Floriane Plard
email: floriane.plard@ens-lyon.fr

Jean-Michel Gaillard

Tim Coulson

A J Mark Hewison

Mathieu Douhard

François Klein

Daniel Delorme

Claude Warnant

AND Christophe Bonenfant

The influence of birth date via body mass on individual 1 fitness in a long-lived mammal 2

Keywords: silver spoon effect, life history traits, IPM, roe deer, body mass

The timing of birth has marked impacts on early life and early development of newborns in many species. Early-born offspring often survive and grow better than late-born offspring, but despite the long-lasting effects of early conditions on life history traits, the influence of birth date on fitness has rarely been investigated for long-lived species. In this study, we analyzed both the short-and long-term effects of birth date on individual life history traits and explored its subsequent impact on individual fitness in a population of roe deer. We considered both the direct effects, as well as the indirect effects of birth date mediated through the effects of body mass, on demographic parameters. We found that in addition to short-term effects on early body growth and survival, birth date generates "silver spoon" effects on adult life history traits of female roe deer. Birth date had long-lasting effects on female adult body mass such that early-born females were, on average, 3 kg heavier as adults than late-born females, although female adult survival was similar between these categories. Based on the observed relationships between birth date, body mass and demographic parameters, we built an integral projection model describing the simultaneous distributions of birth date and body mass to quantify the fitness consequences of birth date. We found that the fitness of early-born females was higher than that of late-born females. These long-lasting effects of birth date on fitness were mostly mediated through the influence of birth date on recruitment and adult body mass. By determining development of newborns during the early stages of life, birth date has a critical influence on each step of an individual's subsequent life history trajectory.

INTRODUCTION

The timing of reproduction is critical in most species because it determines reproductive success of individuals [START_REF] Price | Directional selection and the evolution of breeding date in birds[END_REF]) and thus influences annual population growth. The timing of reproduction has been particularly well studied in the context of climate change [START_REF] Dickey | Climatic effects on the breeding phenology and reproductive success of an arctic-nesting goose species[END_REF][START_REF] Moyes | Advancing breeding phenology in response to environmental change in a wild red deer population[END_REF]) because a mismatch between birth timing and peak resource availability decreases annual recruitment [START_REF] Post | Climate change reduces reproductive success of an Arctic herbivore through trophic mismatch[END_REF]. At the population level, a high degree of birth synchrony may be the result of selection pressure on individuals to minimize this mismatch [START_REF] Rutberg | Adaptive hypotheses of birth synchrony in ruminants: an interspecific test[END_REF] or, alternatively, be linked to a predator-swamping tactic [START_REF] Estes | The significance of breeding synchrony in the wildebeest[END_REF][START_REF] Ims | The influence of birth date via body mass on individual fitness in a long-lived mammal[END_REF]). Indeed, in temperate areas, births are generally characterized by a high degree of seasonality and synchrony each year [START_REF] Bronson | Mammalian reproductive biology[END_REF]). However, within population variation in individual birth date has the potential to influence greatly early survival and body growth. Because body growth affects adult life history traits, birth date is expected to generate long-term effects on fitness (Lindström 1999, Cam and[START_REF] Aubry | Early development, recruitment and life history trajectory in long-lived birds[END_REF]). To date, most studies have focused on the short-term consequences of variation in individual birth date on the early stages of life, while evidence for long-term effects of birth date on adult life history stages and on fitness are lacking for long-lived species [START_REF] Green | Persistent influences of birth date on dominance, growth and reproductive success in bison[END_REF][START_REF] Lindström | Early development and fitness in birds and mammals[END_REF][START_REF] Aubry | Early development, recruitment and life history trajectory in long-lived birds[END_REF]. In this paper, we thus investigated the long-term effects of individual birth date on adult life history traits and individual fitness.

Yearly variation in weather conditions influences both the quality and the quantity of available resources during the period around birth. In temperate and northern areas, early-born young benefit from a longer growth period and attain a higher body mass before the onset of the harsh season than neonates born after the peak of vegetation production (moose Alces alces, [START_REF] Solberg | Annual variation in maternal age and calving date generate cohort effects in moose (Alces alces) body mass[END_REF], bighorn sheep, Ovis canadensis Feder et al. 2008). For example, in bighorn sheep, late-born lambs were 20% lighter in mid-September, at 3 months of age than lambs born one month earlier [START_REF] Feder | Never too late? Consequences of late birthdate for mass and survival of bighorn lambs[END_REF]. For a given individual, being born early in the season also has positive effects on early survival in many species (red deer, Cervus elaphus, [START_REF] Guinness | Factors affecting calf mortality in red deer (Cervus elaphus)[END_REF], western gulls, Larus occidentalis, [START_REF] Spear | Brood size, hatching order and hatching date: effects on four life-history stages from hatching to recruitment in western gulls[END_REF]Nur 1994, mosquitofish, Gambusia affinis, Reznick et al. 2006). For instance, the probability of surviving until the end of the first winter for red deer calves born very early was more than double that of calves born very late in the season [START_REF] Guinness | Factors affecting calf mortality in red deer (Cervus elaphus)[END_REF]. In some cases, young born very early in the season may encounter harsh environmental conditions [START_REF] Wilson | Selection on mothers and offspring: whose phenotype is it and does it matter?[END_REF] or suffer from higher predation [START_REF] Estes | The significance of breeding synchrony in the wildebeest[END_REF]) than young born during the peak of births. However, as a general rule, late-born young have a lower early survival than individuals born early or during the birth peak in most large herbivores and passerine birds.

Because it affects the early body development of young which, in turn, often determines performance during adulthood [START_REF] Douhard | Variation in adult body mass of roe deer: early environmental conditions influence early and late body growth of females[END_REF], date of birth has the potential to generate long-term effects on adult phenotypic traits, but this question has seldom been investigated in long-lived species. Indeed, a link between reproductive timing and fitness components has been demonstrated for short-lived species (hatching date in lizard [START_REF] Sinervo | Interactive effects of offspring size and timing of reproduction on offspring reproduction: experimental, maternal, and quantitative genetic aspects[END_REF], birth date in fish [START_REF] Schultz | The effect of birth date on fitness of female dwarf perch, Micrometus minimetus (Perciformes: Embiotocidae)[END_REF][START_REF] Reznick | On the virtue of being the first born: the influence of date of birth on fitness in the mosquitofish, Gambusia affinis[END_REF]) hatching date and fledging date in birds [START_REF] Visser | Long-term fitness effects of fledging date in great tits[END_REF][START_REF] Sheldon | Natural selection and inheritance of breeding time and clutch size in the collared flycatcher[END_REF][START_REF] Saino | Longevity and lifetime reproductive success of barn swallow offspring are Version preprint Comment citer ce document[END_REF]) and birth date in small mammals [START_REF] Réale | Lifetime selection on heritable life-history traits in a natural population of red squirrels[END_REF][START_REF] Rödel | Family legacies: short-and long-term fitness consequences of early-life conditions in female European rabbits[END_REF]). However, the long-term effects of environmental conditions in early life on adult life history traits are not universal (e.g., Wauters and Dhondt 1995 on European squirrels, [START_REF] Dobson | Increasing returns in the life history of Columbian ground squirrels[END_REF] on Columbian ground squirrels) because some individuals born in poor conditions can compensate for a bad start (Auer 2010 on guppies).

Nevertheless, poor early conditions may also permanently constrain body growth and generate positive covariation among adult life history traits (the "silver spoon effect", [START_REF] Grafen | The influence of birth date via body mass on individual fitness in a long-lived mammal[END_REF]) in long-lived species. For instance, environmental conditions at birth influence both early survival and growth (Gaillard et al. 2003[START_REF] Landete-Castillejos | Calf growth in captive Iberian red deer (Cervus elaphus hispanicus): effects of birth date and hind milk production and composition[END_REF][START_REF] Beauplet | Interannual variation in the post-weaning and juvenile survival of subantarctic fur seals: influence of pup sex, growth rate and oceanographic conditions[END_REF]), but also adult life history traits such as body mass (roe deer, Capreolus capreolus, [START_REF] Pettorelli | Variations in adult body mass in roe deer: the effects of population density at birth and of habitat quality[END_REF], and lifetime reproductive success (red deer, Kruuk et al. 1999, oystercatchers, Haematopus Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 5 ostralegus, [START_REF] Van De Pol | A silver spoon for a golden future: long-term effects of natal origin on fitness prospects of oystercatchers (Haematopus ostralegus)[END_REF].

As a consequence of the long-term effects of early life conditions on life histories, females born early should have high early survival and, when adult, could themselves give birth early in the season. Because birth date and laying date have been found to be heritable [START_REF] Réale | Lifetime selection on heritable life-history traits in a natural population of red squirrels[END_REF][START_REF] Sheldon | Natural selection and inheritance of breeding time and clutch size in the collared flycatcher[END_REF], the young of these early-born females should therefore also have high early survival, so that early-born females should achieve higher fitness than late-born females. Moreover, in species for which adult body mass is positively correlated with juvenile body mass, females born early in the season should also be heavier, and so produce larger average litter sizes, than late-born females. While such relationships are often assumed in studies using laying (or birth) date as a proxy of female quality [START_REF] Sydeman | Repeatability in laying date and its relationship to individual quality for common murres[END_REF][START_REF] Catry | Short-lived repeatabilities in long-lived great skuas: implications for the study of individual quality[END_REF][START_REF] Blums | Individual quality, survival variation and patterns of phenotypic selection on body condition and timing of nesting in birds[END_REF], very few studies have explicitly investigated the relationship between reproductive timing and individual fitness. In the present study, we defined individual fitness as the individual reproductive value early in life [START_REF] Moorad | Individual fitness and phenotypic selection in age-structured populations with constant growth rates[END_REF], see also Methods for a justification of this measure).

In this study, we investigated both the short-term and long-term effects of birth date on individual life history traits and on fitness in an intensively monitored population of long-lived mammals, roe deer. We tested the following hypotheses: (i) as predicted from the match between early growth and resource availability [START_REF] Rutberg | Adaptive hypotheses of birth synchrony in ruminants: an interspecific test[END_REF], early-born fawns should survive better and have higher early body growth than late-born fawns because the former benefit from high quality resources (Plard et al. 2014a), (ii) early-born individuals should be heavier when adults, gain higher annual reproductive success, and live longer than late-born individuals because early conditions have the potential to generate "silver spoon" effects [START_REF] Grafen | The influence of birth date via body mass on individual fitness in a long-lived mammal[END_REF]).

Finally, using the observed relationships between birth date, body mass and demographic parameters, we built an integral projection model [START_REF] Easterling | The influence of birth date via body mass on individual fitness in a long-lived mammal[END_REF] to analyse the link between birth date and fitness. We used an IPM (instead of a classical analysis of lifetime reproductive success) for the following reasons: the IPM allowed us to estimate reproductive 2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 6 value early in life for each birth date and to identify the exact pathways (i.e. survival, reproduction, growth, or inheritance) by which birth date influences fitness. Moreover, by building an IPM based on the distribution of two continuous traits, we could investigate both the direct influence of birth date on fitness through its effect on annual reproductive success and survival, as well as the indirect influence of birth date on fitness through its effect on body mass.

