Marine Cazenave
email: marine.cazenave4@gmail.com

Tracy L Kivell

Marta Pina

David R Begun

Matthew M Skinner

Calcar femorale variation in extant and fossil hominids: Implications for identifying bipedal locomotion in fossil hominins

Keywords: Proximal femur, Extant great apes, X-ray microtomography, Internal bone structure, Calcar femorale occurrence, Calcar femorale morphology

The calcar femorale is an internal bony structure of the proximal femur considered to be functionally related to bipedal locomotion. Among extant primates, the presence of a calcar femorale has been so far documented in extant humans and Pan and, among extinct hominins, in the late Miocene Orrorin, in a Pliocene Australopithecus, and in a Middle Pleistocene Homo specimen. Using high-resolution microcomputed tomography, we investigated the occurrence and morphology (i.e., shape, location, and size) of the calcar femorale in an adult sample of extant humans, Pan troglodytes, Gorilla gorilla, Pongo sp., and Papio ursinus. We also investigated for the first time the occurrence and morphology of a calcar femorale in the adult proximal femoral remains of a late Miocene great ape (Rudapithecus) and five Plio-Pleistocene hominins from Southern and Eastern Africa (Australopithecus and Paranthropus). We took four measurements: periosteal-to-tip maximum length, maximum length excluding cortical thickness, maximum vertical height, and the distance between the most anterior and posterior limits of the root. To allow for intergeneric comparisons, estimated body size was used to standardize all measurements. Nine of 10 extant humans have a well-developed calcar femorale. Among the African apes, 6/10 Pan and 6/10 Gorilla also show a distinct calcar femorale. In Pongo (n = 9), it is only present in one captive individual. None of the five investigated Papio specimens show any trace of this structure.

Only calcar femorale height, which is systematically taller and extends into the lower part of the lesser trochanter, discriminates humans from extant great apes, except for one Gorilla. The calcar femorale was absent in one Paranthropus robustus and variably developed in all other investigated 2 fossils. These results indicate that this structure cannot be considered as a diagnostic feature of habitual bipedal locomotion and emphasize the need for further investigations of its functional role.

Introduction

The calcar femorale (CF) is an osteological feature described in a typical, healthy human femur as a vertical to slightly oblique spur of thickened bone within the femoral neck (Fig. 1). It typically extends from the inferolateral endosteal margin of the femoral neck, posterior to the neutral axis, laterally and distally towards the greater trochanter [START_REF] Griffin | The calcar femorale redefined[END_REF][START_REF] Adam | The internal calcar septum (femoral thigh spur) in computed tomography and conventional radiography[END_REF][START_REF] Corroller | The femoral calcar: A computed tomography anatomical study[END_REF]. The CF was illustrated for the first time in a mid-19 th century human anatomical atlas [START_REF] Pirogov | An Illustrated Topographic Anatomy of Saw Cuts Made in Three Dimensions Across the Frozen Human Body[END_REF], as cited by [START_REF] Glinkowski | The topographic and comparative study of the calcar femorale[END_REF]. Since then, the gross morphology and possible functional significance of the CF, especially in relation to bipedalism, have been discussed in several anatomical studies (e.g., [START_REF] Merkel | Betrachtungen über das Os femoris[END_REF][START_REF] Bigelow | The true neck of the femur: Its structure and pathology[END_REF][START_REF] Dixon | The architecture of the cancellous tissue forming the upper end of the femur[END_REF][START_REF] Harty | The calcar femorale and the femoral neck[END_REF][START_REF] Griffin | The calcar femorale redefined[END_REF][START_REF] Stiles | The calcar femorale. An anatomic, radiologic, and surgical correlative study[END_REF][START_REF] Hammer | The calcar femorale: A new perspective[END_REF] and increasingly considered in clinical research, particularly in relation to osteoporosis and osteoarthritis (e.g., [START_REF] Corroller | The femoral calcar: A computed tomography anatomical study[END_REF]Thakkar et al., 2015;[START_REF] Tetsunaga | Calcar femorale in patients with osteoarthritis of the hip secondary to developmental dysplasia[END_REF][START_REF] Sas | Effect of anatomical variability on stress-shielding induced by short calcar-guided stems: Automated finite element analysis of 90 femora[END_REF][START_REF] Zha | Cementless distal fixation modular stem without reconstruction of femoral calcar for unstable intertrochanteric fracture in patients aged 75 years or more[END_REF].

The CF is thought to be related to the load created by the insertion of the iliopsoas tendon on the lesser trochanter [START_REF] Cunningham | Developmental Juvenile Osteology[END_REF] and experimental and clinical studies have identified that it provides additional mechanical strength for weight transmission [START_REF] Li | A comparison of the stiffness, density and composition of bone from the calcar femorale and the femoral cortex[END_REF][START_REF] Zhang | The role of the calcar femorale in stress distribution in the proximal femur[END_REF]. The CF is proximally rooted at the region of bending of the neck-shaft arch and therefore is thought to act as a transitional structure for load transfer through the hip joint and to prevent the posterior and medial regions of the proximal femur from suffering overload [START_REF] Farkas | An anatomical study of the mechanics, pathology, and healing of fracture of the femoral neck[END_REF]Li andAspden, 1997, 1998;Gao et al., 1999;[START_REF] Wang | Finite element analysis of the biomechanics of human femur (Chin)[END_REF][START_REF] Zhang | The role of the calcar femorale in stress distribution in the proximal femur[END_REF][START_REF] Hammer | The calcar femorale: A new perspective[END_REF]. In addition, it is architecturally connected with the vertical and the trochanteric trabecular bundles [START_REF] Aiello | An Introduction to Human Evolutionary Anatomy[END_REF]. The vertical bundle arises from the superior aspect of the head and radiates inferolaterally towards the inferior cortex of the femoral neck, while the trochanteric bundle radiates towards the medial cortex of the shaft by forming a 'Gothic arch' at the intersection with the arcuate bundle (see Cazenave et al., 2021: Figure 6; [START_REF] Kapandji | The Physiology of the Joints[END_REF]. As reviewed in [START_REF] Kapandji | The Physiology of the Joints[END_REF] and [START_REF] Levangie | Joint Structure and Function: A Comprehensive Analysis[END_REF], these bundles reflect compressive and tensile stress trajectories through the human femoral neck created by axial compressive and bending loads. It is thought that the presence of a CF connected to the two bundles contributes to the strength of the base of the femoral neck in resisting these loads [START_REF] Newell | The calcar femorale: A tale of historical neglect[END_REF][START_REF] Wroblewski | The calcar femorale in cemented stem fixation in total hip arthroplasty[END_REF][START_REF] Hammer | The structure of the femoral neck: A physical dissection with emphasis on the internal trabecular system[END_REF][START_REF] Hammer | The calcar femorale: A new perspective[END_REF][START_REF] Cunningham | Developmental Juvenile Osteology[END_REF]. However, morphostructural variability in the CF in extant humans has been reported across multiple age cohorts [START_REF] Farkas | An anatomical study of the mechanics, pathology, and healing of fracture of the femoral neck[END_REF]. In addition to the occasional absence of this spur of thickened bone in both adult males and females, the CF can vary in adult individuals in shape and dimension, most notably in how far it penetrates within the lesser trochanter and the degree of intra-individual bilateral asymmetry (e.g., Thompson, 1907;[START_REF] Garden | The structure and function of the proximal end of the femur[END_REF][START_REF] Kuperavage | Inferences concerning bipedal locomotion from the internal structure of the BAR 1002'00 and BAR 1003'00 femoral fragments[END_REF][START_REF] Hammer | The structure of the femoral neck: A physical dissection with emphasis on the internal trabecular system[END_REF][START_REF] Hammer | The calcar femorale: A new perspective[END_REF][START_REF] Corroller | The femoral calcar: A computed tomography anatomical study[END_REF][START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF].

