Tony Chevalier
email: tony.chevalier@cerptautavel.com

Thomas Colard

Antony Colombo

Liubov Golovanova

Vladimir Doronichev

Jean-Jacques Hublin

Early ontogeny of humeral trabecular bone in Neandertals and recent modern humans

Keywords: Humerus, Development, Microstructure, Gracilization, Physical activity 1

Trabecular bone ontogeny is well known in modern humans and unknown in Neandertals. Yet bone developmental pattern is useful for interpreting fossils from evolutionary and functional perspectives. Interestingly, microstructure in early ontogeny is supposedly not influenced by high and specific mechanical loading related to the lifestyle of a human group and consequently does not directly depend on the activities of hunter-gatherers. Here, we specifically explored the early growth trajectories of the trabecular bone structure of the humerus, and emphasized in particular how bone fraction (BV/TV) was built up in Neandertals, given specific modern human bone loss after birth and the use of BV/TV in functional studies. Six Neandertals and 26 recent modern humans ranging from perinates to adolescents were included in this study. Six trabecular parameters were measured within a cubic region of interest extracted from the proximal metaphysis of the humerus. We found that the microstructural changes in Neandertals during early ontogeny (<1 year) fit with modern human growth trajectories for each parameter. The specific bone loss occurring immediately after birth in modern humans also occurred in Neandertals (but not in chimpanzee). However, the early childhood fossil Ferrassie 6 presented unexpectedly high BV/TV, while high BV/TV in the Crouzade I adolescent was predictable. These results suggest that Neandertals and modern humans shared predetermined early growth trajectories and developmental mechanisms. We assume that the close relationship between skeletal characteristics in early ontogeny and adults in modern humans also existed in Neandertals.

However, it was difficult to ensure that the high BV/TV in Neandertal early childhood, represented by only one individual, was at the origin of the high BV/TV observed in adults.

2 Consequently, our study does did not challenge the mechanical hypothesis that explains the trabecular gracilization of the humerus during the Holocene.

Introduction

Variation in bone structure is related to variation in lifestyle during human evolution [START_REF] Ruff | Postcranial robusticity in Homo. I: Temporal trends and mechanical interpretation[END_REF][START_REF] Ruff | Gradual decline in mobility with the adoption of food production in Europe[END_REF][START_REF] Ruff | Mechanical determinants of bone form: Insights from skeletal remains[END_REF][START_REF] Carlson | Reconstructing mobility: Environmental, behavioral, and morphological determinants[END_REF][START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF][START_REF] Chirchir | Low trabecular bone density in recent sedentary modern humans[END_REF][START_REF] Ryan | Gracility of the modern Homo sapiens skeleton is the result of decreased biomechanical loading[END_REF][START_REF] Scherf | Patterns of activity adaptation in humeral trabecular bone in Neolithic humans and present-day people[END_REF][START_REF] Sládek | The impact of subsistence changes on humeral bilateral asymmetry in terminal Pleistocene and Holocene Europe[END_REF][START_REF] Holt | Temporal and geographic variation in robusticity[END_REF][START_REF] Chirchir | Trabecular bone fraction variation in modern humans, fossil hominin and other primates[END_REF]. However, the validity and accuracy of the mechanical interpretation of bone structure in past populations is still challenged (Pearson and Liebermann, 2004). While the concept of 'bone functional adaptation' is strongly empirically supported (as reviewed in [START_REF] Ruff | Who's afraid of the big bad Wolff?: "Wolff's law" and bone functional adaptation[END_REF][START_REF] Kivell | A review of trabecular bone functional adaptation: What have we learned from trabecular analyses in extant hominoids and what can we apply to fossils?[END_REF], several issues highlight the limitations of behavioral explanations inferred from the shape, robusticity and microarchitecture of bone (e.g., [START_REF] Lieberman | How and why humans grow thin skulls: Experimental evidence for systemic cortical robusticity[END_REF][START_REF] Schmitt | The relationship between bone mechanical properties and ground reaction forces in normal and hypermuscular mice[END_REF][START_REF] Wallace | Genetic variations and physical activity as determinants of limb bone morphology: An experimental approach using a mouse model[END_REF][START_REF] Wallace | Exercise-induced bone formation is poorly linked to local strain magnitude in the sheep tibia[END_REF]Wallace et al., , 2017a, b;, b;Tsegai et al., 2018a). Furthermore, the impact of mechanical loading can be variably marked as the bone's responsivity to loading stimuli depends on individual-level mechanosensitivity (and species/population-level mechanosensitivity) as well as the stage of ontogeny [START_REF] Robling | Mechanotransduction in bone: Genetic effects on mechanosensitivity in mice[END_REF]Wallace et al., 2017b).

Bone development has a genetic basis [START_REF] Lovejoy | Developmental biology and human evolution[END_REF][START_REF] Wallace | Genetic variations and physical activity as determinants of limb bone morphology: An experimental approach using a mouse model[END_REF], which can generate microstructural signatures related to bone and function before the functional loading that occurs during life [START_REF] Abel | Ontogenetic changes in the internal and external morphology of the ilium in modern humans[END_REF]. The characterization of bone microstructure in the early phases of ontogeny in hominins is one way of assessing the relevance of functional hypotheses based on mature bones to determine the lifestyles of extinct humans. It is essential to determine whether differences present in adulthood already exist during early development. For example, if similar early growth patterns can be demonstrated in Neandertals and modern humans before the initiation of culture-specific physical activities, this would potentially facilitate linking differences in bone structure in adults to such behavioral shifts.

Purportedly, mechanical loadings (patterns and magnitudes) in early ontogeny would not be specific to the lifestyle of a human group, and could be roughly similar in very young individuals in distinct cultural groups, i.e., potential minor differences between human groups in terms of physical behavior did not lead to significant changes in bone structure. In contrast, bone structure in adolescence could reflect magnitude and/or pattern of mechanical loading due to cultural differences in physical behavior. The early bone developmental period is characterized by a relatively high genetic influence and low mechanical loading [START_REF] De Vries | The emergence of fetal behavior. I. Qualitative aspects[END_REF][START_REF] Carter | Skeletal Function and Form: Mechanobiology of Skeletal Development, Aging, and Regeneration[END_REF][START_REF] Lovejoy | Developmental biology and human evolution[END_REF] as reviewed in [START_REF] Land | Fetal and postnatal bone development: Reviewing the role of mechanical stimuli and nutrition[END_REF]. Some trabecular microstructures related to functional bone adaptation are genetically predetermined and therefore develop before the impact of any significant mechanical loading (Cunningham and Black, 2009a, b;[START_REF] Abel | Ontogenetic changes in the internal and external morphology of the ilium in modern humans[END_REF]. Bone strength and structural patterns of both cortical and trabecular bone in immature individuals can also predict or influence adult bone structure [START_REF] Wescott | Population variation in femur subtrochanteric shape[END_REF][START_REF] Wescott | Ontogeny of femur subtrochanteric shape in Native Americans and American blacks and whites[END_REF][START_REF] Cowgill | The ontogeny of Holocene and late Pleistocene human postcranial strength[END_REF][START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF][START_REF] Ireland | Motor competence in early childhood is positively associated with bone strength in late adolescence[END_REF][START_REF] Ireland | Later age at onset of independent walking is associated with lower bone strength at fractureprone sites in older men[END_REF]. For example, functional stimulation is recommended for premature infants and neonates to improve bone development, and impacts bone structure in adulthood (Land and Schoneau, 2008). Later age of onset of independent walking in young children can affect bone strength throughout life [START_REF] Ireland | Later age at onset of independent walking is associated with lower bone strength at fractureprone sites in older men[END_REF]. Consequently, structural dissimilarities early in ontogeny that are maintained between adult Neandertals and modern humans could be accounted for by distinct genetic/hormonal controls and diet, and not, or not only, by distinctive mechanical loading behaviors that occurred during adolescence and/or adulthood. Distinct Neandertal and modern human physical activities during the first two years after birth could also have an impact on adult bone structure. However, there is currently no evidence to suggest immature Neandertals and modern humans differed in their behavior.

