Théo Constant

Sylvain Giroud

Vincent A Viblanc

Mathilde L Tissier

Patrick Bergeron

F Stephen Dobson

Caroline Habold
email: caroline.habold@iphc.cnrs.fr

Marmota Flaviventris

Integrating mortality risk and the adaptiveness of hibernation

Keywords: Life History, activity pattern, time and energy allocation, Trade off, hibernator, temporal organization of activity, predation, Size activity patterns, hibernation, life history, temporal organization of activity, time and energy allocations, trade-off, longevity, mortality risk Ursus arctos Carnivora Ursus maritimus Carnivora Criteria 1 No Ursus thibetanus Carnivora Microcebus murinus marmota Rodentia Marmota monax

Word count: 255

Low mortality rate is often associated with slow life history, and so far, has mainly been assessed through examinations of specific adaptations and lifestyles that limit mortality risk. However, the organization of activity time budgets also needs to be considered, since some activities and the time afforded for performing them may expose animals to higher mortality risks such as increased predation and/or increased metabolic stress, including oxidative stress. We examined the extent of activity time budgets contribution to explaining variation in life history traits in mammals. We specifically focused on hibernating species because of their marked seasonal cycle of activity/inactivity associated with very different mortality risks. Hibernation is considered a seasonal adaptation to prolonged periods of food shortage and cold. This inactivity period may also reduce both extrinsic and intrinsic mortality risks, by decreasing exposure to predators and drastically reducing metabolic rate. In turn, reduction in mortality may explain why hibernators have slower life history traits than non-hibernators of the same size. Using phylogenetically controlled models, we tested the hypothesis that longevity was positively correlated with the hibernation season duration (the time spent between immergence and emergence from the hibernaculum or den) across 82 different mammal species. We found that longevity increased significantly with hibernation season duration, an effect that was particularly strong in small hibernators (< 1.5kg) especially for bats. These results confirm that hibernation not only allows mammals to survive periods of energy scarcity, but further suggest that activity time budgets may be selected to reduce mortality risks according to life history pace.

Contribution to the field

Hibernation is widely considered as an adaptation to a seasonal decrease in food resources and ambient temperatures. If the remarkable physiological aspects of hibernation have been widely studied, fewer studies have focused on its ecological and evolutionary significance. In this study, we investigate whether hibernation, in addition to its survival benefits to face energy stress, may have evolved to limit other risk of mortality. The article is composed of a phylogenetic meta-analysis showing the patterns of association between hibernation season duration and longevity across mammalian hibernators. Our main conclusion is that hibernation season duration has a positive effect on longevity, and this effect is significantly greater small hibernators (<1.5kg) especially for bats, which are assumed to have a higher risk of predation and energy constraints, than larger species. Therefore, our article provides new evidence that hibernation duration may have evolved to limit mortality risk in some species.

Introduction

Energy is the fundamental requirement for life. Its acquisition, storage, and metabolic use shape the diversity of lifestyles in all living organisms [START_REF] Brown | Toward a Metabolic Theory of Ecology[END_REF]. Because energy availability to organisms is limited under natural conditions, in terms of its acquisition in time and space, as well as its quantity and quality, organisms have to simultaneously maximize investments into all biological functions, and to compromise the allocation of metabolic energy among competing demands [START_REF] Hirshfield | Natural selection and the evolution of reproductive effort[END_REF][START_REF] Lack | Population studies of birds[END_REF][START_REF] Reznick | Costs of Reproduction: An Evaluation of the Empirical Evidence[END_REF][START_REF] Williams | Natural Selection, the Costs of Reproduction, and a Refinement of Lack's Principle[END_REF]. For example, demographic characteristics may affect energy investments among biological traits such as growth, reproduction, and somatic maintenance. These trade-offs have led to the evolution of specific life history strategies [START_REF] Stearns | The evolution of life histories[END_REF].

Attributes of life histories generally scale with body size such that large animal species usually take longer to develop and mature, have lower annual reproductive rates, and live longer compared to small species [START_REF] Blueweiss | Relationships between body size and some life history parameters[END_REF][START_REF] Speakman | Body size, energy metabolism and lifespan[END_REF]. However, life history variations that are specific to a particular body size are also observed along a fast-slow continuum [START_REF] Bielby | The Fast-Slow Continuum in Mammalian Life History: An Empirical Reevaluation[END_REF][START_REF] Dobson | Fast and slow life histories of mammals[END_REF][START_REF] Jeschke | The roles of body size and phylogeny in fast and slow life histories[END_REF][START_REF] Jones | Senescence rates are determined by ranking on the fast-slow life-history continuum[END_REF][START_REF] Oli | The fast-slow continuum and mammalian life-history patterns: an empirical evaluation[END_REF]. For a given body size, most species trade off investments between reproduction and self-maintenance. For example, a species with fast life history strategy will exhibit faster growth, earlier reproduction, larger annual reproductive investment, and reduced maximum life span compared to a species with a slow life history strategy, which will typically promote self-maintenance and survival over reproduction (but see [START_REF] Bielby | The Fast-Slow Continuum in Mammalian Life History: An Empirical Reevaluation[END_REF][START_REF] Jeschke | The roles of body size and phylogeny in fast and slow life histories[END_REF].

The evolution of the fast-slow continuum in life history strategies appears to be contingent upon individual mortality risk [START_REF] Healy | Animal life history is shaped by the pace of life and the distribution of age-specific mortality and reproduction[END_REF][START_REF] Martin | Age-related mortality explains life history strategies of tropical and temperate songbirds[END_REF][START_REF] Promislow | Living fast and dying young: A comparative analysis of life-history variation among mammals[END_REF]. Individual mortality can be due to either intrinsic (wear-and-tear of the body) or extrinsic factors, such as

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Time dimension of allocation trade-offs in hibernators 4 predation, disease, or environmental hazards. Species with slow life histories typically exhibit adaptations that limit both sources of mortality [START_REF] Lewis | The naked mole-rat response to oxidative stress: just deal with it[END_REF][START_REF] Macrae | DNA repair in species with extreme lifespan differences[END_REF][START_REF] Munshi-South | Bats and birds: Exceptional longevity despite high metabolic rates[END_REF][START_REF] Wu | Life in the cold: links between mammalian hibernation and longevity[END_REF][START_REF] Blanco | Maximum longevities of chemically protected and nonprotected fishes, reptiles, and amphibians support evolutionary hypotheses of aging[END_REF][START_REF] Healy | Eusociality but not fossoriality drives longevity in small mammals[END_REF][START_REF] Healy | Ecology and mode-of-life explain lifespan variation in birds and mammals[END_REF][START_REF] Holmes | Fly Now, Die Later: Life-History Correlates of Gliding and Flying in Mammals[END_REF][START_REF] Shattuck | Arboreality has allowed for the evolution of increased longevity in mammals[END_REF][START_REF] Sibly | Effects of body size and lifestyle on evolution of mammal life histories[END_REF]Turbill et al., 2011;Wilkinson and South, 2002). Besides these molecular (such as oxidative stress tolerance), physiological/anatomical (such as chemical protection, horns and antlers) or lifestyle (such as arboreality) adaptations that reduce mortality risk, the organization of activity time budgets should be particularly important in shaping the variety of life histories observed in the wild. Mortality rates may change depending on the time allocated to each activity, resulting in trade-offs for which both time and energy can be optimized. For instance, activities that contribute the most to reproductive success are often energy-intensive [START_REF] Alonso-Alvarez | Increased susceptibility to oxidative stress as a proximate cost of reproduction[END_REF] and associated with higher risks of extrinsic mortality [START_REF] Magnhagen | Predation risk as a cost of reproduction[END_REF]. However, the temporal dimension of energy allocation trade-offs in relation to the evolution of life history strategies has been little examined (see [START_REF] Healy | Ecology and mode-of-life explain lifespan variation in birds and mammals[END_REF].

Here, we first examined the extent to which patterns of relative activity and inactivity can explain variation in life history traits in mammals. We specifically focused on hibernating species because of their marked seasonal cycle of activity/inactivity, which is associated with very different risks of mortality. From an intrinsic perspective, hibernation is a period of metabolic depression where energy requirements are reduced to minimal levels compared to the active season [START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF]. It has been suggested that energy restriction slows down the aging process [START_REF] Walford | The Response to Calorie Restriction in Mammals Shows Features Also Common to Hibernation: A Cross-Adaptation Hypothesis[END_REF][START_REF] Masoro | Caloric Restriction and Aging: Controversial Issues[END_REF] and is associated with an enhancement of somatic maintenance [START_REF] Shanley | Calorie Restriction and Aging: A Life-History Analysis[END_REF]. From an extrinsic perspective, even if mortality during hibernation occurs, hibernating mammals are usually hidden in burrows or shelters, which may reduce risks of predation, infections or injuries for several continuous months. As a result, hibernating species generally exhibit lower rates of mortality than similar-sized non-hibernating species during part of the

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Time dimension of allocation trade-offs in hibernators 5 year, resulting in slower life history strategies (Turbill et al., 2011;Wilkinson and South, 2002).

However, previous studies linking hibernation to longevity considered hibernation as a binary trait (if animals hibernate or not), rather than a continuous adaptive response (hibernation season duration) that allows animals to restrict their period of activity during parts of the year. Yet, early data from captivity highlight a positive correlation between longevity and hibernation duration in Turkish hamsters (Mesocricetus brandti) [START_REF] Lyman | Hibernation and longevity in the Turkish hamster Mesocricetus brandti[END_REF]. In addition, a strong negative effect of mean annual temperature on hibernation season duration and annual survival rate, which is highly correlated with longevity (Turbill et al, 2011), has been shown among populations of hibernating rodent species [START_REF] Turbill | Thermal climate-linked variation in annual survival rate of hibernating rodents: shorter winter dormancy and lower survival in warmer climates[END_REF] This raises the question of whether, across hibernating mammals the time spent being inactive (hibernation season duration) influences maximal longevity, a key feature characteristic of fast and slow life history strategies

In the present study, we tested for a positive association between the hibernation season duration and longevity across 82 mammalian species. For this examination, we tested for effect of body size on longevity while controlling statistically for phylogeny. If indeed hibernation season duration is part of a strategy allowing minimizing mortality, we predicted that longer hibernation season duration should be positively associated with species maximum longevity, especially in small mammals (< 1.5 kg) that exhibit greater longevity than non-hibernators of the same size (Turbill et al. 2011). In addition, if metabolic reduction during hibernation slows aging [START_REF] Lyman | Hibernation and longevity in the Turkish hamster Mesocricetus brandti[END_REF][START_REF] Turbill | Seasonal variation in telomere length of a hibernating rodent[END_REF][START_REF] Wu | Life in the cold: links between mammalian hibernation and longevity[END_REF], we predicted that the effect of hibernation season duration on longevity should increase with the percentage of metabolic reduction during hibernation compared to euthermia.