(iii) As long-lasting effects of early life often occur through permanent developmental constraints [START_REF] Lummaa | Early development, survival and reproduction in humans[END_REF], early-born individuals should achieve higher fitness than late-born individuals, mainly because of the positive association between early birth and fast body growth (e.g., [START_REF] Feder | Never too late? Consequences of late birthdate for mass and survival of bighorn lambs[END_REF].

MATERIALS AND METHODS

Study population

The roe deer population at Trois Fontaines inhabits an enclosed 1,360ha forest in north-eastern France (48 • 43'N, 2 • 61'W). The climate is continental, with relatively cold winters and warm summers. This roe deer population has been intensively monitored since 1976 using a Capture-Mark-Recapture (CMR) sampling design. Captured animals are individually marked using collars (numbered or with VHF or GPS devices) and ear tags. The roe deer population at Trois Fontaines is highly productive, mostly because of the rich habitats and soils which promote high forest productivity [START_REF] Pettorelli | Using a proxy of plant productivity (NDVI) to find key periods for animal performance: the case of roe deer[END_REF]. Roe deer mate between mid-July and mid-August. After copulation, embryos cease their development so that their implantation is delayed by a five month diapause. Embryo development starts again in late December-early January (Aitken 1974). Births in roe deer are highly seasonal, synchronous and occur from mid-April to mid-June, with 80% of births taking place between the 5 th and 27 th of May at Trois Fontaines [START_REF] Gaillard | Timing and synchrony of births in roe deer[END_REF] (triplets accounted for only about 2% of the litters), once per year, from 2 years of age onwards.

Date of birth

From the beginning of the birthing season (mid-April), a systematic search for fawns was conducted every year from 1985 to 2012 by the Office National de la Chasse et de la Faune Sauvage [START_REF] Delorme | Intérêt de l'étude de la période juvénile pour le suivi de l'évolution d'une population de chevreuils (Capreolus capreolus)[END_REF]. Between 21 and 63 newborn fawns were found each year through either intensive searches performed by a line of people, or by directed searches targeted towards the fawns of radio-collared females. Fawns were ear-tagged, weighed, sexed and aged using umbilicus characteristics and behavior at marking [START_REF] Jullien | Détermination de l'âge chez le faon de chevreuil (Capreolus capreolus) dans son premier mois de vie[END_REF]. Error when estimating capture age is less than 2 days [START_REF] Gaillard | Timing and synchrony of births in roe deer[END_REF]. Birth date was therefore back-calculated based on the fawn's estimated age at the date it was found. Body mass at capture did not allow us to estimate body mass at birth because early growth rates vary among fawns. Roe deer fawns display a "hider" tactic [START_REF] Lent | Mother-infant relationships in ungulates[END_REF], only coming into close contact with the mother for suckling. However, the mother stays in close proximity to its fawns, remaining spatially isolated from other females. We relied on this behaviour to identify the fawn's mother through direct observation of lactation behavior or by identification of an escaping female in the vicinity of the fawn.

Life history traits

Winter captures of roe deer took place each year between January and mid-March. At each capture, animals were weighed to the nearest 100g. Yearling and adult body masses corresponded respectively to the body mass of a given female at 8 months old (i.e. at first winter capture) and to its median mass as an adult between 4 and 10 years old [START_REF] Pettorelli | Variations in adult body mass in roe deer: the effects of population density at birth and of habitat quality[END_REF].

We assumed that the body mass of a given fawn at first capture measured between early January and early March provides a reliable estimate of its body mass at 8 months of age because body mass remains almost constant over the winter in roe deer [START_REF] Hewison | The influence of density on post-weaning growth in roe deer Capreolus capreolus fawns[END_REF]. The annual reproductive success of each female was assessed by the capture of its marked fawns at 8 months old. As we did not have access to father-fawn filiations, and thus to male reproductive success, we only included females for the analysis of the effect of birth date on adult stage traits. The age of all reproducing females analyzed here was known exactly because they were first captured either as newborns or at 8 months of age during their first winter (identified using tooth eruption sequence, [START_REF] Flerov | Musk deer and deer[END_REF]).

Statistical analysis

To investigate short-and long-term influences of birth date on female life history traits and fitness, we considered both the direct effect of birth date on demographic parameters (survival, reproduction) and the indirect effect of birth date on demographic parameters mediated through its effect on body mass (see Fig. 1). Thus, we tested for both an effect of individual birth date and of body mass (as a yearling or as an adult) on female adult body mass and annual survival.

For early life stage traits, we investigated the short-term influence of individual birth date on early survival (survival from birth to 8 months old) and yearling survival, and yearling body mass. In addition, as heavy mothers give birth earlier than light mothers in roe deer (Plard et al. 2014b), we considered the relationship between maternal body mass and early life history traits of her offspring to control for possible maternal effects. Because these relationships included information on both mother and newborn fawns, we included both male and female newborns and corrected for sex when necessary to increase our sample size. There is no cost of being born very early in the season in roe deer because the earliest born fawns have the highest early survival (Plard et al. 2014a) and the highest yearling body mass, which is highly positively correlated to yearling survival in ungulates (Gaillard et al. 2000b). We thus tested for the linear effect of birth date on yearling and adult body masses, and on adult survival. Moreover, we have previously shown that early survival is linked to birth date through a threshold relationship (Plard et al. 2014a). As we expected a directional effect of body mass on life history traits, we only tested for linear effects of body mass.

Birth date and early life stage traits: The influence of birth date and maternal body mass on individual early survival (proportion of fawns of each litter that survived up to 8 months of age) was investigated using a generalized linear mixed model with a logit link and a binomial distribution. Because there is no difference between the sexes in individual early survival in roe deer (mean survival difference: 0.13, SE: 0.25, p-value: 0.61, see also [START_REF] Gaillard | Early survival in roe deer: causes and consequences of cohort variation in two contrasted populations[END_REF]), we pooled fawns of both sexes in this analysis. We modeled a threshold regression for birth date (with constant survival until the 12 th of May, then decreasing from this birth date onwards, (Plard et al. 2014a)) and a linear regression for maternal body mass on individual early survival.

The influence of birth date and maternal body mass on yearling body mass was modeled using linear mixed models. As males are heavier than females at 8 months of age in roe deer [START_REF] Gaillard | Body mass of roe deer fawns during winter in 2 contrasting populations[END_REF], we included an effect of sex. In models describing individual early survival and yearling body mass, we included cohort as a random effect on the intercept to control for among-year variation in environmental conditions and maternal identity as a random effect to control for the fact that among-female heterogeneity in reproductive performance is partly unrelated to body mass and birth date. The influence of a female's birth date and yearling body mass on its yearling survival (i.e. from 8 to 20 months old) was investigated using a generalized linear mixed model, including cohort as a random effect.

Birth date and adult stage traits: To investigate whether individual birth date has a direct effect on female adult body mass (over and above its indirect effect on adult body mass mediated through its effect on individual yearling body mass), we performed a path analysis [START_REF] Shipley | Confirmatory path analysis in a generalized multilevel contextsinervo[END_REF]. In all models, we included an influence of individual birth date on yearling body mass and tested for direct effects of both individual birth date and yearling body mass on individual adult body mass using a linear model, including only females. The influence of a female's birth date and adult body mass on its adult survival was tested using a capture-mark-recapture (CMR) framework. Models were fitted using capture histories of 110 Ecology,96 (6),1516151715181519152015211522152315241525152615271528, DOI : 10.1890/14-0106.1 10 females in the software ESURGE [START_REF] Choquet | of Springer Series: Environmental and Ecological Statistics, chapter Program E-SURGE: a software application for fitting Multievent models[END_REF]. Based on previous studies in this population (e.g., [START_REF] Thomson | A semi-Markov model to assess reliably survival patterns from birth to death in free-ranging populations[END_REF], recapture probability was modeled for 3 discrete time periods (1986-1999, 2000, 2001-2012).

For each life history trait except adult body mass, the best model was selected using the Akaike Information Criterion (AIC, [START_REF] Burnham | The influence of birth date via body mass on individual fitness in a long-lived mammal[END_REF]. We selected the model with the lowest AIC and highest AIC weight (w i , calculated as a measure of the likelihood that a given candidate model is the best among the set of fitted models). For modeling adult body mass, we used a path analysis in a Bayesian framework [START_REF] Gelman | Data analysis using regression and multilevel/hierarchical models[END_REF] with OpenBUGS and tested for both the influence of yearling body mass and for the direct effect of individual birth date on adult body mass using the Deviance Information Criterion (DIC, following Gelman and Hill 2007, see also [START_REF] Spiegelhalter | Bayesian measures of model complexity and fit[END_REF]. We conducted 20000 simulations and used the first 5000 as burn-in. We checked that the parameters estimated from the models we fitted were not influenced by multi-colinearity problems (especially between birth date and body mass). We compared the values of body mass and birth date estimated from models with and without body mass and birth date included. Estimates were similar in all models. Diagnostic plots displaying the residuals of each selected model are presented in the supplementary material (Fig. A3 andA4). 2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 11 mother. Following standard terminology in the IPM literature [START_REF] Coulson | Modeling effects of environmental change on wolf population dynamics, trait evolution, and life history[END_REF][START_REF] Coulson | Integral projections models, their construction and use in posing hypotheses in ecology[END_REF], we defined inheritance as the part of the trait that is transmitted from mothers to offspring, irrespective of the mechanism of transmission. This measure includes all environmental sources of similarity (e.g., maternal effects, common habitat effects, permanent environment effects) and, is equivalent to "inclusive heritability" (sensu [START_REF] Danchin | Beyond DNA: integrating inclusive inheritance into an extended theory of evolution[END_REF]).

(2) The recruitment function (R(a, t, bm, bd )) describes the number of offspring a mother successfully raised as a function of their birth date and the mother's body mass. (3) The inheritance function for body mass (I bm (a, t, bm |bm, bd )) links an offspring's body mass at 8 months of age (yearling body mass) to its birth date and maternal body mass. (4) The growth function (G(a, t, bm |bm, bd)) describes the individual-level relationship between body mass as a yearling and body mass as an adult, given that individual's birth date. As birth date is unique for a given individual, the growth function included also an implicit developmental function for birth date defined as an identity function. Finally, (5) the survival function (S(a, t, bm, bd)) describes the probability that an individual survives in relation to its birth date and body mass.

