Maxime Dahirel

Loïc Menut

Armelle Ansart

Increased population density depresses activity but does not influence emigration in the snail Pomatias elegans

Keywords: Defence, dispersal syndromes, ecosystem functioning, gastropod, sex-biased dispersal

Dispersal is a key trait linking ecological and evolutionary dynamics, allowing organisms to optimize fitness expectations in spatially and temporally heterogeneous environments. Some organisms can either actively disperse or reduce activity in response to challenging conditions, and both responses may be under a trade-off. To understand how such organisms respond to changes in environmental conditions, we studied emigration (the first step of dispersal) and activity behaviour in the gonochoric land snail Pomatias elegans, a litter decomposer that can reach very high local densities, over most of the range of ecologically relevant densities. We found that crowding had no detectable effect on emigration tendency in this species, contrary to previous results in many hermaphroditic snails. Pomatias elegans is nonetheless able to detect population density; we show they reduce activity rather than increase dispersal in response to crowding. We propose that limiting activity may be more advantageous than moving away in species with especially poor movement abilities, even by land mollusc standards, like P. elegans. Interestingly, emigration and activity were positively correlated independently of density; this dispersal syndrome may reflect an underlying pace-of-life syndrome, and is compatible with a dispersal-dormancy trade-off, which would require further investigation. Additionally, we found snails with heavier shells relative to their size tended to be less mobile, which may reflect physical and metabolic constraints on movement and/or survival during inactivity. We finally discuss how the absence of density-dependent dispersal may explain why P. elegans is often found at very high local densities, and the possible consequences of this behaviour for ecosystem functioning and litter decomposition.

Introduction

Dispersal, i.e. movement potentially leading to gene flow in space, is a key organismal trait influencing and linking ecological and evolutionary dynamics in sometimes complex ways [START_REF] Ronce | How does it feel to be like a rolling stone? Ten questions about dispersal evolution[END_REF][START_REF] Bonte | Costs of dispersal[END_REF][START_REF] Bonte | Dispersal: a central and independent trait in life history[END_REF]. As the cost-benefit balance of dispersal is expected to vary depending on individual phenotype and experienced conditions, dispersal is often highly context-and phenotype-dependent [START_REF] Bowler | Causes and consequences of animal dispersal strategies: relating individual behaviour to spatial dynamics[END_REF][START_REF] Bonte | Costs of dispersal[END_REF][START_REF] Baines | Phenotype-by-environment interactions influence dispersal[END_REF][START_REF] Endriss | The importance of growing up: juvenile environment influences dispersal of individuals and their neighbours[END_REF]. Complexity is even added when one considers that dispersal can be seen as a three-step process (emigration, transfer, immigration), and that different factors can have different levels of influence at each step [START_REF] Matthysen | Multicausality of dispersal: a review[END_REF]. It has been hypothesized (e.g. [START_REF] Matthysen | Multicausality of dispersal: a review[END_REF][START_REF] Bonte | Dispersal: a central and independent trait in life history[END_REF], that this multicausality of dispersal is why even well-studied environmental factors may lead to very different dispersal responses depending on populations and species. To understand the context-dependency of dispersal, it is thus necessary to study a broader range of life histories and contexts, including organisms that may have alternative strategies to escape negative environmental conditions, e.g. dormancy [START_REF] Buoro | Life-history syndromes: Integrating dispersal through space and time[END_REF].

The effects of population density on dispersal, and especially emigration, are likely among the most studied of all dispersal drivers [START_REF] Bowler | Causes and consequences of animal dispersal strategies: relating individual behaviour to spatial dynamics[END_REF][START_REF] Matthysen | Density-dependent dispersal in birds and mammals[END_REF][START_REF] Harman | Frequency of occurrence and populationdynamic consequences of different forms of density-dependent emigration[END_REF]. However, while emigration increasing with density and competition would be a straightforward expectation, and is supported by theory [START_REF] Travis | The evolution of density-dependent dispersal[END_REF][START_REF] Rodrigues | Evolution of positive and negative density-dependent dispersal[END_REF], empirical results are actually much more idiosyncratic both within and among species: positive density-dependent, negative density-dependent and density-independent emigration seem to occur at broadly similar frequencies [START_REF] Jacob | Variability in dispersal syndromes is a key driver of metapopulation dynamics in experimental microcosms[END_REF][START_REF] Harman | Frequency of occurrence and populationdynamic consequences of different forms of density-dependent emigration[END_REF]. Explanations for this variability typically invoke multicausality and interactions with other traits, such as sociality (with more social species/individuals in a species less likely to leave large groups, leading to negative density-dependent dispersalE; [START_REF] Serrano | Social and individual features affecting natal dispersal in the colonial lesser kestrel[END_REF][START_REF] Cote | Social personalities influence natal dispersal in a lizard[END_REF], body size (as larger and smaller individuals do not feel competition with the same intensity; e.g. [START_REF] Baines | Phenotype-by-environment interactions influence dispersal[END_REF], or sex and sex-ratio (as competition for resources and for partners interact; De Meester & [START_REF] De Meester | Information use and density-dependent emigration in an agrobiont spider[END_REF][START_REF] Hovestadt | Gender-specific emigration decisions sensitive to local male and female density[END_REF], among many others. Despite the large amount of studies, we are still far from integrating them into a general understanding of density-dependent dispersal: a possible reason is that invertebrates and especially non-arthropod invertebrates are underrepresented in the published literature [START_REF] Harman | Frequency of occurrence and populationdynamic consequences of different forms of density-dependent emigration[END_REF], meaning we have only explored a narrow part of the trait combinations that may interact with, and shape, the density-dependence of dispersal.

A c c e p t e d m a n u s c r i p t

The land winkle Pomatias elegans (Müller 1774)(Fig. 1A), is a common gastropod species in forests and hedgerows across Europe [START_REF] Kerney | Guide des escargots et limaces d'Europe[END_REF][START_REF] Falkner | Shelled Gastropoda of western Europe[END_REF][START_REF] Welter-Schultes | European non-marine molluscs, a guide for species identification[END_REF].

As a caenogastropod, it has separate sexes, contrary to the majority of land molluscs which are hermaphrodite [START_REF] Barker | Gastropods on land: phylogeny, diversity and adaptive morphology[END_REF]. Its active dispersal capacities are poorly studied but expected to be very low, even by molluscan standards [START_REF] Kramarenko | Active and passive dispersal of terrestrial mollusks: a review[END_REF]; average dispersal distances after 42 days are below 20 cm, according to one of the only (indirect) records available in the peer-reviewed literature [START_REF] Pfenninger | Relationship between microspatial population genetic structure and habitat heterogeneity in Pomatias elegans (O.F. Müller 1774) (Caenogastropoda, Pomatiasidae)[END_REF]. Like many other land snails [START_REF] Cook | Behavioural ecology: on doing the right thing, in the right place at the right time[END_REF][START_REF] Falkner | Shelled Gastropoda of western Europe[END_REF], it is able to survive up to several months under unfavourable conditions by strongly reducing its activity (sometimes down to dormancy; [START_REF] Kilian | Untersuchungen zur Biologie von Pomatias elegans (Müller) und ihrer 'Konkrementdrüse[END_REF][START_REF] Falkner | Shelled Gastropoda of western Europe[END_REF]. P. elegans is often distributed in a very aggregated way across landscapes, and when a population is present, local densities can vary by several orders of magnitude, from less than one to several hundred individuals.m -2 [START_REF] Pfenninger | Relationship between microspatial population genetic structure and habitat heterogeneity in Pomatias elegans (O.F. Müller 1774) (Caenogastropoda, Pomatiasidae)[END_REF]. When it is present, P. elegans plays a key role in ecosystem functioning, by decomposing litter and altering its chemical and physical characteristics [START_REF] Coulis | The fate of condensed tannins during litter consumption by soil animals[END_REF][START_REF] De Oliveira | Snail and millipede complementarity in decomposing Mediterranean forest leaf litter mixtures[END_REF].

We here tested the hypothesis that emigration (the first step of dispersal) and activity were densitydependent in P. elegans. We tested both activity and actual emigration for two reasons. We expected dispersers to be more active than residents, density being equal, in agreement with general predictions on behavioural syndromes [START_REF] Cote | Personality-dependent dispersal: characterization, ontogeny and consequences for spatially structured populations[END_REF][START_REF] Réale | Personality and the emergence of the pace-of-life syndrome concept at the population level[END_REF]. However, the two traits may not be interchangeable, and divergent responses between the two may help understand how P. elegans cope with high densities. For instance, if activity merely is a correlate of dispersal, or viceversa (for instance if dispersal is just a "by-product" of routine movement; [START_REF] Van Dyck | Dispersal behaviour in fragmented landscapes: routine or special movements?[END_REF], we would expect activity to respond to density in the same way as emigration. If reduced activity is also an alternative coping strategy in the face of stress, we may expect instead a depression of activity in response to density [START_REF] Cameron | Intra-and interspecific effects of population density on growth and activity in some helicid land snails (Gastropoda: Pulmonata)[END_REF]. In accordance with the phenotypic compensation hypothesis, which predicts that risk-taking behaviour should be associated with morphological traits limiting vulnerability to predators (e.g. [START_REF] Ahlgren | Individual boldness is linked to protective shell shape in aquatic snails[END_REF], we also expected dispersers/active individuals to be better defended, by having heavier shells. Finally, we accounted for sex differences, given sex-biased dispersal is often observed in other taxa with separated sexes [START_REF] Trochet | Evolution of sex-biased dispersal[END_REF].

Material and methods

We collected Pomatias elegans snails in spring 2017 in leaf litter and at the bottom of calcareous walls [START_REF] Falkner | Shelled Gastropoda of western Europe[END_REF] in Arcais, France (approximate location: 46°17'60"N, 0°41'21"W). They were then kept under controlled conditions (20°C, L:D 16:8) in plastic boxes (25 × 25 × 9 cm, about A c c e p t e d m a n u s c r i p t 500 snails.m -2 ) with a 1 cm layer of soil and a roughly 2-3 cm thick layer of leaf litter collected in urban and suburban woodlots and hedgerows on and close to the University of Rennes Beaulieu campus (predominantly oaks Quercus robur L. 1753 and Q. petraea (Matt.) Liebl. 1784, and beech Fagus sylvatica L. 1753). We provided humidity by watering soil and litter every 10 to 15 days.

Dispersal experiment

In June 2017, we created two-patch systems by adapting the system we developed for slugs in [START_REF] Fronhofer | Bottom-up and top-down control of dispersal across major organismal groups[END_REF], but with a shorter inter-patch gap and longer dispersal period to account for the more limited dispersal capacities of P. elegans (Fig. 1B). We divided 32 × 12 cm 2 transparent plastic boxes (height: 9 cm) in two 12 × 12 cm 2 patches ("release" and "arrival") and one central 10 × 12 cm matrix, using plastified cardboard walls. A 2 × 12 cm 2 slot was left open in each wall to allow snails to leave and enter patches. Both patches were filled with watered soil and litter as in maintenance boxes (see above), while the matrix was kept dry and empty. Experiments were done under controlled conditions (20°C, L:D 16:8).

