Charlotte Depeux
email: charlotte.depeux@gmail.fr

Jean-François Lemaître

Jérôme Lemaître

François-Xavier Moreau

Tiffany Dechaume-Moncharmont

Hélène Laverre

Jean-Michel Pauhlac

Sophie Gaillard

Beltran-Bech

Jean-François Lemaître

Jérôme Moreau

François-Xavier Dechaume-Moncharmont

Tiffany Laverre

Hélène Pauhlac

Jean-Michel Gaillard

Sophie Beltran-Bech

Reproductive senescence and parental effects in an indeterminate grower

Keywords: Reproductive success, aging, Armadillidium vulgare, clutch size, indeterminate growth

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

Introduction

Deciphering the eco-evolutionary roots of inter-individual variation in reproductive success is a long-standing objective in evolutionary biology. Many overall (e.g. temperature, photoperiod) and biotic (e.g. social relationships, population density, predation, food availability) factors can modulate reproductive success in both vertebrates and invertebrates (e.g. [START_REF] Descamps | Age-specific variation in survival, reproductive success and offspring quality in red squirrels: evidence of senescence[END_REF][START_REF] Geister | Effects of temperature on reproductive output, egg provisioning, juvenile hormone and vitellogenin titres in the butterfly Bicyclus anynana[END_REF][START_REF] Schwanz | Chronic parasitic infection alters reproductive output in deer mice[END_REF][START_REF] Bertram | Social factors influencing reproduction in wild lions[END_REF][START_REF] Donelson | Effects of elevated water temperature and food availability on the reproductive performance of a coral reef fish[END_REF]. Within a given population, individual attributes such as age and size are also associated with reproductive performance in most animals [START_REF] Clutton-Brock | Causes of assortative mating in arthropods[END_REF]. However, reproductive success, usually measured as the number of offspring produced at some pre-defined stage, (i.e. conception, birth, or independence of offspring) does not account for the entire process of reproduction. In an evolutionary context where the performance of offspring matters because it shapes Darwinian fitness (at the individual level) and growth (at the population level), a trans-generational approach is required.

Among the factors that influence the fate of offspring, the role of parental age is currently attracting considerable attention (see [START_REF] Lemaître | Reproductive senescence: new perspectives in the wild[END_REF] for a review). Evidence of a decrease in reproductive performance at old age has accumulated in the last decades [START_REF] Nussey | Senescence in natural populations of animals: widespread evidence and its implications for biogerontology[END_REF] and empirical studies have reported evidence of reproductive senescence in a large range of reproductive traits including clutch size [START_REF] Tabata | Sexual attractiveness and reproductive performance in ageing females of a coccoid insect[END_REF], juvenile mass or size [START_REF] Sharp | Reproductive senescence in a cooperatively breeding mammal[END_REF][START_REF] Barks | A multigenerational effect of parental age on offspring size but not fitness in common duckweed (Lemna minor)[END_REF], offspring survival [START_REF] Packer | Reproductive cessation in female mammals[END_REF], offspring reproduction [START_REF] Ducatez | Complex interactions between paternal and maternal effects: parental experience and age at reproducion affect fecundity and offspring performance in a butterfly[END_REF] or offspring lifespan [START_REF] Lansing | A transmissible, cumulative, and reversible factor in aging[END_REF][START_REF] Barks | A multigenerational effect of parental age on offspring size but not fitness in common duckweed (Lemna minor)[END_REF]. Interestingly, when [START_REF] Lansing | A transmissible, cumulative, and reversible factor in aging[END_REF] published his pioneering study demonstrating a negative effect of parental age on offspring lifespan in rotifers, he made no explicit distinction between the maternal and paternal ages. However, most studies of this process (i.e. the so-called Lansing effect) have focused on maternal age [START_REF] Lemaître | Reproductive senescence: new perspectives in the wild[END_REF] while old paternal age should also negatively influence offspring performance [START_REF] Lemaître | Reproductive senescence: new perspectives in the wild[END_REF] as a result of decreasing sperm quality with increasing paternal age [START_REF] Johnson | Are old males still good males and can females tell the difference?[END_REF]. Studies looking at the effect of both maternal and paternal ages as well as their potential interactions are now strongly required [START_REF] Dzyuba | Effect of parental age and associated size on fecundity, growth and survival in the yellow seahorse Hippocampus kuda[END_REF][START_REF] Tidière | Maternal reproductive senescence shapes the fitness consequences of the parental age difference in ruffed lemurs[END_REF].

So far, most studies of parental age effect focused on determinate growers (especially long-lived birds and mammals) [START_REF] Fay | Paternal but not maternal age influences early-life performance of offspring in a long-lived seabird[END_REF], which are supposed to show stronger senescence than indeterminate growers [START_REF] Vaupel | The case for negative senescence[END_REF]. In indeterminate growers the number of offspring produced per reproductive attempt is expected to increase with age, which should counter-select reproductive senescence in terms of fecundity [START_REF] Vaupel | The case for negative senescence[END_REF][START_REF] Jones | Diversity of ageing across the tree of life[END_REF]. For example, in fish populations, older and so bigger individuals generally have a higher reproductive success than younger and smaller individuals [START_REF] Venturelli | Evidence for harvest-induced maternal influences on the reproductive rates of fish populations[END_REF], which might be due to the production of high quality eggs by old females (e.g. [START_REF] Vallin | Maternal effects on egg size and egg buoyancy of Baltic cod, Gadus morhua[END_REF] in the Atlantic cod, Gadus morhua). Age classes largely differ in size in indeterminate growers, which makes mating between individuals from distinct cohorts difficult, if not impossible. In such species, both selective pressure and physical constraints during mating can lead to a pattern of size-(and thus age-) assortative mating (Crespi, 1989). Thus, reproductive senescence can be exacerbated by the cumulative effects of both maternal and paternal ages [START_REF] Lemaître | Reproductive senescence: new perspectives in the wild[END_REF]. While investigations of reproductive senescence are mandatory to refine our current understanding of the evolution of senescence [START_REF] Jones | Senescence is not inevitable[END_REF], such studies are still lacking, especially in invertebrates. Here, we aim to fill this knowledge gap by investigating whether reproductive senescence occurs in an indeterminate grower, the common woodlouse Armadillidium vulgare, and whether advanced parental age negatively influences offspring fitness components.

Materials and Methods

Biological model

The common woodlouse is a terrestrial isopod that can live up to three years [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]. This species exhibits indeterminate growth and as such large size differences across individuals can be observed. For instance, an old individual could be up to ten times larger than a newly mature individual [START_REF] Brody | A cost of reproduction in a terrestrial isopod[END_REF]. Therefore, physical constraints prevent young individuals to mate with old ones, leading to a pattern of assortative mating in both size and age at the population level [START_REF] Lefebvre | Mass drives mating success in Armadillidium vulgare (Crustacea, Oniscidea)[END_REF]. Female parental care is energetically costly in this species as females actively carry and protect their offspring in a marsupium for a month [START_REF] Surbida | Embryo tolerance and maternal control of the marsupial environment in Armadillidium vulgare (Isopoda: Oniscidea)[END_REF]. Offspring develop in this pouch and benefit from a protected environment and nutrients provided by their mother [START_REF] Surbida | Embryo tolerance and maternal control of the marsupial environment in Armadillidium vulgare (Isopoda: Oniscidea)[END_REF]. As the incubation of the offspring takes place in the marsupium, the size of the females is closely associated with the number of offspring produced [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF][START_REF] Dangerfield | Phenotypic variation in the breeding phenology of the woodlouse Armadillidium vulgare[END_REF][START_REF] Durand | Producing offspring in Armadillidium vulgare : Effects of genetic diversity and inbreeding[END_REF].

All individuals of A. vulgare used in our experiment came from a natural population collected in Denmark (Helsingör) in 1982. All the specimens were virgin at the beginning of the study and had been maintained on moistened compost and subjected to Poitiers natural photoperiod (86; 46° 34' 55" N; 0° 20' 10" E) at 20°C with food provided ad libitum (i.e. slices of fresh carrots and dried leaves of linden, Tilia sp). These conditions were repeated across all experiments.

