCO2. # Calculated as C-pCO2 minus Ex-pCO2. $ Calculated as O-pCO2 minus C-pCO2 minus T-pCO2 minus Ex-pCO2. Standard deviation of the differences between two numbers were calculated as the square root of the sum of the squares of the two errors.

	0.6	0.6	0.2	1.6	0.1	1.6	0.1	0.5
	3.4	3.4	2.9	4.1	2.2	4.1	2.2	2.8
	5.6	5.6	4.1	7.3	2.8	7.3	2.8	4.6

Fluxes (mmol m -2 h -1 )

	Water-air CO2 flux calculated with observations	+0.47±0.31 +0.15±0.12 -1.00±0.65 -0.61±0.51
	Water-air flux calculated with conservative mixing	+2.53±1.65 -0.05±0.05 -1.86±0.51 -0.86±0.72
	Deviation of observations from conservative mixing	-2.06±0.97 +0.20±0.07 +0.86±0.48 +0.25±0.51
	Contributions

to the deviation of CO2 fluxes (mmol m -2 h -1 )

	Heating (+) / Cooling (-)	+0.10±0.06 +0.01±0.01 +0.20±0.14 +0.10±0.06
	Invasion (+) / Evasion (-)	-0.21±0.14 -0.04±0.03 +0.03±0.02 +0.01±0.03
	Heterotrophy plus tidal pumping (+) / autotrophy (-)	-1.94±1.31 +0.23±0.20 +0.62±0.41 +0.14±0.45

O-pCO2 and T-pCO2 (E. and F.) versus salinity in the São Francisco Estuary main channel (blue

Acknowledgements

This research was funded by the Brazilian National Council of Research and Technological Development (CNPq) through the Science without border program (PVE 401726-6), the Instituto do Milênio Estuários project (420.050/2005-1) and the National Institute of Science and Technology Transferências de Materiais Continente-Oceano (TCMOcean 573.601/2008-9). Georgenes H Cavalcante was supported by the American University of Sharjah (research Grant FRG19-M-G74). This work is also a contribution to the International Research Project Vulnérabilité des Ecosystèmes Littoraux Tropicaux face à l'Eutrophisation funded by the French National Centre for Scientific Research (CNRS-INEE).

Aug. 2014 Nov. 2015


Éloi Durant
email: eloi.durant@ird.fr

François Sabot

Matthieu Conte

Mathieu Rouard
email: m.rouard@cgiar.org

Panache: a Web Browser-Based Viewer for Linearized Pangenomes

Motivation: Pangenomics evolved since its first applications on bacteria, extending from the study of genes for a given population to the study of all of its sequences available. While multiple methods are being developed to construct pangenomes in eukaryotic species there is still a gap for efficient and user-friendly visualization tools. Emerging graph representations comes with their own challenges, and linearity remains a suitable option for user-friendliness.

Results:

We introduce Panache, a tool for the visualization and exploration of linear representations of gene-based and sequence-based pangenomes. It uses a layout similar to genome browsers to display presence absence variations and additional tracks along a linear axis with a pangenomics perspective.

Introduction

The widespread use of fast and affordable sequencing technologies unveiled how much genomic information was lost when relying on a single and unique reference genome. For instance, it was found that about 10% of additional DNA was not captured by the current human reference genome [START_REF] Sherman | Assembly of a pan-genome from deep sequencing of 910 humans of African descent[END_REF]. By leveraging data of multiple references instead, a new era of genomics emerged: Pangenomics. This is now being applied from bacteria to Eukaryotes and has been increasingly used in more complex genomes such as human and plants. As reviewed in [START_REF] Golicz | Pangenomics Comes of Age: From Bacteria to Plant and Animal Applications[END_REF], some studies handle this approach through a gene or functional annotation lens while others extend it to DNA sequences, especially when the studied organisms are eukaryotes.

Still, more tools are needed to help pangenomics to reach a broader audience within the scientific community (Computational Pan-Genomics Consortium, 2018;[START_REF] Golicz | Towards plant pangenomics[END_REF][START_REF] Tranchant-Dubreuil | Plant Pangenome: Impacts on Phenotypes and Evolution[END_REF] . While recent progress has been made to compute and store pangenomes [START_REF] Garrison | Variation graph toolkit improves read mapping by representing genetic variation in the reference[END_REF][START_REF] Li | The design and construction of reference pangenome graphs with minigraph[END_REF], the tool landscape is particularly barren when it comes to visualization. Tools such as Pan-Tetris [START_REF] Hennig | Pan-Tetris: an interactive visualisation for Pan-genomes[END_REF], PanViz [START_REF] Pedersen | PanViz: interactive visualization of the structure of functionally annotated pangenomes[END_REF] or PanX [START_REF] Ding | panX: pan-genome analysis and exploration[END_REF] were designed for genebased pangenomes and do not scale well to large-scale eukaryotic studies. Indeed, this gene-centric definition does not take into account positions within genomes, thereby blending paralogs together, and ignoring non-coding sequences despite their crucial influence on phenotypes [START_REF] Maston | Transcriptional Regulatory Elements in the Human Genome[END_REF]. The current trend for sequence-based pangenomes is to use graph visualization software like Bandage [START_REF] Wick | Bandage: interactive visualization of de novo genome assemblies[END_REF], a general tool for navigating assembly graphs, but alternatives dedicated to pangenomes and their inner properties are yet to be refined and adopted. For instance Sequence Tube Maps [START_REF] Beyer | Sequence tube maps: making graph genomes intuitive to commuters[END_REF] and MoMi-G [START_REF] Yokoyama | MoMI-G: modular multi-scale integrated genome graph browser[END_REF] both focus on structural variations from individual genomes but lack information on the pangenome itself. As an example, they do not have direct visual cues for the identification of the most represented parts of a pangenome.