Then: The continuous IPM can be approximated as a high dimensional discrete matrix [START_REF] Easterling | The influence of birth date via body mass on individual fitness in a long-lived mammal[END_REF], see also supplementary material, Fig. B2). To minimize the size of the matrix, we All functions required to build the IPM were investigated in the previous analyses (i.e. on birth date and early life stage traits and on birth date and adult stage traits) except the inheritance function for birth date. We thus used the parameters (intercepts and slopes) previously estimated from the selected models of early and adult life history traits (Table B1, Fig. 1) for the survival, growth, inheritance for body mass and recruitment functions and investigated the effects of maternal birth date and body mass on the inheritance function for birth date to build the IPM. For the inheritance function for birth date, we modeled the link between offspring birth date and maternal birth date and body mass using a linear mixed model, including year and maternal identity as random effects on the intercept. Sex was not included as there is no difference in average birth date between male and female roe deer [START_REF] Gaillard | Timing and synchrony of births in roe deer[END_REF].

n(1, t + 1, bm , bd ) = a I bm (a,
As almost all roe deer females give birth to two fawns in the studied population [START_REF] Gaillard | Family effects on growth and survival of juvenile roe deer[END_REF], we considered litter size at birth as a fixed parameter and modeled the recruitment function based solely on estimates of early survival from birth to the onset of winter (at 8 months of age). As the sex ratio at birth of roe deer is close to 0.5 [START_REF] Hewison | Contradictory findings in studies of sex ratio variation in roe deer (Capreolus capreolus)[END_REF], litter size at birth was set to one as only females were considered in the IPM. We modeled both the 2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 13 contribute to future population growth. For a large population living in a constant environment, the sum of the reproductive values over all individuals is proportional to the exponential growth rate r (i.e. Malthusian growth rate) of the population irrespective of its age distribution [START_REF] Fisher | The genetical theory of natural selection[END_REF][START_REF] Engen | Reproductive value and the stochastic demography of age-structured populations[END_REF]. For instance, reproductive values were recently used as a metric of fitness in different contexts (see e.g. [START_REF] Engen | Reproductive value and the stochastic demography of age-structured populations[END_REF][START_REF] Coulson | Integral projections models, their construction and use in posing hypotheses in ecology[END_REF][START_REF] Bouwhuis | The forms and fitness cost of senescence: age-specific recapture, survival, reproduction, and reproductive value in a wild bird population[END_REF]. Moreover, assuming that the population growth rate is constant, reproductive value at birth measures individual fitness [START_REF] Moorad | Individual fitness and phenotypic selection in age-structured populations with constant growth rates[END_REF]. We therefore chose reproductive value in early life to measure fitness and not the more commonly used asymptotic population growth rate (r) [START_REF] Caswell | Matrix population models : construction, analysis, and interpretation[END_REF]. Indeed, the life cycle of roe deer used in our analysis ( 2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 14 birth as the reproductive values at 8 months, which means that we underestimated the influence of birth date on fitness by not accounting for the influence of birth date on early survival (Plard et al. 2014a). To estimate the confidence interval of the individual reproductive values, we performed a bootstrap analysis with 2000 simulations of the datasets used to estimate the different functions of the IPM (except for adult survival that remained constant with respect to body mass and birth date, see results). Second, to investigate the relative influence of each function on the difference in fitness between early-and late-born individuals, we performed a perturbation analysis. We estimated the percentage change in the difference in fitness between early-and late-born females after a 1% change of one of the slopes linking birth date and body mass to the vital rates. Finally, we simulated 60 groups of 1000 yearling females which differed in terms of their birth date and that were monitored until the death for all individuals in the group. For each group, we recorded mean adult body mass, annual reproductive success, and both mean female offspring body mass and life expectancy at 8 months of age as predicted by the IPM. All statistical analyses were performed with the R software from codes (available in the supplementaL material) that we wrote based on [START_REF] Easterling | The influence of birth date via body mass on individual fitness in a long-lived mammal[END_REF] and [START_REF] Coulson | Integral projections models, their construction and use in posing hypotheses in ecology[END_REF].

RESULTS

Birth date and early life stage traits

Early survival of individual fawns was best described by a model that included additive effects of individual birth date and maternal body mass (Table 1A, w i = 0.67, N=256 including 120 different mothers with 2.1 repetitions per mother on average). Fawns born before 12 th May had a probability of 0.52 [0.42;0.61], on average, of surviving to 8 months old. Early survival probability decreased from this date onwards such that fawns born late in the season (May, 31 st , Fig. 2A) had a probability of only 0.32 [0.20;0.48], on average, of surviving to the same age (Table A1A and Fig. 2A). Maternal body mass influenced early survival positively (Table A1A) such that fawns born to mothers weighing 20 kg had, on average a 0.25 [0.14;0.40] probability of surviving, while those born to mothers weighing 28 kg had, on average, a 0.71 [0.54;0.83] probability of surviving (for fawns born at the peak of the birth season). This model accounted for 6% (of which, 1% was due to the effect of birth date) of the observed variation in early survival (note that the proportion of variation explained for a binomial response variable is always much lower than that for a Gaussian variable).

Comment
Individual yearling body mass was best described by a model that included additive effects of maternal body mass and individual birth date (Table 1B, w i = 0.98, N=111 including 67 different mothers with 1.6 repetitions per mother on average) and which explained 26% (of which, 9% was due to the effect of birth date) of the observed variation in yearling body mass.

Early-born fawns (1 st May) were 2.9 kg (20%) [1;4.9 kg] heavier, on average, at 8 months of age than late-born fawns (31 st May) (Table A1B and Fig. 2B). Moreover, for a given birth date, heavy mothers (28 kg) produced offspring that were, on average, 3.1 kg (21.5%) [0.6;5.6 kg] heavier at 8 months of age than light (20 kg) mothers. Nevertheless, our diagnostic plot (Fig. A3) showed that the residuals of the regression tended to increase with the fitted values. This trend was directly linked to the random individual (maternal) effect, suggesting that an unidentified variable related to a maternal effect could influence the inheritance of body mass.

The best model describing individual yearling survival from 1 to 2 years of age included an effect of individual yearling body mass (Table 1C, w i = 0.44, N=113). Heavier yearlings had a higher probability of surviving to 2 years of age than lighter yearlings (0.89 vs. 0.99 for yearlings of 12 kg and 20 kg, respectively, Fig. A4). Adding birth date did not improve the model fit, which indicates that birth date has no detectable effect on yearling survival.

Birth date and adult stage traits

The best model describing individual adult body mass included additive effects of birth date together with the indirect effect of birth date on adult body mass mediated through yearling body mass (Table 1D,N=67). The retained model accounted for 50% of the observed variation in adult body mass, of which 6% was explained by the direct effect of individual birth date (Table A1D). Whether or not we included the two individuals with an adult body mass of 18 kg that appear as outliers in Fig. 3 had no influence on model selection or parameter estimates.

When considering only the direct effect of birth date on adult body mass, early-born individuals (1 st May) reached a body mass that was 6.5% higher than late-born individuals (31 st May) (24.0 kg vs. 22.5 kg, Fig. 3). However, individual birth date influenced adult body mass mainly through its effect on yearling body mass which was a better predictor of adult body mass than birth date (∆DIC = 40.6 if we removed the effect of yearling body mass from the best model vs. ∆DIC = 1.4 if we removed the effect of birth date from the best model, Table 1D). A yearling weighing 12 kg reached an adult body mass of, on average, 20.8 kg, whereas a yearling weighing 20 kg weighed, on average, 25.8 kg as an adult.

As effects of female birth date or body mass on individual adult survival were non-significant (Table 1E, Table A1E, N=110), we used all monitored females to estimate an overall adult survival probability (N=418). The annual survival rate of adult females was 0.824 [0.796;0.850] (Fig. A1).

Birth date and fitness

We built an IPM from the parameter estimates that we generated above for each function (Table B1). For the inheritance function for birth date, offspring birth date was positively related to maternal birth date (Table A2, w i = 0.35, N=89 including 33 different mothers with 2.7 repetitions per mother on average) such that females born early in the season (1 st May) gave birth, on average, 5 [-2;13] days earlier than females born late (31 st May) in the season (Fig. A2). Five per cent of the observed variation in offspring birth date was explained by this model.

The diagnostic plot of this model showed a trend between the residuals and the fitted values (Fig. A3), probably for the same reason as the trend observed in the diagnostic plot of the model for the inheritance function for body mass.

We evaluated the stable distribution of body mass and birth date from the right eigenvector of the projection matrix. The stable distribution of body mass revealed two peaks corresponding to a mean body mass of 16 kg and 23 kg for yearling and adult female roe deer, respectively (Fig. C1A). The stable distribution of yearling birth dates was also in agreement with field observations, with a mean birth date of 135.5 (15-16 th May vs. 16 th May for the observed mean birth date of newborns in this population) and a normal distribution of birth dates (Fig. C1B).

Using this IPM allowed us to estimate reproductive value at 8 months of age, and thereby fitness, of females in relation to their birth date (Fig. C2). We report here the relative change in reproductive value (and not absolute estimations of reproductive value) between early-and late-born individuals because reproductive values should be interpreted as relative values. We estimated that the fitness of early-born females (1 st May) was 1.29 (95% confidence interval:

[1.01 3.47]) higher than that of late-born females (31 st May). The perturbation analysis showed that birth date had both a direct effect (on early survival and inheritance) and an indirect effect (through body mass) on fitness (Fig. 4). Indeed, the difference in fitness between early-and late-born individuals was closely related to the slopes linking birth date to early survival (when we decreased the slope of the recruitment function for birth date by 1%, the difference in fitness between early-and late born females increased by 0.61%) and linking maternal birth date to 2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 18 difference in fitness between early-and late-born females of 0.50% for recruitment, 0.38% for growth and 0.07% for the inheritance function for body mass. Only the survival function contributed relatively little to the difference in fitness between early-and late-born females (Fig. 4). Because birth date had no statistically significant direct effects on survival but was weakly related to yearling survival through its effect on yearling body mass, life expectancy at 8 months of age was slightly higher for early-born individuals than for late-born individuals (6.57 +/-4.91 vs. 6.41 +/-4.93).

DISCUSSION

Using the relationships between birth date, body mass and early and late life history traits, we built an IPM that allowed us to estimate the difference in fitness between early-and late-born females. The fitness of early-born individuals was 1.3 higher than that of late-born individuals.

While the importance of variation in birth timing for life histories has been acknowledged for a long time [START_REF] Bronson | Mammalian reproductive biology[END_REF][START_REF] Lindström | Early development and fitness in birds and mammals[END_REF], the few studies demonstrating long-lasting effects of birth date in mammals are recent and confined to short-lived species (e.g. European rabbit Oryctolagus cuniculus, [START_REF] Rödel | Family legacies: short-and long-term fitness consequences of early-life conditions in female European rabbits[END_REF]. Indeed, [START_REF] Wilson | Selection on mothers and offspring: whose phenotype is it and does it matter?[END_REF] found no evidence for direct effects of birth timing on fitness in the Soay sheep (Ovis aries), the only moderately long-lived mammal studied so far. Our results in a wild population of roe deer suggest that the timing of birth can have a strong long-term influence on life histories, even in long-lived species.