Experimental analyses have demonstrated that, within the limits imposed by a phylogeneticallydetermined adaptive Bauplan and the influence of a number of biological factors (e.g., hormones, cytokines, proteins that regulate bone cells activity; [START_REF] Lovejoy | Morphological analysis of the mammalian postcranium: A developmental perspective[END_REF]Judex et al., 2004;[START_REF] Demissie | Proximal hip geometry is linked to several chromosomal regions: Genome-wide linkage results from the Framingham Osteoporosis Study[END_REF]Havill et al., 2007[START_REF] Havill | Heritability of lumbar trabecular bone mechanical properties in baboons[END_REF][START_REF] Bonewald | Osteocytes, mechanosensing and Wnt signaling[END_REF][START_REF] O'neill | The degree and pattern of phylogenetic signal in primate longbone structure[END_REF][START_REF] Cunningham | Anticipating bipedalism: Trabecular organization in the newborn ilium[END_REF]Estrada et al., 2012;[START_REF] Reissis | Development of fetal trabecular micro-architecture in the humerus and femur[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 | Phalangeal curvature in a chimpanzee raised like a human: Implications for inferring arboreality in fossil hominins[END_REF]Medina-Gomez et al., 2018;[START_REF] Hou | Genetic variants affecting bone mineral density and bone mineral content at multiple skeletal sites in Hispanic children[END_REF], cortical bone thickness distribution and trabecular structure can adapt to the site-specific mechanical loading environment to resist local stresses (e.g., [START_REF] Lanyon | Analysis of surface bone strain in the sheep during normal locomotion[END_REF][START_REF] Skerry | Interruption of disuse by short duration walking exercise does not prevent bone loss in the sheep calcaneus[END_REF][START_REF] Biewener | Adaptive changes in trabecular architecture in relation to functional strain patterns and disuse[END_REF][START_REF] Guldberg | Mechanical stimulation of tissue repair in the hydraulic bone chamber[END_REF][START_REF] Mori | Climbing exercise increases bone mass and trabecular bone turnover through transient regulation of marrow osteogenic 1035 and osteoclastogenic potentials in mice[END_REF][START_REF] Mittra | Interrelationship of trabecular mechanical and microstructural properties in sheep trabecular bone[END_REF][START_REF] Pontzer | Trabecular bone in the bird knee responds with high sensitivity to changes in load orientation[END_REF][START_REF] Carlson | Increased non-linear locomotion alters diaphyseal bone shape[END_REF][START_REF] Chang | Adaptations in trabecular bone microarchitecture in Olympic athletes determined by 7T MRI[END_REF][START_REF] Polk | Knee posture predicted subchondral apparent density in the distal femur: An experimental validation[END_REF][START_REF] Barak | A Wolff in sheep's clothing: Trabecular bone adaptation in response to changes in joint loading orientation[END_REF][START_REF] Wallace | Physical activity engendering loads from diverse directions augments the growing skeleton[END_REF][START_REF] Cresswell | Spatial relationships between bone formation and mechanical stress within cancellous bone[END_REF]Vera et al., 2020). Importantly, several studies have found that variation in cortical and/or trabecular bone structure at different skeletal sites is consistent with predicted differences in locomotorrelated loadings in humans and other primates. Indeed, although the link between the endostructural architecture of the proximal femur and the load transfer and dissipation is more complex than assumed by the first mechanical models (e.g., [START_REF] Fajardo | Nonhuman anthropoid primate femoral neck trabecular architecture and its relationship to locomotor mode[END_REF][START_REF] Ryan | Trabecular bone structure in the humeral and femoral heads of anthropoid primates[END_REF][START_REF] Shaw | Does skeletal anatomy reflect adaptation to locomotor patterns? Cortical and trabecular architecture in human and nonhuman anthropoids[END_REF], trabecular bone variation in the primate femoral head has provided evidence for structural differences across locomotor groups and has identified unique features in humans related to habitual bipedalism [START_REF] Fajardo | Three-dimensional analysis of nonhuman primate trabecular architecture using micro-computed tomography[END_REF][START_REF] Maclatchy | A comparison of the femoral head and neck trabecular architecture of Galago and Perodicticus using micro-computed tomography (microCT)[END_REF]Ryan andKetcham, 2002a, b, 2005;[START_REF] Ryan | Trabecular bone ontogeny in the human proximal femur[END_REF][START_REF] Saparin | Structural adaptation of trabecular bone revealed by position resolved analysis of proximal femora of different primates[END_REF]Ryan andShaw, 2012, 2015;[START_REF] Raichlen | An ontogenetic framework linking locomotion and trabecular bone architecture with applications for reconstructing hominin life history[END_REF][START_REF] Ryan | Humanlike hip joint loading in Australopithecus africanus and Paranthropus robustus[END_REF]Tsegai et al., 2018a;[START_REF] Georgiou | Trabecular architecture of the great ape and human femoral head[END_REF].

Similarly, the asymmetrical distribution of cortical bone thickness within the femoral neck compartment, resulting from an absolutely thinner superior cortex and a thicker inferior cortex, has been shown to similarly characterize bipedal humans and quadrupedal primates, reflecting their stereotypical loadings at the hip joint [START_REF] Lovejoy | Evolution of human walking[END_REF][START_REF] Lovejoy | The natural history of human gait and posture. Part 2. Hip and thigh[END_REF][START_REF] Ohman | Cross sectional geometric properties from biplanar radiographs and computed tomography: functional application to the humerus and femur in Hominoids[END_REF][START_REF] Ohman | Cortical bone distribution in the femoral neck of hominoids: Implications for the locomotion of Australopithecus afarensis[END_REF]Rafferty, 1998;[START_REF] Lovejoy | The Maka femur and its bearing on the antiquity of human walking: Applying contemporary concepts of morphogenesis to the human fossil record[END_REF][START_REF] Ruff | Femoral neck structure and function in early hominins[END_REF][START_REF] Ruff | Limb bone structural proportions and locomotor behavior in A.L. 288-1[END_REF][START_REF] Cazenave | Cortical bone distribution in the femoral neck of Paranthropus robustus[END_REF][START_REF] Pina | Femoral neck cortical bone distribution of dryopithecin apes and the evolution of hominid locomotion[END_REF]. However, compared to the femoral head and neck, other anatomical regions of the proximal femur have received far less attention in extant humans and other primates [START_REF] Miller | Trabecular bone adaptation with an orthotropic material model[END_REF][START_REF] Cazenave | Trabecular organization of the proximal femur in Paranthropus robustus: Implications for the assessment of its hip joint loading conditions[END_REF].

In nonprimate animals, the CF was found to be absent in small samples of hens, dogs, and pigs [START_REF] Glinkowski | The topographic and comparative study of the calcar femorale[END_REF]. Among primates, the presence of a CF has thus far only been investigated in a sample of 10 rhesus macaques, where it was reportedly absent in all individuals [START_REF] Glinkowski | The topographic and comparative study of the calcar femorale[END_REF], and in chimpanzees (Pan troglodytes), where it was identified in 7/10 individuals [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF]. However, in chimpanzees, the CF was found to be distinguishable from the human condition due to both a lower occurrence and because of a more limited vertical extension within the femoral neck [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF]. Recently, it has been reported that some Gorilla specimens also exhibit a calcar femorale (e.g., KUPRI 1492 on the Kyoto University Digital Morphology Museum site; pers. commun. to R. H. Crompton from A. [START_REF] Kuperavage | Inferences concerning bipedal locomotion from the internal structure of the BAR 1002'00 and BAR 1003'00 femoral fragments[END_REF][START_REF] Crompton | StW 573 Australopithecus prometheus: Its significance for an australopith bauplan[END_REF], but neither quantitative evidence nor images have been provided.

Because of the presumed mechanical relationship of the CF to bipedalism, its occurrence (i.e., presence or absence) has also been investigated in some early fossil hominins [START_REF] White | Pliocene hominids from Middle Awash, Ethiopia[END_REF][START_REF] Grine | Fossil Homo femur from Berg Aukas, northern Namibia[END_REF][START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF]. The presence of a CF was reported in the late Miocene Orrorin tugenensis (BAR 1002'00 and 1003'00; [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF] and, because of its relative vertical extension, has been invoked to support the inference of bipedal locomotion in this taxon [START_REF] Senut | First hominid from the Miocene (Lukeino Formation, Kenya)[END_REF][START_REF] Pickford | Bipedalism in Orrorin tugenensis revealed by its femora[END_REF]. A CF has also been identified in an Australopithecus specimen (MAK-VP-1/1; [START_REF] White | Pliocene hominids from Middle Awash, Ethiopia[END_REF]; see also [START_REF] Reed | Proximal femur of Australopithecus africanus from Member 4, Makapansgat, South Africa[END_REF] and in a Middle Pleistocene Homo specimen (Berg Aukas; [START_REF] Grine | Fossil Homo femur from Berg Aukas, northern Namibia[END_REF]. However, to date, no study has evaluated the occurrence of the CF in any extinct nonhuman hominid. Given the variability in occurrence and morphology of the CF observed in extant humans, the very limited data for extant nonhuman primates, and the lack of information on this feature in extinct hominids, a more detailed consideration of the CF in extant and fossil primates may provide greater clarity on its functional significance and whether there are unique aspects of this feature that could potentially be related to bipedalism.