Differences of reproductive performance between young and old parents

We set up two groups of 40 breeding pairs comprising of one male and one female: one group was made up of old parents (36-months-old) and another group was made up of young ones (12months old). The mass of each individual was initially checked to control for variation within age groups. Young individuals weighed (mean ± sd) 0.081g ± 0.027g and old ones 0.293g ± 0.044g.

All 80 breeding pairs were weekly monitored throughout 7 months (i.e. during the entire reproductive season, from January to July) and when females were gravid, they were isolated and placed in boxes with wet paper towel. Only the first clutch produced was used because very few breeding pairs produced a second clutch. We computed the breeding rate according to age (the probability to produce a clutch) for each parental group (i.e. young parents and old parents). We recorded the number of offspring per clutch (hereafter called clutch size). The entire clutch was weighed and then divided by clutch size to obtain the mean offspring mass at birth. The experimental design is presented in Figure 1.

Inter-generational traits: offspring mass, survival and reproductive success

We monitored the offspring during the most critical period of their lives (0-18 months). Indeed, in the wild, only 11% of individuals are able to reach 2-years-old [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]. To estimate survival, we counted offspring from the previous breeding pairs a first time at birth, a second time at 5 months of age, then at 12 months of age and finally at 18 months of age (Figure 1). The number of surviving offspring counted at each time allowed estimating offspring survival from 0 to 5 months of age, offspring survival from 5 to 12 months of age, offspring survival from 12 to 18 months of age and total offspring survival (i.e. from 0 to 18 months of age). Offspring were weighed as 12 (± 1) months old and then as 18 (± 1) months old to obtain the offspring mass at 12 months of age and the offspring mass at 18 months of age. To estimate reproductive success of offspring born from young or old parents, we set up, when they are sexually mature (i.e.1-year-old, [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF], 4 combinations of 20 breeding pairs (avoiding brothers and sisters): (i) offspring from old parents together, (ii) female offspring from old parents with male offspring from young parents, (iii) females offspring from young parents with male offspring from old parents, and (iv) offspring from young parents together. To avoid pseudoreplication issues (sensu [START_REF] Hurlbert | Pseudoreplication and the design of ecological field experiments[END_REF], we analyzed the reproductive status (i.e. reproduction success or failure) of the pair as function of the age group of the female's parents in interaction with the age group of the father' parents.

Statistical analysis

All statistical analyses were performed using the software R 3.6.0 (R Core Development Team 2019). For each model, we followed a model selection procedure to identify the minimal model, or the set of adequate models [START_REF] Anderson | Avoiding Pitfalls When Using Information-Theoretic Methods[END_REF]. Starting from the full model, we ranked all the nested models based on their AIC. Among the top ranked models (delta AIC < 2), we only retained the most parsimonious ones: if one of these models was simply a more complex form of another one listed among the top rank models, for instance because it contained an additional parameter (variable or interaction term), this parameter was ignored and the more complex model was not retained [START_REF] Galipaud | Ecologists overestimate the importance of predictor variables in model averaging: a plea for cautious interpretations[END_REF], Supplementary file 1). For the mean offspring mass at birth, at 12 months of age and at 18 months of age as for offspring survival at 5 months of age, between 5 and 12 months of age and at 18 months of age we first tested for an effect of clutch size. When clutch size had a statistically significant effect (see Supplementary file 2), it was systematically included in the set of considered models (see [START_REF] Berger | How do animals optimize the sizenumber trade-off when aging? Insights from reproductive senescence patterns in marmots[END_REF] for a similar approach).

Breeding rate as binary data has been modelled using a generalized linear model (GLM) with binomial distribution with parent's age ('young parents' vs. 'old parents') as a fixed factor.

Clutch size was modelled using a linear model (LM) with normal distribution with parent's age as a fixed factor. The mean offspring mass at birth was modelled using a linear model (LM) with a normal distribution because only the average clutch mass was recorded (i.e. individual body mass within a clutch could not be measured at birth). Offspring mass at 12 months of age and offspring mass at 18 months of age were analysed with a linear mixed effect models (LMM) with a normal distribution, using parent's age, offspring sex and their interaction as fixed factors and the breeding pairs identity as a random effect to take into account a potential genetic effect. The survival to 5 months of age was modelled with a generalized linear mixed effect model (GLMM) with a binomial distribution, the parent's age and clutch size were the fixed factors, and the breeding pairs identity was the random effect. The offspring survival from 5 to 12 months of age, from 12 to 18 months of age, and the overall survival (i.e. from 0 to 18 months of age) have been modelled as binary data using generalized linear mixed effect models (GLMM) with a binomial distribution, parent's age and offspring sex and their interaction added to the clutch size were the fixed factors and the breeding pairs identity was used as the random effect. The breeding rate of offspring was modelled with binary data using a generalized linear model (GLM), with mother's age and father's age and their interaction as fixed factors.

Finally, reproductive senescence can be masked by the selective disappearance of low quality individuals [START_REF] Nussey | Measuring senescence in wild animal populations: towards a longitudinal approach[END_REF]. For a given age larger females produce more offspring in the common woodlouse and body mass is thus generally thought to be a good indicator of individual quality [START_REF] Durand | Producing offspring in Armadillidium vulgare : Effects of genetic diversity and inbreeding[END_REF]. We tested for such relationships by modelling the breeding rate as binary data using a generalized linear model (GLM) with maternal mass set as a fixed factor within the young and old mothers separately. Then, in absence of repeated data on individuals over the life course, we tested for a putative influence of selective disappearance on our results by testing within the old parents group whether the first 50% of individuals that encounter premature death were lighter than the individuals that survived beyond the median life span. When doing this the effect of mass on survival of the old individual group has been modelled as binary data using a generalized linear model (GLM) with binomial distribution with individual mass, sex and their interaction as fixed factors.

Results

Differences of reproductive performance between young and old parents

Old parents (40 breeding pairs) produced 16 clutches, while young parents (40 breeding pairs) produced 24 clutches. No detectable difference was found concerning the breeding rate. The model with only the intercept was selected, but if we looked the non-selected model concerning the effect of age on the breeding rate, we found that the breeding rate tended to decrease with increasing parental age (estimate ± SE=-0.76 ± 0.45, LR Chisq1=2.86, P=0.09; Table 1; Figure 2). Moreover, old parents produced more and heavier offspring than young parents (estimate ± SE=45.15 ± 12.90, LR Chisq1= 12.62, P<0.001; estimate ± SE=5.63e05g ± 1.77e05g, LR Chisq1=12.62, P=0.001, respectively; Table 1; Figure 3).

Offspring mass, survival and reproductive success

The offspring mass at 12 and 18 months did not differ between young and old parents (estimate ± SE=0.004 ± 0.003, LR Chisq1=1.94, P=0.16; estimate ± SE=0.002 ± 0.004, LR Chisq1=0.11, P=0.73 for 12 and 18 months, respectively; Table 1). The cumulative survival (from 0 to 18 months) was higher for offspring born from young parents than for offspring coming from old parents (estimate ± SE=0.018 ± 0.09, LR Chisq1=3.95, P=0.04; Table 1; Figure 4). More specifically, this difference did not occur for the offspring survival between birth and 5 months of age (estimate ± SE=0.18 ± 0.69, LR Chisq1=0.07, P=0.79; Table 1; Figure 4) but is present for the survival from 5 to 12 months (estimate ± SE=-0.90 ± 0.41, LR Chisq1=4.27, P=0.04; Table 1; Figure 4) and from 12 to 18 months (estimate ± SE=-1.06 ± 0.24, LR Chisq1=8.22, P=0.004; Table 1; Figure 4). Reproductive success of offspring was higher for offspring born from young mothers and young fathers than for offspring born from old fathers or old mothers (0.25 vs. 0 for young and old females, respectively, 0.20 vs. 0.05 for young and old males, respectively; LR Chisq1=15.99, P<0.001 and LR Chisq1=5.06, P=0.02; Table 1; Figure 5).

Selective disappearance

The maternal mass did not influence the breeding rate within both the young mothers (c 2 =0.27, df=1, P=0.59) and the old mothers (c 2 =2.31, df=1, P=0.12). We also did not find any link between body mass and survival in the 3-years-old group (c 2 =1.66, df=1, P=0.20) neither in males nor in females as expressed by the absence of statistically significant interaction between sex and body mass (estimate ± P=0.13).

