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in the Doushantuo fossils [for example, opalinids are multinuclear (32)]. Only volvocalean embryos show so many rounds of palintomy, but the resulting blastomeres are connected by a system of cytoplasmic bridges (35) that are not present in the fossils. The combination of palintomy within a multilayered cyst wall and peanut-shaped germination stages as seen in the fossils conforms to the pattern seen in nonmetazoan holozoans; nonetheless, there are no discrete characters in the Doushantuo fossils that are uniquely holozoan. The animal embryos likely represent nonmetazoan holozoans or possibly even more distant eukaryote branches.
References and Notes
1. S. Xiao, Y. Zhang, A. Knoll, Nature 391, 553 (1998). 2. S. Xiao, Paleobiology 28, 244 (2002). 3. C. Yin, S. Bengtson, Z. Yue, Acta Palaeontol. Pol. 49, 1 (2004). 4. J.-Y. Chen et al., Science 312, 1644 (2006). 5. J. W. Hagadorn et al., Science 314, 291 (2006). 6. L. Yin et al., Nature 446, 661 (2007). 7. P. A. Cohen, A. H. Knoll, R. B. Kodner, Proc. Natl. Acad. Sci. U.S.A. 106, 6519 (2009). 8. J.-Y. Chen et al., Proc. Natl. Acad. Sci. U.S.A. 106, 19056 (2009). 9. J. V. Bailey, S. B. Joye, K. M. Kalanetra, B. E. Flood, F. A. Corsetti, Nature 445, 198 (2007). 10. E. C. Raff et al., Proc. Natl. Acad. Sci. U.S.A. 105, 19360 (2008). 11. R. J. Horodyski, J. Bauld, J. H. Lipps, C. V. Mendelson, in The Proterozoic Biosphere: A Multidisciplinary Study, 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. J. W. Schopf, C. Klein, Eds. (Cambridge Univ. Press, Cambridge, 1992), pp. 185193. J. D. Schiffbauer, S. Xiao, K. S. Sharma, G. Wang, Geology, 10.1130/G32546.1 (2011). Materials and methods are available as supporting material on Science online. S. Xiao, C. Zhou, X. Yuan, Nature 446, E9, discussion E10 (2007). Y.-S. Xue, T.-F. Tang, C.-L. Yu, C.-M. Zhou, Acta Palaeont. Sin. 34, 688 (1995). P. C. J. Donoghue, Nature 445, 155 (2007). J. V. Bailey, S. B. Joye, K. M. Kalanetra, B. E. Flood, F. A. Corsetti, Nature 446, E10 (2007) Reply. S. Xiao, J. W. Hagadorn, C. Zhou, X. Yuan, Geology 35, 115 (2007). P.-J. Liu, C.-Y. Yin, S.-M. Chen, F. Tang, L.-Z. Gao, Acta Geosci. Sin. 30, 457 (2009). Z. Yin et al., Precambr. Res., published online 9 September 2011 (10.1016/j.precamres.2011.08.011). A. Rose, in Cell Division Control in Plants, D. P. S. Verma, Z. Hong, Eds. (Springer, Heidelberg, 2007), pp. 207230. L. Mendoza, J. W. Taylor, L. Ajello, Annu. Rev. Microbiol. 56, 315 (2002). K. V. Mikhailov et al., Bioessays 31, 758 (2009). W. L. Marshall, M. L. Berbee, Protist 162, 33 (2011). M. Pekkarinen, K. Lotman, J. Nat. Hist. 37, 1155 (2003). L. Mendoza, R. A. Herr, S. N. Arseculeratne, L. Ajello, Mycopathologia 148, 9 (1999). A. Franco-Sierra, P. Alvarez-Pellitero, Parasitol. Res. 85, 562 (1999). S. Raghu-Kumar, Bot. Mar. 30, 83 (1987). B. S. Leander, J. Eukaryot. Microbiol. 55, 59 (2008). J. T. Bonner, Integr. Biol. 1, 27 (1998). M. Elbrchter, Helgol. Meersunters. 42, 593 (1988). K. Hanamura, H. Endoh, Zoolog. Sci. 18, 381 (2001). D. P. Molloy, D. H. Lynn, L. Giamberini, Dis. Aquat. Organ. 65, 237 (2005). 34. M. D. Herron, A. G. Desnitskiy, R. E. Michod, J. Phycol. 46, 316 (2010). 35. K. J. Green, D. L. Kirk, J. Cell Biol. 91, 743 (1981). Acknowledgments: We thank S. Xiao, T. Cavalier-Smith, and B. Landfald for discussion; T. Hode and Z. Yue for field-work collaboration; A. Groso for assistance with the srXTM work; and P. Varvarigos and D. Elliott for the use of fig. S7. The work was supported by the Swedish Research Council, Natural Environment Research Council, Ministry of Science and Technology of China, National Natural Science Foundation of China, EU FP7, and the Paul Scherrer Institute. Figured or measured specimens are deposited at the Swedish Museum of Natural History and the Museum of Earth Science, Chinese Academy of Geological Sciences. The srXTM investigations were conducted at the X04SA and X02DA (TOMCAT) beamlines of the Swiss Light Source. The data were visualized and analyzed by using Avizo software. Data are available in the SOM. S.B. and P.C.J.D. designed the research and wrote the paper; T.H. found the nucleic structures, prepared the corresponding visualizations, and wrote the specimen descriptions in the SOM; J.A.C. found the propagule-like structures and performed taphonomic analyses and volumetric measurements; C.Y. and S.B. did the field work; C.Y. provided the additional data from Hubei; and M.S., F.M., S.B. and P.C.J.D. designed the srXTM experiments.

22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.

Supporting Online Material

www.sciencemag.org/cgi/content/full/334/6063/1696/DC1 Materials and Methods SOM Text Figs. S1 to S7 Table S1 References (3667) Movies S1 to S5 8 June 2011; accepted 16 November 2011 10.1126/science.1209537

From Flat Foot to Fat Foot: Structure, Ontogeny, Function, and Evolution of Elephant Sixth Toes
John R. Hutchinson,1 Cyrille Delmer,2 Charlotte E. Miller,1 Thomas Hildebrandt,3 Andrew A. Pitsillides,1 Alan Boyde4 Several groups of tetrapods have expanded sesamoid (small, tendon-anchoring) bones into digit-like structures (predigits), such as pandas thumbs. Elephants similarly have expanded structures in the fat pads of their fore- and hindfeet, but for three centuries these have been overlooked as mere cartilaginous curiosities. We show that these are indeed massive sesamoids that employ a patchy mode of ossification of a massive cartilaginous precursor and that the predigits act functionally like digits. Further, we reveal clear osteological correlates of predigit joint articulation with the carpals/tarsals that are visible in fossils. Our survey shows that basal proboscideans were relatively flat-footed (plantigrade), whereas early elephantiforms evolved the more derived tip-toed (subunguligrade) morphology, including the predigits and fat pad, of extant elephants. Thus, elephants co-opted sesamoid bones into a role as false digits and used them for support as they changed their foot posture. he enlarged radial sesamoid bones of giant panda forefeet (1, 2) are classic examples of evolutionary exaptation (3, 4): co-option of old structures for new functions. It is less widely recognized that such sixth toes or false thumbs have evolved convergently in numerous tetrapods, such as moles and frogs (5, 6). They exist in numerous mammals in a less enlarged state, variably called the prepollex/prehallux (here

inent in both the manus (forefeet) and the pedes (hindfeet) of elephants, where they have been mistaken for sixth digits or otherwise presumed to play a role in foot support (79). Indeed, the recent discovery that moles have developmentally switched their radial sesamoid (prepollex) to a digit-like identity (10) intimates that elephants and other species may have done the same. Here, we report a multidisciplinary anatomical, histological, functional, and phylogenetic analysis (11) of the predigits in elephant feet. We hoped this would illuminate how elephants evolved their characteristic subunguligrade (nearly tip-toed, with only distal toes contacting the ground) foot posture and function, as compared with the plesiomorphic plantigrade (flat-footed, with wrists/ankles contacting the ground) foot posture in many other tetrapods. In 1710, Blair (7) provided the first detailed osteological description of elephants, concluding that they have six toes. The sixth toes (medialmost position; corresponding to digit zero) were later identified as the enigmatic prepollex
1 Department of Veterinary Basic Sciences and Structure and Motion Laboratory, The Royal Veterinary College, Hatfield AL9 7TA and London NW1 0TU, UK. 2Department of Palaeontology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK. 3Leibniz Institute for Zoo and Wildlife Research, im Forschungsverbund Berlin e.V., Postfach 601103, Berlin D-10252, Germany. 4Dental Physical Sciences, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Mile End Road, London E1 4NS, UK.

