Jessica L Joganic
email: jjoganic@wustl.edu

Yann Heuzé

Allometry and advancing age significantly structure craniofacial variation in adult female baboons I

Keywords:

Primate craniofacial growth is traditionally assumed to cease upon maturation, or at least be negligible, while bony remodeling is typically associated with advanced adult age and, in particular, tooth loss. Therefore, size and shape of the craniofacial skeleton (CFS) of young and middleaged adults should be stable. However, research on both modern and historic human samples suggests that portions of the CFS exhibit age-related changes in mature individuals, both related to and independent of tooth loss. These results demonstrate that the age-category "adult" is heterogeneous, containing individuals demonstrating post-maturational age-related variation, but the topic remains understudied outside of humans and in the cranial vault and base. Our research quantifies variation in a sample of captive adult female baboons (N = 97) in an effort to understand how advancing age alters the mature CFS. Craniometric landmarks and sliding semilandmarks were collected from CT scans of adult baboons aged 7-32 years old. To determine if craniofacial morphology is sensitive to aging mechanisms and if any such effects are differentially distributed throughout the cranium, geometric morphometric techniques were employed to compare the shapes of various cranial regions among individuals of increasing age. Unexpectedly, the biggest form differences were observed between young and middle-aged adults, rather than between adults with full dentitions and those with some degree of tooth loss. Shape variation was greatest in masticatory and nuchal musculature attachment areas. Our results indicate that the CFS changes form during adulthood in baboons, raising interesting questions about the molecular and biological mechanisms governing these changes.

Introduction

Bone growth (change in size over time) and development (change in shape over time) are governed by the molecular and hormonal signalling cascades that regulate and coordinate os-teoblastic secretion and osteoclastic resorption of the composite protein-and-mineral matrix forming the molecular structure of bone [START_REF] Enlow | Handbook of facial growth[END_REF]. Consequently, skeletal maturation is acomplexin terpla yofepigenetic,hormonal,bioc hemical,andmec hanicalfactors [START_REF] Biewener | Mechanical loading and bone growth in vivo[END_REF]. Post-natal bone growth occurs via both periosteal expansion (appositional growth) and sutural/synchondroseal/epiphyseal displacement (longitudinal growth).

However, modification to the size and shape of the mature skeleton proceeds differently (e.g., Frost, 1969;[START_REF] Currey | The mechanical adaptations of bones[END_REF], as the growth sites have ossified and longitudinal growth is no longer possible. Additionally, cortical drift (remodeling) can modify the shape of pre-existing bone by altering the relative rates of endosteal resorption and periosteal deposition [START_REF] Boskey | Aging and bone[END_REF]. In this way, bone tissue can adapt to the environmental demands of an organism, helping to ensure that an individual's skeleton is biomechanically optimized over the course of their lifetime. However, the mechanotransductive properties (the ability of cells to sense, process, and respond to mechanical stimuli) of bone cells are altered with increasing age [START_REF] Wu | Effect of aging on cellular mechanotransduction[END_REF], potentially recurrently modifying the remodeling process throughout adulthood.

Fusion of the spheno-o ccipital synchondrosis and eruption of the third molars are traditionally accepted as the craniofacial features in humans and non-human primates that define an individual as being an "adult" (e.g., [START_REF] Bramblett | Non-metric skeletal age changes in the Darajani baboon[END_REF][START_REF] Falk | Endocranial suture closure in rhesu macaques (Macaca mulatta)[END_REF][START_REF] Wang | Fusion patterns of craniofacial sutures in rhesus monkey skulls of known age and sex from Cayo Santiago[END_REF][START_REF] Zihlman | Skeletal and dental growth and development in chimpanzees of the Taï National Park, Côte D'Ivoire[END_REF][START_REF] Poe | Brief communication: testing the usefulness of the basilar suture as a means to determine age in great ape skeletons[END_REF]Franklin and Flavel, 2014;[START_REF] Balolia | Brief communication: the timing of spheno-occipital fusion in hominoids[END_REF]. As it is assumed that processes of skeletal maturation largely function the same in the craniofacial skeleton (CFS) and long bones (e.g., [START_REF] Litsas | Growth hormone and craniofacial tissues. An update[END_REF][START_REF] Wei | Developmental regulation of the growth plate and cranial synchondrosis[END_REF], and because the limbs cease growing when individuals reach adulthood, craniofacial growth in adult individuals is traditionally considered to be nonexistent or negligible [START_REF] Shea | Post-natal craniofacial growth[END_REF][START_REF] Bastir | Craniofacial levels and the morphological maturation of the human skull[END_REF][START_REF] Matros | The aging midfacial skeleton: implications for rejuvenation and reconstruction using implants[END_REF], see also the discussion by G. Northcroft accompanying [START_REF] Hellman | Changes in the human face brought about by development[END_REF]. However, an accumulation of research in the orthodontic, aesthetic plastic surgery, and forensic literature suggests this assumption is invalid (see reviews by Israel, 1973b;[START_REF] Behrents | Growth in the aging craniofacial skeleton[END_REF] This article is protected by copyright. All rights reserved Author Manuscript [START_REF] Mendelson | Changes in the facial skeleton with aging: implications and clinical applications in facial rejuvenation[END_REF][START_REF] Albert | A review of the literature on the aging adult skull and face: implications for forensic science research and applications[END_REF].

There are four main deficits in the literature regarding the study of post-maturational age-related effects in the CFS. First, the vast majority of such research is conducted in humans (although, see [START_REF] Hens | Ontogeny of craniofacial sexual dimorphism in the orangutan (Pongo pygmaeus). i: face and palate[END_REF][START_REF] Wang | Ontogeny and diachronic changes in sexual dimorphism in the craniofacial skeleton of rhesus macaques from Cayo Santiago, Puerto Rico[END_REF][START_REF] Balolia | Sexual dimorphism and facial growth beyond dental maturity in great apes and gibbons[END_REF][START_REF] Minh | Aging-related changes in the skulls of Japanese macaques (Macaca fuscata): effect of the fragmentation on genetic diversity of macaque populations in the Central Vietnam View project[END_REF], so it is difficult to determine what effects dietary shifts and the reorganization of the hominin cranium may have had on any general mammalian patterns of post-maturational craniofacial aging. Second, most research considers only the facial skeleton. There are only a few examples in the clinical literature that include the cranial base or vault (e.g. [START_REF] Zuckerman | Age changes in the basicranial axis of the human skull[END_REF][START_REF] Bondevik | Growth changes in the cranial base and the face: a longitudinal cephalometric study of linear and angular changes in adult Norwegians[END_REF]. Third, research conducted on the subject of aging in the adult CFS deals almost exclusively in linear dimensions and angular measurements. Consequently, a considerable amount of data exists that demonstrates size increase of the CFS in adults, but far less is known about its development and any allometric (co-variation in shape and size) trends. A few of the more recent studies use geometric morphometric methods, which are often preferred for studying shape variation as they preserve the geometry among landmarks (e.g., [START_REF] Bastir | Craniofacial levels and the morphological maturation of the human skull[END_REF][START_REF] Williams | Regional shape change in adult facial bone curvature with age[END_REF], but much more research on post-maturational shape variation is needed. Fourth, very few studies consider the effects of bone resorption independent of tooth loss (although, see [START_REF] Pessa | Relative maxillary retrusion as a natural consequence of aging: combining skeletal and soft-tissue changes into an integrated model of midfacial aging[END_REF][START_REF] Mendelson | Age-related changes of the orbit and midcheek and the implications for facial rejuvenation[END_REF]b e c a u s e ,i ng e n e r a l ,t h em aj o r i t yo fr e s e a r c ho ng r o w t ht r e n d si na d u l tc r a n i a suggests that "craniofacial bony aging, contrary to popular belief, is one of expansion and not of atrophy" [START_REF] Bartlett | Age-related changes of the craniofacial skeleton: an anthropometric and histologic analysis[END_REF].

CFS aging effects in adult humans

Considerable research on age-related growth in the human head has been conducted, starting in 1858 with Sir George Murray Humphry who recognized that bones of the cranial vault increase in thickness with age (cited in [START_REF] Bartlett | Age-related changes of the craniofacial skeleton: an anthropometric and histologic analysis[END_REF]. Additional early work demonstrated that cranial growth continues throughout the life of an individual (To dd, 1924;[START_REF] Hrdlička | The old Americans[END_REF][START_REF] Hellman | Changes in the human face brought about by development[END_REF][START_REF] Israel | Continuing growth in the human cranial skeleton[END_REF]a n d ,i nt h ef a c i a ls k e l e t o na tl e a s t ,i n c r e a s e si n This article is protected by copyright. All rights reserved Author Manuscript rate later in life [START_REF] Hooton | Age changes and selective survival in Irish males[END_REF][START_REF] Lasker | The age factor in bodily measurements of adult male and female Mexicans[END_REF]. A study by Israel (1973a)indicated an overall size increase on the order of 5-7%, although a greater expansion is observed in cranial vault thickness than in global width or length [START_REF] Israel | The dichotomous pattern of craniofacial expansion during aging[END_REF]. These patterns are sexually dimorphic (e.g., Formby et al., 1994;[START_REF] Williams | Regional shape change in adult facial bone curvature with age[END_REF]a n do b s e r v a b l ei n both modern and historic populations (e.g., [START_REF] Herzberg | An anthropologic study of face height[END_REF][START_REF] Ruff | Age differences in craniofacial dimensions among adults from Indian Knoll, Kentucky[END_REF][START_REF] Bartlett | Age-related changes of the craniofacial skeleton: an anthropometric and histologic analysis[END_REF].

