Abstract While there is broad agreement that early hominins practiced some form of terrestrial bipedality, there is also evidence that arboreal behavior remained a part of the locomotor repertoire in some taxa, and that bipedal locomotion may not have been identical to that of modern humans. It has been difficult to evaluate such evidence, however, because of the possibility that early hominins retained primitive traits (such as relatively long upper limbs) of little contemporaneous adaptive significance. Here we examine bone structural properties of the femur and humerus in the Australopithecus afarensis A.L. 288–1 ("Lucy", 3.2 Myr) that are known to be developmentally plastic, and compare them with other early hominins, modern humans, and modern chimpanzees. Cross-sectional images were obtained from micro-CT scans of the original specimens and used to derive section properties of the diaphyses, as well as superior and inferior cortical thicknesses of the femoral neck. A.L. 288–1 shows femoral/humeral diaphyseal strength proportions that are intermediate between those of modern humans and chimpanzees, indicating more mechanical loading of the forelimb than in modern humans, and by implication, a significant arboreal locomotor component. Several features of the proximal femur in A.L. 288–1 and other australopiths, including relative femoral head size, distribution of cortical bone in the femoral neck, and cross-sectional shape of the proximal shaft, support the inference of a bipedal gait pattern that differed slightly from that of modern humans, involving more lateral deviation of the body center of mass over the support limb, which would have entailed increased cost of terrestrial locomotion. There is also evidence consistent with increased muscular strength among australopiths in both the forelimb and hind limb, possibly reflecting metabolic trade-offs between muscle and brain development during hominin evolution. Together these findings imply significant differences in both locomotor behavior and ecology between australopiths and later Homo.

Citation: Ruff CB, Burgess ML, Ketcham RA, Kappelman J (2016) Limb Bone Structural Proportions and Locomotor Behavior in A.L. 288-1 ("Lucy"). PLoS ONE 11(11): e0166095. https://doi.org/10.1371/journal.pone.0166095 Editor: Karen Rosenberg, University of Delaware, UNITED STATES Received: July 25, 2016; Accepted: October 21, 2016; Published: November 30, 2016 Copyright: © 2016 Ruff et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This research was supported by the National Science Foundation (www.nsf.gov) BCS-0642297 and BCS-1316104 to C.B. Ruff, and EAR-0646848, EAR-0948842, and EAR-1258878 to R.A. Ketcham. Institutional support was provided by the University of Oulu and the University of Texas at Austin. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction The acquisition of terrestrial bipedal locomotion was one of the defining events in hominin evolution [1–4]. Although there is anatomical evidence for some form of terrestrial bipedality among hominins at least 6 million years ago [5, 6], there is also accumulating evidence that the form of terrestrial bipedality practiced by some early hominins may have differed from that of modern humans, and that arboreal behavior continued to be an important component of the hominin locomotor repertoire for millions of years [5, 7–19]. There is continuing debate, however, over the functional significance of various postcranial morphological traits observed in early hominins, and whether they constitute primitive retentions of little adaptive importance or indicate major differences in actual locomotor behavior [4, 15, 20]. A key issue in such debates is the degree of developmental plasticity of different skeletal features. As noted by a number of authors, developmentally plastic traits should reflect, at least in part, the actual mechanical loading environment of the animal while it was alive, thus providing direct evidence of its behavior [4, 13, 21–26]. One such trait is the cross-sectional structure of long bone diaphyses, which is known from both experimental and observational studies to be responsive to changes in mechanical loadings during life [27–29]. Thus, for