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RESEARCH ARTICLE Greater Growth of Proximal Metatarsals in Embryos and the of Hallux Position in the Grasping Foot JOÃO FRANCISCO BOTELHO1,2,3,4, DANIEL SMITH-PAREDES1, SERGIO SOTO-ACUÑA1,5, DANIEL NÚÑEZ-LEÓN1, VERÓNICA PALMA4, 1∗ AND ALEXANDER O. VARGAS 1Departamento de Biología, Laboratorio de Ontogenia y Filogenia, Facultad de Ciencias de la Universidad de , Santiago, RM, Chile 2Department of , Faculty of Medical and Health Sciences, University of Auckland, Auckland, 3Instituto Nacional de Ciência e Tecnologia em Estudos Interdisciplinares e Transdisciplinares em Ecologia e Evolução (IN-TREE), Salvador, BA, Brazil 4Departamento de Biología, Laboratorio de Células Troncales y Biología del Desarrollo, Facultad de Ciencias de la Universidad de Chile, Santiago, RM, Chile 5Área de Paleontología, Museo Nacional de Historia Natural, Santiago, RM, Chile

ABSTRACT In early theropod —the ancestors of —the hallux ( 1) had an elevated posi- tion within the foot and had lost the proximal portion of its metatarsal. It no longer articulated with the ankle, but was attached at about mid-length of metatarsal 2 (mt2). In adult birds, the hallux is articulated closer to the distal end of mt2 at ground level with the other digits. How- ever, on chick embryonic day 7, its position is as in early theropods at half-length of mt2. The adult distal location is acquired during embryonic days 8–10. To assess how the adult phenotype is acquired, we produced fate maps of the metatarsals of day 6 embryos injecting the lipophilic tracer DiI. The fates of these marks indicate a larger expansion of the metatarsals at their proximal end, which creates the illusory effect that d1 moves distally. This larger proximal expansion occurs concomitantly with growth and early differentiation of cartilage. Histological analysis of metatarsals shows that the domains of flattened and prehypertrophic chondrocytes are larger toward the proximal end. The results suggest that the distal position of the hallux in the avian foot evolved as a consequence of an embryological period of expansion of the metatarsus toward the proximal end. It also brings attention to the developmental mechanisms leading to

Conflict of interest: None. ∗Correspondence to: Alexander O. Vargas, Departamento de Biología, Laboratorio de Ontogenia y Filogenia, Facultad de Ciencias de la Universidad de Chile, Santiago, RM 7800003, Chile. E-mail: [email protected] Received 15 March 2016; Revised 10 August 2016; Accepted 16 August 2016 DOI: 10.1002/jez.b.22697 Published online in Wiley Online Library (wileyonlinelibrary.com).

C 2016 WILEY PERIODICALS, INC. 2 BOTELHO ET AL.

differential growth between epiphyses and their evolutionary consequences. J. Exp. Zool. (Mol. Dev. Evol.) 00:1–13, 2016. C 2016 Wiley Periodicals, Inc. J. Exp. Zool. (Mol. Dev. Evol.) How to cite this article: Botelho JF, Smith-Paredes D, Soto-Acuña S, Núñez-León D, Palma V, 00:1–13, Vargas AO. 2016. Greater growth of proximal metatarsals in bird embryos and the evolution 2016 of hallux position in the grasping foot. J. Exp. Zool. (Mol. Dev. Evol.) 00B:1–13.

Abbreviations:

