Biology

DOI: 10.1126/science.1211437 , 1699 (2011);334 Science

, et al.John R. Hutchinson Evolution of Elephant ”Sixth Toes” From Flat Foot to Fat Foot: Structure, Ontogeny, Function, and

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in the Doushantuo fossils [for example, opalinids are multinuclear (32)]. Only volvocalean em- bryos 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 with- in 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 nonmeta- zoan holozoans or possibly even more distant eukaryote branches.

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23. K. V. Mikhailov et al., Bioessays 31, 758 (2009). 24. W. L. Marshall, M. L. Berbee, Protist 162, 33 (2011). 25. M. Pekkarinen, K. Lotman, J. Nat. Hist. 37, 1155 (2003). 26. L. Mendoza, R. A. Herr, S. N. Arseculeratne, L. Ajello,

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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.

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 (36–67) 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.

The enlarged radial sesamoid bones of giantpanda forefeet (1, 2) are classic examplesof 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

called predigits), radial/tibial sesamoids, or other terms (such as falciform, accessory scaphoid, or navicular). Whether these sesamoids are ances- trally or convergently evolved in various tetra- pod clades remains to be determined. The latter seems likely, given the absence of similar sesa- moids in most fossil outgroups, yet a cartilag- inous nodular precursor cannot be excluded. Regardless, enlarged sesamoids are quite prom-

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 (7–9). Indeed, the recent discovery that moles have developmen- tally switched their radial sesamoid (prepollex) to a digit-like identity (10) intimates that ele- phants and other species may have done the same. Here, we report a multidisciplinary anatomical, his- tological, functional, and phylogenetic analysis (11) of the predigits in elephant feet. We hoped this would illuminate how elephants evolved their char- acteristic 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 con- tacting the ground) foot posture in many other tetrapods.

In 1710, Blair (7) provided the first detailed osteological description of elephants, conclud- ing that they have six toes. The “sixth toes” (medialmost position; corresponding to digit zero) were later identified as the enigmatic prepollex

1Department 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 Uni- versity of London, Mile End Road, London E1 4NS, UK.

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and prehallux (8, 9, 12) (figs. S1 to S4). Three centuries of sporadic discussion about the iden- tities of predigits in tetrapods have ensued (8, 9), sometimes returning to the question of whether they are actually atavistic digits (9, 13). Consid- ering the characteristic variability (8) and apparent mineralization late in the ontogeny of sesamoids (14), as well as their articulations (15) with meta- carpal 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 pre- vent their preservation, discovery, scholarly descrip- tion, and even museum exhibition. The vexing issue of the homology of elephant predigits re- mains unresolved, complicated by the specializa- tion 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 elec- tron 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 con- version to bone (that is, forming endochondrally) by an unusual ossification mechanism. Histolog- ical examination showed that this initial hyaline cartilage element lacks a preferential orientation of chondrocytes or growth-plate–like 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) struc- ture. The advancing mineralizing fronts and the thickness of the calcified cartilage layers resem- ble 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 ses- amoids have become well mineralized (for ex- ample, 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 exam- ined] or even uninitiated (11/37 feet) in some adult (~20+ years old) individuals (figs. S4 to S6). This singular mode of ossification is endochondral, ex- tending from several seemingly haphazardly po- sitioned centers within the massive cartilaginous precursor. Furthermore, BSE SEM and CT indi- cate that the resultant cancellous (spongy) bone, unlike others in the appendicular skeleton, does

not seem oriented to match any predominant load- ing direction and lacks compact cortices, which could confer greater longitudinal bending stiffness. This indicates an unusually flexible ossified struc- ture 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 func- tion. 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 pre- viously speculated that elephant predigits might function as strut-like weight supports, because they grow with strong positive allometry simi- lar to that of the metapodials (16). This function would be expected to involve a static orientation of the predigits during loading. Alternatively, pre- digits might function as dynamic levers (more

like mobile digits) if they reoriented when loaded, rotating about their joint(s). Animals variably em- ploy similar functions with their true digits (17).

We tested these hypotheses by statically loading cadaveric elephant feet ex vivo and CT- scanning them to examine the effects of applied loads on their orientation (11). Predigits behav- ing 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 apprecia- bly even though its proximal joint allows some mobility, whereas the prehallux rotates caudo- dorsally. Internal motion contributing to this ro- tation is apparent for the prehallux which, once ossified at least, is consistently split into prox- imal (fixed to the first metatarsal and tarsal) and distal (free to move) segments (evident in 8/8 individuals with well-ossified prehalluces; movie

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 digits—the so-called “tib- ial 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 [pre- pollex (dark) and meta- carpal I shown below]; the pes is on the right [prehallux (dark) and me- tatarsal I shown below]. Bottom-row images are from CT scan reconstruc- tions of specimen no. 4 (table S1). See movie S2 for representative mobil- ity of a predigit. Osteo- logical terms are from (25, 26). Labels are as fol- lows: ac, accessorium (pi- siform); ca, calcaneus; D3, third digit; ds, digi- tal sesamoid(s); mc1, metacarpal I; mt, meta- tarsal I; ph, prehallux; pp, prepollex.

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