A c c e p t e d m a n u s c r i p t

We then placed between 1 and 30 snails in each "release" patch, and first closed the patches for 24 h to let the snails habituate. We then let them free to move for 4 days, and recorded which snails stayed in their release patch (hereafter "residents") and which snails emigrated to the arrival patch.

No snails were found in the matrix between the two patches, and no snails died during the experiment. We tested the following group sizes : 1, 2, 3, 4, 5, 10, 15, 20, and30 snails per box, with respectively 30, 20, 10, 9, 6, 4, 3, 3, and2 replicates (total: 371 snails in 87 replicates). We chose these replicate numbers to have at least 30 individuals and 2 replicates per group size. We focused our replication effort more on individuals than boxes (although we did both to some extent), as we were interested in individual traits both as responses and covariates (see Statistical analyses), so a substantial number of individuals at each density was needed. The selected group sizes correspond to initial densities ranging from 69.4 to 2083.3 snails.m -2 (taking only release patch surface area into account), and likely provide a good coverage of natural densities. Indeed, densities of up to 1072 detected snails.m -2 have been found using 25 × 25 cm 2 quadrats in natural populations [START_REF] Pfenninger | Relationship between microspatial population genetic structure and habitat heterogeneity in Pomatias elegans (O.F. Müller 1774) (Caenogastropoda, Pomatiasidae)[END_REF], and a separate study indicates this species has a field detectability in the 0.5 to 0.9 range when hand collected [START_REF] Albano | Field estimates of detectability and site occupancy show that northern Italy forest molluscs are spatially rare and poorly detectable[END_REF], possibly because many individuals may have buried themselves in the substrate at any one time [START_REF] Falkner | Shelled Gastropoda of western Europe[END_REF]. We aimed and managed to ensure balanced sex ratios across densities (in line with natural populationsE; [START_REF] Boycott | On sexual characteristics in the shell and radula of Pomatias elegans (Müller)[END_REF]; see Supplementary Material 1 for a description of the sexing procedure, including both (shell morphology-based) putative sexing before the experiment and (dissection-based) confirmatory sexing after. Tests were spread haphazardly over the month due to logistical constraints (start dates ranging from June 2 to June 26); no reproduction was observed during this interval.

Post-dispersal phenotyping

Within 24h after dispersal tests, we placed each snail on a small (8 cm diameter) clean patch of wet substrate (synthetic upholstery foam) and recorded the latency to activity, snails being defined as active if their operculum was open and both their foot and tentacles were visible out of the shell.

Experiments were done under controlled (20°C) temperature conditions, always during the "light" period. All snails that had not moved after 20 minutes remained inactive, operculum closed, for at least two hours, indicating they were not simply slower individuals, but actually behaved differently from the active ones. Indeed, a limited number was further monitored and remained fully inactive even after 7 hours. We therefore analysed activity as a binary variable (active within 20 minutes/ not active for at least two hours). After sexing by dissection (see Supplementary Material 1), shells were dried at room temperature before being weighed twice (to the nearest 0.1 mg; CP224S scale, A c c e p t e d m a n u s c r i p t Sartorius, Göttingen, Germany). Twenty-four out of 371 shells were partly broken during dissection and could not be weighed.

Statistical analyses

All analyses were done in a Bayesian framework, using the probabilistic language Stan, which uses a variant of Hamiltonian Monte Carlo (NUTS) as an MCMC method to obtain satisfactory posterior samples in a much lower number of iterations than other methods [START_REF] Carpenter | Stan: a probabilistic programming language[END_REF]Stan Development Team, 2018). We created our Stan models using R (R Core Team, 2020) and the brms package [START_REF] Bürkner | brms: an R package for Bayesian multilevel models using Stan[END_REF] as front-ends; this allows models to be specified using R formula syntax.

Scripting, analysis and plotting relied on the tidybayes, bayesplot, and patchwork packages, as well as the tidyverse family of packages [START_REF] Gabry | Visualization in Bayesian workflow[END_REF][START_REF] Kay | tidybayes: tidy data and geoms for Bayesian models[END_REF][START_REF] Pedersen | patchwork: the composer of plots[END_REF][START_REF] Wickham | Welcome to the Tidyverse[END_REF].

We used a multivariate generalized linear mixed model (GLMM) framework [START_REF] Dingemanse | Quantifying individual variation in behaviour: mixedeffect modelling approaches[END_REF], which allowed us not only to draw inferences on dispersal and activity, but also on their correlations with each other and with (relative) shell mass, while handling missing shell mass data by in-model imputation [START_REF] Mcelreath | Statistical rethinking: a Bayesian course with examples in R and Stan[END_REF]. A full write-up of the model is given in Supplementary Material 2. Briefly, we analysed the probability of dispersing and the probability of activity as individual-level binary variables (dispersed/did not disperse; active/not active) using Bernoulli GLMMs with a logit link. We tested whether dispersal or activity state depended on experienced population density, sex and body size (defined as ln(shell height); Fig. 1). The models also included random effects of date, experimental box, as well as a random effect of individual identity. Importantly, the latter was set to be identical (same values) in both the dispersal and activity models. In effect, this means it corresponds to a latent individual "behaviour" variable that would influence both dispersal and activity. This allowed us to estimate the effect of dispersal on activity (and vice-versa), while accounting for the fact both are estimated with uncertainty and the causal path from one to the other is unknown. We can then easily obtain the (logit) activity probability for, for instance, an average disperser by adding (on the logit scale) the average value of the latent behavioural variable for a disperser to the overall average logit(activity probability). The same principle applies to get the average dispersal probability of an active individual. Note that using instead dispersal status as a covariate in a univariate activity model leads to the exact same inferences (Supplementary Material 3); it however prevents us from doing the analyses described in the next few sentences. To be able to separate the effects of relative shell mass from those of body size, we used a third submodel. We fitted a linear mixed model in which ln(Shell mass) was a function of ln(Shell height), sex, and a random effect of individual identity. The use of repeated measures (see A c c e p t e d m a n u s c r i p t "Post-dispersal phenotyping") of shell mass allowed us here to separate individual variation from residual error, and facilitated the estimation of the former. Finally, individual-level random effects were included in a variance-covariance matrix, allowing us to evaluate the correlation between "relative" shell mass (i.e. after accounting for body size and sex) and the latent behavioural variable.

All continuous variables were centred and scaled to unit standard deviation (Supplementary Material 4), and the "sex" variable was converted to a centred dummy numerical variable (-0.5 = "Male"; +0.5 = "Female") [START_REF] Schielzeth | Simple means to improve the interpretability of regression coefficients[END_REF].

We set weakly informative priors by following McElreath (2020)'s suggestions for continuous and proportion variables. We used Normal(μ = 0, σ = 1.5) priors for the dispersal and activity fixed effects intercepts, and Normal(0, 1) priors for all the other fixed effects. We set half-Normal(0, 1) priors for all standard deviations (random effects as well as residual variance of shell mass), and LKJ(η = 2) priors for the random effects correlation matrices.

We ran four chains for 25000 iterations, with the first 5000 of each chain used for warmup. We checked mixing graphically and confirmed chain convergence using the improved ܴ statistic [START_REF] Vehtari | Rank-normalization, folding, and localization: an improved \widehat{R} for assessing convergence of MCMC[END_REF]. The chains were run longer than the default number of iterations (2000), to ensure effective sample size was satisfactory for all parameters (bulk-and tail-ESS sensu [START_REF] Vehtari | Rank-normalization, folding, and localization: an improved \widehat{R} for assessing convergence of MCMC[END_REF]Vehtari et al., > 1000)). All posterior summaries are given as: mean [95% Higher Posterior Density Intervals].

Results

144 snails out of 371 (38.81%) dispersed across all test boxes. There was no detectable effect of box population density (Fig. 2A), sex or shell height on emigration probability (Table 1). Snails exposed to higher population densities were less likely to be active afterwards (Table 1, Fig. 2B). There were no clear effects of sex or size on activity probability (Table 1). The latent behavioural variable was positively associated with both emigration and activity (Supplementary Material 5): accordingly, dispersers were 1.23 [1.02; 1.47] times more likely to be active on average, and conversely active individuals were 1.54 [1.04; 2.16] times more likely to have emigrated (Fig. 3). This activity-emigration correlation was confirmed by analysing activity in a univariate model with dispersal status as a covariate (Supplementary Material 3). After accounting for shell length, sex and measurement error, individual shell mass was negatively correlated with the behavioural latent variable: dispersers/active snails tended to have lighter shells, given their size and sex, than residents/inactive individuals (Fig. 4

Discussion

We studied the relationship between Pomatias elegans dispersal and population density over most of the known range of natural densities [START_REF] Pfenninger | Relationship between microspatial population genetic structure and habitat heterogeneity in Pomatias elegans (O.F. Müller 1774) (Caenogastropoda, Pomatiasidae)[END_REF][START_REF] Albano | Field estimates of detectability and site occupancy show that northern Italy forest molluscs are spatially rare and poorly detectable[END_REF], yet found no evidence of density-dependent emigration. There was however a negative relationship between A c c e p t e d m a n u s c r i p t density and activity. Furthermore, we found that emigration, activity and (relative) shell mass covary in this species, once density variation is accounted for.

We additionally found no evidence of sex bias in dispersal probability or activity. While sex-biased dispersal is abundantly documented in non-molluscan groups [START_REF] Bowler | Causes and consequences of animal dispersal strategies: relating individual behaviour to spatial dynamics[END_REF][START_REF] Trochet | Evolution of sex-biased dispersal[END_REF], there are numerous species with no bias. Broadly, sex-dependent dispersal is predicted to arise when there are sex-differences in spatio-temporal fitness variation due to e.g. sex differences in resource use and/or in local mate competition [START_REF] Hovestadt | Gender-specific emigration decisions sensitive to local male and female density[END_REF][START_REF] Trochet | Evolution of sex-biased dispersal[END_REF][START_REF] Li | Intersexual resource competition and the evolution of sex-biased dispersal[END_REF]. Our results, in light of dispersal theory, imply that male and female P. elegans use resources similarly and that they do not present polygyny or any mating system leading to predictable sex differences in spatio-temporal fitness variation. While sex ratio is, overall, balanced in this species [START_REF] Boycott | On sexual characteristics in the shell and radula of Pomatias elegans (Müller)[END_REF], the other predictions remain to be tested by the collection of further natural history data. We should additionally note that our experimental setup (one measure of dispersal, after 4 days, and groups that were sex-balanced on average) may not have allowed us to detect finer sex differences in dispersal, i.e. in emigration timing [START_REF] Li | Intersexual resource competition and the evolution of sex-biased dispersal[END_REF], or conditional strategies dependent on locally experienced sex-ratio [START_REF] Hovestadt | Gender-specific emigration decisions sensitive to local male and female density[END_REF].