Across a sample of five human populations that differ in geographic region of origin and chronological age, [START_REF] Guagliardo | Craniofacial structure, aging and dental function: their relationships in adult human skeletal series[END_REF]disco v eredan um berofcommonpost-maturationalaging patterns in CFS shape. These included a flattening of the frontal bone profile, increased facial protrusion, widening of various interorbital dimensions, subnasal retraction in relation to the zygomatic arches, and a general increase in the size of both the orbits and the mastoid region. He attributed these changes to a lifetime of strain in response to the stresses of bite forces and exertion of the masticatory muscles.

The majority of research on post-maturational aging in the CFS, especially as it relates to facial rejuvenation surgery and dental orthotics for edentulous individuals, is conducted by clinicians in contemporary humans (see reviews by [START_REF] Mendelson | Changes in the facial skeleton with aging: implications and clinical applications in facial rejuvenation[END_REF][START_REF] Wulc | The anatomic basis of midfacial aging[END_REF]o ri nn o n -h u m a np r i m a t e s ,p a r t i c u l a r l yt h o s ee x p e r i m e n t a l l ym a n i p u l a t e df o r orthodontic research (e.g., [START_REF] Jaul | A cephalometric evaluation of edentulous Rhesus monkeys (Macaca mulatta): A long-term study[END_REF][START_REF] Minh | Aging-related changes in the skulls of Japanese macaques (Macaca fuscata): effect of the fragmentation on genetic diversity of macaque populations in the Central Vietnam View project[END_REF]. According to a review of the aging literature by [START_REF] Albert | A review of the literature on the aging adult skull and face: implications for forensic science research and applications[END_REF], the following changes have been observed in various human skeletal and radiographic samples: (1) horizontal dimensions of the face increase in size, particularly the distance between the cheekbones (i.e., bizygomatic breath);

(2) anterior facial height (chin to nasal bridge) increases, primarily in the lower 4 5 of the face; (3) continued eruption of the teeth increases facial height, while temporomandibular joint (TMJ) arthritis and dental attrition can decrease it; (4) the maxilla is often displaced posteriorly (although, see [START_REF] Levine | Adult facial growth: applications to aesthetic surgery[END_REF]; (5) absolute cranial bone thickness is greater in females and increases until 65 years old; and (6) variation in cranial bones thickness is This article is protected by copyright. All rights reserved

Author Manuscript

greater in males and remains constant over time, yet absolute thickness steadily decreases with age. The authors note that these changes are small (on the order of 1.1-1.6 mm) but noticeable.

The influence of tooth loss

Both tooth loss and subsequent bone resorption are acknowledged to alter the size and shape of the anterior midface, palate, and mandible (e.g., [START_REF] Tallgren | Neurocranial morphology and ageing-a longitudinal roentgen cephalometric study of adult Finnish women[END_REF][START_REF] Jaul | A cephalometric evaluation of edentulous Rhesus monkeys (Macaca mulatta): A long-term study[END_REF][START_REF] Xie | Quantitative assessment of vertical heights of maxillary and mandibular bones in panoramic radiographs of elderly dentate and edentulous subjects[END_REF][START_REF] Sutton | Changes in facial form relative to progressive atrophy of the edentulous jaw[END_REF][START_REF] Dechow | Edentulation alters material properties of cortical bone in the human craniofacial skeleton: functional implications for craniofacial structure in primate evolution[END_REF]. However, it is not until individuals reach an advanced age that the effects of such remodeling on the CFS are usually deemed a potential source of intraspecific variation. Consequently, age is often incorporated as a covariate in regression models or landmarks located in areas expected to be affected by resorption, such as alveolar bone, are omitted from further analysis. Alternatively, individuals demonstrating CFS remodeling are often avoided, if possible. Depending on the organism being studied, this practice can result in a sample comprised of a range of individuals who are somatically mature but that do not yet demonstrate macroscopic signs of aging. Such a sample of wildly varying ages has the potential to bias any resulting inferences that are made.

Additionally, the potential effects of tooth loss and remodeling of alveolar bone on portions of the CFS not directly associated with the oral cavity are rarely considered. There is debate as to whether the same patterns of craniofacial variation are observed in elderly individuals who retain all their teeth (e.g., [START_REF] Herzberg | An anthropologic study of face height[END_REF][START_REF] Pessa | Relative maxillary retrusion as a natural consequence of aging: combining skeletal and soft-tissue changes into an integrated model of midfacial aging[END_REF].

Due to the changing material properties of bone as it ages [START_REF] Colman | Skeletal effects of aging and menopausal status in female Rhesus macaques[END_REF][START_REF] Boskey | Aging and bone[END_REF], one might expect similar aging effects in both elderly individuals who retain all their teeth and their partially edentulous conspecifics. Alternatively, as tooth loss significantly alters the material properties of bone [START_REF] Dechow | Edentulation alters material properties of cortical bone in the human craniofacial skeleton: functional implications for craniofacial structure in primate evolution[END_REF], it may be entirely possible that the difference in craniofacial morphology is so drastic as to be easily accounted for when examining morphological variation in aged individuals. It is also unclear whether This article is protected by copyright. All rights reserved

Author Manuscript

other factors, such as regional muscle atrophy, may affect the aging cranium in both humans and other mammals.

CFS aging effects in other animals

It is possible that these recognized changes in the human adult CFS are related to the fact that the introduction of cooked and less mechanically demanding foodstuffs into our diets has caused facial shortening and the resultant malocclusion issues that plague modern humans [START_REF] Begg | Stone Age man's dentition: with reference to anatomically correct occlusion, the etiology of malocclusion, and a technique for its treatment[END_REF][START_REF] Larsen | Biological changes in human populations with agriculture[END_REF][START_REF] Lieberman | Effects of food processing on masticatory strain and craniofacial growth in a retrognathic face[END_REF]. In addition, the conformation of the CFS has been drastically reorganized in humans to accommodate our greatly encephalized brains [START_REF] Lieberman | Speculations about the selective basis for modern human craniofacial form[END_REF]. Therefore, it is both interesting and informative to look at patterns of CFS post-maturational change in other animals to ascertain whether the trends in size and shape change published in the literature thus far are also unique to humans. If they are, we may need to revisit our use of non-human model organisms in craniofacial research.

Outside of humans, the effects of normal post-maturational aging on the CFS, and in particular the portions of the skull not immediately involved in the oral cavity, are neither well-studied nor often considered a potential source of variation in biological, evolutionary, or anthropological research. The use of non-human primates and other mammals, particularly macaques and rabbits, to explore the response of the CFS to orthodontic and endodontic treatments is popular (e.g., [START_REF] Legan | Craniofacial adaptations after total maxillary osteotomy in Macaca irus:A cephalometric and histologic study[END_REF][START_REF] Jaul | A cephalometric evaluation of edentulous Rhesus monkeys (Macaca mulatta): A long-term study[END_REF][START_REF] Ramfjord | Increased occlusal vertical dimension in adult monkeys[END_REF][START_REF] Rafferty | Botulinum toxin in masticatory muscles: short-and long-term effects on muscle, bone, and craniofacial function in adult rabbits[END_REF]. Consequently, a considerable amount of evidence has been amassed that maxillary growth results from surgical intervention, even in adults, but less research has been done to uncover the natural patterns of CFS change in post-maturational individuals.

Although ontogenetic growth and development of the primate CFS has been the subject of considerable research (e.g., [START_REF] Collard | Ontogeny and homoplasy in the papionin monkey face[END_REF]Mitteroecker et al., 2004aMitteroecker et al., ,b, 2005;;[START_REF] Leigh | Cranial ontogeny of Papio baboons (Papio hamadryas)[END_REF][START_REF] Singleton | Postnatal cranial development in papionin primates: an alternative model for hominin evolutionary development[END_REF], very few studies extend the observational period into adulthood. However, there are three recent studies that provide evidence for post-maturational This article is protected by copyright. All rights reserved

Author Manuscript

changes to the CFS in non-human primates. [START_REF] Minh | Aging-related changes in the skulls of Japanese macaques (Macaca fuscata): effect of the fragmentation on genetic diversity of macaque populations in the Central Vietnam View project[END_REF] present data on captive adult Japanese macaques indicating that cranial length increases more than breadth, the magnitude of post-maturational changes are greater in macaques than in humans, and most changes are observed in male, rather than female, macaques. In contrast, [START_REF] Wang | Ontogeny and diachronic changes in sexual dimorphism in the craniofacial skeleton of rhesus macaques from Cayo Santiago, Puerto Rico[END_REF] demonstrated that CFS development of rhesus macaques is characterized by faster male growth rates during adolescence (2-8 years) and an extended female growth period (8-15 years). Finally, [START_REF] Balolia | Sexual dimorphism and facial growth beyond dental maturity in great apes and gibbons[END_REF] show that hominoid facial dimensions increase in size after the attainment of sexual maturity in male and female orangutans and gorillas, female gibbons, and likely female chimpanzees, although they lacked the power to definitively conclude the trend is visible in Pan as well. In all cases, because the increases were more pronounced between younger adult age categories, the authors conclude that ontogentic trajectories are extended past the age of adolescence and that this may be a feature of female growth and development with deep evolutionary roots.

