the evolutionary origin of the turtle shell and its dependence on...

The Evolutionary Origin of theTurtle Shell and Its Dependenceon the Axial Arrest of theEmbryonic Rib CageTATSUYA HIRASAWA1,JUAN PASCUAL‐ANAYA1, NAOKI KAMEZAKI2,MARI TANIGUCHI2, KANAKO MINE2,AND SHIGERU KURATANI1*1Laboratory for Evolutionary Morphology, RIKEN Center for Developmental Biology, Kobe, Japan2Kobe Municipal Suma Aqualife Park, Kobe, Hyogo, Japan

The skeleton of the turtle shell represents one of the mostenigmatic structures among tetrapods. The shell consists of dorsaland ventral shields, called the carapace and the plastron,respectively. Ever since Cuvier (1799), many researchers haveattempted to compare the osteology of turtles to those of othertetrapods. The ambiguity of the turtles' phylogenetic position andskeletal system classification still fascinates evolutionary biolo-gists and paleontologists. Meanwhile, recent advances inevolutionary developmental biology have shed new light on the

ABSTRACT Turtles are characterized by their possession of a shell with dorsal and ventral moieties: thecarapace and the plastron, respectively. In this review, we try to provide answers to the question ofthe evolutionary origin of the carapace, by revising morphological, developmental, andpaleontological comparative analyses. The turtle carapace is formed through modification ofthe thoracic ribs and vertebrae, which undergo extensive ossification to form a solid bony structure.Except for peripheral dermal elements, there are no signs of exoskeletal componentsontogenetically added to the costal and neural bones, and thus the carapace is predominantlyof endoskeletal nature. Due to the axial arrest of turtle rib growth, the axial part of the embryoexpands laterally and the shoulder girdle becomes encapsulated in the rib cage, together with theinward folding of the lateral body wall in the late phase of embryogenesis. Along the line of thisfolding develops a ridge called the carapacial ridge (CR), a turtle‐specific embryonic structure. TheCR functions in the marginal growth of the carapacial primordium, in whichWnt signaling pathwaymight play a crucial role. Both paleontological and genomic evidence suggest that the axial arrest isthe first step toward acquisition of the turtle body plan, which is estimated to have taken place afterthe divergence of a clade including turtles from archosaurs. The developmental relationshipbetween the CR and the axial arrest remains a central issue to be solved in future. J. Exp. Zool. (Mol.Dev. Evol.) 9999B: XX–XX, 2014. © 2014 Wiley Periodicals, Inc.

How to cite this article: Hirasawa T, Pascual‐Anaya J, Kamezaki N, Taniguchi M, Mine K, KurataniS. 2014. The evolutionary origin of the turtle shell and its dependence on the axial arrest of theembryonic rib cage. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:1–14.

J. Exp. Zool.(Mol. Dev. Evol.)9999B:1–14, 2014

�Correspondence to: Shigeru Kuratani, Laboratory for EvolutionaryMorphology, RIKEN Center for Developmental Biology, 2‐2‐3 Minatojima‐minami, Chuo‐ku, Kobe, Hyogo 650‐0047, Japan.E‐mail: [email protected]

Received 4 February 2014; Revised 25 April 2014; Accepted 7 May 2014DOI: 10.1002/jez.b.22579Published online XX Month Year in Wiley Online Library

(wileyonlinelibrary.com).

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© 2014 WILEY PERIODICALS, INC.

evolutionary origin of the turtle shell (Kuratani et al., 2011). Thereis, however, still much room for investigation to reconstruct theevolutionary process leading to the turtles' unique body plan.

Phylogenetic Position of TurtlesRecently, genome‐wide molecular‐based phylogenetic analyseshave solved the problem of the phylogenetic position of turtles inthe crown‐group amniotes (Shaffer et al., 2013;Wang et al., 2013).The analyses demonstrate that turtles and archosaurs split afterthe diversification of lepidosaurs (Fig. 1A), as previously shown byother molecular‐based analyses (Zardoya and Meyer, '98; Iwabeet al., 2005; Tzika et al., 2011; Chiari et al., 2012; Crawfordet al., 2012). This order of branching is robustly supported by

statistical tests, thereby explicitly rejecting alternative branchingorders.In the classical view, amniote phylogeny was largely based on

generalized morphology and the timing of first appearance in thestratigraphic record. The cranial morphology, in particulartemporal fenestrations, was believed to be the best indicator forhigher‐order phylogeny (Romer, '56, '71; Cox, '69; Kuhn, '69;Keyser and Gow, '81). Because turtles do not possess any temporalfenestra (“anapsid” state), they have been reputed to represent amember of the primitive array of amniotes (Anapsida) survivingacross several mass extinction events. However, recent cladistics‐based studies of amniote phylogeny showed that the mode oftemporal fenestration occasionally represents no more than ahomoplasy (e.g., Cisneros et al., 2004), and that the “anapsid” stategenerally represents symplesiomorphy of the lineages that led todiapsids, including parareptiles and captorhinids (Müller andReisz, 2006; Tsuji et al., 2012). According to the molecular‐basedphylogeny, turtles have lost temporal fenestrae during the earlyevolution, underlining that the “anapsid” state does not representa synapomorphy of a specific clade.There still remains uncertainty as to phylogenetic interrelation-