Discussion

In indeterminate growers, reproductive success should increase with age, leading to an absence or a negligible reproductive senescence [START_REF] Vaupel | The case for negative senescence[END_REF]. At first glance, our results support this prediction as old parents produced clutches containing more offspring than young ones [START_REF] Vaupel | The case for negative senescence[END_REF]. However, old parents tended to produce a smaller number of clutches than young ones and we detected a trans-generational effect of reproductive senescence because parental age negatively influenced both survival and reproduction of offspring. As we had hardly obtained clutches from offspring of old individuals, we were unable to study the quality of the grandchildren.

The effect of the environmental context experienced by the parents (e.g. diet, temperature, pathogen prevalence) on offspring performance has been described for a long time [START_REF] Mousseau | The adaptive significance of maternal effects[END_REF][START_REF] Curley | Epigenetics and the origins of paternal effects[END_REF] but an increasing number of studies have revealed that parental age can also negatively impact offspring survival [START_REF] Priest | The role of parental age effects on the evolution of aging[END_REF][START_REF] Fox | Maternal age affects offspring lifespan of the seed beetle, Callosobruchus maculatus[END_REF] and reproduction [START_REF] Priest | Cross-generational fitness benefits of mating and male seminal fluid[END_REF][START_REF] Bouwhuis | Trans-generational effects on ageing in a wild bird population[END_REF][START_REF] Ducatez | Complex interactions between paternal and maternal effects: parental experience and age at reproducion affect fecundity and offspring performance in a butterfly[END_REF][START_REF] Schroeder | Reduced fitness in progeny from old parents in a natural population[END_REF][START_REF] Lippens | Aging parasites produce offspring with poor fitness prospects[END_REF]. For instance, in Asian elephants (Elephas maximus), older mothers produce offspring with impaired survival [START_REF] Reichert | Maternal age at birth shapes offspring life history trajectory across generations in long lived Asian elephants[END_REF] and in the rotifers Brachionus manjavacas, maternal age negatively affects offspring survival and reproduction (e.g. egg quality) [START_REF] Bock | Maternal age alters offspring lifespan, fitness, and lifespan extension under caloric restriction[END_REF]. These results are in line with ours as we found that the parent's age has a high impact on offspring survival and reproductive prospects. Moreover, we demonstrated the occurrence of a trans-generational effect of parental age across generations. We reported that males from old parents reproduced less than males from young parents and this observed effect in males was stronger in females because females from old parents did not reproduce at all. Several mechanisms could be involved in these trans-generational effects, including oxidative stress, telomere inheritance, epigenetics, or parental care [START_REF] Njajou | Telomere length is paternally inherited and is associated with parental lifespan[END_REF][START_REF] Franklin | Epigenetic inheritance in mammals: Evidence for the impact of adverse environmental effects[END_REF]. Most studies of the physiological basis of senescence in invertebrates (notably in Drosophila melanogaster and Caenorhabditis elegans) have focused on oxidative stress due to its well-described effect on lifespan [START_REF] Golden | Oxidative stress and aging: beyond correlation: Oxidative stress and aging: beyond correlation, T. R. Golden[END_REF]. Parental care could also be impacted by age [START_REF] Lemaître | Reproductive senescence: new perspectives in the wild[END_REF] notably because the ability of acquiring and storing body reserves often decreases with increasing age, at least in vertebrates [START_REF] Skogland | Tooth wear by food limitation and its life history consequences in wild reindeer[END_REF][START_REF] Lecomte | Patterns of aging in the long-lived wandering albatross[END_REF]. Parental care effects are also well documented in invertebrates, in particular in insects [START_REF] Wong | The evolution of parental care in insects: the roles of ecology, life history and the social environment: The evolution of parental care in insects[END_REF] but the few studies investigating the effect of age on parental care in invertebrates have provided mixed results [START_REF] Ivimey-Cook | Disentangling pre-and postnatal maternal age effects on offspring performance in an insect with elaborate maternal care[END_REF]. In some organisms such as viviparous terrestrial isopods, the maintenance of the reproductive system can be very energetically expensive [START_REF] Lardies | The energetics of reproduction and parental care in the terrestrial isopod Porcellio laevis[END_REF]. In the woodlice, females provide nutrients to offspring during the incubation in the marsupium by a fluid called marsupial fluid [START_REF] Surbida | Embryo tolerance and maternal control of the marsupial environment in Armadillidium vulgare (Isopoda: Oniscidea)[END_REF]. Thus, both the quality and the quantity of nutrients provided by females in marsupium fluid could determine offspring quality and thereby their viability. Studies investigating how the marsupial fluid properties change with mother's age in the woodlouse and whether such changes might account for the decrease in reproductive success we reported in old individuals are strongly required. Overall, our knowledge on age-specific allocation to maternal and paternal care is still in its infancy in invertebrates, but we can hypothesise that a decrease in maternal care associated to increased oxidative stress and some epigenetic factors [START_REF] Curley | Epigenetics and the origins of paternal effects[END_REF][START_REF] Guillaumet-Adkins | Epigenetics and oxidative stress in aging[END_REF] could contribute to the trans-generational effect observed in woodlouse.

One of the grey areas of these results is the source of this trans-generational effect. While many studies have focused on the maternal effect, the paternal effect and the interaction between both effects remain relatively unknown. Among the rare studies that have investigated the influence of both maternal and paternal age, Bouwhuis and colleagues (2015) showed that the transgenerational effects are sex-specific in the common tern, Sterna hirundo. In this species, daughters from old mothers had a lower reproductive success than daughters from young mothers and sons from old fathers had a shorter lifespan than sons from young fathers. In the invertebrate Drosophila melanogaster, the effect of parent's age in offspring reproduction differs between sexes: sons were insensitive to maternal age but had reduced reproductive abilities with paternal age when daughters from old mothers showed a decrease in reproductive success [START_REF] Mossman | Age of both parents influences reproduction and egg dumping behavior in Drosophila melanogaster[END_REF]. Offspring produced by both old mothers and fathers show a substantial modification in reproductive behaviour with an extreme egg dumping [START_REF] Mossman | Age of both parents influences reproduction and egg dumping behavior in Drosophila melanogaster[END_REF]. Overall, both advanced maternal and paternal ages could have a negative impact on offspring lifetime reproductive success, which can be exacerbated by the strong positive age assortative mating that occurs in A. vulgare. In our experiment, it was not possible to tease apart the relative contribution of mother and father ages, notably because trials of mating between cross-aged individuals (e.g. 1-year-old individuals* 3-year-old individuals) were unsuccessful. Future experiments would need to investigate the effect of age directly on male or female reproductive traits (e.g. sperm, oocytes), which could play a key role in shaping reproductive senescence patterns [START_REF] Fricke | Male reproductive ageing -a tale of the whole ejaculate[END_REF][START_REF] Monaghan | The deteriorating soma and the indispensable germline: gamete senescence and offspring fitness[END_REF]. Finally, it is important to notice that the survival of older individuals does not seem to be linked to their mass in our experiment, which suggests that the possible selective disappearance of the lightest individuals (in a given age-class) does not impact our results.

Everything else being equal, individuals reaching 3 years of age likely achieve a higher fitness than short-lived individuals, thanks to the much larger number of eggs produced throughout lifetime. However, old individuals suffer from a strong reproductive senescence in terms of offspring quality and why selection did not select for old individuals allocating to offspring quality rather than to offspring quantity can be seen as paradoxical at the light of some recent developments in evolutionary biology of aging, which suggest that reproductive senescence should be absent in indeterminate growing species [START_REF] Vaupel | The case for negative senescence[END_REF]. However, these results can largely be explained by classic evolutionary theories of aging: i) the proportion of individuals reaching 3 years of age in nature (<1%) is far too weak to enable the evolution a new reproductive tactic [START_REF] Medawar | An unsolved problem of biology[END_REF], and ii) the selection for the production of a large quantity of offspring might come at a cost in late life in terms of offspring quality [START_REF] Williams | Pleiotropy, natural selection, and the evolution of senescence[END_REF]. Thus, our findings have two major implications. First, in the particular case of the woodlouse, these results run counter to studies carried out so far, which have viewed oldest individuals as playing a key role in population dynamics through their ability to produce many offspring [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF][START_REF] Dangerfield | Phenotypic variation in the breeding phenology of the woodlouse Armadillidium vulgare[END_REF]. However, our study does not provide information on the consequences of reproductive senescence in terms of population dynamics in the wild. While three-year-old individuals are quite rare (< 1% of a given cohort are reaching 3 years of age) to be expected to play any detectable role in population dynamics, two-years-old individuals are more common in in the wild (i.e. 13% of individuals within a given cohort reach this age) and reproductive senescence in terms of decreased offspring viability we demonstrated in our study might negatively influence population recruitment [START_REF] Paris | Population characteristics of the terrestrial isopod Armadillidium vulgare in California grassland[END_REF]. To better understand the influence of reproductive senescence from a quantitative viewpoint, data on age-specific reproductive success in the wild will be required. Our findings demonstrate that indeterminate growers are likely to show senescence but through different pathways compared to determinate growers.