Research questions

To address the stated deficits in the aging literature and extend what little work has b een done to an additional primate species, we evaluate multivariate age-related changes in craniofacial morphology by quantifying normal variation in the aging CFS from a large number of known-aged and diet-controlled adult female baboons. A major benefit of using this sample is that all individuals were maintained in a similar and constant environment during the entirety of their lives, thereby greatly reducing the effects of dietary and activity pattern variability. Sexual size and shape dimorphism in the CFS is extreme in baboons (e.g., [START_REF] Leigh | Sexual dimorphism in the baboon facial skeleton[END_REF][START_REF] Higgins | Sexual dimorphism and facial growth in papionin monkeys[END_REF][START_REF] Singleton | Patterns of cranial shape variation in the Papionini (Primates: Cercopithecinae)[END_REF]. As the focus of this paper is on age-related variation, we elected to study only one sex and chose females because the presence of a C-P3 honing complex and the variable sagittal crest size in males unnecessarily introduces additional sources of variation. Allometry, or shape variation that is a consequence of differences in the size of individuals, is also an important source of intraspe-This article is protected by copyright. All rights reserved cific variation, particularly in baboons (e.g., Frost et al., 2003;[START_REF] Porto | The evolution of modularity in the mammalian skull I: morphological integration patterns and magnitudes[END_REF][START_REF] Joganic | Additive genetic variation in the craniofacial skeleton of baboons (genus Papio) and its relationship This article is protected by copyright. All rights reserved to body and cranial size[END_REF]. Thus, it will be especially important to determine whether any demonstrated age-related shape variation in the baboon CFS can actually be attributed to allometry.

Using geometric morphometric techniques and multivariate statistics, we address three specific questions. First, are there measurable differences in the patterns of craniofacial variation as individuals age? Second, what role does allometry play in the manifestation of any age-related shape variation? And third, if patterns of CFS size and shape variation are observable across adulthood, are different regions of the skull affected in the same manner?

Based on previous research (e.g., [START_REF] Zuckerman | Age changes in the basicranial axis of the human skull[END_REF][START_REF] Bartlett | Age-related changes of the craniofacial skeleton: an anthropometric and histologic analysis[END_REF][START_REF] Bondevik | Growth changes in the cranial base and the face: a longitudinal cephalometric study of linear and angular changes in adult Norwegians[END_REF], some regions are expected to demonstrate age-related changes (e.g., the midface) while others are expected to remain invariant across age categories (e.g., the basicranium). These analyses will tell us if craniofacial morphology is sensitive to aging mechanisms and if any such effect is differentially distributed throughout the skull. Research Center in San Antonio, Texas. The colony was founded with wild-caught animals from a region of southwestern Kenya overlapping Tsavo East National Park [START_REF] Maples | A preliminary report on classification of the Kenya baboon[END_REF]. Careful breeding to maintain as much of the original genetic diversity as possible has been practiced and has resulted in a seven-generation pedigree preserving 12 founding sire lines. The skulls from a portion (N = 985) of the pedigreed population were curated and CT scanned (0.6-0.75 mm resolution) at Washington University in St Louis and are now housed at Loyola University Chicago (LUC) under the care of Dr. James Cheverud (see [START_REF] Joganic | Additive genetic variation in the craniofacial skeleton of baboons (genus Papio) and its relationship This article is protected by copyright. All rights reserved to body and cranial size[END_REF] for more details).

Methodology

Baboon sample

There are 653 adult females in the LUC baboon collection. Individuals are considered This article is protected by copyright. All rights reserved Author Manuscript adult if they possess a fused spheno-occipital synchondrosis and fully occluded M3's. The average age of this total sample of adult females is 19.83 ± 5.5 years (range: 6.23-33.70).

Four groups were created: individuals in the bottom age quartile (Young Adult, YA), within 0.5 standard deviations of the mean age (Adult, AD), in the top age quartile with full dentitions (Elderly, ER), and in the top age quartile with some degree of maxillary tooth loss (Tooth Loss, TL). A subset of 25 individuals was randomly selected from each group, except in the case of the TL category, for which only 22 individuals could be identified who were missing at least one maxillary tooth and had complete crania (N = 97; Table 1,S u p p . Table S1). The modal number of maxillary teeth missing in TL individuals is 1 (mean: 2.14, range: 1-5), while all individuals in the three other age categories have full maxillary dentitions.

[Table 1 about here.]

Landmarking

Our three research questions deal specifically with the effect of post-maturational aging on differences in CFS shape. Shape is the portion of an object's geometry that is independent of overall size, position in space, or relative orientation [START_REF] Mitteroecker | Advances in geometric morphometrics[END_REF]. Linear distances calculated between landmark pairs are particularly good at capturing variation in size, both relative and overall, but are less useful for quantifying shape differences. Traditional geometric morphometrics utilizes configurations of homologous landmarks strategically placed on an object that are then compared to a reference configuration [START_REF] Bookstein | Combining the tools of geometric morphometrics[END_REF]. This method of studying shape can be problematic as the researcher is limited to using easily-identifiable and biologically homologous traits, such as the intersection between two bones or the point of the chin. Bones of the cranial vault are relatively featureless, making the identification and placement of anatomical landmarks difficult. Semilandmarks (or sliding landmarks, SL), on the other hand, are ideal for studying smooth curves and surfaces.

This article is protected by copyright. All rights reserved

Author Manuscript

For our purposes here, semilandmarks allow us to capture changes in concavity/convexity (e.g., maxillary retrusion and nasal protrusion can alter the relationship between the midface and the upper and lower face as individuals age; [START_REF] Mendelson | Age-related changes of the orbit and midcheek and the implications for facial rejuvenation[END_REF]a n di np r o fi l e( d i fferent CFS regions have been shown to become more or less prominent with age, properties best studied in profile; [START_REF] Bartlett | Age-related changes of the craniofacial skeleton: an anthropometric and histologic analysis[END_REF] of regions displaying a very small number of traditional landmarks.

We examined three anatomical regions of the CFS in our subsample of 97 female baboons: facial skeleton (including the orbits, zygomatic arches, and palate), cranial vault, and cranial base (Fig. 1). These were selected because they are the traditionally recognized modules of the cranium relating to functional and developmental specializations [START_REF] Cheverud | Phenotypic, genetic, and environmental morphological integration in the cranium[END_REF][START_REF] Cheverud | Morphological integration in the saddle-back tamarin (Saguinus fuscicollis) cranium[END_REF].

[Figure 1

about here.]

We used Avizo Standard v 8.1.1 (FEI Visualization Sciences Group) to visualize the CT images, reconstruct the skulls, identify 43 craniometric landmarks (Table 2), and make surface mesh files. Viewbox v 4.0.1.7 (dHAL software, Athens, Greece) was then used to measure curve and surface SL on these surface meshes by applying a thin plate spline deformation to a template landmark configuration [START_REF] Gunz | Semilandmarks: a method for quantifying curves and surfaces[END_REF]. This protocol has been described elsewhere [START_REF] Heuzé | New insights into the relationship between suture closure and craniofacial dysmorphology in sagittal nonsyndromic craniosynostosis[END_REF]but,briefly ,afterbeingman ually placed on a single individual to create a template, the reference landmark configuration is fit to other individuals according to an algorithm that minimizes the bending energy while sliding SL along their identified curves and surfaces.

[Table 2 about for the alveoli, pyriform aperture, orbital margins, and zygomatic temporal processes. The forehead patch was defined as the triangular area bordered by the superior orbital margin and superior temporal lines. Vault patches covered the surface of the neurocranium from the superior temporal line/crest to the nuchal crest. The occiput consisted of the trapezoidal region bordered by the nuchal crest, posterior margin of the foramen magnum, and the mastoid processes. To construct the template, each patch was identified on the surface mesh by highlighting the desired area using the paintbrush tool in Viewbox. Then, we used the functions Distribute on mesh (random) followed by Diffuse points uniformly to associate the SLs with their corresponding patches. Our protocol resulted in a template with a total of 301 landmarks and SL to be identified for each individual (see Fig. 1 and Table 2).