ships among some members of the diapsids. Due to the absence ofmolecular data of extinct taxa, morphology‐based analysis is theonly option to reconstruct these relationships. So far, reconstruc-tion of the phylogenetic position of turtles based onmorphologicalcharacters has varied depending on character lists and taxonsamplings: turtles have been assigned either to an outgroup of alepidosaur‐archosaur clade (Fig. 1B; Lyson et al., 2013a; Neenanet al., 2013), to a group relatedmore closely to lepidosaurs (Fig. 1C;Rieppel and Reisz, '99; Hill, 2005; Liu et al., 2011; Wu et al., 2011),or to a group related more closely to archosaurs (Fig. 1D;Evans, 2009; Evans and Borsuk–Białynicka, 2009). Similarly, thephylogenetic positions of some fossil diapsid groups, in particular,sauropterygians, ichthyosaurs, and thalattosaurs, which second-arily adapted to marine habitats in the Triassic, have been labile inthese morphology‐based analyses. Nevertheless, many analysesdemonstrated that turtles and sauropterygians are likely settled inthe same clade (Fig. 1A; Rieppel and Reisz, '99; Evans, 2009; Evansand Borsuk–Białynicka, 2009; Liu et al., 2011; Wu et al., 2011).Characters available for morphology‐based phylogenetic

analyses have been biased by differential preservation duringdiagenesis, because soft tissues are usually not preserved in fossils.Further refinement of character selection might be a key toreconciling the phylogenetic placements of fossil taxa with thoseof living taxa; however, it is beyond the scope of this paper. Topresent a working hypothesis for the early evolution of turtlemorphology, here we will compare turtles with sauropterygians(Fig. 1A), among other phylogenetic hypotheses.

Endoskeletal Origin of Turtle CarapacesAs described above, the turtle shell consists of a dorsal half calledthe carapace, and a ventral half called the plastron (Fig. 2). The

Figure 1. Phylogenetic position of turtles. (A) The phylogenetictree used in this review. Turtles are placed as a sister group ofarchosaurs. Black solid lines indicate the phylogenetic relationshipof crown groups based on molecular‐based analyses, and gray linesindicate the phylogenetic relationship based on morphology‐basedanalyses with fossil taxa. Arrow “a” indicates the inferred positionon the phylogenetic tree where the axial arrest (and resultant lossof the sternum) took place as a synapomorphy of this clade. Thisevent is recognized as the first step of developmental changestowards the acquisition of the turtle body plan. (B–D) Threealternative hypotheses on the phylogenetic position of turtles.

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origin of the bony plates that fuse to form a solid carapace hasbeen the subject of controversy (reviewed by Rieppel, 2013). Inturtles, the costal and neural plates are contiguous with the axialskeleton, and contact directly with the dermal tissue; whereas, inother vertebrates, the axial skeleton is covered by axial muscles(Fig. 3). Many vertebrates evolved bony tissues that areintramembranously produced within the dermis, namely exoskel-etal elements. The endoskeleton and exoskeleton have differenthistorical continuities in vertebrate evolution, thereby beingdistinguishable (Patterson, '77; Hirasawa and Kuratani, in press).This history‐based classification is fundamentally different fromthe classification of histogenesis, that is, endochondral andintramembranous ossifications.There have been several hypotheses concerning the incorpo-

ration of exoskeletal components into the costal and neural plates.

One hypothesis assumes that these bony plates are comparable toaxial endoskeletal components (Cuvier, 1799; Goette, 1899;Vallén, '42) and thus evolved solely by modification of the axialendoskeleton. In contrast, incorporation of exoskeletal compo-nents into these plates has been proposed repeatedly, assumingeither a scenario in which exoskeletal bony armors, namelyosteoderms, of the ancestral animal were fused with the axialskeleton underneath (Ogushi, '11; Suzuki, '63; Lee, '93, '96;Scheyer et al., 2008; Joyce et al., 2009), or a scenario in whichsuperficially translocated axial skeleton caused a new tissueinteraction in the dermis and induced heterotopically exoskeletalosteogenesis (Gilbert et al., 2001; Cebra‐Thomas et al., 2005). Inthe latter scenario, secretion of bonemorphogenetic protein (BMP)from the rib was hypothesized to induce the dermal cells todifferentiate into osteoblasts to produce exoskeletal bony tissues.

Figure 2. Skeletal morphology of the turtle shell. A–C, Dorsal (A) and ventral (B) views of the carapace and the ventral view of the plastron (C)of the Chinese soft‐shelled turtle (Pelodiscus sinensis) that is frequently employed in evolutionary developmental studies of turtles. D,E:Generalized pattern of the bony shell in modern turtles, in dorsal view (D) and in ventral view (E). Note that peripheral dermal elements aremissing in the P. sinensis (A,B). A–C, Redrawn from Ogushi ('13). endo, endoskeleton; exo, exoskeleton; T1‐10, first to tenth thoracic vertebrae.