The absence of density-dependent emigration was more surprising, as many other land mollusc species have been shown to increase emigration and/or dispersal distances in response to population density [START_REF] Cain | Ecogenetics of a population of Cepaea nemoralis (L.) subject to strong area effects[END_REF][START_REF] Oosterhoff | Variation in growth rate as an ecological factor in the landsnail Cepaea nemoralis (L.)[END_REF][START_REF] Hamilton | The effects of food and density of the movement of Arion ater and Ariolimax columbianus (Pulmonata: Stylommatophora) between habitats[END_REF][START_REF] Livshits | Ecology of the terrestrial snail Brephulopsis bidens (Pulmonata: Enidae): mortality, burrowing and migratory activity[END_REF][START_REF] Baur | Effects of food availability and intra-and interspecific interaction on the dispersal tendency in the land snail Chondrina clienta[END_REF][START_REF] Shachak | Effect of population density on water influx and distribution in the desert snail Trochoidea seetzenii[END_REF][START_REF] Dahirel | Density-dependence across dispersal stages in a hermaphrodite land snail: insights from discrete choice models[END_REF]. In addition, our experimental design included a (supposedly) costly matrix, which should have made the detection of a positive emigration-density link easier, if it exists (as the dispersal-density reaction norm is predicted to become steeper as costs of dispersal increase; [START_REF] Travis | The evolution of density-dependent dispersal[END_REF]. The costs of competition, which can trigger density-dependent dispersal, are well-documented in land molluscs [START_REF] Oosterhoff | Variation in growth rate as an ecological factor in the landsnail Cepaea nemoralis (L.)[END_REF][START_REF] Hamilton | The effects of food and density of the movement of Arion ater and Ariolimax columbianus (Pulmonata: Stylommatophora) between habitats[END_REF][START_REF] Dan | Growth, mortality, and feeding rates of the snail Helix aspersa at different population densities in the laboratory, and the depression of activity of helicid snails by other individuals, or their mucus[END_REF]. However, density-independent emigration also exists in some mollusc species, in at least some contexts (e.g. [START_REF] Hamilton | The effects of food and density of the movement of Arion ater and Ariolimax columbianus (Pulmonata: Stylommatophora) between habitats[END_REF][START_REF] Baur | Effects of food availability and intra-and interspecific interaction on the dispersal tendency in the land snail Chondrina clienta[END_REF]. The traditionally expected positive relationship between density and dispersal can be dampened, or even reversed, if for instance species/individuals benefit from group-living [START_REF] Serrano | Social and individual features affecting natal dispersal in the colonial lesser kestrel[END_REF][START_REF] Bowler | Causes and consequences of animal dispersal strategies: relating individual behaviour to spatial dynamics[END_REF][START_REF] Cote | Social personalities influence natal dispersal in a lizard[END_REF]. In terrestrial gastropods, individuals may aggregate actively in shelters (and get benefits from this aggregation that go beyond the shelter itself, as the presence of conspecifics may create a favourable microclimate), sometimes following trails to do so [START_REF] Cook | Behavioural ecology: on doing the right thing, in the right place at the right time[END_REF]. However, it is unknown whether Pomatias elegans follows trails, and this aggregation behaviour is anyway common to many land molluscs, including species we know show positive density-dependent emigration [START_REF] Cook | Behavioural ecology: on doing the right thing, in the right place at the right time[END_REF], so it is unlikely to explain alone the densityindependence seen in the present study. P. elegans, contrary to most land molluscs has separate sexes, and differences in reproductive systems may drive the evolution of different dispersal strategies [START_REF] Eppley | Moving to mate: the evolution of separate and combined sexes in multicellular organisms[END_REF]. Indeed, all things equal, hermaphrodites are a priori more likely than species with separate sexes to find a mate in small populations [START_REF] Tomlinson | The advantages of hermaphroditism and parthenogenesis[END_REF]. This means the density threshold at which the costs of competition outweigh the benefits of grouping [START_REF] Kim | Simultaneous positive and negative density-dependent dispersal in a colonial bird species[END_REF][START_REF] Fronhofer | Density-dependent movement and the consequences of the Allee effect in the model organism Tetrahymena[END_REF] and the costs of dispersal [START_REF] Bonte | Costs of dispersal[END_REF] is likely lower for hermaphrodites. We hypothesize that context being equal (including e.g. movement costs, or group shelter benefits), this may make hermaphrodites more likely than gonochoric species to exhibit positive density-dependent dispersal over a range of natural densities.

In any case, our study shows that further comparisons between hermaphroditic and gonochoric species sharing ecological and life-history characteristics are crucially needed to clarify the role of reproductive system in shaping context-dependent dispersal.

Alternatively (but not exclusively), the absence of density response in dispersal may reflect the fact Pomatias snails possess other mechanisms to respond to unfavourable densities. Indeed, by contrast with dispersal, activity propensity did decrease with increasing population density (Fig. 2B). Like many other shelled land gastropods, P. elegans can reduce its activity and even enter dormancy when context is unfavourable [START_REF] Cook | Behavioural ecology: on doing the right thing, in the right place at the right time[END_REF][START_REF] Falkner | Shelled Gastropoda of western Europe[END_REF]. Activity reduction in response to increased competition has been observed in several species [START_REF] Oosterhoff | Variation in growth rate as an ecological factor in the landsnail Cepaea nemoralis (L.)[END_REF][START_REF] Cameron | Intra-and interspecific effects of population density on growth and activity in some helicid land snails (Gastropoda: Pulmonata)[END_REF][START_REF] Dan | Growth, mortality, and feeding rates of the snail Helix aspersa at different population densities in the laboratory, and the depression of activity of helicid snails by other individuals, or their mucus[END_REF]. This is generally thought to result from interference (mediated through mucus and/or faeces) by the most competitive individuals, which remain active. However, when the experimental/observational setup also allowed for dispersal, species that reduced activity in response to density (e.g. Cepaea nemoralis, Cornu aspersum, Xerocrassa seetzenii) did however also exhibit density-dependent dispersal [START_REF] Oosterhoff | Variation in growth rate as an ecological factor in the landsnail Cepaea nemoralis (L.)[END_REF][START_REF] Shachak | Effect of population density on water influx and distribution in the desert snail Trochoidea seetzenii[END_REF][START_REF] Dahirel | Density-dependence across dispersal stages in a hermaphrodite land snail: insights from discrete choice models[END_REF].

Given the very limited dispersal capacities of P. elegans [START_REF] Pfenninger | Relationship between microspatial population genetic structure and habitat heterogeneity in Pomatias elegans (O.F. Müller 1774) (Caenogastropoda, Pomatiasidae)[END_REF] even compared to other snails [START_REF] Kramarenko | Active and passive dispersal of terrestrial mollusks: a review[END_REF], the least competitive Pomatias snails may well be more successful reducing activity to wait out more favourable conditions, than by attempting to find better by moving away. Indeed, in snails, starvation, a possible result of inactivity, may trigger entry into actual dormancy [START_REF] Wells | The water relations of snails and slugs: III. Factors determining activity in Helix pomatia L[END_REF], which can be seen as a form of "dispersal in time" [START_REF] Buoro | Life-history syndromes: Integrating dispersal through space and time[END_REF].

The fact there was no effect of body size on activity suggests the loss of activity is not simply an inhibition of activity by competitive individuals, leading to competitive exclusion. Indeed, in that case we would have expected larger individuals to stay active at high densities while smaller less competitive snails are inhibited [START_REF] Dan | Growth, mortality, and feeding rates of the snail Helix aspersa at different population densities in the laboratory, and the depression of activity of helicid snails by other individuals, or their mucus[END_REF]. Given this, and while we need more information to conclude that strongly reduced activity in short-term tests is correlated with longterm dormancy propensity, it is possible that Pomatias snails use dormancy as a general "dispersal in time" mechanism, rather than moving away in response to high densities. Note however that we only studied emigration, the first step of dispersal, and density-dependent responses may also happen during transience [START_REF] Shachak | Effect of population density on water influx and distribution in the desert snail Trochoidea seetzenii[END_REF][START_REF] Poethke | The ability of individuals to assess population density influences the evolution of emigration propensity and dispersal distance[END_REF][START_REF] Dahirel | Density-dependence across dispersal stages in a hermaphrodite land snail: insights from discrete choice models[END_REF]. In addition, we set within-patch conditions to be standard and favourable; food availability may interact with density and shape dispersal (e.g. [START_REF] Endriss | The importance of growing up: juvenile environment influences dispersal of individuals and their neighbours[END_REF].

All three phenotypic traits we studied were linked. First, density being held equal, dispersers were more likely to be active post-dispersal test, and vice versa (Fig. 3, Supplementary Material 3). These results line up with predictions on dispersal syndromes and the pace-of-life hypothesis [START_REF] Cote | Personality-dependent dispersal: characterization, ontogeny and consequences for spatially structured populations[END_REF][START_REF] Réale | Personality and the emergence of the pace-of-life syndrome concept at the population level[END_REF], which predicts among other things a metabolically-underpinned link between activity and risk taking behaviour (such as dispersal). While, again, more extended assessments of activity are needed to confirm that low activity is correlated to true dormancy, the observed syndrome can also be interpreted as a trade-off between dispersal in space and in time [START_REF] Buoro | Life-history syndromes: Integrating dispersal through space and time[END_REF], in which individuals that do not disperse are more likely to be dormant at any point in time, even after accounting for density effects. Second, we found that more mobile snails (higher activity and/or dispersal) had on average lighter shells (Fig. 4, Table 2), compared to inactive/resident snails. This result goes against our predictions based on the phenotypic compensation hypothesis, which states that risk-taking individuals should have better morphological defences against predators [START_REF] Hulthén | Express yourself: bold individuals induce enhanced morphological defences[END_REF][START_REF] Ahlgren | Individual boldness is linked to protective shell shape in aquatic snails[END_REF][START_REF] Kuo | Trait compensation between boldness and the propensity for tail autotomy under different food availabilities in similarly aged brown anole lizards[END_REF].

However, phenotypic compensation is not a hard rule [START_REF] De Winter | Knights in shining armour are not necessarily bold: defensive morphology correlates negatively with boldness, but positively with activity, in wild threespine stickleback, Gasterosteus aculeatus[END_REF], and while thicker/heavier shells are harder to break (see e.g. [START_REF] Rosin | Differential shell strength of Cepaea nemoralis colour morphs-implications for their anti-predator defence[END_REF], variation in shell characteristics depends on many abiotic and biotic parameters [START_REF] Goodfriend | Variation in land-snail shell form and size and its causes: a review[END_REF]. Indeed, thicker shells may also better conserve humidity [START_REF] Goodfriend | Variation in land-snail shell form and size and its causes: a review[END_REF], which may be adaptive for snails more prone to be inactive for extended periods of time. Heavier shells are likely to increase the costs of movement [START_REF] Herreid | Energetics of hermit crabs during locomotion: the cost of carrying a shell[END_REF]; given the already high costs of movement in land molluscs [START_REF] Denny | Locomotion: the cost of gastropod crawling[END_REF], this may explain why mobile individuals have lighter shells, despite the potentially increased predation risk. Experimental manipulations of the costs and benefits of heavier shells (by e.g. testing snails under varying humidity conditions, and/or by adding weights to shells), may help disentangle the underpinnings of this dispersal/shell mass syndrome.