Material and methods

Review criteria

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Time dimension of allocation trade-offs in hibernators 6

We conducted the review using the search engine Google Scholar (https://scholar.google.com) and considered articles up to and including December 2019. We based our survey on the hibernating species listed in [START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF] and Turbill et al (2011), and further identified nine other species, mainly ground squirrels and bats, not mentioned in any of the lists. All the 152 hibernators that were examined in this process are summarized in Electronic Supplementary Materials (see Table S1). We excluded species for which hibernation was restricted to only a few populations or under specific conditions, namely two species, the black-tailed prairie dogs (Cynomys ludovicianus; [START_REF] Gummer | Geographic variation in torpor patterns: the northernmost prairie dogs and kangaroo rats[END_REF][START_REF] Lehmer | Extreme plasticity in thermoregulatory behaviors of free-ranging black-tailed prairie dogs[END_REF] and the polar bears (Ursus maritimus; [START_REF] Amstrup | Polar bear, Ursus maritimus[END_REF]. We did not include non-seasonal hibernating species capable of entering multi-day torpor at any time of the year.

Indeed, for such cases, it is difficult to accurately measure the time spent in hibernation over a year, which may also vary considerably among individuals and between years. Thus, we excluded hibernating elephant shrew species (Elephantulus sp.) and hibernating marsupials with the exception of the mountain pygmy possum (Burramys parvus), reported to be a seasonal hibernator [START_REF] Geiser | Hibernation and daily torpor in Australian mammals[END_REF][START_REF] Lovegrove | Heterothermy in elephant shrews, Elephantulus spp.(Macroscelidea): daily torpor or hibernation?[END_REF].

Longevity and body mass data

Data on maximum longevity, hereafter referred as longevity, and average body mass for the list of hibernating species previously identified were mainly obtained from the AnAge data base (The Animal Aging and Longevity Database; https://genomics.senescence.info/species/search_list.php; Human Ageing and Genomic Resources; Magalhães and Costa, 2009), and complemented these data with information from the PanTHERIA data base (https://ecologicaldata.org/wiki/pantheria; Ecological Archives, Ecological Society of America; [START_REF] Jones | PanTHERIA: a species-level database of life history, ecology, and geography of extant and recently extinct mammals[END_REF] from two reviews on mammalian longevity [START_REF] Heppell | Life Histories and Elasticity Patterns: Perturbation Analysis for Species with Minimal Demographic Data[END_REF]Wilkinson and South, 2002) or from a specific search in I n r e v i e w Time dimension of allocation trade-offs in hibernators 7 Google Scholar combining the following terms: "longevity" OR "life history" AND scientific or common names of species. For this specific search in Google Scholar, we considered both old and new nomenclatures for ground squirrel species, and only selected long-term field studies since they provide a good estimate of maximum longevity. Moreover, we specifically investigated the source of the data (captive vs. wild) and only retained longevity data where the source was available, to control for captivity effects on longevity (see Electronic Supplementary Material, Table S2 for references).

We determined the arboreality lifestyle of the species in order to statistically control (see below section 2.5) for higher longevity [START_REF] Healy | Ecology and mode-of-life explain lifespan variation in birds and mammals[END_REF][START_REF] Kamilar | Is group size related to longevity in mammals?[END_REF][START_REF] Shattuck | Arboreality has allowed for the evolution of increased longevity in mammals[END_REF]. We conducted a review of peer-reviewed scientific journal Mammalian Species (https://academic.oup.com/mspecies) and used Google Scholar. The search criteria were based on combining the following terms: "arboreal" OR "semi-arboreal" OR "climb tree" AND scientific or common names of species. We completed our search by examining the mammalian lifestyle databases of the following articles [START_REF] Healy | Ecology and mode-of-life explain lifespan variation in birds and mammals[END_REF]Hidasi-Neto et al., 2015). Our arboreality factor includes 16 arboreal and semi-arboreal species, which feed, nest, or escape from predators, at least frequently, by climbing into trees (see Electronic Supplementary Materials, Table S2).

Hibernation season duration data

Reviewing strategy

Relatively few studies have investigated hibernation duration with body temperature recorders on wild mammals. However, several studies have estimated hibernation season duration from capturemark-recapture records or direct observations, thus assessing periods of inactivity. Although for some species, the duration of hibernation measured as the period between the first and last torpor bout is

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Time dimension of allocation trade-offs in hibernators 8 probably shorter than the period of inactivity (Siutz et al., 2016;[START_REF] Williams | Phenology of hibernation and reproduction in ground squirrels: integration of environmental cues with endogenous programming[END_REF][START_REF] Young | Hibernating patterns of free-ranging Columbian ground squirrels[END_REF] , these measures should still provide reasonable estimates of the duration of energy savings, thereby allowing species to be compared with each other.

We reviewed the literature to retrieve estimates of the hibernation season duration as the time (in days) spent between immergence and emergence from the hibernaculum or den (with little or no movement outside). The search criteria were based on combining the following terms: "hibernation" OR "hibernation duration" OR "denning" (exclusively for bears) OR "roosting" (exclusively for bats) AND scientific or common names of species. In order to minimize heterogeneity in the scales at which the data were measured (e.g. individual, population), we considered maximum hibernation season duration obtained from same-sex adult groups, either male or female depending on the species (and recorded maximum hibernation season duration from overall population data when more precise data were unavailable).

Inclusion and exclusion criteria

We prioritized studies for which the methodology for estimating hibernation season duration of is described (capture-mark-recapture, direct observation and body temperature recording). These criteria included 64 species. We also included studies based on road kills as an index of activity/inactivity. This criterion has already been used to evaluate changes in hedgehog abundance [START_REF] Bright | The effect of changes in traffic flow on mammal road kill counts[END_REF][START_REF] Morris | Distribution and abundance of hedgehogs (Erinaceus europaeus) on New Zealand roads[END_REF][START_REF] Wembridge | An estimate of the annual number of hedgehog (Erinaceus europaeus) road casualties in Great Britain[END_REF] and was used for 2 species in our data set (Algerian hedgehog (Atelerix algirus) and Southern white-breasted hedgehog (Erinaceus concolor)). Finally, in cases where the above criteria were not available, we included studies for which

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Time dimension of allocation trade-offs in hibernators 9 a precise hibernation period was mentioned but the methodology could not be assessed. This criterion included 16 species (see Electronic Supplementary Material; Table S2, labeled species)

We excluded studies for which periods of extreme inactivity were measured only once in a population. This criterion excluded three maximum hibernation season data: 8 months for the little pocket mouse (Perognathus longimembris; Kenagy and Bartholomew, 1985); 6 months for the longtailed pocket mouse (Chaetodipus formosus; Kenagy and Bartholomew, 1985); and 11 months for the Eastern chipmunk (Tamias striatus; [START_REF] Munro | Extreme suppression of aboveground activity by a food-storing hibernator, the eastern chipmunk (Tamias striatus)[END_REF].

The availability or absence of data (longevity and hibernation season duration) in the literature for the 152 species examined is specified in Electronic Supplementary Materials (see Table S1). In total, our literature search allowed inclusion of 82 hibernating mammal in the analyses including 80 placental mammals, 27 bats, one marsupial (the mountain pygmy possum) and one monotreme (the short-beaked echidna (Tachyglossus aculeatus)). Longevity, body mass and hibernation season duration data for these species are available in Electronic Supplementary Materials (see Table S2).

Phylogenetic data

We downloaded 100 phylogenetic mammalian trees from http://vertlife.org/phylosubsets/ (see [START_REF] Upham | Inferring the mammal tree: Species-level sets of phylogenies for questions in ecology, evolution, and conservation[END_REF], focusing on the 82 species in our data set (see Electronic Supplementary Material, Table S2). These trees were used to construct strict consensus trees for the hibernating species of our study, where the included clades were those present in all the 100 phylogenetic mammalian trees [START_REF] Paradis | ). I n r e v i e w Time dimension of allocation trade-offs in hibernators 26[END_REF]. Because we ran subsequent analyses on different subsets of the data (see section 2 The rationale for eliminating bats from some analyses was to compare the specific effect of hibernation season duration on longevity between bats which have very distinct characteristics (i.e., flight capacity, highly gregarious behavior during hibernation [START_REF] Austad | Mammalian Aging, Metabolism, and Ecology: Evidence From the Bats and Marsupials[END_REF]) and other hibernators.

The metabolic rate during torpor is not known for all hibernating species studied [START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF]. To test the prediction that the effect of hibernation season duration on longevity should increase with metabolic reduction, we compared the effect of hibernation season duration on the longevity of two groups including (all hibernators excluding bats) or excluding (only deep hibernators without bats) species reducing their energy expenditure during hibernation by less than 90% compared to the euthermic state. These comprise Ursidae species and the European badger (Meles meles) that reduce their total energy expenditure from 33 to 75% during hibernation compared to the euthermic state [START_REF] Hellgren | Physiology of Hibernation in Bears[END_REF][START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF][START_REF] Tøien | Hibernation in Black Bears: Independence of Metabolic Suppression from Body Temperature[END_REF][START_REF] Watts | Energetic Cost of Winter Dormancy in Grizzly Bear[END_REF] and small tropical hibernators such as Cheirogaleidae and Tenrecidae species, which show a 70% reduction [START_REF] Dausmann | Energetics of tropical hibernation[END_REF][START_REF] Wein | Effects of ambient temperature on tropical hibernation in the lesser hedgehog tenrec, Echinops telfairi[END_REF]. Thus, the 'deep hibernator' group includes the species capable of reducing their total energy expenditure by about 90% or more during hibernation as compared to the euthermic state and reaching a body temperature during torpor below 10°C (mainly small Holarctic species; [START_REF] Heldmaier | Natural hypometabolism during hibernation and daily torpor in mammals[END_REF] In addition, the analyses of Turbill et al. 2011 indicated a body mass threshold of 1.5 kg, below which the benefits of hibernation (compared to non-hibernation) for longevity increased. To test the effect of hibernation season duration on longevity between hibernators < 1.5kg and larger ones, we used the data set excluding bats and hibernators > 1.5 kg.

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Branch lengths for respective consensus trees were calculated with the 'compute.brlen' function from the 'ape' package based on [START_REF] Grafen | The phylogenetic regression[END_REF] computations, and were used to compute PGLS models with the 'caper' package in R (see section 2.5 below).