Statistical shape analysis

AG e ne ra l i z e dP roc rus te ss upe ri m po s i ti o n [START_REF] Rohlf | Extensions of the Procrustes method for the optimal superimposition of landmarks[END_REF]Goodall, 1991)t or e m o v e the aspects of variation in which we are not interested (differences in overall size, relative orientation, and spatial position) results in shape data in the form of Procrustes shape coordinates (PSCs). To address the research questions, we performed a multivariate multiple regression ("primary shape regression") of the PSCs on centroid size (CS), chronological age (hereafter referred to as age), and number of teeth missing [START_REF] Mitteroecker | Advances in geometric morphometrics[END_REF]. This analysis tests the null hypothesis that post-maturational CFS variation is not structured by cranial size, age, or tooth loss. A Type II MANOVA is then used to test whether each explanatory variable is significant after controlling for the others. Centroid size is a method This article is protected by copyright. All rights reserved

Author Manuscript

of approximating size by calculating the square root of the sum of the squared distances of a set of landmarks from their centroid, the centroid being the mean x,y,z of all coordinates across all landmarks [START_REF] Klingenberg | Size, shape, and form: concepts of allometry in geometric morphometrics[END_REF]. This is important for determining whether CFS shape changes with age simply because adult individuals increase in size with age or because there are shape differences between younger and older adults that cannot be accounted for by size differences among the age categories. All analyses were performed in MorphoJ v 1.06d [START_REF] Klingenberg | MorphoJ: An integrated software package for geometric morphometrics[END_REF]a n dR S t u d i ou s i n gt h eMorpho v2 . 6 [START_REF] Schlager | Morpho and rvcg -shape analysis in R[END_REF] and geomorph v 3.0.6 [START_REF] Adams | Geomorph: software for geometric morphometric analyses[END_REF]p a c k a g e s .

To visualize the shape changes related to CS and age, heat maps were created by deforming the template surface mesh in Avizo [START_REF] Heuzé | Quantification of facial skeletal shape variation in fibroblast growth factor receptor-related craniosynostosis syndromes[END_REF]. The template surface mesh was shifted into shape space by morphing it according to the coordinates of the Procrustes mean shape. The PSCs for an individual at the extreme small end of the multivariate regression of shape on CS were calculated from the regression equation (small PSCs) and the same was done for an individual on the extreme large end (large PSCs). Then, "small" and "large" surface meshes were created by morphing the Procrustes mean shape mesh by the small and large PSCs, respectively. Finally, a displacement field was created using the Vertex Difference function, which calculates vectors between the vertices of each triangle in the reference surface mesh and the corresponding vertices of the homologous triangle in the target surface mesh. The reference surface mesh was then colored according to the magnitude of the vectors, allowing the viewer to discern "hot spots" of change between the two surface meshes. The small surface mesh was selected as the reference and the large as the target, so the visualized displacement field demonstrates regions that experience major change as individuals increase in size. The same procedure was done for age with the reference surface representing a younger adult and the target an older one, thus demonstrating regions where age-related differences are concentrated. It is important to emphasize that the differences represented by the heat maps are not those between an actual young/small individual and an This article is protected by copyright. All rights reserved Author Manuscript old/large one, but represent the extremes of the shape variation vectors that are statistically correlated with age and size, respectively.

1. Growth and allometry. We addressed the question of p ost-maturational growth in female baboons in three ways. First, we performed an ordinary least squares (OLS) regression of CS on age to test the null hypothesis that size of the CFS is static once sexual maturity is achieved. Second, we performed a one-way ANOVA to determine which, if any, of the age-groups differed in CS. Third, we looked at the effect of size on the shape of the cranium as a whole (i.e., allometry) by examining the coefficient of the CS term in the primary shape regression.

2. Age-related shape variation. We examined b oth age effects and the effect of to oth loss on global cranial shape variation. To identify whether shape differences exist among age groups, we calculated the consensus configuration (or mean shape) specific to each group.

For example, a YA mean shape was computed as the coordinate-wise average of the PSCs of the individuals in the youngest age category. These mean shapes were compared between the different age groups using the metric of Procrustes distance, the square root of the sum of the squared Euclidean distances between each landmark in a configuration and its homologous landmark in the sample mean configuration. To test for significant group differentiation, a Procrustes ANOVA was performed, which models the Procrustes distances among specimens, rather than explained covariance among variables (Goodall, 1991). Then, to determine how much of the global variation in CFS shape can be explained adequately by age, we examined the beta coefficient of the age term from the primary shape regression.

Finally, we tested the null hypothesis that tooth loss has no observable effect on the CFS in two ways. We compared the Procrustes distances of the ER and TL groups using ap e r m u t a t i o nt e s tt od e t e r m i n es t a t i s t i c a ls i g n i fi c a n c e . T h i st y p eo fa n a l y s i sc o n s i d e r s tooth loss as a binary variable, as ER individuals have all maxillary teeth present and TL individuals have lost at least one maxillary tooth. To perform a more nuanced analysis of the This article is protected by copyright. All rights reserved Author Manuscript effect of tooth loss on CFS shape, we also coded TL individuals by the number of maxillary teeth missing and performed a multivariate multiple regression ("dental shape regression") using the PSCs as dependent variables and CS, age, and number of teeth lost as independent variables.

3. Region-specific aging effects. PSCs were calculated separately for each of the three craniofacial regions by performing three distinct Generalized Procrustes superimpositions. The resulting alignments are referred to as the vault PSCs (n = 110 landmarks), base PSCs (n =5 0 ) ,a n df a c eP S C s( n=1 4 1 ) . Am u l t i v a r i a t em u l t i p l er e g r e s s i o nw a spe r f o r m e df o re a c h of the regional PSCs ("[region] shape regression"). All three regressions contained the same three explanatory variables as the primary shape regression (CS, age, number of teeth lost).

Procrustes ANOVA was used to test for significant age-group differentiation in the morpho space of each craniofacial region.

Results

The effects of age on size

First, we assessed the impact that aging has on cranial size. There is a 1.8% increase in CS between the YA and AD groups, a 2.8% increase between the YA and ER groups, and a 2.4% increase between the YA and TL groups (Fig. 2). If the three older groups are combined, the pooled mean CS is 2.4% larger than the YA mean CS, and this difference is statistically significant (Welch's t-test: XYA =9 2 7 .5, Xpooled =9 4 9 .9,t = -3.76,P =0 .0004). Age explains 14.5% of the variance in CS (OLS: t =4.2,P < 0.001; Fig. 3).

[Figure 2 This article is protected by copyright. All rights reserved

Author Manuscript

The effects of size on shape

In the primary shape regression, the CS term is highly significant (F =4.37,P =0.001) and accounts for 4.3% of the variance in the PSCs (Table 3; Supp. Fig. S1). Larger individuals have a longer snout, a more narrow midface and shorter posterior facial height, smaller and more ellipsoid orbits, and a neurocranium that is more ovoid in shape. Smaller individuals are more brachycephalic and have large, round orbits and more flaring malars (Supp. Videos 1-4).

[Table 3 about here.]

The effects of age on shape

To determine whether the four groups differed in CFS shape, Procrustes distances between the PSCs of the groups were calculated (Table 4). The YA group is significantly distinct from the other three in the morphospace, which cluster together (Supp. Fig. S2). This result is confirmed with a Procrustes ANOVA. When comparing all four groups, the F-statistic is marginally significant (F =1 .48,P =0 .06), but when pooling the AD, ER, and TL groups, the shape of the YA group is significantly different from that of the older individuals (F =3.02,P =0.006).

[Table 4 about here.]

In the primary shape regression, age accounts for a modest but significant 2.7% (P = 0.005) of the sample variation (see Table 4.Y o u n gi n d i v i d u a l sa r ec h a r a c t e r i z e db yg l o b u l a r neurocrania, wide midfaces, and a more flexed cranial base, while elderly individuals have a more elongated and widened snout, prominent inion, concave nasal bridge with a retruded midface, and a flattened nuchal plane (Supp. Videos 5-8). Although the size and age effects share some commonalities (e.g., both young and small individuals have shorter snouts than This article is protected by copyright. All rights reserved Author Manuscript older and larger individuals; Fig. 4), there are some important distinguishing features.

Overall, the allometric trend gives a sense of a small cranium being stretched in the sagittal plane, primarily in the facial skeleton, to produce a large one. Additionally, the premaxillary dentition is displaced inferiorly, resulting in an increasing degree of klinorhynchy as crania increase in size. In contrast, the age trend is best characterized as an anterior and superior movement of the anterior face in the sagittal plane and a recession of the midface with a concomittant protrusion of the midline upper face.

[Figure 4 about here.] Two methods were applied to determine the effect of tooth loss as quantified in either a binary or an ordinal manner. A Procrustes ANOVA performed only on the ER and TL groups indicates the two are not significantly different (F =0 .64,P =0 .80). This suggests that, in a global sense, whether an individual has lost any teeth has no marked effect on shape of the CFS. The second method, the dental shape regression, provides a more nuanced examination by testing the effect of the number of teeth lost. Although 9.0% of CFS shape variation within the TL category is accounted for by the number of teeth missing, after correcting for multiple comparisons (within-model Bonferroni; [START_REF] Mundfrom | Bonferroni adjustments in tests for regression coefficients[END_REF])t h i s effect is rendered non-significant (α = 0.05 3 =0 .017,P =0 .038; see Table 3). As statistical power may be limited by the small number of individuals within each TL sub-category (range: 1-10, mean: 4.6), future research may yet provide stronger evidence for a link between tooth loss and facial shape. Our results suggest that individuals with a greater number of teeth missing tend to have larger positive regression scores for the dental shape regression (Supp. Fig. S3), which are associated with a more arched palate, a superior and posterior shift of all alveolar landmarks, a more horizontal premaxillary profile, and a slightly wider breadth across the midface and orbits.