Building an IPM based on the distribution of two contemporaneous traits allowed us to identify the relative contribution of each function (i.e. survival, recruitment, growth, and inheritance) to the difference in fitness observed between early-and late-born individuals. We found that the silver spoon effect of birth date in roe deer is generated by both the direct effect of birth date on recruitment and by the indirect effect of birth date on the different vital rates through body mass. The indirect effect of birth date on fitness through body mass is a crucial pathway, because the higher body mass that early-born fawns attained when adult mostly accounts for their higher fitness. Body mass has a strong influence on reproduction in large herbivores and is often directly linked to individual quality (Gaillard et al. 2000a[START_REF] Hamel | Individual quality, early-life conditions, and reproductive success in contrasted populations of large herbivores[END_REF]). As a consequence, compared to body mass, birth date per se only has a minor impact on the demography of the studied roe deer population. An IPM based on body mass only (i.e. without including birth date) would provide a reliable picture of the dynamics of this population but would not provide a mechanistic understanding of the influence of birth date on fitness. The IPM including both birth date and mass highlighted two key findings: (1) birth date plays a determinant role in shaping individual fitness, with early-born females enjoying higher fitness than late-born ones, and (2) the influence of birth date is mostly indirect, with the main pathway involving changes in body mass.

Birth date commonly affects early life history traits in many species [START_REF] Guinness | Factors affecting calf mortality in red deer (Cervus elaphus)[END_REF][START_REF] Reznick | On the virtue of being the first born: the influence of date of birth on fitness in the mosquitofish, Gambusia affinis[END_REF][START_REF] Feder | Never too late? Consequences of late birthdate for mass and survival of bighorn lambs[END_REF][START_REF] Solberg | Annual variation in maternal age and calving date generate cohort effects in moose (Alces alces) body mass[END_REF], including roe deer. Indeed, we found that birth date had a marked influence on early survival, with early-born fawns surviving much better than late-born individuals. Early-born fawns also reach a higher body mass as yearlings than late-born individuals. These short-term effects of birth date probably result from the high food quality available in early spring. The nutritional value of plants is high at the beginning of the growing season and decreases as they mature (Klein 1965, Albon and[START_REF] Albon | Plant phenology and the benefits of migration in a temperate ungulate[END_REF]. Thus, early-born fawns have access to high quality milk, and in large quantities, from their mothers who have access to plentiful, highly digestible food resources. As a consequence, they reach a higher body mass as yearlings than late-born individuals.

Long-lasting impacts of environmental conditions at birth have often been supposed to be mediated through early development [START_REF] Lindström | Early development and fitness in birds and mammals[END_REF][START_REF] Kruuk | Early determinants of lifetime reproductive success differ between the sexes in red deer[END_REF]. For instance, environmental conditions at birth affect both early and late body growth in male and female roe deer in two populations [START_REF] Douhard | Variation in adult body mass of roe deer: early environmental conditions influence early and late body growth of females[END_REF]. Similarly, birth date influences fitness of female roe deer through long-lasting effects on both juvenile and adult body masses in one of these populations. The indirect effect of birth date on adult body mass through yearling body mass is clearly related to the match between birth timing and food resource availability. Fawns of temperate areas that are born early grow for a longer time during the plant growing season and so survive better. The biological interpretation of the direct influence of birth date on adult body mass is more challenging, but the establishment of permanent physiological and metabolic functions during gestation and very early life could explain the direct effect of early conditions on adult life history traits [START_REF] Lummaa | Early development, survival and reproduction in humans[END_REF]. In particular, the development of muscle tissue up until the adult stage is linked to fetal and very early life conditions, because the number of muscle fibers is fixed at birth and strongly influenced by maternal nutrition [START_REF] Du | Fetal programming of skeletal muscle development in ruminant animals[END_REF]. Moreover, although natal dispersal is quite frequent in roe deer of both sexes [START_REF] Gaillard | Population density and sex do not influence fine-scale natal dispersal in roe deer[END_REF][START_REF] Debeffe | Condition-dependent natal dispersal in a large herbivore: heavier animals show a greater propensity to disperse and travel further[END_REF], many fawns remain philopatric and settle on a home range that is contiguous or overlapping with their maternal home range [START_REF] Strandgaard | An investigation of corpora lutea, embryonic development, and time of birth of roe deer (Capreolus capreolus) in Denmark. Vildtbiologisk Station[END_REF]). An alternative explanation for the direct effect involves the correlation between individual quality and habitat quality, which could thus at least partly explain the direct influence of early life on adult body mass. Our results contrast with those on Soay sheep where no direct influence of birth date on fitness was detected and the long-lasting effect of birth date was simply linked to birth mass [START_REF] Wilson | Selection on mothers and offspring: whose phenotype is it and does it matter?[END_REF]. The markedly fluctuating environmental conditions faced by the Soay sheep population [START_REF] Coulson | Age, sex, density, winter weather, and population crashes in Soay sheep[END_REF]) have a strong influence on annual body mass and could mask any direct long-term effect of birth date on fitness.

The influence of birth timing on adult life history traits has been intensively studied in birds. In the intensively studied barn swallow, a short-lived species (< 6 years in this study), hatching date was negatively associated with lifetime reproductive success [START_REF] Saino | Longevity and lifetime reproductive success of barn swallow offspring are Version preprint Comment citer ce document[END_REF]). However, the biological link between reproductive timing and fitness differs between barn swallows and roe deer. In barn swallows, early hatched individuals are longer-lived than late hatched birds and thereby obtain higher lifetime reproductive success through a greater number of reproductive attempts. In contrast, in roe deer, we have shown that birth date has no effect on adult survival which, as a general rule, varies little among years in large mammals (Gaillard . 2000b, Gaillard and[START_REF] Gaillard | Temporal variation in survival of mammals: a case of environmental canalization?[END_REF]. In roe deer, birth timing-related fitness differences among individuals arise because of variation in annual reproductive success rather than in reproductive lifespan. This difference in reproductive performance is likely linked to the 3 kg higher adult body mass of early-born females compared to late-born females. Thus, early-born females are more successful in raising their offspring [START_REF] Andersen | Factors affecting maternal care in an income breeder, the European roe deer[END_REF].

Females which are born the earliest, which are also the heaviest and attain the highest fitness, can be considered as the highest quality individuals [START_REF] Hamel | Individual quality, early-life conditions, and reproductive success in contrasted populations of large herbivores[END_REF]. Indeed, while a variety of mechanisms may link birth or hatching date to fitness, birth date can be considered as an important component of the multi-dimensional complex that describes individual quality (sensu [START_REF] Wilson | What is individual quality? An evolutionary perspective[END_REF]. In roe deer, we have shown that birth date influences the whole life history trajectory of an individual. Nonetheless, one cannot predict an individual's trajectory from its birth date alone. Individual life history trajectories are shaped by both early environmental conditions (including birth date) and current environmental conditions [START_REF] Tuljapurkar | Dynamics heterogeneity in life histories[END_REF]. The relative role of traits that are fixed at birth, such as maternal home range or environmental conditions at birth, versus traits that vary across an individual's lifetime in response to dynamic environmental variations remains to be quantified [START_REF] Tuljapurkar | Dynamics heterogeneity in life histories[END_REF], but see [START_REF] Helle | Life in varying environments: experimental evidence for delayed effects of juvenile environment on adult life history[END_REF] on the bank vole). In our models including both early and late life history traits, birth date explained a maximum of 9% of the observed variation in the life history traits that we studied. The large amount of unexplained variation in these life history traits can be assumed to be linked to either annual environmental conditions or stochastic variation.

Yearly variation in environmental conditions affects annual body condition [START_REF] Mcnamara | State-dependent life histories[END_REF] and the environmental cues used by different species to time their reproduction [START_REF] Reed | Phenotypic plasticity and population viability: the importance of environmental predictability[END_REF]. In a species like roe deer that displays little among-year variation in body mass, body condition [START_REF] Andersen | Factors affecting maternal care in an income breeder, the European roe deer[END_REF] or timing of birth [START_REF] Plard | Parturition date for a given female is highly repeatable within five roe deer populations[END_REF], we expect that birth date and early life conditions would markedly influence individual life history trajectories. On the contrary, in a species that shows large among-year variation in annual body

  Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (

  Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal.

  Birth date and individual fitness. To investigate the influence of birth date on fitness (measured using reproductive value early in life), we used an integral projection model (IPM) to analyse the dynamics of the distributions of two continuous traits over time (Easterling et al. 2000, Ellner and Rees 2006, Coulson 2012): body mass (bm) and birth date (bd), for female roe deer in the population of Trois Fontaines. The contemporaneous distributions of body mass and birth date at year t + 1 according to their age a + 1 (the proportion of individuals of age a + 1 and class (bm, bd) at year t + 1 in the population is defined by n(a + 1, t + 1, bm, bd)) depend on their distributions at year t and on five functions: (1) the inheritance function for birth date (I bd (a, t, bm, bd |bd)) links the offspring's birth date to the birth date and body mass of the Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (

  used a two age-class structure: yearling (1 year old) and adult females[START_REF] Nilsen | A slow life in hell or a fast life in heaven: demographic analyses of contrasting roe deer populations[END_REF], illustrated in the Fig.B1. As adult body mass of an individual female varies little among years in roe deer (e.g.[START_REF] Andersen | Factors affecting maternal care in an income breeder, the European roe deer[END_REF], Plard et al. 2014b), we assumed that an individual's body mass was constant from 2 years old to death and equal to its median adult body mass (between 4 and 10 years old), so limiting the size of the matrix to two age classes. While this assumption may lead to a slight over-estimate of body mass for 2 and 3 year old individuals and for females older than 10 years old, this is unlikely to overly influence our analyses. Roe deer females indeed reach 93% of their adult body mass at 2 years of age and body mass of senescent females is 96% of their adult body mass (a decrease of 1 kg on average,[START_REF] Hewison | Reproductive constraints, not environmental conditions, shape the ontogeny of sex-specific mass-size allometry in roe deer[END_REF]).

  inheritance and the growth functions using a normal probability density. The mean of each of these functions provides the link between the offspring trait and the maternal trait (including the effect of the individual's birth date for the inheritance function for body mass) for the inheritance functions and between an individual's adult body mass and its yearling body mass and its birth date for the growth function. The variances of these functions were estimated from the squared residuals of the mean relationships and were kept constant. We quantified the influence of birth date on fitness by estimating the individual reproductive value of roe deer at 8 months of age in relation to their birth date. Reproductive value was first introduced by Fisher (1930) to measure the extent to which individuals of different ages Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (

  Fig B1) corresponds precisely to the model where maximizing reproductive value is equivalent to maximizing fitness (Caswell 2001). Moreover, using an integral projection model allowed us to generate a given reproductive value for each birth date class, whereas we had only one asymptotic population growth rate for the entire population. As noted by Fisher himself, r is the average individual fitness and is therefore a property of the population. This measure can be biased when used at the individual level because the average of individual measures (individual r) is not equal to the asymptotic population growth rate. Moreover, in contrast to other commonly used measures of individual fitness like lifetime reproductive success (Clutton-Brock 1988, Newton 1989), reproductive values account for how much a given heritable genotype will contribute to future generations (Maciejewski 2014) and for the timing of reproduction. In long-lived species for instance, two individuals that successfully raised the same number of offspring during their lifetime, but at different ages, have the same lifetime reproductive success but different reproductive values when population growth differs from 0. Given the available data, we estimated reproductive values at 8 months of age rather than at birth as commonly done. Indeed, we did not have measures of body mass at birth for most individuals monitored, which were first weighed at 8 months of age and recruited then in our demographic model (see the definition of the recruitment function). We approximated reproductive values at Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (

  citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1

  offspring birth date (a 1% change in the slope of the inheritance function for birth date led to a 0.40% change in the difference in fitness). This difference was also closely related to the indirect effect of birth date on fitness mediated through body mass. Indeed, a 1% change in the slope of the growth function for birth date and in the inheritance function for body mass led to a 0.35% and to a 0.21% change in the difference in fitness between early-and late-born females, respectively. Simulating 60 groups of yearlings born on different days indicated that early-born females (1 st May) weighed 3.3 kg (13%) more as adults than late-born females (31 st May). Moreover a 1% change in the slopes linking body mass and the vital rates led to a change in the Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (

  Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 20

  Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 21 et al

  . (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F.,Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 22 condition(bighorn sheep, Festa-Bianchet 1998) or in species with marked phenotypic plasticity (such as the great tit, Parus major,[START_REF] Nussey | Selection on heritable phenotypic plasticity in a wild bird population[END_REF], the relative influence of current annual environmental conditions on life history trajectories should be higher than in roe deer.Consequently, the magnitude of the long-lasting effects of early conditions for shaping individual life history trajectories and population dynamics is likely to vary as a function of the life history strategy of the species. Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 Table 1. Model selection for the early life stage and adult stage traits. A. Early survival (Recruitment function of the IPM). Influence of maternal body mass (BM M ) and individual birth date (BD, with a threshold date on May 12 th ) on individual early survival (to 8 months of age). B. Yearling body mass (Inheritance function for body mass). Influence of individual birth date (BD) and of maternal body mass (BM M ) on individual yearling body mass. An effect of sex was included in the models because yearling males are heavier than yearling females. C. Yearling survival. Influence of individual birth date (BD) and yearling body mass (BM Y ) on individual yearling survival (Y ). D. Adult body mass (Growth). Influence of individual birth date (BD) and yearling body mass (BM Y ) on female adult body mass. An indirect effect of birth date on adult body mass through its effect on yearling body mass was also included in all models using a path analysis. E. Adult survival. Influence of individual birth date (BD) and adult body mass (BM Ad ) on female adult survival. k indicates the number of estimated parameters, LL is the maximum log likelihood, Dev is the deviance of the model, ∆AIC indicates the difference in the AIC between two competing models, and wi corresponds to Akaike weights. Pd is the estimated number of parameters, D is the deviance of the model and ∆DIC indicates the difference in the DIC between two competing models. The selected models are indicated in bold. 1 represents the constant model (i.e. intercept only). Version preprint Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard, M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015

FIGURE CAPTION Figure 1 .

 CAPTION1 FIGURE CAPTION

Figure 2 .

 2 Figure 2. Influence of individual birth date on individual early survival (A.) and yearling body mass (B.) for roe deer at Trois Fontaines, France. (A.) Data used to fit the model are represented by filled circles whose size is proportional to the number of observed births within 25 classes of birth date (of 2 days from April 17 th to June 5 th ). Males and females were pooled in this analysis (B.) Partial residuals of observed yearling body mass (corrected for maternal body mass and sex) are presented. Males and females are presented in black and grey, respectively, with one point representing one individual. The predictions of the model with their 95% confidence intervals are presented with a grey shade .

Figure 3 .

 3 Figure 3. Long-term direct and indirect (i.e. mediated through body mass) influence of individual birth date on individual adult body mass for female roe deer at Trois Fontaines, France. The dotted line gives the predictions of the direct effect of birth date, only.

Figure 4 .Fig. 1

 41 Figure 4. Percentage change of the difference in fitness between early-and late-born female roe deer generated by a 1% change in the slopes of each function (Rec: Recruitment, Inh_bd: Inheritance for birth date, Inh_bm: Inheritance for body mass, Gro: Growth and Sur:survival).Perturbations of slopes linking birth date and body mass to the early and adult life history traits are presented in black and white, respectively.

  2

  . Birth dates are normally distributed, with a median birth date of May 16 th . Females are monoestrous and give birth to one, two, or occasionally three fawns

	7
	Comment citer ce document :
	Plard, F. (Auteur de correspondance), Gaillard, J.-M., Coulson, T., Hewison, A. J. M., Douhard,
	M., Klein, F., Delorme, D., Warnant, C., Bonenfant, C. (2015). The influence of birth date via
	body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1

TABLE 1 .

 1 ). The influence of birth date via body mass on individual fitness in a long-lived mammal. Ecology, 96 (6), 1516-1528. , DOI : 10.1890/14-0106.1 36

	Early life stage traits
	A. early survival	k	LL	∆AIC	wi
	BM M + BD	5	-181.347	0	0.673
	BD	4	-185.941	7.187	0.018
	BM M	4	-183.140	1.585	0.304
	1	3	-188.334	9.975	0.005
	B. yearling body mass	k	LL	∆AIC	wi
	sex + BM M + BD	7	-238.091	0	0.970
	sex + BD	6	-242.601	7.021	0.029
	sex + BM M	6	-246.0224	13.863	0.001
	sex	5	-250.932	21.682 ≤ 0.001
	C. yearling survival	k	LL	∆AIC	wi
	BM Y + BD	4	-20.757	0.488	0.345
	BM Y	3	-21.513	0	0.441
	BD	3	-22.875	2.724	0.113
	1	2	-23.980	2.934	0.102
	Adult stage traits
	D. adult body mass	P d	D	∆DIC
	BM Y + BD	7	537.400	0
	BM Y	6	540.900	1.400
	BD	6	580.300	40.600
	1	5	585.300	43.500
	E. adult survival	k	Dev	∆AIC	wi
	BM Ad + BD	7	1462.309	3.733	0.080
	BM Ad	6	1462.531	2.055	0.185
	BD	6	1462.323	1.746	0.216
	1	5	1462.576	0	0.518

ACKNOWLEDGEMENTS

We are grateful to the Office National de la Chasse et de la Faune Sauvage and to the many volunteers and colleagues who participated in the roe deer captures in winter and who gave their time to catching and monitoring fawns on the study site of Trois Fontaines. Tim Coulson was funded by an ERC advanced grant. We warmly thank Bernt Erik Saether and the Centre for Biodiversity Dynamics for financial support of the roe deer project. We thank Steve Dobson and two anonymous reviewers for constructive comments on a previous version of the manuscript.

Version preprint

Comment citer ce document : Plard, F. (Auteur de correspondance), Gaillard


Gersende Berthier

Sen Dou

Torren Peakman

Eric Lichtfouse
email: eric.lichtfouse@dijon.inra.fr

Identification of bound alcohols in soil humic acids by GC-MS

Humic acids are complex, partly macromolecular, yellow-brownish substances occurring in soils, waters and sediments. In order to bring some light on their molecular structure, crop humic acids were cleaved by alkaline hydrolysis (KOH). The products were fractionated by thin layer chromatography to give mono-alcohols which were analysed as acetate derivatives by gas chromatography coupled to mass spectrometry. Linear alcohols, sterols, stanols and plant-derived triterpenoid alcohols were identified by co-injection of pure standards and by comparison with literature data. These findings imply that alcohols could have been incorporated into the humic matrix by esterification with carboxylic acids. Furtehrmore, the presence of stanols as hydrogenated counterparts of sterols suggests that a process of hydrogenation is operating in soils.

INTRODUCTION

Humic substances represent a major pool of organic carbon occurring in soil, sediment and waters [1][START_REF] Lichtfouse | Humic Substances[END_REF] . It is generally accepted that humic acids are characterised as polydispersed, acidic, amorphous substances ranging in molecular mass from a few hundred to several thousand daltons. However, the pathways of formation of humic acids remains an enigma mainly because of the lack of structural identification of its network at the molecular level. To unravel the molecular structure of complex, high molecular weight natural organic substances such as kerogen, asphaltene, coal and humic substances, various chemical and physical methods have been applied [3][4][5] . In particular, the breakdown of humic substances by chemical and enzymatic degradation can release substantial amounts of compounds amenable by gas chromatography coupled to mass spectrometry [5][6][7] . In a recent investigations, we identified alkane and alkene biomarkers released by pyrolysis of humin [8][9][10][11][12] . Here, we report the identification of n-alkanols, stanols, sterols and plant-derived triterpenoid alcohols released by mild alkaline degradation of soil humic acids from crop soils.

EXPERIMENTAL

Labware decontamination procedures are described elsewhere 13 . Soils cultivated for 23 years with maize (Zea mays) or wheat (Triticum aestivum) were sampled at Boigneville, France, then dried and sieved to 2 mm. 2 g of humic acids were extracted with 0.1 M sodium hydroxide under nitrogen to prevent oxidation of organic matter, precipitated at pH 1.5 with HCl, dialysed against water to remove molecules smaller than 2000 daltons, freeze-dried, extracted with diethyl ether to remove free organic compounds then refluxed 16 h. in 5 M potassium hydroxide-methanol under nitrogen. After addition of water, alcohols were extracted with diethyl ether (3 x), Na 2 SO 4 -dried then fractionated by thin layer chromatography (TLC) on silica-gel eluting methylene chloride with cholesterol as reference (R F ~ 0.13) to yield a mono-alcohol fraction which was then derivatised with an excess of acetic anhydride and pyridine (50°C, 1 h). The products were fractionated by TLC on silica-gel eluting methylene chloride with acetates of cholesterol, ergosterol and n-hexadecanol as references (R F ~ 0.70) to yield a mono-acetate fraction which was then analysed by GC-MS. GC conditions: HP 5890 series II, on-column injector, 50 m x 0.32 mm i.d. capillary column coated with 0.52 mm 100% polymethyl-siloxane phase (Ultra 1), Helium carrier gas: 0.66 b. head pressure at 50°C (constant flow mode), oven temperature: 50-100°C (15°/min.), 100-310°C (3°/min.), 310°C (60 min.). MS conditions: HP 5989A quadrupole, electronic impact (70 eV), scanning from 50 to 700 amu.

RESULTS AND DISCUSSION

The GC-MS total ion current trace of mono-alcohols, as acetate derivatives, released by alkaline hydrolysis of humic acids from a maize crop soil is presented on The identification of bound alcohols in soil humic acids has several implications. First, a higher plant source 20 can be inferred for most alcohols, e.g. odd-carbon number n-alkanols, campesterol, stigmasterol, β-sitosterol, β-amyrin and α-amyrin. Whereas cholesterol is probably derived at least partly from other organisms, as suggested by the higher β-sitosterol/cholesterol ratio of 8 in maize than in maize soils (value: 2) [START_REF]Quantification by GC-Flame Ionisation Detector[END_REF] . Second, the most likely pathway of incorporation of plant alcohols into soil humic acids could involve esterification of alcohols with carboxylic groups. This hypothesis is in with the high concentration of acidic groups in humic substances 1,5 . Third, we identified C 27 -C 29 stanols (2,5,9) as hydrogenated counterparts of C 27 -C 29 sterols (1,4,8) in hydrolysis products of humic acids. Since stanols are rare in living organisms, their occurrence in soil suggests thier derivation from sterols by hydrogenation into the soil. This assumption is supported by an investigation showing the hydrogenation of 14 C-cholesterol in sediments [START_REF] Gaskell | [END_REF] .

