C Bouvet De Maisonneuve

M A Dungan

O Bachmann
email: bachmano@u.washington.edu

A Burgisser
email: burgisse@cnrs-orleans.fr

Insights into shallow magma storage and crystallization at Volcán Llaima (Andean Southern Volcanic Zone, Chile)

Keywords: Magma storage, Degassing, Crystallization, Melt Inclusions, Strombolian activity

Scoriae produced by four mafic historic eruptions of Volcán Llaima are used to elucidate magma differentiation, recharge, mixing, and eruption triggering. Whole-rock, mineral, and olivine-hosted melt inclusion chemistry suggest that basaltic andesites (~52-53.5 wt.% SiO 2 ) stall at immediately sub-edifice depths (≤4 km beneath the base of the volcano), where they crystallize to large extents and form mush bodies. Melt inclusion trends overlap with the whole-rock data trends defined by the entire volcano up to 53-55% SiO 2 , but more evolved compositions define divergent trends for some elements, with up to 2.2 wt% TiO 2 (increasing Zr) and Al 2 O 3 as low as 12.5 wt% (decreasing Sr) at 57-58 wt% SiO 2 . These more evolved melt compositions are inferred to be the result of shallow evolution of interstitial melt during the formation of crystal mush bodies, as a consequence of degassing and crystallization with a reduced participation of Fe-Ti-oxides. The inferred suppression of Fe-Ti-oxide stability and the modal dominance of plagioclase crystallization are consistent with low fluid-saturation pressures of ~40-70 MPa inferred from average H 2 O contents for mafic arc magmas (dominantly 1-4 wt%) but low CO 2 contents (dominantly 0-300 ppm) in melt inclusions. The broad range in olivine core compositions (Fo 69-83 ) and the absence of correlated degassing and magma evolution trends in historic Llaima magmas suggest that they are stored as multiple dike-like bodies created by a high frequency of magma replenishment relative to the frequency of large eruptions. This temporal-spatial disconnection leads to isolated evolution and degassing of discrete magma batches, followed by remobilization and assembly just prior to eruption. Eruptions are probably triggered by recharge of relatively hot, mafic, and much less degassed magma, in accord with dominantly reversely-zoned olivine crystals, and higher olivine-melt temperatures recorded by the relatively primitive matrix glasses and coexisting olivine rims.

Introduction

Volcán Llaima (38.7° S) is located in the Southern Volcanic Zone of the Andes, about 700 km to the south of Santiago de Chile (Fig. 1). It is one of the most active volcanoes in Chile, with over 50 eruptions since ~1640 AD. Although the explosivity of its eruptions is generally not sufficient to cause lethal hazards at significant distances from the cone (for example, the nearest town of Melipeuco 18 km from the summit), the eruptions can cause significant economic and social disruption due to the destruction of infrastructure by lahars or closure of the national park in which the volcano is located. Even relatively minor eruptive events, such as the Strombolian paroxysms in January, 2008 andApril, 2009, create hazards due to the presence of near-summit glaciers, which are highly susceptible to melting and lahar generation during vigorous fountaining.

Volcán Llaima displays a variety of eruptive behaviors. Violent-Strombolian paroxysms, as on January 1, 2008, often mark the onset of sporadic but prolonged eruptive activity (1-5 years). Paroxysms terminate within hours to a few days and the activity shifts to smaller explosions that typically occur at regular intervals (10-60 minutes), as is observed at Stromboli Volcano, Italy [START_REF] Metrich | Conditions of Magma Storage, Degassing and Ascent at Stromboli: New Insights into the Volcano Plumbing System with Inferences on the Eruptive Dynamics[END_REF][START_REF] Metrich | Crystallization driven by decompression and water loss at Stromboli volcano (Aeolian Islands, Italy)[END_REF]Rosi et al., 2000 and references therein), and last over days or weeks. Lava extrusion may follow explosive activity, and these lavas can be voluminous (e.g. 1955-1957, up to ~0.5-0.8 km 3 based on the thickness and extent of the lava flows). Lavas are the dominant volumetric fraction of erupted magma during the rapid construction of the late Holocene Llaima edifice (≤3-5 ka). Passive degassing with no magma extrusion [START_REF] Kazahaya | Excessive degassing of Izu-Oshima volcano: Magma convection in a conduit[END_REF][START_REF] Shinohara | Excess Degassing from Volcanoes and Its Role on Eruptive and Intrusive Activity[END_REF] frequently occurs between periods of eruptive activity.

Assessing shifts in eruptive styles is crucial for volcanic hazard assessments, and is only possible by identifying how and where magmas are stored beneath the volcano and understanding the triggering mechanisms of eruptions. By combining whole-rock, mineral, and melt inclusion chemistry, we show that magmas at Llaima are stored as crystal mush at very shallow depths where they undergo degassing during crystallization. They probably are stored as many individual batches in a complex of anastamosing dikes.

Eruptions are triggered by the injection of hotter and more mafic magma, which amalgamates contributions from multiple batches just prior to eruption. Magma recharge is essential for long-term eruptive activity, and a high frequency of recharge events is evidently at the root of the "hyperactive" nature of Volcán Llaima, where the average recurrence interval of eruptive activity has been ~5.6 yrs since the beginning of the reliable, high-resolution historical record (~1850 AD; [START_REF] Dzierma | Eruption time series statistically examined: Probabilities of future eruptions at Villarrica and Llaima Volcanoes, Southern Volcanic Zone, Chile[END_REF].

Eruptive history and studied samples

The Llaima edifice can be divided into three stages of eruptive activity (Fig. 1; [START_REF] Naranjo | Geología del volcán Llaima, Región de la Araucanía, Carta Geológica de Chile, Serie Geología Básica[END_REF]. The first and oldest is dominantly effusive, with the construction of the pre-Holocene edifice by lava flows, basaltic-andesitic to dacitic in composition. The eruptive behavior then switched to a period of explosive activity around 13 ky before present with the eruption of a large mafic ignimbrite, the deposits of which can be found up to 20 km from the inferred source. This second era ended around 9.5 ky before present with the Plinian eruption of the most evolved (dacitic) magma found at Llaima. The volcano subsequently shifted to a period of cone-forming, less explosive activity with the eruption of numerous lava flows of basalt to basaltic-andesite, associated with Strombolian activity. In this study we focus on the youngest manifestations (~1850-2008 AD) of the latest stage of cone-building activity.

Four tephra units from the latest Historic eruptions were selected in order to constrain the magma storage conditions and depths over the last 150 years at Volcán Llaima. The tephra units selected were: (1) the 2008 tephra, (2) the 1957 tephra, (3) the upper and (4) the lower tephra layers of the Fissural 3 eruption, which has been dated at ~1850 AD by one 14 C age. The 2008 and 1957 eruptions are products of the central summit vents, whereas the Fissural 3 (~1850 AD) eruption is from the northeastern terminus of a series of two lower-flank fissures on the northeastern flank (Fig. 1).

The 2008 eruption began "tentatively" in May, 2007, but this episode ramped up dramatically on January 1, 2008 with the sudden onset of a violent Strombolian eruption (~12 hrs), which produced the sampled tephra. The eruptive activity then diminished significantly in vigor and minor explosive activity continued through 2008 and part of 2009, with some repose periods. Lava flows were produced in January-February and July, 2008, and then again after the early April, 2009 paroxysm. The 1957 eruption lasted two years and produced three voluminous lava flows on the northern, northeastern, and eastern flanks of the volcano (Fig. 1). Little is known about the temporal evolution of the eruption, but only one tephra unit could be stratigraphically linked to this eruption, and it must correspond to a paroxysm. The Fissural 3 (~1850 AD) eruption occurred from two parallel chains of cones on the northeastern flank of the volcano. Related tephra directly underlie the 1957 tephra layer. Two locally thick tephra layers ("lower" and "upper") could be traced back to the two sets of cones. Voluminous lava flows are associated with the younger set of cones, and are covered with tephra. The studied eruptions produced basaltic andesites (Tables 1 and2) containing about 15-35 vol% plagioclase, 1-4 vol% olivine, and minor clinopyroxene (Fig. 2). Scoriae are generally less crystalline (up to 20-35 vol% phenocrysts) than the subsequent lava flows (up to 55 vol% phenocrysts).

Analytical methods

Mafic scoriae 2-5 cm in diameter were collected from two proximal stratigraphic sections located to the northeast of the volcano (in the direction of prevailing winds, Fig. 1). Samples were gently crushed and olivines were hand-picked under a binocular microscope from the >0.5 mm size fraction. These olivines were mounted in epoxy and polished. Three textural categories of glass were analyzed (Fig. 3a): (1) closed melt inclusions in olivine cores that were isolated from the host melt, (2) open melt inclusions, which consist in embayments at the olivine rims, and (3) matrix glasses attached to olivine grains. Melt inclusions analyzed are at least 40 µ in diameter and reported EPMA, SIMS and LA-ICP-MS measurements correspond to single analyses within an inclusion. Experimental reheating of the inclusions before analysis was not necessary, as only glassy melts were analyzed. Melt inclusions containing vapor bubbles were avoided for H 2 O and CO 2 analysis (except two inclusions which contained a small shrinkage bubble) to ensure accurate measurements of volatile contents. Whole-rock major and trace element compositions of the tephra and the associated lava flows were obtained by X-ray fluorescence analyses at the University of Lausanne (UNIL; Switzerland).

Major element analyses by electron microprobe were performed on glasses (~280 analyses for 185 closed melt inclusions, 42 open melt inclusions, and 50 matrix glasses; including S and Cl) and ~150 host olivines (JEOL 8200 electron microprobe at UNIL). Glasses were analyzed with a 15 µm diameter beam, and variable beam currents: K and Na were analyzed first at 2 nA, Cl, S, and P were analyzed last at 30 nA and the other major elements were analyzed in between at 10 nA. A combination of glass and mineral standards was used. Counting time was 8/4 s (peak/background) for K and Na, 10/5 s for Si and Al, and 20/10 s for the other elements. Host olivines were analyzed at 40 nA with a focused beam, and simple four to six point zoning profiles were obtained for all of them. The relative error on major elements is around 2% for SiO 2 , Al 2 O 3 and CaO, 3% for FeO and MgO, 10% for TiO 2 , 15% for K 2 O, 35% for Na 2 O, and 40% for MnO.

H 2 O and CO 2 contents in ~120 of the glasses analyzed for major elements were measured by secondary ion mass-spectrometry (SIMS) using a CAMECA ims-6f at Arizona State University (ASU; USA). Gold-coated samples were bombarded by a 20 µm diameter primary beam of 16 O -ions, with a 10 nA current, following the technique described in [START_REF] Pan | The Pressure and Temperature-Dependence of Carbon-Dioxide Solubility in Tholeiitic Basalt Melts[END_REF]. The analysis area was pre-sputtered for four minutes to remove surface contamination. Counting cycles were 5 s for both H 2 O and CO 2 , and single analyses are average values of 10 to 30 cycles. Secondary ion intensities were normalized to 18 O and converted to weight concentrations using a basaltic standard with well established H 2 O and CO 2 concentrations (synthesized by Gordon Moore and Kurt Roggensack at ASU and Torsten Vennemann at UNIL; Table 3). Only FTIR measurements were considered for the H 2 O and CO 2 contents of the standards. Errors were obtained from counting statistics, and precision within an analysis session is generally on the order of ±8% [START_REF] Pan | The Pressure and Temperature-Dependence of Carbon-Dioxide Solubility in Tholeiitic Basalt Melts[END_REF]. Background signals for 1 H and 12 C, converted to weight percent H 2 O and ppm CO 2 , are 0.25-0.73 wt% and 20-40 ppm respectively, as measured on nominally volatile-free olivine hosts. High background signals due to epoxy degassing under vacuum (although the epoxy was carefully selected) were not problematic relative to measured H 2 O and CO 2 contents. Reported data are background corrected.