Although P. elegans is overall much less abundant than other macroinvertebrate decomposers at the landscape scale [START_REF] David | Abundance, biomass and functional structure of the saprophagous macrofauna in the litter and soil of Mediterranean oak forests[END_REF], it can locally reach very high densities [START_REF] Pfenninger | Relationship between microspatial population genetic structure and habitat heterogeneity in Pomatias elegans (O.F. Müller 1774) (Caenogastropoda, Pomatiasidae)[END_REF]. Our study shows that increased densities do not trigger increased emigration from crowded into empty habitats in this species. This helps explain the maintenance of this strong spatial heterogeneity and high local densities, as dispersal cannot play its "density-equalizing" role as well. Given decomposition by P. elegans can have marked effects on litter characteristics [START_REF] Coulis | The fate of condensed tannins during litter consumption by soil animals[END_REF][START_REF] De Oliveira | Snail and millipede complementarity in decomposing Mediterranean forest leaf litter mixtures[END_REF], and facilitate decomposition by other co-occurring macroinvertebrates (De

Figure 1 .

 1 Figure 1. (A) Pomatias elegans shell showing the measurements used in the present study. (B) Diagram of the experimental two-patch system, showing (to scale) the release and arrival patches containing litter and soil, and the empty matrix in-between. Top: viewed from above; bottom: side view. Credits: snail photograph -Armelle Ansart; derived snail silhouette -Maxime Dahirel (deposited in phylopic.org); Quercus robur leaves silhouette -Ferran Sayol, available in phylopic.org under Public Domain Dedication 1.0 license.

Figure 2 .

 2 Figure 2. Effect of population density on Pomatias elegans dispersal rate (A) and probability of activity (B) (N = 371 snails). Grey points are observed proportions for each test box, black points averages for each density; black curves give posterior predicted means, grey bands 66, 84 and 95% credible intervals.

Figure 3 .

 3 Figure 3. Relationship between dispersal status and activity post-dispersal. White bars correspond to observed frequencies, grey "eyes" to posterior predictions based on model intercepts and individuallevel random effects, i.e. averaging out the effects of sex, size and population density (black dots: posterior means; segments: 95% credible intervals).

Figure 4 .

 4 Figure 4. Posterior predicted relationship between dispersal status, activity post-dispersal, and mean predicted shell mass, that is relative to the shell mass predicted for a snail of same shell height and sex (black dots: posterior means; segments: 95% credible intervals). Posterior pairwise mean differences are presented in Supplementary Material 6.

Table 1 .

 1 Posterior means [and 95% credible intervals] for the parameters of the multivariate model of dispersal, activity and shell mass. Individual-level random effect covariance matrices are in

Table 2 .

 2 See Supplementary Material 2 for the full write-up of the model, and Supplementary Material 4, for original means and SDs of transformed variables (N = 371 snails, except for Shell mass, N = 347

	snails, each measured twice)
		D i s p e r s a l	Activity probability	Shell mass (ln-
		probability		transformed then
				scaled)
	Fixed effects
	Intercept	-0.54 [-1.04 ; -0.26]	0.74 [0.05 ; 1.49]	-0.01 [-0.09 ; 0.06]
	Population density (scaled)	0.04 [-0.38; 0.46]	-0.55 [-1.01 ; -0.10]	--
	Sex (effect of being female)	-0.16 [-0.79 ; 0.46]	0.23 [-0.42 ; 0.86]	0.18 [-0.02 ; 0.38]
	Body size (shell height, ln-transformed	0.01 [-0.31 ; 0.33]	0.16 [-0.17 ; 0.51]	0.62 [0.52 ; 0.72]
	then scaled)
	Random effects (see Table 2 for individual-level effects)
	Date-level variance	0.13 [0.00 ; 0.59]	0.56 [0.00; 1.75]	--
	Box-level variance	0.52 [0.00; 1.33]	0.38 [0.00; 1.04]	--
	Date-level dispersal-activity correlation	0.13 [-0.69 ; 0.90]	--
	Box-level dispersal-activity correlation	0.03 [-0.74 ; 0.73]	--
	Residual variance	--	--	8.00 ×10 -4	[6.85×10 -
				4		-4
				; 9.23×10	]

Table 2 .

 2 Individual-level variance-covariance matrix between the latent behavioural variable (positively correlated with dispersal and activity, see Fig.3and Supplementary Material 3) and shell mass. Posterior means [95% credible interval] are depicted; random effects variances are given on the diagonal, correlation/covariance on the off diagonal

		Behaviour latent variable	Shell mass
	Behaviour latent variable	0.90 [0.08 ; 1.81]
	Shell mass	-0.39 [-0.67 ; -0.12]/ -0.25 [-0.41 ; -0.09]	0.54 [0.46 ; 0.63]

Acknowledgements

We thank Valérie Briand for her help in obtaining some of the cited scientific articles, as well as the editor and two anonymous reviewers, for helpful comments on a previous version of this manuscript.

Data availability

Data and analysis script are archived on GitHub (https://github.com/mdahirel/pomatias-dispersal-2017) and Zenodo (doi: 10.5281/zenodo.3691977) (version v1.2).


María Sancho-Tomás

Andréa Somogyi

Kadda Medjoubi

Antoine Bergamaschi

Pieter T Visscher

Alexander E S Van Driessche

Emmanuelle Gérard

María E Farias

Manuel Contreras

Pascal Philippot

Geochemical evidence for arsenic cycling in living microbialites of a High Altitude Andean Lake (Laguna Diamante, Argentina)

Arsenic is best known as an environmental toxin, but this element could also serve as a metabolic energy source to certain microorganisms. Moreover, As cycling may have driven microbial life on early Earth prior to oxygenation of the atmosphere. Still, little is known about the arsenic cycling processes occurring in the presence of microorganisms and the possible traces that could be preserved in the rock record. To advance our understanding of this we studied the geochemical proxies of microbial As metabolism in living microbialites from Laguna Diamante, a likely Precambrian ecosystem analogue (Catamarca, Argentina). In this study, we show that the coexistence of As(III) and As(V) strongly supports the presence of active microbially arsenic cycling in these microbialites. In addition, we propose a model by which arsenic metabolic processes can be preserved and interpreted as biosignatures in modern systems as well as in the rock record.

INTRODUCTION

Although arsenic is mainly considered to be highly toxic to organisms in all three domains of life, many microorganisms have evolved to tolerate relatively high concentrations of this metalloid and some even benefit from it as a source of energy for their growth [START_REF] Sun | Biological Chemistry of Arsenic, Antimony and Bismuth[END_REF]. There are several lines of geological evidence, which indicate that As may have played a prominent role on early Earth. For instance, after the early accretionary phase of the Earth, after it had cooled and differentiated, sulfur and chalcophilic elements (e.g., As) were significantly enriched within the mantle and the core relative to the crust due to the sinking of the denser metal(loid)-sulfides. Due to widespread volcanism and geothermal activity characteristic of Archean Earth, As would have been brought back to the Earth's surface (e.g. [START_REF] Cabral | Volcanic red-bed copper mineralisation related to submarine basalt alteration, Mont Alexandre, Quebec Appalachians, Canada[END_REF][START_REF] Witt-Eickschen | The geochemistry of the volatile trace elements As, Cd, Ga, In and Sn in the Earth's mantle: New evidence from in situ analyses of mantle xenoliths[END_REF]. In addition, like many modern hot springs, abundant hydrothermally-influenced systems on the Precambrian Earth presumably contained high concentrations of reduced As(III). Hence, it is likely that prior to the rise of atmospheric oxygen about 2.5 Ga ago, substantial amounts of reduced As were present in the oceans thereby posing a biochemical challenge (but also a potential opportunity) for early emergent life. This is certainly supported by biomolecular studies which have demonstrated that the arsenite oxidase enzyme Aro has a deeply-rooted origin that predates the divergence of Archaea and Bacteria from the last universal common ancestor (Lebrun et al., 2003;Duval et al., 2008). Nonetheless, the role of arsenic cycling in early Earth ecosystems is still not well understood. This could be improved by studying the arsenic mechanisms in microorganisms living today in high arsenic environments and severe conditions.

As cycling in modern sediments and bacteria cultures, implying bacteria mediated As(V) reduction and As(III) oxidation, has been reported for extreme environments, such as the alkaline hypersaline waters of Mono and Searles Lakes, California [START_REF] Oremland | A microbial arsenic cycle in a salt-saturated, extreme environment[END_REF][START_REF] Lis R Van, Nitschke | Arsenics as bioenergetic substrates[END_REF][START_REF] Munoz | Arsenic Biogeochemical Cycling Under Extreme, Archean-like Conditions[END_REF]. Evidence for a complete As cycle have been detected in High-altitude Andean Lakes such as La Brava, Diamante and Socompa Lakes [START_REF] Farías | The Discovery of Stromatolites Developing at 3570 m above Sea Level in a High-Altitude Volcanic Lake Socompa, Argentinean Andes[END_REF][START_REF] Kurth | Arsenic metabolism in high altitude modern stromatolites revealed by metagenomic analysis[END_REF]. Sequences for arsenite oxidases and respiratory arsenate reductase have been identified in haloarchaea from Diamante and Tebenchique Lakes, suggesting that in addition to detoxification processes, these microbes could also use arsenic redox reactions to obtain energy [START_REF] Lara | Enrichment of arsenic transforming and resistant heterotrophic bacteria from sediments of two salt lakes in Northern Chile[END_REF][START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF][START_REF] Ordoñez | Haloarchaea from the Andean Puna: Biological Role in the Energy Metabolism of Arsenic[END_REF]. However, very few biogeochemical data supporting this claim have been reported so far.

Recently, Chi Fru and coworkers indicated that the concentration of As(V) increased rapidly during the Great Oxidation Event, which may have enabled the expansion of a global As cycle [START_REF] Fru | The rise of oxygen-driven arsenic cycling at ca. 2.48 Ga[END_REF]. In a previous study, Sforna et al. reported the discovery of 2.7-billion-year-old stromatolites from Tumbiana, in which fossilized microorganisms and biogenic CaCO 3 minerals were associated with high concentrations of As [START_REF] Sforna | Evidence for arsenic metabolism and cycling by microorganisms 2.7 billion years ago[END_REF]. Based on hierarchical length-scale information of the element distribution and inter-element correlations, Sforna and colleagues argued that Tumbiana stromatolites were formed by microbial mats thriving in an anoxic, salt-saturated lake setting, and, importantly, in which As(III) oxidation and As(V) reduction metabolic pathways were taking place [START_REF] Sforna | Evidence for arsenic metabolism and cycling by microorganisms 2.7 billion years ago[END_REF].