Statistics

We tested for a significant relationship between hibernation season duration and species maximum longevity, using phylogenetic generalized least squares (PGLS) models using the 'ape 5.0', 'apTreeshape 1.5' and 'caper 1.0' packages in R v. 3.6.2 (Orme et al., 2013;[START_REF] Paradis | ). I n r e v i e w Time dimension of allocation trade-offs in hibernators 26[END_REF][START_REF] Paradis | ape 5.0: an environment for modern phylogenetics and evolutionary analyses in R[END_REF]R Core Team 2014). We thus statistically "controlled" for the influence of the phylogenetic relationships among species on the variables before evaluating relationships. In addition, the relative effect of the phylogenetic tree on the linear model could be estimated as a λ parameter, ranging between 0 (covariation among species measurements is independent of co-ancestry) and 1 (covariance entirely explained by co-ancestry). Testing the models with λ = 0 allowed comparison to λ-positive models, and thus the extent to which phylogeny influenced analyses of the models examined.

In addition to phylogeny, our model evaluated the influences of average body mass (of adults) of the different species, bats and arboreality lifestyle, and the fact that some data were acquired from captive and wild populations (see below).

We ran PGLS models for the 4 different conditions listed above (Table 1). Longevity was our dependent variable in all models, and hibernation season duration and species average body mass were independent variables. In all models, body mass and longevity were log-transformed to normalize their distributions, and all independent variables were standardized (using z-scores), so that their coefficients are directly comparable as estimates of effect sizes [START_REF] Abdi | Z-scores[END_REF]. In the original models, we included I n r e v i e w the interaction hibernation season duration x body mass to test for the possibility that the effect of hibernation season duration on longevity was more important for species of small body mass (see Fig 2 in Turbill et al., 2011), as well as a 'captive/wild' factor to account for captivity-related variation in longevity [START_REF] Tidière | Comparative analyses of longevity and senescence reveal variable survival benefits of living in zoos across mammals[END_REF]. However, these factors were parsimoniously dropped in the final models based on Akaike's Information Criterion (AIC). Among the models within ΔAIC <2 (ΔAICi=AICi-AICmin), we kept the model with the lowest number of terms (see Electronic Supplementary Materials Table S3). Nevertheless, body mass was retained throughout our models, because of the dominance that it shows as a primary axis of energetics and life history [START_REF] Brown | Toward a Metabolic Theory of Ecology[END_REF][START_REF] Stearns | The evolution of life histories[END_REF]. In order to control for higher longevity due to particular lifestyles, we added a 'bat (yes/no)' factor in the full model (Turbill et al., 2011;Wilkinson and South, 2002) and an 'arboreality (yes/no)' factor in each model [START_REF] Healy | Ecology and mode-of-life explain lifespan variation in birds and mammals[END_REF][START_REF] Kamilar | Is group size related to longevity in mammals?[END_REF][START_REF] Shattuck | Arboreality has allowed for the evolution of increased longevity in mammals[END_REF].We limited the number of additional predicators in order to maintain sufficient statistical power with respect to the sample size (table 2 ; Mundry, 2014).

For the final models, the level of covariation in maximum longevity among species was estimated by maximum likelihood (λML).

Within bats (individuals from wild populations only), we were not able to estimate the effects of body mass and hibernation season duration on longevity, while controlling statistically for phylogeny.

We had too few species of bats (N = 27; [START_REF] Münkemüller | How to measure and test phylogenetic signal[END_REF] for properly evaluating the phylogenetic signal (the lower CI bound for the phylogenetic signal could not be estimated; see Electronic Supplementary Material, Fig S2). Thus, we present simple linear regressions for this group later indicated as model 5 (Table 1 and2).

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Results

The characteristics of the models and data used are summarized in Table 1. For each model, hibernation season duration and longevity were similar in range between the different datasets (Table 1). Naturally, the range of body mass was much smaller when considering only deep hibernators, small species and bats.

Model 1(N = 82):

Accounting for the effect of phylogeny, variation in longevity was positively associated with hibernation season duration and body mass across all hibernating mammals (Table 2, Fig 1 and2). On average, bats had significantly longer lifespans (79%, ͞ x = 21 years, N = 27) as well as species with an arboreal lifestyle (11%, ͞ x =13 years, N =16), than other mammals (͞ x = 11.8 years, N = 55) (Table 2).

Model 2 (N = 55):

Removing hibernating bats from the analyses showed that hibernation season duration had a significant positive effect on longevity (Table 2, Fig 1 and2). The positive effect of body mass and an arboreal lifestyle on longevity remained, mammals with a higher body mass or an arboreal lifestyle exhibiting significantly longer lifespan (Table 2, Fig 1 and2).

Models 3 and 4 (N= 46 and N = 44 respectively):

When only deep hibernators (model 3) and small hibernators < 1.5 kg (excluding bats) were considered (model 4), we found a positive effect of hibernation season duration on longevity (Table 2, Fig 1 and2). The positive effect of arboreality lifestyle on longevity only remained for small hibernators (model 4).

Among the above models, the effect of hibernation season duration was slightly higher for small hibernators < 1.5 kg and highly significant (Table 2, Fig 1 and2). These models showed a strong influence of phylogeny on the results (Table 2). Ignoring the effect of phylogeny by constraining λ to 0 removed the effect of hibernation season duration on longevity (Table 2).

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Time dimension of allocation trade-offs in hibernators 14 Model 5 (N = 27): Because our sample size for bats alone was too small, we could not perform an analysis controlling for phylogeny. Instead, we ran simple linear models to test for the effects of hibernation season duration and body mass on longevity. Here as well, hibernation season duration, but not body mass, was positively and significantly related to maximum longevity (Table 2, Fig 1 and2). Hibernation season duration had an effect on bat longevity up to two times higher than in previous models excluding bats. This significant effect without accounting for phylogeny was probably due to a lower level of phylogenetic differences among bat species, compared to other models including up to eight different orders.

Discussion

Influence hibernation season duration on longevity

Our purpose was to examine the extent to which the activity time budget explains variation in life history traits in mammals. We investigated this question in hibernating species because of their marked seasonal cycle of activity/inactivity, which is associated with very different risks of mortality (Turbill et al., 2011). While controlling for phylogeny, our study highlighted a positive influence of hibernation season duration on longevity in mammalian hibernators. These results were in agreement with an early study that tested the effect of hibernation duration on longevity in captive Turkish hamsters [START_REF] Lyman | Hibernation and longevity in the Turkish hamster Mesocricetus brandti[END_REF]. In agreement with [START_REF] Dobson | A lifestyle view of life-history evolution[END_REF] and [START_REF] Sibly | Effects of body size and lifestyle on evolution of mammal life histories[END_REF], the two major axes of life histories of mammalian hibernators are body mass and lifestyle, with lifestyle contributing to the slow-fast continuum. The bat lifestyle (e.g. aerial) had the greatest influence on longevity, with a positive effect on longevity that was three times greater than the arboreality lifestyle.

The effect of hibernation season duration, in addition to these lifestyles, appeared to be roughly onethird of the effect of body mass on longevity (see model estimates in Table 2).

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Time dimension of allocation trade-offs in hibernators [START_REF] Kryštufek | Cricetus cricetus (Rodentia: Cricetidae)[END_REF] Our results show that the effects of hibernation season duration on longevity were consistent across a wide range of body sizes, and became stronger with the limitation in body mass to small species (species < 1.5 kg), especially for bats (Figure 1) . Interestingly, hibernation season duration appeared to be more important than body mass in explaining longevity in the latter species. These results support the idea that hibernation (1) is an efficient strategy that limits mortality in periods of energy scarcity for some larger species facing strong energy constraints during part of the year (e.g., Marmota species), and ( 2) may be an especially effective strategy for small mammals that are expected to suffer from both higher predation rate [START_REF] Cohen | Body Sizes of Animal Predators and Animal Prey in Food Webs[END_REF][START_REF] Sinclair | Patterns of predation in a diverse predatorprey system[END_REF], and increased loss of energy expenditure during winter [START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF]. In either case, increased hibernation season duration may increase both annual survival rates [START_REF] Turbill | Thermal climate-linked variation in annual survival rate of hibernating rodents: shorter winter dormancy and lower survival in warmer climates[END_REF] and overall longevity. Note that in some cases, the lack of relationship between body mass and longevity in our study could also be due to a smaller range of body mass variation than reported in other studies (for instance in bats; Wilkinson and South, 2002).

In our study, the effect of hibernation season duration on the longevity for bats was twice that of small non-flying terrestrial mammals, though this result should be considered with caution since we were not able to control for phylogeny when considering only bats (Table 2, Fig 2 and3). Bats stand somewhat apart from other mammals, distinguished notably by their ability for sustained flight, an important lifestyle characteristic [START_REF] Sibly | Effects of body size and lifestyle on evolution of mammal life histories[END_REF]. Flying is an energy-intensive activity, considerably more than terrestrial locomotion [START_REF] Thomas | The Physiology and Energetics of Bat Flight[END_REF][START_REF] Tucker | Respiratory Exchange and Evaporative Water Loss in the Flying Budgerigar[END_REF]. [START_REF] Thomas | The Physiology and Energetics of Bat Flight[END_REF] estimated that the greater spear-nosed bat (Phyllostomus hastatus) increases its resting metabolic rate more than 34 times during flight, while rodents of similar size increase it only by 8-fold during terrestrial locomotion. Thus, in bats the reduction of metabolism during hibernation is particularly important compared to their period of activity (Wilkinson and South, 2002).

I n r e v i e w

The marked effect of hibernation season duration on longevity in bats may also be explained by some extreme physiological adaptations to hibernation having evolved in response to specific ecological and anatomical constraints [START_REF] Willis | Trade-offs Influencing the Physiological Ecology of Hibernation in Temperate-Zone Bats[END_REF]. For instance, several bat species are capable of very long torpor bouts (up to 60 days; reviewed in [START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF], perhaps in response to their limitation on accumulation of internal or external energy reserves [START_REF] Willis | Trade-offs Influencing the Physiological Ecology of Hibernation in Temperate-Zone Bats[END_REF]. In addition, the little brown bat (Myotis lucifugus), for instance, is capable of performing "heterothermic arousals", corresponding to shallow torpor bouts (Tskin > 20°C), during arousal phases, and thus reduce the cost of euthermia [START_REF] Czenze | Thrifty Females, Frisky Males: Winter Energetics of Hibernating Bats from a Cold Climate[END_REF][START_REF] Jonasson | Hibernation energetics of free-ranging little brown bats[END_REF]. This particular adaptation may be present in other bat species as well. Finally, bats are highly gregarious during hibernation and can cluster in colonies of up to thousands of individuals (Clawson et al., 1980). Huddling could enable them to reduce energy costs and water loss during hibernation, making hibernation a particularly profitable strategy [START_REF] Boratyński | Clustering behavior in wintering greater mouse-eared bats Myotis myotis -the effect of microenvironmental conditions[END_REF][START_REF] Boratyński | Huddling reduces evaporative water loss in torpid Natterer's bats, Myotis nattereri[END_REF][START_REF] Boyles | Thermal Benefits of Clustering during Hibernation: A Field Test of Competing Hypotheses on Myotis sodalis[END_REF][START_REF] Gilbert | One for all and all for one: the energetic benefits of huddling in endotherms[END_REF].