called predigits), radial/tibial sesamoids, or other terms (such as falciform, accessory scaphoid, or navicular). Whether these sesamoids are ancestrally or convergently evolved in various tetrapod clades remains to be determined. The latter seems likely, given the absence of similar sesamoids in most fossil outgroups, yet a cartilaginous nodular precursor cannot be excluded. Regardless, enlarged sesamoids are quite promSCIENCE VOL 334

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and prehallux (8, 9, 12) (figs. S1 to S4). Three centuries of sporadic discussion about the identities of predigits in tetrapods have ensued (8, 9), sometimes returning to the question of whether they are actually atavistic digits (9, 13). Considering the characteristic variability (8) and apparent mineralization late in the ontogeny of sesamoids (14), as well as their articulations (15) with metacarpal I/tarsal I and metatarsal I (Fig. 1 and movie S1), the prepollex/prehallux of elephants must correspond to the radial/tibial sesamoids of other tetrapods. The late mineralization and confounded scientific history of predigits have tended to prevent their preservation, discovery, scholarly description, and even museum exhibition. The vexing issue of the homology of elephant predigits remains unresolved, complicated by the specialization of paenungulate outgroups (such as Sirenia and Hyracoidea). Despite early studies, it remains unclear whether elephant predigits are never more than cartilaginous rods, as current literature assumes (8, 9, 12), or whether they become true bones at some point in ontogeny. We used a combination (11) of dissection, computed x-ray tomography (CT) scans, histology, and backscattered electron scanning electron microscopy (BSE SEM) to address this question (Fig. 2 and figs. S5 to S13). Using this combination of methods, we found that elephant predigits initially form as massive, purely cartilaginous rods and that these can become further stiffened through a slow conversion to bone (that is, forming endochondrally) by an unusual ossification mechanism. Histological examination showed that this initial hyaline cartilage element lacks a preferential orientation of chondrocytes or growth-platelike stratification (fig. S13). Imaging with BSE SEM and CT in addition to histology revealed that patches of this cartilage calcify and are resorbed and replaced by bone that subsequently models to a foam- or honeycomb-like cancellous (spongy bone) structure. The advancing mineralizing fronts and the thickness of the calcified cartilage layers resemble those seen in mature articular cartilage. Together, our analyses not only show that the cartilaginous predigits are slowly replaced by bone during late ontogeny, but that this bone is unusual in its development [Fig. 2, supporting online material (SOM) text, and figs. S5 to S13]. Ossification typically begins years after other sesamoids have become well mineralized (for example, the proximal digital sesamoids, at ~3 to 7 years of age), and it occurs in a large cartilage structure surrounded by a fat pad rather than by tendon or ligament. Such ossification can remain incomplete [in 10 out of 37 (10/37) feet examined] or even uninitiated (11/37 feet) in some adult (~20+ years old) individuals (figs. S4 to S6). This singular mode of ossification is endochondral, extending from several seemingly haphazardly positioned centers within the massive cartilaginous precursor. Furthermore, BSE SEM and CT indicate that the resultant cancellous (spongy) bone, unlike others in the appendicular skeleton, does not seem oriented to match any predominant loading direction and lacks compact cortices, which could confer greater longitudinal bending stiffness. This indicates an unusually flexible ossified structure that nevertheless is stiffer than the surrounding fat pad or cartilage, although even cartilaginous enlarged predigits should provide some support. We used an indirect approach to solve the difficult question of how elephant predigits function. Elephant predigits are deeply embedded in the digital cushions or fat pads of the feet, thus their positions and motions are obscured. The thick keratinized skin of elephant feet prevents ultrasound or x-ray imaging at safe intensities, thus preventing in vivo investigation. We previously speculated that elephant predigits might function as strut-like weight supports, because they grow with strong positive allometry similar to that of the metapodials (16). This function would be expected to involve a static orientation of the predigits during loading. Alternatively, predigits might function as dynamic levers (more Fig. 1. Foot anatomy in humans and elephants, with sesamoids shown in white. (Top) Diagram of human manus and pes (for comparison). Dotted lines for the prepollex and prehallux show rough approximations of where these structures would lie in humans, but they are normally absent. These predigits are not to be confused with the paired digital sesamoids, which elephants and humans have more distally in their digitsthe so-called tibial sesamoid in humans is one of these. (Middle and bottom) Elephant foot anatomy in medial view of right feet. The manus is on the left [prepollex (dark) and metacarpal I shown below]; the pes is on the right [prehallux (dark) and metatarsal I shown below]. Bottom-row images are from CT scan reconstructions of specimen no. 4 (table S1). See movie S2 for representative mobility of a predigit. Osteological terms are from (25, 26). Labels are as follows: ac, accessorium (pisiform); ca, calcaneus; D3, third digit; ds, digital sesamoid(s); mc1, metacarpal I; mt, metatarsal I; ph, prehallux; pp, prepollex. VOL 334 like mobile digits) if they reoriented when loaded, rotating about their joint(s). Animals variably employ similar functions with their true digits (17). We tested these hypotheses by statically loading cadaveric elephant feet ex vivo and CTscanning them to examine the effects of applied loads on their orientation (11). Predigits behaving in a weight-supporting role should maintain a constant orientation, whereas predigits acting as dynamic levers should display joint mobility (movie S2) that reorients them with increasing load. Our reconstructions (Fig. 3) reveal that the prepollex and prehallux act differently when loaded: The prepollex does not move appreciably even though its proximal joint allows some mobility, whereas the prehallux rotates caudodorsally. Internal motion contributing to this rotation is apparent for the prehallux which, once ossified at least, is consistently split into proximal (fixed to the first metatarsal and tarsal) and distal (free to move) segments (evident in 8/8 individuals with well-ossified prehalluces; movie
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S1). The prehallux thus has a proximal portion that statically transfers load to the tarsus (analogous to the whole prepollex), and a distal, mobile, lever-like portion. Such segmentation was not apparent in any of our prepollex specimens, which behaved as simple struts. This difference in prepollex and prehallux mobility and function Fig. 2. Histology of elephant predigit, from specimen no. 2 (table S1) prepollex. (A) Toluidine blue histology of bone:cartilage interface [proximal slab 4 (fig. S6); cartilage, dark blue, bone, pale blue, bone marrow space, white; width = 1200 mm]; see also fig. S13. (B) BSE SEM macerated slab 1 (width = 34 mm). The large space in the right central area (see also fig. S9) was occupied by cartilage and shows the endochondral mineralization front [higher magnification in (C), width = 1204 mm]. (D) BSE SEM of polymethylmethacrylate-embedded slab 0 (width = 28 mm; see also fig. S7) with a pseudocolor lookup table. The lowest backscattering coefficient (top) is level from the monobrominated standard and highest at 255 from the monoiodinated dimethacrylate standard (27); the densest phase is calcified cartilage. (E) Higher-magnification gray image of the calcified cartilage:bone interface (width = 900 mm). Enlarged versions of images (B) to (E) are in figs. S8 and S10 to S12. may relate to the more upright manus and more horizontal pes bone orientations (Fig. 1). Both types of predigits, however, are particularly well suited to stiffen the highly compliant fat pad against excessive deformation. Furthermore, the predigits tight syndesmotic articulations with the carpus and tarsus indicate that they also are able to transfer loading proximally from the sole of the fat pad to those bones, partly bypassing the digits. Therefore, the enlarged predigits render elephant feet functionally plantigrade while the true digits remain in subunguligrade orientations. Indeed, the predigits may allow elephants to effectively reduce the degrees of freedom in their

Fig. 3. Passive motion of elephant predigits under loading. Right cadaveric manus (top row) and pes (bottom row) specimens under minimal (left) and maximal (right) loads are shown. In the manus, the prepollex does not move noticeably relative to the vertical, whereas the metacarpal dorsiflexes up to 13 at maximal load. In the pes, the distal segment of the prehallux rotates around the static proximal segment, dorsiflexing up to 17 as the metatarsal dorsiflexes up to 10. Bones (Fig. 1) are colored to match movies S1, S3, and S4. Predigits are aquamarine color. Specimen numbers from table S1 are no. 3 (manus) and no. 5 (pes). Labels are as follows: MC3, metacarpal 3; MT3, metatarsal 3; ph, prehallux; pp, prepollex.