EVOLUTIONARY ORIGIN OF THE TURTLE SHELL 3

Although theoretically attractive, there has been no directevidence so far to support this idea. On the contrary, anexperiment in which periosteal cells were grafted heterotopicallyinto rabbit dermis showed that BMP signaling is not able to inducedifferentiation of the dermal cells into osteoblasts (Nishimuraet al., '97). In this experiment, BMP signaling from periostealgrafted cells but not differentiation of the dermal cells intoosteoblasts was observed, and the heterotopic bones in the dermiswere produced by osteoblasts derived exclusively from the graftedperiosteal cells. Furthermore, in normal development of amniotes,there have been no reports that show dermally‐derived osteoblastsinduced by endoskeletal bones. Without differentiation of dermal

cells into osteoblasts, it is not possible that exoskeletalcomponents incorporate into the costal and neural plates of theturtle.The results of a detailed study of the embryonic development of

the costal and neural plates of a soft‐shelled turtle species indicatethat these plates most likely evolved solely by modification of theaxial endoskeleton (Hirasawa et al., 2013). In embryonicdevelopment, the extensive outgrowths of bony tissues from therib cartilage, named the bony trabeculae, which later develop intothe costal and neural plates, are produced within the periosteum atthe level where the axial muscle anlagen are initially distributed.The axial muscles covering the rib cage are not differentiated inturtles, but the boundary between this subdermal layer and theoverlying dermis is retained when the bony trabeculae develop.Therefore, it is most likely that the costal and neural plates aregenerated without contributions from exoskeletal components.Similar bony outgrowths from ribs are recognizable in the chicken,thereby corroborating the axial endoskeletal nature of costal andneural plates (Hirasawa et al., 2013). Since the costal and neuralplates are not covered with muscles, the outer parts of the bonesincorporate collagen fiber bundles of the dermis in later stages(Scheyer et al., 2008). This mode of bone formation (“metaplasticbone formation”; Haines and Mohuiddin, '68) is also seen in theendoskeleton of the chicken, namely the distal part of the distalphalanx of the foot (Hirasawa et al., 2013), thus this is not a turtle‐specific mode of endoskeletal development. In the evolutiontowards the turtle, the costal and neural plates were acquired by anincrease in the existing mode of endoskeletal development, inwhich the periosteum surrounding the rib cartilage becameexpanded radically to invade the intercostal spaces (Hirasawaet al., 2013).Despite the mode of endoskeletal development common to the

costal and neural plates of turtles and the rib cage of non‐turtleamniotes, there is a fundamental difference associated with theturtle rib cage—its dorsally arrested position (Fig. 4). In mostamniotes, the thoracic ribs are curved ventrad to surround theheart and lungs, and this is a key adaptation for aspirationbreathing (Perry, '89; Brainerd and Owerkowicz, 2006) and bodysupport on the ground (Fujiwara et al., 2009). Thus, these ribs growsecondarily into the lateral body wall (Nagashima et al., 2007). Bycontrast, in turtles, the ribs are laterally projected immovably, andthus turtles have both a unique ventilatory mechanism (Gans andHughes, '67; Gaunt andGans, '69; Landberg et al., 2003, 2009) anda unique mode of body support by the shoulder girdle (Nagashimaet al., 2009). The laterally projected ribs were present even in thestem turtle,Odontochelys semitestacea, in which the carapace doesnot form a closed‐shell structure (Li et al., 2008; Hirasawaet al., 2013), suggesting that it is likely a key synapomorphy of thewhole turtle group. However, much about the developmental basisbehind this putative key synapomorphy remains unclear.The ventral hypaxial muscles of turtles, which contribute to

lung ventilation (Gans and Hughes, '67; Gaunt and Gans, '69;

Figure 3. Muscle attachment sites of the ribs in non‐turtlediapsids. A,B: The rib of crocodilians in cranial (A) and caudal (B)views, based on the 12th presacral (third thoracic) rib of theAmerican alligator, Alligator mississippiensis. C,D: The rib of birds incranial (C) and caudal (D) views, based on the 19th presacral (fifththoracic) rib of the Darwin's rhea, Rhea pennata. Dark gray, bonypart; light grey, cartilaginous part.


4 HIRASAWA ET AL.

Landberg et al., 2003, 2009), have been called terms applicableexclusively to turtles, for example, M. diaphragmaticus, M.transversus abdominis, and M. obliquus abdominis (Gaunt andGans, '69). Although some classical studies suggest homologiesbetween these muscles and the lateral body wall muscles of otheramniotes (Ogushi, '13; Nishi, '38), these correspondences have notyet been explicitly resolved (Gasc, '81).In this study, we describe data pertaining to the comparative

anatomy and embryology of turtle and non‐turtle diapsid ribcages, focusing not only on the skeletal system but also on themuscular system, with the aim of achieving a better understandingof the origin of the turtle shell. First we describe the anatomy andembryonic development of rib cages in turtles and other diapsids,and then we identify the synapomorphies in the rib cagedevelopment of turtles. Lastly, with consideration to fossil taxa,we put forward an updated hypothesis for the evolution of theturtle shell, with intermediate stages bridging the gap between theturtle and the typical amniote trunks, in a more inclusive cladethan the turtles.