Trabecular bone patterns during development are well known in modern humans [START_REF] Ryan | Trabecular bone ontogeny in the human proximal femur[END_REF][START_REF] Reissis | Development of fetal trabecular microarchitecture in the humerus and femur[END_REF]Zeininger, 2013;[START_REF] Colombo | La micro-architecture trabéculaire de l'os en croissance: Variabilité tridimensionnelle normale et pathologique analysée par microtomodensitométrie[END_REF][START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF][START_REF] Milovanovic | Region-dependent patterns of trabecular bone growth in the human proximal femur: A study of 3D bone microarchitecture from early postnatal to late childhood period[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF][START_REF] Saers | Ontogeny and functional adaptation of trabecular bone in the human foot[END_REF][START_REF] Colombo | Trabecular analysis of the distal radial metaphysis during the acquisition of crawling and bipedal walking: a preliminary study[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF], and chimpanzees (Tsegai et al., 2018b). By contrast, nothing is known of Neandertal trabecular bone patterns during ontogeny. Humeral development in recent modern humans shows remarkable bone loss at a very early stage (<1 year) in comparison with a high bone volume/total volume (BV/TV) in newborns [START_REF] Colombo | La micro-architecture trabéculaire de l'os en croissance: Variabilité tridimensionnelle normale et pathologique analysée par microtomodensitométrie[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]. This growth trajectory appears to be unique to humans in upper and lower limb bones and vertebrae [START_REF] Colombo | La micro-architecture trabéculaire de l'os en croissance: Variabilité tridimensionnelle normale et pathologique analysée par microtomodensitométrie[END_REF][START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF], and in distinct regions within a bone [START_REF] Milovanovic | Region-dependent patterns of trabecular bone growth in the human proximal femur: A study of 3D bone microarchitecture from early postnatal to late childhood period[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF], and thus has not been reported for other mammals [START_REF] Tanck | Increase in bone volume fraction precedes architectural adaptation in growing bone[END_REF]Tsegai et al., 2018b). After one year of age in humans, BV/TV reportedly increases as a consequence of increase in trabecular thickness (Tb.Th; [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]; this change is not due to an increase in trabecular number as this has been shown to decrease (as reflected by a decrease in connectivity density, Conn.D; [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]. In this respect, the modern human BV/TV growth trajectory takes the form of a U-Curve before ~4 years of age [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]. From six months to 2-2.5 years old, Conn.D clearly decreases and Tb.Th increases slightly. These two parameters do not present the U-Curve observed in BV/TV, but changes in Conn.D and Tb.Th are related to an inverse U-Curve in trabecular spacing (Tb.Sp; [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]but see Saers et al., 2019).

Interestingly, in modern humans, the degree of anisotropy (DA) and Tb.Sp show similar values and trends for humeri and femora [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF] from two years of age onwards, despite the involvement of the latter in bipedalism; this is in contrast with what has been reported for BV/TV, Tb.Th and primary Young's modulus [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]. In humans, the primary Young's modulus shows some similarities with the growth trajectory for humeral BV/TV, which is confirmed by their significant relationship [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF], although the Spearman's correlation is low (r = 0.403). Thus, BV/TV can be a rough indicator of trabecular bone stiffness. Collectively, these results support the assumption that complex patterns of trabecular bone growth impact bone mass, organization and stiffness. Remarkable changes in some trabecular bone parameters are observed during early modern human ontogeny.

However, some of the trabecular bone parameters exhibit few changes after 4-8 years of age, depending on the bone [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF][START_REF] Milovanovic | Region-dependent patterns of trabecular bone growth in the human proximal femur: A study of 3D bone microarchitecture from early postnatal to late childhood period[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF].

The lack of data on trabecular bone development in Neandertals presents a significant obstacle to interpreting structural differences between Neandertal and modern human adults.

For example, the evolutionarily recent gracilization1 of the upper limb has been reported to result primarily from a decrease in physical activity [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF][START_REF] Chirchir | Low trabecular bone density in recent sedentary modern humans[END_REF]. This gracilization during human evolution corresponds to a decrease in BV/TV in recent modern human adults. However, we do not know when this difference occurred during ontogeny. It would be useful to determine whether a high BV/TV is present during early Neandertal ontogeny. If so, this would indicate that the high BV/TV reported for adult Neandertals is not related solely to lifestyle.

Here we describe for the first time the ontogenetic trajectories of trabecular bone parameters for the Neandertal humerus to determine whether trabecular bone microarchitectural differences were present between Neandertals and modern humans, and, if so, when they appeared and which of the bone parameters are most distinct. We specifically focused on early ontogeny as noticeable modifications in modern human bone microstructure occur during the early stages of development [START_REF] Gosman | Development of cortical bone geometry in the human femoral and tibial diaphysis[END_REF][START_REF] Colombo | La micro-architecture trabéculaire de l'os en croissance: Variabilité tridimensionnelle normale et pathologique analysée par microtomodensitométrie[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF], and the ages of most of the Neandertals in this study fall within this age bracket.

We predicted that early humeral development will be similar for these two closely related species, but that there will be differences between adolescents since differences are present in adults [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF]. This latter prediction was supported by the behavior of adolescent hunter-gatherers. From the age of 10-12 years, hunter-gatherer and non-urban children can undertake tasks similar to those carried out by adults [START_REF] Moberg | Housefold production and the value of children: A microeconomic approach to third world population trends[END_REF][START_REF] Bradley | Women's power, children's labor[END_REF]Hewlett and Hewlett, 2012), which can affect bone structure [START_REF] Cowgill | The ontogeny of recent and Late Pleistocene human postcranial robusticity[END_REF].

Microstructural studies of extinct humans frequently focused on the best-preserved adult bones: humerus, femur and tibia [START_REF] Ryan | Gracility of the modern Homo sapiens skeleton is the result of decreased biomechanical loading[END_REF][START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF][START_REF] Chirchir | Low trabecular bone density in recent sedentary modern humans[END_REF][START_REF] Scherf | Patterns of activity adaptation in humeral trabecular bone in Neolithic humans and present-day people[END_REF][START_REF] Chirchir | Trabecular bone fraction variation in modern humans, fossil hominin and other primates[END_REF]. The discovery of several Neandertal humeri from the early period of development [START_REF] Bordes | Découverte d'un squelette d'enfant moustérien dans le gisement du Roc de Marsal, commune de Campagne-en-Bugue (Dordogne)[END_REF]Včlek, 1972;[START_REF] Lumley De | Les Anténéandertaliens et Néandertaliens du bassin méditerranéen occidental européen[END_REF][START_REF] Heim | Les Enfants Néandertaliens de La Ferrassie[END_REF][START_REF] Akazawa | The Neanderthal remains from Dederiyeh Cave, Syria: Interim report[END_REF][START_REF] Rak | A Neandertal infant from Amud Cave, Israel[END_REF][START_REF] Golovanova | Mezmaiskaya Cave: A Neanderthal occupation in the northern Caucasus[END_REF][START_REF] Akazawa | Neanderthal Burials. Excavations of the Dederiyeh Cave, Afrin, Syria[END_REF][START_REF] Maureille | A lost Neanderthal neonate found[END_REF] prompted us to preferentially study this non-weight-bearing bone. We assume that the bone microstructure of the humerus during human development depends to a far greater extent on factors other than mechanical loading in comparison with the femur, and this lack of knowledge of humeral development is likely to be a source of error in functional interpretations of adults. We therefore address potential singularities and explanations for the observed trabecular bone pattern in Neandertal, given previous animal and modern human studies, and finally we questioned the impact of our results on the functional hypothesis associated with recent evolutionary humeral gracilization.