Trace element abundances in ~150 of the studied glass inclusions were analysed by laser ablation inductively coupled plasma mass-spectrometry (LA-ICPMS) at UNIL, using an UP193-FX ArF 193 nm excimer ablation system (New Wave Research, USA) interfaced to an Element XR sector field ICPMS (ThermoFisher Scientific, Germany). Operating conditions of the laser included an on-sample fluence of 4.7 J/cm 2 , 5 Hz repetition rate and 25-50 μm pit sizes. Helium was used as a cell gas. The acquisition times for the background and the ablation interval amounted to ~ 70 and 40-60 s, respectively. All isotope masses were scanned using four acquisition points per peak, the mass window having been set at 20% of the peak width. Dwell times per point were 5 ms for 42 Ca, 88 Sr and 137 Ba and 10 ms for other isotopes. The ThO + /Th + and Ba ++ /Ba + ratios were optimised to ~ 0.1 % and 2.0 %, respectively. The NIST 612 synthetic glass was used for external standardisation. The average element abundances were taken after [START_REF] Pearce | A compilation of new and published major and trace element data for NIST SRM 610 and NIST SRM 612 glass reference materials[END_REF]. 42 Ca served as an internal standard. Intensity vs. time data were reduced using SILLS [START_REF] Guillong | SILLS: A MATLAB-based program for the reduction of laser ablation ICP-MS data of homogeneous materials and inclusions[END_REF].

Assessing post-entrapment modification

Prior to interpretation as recorders of magmatic processes, melt inclusions must be assessed with regard to the possibility that their compositions have been modified by secondary or post-entrapment processes. Such processes include boundary-layer melt entrapment during crystal growth, post entrapment crystallization (or melting) of the inclusion walls, FeO loss by coupled Fe-Mg diffusion through the host, and water loss by proton or molecular diffusion through the host olivine. These processes have been addressed in detail by [START_REF] Danyushevsky | Re-equilibration of melt inclusions trapped by magnesian olivine phenocrysts from subduction-related magmas: petrological implications[END_REF]Danyushevsky et al. ( , 2002aDanyushevsky et al. ( , and 2002b) and a review of the ways to test for and correct them can be found in [START_REF] Kent | Melt Inclusions in Basaltic and Related Volcanic Rocks[END_REF].

Diffusion-related chemical boundary layers may form in melt surrounding rapidly grown crystals due to the consumption of compatible elements and the exclusion of incompatible elements by the crystallizing mineral. If modified boundary-layer melt is trapped as a melt inclusion, it will not be representative of the host magma composition. Boundary layer entrapment has been shown to be negligible for melt inclusions larger than 50 µm [START_REF] Lu | Diffusional Gradients at the Crystal/Melt Interface and Their Effect on the Composition of Melt Inclusions[END_REF], which is the case for the vast majority of the melt inclusions analyzed in this study. In addition, we detect no correlation between melt inclusion diameter and Mg#, Fe-or Mg-content (Fig. 1 in supplementary material). We infer that diffusion-related boundary layer processes did not significantly affect these melt inclusions.

Post-entrapment modification of a melt inclusion by equilibration with the host crystal will affect its composition. Assessing equilibrium between melt inclusions and their olivine hosts requires knowledge of the oxidation state of the melt at the time of entrapment (i.e. the exact FeO content of the melt inclusion).

Due to the absence of analyzable Fe-Ti-oxide pairs in Llaima magmas, we do not have good constraints on their oxidation states. Melt inclusions analyzed in this study define a good positive correlation between the Mg# of the inclusion and the Fo content of the olivine, suggesting equilibrium, but they appear to span a range in oxidation states between QFM and NNO (Fig. 4). This could either represent a real variability of the oxidation state of the melt inclusion population due to variable degrees of degassing (loss of some S species such as H 2 S would cause oxidation of the melt; [START_REF] Kelley | Water and the Oxidation State of Subduction Zone Magmas[END_REF], or the spread could be due to post-entrapment crystallization or melting of inclusion walls, which would shift the inclusions away from equilibrium in opposite directions.

Post-entrapment melting of inclusion walls is a process that has not yet been invoked in melt inclusion studies, but we believe it could be occurring in Llaima magmas due to their shallow storage at relatively low temperatures, and their interaction with hotter recharge magmas just prior to eruption, as discussed below. For simplicity, we chose to consider that the studied melt inclusions were in equilibrium with the olivine hosts at an oxygen fugacity of NNO. Any deviation from equilibrium was attributed to either postentrapment crystallization (PEC, max 7 vol%) or post-entrapment melting (PEM, max 5 vol%; Table 1 in supplementary material). To attest the reliability of this approach, the same procedure was repeated for an assumed oxygen fugacity of QFM. This yielded minimally different results (max 8 vol% PEC and 4 vol% PEM; Table 1 in supplementary material). Initial compositions of inclusions that were not in equilibrium with the host olivine were recalculated by incremental (0.01 wt %) addition or removal of olivine to the inclusion until achieving equilibrium with the host mineral, using the PETROLOG 2.1 software (Danyushevsky et al., 2002a). The amount of post-entrapment modification experienced by the most affected of these inclusions has a negligible effect on all elements other than Fe and Mg, and sometimes Si. All data shown in figures and discussed in the text are corrected values considering an oxygen fugacity of NNO. H 2 O-CO 2 -saturation pressures were calculated using the post-entrapment corrected values of volatile contents.

Melt inclusions may also be affected by diffusive exchange with the host olivine; i.e. Fe loss by coupled Fe-Mg exchange, and/or water gain or loss by proton or molecular diffusion through the host, at relatively short timescales (Danyushevsky et al., 2002a;[START_REF] Gaetani | Modeling the major-element evolution of olivine-hosted melt inclusions[END_REF][START_REF] Hauri | SIMS analysis of volatiles in silicate glasses, 2: isotopes and abundances in Hawaiian melt inclusions[END_REF][START_REF] Portnyagin | Experimental evidence for rapid water exchange between melt inclusions in olivine and host magma[END_REF]. Fe loss was tested for on a plot of total Fe (FeO*) versus MgO by comparing the melt inclusion data to the whole-rock data (Fig. 5). Fe-loss would move the melt inclusion data vertically downwards, below the whole-rock trend. Since this is not the case here, we infer that no Fe was lost.

Water diffusion is more difficult to evaluate. Water exchange by proton diffusion between the matrix melt and the melt inclusion is rapid and accommodated by a change in the oxidation state of the inclusion (Danyushevsky et al., 2002a). Vacancy diffusion through olivine was observed to occur on similarly fast time scales [START_REF] Gaetani | Mechanisms and timescales for reequilibration of water in olivine-hosted melt inclusions[END_REF], promoting rapid re-equilibration of the melt inclusion oxidation state with the ambient, and therefore leaving no sign of water re-equilibration. In the case of Llaima, the low CO 2 contents in melt inclusions strongly imply entrapment at shallow depths, since the migration of CO 2 through host minerals (apart from fractures) is generally considered to be of no consequence. Shallow magma storage and crystallization is also suggested by the mineral assemblage and element fractionation trends, as discussed below. Considering that the melt inclusions were trapped at relatively shallow depths, we do not believe that they experienced much re-equilibration of their H 2 O contents, because they probably have not travelled much -if at all -between the level of entrapment and the level of storage prior to eruption. The consequences of such minor re-equilibration, a slight under-estimation of the H 2 O-CO 2saturation pressure, would not change the interpretation below.

Results

Melt inclusions and their olivine hosts

Olivine crystals are generally euhedral to subhedral. Some crystals are broken, others are slightly rounded or resorbed, but the main deviation from euhedral shapes is due to crystals aggregating to each other and merging into one grain with a complex shape (Fig. 3b or d). Very thin melt films persist between some grains, enabling us to identify individual crystals in the aggregate. Most olivines host a few large inclusions and/or embayments (Fig. 3a andd), but some crystals contain numerous small wormy inclusions often containing small vapor bubbles (Fig. 3e andf). These crystals probably reveal complex histories of repeated crystallization/melting cycles, as observed at Stromboli [START_REF] Metrich | Conditions of Magma Storage, Degassing and Ascent at Stromboli: New Insights into the Volcano Plumbing System with Inferences on the Eruptive Dynamics[END_REF][START_REF] Metrich | Crystallization driven by decompression and water loss at Stromboli volcano (Aeolian Islands, Italy)[END_REF]. Closed melt inclusions are rounded, wormy, or inverse crystal shapes (Fig. 3g-i). Small euhedral plagioclase crystals were found inside a few inclusions (Fig. 3c). Embayments are either long, narrow tubes or rounded inclusions that remained in communication with the external melt via a "bottleneck" (Fig. 3j-k). Matrix glass usually contains small plagioclase microlites (Fig. 3l), except between two closely neighboring crystals.

Four to six point zoning traverses across olivine host crystals (Fig. 6a) record diverse core compositions and zoning trends in each sample, with cores ranging from Fo 84-67 . It is difficult to define an "equilibrium" population. Rim compositions span a slightly narrower range (Fo 81-75 ), focused around Fo 78- 80 in all four units (Fig. 6b). Zoning profiles record non-zoned, normally zoned, and reversely zoned olivines. Reversely-zoned olivines seem to be the most common.

Major elements

Whole-rock compositions of the four studied units cluster in a narrow range of compositions around evolved basalt to basaltic andesite. The glasses define larger ranges in composition from slightly more mafic up to andesite, with SiO 2 contents ranging from 49-57 wt% compared to 51-54 wt% for the wholerock analyses (Fig. 7, Table 2, Table 1 in supplementary material). The melt inclusion data for the four units overlap with each other and with the whole-rock data from the entire volcano up to ~53.5 wt% SiO 2 .

More evolved melt inclusions diverge from the whole-rock trend towards lower Al and higher Ti contents (down to 13 wt % Al 2 O 3 and >2 wt% TiO 2 compared to 16 wt% Al 2 O 3 and 1.4-1.6 wt% TiO 2 in Llaima andesitic magmas with 56 wt% SiO 2 ). The most evolved compositions are represented by closed melt inclusions, whereas the open melt inclusions and matrix glasses are closer to whole-rock compositions.

Similar observations can be made from plots of total Fe (FeO*) versus MgO (Fig. 5), where closed melt inclusions are in equilibrium with rather evolved olivines (down to Fo 71 ), whereas open melt inclusions and matrix glasses are in equilibrium with less evolved olivine compositions (Fo 77 or greater), and wholerocks with even less evolved olivine compositions (up to Fo 83 ). There are no systematic differences between the studied units.