CONCLUSION

Several classes of alcohols have been identified by GC-MS as bound moieties of soil humic acids using alkaline degradation. They are probably incorporated in the humic matrix by esterification with carboxylic groups. Most alcohols are typical of plant substances whereas cholesterol and cholestanol are probably inherited at least parlty from other organisms. Furthermore, the presence of stanols in humic substances could be explained by hydrogenation of their sterols counterparts.

Figure 1 .

 1 Three classes of alcohols were identified in hydrolysis products of humic acids by mass spectrometry and by comparison with pure standards: linear alkanols, sterols, stanols and triterpenoid alcohols (Figure2). The spectra of all compounds show low relative intensity of the molecular ion and high relative intensity of the M-60 peak corresponding to the easy removal of acetic acid. Sterols give a high m/z 255 peak due to the cleavage of the side-chain. The m/z 215 base peak of stanols is explained by the cleavage of the 5-carbon ring. β-and α-amyrin are easily characterised by a m/z 218 base peak. Compounds 1,2,4,6-8,10 and 11 were identified by co-injection of pure standards. The structure of the other compounds were assigned on MS grounds and by comparison with literature data[14][15][16][17][18][19] .

Figure 1 .

 1 Figure 1. GC-MS total ion current of alcools (as acetate derivatives) released by alkaline hydrolysis of humic acids from a maize crop field. A similar fingerprint was obtained by analysis of humic acids from wheat crop soils. C 16 -C 30 refer to n-alkanols. 1 Cholest-5-en-3β-ol (cholesterol) , 2 5α-cholestan-3β-ol, 3 (3β, 22E)-ergosta-5,22-dien-3-ol (brassicasterol), 4 (24R)-ergost-5-en-3β-ol (campesterol), 5 (5α, 24R)-ergostan-3β-ol, 6 (3β, 22E)-stigmasta-5,22dien-3-ol (stigmasterol), 7 (3β,5α,22E)-stigmast-22-en-3-ol, 8 stigmast-5-en-3β-ol (β-sitosterol), 9 olean-12-en-3β-ol (β-amyrin), 10 5α-stigmastan-3β-ol 11 urs-12-en-3β-ol (α-amyrin).

Figure 2 .

 2 Figure 2. Molecular structure of alcools released by alkaline hydrolysis of humic acids.


Louis Hollande

Abdus Samad

Jaufurally §a

Paul-Henri Ducrot
email: ducrot@versailles.inra.fr

Florent Allais
email: florent.allais@agroparistech.fr

Prof F Allais

ADMET polymerization of biobased monomers deriving from syringaresinol

INTRODUCTION

The collapse of fossil resources and rising prices may have been the first trigger for a biobased economy. Nevertheless, today, industries, customers and regulators (e.g., REACH) are increasingly demanding for eco-friendly and bio-based chemicals issued from sustainable industrial processes. In this context, the production of new platform chemicals from biomass through green processes is an alluring strategy for a sustainable development. Indeed, biomass offers a wide range of molecules that can be used to access valuable synthons such as polyols, furans, fatty acids, aliphatic alkanes/alkenes or aromatics/phenolics to name a few. [START_REF] Bozell | Green Chemistry -Frontiers in Benign Chemical Synthesis and Processes[END_REF] Out of these sustainable and valuable chemical feedstocks, sinapic acid, from the ancient greek σιναπι (sinapi), a major compound isolated from Brassicaceae seeds and also one of the three major p-hydroxycinnamic acids found in lignocellulosic biomass, is one of the most promising substitute to fossil phenolics. It is present in relatively large quantities in the form of sinapine (choline ester of sinapic acid) in the seeds of oil plants from the Brassicaceae family. [START_REF] Niciforovic | [END_REF] In order to offer some new opportunities of valorization of this compound, and having dedicated ourselves to the synthesis and valorization of lignin-derived phenolics and more particularly p-hydroxycinnamic acids and derivatives, [3][4][5][6][7][8][9][START_REF] Pion | ADMET polymerization of biobased monomers deriving from syringaresinol[END_REF][11][12][13][14] we recently optimized a chemoenzymatic synthetic pathway allowing the ABSTRACT Renewable α,ω-dienes have been prepared from syringaresinol, a naturally occurring bisphenol deriving from sinapyl alcohol, and further studied as monomers in ADMET polymerizations. Polymerization was optimized according to catalyst loading and reaction conditions (in mass vs. in solvent), and led to polymers with molecular weight up to 14.1 kDa. Thermal analyses of these new polymers showed excellent thermal stabilities (257-360 °C) and tunable T g (18-70 °C) depending on the structure of the starting α,ω-diene monomer.

KEYWORDS: ADMET polymerization, syringaresinol, bisphenol

Comment citer ce document : Hollande, L. (Co-premier auteur), Jaufurally, A. S. (Co-premier auteur), Ducrot, P. H. (Auteur de correspondance), Allais, F. (Auteur de correspondance) (2016). ADMET polymerization of biobased monomers deriving from syringaresinol. RSC Advances, 6 (50), 44297-44304. DOI : 10.1039/c6ra06348a 2 synthesis of syringaresinol from sinapic acid (or syringaldehyde) in very high yield and purity. 15 The work reported here presents the use of syringaresinol as precursor for the synthesis of novel α,ω-dienes monomers incorporating the two aromatic rings of syringaresinol linked through a cis-fused bisfuranic moiety. These monomers were then submitted to acyclic diene metathesis (ADMET) polymerization. Indeed, thanks to the ease of handling and high functional group tolerance of the Ru-based catalysts used in ADMET, this method is a very useful approach for the construction of well defined polymer architectures 16 and allowed the synthesis of renewable polymers, such as polyesters, polyethers, polyamides and many others, very promising for commercial applications. 12,[17][18][19][20][21][22][23][24][25][26][27] In spite of extensive research to develop commercial ferulic acid-, 12,17 eugenol-, 22 vanillin- 23 and bisvanillin-24 renewable polymers, to the best of our knowledge, there is no example of ADMET polymerization involving sinapic acid derivatives (i.e., syringaresinol, syringaldehyde) as diene substrates.

The structure and thermal properties of these novel polymers were thereafter studied in order to evaluate their potential in industrial applications.

EXPERIMENTAL

Materials and methods

All reagents were purchased from Sigma-Aldrich or Tokyo Chemical Industry Co and used as received. Solvents were purchased from ThermoFisher Scientific, DMF was dried on a mBraun SPS 800 system. Deuterated chloroform (CDCl 3 ) was purchased from Eurisotop. Evaporations were conducted under reduced pressure at temperature below 40 °C. Column chromatographies were carried out with an automated flash chromatography (PuriFlash 4100, Interchim) and pre-packed INTERCHIM PF-30SI-HP (30 µm silica gel) columns. FT-IR and UV analyses were performed on Cary 630 FTIR and Cary 60 UV-Vis from Agilent technologies, respectively. NMR analyses were recorded on a Bruker Fourier 300. [START_REF] Bozell | Green Chemistry -Frontiers in Benign Chemical Synthesis and Processes[END_REF] H NMR spectra of samples were recorded in CDCl 3 at 300 MHz (residual CHCl 3 signal at δ = 7.26 ppm). 13 C NMR spectra of samples were recorded at 75 MHz (CDCl 3 signal at δ = 77.16 ppm). Thermo-gravimetric analyses (TGA) were recorded on a Q500, from TA. Around 5 mg of each sample was heated at 10 °C.min -1 from 50 to 500 °C under nitrogen flow (60 mL.min -1 ). Differential scanning calorimetry (DSC) thermograms were obtained using a DSC Q20, from TA, under inert atmosphere (N 2 ). Around 5 mg were weighed in a pan which was then sealed and submitted to the following heat/cool/heat cycle: equilibration at -60 °C and hold for 5 min, heating from -60 °C to 200 °C at 10 °C.min -1 and hold for 5 min, cooling from 200 °C to -60 °C at 10 °C.min -1 . Gel Permeation Chromatography (GPC) was performed at 40 °C on an Infinity 1260 system from Agilent Technologies with a quadruple detection (IR, UV, MALS, viscosimetry) and two PL-Gel 5 mm Mixed D column (300 mm × 7.5 mm) in THF (flow rate 1 mL.min -1 ) using polystyrene calibration. HRMS were recorded by the PLANET platform at URCA on a Micromass GC-TOF.

Synthesis of (+/-)-syringaresinol

Sinapyl alcohol (4 g, 19 mmol, 1 eq) was dissolved in acetonitrile (80 mL) in a triple-neck round bottom flask equipped with a cooling system and citrate/phosphate buffer (320 mL) at pH 5 were added (0.05 M). A solution containing 35.2 mg of laccase from Trametes versicolor (0.1 U/mg of substrate) in 50 mL of citrate/phosphate buffer was added dropwise to the monolignol solution at a rate of 9 mL/hour with a syringe pump. The reaction mixture was stirred with a magnetic stirrer in the presence of O 2 (air) at 50 °C for 470 min in darkness. At the end of the reaction, the product was extracted with dichloromethane (2 x 150 mL) and ethyl acetate (2 x 150 mL). The

General procedure for the synthesis of α α α α,ω ω ω ωdiene monomers

Syringaresinol (1 equiv) and K 2 CO 3 (5 equiv) were dissolved in dry DMF (2 M) under nitrogen. Bromo-alkene (4 equiv) was then added, and the mixture was stirred and heated at 80 °C for 12 h. Reaction was quenched with water (v(H 2 O) = v(DMF)) and the aqueous layer was extracted three times with ethyl acetate (3 x v(H 2 O)). Organic layers were combined, washed with brine, dried over anhydrous MgSO 4 , filtered and concentrated. Crude product was purified by flash chromatography on silica gel Cyclohexane:AcOEt (from 95/5 to 50/50) to yield the corresponding α,ω-diene monomer.

Bis-Allyl

General Procedure for ADMET polymerizations

Mass polymerizations

Monomer (500 mg) and p-benzoquinone (2 eq. relative to Hoveyda-Grubbs II catalyst) were stirred at 100 °C, under vacuum for 5 min. Hoveyda-Grubbs II catalyst (0.1-10 mol%) was added and the reaction was continued, under vacuum, for 4 h at 100 °C. Reaction was quenched with 1 mL of THF and 4 drops of vinyl ethyl ether. Product was concentrated and further dissolved in THF (1 mL) and precipitated in cold methanol (50 mL).

Solvent polymerization

Monomer (1 eq.) and p-benzoquinone (2 eq. relative to Hoveyda-Grubbs II catalyst) were dissolved in dry DCM (2 M). Hoveyda-Grubbs II catalyst (1-10 mol%) was added and the reaction was stirred, under nitrogen, at 40 °C for 4 h. Reaction was quenched with 1 mL of THF and 4 drops of vinyl ethyl ether. Product was concentrated and a sample was taken for GPC analyses. The remainder was dissolved in THF (1 mL) and precipitated in cold methanol (50 mL).