Figure's Captions

Figure 1 :

 1 Figure 1: Experimental Design.

Figure 2 :

 2 Figure 2: Breeding rate according to individual's age.

Figure 3 :

 3 Figure 3: Number (A) and Mass (B) of offspring per clutch according to their parent's age.

Figure 4 :

 4 Figure 4: Offspring survival according to their parent's age.Mean proportion of offspring surviving (+/-bootstrapped 95% confidence interval) at 5 months of age, 12 months of age, and 18 months of age. Each data point corresponds to one clutch and was plotted as grey dots with a slight jitter added to distinguish adjacent points.

Figure 5 :

 5 Figure 5: Graphical display of the interactive effect of offspring sex and parental age on offspring reproductive success.

Acknowledgments

This work was supported by the French ministry of Education, the 2015-2020 State-Region PlanningContract and European Regional Development Fund and intramural funds from the Centre Nationalde la Recherche Scientifique and the University of Poitiers and by a grant from the Agence Nationalede la Recherche (ANR-15-CE32-0002-01). We would like to thank Sylvine Durand, Cybèle Prigot, Ophélie Trainson, Clémentine Préau and Marius Bredon for our constructive exchanges about that work as well as Alexandra Lafitte for technical assistance. We would also like to thank Richard Cordaux and Xavier Bonnet for their insightful comments.

Conflict of Interest

The authors declare that there is no conflict of interest.


Aline Roc
email: aline.roc@inria.fr

Fabien Lotte

Reviewing open challenges in Mental Tasks (MT-)based BCI user training

. We conducted a literature review on several aspects of MI-BCI user-training, e.g. environment, cognitive support, exercises. Here, we highlight the elements that we identified as not sufficiently explored or formalized yet.

Concerning the training environment, these challenges include:

a) balancing between a relevant control task [START_REF] Vlek | Bci and a user's judgment of agency[END_REF], a motivating environment, and the associated risk of imposing cognitive overload [START_REF] Mladenović | The impact of flow in an eeg-based brain computer interface[END_REF][START_REF] Enriquez-Geppert | Eeg-neurofeedback as a tool to modulate cognition and behavior: a review tutorial[END_REF][START_REF] Emami | The effects of visual distractors on cognitive load in a motor imagery brain-computer interface[END_REF]; b) studying social aspects of training, e.g. feedback content [START_REF] Pillette | A physical learning companion for mental-imagery bci user training[END_REF].

Concerning instructions and cognitive support, it includes:

a) introducing the BCI to foster users' understanding and engagement; b) evaluating the learnability/memorability of the MI-BCI skills and instructions [START_REF] Loup-Escande | User-centered bci videogame design[END_REF]; c) assessing users' strategies [START_REF] Bismuth | Relieving peripheral neuropathic pain by increasing the power-ratio of low-β over highβ activities in the central cortical region with eeg-based neurofeedback: Study protocol for a controlled pilot trial (smrpain study)[END_REF]; d) selecting user-centered task(s) [START_REF] Eliseyev | Personalized adaptive instruction design (paid) for brain-computer interface using reinforcement learning and deep learning: simulated data study[END_REF]; e) providing guidance based on user-only metrics instead of classification accuracy [START_REF] Lotte | Defining and quantifying users' mental imagery-based bci skills: a first step[END_REF].

Current challenges for MI-BCI exercise design include:

a) implementing varied exercises instead of the only same one [START_REF] Chavarriaga | Heading for new shores! overcoming pitfalls in BCI design[END_REF]; b) studying indirect/incidental MI-BCI training tasks, e.g. mindfulness meditation [START_REF] Tan | Effect of mindfulness meditation on brain-computer interface performance[END_REF], physical preparation [START_REF] Wriessnegger | Frequency specific cortical dynamics during motor imagery are influenced by prior physical activity[END_REF]; c) assessing online learners' states [START_REF] Felton | Mental workload during brain-computer interface training[END_REF] and progress to provide adapted/adaptive environment (e.g. manipulating timing [START_REF] George | Freeze the bci until the user is ready: a pilot study of a bci inhibitor[END_REF]); d) exploring the suitable parameters of trials (e.g. order, duration, number [START_REF] Rozand | A prolonged motor imagery session alter imagined and actual movement durations: potential implications for neurorehabilitation[END_REF]) and their influences on users' states and performances.


Z Fekiacova

D Montagne

A Duvivier

Abel Guihou

P Deschamps

Sophie S Cornu

Evolution of Retisol impacted by artificial drainage: What can we learn from stable Fe isotope ratios?

Keywords: Fe isotopes, redox processes, eluviation, drainage, mineral Highlights

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

INTRODUCTION

Iron is one of the most dynamic major elements in soils due to its sensitivity to redox conditions. In the form of Fe oxide it also represents one of the most reactive mineral phases in soils (e.g., [START_REF] Cornell | The Iron Oxides[END_REF][START_REF] Schwertmann | Solubility and dissolution of iron oxides[END_REF]. Elucidating the dynamics of Fe oxides is therefore a key issue for the evolution of soils. A potentially powerful tool to study Fe mineral transformations in soil is the isotopic composition of Fe, as many soil processes (e.g., dissolution, precipitation, lateral and vertical translocation and organic complexation, biological activity such as microbial activity and plant uptake) have been shown to fractionate Fe isotopes. For example, mineral dissolution favors light Fe isotopes in the dissolved fraction (e.g., [START_REF] Beard | Iron isotope biosignatures[END_REF], colloids with low  56 Fe values (heavy Fe depleted) play an important role in Fe release from soil [START_REF] Kusonwiriyawong | Isotopic variation of dissolved and colloidal iron and copper in a carbonatic floodplain soil after experimental flooding[END_REF] and isotopically light Fe is preferentially taken up from the soil Fe pool by strategy I plants, while limited fractionation favoring heavy Fe isotopes was observed during Fe uptake by strategy II plants (e.g., [START_REF] Kiczka | Iron Isotope Fractionation during Fe Uptake and Translocation in Alpine Plants[END_REF]Guelke and von Blanckenburg, 2007). However, there are still significant gaps in our knowledge of Fe isotope fractionations and experimental studies show that the amplitudes and directions of the Fe isotopic fractionations are variable and that their extents can overlap (e.g., [START_REF] Anbar | Iron stable isotopes: beyond biosignatures[END_REF][START_REF] Balci | Iron isotope fractionation during microbially stimulated Fe(II) oxidation and Fe(III) precipitation[END_REF][START_REF] Beard | Iron isotope biosignatures[END_REF][START_REF] Brantley | Fractionation of Fe isotopes by soil microbes and organic acids[END_REF][START_REF] Bullen | Demonstration of significant abiotic iron isotope fractionation in nature[END_REF][START_REF] Icopini | Iron isotope fractionation during microbial reduction of iron: The importance of adsorption[END_REF][START_REF] Johnson | Isotopic fractionation between Fe(III) and Fe(II) in aqueous solutions[END_REF][START_REF] Mathews | Kinetic iron stable isotope fractionation between iron (-II) and (-III) complexes in solution[END_REF][START_REF] Skulan | Kinetic and equilibrium Fe isotope fractionation between aqueous Fe(III) and hematite[END_REF]. For example, both a rapid non-biological oxidative precipitation of ferrihydrite and a biological extracellular reductive dissolution have been shown to produce a similar range of Fe isotopic fractionations (from ~ -1.5 to -0.5 ‰, [START_REF] Anbar | Iron stable isotopes: beyond biosignatures[END_REF]. The knowledge gaps are even larger when considering processes controlling the isotopic fractionations in natural soils. Despite evident Fe redistributions, the variations of Fe isotopic composition recorded in bulk soil profiles are small (Δ 56 Fe profile ≤ 0.15‰, with Δ 56 Fe profile =  56 Fe max - 56 Fe min within a given soil profile), with the exception of soils with dominant organic matter-controlled redox processes where Δ 56 Fe profile can reach up to 0.8 ‰ (e.g., [START_REF] Fekiacova | Can Fe isotope fractionations trace the pedogenetic mechanisms involved in podzolization?[END_REF][START_REF] Fekiacova | Inferences from the vertical distribution of Fe isotopic compositions on pedogenetic processes in soils[END_REF][START_REF] Poitrasson | Limited iron isotope variation in recent lateritic soils from Nsimi, Cameroon: implications for the global Fe geochemical cycle[END_REF][START_REF] Thompson | Rayleigh fractionation of iron isotopes during pedogenesis along a climatic sequence of Hawaiian basalts[END_REF]Wiederhold et al., 2007a[START_REF] Wiederhold | Iron isotope fractionation during pedogenesis in redoximorphic soils[END_REF]. Different hypotheses could explain this apparent contradiction: (i) the soil formation processes do not fractionate Fe isotopes (e.g., a complete dissolution of Fe bearing mineral phases, transport of Fe with the particular phase, etc); (ii) the isotopic fractionations induced by the different soil processes compensate each other; (iii) the mobilization of Fe is too slight (small losses relative to the large stocks) to induce a measurable change in the bulk soil Fe isotopic fractionation.