This article is protected by copyright. All rights reserved

Author Manuscript

Size and age effects by region

According to the region-specific Procrustes ANOVA, there were no group differences in shape of the vault (F =0 .89,P =0 .62) or base (F =1 .16,P =0 .23), but the face PSCs differed significantly among the age categories (F =2.10,P =0.008). Using Procrustes distance as ametric,Y Aindividualsareseparatefromallothergroupsinthefacialmorphospace(Supp. Table S2).

Centroid size explains 2.8% (P =0.007) of the total variation in vault PSCs (see Table 3), with a contrast between dolicocephalic large individuals and brachycephalic small ones. Age accounts for a marginally insignificant 1.8% (P =0 .059) of the variation, with the vaults of older animals demonstrating increased postorbital constriction, a protruding glabellar region, an anterior flattening, and a particularly pointy inionic region.

Only CS has a significant effect on cranial base shape variation. A modest 2.5% (P = 0.006) of the total cranial base variation is size-related and an insignificant 1.4% (P =0.12) is age-related. The major effect of allometry is a vertical compression of the basicranium in larger individuals. In contrast, age has the effect of rotating the basicranium with the fulcrum essentially located at the landmark OPI. The result is an occipital bone that is tilted backward along its superior margin at the nuchal crest, external auditory meati that extend posteriorly, a foramen magnum that is displaced superiorly at its anterior margin, and a less flexed posterior cranial base (i.e., a more horizontally positioned basioccipital).

Finally, the facial skeleton is affected to varying degrees by all three independent variables. Of the total facial variation, CS accounts for 9.9% (P =0 .001), age for 3.8% (P =0 .001), and tooth loss for 1.3% (P =0 .066). Older individuals have a longer snout that is also wider across the anterior palate than their younger conspecifics. Large individuals also have a longer snout, but there is no difference in width when compared to smaller individuals. Both advanced age and increased size are associated with a more narrow upper face, but this is due to a disproportionate decrease in the width of the fronto-zygomatic This article is protected by copyright. All rights reserved

Author Manuscript

process in older individuals, whereas it is the width of the orbit itself that is reduced in large individuals. The glabellar and supraorbital regions are only variable along the age axis, with both increasing in prominence in older individuals. The maxillary dentition is placed more anteriorly in large and old individuals relative to small and young ones. However, the allometric trend affects the entire oral cavity, displacing both the palate and dentition forward, while age alters the depth of the palate, the relationship between the palate and maxillary teeth, and the angle between the palate and the bones comprising the posterior nasopharynx.

Discussion

Much research has been conducted to describe patterns of growth and development in ontogenetic series of non-human primates, but the effect of post-maturational aging on the CFS remains understudied. Researchers are well aware that cranial morphology changes, often drastically, before individuals reach sexual maturity (e.g., in Papio: [START_REF] Leigh | Sexual dimorphism in the baboon facial skeleton[END_REF][START_REF] Collard | Ontogeny and homoplasy in the papionin monkey face[END_REF][START_REF] Singleton | Postnatal cranial development in papionin primates: an alternative model for hominin evolutionary development[END_REF]. Consequently, such ontogenetic patterns are typically accounted for, or at least acknowledged, in studies of both extant and fossil taxa. However, research on modern and historic geriatric human samples and on a select few non-human primate populations indicates that portions of the CFS demonstrate age-related changes in mature individuals. Our data add to this body of literature by demonstrating age-related craniofacial variation in a sample of sexually mature female baboons.

We suggest the combination of our data and those presented in the literature lends support to the idea that there exists in primates a more general post-maturational aging pattern that has heretofore been inadequately studied. Whether such a pattern extends to other mammalian orders largely remains to be seen, although there is some evidence for variation in both adolescent and post-maturational growth patterns of the carnivoran CFS, with some felid genera developing past the age of sexual maturity (e.g., Segura and Prevosti, This article is protected by copyright. All rights reserved Author Manuscript 2012; [START_REF] Segura | Cranial ontogeny in the Puma lineage, Puma concolor, Herpailurus yagouaroundi, and Acinonyx jubatus (Carnivora: Felidae): a three-dimensional geometric morphometric approach[END_REF][START_REF] Segura | A three-dimensional skull ontogeny in the bobcat (Lynx rufus) (Carnivora: Felidae): a comparison with other carnivores[END_REF]. Surprisingly, our data do not document a significant degree of tooth loss-related modification of the midface and palate. Primarily, we observed morphological change near muscle attachment sites of the CFS and in individuals who retain all their teeth. These results suggest that the age-category "adult" is heterogeneous and we should be aware that it encompasses individuals demonstrating the effects of age on their CFS, regardless of their dental status.

Allometric patterns and how they differ from age-related ones

When visualizing the age-and size-related changes in the female baboon CFS, it appears that some of the regions experiencing the biggest shape changes are the same in both cases (see Fig. 4). However, there are subtle differences between the two. For example, the patterns of large-scale increase in facial length differ when considering solely age-or sizerelated variation. The snout of older individuals is primarily elongated in the sagittal plane only. In contrast, allometry displaces the premaxilla obliquely, both forward and downward.

Additionally, with the major exception of the neurocranium, which is more globular in small females and narrows with increasing size, form differences associated with allometry are almost exclusively the result of increase in size of various cranial dimensions (i.e., osteoblastic activity on ectocranial surfaces coupled with osteoclastic activity on endocranial ones). The same cannot be said for age-related shape variation, which appears to result from osteoclastic activity on both endo-and ectocranial surfaces. The most obvious example of this difference is the midfacial remodeling of older females, which produces a marked concavity that is not seen in younger individuals and does not differ between small and large individuals.

As many of the regions showing localized differences between small and large crania are where major muscles attach (e.g., nuchal crest, inferior zygomatic arch, temporal surface, and pterygoid plates), it seems reasonable to suggest that an allometric increase in muscle mass with increasing body size (e.g., [START_REF] Alexander | Allometry of the leg muscles of mammals[END_REF][START_REF] Pollock | Allometry of muscle, tendon, and elastic energy storage capacity in mammals[END_REF] This article is protected by copyright. All rights reserved Author Manuscript may contribute to size-related variation in the CFS. Because the variance in adult male baboon body weight (27.01 ± 4.72 kg, N =2 4 7 ; [START_REF] Leigh | Growth and development of baboons[END_REF]i ss i g n i fi c a n t l yg r e a t e r( F = 0.513,P < 0.0001) than that of females (15.96 ± 3.38 kg, N =215; [START_REF] Leigh | Growth and development of baboons[END_REF], we would expect allometry to account for a larger proportion of craniofacial variation in a dataset of adult male baboons that was analyzed in a comparable manner to our present analysis of adult female baboons. Additionally, given that (a) numerous studies have identified sexual dimorphism in patterns of age-related variation of human and non-human primate CFS (e.g., [START_REF] Wang | Ontogeny and diachronic changes in sexual dimorphism in the craniofacial skeleton of rhesus macaques from Cayo Santiago, Puerto Rico[END_REF][START_REF] Williams | Regional shape change in adult facial bone curvature with age[END_REF][START_REF] Balolia | Sexual dimorphism and facial growth beyond dental maturity in great apes and gibbons[END_REF]and(b)sexualdimorphism in both cranial size and shape is extremely large in baboons (e.g., [START_REF] Leigh | Sexual dimorphism in the baboon facial skeleton[END_REF][START_REF] Higgins | Sexual dimorphism and facial growth in papionin monkeys[END_REF][START_REF] Singleton | Patterns of cranial shape variation in the Papionini (Primates: Cercopithecinae)[END_REF], we could expect to identify different patterns of age-related variation in adult male baboon crania than those we have observed in females.