MATERIALS AND METHODSIn addition to conducting a literature survey, we examined theembryos of four turtle species, namely the Chinese soft‐shelledturtle, Pelodiscus sinensis (Cryptodira: Trionychoidea: Trionychi-dae), the red‐eared slider turtle, Trachemys scripta (Cryptodira:Testudinoidea: Emydidae), Reeve's pond turtle, Chinemys reevesii(Cryptodira: Testudinoidea: Geoemydidae), and the loggerhead seaturtle, Caretta caretta (Cryptodira: Chelonioidea), to confirm thesynapomorphies of the crown group turtles. For outgroupcomparison, embryos of the chicken, Gallus gallus (Archosauria:Aves), the American alligator, Alligator mississippiensis (Arch-osauria: Crocodilia), and the Madagascar ground gecko, Paroe-dura pictus (Lepidosauria: Squamata) were examined.We applied the embryonic stage tables of Tokita and Kuratani

(2001) for turtles (TK stage) with consultation with other papers(Yntema, '68; Kuratani, '99; Greenbaum, 2002), and the tables ofHamburger and Hamilton ('51) for the chicken (HH stage), thetables of Ferguson ('85) for the alligator (FG stage), and thetables of Noro et al. (2009) for the gecko (NR stage).

Figure 4. Anatomy of the rib cage in non‐turtles and in turtles. A: Transverse section of the rib cage in non‐turtle diapsids; (B), transversesection of the rib cage in turtles; (C), ventral view of the rib cage in the American alligator, Alligator mississippiensis; (D), ventral view of the ribcage (or the carapace) in the Chinese soft‐shell turtle, Pelodiscus sinensis. DI,M. diaphragmaticus; ICN, intercostal nerve; IEd, M. intercostalesexterni dorsales; RA, rami anteriores; Td, M. transversus dorsalis



We studied A. mississippiensis legally, by transportingembryonic samples with the permission of CITES (CertificateNo. 11US37892A/9), as well as by dissecting an adult specimenfrom the museum collection of the Kanagawa Prefectural Museumof Natural History, Odawara, Japan (KPM‐NFR000016).

RESULTS AND DISCUSSION

Axial Arrest of the Rib Cage—A Shared Feature of TurtlesThe rib cage of amniotes consists of vertebrae, ribs, and epaxialand hypaxial muscles. In non‐turtle amniotes, part of the ribsextends ventrally to connect with the sternum and surround theheart and lungs. However, rib morphology and the pattern of ribsegmentation in particular varies among taxa: in most mammals,the thoracic ribs are connected ventrally to the rib cartilages(Parker, 1868; Remane, '36); in birds, the ribs are composed of twopieces (vertebral and sternal), and the vertebral ribs articulatemovably with the sternal ribs (Parker, 1868; Remane, '36; Bellairsand Jenkin, '60); and in many lepidosaurs and crocodilians, thevertebral segment of each rib is connected ventrally to thecartilaginous intermediate segment, which is further articulatedwith the sternal segment of the rib (Günter, 1867; Parker, 1868;Remane, '36; Romer, '56; Hoffsteter and Gasc, '69). By contrast, inturtles, each rib consists of a single element, which is not curvedventrally.The muscles attached to the ribs of turtles are poorly developed

and highly specialized in comparison with those of otheramniotes. In most turtle species, the intercostal space is filledwith the bony costal plate, which is an extension of the ribs per se(Hirasawa et al., 2013), and correspondingly the intercostalmuscles are not developed. An exception among living turtles canbe found in Dermochelys, in which the intercostal space remainsopen in mature individuals (Nishi, '38). In this species, there is athin connective tissue spanning the intercostal space. Similarconnective tissue sheets are present in the Cheloniidae (Gasc, '81).With the exception of these connective tissues inDermochelys andthe Cheloniidae, the hypaxial muscles of turtles attach only to themedial surface of the rib cage and extend ventrally (Ogushi, '13;George and Shah, '54; Gans and Hughes, '67; Gaunt andGans, '69;Bramble and Hutchison, '81).In non‐turtle diapsids, the M. serratus ventralis, the M.

iliocostalis, and the hypaxial muscles attach to the rib shaft inthe thoracic region. Among these muscles, the M. serratusventralis arises from, and theM. iliocostalis inserts into, the lateralsurface of the rib shaft (Fürbringer, '02; Ghetie, '76; Frey, '88; Conget al., '98; Yasuda, 2002; Tsuihiji, 2007; Fig. 3). There are sharedtopographical features among the superficial layers of thehypaxial muscles (Figs. 3 and 4A). Specifically, the M. obliquusexternus arises from the lateral surface of the rib shaft or the fasciaoverlying the ribs, and theMm. intercostales externi (including theM. scalenus, the M. levatores costarum and the M. appendico-costalis of birds (Codd et al., 2005; Tsuihiji, 2007)) arise from the

cranial edge of the rib shaft and insert into the caudal edge of therib shaft and the ventral edge of the uncinate process(Maurer, 1896; Fürbringer, '02; Frey, '88; Murakami, '88; VandenBerge and Zweers, '93; Fig. 3). The above muscles are all locatedsuperficial to the main trunk of the intercostal nerve (Fig. 4A).By contrast with the muscles described above, comparison of