Calculations have shown that arsenic cycling may play a role in microbialite formation (i.e. CaCO 3 precipitation) [START_REF] Visscher | The role of arsenic cycling in carbonate precipitation in microbialites through time[END_REF]. Microbially-mediated CaCO 3 precipitation has two components. Firstly, the metabolism of the microbial community influences the geochemical environment through which the speciation of calcium and carbonate ions is altered (i.e. change the alkalinity). Secondly, the functional groups in the exopolymeric substance (EPS) matrix influences the mineral product formation through cation binding and successive release by providing preferential mineral nucleation sites [START_REF] Dupraz | Microbial lithification in marine stromatolites and hypersaline mats[END_REF]. Other elements (e.g. metal(loid)s) may be incorporated in CaCO 3 solids. For example, calcite, the most stable polymorph of CaCO 3 , is capable of retaining trace elements via adsorption (surface) reactions as well as through co-precipitation reactions [START_REF] Alexandratos | Arsenate uptake by calcite: Macroscopic and spectroscopic characterization of adsorption and incorporation mechanisms[END_REF]. Furthermore, certain types of EPS components (e.g. agarose or the organic matrix extracted from marine organisms) can interfere the growth of calcite crystals [START_REF] Li | Visualizing the 3D Internal Structure of Calcite Single Crystals Grown in Agarose Hydrogels[END_REF][START_REF] Cho | Direct observation of mineral-organic composite formation reveals occlusion mechanism[END_REF]. Consequently, physicochemical incorporation of metal(loid)s in polymers (such as EPS) is a viable mechanism by which other trace metal(loid)s including As can become incorporated in crystals, specifically calcite [START_REF] Huerta-Diaz | Millimeter-scale resolution of trace metal distributions in microbial mats from a hypersaline environment in Baja California, Mexico[END_REF][START_REF] Sforna | Patterns of metal distribution in hypersaline microbialites during early diagenesis: Implications for the fossil record[END_REF]. Arsenic could be also incorporate in the calcite lattice by the substitution of a CO 3 2-and AsO 3 3-as it has been observed in the As-bearing travertine rocks [START_REF] Bardelli | Arsenic uptake by natural calcite: An XAS study[END_REF]. Through these different mechanisms, As can be retained and preserved in the rock record. However, to date, the link between CaCO 3 biomineralization and As has not been clarified.

The goal of this study is to identify geochemical proxies of As-based metabolisms and its preservation in living microbialites of Laguna Diamante, which are considered to be likely analogues of early Earth. To achieve this, we have characterized at multiple length scales the geochemical imprint of microbially-mediated arsenic cycling in present day samples, from superficial, biologically active, organic-rich zone to the internal carbonate-rich area in advanced stages of lithification. We combined powder X-ray diffraction (PXRD), scanning electron microscopy with energy dispersive Xray analysis (SEM-EDX) and Confocal Laser Scanning Microscopy (CLSM) with synchrotron scanning techniques, including X-ray micro-fluorescence (µXRF), X-ray Absorption Near Edge Structure (XANES) imaging and punctual XANES analyses [START_REF] Somogyi | Optical design and multi-length-scale scanning spectro-microscopy possibilities at the Nanoscopium beamline of Synchrotron Soleil[END_REF].

MATERIALS AND METHODS

Sample collection

The hypersaline shallow Laguna Diamante is located in the caldera of the Galán Volcano in Catamarca, Argentina, at approximately 4590 meters above sea level (Fig. 1a). Samples were in January 2015 from the shallow part of the Laguna Diamante. Round to oval-shaped rocks, around 1cm in diameter were partially exposed to the atmosphere but during daily high-wind events were completely submersed during part of the day. The rocks were covered by semi-continuous purple layer with building-up tubular-shaped microbialitic buildups (Fig. 1b andc). The samples were immediately wrapped in PVDC plastic, transported to the lab in plastic containers and stored in the dark at 4 °C.

Water chemistry

The pH and the temperature were measured using a Metter-Toledo GoFive handheld pH/temperature probe. The conductivity and the salinity were determined with a Fisher Accumet AP75 handheld conductivity/temperature probe. Three replicas of water samples were taken ca. 5 cm above the mat and stored in glass vials (4-ml vials either under in situ pH conditions, acidified for analysis of major ions or fixed in 1.25 mM EDTA 87 mM acetic acid to determine the arsenic concentration). The water samples were kept refrigerated while being transported to the laboratory. Major cation (NH 4 + , K + , Mg 2+ and Ca 2+ ) and anion (SO 4 2-and NO 3 -) concentrations were determined by ion chromatography (Dionex ICS 3000) [START_REF] Pace | Formation of stromatolite lamina at the interface of oxygenic-anoxygenic photosynthesis[END_REF]. The physicochemical composition of the water, given in Table 1, represents the average composition of the three water samples.

Mineralogical and elemental analysis

X-ray diffraction (PXRD). The mineral composition was determined by powder X-ray diffraction in a chamber with controlled temperature and humidity. Finely grounded powder of fresh samples were analyzed using a Panalytical X'per MPD diffractometer with copper anode (Cu Ka radiation). The diffraction patterns were collected from 5 to 80º (2θ) at 100% humidity and decreasing humidity (until 50% relative humidity). The patterns thus obtained were analyzed employing the program XPowder (Martin, 2004) with the ICDD-PDF2 database. The results are shown in Fig. SI1.

Microscopy SEM-EDX. The thin section of Fig. 3a was observed under vacuum using a Zeiss EVO SEM equipped with an Energy Dispersive Spectrometer (Bruker) using a voltage of 15 keV and a current beam of 2 nA (Fig. 2). Spectra were analyzed using the software Quantax 200 (Bruker).

Major and trace element analysis. The major and trace element composition of the portion of microbialite investigated using synchrotron X-ray microfluorescence were analyzed at the "Services d'Analyses des roches et des Minéraux" (SARM) du Centre de Recherches Pétrographiques et Géochimiques de Nancy, France (Table 2). Major and trace elements were measured by ICP-OES and ICP-MS, respectively. The uncertainties are 5% for concentrations greater than 10 ppm and 15% for concentrations between 10 and 1 ppm (See [START_REF] Carignan | Routine Analyses of Trace Elements in Geological Samples using Flow Injection and Low Pressure On-Line Liquid Chromatography Coupled to ICP -MS: A Study of Geochemical Reference Materials BR, DR -N, UB-N, AN-G and GH[END_REF] for a detail description of the experimental protocols use at SARM for major and trace element analyses).

Confocal laser scanning microscopy analysis. The thin section of Fig. 3a was examined using a FluoView FV1000 CLSM with spectral resolution of 2 nm and spatial resolution of 0.2 m (Olympus Tokyo, Japan). An additional section of the fresh microbialite was also inspected. For examination of the later, an oil immersion objective UPLSAPO 60XO (Olympus; 60 magnification, numerical aperture 1.35) was used. Fluorescence stack images for both samples were obtained with a concomitant excitation at wavelengths of 405, 488 and 543 nm by collecting the emitted fluorescence between 425-475, 500-530 and 560-660 nm. Fluorescence spectra were acquired by collecting the emitted fluorescence between 435 and 796 nm, with bandwidth analyses of 10 nm and step size of 5 nm. The images were visualized and processed using the F10-ASW FLUOVIEW software (Olympus) (Fig. S4). The samples were investigated following the protocol described by [START_REF] Gérard | Specific carbonate-microbe interactions in the modern microbialites of Lake Alchichica (Mexico)[END_REF].

Synchrotron analysis

For synchrotron analysis, microbialites were progressively dehydrated in a series of ethanol baths (30%, 50%, 70%, 90% and 100%) and then gradually impregnated with hard grade LR-white resin (for details on the protocol, see [START_REF] Gérard | Specific carbonate-microbe interactions in the modern microbialites of Lake Alchichica (Mexico)[END_REF]). This resin has a strong resistance to prolonged light exposure inherent to imaging and is free of metallic impurities. Petrographic thin sections were cut with a diamond wire saw and polished down to homogenous thickness of ~100 µm (Fig. 3a). All synchrotron analyses were performed at the Nanoscopium beamline (SOLEIL synchrotron facility, France) [START_REF] Somogyi | Optical design and multi-length-scale scanning spectro-microscopy possibilities at the Nanoscopium beamline of Synchrotron Soleil[END_REF]. The sample was raster scanned in a focused X-ray beam while measuring the emitted secondary radiation and/or the transmitted beam at each position. The spatial resolution is determined by the beam-size and the experimental conditions and was between µm and 500 nanometers during the recent measurements.

Synchrotron radiation X-ray micro-Fluorescence (SR-µXRF).

A monochromatic incident X-ray beam was focused into a 300 x 300 nm 2 FWHM spot (Full Width at Half Maximum) at the sample position by using Kirckpatrick-Baez (KB) mirrors. The incident beam energy, 12 keV, was chosen by a doublecrystal Si(111) monochromator, just above the As K-edge for XRF imaging. At this energy, the measured photon flux in the 300 x 300 nm 2 -sized focused beam was ~2×10 10 photons/s. The sample was mounted onto a 3-axis sample positioning stage. The X-ray fluorescence radiation emitted by the sample was recorded by two identical silicon drift detectors of 50 mm 2 active surfaces (KETEK H50, KETEK GmbH) used with XMAP (XIA LLC) fast digital multichannel analyzer cards. The detectors were placed at backscattering geometry at ~100° compared to the incoming X-ray beam (at ~10° compared to the sample surface) [START_REF] Somogyi | Optical design and multi-length-scale scanning spectro-microscopy possibilities at the Nanoscopium beamline of Synchrotron Soleil[END_REF]. Elemental distribution maps (Fig. 3c-e andSI2) were reconstructed from the XRF spectra by using preselected spectral regions of interest (ROIs) and summing the intensities measured by the two detectors [START_REF] Bergamaschi | MMX-I: dataprocessing software for multimodal X-ray imaging and tomography[END_REF]. A deconvolution procedure using PyMCA [START_REF] Solé | A multiplatform code for the analysis of energy-dispersive X-ray fluorescence spectra[END_REF] was applied in case of overlapping X-ray peaks. The transmitted X-ray beam was recorded by a 300 µm-thick Si diode placed behind the sample, providing information about the morphology of the sample.

2D elemental distribution and transmission contrast maps were measured. In a first step, a large field of view (H x V = 720 µm x 700 µm) 1 µm x 1 µm resolution map was collected with 10 ms dwell time per point (Fig. 3b-e); for the second step, a medium field of view (H x V = 450 µm x 170 µm) 0.5 µm x 0. 5 µm resolution map was measured with 15 ms dwell time per point (Fig. SI2). In order to compare the elemental distribution maps measured with different dwell times, the intensity maps were normalized by the incident X-ray intensities (I 0 ) recorded for each pixel. Further data treatment was performed in Matlab.

X-ray absorption near edge structure (XANES).

Seven different pellets and mineral standards were used for calibrating the XANES analysis of arsenic. These include: arsenopyrite (AsFeS), orpiment (As 2 S 3 ), fetiasite (As(III)-fet, (Fe(II,III)Ti) 3 (As(III) 2 O 5 )O 2 ; As 2 O 3 pellet (Sigma-Aldrich), arsenobetaine (Asbet, C 5 H 11 AsO 2 ), legrandit (As(V)-leg, Zn 2 (As(V)O 4 )(OH)•(H 2 O)) and As 2 O 5 pellet (Sigma-Aldrich).