The remaining hot-spot not related to a muscle attachment site on the size-related panel of Figure 4 is the anterior rostrum. This is the same region that is highly sexually dimorphic in baboons (e.g., [START_REF] Leigh | Cranial ontogeny of Papio baboons (Papio hamadryas)[END_REF][START_REF] Collard | Ontogeny and homoplasy in the papionin monkey face[END_REF][START_REF] Singleton | Patterns of cranial shape variation in the Papionini (Primates: Cercopithecinae)[END_REF]. As males are also significantly larger than females in body size (e.g., [START_REF] Leigh | Growth and development of baboons[END_REF], it is not surprising that the crania of larger females appear more "male-like" in having a relatively long snout. This supports the suggestion that the extreme prognathism of Papio and its sister taxon Mandrillus,isrelatedtotheirincreasedbodysizerelativ etosmaller-bodiedpapionins,suc h as Macaca, Cercocebus, and Lophocebus.W ew e r ea b l et ot e s tf o rc o -v a r i a t i o nb e t w e e nb o d y and cranial size in our sample of female baboons because longitudinal body weight records were collected opportunistically from birth to 15 years of age (see [START_REF] Joganic | Additive genetic variation in the craniofacial skeleton of baboons (genus Papio) and its relationship This article is protected by copyright. All rights reserved to body and cranial size[END_REF],f o r more details). Female adult body weight explains a significant 14.8% (OLS: F =17.7,P < 0.0001) of the variation in overall CFS centroid size and a varying amount of the variation within each region (face: R 2 =0 .171,P < 0.0001; base: R 2 =0 .124,P =0 .0003; vault: R 2 =0 .054,P =0 .02). In female baboons at least, these findings are consistent with the This article is protected by copyright. All rights reserved hypothesis of [START_REF] Joganic | Additive genetic variation in the craniofacial skeleton of baboons (genus Papio) and its relationship This article is protected by copyright. All rights reserved to body and cranial size[END_REF], which posits that larger-bodied papionin species tend to have similar-looking longer faces because approximately 15% of the genetic variation underlying morphological variation in both body size and CFS shape is shared.

Age-effects are global but differ by region in magnitude

According to Israel (1973a), human crania exhibit "virtual magnification," whereby the percent size increase (5-7%) is uniform and symmetrical across regions as adults age. The only exceptions he identified were in the sizes of the sella turcica (9-22%) and paranasal sinuses (7-12%) and in cranial vault thickness (1-11%). All other dimensions demonstrated less gain (0-5%). We found a subtly different pattern in our sample of female baboons (Supp. Fig. S4). The centroid size of both the cranial vault and base increased 1.8% and 1.5%, respectively, while in the facial skeleton it increased 3.3%. Even though the differences are slight, we suggest they may reflect what we know of baboon biology. Most of the ontogenetic change that occurs late in the CFS is primarily in the face, particularly the lengthening of the snout, especially in males (e.g., [START_REF] Collard | Ontogeny and homoplasy in the papionin monkey face[END_REF][START_REF] Leigh | Cranial ontogeny of Papio baboons (Papio hamadryas)[END_REF]. Although the focus in the literature is primarily on the effect that this phenomenon has on the male CFS, our results suggest that it may be relevant when considering female cranial morphology as well.

Most of the age-related changes we observed in our sample are concentrated in the facial skeleton, although the nuchal region is affected also (Fig. 5a-5d ). As sutural fusion and subsequent obliteration likely prevent depositional growth at sutural margins, especially in the cranial vault and basicranium (see [START_REF] Opperman | Cranial sutures as intramembranous bone growth sites[END_REF] for a review), the size and shape variation associated with increasing age in our sample are likely the result of appositional growth along ectocranial surfaces. In some regions, such as the midsagittal profile of the superior neurocranium, this is coupled with endocranial resorption that serves to maintain the same cranial vault thickness in YA and older age groups. Other areas, such as the supraorbital and inionic regions, experience a disproportionate degree of ectocranial deposi-This article is protected by copyright. All rights reserved

Author Manuscript

tion relative to endocranial resorption (remodeling), resulting in a thickening of the involved cranial bones (see Fig. 5e).

[Figure 5 about here.]

Potential role for extended ontogenetic trajectories

Previous research in baboons has suggested skeletal growth, including upper facial height and cranial length, generally ceases in females by the age of 5 years [START_REF] Leigh | Growth and development of baboons[END_REF], around the time when sexual maturity is achieved [START_REF] Altmann | Physical maturation and age estimates of yellow baboons, Papio cynocephalus, in Amboseli National Park, Kenya[END_REF]. Our data provide an opportunity to more thoroughly test this hypothesis, as well as to examine potential changes in shape that may occur in the CFS of female baboons after this age. We found that female baboon cranial shape changes and its size increases between young adulthood (ages 7-16 years) and older age (> 18 years).

Our results raise interesting questions about the potential for various intrinsic and extrinsic factors to pattern variation in the post-maturational CFS. Due to large canine size, especially in males, canine development is relatively extended in baboons. Research on mandibular teeth in captive baboons by [START_REF] Swindler | Dental development of the permanent mandibular teeth in the baboon, Papio cynocephalus[END_REF]d e m o n s t r a t e st h a t time from initial crown calcification to apical closure in canines is ∼5.5 years, while for all other tooth types it ranges from 2.4-3.7 years. However, despite this prolonged canine development, the size and shape changes we have observed in the YA female face cannot be linked to anterior dental development because maxillary canine length has reached its adult size by 5 years of age in captive females (Table 5)a n da p i c a lc l o s u r e ,a tl e a s to ft h e mandibular canine, occurs by 6.3 years of age [START_REF] Swindler | Dental development of the permanent mandibular teeth in the baboon, Papio cynocephalus[END_REF]. Although age at apical closure for the maxillary dentition is unknown, age at eruption for the maxillary (4.1 years) and mandibular (3.9 years) canines differs by very little [START_REF] Phillips-Conroy | Dental eruption schedules of wild and captive baboons[END_REF], so it stands to reason that apical closure of the maxillary canine is also not more than a few months later than that of the mandibular canine.

This article is protected by copyright. All rights reserved

Author Manuscript

[Table 5 about here.] Size and shape change of the CFS of young adult female baboons also cannot be attributed to the appearance of the last tooth to erupt, the M3, which occurs at ages 6.2 (mandibular) and 7.1 (maxillary) years in captive females [START_REF] Phillips-Conroy | Dental eruption schedules of wild and captive baboons[END_REF], because eruption proceeds in a very rapid manner. In captive males, the mandibular M3 erupts at 6.3 years and apical closure is achieved by 6.5 years [START_REF] Swindler | Dental development of the permanent mandibular teeth in the baboon, Papio cynocephalus[END_REF].

Although this M3 completion time is only for mandibular teeth in males, there is no evidence to suggest estimates of the length of time between eruption and apical closure in maxillary teeth differs from that of mandibular teeth. Additionally, evidence from humans, macaques, and baboons demonstrates that the general trend is for teeth to develop slightly earlier in males but more rapidly in females, such that female dental development is actually completed earlier than that of males (e.g., Garn et al., 1958;[START_REF] Swindler | Dental development of the permanent mandibular teeth in the baboon, Papio cynocephalus[END_REF][START_REF] Swindler | Sex differences in permanent mandibular tooth development in Macaca nemestrina[END_REF]. Finally, although [START_REF] Swindler | Dental development of the permanent mandibular teeth in the baboon, Papio cynocephalus[END_REF]d i dn o th a v ee n o u g h individuals to provide estimates of variation in age at apical closure for the mandibular M3, the range of standard deviation estimates for the other tooth types is 0.13-0.54 years.

Therefore, all forces that may be exerted by dental eruption on the CFS cease before individuals are ∼7.5 years old. Yet, the female baboon CFS increases in size by 2.4% and changes shape between the ages of 7 and 16.

The fact that the greatest differences are between the YA group and all older baboons lends indirect support to the idea that growth in the CFS may not cease upon achieving sexual maturity. It is interesting to note that the late-stage ontogenetic patterns in Pan cranial development identified by Mitteroecker et al. (2004b)( t oi n c l u d eas u p e r i o rs h i f to f the nuchal plane, an elongation of the anterior midface, and an enlargement of the torus supraorbitalis) appear remarkably similar to the differences between YA and older baboons that we have observed. Furthermore, [START_REF] Balolia | Sexual dimorphism and facial growth beyond dental maturity in great apes and gibbons[END_REF]d e m o n s t r a t ec o n t i n u e dg r o w t ho f the CFS in young adults of multiple species of non-human primates, which they interpret This article is protected by copyright. All rights reserved

Author Manuscript

to be an extension of ontogenetic trajectories into adulthood. However, without a complementary ontogenetic sample in which to measure growth vectors to determine if the shape changes prior to achieving sexual maturity parallel those following it, we cannot definitively determine if the patterns we have observed are the result of ontogeney extending into "adulthood."