the deeper layers of hypaxial muscles in non‐turtle diapsids is notstraightforward in terms of the muscles' positions relative to theintercostal nerve. The Mm. intercostales interni (including the M.costosternalis of birds in Vanden Berge and Zweers, '93; Fig. 3)arise from the cranial edge and insert into the caudal edge of therib shaft. The Mm. intercostales interni usually span laterally tothe main trunk of the intercostal nerve (Murakami, '88), but adorsal (i.e., proximal) part of theMm. intercostales interni in birdsspans medially to the intercostal nerve (unpublished data fromdissection by Hirasawa; see also Maurer, 1896 and Nishi, '38 forthe classification of some dorsalMm. intercostales interni of othernon‐turtle diapsids). The M. transversus constitutes the deepestlayer of the hypaxial muscles, and it arises from themedial surfaceof the rib shaft, running medially to the main trunk of theintercostal nerve (de Wet et al., '67; Ghetie, '76; Murakami, '88;Yasuda, 2002; Fig. 3A,B). In birds, the M. costoseptalis (or M.costopulmonaris) also arises from the medial surface of the ribshaft, and inserts into the postpulmonary septum (de Wetet al., '67; Duncker, '79; Vanden Berge and Zweers, '93; Fig. 3C,D). The above comparison of the deeper hypaxial muscles amongnon‐turtle diapsids indicates that muscles running medially to themain trunk of the intercostal nerve attach to the medial surface ofthe rib shaft, except for its dorsal (proximal) part (Figs. 3 and 4A,C), although specific homologization remains uncertain (see alsoNishi, '38).The outgroup comparison with mammals substantiates the

conservation of deeper layers of hypaxial muscles attached to themedial surface of the rib shaft. In mammals, the M. transversusthoracis, the Mm. intercostales intimi (innermost intercostalmuscles) and the Mm. subcostales run medially to the intercostalnerve and attach to the medial surface of the rib shaft (Eisler, '12;Nishi, '38; Fujita, '63).Thus, the muscles attached to the medial surface of the ribs in

turtles are likely comparable to the transversus (i.e., deeper layer)muscles, because they both run medially to the intercostal nerve(Fig. 4B,D). It is conceivable that the trunk muscles of turtles differfrom those of other amniotes simply in the absence of the epaxialand superficial hypaxial muscles, which leads to the ribs of turtlesnot being covered with body wall muscles.Collectively, the extraordinary features seen in the rib cage of

turtles can be summarized as two unique anatomical features: thedorsally‐held ribs and the absence of the epaxial and superficialhypaxial muscles. In the embryonic development of turtles, theabove two apomorphic features become recognizable histologi-cally from the late pharyngula stage when the dermomyotomebegins to extend ventrally into the somatopleure (approximately


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TK stage 14 in P. sinensis; Tokita and Kuratani, 2001; Fig. 5A–D).During this period, the trunk of each turtle embryo exhibitsanother apomorphic feature on its flank, that is, the carapacialridge (CR, Fig. 5A–D; Burke, '89). The CR protrudes at the level justdorsal to the boundary between the axial part of the embryonicbody and the lateral body wall, and the dermis within the CR isderived from the ventrolateral portion of the dermomyotome(Nagashima et al., 2007). The CR initially develops along the flankbetween the fore‐ and hind limbs, and subsequently extendscraniodorsally and caudodorsally, thereby forming a “ring” thatsurrounds the carapacial primordium. The rib primordium ofturtles does not enter the body wall beyond the CR (Burke, '89;Gilbert et al., 2001; Nagashima et al., 2007), and keeps growinglaterally within the axial domain of the embryonic body. Thispattern of growth results in the inward folding of the lateral bodywall to encapsulate the shoulder girdle inside the rib cage(“Folding theory”; Nagashima et al., 2009).The postcranial musculoskeletal system of vertebrates consists

of primaxial and abaxial elements, which are distinguishable bytheir cell lineages, specifically whether somite‐derived cellscontribute to the connective tissue (primaxial) or not (abaxial)(Nowicki et al., 2003; Burke and Nowicki, 2003). Duringdevelopment, somite derivatives extensively invade the bodywall of vertebrates to pattern themselves in a rather cell‐autonomous fashion. The boundary between the primaxial andabaxial domains is termed the “lateral somitic frontier,” andreflects themodified boundary once found between the somite andlateral plate in younger embryos (Burke and Nowicki, 2003). As faras the skeletal components are concerned, therefore, the lateralsomitic frontier of turtles uniquely remains at the originalposition, namely at the proximal junction of the lateral body wallof the embryo. We previously termed this embryonic phenomenon

the “axial arrest” and suggested that it is the key to understandingturtle morphology (Nagashima et al., 2007). The axial arrest of theribs subsequently gives rise to a dissociation of the ribs and thehypaxial muscles distally, because the trunk muscles attach onlyto the medial surfaces of the ribs in turtles (Fig. 4B). The latterdevelopmental pattern gives an impression that the distal tip of therib primordium becomes protruded from the developing muscleplate at the level of the axial/lateral body wall junction ofthe turtle embryo. Although this pattern is less conspicuous inthe soft‐shelled species, P. sinensis, compared with the othercryptodire species (e.g., Gilbert et al., 2001), the pattern in P.sinensis is topographically identical to the patterns in the otherturtle species that we examined (Fig. 5A–D). Therefore, regardlessof the variety of adult shell morphologies, the axial arrest appearsto be a synapomorphy that characterizes turtle embryogenesis.According to analyses of mice and chickens, the distal

elongation of the ribs depends on the development of themyotomes (reviewed by Nagashima et al., 2012). For instance, inmice with deficiencies in myotome development, the ribs failedto extend distally (Braun et al., '92; Hasty et al., '93; Tremblayet al., '98), and cell‐lineage tracing experiments in chickensconfirmed that the lateral part of the sclerotome, which issandwiched between the adjoining myotomes, develops into thedistal part of the rib extending ventrally (Kato and Aoyama, '98;Huang et al., 2000; Olivera‐Martinez et al., 2000; Evans, 2003).Based on these findings, we expect that the axial arrest of the ribreflect a turtle‐specific developmental mode of the ventral part ofthe myotome.To date, myotome development has been compared most

frequently between the soft‐shelled turtle, P. sinensis, and thechicken. In the P. sinensis embryo, Pax3, a master regulatory genefor myogenic cells (Tajbakhsh et al., '97), is expressed by the cells