Materials and methods

Samples

The sample of fossil and modern humans (MH) is presented in Table 1 and Figure 1.

The adolescent Crouzade I is a left humerus (SOM Fig. S1) from a Mousterian layer (C8) from Crouzade Cave (France), dated to 49,776-44,805 cal BP ( 14 C; [START_REF] Saos | The Middle and Upper Palaeolithic at La Crouzade cave (Gruissan, Aude, France): New excavations and a chronostratigraphic framework[END_REF]. The external morphology of the humerus was previously studied by [START_REF] Lumley De | Les Anténéandertaliens et Néandertaliens du bassin méditerranéen occidental européen[END_REF]. The distal metaphysis and epiphysis are missing. The proximal extremity is slightly eroded, and multiple cracks run longitudinally through this bone. The proximal epiphysis is not fused.

Insert Table 1 and Figure 1 here

The unpublished Crouzade IX (n°272) is a right humerus (Fig. 1; SOM S1; SOM Fig. S2), excavated in a Mousterian layer (C7), from Crouzade Cave (France), dated to around 41,000 +/-2,000 by combined dating methods (electron spin resonance/uranium-thorium, ESR/U-Th; [START_REF] Saos | The Middle and Upper Palaeolithic at La Crouzade cave (Gruissan, Aude, France): New excavations and a chronostratigraphic framework[END_REF]. It is distally broken just at the proximal margin of the olecranon fossa.

The Mezmaiskaya 1 humeri were found in a Middle Paleolithic deposit in Mezmaiskaya Cave, in the north-western Caucasus (Russia), dated to 60-70,000 BP by ESR estimates [START_REF] Skinner | ESR dating at Mezmaiskaya Cave, Russia[END_REF] and 85,930-101,304 BP by mutation rate estimates (see Supplementary Information S.5 and Table S5.5 in Hajdinak et al., 2018). They belong to an almost-complete skeleton of an infant who most probably died during the first two weeks after birth based on dental and postcranial dimensions [START_REF] Weaver | Neonatal postcrania from Mezmaiskaya, Russia, and Le Moustier, France, and the development of Neandertal body form[END_REF][START_REF] Ponce De León | Neanderthal brain size at birth provides insights into the evolution of human life history[END_REF][START_REF] Gunz | A uniquely modern human pattern of endocranial development. Insights from a new cranial reconstruction of the Neandertal newborn from Mezmaiskaya[END_REF]. Additional information can be found in [START_REF] Golovanova | Mezmaiskaya Cave: A Neanderthal occupation in the northern Caucasus[END_REF], Ponce de [START_REF] Ponce De León | Neanderthal brain size at birth provides insights into the evolution of human life history[END_REF], [START_REF] Golovanova | Les hommes de Néandertal du Caucase du Nord: Entre l'Ouest et l[END_REF] and [START_REF] Weaver | Neonatal postcrania from Mezmaiskaya, Russia, and Le Moustier, France, and the development of Neandertal body form[END_REF].

The Le Moustier 2 humerus was found in a Mousterian layer (H) in Le Moustier lower rock shelter (France; [START_REF] Weaver | Neonatal postcrania from Mezmaiskaya, Russia, and Le Moustier, France, and the development of Neandertal body form[END_REF]. It belongs to a well-preserved skeleton (see also [START_REF] Maureille | A lost Neanderthal neonate found[END_REF].

The Ferrassie 4bis humerus came from the La Ferrassie site (France). This bone is not completely preserved and bears some damage on both metaphyses.

The Ferrassie 6 humerus also came from the La Ferrassie site (France). This humerus presents with a relatively well-preserved proximal metaphysis, but the diaphysis is distorted by flattening and the distal metaphysis is missing. The MH humeri ( [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]. Sixteen humeri belong to individuals 1.5 years old or less.

Age estimates of the study sample

The Crouzade I humerus was considered as an adult by [START_REF] Lumley De | Les Anténéandertaliens et Néandertaliens du bassin méditerranéen occidental européen[END_REF], and as an adolescent by [START_REF] Trinkaus | Neanderthal mortality pattern[END_REF]. The unfused proximal epiphysis was consistent with that of an adolescent [START_REF] Cunningham | Developmental Juvenile Osteology[END_REF]. We also took the overall size into account by measuring bone length and maximal breadth of the proximal metaphysis. Preserved bone length was 186 mm and the maximal breadth of the proximal metaphysis was 37 mm. In our MH sample, three adolescents were of known age. One adolescent was 13 years old and the other two were 14 years old. The maximal breadth of the 13-year-old proximal metaphysis was 35.7 mm; the two 14-year-old proximal metaphyses were 36.6 mm and 33.0 mm. Thus, the breadth of the Crouzade I proximal metaphysis was slightly greater than that of individuals of 13-14 years. Furthermore, the humeral midshaft total area (TA) of Crouzade I (203.9 mm²) corresponded to the mean value for recent 14-16-year-old MHs (204.3 mm 2 ), as reported by [START_REF] Cowgill | The ontogeny of recent and Late Pleistocene human postcranial robusticity[END_REF]. Based on these data, we attributed Crouzade I to the adolescent group (in the same way as [START_REF] Trinkaus | Neanderthal mortality pattern[END_REF]. We chose to plot Crouzade I at the age of 15 years on our graphs, corresponding to the middle of the later age category (14-16).

Anatomical comparisons of Crouzade IX with fetal and infant individuals allowed us

to propose an estimated maximal length of 80 mm (from a preserved length of 75 mm; see SOM S1 and SOM Fig. S3 for details). This humeral length corresponded to a three-monthold individual (0.25 years old; SOM Table S2), according to [START_REF] Maresh | Measurements from roentgenograms[END_REF]see table 10-9 in Cunningham et al., 2016, adapted from Maresh, 1970).

We assigned an age at death for Mezmaiskaya 1 of <1 week (approximately 0.016 years; SOM S1; SOM Table S1), as estimated using humeral length following the formula of [START_REF] Scheuer | The estimation of late fetal and perinatal age from limb bone length by linear and logarithmic regression[END_REF].

Weaver et al. ( 2016) suggested an age no older than four months for Moustier 2.

Given our predictions relating to humeral length, and the humeral length of Le Moustier 2 (64 mm; [START_REF] Weaver | Neonatal postcrania from Mezmaiskaya, Russia, and Le Moustier, France, and the development of Neandertal body form[END_REF], which was slightly shorter than Mezmaiskaya 1 (68 mm; [START_REF] Weaver | Neonatal postcrania from Mezmaiskaya, Russia, and Le Moustier, France, and the development of Neandertal body form[END_REF], we attributed an age of 0 years old approximately (-0.019 years) to this individual (SOM S1; SOM Table S1).

Despite damage, bone length of Ferrassie 4bis could be directly measured (73.4 mm; [START_REF] Heim | Les Enfants Néandertaliens de La Ferrassie[END_REF]. According to [START_REF] Heim | Les Enfants Néandertaliens de La Ferrassie[END_REF], the age of Ferrassie 4bis was estimated at 12 days old. However, we assigned an approximate age of 0.125 years (6.5 weeks) using the Maresh (1970) method of prediction based on a large sample of known age (SOM Table S2).