Temperature was estimated on the basis of Mg partitioning for each glass analyzed, using the olivineliquid thermometer of [START_REF] Beattie | Olivine-Melt and Ortho-Pyroxene-Melt Equilibria[END_REF]. This thermometer can be used without an olivine composition, as it retrieves the temperature at which a liquid would become saturated with olivine at a given pressure (50 MPa in our case). However, it does not take into account the amount of H 2 O dissolved in the melt, and therefore overestimates the temperature of all the closed melt inclusions and part of the open melt inclusions. [START_REF] Medard | The effect of H2O on the olivine liquidus of basaltic melts: experiments and thermodynamic models[END_REF] developed an expression to correct temperatures for the presence of H 2 O in the melt. We used this expression for melt inclusions in which H 2 O contents were measured in order to evaluate the temperature overestimation of the [START_REF] Beattie | Olivine-Melt and Ortho-Pyroxene-Melt Equilibria[END_REF] thermometer. Without considering the effect of dissolved H 2 O in the melt, closed melt inclusions display a relatively wide range in temperatures from ~1000°C to ~1170°C, whereas matrix glass temperatures cluster around 1130-1170°C similarly to open melt inclusions (Fig. 8). When correcting the temperature for the presence of dissolved H 2 O in the melt, closed and open melt inclusion temperatures are lowered by 60-110°C and range from ~900-1100°C for the closed melt inclusions and ~1000-1100°C for the open melt inclusions. Matrix glasses being essentially degassed, their temperatures remain globally unchanged. These matrix temperatures, however, may not be representative of pre-eruptive temperatures because H 2 O was lost upon eruption and not prior to eruption. In order to more accurately compare melt inclusion and matrix temperatures, we chose to consider the temperature distribution on an H 2 O-free melt basis.

Volatiles

Llaima glasses have H 2 O contents of 0-4 wt% (one outlier at 4.4 wt%; Fig. 9, Table 1 in supplementary material), and low CO 2 contents of 0-400 ppm (one outlier at 511 ppm). Matrix glasses are degassed, but open inclusions usually still contain H 2 O and CO 2 , in some cases as much as in closed inclusions. H 2 O-CO 2 -saturation pressures were estimated with the model of [START_REF] Papale | The compositional dependence of the saturation surface of H2O+CO2 fluids in silicate melts[END_REF], wherein FeO and Fe 2 O 3 were recalculated for an oxygen fugacity buffered at NNO. Strikingly low entrapment pressures are obtained, primarily due to low CO 2 contents. These rarely reach values of 100 MPa, and only a few inclusions indicate pressures up to 150 MPa (Fig. 10). Assuming an average density of 2200 kg/m 3 [START_REF] Finn | Gravity Evidence for a Shallow Intrusion under Medicine Lake Volcano, California[END_REF] and a volume of ~250 km 3 for the volcanic edifice (a right circular cone ~2.4 km high and ~10 km in radius at its base), the pressure at the base of the edifice is on the order of 18 MPa. Estimated H 2 O-CO 2 -saturation pressures thus yield shallow entrapment depths of 0-4 km below the base of the edifice with only a few inclusions recording depths approaching 6 km. Only the 1957 inclusions seem to define a coherent degassing trend over a large range of pressures. H 2 O and CO 2 contents do not correlate with major element concentrations (coefficients of determination from linear regressions, R² < 0.2; Table 4, Fig. 11), nor do H 2 O-CO 2 -saturation pressures correlate with a fractionation index such as K 2 O (R² ≤ 0.1, except maybe for the 1957 inclusions; Table 4, Fig. 10).

Sulfur and Chlorine contents (up to 1300 ppm Cl and 1500 ppm S; Table 1 in supplementary material) are in the range of values observed at many arc volcanoes (up to 2000 ppm Cl and 2500 ppm S; [START_REF] Wallace | Volatiles in subduction zone magmas: concentrations and fluxes based on melt inclusion and volcanic gas data[END_REF]. They are weakly positively correlated with each other (R² = 0.3-0.4; Fig. 12), but are strongly correlated when normalized to a fractionation index such as K 2 O (R² ≥ 0.6). The S/Cl ratio varies negatively with K 2 O, suggesting that S is preferentially degassed as the magma evolves, as previously suggested by [START_REF] Sisson | H2O in basalt and basaltic andesite glass inclusions from four subduction-related volcanoes[END_REF]. No simple correlations can be defined between S or Cl and H 2 O or CO 2 . Some closed melt inclusions display very low S (even below detection limit) but H 2 O, CO 2 , and Cl contents that are not anomalous. Careful examination of these inclusions revealed healed cracks from which S and small amounts of the other volatiles may have leaked out of the inclusion prior to or during eruption. S, therefore, could be a good proxy for assessing volatile leakage through cracks. Twenty-six of the studied inclusions have been compromised by volatile leakage. Their major and trace element compositions have been kept in the data set, although their volatile contents were excluded.

Trace elements

We focus on Sr and Zr contents and ratios to evaluate crystallization processes, Zr being an incompatible element and Sr being a compatible element in plagioclase (the dominant crystallizing phase at Llaima). Sr contents of the glasses range from 330-590 ppm, and Zr contents range from 50-150 ppm.

Sr/Zr ratios vary over a large range (from 2-10) and are strongly correlated with both TiO 2 and Al 2 O 3 (Fig. 13, Table 2, Table 1 in supplementary material). The trends defined by the four units overlap, and are mainly defined by closed melt inclusion populations. Sr/Zr ratios of open melt inclusions and matrix glasses are identical and define a narrower range (3.5-7), similar to the whole-rock Sr/Zr ratios (4.5-7).

Whole-rocks, however, display slightly higher Al 2 O 3 and lower TiO 2 contents than the matrix glass and open melt inclusions.

Discussion

Magma storage

Major elements, trace elements, and volatile contents all suggest that Llaima magmas are stored at immediately sub-edifice depths. Glass compositions extend the whole-rock trend toward lower Al and higher Ti contents (Fig. 7). Major element models of a fractionation trend leading to the combination of high TiO 2 (suppression of FeTi-oxide stability) and anomalously low Al 2 O 3 (expansion of plagioclase stability at the expense of ferromagnesian phases) support extensive crystallization and degassing at shallow storage depths, in relatively dry conditions. Trace elements (e.g. Sr/Zr) record the same conditions. The trend toward low Sr/Zr and the strong correlation of this ratio with Al 2 O 3 and TiO 2 (Sr/Zr increasing with Al 2 O 3 and decreasing with TiO 2 ; Fig. 13) are consistent with voluminous plagioclase crystallization and suppression of FeTi-oxide stability during shallow magma evolution. Due to the high plagioclase contents of Llaima magmas, and the absence of FeTi-oxides, whole-rock compositions are shifted towards higher Al 2 O 3 and lower TiO 2 contents relative to glasses with the same Sr/Zr. Finally, extremely low H 2 O-CO 2 -saturation pressures around 40-70 MPa are recorded by the melt inclusions (Figs. 9 and 10). In conclusion, a significant fraction of erupted Llaima magmas has been stored at depths as shallow as 0-4 km below the base of the edifice.

The closed melt inclusion trend toward low Sr/Zr and the strong correlation of this ratio with Al 2 O 3 and TiO 2 is reproduced by a simple Rayleigh fractionation model (FC; Fig. 13b). Olivine and plagioclase were assumed to crystallize in proportions 1:9, and the distribution coefficients used for crystallization modeling are reported in Table 5. By modeling the melt inclusion composition trend, we are not looking at a true fractional crystallization process in which crystals are physically separated from the liquid, but rather at the evolution of the interstitial liquid composition during crystallization. Up to 55 wt% crystallization (i.e. 54 vol% crystallization considering densities of 2640 kg/m 3 , 2680 kg/m 3 , and 3320 kg/m 3 for the liquid, plagioclase and olivine respectively) is required to reproduce the composition of the most evolved melt inclusions from the least evolved liquid composition (highest Sr/Zr), which may have already experienced some crystallization. These maximum crystallinities are higher than crystallinities observed for the tephra (up to 30 vol% crystals). Assimilation could have been taking place during magma storage and crystallization, as suggested by a U-series study conducted on Llaima magmas [START_REF] Reubi | Assimilation of the plutonic roots of the Andean arc controls variations in U-series disequilibria at Volcan Llaima, Chile[END_REF]. To ensure assimilation is not significantly affecting the Sr/Zr ratios or the amount of crystallization implied, we calculated an assimilation-fractional crystallization (AFC) trend, following the method of [START_REF] Depaolo | Trace-Element and Isotopic Effects of Combined Wallrock Assimilation and Fractional Crystallization[END_REF]. We considered the assimilation of 10 wt% of a granite from the surroundings (Table 5; [START_REF] Lucassen | Distinguishing crustal recycling and juvenile additions at active continental margins: the Paleozoic to recent compositional evolution of the Chilean Pacific margin (36-41°S)[END_REF], and kept all the other parameters identical to the FC model. The AFC trend almost overlaps with the FC trend, yielding similarly high amounts of crystallization (up to 55 wt% ~ 54 vol%; Fig. 13b) and implying once more that magmas at Llaima are stored in a highly crystalline state. Storage at high crystallinity is consistent with thermal models of magma reservoirs that imply extended residence at ≥ 50-60 vol% crystals [START_REF] Huber | Homogenization processes in silicic magma chambers by stirring and mushification (latent heat buffering)[END_REF][START_REF] Koyaguchi | A two-stage thermal evolution model of magmas in continental crust[END_REF]. The fact that magmas are not eruptible at crystal contents ≥ 55-60 vol% (e.g., [START_REF] Marsh | On the crystallinity, probability of occurrence, and rheology of lava and magma[END_REF][START_REF] Vigneresse | Rheological Transitions During Partial Melting and Crystallization with Application to Felsic Magma Segregation and Transfer[END_REF] implies some magma remobilization process prior to eruption, and is in agreement with the lower crystal fractions measured in the erupted products.

The prolongation of the whole-rock trend by evolved melt inclusion compositions is defined primarily by the closed melt inclusion population (Fig. 7), which includes compositions that are not representative of any erupted magma. We infer that the closed inclusions represent the evolved interstitial liquid produced by extended fractional crystallization of magma during the formation of a mush. This is in agreement with: (1) The observation of a high volume fraction of plagioclase crystallization. (2) The widespread occurrence at Llaima of very crystal-rich erupted mafic magmas, particularly as lava flows (up to 55 vol% crystals). (3) The observation of olivine and olivine-plagioclase multi-crystal aggregates, wherein solid-solid grain boundaries suggest that they were highly concentrated during storage and that they were evolving toward completely solid intrusions. And (4) the presence of small euhedral plagioclase crystals inside open melt inclusions (Fig. 3c) which have the same evolved compositions as the closed melt inclusions without such plagioclase crystals. The latter observation also implies that crystals were densely packed prior to eruption.

We call particular attention to the divergence of the evolved extension of the melt inclusion trend from the Llaima whole-rock trend and emphasize that the melt inclusions with the highest TiO 2 and lowest Al 2 O 3 (at 56-58 wt.% SiO 2 ) do not correspond to any erupted andesitic magma compositions. Such melts may be present in many crystal-rich Llaima magmas as mixing components related to immediately preeruptive crystal mush remobilization, but their exceptional compositions strongly support the conclusion that they formed as interstitial liquids during mush formation at shallow depths (≤4 km beneath the base of the edifice), rather than in volumetrically significant magma chambers wherein crystals were being physically separated from eruptible volumes of evolved magmas.