To address these knowledge gaps, we use high-resolution microcomputed tomography ( CT) to evaluate and compare CF occurrence and three-dimensional (3D) morphology (i.e., shape, location, extension within the lesser trochanter, absolute and standardized horizontal length and vertical height, and root width) in adult femora representing extant humans, extant great apes (Pan, Gorilla, and Pongo), and one species of cercopithecine monkey (Papio). We then evaluate CF occurrence and morphology in the adult femoral remains of a late Miocene great ape (Rudapithecus) and five Plio-Pleistocene hominins from Southern and Eastern Africa representing Australopithecus and Paranthropus. We expand on previous studies by not only assessing CF occurrence and morphology in a broader taxonomic extant and extinct primate sample, but also by quantifying additional aspects of the CF, i.e., CF length excluding the cortex to assess the influence of variation in cortical bone thickness, and CF maximum vertical height and root thickness to determine whether the degree of development of such parameters is unique to habitual bipeds.

Based on the body of evidence available thus far, we hypothesize that 1) extant humans and great apes differ in CF occurrence and morphology, and both differ from the pattern observed in Papio;

2) extinct hominins are more similar in CF occurrence and morphology to that of extant humans compared with other extant apes, and 3) the Miocene ape Rudapithecus is most similar to the CF morphology to extant apes. Below we outline our predictions.

Assuming the CF is primarily, though not necessarily uniquely related to mechanical loading during bipedal behaviors [START_REF] Farkas | An anatomical study of the mechanics, pathology, and healing of fracture of the femoral neck[END_REF][START_REF] White | Pliocene hominids from Middle Awash, Ethiopia[END_REF][START_REF] Reed | Proximal femur of Australopithecus africanus from Member 4, Makapansgat, South Africa[END_REF][START_REF] Grine | Fossil Homo femur from Berg Aukas, northern Namibia[END_REF][START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF][START_REF] Hammer | The calcar femorale: A new perspective[END_REF], and given the results of human and Pan comparisons presented in [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF], we predict that the CF will be more frequently expressed, absolutely and relatively longer, more vertically extended, and characterized by a thicker root, in our sample of extant humans compared to the extant ape and Papio samples.

Although extant great apes differ in their type and frequency of locomotor and postural behaviors (primarily knuckle walking in the African apes vs. specialized suspensory behaviors in orangutans; e.g., [START_REF] Hunt | Positional behavior in the Hominoidea[END_REF][START_REF] Doran | The comparative positional behavior of the African apes[END_REF]Thorpe and Crompton, 2006; see also Almécija et al., 2021 for a review), they share a high degree of hip joint mobility with enhanced abduction and lateral rotation movements [START_REF] Isler | Gait parameters in vertical climbing of captive, rehabilitant and wild Sumatran orang-utans (Pongo pygmaeus abelii)[END_REF][START_REF] Hammond | 3D analysis of hip joint mobility and the evolution of locomotor abilities in Miocene hominoids[END_REF][START_REF] Finestone | Great ape walking kinematics: Implications for hominoid evolution[END_REF]. Moreover, great apes show similar patterns of trabecular bone distribution within the femoral head [START_REF] Ryan | Unique suites of trabecular bone features characterize locomotor behavior in human and non-human anthropoid primates[END_REF][START_REF] Georgiou | Trabecular architecture of the great ape and human femoral head[END_REF] and similar arrangement of cortical and trabecular bone at the neck/shaft junction, suggesting that Pan, Gorilla, and Pongo experience generally comparable load transfer at the hip joint (Rafferty, 1998;[START_REF] Cazenave | Cortical bone distribution in the femoral neck of Paranthropus robustus[END_REF][START_REF] Pina | Femoral neck cortical bone distribution of dryopithecin apes and the evolution of hominid locomotion[END_REF].

We therefore predict a similar degree of development of the CF (i.e., occurrence, shape, extension into the lesser trochanter, absolute and relative vertical length, height and root width) within the three great apes. In contrast, Papio, which engages primarily in terrestrial, pronograde quadrupedal walking and running [START_REF] Rose | Positional behaviour of olive baboons (Papio anubis) and its relationship to maintenance and social activities[END_REF], has a lower hip joint mobility than that of great apes [START_REF] Hammond | 3D analysis of hip joint mobility and the evolution of locomotor abilities in Miocene hominoids[END_REF], which is reflected in a more asymmetrical distribution of cortical bone in the femoral neck (Rafferty, 1998;[START_REF] Pina | Femoral neck cortical bone distribution of dryopithecin apes and the evolution of hominid locomotion[END_REF]. Thus, we predict that the CF should not be present in Papio.

Australopithecines are considered habitual bipeds when terrestrial [START_REF] Ward | Postural and locomotor adaptations of Australopithecus species[END_REF], although likely somewhat distinct from humans in their bipedal biomechanics and loading environment at the hip joint (e.g., [START_REF] Ruff | Femoral neck structure and function in early hominins[END_REF][START_REF] Ryan | Humanlike hip joint loading in Australopithecus africanus and Paranthropus robustus[END_REF][START_REF] Cazenave | Cortical bone distribution in the femoral neck of Paranthropus robustus[END_REF][START_REF] Cazenave | Trabecular organization of the proximal femur in Paranthropus robustus: Implications for the assessment of its hip joint loading conditions[END_REF]. It is thus expected that australopithecines express a CF configuration (i.e., shape, location, extension within the lesser trochanter, absolute and standardized horizontal length and vertical height, and root width) similar to the extant human condition. In contrast, Rudapithecus, the only fossil ape analyzed in this study, has postcranial morphology suggesting frequent use of forelimb-dominated suspensory behaviors [START_REF] Morbeck | Miocene hominoid discoveries from Rudabánya[END_REF][START_REF] Begun | New catarrhine phalanges from Rudabánya (Northeastern Hungary) and the problem of parallelism and convergence in hominoid postcranial morphology[END_REF][START_REF] Kivell | New primate carpal bones from Rudabánya (late Miocene, Hungary): Taxonomic and functional implications[END_REF][START_REF] Ward | A late Miocene hominid partial pelvis from Hungary[END_REF][START_REF] Almécija | Fossil apes and human evolution[END_REF]. Thus, if a CF is present in Rudapithecus, we expect its morphology to be most similar to that of extant great apes.

Materials and methods

Samples

Our extant primate sample consists of 44 femora from adult males and females of modern humans (n = 10), Pan troglodytes (n = 10), Gorilla gorilla (n = 10), Pongo sp. (n = 9), and Papio ursinus (n = 5). All specimens were free from obvious signs of pathology. All nonhuman primate specimens were wild caught, with the exception of two captive Pongo specimens. Six P. troglodytes and one Pongo specimens were of unknown provenance. Details of the extant sample composition are provided in Supplementary Online Material (SOM) Table S1.

We included one fossil great ape specimen representing Rudapithecus hungaricus (RUD 234) from the late Miocene site of Rudabánya (Hungary), housed at the Mining and Geological Survey of Hungary, Budapest [START_REF] Begun | European Miocene hominids and the origin of the African ape and human clade[END_REF][START_REF] Ward | A late Miocene hominid partial pelvis from Hungary[END_REF]; SOM Table S2). The Plio-Pleistocene hominin sample includes the following specimens: StW 99 and 367 from Sterkfontein Member 4, South Africa, both attributed to Australopithecus africanus and curated at the Evolutionary Studies Institute of the University of the Witwatersrand, Johannesburg, South Africa [START_REF] Harmon | Size and shape variation in the proximal femur of Australopithecus africanus[END_REF][START_REF] Ruff | Femoral neck structure and function in early hominins[END_REF][START_REF] Zipfel | Hominin Postcranial Remains from Sterkfontein, South Africa, 1936-1995[END_REF]Pickering et al., 2021); SK 97 from Swartkrans Member 1, South Africa representing Paranthropus robustus curated at the Ditsong National Museum of Natural History, Pretoria, South Africa [START_REF] Robinson | Early Hominid Posture and Locomotion[END_REF][START_REF] Susman | Recently identified postcranial remains of Paranthropus and early Homo from Swartkrans Cave, South Africa[END_REF][START_REF] Cazenave | Cortical bone distribution in the femoral neck of Paranthropus robustus[END_REF][START_REF] Cazenave | Trabecular organization of the proximal femur in Paranthropus robustus: Implications for the assessment of its hip joint loading conditions[END_REF]; and KNM-ER 738 and 1465 from Koobi Fora, Kenya, both likely representing Paranthropus boisei curated at the National Museums of Kenya, Nairobi (Wood and Costantino, 2007;[START_REF] Wood | The Omo-Turkana Basin fossil hominins and their contribution to our understanding of human evolution in Africa[END_REF][START_REF] Ward | Associated ilium and femur from Koobi Fora, Kenya, and postcranial diversity in early Homo[END_REF][START_REF] Ruff | Limb bone structural proportions and locomotor behavior in A.L. 288-1[END_REF]; SOM Table S2). We also assessed additional fossil specimens including four Miocene apes (including one Dryopithecus fontani, two Hispanopithecus laietanus, and another R. hungaricus femora) and five Plio-Pleistocene hominins (representing three additional A. africanus, one P. robustus and one P. boisei or Homo sp.); however, their poor internal preservation prevented the possibility to reliably quantify potential CF morphology (SOM Table S2; SOM Fig. S1).