An age of 2.4 years was predicted for Ferrassie 6 on the basis of humeral length [START_REF] Cowgill | The ontogeny of Holocene and late Pleistocene human postcranial strength[END_REF]. Based on our MH sample of known age, we noted that the metaphyseal breadth of Ferrassie 6 (23.7 mm) was slightly larger than that of a MH 2-year-old individual (22.3 mm, HA-25939), which would consistent with the predicted age of 2.4 years.

For the medieval sample, we estimated ages from humeral length using the Maresh (1970) and [START_REF] Scheuer | The estimation of late fetal and perinatal age from limb bone length by linear and logarithmic regression[END_REF] methods (SOM S1; SOM Tables S1,S2).

Micro-CT acquisition

The sample of perinate to 15-years-old Neandertal humeri was scanned with a voxel size of 11.5-35 µm at the ISIS4D X-ray platform (In Situ Innovative Set-ups, University of Lille, France), AST-RX platform (MNHN, Paris, France) and Max Planck Institute for Evolutionary Anthropology (Leipzig, Germany). The sample of MH fetuses to 14-year-olds was scanned with a voxel size of 5.5-20 µm at ISIS4D and MNHN. Micro computed tomographic (microCT) information is summarized in Table 1 and SOM S1.

Our protocol for selecting regions of interest allowed us to include all the humeri of the sample and to extract homologous volumes of interest (VOIs). Based on the state of preservation of the fossils, we visually identified three main trabecular regions (medial, central and lateral). The central and the medial regions were the best preserved in our fossil sample. However, the central region frequently showed an unexpected loss of bone in some individuals aged 3 years to adolescent age (SOM Fig. S4). Thus, we restricted our measurements to the medial region. The trabecular parameters were measured from a cubic VOI located in the medial part of the humeral proximal metaphysis (Fig. 2), using Avizo v.

7.1.0 (FEI Visualization Sciences Group, Hillsboro). We scaled the VOI to maximal metaphyseal breadth (MMB). We located and selected the largest VOI within this medial region without including any parts of the central region. The edge length of the VOI was 20% of the MMB. The distance between the VOI and the medial border corresponded to 25% of the MMB. The VOI was centered on the MMB axis; the superior part of the VOI was positioned at the same level as the medial border of the surgical neck of the humerus (Fig. 2).

Very slight shifts of the VOI were required to avoid the inclusion of cortical bone for two individuals (one early childhood, Ferrassie 6, and one adolescent, Crouzade I). These very slight shifts corresponded to a thickness of about 1-2 trabeculae (0.1-0.2 mm).

Insert [Figure 2] here Five microarchitectural variables were measured with the BoneJ plugin [START_REF] Doube | BoneJ: Free and extensible bone image analysis in ImageJ[END_REF] for ImageJ v. 1.51q [START_REF] Schneider | NIH Image to ImageJ: 25 years of image analysis[END_REF]: bone fraction (BV/TV), i.e., bone volume (BV, mm 3 ) divided by total volume (TV, mm 3 ); trabecular thickness (Tb.Th, mm), i.e., mean trabecular thickness; trabecular spacing (Tb.Sp, mm), i.e., mean trabecular spacing; degree of anisotropy (DA) as an index of directionality; and connectivity density (Conn.D, mm -3 ), which refers to the number of connected structures. Conn.D can be informative about the number of trabeculae (Tb.N; [START_REF] Odgaard | Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF], which could not be measured directly with the BoneJ plugin. Tb.N (mm -1 ) could be predicted from formulas related to the structural trabecular model, parallel plate or cylindrical rod models [START_REF] Parfitt | Relationships between surface, volume, and thickness of iliac trabecular bone in aging and in osteoporosis[END_REF][START_REF] Parfitt | Bone histomorphometry: Standardization of nomenclature, symbols, and units[END_REF]. These models refer to the trabecular bone composed of plate-like and rod-like trabeculae. We chose to apply the formula using the parallel plate model [1/(Tb.Th + Tb.Sp)] [START_REF] Parfitt | Bone histomorphometry: Standardization of nomenclature, symbols, and units[END_REF]Tsegai et al. (2018b). In the BoneJ plugin [START_REF] Doube | BoneJ: Free and extensible bone image analysis in ImageJ[END_REF], the automatic optimal threshold for segmentation with visual checking for approval was used before quantification. The Gaussian filter was applied when noise disturbed segmentation and the Purify function was run to correctly measure connectivity [START_REF] Odgaard | Quantification of connectivity in cancellous bone, with special emphasis on 3-D reconstructions[END_REF].

Effect of voxel size on 3D measurements

Resolution depends on the size of the scanned region, i.e., size of the field of view (FOV). The smaller the FOV, the smaller the size of the voxels, i.e., the higher the spatial resolution [START_REF] Bushberg | The Essential Physics of Medical Imaging[END_REF]. For this study, we tried to use the best possible resolution with regard to the size of the proximal humeral metaphysis, as it changes during ontogeny.

This was not always possible as some fossil and recent human humeri were not accessible for performing new scans at the required spatial resolution. Micro-CT analysis of trabecular bone depends on scan resolution as differences in voxel size can greatly influence estimates of architectural parameters [START_REF] Kim | The effect of microcomputed tomography scanning and reconstruction voxel size on the accuracy of stereological measurements in human cancellous bone[END_REF]Gosman and Ketcham, 2009). Thus, four Neandertals (Moustier 2, Mezmaiskaya 1, Ferrassie 4bis, Ferrassie 6) and five modern humans (S23, S302, S269, S22, S62) required correction for low spatial resolution.

In the literature, two options were available to quantify the effect of voxel size on 3D measurements before determining whether it was necessary to correct the measures to enable the use of material scanned at lower spatial resolutions. In one option, the influence of voxel size on trabecular properties was quantified by artificially reducing voxel size (re-sampling)

with a software program [START_REF] Ryan | Gracility of the modern Homo sapiens skeleton is the result of decreased biomechanical loading[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF]. The initial values measured at the best resolution were compared with those obtained after re-sampling. In another study, the same individuals were scanned at two distinct resolutions (re-scanning) and the authors measured the influence on trabecular properties (Gosman and Ketcham, 2009). In both cases, if the differences are significant, trabecular properties can be corrected with a linear regression equation [START_REF] Ryan | Gracility of the modern Homo sapiens skeleton is the result of decreased biomechanical loading[END_REF] or correction factor (CF; Gosman and Ketcham, 2009). The bones can also be re-sampled to the largest voxel size in the bone sample [START_REF] Hill | A quantitative method for the evaluation of threedimensional structure of temporal bone pneumatization[END_REF]. The CF of Gosman and Ketcham (2009) corresponded to the difference in measurements obtained from bones scanned at low and high resolution.

However, [START_REF] Kim | The effect of microcomputed tomography scanning and reconstruction voxel size on the accuracy of stereological measurements in human cancellous bone[END_REF] scanned at two different resolutions (high and low, i.e., rescanning), and compared the low-resolution measurements obtained by re-scanning and resampling. They noted that re-sampling from high to low resolution could generate significant measurement errors compared to re-scanning at the same low resolution [START_REF] Kim | The effect of microcomputed tomography scanning and reconstruction voxel size on the accuracy of stereological measurements in human cancellous bone[END_REF].