Melt inclusion populations from single eruptive events do not seem to record magma evolution along single-stage ascent and decompression paths. H 2 O and CO 2 contents in melt inclusions do not correlate systematically with any major or trace element index of progressive magma evolution (Fig. 11,Table 4).

H 2 O-CO 2 degassing trends are not observed (except perhaps for the 1957 unit; Fig. 9), and H 2 O-CO 2saturation pressures do not correlate with a fractionation index such as K 2 O (Fig. 10). Pressure (first boiling) is the primary factor controlling dissolved H 2 O-CO 2 contents; variations in temperature and crystallinity (and hence melt composition) for the four units studied are not significant enough to affect the solubilities. One way of explaining the general lack of correlated degassing and magma evolution trends is to consider magma storage in multiple dike-shaped bodies (Fig. 14). Each body has a slightly different composition, depending on its degree of crystallization amongst other parameters, but all bodies undergo degassing in a similar way (according to the vertical pressure in the dyke body). This situation would not yield a unique correlated degassing and magma evolution trend, rather as many trends as there are bodies. Individual trends are not distinguishable within our dataset, however we do see that at a H 2 O-CO 2 -saturation pressure of about 40-50 MPa, magmas display a range in K 2 O of ~0.8 wt% suggestive of multiple sources. Interpretation of the data may be complicated by CO 2 fluxing from an underlying magma undergoing degassing. However, the perspective of multiple bodies is also required by wide ranges of olivine core compositions (Fo 84-67 , Fig. 6a), which can only be explained by multiple magma sources.

Magma recharge and eruption triggering

The high frequency of historic eruptions at Llaima (one eruption with a VEI ≥ 2 every ~5.6 yrs years; Dzierma and Wehrmann, 2010) implies a high frequency of magma replenishment events. Magma recharge is a potential triggering mechanism, and the recharge magma is likely to be hotter and more mafic than the resident magma. Matrix glasses are commonly slightly more mafic than the closed inclusions, and associated olivine-liquid equilibrium temperatures are amongst the highest registered (1130-1170°C compared to 1000-1170°C for the closed melt inclusions without considering the presence of dissolved H 2 O in the melt, Fig. 8). Olivine zoning profiles record essentially normally zoned and reversely zoned olivines (Fig 6) with reversely zoned olivines being the most common. High Sr/Zr ratios were measured in closed melt inclusions from normally zoned olivines, which is in agreement with a hotter, less evolved recharge magma that has not experienced much crystallization. Conversely, low Sr/Zr ratios were measured in reversely zoned olivines, which originated from the cooler, more evolved resident mush. Olivine rim compositions cluster around Fo 78-80 (Fig 6b) indicating assembly and partial homogenization between reversely zoned crystals originating from the cooler resident magma, and normally zoned crystals derived from the hotter recharge magma.

Little time has elapsed between recharge and eruption, as olivines have not re-equilibrated entirely with the new magma (short diffusion profiles) and a diverse olivine population is preserved in scoria samples.

In contrast, open inclusions and matrix glasses have had time to homogenize. They define a narrower range in major element compositions than do closed inclusions, and form relatively tight clusters in terms of Sr/Zr (Fig. 13). These have intermediate values with respect to the closed inclusion trend, and can be interpreted by diffusive mixing and homogenization of the high-Sr/Zr recharge magma with the low-Sr/Zr evolved interstitial melt in the resident mush. Equilibration of the melt composition over short time scales is possible because the diffusion of elements in melt is five to six orders of magnitude faster (diffusivity of Zr in melt is ~10 -10 -10 -11 m 2 /s; [START_REF] Latourrette | Self diffusion of Mg, Ca, Ba, Nd, Yb, Ti, Zr, and U in haplobasaltic melt[END_REF] than in crystals (diffusivity of Fe-Mg in olivine is ~10 -16 m 2 /s; [START_REF] Costa | Time Scales of Magmatic Processes from Modeling the Zoning Patterns of Crystals[END_REF].

A complex of anastamosing dikes (Fig. 14) would reduce heat loss in this shallow mush environment.

Each new recharge would either intrude partially solidified preexisting dikes or form a new adjacent dike, or contribute to both. When recharge magmas do not act as triggers, a certain fraction of them will be trapped as partly isolated dikes within a zone of concentrated injections, and progressively evolve into mushes. This scenario would lead to the focalization of upward gas percolation through partly molten mush bodies. Remobilization through partial melting and/or mixing with a crystal-poor magma is required to decrease the crystallinity to about 30 vol% prior to eruption as tephra, and could be explained by this process.

Volcán Llaima appears to be a strongly fault-controlled system. It displays two summit craters aligned north-south, parallel to the north-south trending Liquiñe-Ofqui fault bounding the edifice to the east. In addition, several sets of fissural vents are aligned NNE-SSW on the western flank, and east-west on the northeastern flank. Considering the geographical distribution of vents, and the range in depths suggested by the H 2 O-CO 2 -saturation pressures recorded by melt inclusions, dike-like reservoirs at Llaima would be approximately 3-4 km deep, ~5km long and a few tens of meters wide, yielding a volume of ~0.75-1 km 3 per reservoir (Fig. 14). Assuming that the entirety of a reservoir is not tapped upon eruption due to higher crystallinities on the walls, magma remobilization in three to four of such reservoirs is more than sufficient to explain the observed erupted volumes (i.e. 0.5-0.8 km 3 for the 1955-57 eruption). A strongly fault-controlled conduit system would contribute to explain the observed chemical characteristics at Llaima and enhance the proposed mush remobilization scenario. Mixing of diverse magma batches, probably in multiple stages prior to eruption would lead to the observed range in olivine core compositions as well as the poorly correlated relations between melt compositions, volatile contents, and the entrapment pressures calculated for diverse trapped melts present in single, paroxysm-related tephra deposits.

We suggest that petrologic constraints and the historical record of activity are consistent with a high, long-term magma recharge flux consisting in part of frequent but small-volume events. These are essential to maintain the bulk reservoir of many crystal mush-filled dikes at shallow, upper crustal levels. This mush-rich zone could act as an impediment to eruptions until the mass and vigor of a recharge event, or a group of multiple closely spaced events, is able to remobilize enough of the mush plug to allow a gas-rich mixture of recharge magma plus mush to open the conduit to the surface and then continue the eruption. In this scenario, the VEI of the initial activity would be directly proportional to the volume and thermomechanical state of the mush plus the amount of gas furnished by short-term recharge input. In this sense, the volatile contents of the melt inclusions that we have measured are decoupled from eruptive vigor in that most of them represent melts that were trapped at shallow depths, after significant degassing and crystallization of previous replenishment magmas, rather than the necessarily large magma aliquot needed to trigger a paroxysmal eruption. The dynamics of Violent Strombolian eruptions at Llaima are related to the high gas flux of recharge magmas, which is recorded by only a very small fraction of the melt inclusions that are erupted. The maximum value of 4.5 wt% water that we have measured in only one melt inclusion may or may not accurately record the maximum water content of recharge magmas. However, monitoring gas fluxes at the vent and/or around the volcano may actually be effective in predicting the size of an upcoming event during period of unrest.

The notion that the mush zone could be lying at a perched and "vulnerable" state following an initial period of many small replenishment events is consistent with the onset of a violent but short phase of activity on January 1, 2008. This paroxysm was not accompanied by a large or temporally extended seismic crisis (Hugo Moreno, personal communication 2010). The first surface manifestation of activity in May, 2007 does, however, provide an indication of when the mush plug began to increase in melt fraction and it marks the beginning of another seven months of time for the system to reach a state wherein mush bodies could be extensively remobilized. This interpretation is supported by an InSAR study of Volcán Llaima that identified a period of uplift from May, 2007 to November, 2008, following a subsidence period started in November, 2003 [START_REF] Bathke | Inflation and deflation at the steep-sided Llaima stratovolcano (Chile) detected by using InSAR[END_REF]. Through inverse modeling of both periods, a deflating and inflating magma body was inferred, located at a depth of 4-12 km, which is in agreement with the injection of a recharge magma just at the base of the mush inferred from this study (≤4 km below the base of the edifice). In addition, [START_REF] Bathke | Inflation and deflation at the steep-sided Llaima stratovolcano (Chile) detected by using InSAR[END_REF] estimated an inflation rate ~3 times faster than the deflation rate, meaning a magma supply to the reservoir which is much faster than magma withdrawal, which is also in agreement with the high, long-term magma recharge flux suggested in this study.

More detailed geophysical studies aimed at identifying the number and depths of magma reservoirs beneath Volcán Llaima are essential to verify the proposed storage system. Melt inclusions only contain information from the time of entrapment onwards; i.e. once the magma has started crystallizing. [START_REF] Saito | Magma plumbing system of the 2000 eruption of Miyakejima volcano, Japan, deduced from volatile and major component contents of olivinehosted melt inclusions[END_REF] studied the plumbing system of Miyakejima volcano (Japan) prior to the 2000 eruption.

Geophysical studies reveal the presence of both deep and shallow magma reservoirs, whereas the volatile contents of both olivine-and plagioclase-hosted melt inclusions exclusively record entrapment at the depth of the shallow reservoir. It is the combination of both types of studies (geophysical and petrological) that permitted identification of the timing and location of magma mixing prior to the 2000 eruption. The compositions of the Miyakejima melt inclusions are similar to those from Llaima in many respects, and both systems are highly active and dominantly mafic, but the interpretation of magma evolution and ascent for Miyakejima [START_REF] Saito | Magma plumbing system of the 2000 eruption of Miyakejima volcano, Japan, deduced from volatile and major component contents of olivinehosted melt inclusions[END_REF] differs in many details from that which we infer for Llaima. We do not find it surprising that magmatic systems that are characterized by the eruption of evolved mafic magmas are dominantly recording an overprint by shallow processes.

What can we say about individual eruptions?

The 2008 eruption is probably the most representative of Llaima"s eruptive behavior. It was erupted from the summit vent, and was of small to moderate volume, like most of Llaima eruptions documented in the historical record [START_REF] Naranjo | Geología del volcán Llaima, Región de la Araucanía, Carta Geológica de Chile, Serie Geología Básica[END_REF]. The tephra deposits of such eruptions are generally poorly preserved due to their limited thickness and restricted lateral extents, which is why we have studied only one of these common eruptions. After two years of activity, the amount of erupted magma during the 2008-2009 eruption is a small fraction of the 1955-1957 eruptive volume, and is also substantially less than the Fissural 3 (~1850 AD) eruption and the other large historic events. This is the only historic eruption, which has been sampled in detail during eruption on the basis of temporal control (25 wholerock samples). The tephra erupted on January 1, 2008 (~51.3-51.7 wt% SiO 2 ; 1.01-1.05 wt% TiO 2 ; Fig. 2 in supplementary material) and the February, 2008 lava (~52.0-52.1 wt% SiO 2 ; 1.11-1.13 wt% TiO 2 ) are two distinct but apparently internally homogeneous magma batches. The 2009 lava is intermediate in composition between these two (~51.7-52.0 wt% SiO 2 ; 1.05-1.06 wt% TiO 2 ). Melt inclusions record shallow magma storage as crystal mush, in a context of anastamosing dikes, as discussed above.