Microcomputed tomography

Details on the different microtomographic (μCT) equipment and the scanning resolutions are provided in SOM Tables S1 andS2 for the extant and the fossil specimens, respectively. Four S2).

Previous quantitative analyses of the CF in humans and Pan indicate that, when present, this spur of thickened bone measures several centimeters vertically and transversely (e.g., [START_REF] Kuperavage | Inferences concerning bipedal locomotion from the internal structure of the BAR 1002'00 and BAR 1003'00 femoral fragments[END_REF][START_REF] Corroller | The femoral calcar: A computed tomography anatomical study[END_REF][START_REF] Tetsunaga | Calcar femorale in patients with osteoarthritis of the hip secondary to developmental dysplasia[END_REF][START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF]. Accordingly, potential measurement bias of the CF due to differences in CT resolution or technical differences across scanning facilities is negligible (for comparative measurements of the -scale sized trabecular struts see [START_REF] Cazenave | Trabecular organization of the proximal femur in Paranthropus robustus: Implications for the assessment of its hip joint loading conditions[END_REF]. For instance, if we consider a CF length of 7.76 mm in a Pan individual scanned with a resolution of 80 (i.e., the lowest resolution used in the present study), a measurement error of 1 pixel would represent only 1.03% of the length of the CF (i.e., 0.08 mm).

All specimens were virtually rotated to the same anatomical position in Avizo v. 9.0.0 (Visualization Sciences Group Inc., Bordeaux) by using the neck axis and the principal axis of the proximal shaft portion as anatomical references. Left-side specimens were flipped so all specimens were analyzed as right femora.

Measurements and analyses

To assess the occurrence and, when present, the variation in morphology of the CF across our sample, we placed a transverse cross section at the level of the upper margin of the lesser trochanter and then virtually moved proximally towards the dorsomedial neck region and distally across the lesser trochanter. When the presence of the CF was identified, we placed a parasagittal cross section at the level of the center of the lesser trochanter and virtually moved mediolaterally to delimit the extent of the CF. In the specimens showing at least a trace of the bony spur, we characterized the shape of the CF according to the three categories established by Le Corroler et al. ( 2011): 1) a relatively short and thick ridge-like CF, 2) a spur-type CF, or 3) a relatively thin and long septum-like CF.

To test our predictions of a longer and more vertically extended CF with a wider root in extant and fossil hominins compared with the extant and fossil great apes, and of a similar degree of development (i.e., vertical extension, absolute and relative vertical length and height, and root width thickness) among the great ape taxa, we automatically segmented the cortex from the trabecular bone using the software Dragonfly v. 2020.2.0.941 (Object Research Systems Inc., Montreal) and the method developed by [START_REF] Buie | Automatic segmentation of cortical and trabecular compartments based on a dual threshold technique for in vivo micro-CT bone analysis[END_REF]. The CF was virtually extracted from the cortex for 3D rendering. Due to preservation, the CF from fossil specimens could not be automatically segmented and was completed manually instead. We first qualitatively compared the extent of the CF into the lesser trochanter and we took the following CF measurements: maximum length, maximum vertical height and maximum root width.

We used two methods to assess CF maximum length. In all extant specimens, the distance between the periosteal surface and the most internal point (the tip) of the CF (PT MaxLength) was automatically computed using the 'Surface Distance' module in Avizo v. 9.0.0 and then rendered in 3D using a chromatic scale where the outer contour-tip distance increases from dark blue (thin) to red (thick; Fig. 2a). However, for the six fossil specimens, we could not automatically quantify the distance from the periosteal surface due to poor preservation. In these specimens, we manually measured CF maximum length (CF MaxLength) from the base lying on the endosteal contour (the root) to the tip of its most lateral extension, thus excluding the cortical thickness (Fig. 2b). Given that multiple factors have been shown in humans to affect cortical thickness variation throughout life (e.g., [START_REF] Gocha | Spatial variation in osteon population density at the human femoral midshaft: Histomorphometric adaptations to habitual load environment[END_REF], we also quantified CF MaxLength (length excluding the cortical bone) in all extant specimens for direct comparison with the fossil specimens. We found a high Spearman's rank correlation coefficient between PT MaxLength and CF MaxLength across the extant sample (r = 0.91, p < 0.0001). Calcar femorale maximum vertical height (CF MaxHeight) was measured as the vertical distance between the uppermost and lowest extracted slices bearing evidence of a CF-related spur of thickened bone, i.e., between the parallel planes orthogonal to the principal axis of the proximal shaft where the CF merges with the cortical bone of the posterior wall of the neck and the endosteal surface of the posterior wall of the diaphysis (Fig. 2c). Finally, in all cases where the most anterior and posterior limits of the CF root on the endosteal contour were clearly identifiable (n = 18 specimens), we also measured maximum CF root width on the related transverse cross section (Fig. 2d).

To allow for comparisons among extant taxa that differ in body size, all measurements were standardized by body mass. Body mass was estimated for each extant individual using the superoinferior diameter of the femoral head and the equations provided in [START_REF] Ruff | Lower limb articular scaling and body mass estimation in Pliocene and Pleistocene hominins[END_REF] for the extant human sample and in [START_REF] Ruff | Lower limb articular scaling and body mass estimation in Pliocene and Pleistocene hominins[END_REF] for the extant great ape taxa. Interspecific comparisons were completed on both the absolute and standardized mean values of each taxon.

However, due to poor preservation of the femoral head, measurements of the fossils could not be standardized. Thus, all fossil results are presented and compared as absolute values only.

Given that cortical bone thickness at the base of the neck in extant apes and Neotropical monkeys has been shown to be influenced by loading conditions at this anatomical site [START_REF] Ohman | Cross sectional geometric properties from biplanar radiographs and computed tomography: functional application to the humerus and femur in Hominoids[END_REF][START_REF] Ohman | Cortical bone distribution in the femoral neck of hominoids: Implications for the locomotion of Australopithecus afarensis[END_REF]Rafferty, 1998), we additionally tested whether CF size correlates with variation in cortical thickness at this specific site. We measured cortical bone thickness (CBT) at the level of the most anterior (CBTa) and most posterior (CBTp) limits of the CF root in our extant sample (SOM Fig. S2) and used the average between the two as our estimate of CBT. We tested the possible influence of CBT variation on the CF parameters (i.e., CF length, vertical height, and root width) using a partial Spearman's correlation test controlling for body mass between CBT and each CF measurement. We also tested for the human, Pan, and Gorilla samples the possible influence within each species of body mass on CF length and vertical height using Spearman's correlation test. Given the sample size (n = 18), we could not test the possible influence of body mass on CF root width for the entire sample or all CF parameters in Pongo (n = 1).

All measurements used in this study were taken by one observer (M.C.). To examine the accuracy of the non-automated variables (i.e., CF MaxLength and CF MaxHeight), M.C. measured these variables twice in 50% of the specimens, another observer also measured these same variables in 50% of the specimens and intra-and interobserver tests were run, calculating the intraclass correlation coefficient (ICC) between the two sets of measurements. The results show an intraobserver ICC of 0.98 and 0.99 and an interobserver ICC of 0.78 and 0.85 for CF MaxLength and CF MaxHeight, respectively, indicating high reproducibility of the measurements.