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60 my 30 my 20 my 50 my 10 my 40 my Fig. 4. Evolution of proPaleP PLIO IV boscidean foot posture. Paleocene Eocene Oligocene Miocene Plio A stratigraphically timecalibrated axis is shown Sirenia at top, using the phylogenetic tree from (2830), with clades Proboscidea, Erytherium Elephantiformes, and Elephantoidea labeled at Proboscidea nodes; the Sirenia (seaNumidotherium cows; manatees and du(1m) gongs) extant outgroup is shown. Manus (on left) Barytherium and pes (on right) speci(2m) mens are shown in approximate osteologically Deinotheriinae neutral poses in lateral (3m) view (more explanation Phiomia and images are in the (<2m) SOM text and figs. S14 to Increasing Elephantiformes S16). Movies S3 and S4 Mammut terrestriality americanum show three-dimensional & gigantism (3m) foot reconstructions and (>2m) Elephantoidea predigit articular surfaces Gomphotherium (where present). A shift (3m) from a relatively more plantigrade manus and Elephantidae pes in Numidotherium (3m) and Barytherium to more subunguligrade feet in later taxa is evident, especially when articular surfaces are compared. Shoulder heights (top of scapula) for each genus are roughly estimated in parentheses, as a proxy for body size changes. Representative skeletons of Barytherium (top) and Deinotherium (bottom) are shown with approximate relative size differences.

feet, by providing a more passive stabilizing support that reduces need for more active and massive muscular tissues, analogous to the reduction of toes in other ungulate groups (18). Yet the persistence of musculotendinous structures anchored to these sesamoids [such as the abductor pollicis (9)] indicates some retained ability to control their position or caudolateral motion, so the predigits are not entirely passive structures. There is a smooth ridge on the caudomedial surface of metacarpal I with which the prepollex articulates, as well as a mobile ball-and-socket like joint on the distal end of tarsal I and a ridge on the caudomedial side of metatarsal I that both articulate with the prehallux (15). These features, found even in juvenile elephants that lack ossified predigits, are thus osteological correlates of the presence of predigits (Fig. 1) that might be identifiable in fossils. Their presence in any skeletal specimen would corroborate the existence of enlarged predigits (cartilaginous or ossified). Our survey of the fossil record of the clade Proboscidea revealed some evidence of predigits in extinct forms (11), which also clarifies how elephant foot posture and function evolved. Unfortunately the most basal proboscideans (such as Barytherium and Numidotherium) lack sufficiently well-preserved metapodials (and thus potential evidence of predigit articulations) to more

directly test whether they had large predigits. However, their preserved proximal carpal and tarsal elements show that the feet were quite plantigrade, leaving little space for an expanded digital cushion or predigits (Fig. 4, movies S3 and S4, and SOM text). Furthermore, the articulations of more distal foot bones indicate the presence of relatively dorsiflexed and more splayed (abducted) toes; not as adducted as in later Proboscidea and consistent with a more amphibious lifestyle. Hence we infer that basal proboscideans, like many of their amphibious or wholly aquatic tethytherian outgroups [Sirenia and Embrithopoda (19)] were more plantigrade than extant elephants, as is ancestral for tetrapods. We therefore hypothesize that the evolution of more subunguligrade toes in elephants is linked with the expansion of the manual and pedal digital cushions and their supporting predigits. In this scenario, the predigits increasingly adopted the supportive roles that were played by the carpals (e.g., pisiform) and tarsals (e.g., calcaneus) in more plantigrade basal Proboscidea. Representative elephantiform and deinothere taxa along the phylogeny (Fig. 4) before Elephantidae support this hypothesis (SOM text, figs. S14 to S16, and movies S3 and S4): All well-preserved taxa exhibit smaller proximal carpal/tarsal bones and foot bone articulations that are more consistent with increased VOL 334 SCIENCE

dorsiflexion of the toes, and thus a more subunguligrade toe posture relative to the ancestral condition for Proboscidea. All of these taxa display osteological correlates for the articulation of predigits in the manus and pes. Thus, we conclude that the predigits have served to stiffen the expanded fat pad and maintain a plantigrade-like foot function, transferring loads from the substrate to the carpus/tarsus, since early in elephantiform evolution. Extant elephants have remarkable feet that combine advantages of plantigrady [such as the potential for damping impacts at heelstrike (20), larger foot surface area and thus moderated pressures (21), large translations of the center of pressure during the stance phase involving pronounced heelstrike, dynamic gearing, and toeoff dynamics (17)] with those of digitigrady or subunguligrady [such as reasonable mechanical advantage of the toes to keep supportive tissue stresses at safe levels (22), or even potential benefits to metabolic economy from elastic energy storage (23)]. These changes occurred while early elephantiforms attained gigantism (>2000 kg of body mass or shoulder height >2 m) in the Eocene epoch (~40 million years ago, Fig. 4) and occupied a wider range of terrestrial habitats, becoming less amphibious around the node joining Deinotheriinae and Elephantiformes (Fig. 4). Hence, there is probably a link between the in-