In order to examine the Fe isotope fractionations induced by individual processes in soils, we need to isolate the minerals formed by these processes to analyze their isotopic signatures.

However, the available sorting methods commonly used for this purpose in Earth Sciences, namely chemical and physical separations (e.g., [START_REF] Borggaard | Phase identification by selective dissolution techniques[END_REF][START_REF] Chao | Extraction Techniques for Selective Dissolution of Amorphous Iron Oxides from Soils and Sediments[END_REF][START_REF] Filgueiras | Chemical sequential extraction for metal partitioning in environmental solid samples[END_REF][START_REF] Force | Solid-Phase Iron Characterization During Common Selective Sequential Extractions[END_REF] present major drawbacks. Chemical extractions induce an uncontrolled bias in the Fe isotopic composition due to incomplete dissolution of the targeted phase and/or the complexing nature of some of the reagents (e.g., Wiederhold et al., 2007a). Physical separations are generally not suitable when the size of minerals contained in the soil matrix is very small and are reduced, in the best case, to the analysis of manually isolated soil features, which are indicative of one or a small number of pedological processes. This approach was used by [START_REF] Wiederhold | Iron isotope fractionation during pedogenesis in redoximorphic soils[END_REF] who observed a systematic enrichment in light Fe isotopes in the Fe-enriched, and enrichment in heavy Fe isotopes in the Fe-depleted soil features, suggesting a link between pedogenic distribution of Fe and Fe isotopic fractionation.

In the present work, in order to elucidate the apparent contradiction between Fe isotope fractionating soil processes and unfractionated bulk soil Fe isotopic profiles, and test the associated hypotheses, we selected a soil sequence characterized by (i) high Fe losses along the soil profile (ii) contrasted redoximorphic features (designated as "soil volumes" hereafter) characterized by strong differences in Fe oxide content and (iii) an pronounced lateral differentiation induced by drainage. The chosen soil sequence consisted of a drainageinfluenced sequence of Retisols (IUSS Working Group WRB, 2015) extensively characterized by [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. This soil type is characterized by a process of morphological degradation and subsequent clay translocation. The morphological degradation consists in the dissolution of Fe-oxides from the parental ochre material, producing residual pale-brown volumes (PB volumes) and white-grey volumes (WG volumes), and in the precipitation of the released Fe and Mn in black volumes (B volumes) (Cornu et al., 2012a;[START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF].

These transformations reflect changes in the mineralogical composition [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF] demonstrated that drainage of these soils increased the morphological degradation in the vicinity of the drain by enhancing eluviation and decreasing the occurrence of reducing phases [START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF]. We combined the isotopic analyses of soil volumes with mineralogical analyses and mass balance calculations to estimate the Fe isotopic signatures of the different mineral phases. Our aim was to (i) shed light on the fluxes and Fe isotope signatures variability along the soil sequence at three different scales: the horizon, the soil volume and the mineral scale and (ii)

decipher the impact of the different soil processes on the Fe isotope signatures.

MATERIAL AND METHODS

Studied site

The studied site is located on the Yonne plateau, France where Albeluvisol/Retisol (IUSS Working Group WRB, 2007/ IUSS Working Group WRB, 2015) developed in Quaternary noncalcareous loess deposited above Eocene clays [START_REF] Baize | Notice de la carte des sols de l'Yonne à 1/50000, feuille Joigny[END_REF]. The soils have been cultivated over the last 200 years and a subsurface drainage was installed during 16 years. The soils form four horizons: a silty, brown ploughed surface horizon; a silty grey E-horizon; and two horizons composed of a complex mixture of several soil volumes of distinctive colors (Figure 1). In the upper of these two horizons, called E&Bt-horizon hereafter, the most abundant volumes are silty and of a white-grey (10YR8/2 to 10YR7/1, called WG volumes hereafter) to pale-brown (10YR7/4, called PB volumes hereafter) color. In the lower horizon called Bt-horizon hereafter, there are clayey volumes of ochre color (10YR5/6 to 10YR5/8, called O volumes hereafter). Black concretions and impregnations occur (called B volumes hereafter) in the core of the ochre volumes, in both Bt-and E&Bt-horizons (Cornu et al., 2012a). Bulk soil samples were collected from the different horizons at different distances along a 4-m trench dug perpendicularly to a drain. The different soil volumes were extracted from the E&Bt-and Bt-horizons (Figure 1). Their physical properties -mineralogical and chemical composition, abundance and morphology -were extensively characterized (e.g., [START_REF] Cornu | Image-analytically derived conceptual model of Albeluvisol morphological degradation induced by artificial drainage in France[END_REF][START_REF] Cornu | Evidence of short-term clay evolution in soils under human impact C[END_REF][START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF][START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF][START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. Further methodological details can be found in [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF].

The soil profile 400 cm from the drain (P400) was shown to represent the Retisol differentiation with a negligible influence of the drain [START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF][START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF], while differences observed between the soil profiles at 400 cm (P400) and 60 cm (P60) distance from the drain were interpreted as reflecting the impact of drainage on the soil, i.e., the effect of enhanced eluviation and a decline in reducing processes [START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF]. These two profiles were further characterized for Fe isotope measurements.

Sample preparation for Fe isotope analysis

For bulk soil and soil volume analyses, samples were dried at 40°C and sieved to <2 mm.

For concentration and isotopic analyses, an aliquot of each sample was ground to fine powder using an agate mortar. For each sample, between 0.15 and 0.20 g of powdered soil was calcinated at 400°C for 4 hours in order to eliminate organic matter. Then, the samples were dissolved in a mixture of concentrated HF-HNO 3 , followed by concentrated HCl, at ~ 120-130°C, during 48 hours. After drying down, the samples were taken up in 7N HCl-0.001% H 2 O 2 for Fe separation and purification. Iron was separated and purified by anion exchange chromatography using the AG MP1, 100-200 mesh, chloride form, according to the protocol of [START_REF] Maréchal | Precise analysis of copper and zinc isotopic compositions by plasma-source mass spectrometry[END_REF]. All reagents were ultrapure distilled or Seastar© quality and overall procedural blanks contained negligible quantities of Fe (< 15 ng) compared to the total dissolved sample Fe content. Yield (recovery) of the total liquid ion exchange resin chromatography procedure was verified and within 95 ± 5%. The reference material CCRMP Till-1 was analyzed with each batch of samples and Fe concentration analyses were in agreement with the certified values (± 0.1%).