No interobserver tests were performed for CF root width, but repeated intraobserver measurements provided a highly satisfactory ICC of 0.93.

All statistical analyses and plots were performed in RStudio v. 1.2.5033 running with R v. 3.4.4 (R Core Team, 2018). Plots were generated using ggplot2 [START_REF] Wickham | ggplot2: Elegant Graphics for Data Analysis[END_REF]. Due to small sample sizes, only nonparametric tests were performed. Tests of significant interspecific differences in PT MaxLength, CF MaxLength, and CF MaxHeight (both absolute and standardized values) were conducted using the pairwise Wilcoxon rank sum test (alpha = 0.05). Given the small sample size, interspecific differences in maximum CF root width could not been carried out and males and females were combined for the analyses.

Results

Calcar femorale in the extant primate sample

Nine of the 10 extant humans (3 males and 6 females) showed the presence of a thick, ridgelike CF; a CF was absent in a 30-49 year-old male (Fig. 3a). Among the great apes, 6/10 Pan (4 males and 2 females) and 6/10 Gorilla (4 males and 2 females) expressed a bony spur ranging from a only ridge-like trace of this spur to a distinct septum-like structure. In Pongo, a CF was unambiguously found in one of the two captive males (Fig. 3e; SOM Table S1) and a CF ridgelike structure was also present in one wild Pongo female (Fig. 3d). In contrast, none of the Papio specimens showed a CF or a CF-like structure (Fig. 3; SOM Fig. S3).

In all specimens and taxa where the CF was present, it was located within the region spanning from the posteroinferior surface of the femoral neck to the lesser trochanter (Fig. 4). However, we observed intra-and interspecific variation in CF shape, extent, and size (both mediolaterally and proximodistally). In nearly all cases, the CF progressively thinned medially towards the tip, where it merged into the surrounding trabecular network with a tree-like pattern (SOM Fig. S3).

However, CF thickness and density were variable, with some individuals, most notably in humans, displaying a plate-like structure resulting from the merging of thickened trabeculae rather than a true spur of dense bone. In no case in our human sample did the CF extend to the posterior wall of the greater trochanter (Fig. 3; SOM Fig. S3). In some Pan and Gorilla (SOM Fig. S4), as well as in the Pongo individual displaying the CF (Fig. 3e), a variably-developed bone reinforcement emerging from the lateral margin of the lesser trochanter and radiating medially towards the CF was observed in the transverse slices. We did not observe a similar feature in our sample of human femora.

In extant humans, the CF systematically extended into the distal part of the lesser trochanter (Fig. 3). By contrast, in all Pan, 5/6 Gorilla and in the Pongo individual where it was unambiguously expressed, the distal end of the CF was typically confined to the proximal half of the lesser trochanter compartment, or occasionally near its middle portion. However, a typically human-like condition was found in one female Gorilla, whose distal extension was deep within the lesser trochanter (Fig. 3a; SOM Fig. S5).

Quantitative analyses revealed that humans had the absolutely greatest PT MaxLength, CF MaxLength, and CF MaxHeight (Fig. 5; Table 1). However, for PT MaxLength and CF MaxLength, Gorilla showed the greatest range of variation and also had the highest individual values. For all three variables, the differences between humans and Pan were statistically significant, while humans and Gorilla differed significantly only in CF MaxHeight (Fig. 5; Table 1; SOM Table S3). Pan and Gorilla also differed significantly only in CF MaxHeight (Fig. 5; Table 1; SOM Table S3). Relative to body size, humans had significantly larger PT MaxLength and CF MaxLength compared with Gorilla, and significantly higher CF MaxHeight compared with Pan and Gorilla (Fig. 5; Table 1; SOM Table S3). The Pongo specimen values were closer to those of Gorilla or intermediate between Pan and Gorilla (Fig. 5; Table 1). Calcar femorale root width was absolutely larger in Gorilla (Fig. 2c) and narrower in the captive Pongo individual, the latter falling below the ranges of the three other taxa. However, human and great apes overlapped in both absolute and standardized values, with humans and Gorilla showing the greatest degree of overlap (SOM Table S4).

Spearman's correlation results revealed that PT MaxLength, CF MaxLength, CF MaxHeight, and CF root width were uncorrelated with CBT when controlling for body mass (SOM Fig. S3; SOM Tables S5 andS6). The distance between the periosteal surface and the most internal point of the CF (PT MaxLength), CF MaxLength, and CF MaxHeight were also uncorrelated with estimated body mass (SOM Table S7) in extant humans, Pan, and Gorilla. There was, however, a significant positive correlation between PT MaxLength and CBT, which is not surprising given that this measurement includes the cortical thickness (SOM Table S6).

Calcar femorale occurrence in the fossil hominid sample

The CF was present in five of the six specimens comprising our fossil sample: RUD 234 (Rudapithecus), StW 99 and 367 (A. africanus), and KNM-ER 738 and 1465 (likely P. boisei; Fig. 6). Conversely, we observed no trace of a CF on the endosteal contour of SK 97 (P. robustus), although possible strengthening of the trabeculae projecting towards the greater trochanter could not be confidently ascertained because of the incomplete preservation in this specimen (Fig. 6; SOM Fig. S6). Due to preservation quality, 3D rendering of the CF was possible only in RUD 234 and KNM-ER 738 (Fig. 7).

Comparison of the calcar femorale between the extant and fossil samples

None of the fossils showed distinct evidence of a bony reinforcement at the lateral margin of the lesser trochanter comparable to that found in some Pan and Gorilla specimens, and in the Pongo individual displaying the CF (Fig. 3; SOM Fig. S4).

RUD 234 showed a well-developed CF (Figs. 6 and7), similar in structure to some Pan (Fig. 3; SOM Fig. S3) but extended more transversely. Indeed, CF MaxLength in RUD 234 slightly exceeded that of the CF in Pongo and fell within the upper end of the interquartile range of the Gorilla sample, outside of the range above Pan, and in the lower range (higher than the minimum value) of humans (Fig. 5; Table 2). RUD 234 also had a longer CF MaxLength relative to the other fossils. RUD 234 CF MaxHeight was found in the upper range (lower than the maximum value) of Pan, and was in the lower range of Gorilla, and lower than Pongo as well as the other three fossils for which CF MaxHeight could be assessed (Fig. 5; Table 2).

StW 99 and StW 367 (A. africanus) both displayed a short CF MaxLength, most comparable to that of Pan; StW 99 fell below the 25 th percentile of Pan and in the lower end of the interquartile range of Gorilla while StW 367 fell below the 25 th percentile of Pan and Gorilla (Fig. 5; Table 2).

For CF MaxHeight, StW 99 fell well within the upper end of the range of variation of Gorilla and the lower range of extant humans, but well above the range of variation found in Pan (Fig. 5; Table 2). The distal end of the CF in StW 99 was not confined within the proximal portion of the lesser trochanter but extended at least to its mid portion, as seen in humans.

In KNM-ER 738, CF MaxLength fell in the upper end of the interquartile range of Pan and lower end of the interquartile range of Gorilla, lower than the minimum value of extant humans (Fig. 5; Table 2). Calcar femorale MaxLength in KNM-ER 1465 is slightly longer than in KNM-ER 738, falling in the lower end of the interquartile range of Gorilla near the median value and at the 75 th percentile of Pan, in the lower range of variation of extant humans (Fig. 5; Table 2). In KNM-ER 738, CF MaxHeight fell within the lower range of variation for Gorilla and in the upper range for Pan (Fig. 5; Table 2). In this specimen, the CF reached the upper region of the lesser trochanter (Fig. 7), while in KNM-ER 1465 the CF was longer and extended more distally (Fig. 5;

Table 2), similar to the condition found in StW 99.

In the five fossils displaying a CF, the CF root width was broad, particularly in the two A. africanus specimens, overlapping with the Pan and Gorilla and broader than that of our extant human sample (SOM Fig. S7).

Discussion and conclusions

This study explored the occurrence and, when present, 3D morphology of the CF in extant humans, great apes, Papio, and a sample of fossil hominids. We found that a CF is most commonly found in extant humans, but that there was substantial variation in CF occurrence and morphology across our sample, including within humans. These results add to previous studies that have called into question the functional significance of the CF.