The 1955-57 eruption of Llaima is the most voluminous of the six large historic eruptions. Analyses of 22 lava samples from three lava flows on different sectors of the cone define a relatively narrow range of compositions (~51.7-52.5 wt% SiO 2 ; 0.99-1.05 wt% TiO 2 ). H 2 O-CO 2 data from 1957 melt inclusions are the only set of analyses that appear to approximate evolution along a closed-system degassing trend. This may reflect the relative importance of combined ascent (decompression) and degassing of magma prior to eruption. A potential correlation between H 2 O-CO 2 -saturation pressures and a fractionation index such as K 2 O for the same unit (Fig. 9), suggests that the magma evolved by essentially plagioclase crystallization while ascending and degassing. The data from the 1957 tephra span a narrow range in temperature on an H 2 O-free melt basis (1110-1150°C; Fig. 8), with similar values for both melt inclusions (1110-1140°C) and matrix glass (1120-1150°C). All of these observations tend to suggest that the magma behaved primarily as a single coherent batch, evolving and degassing while ascending. A relatively limited participation of crystal mush bodies in an advanced state of solidification is consistent with the occurrence of significant lava-forming eruptions in 1945 and 1949, as it implies that this 10 year period was characterized by an unusually high rate of voluminous magma recharge/pulses. Olivines record a variety of core compositions, implying that many of them were entrained from multiple sources and incorporated into ascending magma. The large volume and relative compositional simplicity of this eruptive episode may signal that the fraction of recharge magma in the erupted products was unusually large for Llaima.

Closed melt inclusions from the lower Fissural 3 (~1850 AD) unit are slightly more evolved and they record slightly cooler olivine-melt temperatures than glasses from the other units (Fig. 8). The lower Fissural 3 (~1850 AD) magma may have experienced a slightly longer repose period before its extrusion, compared to the other magmas. This hypothesis is in agreement with lower 226 Ra/ 230 Th, suggesting longer residence time or mingling with an older (few kyr) mush [START_REF] Reubi | Assimilation of the plutonic roots of the Andean arc controls variations in U-series disequilibria at Volcan Llaima, Chile[END_REF]. Mafic magma recharge events that trigger eruptions on the lower flanks of Llaima, such as the Fissural 3 (~1850 AD) event, may encounter a more heterogeneous and partly more evolved family of mush bodies. This would be consistent with the relatively large range of compositions present in this eruption (~51.8-53.0 wt.% SiO 2 , and one at 54.3; 1.02-1.20 wt.% TiO 2 ). Flank lavas tend to be more heterogeneous at the level of single eruptions than are those erupted from the summit vents, due to lower long-term magma fluxes away from the main ascent route for magmas (Fig. 14).

Although subtle distinctions between individual eruptions are observed, general trends dominate.

Crystallinities, whole-rock and melt inclusion compositions, and volatile contents are very similar for the four studied units. Olivine rims converge toward the same Fo content (Fo 77-79 ), and matrix temperatures are very similar (~1130°C). The volcano seems to be repeating itself from one eruption to the next and magmas are at similar conditions just prior to eruption. These observations are important for hazard assessment as they make Volcán Llaima a potentially predictable volcano.

Conclusions

Volcán Llaima has produced fundamentally similar magmas in terms of whole-rock compositions (evolved basalt to basaltic andesite), mineralogy (plagioclase, olivine, and minor clinopyroxene), and crystallinity (30-55 vol% crystals) during the growth of the late Holocene edifice. The melt inclusions analyzed in this study are in part within the same range of compositions as whole-rock samples from the entire late Holocene edifice and they are similar from one eruption to the next. Matrix glass compositions, Fo-contents of olivine rims, and calculated olivine-melt temperatures are almost identical for all four studied eruptions. The volcano appears to have been operating in a repeatable state and erupting on short time scales for at least 1000 years. If Llaima is locked into a highly repetitive eruptive mode, including the processes that operate in its sub-volcanic plumbing-system, it offers volcanologists the opportunity to render it a predictable volcano when seismic monitoring results are integrated with these and subsequent petrologic insights.

Characterization of a large number of olivine-hosted melt inclusions from four historical tephra units at Volcán Llaima (2008 and 1957 summit eruptions, Fissural 3 (~1850 AD) eruption) reveals that magmas are stored at very shallow depths (dominantly ≤4km, 40-70 MPa), and that they undergo intense degassing and crystallization. Llaima magmas are most probably stored as many individual batches of crystal mush, perhaps in anastamosing dikes. This type of magma storage and solidification is probably common at many dominantly mafic arc volcanoes, but Llaima may be unusual in its capacity to maintain crystal mush bodies in a perched thermal state so that they retain sufficient melt fractions to render them frequently susceptible to remobilization and recycling in subsequently erupted magmas. We offer this working hypothesis as a potential framework for evaluating the eruptive mechanisms (through numerical modeling for example, in progress) and eventually understanding the shifts in eruptive styles at Llaima.

Acknowledgements

This project was supported by the Swiss NSF grant #20_125019 to Dungan. We thank Jose-Antonio Naranjo, Hugo Moreno, Daniel Selles and the SENAGEOMIN for their help in the field and for providing access to the volcano during the period of eruptive activity in 2008. We thank Richard Hervig and Linda Williams for providing access to the SIMS laboratory and for their help during analysis, as well as Alexey Ulianov for providing access to the LA-ICP-MS laboratory and for his help running the instrument. We would also like to thank O. Reubi and L. Cooper for their useful advices and the fruitful scientific conversations we have had, as well as Tom Sisson and Genji Saito for their constructive reviews. 4: Coefficients of determination obtained from linear regressions to evaluate the degree of correlation between volatiles and major elements and between different volatile species. SatP is the H 2 O-CO 2 saturation pressure obtained using the model of [START_REF] Papale | The compositional dependence of the saturation surface of H2O+CO2 fluids in silicate melts[END_REF] considering a temperature of 1130°C, UF3 = Upper Fissural 3 (~1850), and LF3 = Lower Fissural 3 (~1850).

Unit

Olivine

Ref.

Plagioclase

Ref.

D Ti

0.08 [START_REF] Pedersen | Basaltic Glass with High-Temperature Equilibrated Immiscible Sulfide Bodies with Native Iron from Disko, Central West Greenland[END_REF]) 0.197 (Bindeman et al., 1998) D Al 0.015 [START_REF] Beattie | Systematics and Energetics of Trace-Element Partitioning between Olivine and Silicate Melts -Implications for the Nature of Mineral Melt Partitioning[END_REF] 1.63 [START_REF] Higuchi | Partition of Trace Elements between Rock-Forming Minerals and Host Volcanic Rocks[END_REF]) D Sr 0.014 [START_REF] Arth | Behavior of Trace-Elements during Magmatic Processes -Summary of Theoretical Models and Their Applications[END_REF] 1.83 [START_REF] Arth | Behavior of Trace-Elements during Magmatic Processes -Summary of Theoretical Models and Their Applications[END_REF]) D Zr 0.012 [START_REF] Arth | Behavior of Trace-Elements during Magmatic Processes -Summary of Theoretical Models and Their Applications[END_REF] 0.048 [START_REF] Arth | Behavior of Trace-Elements during Magmatic Processes -Summary of Theoretical Models and Their Applications[END_REF]) Proportion 0.1 0.9 (T-00-56;[START_REF] Lucassen | Distinguishing crustal recycling and juvenile additions at active continental margins: the Paleozoic to recent compositional evolution of the Chilean Pacific margin (36-41°S)[END_REF]). All concentrations are in wt% except for Sr and Zr which are in ppm. Full and dashed lines correspond to olivine-liquid equilibrium for an Fe-Mg exchange coefficient k D of 0.3±0.03 [START_REF] Putirka | Thermometers and barometers for volcanic systems[END_REF]. Data considering an oxygen fugacity of QFM plots at slightly lower Mg# than the data considering an oxygen fugacity of NNO. In this study we assumed an oxygen fugacity of NNO and melt inclusion compositions were corrected considering that they were shifted out of equilibrium due to post-entrapment crystallization (PEC; shifted to the left of the equilibrium curves) or post-entrapment melting (PEM; shifted to the right of the equilibrium curves). for K 2 O and other major elements, and they all yield values lower than 0.2 for all four units (Table 4).

Figure Captions

We thereby infer that magmas were stored in several batches, experiencing multiple degassing and crystallization paths. Assimilation does not seem to affect Sr/Zr ratios; both FC and AFC models imply up to ~55 wt% (i.e. ~54 vol%) crystallization. Model parameters are reported in Table 5.

Figure 14: Schematic view of the dike complex acting as a reservoir for Llaima magmas. This geometry would account for the observed diversity in olivine core compositions, the absence in correlation between degassing and magma fractionation trends, and is in agreement with the faultcontrolled distribution of vents. We believe that individual dikes are about 3-4 km deep, ~5km long and a few tens of meters wide. Magma extrusion from multiple dikes would account for the erupted magma volumes observed. Table 1: Major element, volatile, and trace element composition of all the melt inclusions analyzed. The file contains four spreadsheets, one per tephra unit studied (2008, 1957, UF3, LF3).

Supplementary Material

N S

Figure 1 :

 1 Figure1: Geological map of Volcán Llaima modified after[START_REF] Naranjo | Geología del volcán Llaima, Región de la Araucanía, Carta Geológica de Chile, Serie Geología Básica[END_REF]

Figure 2 :

 2 Figure 2: Top: Mode composition obtained by point-counting on scanned thin sections. The

Figure 3 :

 3 Figure 3: (a) Typical olivine displaying the three types of glasses analyzed, i.e. closed melt

Figure 4 :

 4 Figure 4: Mg# of the analyzed glasses considering an oxygen fugacity of NNO (colored symbols)

Figure 5 :

 5 Figure 5: MgO versus total Fe (FeO*) contents of glass (colored symbols), whole-rocks from the

Figure 6 :

 6 Figure 6: (a) Olivine zoning profiles (4-6 points) for the 2008, 1957, Upper Fissural 3 (UF3), and

Figure 7 :

 7 Figure 7: Major element variation plots comparing melt inclusion and matrix glass trends (colored

Figure 8 :

 8 Figure 8: Temperature versus Mg number (Mg#) for the 2008, 1957, Upper Fissural 3 (UF3), and

Figure 9 :

 9 Figure 9: H 2 O versus CO 2 plots for the 2008, 1957, Upper Fissural 3 (UF3), and Lower Fissural 3 (LF3) units. Error bars are generally smaller than symbol size, and the few that are not are never larger

Figure 10 :

 10 Figure 10: H 2 O-CO 2 -saturation pressures obtained using the model of Papale et al. (2006) versus K 2 O, a fractionation index, for the 2008, 1957, Upper Fissural 3 (UF3), and Lower Fissural 3 (LF3) units. No simultaneous degassing and evolution trends are visible, except maybe for a slight

Figure 11 :

 11 Figure 11: H 2 O (left) and CO 2 (right) versus K 2 O plots showing no correlation between major elements and volatiles. Coefficients of determination obtained from linear regressions were estimated

Figure 12 :

 12 Figure 12: (a) Chlorine versus Sulfur plot showing a mild positive correlation. Average errors for

Figure 13 :