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creasing demands of supporting and moving greater weight on land and the benefits of having more upright toe bones but directing some loads away from the toes with the predigits and fat pad, which resulted in the peculiar compromise that persists in the feet of extant elephants. The recognition of elephant predigits as enlarged sesamoids that perform digit-like functions fuels inspiration for examining the evolution of foot function, terrestriality, and gigantism in other lineages. Sauropod dinosaurs had expansive foot pads, particularly in their pedes (24); however, no evidence of predigits has been found. Considering that the predigits form on the medial border of the feet, they would tend to be lost if digit I is lost or reduced, as it was in early perissodactyls and artiodactyls. This loss might limit foot pad expansion and thereby explain why rhinos and hippos seem to lack predigits [but see (18) for a possible rudimentary pollex in hippos] and have less expanded foot pads than elephants do (8). Regardless, the previously misunderstood and neglected predigits of elephants now deserve recognition as a remarkable case of evolutionary exaptation (4), revealing how elephants evolved their specialized foot form and function.
References and Notes
1. 2. 3. 4. D. D. Davis, Fieldiana 3, 1 (1964). H. Endo et al., J. Anat. 195, 295 (1999). S. J. Gould, Nat. Hist. 87, 20 (1978). S. J. Gould, E. S. Vrba, Paleobiology 8, 4 (1982). 5. M. Fabrezi, Zool. J. Linn. Soc. 131, 227 (2001). 6. M. R. Snchez-Villagra, P. R. Menke, Zoology 108, 3 (2005). 7. P. Blair, Philos. Trans. 27, 53 (1710). 8. H. Neuville, Arch. Mus. Natl. Hist. Nat. Paris 13, 6e Serie, 111 (1935). 9. K. von Bardeleben, Proc. Zool. Soc. 1894, 354 (1894). 10. C. Mitgutsch et al. Biol. Lett., 10.1098/rsbl.2011.0494 (2011). 11. Materials and methods are available as supporting material on Science Online. 12. G. E. Weissengruber et al., J. Anat. 209, 781 (2006). 13. F. Galis, J. J. M. van Alphen, J. A. J. Metz, Trends Ecol. Evol. 16, 637 (2001). 14. J. Prochel, P. Vogel, M. R. Snchez-Villagra, J. Anat. 205, 99 (2004). 15. J. R. Hutchinson, C. E. Miller, G. Fritsch, T. Hildebrandt, in Anatomical Imaging: Towards a New Morphology, R. Frey, H. Endo, Eds. (Springer, Berlin, 2009), pp. 2338. 16. C. E. Miller, C. Basu, G. Fritsch, T. Hildebrandt, J. R. Hutchinson, J. R. Soc. Interface 5, 465 (2008). 17. D. R. Carrier, N. C. Heglund, K. D. Earls, Science 265, 651 (1994). 18. A. B. Clifford, J. Vertebr. Paleontol. 30, 1827 (2010). 19. N. Court, Palaeontogr. Abt. A 226, 125 (1993). 20. D. E. Lieberman et al., Nature 463, 531 (2010). 21. F. Michilsens, P. Aerts, R. Van Damme, K. DAot, J. Zool. (London) 279, 236 (2009). 22. A. A. Biewener, Science 250, 1097 (1990). 23. M. N. Scholz, M. F. Bobbert, A. J. van Soest, J. R. Clark, J. van Heerden, J. Exp. Biol. 211, 3266 (2008). 24. M. F. Bonnan, in Thunder-Lizards: the Sauropodomorph Dinosaurs, K. Carpenter, V. Tidwell, Eds. (Indiana Univ. Press, Bloomington, IN, 2005), pp. 346380. 25. M. M. Smuts, A. J. Bezuidenhout, Onderstepoort J. Vet. Res. 60, 1 (1993). 26. M. M. Smuts, A. J. Bezuidenhout, Onderstepoort J. Vet. Res. 61, 51 (1994). 27. A. Boyde, R. Travers, F. H. Glorieux, S. J. Jones, Calcif. Tissue Int. 64, 185 (1999). 28. C. Delmer, Acta Palaeontol. Pol. 54, 561 (2009). 29. E. Gheerbrant, Proc. Natl. Acad. Sci. U.S.A. 106, 10717 (2009). 30. E. Gheerbrant, P. Tassy, C. R. Palevol. 8, 281 (2009). Acknowledgments: We thank the staff of the Structure and Motion Laboratory of the Royal Veterinary College for assistance and three anonymous reviewers for constructive criticism. Many individuals assisted with the collection of the cadaveric data; we particularly thank the European-based zoos that provided the specimens and G. Fritsch for CT scans done in Germany. O. Cosar, R. Weller, A. Wilson, and K. Jespers assisted with the ex vivo loading experiments. J. Molnar assisted with Figs. 1 to 4 and the movies. This project was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) (grants BB/C516844/1 and BB/H002782/1 to J.R.H.). Additionally, A.A.P. appreciates funding from Arthritis Research UK and the BBSRC, and A.B. was supported by the Veterinary Advisory Committee of the UK Horserace Betting Levy Board. The data reported in this paper are tabulated in the SOM. The authors declare no conflicts of interest.

Supporting Online Material

www.sciencemag.org/cgi/content/full/334/6063/1699/DC1 Materials and Methods SOM Text Figs. S1 to S16 Tables S1 to S3 References (3141) Movies S1 to S4 20 July 2011; accepted 8 November 2011 10.1126/science.1211437

Global Seabird Response to Forage Fish DepletionOne-Third for the Birds

Philippe M. Cury,1* Ian L. Boyd,2* Sylvain Bonhommeau,3 Tycho Anker-Nilssen,4 Robert J. M. Crawford,5 Robert W. Furness,6 James A. Mills,7 Eugene J. Murphy,8 Henrik sterblom,9 Michelle Paleczny,10 John F. Piatt,11 Jean-Paul Roux,12,13 Lynne Shannon,14 William J. Sydeman15 Determining the form of key predator-prey relationships is critical for understanding marine ecosystem dynamics. Using a comprehensive global database, we quantified the effect of fluctuations in food abundance on seabird breeding success. We identified a threshold in prey (fish and krill, termed forage fish) abundance below which seabirds experience consistently reduced and more variable productivity. This response was common to all seven ecosystems and 14 bird species examined within the Atlantic, Pacific, and Southern Oceans. The threshold approximated one-third of the maximum prey biomass observed in long-term studies. This provides an indicator of the minimal forage fish biomass needed to sustain seabird productivity over the long term. ublic and scientific appreciation for the role of top predators in marine ecosystems has grown considerably, yet many upper trophic level (UTL) species, including seabirds, marine mammals, and large predatory fish, remain depleted owing to human activities (14). Fisheries impacts include direct mortality of exploited species and the more subtle effects of altering trophic pathways and the functioning of marine ecosystems (5). Specifically, fisheries for lower trophic level (LTL) species, primarily small

impacts on food webs because numerical relationships between predators and prey are often unknown, even for commercially valuable fish (9, 10). Ecosystem models and ecosystem-based fisheries management, for which maintaining
1 Institut de Recherche pour le Dveloppement, UMR EME-212, Centre de Recherche Halieutique Mditerranenne et Tropicale, Avenue Jean Monnet, BP 171, 34203 Ste Cedex, France. 2 Scottish Oceans Institute, University of St Andrews, St Andrews KY16 8LB, UK. 3Ifremer, UMR EME 212, Centre de Recherche Halieutique Mditerranenne et Tropicale, Avenue Jean Monnet, BP 171, 34203 Ste Cedex, France. 4Norwegian Institute for Nature Research, Post Office Box 5685 Sluppen, NO-7485 Trondheim, Norway. 5Branch Oceans and Coasts, Department of Environmental Affairs, Private Bag X2, Rogge Bay 8012, South Africa. 6College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK. 710527 A Skyline Drive, Corning, NY 14830, USA. 8British Antarctic Survey, High Cross, Madingley Road, Cambridge CB3 0ET, UK. 9Baltic Nest Institute, Stockholm Resilience Centre, Stockholm University, SE-106 91 Stockholm, Sweden. 10Fisheries Centre, Aquatic Ecosystems Research Laboratory (AERL), 2202 Main Mall, The University of British Columbia, Vancouver, BC, Canada V6T 1Z4. 11 U.S. Geological Survey, Alaska Science Center, 4210 University Drive, Anchorage, AK 99508, USA. 12Ecosystem Analysis Section, Ministry of Fisheries and Marine Resources, Lderitz Marine Research, Post Office Box 394, Lderitz, Namibia. 13 Animal Demography Unit, Zoology Department, University of Cape Town, Private Bag X3, Rondebosch, Cape Town 7701, South Africa. 14Marine Research Institute and Zoology Department, University of Cape Town, Private Bag X3, Rondebosch, Cape Town 7701, South Africa. 15Farallon Institute for Advanced Ecosystem Research, Post Office Box 750756 Petaluma, CA 94952, USA.

coastal pelagic fish (e.g., anchovies and sardines), euphausiid crustaceans (krill), and squid (hereafter referred to as forage fish), threaten the future sustainability of UTL predators in marine ecosystems (6, 7). An increasing global demand for protein and marine oils contributes pressure to catch more LTL species (8). Thus, fisheries for LTL species are likely to increase even though the consequences of such activity remain largely unknown at the ecosystem level. It remains challenging, however, to assess fishing SCIENCE VOL 334

*To whom correspondence should be addressed. E-mail: philippe.cury@ird.fr (P.M.C.); ilb@st-andrews.ac.uk (I.L.B.)