Extant hominoids

Based on previous studies [START_REF] Farkas | An anatomical study of the mechanics, pathology, and healing of fracture of the femoral neck[END_REF][START_REF] White | Pliocene hominids from Middle Awash, Ethiopia[END_REF][START_REF] Reed | Proximal femur of Australopithecus africanus from Member 4, Makapansgat, South Africa[END_REF][START_REF] Grine | Fossil Homo femur from Berg Aukas, northern Namibia[END_REF][START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF][START_REF] Hammer | The calcar femorale: A new perspective[END_REF], we predicted that, compared to great apes, the CF in humans would be expressed more frequently, and absolutely and relatively longer and more vertically extended, with a thicker root width. However, only its higher frequency (90% in humans vs. 60-65% in the Pan and Gorilla samples) and absolute and standardized average height of the CF clearly distinguish humans from other extant hominids. Indeed, contrary to our expectation, no other quantitative CF variables measured in our study unambiguously discriminate between humans and great apes. Although our hominoid sample sizes were relatively small (n = 9-10) and larger samples may provide greater discrimination in CF morphology across taxa, it is notable that we nonetheless found substantial intraspecific variability in each hominoid taxon, including humans. In addition, the extent to which geometric differences in the proximal femur (i.e., neck shaft angle, superoinferior length of the greater trochanter, projection of the head relative to the greater trochanter; [START_REF] Harmon | The shape of the hominoid proximal femur: A geometric morphometric analysis[END_REF] across hominoids contribute to the distal extension of the CF in humans requires further investigation.

Whatever the cause(s) responsible for the expression of a spur of vertically and transversely thickened bone within the femoral neck, and whatever its functional significance [START_REF] Farkas | An anatomical study of the mechanics, pathology, and healing of fracture of the femoral neck[END_REF]Li andAspden, 1997, 1998;Gao et al., 1999;[START_REF] Wang | Finite element analysis of the biomechanics of human femur (Chin)[END_REF][START_REF] Zhang | The role of the calcar femorale in stress distribution in the proximal femur[END_REF][START_REF] Hammer | The calcar femorale: A new perspective[END_REF], our results make clear that presence vs. absence of a CF does not distinguish humans from great apes. As noted above, variation in the expression of the CF was also observed in our extant human sample (e.g., one individual did not show any trace of a CF), supporting previous studies identifying variability in shape and proportions of the CF in humans (e.g., Thompson, 1907;Glinkowki and Ciszek, 1989;[START_REF] Corroller | The femoral calcar: A computed tomography anatomical study[END_REF][START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF].

Additional comparative study of the ontogeny of CF expression in humans and extant great apes would provide much-needed information on when the CF first appears during growth and patterns of age-related strengthening, notably with respect to the onset of independent locomotion. This approach which has already provided valuable information when applied to the endostructural organization of the proximal femur (e.g., [START_REF] Ryan | Trabecular bone ontogeny in the human proximal femur[END_REF][START_REF] Raichlen | An ontogenetic framework linking locomotion and trabecular bone architecture with applications for reconstructing hominin life history[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]Tsegai et al., 2018b). In this context, investigation of the role of a growing iliopsoas tendon in the ontogeny of CF expression and morphology [START_REF] Cunningham | Developmental Juvenile Osteology[END_REF] should be particularly informative given that in humans, the CF is thought to be related to the load created by the insertion of this musculotendinous complex [START_REF] Tatu | Descriptive anatomy of the femoral portion of the iliopsoas muscle. Anatomical basis of anterior snapping of the hip[END_REF] on the lesser trochanter [START_REF] Cunningham | Developmental Juvenile Osteology[END_REF], which has a fulcrum at the femoral head to increase its moment arm [START_REF] Hogervorst | Evolution of the human hip. Part 2: Muscling the double extension[END_REF].

Our second prediction was that extant great ape taxa would show a similar degree of CF occurrence given their shared high degree of hip joint mobility [START_REF] Isler | Gait parameters in vertical climbing of captive, rehabilitant and wild Sumatran orang-utans (Pongo pygmaeus abelii)[END_REF][START_REF] Hammond | 3D analysis of hip joint mobility and the evolution of locomotor abilities in Miocene hominoids[END_REF][START_REF] Finestone | Great ape walking kinematics: Implications for hominoid evolution[END_REF] and their similar patterns of cortical and trabecular bone distribution in the proximal femur, which suggest similar loading conditions at the hip joint (Rafferty, 1998;[START_REF] Ryan | Unique suites of trabecular bone features characterize locomotor behavior in human and non-human anthropoid primates[END_REF][START_REF] Georgiou | Trabecular architecture of the great ape and human femoral head[END_REF][START_REF] Pina | Femoral neck cortical bone distribution of dryopithecin apes and the evolution of hominid locomotion[END_REF]. This prediction was partially supported by our results. Our study confirmed the frequent presence of a CF in Pan [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF] and, for the first time, also showed that a CF commonly occurs in Gorilla (cf. [START_REF] Crompton | StW 573 Australopithecus prometheus: Its significance for an australopith bauplan[END_REF]. However, a CF has only been identified in one captive Pongo (while it is absent in another captive individual from the same zoo).

Within the three great ape taxa we also expected to detect a comparable shape, extension into the lesser trochanter, absolute and relative vertical length, height and root width of the CF. This prediction is not supported as we found a high degree of variation between the African apes.

Compared to the more common human condition, Pan shows a less proximodistally developed CF. [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF] also found a degree of vertical extension comparable to the human pattern in two of seven Pan bearing a CF. However, one Gorilla included in our sample shows a human-like CF with a distal end that extends to the middle-lower part of the lesser trochanter.

Interestingly, compared to Pan, the Gorilla sample is more similar to the human condition in PT MaxLength and CF MaxLength. However, when standardized by body mass, humans and Pan overlap in CF transverse length, while CF transverse length in Gorilla is absolutely shorter compared to Pan and Pongo. In all cases, the degree of CF transverse extension is uncorrelated with both cortical bone thickness variation at the lesser trochanter cross-sectional level and with body mass. Finally, we found a similar extent of variation in Pan (see also [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF] and in Gorilla.

The variation in CF morphology observed between African apes and Pongo might reflect documented differences in climbing frequency and kinematics. Specifically, Pan and Gorilla vertically climb and descend less frequently [START_REF] Hunt | Positional behavior in the Hominoidea[END_REF][START_REF] Doran | The comparative positional behavior of the African apes[END_REF]Thorpe and Crompton, 2006) and have lower ranges of hip extension, abduction, and external rotation [START_REF] Isler | on the site-specific regulation of trabecular and cortical bone morphology[END_REF][START_REF] Hammond | 3D analysis of hip joint mobility and the evolution of locomotor abilities in Miocene hominoids[END_REF][START_REF] Hammond | In vivo baseline measurements of hip joint range of motion in suspensory and nonsuspensory anthropoids[END_REF] compared with that of Pongo. In addition, although the cortical and trabecular distribution patterns are generally similar across great apes at the proximal femur [START_REF] Ryan | Unique suites of trabecular bone features characterize locomotor behavior in human and non-human anthropoid primates[END_REF][START_REF] Georgiou | Trabecular architecture of the great ape and human femoral head[END_REF]Georgiou et al., , 2020)), Pongo is distinct in having a lower trabecular bone density in the femoral head [START_REF] Georgiou | Trabecular architecture of the great ape and human femoral head[END_REF], suggesting subtle differences in loading magnitude or direction at the hip joint, which may explain variation in CF expression between African apes and Pongo. If the lower frequency of a CF in Pongo is related to its arboreal suspensory specializations, then we would expect a similar condition in hylobatids given their shared hip range of motion related to their suspensory behaviors [START_REF] Hammond | 3D analysis of hip joint mobility and the evolution of locomotor abilities in Miocene hominoids[END_REF][START_REF] Hammond | In vivo baseline measurements of hip joint range of motion in suspensory and nonsuspensory anthropoids[END_REF]. In a preliminary assessment of two adult Hylobates lar specimens (1 male and 1 female) we found no evidence of a CF (SOM Fig. S8), but further examination with a larger sample is needed to confirm this finding. The possible influence of captivity-related mechanical/environmental constraints on CF expression in extant great apes is currently unknown but the variable presence of a CF in captive Pongo coupled with the complete absence in wild Pongo individuals, as shown in this study, emphasizes the need for further investigation.