On the other hand, if future research demonstrates that the developmental trajectories of ontogenetic and adult samples of baboons are divergent, it is less likely that changes in the adult CFS are the result of either intrinsic growth mechanisms or the influence of adaptive growth centers that accommodate change among adjacent cranial components during ontogeny. Rather, it is more likely that factors that respond to extrinsic environmental cues, such as ambient temperature, stress and strain from muscular activity, and idiosyncratic behaviors, may play more pivotal roles in structuring adult craniofacial variation. It is also possible that, as has been shown in human research (e.g., [START_REF] Crothers | Vertical height differences in subjects with severe dental wear[END_REF][START_REF] Johansson | Covariation of some factors associated with occlusal tooth wear in a selected high-wear sample[END_REF], both dental wear and intra-population variation in dental wear could contribute to observable differences in the CFS of adult baboons. However, it may be difficult to disentangle the effects related to dental wear and those related to increased usage of masticatory musculature, which also has been shown to pattern CFS shape and is often the cause of excessive dental wear (e.g., [START_REF] Krogstad | Dento-facial morphology in patients with advanced attrition[END_REF][START_REF] Waltimo | Bite force and dentofacial morphology in men with severe dental attrition[END_REF][START_REF] Kiliaridis | Masticatory muscle influence on craniofacial growth[END_REF][START_REF] Kiliaridis | Craniofacial morphology, occlusal traits, and bite force in persons with advanced occlusal tooth wear[END_REF]. of each new permanent post-canine tooth, the maxilla is repeatedly displaced anteriorly [START_REF] Oyen | Palatal Growth in Baboons (Papio cynocephalus anubis)[END_REF]. This lengthens the load arm of the TMJ, thus theoretically decreasing the biomechanical efficiency of the entire masticatory apparatus. However, subsequent research This article is protected by copyright. All rights reserved

The effects of mastication on the aging baboon cranium

Author Manuscript

demonstrates that very little strain is actually experienced in the circumorbital region (e.g., [START_REF] Hylander | Function of the supraorbital region of primates[END_REF][START_REF] Ross | Bone strain gradients and optimization in vertebrate skulls[END_REF], although [START_REF] Dechow | Edentulation alters material properties of cortical bone in the human craniofacial skeleton: functional implications for craniofacial structure in primate evolution[END_REF]d i do b s e r v e thinner cortical bone with reduced directional orientation across the cranium and including the browridge, suggesting that masticatory forces do play a role in maintaining CFS bone mass. [START_REF] Dechow | Occlusal force and craniofacial biomechanics during growth in Rhesus monkeys[END_REF] directly tested Oyen's model by conducting biomechanical and comparative anatomical analyses in immature and adult macaques, phylogenetic cousins of baboons. They were able to demonstrate that the mechanical advantage of the masticatory muscles considered (masseter and temporalis) actually increased 7-14% for molar biting between juvenile and sexually mature individuals. As their muscle force data suggest that adult macaques actually have more efficiently placed masseters, they argue that the biomechanial model of [START_REF] Oyen | Palatal Growth in Baboons (Papio cynocephalus anubis)[END_REF]i si n v a l i d . [START_REF] Dechow | Occlusal force and craniofacial biomechanics during growth in Rhesus monkeys[END_REF]s u g g e s t their unexpected results are either due to a disproportionate growth in the anterior portion of the muscle or a mechanically advantageous shift in how the teeth are positioned relative to muscle attachment sites.

We observed b oth an anterior displacement of the teeth relative to the zygomaxillary suture (the anterior-most extent of the superficial masseter origin) and a posterior displacement of the zygomatic bone itself with little relative movement of the glenoid fossa (see Fig. 5f ). According to the biomechanical model presented by Dechow and Carlson (1990, Fig. 2), these changes in facial architecture as adults age should have an effect on masticatory efficiency. The result would be an increased molar biting moment (load) arm and a decreased masseter muscle moment (lever) arm. As mechanical advantage of a muscle is defined as the ratio between lever and load arms, the change in facial configuration from the YA to older age categories should have the effect of decreasing superficial masseter efficiency, as suggested by [START_REF] Oyen | Palatal Growth in Baboons (Papio cynocephalus anubis)[END_REF]. An additional issue to be considered is that increasing tooth wear and the potential for changes in manidbular ramus height as individuals age can alter masseter orientation, thus affecting its action and potentially its efficiency, as well. Although

This article is protected by copyright. All rights reserved Author Manuscript anatomical arrangement of macaque and baboon masticatory musculature is incredibly similar (JLJ, pers. obs.; [START_REF] Schwartz | Morphology of the head and neck of the macaque monkey: the muscles of mastication and the mandibular division of the trigeminal nerve[END_REF], the baboon facial skeleton is much more prognathic than that of macaques, which may explain the discrepancy between the results of [START_REF] Oyen | Palatal Growth in Baboons (Papio cynocephalus anubis)[END_REF]a n dt h o s eo fDechow and [START_REF] Dechow | Occlusal force and craniofacial biomechanics during growth in Rhesus monkeys[END_REF].

Our morphological data suggest that, even though the age-effects we have observed in adult female baboons are small in magnitude, the associated CFS size and shape changes occur in regions with the potential to impact masticatory behavior. However, biomechanical analyses on the baboon masticatory system need to be conducted to test whether muscular advantage changes over the course of both adolescent growth and post-maturational size and shape change. If future research is able to demonstrate that even subtle morphological change in adult craniofacial structure, such as the shape variation we have identified in our data, potentially alters masticatory effeciency, then the resulting modification to biomechanical loading regimes could induce additional changes in the CFS, as [START_REF] Oyen | Craniofacial growth in olive baboons (Papio cynocephalus anubis): browridge formation[END_REF]suggest(seealsoKiliaridis 1995). In fact, the subtle decrease in orbital circumference that is visible along the superior orbital margin may be a manifestation of changes in the masticatory complex. Alternatively, it could be related to the fact that the frontal sinus enlarges with age, which may influence age-related changes in the shape of the orbits [START_REF] Fatu | Morphometric evaluation of the frontal sinus in relation to age[END_REF]. Much more work needs to be done to determine whether such intraindividual variation has the potential to affect evolutionary fitness over the course of an individual's lifetime.

Finally, modifications in craniofacial configuration are not only observed in relation to morphological features associated with mastication. The other main feature that distinguishes YA and older crania is the increasing prominence of the inionic region as individuals age (see Fig. 5a). Interestingly, despite having absolutely larger muscles, large individuals do not have an appreciably more developed inion than their smaller conspecifics. This indicates that allometric shape differences may help larger animals withstand the increased force gen-This article is protected by copyright. All rights reserved Author Manuscript erated by larger muscles, but not necessarily their repetitive actions. As an indirect method of testing this hypothesis, we compared the mean difference in shape between five each of the smallest YA and older individuals and between five each of the largest YA and older individuals. Shape was quantified as an individual's regression score from the multivariate regression of PSCs on age. Although the larger individuals are located in a different area of the morphospace (average regression scores for small: XYA = -0.0248, Xpooled = -0.0065; large: XYA = -0.0066, Xpooled =0 .0130; Supp. Fig. S5), the amount of shape difference between the younger and older individuals (small: 0.0183, large: 0.0196) is essentially the same in both size categories.

These results, coupled with the observed pattern of shape change in the adult CFS, suggest that bone remodeling in response to cyclical bone strain from repetitive muscle use over the course of an individual's life is potentially a major factor in patterning age-related changes in craniofacial form in adult female baboons, regardless of the size of either the individual or their masticatory muscles. Given that age-related shape change appears to be concentrated at muscle attachment sites, including the nuchal region, we propose that future research should aim to further our knowledge of cranial-postcranial integration. Vertebral anatomy and its relationship to basicranial anatomy and posture in primates is the subject of some research (e.g., [START_REF] Russo | Foramen magnum position in bipedal animals[END_REF][START_REF] Nalley | Functional morphology of the primate head and neck[END_REF][START_REF] Villamil | Phenotypic integration of the cervical vertebrae in the Hominoidea (Primates)[END_REF], but our results demonstrate that its effect on cranial vault anatomy via the link with the nuchal musculature should be assessed as well.

Conclusion

We have demonstrated that the size and shape of the CFS in female baboons change moderately past the age of sexual maturity and into early adulthood. Contrary to expectations based on previous knowledge about the effects of tooth loss and accompanying alveolar resorption, it is actually the YA category that is most different from the other age groups.

This article is protected by copyright. All rights reserved

Author Manuscript

Younger and older adults differ most in regions related to muscle attachment sites, suggesting a possible role for repetitive masticatory stress in patterning bone remodeling in the adult CFS. The facial skeleton experiences the greatest amount of age-and size-related change. A complementary and detailed geometric morphometric study of post-maturational modifications in the human CFS could provide insight into mechanisms of aging that are shared or differ across primate taxa. Maximum: top whisker and number above it, 3rd quantile: top hinge, median: waist, confidence interval of the median: notch, mean: boldface number above the waist, standard deviation of the mean: italic typeface number below the waist, 1st quantile: bottom hinge, minimum: bottom whisker and the number below it. The confidence interval of the median is calculated as: median ± 1.58 × IQR √ n . In other words, these are areas that change the most with increasing age and size. Because the color scales are relative, the magnitude of the differences should not be compared between the allometric and age-related meshes.