 13 Figure 13: Sr/Zr vesus Al 2 O 3 (left) and TiO 2 (right) showing clear positive and negative correlations respectively. Closed melt inclusions are marked with closed symbols, open inclusions

Figure 1 :

 1 Figure 1: Melt inclusion diameter versus Mg#, FeO and MgO content of the melt. No trend can be

Figure 2 :

 2 Figure 2: Comparison of 2008 tephra, 2008 and 2009 lava whole-rock compositions. The tephra

Table 2 :

 2 Average whole-rock compositions of lava and tephra. Sr and Zr contents are in ppm, the other concentrations are given in wt%. F3 = Fissural 3 (~1850), Av. is the average, StdDev is the standard deviation, and n is the number of samples analyzed.

			n SiO2 TiO2 Al2O3 FeO* MnO MgO CaO Na2O K2O P2O5 Sr Zr
	2008 Av.	25 51.80 1.07 17.87 10.09 0.16 5.69 9.67 2.78 0.63 0.20 76 452
	2008 StdDev	0.25 0.05 0.17 0.11 0.00 0.19 0.16 0.06 0.05 0.01 7 4
	1957 Av.	21 52.04 1.02 17.82 9.81 0.16 5.70 9.58 3.03 0.64 0.19 72 463
	1957 StdDev	0.18 0.02 0.12 0.05 0.00 0.10 0.14 0.09 0.03 0.01 2 4
	F3	Av.	30 52.41 1.14 17.68 10.18 0.17 5.23 9.24 3.05 0.71 0.21 87 445
	F3	StdDev	0.41 0.06 0.35 0.22 0.00 0.25 0.26 0.16 0.07 0.02 9 9
	Sample	P -MPa T -°C	H2O (wt%)	H2O (wt%)	CO2 (ppm)	CO2 (ppm)
	No.				manometry	FTIR	manometry	FTIR
	A-34		410	1200	5.5 (0.1)	5.2	2655 (195)	2270
	A-36		740	1200	6.3 (0.2)	6.0	7050 (230)	6210
	A-51		410	1200	3.2 (0.3)	4.1	2545 (470)	2780
	A-68		620	1200	2.0 (0.3)	2.1	5150 (420)	5720
	A-72		420	1200	2.3 (0.3)	2.7	2410 (380)	2725
	A2-93		420	1200	1.6 (0.3)	1.8	6710 (480)	5240

Table 3 :

 3 Run conditions, manometry, and FTIR results for dissolved H 2 O and CO 2 contents in the standards. FTIR data exclusively were used for the calibration curves. Samples were synthesized and characterized by Gordon Moore, Kurt Roggensack (ASU) and Torsten Vennemann (UNIL)

		SiO2	TiO2	Al2O3	FeO*	MgO	CaO	Na2O	K2O	Total
	A	49.30	0.97	15.50	10.40	8.30	12.55	2.40	0.26	99.68
	A2	45.80	0.90	14.10	9.62	7.80	18.54	2.00	0.26	99.02
		Basalt glass starting compositions for the standards, as determined by electron microprobe

Table 5 :

 5 Distribution coefficients and phase proportions used for Rayleigh fractionation and assimilationfractional crystallization modeling(AFC -DePaolo, 1981).

	SiO2	TiO2	Al2O3	FeO*	MgO	CaO	Na2O	K2O	Sr	Zr
	69.7	0.5	14.9	2.7	0.86	2.14	4.11	4.2	213.1	18.71
	Composition of the assimilated granite


F Bard
email: bard@emse.fr

D Garcia

G Févotte

SEMI-BATCH CRYSTALLIZATION OF GYPSUM FROM CALCITE AND SULFURIC ACID

Keywords: crystallization process for sustainability, crystal growth, modeling of solubility and of kinetic properties

Figure 1: SEM image of gypsum overgrown at the surface of a calcite particle.

One of the most important environmental issues for decades to come is the treatment of industrial wastewaters containing salts (sulfates, chlorides, sodium, magnesium ...) which can lead to strong perturbations affecting the fauna and flora when highly concentrated at the discharge point. Different processes used to separate the dissolved salts from the liquid matrix may be encountered depending on the nature and the composition of the effluent. One major objective in the R&D sectors of water and environment is to optimize such processes, while another is to promote the treated wastewaters by recycling the cleared liquid phase or reusing some compounds they contain in other production processes. The latter is of great interest, both to limit the amount of waste and to save natural resources including rare metals but also less noble materials whose amount consumed each year is very important, but the one available not unlimited. Gypsum, the most important raw material of plaster's industry, is a rather interesting example from this point of view as world's known resources are estimated at 2.34 billion tons, which roughly represents 25 years of operation. The industry of plaster will therefore meet in the coming decades a decrease in resources for the commodity, especially in some countries where gypsum deposits will eventually become out of reach or completely depleted.

This study thereby focuses on the precipitation of gypsum in a semi-batch laboratoryscale installation modeling the neutralization of a sulphuric acid solution by injection of a diluted limestone suspension. The generation of the supersaturation of the calcium sulphate is then obtained through the gradual dissolution of calcite particles providing accumulation of calcium ions in the solution. Particular attention is paid to the direct conversion of calcite into gypsum beginning with the rapid formation of an overlayer of gypsum instantaneously nucleated on the surface of calcite particles (see figure 1) and delaying the dissolution process as previously mentioned by Booth et al [START_REF] Booth | Gypsum overgrowths passivate calcite to acid attack[END_REF] . This phenomenon is of first importance with regards to the process as it highly impacts both of the size and shape of the precipitated gypsum crystals through the kinetic control it exerts on the liberation of calcium ions in solution. Effects of temperature (20°C, 40°C and 60°C), concentration of sulphate (0.15M, 0.30M and 0.60M), mean particle size of calcite powder (25µm, 35µm and 55µm) and inlet flow of calcite suspension (10ml/min, 5ml/min and 2.5ml/min) are investigated. Temperature has been found to increase the supersaturation while being decreased. At 20°C and 60°C, formation of gypsum occurs preferentially by precipitation onto the surface of calcite particles rather than from solution (Figure 2). Acid concentration has been found to increase the supersaturation while being decreased. Starting with a high sulphate concentration provide a quasi-exclusive precipitation on the surface of calcite particles while starting with lower acid concentrations favours precipitation from solution (Figure 3). The smaller the calcite particles, the higher the peak of supersaturation reached before precipitation occurs. The use of smaller particles of calcite in the feeding suspension also leads to a solid product in which the particles precipitated from the solution prevail (Figure 4). The slower the inlet flow of suspension, the closer the supersaturation to equilibrium concentration. Decreasing the inlet flow results in favouring the precipitation of gypsum onto the surface of calcite particles (Figure 5). The higher supersaturation level reached by decreasing calcite particle size finds its origin clearly in the increase in calcite reactive surface for a given mass of calcium carbonate which provides quicker liberation of calcium, and the lower supersaturation level reached by decreasing the inlet flow of suspension may find a reasonable explanation in a lower rate of injection of calcium towards the rate of dissolution of calcite. However, modifications of the behaviour of the mixture induced by temperature and acid concentration appear to be more difficult to explain without taking in account the conversion mechanism of calcite particles into gypsum.

The present study aims to purpose a reaction -diffusion mechanism for calcite particles based on calcium mass balance equation (1) which might explain the delaying role played by the gypsum layer in the liberation of calcium in solution. Equation ( 1) is based on equality at calcite/gypsum layer interface between the fluxes of calcium liberated by the dissolution of calcite in one hand and consumed by gypsum precipitation and diffusion trough the gypsum layer in the other hand. The dissolution term in the left member is expressed as linear function of proton concentration as mentioned by Chou et al [START_REF] Chou | Comparative study of the kinetics and mechanism of dissolution of Carbonate minerals[END_REF] . The first term of the second member shows the second order dependence of gypsum growth with absolute supersaturation in calcium as depicted in [START_REF] Brandse | The influence of sodium chloride on the crystallization rate of gypsum[END_REF] .

Figures 2

 2 Figures 2 to 5 expose total calcium concentration vs. total sulphate concentration curves reflecting the generation of the supersaturation during the injection of the calcite suspension. Additional experimental measurements are conducted to get the concentration of calcium at different concentration of sulphate in conditions of equilibrium with gypsum. Final particle size distributions are also exposed aside. Temperature has been found to increase the supersaturation while being decreased. At 20°C and 60°C, formation of gypsum occurs preferentially by precipitation onto the surface of calcite particles rather than from solution (Figure2). Acid concentration has been found to increase the supersaturation while being decreased. Starting with a high sulphate concentration provide a quasi-exclusive precipitation on the surface of calcite particles while starting with lower acid concentrations favours precipitation from solution (Figure3). The smaller the calcite particles, the higher the peak of supersaturation reached before precipitation occurs. The use of smaller particles of calcite in the feeding suspension also leads to a solid product in which the particles precipitated from the solution prevail (Figure4). The slower the inlet flow of suspension, the closer the supersaturation to equilibrium concentration. Decreasing the inlet flow results in favouring the precipitation of gypsum onto the surface of calcite particles (Figure5). The higher supersaturation level reached by decreasing calcite particle size finds its origin clearly in the increase in calcite reactive surface for a given mass of calcium carbonate which provides quicker liberation of calcium, and the lower supersaturation level reached by decreasing the inlet flow of suspension may find a reasonable explanation in a lower rate of injection of calcium towards the rate of dissolution of calcite. However, modifications of the behaviour of the mixture induced by temperature and acid concentration appear to be more difficult to explain without taking in account the conversion mechanism of calcite particles into gypsum.

Figure 2 :

 2 Figure 2: Total calcium vs. Total sulphate concentrations measured by ICP-AES (on the left) and final particle size distribution measured by laser granulometer (on the right) in experiments conducted at 20°C (squares), 40°C (dots) and 60°C (triangles). Empty symbols represent concentrations measured in conditions of equilibrium with gypsum.

Figure 3 :

 3 Figure 3: Total calcium vs. Total sulphate concentrations measured by ICP-AES (on the left) and final particle size distribution measured by laser granulometer(on the right) in experiments conducted with 0.15M (squares), 0.30M (dots) and 0.6M (triangles) initial sulphate concentrations. Empty symbols represent concentrations measured in conditions of equilibrium with gypsum.

Figure 4 :

 4 Figure 4: Total calcium vs. Total sulphate concentrations measured by ICP-AES (on the left) and final particle size distribution measured by laser granulometer (on the right) in experiments conducted with 25µm (squares), 35µm (triangles) and 55µm (dots) mean particle size of calcite powder. Empty symbols represent concentrations measured in conditions of equilibrium with gypsum.

Figure 5 :

 5 Figure 5: Total calcium vs. Total sulphate concentrations measured by ICP-AES (on the left) and final particle size distribution measured by laser granulometer(on the right) in experiments conducted with 10ml/min (dots), 5ml/min (squares) and 2.5ml/min (triangles) inlet flow of calcite suspension. Empty symbols represent concentrations measured in conditions of equilibrium with gypsum.