Comparisons between of models either including (model 2) or excluding (model 3) species with lowest metabolic reduction during hibernation did not reveal significant differences. These results suggest that the effect of hibernation season duration on longevity remains consistent whatever the rate of metabolic reduction reached during hibernation compared to the active state (between 70 to 90%). This finding should pave the way for future studies to specifically test this effect of metabolic reduction during hibernation.

Interestingly, in all PGLS models, removing the effect of phylogeny by constraining λ to 0 also removed the effect of hibernation season duration on longevity. This suggests that the effect of hibernation season duration on longevity is masked by the phylogenetic pattern. Thus, hibernation season duration might be a stronger explanation of variation within species or between closely related species, as shown in Turkish hamsters [START_REF] Lyman | Hibernation and longevity in the Turkish hamster Mesocricetus brandti[END_REF]. For instance, studies comparing I n r e v i e w populations of golden-mantled ground squirrels (Callospermophilus lateralis) and Columbian ground squirrels (Urocitellus columbianus) living along an altitudinal gradient show that populations with longer hibernation season duration generally have higher annual survival and longevity [START_REF] Bronson | Altitudinal Variation in the Life History of the Golden-Mantled Ground Squirrel (Spermophilus Lateralis)[END_REF][START_REF] Dobson | Interpretation of intraspecific life history patterns: evidence from Columbian ground squirrels[END_REF][START_REF] Murie | Annual Variation of Spring Emergence and Breeding in Columbian Ground Squirrels (Spermophilus columbianus)[END_REF].

Although our results highlight an association between hibernation season duration and longevity, they do not provide a causal mechanism through which such an association might arise. Periods of prolonged inactivity are likely to increase longevity through the integration of multiple factors affecting both intrinsic and extrinsic mortality, as discussed below.

Factors affecting extrinsic and intrinsic mortality and the evolution of hibernation

Energetic stress, when energy demand is greater than energy availability in the environment, has a proximate role in the regulation of hibernation pattern [START_REF] Vuarin | Field evidence for a proximate role of food shortage in the regulation of hibernation and daily torpor: a review[END_REF]. However, few studies have focused on the causal link between energetic stress, and the timing of hibernation immergence and emergence (e.g., [START_REF] Humphries | The energetics of autumn mast hoarding in eastern chipmunks[END_REF]. Thus, the hypothesis that hibernation occurs primarily as a response to an energetic stress has not been completely studied (focusing on torpor bouts frequency, depth and duration). To the best of our knowledge, the only study having measured both energy availability in the environment and individual energy expenditure before immergence in hibernation shows in eastern chipmunks (Tamias striatus), that immergence occurs while food is still plentiful and climatic conditions are still favorable for maximizing energy storage [START_REF] Humphries | The energetics of autumn mast hoarding in eastern chipmunks[END_REF]. Other observations also suggest that immergence into hibernation while food is still available seems common, at least in sciurids (Humphries et al., 2003) and for the little pocket mouse (French,

I n r e v i e w

Time dimension of allocation trade-offs in hibernators 18 2004). Thus, food availability and ambient temperature alone may not be sufficient to explain the phenology of immergence.

Other evidence suggests that hibernation is not initiated solely in response to deficiencies in energy, water, or poor food quality. Many observations suggest that early immergence (before energetically stressful periods start) occurs when the benefits of reproduction are low. For instance, in years of low beech seed abundance, the edible dormouse (Glis glis) skips reproduction, quickly accumulates fat reserves, and is able to hibernate for up to 11 months [START_REF] Hoelzl | How to spend the summer? Free-living dormice (Glis glis) can hibernate for 11 months in nonreproductive years[END_REF]. This occurs even if food in the environment is sufficient to allow the edible dormouse to remain active but not to reproduce. Similarly, Eastern chipmunks skip reproduction and cease foraging for almost a full year when food availability is particularly low [START_REF] Munro | Extreme suppression of aboveground activity by a food-storing hibernator, the eastern chipmunk (Tamias striatus)[END_REF]. It seems that at that time, chipmunks rely on large amounts of food hoarded during the preceding year; but there is no evidence of torpor expression. This kind of behaviour is also observed in several hibernating ground squirrel species.

Females that fail to reproduce may immerge up to several weeks before the others [START_REF] Bintz | Lipid synthesis and deposition by adult Richardson's ground squirrels in the natural environment[END_REF][START_REF] Choromanski-Norris | Seasonal Activity Cycle and Weight Changes of the Franklin's Ground Squirrel[END_REF][START_REF] Michener | Effect of age and parity on weight gain and entry into hibernation in Richardson's ground squirrels[END_REF][START_REF] Neuhaus | Timing of Hibernation and Molt in Female Columbian Ground Squirrels[END_REF]. An experiment in semi-natural conditions shows that female European ground squirrels (Spermophilus citellus) that were separated from males (and thus did not breed) entered into hibernation 4 to 6 weeks before females that were not separated [START_REF] Millesi | Seasonal timing and pre-hibernation fattening in breeding and non-breeding European ground squirrels[END_REF]. These results support the view that hibernation phenology is influenced by a trade-off between reproduction and survival, where hibernation seems to provide benefits other than surviving periods of energetic stress. This trade-off may also explain differences (up to 1 month) in the timing of immergence and emergence between gender and age observed in rodents (Siutz et al., 2016;[START_REF] Sheriff | Phenological variation in annual timing of hibernation and breeding in nearby populations of Arctic ground squirrels[END_REF]Kawamichi, 1989 ;[START_REF] Kart Gür | Age and sex differences in hibernation patterns in free-living Anatolian ground squirrels[END_REF][START_REF] Snyder | Seasonal Changes in the Weights of Woodchucks[END_REF][START_REF] Holekamp | Seasonal variation in body weight, fat, and behavior of California ground squirrels (Spermophilus beecheyi)[END_REF] and bats (Decher and Choate, 1995;Norquay and Willis, 2014;Stebbings, 1970;[START_REF] Thomson | Myotis sodalis[END_REF].

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Time dimension of allocation trade-offs in hibernators 19 So far, most studies have focused mainly on temperate hibernating species. However heterothermy that occurs during daily torpor and hibernation, is taxonomically and geographically widespread [START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF]. A surprisingly large proportion of mammals, including a monotreme, several marsupials, and placental species regularly enter daily torpor and seasonal hibernation in the southern hemisphere [START_REF] Grigg | Hibernation by Echidnas in Mild Climates: Hints about the Evolution of Endothermy? In Life in the Cold[END_REF]. For some species in these regions, the use of torpor may not be related to low environmental temperatures or limited food availability [START_REF] Nowack | Variable climates lead to varying phenotypes: 'weird' mammalian torpor and lessons from non-Holarctic species[END_REF]. For example, the short-beaked echidna hibernates [START_REF] Grigg | Hibernation in a monotreme, the echidna (Tachyglossus aculeatus)[END_REF]) while ants and termites, which constitute the main part of its diet, remain available throughout the year [START_REF] Grigg | Hibernation by Echidnas in Mild Climates: Hints about the Evolution of Endothermy? In Life in the Cold[END_REF].

Short-beaked echidnas are heavily armored, perhaps rendering avoidance of predation an unlikely adaptive benefit for hibernation. These observations suggest that some species may use hibernation because of the energy advantages provided by lack of activity, even though it is not an energy necessity for survival during a period of energetic stress [START_REF] Grigg | Hibernation by Echidnas in Mild Climates: Hints about the Evolution of Endothermy? In Life in the Cold[END_REF]. Such case studies broaden the scope of possibilities for understanding the evolution of hibernation [START_REF] Grigg | Hibernation by Echidnas in Mild Climates: Hints about the Evolution of Endothermy? In Life in the Cold[END_REF][START_REF] Ruf | Survival, Aging, and Life-History Tactics in Mammalian Hibernators[END_REF], and open up exciting perspectives for future research.

Conclusion

Hibernation is considered an adaptation to seasonal, hence predictable decrease in food resources and ambient temperatures. However, hibernation is also observed in mild climates and when ambient conditions are still favorable for activity [START_REF] Nowack | Variable climates lead to varying phenotypes: 'weird' mammalian torpor and lessons from non-Holarctic species[END_REF]. If remarkable physiological aspects of hibernation have been widely studied, fewer studies have focused on its ecological and evolutionary significance in the field. Our study provides evidence that there may be a relationship between activity time budgets, and hence the time dimension of allocation trade-off, and life history traits.

I n r e v i e w

Time dimension of allocation trade-offs in hibernators 20

Our phylogenetic analyses shows that variations in hibernation season duration can partially explain variations in longevity in hibernators. The models show a strong influence of phylogeny on this relationship and highlight the need for in-depth studies at an inter-and intra-population scales. For example, future studies may attempt to consider activity time budgets in the context of the pace of life syndrome by examining variations in hibernation with other physiological and behavioral traits. Our results, combined with information available in the literature, suggest that, in addition to its survival benefits during a period of energetic stress, hibernation season duration may have evolved to reduce the effects of other sources of extrinsic and intrinsic mortality.

Table 1. Summary of models and datasets. The category known as 'deep hibernator' includes the species capable of reducing their total energy expenditure by about 90% or more during hibernation as compared to the euthermic state and reaching a body temperature during torpor below 10°C (mainly small Holarctic species; [START_REF] Heldmaier | Natural hypometabolism during hibernation and daily torpor in mammals[END_REF]. Crosses indicate group(s) included in each model. Table 2. Z-standardized model estimates (β) for the effects of hibernation season duration and body mass on species maximum longevity. The phylogenetic effect is estimated by λML. Both body mass and longevity were log-transformed before the analyses. For comparison, we have provided estimates for λ constrained to zero (no effect of phylogeny). The model for bats was a simple linear model not controlling for phylogeny, due to limited sample size in this group. In the model with all hibernators, the NA value in the confidence interval for λ ML indicates that the caper package could not calculate the full confidence interval. An NA value is considered as 0. I n r e v i e w squares highlight species reducing their energy expenditure during hibernation by less than 90% compared to the euthermic state, green item highlight species below 1.5kg, and black circle highlight all remaining hibernators above 1.5kg in the data set. Please note that some dots referring to bat species are superimposed because the corresponding species have exact same hibernation season durations and longevities. [START_REF] Jones | PanTHERIA: a species-level database of life history, ecology, and geography of extant and recently extinct mammals[END_REF]. We mention the new nomenclature for ground squirrel species, which may not correspond to the nomenclature of the lists presented. Some species were first excluded according to the exclusion criteria listed in the publication. We then investigate the longevity of hibernators according to the methods described in the publication. Only species for which we have found both the hibernation season duration and longevity were included in the study. All references for longevity and hibernation duration data are presented in Table S2.