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www.sciencemag.org/cgi/content/full/334/6063/1699/DC1

Supporting Online Material for

From Flat Foot to Fat Foot: Structure, Ontogeny, Function, and Evolution Of Elephant Sixth Toes
John R. Hutchinson, Cyrille Delmer, Charlotte E. Miller, Thomas Hildebrandt, Andrew A. Pitsillides, Alan Boyde
Published 23 December 2011, Science 334, 1699 (2011) DOI: 10.1126/science.1211437 This PDF file includes: Materials and Methods SOM Text Figs. S1 to S16 Tables S1 to S3 References (3141) Captions for Movies S1 to S4 Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/334/6063/1699/DC1) Movies S1 to S4

Supporting Online Material For the paper by J. R. Hutchinson, C. Delmer, C. E. Miller, T. Hildebrandt, A. A. Pitsillides and A. Boyde, From flat foot to fat foot: the structure, ontogeny, function, and evolution of elephant sixth toes. Materials and Methods Gross Anatomy and Dissections: We obtained 63 fore and hind feet of 29 individual Asian (Elephas maximus Linnaeus 1758) and African (Loxodonta africana Blumenbach 1797; i.e., bush/savannah) elephant cadavers, from neonatal to adult (tables S1,S2). These were sourced from EU (mainly UK) zoos and safari parks after natural mortality or euthanasia for reasons unrelated to this study. Pathologies were observed in some specimens, but these did not relate to the factors described here and are the basis of another study. Selected feet were dissected to determine the relationships of the predigits with surrounding tissues, to manually investigate predigit mobility and joints (Movie S2), and to remove the predigits for imaging and histology. Three-dimensional (3D) Reconstructions: All specimens were scanned using computed tomography (CT), using a GE LightSpeed Ultra 8detector scanner; typical settings: 2.5-5mm axial slices, x-ray beam intensities of 120 kVp and 100 mA, 512x512 pixel and ~1.067 pixels/mm images, bone algorithm). A laser surface scanner was used for some fossil specimens (see table S3). Bone; including predigit; positions and mineralization patterns then were visually inspected by segmenting the CT DICOM slices in Mimics 15.0 (Materialize, Inc.; Leuven, Belgium) software following previous methods (15), with semi-automatic thresholding (bone default cutoff; 226 Hounsfield units), to create 3D images of each bone element (Movie S1). Histology and Imaging: Four adult and two juvenile elephant predigits (tables S1,S2) were inspected with the following methods. The results from these analyses consistently matched those from our CT imaging (above). Hence we consider the general qualitative conclusions drawn about predigit structure and ontogeny robust enough to broadly apply to elephants. Slabs selected for SEM were either macerated in 4% alkaline bacterial pronase for 5 days and then 8h in 1% KOH both at 50C - to remove all non-mineralized tissues (including hyaline cartilage), washed, dried from ethanol and carbon coated; or embedded in PMMA, and the blocks trimmed and polished to 1um diamond and carbon coated. Both types of preparation were imaged using backscattered electrons at 20kV in an SEM and also by 30kV point projection digital microradiography. To standardize the BSE signal from the polished samples, halogenated dimethacrylate standards were employed (27). Selected segments of the samples (fig. 2 and fig. S13) were decalcified for up to 14 days in 10% EDTA/TRIS-HCl (pH 7.4) at 4 C, depending on degree of mineralization and size of specimen, with the solution refreshed at least twice each week. After decalcification, samples were processed and embedded by standard methods in paraffin. Transverse 8m sections of individual 1

predigits were cut across the entire element and stained with Toluidine blue (0.1% in 0.1M solution of acetate buffer, pH 5.6) or with 0.1% Safranin O with 0.02% Fast Green to study proteoglycan content. Functional analysis ex vivo: We loaded two manus and two pes specimens from different individual adult cadavers (sources above) with a custom-made wooden-framed loading jig to apply vertical loads to the centre of the proximal carpals/tarsals at zero (i.e., only foot weight) and near-maximal locomotor loads of approximately one body weight per limb (31). The custom loading jig used a 10-tonne capacity car jack (Ferm HB-10; Ferm B.V., Genemuiden, Netherlands) outfitted with a customized pressure gauge that was calibrated to quantify axial forces along its distal steel loading bar, which was rigidly inserted into drilled holes in the proximal bones. We then CT-scanned the statically loaded foot once per loading condition, and segmented the images as above to examine the positional changes of foot bones with loading (fig. 3). The predigit, metapodial III and the loading bar were segmented, as above, in Mimics software. 3D vectors were then placed along their central shafts to represent segment axes. Two-dimensional angles of the predigit and metapodial III were measured in the mediolateral and craniocaudal planes, with respect to the orientation of the loading bar, and hence the applied load. Angular changes were calculated between minimum and maximum loading conditions. While quantitative, this simple analysis of limited specimens was merely intended to give generally descriptive indications of the passive loading behavior of elephant predigits, as the first functional analysis of these structures. Repeated measurements (five repetitions of the entire 3D data analysis procedure) for one manus specimen showed maximal differences of predigit angle changes (loaded v. unloaded) of 2.1-4.2 (from 3D, mediolateral and craniocaudal perspectives). Thus in our results (fig. 3; main text), we conservatively considered any differences between loading conditions of <5 predigit angle to be inconsequential; potentially just measurement error; and simply report those angle changes in our specimens that exceeded this threshold. Fossils and phylogeny: We surveyed museum specimens (table S3) and literature for all basal proboscidean taxa (especially Numidotherium and Barytherium) continuing into Elephantiformes (including the basal taxa Phiomia, Gomphotherium and Mammut), and integrated these data into a phylogenetic context (fig. 4). Detailed descriptions are in the supporting online text and figures.

Supporting Online Text and Figures Dissections Tables S1 and S2 outline the specimens we used. Additional images with descriptions follow below as figs. S1-S4.

Figure S1. Right feet of elephants in medial view with skin removed to show the superficial surface of predigit regions. A, Manus (some superficial tendons removed) of specimen #5 (table S1); B, Pes of specimen #10 (table S1). Not to scale. The arrows are just above, and run parallel to, the dorsal border of the predigits, showing the proximodistal axis. See also Movie S2.

Figure S2. Right feet of elephants in caudal view showing exposed predigit (still embedded in part of foot pad, especially in B); otherwise as in fig. S1. 3

Figure S3. Dissected elephant predigits and first metapodials. A, Left prepollex and metacarpal I (showing associated musculature) of specimen #15 (table S1) in oblique lateral view; B, Right prehallux and metatarsal I of specimen #10 (table S1) in oblique dorsolateral view (proximal articular surface of metatarsal I visible at bottom right). Not to scale. Arrow is just above, and runs parallel to, the dorsal border of the predigit, showing the proximodistal axis.

Figure S4. Bandsawn frozen specimen of the left pes of a 38 year old female Asian elephant (not tables S1,S2; these images only), in oblique lateral (A) and cross-sectional views (B). Red arrows denote the prehallux, mainly exhibiting white cartilage but with an ossifying caudal border visible in A (location of arrow; pink tissue). Also note intrinsic musculature around proximal/cranial margin of prehallux in both images.

Histology and Imaging Additional images with descriptions follow below as figs. S5-S13.

Figure S5. X-ray image from the right manus of specimen #15 (see fig. S3A for photograph). The first metacarpal is the bright white object on the bottom right; the fainter object above it is the prepollex, which is largely cartilaginous but shows some small centers of ossification (higher contrast areas).

Figure S6. Series of photographs showing sectioning of right prepollex of specimen #2 (table S1) in preparation for histology and imaging (fig. 2). Pink/red ossified regions contrast with white cartilage islands. White numbers show section locations. Scale bars show mm lines.

Figure S7. Right prepollex, section 0 from fig. S6. A, enlarged photograph; B, 3x magnification Faxitron point projection digital x-ray image of PMMA embedded block of same specimen as prepared for SEM, width of field = 27mm.

Figure S8. Right prepollex, section 1 (same specimen as in fig. S7), BSE SEM montage of macerated specimen, width of field = 34mm (cf. fig. 2B; smaller image). Large space in right central area of specimen - which was occupied by cartilage - shows endochondral mineralization front (shown at higher magnification in figs. S9-11).

Figure S9. Right prepollex, section 1 (same specimen as in figs. S6-S8), showing magnified view of endochondral mineralization front, width of field = 4.45mm (cf. fig. S8). Center of field shown at higher magnification in fig. S10.

Figure S10. Right prepollex, section 1 (same specimen as in figs. S6-S9), showing detailed view of cartilage mineralization front; width of field = 1.78mm (cf. fig. 2C; smaller image).

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Figure S11. Right prepollex, section 0 (same as in figs. S6,S7; cf. fig. 2D; smaller image), showing BSE image of density variations in block surface (scale: 1=lowest; 8=highest: see fig. S12 legend for more details). Field is 27.95 x 23.39mm. The calcified cartilage (pink and grey) has been partially resorbed on the opposite surface, and served as a scaffold for bone deposition. Bone is seen as red, orange and yellow colors towards the bottom of the image, where the black regions are bone marrow.