Finally, our prediction that none of the Papio specimens included in our sample would show evidence of a CF was supported. This result is consistent with results previously reported for rhesus macaques [START_REF] Glinkowski | The topographic and comparative study of the calcar femorale[END_REF]. In general, future research examining CF presence and patterns of variation in larger samples of extant apes, with special attention to the degree of expression of this feature between sister taxa that vary in the frequency of different locomotor behaviors (e.g., P. troglodytes vs. P. paniscus; G. gorilla vs. G. beringei ;[START_REF] Hunt | Positional behavior in the Hominoidea[END_REF][START_REF] Crompton | Arboreality terrestriality and bipedalism[END_REF] may shed light on the variation of the CF expression and its potential functional significance. Moreover, although we did not quantitatively evaluate sex differences in the CF in this study, future investigations should also examine the possible influence of sexual dimorphism on CF expression.

Fossil hominins and Rudapithecus

We predicted that the fossil hominins (Australopithecus and Paranthropus) in our sample would display a CF morphology more similar to extant humans given that they are all considered habitual bipeds when terrestrial. We also expected the orthograde suspensory Rudapithecus to show a CF morphology more similar to that of great apes. Both predictions were generally supported, but with some notable exceptions. The results of our high-resolution analyses of the occurrence and 3D shape variation of the CF in a selected sample of hominid representatives (Rudapithecus, Australopithecus, Paranthropus) are in line with our results for our extant hominid sample. This is also the case for the evidence provided by previous studies of fossil hominin femora [START_REF] White | Pliocene hominids from Middle Awash, Ethiopia[END_REF][START_REF] Grine | Fossil Homo femur from Berg Aukas, northern Namibia[END_REF][START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF]. Indeed, although not all CF variables could be assessed due to variable preservation, the CF was present in all investigated fossil hominid specimens except SK 97 (P. robustus). However, CF morphology (i.e., shape, location, extension within the lesser trochanter, absolute horizontal length, absolute vertical height, and root width) in fossil hominids varied to an extent comparable to that observed in our extant hominid sample and the CF dimensions do not always fall within the human range of variation. Specifically, among the fossil hominins, a human-like CF vertical extension is found in A. africanus StW 99 and P. boisei KNM-ER 1465, with both specimens showing a CF confined within the proximal portion of the lesser trochanter, while other fossils have a shorter distal extension.

A previous study has reported the presence of a CF in the two partial femora of Orrorin, BAR 1002'00 and 1003'00, but only the latter, less heavily mineralized specimen could be assessed quantitatively [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF]. Axial virtual cross sections obtained from a medical CT scan showed the presence of a distinct CF. However, the reported absolute length of the Orrorin CF (1.58 cm; [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF] is most similar to the mean values we have documented in humans and Gorilla. Moreover, its observed distal extension to the middle of the lesser trochanter [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF] is intermediate between the average conditions we measured in Pan and humans and, most similar to the morphology displayed in a female Gorilla (SOM Fig. S5) and a captive male Pongo (Fig. 3).

The variation in CF occurrence and expression documented in the fossil hominin sample here, in combination with previous studies (i.e., Orrorin, Australopithecus, Middle Pleistocene Homo), may reflect variation in the biomechanics and/or frequency of bipedal (or climbing) locomotion of these hominin taxa. Alternatively, this variation, including the notable absence of a CF in SK 97, may simply represent 'normal' variation in CF occurrence and expression that has been documented here and in previous studies [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF] among humans and other great apes, further emphasizing that presence/absence of a CF is not a reliable indicator of bipedalism.

A comprehensive analysis of the degree of development of the CF in hominin taxa that also combines cortical thickness topographic distribution and trabecular structural organization at the proximal femoral end (e.g., [START_REF] Ohman | Cortical bone distribution in the femoral neck of hominoids: Implications for the locomotion of Australopithecus afarensis[END_REF][START_REF] Ruff | Femoral neck structure and function in early hominins[END_REF]Kivell, 2016;[START_REF] Ruff | Limb bone structural proportions and locomotor behavior in A.L. 288-1[END_REF][START_REF] Cazenave | Cortical bone distribution in the femoral neck of Paranthropus robustus[END_REF][START_REF] Cazenave | Trabecular organization of the proximal femur in Paranthropus robustus: Implications for the assessment of its hip joint loading conditions[END_REF][START_REF] Ryan | Humanlike hip joint loading in Australopithecus africanus and Paranthropus robustus[END_REF]Georgiou et al., 2020) may reveal whether CF variation reflects individual differences in mechanical strength across the different compartments, and therefore, individual differences in the loading environment of the hip joint.

Importantly, we also detected a Pan-like CF in Rudapithecus RUD 234, but more transversely extended, as observed in Gorilla. This finding is especially interesting given that short-distance bipedal walking and stance behaviors are variably displayed by all great apes, including occasionally by Pongo when moving on flexible branches (Thorpe et al., 2007;[START_REF] Crompton | Arboreality terrestriality and bipedalism[END_REF]. In addition, the external postcranial morphology of the stem hominid dryopithecin Danuvius guggenmosei [START_REF] Böhme | A new Miocene ape and locomotion in the ancestor of great apes and humans[END_REF] has been interpreted as reflecting habitual bipedal postures (but see [START_REF] Williams | Reevaluating bipedalism in Danuvius[END_REF]. Thus, the potential that Rudapithecus may have engaged in similar bipedal postures than that of great apes cannot be excluded, although bipedalism is a minor component of the Pan locomotor repertoire (0.4-8.0% of locomotor time; [START_REF] Hunt | Positional behavior in the Hominoidea[END_REF]. The results of our study thus suggest that CF morphology in extant great apes and Rudapithecus may represent a bone functional adaptation to different orthograde locomotor behaviors likely related to a more extended hip joint such as during vertical climbing or extended-limb clambering [START_REF] Isler | on the site-specific regulation of trabecular and cortical bone morphology[END_REF][START_REF] Böhme | A new Miocene ape and locomotion in the ancestor of great apes and humans[END_REF], rather than bipedalism specifically.

Potentially valuable information about the functional link between the CF and bipedalism could derive from the analysis of the proximal femora of Sansuke, an adult Macaca fuscata that regularly engaged for eight years in bipedal performance [START_REF] Nakatsukasa | Postcranial skeleton of a macaque trained for bipedal standing and walking and implications for functional adaptation[END_REF]. The femora of this individual have not yet been investigated for the presence of a CF, but previous research has shown that cortical bone thickness distribution and trabecular organization within the innominate, the femur, and the proximal tibia show distinct adaptive responses to the more compressive loads imposed by bipedal locomotion [START_REF] Nakatsukasa | Skeletal response to bipedalism in macaques, with emphasis on cortical bone distribution of the femur[END_REF]Volpato et al., 2008;[START_REF] Mazurier | The inner structural variation of the primate tibial plateau characterized by high-resolution microtomography. Implications for the reconstruction of fossil locomotor behaviours[END_REF]. If the CF, or at least a CF-like structure plays an active role in transferring loads incurred during bipedal gait from the femoral head through the neck to the shaft (see review in In conclusion, this study demonstrated that humans are distinguished from extant great apes in the more frequent occurrence of a CF, and a CF that is distinct in its greater proximodistal extension to within the lesser trochanter. However, an increasing body of comparative evidence from extant and extinct hominid taxa shows that the CF is a highly variable, possibly ecophenotypic (i.e, modified during life by environmental factors) structure, even in humans. As already suggested by its previous identification in some Pan femora [START_REF] Kuperavage | Earliest known hominin calcar femorale in Orrorin tugenensis provides further internal anatomical evidence for origin of human bipedal locomotion[END_REF], the presence of a CF-like structure and even human-like CF in some great apes makes clear that this feature cannot be reliably considered as a 'magic trait' (sensu [START_REF] Stern | Total morphological pattern" versus the "magic trait": Conflicting approaches to the study of early hominid bipedalism[END_REF] to identify habitual bipedal locomotion in hominins. In light of our results, several future investigations have been suggested that may elucidate the possible functional influence on the degree of expression of the CF. Higher resolution imaging and quantitative assessment of the CF in Orrorin BAR 1003'00, and the analysis of the partial femora representing the late Miocene taxa Danuvius [START_REF] Böhme | A new Miocene ape and locomotion in the ancestor of great apes and humans[END_REF] and Sahelanthropus [START_REF] Macchiarelli | Nature and relationships of Sahelanthropus tchadensis[END_REF] would further contribute the understanding of the evolutionary history of this trait.