I n r e v i e w

I n r e v i e w

*Note that longevity (12.5 years) and hibernation season duration (90 day) of Zaedyus pichiy are known (Magalhaes and Costa, 2009;[START_REF] Superina | Zaedyus pichiy (Cingulata: Dasypodidae)[END_REF] but by including this species the binary condition of the phylogenetic tree was not satisfied. A rooted tree is considered binary if all nodes (including the root node) have exactly two descendant nodes. This condition was tested using the 'ape 5.0' packages in R v.

  .5 below), we constructed separate phylogenetic trees on (1) the full hibernator data set (N = 82 species), (2) the data set excluding bats (N = 55 species), (3) the data set with only deep hibernator (see below) I n r e v i e w Time dimension of allocation trade-offs in hibernators 10 excluding bats (N = 46 species) and (4) the data set excluding bats and hibernators > 1.5 kg (N = 44 species) (see Electronic Supplementary Material, Fig S1).

Figure 1 .

 1 Figure 1. Effects of hibernation season duration and body mass on longevity. Z-standardized model coefficients are presented ± 95% Confidence Interval (1.96xSE). Both body mass and longevity were log-transformed before the analyses. The effect sizes are presented for PGLS models for all mammals (N = 82), mammals without bats (N = 55), deep hibernators without bats (n=46) and small mammals without bats (N = 44). For comparison, effect sizes from a simple linear model not accounting for phylogeny are presented for bats only (N = 27).

Figure 2 .

 2 Figure 2. Relationship between hibernation season duration (standardized) and longevity (log-transformed and standardized). The regression lines are presented for PGLS models for all mammals (bold black line, N = 82, p=0.015), all hibernators without bats (black line, N = 55, p=0.011), deep hibernators without bats (dashed line, N = 46, p=0.028) and small hibernators without bats (green line, N = 44, p=0.005). For comparison, effect sizes from a simple linear model not accounting for phylogeny are presented for bats only (red, N = 27, p=0.036). Full red circles highlight bat species, the

Table S1 :

 S1 Summary of the hibernators selected in this study among the 152 listed hibernators.

Species (1) in black are listed in

Turbill et al (2011)

,

[START_REF] Körtner | Rhythmicity of torpor in a marsupial hibernator, the mountain pygmy-possum (Burramysparvus), under natural and laboratory conditions[END_REF]

in dark blue represent the other species listed by

[START_REF] Ruf | Daily torpor and hibernation in birds and mammals[END_REF]

and (

3

) in light blue represent other known hibernators. AnAge refers to AnAge data base (The Animal Aging and Longevity Database; https://genomics.senescence.info/species/search_list.php; Human Ageing and Genomic Resources;

Magalhães and Costa, 2009)

, and PanTHERIA refers to the PanTHERIA data base (https://ecologicaldata.org/wiki/PanTHERIA; Ecological Archives, Ecological Society of America;

Acknowledgments

The authors are grateful to François Criscuolo for his help in the phylogenetic analyses. Two anonymous reviewers provided excellent suggestions for analysis and manuscript improvements. F.S.D. thanks the Institute of Advanced Studies of the University of Strasbourg for their financial support through an USIAS fellowship, and the Région Grand Est and the Eurométropole de Strasbourg I n r e v i e w

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

Rhinolophus ferrumequinum

Rhinolophidae 225 54 22.875 3 30.5

Tamias amoenus

Sciuridae 150 80 50.63 17 5.17 81 Y e s

Plecotus austriacus

Vespertilionidae 180 152 12 3 25.5 3 N o 4 Wild

Table S2: Data on hibernation season duration, body mass, longevity and arboreality lifestyle of all mammals studied in the different models.

Hibernation season duration with (*) corresponds to publications for which the methodology used to determine this duration could not be verified.

The "Wild or Captivity" parameter refers to the source of longevity data. The "arboreality" parameter distinguishes between species considered arboreal and semi-arboreal (Yes) and non-arboreal (No). Brown bear (Ursus arctos) and American black bear (Ursus americanus) have been defined as non-arboreal, as in Healy et al, 2014, as the majority of adults are losing this lifestyle 153 contrary to Asian black bear (Ursus thibetanus) 124 . Bats were considered to be non-arboreal even though it nests in trees as nesting in a protected area is a common characteristic of all bats and therefore already taken into account in the "bat (yes/no)" factor. Lambda (λ) correspond to the relative effect of the phylogenetic tree on the linear model, ranging between 0 (covariation among species measurements is independent of co-ancestry) and 1 (covariance entirely explained by co-ancestry). Location of the maximum likelihood estimate of λ is symbolised by a red line, upper 95% CI is symbolized by a dashed red line. Note that the lower 95% CI can not be estimated.

I n r e v i e w S3: Model selection for the effects of hibernation season duration (zhiber), body mass (zlogmass), the interaction zhiber x zlogmass, bats and arboreality lifestyle and the effect of captivity (C_W; captive vs. wild) on longevity in hibernating mammals.


David Genevieve

Delphine Héron

Vincent El Ghouzzi

Catherine Prost-Squarcioni

Martine Le Merrer

Aurélia Jacquette

Damien Sanlaville

Florence Pinton

Nathalie Villeneuve

Gabriel Kalifa

Exclusion of the dymeclin and PAPSS2 genes in a novel form of spondyloepimetaphyseal dysplasia and mental retardation

Keywords: Novel syndrome with SEMD and mental retardation Spondyloepimetaphyseal dysplasia, mental retardation, autosomal recessive

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

alpha kinase 3 and regulating the synthesis of unfolded proteins in the endoplasmic reticulum [START_REF] Zhang | The PERK eukaryotic initiation factor 2 alpha kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas[END_REF] , ii) Dyggve-Melchior-Clausen syndrome [START_REF] Dyggve | Morquio-Ullrich's disease: an inborn error of metabolism?[END_REF] (DMC, MIM 223800) due to mutations in the dymeclin gene encoding a protein of hitherto unknown function and iii) lysosomal storage disorders [START_REF] Maroteaux | Maladies osseuses de l'enfant[END_REF] . Here, we report on what we believe to be a novel autosomal recessive syndrome of SEMD and MR in two Pakistani sisters.

PATIENTS REPORT

Patient 1, a girl, was born to first-cousin Pakistani parents after term pregnancy and normal delivery (birthweight 3 Kg, height and OFC were unknown). Hypotonia, nystagmus with poor vision and feeding difficulties were noted in the neonatal period. She was able to control her head at 11 months and to sit unaided at 2 years but her gait was unstable. At 8 ½ years, she had no speech, ataxia and was unable to walk unaided (weight = 18.8 Kg; -2 SD ; height = 104 cm, -4.5 SD and OFC = 46.5 cm , -5 SD). No abnormal body proportion was observed. Facial dysmorphism included coarse facies, low anterior and posterior hairline, brachycephaly, simple and flat ears, small and well shaped eyebrows, synophrys, bilateral epicanthi, broad nose, full lips with eversion of the lower lip, short neck, and hirsutism of A back and legs (Figure 1A). At the last examination (11 years), she could walk unaided but with ataxia and her coarse facial appearance had worsened.

Patient 2, her younger sister, was born after an uneventful pregnancy (birthweight = 2500 g, length and OFC were unknown). She could sit at 2 years of age and walk unaided at 3 ½ years. She was first seen at 32 months of age for hypotonia with trunkal and gait ataxia (weight = 9.8 Kg, -2.5 SD; length = 83.5 cm, -2 SD; and OFC = 43 cm, -5 SD). She also had repetitive behaviour and no speech. Facial features included brachycephaly, low anterior and posterior hairline, bilateral epicanthus, simple and flat ears with everted lobules, broad nose and full lips with eversion of the lower lip. She also had abnormal pigmentary skin changes on the legs along the Blaschkolines. No abnormal body proportion was observed. At 5 years of age, her coarse facial appearance and failure to thrive had worsened (weight = 11 Kg, -3.5 SD; length = 90 cm, -4 SD; and OFC = 45 cm, -5 SD -Figure 1B).

METHODS

Molecular studies :

DNA was extracted from peripheral blood lymphocytes according to standard techniques [START_REF] Sambrook | Molecular cloning: a laboratory manual[END_REF] . Because of non informative haplotype analysis of the DMC locus, direct sequencing of the DMC gene, dymeclin, was performed using primers previously described 8 .

Intragenic polymorphic microsatellite marker was used to analysis the PAPSS2 (3-primealpha phosphoadenosine 5-prime-phosphosulphate synthase 2) gene, which accounts for the SEMD Pakistani type (PAPSS2 5' GGC AGT AAG TTT GCA GTA GA; PAPSS2 3' GTC TAC AAC CCA AGT CT).

Electron microscopy and ultrastructural analyses:

A 4mm punch skin biopsy including dermis was performed in both sisters. The sample was fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium-tetroxyde, dehydrated in a graded ethanol series and embedded in epon 812. Semi-thin sections (1µm) were stained with toluidine blue. Ultra-thin sections were selected, contrasted with uranyl acetate and lead citrate and examined with a Philips CM 10 transmission electron microscope.

RESULTS

Skeletal X-rays of patient 1 showed radial incurvation and spondyloepimetaphyseal dysplasia including flat vertebral bodies with irregular vertebral plates and posterior scalloping, irregular and flared femoral superior and inferior, radial and ulnar as well as fibulae and tibial superior metaphyses with vertical striations, small and irregular femoral C D 3-4 superior and inferior as well as tibial superior epiphyses, small carpal bones, flat acetabular roofs, short femoral neck, disproportionate long fibulae, and narrow iliac wings without lacy pelvis iliac crest (Figure 2A).

In patient 2, skeletal X-rays showed similar, but less severe, bone involvement including radial incurvation and spondyloepimetaphyseal dysplasia with flat vertebral bodies and posterior scalloping, irregular vertebral plates, irregular and flared femoral superior and inferior, radial and ulnar as well as fibulae and tibial superior metaphyses with vertical striations, small and irregular femoral superior and inferior as well as tibial superior epiphyses with small carpal bones, flat acetabular roofs, short femoral neck, disproportionate long fibulae, and narrow iliac wings without lacy pelvis iliac crest (Figure 2B).