Figure 5. Axial arrest of the ribs in the embryonic development of turtles. A: Chinese soft‐shell turtle, Pelodiscus sinensis (Trionychoidea); (B)red‐eared slider turtle, Trachemys scripta (Testudinoidea: Emydidae); (C) Reeve's pond turtle, Chinemys reevesii (Testudinoidea:Geoemydidae); (D) loggerhead sea turtle, Caretta caretta (Chelonioidea).



of the dorsomedial and ventrolateral lips of the dermomyotome,and cells of the ventrolateral lip later become distributed ventrallyto form the Pax3‐positive ventrad myotome extension, as in thechicken embryo. However, the number of Pax3‐expressing cells inthe P. sinensis embryo is smaller than that in the chicken embryo(Kawashima‐Ohya et al., 2011). Also, in the P. sinensis embryo, thecells of the ventrad myotome extension express regulatory genesfunctioning in muscle differentiation (Myf5, MyoD, Myogenin,and MRF4) as in the embryos of other amniotes, but these cellstend to deviate from the segmentation pattern and are fewer innumber than those of the chicken embryo (Kawashima‐Ohyaet al., 2011). So, as a whole, the development of the myotomeswithin the lateral body wall of P. sinensis and other amniotes iscomparable in terms of gene expression, but the former is smallerin terms of the size of the cell population. In a histologicalcomparison of a wide range of taxa, it appears that there is ageneral difference between turtle and non‐turtle diapsid species interms of the size of the cell population of the myotome extendingventrad (Figs. 5A–D and 6A–C; see also Maurer, 1896 for Lacertaagilis). Thus, the above noted feature of muscle development inP. sinensis is widespread among turtles, and is tightly linked withthe axial arrest of the embryonic rib cage.The ventral part of each myotome, surrounded by the

somatopleure, develops into multiple layers of ventrolateralbody wall muscles in the chicken (Christ et al., '83; Murakami andNakamura, '91; Burke and Nowicki, 2003; Nowicki et al., 2003).The smaller cell population of myogenic cells observed in turtleembryos compared with non‐turtle diapsid embyos might relate tothe turtles possessing a single‐layer ventrolateral body wallmuscle, which is putatively comparable to the transversus musclesof non‐turtle diapsids (see above). In P. sinensis embryos,the ventral myogenic cells tend to lose the segmental pattern

(Nagashima et al., 2009; Kawashima‐Ohya et al., 2011), as doesthe transversus muscle primordium in non‐turtle diapsids(Maurer, 1896; Burke and Nowicki, 2003).

Searching for the Genetic Basis for the Axial Arrest of the Rib CageIt is natural to presume a developmental link between the axialarrest and the formation of the CR, since both take place at thesomite/lateral plate boundary (Figs. 5 and 7). Although the role ofthe CR in embryonic development remains to be resolved, theresults of previous experimental studies suggest that the CR isassociated with the marginal growth of ribs in the late phase ofdevelopment to promote folding of the body wall (Nagashimaet al., 2007, 2009). It is also possible that the craniodorsal part ofthe “ring” of CR (i.e., the cranial edge of the future carapace) isrelevant to the development of the rib cage, because turtles are theonly amniotes that show the brachial plexus position at anexceptional axial level relative to the cervico‐thoracic transitionof ribs (Hirasawa and Kuratani, 2013). So far, this deviation hasnot been explained by the cranio‐caudal pattern of the Hox codein P. sinensis (Ohya et al., 2005).Several studies have reported key developmental genes ex-

pressed in the CR (Loredo et al., 2001; Vincent et al., 2003; Kurakuet al., 2005; Moustakas, 2008; Wang et al., 2013). In this context,the histological similarity between the CR and the apicalectodermal ridge (AER) of the limb bud has drawn the attentionof researchers: the seemingly deviated growth of the turtle ribsmight be ascribed to the AER‐like patterning function of the CR(Burke, '89). In the development of the limb bud, Wnt/b‐cateninsignaling pathway from the limb ectoderm is necessary andsufficient to induce the AER, which is the main signaling centerfor limb outgrowth mediated by FGF signaling (reviewed byFernandez‐Teran and Ros, 2008; Tanaka, 2013). Thus, it is possible

Figure 6. Typical development of the ribs in amniotes. A: Chicken, Gallus gallus (Archosauria: Aves); (B) American alligator, Alligatormississippiensis (Archosauria: Crocodilia); (C) Madagascar ground gecko, Paroedura pictus (Lepidosauria: Squamata).