Some variables, such as BV/TV and Tb.Th, two key variables in our study, were not greatly influenced by re-sampling, whereas they were expected to vary greatly based on variation in spatial resolution [START_REF] Kim | The effect of microcomputed tomography scanning and reconstruction voxel size on the accuracy of stereological measurements in human cancellous bone[END_REF]. Re-sampling at low resolution tends to preserve the value obtained from the high-resolution scan and therefore does not change the value as much as it should at low resolution, e.g., the BV/TV does not increase as much as it should [START_REF] Kim | The effect of microcomputed tomography scanning and reconstruction voxel size on the accuracy of stereological measurements in human cancellous bone[END_REF]. In this study, we compared the error of prediction of the BV/TV estimated from resampling and re-scanning (i.e., when we apply CF obtained from re-scanning). Based on our preliminary analysis (SOM S1), re-sampling errors were higher than the errors associated with the use of CFs calculated with re-scanning. Thus, we deemed the use of a CF from rescanning to be the best, although more expensive solution.

The CFs of the five trabecular variables used in this study were determined by rescanning 2-3 times eight modern humans covering periods of major structural change during growth (Table 2). Because the humerus both increases in size and undergoes microarchitectural changes during growth [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF], it was necessary to obtain CFs corresponding to the ages of each individual requiring correction. The details of our correction method are explained in SOM S1 and the corrected values for the nine individuals are presented in Table 3. 3] here

Insert [Table 2] and [Table

Statistical analysis

The relationship between humeral structural parameters and age was determined using the nonparametric Spearman's rank-order correlation. Locally weighted polynomial regressions (LOESS curve fitting) were plotted for each variable. LOESS was well suited to tracking the ontogenetic trends of our variables which presented a nonlinear relationship with age (Fig. 3). Correlations were considered statistically significant if p < 0.05. Principal component analysis (PCA) was used to identify patterns of change in trabecular microstructure during ontogeny. PCA was calculated from log10-transformed trabecular variables to equalize proportional changes in raw data. We used the correlation matrix given that variables were measured in different units. In PCA, the minimal spanning tree (MST) connects nearest neighbors (i.e., individuals) with respect to the original distances present in the highdimensional space including all components, and not in the two-dimensional distances comprised of two principal components [START_REF] Hammer | Paleontological Data Analysis[END_REF]. We thus used MST as a way to objectively describe our PCA. All statistical analyses were performed using PAST v.

2.14 [START_REF] Hammer | PAST: Paleontological statistics software package for education and data analysis[END_REF].

Insert [Figure 3] here

Results

In general, BV/TV, Tb.N and Conn.D decreased and Tb.Th and Tb.Sp increased significantly with age in modern humans with a critical period between the perinatal period and about one year of age (Table 4; Fig. 3). DA showed high variation in perinates compared to modern humans between ~1.5-14 years of age. After one year of age (n = 15), only Tb.Sp and Tb.N were significantly correlated with age (Table 5). During early ontogeny, all trabecular variables were significantly correlated (Table 4) except DA. These close relationships between trabecular parameters changed after one year (Table 5).

Neandertals followed a similar pattern in comparison with modern humans during early ontogeny (<1.5 years). Due to the presence of only two Neandertals older than 1.5 years, i.e., the 2.4-year-old and another one of 15, we could not specify the overall ontogenetic similarities between Neandertals and modern humans. We note, however, that for certain variables (e.g., BV/TV, Tb.Th), these two older Neandertals deviated from modern human ontogenetic trajectories.

Insert [Table 4] [Table 5] here

In modern humans, BV/TV declined dramatically between the perinatal/neonatal period and the age of 6 months (Fig. 3), after which BV/TV remained relatively constant until the age of 14. We observed a slight increase in BV/TV until six years of age, followed by a slight decline. In Neandertals, the decline from birth to three months was similar to the decline observed in modern humans. However, BV/TV for la Ferrassie 6 (2.4 years old) and the adolescent Crouzade I, were notably different from expected values for modern humans, as they displayed very high values (Fig. 3).

Tb.Sp in modern humans increased with age, particularly from the perinatal period to one year of age, after which the increase was less marked. Neandertals exhibited a growth trajectory similar to modern humans, with the adolescent Crouzade I displaying a slightly lower Tb.Sp compared to modern humans of similar age (Fig. 3). However, Tb.Sp was very low at 2.4 years of age (Ferrassie 6) in Neandertals.

There was a marked decline in Tb.N in modern humans from the perinatal period to one year of age followed by a slight but steady decrease until 14 years. Neandertals fit with the modern human growth trajectory.

Tb.Th in modern humans showed marked increases from the perinatal period to ~1-1.5 years but with a high degree of variation. From 6-14 years of age, Tb.Th values remained roughly constant. Neandertals seemed to display a greater increase in Tb.Th than modern humans. However, only two individuals of more than one year of age were present in our sample. The most dramatic difference was observed for the adolescent Crouzade I (Fig. 3).

Variation in DA in modern humans was high from the perinatal period to two years of age. Only a few perinate individuals showed a highly oriented trabecular structure denoted by a high degree of anisotropy. After six years of age, DA values were closer to the mean value for the early period of ontogeny and variation decreased. Prior to ~3 years of age, Neandertals showed a high degree of variation in DA, similar to modern humans. Neandertals fell within the lower DA range for modern humans. The adolescent Crouzade I showed the expected value for modern humans (Fig. 3).

There was an impressive decrease in Conn.D from the perinatal stage to one year of age, after which we noted a slight trend towards decreasing values. The Neandertal growth pattern was the same as for modern humans.

BV/TV showed significant and moderately high correlations with all microstructural parameters during ontogeny except DA (Table 4). However, after one year of age, the number of significant correlations decreased, with only Tb.Th and Tb.Sp significantly (p < 0.05) correlated with BV/TV, and Tb.Sp significantly (p < 0.05) correlated with Tb.N and Conn.D.

We note that the nearly perfect correlation between Tb.N and Tb.Sp during ontogeny (Table 4) was due to the fact that Tb.N was predicted from an equation that included Tb.Sp (as noted above).

PCA (Fig. 4A) displayed a clear separation between distinct growth periods. The first component (PC1, 69.2% of variance) was influenced by all variables except DA (Fig. 4B). In general, microarchitectural changes in the proximal metaphysis during ontogeny could be observed along PC1 from perinate/neonate (on the right) to individuals 1.5 years of age and older (on the left). Younger individuals with positive values along PC1 tended to have high values for BV/TV, Tb.N and Conn.D, while older individuals with negative values tended to have higher Tb.Th and Tb.Sp values (Fig. 4). For DA, we observed high degrees of variation for the younger individuals, who were widely dispersed along PC2, and low degrees of variation for the older individuals (Fig. 4A). The '6 months-1 year' group was an

Discussion

The microstructural changes in Neandertals during early ontogeny followed the modern human growth trajectories for all variables. Notably, Neandertals experienced a decline in BV/TV immediately after birth, which was previously recognized as specific to modern humans (Tsegai et al., 2018b). However, while a high BV/TV could be expected for the adolescent Crouzade I, given that high BV/TV was found in Neandertal adults [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF], the very high BV/TV in early childhood for Ferrassie 6 was surprising. In our young Neandertal sample, Ferrassie 6 was the only one with any trabecular parameter that deviated markedly from the ontogenetic trajectory of modern humans. BV/TV is central to mechanical hypotheses relative to recent evolutionary gracilization [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF][START_REF] Ryan | Gracility of the modern Homo sapiens skeleton is the result of decreased biomechanical loading[END_REF] and for defining the special feature of human trabecular development with bone loss after birth [START_REF] Colombo | La micro-architecture trabéculaire de l'os en croissance: Variabilité tridimensionnelle normale et pathologique analysée par microtomodensitométrie[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]Tsegai et al., 2018b). Ferrassie 6 was distinguished from modern humans by an unexpectedly high value for this parameter. In this regard, the BV/TV and related trabecular parameters, which were sometimes strongly correlated as we have shown in this study, require further discussion to understand the implications of these trabecular similarities and what conclusions can be drawn from the high BV/TV of Ferrassie 6.