Molecular studies allow us to exclude the Dymeclin gene by direct sequencing and the PAPSS2 gene by linkage analysis (Figure 3). Electron microscopy analysis of the skin biopsy of affected children revealed an abnormally large number of small vacuoles near the cytoplasmic membrane in fibroblasts, macrophages (Figure 4 A -C) and cells of the duct canal portion of the sweat glands (Figure 4 D -E). Moreover, large vacuoles containing either myelinic or granular osmiophilic bodies were observed in most cutaneous cell types studied, particularly in fibroblasts, macrophages, keratinocytes, melanocytes, secretory and duct canal portions of the sweat glands. Other features included dilated endoplasmic reticulum in fibroblasts while Golgi apparatus and mitochondria were normal. The radiological features were reminiscent of the SEMD Pakistani type (especially vertebral anomalies and epiphyseal dysplasia) [START_REF] Ahmad | Distinct, autosomal recessive form of spondyloepimetaphyseal dysplasia segregating in an inbred Pakistani kindred[END_REF] and the combination of these finding with the ethnic origin of the family and the likely autosomal recessive mode of inheritance prompted us to study and eventually exclude the PAPSS2 locus [START_REF] Ul Haque | Mutations in orthologous genes in human spondyloepimetaphyseal dysplasia and the brachymorphic mouse[END_REF] .

Metaphyseal striations is a rare radiological feature observed in several skeletal conditions but only a few of them are associated with mental retardations. Among them, SPONASTRIME dysplasia (MIM: 271510) is characterised by Spondylar (i.e. platyspondyly and irregular vertebral end-plates), nasal anomalies, striations of metaphyses and epiphyseal dysplasia. Mental retardation has been described in two families [START_REF] Camera | Sponastrime dysplasia: report on two siblings with metal retardation[END_REF][14] but the absence of vertebral body abnormalities has suggested that these patients have a different condition [START_REF] Langer Lo | Sponastrime dysplasia: five new cases and review of nine previously published cases[END_REF] .

Moreover, the combination of dysmorphic and radiological features, observed in SPONASTRIME dysplasia or related condition are clearly distinct from this new SEMD form reported here.

Despite negativity of extensive blood and urinary metabolic screening, the progressivity of organ involvement and electron microscopy studies of the skin (abnormally E 2 8 large number of small vacuoles in fibroblasts, macrophages, keratinocytes, melanocytes, secretory and duct canal portion of sweat glands) observed in our patients, may argue in favour of a novel hitherto unknown metabolic disorder.

We suggest therefore that the combination of features observed in this sibship corresponds to a novel form of autosomal recessive SEMD with MR, especially as severity of symptoms worsened in the course of the disease. F

LEGENDS OF FIGURES

1 1 . 1 -

 11 Exclusion of the Dymeclin, and PAPSS2 genes in a novel form of spondyloepimetaphyseal dysplasia and mental retardation. D. Geneviève 1 , D. Héron 2 , V. El Ghouzzi 1 , C. Prost-Squarcioni 3 , M. Le Merrer 1 , A. Jacquette 2 , D. Sanlaville 1 , F. Pinton 4 , N. Villeneuve 4 , G. Kalifa 5 , A. Munnich 1 , V. Cormier-Daire Département de Génétique et Unité INSERM U393, Hôpital Necker-Enfants Malades, Paris, France. 2-Département de Génétique, Hôpital de la Pitié-Salpêtrière, Paris, France 3-CNRS UPRES 3410, Faculté de Médecine, Hôpital Avicenne, Bobigny, France 4-Service de Neuropédiatrie, Hôpital Saint Vincent de Paul, Paris, France 5-Service de Radiologie, Hôpital Saint Vincent de Paul, Paris, France Correspondance to: Valérie Cormier-Daire Département de Génétique, Hôpital Necker-Enfants Malades, 149, rue de Sèvres 75743 Paris Cedex, France TEL: 00 33 1 44 49 51 63 FAX: 00 33 1 44 49 51 50 E-mail: cormier@necker.fr INTRODUCTION Spondyloepimetaphyseal dysplasias (SEMD) represent a heterogeneous group of conditions composed of at least fifteen well defined entities. These various forms of SEMD have been characterized based on clinical, radiological and molecular features but, only few of them are associated with MR 1 namely i) Wolcott-Rallison syndrome 2-3 (WR, MIM 226980) due to mutations in the EIF2AK3 gene encoding a translation initiation factor 2-

  LABORATORY INVESTIGATIONSHeart and kidney ultrasounds, brain MRI, fundus examination, evoked visual potential, electromyogram, standard and high resolution blood karyotype and FISH analyses of the telomeres were normal on the two sibs. Plasma and urinary aminoacids and organic acids, plasma T3, T4,TSH, protein glycosylation tests (western blot analysis) and blood and fibroblast lysosomal enzymes ( β-Glucosidase, β-Galactosidase, Hexosaminidase A and B, α-L-Iduronidase , α-Glucosaminidase, α-Galactose 6 sulfatase, Arylsulfatase A and B, α-and β-Mannosidase, α-Fucosidase, α-Neuraminidase and α-Nacetylgalactosaminidase) were unremarkable. Molecular analysis of the MECP2 gene was also normal. Electroretinogram of patient 1 detected an abnormal macular signal but normal peripheral retina.

  on SEMD, severe MR, microcephaly, facial dysmorphism and ataxia in two Pakistani sisters born to first-cousin parents. Severity of the clinical and radiological manifestations worsened in the course of the disease in the two sibs. The association of SEMD and MR prompted us to consider the diagnosis of DMC and Smith-McCort syndrome 8-10 but molecular genetic studies and distinct radiological features (ie lacy pelvis iliac crest and double vertebral humps) ruled out this diagnosis. Similarly, the absence of insulindependent diabetes mellitus and distinctive radiological features ruled out the diagnosis of WR syndrome as well.

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ACKNOWLEDGEMENTS

We thank Jean-Paul Monnet and Nicole Lièvre for their technical assistance for electron microscopy. Financial support from the association "Vaincre les maladies lysosomales" is gratefully acknowledged.


Yannicke Dauphin
email: yannicke.dauphin@u-psud.fr

Jean-Pierre Cuif

Hiram Castillo-Michel

Corinne Chevallard

Bastien Farre

Anders Meibom

Unusual Micrometric Calcite-Aragonite Interface in the Abalone Shell Haliotis ~Mollusca, Gastropoda!

Keywords: biominerals, mollusk shells, aragonite, calcite, FTIR, NanoSIMS

Species of Haliotis ~abalone! show high variety in structure and mineralogy of the shell. One of the European species ~Haliotis tuberculata! in particular has an unusual shell structure in which calcite and aragonite coexist at a microscale with small patches of aragonite embedded in larger calcitic zones. A detailed examination of the boundary between calcite and aragonite using analytical microscopies shows that the organic contents of calcite and aragonite differ. Moreover, changes in the chemical composition of the two minerals seem to be gradual and define a micrometric zone of transition between the two main layers. A similar transition zone has been observed between the layers in more classical and regularly structured mollusk shells. The imbrication of microscopic patches of aragonite within a calcitic zone suggests the occurrence of very fast physiological changes in these taxa.

INTRODUCTION

Mollusks are organisms characterized by important internal interfaces at a variety of levels ~Boggild, 1930; Taylor et al., 1969!. The first interface level is defined at the macroscale where mantle cells are in contact with the carbonate shell they form ~Fig. 1a!. A second mesoscale interface exists within the shells, which are composed of several layers of variable structure and/or mineralogy ~Fig. 1b!. At the microscale, an organo-mineral interface level exists within each layer where crystalline units are surrounded by organic envelopes ~Fig. 2c, arrow!. Within an individual crystalline unit, another organo-mineral interface exists because rounded nanogranules are surrounded by a cortex probably composed of a mixture of amorphous calcium carbonate and organics ~Fig. 1d, star!. Nanogranules are themselves a mixture of organic and minerals, as shown by color variations in atomic force phase image ~Fig. 1c!. And finally, a molecular-level interface has been shown: the organic matrices extracted from the crystalline units are a mixture of proteins, sugars ~Figs. 1e, 1f!, and lipids ~Farre et al., 2011!.

The shells produced by mollusks are made of calcium carbonate in the form of aragonite and/or calcite. In most shells, calcite and aragonite are arranged in regular layers, the limits of which are parallel to the outer surface of the shell. Because of their spiral shell morphology, gastropods are not the most studied shells. However, some species have shells with a reduced coiling in which only the apex has a spiral shape. Among them is Haliotis ~abalone!, a popular seafood, with a shell up to 25 cm long, living in shallow water underneath weed-covered rocks and boulders to depths of about 40 m in oceans worldwide. As an aside, this slow moving gastropod is easy to catch so that overfishing, based exclusively on the extraction of wild stocks, has threatened the species in several countries ~Japan, Australia, California, South Africa, France, etc.!. An official ban on ormer fishing has been imposed in the countries mainly concerned because of the severe drop in the yields ~Clavier, 1982!.

Haliotis comprises more than 100 species, each with a specific microstructural arrangement. The shell is usually bilayered with an inner aragonitic nacreous layer and an outer prismatic layer. Depending of the species, the outer layer is either calcite or aragonite. This unusual variability was noticed as early as 1930 ~Boggild, 1930!: they "possess, in some instances, a calcitic layer inserted between two aragonitic ones. . . The calcite has a rather regularly prismatic structure." Kessel ~1935!, using specific stains, noted that in Haliotis tuberculata, aragonite and calcite are not separated into distinct layers and that numerous aragonitic granules are imbricated in calcitic prisms. The imbricated structure of H. tuberculata as well as Haliotis lamellosa was confirmed by scanning electron microscope ~SEM! observations [START_REF] Mutvei | Observations sur l'organisation de la couche externe du test des Haliotis ~Gastropoda!: Un cas exceptionnel de variabilité minéralogique et microstructurale[END_REF][START_REF] Dauphin | Mineralogy, chemistry and ultrastructure of the external shell layer in ten species of Haliotis, with reference to H. tuberculata ~Mollusca, Archaeogastropoda![END_REF] the inner nacreous layer is aragonitic, whereas the middle layer is mainly prismatic calcite, the outer layer being prismatic aragonite. A similar arrangement also exists in Haliotis discus and Haliotis roei ~Fig. 2!. Thus, the two prismatic layers have different mineralogies, while the two aragonitic layers have distinct microstructures. Only in situ localized analyses are suitable to improve our knowledge of the structure and composition of individual layers, and to understand the biomineralization mechanisms of such com-plex shells. Electron microprobe analyses have shown that the magnesium ~Mg! contents of the calcitic layers are low but differ ~from 800 ppm in Haliotis kamtschakana to 3,800 ppm in Haliotis rotundata!. Analyses of ten species have also shown that strontium ~Sr! contents of the aragonitic prismatic and nacreous layers are also low and differ-ent in a given species ~Dauphin et al., 1989!, but both calcite and aragonite compositions in these two species fall within the broad range of compositions defined by mollusk shells in general.