Isotopic analyses

Iron isotope analyses were performed using MC-ICP-MS Neptune Plus at CEREGE in medium-resolution. Prior to measurements, pure Fe fractions were diluted and doped with Cu (VWR ICPMS solution) to concentrations of 1 µg/g of both analytes which resulted in total Fe and Cu ion beams around 1:1. Iron ( 54 Fe, 56 Fe, 57 Fe and 58 Fe) and Cu ( 63 Cu and 65 Cu) isotopes

were measured in wet plasma conditions using a thermoregulated spray chamber (Isomist from

Glass expansion regulated at 10°C) and in multi-static mode (magnet jump alternating between Fe and Cu isotopes). The instrumental mass discrimination was corrected both via Cu isotopes and standard-sample-standard bracketing with IRMM-524a (also diluted and doped with Cu to match the concentration of the samples). Iron isotope results are reported relative to the IRMM-524a standard using the conventional δ notation, in ‰ (Eq. 1). Thus,  56 Fe<0‰ reflects enrichment in light 54 Fe isotopes, while  56 Fe>0‰ reflects enrichment in heavy 56 Fe isotopes (Eq. 1).

 56 Fe = [( 56 Fe/ 54 Fe) sample / 56 Fe/ 54 Fe) IRMM524a -1]*1000

(1) The analytical reproducibility (2SD) calculated on the basis of repeated measurements of the IRMM-524a standard was 0.07 and 0.11‰ (N=78) for δ 56 Fe and δ 57 Fe, respectively. In a δ 57 Fe vs. δ 56 Fe diagram, all soil sample measurements plot along a line with a slope which is equal, within analytical error, to the theoretical value of ln(M57/M54)/ln(M56/M54)=1.48 (e.g., Beard and Johnson, 2004;[START_REF] Craddock | Iron isotopic compositions of geological reference materials and chondrites[END_REF], indicating mass-dependent fractionation and no influence of isobaric interferences. Data accuracy was monitored by repeated analyses of the rock standards with known Fe isotopic compositions, with each batch of samples.

Mass balance

We used the isotopic data together with the available information on Fe concentrations and volume abundances (Table 1) of Fe-bearing mineral phases in the individual volumes [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF] for the mass balance calculations. At the scale of the horizon, we set up a similar mass balance to verify that the sum of volumes corresponds to the bulk soil (Eq. 2) and that no bias was introduced into the Fe isotopic compositions during the volume separation and analyses.

 where Fe stands for Fe concentration,  56 Fe value for Fe isotopic compositions, Ab for the abundance of a given volume (% of the picture surface, [START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF], subscripts "O", "B", "PB" and "WG" for ochre, black, pale-brown and white-gray volumes, respectively, and "E&Bt" for the bulk E&Bt 35-45 cm -horizon. The calculated  56 Fe values were in agreement, within the analytical error with the analyzed  56 Fe values. Furthermore, the mass balance approach allowed us to estimate the isotopic compositions of the Fe fluxes (losses/gains) (i) at the scale of a horizon and (ii) among the volumes. These fluxes are considered to represent the Fe concentrations and the Fe isotopic signatures in the solution resulting from the differentiation of the of the studied Retisol. The aim of this calculation was to evaluate the fractionation of the entire process involving, on the one hand, the dissolution of Fe-oxides, i.e. release of Fe into solution, and on the other hand, the precipitation of Mn oxides containing Fe, i.e., removal of Fe from the solution.

We calculated Fe fluxes and their isotopic signatures linked to the studied Retisol differentiation at a scale of horizons and among the soil volumes, using the Brimhall et al.

(1991) mass balance approach (Eq. 3), adapted for soil volumes by [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. m Fe, flux = 1/1000 × ( reference × C Fe, reference × Th ×  Fe, product )/( imm,product -1)

where  corresponds to the bulk density in g cm 3 , C to the concentration of Fe in g kg, Th to the thickness of the horizon in cm, and  and  to strain and the open-system mass-transport function, respectively, as defined by [START_REF] Brimhall | Quantitative geochemical approach to pedogenesis: importance of parent material reduction, volumetric expansion, and eolian influx in lateritization[END_REF]. To compute these functions, quartz was selected as the immobile element, subscript "imm" (Montage et al., 2008). The subscripts "reference" and "product" refer to the soil volume taken as a reference, i.e., the parent material, and to the product of the transformation, respectively. For example, at the scale of a horizon, the flux of Fe lost at P400 was related to the natural differentiation of the E&Bt 400 horizon from the reference O volume while at P60, it was related to the differentiation of the E&Bt 60 from the reference E&Bt 400, due to the drainage. Furthermore, among the volumes, in the calculation of the flux related to the reference O volume dissolution, the O volume is the reference while B-and PB volumes are products of the transformation. In the calculation of the Fe flux linked to the formation of the WG volume, the PB volume is the reference and the WG volume is the product of the transformation. Since the soil volumes consist of variations of proportions of different minerals (goethite, ferrihydrite, Mn-oxide, clay minerals; [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF] we used the mass balance approach to calculate the theoretical  56 Fe values of these mineral phases (Eq. 4):

 56 Fe Vol = ( 56 Fe Clay ×Fe Clay +  56 Fe FH ×Fe FH +  56 Fe GT ×Fe GT +  56 Fe MnOx ×Fe MnOx )/Fe vol (4) where the subscripts "Vol", "Clay", "FH", "GT" and "MnOx" stand for the volume type, clay minerals, ferrihydrite, goethite and Mn oxides, respectively. Doing this for each volume, we obtained four equations. By solving this linear system, where  56 Fe values of the four mineral phases (clay minerals, ferrihydrite, goethite and Mn oxides) were unknown, the theoretical  56 Fe values for these minerals were determined.

RESULTS AND DISCUSSION

No Fe isotope fractionations in bulk soils but large isotopic contrasts between soil volumes

Iron concentrations in both soil profiles varied from 15.5 ± 0.8 to 26.4 ± 1.3 g kg -1 in P60 and from 16.1 to 27.4 ± 1.4 g kg -1 in P400 and showed an evolution with depth typical of Retisols, with the upper horizons, Ap and E&Bt, depleted in Fe compared to the deep Bthorizons (Figure 2 a, b). This evolution was a result of clay translocation and subsequent strong bleaching due to the periodic saturation and reduction of iron compounds (e.g., [START_REF] Pedro | Two routes in genesis of strongly differentiated acid soils under humid, cool-temperate conditions[END_REF]. Bulk soil Fe isotopic compositions remained constant within the analytical error, close to  56 Fe = 0 ± 0.05‰, throughout both soil profiles (Figure 2 c,d). These observations corroborate literature data on bulk soils, where a large range of Fe concentrations contrasts with minor isotopic variability (e.g., [START_REF] Emmanuel | A preliminary mixing model for Fe isotopes in soils[END_REF]Fantle and De Paolo, 2004;[START_REF] Fekiacova | Can Fe isotope fractionations trace the pedogenetic mechanisms involved in podzolization?[END_REF][START_REF] Fekiacova | Inferences from the vertical distribution of Fe isotopic compositions on pedogenetic processes in soils[END_REF][START_REF] Poitrasson | Limited iron isotope variation in recent lateritic soils from Nsimi, Cameroon: implications for the global Fe geochemical cycle[END_REF]Wiederhold et al., 2007a[START_REF] Wiederhold | Iron isotope fractionation during pedogenesis in redoximorphic soils[END_REF].

Contrasting with the absence of isotopic fractionation in the bulk soil samples along the soil profiles, the soil volumes sampled in the E&Bt-horizons were heterogeneous in both Fe concentrations (Figure 2 a,b) and isotopic compositions (Figure 2 c,d). Black (B) volumes were enriched in Fe (45 ± 2 g kg -1 and 33 ± 2 g kg -1 for B 400 and B 60 , respectively) compared to ochre (O) volumes (37 ± 2 g kg -1 and 33 ± 2 g kg -1 for O 400 and O 60 , respectively), while pale-brown (PB) and white-grey (WG) volumes were depleted in Fe (15 ± 0.7 g kg -1 and 12 ± 0.6 g kg -1 for PB 400 and PB 60 , respectively and 10 ± 0.5 g kg -1 and 8 ± 0.4 g kg -1 for WG 400 and WG 60 , respectively), as already described in [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. Furthermore, B volumes were enriched in light Fe isotopes (-0.49 ± 0.05‰ and -0.39 ± 0.05‰ for B 400 and B 60 , respectively), while WG volumes were enriched in heavy Fe isotopes (0.29 ± 0.06‰ and 0.15 ± 0.03‰ for WG 400 and WG 60 , respectively). These contrasts in Fe isotopic compositions between individual soil volumes provide evidence of fractionating processes that occur during the internal horizon differentiation. However, the resulting bulk soil isotopic signatures were similar across horizons, which suggests, given that there is a loss of Fe from a horizon (for E&Bt 400 : -0.65 g cm -2 ; for E&Bt 60 : -0.57 g cm -2 ), that the processes-related isotopic fractionations compensate each other (directions and/or amplitudes) and generate a homogenous integrated signal for a bulk soil sample, over the profile. This interpretation corroborates the hypotheses proposed in the previously published literature to explain a bulk soil Fe isotopic homogeneity (e.g., Wiederhold et al., 2007a[START_REF] Wiederhold | Iron isotope fractionation during pedogenesis in redoximorphic soils[END_REF].