Trabecular bone loss immediately after birth in Neandertals and modern humans

In this study, we showed that the dramatic bone loss (as measured by a decrease in BV/TV) that occurs during the first weeks to months after birth in modern humans also occurred in Neandertals. We thus confirm previous results for modern humans [START_REF] Colombo | La micro-architecture trabéculaire de l'os en croissance: Variabilité tridimensionnelle normale et pathologique analysée par microtomodensitométrie[END_REF][START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF][START_REF] Milovanovic | Region-dependent patterns of trabecular bone growth in the human proximal femur: A study of 3D bone microarchitecture from early postnatal to late childhood period[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF] and provide novel findings for Neandertals. In this study, bone loss was associated with a decline in Tb.N and Conn.D in modern humans and Neandertals. By contrast, BV/TV and Tb.N reportedly do not change significantly between age groups during humeral growth in chimpanzees, nor do Tb.Sp and Tb.Th (Tsegai et al., 2018b;Conn.D was not included in their study). Some trends in chimpanzees have been observed (i.e., an increase in BV/TV, Tb.Th, Tb.Sp and a decrease in Tb.N and DA; Tsegai et al., 2018b), but no bone loss occurs just after birth. Consequently, modern humans and Neandertals shared an early developmental pattern for trabecular bone that differs from that of chimpanzees, with, in particular, a distinct balance of bone modeling (resorption versus deposition) for one year after birth, probably driven by slow deposition and constant resorption rates in modern humans [START_REF] Parfitt | Structural and cellular changes during bone growth in healthy children[END_REF][START_REF] Reissis | Development of fetal trabecular microarchitecture in the humerus and femur[END_REF] and Neandertals.

According to [START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF], two hypotheses can be formulated to explain this bone loss, one related to the loading environment, the other related to development. With regard to the first hypothesis, we could postulate a similar change in the loading environment from an intrauterine to extrauterine environment in modern humans and Neandertals. It has been hypothesized that mechanical stimuli are higher in utero than for several months after birth due to fetal kicking and resistance against the intrauterine cavity (Land and Schoneau, 2008). In contrast to quadrupedal mammals (e.g., the pig, [START_REF] Oakley | Ontogeny of spontaneous locomotor activity in rabbit, rat and guinea pig[END_REF]; see Table S2 in [START_REF] Garwicz | A unifying model for timing of walking onset in humans and others mammals[END_REF], the human newborn is functionally unable to move around independently and has a very limited range of motion (Young and Shapiro, 2018). Less mechanical loading of the humerus at birth in humans could result in distinct early growth trajectories in trabecular parameters in comparison to the pig, which is a fully independent quadruped at birth [START_REF] Sacher | Relation of gestation time to brain weight for placental mammals: Implications for the theory of vertebrate growth[END_REF], and which shows no decline in BV/TV immediately following birth [START_REF] Tanck | Increase in bone volume fraction precedes architectural adaptation in growing bone[END_REF]. However, chimpanzees, like humans, do not locomote independently just after birth [START_REF] Doran | The ontogeny of chimpanzee and pygmy chimpanzee locomotor behavior: A case study of paedomorphism and its behavioral correlates[END_REF]Young and Shapiro, 2018), yet as noted above (Tsegai et al., 2018b), their humeral BV/TV does not decline after birth as it does for pigs. Moreover, we currently have no data to suggest that the human humerus supports less mechanical loading for six months after birth (i.e., during the most critical period of bone loss) than in the intrauterine environment.

With regard to the second hypothesis of [START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF], [START_REF] Reissis | Development of fetal trabecular microarchitecture in the humerus and femur[END_REF] showed that BV/TV is not bone-specific (or sex-or site-specific) during human fetal development. BV/TV (reported as 'Tb.Th/transect length' in their study), as well as Tb.Th and Tb.N, are reportedly similar during human fetal development between four and nine months in humeri and femora. They deduced a generalized predetermined growth trajectory.

Based on the same arguments that Reissis and Abel (2012) advanced for human fetal bones, similarities in trabecular bone development between the humerus and femur after birth and before the onset of independent bipedal walking (<1 year; [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF] would more likely result from genetic rather than mechanical influence. In other words, if movements and muscular contractions in early ontogeny affect bones, then there should be differences between the trabecular bone structure of the humerus and femur, two bones belonging to two distinct functional units. However, this is not what they observed. According to [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF], during the first year after birth, the initial primary trabecular bone is rapidly replaced by secondary trabecular bone. This event is associated with a new microstructural organization, more similar to the one observed in adults, with a columnar structure progressively transformed into a trabecular structure [START_REF] Martin | Skeletal Tissue Mechanics[END_REF]Carter and Beaupre, 2001;[START_REF] Currey | Bones: Structure and Mechanics[END_REF]. The early phase of this trabecular development, marked by a decline in 20 BV/TV [START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF][START_REF] Milovanovic | Region-dependent patterns of trabecular bone growth in the human proximal femur: A study of 3D bone microarchitecture from early postnatal to late childhood period[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF] results from (1) a primary trabecular bone formed by endochondral ossification, which reflects cartilage organization, followed by (2) a reorganization of the trabecular microstructure related to the development of the secondary trabecular bone [START_REF] Carter | The role of mechanical loading histories in the development of diarthrodial joints[END_REF][START_REF] Carter | Relationships between loading history and femoral cancellous bone architecture[END_REF][START_REF] Carter | Musculoskeletal ontogeny, phylogeny, and functional adaptation[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]. This critical event in humans is not dictated by the mechanical environment. Here, we showed that the decline of BV/TV in modern humans and Neandertals was related to a decline in Tb.N, Conn.D and a reduction in the variation of DA. Thus, the microstructural reorganization in modern humans and Neandertals could potentially be initiated by similar predetermined developmental mechanisms reflecting a shared genetic blueprint.

Relevance of the high bone fraction in Ferrassie 6

Following the bone loss in Neandertals and modern humans observed during the neonatal (birth-0.5 year) and infancy (0.5-1 year) periods, some of the similarities between these two groups seemed to decline. In particular, Neandertals, as represented by Ferrassie 6, underwent a dramatic change in BV/TV at 2.4 years of age. Ferrassie 6 showed a very high BV/TV, almost as high as the maximum values for newborn Neandertals and modern humans and higher than that of the Neandertal adolescent (Crouzade I). Whereas the high BV/TV of Crouzade I might be expected, based on the results of [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF] with Neandertal adults, the high BV/TV for Ferrassie 6 was unexpected given the modern human growth trajectory.

Unfortunately, Ferrassie 6 is the only Neandertal specimen at this ontogenetic stage. In the absence of additional evidence, we cannot determine whether Ferrassie 6 is representative or an outlier at this stage of ontogeny. Assuming the age estimates are reasonable, it is important to note that Ferrassie 6 was in a different phase of development compared with younger Neandertals (Fig. 3). Although the BV/TV of Ferrassie 6 is high, it fell within the modern human range for all developmental stages. [START_REF] Hébert | Comparative three-dimensional structure of the trabecular bone in the talus of primates and its relationship to ankle joint loads generated during locomotion[END_REF] underscored the possible impact of diagenesis on the BV/TV in Eocene primates by increasing the Tb.Th postmortem. However, such structural change is only clearly observed for two Eocene primates and we did not notice any structural evidence in Ferrassie 6 suggesting modification due to taphonomic processes. We also note that the other young Neandertals did not present high BV/TV or Tb.Th values compared to modern humans. The very high BV/TV of Ferrassie 6 is due to the combination of its trabecular characteristics (high Tb.Th, low Tb.Sp and slightly high Tb.N), consistent with modern human variation (Fig. 3). Thus, the high BV/TV is not specifically due to a very high Tb.Th, as would be expected if diagenesis was the cause.