  )

	S r k d			Reactive surface Rate constant for calcite dissolution	m 2 m.s -1
	s H C +	Proton concentration at calcite/gypsum interface	mol.m -3
	k g			Rate constant for gypsum precipitation	m 4 .s -1 .mol -1
	s Ca C + 2	Calcium concentration at calcite/gypsum interface	mol.m -3
	C	* Ca	2+	Calcium concentration at equilibrium with gypsum	mol.m -3
	b Ca C + 2	Calcium concentration in the bulk solution	mol.m -3
	eff Ca D + 2	Calcium coefficient for effective diffusion in gypsum layer m 2 .s -1
	e			thickness of the gypsum layer	m


Lucía Fuentes

Kristiaan Wouters

Sarah Anissa Hannou

Céline Cudejko

Elena Rigamonti

Hèrvée Thérèse

Bruno Mayi

François Derudas

Giulia Pattou

Bart Chinetti-Gbaguidi

Staels

T H Mayi

B Derudas

F Pattou

G Chinetti-Gbaguidi

B Staels
email: bart.staels@pasteur-lille.fr

R Paumelle

Downregulation of the tumour suppressor p16 INK4A contributes to the polarisation of human macrophages toward an adipose tissue macrophage (ATM)-like phenotype*

Keywords:

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

Downregulation of the tumour suppressor p16INK4A contributes to the polarisation of human macrophages

toward an adipose tissue macrophage (ATM)-like phenotype.

Introduction

Macrophage infiltration into adipose tissue (AT) reflects the low-grade inflammatory state characterising obesity and insulin resistance [START_REF] Fuentes | Inflammatory mediators and insulin resistance in obesity: role of nuclear receptor signaling in macrophages[END_REF]. Obesity-induced adipocyte hypertrophy causes adipocyte inflammation and cell death triggering macrophage accumulation. These AT macrophages (ATMs) accumulate in AT, surrounding dead adipocytes and forming so-called crown-like structures (CLS) [START_REF] Cinti | Adipocyte death defines macrophage localization and function in adipose tissue of obese mice and humans[END_REF]. Macrophages exhibit different activation states allowing them to exert specialised functions: classically activated (M1) macrophages produce high levels of inflammatory mediators such as TNF, IL-6 and cyclo-oxygenase 2 (COX2), while alternatively activated (M2) macrophages produce anti-inflammatory cytokines [START_REF] Martinez | Macrophage activation and polarization[END_REF]. In vitro, monocyte differentiation into M2 macrophages is induced by stimulation with T helper 2 (Th2) cytokines, such as IL-4 or IL-13, or into M1 macrophages by stimulation with IFN or lipopolysaccharide (LPS) [START_REF] Porcheray | Macrophage activation switching: an asset for the resolution of inflammation[END_REF]. M2 markers include mannose receptor, C type 1 (CD206), the haptoglobinhaemoglobin scavenger receptor (CD163) and the chemokine chemokine (C-C motif) ligand 18

(pulmonary and activation-regulated) (CCL18 [also known AMAC1]) [START_REF] Porcheray | Macrophage activation switching: an asset for the resolution of inflammation[END_REF]. In obesity, inflammatory cytokine production by ATMs increases significantly. Based on animal studies it was proposed that obesity promotes the recruitment of M1 macrophages into AT and/or induces a shift of M2 resident macrophages toward the M1 state [START_REF] Lumeng | Obesity induces a phenotypic switch in adipose tissue macrophage polarization[END_REF]. Indeed, mouse ATMs form a heterogeneous population including both M1 and M2 subsets [START_REF] Prieur | Differential Lipid Partitioning Between Adipocytes and Tissue Macrophages Modulates Macrophage Lipotoxicity and M2/M1 Polarization in Obese Mice[END_REF]. However, in humans, ATMs have a more complex phenotype, possibly depending on their localisation within the AT [7;8].

Notwithstanding, the molecular mechanisms determining this particular polarisation remain elusive.

p16 INK4A , encoded by the CDKN2A locus, is a cell cycle inhibitor which blocks cyclin D/cyclindependent kinase 4 (CDK4) kinase activity by binding to the catalytic subunit of CDK4, thereby preventing retinoblastoma protein phosphorylation and subsequent liberation of the E2F1 transcription factor. Hence, the transcription of genes required for progression to the S phase is restrained [START_REF] Sharpless | INK4a/ARF: a multifunctional tumor suppressor locus[END_REF]. Historically, p16 INK4A has been studied in relation to cancer and many human tumours display altered p16 INK4A levels [START_REF] Nobori | Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers[END_REF]. Because of its role in cell cycle exit, p16 INK4A plays a role in senescence. For instance, mammary epithelial cells, fibroblasts and T lymphocytes accumulate p16 INK4A during ageing [START_REF] Huschtscha | p16(INK4a) and the control of cellular proliferative life span[END_REF]. More recently, p16 INK4A was shown to be absent in induced pluripotent stem cells, but to be re-induced during cellular reprogramming, highlighting a role of p16 INK4A in cell fate determination [START_REF] Li | The Ink4/Arf locus is a barrier for iPS cell reprogramming[END_REF]. However, little is known about whether p16 INK4A exerts functions unrelated to cell cycle control. Interestingly, a region near the CDKN2A locus has been associated with type 2 diabetes development [START_REF] Saxena | Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels[END_REF]. As macrophage infiltration and inflammation are key events of type 2 diabetes and obesity, the present study aimed to investigate the connection between p16 INK4A levels in human ATMs and their particular phenotype.

Methods

Human ATM purification Human ATMs were purified from visceral AT biopsies as described by 1). RNA levels were normalised to cyclophilin A mRNA.

Proteins were determined by western blot analysis using a monoclonal antibody against human

Results

Human p16 INK4A expression is lower in ATMs than in MDMs or AMs Expression of p16 INK4A in

human ATMs was compared with MDMs differentiated from monocytes from the same obese donors. Confirming previous reports on the human ATM phenotype [START_REF] Zeyda | Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production[END_REF], mRNA expression of typical M2 markers (AMAC1 [also known as CCL18] and IL10) and of the inflammatory cytokine TNF was higher in ATMs compared with MDMs (data not shown). ATMs expressed significantly lower p16 INK4A mRNA levels than MDMs as well as CD68-positive atherosclerotic plaque macrophages (AMs) (Fig. 1a). This observation suggests that p16 INK4A expression is specifically decreased in ATMs. Interestingly, in vitro macrophage differentiation led to the induction of p16 INK4A protein levels (Fig. 1b), whereas this induction was inhibited in macrophages differentiated in the presence of IL-4, leading to M2 polarisation (Fig. 1c). All observed changes in p16 INK4A levels occurred without alterations in cell cycle progression (data not shown).

As peroxisome proliferator-activated receptor (PPAR)α activation induces p16 INK4A expression in smooth muscle cells [START_REF] Gizard | PPAR alpha inhibits vascular smooth muscle cell proliferation underlying intimal hyperplasia by inducing the tumor suppressor p16INK4a[END_REF] and as PPARγ activation enhances M2 polarisation [START_REF] Bouhlel | PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[END_REF], we investigated whether PPARγ activation with GW929 affects p16 INK4A expression. GW929 treatment of mature MDM macrophages resulted in a very mild inhibition of p16 INK4A expression while GW929 treatment had no effect on p16 INK4A levels in mature M2 macrophages (ESM Fig. 1a). Thus, p16 INK4A is not induced by PPARγ activation in human macrophages. Moreover, enhancing M2 polarisation by adding GW929 to monocytes at the start of differentiation [START_REF] Bouhlel | PPARgamma activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties[END_REF] did not further decrease p16 INK4A expression (ESM Fig. 1b), suggesting that downregulation of p16 INK4A is mainly IL-4 dependent.

These data show that p16 INK4A expression is low in human ATMs and that in vitro differentiation of primary monocytes into M2 macrophages mimics this effect.

Silencing of p16 INK4A in MDMs induces a phenotype resembling human ATMs As ATMs display low p16 INK4A expression compared with MDMs and as M2 polarisation lowers p16 INK4A levels, we determined whether a direct link exists between p16 INK4A levels and macrophage polarisation.

Silencing p16 INK4A in MDMs by approximately 60% using siRNA (Fig. 2a) increased mRNA expression of the M2 markers CD206 (also known as MRC1) and AMAC1, while the expression of CD163, an M2 marker typically downregulated when macrophages are differentiated in vitro with IL-4 [START_REF] Porcheray | Macrophage activation switching: an asset for the resolution of inflammation[END_REF], decreased (Fig. 2c).

Activation of MDMs with LPS increased p16 INK4A protein levels (Fig. 2b), suggesting a role for p16 INK4A in the inflammatory response. To determine whether p16 INK4A knockdown also modulates the inflammatory response of macrophages, MDMs from healthy donors were treated with LPS after p16 INK4A silencing. Although p16 INK4A knockdown induced an M2-phenotype (Fig. 2c), the expression of the inflammatory response genes TNF, COX2 and IL6 was more strongly induced by LPS (Fig. 2d). Furthermore, LPS-induced TNF secretion was higher in cells transfected with p16 INK4A siRNA (Fig. 2e). The production of high levels of inflammatory cytokines upon activation, despite being M2 polarised, resembles the human ATM phenotype [START_REF] Zeyda | Human adipose tissue macrophages are of an anti-inflammatory phenotype but capable of excessive pro-inflammatory mediator production[END_REF].

Importantly, p16 INK4A overproduction by adenovirus-mediated gene transfer (Fig. 3a) showed an expression pattern opposite to the one observed with p16 INK4A silencing, i.e. downregulation of CD206 and AMAC1, upregulation of CD163 (Fig. 3b) and a decreased response to LPS (Fig. 3c).

Together, these data indicate that downregulation of p16 INK4A levels results in an M2-like phenotype associated with an exacerbated response to inflammation, reflecting the phenotype of ATMs, while p16 INK4A overproduction inhibits the ATM phenotype.

p16 INK4A interferes with the TLR4 response upstream of NFκB without affecting TLR4 levels A major pathway involved in regulating M2 genes involves STAT6 signalling [START_REF] Martinez | Macrophage activation and polarization[END_REF]. However, modulation of p16 INK4A levels did not affect phosphorylation of STAT6 under basal conditions or after IL-4 treatment (data not shown). LPS signalling involves TLR4-mediated phosphorylation of p38 and inhibitor of nuclear factor kappa-B kinase (IKK) α,β [START_REF] Carpenter | Recent insights into the structure of Toll-like receptors and posttranslational modifications of their associated signalling proteins[END_REF]. The latter phosphorylates nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα), leading to IκBα degradation and subsequent release and nuclear translocation of nuclear factor of  light polypeptide gene enhancer in B cells (NFB), thereby initiating the transcriptional activation of inflammatory genes [START_REF] Carpenter | Recent insights into the structure of Toll-like receptors and posttranslational modifications of their associated signalling proteins[END_REF]. Subsequent to its activation, TLR4 protein is degraded [START_REF] Liew | Negative regulation of toll-like receptor-mediated immune responses[END_REF]. Although p16 INK4A overproduction decreased the response to LPS (Fig. 3c), this effect was unrelated to TLR4 protein levels in basal or in LPS-stimulated conditions, the latter resulting in the complete degradation of TLR4 (Fig. 4), pointing to an effect downstream in this pathway.