Iron isotopic contrasts between soil volumes -evidence of internal horizon reorganization

during the Retisol differentiation (the P400 soil profile) At a distance of 400 cm from the drain, the O volume is characterized by high Fe concentrations and  56 Fe values close to 0‰ (Figure 3a), similar to the bulk  56 Fe values. As already mentioned above, the O volume corresponds to the initial Bt-horizon before morphological degradation [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. The PB and WG volumes, formed by dissolution of the Fe oxides of the O volume [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF], have lower Fe concentrations and higher  56 Fe values (Figure 3a). Heavy  56 Fe values in the Fe-poor soil features have already been observed by [START_REF] Wiederhold | Iron isotope fractionation during pedogenesis in redoximorphic soils[END_REF]. Experimental dissolutions of pure minerals showed that at the scale of a mineral, the heavy Fe-enriched residual phase reflects a preferential removal of the light Fe isotopes during mineral dissolution. For example,

 56 Fe values of up to -1.7‰ have been observed in the solution (e.g., [START_REF] Wiederhold | Iron isotope fractionation during proton-controlled and reductive dissolution of goethite[END_REF].

Nevertheless, the studied soil volumes correspond to mixtures of several mineral phases [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. We calculated Fe fluxes from the O to PB and PB to WG volumes and their Fe isotopic signatures were not significantly different from 0 ‰ (-0.02 ‰ and -0.03 ‰, for Fe flux from O and PB, respectively; Figure 4), suggesting no fractionation related to the dissolution of their Fe-oxides in these volumes. Thus, the heavy  56 Fe values of the residual volumes (PB, WG) are likely to reflect the Fe isotopic signatures of the residual mineral phases (silicates). At the scale of soil volumes in these Retisols, we propose that the observed isotopic contrast may result from a selective removal of a particular mineral phase. Dissolution is the main process of morphological differentiation in this Retisol [START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF]) and this explanation is consistent with the  56 Fe value close to 0‰ calculated for the total Fe lost during the transformation of the reference (parent) O volume to the E&Bt 400 horizon ( 56 Fe = 0.06‰). Furthermore, a light Fe pool is immobilized within the B volume by precipitation of Mn oxides containing Fe and of ferrihydrite, in the core of the remaining O volume. The B volumes formed have negative  56 Fe values (-0.49 ± 0.05‰ and -0.39 ± 0.05‰ for B 400 and B 60 , respectively; Table 1). The precipitation of Mn oxides may be a fractionating process that preferentially incorporates light Fe isotopes in the solid phase. The calculated Fe flux with a negative  56 Fe value (-2.94 ‰; Figure 4) corroborated this explanation. These results seem to contradict the isotopic theory (e.g., [START_REF] Bigeleisen | Calculation of equilibrium constants for isotopic exchange reactions[END_REF][START_REF] Urey | The thermodynamic properties of isotopic substances[END_REF]) and experimental studies (e.g., [START_REF] Wiesli | Experimental determination of Fe isotope fractionation between aqueous Fe(II), siderite and "green rust" in abiotic systems[END_REF][START_REF] Johnson | Isotopic fractionation between Fe(III) and Fe(II) in aqueous solutions[END_REF][START_REF] Skulan | Kinetic and equilibrium Fe isotope fractionation between aqueous Fe(III) and hematite[END_REF][START_REF] Bullen | Demonstration of significant abiotic iron isotope fractionation in nature[END_REF], which predict that, at the scale of a mineral, heavy isotopes partition preferentially into a stronger bonding environment and during precipitation the heavy Fe isotopes are preferentially immobilized in the solid phase. However, in the soils, rapid precipitation with a dominant kinetic isotopic effect could explain the observed lighter isotopic signatures in the Mn oxides (e.g., [START_REF] Skulan | Kinetic and equilibrium Fe isotope fractionation between aqueous Fe(III) and hematite[END_REF]. The observed isotopic fractionations support the model of the internal mineralogical reorganization of the E&Bt 400 horizon (Cornu et al., 2012a). Furthermore, these fractionations are in agreement with the redox changes, which are dominant mechanisms controlling the horizon evolution [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. Lastly, despite the fact that light Fe flux is recorded within the B volumes, its size is low and does not impact the global isotopic signature of the flux and of the bulk soil at the horizon scale.

Iron isotopic contrasts between soil volumes -evidence of enhanced morphological degradation by eluviation induced by drainage

From the E&Bt 400 to E&Bt 60 horizons, we also observed significant shifts of Fe concentrations in the volumes (Figure 3b) due to the impact of the drain on the E&Bt-horizon [START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF]. Iron concentrations decrease with decreasing distance from the drain (Table 1). In contrast, we observed only small (PB and WG volumes) or non-significant, i.e., smaller than the analytical error (O and B volumes), isotopic differentiation with decreasing distance from the drain (Table 1; Figure 3b). Thus, drainage, which has been shown to reduce drastically reducing conditions and enhance eluviation close to the drain [START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF], results for Fe in a dominant physical mobilization that does not likely induce further Fe isotope fractionations in soil volumes.

Iron isotopic signatures of the mineral phases

While physical separations of the minerals from the soil matrix are impossible, we can calculate the theoretical  56 Fe values for the main mineral phase of each volume (eq. 4). The calculation suggested that goethite and ferrihydrite have  56 Fe values close to 0‰, regardless of the distance from the drain (Figure 5). This unfractionated isotopic signature is in agreement with the calculated  56 Fe values close to 0‰ of the Fe fluxes induced by their dissolution (Figure 4). Both minerals represent the main Fe-bearing mineral phases of the ochre volume (Table 1), whatever the distance from the drain [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF], and thus fix the isotopic signature of this volume.

The Mn oxides containing Fe, which is the main mineral phase of the black volumes [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF], are markedly enriched in light Fe isotopes (-8.83 ‰ and -6.01 ‰ for Mn-ox 400 and Mn-ox 60 , respectively; Figure 5). In contrast, the clay minerals, the main mineral phase of the WG volumes, have positive  56 Fe values (1.31 ‰ and 0.60 ‰ for Clay 400 and Clay 60 , respectively; Figure 5) indicating enrichment in heavy Fe isotopes. The  56 Fe values of both Mn oxides and clay minerals differ significantly depending on the distance from the drain and the minerals from the farthest profile (P400) appear to be more strongly fractionated than the minerals from the profile close to the drain (P60) (Figure 5). We propose two hypotheses to explain the calculated, less fractionated, Fe isotopic signatures of Mn oxides in the soil close to the drain (P60). First, decreasing isotopic fractionation with decreasing distance from the drain may reflect the progressive change in the dominant process controlling the mineralogy and Fe budget in these soils. While far from the drain (P400), dominant redox changes due to temporal water saturation remain active, this process disappears in the vicinity of the drain (P60). Since the redox process fractionates Fe isotopes, the larger isotopic fractionations observed at P400 may reflect the larger number of redox cycles experienced far from the drain (e.g., [START_REF] Schuth | Redox-dependent fractionation of iron isotopes in suspensions of a groundwater-influenced soil[END_REF]. However, we suggest that the fractionation observed between the volumes mostly results from precipitation rather than dissolution. Also, a higher abundance of B volumes at P60 compared to P400 indicated that the precipitation of Mn oxides occurs more in soils that are 60 cm rather than 400 cm from the drain [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]). This can be interpreted as precipitation of a younger generation of Mn oxides at P60, induced by drainage. [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF] further hypothesized that the additional Mn input at P60 was partially due to dissolved elements brought by lateral water flux from positions farther from the drain, and that Mn oxides precipitated from the soil solution progressively. Such a progressive precipitation could induce a progressive removal of light Fe isotopes from the soil solution by co-precipitation and/or adsorption in the Mn oxides. However, this hypothesis needs to be verified, for example by analyzing the Fe isotopic compositions of the soil solution at different distances from the drain. Second, the source of Fe immobilized in the Mn oxides changes with decreasing distance from the drain. It has been demonstrated that the Mn oxides form progressively by diffuse impregnation of different volumes [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF][START_REF] Stolt | Strongly contrasting redoximorphic patterns in Virginia valley and ridge paleosols[END_REF]. At P400, the B volumes form within the core of the O volume by precipitation of the light Fe released by dissolution of Fe-oxides from the O volume. The Fe released by dissolution from the more permeable PB volume [START_REF] Frison | Soil hydraulic properties in relation to local rapid soil changes induced by field drainage: a case study[END_REF], is lost from the horizon. In contrast, at P60 the abundance of O volume decreased significantly thus the B volumes are more connected to the PB volumes (Cornu et al., 2012a). Consequently, further development of the B volumes must result from the Mn oxide precipitation with Fe originating from the dissolution of Fe from these PB volumes. However, the dissolution of Fe oxides from the PB volumes also results in an Feflux with an unfractionated  56 Fe value (Figure 4). The flux of Fe originating from the PB volumes has  56 Fe values close to 0‰, as suggested by our mass balance calculation and should not modify the  56 Fe value of the precipitating Mn oxides. Thus, this second hypothesis cannot explain the observed lower  56 Fe values of Mn oxides in the soil close to the drain (P60).