Implications of high bone fraction in Neandertal early childhood

It is possible that the BV/TV of Ferrassie 6 reflects the normal condition and it is important to discuss the consequences of this. In this respect, this BV/TV could be specific to Neandertal development and reflect the structural complexity of BV/TV linked to variable trabecular parameters. In fact, based on our results, one value of BV/TV at different stages of development may not relate to the same trabecular bone structure (e.g., high BV/TV can result from a distinct combination of trabecular parameters).

Our results merely confirm the suggestion of [START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF] that in modern humans, bone loss during the first year of life is never recovered (Table 6). Neandertals and modern humans differ in that the humeral BV/TV of Ferrassie 6 suggests that term bone mass was recovered in the Neandertal humerus. In contrast, the high BV/TV at birth never seems to recoverfor the proximal humerus [START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF], proximal femur [START_REF] Ryan | Trabecular bone ontogeny in the human proximal femur[END_REF], proximal tibia (Gosman and Ketcham, 2009) and vertebrae [START_REF] Acquaah | Early trabecular development in human vertebrae: Overproduction, constructive regression, and refinement[END_REF] in modern humans. However, given previous studies, the high BV/TV in modern humans observed at birth and which disappears during the early months of life could increase later in the proximal femur [START_REF] Milovanovic | Region-dependent patterns of trabecular bone growth in the human proximal femur: A study of 3D bone microarchitecture from early postnatal to late childhood period[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF] and under the posterior talar facet of the calcaneus [START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF]Table 6). Substantial increases in mechanical loads, which are more apparent in the femur and parts of the calcaneus during growth and associated with unassisted bipedal walking, could be a factor favoring the dramatic increase in BV/TV (as observed in the humerus of Ferrassie 6), even if other results from the femur [START_REF] Ryan | Trabecular bone ontogeny in the human proximal femur[END_REF] do not confirm this assertion. Clearly, the explanation given for the increase in femoral BV/TV cannot account for the high humeral BV/TV of Ferrassie 6. There is no obvious behavior or behavioral shift that would account for the high BV/TV of Ferrassie 6

and, thus, we speculate that this characteristic seems more likely to be related to nonmechanical factors, such as genetic or hormonal factors and diet.

Insert [Table 6] here

Caution is required in interpreting BV/TV values given that this variable is both region-and bone-dependent [START_REF] Griffin | Comparative forefoot trabecular bone architecture in extant hominids[END_REF][START_REF] Colombo | La micro-architecture trabéculaire de l'os en croissance: Variabilité tridimensionnelle normale et pathologique analysée par microtomodensitométrie[END_REF][START_REF] Ryan | Structural and mechanical changes in trabecular bone during early development in the human femur and humerus[END_REF]Saers et al., 2019), and depends as well on distinct microstructural parameters that contribute to the amount of bone. During prenatal development of the humerus and femur, BV/TV is constant but Tb.Th increases and Tb.N decreases [START_REF] Reissis | Development of fetal trabecular microarchitecture in the humerus and femur[END_REF]. In contrast, [START_REF] Salle | Human fetal bone development: Histomorphometric evaluation of the proximal femoral metaphysis[END_REF] showed an increase for BV/TV and Tb.Th in the proximal femur during the gestational period, and no significant change for Tb.N. In this study, before 1.5 years, variation in BV/TV in Neandertals and modern human humeri was strongly related to variation in Tb.N (i.e., high BV/TV means high Tb.N). At 2.4 years, the high BV/TV of Ferrassie 6 is related to a high Tb.Th, low Tb.Sp and slightly high Tb.N, compared to modern humans of the same age and older, while the high BV/TV of Crouzade I (adolescent) is mainly due to a very high Tb.Th.

Thus, the BV/TV of the young Neandertal (Ferrassie 6) is structurally distinct from that of the adolescent Neandertal (Crouzade I). Furthermore, the relationship between microstructural parameters varies with age, related to a specific growth event before about 1.5 years and a distinct growth pattern after 1.5 years.

In a wider taxonomic and non-ontogenetic context, [START_REF] Barak | Of mice rats and men: Trabecular bone architecture in mammals scales to body mass with negative allometry[END_REF] highlighted two distinct mechanisms for achieving variation in BV/TV. The main contributing microstructural factor is Tb.N in rodents (mice and rats). Although Tb.Th mainly explains the variation in humans, but Tb.N is also a significant contributing factor. These findings confirm the need to include distinct trabecular parameters when analyzing BV/TV.

Finally, we cannot claim that the high value of BV/TV in Ferrassie 6 could have predicted the value in adolescents and adults because the structural pattern related to the BV/TV is distinct in Ferrassie 6 and Crouzade I and we do not know if developmental processes act on the conservation of the BV/TV, whatever the underlying structural characteristics.

The mechanical hypothesis for gracilization and Neandertal development

In the current study, BV/TV was similar in Neandertals and modern humans during early ontogeny (<1 year). Early morphological divergence between Neandertals and modern humans prior to the onset of different loading behaviors would have suggested that the same divergence in adults would be unlikely to be related to mechanical factors. Therefore, our results do not call into question the mechanical hypothesis as an explanation for the recent gracilization of the upper limb in humans [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF]. We cannot claim that high BV/TV in Neandertals appeared before the practice of specific cultural physical activity despite the high BV/TV of Ferrassie 6 (2.4 years). In contrast, the BV/TV of the adolescent Crouzade I (15 years) corroborates the finding for the adult of [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF] of a high BV/TV in Neandertals compared to recent modern humans, despite the non-homologous VOI.

Our findings thus underscore (1) strong similarities in the early developmental pattern of humeral trabecular bone between Neandertals and modern humans; and (2) the lack of data to support a common origin for the high BV/TV of Ferrassie 6 and Crouzade I. Consequently, although it does not seem appropriate to link the very high BV/TV at 2.4 years in Ferrassie 6 with a culturally-specific and intensive physical activity (i.e., hunter-gatherer activity), the high BV/TV and Tb.Th in Crouzade I could be interpreted as the result of high mechanical loads based on the hypothesis of [START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF].

Genetically determined bone structure may be influenced by past activity (Wallace et al., 2010); indeed, the structure observed does not necessarily reflect direct bone adaptation to mechanical stimuli. The results of Wallace et al. (2010) on mice suggest an explanation other than bone plasticity for the high BV/TV of Ferrassie 6 and Crouzade I. However, such a hypothesis would work better for Ferrassie 6 than for Crouzade I to explain high BV/TV because the more the individual grows, the more their bone is likely to adapt to new mechanical loads that could supplant genetic functional adaptation. In our study, it was not possible to discern the impact of the potential genetic assimilation to the plastic response to activity levels of the adolescent Crouzade I. It is important to note that Wallace et al. (2010) presented distinct mechanisms (e.g., genetic assimilation, pleiotropy) for what they call 'evolutionary signals', but they noted that none of them is an indisputable hypothesis.