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Figure S12. A, Right prepollex, section 0 (same as in figs. S6,S7,S11), BSE image showing density variations. Hyaline cartilage is in the black area at the top of the image and is calcifying along (and in places, ahead of) a mineralization front (cf. fig. 2E; smaller greyscale image). The calcified cartilage (pink and grey) has been partially resorbed on the opposite surface, and served as a scaffold for bone deposition. Bone is seen as red, orange and yellow colors towards the bottom of the image and the black regions here are bone marrow. B, further explanation of the pseudo-color look-up table. The grey signal level represents electron backscattering, which increases with increasing mineral concentration in the calcified tissue. For quantitative BSE imaging, signal levels are calibrated using halogenated dimethacrylate standards (27). The grey level obtained from the monobrominated compound is adjusted to zero and that from the monoiodinated is adjusted to 255. The color look-up table shows zero as black, 1-32 as dark blue, 33-64 lighter blue, 65-96 green, 97-128 yellow, 129-160 orange, 161-192 red, 193-224 pink, 225-254 grey, 255 white.

12

Figure S13. Safranin-O and Fast green stained transverse section of decalcified prepollex from specimen #10 (table S1) showing an almost entirely cartilaginous element (red), and detail showing disorganized chondrocyte orientation but some evidence of hypertrophy and chondrocyte cluster formation near non-cartilaginous tissues (blue-green).

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Fossil Evidence Known autopodium (foot; manus and pes) fossils from basal proboscideans: Autopodial elements of early proboscideans are extremely rare in the fossil record. The oldest proboscideans, Erytherium, Phosphatherium and Daouitherium are known only from craniodental remains (except for one phalanx referred to Phosphatherium) found isolated in marine sediments. Discoveries of foot elements for these taxa, basal tethytherian outgroups, and more material of later taxa mentioned below will be critical for testing the inferences made in this study. Anatomical terminology follows (25,26), with alternative comparative terms in parentheses when first used. The oldest well documented early proboscidean is Numidotherium koholense, from the late early-middle Eocene, for which numerous postcranial elements together with several skulls were collected in Algeria during the 1980s. This material includes two partial manus, possibly from the same individual, together with scattered pes remains, mainly tali (astragali) and calcanei (calcanea). Similarly, two partial manus of Barytherium were found in Dor El Talha, Libya, during the 1960s, together with a few tali. A potential talus and calcaneus have been referred to the amphibious Moeritherium: a talus (Yale Peabody Museum specimen YPM 24186); noted by (32); and a calcaneus (YPM 24125); referred to Moeritherium but apparently unpublished. The two elements seem to be from different individuals; if not species; because they do not articulate well (Delmer, pers. obs.; 33). The talus bears a tuberculum mediale quite like that of other early proboscideans, but bears no diagnostic features allowing its confident assignment to Moeritherium. Thus because no unambiguous remains of autopodia exist for many basal taxa, Numidotherium koholense and Barytherium grave are the only two taxa that give us some indication of the manus and pes morphology of basal proboscideans. The morphology of deinotheres, the only other non-elephantiform proboscideans, is very well understood based on almost complete skeletons from Europe (34-36). Yet their postcranial anatomy is strikingly derived; similar to that in elephantoids (e.g., fig. 4). Most of their postcranial elements, if found isolated, are not easily distinguishable from coeval proboscideans such as Gomphotherium. The morphology of the manus of early Elephantiformes is fairly well known. It is exemplified by a semi-complete associated manus of Phiomia from the Fayum region of Egypt, but the pes of Phiomia remains still poorly known, essentially from its talus and calcaneus (like Palaeomastodon). However, the remains of all known deinothere and elephantiform manus and pedes are sufficiently similar to draw robust inferences about the ancestral morphology for this clade. Movies S3,S4 show the manus and pes morphology for some of the best preserved specimens of representative taxa along this lineage (see also fig. 4).

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Figure S14. Right manus of Barytherium grave (table S3), in dorsal view, showing plantigrade orientation and other features noted in the text. Not to scale. Morphology of the autopodia in basal Proboscidea: The autopodia of Numidotherium and Barytherium, although quite different from those of Elephantiformes, display characteristics that still link them to the seriated (taxeopode) anatomical structure that is typical of Proboscidea and its close relatives, particularly the following four traits. First, The intermedium (lunar/semilunar) does not articulate with carpal IV (unciform/hamate), and carpal III (magnum) does not articulate with the ulnar carpal (trichetrum/cuneiform). The intermedium of Numidotherium koholense has a proximal articulation that is almost solely with the radius (the ulna is almost excluded from this joint), as in other more derived proboscideans. Barytherium, however, has an intermedium with a strong contact for the ulna, quite similar to that in Arsinoitherium, but this is probably an autapomorphy (33). Second, the articulations between the metacarpals (Mc) and distal row of carpals are identical to those in later proboscideans: Mc II articulates mainly with carpals II and III, Mc III with carpals III and IV, and Mc IV and V with carpal IV. Third, the talus articulates only with the centrale (navicular). It also displays a well developed tuberculum mediale, identified as a diagnostic character for Proboscidea (32). Finally, the tuber calcanei also exhibits a strong mediolateral widening that is also found in Elephantiformes. However, overall, the manus and (fragmentary) pes of Numidotherium and Barytherium (fig. S14; Movies S3,S4) are strikingly different from those of deinotheres and Elephantiformes (Movies S3,S4) in the following seven features. First, Carpal III has a strong caudodistal extension, which creates a strong craniodistal orientation of its articular facet for Mc II and III. In deinotheres and Elephantiformes, that facet is much more parallel to that of the proximal 15

articulation. In association with the related strong caudal extension of the proximal articular surfaces of the metacarpals observed in Barytherium and Numidotherium, such a disposition supports the inference that both taxa have almost plantigrade manus, very dissimilar from the stereotypically vertical (digitigrade/subunguligrade) skeletal orientation of the manus in deinotheres and Elephantiformes. Second, the metacarpals are much more flattened craniocaudally, and proportionally wider than in deinotheres and Elephantiformes; especially in Barytherium. They therefore seem to have been mediolaterally splayed (abducted) more widely, confirmed by their articulations. In conjunction with the plantigrady of the manus, this could have facilitated swimming as well as locomotion over soft ground such as swamps. It is unclear whether this state is plesiomorphic for Proboscidea as one might suspect. The feet of basal taxa within outgroup clades such as Pezosiren (Sirenia), Ashoroa (Desmostylia), and Anthracobune are either already highly specialized, insufficiently described or unpreserved. This possible amphibious specialization is, however, congruent with recent isotope analysis of the enamel of Barytherium that concluded that its diet was mainly composed of aquatic plants (35). In later Proboscidea, the metacarpals become strongly adducted, forming a near-vertical colonnade (12,15,16,25). Third, the Mc V of Numidotherium and Barytherium is massive, very asymmetrical and has an almost completely lateral orientation. It bears a strong proximolateral articular facet for the ulnare. Considering this and the massive development of the accessory carpal (pisiform/accessorium; below), we infer that there was a lateral bias for forefoot forces in basal Proboscidea. This condition is reduced in Elephantiformes, with support seemingly becoming more balanced toward the middle of the feet (but see 16). Fourth, the accessory carpal of Barytherium is the largest carpal of the manus. It displays a very strong contact for the ulnare, but an even larger contact, almost vertically oriented, for the ulna. Considering the almost plantigrade posture of the manus, this intimates that the accessory carpal of Barytherium was the major support for the caudal border of the manus. Thus it likely contributed to limb support in a similar way as the prepollex and foot pad do in deinotheres and Elephantiformes. No accessory carpal is known for Numidotherium, but the size, shape and concavity of the facet for the accessory carpal on the ulnare suggest that its morphology was similar to that of Barytherium. In later Proboscidea, the accessory carpal is comparatively smaller and does not have potential to contact the ground, serving more as a lever/attachment point for carpal and digital flexor muscles. Fifth, the intermedium in Barytherium and Numidotherium displays a craniomedially positioned, triangular facet interpreted as a contact for a free centrale (38). It however lacks a contact for carpal II (trapezoid), as deinotheres and Elephantiformes have, which is congruent with the hypothesis that strict taxeopody/serial arrangement of the carpals is a synapomorphy of Paenungulata (39). In this case, the contact between the intermedium and carpal II displayed by deinotheres and Elephantiformes must have appeared later in the history of the clade. Sixth, the main difference between the calcaneus of Barytherium and Numidotherium v. Phiomia, deinotheres and elephantoids is that the ectal and sustentacular facets of the calcaneus are perpendicular to the facet for tarsal IV (cuboid) in the latter taxa, whereas they are slightly cranially inclined in the former taxa (apparently plesiomorphic for tethytheres). The fibular facet is somewhat smaller in Barytherium and Numidotherium.