Polymer obtained from SYR-All (P1) (

RESULTS AND DISCUSSION

α,ω-diene monomers SYR-All, SYR-But, SYR-Hex and SYR-Dec were prepared by conducting Williamson etherification of syringaresinol with four bromo-alkenes (3-bromoprop-1-ene, 4, bromobut-1-ene, 6-bromohex-1-ene and 10bromodec-1-ene) in DMF at 80 °C, for 12 hours under nitrogen, in presence of potassium carbonate (Scheme 1). These four α,ω-diene monomers were used to study the impact of the chain length of the olefin on the polymerizations and the thermal properties of the resulting polymers.

ADMET polymerization

We first studied the reactivity of the four syringaresinol-derived α,ω-diene in ADMET polymerization. Though it has been reported that ADMET polymerizations are favored when carried out under bulk conditions (aka in mass), 28,12 we also performed polymerizations in solvent to verify if this also applied to syringaresinol-based α,ω-diene monomers (Scheme 1). For ADMET reactions conducted in mass at 100 °C, continuous vacuum (ca. 20 mbar) was applied to guarantee the efficient removal of ethylene and thus shift the equilibrium towards polymerization. The latter temperature was chosen as it has been already shown that, in the ADMET polymerization of rigid IDF-based α,ω-dienes 12 incorporating two dihydroferulate esters of isosorbide, heating the reaction mixture at 100 °C significantly improves the molecular weights by lowering the viscosity of the reaction medium thus facilitating both ethylene removal and stirring. Dichloromethane, previously reported as a suitable solvent for ADMET, 29 was used for in solvent polymerizations. Solvent reactions were performed at 40 °C under a low and steady nitrogen flow to remove ethylene but not dichloromethane. In both reaction conditions, 1,4-benzoquinone (2 eq. relative to catalyst) was used to limit olefin isomerization. 30 Finally, Hoveyda-Grubbs second generation catalyst (HG-II), with loadings varying from 0.1 to 10 mol%, was the only catalyst tested in this study as previous works by Abbas, 31 of acrylates-, vanillin-and ferulic acid-based α,ω-dienes, respectively. All ADMET reactions were run for 4 hours and followed by GPC in order to identify the best conditions (nature of the α,ω-diene monomer, in mass vs. in solvent, catalyst loading) (Figure 2). Table 1 summarizes the results of these polymerizations. The 1 H and 13 C NMR spectra of all polymers are displayed in the Supporting Information.

Structural analysis

In addition to GPC, 1 H NMR spectrometry of the resulting crude polymerization mixtures was also performed not only to confirm the polymers structures but also to reveal potential undesired side reactions such as ring-closing metathesis (RCM) or olefin isomerization (Figure 1). and their corresponding polymers obtained in mass reveal the decrease of intensity of the signals at 5.0-5.2 and 5.8-5.9 ppm corresponding to the terminal olefin protons along with the formation of the internal double bound whose signal is at 5.5 ppm (See Supporting Information). ADMET polymerizations performed in solvent led to short oligomers (Table 1, Entries 4-6 and 13-15) and higher dispersity (Table 1, Entries 14 and 15), whereas in mass ADMET polymerizations gave higher molecular weights as proven by the GPC results (Table 1, Figure 2). Detailed analyses of 1 H and 13 C NMR spectra show no evidence of isomerization of the double bond (Supporting information).

On the basis of the results reported in Table 1 and Figure 1, it was concluded that in mass procedure with 1 mol% HG-II, at 100 °C for 4 hours was the best procedure for ADMET polymerization for all monomers. In such conditions, syringaresinol-based polymers were obtained with M n in the range of 4.5-14.1 kDa.

It is also noteworthy that ADMET polymerization of monomers bearing allyl moieties only provides low molecular weight oligomers. Furthermore, no RCM product was observed in the case of SYR-All, probably because of the ring strain of syringaresinol that prevents intramolecular ring closing metathesis.

Because in solvent ADMET conditions proved unsuitable for our monomers, only results for polymers obtained in mass are being considered in the following discussion (Table 1, Entries 1-3, 7-12 and 16-18). As previously observed in the case of isosorbide based α,ωdienes, 12 the rigid bicyclic structure of syringaresinol and its relatively high viscosity Table 1. Analytical data (GPC, TGA and DSC) of the synthesized polymers via ADMET In mass ); e DSC data recorded at 10 °C.min -1 under nitrogen (60 mL.min -1

), value determined at the 2 nd heating scan Scheme 2. Syringaresinol-and ferulic acid-derived 12 polymers via ADMET restricts the polymerization to a degree of polymerization (DP n ) up to ca. 20 (Table 1, Entries 16-18). As expected, because of decreasing viscosity, the longer the α,ω-dienes the higher the M n (Table 1, Entries 3, 9, 12 and 18). Interestingly, while molecular weights (M n ) of polymers increase by 98% when but-1-ene was replaced by hex-1-ene (P3 vs. P2, Table 1, Entries 10 vs. 7), they increase only by 46% when hex-1-ene is replaced by dec-1-ene (P4 vs. P3, Table 1, Entries 16 vs. 10). Increasing the catalyst loading above 1 mol% resulted in similar to slightly higher molecular weights. In the other hand, lower catalyst loadings (e.g., 0.1-0.5 mol%) did not lead to higher molecular weight, neither did longer reactions times (8 hours vs. 4 hours).

In terms of reactivity, syringaresinol-based α,ωdiene proves as efficient as recently published IDF-Hex and IDF-Dec α,ω-dienes which give similar molecular weights when submitted to ADMET polymerization with HG-II (Scheme 2). 12 Thermal properties

Table 2. Thermostability of syringaresinol-based monomers

The thermal properties of monomers and polymers described in Table 1 were investigated by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC).

TGA analyses of the monomers revealed a thermostability (T d 50% ) in the range of 333-392 °C (Table 2). Furthermore, the nature of the alkene impacts the degradation temperature (T d 50% ). Indeed replacing but-1-ene by dec-1-ene strongly increases their thermostability by 58 °C (Table 2).

Thermal analyses of polymers P1-P4 showed significant differences in T d(5%) and glass transition temperature (T g ) depending on the alkene length (C 4 , C 6 and C 10 ) (Figure 3 &4). Because of the presence of aromatic moieties and the rigidity of syringaresinol, all polymers exhibit a thermostability in the range of 257-360 °C (T d(5%) ), the dec-1-ene based ones being the most stable (Table 1, Entries 16, 17 and 18). DSC analyses also reveal that a decrease of the alkene chain length (from decene to butene) results in an inversely proportional increase of the T g (P4<P3<P2<P1). Varying the nature of the α,ω-diene monomers thus provides T g in the range of 18 to 70 °C (Figures 4 &5). Compared to IDF-Hex α,ω-diene monomerbased polymer 12 that exhibits a T g of 18 °C, that of P3 deriving from SYR-Hex monomer is of 39 °C (Scheme 2). Such an increase could be explained by not only the shorter distance between the two phenols in the α,ω-diene monomer (12 atoms vs. 4 carbon atoms), but also by the presence of extra methoxy groups on the aromatic rings that increases the conformational barriers for chain motion. [START_REF] Nguyen | ADMET polymerization of biobased monomers deriving from syringaresinol[END_REF] Finally, it is noteworthy to mention that all polymers are amorphous and do not show melting points (T m ) on DSC.

CONCLUSIONS

In summary, syringaresinol-based α,ω-diene monomers were obtained in very good yields through a chemo-enzymatic synthetic pathway and were successfully polymerized via ADMET in presence of second generation Hoveyda-Grubbs catalyst. Mass polymerization resulted in polymers with M n as high as 14.1 kDa. Thermal analyses through TGA and DSC demonstrated that these polymers are thermostable up to 257-360 °C and, more importantly, that their T g can be easily tuned by adjusting the alkene length of the α,ω-diene monomers (from 18 to 70 °C). Prepared from renewable feedstocks, these polymers could be envisaged as sustainable substitutes to conventional petro-based polyesters.

11

  Comment citer ce document : Hollande, L. (Co-premier auteur), Jaufurally, A. S. (Co-premier auteur), Ducrot, P. H. (Auteur de correspondance), Allais, F. (Auteur de correspondance) (2016). ADMET polymerization of biobased monomers deriving from syringaresinol. RSC Advances, 6 (50), 44297-44304. DOI : 10.1039/c6ra06348a 4 (C ɣ,ɣ' ), 86.0 (C α ), 103.0 (C 2 ), 114.41 (C 11 ), 136.4 (C 1 ), 136.8 (C 4 ), 138.9 (C 10 ), 153.7 (C 3

5 Scheme 1 .

 51 Scheme 1. Synthesis and ADMET polymerization of the four syringaresinol derived α,ω-diene monomers

6 Figure 1 . 1 HFigure 2 .

 6112 Figure 1. 1 H NMR spectra of SYR-All, SYR-But, SYR-Hex and SYR-Dec and their corresponding oligomers and polymers (P1, P2, P3 and P4)

  1 H NMR spectra of monomers (SYR-Comment citer ce document : Hollande, L. (Co-premier auteur), Jaufurally, A. S. (Co-premier auteur), Ducrot, P. H. (Auteur de correspondance), Allais, F. (Auteur de correspondance) (2016). ADMET polymerization of biobased monomers deriving from syringaresinol. RSC Advances, 6 (50), 44297-44304. DOI : 10.1039/c6ra06348a 7 All (A), SYR-But (B), SYR-Hex (C), SYR-Dec (D))

a

  HG-II (1, 5 or 10 mol%), 1,4-benzoquinone (2 equivalent relative to HG-II), DCM (2 M), 40 °C, 4 hours; b HG-II (1, 5 or 10 mol%), 1,4-benzoquinone (2 equivalent relative to HG-II), vacuum, 100 °C, 4 hours;c Calculated with the residual monomer amounts determined from the corresponding peak area on SEC traces of crude reaction mixtures prior precipitation;

9 Figure 3 .Figure 4 .

 934 Figure 3. TGA analyses for polymers P1, P2, P3, and P4 (under nitrogen, 10 °C.min -1 )

Figure 5 .