As useful as graph representations may be, they can easily be overloaded with content, resulting in a 'hairball' effect that is hard to read and explore [START_REF] Yoghourdjian | Scalability of Network Visualisation from a Cognitive Load Perspective[END_REF].

Linear representations of genomes have their own weaknesses [START_REF] Nielsen | Representing the genome[END_REF] but are widely used in a variety of genome browsers, and most efficient when it comes to exploration tasks. Here we introduce Panache, the PANgenome Analyzer with CHromosomal Exploration, a web browser-based viewer which renders interactive linear representations of pangenomes one panchromosome at a time.

Features

Panache is designed to display a linear representation of pangenomes. This representation is based on the idea that every genome within a pangenome can be divided into multiple blocks, with each block potentially shared with other genomes. Such blocks could either DNA sequences (extracted from the nodes of a graph pangenome for example) or genes.

Blocks from all genomes could be laid out and ordered along a single string, which would then serve as a flattened pan-reference, as illustrated in Figure 1A. One could also imagine ordering the blocks according to an existing genome instead, using its own linear coordinate system as a reference. Every pangenomic block can therefore be represented in an easy-to-browse visualization, with additional tracks of summarized information such as a block presence/absence status or whether it belongs to the core genome (most present blocks) or the variable genome (also referred to as dispensable genome).

While a graph representation might give a better sense of structural variations or of a genome's full sequence within a pangenome, a linear representation allows more reproducibility when exploring data thanks to its fixed and ordered coordinate system. Moreover, missing information can be visually hinted even when not directly available. For example, an additional track can specify which blocks are repeated elsewhere in the pangenome.

The tool comes with a variety of navigation related functionalities: choosing which panchromosome to display and browsing through it, modifying display size, jumping automatically to areas enriched in absent blocks. It also allows modifications to the core/variable threshold and hovering over visual elements to display additional information such as functional annotation pop-up windows. All available functionalities are further detailed within Panache's documentation.

Figure 1B illustrates how Panache displays linear pangenome data using a pangenome generated in banana [START_REF] Rijzaani | The pangenome of banana highlights differences between genera and genomes[END_REF]. A set of 34,878 genes from 15 genotypes have been grouped into chromosomes and positioned linearly on a panreference. Here a user can quickly identify lines with missing genes, and which genes belong to the core genome (in orange) or to the variable genome (in blue). Details about the individual gene annotations can be accessed by hovering over the beeswarm plot on top of the presence/absence matrix.

Implementation

Panache is a JavaScript application built with Vue.js 2 and additional librairies, namely D3.js v5 (enabling linkages between data and SVGs) and Vuex. For easy deployment, we have created a docker instance that can run Panache through nginx but the production files are available for deployment through other means.

Panache can take two types of files as input. The main file is a presence/absence matrix file in a BED-like format. Each line stores information about one pangenomic block (either a gene or a sequence) detailed through multiple columns, starting with linear position data and ending with the presence/absence information within every genome. An optional GFF file of annotations on the linear pangenomic coordinates may be loaded in addition to the matrix file. Panache can be used to display graph based pangenomes if they are previously linearized with tools such as BioGraph.jl (https://github.com/nguyetdang/BioGraph.jl).

Example files for trial and details about how to format datasets are provided on the GitHub repository.

Discussion

Panache offers an innovative web interface for the linear representation of pangenomes, explorable through a web interface, making the exploration and use of pangenomes easier.

As a lightweight application, it can be embedded easily as an iframe to complement other interfaces in existing genome information systems. It has already been tested with open access data sets [START_REF] Golicz | The pangenome of an agronomically important crop plant Brassica oleracea[END_REF][START_REF] Rijzaani | The pangenome of banana highlights differences between genera and genomes[END_REF] and proved to be an effective alternative to existing tools by highlighting inherent pangenomic properties.

However, as a visualization tool, results are highly dependent on the methods used to create and linearize the pangenomes. Further work on pangenome representations, and particularly on structural variations, is still needed to enhance existing representations.

Current plans for Panache include native support of graph files such as GFA (https://github.com/GFA-spec/GFA-spec) as an input and faster display technologies like

Figure 1 .

 1 Figure 1. Panache offers a linear representation of pangenomes, with blocks information detailed through multiple tracks much like classic genome browsers. A) Linearized pangenomes represent chains of present/absent pangenomic blocks on a single string. B) Panache's interface for browsing through the Presence/Absence matrix and navigating through panchromosomes.

Acknowledgements

The authors wish to thank Romain Basset and Mel Florance for their help on Vue.js and improvements brought to the code, as well as Dr. Eric Ganko and Steve Graham for their useful feedback. The authors acknowledge the ISO 9001 certified IRD itrop HPC (member of the South Green Platform) at IRD montpellier for providing HPC resources that have contributed to the research results reported within this paper. URL: https://bioinfo.ird.fr/http://www.southgreen.fr.

Funding

This work was supported by funding from Agropolis Fondation's 'GenomeHarvest' project (ID 1504-006), CIFRE doctoral contract 2018/1475, Syngenta and the CGIAR Research Program, Roots, Tubers and Bananas.

The authors declare to have no conflict of interest.