XANES spectra were collected by scanning the energy at 0.5 eV steps around the K-edge of As in Xray fluorescence and transmission modes. The beam energy was calibrated by defining the edge position of the As 2 O 5 standard at 11.8745 keV. The inflections points of the As K-edge XANES spectrum remained stable within 0.5 eV during this measurement period. To ensure the absence of photo-oxidation/reduction during synchrotron measurements, several As-XANES spectra were collected successively as a function of time in some selected points of the sample. No changes in these successive XANES spectra have been detected.

In order to determine the spatial variation of arsenic speciation in the sample, several "characteristics" X-ray energies were defined for XANES mapping, which correspond to the white lines of the XANES spectra of the FeAsS, As 2 S 3 , As 2 O 3 , Asbet and As 2 O 5 standards (Fig. SI3). In addition, three energy points were selected, corresponding to the pre-and far away As K-edge, and a medium energy point between Asbet and As 2 O 5 . The resulting excitation energies (11.8653, 11.8684, 11.8696, 11.8714, 11.8727, 11.8745 and 11.8819 keV) allowed tracking the variations of arsenic speciation variation in the sample. For each incident X-ray energy, a XRF map was obtained, representing a total of 7 X-ray maps for the selected zone of measurement. The obtained As speciation maps were treated with Matlab software. Only As 2 O 3 , [As(III)], and As 2 O 5 , [As(V)], dominant regions are shown here for clarity (Fig. 5). Micro-XANES measurements were also performed in several spots of the mat sample.

Principal Component Analysis (PCA) and clustering. The elemental intensities obtained from the XRF spectra were further analyzed by Principal Component Analysis (PCA) for data reduction and to identify quantitatively the dominant modes of metal associations and spatial distributions. The principal components are linear combinations of the original elemental intensities, along which the variation in the data is maximal. Since PCA is designed to identify directions with the largest variation, it is not adapted for the identification of groups describing small sample variance. In the present case, the method was well-suited for discarding several variables and obtaining a final multivariate analysis with the chemical elements most linked with arsenic (As, Ca and Fe). The principal components for the As, Ca and Fe stack of the higher resolution maps were determined and cluster analysis was performed on the dataset based on the components with the higher contribution of arsenic (Fig. 4b). Cluster analyses provides an "assumption-free" analysis of the distribution of chemical components in the sample, by classifying pixels according to their spectral similarity [START_REF] Etschmann | Speciation mapping of environmental samples using XANES imaging[END_REF]. Scatterplots were derived from the clustering analysis (Fig. 4c).

The ratios of As(V)/As(III) and As/Ca were calculated by dividing the As(V) intensity counts by the As(III) counts and the As intensity counts by the Ca counts, respectively (Fig. 4d). The ratios were calculated for the different clusters areas, indicated in different colors in figure 4b.

RESULTS

Microbialite environment

Laguna Diamante is located inside the Galan Volcano caldera (Fig. 1a), in Catamarca, Argentina (26°00'51.04 'S, 67°01'46.42'O). This is one of the largest uncovered volcano calderas of the world, measuring 34 km by 20 km, and is surrounded by mountain ranges > 6000 meters in altitude. Galan Volcano reaches 5912 m above sea level and hosted extensive volcanic activity from the upper Miocene to the Pleistocene (7 to 4 Ma) [START_REF] Soler | Geology of the Vilama caldera: A new interpretation of a large-scale explosive event in the Central Andean Plateau during the Upper Miocene[END_REF][START_REF] Ruggieri | Environmental geochemistry of ancient volcanic ashes[END_REF]. This environment is strongly influenced by hydrothermal sources, large temperature fluctuations (water temperature fluctuates daily from 1.2ºC to 21.2ºC, strong UV radiations (maximum UV-AB reaches ca. 71.3 Wm -2 ), hypersaline, alkaline (pH=9.1-10.7) water conditions and a high arsenic concentration (2.27 ± 0.88 mM).

Microbialite characterization

Laguna Diamante microbialites occur as partially submerged calcareous formations, showing tubeforming thrombolitic structures of several centimeters in size (insert in Fig. 1c). The microbialite investigated in this study was sampled near the shoreline and intermittently exposed to air. The horizontal cross section of the Laguna Diamante microbialites revealed a concentric layered structure with three distinct zones: an external, pink crystalline surface; a medium, organic-rich part, composed of two dominant colored layers, purple and green, and an internal part primarily composed of clotted carbonate (Fig. 1d).

PXRD analyses showed that calcite is the main phase and gaylussite (Na 2 Ca(CO 3 ) 2 -5 H 2 O), phyllosilicates and quartz occur as secondary phases (Fig. S1). Gaylussite, which corresponds to the pink layer, covers the tubular structure of the microbialites and is likely formed through efflorescence from the alkaline (9-10 pH) lacustrine water, similar to those found in lake Magadi, in Kenia (pH>9.5, [START_REF] Jones | Hydrochemistry of the Lake Magadi basin, Kenya[END_REF]) and Mono Lake, in USA (pH>9.5 [START_REF] Bischoff | Gaylussite formation at mono lake, california[END_REF]). Importantly, no traces of this mineral are found inside the microbialites. The internal part of the microbialite is mainly composed of calcite. SEM images showed that calcite typically appeared either as large >50 μm (Fig. 2a), or small aggregates, ~3-5 μm (Fig. 2b). Secondary phases detected included phyllosilicates and quartz that were most likely derived from weathering of the volcanic rocks, which form the local basement of Laguna Diamante. Halite was detected only when the humidity in the PXRD chamber was decreased and halite precipitated due to evaporation of the residual water trapped in the fresh sample (Fig. SI1).

Intercorrelation of arsenic and other elements

In order to determine the distribution of arsenic within the microbialite, we performed X-ray synchrotron radiation analyses in a selected area of the sample that included both organic-rich and carbonate-rich regions (the studied area is marked with an orientative black box in Fig. 1d and the exact measured area is shown in Fig. 3a). The selected area showed contrasted organic-rich (left side) and carbonate-rich (right side) regions (Fig. 3a). This can be better seen in the transmission image (Fig. 3b), where the low transmission region in the upper-right part of the sample is enriched in Ca (Fig 3d), attributed to carbonate minerals. This Ca-rich region appears linked to As (Fig. 3c). This is in contrast with the organic-rich region, indicated by the high transmission signal (center-left side of Fig. 3b). In this organic-rich zone, As and Ca show a heterogeneous distribution characterized by disseminated small (< 10 μm) hotspots (Fig. 3b andc). Complementary ICP-MS analyses highlighted the presence of Fe. Iron occurred as heterogeneously distributed hotspots in the organic-rich region (Fig. 3d). Other relatively abundant low Z elements detected by ICP-MS such as Na and Mg, or high Z elements, such as Sr and Rb, could not be detected with the experimental protocol used at Nanoscopium (see Mat&Met).

In order to better understand the chemical behavior of As in the organic-rich zone, the area marked by a yellow frame in Fig. 3c was analyzed with a 0.5 x 0.5 µm spatial resolution mapping, using 15 ms/pixel integration time (Fig. SI2, Fig. 4). The RGB image of As (green), Ca (red) and Fe (blue) of Fig. 4a shows that As occurs as a disseminated compound in irregular globular domains about 3 to5 µm in diameter (these domains will be referred as globules or globular distribution in the following text)). These globules are devoid, or contain only minor amounts of Fe and Ca. Noteworthy, the top right corner of Fig. 4a shows that As and Ca are correlated in the carbonate-rich region.

In order to gain detailed insight on the spatial distribution and the degree of co-localization of As with Ca and Fe, clustering and scatter plots were combined with PCA (Fig. 4b andc). The obtained clustering image highlighted three types of As distribution: (i) a globular distribution of As with minor contribution of Ca and Fe (labeled {As1} and marked in green), (ii) areas attributed to carbonate (labeled {As2} and marked in dark red), with a high correlation between As and Ca (indicated by the 2D plot), and (iii) disseminated As showing a diffuse pattern in the RGB image (Fig. 4a). These are represented by the light blue pixels of {As3}, where Fe and Ca are both correlated with As. The distribution labeled {As4} and marked in orange correspond to Ca, with no As or Fe correlation. In addition, the calculated As/Ca ratio for the three major distributions of arsenic showed that the irregular globules {As1} contain the highest As/Ca ratio, while the carbonate area {As2} exhibits the lowest As/Ca ratio (Fig. 4d).

Speciation of arsenic

The speciation maps of As(III) and As(V) revealed that the As(III) distribution exhibited a more diffuse pattern (Fig. 5a), while the As(V) distribution was predominantly defined by a globular pattern (Fig. 5b). The occurrence of organic material in the globular distribution was confirmed by CLSM analysis. The fluorescence emission of the globules with a maximum at 470 nm appeared similar to the one obtained in the organic area of the fresh microbialite (Fig. S4). This suggests that the main component forming the globules comprised organic material.

To better evaluate the difference between As(III) and As(V) distributions, several XANES points were collected in both the diffuse and globular regions (Fig. 5c). The XANES spectra collected in the diffuse regions showed a higher contribution of As(III) than As(V) (Fig. 5c,pt1), while the XANES spectra collected in the globules showed a higher contribution of As(V) than As(III) (Fig. 5c,pt2). This is also confirmed by the calculated As(V)/As(III) ratio (Fig. 4d), i.e. the As(V)/As(III) ratio decreases from the globular trend (labeled {As1} and marked in green) to the more diffuse trend (labeled {As3} and marked in light blue). No As(0) was identified in the XANES analysis.

DISCUSSION

The presence of organic-rich globules containing mainly arsenic ({As1}, Fig. 4) accumulated in specific zones of the microbial mats can be explained by different mechanisms. Investigations in modern sediments and bacterial cultures have addressed that prokaryotes are able to assimilate, methylate, detoxify and respire arsenic [START_REF] Stolz | Arsenic and selenium in microbial metabolism[END_REF]. Prokaryotes have developed strategies to detoxify arsenic, such as the ars gene [START_REF] Páez-Espino | Microbial responses to environmental arsenic[END_REF] and the methylation of As species [START_REF] Bentley | Microbial methylation of metalloids: arsenic, antimony, and bismuth[END_REF], but also to actively use arsenic compounds in their metabolism for obtaining energy or to support photosynthetic carbon fixation [START_REF] Oremland | A microbial arsenic cycle in a salt-saturated, extreme environment[END_REF][START_REF] Lis R Van, Nitschke | Arsenics as bioenergetic substrates[END_REF]. In addition, arsenic compounds expelled from microbial activities can likely interact with the functional groups of the extrapolymeric matrix (EPS) [START_REF] Braissant | Exopolymeric substances of sulfate-reducing bacteria: interactions with calcium at alkaline pH and implication for formation of carbonate minerals[END_REF]. These functional groups deprotonate as a function of pH, and will lead to a higher overall negative charge for EPS in alkaline conditions, such as those in Laguna Diamante. Positively charged ions, such as Ca 2+ and Mg 2+ , can form bidentate bonds with low-molecular-weight organic carbon [START_REF] Braissant | Characteristics and turnover of exopolymeric substances in a hypersaline microbial mat[END_REF], but also with negatively charged ions, such as arsenic oxyanions. In addition to this mechanism, active element cycling may also create globules of metal(loid) compounds in the organic matrix (whether this comprises EPS or microbial cells). The local environment plays then a crucial role for the metal(loid)s distribution, creating micro zones with oxidized or reduced elements. For instance, accumulation of As(III) or As(V) through detoxification reactions or bioenergetics reactions (see below), such as anoxygenic phototrophy, chemolithoautotrophic oxidation (e.g. of arsenate and sulphide) or arsenite oxidation may result in the incorporation of arsenic in the EPS.