In complex shells such as H. tuberculata or H. roei, bulk separation of the distinct shell components is not possible, making a reliable investigation of their respective chemical and biochemical compounds very difficult by conventional methods. In situ characterizations of biochemical compounds have proven efficient methods in previous studies. Synchrotron-based XANES mapping, for instance, has demonstrated that sulfur is mainly associated with sulfated sugars, and that calcite layers have higher contents ~Dauphin et al., 2005!. The focus of the current investigation is to determine the spatial and chemical relationship between aragonite and calcite at a microscale in the outer layer of Haliotis.

MATERIALS AND METHODS

Material

Haliotis ~Gastropoda, Orthogastropoda, Pleurotomaroidea, Haliotidae! has more than 100 species and a worldwide distribution.

H. tuberculata Linnaeus, 1758 were obtained from the Station Biologique de Dinard ~France! in the English Channel. H. tuberculata is present in the Channel Isles but absent from the coasts of Britain and Ireland. H. lamellosa Lamarck, 1822 is usually considered as a subspecies of H. tuberculata. Shells were collected in the Mediterranean Sea.

H. tuberculata has a flattened, oval shell, up to 9 cm long and 6.5 cm large, slightly coiled at the apex ~Fig. 3!. A row of round respiratory holes runs along the bottom margin of the shell. The outer, larger ~from five to seven! holes are open while the older are closed. The outer surface of the shell is greenish-brown to red ~Figs. 3a, 3c!. The inner surface is lined with an iridescent nacreous layer ~ϭ motherof-pearl! ~Figs. 3b, 3d!.

Optical Microscopy

Thin sections were observed under crossed Nicols in transmitted light. The epifluorescence signal of polished surfaces was observed under ultraviolet ~UV! light using a Zeiss Standard microscope ~Carl Zeiss, Jena, Germany! equipped with Neofluor fluorine objectives, a Zeiss mercury lamp, excitation filter ~365 nm!, and transmission cut-off filter ~400 nm!.

A second set of observations was conducted at the LIONS laboratory ~CEA Saclay, DSM/IRAMIS/SIS2M! on an Olympus FV1000 confocal microscope ~Olympus Corporation, Tokyo, Japan!. All the samples examined had been previously sectioned and polished to a smooth surface. A 60ϫ oil-immersion objective was used in order to achieve the best achievable spatial resolution ~;200 nm horizontal resolution!. Two different excitation wavelengths were used, namely the 488 nm laser line of an Argon laser and the 633 nm line of a He-Ne laser, and the emitted fluorescence signal was collected in the range 515-545 nm and above 650 nm, respectively.

SEM

A Philips 505 SEM, a Philips XL30, and a Phenom Pro X ~Université Paris Sud! were used to obtain images of the features within the etched and polished samples. Various etching and/or fixative procedures were used to reveal the details of the microstructures of the prismatic layers. Bacteria were used to remove the organic matrices, formic and acetic acids were used to etch the carbonate. Actually, bacterial etchings partially dissolved the minerals, whereas acids partially etched the organic matrices. So some fixatives such as glutaraldehyde, or fixative-etching agent such as chromium sulfate were used to fix the organic matrix. Details of the etching procedures are given in the figure captions.

Fourier Transform Infrared Microscopy (FTIR)

The synchrotron infrared microscopy was performed on the FTIR end station of the ID21 beam line at the European Synchrotron Radiation Facility ~ESRF!. This beam line is equipped with a Continuum IR microscope ~Thermo Nicolet, Thermo Fisher Scientific, Inc., Waltham, MA, USA! coupled to a Nexus FTIR bench ~Thermo Nicolet!. The microscope can be operated in confocal or reflection mode, where the focusing Schwarzschild objective and the collection Schwarzschild objective have a magnification of 32ϫ ~NA ϭ 0.65!. Due to its reduced source size and high collimation properties, a synchrotron infrared source is more efficiently coupled to the low acceptance of the confocal microscope, while its higher spectral luminescence ~brightness! allows signal-to-noise ratios to be kept at diffraction-limited resolutions. The microscope is equipped with a computer-controlled x/y stage allowing acquisition of profiles or maps of the sample. Maps were scanned with steps of 4 mm in both directions, with an aperture size of 5 ϫ 5 mm 2 . Spectra were collected in reflection mode, with a resolution of 8 cm Ϫ1 . For each spectrum, 100 scans were accumulated in the wavenumber range 4,000-700 cm Ϫ1 . Polished and cleaned surfaces were prepared as for the first step of SEM observations, without etching. The final format of the data were recorded as absorbance values.

NanoSIMS

Samples were cut perpendicular to the interface between the nacre and the prismatic layer, mounted in epoxy ~Körapox! and polished to a 0.25 mm finish using diamond paste. The samples were then gold coated. Chemical mapping was carried out with the Cameca NanoSIMS N50 at the Muséum National d'Histoire Naturelle in Paris, following established procedures for biogenic carbonates ~Meibom et al., 2004, 2008;[START_REF] Brahmi | Skeletal growth, ultrastructure and composition of the azooxanthellate scleractinian coral Balanophyllia regia[END_REF][START_REF] Kopp | Calcareous sponge biomineralization: Ultrastructural and compositional heterogeneity of spicules in Leuconia johnstoni Carter 1871[END_REF] Briefly, using a focused primary beam of O Ϫ to a spot size of about 250 nm, secondary ions of 24 Mg ϩ , 44 Ca ϩ , and 88 Sr ϩ were sputtered from the sample surface and detected simultaneously ~in multicollection mode! using electron multipliers. The mass-resolving power was ;4,000 ~M/ DM !, sufficient to resolve all isobaric interferences. Chemical maps were obtained by rastering the primary beam across a selected and presputtered sample surface.

RESULTS

Structure

Three structural types build the shell of H. tuberculata. These structures are not regular layers with boundaries parallel to the inner and/or outer surface of the shell. UV optical images show the different auto-fluorescence of the three structures ~Figs. 4a!. Despite the fact that nacre and the outer layers are aragonite, intensities and colors differ. Confocal laser images also show the distinct response of the structures ~Figs. 4b-4e!. Growth layers are clearly visible in the outer aragonitic prisms, whereas the nacreous layer seems to be homogeneous ~Fig. 4b!. A green excitation wavelength emphasizes the shape of the calcitic prisms, the irregular distribution of the aragonitic prisms ~Figs. 4c, 4d!, and the organic envelopes of the calcitic prisms ~Fig. 4d!. Larger magnifications show the granular structure and growth layers in the aragonitic layer ~Fig. 3e!. The small darker zone may correspond to calcitic prisms embedded in the aragonitic zone ~Fig. 4e!. Transmitted crossed Nicols images of thin sections show the large size of the calcitic prisms and the small aragonitic units ~Figs. 4f, 4g!. A larger magnification shows that calcite and aragonite are produced simultaneously at a microscale ~Fig. 4g!.

SEM confirms the irregular distribution of the calcite and aragonite in the outer part of the shell ~Figs. 5a-5c!. The outermost part of the shell is an irregular arrangement of small aragonitic prisms or spherulites, containing patches of calcitic prisms ~Figs. 5a-5g!. The individual spatial distribution of calcite and aragonite, as well as the size of the calcitic patches, vary substantially. However, the most external part of the shell is always aragonite. The calcite is between prismatic-spherulitic aragonite and the inner aragonitic nacre, but has a very irregular thickness and presence ~Figs. 5a-5c!. The structural differences between aragonite and calcite of the external zone of the shell observed in fractured surfaces and polished sections show that small aragonitic spherulites are also dispersed in large calcite crystals ~Figs. 5d, 5e!. Etched and fixed sections or fractures show the organic framework of the aragonitic spherulites ~Fig. 5f! and of the calcitic prisms. The inner structures of the aragonitic outer layer, the calcitic prismatic layer, and the nacreous layer are shown, respectively, in Figures 5g-5i.

Bulk Composition

FTIR maps have been acquired on a vertical section where aragonite and calcite are not in regular layers ~Fig. 6!. Spectra of the calcite and aragonite groups are characterized by three major bands attributed to the carbonate ion CO 2-3 : n3 at 1,429 cm Ϫ1 , the n2 doublet 877-848 cm Ϫ1 , and n4 at 713 cm Ϫ1 for the calcite group; n3 at 1,471 cm Ϫ1 and two doublets: n2 at 858-844 cm Ϫ1 and n4 at 713-700 cm Ϫ1 for the aragonite group ~White, 1974!. Map at 875 cm Ϫ1 displays the aragonitic and calcitic zones of the shell ~Fig. 7!. The characteristic n2 absorption bands of calcite and aragonite can be observed in the infrared spectra extracted from a region where the map at 875 cm Ϫ1 shows a transition from calcite to aragonite in the shell structure ~Fig. 7!. In such organo-mineral assemblages, a precise assignment of FTIR bands is not possible because of the overlapping of bands. The 883 cm Ϫ1 band corresponds to various organic components with C-H and CH 2 bonds. Despite this vague assignment, the 883 cm Ϫ1 map clearly shows the difference in aragonite and calcite, whatever the structure ~Fig. 7!. Sugar bands are between 950 and 1,150 cm Ϫ1 , so that it can be suggested that the map at 1,148 cm Ϫ1 is related to these components. Again, aragonite and calcite are different, but nacre and outer aragonite are not identical. The calcitic patches have a high content, whereas the nacre has a low content. This "sugar" map is very similar to that at 1,228 cm Ϫ1 , a wavelength related to SO 42 -or amide III. Amide II ~1,156 cm Ϫ1 ! and amide I ~1,607, 1,649 cm Ϫ1 ! maps also show the differences in the organic components of the structures ~Fig. 7!. Amide I and the 1,720 cm Ϫ1 band ~COOH! maps are similar. The 1,720 cm Ϫ1 band is strong in aspartic acid, an amino acid usually in high concentrations in biogenic carbonates.

Chemical Maps

Previous electron microprobe quantitative analyses and distribution maps have shown that despite seemingly higher Sr contents in aragonitic layers ~nacre and prisms!, the differences are not significant [START_REF] Dauphin | Mineralogy, chemistry and ultrastructure of the external shell layer in ten species of Haliotis, with reference to H. tuberculata ~Mollusca, Archaeogastropoda![END_REF][START_REF] Dauphin | Microstructures and compositions of multilayered shells of Haliotis ~Mollusca, Gastropoda![END_REF]!. The Mg content is more indicative of the mineralogy, especially in H. tuberculata ~Fig. 8!. As shown in Figure 8, the nacreous layer of H. tuberculata has the lowest Sr content, and the highest Mg content is found in the calcitic layer. Thus, Mg/Ca and Sr/Ca ratios in this species have been selected for chemical maps.