Furthermore, the calculations predicted a shift of  56 Fe values of the silicates from P400 towards P60 (Figure 5). The silicate fraction (i.e., particles <2µm, lutum) of these Retisols is composed mostly of quartz, feldspars and clay minerals (i.e., kaolinite, illite, smectite and chlorite). Quartz and feldspars do not contain Fe as a major element, but Fe can be present in the clay minerals due to the substitution of Al and Mg. [START_REF] Cornu | Evidence of short-term clay evolution in soils under human impact C[END_REF] showed that the proportions of different clay minerals changed with the distance from the drain, i.e., the concentration of smectites decreased compared to the illite and interlayered clays. Furthermore, the presence of chlorites increased in the vicinity of the drain [START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF]. We hypothesized that the calculated shift in the  56 Fe values from P400 to P60 may result from the selectivity of the eluviation process and reflects the described mineralogical changes in the proportions of different clay minerals. To verify this hypothesis, it would be necessary to analyze the Fe isotopic composition of the individual clay types. Such analyses could be partially achieved by particle size fractionation of the < 2 µm fraction [START_REF] Hubert | Unraveling complex < 2 mu M clay mineralogy from soils using X-ray diffraction profile modeling on particle-size subfractions: Implications for soil pedogenesis and reactivity[END_REF].

Given that such a particle-size separation remains challenging due to the extremely small mineral size of the individual clays, verification of this hypothesis remains an open question for future research.

CONCLUSION

In this work we examined the Fe isotope fractionations related to processes occurring in a drainage-influenced sequence of Retisol, a soil type characterized by clay migration and the formation of redoximorphic features resulting from the strong Fe dynamics. We aimed at identifying (i) the Fe fluxes and their isotopic variability between soil horizons and between individual soil volumes and (ii) the isotopic signatures of the individual mineral phases, by combining the isotopic analyses, mineralogical data and mass balance calculation. We showed that, as classically seen in the literature, the bulk soils have unfractionated Fe isotopic signatures, constant with depth, irrespective of the distance from the drain. However, the data on the different soil volumes suggest that some processes, and particularly precipitation, contributing to the internal horizon reorganization can produce large isotopic fractionations.

Furthermore, we shed light on the Fe isotopic compositions of the individual mineral phases present in the soil volumes, showing that clay minerals and newly precipitated Mn oxides exhibit large isotopic fractionation. Our results suggest that while the residues of dissolution, represented by the morphologically degraded soil volumes, are rich in heavy Fe isotopes, the dissolution process associated with the reduction of Fe oxides is, in these Retisols, not a fractionating process. The apparent fractionation producing this heavy residue is likely to result from the selective removal of the Fe oxides, resulting in the accumulation of isotopically heavy clay minerals. In contrast, the process of Mn oxide precipitation incorporated light Fe isotopes.

This last result appears to contradict the isotopic theory and remains to be explained. A dominant kinetic effect during rapid precipitation could explain the finding.

We suggest that further research should be directed towards better understanding the Fe isotopic fractionation by clay minerals and Mn oxides. Particle-size fractionation of clay minerals combined with sequential extractions should be performed in order to better constrain the isotopic signature of a single clay family. Spectroscopic approaches (Mössbauer, XAS, etc.) combined with laboratory experiments would help to shed light on the Fe isotopes fractionation during Mn oxide precipitation.

Table and figure captions

Table 1:

Iron concentrations and isotopic compositions of the bulk soils and individual volumes.

Figure 1 :

 1 Figure 1: a) The different soil volumes of the E&Bt-horizon (photo from Cornu et al., 2012b) and corresponding colors used in the conceptual model. Ochre volume (O volume) corresponds to the parental material. Pale-brown volume (PB volume) formed by morphological degradation, i.e., dissolution of Fe-oxides, from the O volume. Further degradation, i.e., dissolution of Feoxides of the PB volume, produces white-grey volumes (WG volumes). Black volumes (B volumes) form by precipitation of the Fe and Mn released from this dissolution. b) Conceptual model of the internal horizon reorganization (after Cornu et al., 2012b) representing differentiation of the parental O volume (Bt-horizon) into E&Bt-horizon at the positions P400 and P60. Abbreviations: O=ochre volume, PB=pale-brown volume, WG=whitegrey volume, B=black volume, P400 and P60 correspond to positions 400 cm and 60 cm from the drain, respectively.

Figure 2 :

 2 Figure 2: Evolution with depth of Fe concentrations (a, b) and Fe isotopic compositions (c, d) in the Retisol profiles at different distances from the drain, 400 cm (a, c) and 60 cm (b, d). Ap, E&Bt, Bt correspond to the horizons, O, B, PB, WG to the soil volumes. The analytical error of the Fe concentration analyses is ± 5%. The analytical error of the  56 Fe values is 2SD

Figure 3 :

 3 Figure 3: Relationship between Fe concentrations and  56 Fe values of the individual volumes 400 cm (a)

Figure 4 :

 4 Figure 4: Conceptual model of Fe fluxes during Retisol differentiation, i.e., during the transformation from the reference O volume (parent material of E&Bt) to the E&Bt horizon, 400 cm from the drain. O, PB, WG and B correspond to ochre, pale-brown, white-grey and black volumes, respectively, 400 cm from the drain. Data within the rectangles correspond to the analyzed Fe concentrations (Montagne et al. 2008), Fe isotopic compositions (this work) and calculated Fe stock in the actual volumes, in the E&Bt-horizon, 400 cm from the drain. Data with arrows represent the calculated Fe concentration and its  56 Fe values in the Fe fluxes related to the

Figure 5 :Figure 3

 53 Figure 5: Theoretical  56 Fe values calculated (Eq. 4) for the individual mineral phases present in the soil

  56 Fe E&Bt = ( 56 Fe O ×Fe O ×Ab O +  56 Fe B ×Fe B ×Ab B +  56 Fe PB ×Fe PB ×Ab PB +  56 Fe WG ×Fe WG ×Ab WG ) / Fe E&Bt (2)

Table 1

 1 * data from[START_REF] Montagne | Impact of drainage on soil-forming mechanisms in a French Albeluvisol: input of mineralogical data in mass-balance modelling[END_REF] Analytical errors for Fe concentrations equals to 5 %. Volume abundances are relative abundances of the different volumes in % of the picture surface[START_REF] Montagne | Changes in the pathway and the intensity of albic material genesis: Role of agricultural practices[END_REF] na -not analysed

Acknowledgments

The authors wish to thank B. Angeletti for support with Q-ICPMS analyses. This research benefited from the support of the INRAE and of the EQUIPEX ASTER-CEREGE project.

We also thank the reviewers for their constructive comments and suggestions, which have helped to improve this manuscript.