Taylor, the Associate Editor, and to three anonymous reviewers for constructive critique that considerably improved this manuscript. We acknowledge the DST-NRF for financial support (Grant # UID23456) to establish the MIXRAD microfocus X-ray tomography facility at Necsa. 12.2 (2.2) 8.4-13.9

9.9 (1.8) 6.3-11.9

35.9 (7.9) 22.3-46.2 Pan (n = 6) 7.8 (1.5) 5.9-10. Abbreviations: CF MaxLength = length from the endosteal contour (see also Fig. 2b), CF MaxHeight = maximum height (see also Fig. 2c).

  individuals of the extant human sample and one Pan femur were scanned using a Nikon XT H225/320L industrial XCT system at the Evolutionary Studies Institute of the University of the Witwatersrand (Johannesburg, South Africa) using 70 kV, 120 μA, and 2000 projections. The remaining human specimens and one Papio specimen were scanned at the microfocus X-ray tomography facility (MIXRAD) of the South African Nuclear Energy Corporation SOC Ltd (Necsa, Pelindaba), using a Nikon XTH 225 ST (Metris) equipment and 100 kV, 100 μA, and 1000-2000 projections. Seven Pan specimens were scanned at the AST-RX: X-ray tomography technical platform by the v|tome|x L 240-180 equipment housed at the Muséum National d'Histoire Naturelle (Paris, France) using 100 kV, a current ranging from 500 to 610 μA, and 2400-2900 projections. The two remaining Pan specimens, the Pongo sample, and all fossil specimens were scanned with the BIR ACTIS 225/300 industrial microfocus X-ray tomography scanner at the Department of Human Evolution in the Max Planck Institute for Evolutionary Anthropology (Leipzig, Germany), using 130-140 kV, 100-140 μA, and 2500-2790 projections. The Gorilla sample was scanned with a Nikon XT 225 ST Metris scanner at the Cambridge Biotomography Centre of the Department of Zoology, University of Cambridge (Cambridge, UK) at 145-170 kV, 110-160 μA, and 2000 projections. Details about the scanning procedures used to image the additional nine fossil specimens that were excluded from the study due to poor preservation are provided in SOM Table S2. Femur size varied greatly across our comparative sample, ranging from the smallest Papio to the largest Gorilla. Thus, scan resolution varied as well due to the constraints of the CT systems and taking into account the specific geometric characteristics of the analytical platforms. The resolution range for each taxonomic sample was as follows: humans, 50-70 m; Pan, 30-80 m; Gorilla, 45-75 m; Pongo, 28-29 m; Papio, 45-67 m. The fossil specimens were scanned at resolutions ranging from 30 to 91 m (SOM Table

  [START_REF] Hammer | The calcar femorale: A new perspective[END_REF]), Sansuke's proximal femora should show at least traces of a cortico-trabecular strengthening extended across this region compared to wild M. fuscata individuals.

  M.C. is funded by the Fyssen Foundation. M.P. is funded by European Commission under the Marie Skłodowska-Curie Individual Fellowship Programme (H2020-MSCA-IF-2018-837966). This project has received funding from the European Research Council (grant agreement No. 819960).

Figures captions Figure 1 .

 captions1 Figures captions

Figure 2 .

 2 Figure 2. Micro-CT-based measurements (black lines) of the calcar femorale (CF) in a virtually-

Figure 3 .

 3 Figure 3. Micro-CT-based transverse (upper left) and parasagittal (right) virtual sections

  Abbreviations: PT MaxLength = length from the periosteal contour (see also Fig.2a), CF MaxLength = CF maximum length from the endosteal contour (see also Fig.2b), CF MaxHeight = maximum height (see also Fig.2c). a Absolute values were standardized by body mass estimated for each individual from their femoral head diameter by using the equations provided by[START_REF] Ruff | Lower limb articular scaling and body mass estimation in Pliocene and Pleistocene hominins[END_REF] for humans and Burgess et al. (2018) for extant apes.

Table 2

 2 Absolute values (in mm) of calcar femorale maximum length and height measurements for the fossil hominid sample.

	Taxon/
	specimen	CF MaxLength	CF MaxHeight
	Rudapithecus hungaricus RUD 234	8.5	14.9
	Australopithecus africanus StW 99 StW 367	5.6 4.8	28.7 -
	Paranthropus boisei? KNM-ER 738 KNM-ER 1465	6.2 7.5	18.5 26.4

Acknowledgments

For access to fossil and comparative materials, we are grateful to the curatorial staff of the Ditsong National Museum of Natural History, Pretoria; the Evolutionary Studies Institute at the University of the Witwatersrand, Johannesburg; the Institut Català de Paleontologia Miquel Crusafont, Sabadell; the McGregor Museum of Kimberley; the Mining and Geological Survey of Hungary, Budapest (MBFSZ); the Museum National d'Histoire Naturelle (MNHN), Paris; the National Museums of Kenya (KNM), Nairobi; the Pretoria Bone Collection at the Department of Anatomy of the University of Pretoria. We acknowledge C. Boesch for access to the Pan sample at the Max Planck Institute for Evolutionary Anthropology (MPI), Leipzig; L. Hutten to the baboon sample from the University of Cape Town; I. Livne to the Gorilla sample of the Powell-Cotton Museum; A. Van Heteren to the Pongo sample of the Zoologische Staatssammlung München. For the extant human comparative sample, ethical clearance was obtained from the Faculty of Health Sciences Research Ethics committee of the University of Pretoria (ref. no. 39/ 2016). We especially acknowledge J.-J. Hublin (Leipzig), E. L'Abbé (Pretoria), D. Morris (Kimberley), A. Oettlé (Pretoria), F. Thackeray (Johannesburg), K. Palotás (Budapest) and B. Zipfel (Johannesburg). We thank M. Bellato for scanning the Pan sample at the AST-RX Plateau d'Accès Scientifique à la Tomographie à Rayons X of the UMS 2700 2AD CNRS-MNHN, Paris (data kindly shared by R. Macchiarelli). We thank F. de Beer (Pelindaba), J. Hoffman (Pelindaba), L. Bam (Pelindaba), K. Jakata (Johannesburg), K. Smithson (University of Cambridge), and D. Plotzki (MPI) for CT scanning at Necsa, Wits, Cambridge and MPIEVA, respectively, and the staff of industrial-CT YXLON Compact at the Human Evolution Lab. (LEB) at the Universidad de Burgos. For availability to run independent measures for interobserver error assessment, we thank A. Bardo (Canterbury). For discussion, we thank A. Beaudet (Cambridge), C. Dunmore (Canterbury), S. Syeda (Canterbury), C. Theye (Pretoria), Z. Tsegai (Leipzig). Finally, we are grateful to Andrea Thakkar, C.J., Thakkar, S., Kathalgere, R.T., Kumar, M.N., 2015. Calcar femorale grafting in the hemiarthroplasty of the hip for unstable inter trochanteric fractures. Indian J. Orthop. 49, 602-609. Thompson, R., 1907. The relationship between the internal structure of the upper part of the femur and fractures through the base of the neck of the femur. J. Anat. Physiol. 42, 60-68. Thorpe, S.K.S., Crompton, R.H., 2006. Orangutan positional behavior and the nature of arboreal locomotion in Hominoidea. Am. J. Phys. Anthropol. 131, 384-401. Thorpe, S.K.S., Holder, R.L., Crompton, R.H., 2007. Origin of human bipedalism as an adaptation for locomotion on flexible branches. Science 316, 1328-1331. Tsegai, Z.J., Skinner, M.M., Pahr, D.H., Hublin, J.-J., Kivell, T.L., 2018a. Systemic patterns of trabecular bone across the human and chimpanzee skeleton. J. Anat. 232, 641656.

Tsegai, Z.J., Skinner, M.M., Pahr, D.H., Hublin, J.-J., Kivell, T.L., 2018b. Ontogeny and variability of trabecular bone in the chimpanzee humerus, femur and tibia. Am. J. Phys.

Anthropol. 167, 713-736. Vera, M.C., Ferretti, J.L., Abdala, V., Cointry, G.R., 2020. Biomechanical properties of anuran long bones: correlations with locomotor modes and habitat use. J. Anat. 236, 1112Anat. 236, -1125. .

their original side, all specimens are imaged as right femora. See SOM Figure S3 for the additional extant specimens. Scale bar = 10 mm. robustus (SK 97), and likely P. boisei (KNM-ER 738 and 1465). The white arrows indicate the CF. We observed no trace of a CF in SK 97, although possible strengthening of the trabeculae projecting towards the greater trochanter could not be confidently ascertained because of the incomplete preservation in this specimen. Independent of their original side, all specimens are imaged as right femora (for their original side, see SOM Table S2). Scale bar = 10 mm.