Indeed, LPS-induced phosphorylation of p38 and of IKKα,β was less pronounced in cells overproducing p16 INK4A (Fig. 4). In line, IκBα, which inhibits the nuclear translocation of NFκB [START_REF] Carpenter | Recent insights into the structure of Toll-like receptors and posttranslational modifications of their associated signalling proteins[END_REF], was less degraded on LPS treatment (Fig. 4). Notably, under unstimulated conditions, p16 INK4A overexpression induced lower levels of anti-inflammatory IκBα (Fig. 4). Obesity is associated with increased levels of NEFAs, such as palmitate [START_REF] Garaulet | Site-specific differences in the fatty acid composition of abdominal adipose tissue in an obese population from a Mediterranean area: relation with dietary fatty acids, plasma lipid profile, serum insulin, and central obesity[END_REF], which are known to act as TLR4 ligands [START_REF] Nguyen | A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK-dependent pathways[END_REF]. In line with the observations on LPS-treatment, MDM overproducing p16 INK4A displayed decreased phosphorylation of p38 and IKKα,β after stimulation with palmitate (ESM Fig. 2). These results suggest that p16 INK4A directly interferes with TLR4-NFκB signalling, resulting in an altered inflammatory profile.

Discussion

We show that ATMs from obese patients as well as IL4-polarised human M2 macrophages in vitro are characterised by low p16 INK4A expression compared with, respectively, MDMs or AMs and to LPS-induced M1 macrophages in vitro. Moreover, siRNA-mediated downmodulation of p16 INK4A in MDMs induced an M2 phenotype while increasing the inflammatory response to LPS, thus inducing an ATM-like phenotype. Overproduction of p16 INK4A resulted in the opposite phenotype, providing proof that the effects are p16 INK4A specific.

Although the dominant phenotype of human ATMs is a mixed M1/M2 phenotype [8;21], recent reports describe the existence of different macrophage subpopulations in obese AT [7;22].

Compared with resident ATMs, which are negative for the M1 marker CD11c and positive for the M2 marker MR, crown-like structure (CLS)-ATMs display a mixed profile of polarisation markers (i.e. they are positive for both CD11c and MR) and produce more pro-inflammatory mediators than resident parenchymal ATMs [START_REF] Wentworth | Pro-inflammatory CD11c+CD206+ adipose tissue macrophages are associated with insulin resistance in human obesity[END_REF], thus resembling the phenotype observed on downmodulation of p16 INK4A levels by siRNA. Interestingly, these CLS macrophages express lower levels of CD163 than resident ATMs [START_REF] Wentworth | Pro-inflammatory CD11c+CD206+ adipose tissue macrophages are associated with insulin resistance in human obesity[END_REF]. Combined with our results that inhibition of p16 INK4A levels leads to a mixed phenotype characterised by low CD163 expression, these observations suggest that p16 INK4A may be particularly involved in the differentiation of blood monocytes to CLS-ATMs during obesity. Therefore, it would be interesting to determine the ATM subpopulation in which p16 INK4A is silenced and whether its expression levels correlate with CLS formation, AT inflammation or metabolic disturbances.

Our data show that p16 INK4A in human macrophages interferes with TLR4-NFB signalling, identifying a mechanism via which p16 INK4A regulates the inflammatory status of these cells. In line, it has been previously shown that local transfer of p16 INK4A into bone joints suppresses proinflammatory cytokines in a mouse model of rheumatoid arthritis [START_REF] Nasu | Adenoviral transfer of cyclin-dependent kinase inhibitor genes suppresses collagen-induced arthritis in mice[END_REF] and that the E2F1 transcription factor, a target of p16 INK4A , is required for the transcriptional activation of certain NFB target genes [START_REF] Chen | E2F-1 regulates nuclear factor-kappaB activity and cell adhesion: potential antiinflammatory activity of the transcription factor E2F-1[END_REF]. Moreover, the NFBpathway has been shown, at least in mice, to play a role in ATM phenotype and function. Mice with a luciferase construct driven by a NFresponsive promoter show signal enrichment in ATM clusters during diet-induced obesity [START_REF] Chiang | The protein kinase IKKepsilon regulates energy balance in obese mice[END_REF] and modulation of haematopoietic levels of Ikkβ affects the development of obesity and insulin resistance [26;27]. In parallel, the pro-inflammatory phenotype found in human ATMs involves many NFB target genes, such as TNF, IL6, MCP1 (also known as CCL2), IL8 and COX2 [START_REF] Bourlier | Remodeling phenotype of human subcutaneous adipose tissue macrophages[END_REF].

Overproduction of p16 INK4A resulted in decreased IKKα,β phosphorylation. Moreover, phosphorylation of p38, another component of the LPS-induced signalling pathway converging with NFκB signalling at the level of TNF receptor-associated factor 6 (TRAF-6) [START_REF] Carpenter | Recent insights into the structure of Toll-like receptors and posttranslational modifications of their associated signalling proteins[END_REF], was diminished. Although p16 INK4A has been shown to induce anti-proliferative responses via negative cross-talk with the p65 subunit of NFκB [START_REF] Wolff | INK4 cell cycle inhibitors direct transcriptional inactivation of NF-kappaB[END_REF], our results thus indicate that p16 INK4A interferes upstream of the p65 subunit of NFκB, possibly at the level, or upstream, of TRAF6.

Notably, TLR4 levels were unaffected by p16 INKa overproduction.

Interestingly, the main pathway controlling M2 polarisation, i.e. STAT6 phosphorylation, was unchanged on modulation of p16 INK4A levels (data not shown). However, under basal conditions, overproduction of p16 INK4A also exerted effects on the NFB pathway, as shown by lower IκBα protein levels. IκBα is known to be anti-inflammatory [START_REF] Carpenter | Recent insights into the structure of Toll-like receptors and posttranslational modifications of their associated signalling proteins[END_REF] and inhibition of IκBα phosphorylation and subsequent degradation by adiponectin promotes M2 polarisation [START_REF] Lovren | Adiponectin primes human monocytes into alternative antiinflammatory M2 macrophages[END_REF],

suggesting that the anti-inflammatory effects of IκBα can enhance alternative polarisation. The lower IκBα levels in basal conditions after p16 INK4A overexpression may thus, at least in part, contribute to the decreased expression of M2 genes. Alternatively, NFB has been shown to maintain the M2 polarisation status in tumour-associated macrophages [START_REF] Hagemann | Re-educating" tumor-associated macrophages by targeting NF-kappaB[END_REF], suggesting that a decrease in NFB signalling itself could also contribute to the decreased M2 profile of macrophages overexpressing p16 INK4A .

Silencing of the p16 INK4A locus is associated with an increased risk of cancer development. For example, in certain haematological malignancies p16 INK4A loss of function leads to uncontrolled cell proliferation [START_REF] Hackanson | The silence of the genes: epigenetic disturbances in haematopoietic malignancies[END_REF]. However, human MDMs are terminally differentiated cells, which means they are in a cell state tightly linked to cell cycle withdrawal. In line, the mature macrophages were in the G0 phase regardless of their polarisation state (data not shown). Therefore, we propose a direct association between p16 INK4A expression and macrophage phenotype, independent of cell cycle control. In parallel with several cancers such as lung cancer and the haematopoietic cancer leukaemia, where the CDKN2A/B genomic methylation status regulating p16 INK4A is used as an early diagnostic marker [START_REF] Hackanson | The silence of the genes: epigenetic disturbances in haematopoietic malignancies[END_REF], p16 INK4A levels in ATMs might serve as a diagnostic marker of ATM function.

Although studies in mice suggest that an age-dependent increase of p16

  p16 INK4A (Beckton Dickinson), signal transducer and activator of transcription 6, interleukin-4 induced (STAT6) and phosphorylated STAT6 (9362 and 9361), phospho-IKKα,β (2681), IκBα (9242), phospho-P38 (9211) (Cell Signaling), Toll-like receptor 4 (TLR4) (sc-10741), and βactin (sc-1616) (Santa Cruz). Levels of TNF protein in culture media were measured using an ELISA (R&D Systems, Lille, France). Statistical analysis Data are from representative experiments. Each set of experiments was repeated at least three times with similar results. Statistical significance was determined by the Student's t test. Values are reported as means ± SD. Values of p<0.05 were considered significant and different levels of significance are represented by asterisks: *p<0.05, **p<0.01 and ***p<0.001.

Fig. 2 Fig. 3 Fig. 4

 234 Fig. 2 Inhibition of p16 INK4A levels in MDMs induces a phenotype resembling that of human ATMs. Western blot of p16 INK4A protein in MDMs transfected with either control scrambled or

  INK4A levels hamper beta cell proliferation, resulting in impaired glucose homeostasis[START_REF] Krishnamurthy | p16INK4a induces an age-dependent decline in islet regenerative potential[END_REF], it remains obscure whether p16 INK4A plays a similar role in human pancreatic function. Our data show a novel role of p16 INK4A in another cell type, i.e. human ATMs, which are important during the development of type 2 diabetes[START_REF] Fuentes | Inflammatory mediators and insulin resistance in obesity: role of nuclear receptor signaling in macrophages[END_REF]. As in pancreatic beta cells, increased p16 INK4A levels causes the agedependent decline in proliferation of haematopoietic stem cells[START_REF] Janzen | Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a[END_REF], which give rise to immune cells. As our results show that p16 INK4A overproduction inhibits the phenotype of non-Fig. 1 p16 INK4A levels are low in ATMs and M2 macrophages. a Human ATMs were purified from visceral AT biopsies; monocytes purified from whole blood of the same obese donors (n=9) were differentiated for 7 days into MDMs. CD68 + AMs were isolated from human carotid plaques (n=6) by laser-capture microdissection. mRNA was isolated and quantitative RT-PCR was performed to measure mRNA expression levels of p16 INK4A in the cell types. Statistically significant differences are indicated (t test; *p0.05, **p<0.01 compared with ATM). b p16 INK4A protein was detected by western blot at different time points during macrophage differentiation from blood monocytes of healthy donors. c p16 INK4A protein was detected by western blot at different time points during alternative macrophage differentiation of blood monocytes from healthy donors in the presence of IL-4

	proliferating ATMs, age-related increases of p16 INK4A levels may disturb normal ATM function,
	possibly leading to disturbed AT homeostasis. As the locus encoding p16 INK4A has been
	associated with the risk of type 2 diabetes [13], our results suggest that p16 INK4A levels in ATMs
	may partially explain the link between type 2 diabetes and the linkage disequilibrium found with
	the CDKN2A locus.
	In conclusion, this study indicates that downmodulation of p16 INK4A is an important event during
	macrophage maturation into ATMs, acting as a modulator of the cellular inflammatory status.

Acknowledgements

This work was supported by the EU FP6 grant (EIF 040851 to L. Fuentes), EU FP7 grants (PIEF-GA-2009-23522 to K. Wouters and grant no. 201608 to T. H. Mayi), an EFSD/GlaxoSmithKline Research Grant 2009 (K. Wouters and B. Staels), the Spanish Government MEC (Ministerio de Educación y Ciencia to L. Fuentes) and the Fondation pour la Recherche Médicale (DCV20070409276 to B. Staels). M. Ploton, M. Daoudi and J. Vanhoutte are acknowledged for technical support.

Contribution statement

All authors participated in the conception and design, or analysis and interpretation of the data, contributed to drafting and revising the manuscript and the final approval of the version to be published.

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.