d1, digit 1 (hallux); mt1, metatarsal 1

INTRODUCTION Birds are remarkable in that they have lost grasping abilities in their , while evolving a foot with an opposable hallux (digit 1 [d1]), an impressive morphological allowing them to perch on trees, capture prey or manipulate , among other functions (Raikow, ’85; Sereno and Rao, ’92; Abourachid and Höfling; 2012, Sustaita et al., 2013). The avian hallux is quite different from any other digit. It has an extremely short metatarsal 1 (mt1) without a joint connecting it to the ankle but rather it tapers toward its proximal (upper) end. Despite this, the phalanges of the hallux are at level with the other digits, because mt1 attaches medioventrally near the distal end of metatarsal 2 (mt2) by means of a rigid, nonsynovial articulation. Importantly, mt1 has a twisted shape, which is key in conferring the hallux with an opposite orientation to the other foot digits (Middleton, 2001). The twisted shape of mt1 is brought about by embryonic muscular activity (Botelho et al., 2015). Intriguingly, during early embryonic stages, the hallux is found at a more proximal position at mid-length of mt2 (Fig. 1A) (Heilmann, ’26). The distal position of the hallux in the adult foot is acquired between embryonic days 8–10 (stages HH34–HH36 (Hamburger and Hamilton, ’51; Vargas et al., 2008)). The developmental mechanisms involved in establishing the distal position of the hallux are therefore key to attain a func- tional grasping foot, with a hallux that is at level with the other digits. A possible mechanism is that d1 moves distally along mt2 during development. Alternatively, it is possible that embryonic mt2-4 grow asymmetrically, with greater elongation at the prox- imal ends, thus creating the illusion that d1 has moved distally (Fig. 1B). Metatarsals are long that undergo endochondral ossi- Figure 1. The development of digit 1 position in birds. (A) Embry- fication, a process in which a cartilaginous or “tem- onic development of the chicken foot shows a proximally placed plate” is replaced from within by (Fell, ’25; Kronenberg, mt1 at HH30 (day 6 of incubation), at about mid-length of mt2 2003). Bone replacement begins at mid-length of a bone (the between HH32 and HH34 (days 7 and 8), and reaching its final diaphysis) and is preceded by cartilage maturation. Distinct distal position at HH36 (day 10). (B) Two hypotheses are proposed chondrocytes progressing through different stages of matura- for the development of the distal position of the hallux: either the tion characterize different domains within the template, with hallux moves to a more distal position, or mt2-4 grow more to- more mature cells closer to the diaphysis. A domain of immature, wards proximal, creating the illusion that the hallux moves toward round stem chondrocytes is situated at the ends of the template, distal. adjacent to the joints (the epiphysis). From here, there is a zone of flattened, maturing chondrocytes arranged in columns that

J. Exp. Zool. (Mol. Dev. Evol.) EVOLUTION OF THE DISTAL POSITION OF AVIAN HALLUX 3 extend toward the diaphysis. When flattened chondrocytes exit metatarsal 3 (mt3), as it is deflected only in the dorsoventral the cell cycle, they grow in size generating a zone of hyper- plane, which makes it possible to have both epiphyses in a sin- trophic chondrocytes closer to the diaphysis. Those hypertrophic gle cutting plane. To avoid typical wrinkling associated with re- cells eventually enter apoptosis, leaving their extracellular ma- traction and shrinkage of paraffin sections of long cartilages, we trix as a scaffold for osteoblast and vascular invasion at the cen- cut 45 μm thick sections in a sliding microtome equipped with a ter of the element, where bone replacement begins. Lengthwise frozen stage. Embryos legs were fixed in 4% PFA for 48 hr at° 4 C, growth depends on the dynamics of chondrocyte proliferation, washed in PBS, and then cryoprotected in 30% sucrose. Floating differentiation, and deposition of extracellular matrix in the epi- sections were mounted in gelatin-coated slides and stained with physeal growth plates (Wilsman et al., ’96a, ’96b; Hall, 2005). Cresyl Violet. In this study, we first analyzed landmarks in morphology and protein expression to assess whether there is asymmetric elonga- Section Immunolabeling tion of mt2–4, or movement of the hallux. We also fate mapped Embryos were sectioned either in a paraffin microtome, for anti- the destiny of early metatarsal cells to test whether the proximal collagens immunolabeling, or a sliding microtome equipped with region of early metatarsals is precursor to a larger domain of a freezing stage for immunolabeling of the mitosis marker phos- late-stage metatarsals. We additionally looked for asymmetries pho histone-3 (PH3) (Rapacioli et al., 2012). After epitome re- in the pattern of cell proliferation and cartilage maturation by trieval in buffer citrate, sections were incubated overnight at 4°C studying histological sections of the metatarsals using immuno- with primary in the following concentrations: anti- histochemistry and traditional skeletal staining. Our results pro- collagen II (II-II6B3s, Developmental Studies Hybridoma vide compelling evidence for asymmetric elongation, increased Bank (DSHB), 1:40), anti-collagen type X (X-AC9, DSHB, 1:20), at the proximal region of mt2–4, which coincides with asym- and anti-PH3 (06-570, Upstate, 1:500). Sections labeled with metric domains of chondrocytes entering cartilage maturation. antibodies against collagen were washed in PBST, incubated Importantly, evolution of hallux position in the lineage leading with HRP-conjugated anti-mouse secondary antibodies at RT for to birds shows a similar distal shift occurred in the –bird 2 hr, washed again with PBST, and then revealed using 3,3’- transition, near the evolution of arboreal and specializa- diaminobenzidine. Sections labeled with PH3 were incubated tions. We review and discuss the paleontological evidence doc- with anti-mouse Alexa-568 (Jackson ImmunoResearch, USA). umenting evolutionary changes in hallux position, and assess their consistency with a role for embryonic asymmetric elonga- Whole-Mount Immunolabeling tion in the development and evolution of a distal hallux with Legs were washed in cold PBS for 15 min and immersed in a grasping capability. modified version of Clarity fixative (PFA 4%, 2% acrylamide, 1% VA-044 initiator in PBS) for 2 days in movement at 4°C MATERIALS AND METHODS (Tomer et al., 2014). The air in the tube was replaced by nitrogen in a desiccation chamber and polymerized at 37°C in movement Skeletal Staining for 2 hr. Embryos were cleaned in 4% SDS pH 8.5 for 1 week. ° Fertilized broiler chicken were incubated at 37.5 Cand Primary anticollagen type IX (2C2, DSHB, 1:20) was 70% humidity in an incubator with automatic rotating shelves. incubated at 37ºC for 48 hr in 5% donkey serum and 5% DMSO. Cartilage and bone staining followed the protocol described by Secondary antibody (anti-mouse Alexa-568) was diluted in 5% Botelho et al. (2014). Embryos were anesthetized in ice, embry- donkey serum, 5% DMSO, and incubated at 37ºC for 24 hr. Legs onic legs were fixed in methanol for 2 days, stained in Alcian were counterstained with Hoeschst and photographed using a Blue for 8 hr, washed in phosphate-buffered saline (PBS), and fluorescent stereomicroscope. then stained for 2 hr in Alizarin Red (0.04% in 0.5% KOH). This short incubation in Alcian Blue results in light staining of the Cell Labeling cartilage, but avoids acidic decalcification, allowing Alizarin Red Fertilized eggs were horizontally incubated at 37°C and 70% hu- staining of the onset of ossification (see also Namba et al. (2010)). midity. After 2 days, 3.5 mL of white was removed through All procedures were in accordance with Chilean legisla- a hole over the with the aid of a 5-mL plastic syringe, and tion and were approved by Institutional Animal Care and Use then incubated for 4 more days. At day 6, an elliptic window Committees. was opened in the upper side of the egg. The right legs of embryos at stage HH29 were exposed through small holes in the Histology amniotic sac. To avoid movement, legs were immobilized with The three middle avian metatarsals (2, 3, and 4) are not perfectly a tungsten wire ring sustained by a micromanipulator. DiI was parallel to each other, being distally deflected in the dorsoventral pressure injected using glass micropipettes, pulled and broken and anteroposterior directions. In to compare the whole back to produce tips having internal diameters of 15–20 μm developing cartilage, we chose to follow sagittal section of the (Salinas-Saavedra et al., 2014). Injections sites were

J. Exp. Zool. (Mol. Dev. Evol.) 4 BOTELHO ET AL. photographed, eggs were sealed with adhesive tape, and Cartilage Differentiation within Metatarsal 3 is Asymmetric then reincubated immobile for 3 days. Both legs were fixed in At HH29, the mt3 cartilage is entirely formed by rounded, un- 4% PFA for 24 hr at 4°C. Embryos were cleared in 30% sucrose differentiated chondrocytes. Cells at the center of mt3 become and photographed using a fluorescent stereomicroscope. polyhedral at HH31, indicating the onset of chondrocyte matu- ration (Fig. 4A, HH31). As observed using PH3, at stage HH31, cells undergoing cell division are uniformly distributed in the RESULTS entire mt3 (Fig. 4B). At following stages (HH32 and HH33), cell A Distally Positioned Hallux is Acquired during Elongation of mt3 proliferation disappears at mid-shaft, yet the proximal and distal We measured elongation of mt3 and found that distal placement regions do not show any striking differences in size or number of d1 occurs concomitant and proportional to its growth. During of proliferating cells (Fig. 4B, green boxes). Those nonprolifer- stages HH31 to HH36, the period when d1 changes in position ating chondrocytes in the mid-shaft become flattened: each cell in relation to other digits, the total length of mt3 increases al- elongates in a direction perpendicular to the long axis of the car- most 3.5-fold (from 1.46 ± 0.14 mm to 5.07 ±0.13 mm, n = 10) tilage. Thereafter, cells at the center of the domain of flattened (Fig. 2A). If the position of the distal end of mt1 in relation to mt3 chondrocytes show hypertrophism (Fig. 4A, box 1), separating is used as a landmark, the region distal to the hallux would in- a larger domain of flattened cells in the proximal side from a crease only 0.47 mm between stages HH31 and HH36 (from 0.94 smaller domain in the distal side (Fig. 4A, boxes 2). Hypertrophic ± 0.1mmto1.41± 0.25 mm) (Fig. 2A, blue rectangles), com- cells express collagen type X, and increase their size many fold, pared to 3.14 mm at the same period in the proximal side (from presenting big vacuoles in the cytoplasm and narrower extra- 0.51 ± 0.08 mm to 3.65 ± 0.14 mm). Morphological landmarks cellular matrix (Fig. 4A and C). Importantly, the region entering are coherent with the hypothesis that the hallux does not move hypertrophy and expressing collagen type X is notoriously dis- in relation to mt2–4 during their fast growth. The position of d1 placed toward distal (Fig. 4C, black box). At HH35, a domain of is kept aligned with a medial deflection of mt2 (Fig. 2B, black terminally hypertrophic cells is displaced distally (Fig. 4A, box arrowhead). The onset of ossification of mt2–4 also shows that 3) and is surrounded by a large domain of prehypertrophic cells d1 is maintained close to the distal borders of the bone collars proximal to it (Fig. 4A, box 4), but a small domain of prehyper- of mt2–4 (Fig. 2B). During these stages, mt2–4 conspicuously trophic cells distal to it (Fig. 4A, box 5), suggesting that most of change their shape: they become narrower toward the proximal the transition from flattened to hypertrophic chondrocytes first end as compared with the distal end. takes place in the proximal side of the cartilage. Additional landmarks at earlier stages are provided by protein expression patterns. Collagen type IX is expressed only in early DISCUSSION embryonic cartilages. Antibodies against collagen type IX ceased to be expressed near mid-length of mt2–4, predicting the region The Development of Hallux Position in Birds where ossification will begin, long before it actually occurs. At Our cell-labeling experiments confirm, the suggestion by molec- these early stages, the hallux is found near the distalmost border ular and morphological landmark analysis, that the proximal of the region lacking collagen type IX expression, the same po- region of early metatarsals undergoes a considerably larger sition in which it is observed thereafter in relation to the early lengthwise expansion, much greater than the distal region (Fig. bone collars of mt2–4 (Fig. 2C). This suggests the hallux remains 1A). This combined with the fixed position of the hallux ex- largely fixed in this position during ontogeny. plains how it becomes distally allocated within the foot, dis- carding any alternative explanation for movement of the hallux (Fig. 1B), which in itself would have required a complex mech- Cell Labeling Shows Asymmetric Growth of Metatarsals anism capable of transporting an entire embryonic digit along To confirm the evidence of differential growth suggested by mt2, effectively moving it through tissue. landmarks, we labeled cells with DiI injections at different prox- Modern knowledge about the mechanisms of long bone imodistal positions of early mt3 at HH30 (embryonic day 6.5) growth originated from simple experiments carried out in the and observed their position at HH35 (embryonic day 9). Chon- 18th century. The first evidence that longitudinal growth occurs drocytes labeled in the central and distal portions of mt3 were in the epiphysis was published by Hales (1727). Hales pierced two always found at the distal region of mt3 at later stages (Fig. 3A small holes through the center of the metatarsus of a posthatch- and B). Chondrocytes labeled in the proximal portion of mt3 ing chick, separated by a certain distance. Two months later, were found in the center and proximal region of the cartilage he found the two marks separated by that same distance, even (Fig. 3A and B). The results confirm that most of the early mt3 though “the bone was in that time grown an inch more in length”. is precursor to the distal region of later stages, with the prox- This experiment became famous for showing that lengthwise imal region of early mt3 expanding into a considerably larger growth does not occur at the center of the bone (diaphysis) but portion. exclusively at its ends (epiphyses). Less famous is Hales’ remark

J. Exp. Zool. (Mol. Dev. Evol.) EVOLUTION OF THE DISTAL POSITION OF AVIAN HALLUX 5

Figure 2. Asymmetric metatarsal growth and the distal position of mt1. (A) Quantification of mt3 growth at the regions proximal (white) and distal (blue) to the proximal end of d1 shows that metatarsal lengthening between HH31 and HH36 is coherent with the hypothesis that asymmetrical growth causes the development of the distally positioned hallux. (B) Morphological landmarks, such as the medial deflection of mt2 (black arrowhead) and the collar of bone at the diaphysis at the onset of ossification (revealed by red staining with Alizarin Red), show that d1 does not move distally, but rather stays at the same position. (C) Developmental series of feet from embryos between HH31 and HH35 stained with Hoechst and immunostained with antibody against Col IX. The proximal end of d1 (white arrowheads) remains in line with the zone that first stops producing Col IX, at HH31-HH32, the same region where ossification will begin at HH34 (scale:1mm). that the metatarsal bone growth “was mostly at the upper end”. Remarkably, we found that this greater expansion of the proxi- Therefore, lengthwise metatarsal growth is asymmetric at late- mal metatarsals does not correlate with strong differences of cell posthatching stages. proliferation between the proximal and distal regions. Recent Here, we have documented that considerable asymmetric research in vivo on the growth of chicken and metacar- elongation occurs in early embryonic cartilage of bird mt2–4. pus (the segment in the that is serially homologous to the

J. Exp. Zool. (Mol. Dev. Evol.) 6 BOTELHO ET AL.

Figure 3. Cell labeling shows differential growth between proximal and distal metatarsal regions. (A) Cells labeled by DiI injections in the proximal region of mt3 at HH30 (blue arrowheads) are found at the center of the element at HH34. In contrast, cells labeled by injections made at the center or distal end of mt3 at HH30 (green arrowheads) are found at the distal region of the element. (B) The fate of 17 injections in different parts of mt2-3 show the asymmetrical growing of metatarsals between stages HH30-35. Blue numbers: proximal injections; red numbers: central injections; green numbers: distal injections.

metatarsus) has clarified that, around HH34-36, the key contrib- pericentric inversion of chromosome 2. While the effect of this utor to cartilage elongation is not cell proliferation, but the pro- mutation has only been characterized as a shortening of mt2–4, cesses of cartilage differentiation and maturation at the early we note that the hallux is found in an elevated, dinosaur-like epiphyses, especially extracellular matrix deposition (Li et al., position (Fig. 1 in Smyth et al. (2000)), which suggests abnormal 2015). The expansion of extracellular matrix allocates or “dis- symmetric growth of mt2–4 in this mutant. places” cells that are near the diaphysis towards the epiphysis. (Li et al., 2015). The fact we discovered an asymmetric pattern The Evolution of Hallux Position in the Dinosaur–Bird Transition of cartilage differentiation and chondrocyte displacement within Despite current grasping capability, the evolution of the hal- the metatarsals toward their proximal end suggests that greater lux was not a straightforward story of adaptation for this cur- elongation in the proximal region may occur as in metacarpals, rent function. In early dinosauromorphs and dinosaurs, without differential cell proliferation. In vivo quantitative anal- digit V was reduced to a mere metatarsal vestige, and the hal- ysis may further help test this hypothesis and provide more in- lux shortened considerably (Gauthier, ’86; Langer, 2004; Nesbitt formation about the mechanisms underlying greater proximal et al., 2009; Langer et al., 2010; Carrano et al., 2012; Sereno elongation of the metatarsus. et al., 2012). The short hallux was held in an elevated position, Epiphyseal growth rates are known to be highly variable. where it no longer carried weight, but mt1 still articulated with Each growth plate within an individual bone has its own tem- the ankle (Fig. 5), as in (Novas, ’94) and Tawa poral dynamics of chondrocyte proliferation and differentiation (Nesbitt, et al., 2009). The elevated hallux in basal theropods is (Wilsman et al., ’96b; Hall, 2005; Farnum et al., 2008; Cooper not unlike the reduced (d1) in the of digiti- et al., 2013), and differences can also be found when comparing grade such as canids and felids, which is also associ- homologous epiphyses in different (Cubo et al., 2000). ated with cursoriality (Lull, ’04). However, epiphyseal growth is mostly studied at postnatal stages In early neotheropod dinosaurs, the proximal portion of (Farnum et al., 2007) and very little is known about earlier em- mt1 was lost, acquiring its proximally tapering, tear-like shape bryonic stages (Hall, 2005). Our new data suggest that different (Gauthier, ’86; Nesbitt, 2011) (Fig. 5). The absence of a proximal growth rates are defined at much earlier, embryonic stages. Fur- articulation to the ankle makes it difficult to establish the exact ther insight on genes affecting metatarsal elongation could be position of the hallux in most neotheropods. Undistorted, derived from the chicken mutant Shankless associated with a articulated specimens are not abundant. Osteologically, birds

J. Exp. Zool. (Mol. Dev. Evol.) EVOLUTION OF THE DISTAL POSITION OF AVIAN HALLUX 7

Figure 4. Asymmetric histology of metatarsal elements. Metatarsals are initially composed of immature chondrocytes (HH30). As mat- uration proceeds, a region of irregularly shaped chondrocytes is visible at the diaphysis (HH31, black box 1), thereafter separating two regions of flattened chondrocytes at both ends of the element (HH32, black boxes 2). Later, hypertrophic cells are visible at thediaphysis (HH35, black box 3), flanked by prehypertrophic cells. The region of prehypertrophic cells at the proximal side of the diaphysis (blackbox 4) is larger than its distal counterpart (black box 5). (B) Proliferating chondrocytes in the mt3 are visualized by immunostaining of PH3. Proliferation is homogenous at early stage HH31, and then becomes restricted to both epiphyses at HH32 and HH33 (green dotted boxes), although no striking difference can be seen between both ends. (C) Collagen type IIa (Col IIa) and collagen type X (Col X) immunolabeled sagittal sections of mt3 confirms that differentiating chondrocytes are displaced to the distal side of the element. Scale: 100 μm.

J. Exp. Zool. (Mol. Dev. Evol.) 8 BOTELHO ET AL.

Figure 5. The evolution of hallux position in theropods. The ancestral state for Dinosauria is a hallux shorter than other , but proximally connected to distal tarsals (blue). In , mt1 reduced, losing its proximal epiphysis, and articulated at the mid-shaft position of mt2 in non- (green). In several Maniraptora (such as , , , , and some ), mt1 articulates closer to the distal end of mt2 (red).

J. Exp. Zool. (Mol. Dev. Evol.) EVOLUTION OF THE DISTAL POSITION OF AVIAN HALLUX 9 present a fossa metatarsi I on mt2 that marks the contact with et al., ’98), (Sternberg, ’32), (Balanoff and mt1 (Baumel et al., ’93), but this mark is not always evident in Norell, 2012), and (Norell et al., ’95; Clark et al., ’99). ancient dinosaurs (Hattori, 2016). Even so, sufficient evidence The enigmatic have been proposed to be is available to understand the evolution of hallux position. stem avialans (Lee and Worthy, 2012; Godefroit et al., 2013a; The topic is well known from classic debates in the 1970s Lee et al., 2014), but also, alternatively, the to the and 1980s, when the dinosaurian affinities of Avialae (Hu et al., 2009; Xu et al., 2011b), (Ag- were still in question. Hecht (’76) and Tarsitano and Hecht nolín and Novas, 2013), and the basal-most paravians (Xu et al., (’80) argued for the lack of perching capability in dinosaurs, 2015). Forms like Epidendrosaurus (Zhang et al., 2002) and Epi- pointing out that in articulated specimens of dexipteryx (Zhang et al., 2008) present a distally articulated mt1, and , the hallux was nonopposable, and at similar to birds. an elevated position, at mid-length of mt2. This pattern was Within , a distally placed hallux has been reported in thereafter confirmed by other authors studying the foot and Archaeopteryx (Owen 1862; Mayr et al., 2005) and other stem hallux of nonavian theropods (reviewed in Middleton, 2001; avialans such as (Zhou and Zhang 2002; O’Connor Hattori, 2016). Specimens of nonmaniraptoran neotheropods et al., 2011a), Jixiangornis (Ji et al., 2002), such as (Welles, ’84), (Rowe, ’89; (Chiappe et al., ’99), and also in clades closely related to Avialae Tykoski, 2005), Compsognathus (Peyer, 2006), (Car- such as (Xu et al., 2011b), Anchiornis (Xu et al., rano et al., 2005), Procompsognathus (Sereno and Wild, ’92), 2009), and (Godefroit et al., 2013b). Articulated (Chen et al., ’98), (Coria et al., of unquestioned troodontids, such as Mei (Xu and Norell, 2002), (Choiniere et al., 2014), (Lambe, ’17; 2004), (Xu and Wang, 2004), (Rus- Matthew and Brown, ’23), (Madsen, ’76), sell and Dong, ’93), Linhevenator (Xu et al., 2011a), (Rus- (Liyong et al., 2012), and (Kobayashi and Bars- sell, ’69), (Norell et al., 2009), and Talos (Zanno bold, 2005) support the view that the ancestral state before et al., 2011), also have a distally articulated hallux. Within Dro- Maniraptora was that the hallux articulated at about mid-length maeosauridae, the hallux articulated distally in (Xu the medioplantar side of mt2 (Fig. 5). Importantly, ichnological et al., 2000, 2003), (Zheng et al., 2010), Zhenyuan- evidence endorses this interpretation, confirming that the hallux long (Lü and Brusatte, 2015), and (Xu et al., of nonmaniraptoran theropods did not carry weight (Gatesy ’99b), but at mid-length of mt2 in (Ostrom, ’69) et al., ’99; McCrea et al., 2014). and (Norell and Makovicky, ’97, ’99, Hattori, 2016). The position of the hallux in Maniraptora is very variable, A hallux articulated at mid-length has also been reported in the and the current lack of consensus on the phylogenetic relations unusual taxon Balaur (Brusatte et al., 2013; Cau et al., 2015). makes it difficult to determine how many times a distally artic- In conclusion, the distally displaced hallux of birds had defini- ulated hallux evolved (Hu et al., 2009; Xu et al., 2011b, 2015; tively already evolved in Avialae and their closest relatives, Turner et al., 2012; Agnolín and Novas, 2013; Godefroit et al., but an earlier origin within Maniraptora cannot be discarded 2013a; Brusatte et al., 2014). Alvarezsaurids were once consid- (Fig. 5). It also remains possible that it evolved independently ered basal Avialae, but are now thought to have a more basal po- in more than one lineage of Maniraptora, including more than sition within Maniraptora (Sereno, 2001; Choiniere et al., 2010; one lineage of Paraves. Importantly, distal placement of the hal- Godefroit et al., 2013a; Brusatte et al., 2014). The articulated lux occurred before it became a fully opposed digit: Changes in feet of the alvarezsaurids Kol (Turner et al., 2009), Albinykus the shape of mt1 that confer an opposable hallux occurred first (Nesbitt et al., 2011), and Parvicursor (Suzuki et al., 2002) have in basal (which have an “L”-shaped, partially twisted distally articulated halluces. In Therizinosauroidae, a with mt1) before the acquisition of a fully twisted mt1 in Ornithuro- feet, some species re-evolved a fully formed proxi- morpha (Middleton, 2001; O’Connor et al., 2011a; Botelho et al., mal epiphysis of mt1 articulated to the ankle, a remarkable case 2015). of reversal (Zhang et al., 2000; Zanno et al., 2009). The mt1 of the basal therizinosaurs (Kirkland et al., 2005) and Beip- A Developmental Mechanism for the Evolution of Hallux Position in iaosaurus (Xu et al., ’99a) still lacked the proximal epiphysis, Birds and articulated at about half-length of mt2 at the medioplan- The elevated position of the hallux in the foot of adult non- tar side (Fig. 5). Several phylogenetic analyses recover Ovirap- avian theropods bears a striking resemblance to the condition torosauria as the sister clade of Paraves, composing a clade called of the chicken embryo at stage HH32. We propose that until (Godefroit et al., 2013a, Brusatte et al., 2014) (Fig. this stage, the development of the foot in the chicken represents 5). There is abundant fossil evidence that the hallux in Ovirap- a common pattern for all Neotheropoda (which includes both torosauria articulated at about half-length of mt2, as observed ancient forms and modern birds). Thereafter, development fol- in Similicaudipteryx (Xu et al., 2010), (Ji et al. lowed different trajectories in different clades: In nonmanirap- ’97, ’98), Ganzhousaurus (Wang et al., 2013), (Ji toran neotheropods, the position of the hallux would remain at

J. Exp. Zool. (Mol. Dev. Evol.) 10 BOTELHO ET AL. mid-length of mt2 until the adult, whereas in Avialae and their ACKNOWLEDGMENT close relatives, it becomes distally allocated. We are thankful to Martin Wild for his support at the Uni- Because of early elevated hallux position in bird embryos, our versity of Auckland and Matteo Fabbri for his comments on new data on subsequent development suggest that asymmetric the manuscript. This research was funded by Fondecyt grants elongation (greater at the proximal end) was key in the evolu- 1150906 to AOV, Fondecyt grant 1110237, and Human Frontier tionary origin of a foot with a distally placed hallux. Early (non- Science Program. avialan) neotheropods would have presented a primitive pattern of symmetric elongation, leading to an adult with a hallux in ele- vated position. Since most Paraves, including basal Avialae, have LITERATURE CITED considerably longer metatarsi than other dinosaurs, it seems rea- Abourachid A, Höfling E. 2012. 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Cambridge, bine long, slender metatarsi with an elevated hallux. Therefore, MA: Publications of the Nuttall Ornithological Club. regardless of whether specific taxa show metatarsi with longer or Botelho JF, Ossa-Fuentes L, Soto-Acuna S, et al. 2014. New devel- shorter adult proportions, the evolution of hallux position shows opmental evidence clarifies the evolution of wrist bones inthe a clear phylogenetic pattern, being elevated in most theropods dinosaur–bird transition. PLoS Biol 12:e1001957. (symmetric growth), and distal in Avialae and their close rel- Botelho JF, Smith-Paredes D, Soto-Acuña S, et al. 2015. Skeletal plas- atives (asymmetric growth). This is consistent with the notion ticity in response to embryonic muscular activity underlies the de- that in all taxa hallux position was defined at early embryonic velopment and evolution of the perching digit of birds. 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