example, changes in inter-limb bone diaphyseal strength proportions accurately reflect changes in locomotor behavior during development in humans, gorillas, and chimpanzees [30–32]. Limb bone strength proportions in early Homo erectus (KNM-ER 1808 and KNM-WT 15000) are similar to those in modern humans [33], supporting other evidence for completely modern terrestrial locomotor behavior in this taxon [34]. In contrast, strength proportions in H. habilis (sensu stricto, OH 62) are more chimpanzee-like, suggesting a significant arboreal component in its locomotor repertoire [13]. To date, in part because of the fragmentary nature of most early hominin fossils, no similar analysis of an australopith individual has been carried out. Instead, analyses have focused on indirect assessments of limb bone proportions, through resampling techniques or comparisons between elements from different individuals, or between functionally disparate characteristics, such as articular and length proportions [17, 20, 35–37], which are difficult to interpret. Another issue with such analyses is that long bone length and articular size appear to be more genetically canalized than diaphyseal cross-sectional structure [31, 38–41], thus leading to familiar arguments regarding their effectiveness in reconstructing actual behavior during life [4, 20]. Diaphyseal external breadths or circumferences, included in some previous studies [37, 42], are also less effective than true cross-sectional properties in distinguishing between locomotor groups [13, 43]. The Australopithecus afarensis specimen A.L. 288–1 ("Lucy") is one of the very few early hominin (3.2 Myr) individuals to preserve relatively complete upper and lower limb bone elements [44] (Fig 1). A.L. 288–1 is a young adult female who had reached skeletal (all epiphyses fused) and dental (m3 erupted and in occlusion) maturity at death. It has long been recognized that her humerofemoral length proportion is intermediate between that of modern humans and chimpanzees, although this has led to different interpretations of her locomotor capabilities [45–49], and again is subject to the argument of primitive retention versus contemporary function [20]. Although diaphyseal cortices of the left femur and humerus of A.L. 288–1 are relatively well preserved (see below), no radiographic study of internal bone structure of these elements has been carried out to date (two earlier studies estimated some section properties from casts and photographs of natural breaks [50, 51]; these studies are discussed later in this paper). In this study, we use micro-CT to derive cross-sectional geometric properties of the humeral and femoral diaphyses and femoral neck of A.L. 288–1. Together with limb bone articular properties, these data are used to investigate inter- and intra-limb structural proportions of A.L. 288–1 in comparison to those of modern humans, chimpanzees, and other fossil hominin specimens, and to reconstruct locomotor behavior in early hominins. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Composite photograph of original fossil specimens comprising A.L. 288–1. Scale bar is 50 mm. https://doi.org/10.1371/journal.pone.0166095.g001 We also use these data to address the general issue of relative muscular strength in early hominins, which has important implications for interpreting metabolic trade-offs, energy allocation, and ecology [52–56]. Because external articular size is less developmentally plastic than diaphyseal cross-sectional geometry [39, 41, 57], variation in diaphyseal strength to articular size should reflect to some extent relative muscular loadings during life (see S1 Text, S1 Fig). In addition, relative articular size is related in a long-term evolutionary timeframe to joint reaction force (JRF) and joint mobility [43, 58, 59]. These factors are also considered in our interpretations of variation in articular and shaft strength proportions in A.L. 288–1 and comparative samples. Our results indicate that A.L. 288–1, and australopiths in general, show differences in limb bone structural proportions from those of modern humans and Homo erectus that indicate significant differences in both locomotor behavior and relative muscularity.

Discussion It is clear that A.L. 288–1 and australopiths in general show many postcranial adaptations to terrestrial bipedality and probably walked in a basically human-like manner when on the ground [4, 26, 44, 88–92]. The results of the present study reinforce this view. A.L. 288–1's knee to elbow size proportion is distinctly human and non-ape-like, indicating an adaptation to greater loading of the hind limb than in apes. Her femoral neck structure implies a hip abductor mechanism during bipedal gait that was broadly similar to that in modern humans. However, we found that A.L. 288–1 also exhibits morphological features that imply substantial differences in locomotor behavior from that in modern humans or early Homo. Lucy’s femoral/humeral diaphyseal strength proportion indicates greater muscular loading of her upper limb relative to her lower limb than is characteristic of either modern humans or Homo erectus, and more similar to that of chimpanzees. While other behavioral explanations are conceivable (such as increased upper limb use related to food procurement or defense), given the range of morphological evidence throughout her skeleton that is consistent with greater arboreality [10, 19, 93], the most likely explanation is that Lucy climbed trees with a greater reliance on her upper extremity much more frequently than modern humans or early Homo (with the exception of H. habilis sensu stricto [13]). It is even possible that adaptations for terrestrial bipedality, such as restructuring the foot in Au. afarensis [4], necessitated greater compensatory muscular force in the upper limb to facilitate frequent climbing, which could account in part for her relatively increased humeral strength. The developmental plasticity of long bone diaphyseal structure negates the argument that this proportional difference in strength could be just a primitive retention from a more arboreal ancestor, and the degree of adaptation observed argues against its being an infrequent and minor component of the locomotor repertoire. These results also support the view that other potentially more genetically constrained morphological features observed in Au. afarensis that could facilitate arboreal behavior, such as longer and more curved toes and fingers, a higher intermembral index, and a more cranially oriented glenoid fossa [10, 18, 19, 46], were maintained by stabilizing selection for arboreality rather than simply not being selected against [4]. Our interpretation of the functional significance of inter-limb strength proportions is supported not only by direct experimental evidence for bone developmental plasticity in general [27], but also by comparisons of limb bone strength proportions between and within hominoids in relation to locomotor behavior. Among great apes, forelimb to hind limb strength proportions parallel the degree of arboreality, from orangutans to chimpanzees to Western lowland gorillas to mountain gorillas, the most terrestrial nonhuman hominoid [43, 94]. Furthermore, within both mountain gorillas and common chimpanzees, ontogenetic declines in arboreal locomotion are associated with declines in forelimb to hind limb strength [31, 32]. It should be noted that these trends are not simply a function of differences or changes in overall limb bone size, e.g., length, as lowland and mountain gorillas have similar limb length proportions but different strength proportions, and ontogenetic changes in limb length proportions in mountain gorillas do not parallel those in strength proportions [31]. The same is true for growing humans, who show much greater increases in femoral/humeral strength proportions than in length proportions, including a sharp reversal in strength proportions at the initiation of walking that has no counterpart in length changes [30, 95]. Infant (pre-walking) humans are in fact similar to quadrupedal primates in limb bone strength proportions, even though the hind limb already shows evidence of relative elongation, and only develop typically human strength proportions with the increased mechanical loading of the lower limb generated by bipedal gait [30]. Another interesting "natural experiment" in limb strength proportions among humans is provided by gymnasts, who habitually load their upper limbs at several times body mass during practice and performance [96], and show much greater increases in upper limb bone strength than in lower limb bone strength over non-gymnast controls [96, 97]. Exactly how upper limb bone loading is increased during arboreal locomotion among hominoids has not yet been quantified, although it seems probable that climbing and forelimb suspensory behavior produce relatively large mechanical loads on the forelimb bones. Although the forelimb is also loaded during terrestrial quadrupedal locomotion in nonhuman hominoids, peak vertical forces are much higher on the hindlimb [98]. A relatively longer humerus and shorter femur in A.L. 288–1 compared to modern humans [45–49] could also theoretically contribute to higher bending loads in the humerus in A.L. 288–1, by increasing bending moment arms. However, limb muscle moment arms in chimpanzees and modern humans, scaled for body size, are not systematically different and do not covary consistently with limb length differences [99]. Also, as discussed above, ontogenetic changes in bone length proportions are not paralleled by similar changes in bone strength proportions. Thus, it seems unlikely that limb length differences alone could account for proportional differences in strength between A.L. 288–1 and modern humans. Other purportedly developmentally plastic traits such as phalangeal curvature have also been argued to support the argument for more arboreal behavior in australopiths and other early hominins [5, 18, 19, 77, 100]. However, it should be noted that the inference of developmental plasticity of phalangeal curvature is based only on comparative ontogenetic studies [21, 101, 102], and in addition to possible direct mechanical effects there is also likely a strong genetic component to curvature [102]. There are also undoubtedly genetic effects on long bone cross-sectional geometry (e.g., [103, 104]); however, there is also abundant experimental evidence that long bone strength is modified by behavioral use during life [27]. It should also be noted that genetic differences in skeletal mechanosensitivity to applied loadings, demonstrated in some modern inbred strains (e.g., [105]), cannot account for inter-limb proportional differences, since skeletal elements from the same individual are being compared. Thus, the present results provide the strongest evidence to date for increased mechanical loading of the upper relative to the lower limb in an australopith, which implies a significant difference in its locomotor repertoire. While direct femoral to humeral strength comparisons are not possible in other associated specimens, the very strong upper limb bones characteristic of australopiths generally ([77, 106, 107], and present study) are also consistent with this interpretation. OH 62, attributed to H. habilis (sensu stricto) and dated to about 1.8 Myr [108], also shows increased upper relative to lower limb bone strength compared to modern humans [13]. Although not included in the present study because of uncertainties regarding the exact placement of available cross sections in OH 62, for the most reasonable reconstructions it clearly is more similar to chimpanzees in strength proportions, falling in a position similar to that of A.L. 288–1, if not even more chimpanzee-like [13]. The attribution of OH 62 to H. habilis has been questioned, based on comparisons to Au. sediba [109], but regardless of its phylogenetic position, it provides evidence that increased upper limb loading, and by implication, frequent arboreal behavior, remained an important part of the locomotor behavior of at least some hominin lineages for millions of years after the first evidence for terrestrial bipedality [4–6]. Two earlier studies included some limited analyses of femoral and humeral diaphyseal cross sections of A.L. 288–1 taken at approximately midshaft [50, 51]. However, in each case the sections were derived from measurements of natural breaks on casts, supplemented with linear measurements on the original specimens, i.e., no radiography or CT imaging was performed. Although the humerus was included in one of these studies [50], there is no natural break near midshaft on either humerus of A.L. 288–1 (Fig 2), so it is not clear how this section was obtained. Furthermore, cross-sectional properties for A.L. 288–1 in this study [50] were calculated using an approximate eccentric ellipse model based on linear breadth measurements. It is not surprising, then, that cross-sectional properties in both studies ([50, 51] and Jungers, pers. comm.) vary by up to almost 30% from those obtained here (Table 1). The current properties, derived directly from micro-CT scans at well-defined locations, should be used in any future analyses. A.L. 288–1's femur is also strong relative to either hip or knee articular size, as is the Stw 99 (Au. africanus) femur (relative to hip size). The overall greater robusticity of limb bone diaphyses in early hominins relative to modern humans has been previously noted [4]. The present study results indicate that this is specifically characteristic of australopiths and not to the same extent of early Homo. This difference implies a significant reduction in muscular loadings of both the humerus and femur in H. erectus relative to australopiths, if diaphyseal strength/articular size can be taken to represent relative muscular strength during life (S1 Text). This interpretation assumes that other factors that may influence relative articular size, including joint reaction force (relative to body size) and joint excursion [43, 58, 59], were similar between the groups compared. Although possibly somewhat variable in certain respects [110], joint excursion in the limbs is likely to have been relatively similar among all early hominins [111]. Joint reaction forces relative to body mass probably did vary, at least in the hip (see below). However, because of its much greater developmental plasticity, diaphyseal strength relative to articular size should still reflect in-vivo muscular loadings relative to joint reaction forces, including muscles not primarily involved in joint stabilization (such as the gluteal abductors). If australopiths were indeed characterized by greater applied muscular forces in their limbs than H. erectus or modern humans, this observation has interesting implications for hominin evolutionary scenarios involving metabolic trade-offs between different organ systems [52–56, 112]. Muscular strength relative to body size is substantially greater in chimpanzees than in modern humans including even trained athletes (see S2 Text). The present results imply that this characteristic may also have been true of australopiths. There is evidence for marked evolutionary divergence between modern