Using the NanoSIMS, more detailed distribution maps have been done in the outer layers, where aragonite and calcite were mixed. They clearly show the negative correlation between Mg and Sr in three mapped zones of H. tuberculata ~Fig. 9!. Mg/Ca maps are less distinct than those of Sr/Ca. Because of the irregular structures and thickness of the calcitic and aragonitic zones, it is difficult to know the actual origin of this phenomenon. However, Sr/Ca maps in the aragonite are not uniform ~Fig. 9e!. Detailed maps show aragonitic spherolites of about 1 mm in diameter ~Figs. 9c, 9d!.

DISCUSSION

Electron microprobe analyses have previously shown that the shells of some Haliotis species comprise aragonite and calcite that are irregularly dispersed and mixed ~Dauphin phin et al., 2008, 2012;[START_REF] Farre | Shell layers of the black-lip pearl oyster Pinctada margaritifera: Matching microstructure and composition[END_REF][START_REF] Nouet | Biochemical change at the setting-up of the crossedlamellar layer in Nerita undata ~Mollusca, Gastropoda![END_REF] In such shells, the main layers are easy to separate for various analyses, whereas the structure and composition of the transition layer is only known using in situ analyses. The efficiency of such techniques allows us to improve our knowledge of the complex and irregular shell of abalone.

The comparison of elemental chemical composition, fluorescence images, FTIR, and NanoSIMS maps shows the good correlation between structure mineralogy and biochemical composition at a microscale. The difference between the two types of aragonitic structures ~nacre-prisms! is also confirmed.

In shells with more typical structure ~two main layers!, growth lines show that the mantle is able to produce simultaneously two different mineralizing organic matrices, with a transitional stage. In the most studied model ~nacreprism!, the macromolecular assemblage for the prisms is first formed, then the nacreous macromolecular assemblage is done. There is no reverse production, from nacre to prism in these shells. In the prismatic layer, the presence of calcite and aragonite is not rare but the ratio calcite/aragonite is highly variable according to the species ~Dauphin et al., 1989!. Calcitic prisms are always larger than aragonitic units. Some proteins have been localized in the mantle cells. According to these data, "cell clusters in the front edge of the mantle could be able to synthesize both aragonite and calcite matrix proteins" ~Jolly et al., 2004!. However, the growth-regulating mechanisms for such complex arrangements are not yet deciphered. Changes in mineralogy, crystallinity, and structure are normal in mollusks, but they usually follow a well-defined sequence. Nevertheless, the distribution of proteins probably involved in the biomineralization processes is both time and location dependent in H. tuberculata [START_REF] Jardillier | A morphological and structural study of the larval shell from the abalone Haliotis tuberculata[END_REF][START_REF] Auzoux-Bordenave | Ultrastructure, chemistry and mineralogy of the growing shell of the European abalone Haliotis tuberculata[END_REF] Mixed structures and mineralogies have been obtained in experiments involving mollusk shell repair and regeneration [START_REF] Muzii | Calcite deposition during shell repair by the aragonitic gastropod Murex fulvescens[END_REF][START_REF] Saleuddin | Shell regeneration in Helix pomatia[END_REF][START_REF] Meenakshi | An ultrastructural study of shell regeneration in Mytilus edulis ~Mollusca: Bivalvia![END_REF][START_REF] Meenakshi | Studies on shell regeneration-I. Matrix and mineral composition of the normal and regenerated shell of Pomacea paludosa[END_REF][START_REF] Chan | Acid phosphatase in the mantle of shell-regenerating snail Helisoma duryi duryi[END_REF][START_REF] Uozumi | Organic membrane-shell" and initial calcification in regeneration[END_REF][START_REF] Reed-Miller | Scanning electron microscopy of the regenerated shell of the marine archaeogastropod[END_REF]!. In most cases, the structure and mineralogy of the repaired shells do not correspond to those of the normal shell. The first stages do not involve mineral deposits. Moreover, the organic contents of the regenerating membrane are unusual, and cellular activities are higher. Meenakshi et al. ~1974! observed that the repaired shell of a given species is dependent on the substrata inserted in the damaged zone. While structure and mineralogy are mixed throughout the life in the normal shell of H. tuberculata, shell repair is a sequential process.

Complex and irregular structures have been also described in cultured pearls ~Cuif et al., 2011!. As for regeneration experiments, irregularities are said to be the results of the stress induced by the grafting process: the small piece of mantle cut in another oyster, incision of the gonad to insert the nucleus and the mantle tissue. Moreover, in cultured pearls, the final structure is, in the end and in most cases, the nacreous layer.

CONCLUSION

The shell of H. tuberculata is irregularly layered: the inner layer is the aragonitic nacre, but the outer part of the shell is a mixture of prismatic calcite and spherulitic-prismatic aragonite. Such microscale mixings are unique in mollusk shells. Previous observations have shown that the structure, mineralogy, and elemental composition of these three phases differ. In such samples devoid of regular layers, only in situ analyses are available because of the complex arrangement and small sizes of the structural units. Additional analyses using confocal microscopy and infrared spectrometry maps at a microscale show that the organic components of these three phases differ. These results are concordant with what is known from other mollusks, the shell layers of which are easy to separate. Moreover, NanoSIMS chemical maps show that the chemical contents of the aragonite patches embedded in the calcite are not homogeneous. Again, similar results have been observed between two layers with different mineralogy in regularly layered mollusk shells. Thus, whatever the scale of observation, changes in the mineralizing process of the mantle cells are always progressive. The change in the secretion process from calcite to aragonite layers was estimated at ;6 days ~Dauphin et al., 2008!. In Haliotis, the physiological changes in the mantle cells must be faster, the size of the aragonitic patches being small ~sometimes 1 mm!. Throughout the lifespan of Haliotis, it seems that the same cells are able to synthesize and to very quickly switch from matrix components inducing calcite and aragonite.

Figure 1 .

 1 Figure 1. Multiscale interfaces in mollusk shells. a: Inner view of the pallial cavity of Pinctada showing the macroscale interface between the mantle cells and the shell. b: Vertical section of a shell of Pinctada showing the interface between two different structural and mineralogical layers. c: Section of a nacreous layer of Haliotis showing the microscale interface between the tablets and their organic envelopes ~arrow!. d: Nanoscale interfaces between the rounded granules with a cortex ~star!, and within the organo-mineral granules; Neotriginia shell.

Figure 2 .

 2 Figure 2. Vertical shell sections of four Haliotis species, showing the irregular layering of the calcite ~black! and aragonite ~white! "prismatic" layer in the outer part of the shell. In all species, the inner layer is the aragonitic nacre. Redrawn from Mutvei et al. ~1985!.

Figure 3 .

 3 Figure 3. Juvenile ~a, b! and adult ~c, d! shells of Haliotis tuberculata. The outer surfaces of these cultivated samples are unusually clean, devoid of encrusting epibionts. a, c: Outer surfaces. b, d: Inner surface showing the nacreous layer.

Figure 4

 4 Figure 4. a: Fluorescence image ~UV excitation! showing the differential response of the three structures. b: Laser confocal image showing differences in the three structures of the shell of Haliotis tuberculata, and the banding pattern of the prismatic-spherulitic aragonite; laser wavelength 633 nm. c: Laser confocal image: detail showing the calcitic prisms and the "granular" aspect of the aragonitic outer layer; laser wavelength 488 nm. d: Laser confocal image showing the organic envelope surrounding the calcitic prisms and the growth layer in the external aragonite; laser wavelength 488 nm. e: Detail showing a small patch of calcite in the aragonitic layer; laser wavelength 488 nm. f: Polarized crossed Nicols image showing the small aragonitic units and the large calcitic prisms. g: Polarized cross Nicols image showing the various size of the small aragonitic units within a calcitic crystal. PC, prismatic calcite; PA, prismatic-spherulitic aragonite; N, nacreous layer.

Figure 5 .

 5 Figure 5. Microstructures of Haliotis tuberculata. a: Vertical fracture showing the inner calcitic patches ~PC! and the outer aragonitic prismatic-spherulitic layer ~PA!. b, c: Polished, fixed, and etched surfaces showing the irregular arrangement of calcite and aragonite ~PA! in the outer part of the shell; N: inner nacreous layer chromium sulfate for 90 s. d, e: Surface showing the microscale interplay between calcite and aragonite-polished, fixed, and etched surface, formic acid 0.1% ϩ glutaraldehyde 3% for 25 s. Back scattered electron image. f: Fracture in the outer aragonitic layer showing the organic matrices as filaments. g: Fracture showing the small aragonitic units. h: Polished and etched surface showing the inner structure of the calcitic prisms fixed with glutaraldehyde for 30 min at 508C, critical point drying. i: Columnar nacreous layer. PC, prismatic calcite; PA, prismatic-spherulitic aragonite; N, nacreous layer.

Figure 6

 6 Figure 6. a: Polished surface showing the thick aragonitic nacreous layer, and the position of Fourier transform infrared microscopy maps ~black rectangle! shown in Figure 7; the white line ~b! corresponds to the spectra shown in ~b! from calcite to aragonite. b: Spectra: IR absorption spectra showing the transition from aragonite to calcite extracted from the region labeled in the top optical image.

Figure 7 .

 7 Figure 7. Top: Image of the mapped zone of the shell. Color images: Fourier transform infrared microscopy maps showing the compositional difference in mineralogy ~875 cm Ϫ1 !. Other maps show the differences in organic matrices between calcite and aragonite, and also between the aragonitic prismatic-spherulitic layer and the nacre.

Figure 8 .

 8 Figure 8. Elemental chemical composition ~Mg, Sr! of the three structures in three species of Haliotis. Despite the common mineralogy ~aragonite!, nacre, and spherulitic-prismatic layers are not identical; 883 cm Ϫ1 band is assigned to C-H and CH 2 bonds, 1,148 cm Ϫ1 to sugars, 1,228 cm Ϫ1 to SO 42-or amide III, 1,156 cm Ϫ1 to amide II, 1,607 cm Ϫ1 and 1,649 cm Ϫ1 to amide I, and 1,720 cm Ϫ1 to aspartic acid.

Figure 9 .

 9 Figure 9. NanoSIMS images showing the microscale interplay between the prismatic calcite ~high Mg/Ca! and the spherulitic aragonite ~high Sr/Ca! in three different zones of the outer layers. PA, prismatic aragonite; PC, prismatic calcite.

ACKNOWLEDGMENTS

This work was supported by ESRF Grant ~EC 792! and by European Research Council Advanced Grant 246749 BIOCARB.