Laguna Diamante microbialites grow in a hypersaline alkaline high-altitude lake with severe conditions, such as high UV radiation, low O 2 pressure and very low rainfall (table 1). In such an environment, the role of photosynthesis to convert inorganic carbon into organic biomass is usually the dominant metabolic pathway [START_REF] Knoll | The evolution of ecological tolerance in prokaryotes[END_REF]. In addition, the Diamante microbial metagenome suggests that methanogenesis is also an important metabolic process in this environment [START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF]. Methanogenic metabolism has also been observed in other high-altitude Andean lakes [START_REF] Maldonado | Culture-dependent andindependent methods reveal dominance of halophilic Euryarchaeota in high-altitude Andean lakes[END_REF] and most likely also occurred in Precambrian lakes with similar conditions, such as the 2.7 billion-year-old anoxygenic Tumbiana lake [START_REF] Eigenbrode | Late Archean rise of aerobic microbial ecosystems[END_REF][START_REF] Sforna | Evidence for arsenic metabolism and cycling by microorganisms 2.7 billion years ago[END_REF][START_REF] Lepot | Extreme 13C-depletions and organic sulfur content argue for S-fueled anaerobic methane oxidation in 2.72 Ga old stromatolites[END_REF]. Methanogenic respiration produces methane, which can be incorporated as organic carbon into biomass through oxidation. Together with methane oxidation, nitrification (i.e. the oxidation of ammonia to nitrite and nitrate) is also likely taking place in Laguna Diamante microbialites, similar to what has been observed for related Andean lakes, such as Socompa and Titicaca Lake [START_REF] Vincent | Nitrate cycling in Lake Titicaca (Peru-Bolivia): the effects of high-altitude and tropicality[END_REF][START_REF] Farías | The Discovery of Stromatolites Developing at 3570 m above Sea Level in a High-Altitude Volcanic Lake Socompa, Argentinean Andes[END_REF]. Methanotrophic and nitrification-denitrification reactions need strong oxidants, such as oxygen, ferric iron, sulphate or any other available electron acceptor. These oxidants are potentially delivered by photosynthetic reactions. However, the precise nature of these possible candidates has not yet been identified. Similar as in other high-altitude lakes, the water column of Laguna Diamante contains low saturation levels of oxygen due to its high altitude, resulting in limited oxygen availability to be used as electron acceptor for methanotrophic or nitrification-denitrification pathways (i.e. Vincent et al., 1985, Visscher et al., in prep). Anoxygenic photosynthesis, using iron as an electron donor, cannot be significant since no ironbearing minerals were detected in the microbialites of Laguna Diamante. On the other hand, anoxygenic phototrophs using reduced sulfur as electron donor, are common in hypersaline environments [START_REF] Visscher | Competition between anoxygenic phototrophic bacteria and colorless sulfur bacteria in a microbial mat[END_REF][START_REF] Dupraz | Stromatolitic knobs in Storr's Lake (San Salvador, Bahamas): a model system for formation and alteration of laminae[END_REF][START_REF] Pace | Formation of stromatolite lamina at the interface of oxygenic-anoxygenic photosynthesis[END_REF]. Several anoxygenic phototrophs that use sulfur can also use other electron donors, such as As(III). For instance, phototrophic sulfide oxidizers of Diamante, such as Chloroflexus aurantiacus, have been reported to contain arsenite oxidation gene, Aio [START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF], which may produce As(V). Some anoxyphototrophs can use sulfide for their metabolic activities, but other electron donors, such as As(III). In this case, As(III) may be a potential electron donor for anoxygenic photosynthesis and denitrification in Laguna Diamante microbialites. The arsenite can be used instead of sulfide as electron donors by some purple sulfur bacteria that live in As-rich environments. This has been observed in Ectothiorhodospira sp. in a Hot Spring Photosynthetic Biofilm at Mono Lake [START_REF] Hoeft | Coupled Arsenotrophy in a Hot Spring Photosynthetic Biofilm at Mono Lake, California[END_REF]. As(V), in turn, is then available as an electron acceptor for chemolithoautotrophic or chemoorganoheterotrophic anaerobes capable of As(V) respiration, for which also a gene, Arr, has been detected in Laguna Diamante microbialites [START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF]. Thus, arsenic cycling is certainly feasible in Laguna Diamante microbialites. Evidence for arsenic cycle has been reported in other high-altitude lakes [START_REF] Lara | Enrichment of arsenic transforming and resistant heterotrophic bacteria from sediments of two salt lakes in Northern Chile[END_REF][START_REF] Farías | The Discovery of Stromatolites Developing at 3570 m above Sea Level in a High-Altitude Volcanic Lake Socompa, Argentinean Andes[END_REF][START_REF] Kurth | Arsenic metabolism in high altitude modern stromatolites revealed by metagenomic analysis[END_REF][START_REF] Ordoñez | Haloarchaea from the Andean Puna: Biological Role in the Energy Metabolism of Arsenic[END_REF][START_REF] Sancho-Tomás | Distribution, redox state and (bio)geochemical implications of arsenic in present day microbialites of Laguna Brava, Salar de Atacama[END_REF] and in similar extant environments, such as Mono Lake and Searles Lakes [START_REF] Oremland | A microbial arsenic cycle in a salt-saturated, extreme environment[END_REF][START_REF] Kulp | Dissimilatory arsenate and sulfate reduction in sediments of two hypersaline, arsenic-rich soda lakes: Mono and Searles Lakes, California[END_REF][START_REF] Kulp | Arsenic(III) Fuels Anoxygenic Photosynthesis in Hot Spring Biofilms from Mono Lake, California[END_REF][START_REF] Lis R Van, Nitschke | Arsenics as bioenergetic substrates[END_REF][START_REF] Edwardson | Metatranscriptomic analysis of prokaryotic communities active in sulfur and arsenic cycling in Mono Lake, California, USA[END_REF][START_REF] Saunders | Complete arsenic-based respiratory cycle in the marine microbial communities of pelagic oxygen-deficient zones[END_REF]. Remarkably, the sequences for arsenite oxidases and respiratory arsenate reductase from Laguna Diamante microbialites have been identified in haloarchaea [START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF] and further research performed with Haloarcheae isolated from the same biofilm demonstrated the expression of this AioA gene under As growth [START_REF] Ordoñez | Haloarchaea from the Andean Puna: Biological Role in the Energy Metabolism of Arsenic[END_REF]. Divergence in the sequences of haloarchaea AioA and ArrA genes and the wide geographical distribution of the species that harbour them, could indicate a long evolutionary history of these enzymes in haloarchaea. It is thus likely that arsenic metabolisms may have been essential on the anoxic Precambrian Earth. This notion is supported by the evidence of a complete arsenic cycle taking place in 2.7 billion-year-old stromatolites from Tumbiana, Australia [START_REF] Oremland | Arsenic in the Evolution of Earth and Extraterrestrial Ecosystems[END_REF][START_REF] Sforna | Evidence for arsenic metabolism and cycling by microorganisms 2.7 billion years ago[END_REF].

Detoxification reactions can also expel arsenic into the EPS. This mechanism cannot be excluded as genes related to detoxification mechanisms, particularly the Ars operon, were detected in the Diamante microbial metagenome [START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF]. The Ars operon promote a multistep process of methylation and redox reactions followed by formation of organoarsenic intermediates, like arsenosugars or arsenolipids [START_REF] Bentley | Microbial methylation of metalloids: arsenic, antimony, and bismuth[END_REF] to eliminate intracellular arsenic [START_REF] Páez-Espino | Microbial responses to environmental arsenic[END_REF] and it may also locally release several arsenic compounds. Although the involved reactions are still not well understood [START_REF] Couture | Sorption of Arsenite, Arsenate, and Thioarsenates to Iron Oxides and Iron Sulfides: A Kinetic and Spectroscopic Investigation[END_REF][START_REF] Huertas | Metals in Cyanobacteria: Analysis of the Copper, Nickel, Cobalt and Arsenic Homeostasis Mechanisms[END_REF], it has been suggested that methylation reactions may be used as an (supplementary) energy source [START_REF] Yamamura | Microbiology of inorganic arsenic: From metabolism to bioremediation[END_REF]. Methylation and excretion of arsenic have been estimated to originate during the Archaean eon [START_REF] Chen | The Great Oxidation Event expanded the genetic repertoire of arsenic metabolism and cycling[END_REF]. Together with the Ars operon, other detoxifying genes such as ArsB and Acr3 arsenite efflux pumps have also been detected in the Diamante microbial metagenoma [START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF]. Hence, detoxification process is likely to occur in the studying microbialites. Detoxifying genes are present in most of the organisms that live in As-rich environments, from humans [START_REF] Schlebusch | Possible positive selection for an arsenic-protective haplotype in humans[END_REF] to microbial living in shallow submarine hydrothermal ecosystems along the Hellenic Volcanic [START_REF] Fru | Arsenic and high affinity phosphate uptake gene distribution in shallow submarine hydrothermal sediments[END_REF]. In any case, the products of both bioenergetics and detoxification reactions will eventually be expelled from the cell and accumulate in the EPS.

When the surface biofilm grows upward, the deeper layers of the microbialite undergo early and slow heterotrophic degradation and ultimately diagenetic alteration, leading to the fossilization of the microbialite [START_REF] Dupraz | Microbial lithification in marine stromatolites and hypersaline mats[END_REF]. It is in this process the imprint of specific metabolic activity can be preserved. A preliminary study of processes taking place in microbialites from Bahamas during diagenesis evaluated the metal(loid)s association and distribution within an extant stromatolite down to relic layers (~2 kyr) [START_REF] Sforna | Patterns of metal distribution in hypersaline microbialites during early diagenesis: Implications for the fossil record[END_REF]. The ongoing changes in the elemental distribution patterns through early diagenesis were traced from the centimetre to the micrometer scale. Remarkably, the metal(loid)s distribution at the bottom of the mat was both homogeneous (due to passive absorption of elements within the mat) and heterogeneous. In particular, the Asbearing organic globules embedded in carbonate grains were not associated with any other metal. The As globules were attributed to As-metabolizing microorganisms, showing the potential of this element to be interpreted as a biosignature in modern system as well as in the rock record [START_REF] Sforna | Patterns of metal distribution in hypersaline microbialites during early diagenesis: Implications for the fossil record[END_REF]. Although difficult to constrain the processes involved (probably both, energetic and detoxification pathways), we propose below a potential mechanism of the transformation of the arsenic fingerprints, from the organic-rich, irregular arsenic globules in the newly formed surface layer to the early diagenetic stage (Fig. 6). This might be useful for identifying past biologically activity in the ancient rock record.