8 HIRASAWA ET AL.

that the CR was acquired by partial co‐option of the generegulatory network that functioned in the limb bud formation inturtle ancestors, as has been speculated previously (Kurakuet al., 2005; see also Capdevila and Belmonte, 2001 for theconcept). Several candidate‐gene approaches have identifiedapparently CR‐specific expression patterns of some regulatorygenes. For example, the expression ofMsx1 has been demonstratedin the CR of relatively young embryos of Emys orbicularis (at leastat Yntema stage 15; Vincent et al., 2003) and in the CR of laterstages of Trachemys embryos (around Yntema or Greenbaum stage18–19) epidermal expression ofMsx2 and Shh, and mesenchymalexpression of Fgf10,Gremlin, Bmp4, and Pax1, have been reported(Loredo et al., 2001; Moustakas, 2008). Expression of these genes,however, has not been detected in P. sinensis. Instead, by acomprehensive screening of CR‐specific genes based on atechnique called Megasort, Sp5, cellular retinoic acid–bindingprotein‐1 (Crabp‐1), adenomatous polyposis coli down‐regulated 1(Apcdd1), and lymphoid enhancer‐binding factor (Lef1) genes werefound to be expressed in the CR in P. sinensis (Kuraku et al., 2005).The transcription factor encoded by Lef1, together with its co‐factorb‐catenin, is involved in the regulation of the canonicalWntsignaling pathway (Novak and Dedhar, '99). After the develop-mental perturbation that eliminates the CR, these CR‐marker genescease to be transcribed (Kawashima‐Ohya et al., 2011). Further-more, translocation of b‐catenin into the nucleus was detected inthe CR ectoderm (Kuraku et al., 2005) and a recent genome‐wideanalysis identified the expression of Wnt5a in the CR (Wanget al., 2013). Additional evidence for a role of the Wnt/b‐cateninpathway in the CR growth is that ectopic expression of dominant‐negative Lef1, which lacks theb‐catenin‐binding domain, resultedin the partial arrest of carapacial growth at its margin, consistentwith the function of the CR as the marginal growth zone of thecarapacial primordium (Nagashima et al., 2007). These compre-hensive approaches have likely identified genes that are quite

relevant to the developmental mechanism of turtle‐specificmorphological patterns.The reason for the lack of similarities between CR gene

expression patterns in P. sinensi and Trachemys is not fullyunderstood. One plausible explanation would be that theseexpression patterns are species‐specific oddities. Exoskeletalbones, named peripherals, and scutes develop at the CR inhard‐shelled turtles, including T scripta and E. orbicularis, but notin soft‐shelled turtles, such as P. sinensis. Thus, gene expressionpatterns observed at the CR of hard‐shelled species may notnecessarily be involved in the patterning function of the CR, butrather in local histogenesis, which is not entirely relevant tothe turtle body plan. Because Shh and BMPs are known to playcentral roles in the differentiation of integumentary structures(Harris, 2002; McKinnell et al., 2004; Milinkovitch et al., 2013), itcan be assumed that they regulate the integumentary developmentin hard‐shelled turtles, but not in soft‐shelled turtles, which mayexplain the lack of these gene transcripts in the CR of P. sinensis.Since the carapace of P. sinensis has secondarily lost dermalintegmentary structures from its periphery (Fig. 2), the funda-mental role of the CR, and its possible relation to thedevelopmental basis for axial arrest, can be explored withoutobstruction by integumentary development in this species. Thus,P. sinensis has clear advantages over hard‐shelled turtles for thistype of study. However, further systematic comparative analysesbetween hard‐shelled and soft‐shelled turtles will be needed tounravel the genomic changes explaining the evolutionary originof the turtle shell in the last common ancestor.In summary, the above lines of evidence suggest that the

canonical Wnt signaling pathway might play a role in theformation of the CR as an upstream factor. Although Wnt5a is agood candidate triggering the Wnt pathway, it remains to beelucidated how this turtle‐specific regulation of Wnt5a wasachieved in the turtle ancestor.

Figure 7. The axial arrest of the rib cage and the carapacial ridge in the turtles. A: Axial arrest of the rib cage and the initial development ofthe carapacial ridge (CR); (B) subsequent lateral growth of the CR; (C) completion of the turtle shell.



New Look at the Fossil Record—Signs of the Axial ArrestAssuming the close phylogenetic relationship between the turtlesand the sauropterygians as a working hypothesis, a synapomor-phy is recognizable in their skeletons, that is, the absence of thesternum. It has long been an enigma for paleontologists that notrace of the sternum is found in sauropterygian fossils (Nichollsand Russell, '91).In the embryonic development of amniotes, the sternum