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Finally, the central (navicular) facet of the talus is almost perpendicular to the tibial facet in Barytherium. This is less true for Numidotherium, but still more than in Phiomia, deinotheres or elephantoids, in which it is more cranioventrally and mediodistally inclined.

A B Figure S15: Osteological correlates (joint surfaces) for the articulation of the prepollex in fossil proboscideans; metacarpal I in proximal view. A, Phiomia sp. (right side); B, Gomphotherium sp. (right side). Arrows note a palmar ridge on Mc I that is the articular surface. Not to scale. Specimen details in table S3.

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A B Figure S16: Osteological correlates (joint surfaces) for the articulation of the prehallux in a representative fossil elephantiform; tarsal I (above) and metatarsal I (below) of Gomphotherium sp. in caudal (A) and medial (B) views. Arrows note articular surfaces for the prehallux. Not to scale. Specimen details in table S3. Deinotheres and Elephantiformes: evidence for the ancestral presence of an expanded foot pad and predigits: Although it is much more gracile than the contemporaneous Barytherium, Phiomia displays a manus with all the typical characteristics of the subunguligrade and taxeopod elephantine structure (Movie S3); even its specific articulations (cranial contact between the intermedium and carpal II) that have prompted descriptions of the taxeopody of proboscideans as non-strictly serially arranged (40). Although still displaying some plesiomorphic traits (e.g., very flat ulnare with an extended lateral hook), its manus displays the same basic arrangement as the manus of an extant elephant or other elephantiform outgroups (Movie S3), with markedly similar contacts. It also lacks a free centrale. With very few differences (mainly the shape of carpal III; as previously emphasized (41)), carpal III of deinotheres is proportionally thicker than that of elephantoids, especially in its caudal half), the same features are exhibited by the manus of deinotheres. Most importantly, the first metacarpal of all deinothere/elephantiform taxa inspected has a tuberosity located caudal (palmar) to its proximal articulation, with a shape and orientation 18

similar to that observed in later elephantoids and extant elephants (fig. S15). In Phiomia and deinotheres, this tuberosity, which we have identified as the location where the prepollex articulates with the rest of the manus in extant elephants (15) (see main text), indicates a contact with another element that is caudolaterally oriented (i.e., into the fat pad). That element could only be the radial sesamoid bone; i.e., prepollex. Similar features for the articulation of the prehallux (tibial sesamoid bone) with metatarsal I and tarsal I are evident in the same taxa (fig. S16; Movie S4). The above observations, together with the subunguligrade disposition of the manus and pes, support the hypothesis that all deinotheres and Elephantiformes possessed predigits and expanded foot pads in their more upright manus and pedes. Our inspections of numerous specimens of Mammuthus and Palaeoloxodon (not shown) as well as our extant samples of Elephas and Loxodonta (tables S1,S2) confirm that this pattern is plesiomorphic for Elephantidae.

Conclusions: We infer that the early proboscideans Barytherium and Numidotherium had an almost completely plantigrade manus and pes, with an enlarged accessory carpal and a plantarflexed calcaneus contributing to the support of the body. This foot orientation most parsimoniously represents the ancestral condition for Proboscidea (fig. 4; Movies S3,S4). It does not leave space for the presence of an enlarged foot pad similar to that observed in extant elephants. However, as in essentially all other tetrapods with autopodia, basal proboscideans should still have had some compliant (but small and superficial) foot pads that were the plesiomorphic predecessors to the giant fatty-fibrous pads of later Proboscidea. These probably extended over the entire plantigrade foots palmar/plantar surface including the heel. The enlarged predigits, and therefore the expanded foot pads, should have appeared early in the history of Proboscidea, in the mid-late Eocene (fig. 4). This is because the basal elephantiform Phiomia bears evidence of the enlarged prepollex and a derived manus morphology suggesting the presence of an expanded foot pad. Furthermore, deinotheres have osteological correlates on their Mc I and Mt I (and tarsal I) showing the attachments of the prepollex and prehallux. The more subunguligrade manus and pes postures of all these later Proboscidea (fig. 4; Movies S3,S4) should have evolved in conjunction with this change. Thus foot posture, enlarged sesamoids (predigits) and expanded foot pads seem to have coevolved in Proboscidea. It is interesting to note that Phiomia was not the largest proboscidean representative in the Eocene, and that the latter, Barytherium, lacks this derived foot posture. Perhaps foot pad evolution was more greatly influenced by the habitat or ecology of proboscideans rather than just increasing body size. Regardless, a gradient toward decreasing amphibious habits and increasing terrestriality extends from basal Proboscidea (and other tethytheres) to Phiomia and deinotheres as well as Elephantoidea. Across this trend, body size changed frequently.

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Acknowledgements for specimen access We thank Bernard Marandat, Universite Montpellier II, France, for access to manus and pes elements of Numidotherium koholense; William Sanders, University of Michigan, for providing the cast of the calcaneum of Barytherium grave; the Museum dHistoire Naturelle of Toulouse France, for access to the Prodeinotherium bavaricum pes, and Docteur Eric Jouan, from the Villeneuve St Georges Hospital, France, for providing the CT-scan of the same pes; the Palaeontology department of the Natural History Museum, London for access to specimens and the use of their Konica Minolta laser scanner; Ursula Gohlich, Natural History Museum of Vienna, Austria, for access to the pes of Gomphotherium cf. steinheimensis; Dr Reinhart Ziegler, Staatliches Museum fur Naturkunde, Stuttgart, for access to the manus of Gomphotherium sp.; and Dr Gertrud Rossner, Bayerische Staatssammlung fr Palontologie und Geologie und Geobio-Center der Ludwig-Maximilians-Universitt Mnchen, Germany, for access to a manus of Phiomia sp.

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Specimen 1 1 2 2 3 4 4 5 5 6 7 7 8 9 9 10 10 11 11 12 12 13 13 14 15 15

Species Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian African African African Asian Asian Asian Asian Asian Asian Asian Asian African African Asian Asian Asian

Element manus pes manus pes manus manus pes manus pes pes manus pes manus manus pes manus pes manus pes manus pes manus pes manus pes manus

left/right L,R L,R R L R L,R L,R L L R L R R L,R L,R L,R L,R L,R L,R L,R R R R L R L

Body mass (kg)

3400 3400

2500? 2500? 3550 3550 3920 3920 3690 3690

Age (yr) 20+ 20+ 20+ 20+ ~20 26 26 50 50 18 24 24 28 40 40 35 35 55 55 35 35 20+ 20+ 40+ 50+ 50+

Gender

F F M M F F M F F F F F F F F

MRI y y n n y y y n y n n n y y y

dissected y y y y y y y y y y y y n y y y y

ex vivo loaded n n y n y n y n y n n n n n n n n

cleaned bones y y y y y y y y y y y y n y y y y

n n y

n n

n n

n n

F F F

n y y n y

predigits ossified y y y y n y y y n y n p n p p n p y y n p n p n n p

CT predigits y y n n y y y y y y y y n y y y y

predigit histology

y y

n n

n y y y y

n n

n n

Table S1. Adult elephant specimens examined in this study. Body mass with ? indicates an uncertain estimate from incomplete measurements. The columns from MRI? to the far right column indicate, respectively, whether (y/n= yes/no) the specimens anatomy was inspected with an MRI scan, had a complete dissection (i.e. disarticulated), was used for ex vivo loading for functional analysis (11), had its bones defleshed and bleached to check skeletal morphology, determined to have an ossified (p= partially; y= extensively) prepollex (manus) or prehallux (pes), had its predigit(s) removed and CT scanned at higher resolution to check ossification patterns, or had more detailed histology and imaging techniques applied to its predigit(s) (11). Blank entries indicate that a procedure or check has not yet been applied to the specimen or was inconclusive, whereas n entries for procedures indicate that the specimen was destructively analyzed before such procedures could be applied.