During the first step, arsenic cycling occurs through microbe-mediated reactions (Fig. 6a), which releases different As compounds in the environment. During a second step, these As species bound to the organic matrix, whether interacting with the functional groups of the EPS or with microbial cells, and form irregular globules (Fig. 6b) such as those shown in Figure 4, {As1}. Once released into the extracellular matrix (EPS), the distribution of the arsenic species may vary depending on the mobility of the released arsenic species and the chemical conditions of the extracellular medium. As such, the As(III) distribution displayed a more diffuse pattern (Fig. 5a) compared to As(V), which shows a globular morphology (Fig. 5b). Arsenite is considered to be more mobile than arsenate [START_REF] Smedley | Chapter 1 Source And Behaviour Of Arsenic In Natural Waters[END_REF] because As(III) exists predominately as a neutral species at mildly basic conditions (PKa of H 3 AsO 3 = 9.32, 12.10, 13.41) [START_REF] Sø | Sorption and desorption of arsenate and arsenite on calcite[END_REF][START_REF] Yokoyama | Differences in the immobilization of arsenite and arsenate by calcite[END_REF]. Contrary, As(V), is a stronger acid (PKa = 2.24,6.96,11.50), being more readily immobilized in the microbial mat through interactions with the functional groups of EPS, such as carboxyl, phosphate, amino, or sulfur groups [START_REF] Braissant | Exopolymeric substances of sulfate-reducing bacteria: interactions with calcium at alkaline pH and implication for formation of carbonate minerals[END_REF]. During the third step of the proposed mechanism, precipitation of CaCO 3 takes place (Fig. 4,{As2}). The carbonate mineral replacing the EPS [START_REF] Dupraz | Microbial lithification in marine stromatolites and hypersaline mats[END_REF] then incorporates As (Fig. 6c).

Microbial metabolism plays an important role in carbonate precipitation, mainly through inducing pH changes [START_REF] Dupraz | Microbial lithification in marine stromatolites and hypersaline mats[END_REF]. Processes, such as anoxygenic phototrophy and sulfate reduction (and by extension also arsenate reduction) contribute to CaCO 3 precipitation (Dupraz et al., 2009). Laboratory studies of the incorporation of As in the calcite structure have shown that abiotic incorporation processes result in predominantly As(V) uptake (e.g. [START_REF] Sø | Sorption and desorption of arsenate and arsenite on calcite[END_REF][START_REF] Yokoyama | Differences in the immobilization of arsenite and arsenate by calcite[END_REF]). This can be explained on the basis of the different chemical nature of arsenite and arsenate oxyanions [START_REF] Sø | Sorption and desorption of arsenate and arsenite on calcite[END_REF][START_REF] Yokoyama | Differences in the immobilization of arsenite and arsenate by calcite[END_REF]. The first one exists predominantly as a neutral species while the second one is a stronger acid, forming a negative oxyanion that favours not only the interactions with EPS but also the adsorption and incorporation of As(V) into the calcite structure [START_REF] Yokoyama | Differences in the immobilization of arsenite and arsenate by calcite[END_REF][START_REF] Renard | Interactions of arsenic with calcite surfaces revealed by in situ nanoscale imaging[END_REF]. The presence of As(III) in the calcite structure has been related with the occurrence of biological process, for example, a significant fraction of As(III) has been detected in natural calcite from travertine deposits of Central Italy (Di [START_REF] Benedetto | Arsenic incorporation in natural calcite lattice: Evidence from electron spin echo spectroscopy[END_REF][START_REF] Bardelli | Arsenic uptake by natural calcite: An XAS study[END_REF]. More recently, the incorporation of As(III) was detected in biogenic calcite precipitated from bacteria cultures but not in the abiotic control incubations [START_REF] Catelani | Arsenic uptake in bacterial calcite[END_REF]. Hence, the presence of As(III) in the globules ({As1} in fig. 4) and in the high-carbonate areas ({As2} in fig. 4) strongly indicates that the incorporation of As into the structure is microbial mediated (Fig. 5e). Finally, the EPS is gradually replaced by micrite (Fig. 6d) [START_REF] Dupraz | Microbial lithification in marine stromatolites and hypersaline mats[END_REF], which then can incorporate the remaining more diffuse and mobile As(III) excreted by the arsenic respiring bacteria. This is supported by the increase of As(III) in the Ca-rich globules/carbonate zone compared to the organic-rich globules (see As(V)/As(III) ratio of Fig. 4e).

CONCLUDING REMARKS

The co-occurring presence of two oxidation states, As(III) and As(V), discovered in this study represents the first geochemical evidence of microbial-mediated arsenic cycling in Laguna Diamante microbialites, supporting the metagenomic analysis published by Rascovan and co-workers [START_REF] Rascovan | Metagenomic study of red biofilms from Diamante Lake reveals ancient arsenic bioenergetics in haloarchaea[END_REF]. We propose a mechanism for preserving the As-microbial mediated cycle in the carbonate structure of microbialites from initial lithification to early diagenesis. This might be extended to the fossil record, where further studies may lead to development of an arsenic fingerprint for detection of life in the fossil record from the early Earth and, possibly, from other planets such as Mars. versus Ca (left) and As versus Fe (right) intercorrelation diagrams: {As1, green} corresponds to the globular distribution of As and it is partially correlated with Ca; {As2,red} corresponds with the carbonate areas and reflects the spatial overlap between As and Ca; {As3, light blue} corresponds with the diffused distribution of As and it is partially correlated with Fe and Ca; and {As4, orange} corresponds with the areas where Ca is mostly alone with no or only minor contributions of As or Fe. (d) As(V)/As(III) and As/Ca ratios of the pixel counts in the specific areas inferred by PCA and clustering analysis, showed in panel b: {As1} in green, {As2} in dark red, and {As3} in light blue. Figure 6. A tentative model of the transformation of arsenic fingerprints from an organic-rich active metabolic area up to the early diagenesis stage. (a) Arsenic cycling is promoted due to microbial activities. (b) Arsenic species interact with the organic matrix accumulating in zones with a higher concentration of organic material. The dotted circle marks the As-EPS globular distribution with higher contribution of As(V) (e.g. {As1}, Fig. 4), while a more diffuse trend with higher contribution of As(III) (e.g. {As3}, Fig. 4) is located around the dotted circle. The organic material (EPS) is shown as white background. (c) Precipitation of CaCO 3 takes place in the globular distribution and arsenic is retained in calcite precipitates (e.g. {As2}, Fig. 4). (d) Finally, EPS is gradually replaced by micrite (e.g. top right corner of Fig. 4b,{As2}).

FIGURE and TABLE CAPTIONS

Table 1. Physicochemical properties of the water column at the sample locality. Measurements shown indicate the minimum and maximum values observed during a 24-hr period. The lowest values for pH, temperature of the air and water and light intensity were measured just before sunrise, around 6am in the morning. The maximum values reflect observations made early-to midafternoon during peak photosynthetic activity. Journal Pre-proof Supp. info Figure SI1. Power X-ray diffraction patterns obtained from Lake Diamante microbialites, showing predominantly calcite with minor quantities of gaylussite, muscovite and quartz (fresh sample, in blue). Halite precipitates when the relative humidity is decreases in the sample chamber (RH 50%, in red) confirming that this mineral is not formed inside of living microbialites 3c showing the distribution patterns of As, Ca and Fe. The colored scale indicates the square root of the XRF intensities: the background intensity (in blue, below detection limit) and the maximum measured intensity (in red). Dimensions of the map is 450 µm x 175 µm. Mapping was performed using a 0.5 µm x 0.5 µm incremental step.

Figure 1 .

 1 Figure 1. Site location and view of the studied microbialite from Diamante Lake (Argentina). (a) Aerial view of Diamante Lake showing the Galán peak. The sampling site is marked by a red arrow. (b) Closer view of the sampling site. (c) Overview of the tubular build-up microbialites (corresponds with the black box shown in image b). Inset: Photograph of the living microbialite analyzed in this study, the arrow indicates the grow direction. (d) Zoom in of the area outlined in (c) showing the cross section of the Diamante microbialites. The analyzed area of Fig. 3a is marked by a black box.

Figure2.

  Figure2. SEM images (left) and EDX analysis (right) of the area investigated using synchrotron techniques. Calcite precipitates, showing the As-Ca intercorrelation {As2} in the carbonate layer (a) and in the globules (b). Image (b) corresponds with the white rectangle in Fig. 2b. The arrows indicate the location where the X-ray beam was focused.

Figure 3 .

 3 Figure 3. Large field of view multi-technique maps of the sample area marked by the red box in the 100 µm-thick polished section (a), showing the transmission image (b) and the distribution of As, Ca and Fe (c, d and e). The yellow square in (c) indicates the area where the high resolution map was performed. The color scale indicates the square root of the XRF intensities where blue represents the background intensity (i.e. below detection limit) and red represents the maximum measured intensity (see main text for details).

Figure 4 .

 4 Figure 4. (a) RGB image of the distribution of As(green), Ca(red) and Fe(blue) in the area indicated in Fig. 3c. Blue arrows indicate the Fe regions. (b) Image of the four main clusters of PCA score results characteristics for the elemental variation of As, Ca and Fe. Dark blue represents the background. As

Figure 5 .

 5 Figure 5. XANES maps of (a) an As(III) dominanted region and (b) an As(V) dominanted region, with an inset view of the globular sample region marked by the white rectangle for (d) As(III) contribution and (e) As(V) contribution. Dark blue indicates the background. (c) XANES spectra at the As k-edge obtained in two representative areas where either As(III) (pt1, As-diffused area, {As3}) or As(V) (pt2, As-globular area, {As1}) is most abundant. The three black curves correspond to As 2 O 3 and As 2 O 5 standards.

Figure SI2 .

 SI2 Figure SI2. XRF maps of the boxed area in Fig.3cshowing the distribution patterns of As, Ca and Fe. The colored scale indicates the square root of the XRF intensities: the background intensity (in blue, below detection limit) and the maximum measured intensity (in red). Dimensions of the map is 450 µm x 175 µm. Mapping was performed using a 0.5 µm x 0.5 µm incremental step.

  Figure1

Figure 4 Figure 5

 45 Figure 4

Figure 6

 6 Figure 6

Table 2 .

 2 Bulk major and trace element concentrations of Laguna Diamante microbialites.

ACKNOWLEDMENTS

This work has received funding from the European Union's Horizon 2020 Research and Innovation programme under the Marie Skłodowska -Curie Grant Agreement N o 701662 and the Programme National de Planétologie. PTV Acknowledges support from US National Science Foundation grant EAR 1561173.

Figure SI3

. XANES spectra of the measured standards. The X-ray energy points defined for XANES mapping correspond to the energy at the maximum intensity of: FeAsS, As 2 S 3 , As 2 O 3 , Asbet and As 2 O 5 and to a medium energy point between Asbet and As 2 O 5 . Two additional energy points were selected, corresponding to the pre-and far away As K-edge. Journal Pre-proof

Journal Pre-proof

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: