How the pterosaur got its wings.

Biol. Rev. (2014), pp. 000–000. doi: 10.1111/brv.12150

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How the pterosaur got its wings Masayoshi Tokita∗ Department of Organismic and Evolutionary Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, U.S.A.

ABSTRACT Throughout the evolutionary history of life, only three vertebrate lineages took to the air by acquiring a body plan suitable for powered flight: birds, bats, and pterosaurs. Because pterosaurs were the earliest vertebrate lineage capable of powered flight and included the largest volant animal in the history of the earth, understanding how they evolved their flight apparatus, the wing, is an important issue in evolutionary biology. Herein, I speculate on the potential basis of pterosaur wing evolution using recent advances in the developmental biology of flying and non-flying vertebrates. The most significant morphological features of pterosaur wings are: (i) a disproportionately elongated fourth finger, and (ii) a wing membrane called the brachiopatagium, which stretches from the posterior surface of the arm and elongated fourth finger to the anterior surface of the leg. At limb-forming stages of pterosaur embryos, the zone of polarizing activity (ZPA) cells, from which the fourth finger eventually differentiates, could up-regulate, restrict, and prolong expression of 5′ -located Homeobox D (Hoxd) genes (e.g. Hoxd11, Hoxd12, and Hoxd13) around the ZPA through pterosaur-specific exploitation of sonic hedgehog (SHH) signalling. 5′ Hoxd genes could then influence downstream bone morphogenetic protein (BMP) signalling to facilitate chondrocyte proliferation in long bones. Potential expression of Fgf10 and Tbx3 in the primordium of the brachiopatagium formed posterior to the forelimb bud might also facilitate elongation of the phalanges of the fourth finger. To establish the flight-adapted musculoskeletal morphology shared by all volant vertebrates, pterosaurs probably underwent regulatory changes in the expression of genes controlling forelimb and pectoral girdle musculoskeletal development (e.g. Tbx5), as well as certain changes in the mode of cell–cell interactions between muscular and connective tissues in the early phase of their evolution. Developmental data now accumulating for extant vertebrate taxa could be helpful in understanding the cellular and molecular mechanisms of body-plan evolution in extinct vertebrates as well as extant vertebrates with unique morphology whose embryonic materials are hard to obtain. Key words: pterosaurs, wing, fingers, bone, muscle, wing membrane, bats, birds, evolution, development. CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. A unique body plan for flight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Pterosaur wing anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Comparisons with the wings of bats and birds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Potential cellular and molecular mechanisms underlying pterosaur wing evolution . . . . . . . . . . . . . . . . . (1) Elongation of the wing finger by SHH, 5′ HoxD and BMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Wing membrane and elongation of the wing finger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Development of the muscles associated with the wing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Modular evolution in pterosaur wing bones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Supporting information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Address for correspondence (Tel: (617) 496-9245; E-mail: [email protected]).

Biological Reviews (2014) 000–000 © 2014 The Author. Biological Reviews © 2014 Cambridge Philosophical Society

Masayoshi Tokita

2 I. INTRODUCTION Throughout the evolutionary history of vertebrates, only three lineages advanced to the air by acquiring a body plan suitable for powered flight: birds, bats, and pterosaurs. Birds are considered to be derived from theropod dinosaurs; Archeopteryx lithographica, a basal member of Avialae, that possessed intermediate anatomical features between ancestral dinosaurs and descendant birds has been found from the late Jurassic [around 150 million years ago (Mya)] in Europe (Benton, 2005; Gill, 2006; Dyke & Kaiser, 2011; Foth, Tischlinger & Rauhut, 2014). Unlike birds, no mammalian fossils with an intermediate morphology linking bats to gliding or flightless ancestors have been found, although it is almost universally accepted that bats evolved from small arboreal mammals (Speakman, 2001; Gunnell & Simmons, 2005; Altringham, 2011). The earliest known fossil bat, Onychonycteris finneyi, from the early Eocene (around 52.5 Mya), was clearly capable of powered flight, possessing a forelimb that was morphologically similar to that of extant bats (Simmons et al., 2008). A recent molecular phylogeny indicates that bats diverged from a lineage belonging to the group Laurasiatheria that includes moles, dogs, cats, horses, cows, and whales, around the end of the Cretaceous (66.5 Mya) (Meredith et al., 2011) to early Paleogene (59.1 Mya) (dos Reis et al., 2012). The oldest pterosaur fossil was found in Europe and dates to the late Triassic period (210–225 Mya) (Unwin, 2006; Witton, 2013). The lineage became extinct at the end of the Cretaceous, together with dinosaurs (Unwin, 2006; Witton, 2013). Although the phylogenetic position of pterosaurs within reptiles still remains controversial (Unwin, 2006), a close relationship with a lineage of Archosauria such as the Late Triassic Scleromochlus, a member of the clade that includes birds and dinosaurs (Ornithodira), has been suggested (Hone & Benton, 2007; Witton, 2013). However, as for bats, transitional fossils with intermediate anatomical features linking pterosaurs with ancestral reptiles remain to be found. The scenario that pterosaurs evolved from an arboreal tetrapod with gliding (parachuting) ability was inferred from data on limb functional morphology and has been widely accepted (Bennett, 1997, 2008; Unwin, 2006; Witton, 2013). Although they are extinct and few fossils tell us about pterosaur embryogenesis and the early phase of their growth (Bennett, 1995, 2007; Chiappe et al., 2004; Ji et al., 2004; Wang & Zhou, 2004; Chinsamy, Codorniú & Chiappe, 2008; Delfino & Sánchez-Villagra, 2010; Prondvai et al., 2012; Manzig et al., 2014), because pterosaurs were the earliest vertebrate lineage capable of powered flight and included the largest volant animal in the history of the earth, understanding how they evolved their flight apparatus, the wing, is an important issue in evolutionary biology.

Furthermore, it has been pointed out that the integration of palaeontology, which can provide information about phenotypic evolution on a geological timescale by analysing extinct organisms, and developmental biology, which can add information on how an evolutionary change happened at a mechanistic level by examining extant organisms, will allow us to understand more deeply the morphological evolution of animals (Sánchez-Villagra, 2012; Thewissen, Cooper & Behringer, 2012; Urdy et al., 2013; Wilson, 2013). From this point of view, several authors have attempted to infer potential cellular and molecular mechanisms underlying phenotypic evolution in extinct vertebrates. These include: studies on vertebral numbers/identity in fossil amniotes (Müller et al., 2010), extinct mammals (Luo et al., 2007), and cetaceans including Eocene whale species (Thewissen et al., 2012); reports on scale-pattern diversification and evolution of craniofacial, trunk and fin morphology in a Triassic basal actinopterygian fish (Schmid & Sánchez-Villagra, 2010; Schmid, 2012); and the evolution of limb skeletal morphology in Mesozoid ichthyosaurs (Maxwell, Scheyer & Fowler, 2014). Herein, I speculate about the potential developmental basis of pterosaur wing evolution given recent advances in the developmental biology of other flying vertebrates as well as of non-volant vertebrates.

II. A UNIQUE BODY PLAN FOR FLIGHT (1) Pterosaur wing anatomy Here, I briefly overview the major morphological characteristics of the pterosaur wing and compare it to those of bats and birds. The pterosaur wing is a complex composed of the forelimb skeleton, muscles, and wing membranes (a smaller propatagium and larger brachiopatagium) (Fig. 1A, F). Another wing membrane, called the uropatagium, stretches between the hindlimb and tail; further discussion of this membrane is omitted due to its potential lesser contribution to powered flight and its reduction in multiple pterosaur lineages (reviewed by Dyke, Nudds & Rayner, 2006). Pterosaurs have only four fingers in the forelimb, one less than the ancestral condition of five digits in both the fore- and hindlimbs of amniotes. Judging from the morphological characteristics of the fingers, especially the number of phalanges that compose each finger, the four fingers of pterosaurs are considered to be homologous to the first four fingers of the amniote forelimb, i.e. digits I, II, III, and IV from anterior to posterior (Fig. 1) (Bennett, 2008; Witton, 2013). Thus, the ancestor of pterosaurs lost the fifth finger, corresponding to our little finger, during their evolution (Bennett, 2008). The loss of the fifth finger is not a rare event in amniote evolution. For example, Herrerasaurus, an ancestral lineage of theropod dinosaurs, had only a vestigial fifth


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Fig. 1. Comparison of autopod (hand) skeletal morphology and basic structure of wings. (A) The pterodactyloid pterosaur Pteranodon, (B) the theropod dinosaur Herrerasaurus, (C) the theropod Coelophysis, (D) a microchiropteran bat, and (E) the chicken (Gallus gallus). Note the disproportionally elongated phalanges of the fourth finger in the pterosaur (A). Fingers labelled with the same number are anatomically homologous. Yellow: carpals, ulnare and radiale; green: metacarpals; pink: phalanges. (F) Simplified illustration of the body and forelimb skeleton of the pterosaur, Pteranodon. (G) Forelimb of a microchiropteran bat. (H) Forelimb of the chicken. Skeletal muscles attached to the forelimb skeletons were omitted for simplicity. Note that two wing membranes: the propatagium (pp, shown in blue) and the brachiopatagium (bp, shown in cream) in the pterosaur correspond spatially to the propatagium and the plagiopatagium (plp) in bats, respectively. Unlike pterosaurs and bats, birds do not have wing membranes in their forelimb. Abbreviations: c, coracoid; ca, carpals; cp, chiropatagium; h, humerus; mca, metacarpals; ph, phalanges; prf, primary flight feathers; pt, pteroid; r, radius; sc, scapula; scf, secondary flight feathers; trf, tertial flight feathers; u, ulna; up, uropatagium.

metacarpal posterior to the fourth finger (Fig. 1B), and Coelophysis were completely lacking a fifth finger (Fig. 1C) (Wagner & Gauthier, 1999; Vargas et al., 2008; Towers et al., 2011). One of the most noticeable features of the pterosaur wing is the disproportionally elongated fourth finger, also called the ‘wing finger’.

All four phalanges composing the fourth finger are extremely elongated. By contrast, the anterior three fingers (=digits I, II, and III) remain short because of the truncated state of each phalanx of these fingers. In addition, the metacarpals and phalanges of these three fingers became thinner. Although no quantitative and


Masayoshi Tokita

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A

M. trapezius

Ligamentum extensor digiti alae M. extensor digitorum brevis

M. scapulohumeralis anterior M. deltoides scapularis

M. flexor digitorum brevis

M. extensor pteroideus

M. flexor carpi ulnaris M. biceps

M. flexor digitorum longus (ulnar head)

M. flexor digitorum longus M. extensor digiti quarti brevis M. extensor carpi ulnaris M. teres major

M. extensor carpi radialis

M. latissimus dorsi M. extensor digitorum longus M. triceps (lateral head) M. triceps (scapular head)

B

M. rhomboideus superficialis

C

M. deltoideus minor M. deltoideus major

M. omocervicalis M. occipito-pollicalis M. supraspinatus M. spinodeltoideus

M. proscapulohumeralis

M. pectoralis

M. extensor metacarpi radialis Tendon of tensor patagii longus

M. extensor digitorum communis

M. levator scapulae

M. extensor pollicis longus M. extensor pollicis brevis

Tendon of tensor patagii brevis

M. acromiodeltoideus M. rhomboideus

M. biceps brachii M. brachio-radialis

M. trapezius

M. brachialis

M. extensor carpi radalis longus M. extensor carpi radalis brevis

M. interosseus dorsalis + M. interosseus palmaris M. triceps M. dorsalis scapulae M. rhomboideus profundus M. latissimus dorsi

M. triceps

M. flexor metacarpi radialis M. anconeus

M. abductor pollicis longus

M. teres major

M. flexor metacarpi posterior M. flexor digiti III + M. flexor brevis digiti III

M. serratus anterior M. latissimus dorsi

M. extensor M. supinator pollicis brevis M. extensor carpi ulnaris M. extensor digitorum communis

M. coraco-cutaneus

Fig. 2. A comparison of forelimb and pectoral girdle muscle morphology between pterosaurs, birds, and bats (dorsal view). (A) Reconstruction of forelimb and pectoral girdle muscles of a pterosaur, Anhanguera sp. Based on Bennett (2003, 2008). Ligamentum extensor digiti alae (shown in green) were suggested to allow automatic extension of the wing finger during flight and prohibit hyperextension of the elbow (Prondvai & Hone, 2008). (B) Forelimb and pectoral girdle muscles of a bird, Crotophaga sulcirostris. Redrawn from Berger (1954). (C) Forelimb and pectoral girdle muscles of a bat, Rousettus aegyptiacus. Based on Norberg (1972). Muscles used in the wing upstroke are labelled in red; downstroke muscles are in purple. Note that some flight muscles are not shown due to their arrangement beneath other muscles. See Table 1 for a complete list of flight muscles in these animals.

comparative data exist, it appears that these anterior three fingers of pterosaurs were not substantially shortened from the ancestral condition seen in potential ancestral taxa such as Scleromochlus, Lagosuchus, and Euparkeria (Witton, 2013). In the Pterodactyloidea, the fourth metacarpal proximal to the phalanges of the fourth finger also became elongated (Andres, Clark & Xu, 2014). Interestingly, the length of the stylopod and zeugopod elements in the forelimb (humerus and radius + ulna, respectively) is only slightly larger than that of homologous elements in the hindlimb (femur and tibia + fibula, respectively) in pterosaurs (Dyke et al., 2006; see figures in Unwin, 2006; Witton, 2013). The smaller and narrower propatagium stretched from the anterior side of the shoulder and arm to the hand (Elgin, Hone & Frey, 2011; Witton, 2013). In the

middle portion (the proximal part of the hand: carpal region), this membrane was supported by the pteroid bone, unique to this clade. Well-preserved pterosaur fossil impressions indicate the presence of a muscle complex within the propatagium (Wollenhofer, 1975; Thewissen & Babcock, 1992). To achieve distal wing extension with lower energy consumption, pterosaurs may have employed a propatagial ligamentous system (in addition to a tendinous extensor muscle system) that automatically opened and closed their wings when the elbow was moved (Prondvai & Hone, 2008) (Fig. 2). The larger and wider brachiopatagium stretched from the posterior surface of the arm and wing finger to the anterior surface of the ankle (Kellner et al., 2010; Elgin et al., 2011). Histological analyses of the wing membranes in well-preserved pterosaur fossils revealed


Pterosaur wing developmental mechanisms A

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M. sternocleidomastoideus

M. extensor digitorum brevis M. extensor pteroideus

M. coracobrachialis

M. flexor digitorum brevis

M. supracoracoideus M. biceps M. flexor carpi ulnaris

M. humeroradialis M. brachialis

M. flexor digitorum longus M. extensor digiti quarti brevis M. triceps (coracoid head) M. subscapularis

M. extensor digitorum longus

M. scapulohumeralis posterior

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M. flexor carpi radiaris

M. triceps (medial head)

M. pectoralis

M. tensor patagii brevis M. coracobrachialis anterior + M. tensor patagii longus M. extensor metacarpi radialis M. deltoideus major

C M. trapezius

M. abductor pollicis

M. omocervicalis

M. flexor pollicis

M. pronator brevis

M. occipito-pollicalis

M. abductor indicis

M. clavodeltoideus M. subscapularis

M. pectoralis (anterior division) M. biceps brachii

M. serratus anterior M. subclavius

M. extensor carpi radalis longus M. brachialis

M. pronator teres

M. brachio-radialis

M. flexor metacarpi posterior

M. flexor digiti III + M. flexor brevis digiti III M. flexor digitorum sublimus M. triceps

M. triceps

M. flexor carpi ulnaris M. expansor secundariorum

M. coracobrachialis posterior M. sternocoracoideus M. pectoralis M. supracoracoideus

M. bicipitoplagiopatagialis M. latissimus dorsi M. serratus anterior M. pectoralis abdominalis M. pectoralis

M. flexor carpi radialis M. palmaris longus M. flexor carpi ulnaris

M. coraco-cutaneus

Fig. 3. A comparison of forelimb and pectoral girdle muscle morphology between pterosaurs, birds, and bats (ventral view). (A) Reconstruction of the forelimb and pectoral girdle muscles of a pterosaur, Anhanguera sp. Based on Bennett (2003, 2008). The ligamentum extensor digiti alae is omitted in this illustration. (B) Forelimb and pectoral girdle muscles of a bird, Crotophaga sulcirostris. Redrawn from Berger (1954). (C) Forelimb and pectoral girdle muscles of a bat, Rousettus aegyptiacus. Based on Norberg (1972). Muscles used in the wing upstroke are labelled in red; downstroke muscles are in purple. Note that some flight muscles are not shown due to their arrangement beneath other muscles. See Table 1 for a complete list of flight muscles in these animals.

that the brachiopatagium was a multilayered structure (Frey et al., 2003; Kellner et al., 2010; reviewed by Unwin, 2006; Witton, 2013). The uppermost layer comprised a network of structural wing fibres, including pterosaur-specific and stiff ‘actinofibrils’ distributed in the distal region of the wing. The lowest layer consisted of blood vessels. The intermediate layer between the fibre and blood-vessel layers was composed of muscles and connective tissues like fascia. Scanning electron microscopic examination revealed that the muscular tissue was striated muscle (Unwin, 2006). Although the function of the muscles distributed within the wing membranes still remains unclear (Kellner et al., 2010), pterosaurs may have used these muscles to control the shape of the wing during flight (Unwin, 2006). Careful examination of bone–muscle correlates suggests that the architectural pattern of pectoral and forelimb muscles of pterosaurs was similar to that of crocodiles and lizards, indicating the retention of a basic tetrapod pattern rather than the development

of an unusual, derived myology (Bennett, 2003, 2008) (Figs 2 and 3; see online Fig. S1). Because, in pterosaurs, the pectoral girdle and forelimbs represent a disproportionate percentage (>40%) of their mass, it seems probable that their pectoral girdle and forelimbs were more muscular than suggested previously (reviewed by Witton & Habib, 2010). Flapping of the wing probably was accomplished by several groups of muscles around the chest and back (rather than the two primary muscles in birds; see Section II.2). Muscle reconstructions suggest that the arm was lifted by large muscles anchored to the scapula and back (e.g. the M. latissimus dorsi and M. deltoides scapularis) and lowered by those attached to the sternum and the coracoid (e.g. the M. pectoralis and M. supracoracoideus) (Table 1). (2) Comparisons with the wings of bats and birds Unlike pterosaurs, bats have five fingers in their forelimb (Fig. 1D). The metacarpals and phalanges of the


Masayoshi Tokita

6 Table 1. Muscles involved in flapping of the wing in bats, pterosaurs, and birds

Bats (Norberg, 1972)

Pterosaurs (Bennett, 2003)

Birds (Raikow, 1985)

Upstroke (elevation of humerus)

Downstroke (depression of humerus)

M. acromiodeltoideus M. infraspinatus M. latissimus dorsi M. spinodeltoideus M. supraspinatus M. teres major M. deltoides scapularis M. latissimus dorsi M. scapulohumeralis anterior M. teres major M. supracoracoideus

M. clavodeltoideus M. coraco-brachialis M. pectoralis M. subscapularis — — M. coracobrachialis M. pectoralis M. supracoracoideus — M. pectoralis

posterior four fingers (digits II, III, IV, and V) are highly elongated and connected to each other by an interdigital wing membrane called the chiropatagium (Fig. 1G). The first finger of bats is inconspicuous, like the anterior three fingers of pterosaurs. Bats have another two major wing membranes. The propatagium is stretched from the anterior side of the shoulder and the arm to the hand with comparable topology to the propatagium of pterosaurs. A unique muscle complex called the M. occipito-pollicalis, which controls the shape of the leading edge of the wing, is located within the propatagium (Thewissen & Babcock, 1991, 1992; Altringham, 2011; Tokita, Abe & Suzuki, 2012) (Figs 2 and 3). The plagiopatagium is stretched from the posterior side of the arm and the posterior most fifth finger to the anterior side of the upper and lower legs. Bats have arrays of muscles embedded in the plagiopatagium: the M. plagiopatagiales, the M. coraco-cutaneus, and the M. humeropatagialis (Norberg, 1972; Tokita et al., 2012) (Figs 2 and 3). A recent electrophysiological study suggested that the M. plagiopatagiales may provide a mechanism for bats to increase wing stiffness and thereby reduce passive membrane deformation (Cheney et al., 2014). The bat flight musculature distributed over the pectoral girdle and the arm is architecturally complex and flapping of their wing is achieved by the action of multiple muscles (Norberg, 1972; Altringham, 2011) (Table 1, Figs 2 and 3). The wing of birds is morphologically distinct from those of pterosaurs and bats (Fig. 1E). In birds, the forelimb skeleton is not always longer than the hindlimb skeleton and may even be shorter, unlike the conditions in pterosaurs and bats. Bird wings have a series of feathers, instead of wing membranes, to obtain lift (Fig. 1H). The majority of birds have only three morphologically highly modified fingers in their wing [some avian species have only two fingers (e.g. penguins and kiwis Apteryx spp.) or one finger (e.g. emu Dromaius novaehollandiae)] (Parker, 1891; Wagner & Gauthier, 1999; de Bakker et al., 2013; reviewed by Seki et al., 2012). Recent developmental biology studies support the idea that these three fingers are homologous with the anterior

three fingers (digits I, II, and III) of five-fingered amniotes (Vargas et al., 2008; Tamura et al., 2011; Towers et al., 2011; Wang et al., 2011; Salinas-Saavedra et al., 2014). The flight musculature of birds is very large relative to their total body mass (Dial, Kaplan & Goslow, 1988; Newman, Mezentseva & Badyaev, 2013) but its architectural pattern is simple, consisting of only two major muscles (Raikow, 1985; Dial et al., 1988) (Table 1, Figs 2 and 3). The M. pectoralis originates on the ventral part of the keel (carina) of the sternum, the clavicle, the coracoid, and the ribs and inserts on the proximal part of the humerus (Raikow, 1985). The M. pectoralis of birds acts during the downstroke of the wing. The M. supracoracoideus originates on the dorsal part of the sternal keel and adjacent body of the sternum, and sometimes also on the coracoid and the clavicle. The muscle arcs over the glenoid passing through the dorsal aspect of the enlarged biceps tubercle of the coracoid, which is the origin of the M. biceps brachii, and attaches on the dorsal surface of the humerus (Raikow, 1985; Bennett, 2003). This pulley-like system elevates the wing (Raikow, 1985; Bennett, 2003). Several muscles located in the dorsal aspect of the pectoral girdle such as the M. deltoideus major also contribute to elevation of the humerus, although their contribution is considerably smaller than that of the M. supracoracoideus (Raikow, 1985). In pterosaurs, on the other hand, the M. supracoracoideus originates on the anterior surface of the coracoid and inserts on the deltopectoral crest of the humerus. The shape of their coracoid shows that the pterosaur M. supracoracoideus did not arc over the glenoid to attach on the dorsal surface of the humerus (Bennett, 2003). Thus, the pterosaur M. supracoracoideus presumably acted in depression, flexion, and medial rotation of the arm as in extant reptiles (Bennett, 2003), and the role of the M. supracoracoideus in elevation of the arm is a novel feature acquired in birds (Ostrom, 1976; Bennett, 2003). Mammals, including bats, do not possess a M. supracoracoideus in their pectoral girdle, instead having the M. supraspinatus and M. infraspinatus that appear to be homologous with the


Pterosaur wing developmental mechanisms M. supracoracoideus in reptiles and birds (Diogo et al., 2009; Diogo & Abdala, 2010). The above descriptions show that the most significant morphological features of the pterosaur wing not present in the wing of bats and birds are disproportionally elongated phalanges of the fourth finger and a brachiopatagium stretching from the posterior surface of the arm and elongated fourth finger to the anterior surface of the leg.

III. POTENTIAL CELLULAR AND MOLECULAR MECHANISMS UNDERLYING PTEROSAUR WING EVOLUTION (1) Elongation of the wing finger by SHH, 5′ HoxD and BMP The cellular and molecular mechanisms underlying the determination of amniote digit identity are well understood. It is well known that the zone of polarizing activity (ZPA), an area of mesenchyme located in the posterior region of the limb bud, and Sonic hedgehog (Shh) expressed in the ZPA play a fundamental role in this process (Harfe et al., 2004; Zhu et al., 2008; Tamura et al., 2011; Towers et al., 2011; Chinnaiya, Tickle & Towers, 2014; Li et al., 2014; reviewed by Zeller, López-Ríos & Zuniga, 2009; Suzuki, 2013). The most anterior digit of both the fore- and hindlimbs is formed independently of Shh (Fig. 4). The second and third digits are specified depending on the concentration of SHH protein diffused from the ZPA. Lower concentrations of SHH diffusing into the anterior domain of the limb bud specify the digit progenitor in this region as digit II. By contrast, higher concentrations of SHH protein located just anterior to the ZPA specifies the digit progenitor in this region as digit III. Both digits IV and V originate from descendants of the ZPA cells. Differential specification of digits IV and V seems to be defined by the differential length of exposure to SHH protein (Harfe et al., 2004). In pterosaurs, the fifth finger, corresponding to our little finger, was not present. However, it is highly probable that in early limb developmental stages [e.g. limb-budor hand-plate (paddle)-forming stages], the progenitors of all five digits appeared as a series of mesenchymal cells (Fig. 4). Although the progenitors of the anterior four fingers (digits I–IV) continued to develop, that of the fifth finger (digit V) presumably degenerated during its development as in the hindlimb of chicken (Welten et al., 2005) and crocodile (de Bakker et al., 2013) embryos whose adults have only four toes. Because the three anterior fingers of pterosaurs are morphologically similar and comparable to those of potential ancestral lineages such as the late Triassic ornithodiran Scleromochlus, they might exploit a developmental program shared with their ancestors to acquire its terminal morphology. By contrast, because

7 the fourth finger (wing finger) of pterosaurs is morphologically so divergent from their three anterior fingers and the corresponding finger of potential ancestral lineages, this finger likely exploited a novel developmental program not shared with their ancestors to acquire its terminal morphology, i.e. four extremely elongated phalanges. Because the wing finger appears to be already extremely elongated compared to the other three fingers in late-stage pterosaur embryos (Ji et al., 2004; Wang & Zhou, 2004), it is possible that such elongation was brought about by certain changes early in long-bone development, which is a complex process consisting of a series of cellular events: condensation of mesenchymal cells, proliferation and differentiation of chondrocytes, and growth plate development and ossification (reviewed by Egawa et al., 2014). What molecular mechanisms might play a role in the morphogenesis of the highly unusual fourth finger of pterosaurs? Because the fourth finger differs from the three anterior fingers with respect to its cellular origination, i.e. this finger is derived from the ZPA cells themselves, Shh could be one of the key players in this process. In bats, in which the wing is supported by four greatly elongated posterior fingers, the ZPA, marked by Shh expression, is extended anteriorly at the bud stage (stage 14) and Shh expression is uniquely reinitiated in the forelimb at a later embryonic stage (stage 16) (Hockman et al., 2008; Cooper, Cretekos & Sears, 2012) (Fig. 4). In stage 16 bat embryos, Shh is expressed in interdigital tissue (interdigit) with an anterior–posterior gradient; the greatest concentrations are found adjacent to the primordia of the third, fourth, and fifth fingers, which eventually achieve exceptional lengths, forming the bones that support the wing. Furthermore, in bat embryos, the apical ectodermal ridge (AER), a thickened ectodermal structure located at the distal tip of embryonic limb primordia and marked by fibroblast growth factor 8 (Fgf8) expression, is widened in a dorsal–ventral direction (Cretekos et al., 2007) and Fgf8 expression is uniquely up-regulated in the interdigits at a later embryonic stage (stage 16) (Weatherbee et al., 2006; Hockman et al., 2008) and may contribute to maintain a Shh–Fgf feedback loop to allow for the proliferation of the interdigits into more advanced stages of wing membrane development (Weatherbee et al., 2006; Hockman et al., 2008). It could be that these interdigits act as signalling reservoirs that also direct the extreme lengthening of the posterior finger bones (Cooper et al., 2012). Although the length of progenitors of the three posterior fingers that first appear as mesenchymal condensations are equivalent in bats and mice during the incipient phases of limb development, the relative lengths of these fingers increase dramatically in bats relative to those in mice through increased rates of chondrocyte proliferation in the growth plate during chondrogenesis (Sears et al., 2006; Cooper et al.,


Masayoshi Tokita

8

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Fig. 4. One scenario for pterosaur forelimb development and comparison to limb development in other amniotes (mouse, chick, and bat). (Top) At the early limb bud stage, there are five digit progenitors (grey circles and P1–5). P4 and P5 are located inside the zone of polarizing activity (ZPA; light blue). Dotted circle indicates a digit progenitor that does not give rise to a digit. (Upper middle) At the digit-specification stage, a distinct molecular mechanism determines digit (D) identity. The fourth and fifth digits originate from descendants of the ZPA cells that express Shh. The second and third digits are specified depending on the concentration of SHH protein diffused from the ZPA. The first digit is formed independently of the function of Shh. In the chick forelimb, P4 whose position has been shifted from inside the ZPA to outside the ZPA (arrow) is specified as D3. In the bat forelimb, the ZPA is extended anteriorly and the primordium of the plagiopatagium (plagiopatagium bud) where Fgf10 and Tbx3 are expressed (cream) is formed posterior to the forelimb bud. In the pterosaur forelimb, the progenitor of the fifth digit is degenerated and the primordium of the brachiopatagium (brachiopatagium bud) where Fgf10 and Tbx3 are potentially expressed as in the corresponding structure in bats is formed posterior to the forelimb bud. (Lower middle) Lateral view of late-stage embryos where the basic architectural pattern of the limb has been established. (Bottom) Higher magnification of the limbs at this late stage. In the bat forelimb, the plagiopatagium bud gives rise to the plagiopatagium (cream) stretched from the posterior surface of the arm and the fifth finger to the anterior surface of the leg. In addition, the interdigits remain and give rise to the chiropatagium where Shh, Hoxd11–13, Fgf8, and Tbx3 are expressed (pink). These wing membranes may facilitate elongation of the second, third, fourth, and fifth finger (arrows) in this animal. In the pterosaur forelimb, the brachiopatagium bud gives rise to the brachiopatagium (cream) stretched from the posterior surface of the arm and the fourth finger to the anterior surface of the leg. This wing membrane might facilitate elongation of the fourth finger (arrow) in this animal. Data on the mouse and chick were derived from Tamura et al. (2011) and for the bat from Weatherbee et al. (2006), Hockman et al. (2008), Tokita et al. (2012) and Wang et al. (2014). See text for details of cellular and molecular mechanisms of finger bone development.

2012). Moreover, up-regulation of bone morphogenetic protein 2 (Bmp2) in the perichondrium, a dense fibrous connective tissue surrounding the growth plate, of these fingers was observed and the involvement of BMP signalling is thought to play a key role in the

elongation of bat fingers (Sears et al., 2006; Cooper et al., 2012). Recent transcriptome analysis (mRNA-seq) identified 5′ -located Homeobox D (Hoxd) genes (especially, Hoxd11, Hoxd12, and Hoxd13) as candidates that facilitate the elongation of bat finger bones by regulating


Pterosaur wing developmental mechanisms endochondral skeletal growth (Wang et al., 2014). It has been known that 5′ Hoxd genes show nested expression along the anterior–posterior axis in the autopod, and each expression pattern is correlated with condensed cartilage of the digit primordium involved in the determination of digit identity (Suzuki, 2013). Also, 5′ Hoxd genes regulate the length of digit bones in a dose-dependent manner (Chen et al., 2005; Ray & Capecchi, 2008; Villavicencio-Lorini et al., 2010; Suzuki, 2013; González-Martín, Mallo & Ros, 2014; Wang et al., 2014). In bats, the expression of all 5′ Hoxd genes is extremely high and prolonged, compared to mice, in the perichondrium and the interdigits of the embryonic forelimb where the second to fifth fingers form and grow (Wang et al., 2014). It is known that the expression of 5′ Hoxd genes influences BMP (Suzuki, Ueno & Kuroiwa, 2003; Williams et al., 2005; Salsi et al., 2008; Villavicencio-Lorini et al., 2010) and SHH (Zákány, Kmita & Duboule, 2004; Capellini et al., 2006; Tarchini, Duboule & Kmita, 2006; Sheth et al., 2013) signalling during limb development, and that SHH signalling is necessary for 5′ Hoxd expression in the autopod (Chiang et al., 2001; Ros et al., 2003). Shh may also directly regulate the expression of Bmp2 in the interdigits and the perichondrium of finger bones (Hockman et al., 2008). Taken together, these data imply that in bat embryos, Shh in the anteriorly extended ZPA induces ectopic and more prolonged expression of 5′ Hoxd genes in the posterior region of the hand plate, which in turn regulates later expression of Shh in the interdigits and Bmp2 in both the interdigits and the perichondrium to direct the extreme lengthening of the posterior finger bones that support the bat wing. Pterosaurs did not have an interdigital wing membrane between the fingers, comparable to the chiropatagium of bats that may allow the elongation of finger bones (Fig. 1). This is consistent with the lack of elongation of the anterior three fingers of pterosaurs. One potential molecular mechanism underlying the development of extremely elongated phalanges of the fourth fingers is that the ZPA cells from which the fourth finger eventually differentiates up-regulated, restricted, and prolonged expression of 5′ Hoxd genes around the ZPA through pterosaur-specific exploitation of SHH signalling. These up-regulated 5′ Hoxd genes could then have influenced downstream molecules such as BMPs that might increase the overall rate of chondrocyte maturation through increased rates of both chondrocyte proliferation and differentiation of wing finger phalanges. There are other candidate molecules that could potentially influence the elongation of pterosaur wing finger phalanges. For example, paired related homeobox 1 (Prx1) is known as an essential regulator of long bone elongation during limb development and unique expression of Prx1 in the perichondrium cells of developing forelimbs contributes to wing bone

9 elongation in bats, through increasing chondrocyte proliferation as well as accelerating chondrocyte differentiation (Cretekos et al., 2008). In mice, insulin-like growth factor (IGF) signalling regulates the length of long bones such as the tibia and metatarsals through chondrocyte size regulation (Wang, Zhou & Bondy, 1999). It is likely that IGF1 signalling facilitates the elongation of leg skeletons of Egyptian jerboa (Jaculus jaculus), a small bipedal rodent with greatly elongated hindlimbs (Cooper et al., 2013), and forelimb skeletal growth in the marsupial opossum (Monodelphis domestica) whose newborns have enlarged forelimbs twice the size of their smaller hindlimbs (Sears et al., 2012). In chicken embryos, overexpression of IGF binding protein 2 (IGFBP2) in limb buds leads to shortening of the long bones through a reduction of chondrocyte proliferation in the cartilaginous precursor (Fisher et al., 2005). Also, hairy2, a downstream target of Notch signalling is expressed in the forelimb of chicken embryos and its cyclic expression in autopod chondrogenic precursor cells may determine the formation time of each autopod limb skeletal element (Pascoal et al., 2007). It is known that hairy2 expression in the limb depends on joint AER/FGF and ZPA/SHH signalling (Sheeba, Andrade & Palmeirim, 2012). Furthermore, WNT/𝛽-catenin signalling is sufficient and necessary for the formation of synovial joints, which define the relative size (length) of discrete skeletal elements, in both mice and chicken limbs (Hartmann & Tabin, 2001; Guo et al., 2004; Kan & Tabin, 2013), through induction of the BMP family member gene Gdf5, whose overexpression causes excessive chondrocyte differentiation and cartilage outgrowth (Tsumaki & Tanaka, 1999). If the oscillation period of hairy2 expression or timing of Wnt expression is altered in the chondrogenic precursor cells of the fourth finger of pterosaur embryos, the length of each phalanx of this finger could already be substantially elongated, compared to those of the three anterior fingers, in middle/late stage embryos. In that case, the contribution of postnatal growth of skeletal elements that is observed in finger-bone elongation of bats (Farnum, Tinsley & Hermanson, 2007; Young, 2013) would not be a primary mechanism in establishing the extremely elongated phalanges of the pterosaur fourth finger. Interestingly, cetaceans (whales and dolphins) possess highly elongated second and third fingers within their flippers, due to hyperphalangy (numerous finger bones). It is likely that persistence of the AER at the distal tip of the second and third fingers produces excessive phalanges in these fingers (Richardson & Oelschläger, 2002; Sanz-Ezquerro & Tickle, 2003). The outgrowth of the limb along its proximal to distal axis is dependent upon FGF signalling (Fgf4, -8, -9 and -17) from the AER which moderates the initial size of the limb bud, the rates of cell survival and proliferation, and the initial size of the chondrogenic precursors (Sears, 2008). Because


Masayoshi Tokita

10 the number of phalanges of the pterosaur fourth finger was unchanged from the condition in their ancestors, persistence of the AER at the distal tip of their fourth finger is not a probable mechanism. Instead, because Fgf8 is ectopically expressed in the interdigits [but not in more posteriorly located primordium of the plagiopatagium (plagiopatagium bud)] in bat embryos, such a spatiotemporal change of Fgf8 expression in autopod precursors might cause the embryonic enlargement of pterosaur wing finger bones. In any case, the potential factors discussed above must function specifically in the precursor of the fourth finger in pterosaur embryos through an unknown pterosaur-specific regulatory mechanism. (2) Wing membrane and elongation of the wing finger Another fascinating idea regarding the cellular and molecular mechanisms underlying pterosaur wing finger development is that the primordium of the brachiopatagium (hypothetical brachiopatagium bud), that could be formed as an epithelial lateral budding at the trunk just posterior to the forelimb bud of the embryo, regulated the elongation of the finger (Fig. 4). It is known that the spatially equivalent wing membrane, i.e. the plagiopatagium, is initiated as a laterally enlarged epithelial structure, immediately posterior to the forelimb bud in stage 15 bat embryos (Schumacher, 1932; Tokita, 2006), and its primordium expresses Fgf10 in the mesenchyme and its receptor gene Fgfr2IIIb in the epidermis, implying that this structure exploits FGF signalling for its outgrowth (Tokita et al., 2012). In later stages (after stage 17), the further enlarged plagiopatagium bud merges with the posterior surface of the forelimb bud (Schumacher, 1932; Giannini, Goswami & Sánchez-Villagra, 2006; Tokita, 2006). It is known that Fgf10 is necessary for 5′ Hoxd-dependent control of AER-Fgf s expression (Sheth et al., 2013), regulates mammalian palatal outgrowth in interaction with SHH signalling (Lan & Jiang, 2009; Zhou et al., 2013), regulates the size and shape of Meckel’s cartilage in mammals (Terao, Takahashi & Mitani, 2011), and is expressed in chondrocytes and possibly regulates the growth of the limb skeleton through promoting chondrocyte proliferation (Sekine et al., 1999). Interestingly, within the pterosaur brachiopatagium, muscular tissues were distributed between a dorsal layer composed of structural fibres and a ventral layer enriched by blood vessels (Unwin, 2006; Witton, 2013) (details of wing membrane muscles are given in Section III.3). This anatomical pattern is comparable to that observed in the bat plagiopatagium and supports the hypothesis that the embryonic pterosaur wing enlarged laterally through global tissue–tissue interactions (e.g. between bone and muscle) as in the embryonic bat wing (Tokita et al., 2012). These data from studies of extant amniote species suggest that the brachiopatagium tissue, and more specifically FGF10 signalling potentially involved

in brachiopatagium outgrowth, might influence the outgrowth of the fourth finger phalanges located just anterior to this wing membrane in pterosaur embryos. Recently, Wang et al. (2014), using mRNA-seq analysis, identified T-box 3 (Tbx3), in addition to 5′ Hoxd genes, as the candidate that regulates elongation of bat finger bones. In bat embryos, expression of Tbx3 is up-regulated and prolonged in the interdigits and the perichondrium of the posterior domain of the hand as well as in the plagiopatagium bud (Wang et al., 2014). In chicken embryos, Tbx3 is expressed in the interdigit between the third and fourth toes as well as in the connective tissue posterior to the fourth toe and specifies posterior digit identity (Suzuki et al., 2004; Suzuki, 2013). Also, Tbx3 can promote cell proliferation in a variety of cell lines and organs (Carlson et al., 2002; Ito et al., 2005; Renard et al., 2007; Esmailpour & Huang, 2012), implying a role in promoting the growth of wing membranes in bats. Furthermore, Tbx3 can suppress osteoblast differentiation (Govoni et al., 2009), suggesting a possible contribution to elongating the posterior fingers by promoting chondrocyte proliferation and delaying osteoblast differentiation. In fact, Tbx3 up-regulates expression of Bmps, including Bmp2, which is known to stimulate cartilage proliferation and differentiation and increase digit length in the bat embryonic forelimb (Sears et al., 2006), in interdigits (Suzuki et al., 2004). If the pterosaur brachiopatagium bud expressed Tbx3 as in the bat plagiopatagium bud, the phalanges of their fourth finger located just anterior to this structure could be elongated due to activation of BMP signalling. There are several mammalian lineages that are capable of gliding using well-developed gliding membranes between the fore- and hindlimbs (e.g. marsupial flying phalangers, dermopteran colugos, and rodentian flying squirrels) (Goldingay & Scheibe, 2000; Feldhamer et al., 2007; Diogo, 2009; Jackson, 2012). Interestingly, in flying squirrels, the gliding membrane is anteriorly supported by an elongated skeletal structure called the styliform cartilage that is located posterior to the fifth finger (Thorington, Darrow & Anderson, 1998). By contrast, the fifth finger to which the gliding membrane is connected is not so elongated in flying phalangers (Johnson-Murray, 1987) and colugos (Jackson, 2012; M. Tokita, personal observations). Unfortunately, embryogenesis of these gliding mammals remains poorly understood. However, recently, the marsupial sugar glider (Petaurus breviceps) has been used as a new model organism for understanding gliding membrane development, and breeds readily in captivity (Tzika & Milinkovitch, 2008). Investigating the embryonic development of gliding mammalian species would be helpful to understand better the role of lateral gliding membranes in skeletal formation and growth and to investigate further the hypothesis that the brachiopatagium facilitated the elongation of the pterosaur wing finger located just anterior to it.


Pterosaur wing developmental mechanisms (3) Development of the muscles associated with the wing Musculoskeletal specialization for flight, such as a robust pectoral girdle and large plate-like sternum to which the massive pectoralis muscle attaches, evolved independently in pterosaurs and other flying vertebrates, i.e. birds and bats. Formation of the vertebrate musculoskeletal system, which comprises muscles, tendons, and bones, is a complex process and requires precise connectivity among these tissues; this is crucial for efficient movement and stability of the entire organism. The musculoskeletal systems of the forelimb and pectoral girdle are composed of multiple tissues of diverse developmental origins. The axial skeletal elements to which the pectoral girdle is anchored arise from the somites (Shearman & Burke, 2008). The forelimb skeleton (e.g. humerus, metacarpals), the pectoral girdle, and the sternum as well as the tendons associated with these skeletal elements are derived from the lateral plate mesoderm (Rallis et al., 2003; Shearman & Burke, 2008; Schweitzer, Zelzer & Volk, 2010; Valasek et al., 2011). Interestingly, the scapula of tetrapods is derived from both somites and lateral plate mesoderm, showing its mixed mesodermal origin (Huang et al., 2000; Valasek et al., 2010; Piekarski & Olsson, 2011; Shearman, Tulenko & Burke, 2011; Ericsson, Knight & Johanson, 2012). Skeletal muscles associated with the appendicular skeleton and the sternum are exclusively derived from the somites (Galis, 2001; Murphy & Kardon, 2011; Valasek et al., 2011). In mouse embryos, Tbx5 is expressed in lateralplate-derived limb mesenchyme and muscle connective tissues (Hasson et al., 2010), as well as in the tissue of the proximal pectoral girdle region and the ventral midline region of the thorax (Valasek et al., 2011). Inactivation of Tbx5 causes loss of forelimb and pectoral girdle skeletons (scapula and clavicle), as well as loss of the sternum (Rallis et al., 2003). Interestingly, the pectoral girdle muscles, including the M. pectoralis, are developed to a limited extent and mis-patterned due to the absence of their skeletal attachment in the Tbx5 mutant (Valasek et al., 2011). These results imply that Tbx5 regulates development of the musculoskeletal system of the forelimb and the pectoral girdle, and how Tbx5 is expressed in the connective tissues of the region determines the morphology in the adult form. Regulatory changes in Tbx5 expression might underlie the convergent evolution of the robust pectoral girdle and sternum, as well as of the large pectoralis muscle, for powered flight in three distantly related flying vertebrate lineages. In vertebrates, the architectural pattern of the skeletal muscles is regulated by extrinsic cues from surrounding connective tissues to adopt specific arrangements of muscle fibres in the adult (Kardon, Campbell & Tabin, 2002; Evans et al., 2006; Shearman & Burke, 2008; Tokita & Schneider, 2009; Hasson, 2011; Huang et al., 2013; Tokita et al., 2013b). In mice, the M. rhomboideus and

11 M. levator scapulae insert predominantly on the ‘primaxial’ domain of the scapula where muscles are patterned by the somite-derived connective tissues (Valasek et al., 2010; Shearman et al., 2011). In chickens, those muscles extend their insertion to the ‘abaxial’ domain of the scapula where muscles are patterned by the lateral-plate-derived connective tissues and this extensive insertion of pectoral girdle muscles onto an abaxial element may represent a significant adaptation for flight (Shearman et al., 2011). I speculate that a similar type of change in the mode of cell–cell interaction (between connective and muscular tissues) occurred early in pterosaur evolution to produce a musculoskeletal morphology adapted for flight. Although pterosaurs shared some anatomical characteristics in flight apparatus with birds and bats (e.g. a robust pectoral girdle and accompanying massive pectoralis muscle), they had some wing musculoskeletal features not present in either birds or bats. It appears that pterosaurs retained the architectural pattern of pectoral girdle and forelimb muscles found in non-avian tetrapods, including lizards (e.g. Varanus spp.), crocodiles (e.g. Crocodylus spp.) and mammals (e.g. Didelphis spp.) (Bennett, 2003). Unlike birds in which flapping of the wing is accomplished by two major flight muscles anchored to the sternum (M. pectoralis for the downstroke and M. supracoracoideus for the upstroke), pterosaurs may have used several muscles anchored to the scapula and back for elevation of the wing, and those attached to the sternum and the coracoid for depression of the wing (Table 1, Figs 2 and 3). In chickens and mice, more superficial pectoral girdle muscles such as the M. pectoralis, M. latissimus dorsi, and M. deltoideus develop by the ‘In–Out’ mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax (Valasek et al., 2011). Because development of these superficial girdle muscles depends upon mesenchymal epithelial transition factor (MET)/hepatocyte growth factor (HGF) (scatter factor, SF)-mediated migration of muscle precursor cells delaminated from the dermomyotome of somites, they disappear in Met null or Splotch (Pax3 mutant) mice (Prunotto et al., 2004). On the other hand, deeper pectoral girdle muscles such as the M. rhomboideus profundus, M. serratus anterior, and M. levator scapulae are induced by the forelimb field, which promotes myotomal extension directly from the somites, and are not affected by the Met mutation (Valasek et al., 2010). These data indicate that the superficial and the deep pectoral girdle muscles belong to two distinct developmental modules. It is possible that birds acquired well-developed superficial pectoral girdle muscles (M. pectoralis and M. supracoracoideus) through independently altering the developmental program of these muscles. Although an exact reconstruction of the soft tissue anatomy of Mesozoic


Masayoshi Tokita

12 extinct reptiles is difficult (Bennett, 2003, 2008; Prondvai & Hone, 2008), it would be an interesting scenario if pterosaurs employed multiple and enlarged deeper pectoral girdle muscles (e.g. the M. subscapularis, M. levator scapulae) to rotate the wing and stabilize the girdle to allow the construction of a powerful flight apparatus, through independently modifying the developmental programs of those muscles. Several molecules or pathways that are activated in limb mesenchyme/connective tissue and regulate the pattern of adjacent muscles have been identified: Tbx4 and Tbx5 (Hasson et al., 2010), Hox11 (Swinehart et al., 2013), SHH signalling (Anderson et al., 2012; Hu et al., 2012), transforming growth factor-𝛽 (TGF-𝛽) signalling (Pryce et al., 2009), and WNT/𝛽-catenin signalling (Kardon, Harfe & Tabin, 2003). Because pterosaurs possessed forelimb and pectoral girdle muscles whose architectural pattern was similar to those in primitive tetrapods rather than those in birds, they might have exploited in large part the plesiomorphic developmental program acquired in basal tetrapods, to build their flight apparatus. Therefore, understanding the molecular nature of this plesiomorphic developmental program and to what degree non-avian tetrapods share the program is important. A comparison of the expression pattern of the aforementioned molecules that pattern limb skeletal muscles, as well as the genes that regulate morphogenesis of the pectoral girdle skeletons: Hoxc6 and Pbx1 for coracoid morphogenesis, Pax1 for acromion patterning, Emx2 for scapular blade condensation, Alx1, Alx4, Tbx15 and Gli3 for scapular blade patterning (Oliver et al., 1990; Capellini et al., 2010; reviewed by Hübler et al., 2013), among a variety of tetrapod lineages including lizards, crocodiles, and non-mouse mammalian species would be a useful initial step. To provide a broader area for the attachment of the flight muscles in a limited space, pterosaurs acquired certain traits on their pectoral girdle and forelimb skeleton. For example, the deltopectoral crest, a bone eminence in the proximal region of the humerus, was considerably enlarged and anchored powerful flight musculature extending from the shoulders to the forelimb. The formation of bone eminences on the limb skeleton is initiated by tendon cells that express the Scx gene, and the Scleraxis (Scx) transcription factor protein up-regulates Bmp4 expression in the tendon cells, and finally BMP4 protein secreted from the tendon cells binds its receptor on the cell surface of adjacent chondrocytes and induces bone eminences there (Blitz et al., 2009; Schweitzer et al., 2010). Bone eminences originate from a field of progenitor cells located outside the primary cartilage and these progenitor cells express both Sox9 and Scx (Blitz et al., 2013). It is reported that the late-stage embryos of pterosaurs had disproportionally elongated forelimb skeletons that appeared functional for flight, due to their allometric growth in

embryogenesis (reviewed by Unwin, 2006). I speculate that the enlarged deltopectoral crest developed on the pterosaur humerus was brought about by a regulatory change in Scx expression (e.g. a longer period and greater levels of expression) in the tendon cells of the embryos. Besides the flight muscles associated with the pectoral girdle and the forelimbs, pterosaurs had arrays of striated muscles within their lateral wing membrane (brachiopatagium). Tokita et al. (2012) investigated the development of cutaneous muscles distributed within the wing membranes of bats and suggested that these muscles are patterned by connective tissues within the wing membranes, which uniquely express Fgf10. A possible scenario is that the muscles within the pterosaur brachiopatagium were derived from somite-derived forelimb muscle precursors through patterning by the laterally located brachiopatagium bud filled with connective tissues. This would occur in a similar fashion to that of the development of the M. plagiopatagiales, M. humeropatagialis and M. coraco-cutaneous within the plagiopatagium of bats. Interestingly, muscle-like tissues have been found within the propatagium of well-preserved pterosaur fossils (Wollenhofer, 1975). The spatial pattern of the muscle showed a superficial resemblance to the M. occipito-pollicalis located in the anterior part of the propatagium of bats (Thewissen & Babcock, 1991, 1992). If a muscle-like complex found in the propatagium of pterosaur fossils was truly muscle, it might have arisen from a cranial paraxial mesoderm-derived muscle precursor (including that giving rise to the M. depressor mandibulae possessed by amphibians, reptiles and birds and the M. digastricus, and the M. stylohyoideus in mammals, that are innervated by the facial nerve) (Thewissen & Babcock, 1991), or from the cucullaris muscle complex (M. sternocleidomastoideus and M. trapezius of amniotes, which are innervated by the accessory nerve) precursor that is in large part derived from the occipital lateral plate mesoderm (Diogo & Abdala, 2010; Theis et al., 2010; Ericsson et al., 2012). If the muscle was the derivative of cranial muscles, it might be patterned by the connective tissues distributed within their propatagium, being a convergence with M. occipito-pollicalis development in bats (Tokita et al., 2012).

IV. MODULAR EVOLUTION IN PTEROSAUR WING BONES The most noticeable morphological feature shared by all pterosaur species is the extremely elongated phalanges of the fourth finger that reinforce the distal part of the brachiopatagium. In species belonging to early groups of pterosaurs such as Eudimorphodontidae, Rhamphorhynchidae, and Anurognathidae, the metacarpals of each finger are much shorter and less


Pterosaur wing developmental mechanisms variable in length compared to the (distally located) phalanges of the fourth finger. At the later origin of the Pterodactyloidea, a drastic change occurred in the metacarpals, which changed from being the shortest and least variable in length of the major wing elements in non-pterodactyloids to being the longest and most variable in length (Witton, 2013; Andres et al., 2014). It appears that this increased variation in metacarpal length relates functionally to radiation into new and more varied environments, including terrestrial environments (Andres et al., 2014). Recent comparative morphometric studies of avian phalanges (Kavanagh et al., 2013) and amniote limbs (Young, 2013) revealed that phalanges and metapodials (i.e. metacarpals in the forelimb and metatarsals in the hindlimb) belong to two independent developmental modules so that they are capable of independent transformation. This implies that the morphological change in the metacarpals of pterodactyloids could be brought about independently by altering the proliferation rate of metacarpal chondrogenic precursors or the growth rate of ossifying metacarpals in later ontogenetic periods. However, details of the molecular mechanism that released a developmental constraint on the metacarpals at the origin of pterodactyloids remain unknown.

V. PERSPECTIVES This article focused on the evolution and development of the pterosaur wing. However, this extinct reptilian lineage possesses other unique morphological features that deserve further investigation from an Evo-Devo perspective. For example, some pterodactyloid lineages such as Pteranodon (Pteranodontidae) and Quetzalcoatlus (Azhdarchidae) lost their teeth completely and instead acquired a large and extensive keratinous beak (Frey et al., 2003; Unwin, 2006; Witton, 2013). To speculate on the cellular and molecular mechanisms underlying toothless beak evolution in these pterosaur taxa, data on beak development obtained from toothless extant amniotes [birds (reviewed by Louchart & Viriot, 2011) and turtles (Abramyan et al., 2013; Tokita, Chaeychomsri & Siruntawineti, 2013a)] could be helpful. Furthermore, it is known that the head and body of some pterosaurs were covered with an epithelial appendage called pycnofibres, indicating endothermy in this extinct reptilian lineage (Witton, 2013). To understand how pycnofibres were acquired and formed in pterosaurs, data on mammalian hair and avian feather development may be useful (Lin et al., 2006; Dhouailly, 2009). Although molecular-level analyses, including DNA/genome sequencing, of this extinct reptilian lineage is currently impossible, the genome size of pterosaurs has been estimated from measuring osteocyte lacunae; contraction of genome size was observed in all three volant amniote lineages

13 (Organ & Shedlock, 2009). Genomic data from the two extant flying amniotes [birds (International Chicken Genome Sequencing Consortium, 2004; Warren et al., 2010) and bats (Seim et al., 2013; Zhang et al., 2013)] could be useful to allow well-founded inferences about developmental aspects of flight-adapted pterosaur body plan evolution.

VI. CONCLUSIONS (1) At limb-forming stages of pterosaur embryos, the ZPA cells from which the fourth finger eventually differentiates might up-regulate, restrict, and prolong expression of 5′ Hoxd genes around the ZPA through pterosaur-specific exploitation of SHH signalling. The 5′ Hoxd genes then influence downstream BMP signalling that facilitates chondrocyte proliferation in vertebrate long bones. (2) The primordium of the brachiopatagium (brachiopatagium bud) that could be formed posterior to the forelimb bud of pterosaur embryos potentially expressed Fgf10 and Tbx3, as in bat embryos, and might have facilitated elongation of the phalanges that constitute the pterosaur wing finger. (3) Pterosaurs had a robust pectoral girdle and large plate-like sternum to which the massive pectoralis muscle attached, similar to the other volant vertebrates: birds and bats. To acquire such musculoskeletal morphology adapted for flight, they might have undergone regulatory changes in the expression of genes controlling forelimb and pectoral girdle musculoskeletal development (e.g. Tbx5), and certain changes in the mode of cell–cell interaction between muscular and connective tissues early in their evolution. (4) In the later group of pterosaurs, Pterodactyloidea, the metacarpals became the longest and most variable in length of the major wing elements. This morphological change might have been brought about by independently altering the proliferation rate of metacarpal chondrogenic precursors or the growth rate of ossifying metacarpals in later ontogenetic periods. (5) It should be noted that developmental data recently accumulating for extant vertebrate taxa may be helpful in understanding the cellular and molecular mechanisms of body plan evolution in extinct vertebrates as well as extant vertebrates with unique morphology whose embryonic materials are hard to obtain.

VII. ACKNOWLEDGEMENTS I appreciate the critical and constructive comments of two anonymous reviewers that helped to improve the manuscript. I also thank A. Cooper and W. Foster for their editorial assistance. This work was supported


Masayoshi Tokita

14 in part by JSPS Postdoctoral Fellowships for Research Abroad (23-771), and the UEHARA Memorial Foundation Research Fellowship.

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IX. SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article. Fig. S1. Reconstruction of the pectoral girdle muscles of a pterosaur, Anhanguera sp. (A) Left lateral view of deep pectoral girdle muscles. (B) Left lateral view of superficial and deep pectoral girdle muscles. The muscles for the wing upstroke are labelled in red; downstroke muscles are in purple. Redrawn from Bennett (2003).

(Received 26 May 2014; revised 10 September 2014; accepted 1 October 2014 )


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