Specimen 16 16 17 18 18 19 19 20 21 22 22 23 23 24 24 25 26 27 27 28 29

Species Asian Asian African Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian Asian

Element manus pes pes manus pes manus pes pes manus manus pes manus pes manus pes manus pes manus pes manus manus

left/right L,R L,R L L,R L,R L R R R L L L L L L R L R,L R R R

Body mass (kg) 116? 116? 500? 838 838 110 110 116? 452 452 833 833 950 950

Age (yr) 0.1 0.1 7.5 2.5 2.5 0.17 0.17 2 to 4 0.1 1.5 1.5 2 2 2.5 2.5 3.5 12 0.1 0.1 0.1 0.1

Gender

F F F F F

M M F F M M

MRI y y n y y n n y n n n

dissected y y y y y n n y n y y

ex vivo loaded

cleaned bones

n n n n n n n n n

y y y n n y n y y

predigits ossified n n n n n n n n n n n n n n n n p n n n n

CT predigits

predigit histology

n n n n n n n n

n y y n n n n n

Table S2. Juvenile (12 years or younger) elephant specimens examined in this study. Body mass with ? indicates an uncertain estimate from incomplete measurements. The columns from MRI? to the far right column indicate, respectively, whether (y/n= yes/no) the specimen was checked with an MRI scan, had a complete dissection, was used for ex vivo loading for functional analysis (11), had its bones defleshed and bleached to check skeletal morphology, determined to have an ossified (p= partial; y= extensive) prepollex (manus) or prehallux (pes), had its predigit(s) removed and CT scanned at higher resolution to check ossification patterns, or had more detailed histology and imaging techniques applied to its predigit(s) (11). Blank entries indicate that a procedure or check has not yet been applied to the specimen or was inconclusive, whereas n entries for procedures indicate that the specimen was destructively analyzed before such procedures could be applied.

Taxon Numidotherium koholense Numidotherium koholense Numidotherium koholense Numidotherium koholense Numidotherium koholense Numidotherium koholense Numidotherium koholense Numidotherium koholense Numidotherium koholense Barytherium grave Barytherium grave Barytherium grave Barytherium grave Barytherium grave Barytherium grave Barytherium grave Barytherium grave Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Phiomia sp. Palaeomastodon beadnelli Deinotherium sp. Deinotherium sp. Deinotherium sp. Deinotherium sp. Deinotherium sp. Deinotherium sp. Deinotherium sp. Deinotherium sp. Deinotherium sp. Prodeinotherium bavaricum Prodeinotherium bavaricum Gomphotherium sp. Gomphotherium cf. steinheimensis Mammut americanum Mammut americanum

Collection # UO-K 065 UO-K 066 UO-K 068 UO-K 070 UO-K 076 UO-K 072 UO-K 075 UO-KX 18 UO-KA 1-31 NHMUK. M84010 NHMUK. M84011 NHMUK. M84012 NHMUK. M84013 NHMUK. M84014 NHMUK. M84015 NHMUK. M84016 UM. 13973 BSP.1905.XIII 502 BSP.1905.XIII 501 BSP.1905.XIII 503 BSP.1905.XIII 504 BSP.1905.XIII 505 BSP.1905.XIII 506 BSP.1905.XIII.b.3a BSP.1905.XIII 507 BSP.1905.XIII 508 BSP.1905.XIII 509 NHMUK. M8513 NHMUK. M8484 NHMUK. M9261 NHMUK. M9261 NHMUK. M9261 NHMUK. M9261 NHMUK. M9261 NHMUK. M9261 NHMUK. M9261 NHMUK. M9261 NHMUK. M9261 MNHT.PAL.2010.0.3 uncatalogued SMNS 47443 BSP 1971 I 275 PRI 49820 PRI 49820

Original/cast Cast Cast Cast Cast Cast Cast Cast Cast Cast Original Original Original Original Original Original Original Cast Original Original Original Original Original Original Original Original Original Original Original Original Cast Cast Cast Cast Cast Cast Cast Cast Cast Original Original Original Original Original Original

Element Ulnare Intermedium Carpal IV Carpal III Carpal II Metacarpal III Metacarpal V Talus Calcaneus Carpal III Accessorium Metacarpal III Metacarpal V Metacarpal IV Intermedium Talus Calcaneus Intermedium Ulnare Radiale Carpal IV Carpal III Carpal II Metacarpal I Metacarpal II Metacarpal III Metacarpal IV Talus Calcaneus Carpal III Carpal IV Carpal II Intermedium Ulnare Carpal I Metacarpal I Metacarpal III Metacarpal IV Pes Metacarpal I Manus Pes Manus Pes

Side R R R L L L R L R R R R R R R R L R R R R R R R R R R R L R R L L L L L R L L R L L L

Scan Method Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan CT scan CT scan CT scan CT scan CT scan CT scan CT scan CT scan CT scan CT scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan Laser scan CT scan CT scan CT scan CT scan CT scan CT scan

Equipment Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Konica Minolta VI-910 digitizer Polhemus Fastscan Polhemus Fastscan Polhemus Fastscan Polhemus Fastscan Polhemus Fastscan Polhemus Fastscan Polhemus Fastscan Polhemus Fastscan Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Marconi Medical Systems Polhemus Fastscan Polhemus Fastscan Konica Minolta VI Konica Minolta VI Konica Minolta VI Konica Minolta VI Konica Minolta VI Konica Minolta VI Konica Minolta VI Konica Minolta VI Konica Minolta VI Philips\Mx8000 GE LightSpeed 16 Marconi Medical Systems GE LightSpeed 16 TOSHIBA\Acquilion\LB TOSHIBA\Acquilion\LB

Resolution/Lens Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm

0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel 0.742 mm per pixel

Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm Minolta lens TELE 25mm 0.627 mm per pixel 0.813 mm per pixel 0.742 mm per pixel 0.813 mm per pixel 0.175-0.456 mm per pixel 0.112-0.781 mm per pixel

Scanning Location NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK NHM, London, UK Villeneuve St Georges Hospital, France RVC, Hatfield, UK RVC, Hatfield, UK RVC, Hatfield, UK Cayuga Medical Center, USA Cayuga Medical Center, USA

Table S3. Fossil specimens examined. For further details see supplementary online text: Fossil Evidence. All specimens except Palaeomastodon were used in Movies S3,S4.

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Movie S1. Foot anatomy of an extant elephant (Elephas maximus; specimen #4 in table S1). Manus, then pes anatomy are shown, with bones labeled using terms from (25, 26) (alternative comparative terms in parentheses). Movie S2. Prehallux mobility in a cadaveric hindfoot. Right pes of specimen #5 in table S1 shown in medial view. Outlines at beginning show rough positions of some anatomical structures (see movie S1); labels: c = centrale; ca = calcaneus, D2 = distal end (ungual/nail) of digit II, mt1,2,3 = metatarsals I-III, ph = prehallux, t1 = tarsal I. Some tissue around the proximal prehallux has been cut and part of the fat pad has been removed around the distal prehallux so overall mobility is exaggerated, but the video is representative of potential caudodorsal mobility of the predigits under loading (fig. 2). Movie S3. Evolution of manus anatomy in Proboscidea. Rotating movies of 3D forefoot anatomy in representative taxa along the line to extant elephants (fig. 4); introductory image explains the format. Still images in caudal and cranial views show some major structures as per movie S1 (MC = metacarpals). Specimens and scanning modalities are listed in table S3. Movie S4. Evolution of pes anatomy in Proboscidea. Rotating movies of 3D hindfoot anatomy in representative taxa along the line to extant elephants (fig. 4); introductory image explains the format. Still images in caudal and cranial views show some major structures as per movie S1 (MT = metatarsals). Specimens and scanning modalities are listed in table S3.