Figure 5: Detailed depictions of age-related changes in the baboon CFS.I ne a c h panel, two surface meshes are superimposed. They were obtained by morphing the mean shape by the fitted regression coordinates for the youngest (pink mesh) and oldest (gray mesh) individuals from the multivariate regression of Procrustes shape coordinates (PSCs) on age. In panels ab , d and f,t h ey o u n gm e s hi sp a r t i a l l yt r a n s p a r e n t ;i np a n e lc,i ti s the old mesh; and in panel e,bo t hm e s h e sa r ef u l l yo p a q u e . T h es y m bo l si ne a c hp a n e la r e as follows: (a)G r e e nt r i a n g l e s ,n u c h a ll i g a m e n ta n dm e d i a lt r a p e z i u sa t t a c h m e n t ;o r a n g e triangles, deep masseter attachment; magenta arrows, primary This article is protected by copyright. All rights reserved

Author Manuscript

Tables Table 1: This article is protected by copyright. All rights reserved

Author Manuscript

Table 2: Anatomical landmarks. Names, abbreviations, descriptions, and characteristics of the craniometric landmarks measured on female baboon crania. This article is protected by copyright. All rights reserved

Landmark

Author Manuscript

Table 3: Shape regression statistics. Type II MANOVA results from primary, dental, and region-specific shape regressions of Procrustes shape coordinates (PSCs) on centroid size (CS), age, and the number of teeth missing. The coefficient of determination (R 2 ), F -statistic, and P -value are provided for each independent variable included in the models. Values indicated in bold typeface are significant at an α = 0.05 3 =0 .017, a Bonferroni correction applied to account for multiple comparisons within each model [START_REF] Mundfrom | Bonferroni adjustments in tests for regression coefficients[END_REF]

  Al a r g ec o l o n yo fb a b o o n s( Papio ssp.) is maintained by the Southwest National Primate

  here.] The template we constructed consisted of four curves and six surfaces. An open curve of 15 SL was fit to each orbital margin (supraorbital notch to a point on the medial orbital This article is protected by copyright. All rights reserved Author Manuscript margin at the level of nasion) and an open curve of 9 SL to each zygomatic arch (frontozygomatic foramen to superior zygotemporal suture) for a total of 48 curve SL. The 210 surface SL were divided among six patches located on the left and right face (30 SL each), forehead (30 SL), left and right vault (40 SL each), and occiput (40 SL). Facial patches covered the entirety of the anterior surfaces of the malars, nasals, maxillae, and premaxillae, except

  about here.] [Figure 3 about here.]

  [START_REF] Oyen | Craniofacial growth in olive baboons (Papio cynocephalus anubis): browridge formation[END_REF]d e v e l o p e dam o d e lf o rPapio browridge formation positing that cyclical deposition of supraorbital bone as a response to recurrently increased masticatory stress produces the distinctive torus seen in adult baboons, particularly males. With the eruption

Figure 1 :

 1 Figure1: Craniometric landmarks, semilandmarks, and cranial regions.L o c ations of the craniometric landmarks color-coded by type: anatomical (black); orbital and zygomatic curve semilandmarks, SL (white); facial, vault, and occipital surface SL (pink); frontal surface SL (gold). Only the anatomical landmarks are identified and the corresponding abbreviations are provided in Table2.D e s c r i p t i o n so fa n a t o m i c a lf e a t u r e su s e dt od e fi n e the surface SL patches are provided in the text. The three anatomical regions are indicated by colored shading: face (green), base (yellow), and vault (lavender).

Figure 2 :

 2 Figure 2: Centroid size boxplot.C o m p a r i s o no fc e n t r o i ds i z ed i s t r i b u t i o na c r o s sa g e groups.Maximum: top whisker and number above it, 3rd quantile: top hinge, median: waist, confidence interval of the median: notch, mean: boldface number above the waist, standard deviation of the mean: italic typeface number below the waist, 1st quantile: bottom hinge, minimum: bottom whisker and the number below it. The confidence interval of the median is calculated as: median ± 1.58 × IQR √ n .

Figure 3 :

 3 Figure 3: Growth maturation. Centroid size regressed on chronological age. Individuals are color-coded by age category, as indicated by the key in the top left corner. The OLS regression line is indicated with a solid black line and its formula and associated regression statistics are provided in the bottom right corner.

Figure 4 :

 4 Figure4: Vertex difference surface morphs. Heat maps of the distances between vectors comprising the triangular surface mesh of a small individual and the corresponding vectors of a large one (left panels) and between those of a young adult and the corresponding ones of an older individual (right panels). Regions shown in red are further apart between the two meshes than those shown in blue. In other words, these are areas that change the most with increasing age and size. Because the color scales are relative, the magnitude of the differences should not be compared between the allometric and age-related meshes.

  This article is protected by copyright. All rights reserved Author Manuscript

FM1

  The region of the cranium in which the landmark is located: vault (V), base (B), face (F). 2 Whether the landmark is located in the midline (M) or bilaterally (B).

  Sample demographics. Sample composition of the different age categories, the age ranges of included individuals in years, and the human equivalent age range, based on the worldwide average female human life expectancy of 73.1 years (United Nations, 2017).

	Age Category	N	Mean (SD)	Min	Max	Human
	Young Adult (YA)	25	11.56 (3.0)	7.12	16.20	15.7-35.8
	Adult (AD)	25	20.00 (1.3)	17.97	22.55	39.7-49.9
	Elderly (ER)	25	27.23 (2.5)	23.76	31.69	52.5-70.0
	To oth Loss (TL)	22	26.92 (2.3)	23.74	32.47	52.5-71.8

Table 2 -

 2 Continued from previous page

	Landmark	Symbol Description	Region 1 Located 2
	Nasion	NAS	Intersection of the internasal su-
			ture and the frontal bone

Table 4 :

 4 . Procrustes distance matrix. Intergroup Procrustes distances (above the diagonal) and their significance based on permutation tests (below the diagonal). Distances that are significantly large at α =0.05 and their corresponding P-values are shown in bold typeface.

	Shape Reg.	Centroid Size R 2 FPR 2	Age FPR 2	To oth Loss FP
	Primary	0.043	4.37 0.001 0.027	2.77 0.005 0.010	1.02 0.317
	Dental	0.082	1.90 0.041 0.036	0.83 0.392 0.090	2.08 0.038
	Vault	0.028	2.76 0.007 0.018	1.73 0.059 0.005	0.45 0.939
	Base	0.025	2.49 0.006 0.014	1.37 0.121 0.007	0.69 0.728
	Face	0.099 10.97 0.001 0.038	4.21 0.001 0.013	1.46 0.066

This article is protected by copyright. All rights reserved

Author Manuscript

This article is protected by copyright. All rights reserved Author Manuscript

Table 5 :

 5 Female baboon canine growth. Maximum buccal length (mm) of the maxillary canines as measured from canine tip to gum line on anesthetized female baboons, from[START_REF] Leigh | Growth and development of baboons[END_REF].

	Age (yrs)	N	Length (SD)	Age (yrs)	N	Length (SD)
	32	2 . 0 5 ( 2 . 9 )	1 04	9 . 6 5 ( 1 . 9 )
	42 1	8 . 3 0 ( 2 . 4 )	1 15	1 1 . 9 4 ( 1 . 4 )
	52 8	1 0 . 3 7 ( 1 . 5 )	1 22	9 . 1 0 ( 1 . 3 )
	61 7	1 0 . 2 5 ( 0 . 7 )	1 62	8 . 8 0 ( 1 . 4 )
	78	1 0 . 7 3 ( 0 . 6 )	1 92	9 . 6 0 ( 0 . 1 )
	85	1 1 . 6 5 ( 1 . 2 )	2 02	1 2 . 7 0 ( 0 . 7 )
	93	1 0 . 7 2 ( 1 . 6 )

Acknowledgements

Funding for specimen curation at Washington University in St. Louis and CT scan acquisition at the Washington University School of Medicine Mallinckrodt Institute of Radiology was provided by NSF BCS-0725068 and BCS-0523637. This study received financial support from the French State in the framework of the "Investments for the Future" Program, IdEx Bordeaux (ANR-10-IDEX-03-02). Anthony Comuzzie and Michael Mahaney provided access to the body weight data from the Southwest National Primate Research Center. Frédéric Santos and Kathryn Trinkaus provided valuable statistical advice and the comments of David Strait and four anonymous reviewers greatly improved early versions of the manuscript. Neither author has a conflict of interest to declare.

Formby WA, Nanda RS, Currier GF, 1994. Longitudinal changes in the adult facial profile. Am J Orthod Dentofac Orthop 105, 464-476. Franklin D, Flavel A, 2014. Brief communication: timing of spheno-occipital closure in modern western Australians. Am J Phys Anthropol 153, 132-8. Frost HM, 1969. Tetracycline-based histological analysis of bone remodeling. Calcif Tissue Res 3, 211-37. Frost SR, Marcus LF, Bookstein FL, Reddy DP, Delson E, 2003. Cranial allometry, phylogeography, and systematics of large-bodied papionins (Primates: Cercopithecinae) inferred from geometric morphometric analysis of landmark data. Anat Rec A Discov Mol Cell Evol Biol 275, 1048-72. Garn SM, Lewis AB, Koski K, Polacheck DL, 1958. The sex difference in tooth calcification. J Dent Res 37, 561-7. Goodall C, 1991. Procrustes methods in the statistical analysis of shape. J R Stat Soc Ser B 53, 285-339.

This article is protected by copyright. All rights reserved

Author Manuscript Author Manuscript

Author Contributions Jessica Joganic: project conception and design, data acquisition and analysis, results interpretation, manuscript drafting and revision. Yann Heuzé: project conception and design, results interpretation, manuscript revision.