Limitation of the study

First, although we have taken the appropriate precautions to provide valid data, the results may have been impacted by the different scanning protocols and the use of correction factors for some individuals scanned at low a resolution, notably human fossils. Future studies will be necessary to reinforce our correction method by re-scanning since human fossils must be protected as much as possible from any invasive method (in this case X-rays). Secondly, in this study, we referred to the influence of diagenesis and potential inflating of bone trabecular properties of fossils [START_REF] Hébert | Comparative three-dimensional structure of the trabecular bone in the talus of primates and its relationship to ankle joint loads generated during locomotion[END_REF]. This is an aspect that must be kept in mind particularly when studying trabecular bone structures of fossils. However, currently, no strong evidence is available to highlight this taphonomic process. Thirdly, increased sample sizes are needed for both MHs and Neandertals. For the former, our perspective is to complete the sample and integrate human groups from different chronological periods; for the latter, for which we had only two individuals in the 2.5-year to 15-year age group, only new discoveries or unpublished specimens will bridge the gaps.

Conclusions

The comparative analysis of trabecular growth trajectories in Neandertals and modern

  intermediate group similar to the '>1.5 years' group for PC1, but also widely dispersed along PC2. This group preserved the high degree of variation in DA recorded in younger individuals and had a high Tb.Sp and low Conn.D. All of the Neandertal individuals matched their corresponding age groups except Crouzade IX, which fell outside the range of its age group. This was due to its low Conn.D, low Tb.N, high Tb.Sp and moderately high Tb.Th. The trabecular microstructure of Crouzade IX (3 months old) corresponded to modern humans in the 6 months-1 year range and >1.5 years group. Insert [Figure 4] here

  humans offered a new opportunity to discern similarities in their developmental pattern and discuss the biomechanical interpretation of their bone microstructures. Ultimately, overall growth trends were similar for both taxa. In particular, notable bone loss after birth and decreases in Tb.N and Conn.D revealed similar predetermined developmental mechanisms (high modeling with relatively low bone deposition and structural re-organization) in Neandertals and modern humans, distinguishing them from chimpanzees. However, an unexpected value for Neandertal BV/TV was observed during early childhood. The unique microstructural pattern of Ferrassie 6, marked by high BV/TV in early childhood and likely not based on mechanical factors, could have influenced the adult pattern in addition to subsequent changes in the loading environment during growth. However, even if this individual reflected the normal conditions of Neandertals at this age, the impact on adult bone structure remains uncertain, considering how its bone fraction was built. Finally, the mechanical hypothesis, which explains humeral gracilization by bone loss (i.e., decrease in and Martin Friess (UMR 7206) for authorization to scan fossil specimens and for access to the micro-CT scan archives of the MNHN (Paris, France); Henry de Lumley (Institut de Paléontologie Humaine, Paris) and Marie-Antoinette de Lumley (UMR 7194 HNHP) for access to the Crouzade specimens; the Max Planck Institute for Evolutionary Anthropology (J.-J. Hublin and H. Temming) and the ANO Laboratory of Prehistory for access to Mezmaiskaya micro-CT scan (L.V. Golovanova and V.B. Doronichev). We would like to extend special thanks to Hélène Coqueugniot and Olivier Dutour (PACEA, University of Bordeaux) who funded and obtained approvals to scan the modern human sample curated at the MNHN for an earlier project, and Pierre Marchandise (University of Lille) for his cautious help with microCT acquisitions. Thanks are also due to three anonymous reviewers and the editors for insightful comments and Louise Byrne for English editing. Young, J.W., Shapiro, L.J., 2018. Developments in development: What have we learned from primate locomotor ontogeny? Am. J. Phys. Anthropol. 165, 37-71. Zeininger, A.D., 2013. Ontogeny of bipedalism: Pedal mechanics and trabecular bone morphology. Ph.D. Dissertation, University of Texas at Austin.

Figure 1 .

 1 Figure 1. Sample of fossil humeri. Moust. 2 = Moustier 2; Mez. 1 = Mezmaiskaya 1; Fer. 4bis = Ferrassie 4bis; Crouz. IX = Crouzade IX; Fer. 6 = Ferrassie 6; Crouz. I = Crouzade I.

Figure 2 .

 2 Figure 2. Selection of the cubic volume of interest (VOI) for measurements of 3D humeral trabecular variables. MMB = maximal metaphyseal breadth. The edge of the cubic VOI measures 20% of the MMB and it is located at 25% (with respect to the MMB) of the medial border at the same level of the neck border.

Figure 3 .

 3 Figure 3. Age-related variation of humeral trabecular variables during ontogeny in modern humans and Neandertals. Modern humans: red; Neandertals (blue): (1) Moustier 2, (2) Mezmaiskaya 1, (3) Ferrassie 4bis, (4) Crouzade IX, (5) Ferrassie 6, (6) Crouzade I. Locally weighted polynomial regressions (LOESS) are plotted for each variable with a smoothing range of 0.5-0.7.

Figure 4 .

 4 Figure 4. Principal component analysis (PCA) for the trabecular bone of the humeral proximal metaphysis. Six trabecular variables are included in (A): BV/TV, Tb.Th, Tb.Sp, DA, Tb.N and Conn.D. Minimal spanning tree connects nearest neighbors in the high-dimensional space. Contributions of each variable are presented in (B) (top): spatial influence of each variable; (middle): degree of contribution (loading) of each variable to the component 1 (CP1); (bottom): degree of

Table 1

 1

	) came from two

To be consistent with various studies, we used the term 'gracilization' (i.e., decrease in robusticity) as synonymous with 'decrease in bone fraction' or 'decrease in trabecular bone density' although we do not think this term is strictly appropriate (see Supplementary Online Material [SOM] S1).

Acknowledgments

This work was developed as part of the BQR DéHom led by Tony Chevalier and funded by the University of Perpignan, and the 'Projet Collectif de Recherche'(PCR: Le monde moustérien méditerranéen entre Rhône et Pyrénées) led by Thibaud Saos (UMR 7194 HNHP). Our thanks go to: Grégory Hauss and Jerôme Hosdez, research engineers at the ISIS4D X-ray platform (University of Lille, France), funded by International Campus on Safety and Intermodality in Transportation (CISIT), the Haut-de-France Region, the European Community and the National Center for Scientific Research (CNRS); Marta Bellato, technical supervisor and operator at AST-RX, plateforme d'Accès Scientifique à la Tomographie à Rayons X, UMS 2700 2AD Acquisition et Analyse de Données pour l'histoire naturelle CNRS-MNHN, Paris; Antoine Balzeau and Dominique Grimaud-Hervé (UMR 7194 HNHP),

BV/TV) during human evolution is not challenged by results of our study. Furthermore, on account of its high BV/TV and Tb.Th, the adolescent Crouzade I showed the habitual expected structural characteristics when mechanical stresses model the trabecular bone microstructure in distinct taxa and bones (e.g., [START_REF] Barak | A Wolff in sheep's clothing: Trabecular bone adaptation in response to changes in joint loading orientation[END_REF][START_REF] Chirchir | Recent origin of low trabecular bone density in modern humans[END_REF][START_REF] Ryan | Gracility of the modern Homo sapiens skeleton is the result of decreased biomechanical loading[END_REF][START_REF] Saers | Trabecular bone structural variation throughout the human lower limb[END_REF][START_REF] Saers | Baby steps towards linking calcaneal trabecular bone ontogeny and the development of bipedal human gait[END_REF][START_REF] Doershuk | Complex variation of trabecular bone structure in the proximal humerus and femur of five modern human populations[END_REF]. The adult microstructural pattern is probably significantly determined by a complex interaction between nonmechanical factors in early ontogeny and mechanical factors modeling bone in adolescents and young adults. However, supplementary fossils are required to interpret the trabecular pattern in early childhood in Neandertals and the consequences of non-mechanical factors in adult Neandertal trabecular microstructure.

Declaration of competing interest

The authors declare no conflict of interest. contribution (loading) of each variable to the component 2 (CP2). A synthesis by age group is presented around the graph with the main microstructural characteristics. The range of red number refers to the individuals included in each category.