develops just distal to the distal ends of the developing ribsto become the ventral seam of the rib cage (Gladstone andWakeley, '32; Chen, '52a; Rodríguez‐Vázquez et al., 2013),although it develops even if the distal part of the ribs is removed(Chen, '52b, '53; Braun et al., '92) and is considered to be a pectoralgirdle element rather than an axial element (Seno, '61; Valaseket al., 2011). Developmental perturbations of the distal growth ofthe ribs often accompany the absence or deformation of thesternum, suggesting that the distal ribs and the sternum aredevelopmentally coupled with each other (as a module), althoughthese structures are derived from different mesodermal celllineages. Assuming that this coupling is shared among amniotes,the axial arrest would lead to the loss of sternum in addition to theloss of distal ribs, as seen in the turtle. The absence of the sternumin the turtles and sauropterygians, therefore, appears to be at leastpartly related to the axial arrest of the embryonic rib cage (Fig. 7B).This inference is consistent with the fact thatmany sauropterygianspecies had laterally projected ribs and a shoulder girdle locatedventral to the rib cage (Huene, '36; Nicholls and Russell, '91;O'Keefe et al., 2011; Hirasawa et al., 2013). Thus, the loss of thesternum (axial arrest of the ribs) is likely a synapomorphy for theclade that includes sauropterygians and turtles, and this eventappears to have taken place possibly only once in the commonancestor of these lineages after the split from the crown grouparchosaurs (Fig. 1A, arrow “a”). From the fossil record, the latterdivergence dates back to at least�245 million years ago (Anisian,or early Middle Triassic; Rieppel, 2000; Liu et al., 2011).The axial, endoskeletal part of the turtle carapace is surrounded

marginally by the peripherals, which connect ventrally to theexoskeletal bones that form the plastron (Fig. 2D). In contrast, innon‐turtles, the clavicular girdle and the gastralia are theexoskeleton homologs of the bony elements of the plastron,and are separated by the ventral part of the rib cage (e.g., thesternum). Among living amniotes, the widespread distribution ofthe exoskeleton (plastron) on the ventral side of the trunk is seenonly in turtles, and is likely linked to the axial arrest of theembryonic ribs. In many sauropterygian species, well‐developedgastralia were distributed cranially to reach to the pectoral girdle,and they filled the space where the sternum is distributedin most amniotes (Smith and Vincent, 2010; Vincent, 2011).This morphological feature is also reminiscent of the ventralexoskeleton of turtles.Finally, it is worth noting that the stem turtle Odontochelys

possessed a large plastron relative to its rib length (Fig. 8; Li

et al., 2008), indicating that the width of the plastron exceededthat of the carapace. It is conceivable that evolution of theplastron, reflecting the axial arrest, preceded evolution of thesolid structure of the carapace. The evolutionary origin ofthe peripherals remains unclear, and three possibilities shouldbe investigated: evolution from the osteoderm, a dorsal invasionof the lateral part of the plastron, or an innovation.The above scenario for turtle evolution is well in accordance

with recent genome‐wide analyses. Molecular clock analysis ofmore than 1000 genes (Wang et al., 2013) estimates the time ofdivergence of turtles from archosaurs to be 268–248 million yearsago (Late Permian to Early Triassic). This estimate is very closeto the geologic age of the early evolution of above fossil taxa,

Figure 8. Stem turtle Odontochelys semitestacea from the UpperTriassic Falang Formation, Guizhou Province, China. Paratypespecimen (IVPP V 13240) in ventral view.


10 HIRASAWA ET AL.

although whether the Late Permian Eunotosaurus diverged fromthe turtle clade (Lyson et al., 2013a,b) or not (parareptiles,outgroup of the Neodiapsida; Tsuji et al., 2012) is an intriguing andimportant question that is still to be resolved (reviewed byCarroll, 2013). Notably, this divergence does not automaticallyimply the acquisition of the turtle body plan, but rather the loss ofsternum and the axial arrest of the ribs in the common ancestor ofturtles and sauropterygians. This hypothesis, which assumes theearly origin of the axial arrest, is consistent with the step‐wiseevolution of the turtle body plan (Nagashima et al., 2009; reviewedby Kuratani et al., 2011). Most probably, the marginal growth ofthe carapacial primordia, leading to the folding of the lateral bodywall and encapsulation of the shoulder girdle, relies primarily onthe function of the CR that was acquired later than theevolutionary origin of the axial arrest. We propose that thisdevelopmental change had not yet occurred in Odontochelys,which had a complete plastron and an incomplete carapace, andwould occur later in the turtle lineage to produce themorphologically‐complete turtles we see today.Note added in proof: Regarding our discussion about the

differences on gene expression patterns reported so far betweensoft‐shelled and hard‐shelled turtles, during the proof edition ofthis manuscript an article by Nagashima H, Shibata M, TaniguchiM, et al. (2014) has been published. Nagashima et al. show theexpression of Wnt5a and APCDD1 in the CR of a hard‐shelledturtle (Trachemys scripta), therefore corroborating that the Wntsignaling pathway is conserved in the turtle lineage.

ACKNOWLEDGMENTSWe thank Ruth Elsey (Rockefeller Wildlife Refuge, USA) and NeilShubin (University of Chicago, USA) for support in samplingalligator embryos; the Animal Resource Unit of the Center forDevelopmental Biology, RIKEN, Japan, for the gift of geckoembryos; Zhonghe Zhou, and Chun Li (Institute of VertebratePaleontology and Paleoanthropology, China) for access to thefossil specimens; Sou Taru (Kanagawa Prefectural Museum ofNatural History, Odawara, Japan) for support in dissectingalligator adult specimens; and Hiroshi Nagashima (NiigataUniversity, Japan) and Naoki Irie (University of Tokyo, Japan)for discussions. We also thank the two anonymous reviewers forcomments that improved the manuscript.

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14 HIRASAWA ET AL.

the evolutionary origin of the turtle shell and its dependence on...

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