humans and chimpanzees in metabolite concentrations specifically in the brain and skeletal muscle [52]. Based on these results, the authors speculated that "While the molecular mechanism linking metabolic divergence with changes in muscular strength on the human evolutionary lineage cannot be determined based on our observations alone, we hypothesize that metabolic evolution of human muscle and brain metabolites may have occurred in parallel … Our results indicate that the reallocation of energy to energetically costly human brains may have required further decrease in energy expense in the skeletal muscle, at least during peak performance" ([52]: 6). Other authors have proposed similar scenarios based on comparative studies within vertebrates more broadly [53–55]. Recent work demonstrating a higher basal metabolic rate in modern humans than in great apes (as well as greater body fat storage) [113] indicates that certain aspects of these scenarios may need to be modified, i.e., humans may be able to afford a relatively larger brain in part because of our higher metabolic rate. However, other factors, including locomotor energetic efficiency, were also likely important [113]. Thus, the marked increase in relative brain size of early Homo, especially H. erectus, compared to australopiths [114] may have been facilitated in part by reductions in energy allocated to skeletal muscle. If true, these observations could have other behavioral implications. For example, the capacity for increased peak power output of muscles in certain situations, such as defense against or escape from predators, or during inter-taxon aggression, may have had important selective advantages prior to the development of effective weapons [115]. The development of more efficient tools for food procurement or processing may also have decreased selective pressures for muscular strength. Increased muscular strength in extant great apes relative to modern humans is apparently attributable to a difference in specific force generated per volume of muscle tissue, rather than an increase in muscle mass in apes, although the cellular and molecular mechanisms involved have yet to be identified [56] (S2 Text). Thus, despite their implied greater muscular strength, australopiths may not necessarily have been characterized by large muscles relative to body mass. As noted earlier, in terms of terrestrial locomotion, A.L. 288–1 (the best represented individual of Au. afarensis) shows many skeletal adaptations for bipedality [4]. Some aspects of Lucy’s proximal femoral morphology, however, indicate that her bipedal gait may have differed slightly from that of later Homo. A relatively small femoral head in australopiths in general has long been observed, although the mechanical significance of this observation has been debated [116–122]. One issue in this regard concerns which overall "size" parameter to use for evaluating relative femoral head size [51, 117, 120, 122]. The present analyses show that compared to modern humans or H. erectus, A.L. 288–1 has a small femoral head, but not a small knee, relative to distal humeral articular breadth. This observation suggests that overall lower limb mechanical loading was not reduced, i.e., that A.L. 288–1 was bipedal when on the ground, but that the joint reaction force on the femoral head was reduced relative to lower limb loadings as a whole (also see evidence for a relatively modern ankle joint and calcaneus in Au. afarensis [90, 91]). In contrast, a biomechanical model of the pelvis and proximal femur based on modern humans predicts that hip JRF would have been increased in A.L. 288–1 (see S3 Text, S2 Fig). It was hypothesized that she may have laterally deviated her body center of mass (COM), i.e., leaned over her stance limb, to reduce hip JRF during walking [51]. Note that this argument does not imply a developmentally plastic response to reduced hip joint loading, given the evidence for genetic constraint on articular size [39, 41, 57], but rather was more likely a long-term evolutionary (genetic) adaptation to reduced hip joint loadings in australopiths (and possibly other early hominins [5]) in conjunction with a different gait pattern. The present new results for A.L. 288–1 are consistent with this hypothesis. Her superior and inferior femoral neck cortical breadths are asymmetric and more similar to modern humans than to nonhuman hominoids, but are also somewhat intermediate in asymmetry. This difference implies a more vertically oriented hip JRF than in modern humans, which would result from lateral deviation of the body COM during stance [14]. Lucy’s proximal femoral diaphysis is mediolaterally buttressed, but less so than in early Homo femora, even though her pelvic and femoral neck morphology would predict greater buttressing (S2 Fig). (Allometric scaling is unlikely as an explanation for this observation because large australopith specimens show the same pattern.) This observation is again consistent with a reduced hip JRF [51]. The morphology of other australopith femora is similar to that of A.L. 288–1, suggesting a similar gait modification. If australopiths did indeed employ greater lateral deviation of the body COM over the stance limb during bipedal gait, it would likely have involved a substantial increase in metabolic cost of locomotion (S3 Text). Together with other morphological attributes, such as a relatively short hind limb [51, 65], this difference in turn may have limited long-distance terrestrial mobility in australopiths.

Conclusions Although bipedal when on the ground, the limb bone structural proportions of A.L. 288–1 provide evidence for substantially more arboreal, i.e., climbing behavior than either modern humans or Homo erectus. The frequency and magnitude of force required to stimulate bone modeling and remodeling of this kind [27] implies that this behavior was adaptively significant and not a trivial component of the locomotor repertoire [4, 89]. Possible reasons for using the trees more often include foraging for food and escape from predators. Furthermore, there is evidence that terrestrial bipedal gait in A.L. 288–1 may have differed in subtle but important ways from that of later Homo, decreasing locomotor efficiency when on the ground and limiting terrestrial mobility. Overall muscular strength relative to body size was likely greater than in Homo, perhaps reflecting less reliance on technology for food procurement/processing and defense. Where possible to evaluate, the same morphological attributes are present in other australopith specimens as well as H. habilis sensu stricto, i.e., OH 62 [13]. Overall these observations imply fundamental differences in ecology and behavior between australopiths and Homo erectus. It is likely that a number of different forms of terrestrial bipedality were practiced by early hominins, and that arboreal behavior remained an important part of the locomotor repertoire in particular taxa for millions of years.

Acknowledgments We thank the Authority for Research and Conservation of Cultural Heritage and the National Museum of Ethiopia of the Ministry of Tourism and Culture for permission to scan, study, and photograph Lucy; A. Admassu, K. Ayele, J. A. Bartsch, Y. Beyene, Y. Desta, R. Diehl, R. Flores, R. Harvey, G. Kebede, R. Lariviere, J. H. Mariam, S. M. Miller, L. Rebori, B. Roberts, J. Ten Barge, J. M. Sanchez, D. Slesnick, D. Van Tuerenhout, S. Wilson, M. Woldehan, and M. Yilma for facilitating and assisting with the scanning; T. Getachew and M. Endalamaw for assisting with the photography, and S. Mattox for the photograph in Extended Data Fig 3; A. Witzel for assistance with 3D printing; and Paleoanthropology Lab Fund, College of Liberal Arts UT Austin, and Houston Museum of Natural Science for financial and logistical support, and Owen-Coates Fund of the Geology Foundation of UT Austin for publication costs. The University of Texas High-Resolution X-ray CT Facility was supported by U.S. National Science Foundation grants EAR-0646848, EAR-0948842, and EAR-1258878 (www.nsf.gov). We would also like to thank L. Rodriguez and J. M. Carretero for making available unpublished data on several Atapuerca specimens and Gombore MK3; B. Holt, M. Niskanen, V. Sládek, and M. Berner for collaboration in obtaining the modern European comparative data set, which was supported by NSF (www.nsf.gov) grant BCS-0642297; J-A. Junno for collaboration on obtaining the chimpanzee comparative data set, which was supported by NSF grant (www.nsf.gov) BCS-1316104, with institutional support from the University of Oulu; and J-J. Hublin and C. Boesch for access to chimpanzee specimens in their care. The paper benefited from the comments of three anonymous reviewers.

Author Contributions Conceptualization: CBR JK RAK. Data curation: JK RAK. Formal analysis: CBR MLB. Funding acquisition: CBR JK RAK. Investigation: CBR JK RAK. Methodology: CBR JK RAK MLB. Project administration: CBR JK. Resources: CBR JK RAK. Software: CBR JK RAK MLB. Supervision: CBR JK. Visualization: CBR JK RAK MLB. Writing – original draft: CBR. Writing – review & editing: CBR JK RAK.