 5 Figure 5. Alkene chain-length dependence of the glass transition temperature (T g )

  -O-syringaresinol (SYR-All) ES + ): m/z calcd for C 28 H 34 O 8 Na [MNa] + : 521.2151; found: 521.2142. 1 H NMR (300 MHz, CDCl 3 , δ): 3.10 (m, 2H, H β ), 3.85 (s, 12H, H 5 ), 3.91 (dd, 2H, J = 3.6 Hz H ɣ ), 4.33 (m, 2H, H ɣ' ), 4.48 (d, 4H, J = 7 Hz, H 6 ), 4.73 (d, 2H, J= 4 Hz, H α ), 5.15 (m, 2H, H 8 ), 5.32 (m, 2H, H 8 ), 6.10 (m, 2H, H 7 ), 6.58 (s, 4H, H 2 ). 13 C NMR (75 MHz, CDCl 3 , δ): 54.6 (C β ), 57.3 (C 5 ), 71.9 (C 6 ), 74.2 (C ɣ,ɣ' ), 85.9 (C α ), 102.87 (C 2 ), 117.7 (C 8 ), 134.5 (C 7 ), 136.1 (C 1 ) , 136.7 (C 4 ), 153.63 (C 3 ). TOF MS, ES + ): m/z calcd for C 30 H 38 O 8 Na [MNa] + : 549.2464; found: 549.2474. 1 H NMR (300 MHz, CDCl 3 , δ): 2.49 (q, 4H, J = 6.9 Hz and 13.8 Hz, H 7 ), 3.10 (m, 2H, H β ), 3.84 (s, 12H, H 5 ), 3.90 (m, 2H, J = 3.6 Hz, H ɣ ), 3.99 (t, 4H, J = 7,2 Hz, H 6 ), 4.30 (m, 2H, H ɣ' ), 4.72 (d, 2H, J = 4 Hz, H α ), 5.09 (m, 4H, H 9 ), 5.87 (m, 2H, H 8 ), 6.6 (s, 4H, H 2 ). 13 C NMR (75 MHz, CDCl 3 , δ): 34.5 (C 7 ), 54.3 (C β ), 56.2 (C 5 ), 71.9 (C 6 ), 72.4 (C ɣ,ɣ' ), 86.0 (C α ), 102.9 (C 2 ), 116.4 (C 9 ), 134.9 (C 8 ), 136.6 (C 4,1 ), 153.6 (C 3 ).

	White (75%). FTIR (neat): powder ν max : 1501 (C=C Ar). HRMS: (TOF MS, Bis-Butene-O-syringaresinol (SYR-But) Viscous (78%). (neat): ν max : oil FTIR 1501 (C=C Ar). HRMS: (Bis-Hexene-O-syringaresinol (SYR-Hex) Viscous oil (71%). FTIR (neat): ν max : 1501 (C=C Ar). HRMS: (TOF MS, ES + ): m/z calcd for C 34 H 46 O 8 Na [MNa] + : 605.3090; found: 605.3098. 1 H NMR (300 MHz, CDCl 3 , δ): 1.54 (m, 4H, H 8 ), 1.7 (qt, 4H, J = 6.9 Hz, H 7 ), 2.1 (q, 4H, J = 7.2 Hz, H 9 ), 3.11 (m, 2H, H β ), 3.84 (s, 12H, H 5 ), 3.84 (m, 2H, H ɣ ), 3.93 (m, 4H, H 6 ), 4.31 (m, 2H, H ɣ' ), 4.73 (d, 2H, J = 4.2 Hz, H α ), 4.94 -5.04 (m, 4H, H 11 ), 5.84 (m, 2H, H 10 ), 6.58 (s, 4H, H 2 ). 13 C
	NMR (75 MHz, CDCl 3 , δ): 25.1 (C 8 ), 29.5 (C 7 ),
	33.5 (C 9 ), 54.3 (C β ), 56.2 (C 5 ), 71.9 (C 6 ), 73.21

  ).

	Bis-Decene-O-syringaresinol (SYR-Dec)
	Viscous oil (77%). FTIR (neat): ν max : 1502 (C=C
	Ar). HRMS: (TOF MS, ES + ): m/z calcd for
	C 42 H 62 O 8 Na [MNa] + : 717.4342; found: 717.4351.
	1 H NMR (300 MHz, CDCl 3 , δ): 1.30 -1.44 (m,
	20H, H 8,9,10,11,12 ), 1.73 (qt, 4H, J = 7.8 Hz, H 7 ),
	2.02 (q, 4H, J = 7.1 Hz, H 13 ), 3.1 (m, 2H, H β ), 3.83
	(s, 12H, H 5 ), 3.92 (m, 6H, H ɣ,6 ), 4.29 (m, 2H, H ɣ' ),
	4,72 (d, 2H, J = 4.2 Hz, H α ), 4.89 -5.28 (m, 4H,
	H 15 ), 5.75 (m, 2H, H 14 ), 6.55 (s, 4H, H 2 ). 13 C NMR
	(75 MHz, CDCl 3 , δ): 25.8 (C 8 ), 28.9 -30.1
	(C 7,9,10,11,12 ) , 33.8 (C 13 ), 54.3 (C β ), 56.1 (C 5 ),
	71.9(C 6 ), 73.51 (C ɣ,ɣ' ), 86.0 (C α ), 103.0 (C 2 ), 114.1
	(C 15 ), 136.3 (C 1 ), 136.9 (C 4 ), 139.2 (C 14 ), 153.7
	(C 3 ).

Polymer obtained from SYR-Hex (P3) (1 mol% HG-II, 2

  1 mol% HG-II, 2 mol% 1,4-benzoquinone, 100 °C): 1 H NMR (300 MHz, CDCl 3 , δ): 3.10 (m, 2H, H β ), 3.84 (s, 12H, H 5 ), 3.90 (m, 2H, H ɣ ), 4.30 (m, 2H, H ɣ' ), 4.49 (m, 4H, H 6 ), 4.74 (m, 2H, H α ), 6.05 (m, 2H, H 7 ), 6.58 (s, 4H, H 2 ). 13 C NMR (75 MHz, CDCl 3 , δ): 54.7 (C β ) , 56.6 (C 5 ) , 72.3 (C 6 ), 73.5 (C ɣ ), 86.3 (C α ), 103.2 (C 2 ), 130.2 (C 7 ), 136.6 (C 1 ) , 137.1 (C 4 ) , 153.9 (C 3 ). mol% 1,4-benzoquinone, 100 °C): 1 H NMR (300 MHz, CDCl 3 , δ): 1.50 (m, 4H, H 8 ), 1.71 (m, 4H, H 7 ), 2.03 (m, 4H, H 9 ), 3.10 (m, 2H, H β ), 3.83 (s, 12H, H 5 ), 3.95 (m, 6H, H ɣ,6 ), 4.29 (m, 2H, H ɣ' ), 4.73 (d, 2H, J = 3,9 Hz, H α ), 5.43 (m, 2H, H 10 ), 6.55 (s, 4H, H 2 ).

	Polymer obtained from SYR-But (P2) (1 mol%
	HG-II, 2 mol% 1,4-benzoquinone, 100 °C): 1 H
	NMR (300 MHz, CDCl 3 , δ): 2.47 (m, 4H, H 7 ), 3.10
	(m, 2H, H β ), 3.83 (s, 12H, H 5 ), 3.95 (m, 6H, H ɣ,6 ),
	4.29 (m, 2H, H ɣ' ), 4.72 (d, 2H, J = 2.7 Hz, H α ),
	5.60 (m, 2H, H 8 ), 6.55 (s, 4H, H 2 ). 13 C NMR (75
	MHz, CDCl 3 , δ): 34.4 (C 7 ), 54.3 (C β ), 56.2 (C 5 ),
	71.9 (C 6 ), 72.9 (C ɣ ), 86.0 (C α ), 102.9 (C 2 ), 128,2
	(C 8 ), 136.6 (C 4,1 ), 153.6 (C 3 ).

  Comment citer ce document : Hollande, L. (Co-premier auteur), Jaufurally, A. S. (Co-premier auteur), Ducrot, P. H. (Auteur de correspondance), Allais, F. (Auteur de correspondance) (2016). ADMET polymerization of biobased monomers deriving from syringaresinol. RSC Advances, 6 (50), 44297-44304. DOI : 10.1039/c6ra06348a

	recorded at 10 °C.min -1 under nitrogen (60 mL.min -1
		b	1	SYR-All		2.0	2.6	4	1.3	59	283	352	65
	2	In mass b	5	SYR-All	P1	2.2	2.9	4	1.3	73	266	327	70
	3	In mass b	10	SYR-All		2.8	3.9	6	1.4	80	263	343	68
	4	In solvent a	1	SYR-All		1.4	1.6	2	1.1	/	/	/	/
	5	In solvent a	5	SYR-All	P1	1.6	2.1	3	1.3	/	/	/	/
	6	In solvent a	10	SYR-All		2.2	2.8	4	1.2	/	/	/	/
	7	In mass b	1	SYR-But		4.5	7.7	9	1.7	93	283	376	58
	8	In mass b	5	SYR-But	P2	5.1	8.5	10 1.7	88	274	369	60
	9	In mass b	10	SYR-But		6.7	13.1	13 1.9	89	272	339	61
	10	In mass b	1	SYR-Hex		8.9	18.3	15 2.1	94	308	392	39
	11	In mass b	5	SYR-Hex	P3	8.3	15.5	14 1.9	90	268	342	40
	12	In mass b	10	SYR-Hex		8.9	16.8	15 1.9	86	257	324	38
	13	In solvent a	1	SYR-Hex		1.4	1.5	2	1.1	/	/	/	/
	14	In solvent a	5	SYR-Hex	P3	8.4	30	14 3.6	/	/	/	/
	15	In solvent a	10	SYR-Hex		8.2	26.1	14 3.2	/	/	/	/
	16	In mass b	1	SYR-Dec		13.0	25.5	19 2.0	97	360	393	20
	17	In mass b	5	SYR-Dec	P4	12.7	28.6	19 2.2	95	349	392	19
	18	In mass b	10	SYR-Dec		14.1	32.0	21 2.3	82	347	394	18
	8

ACKNOWLEDGEMENTS

The authors are grateful to the Region Champagne-Ardenne, the Conseil Général de la Marne and Reims Métropole for their financial support.


D De Guibert

F Martin

A Kharlamova

A Kerjouh

C Le

Floch Fouéré

G Delaplace

R Jeantet

Profil

Dried whey protein fractal aggregates for substituting texturizing additives in dairy products

.02

Science & Technology of Milk & Egg

Rennes A multidisciplinary and multiscale approach, reinforced by two high-calibre facilities:

Dairy Platform Biological Resource Centre q Structuration / destructuration mechanisms of food matrix:

from structural characterisation to digestion q Dairy processing and cheese making: toward sustainable dairy systems q Microbial interaction: food matrix and host cell

Please visit http://www6.rennes.inra.fr/stlo_eng

Thank you for your attention

.013

  , Martin F, Hennetier M, Gu YY, Le Floch Foueré C, Delaplace G, Jeantet R. 2020. Flow process and heating conditions modulate the characteristics of whey protein aggregates. J Food Eng 264, article 109675, 1-10 International Hydrocolloids Conference 2020 / Domitille de Guibert (INRAE) 4 March 2020Research questionWhat is the impact of the concentration and drying operations on the cold gelation functionality of fractal aggregates

83 standing fellow workers

	CONTEXT
	.03
	.03

International Hydrocolloids Conference 2020 / Domitille de Guibert (INRAE) 4 March 2020 Background & RQ Strategy Results / discussion Conclusion Industrial expectation

value to milk protein (technofunctional interest)

	Consumer expectation	• Target regular products properties by using additives • Use less additives as possible (Clean label) • Add PROFIL 2014-2019 Joelle LEONIL

Fractal aggregates : Whey protein aggregates Adapted from Nicolai 2011 WPI, 80°C, pH 8, NaCl

	• Good organoleptic quality
	• More natural and healthy
	products