The Development and Evolution of the Turtle Body Plan

AMER. ZOOL., 31:616-627 (1991)

The Development and Evolution of the Turtle Body Plan:Inferring Intrinsic Aspects of the Evolutionary

Process from Experimental Embryology1

A N N CAMPBELL BURKE2

Museum of Comparative Zoology, Harvard University,Cambridge, Massachusetts 02138

SYNOPSIS. The body plan of turtles is unique among tetrapods in the presence of the shell.The structure of the carapace involves a unique relationship between the axial and theappendicular skeletons. A common developmental mechanism, an epithelial-mesenchymalinteraction, has been identified in the early stages of carapace development by means ofbasic histological and immunofluorescence techniques. By analogy to other structures ini-tiated by epithelial-mesenchymal interactions, it is hypothesized that carapace developmentis dependent on this interaction in the body wall. Surgical perturbations were designed totest the causal connection between the epithelial-mesenchymal interaction in the body walland the unusual placement of the ribs in turtles. By comparison to data available on bodywall formation in avian embryos, these experiments also shed light on the segregation ofsomitic and lateral plate cell populations and the embryonic origin of the scapula in turtles.

This study specifically addresses the ontogeny of a unique tetrapod body plan. The onto-genetic information can be used to make inferences about the phytogeny of this body planand how it could have evolved from the more typical primitive tetrapod. On a more generallevel this study explores the potential role of common developmental mechanisms in thegeneration of evolutionary novelties, and the developmental incongruities between homol-ogous skeletal elements in different groups of tetrapods.

INTRODUCTION

The turtle body plan represents a classicproblem of both ontogeny and phylogeny.The anatomy of the shell, composed of adorsal carapace and ventral plastron, isunique among tetrapods. It is the basal syn-apomorphy for the order Chelonia and themonophyly of this group has never beenquestioned.

The nature of the carapace and plastronhas intrigued morphologists for hundreds ofyears. The plastron, or lower shell, is com-posed entirely of dermal bones, and will notbe discussed here. The carapace is a com-posite of the endochondral axial skeleton(the thoracic vertebrae and ribs) and a spe-cialized dermis that overlaps and surroundsthem. In many forms, this dermis ossifiesto a greater or lesser extent in a character-istic bony mosaic (Owen, 1849; Zangerl,1969).

1 From the Symposium on Experimental Approachesto the Analysis of Form and Function presented at theCentennial Meeting of the American Society of Zool-ogists, 27-30 December 1989, at Boston, Massachu-setts.

2 Current address: Edgerton Research Lab, NewEngland Aquarium, Central Wharf, Boston, MA 02110.

An association of dermal bones and endo-skeletal elements is typical in the cranialskeleton of vertebrates, but very rarepostcranially. Even so, this associationwould not be so remarkable if it did not alsoalter the position of the ribs relative to thelimbs and girdles. The dermal carapaceoverlaps the pectoral and pelvic girdlesanteriorly, posteriorly and laterally. The ribs,because of their close association with thisdermis, maintain a dorsal position and thegirdle elements are situated ventrally, deepto the axial elements. This is in strong con-trast to other tetrapods (Fig. 1).

How novel morphologies are produced isessentially an evolutionary question. How-ever, ontogeny generates the variation wesee in phylogeny and is thus an intrinsicaspect of the evolutionary process. Sinceevery change in phenotype is the result ofsome change in developmental pattern, thestudy of ontogeny can potentially tell usmore about the evolution of a particularmorphology than the adult morphologyitself. A broad comparative approach is cru-cial for such evolutionary studies. To date,much of the developmental data in the lit-erature has been gathered from a limitednumber of organisms. Reptiles are under-

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TURTLE DEVELOPMENT AND EVOLUTION 617

represented for numerous reasons, not leastof which are the practical difficultiesinvolved in working on animals with sea-sonal reproduction and relatively smallnumbers of eggs. Many of these difficultiescan be overcome, however, and the datagenerated have added greatly to our knowl-edge of both process and pattern in verte-brate evolution (Billett et al., 1985).

In this review I describe how experimen-tal techniques have contributed to ourknowledge of turtle development, and howthis knowledge can be used to understandthe evolution of novel forms. I will also pointout how experimental data from two widelydivergent tetrapod embryos, turtles andbirds, can bring many of our implicitassumptions about homology into questionwhile it expands our knowledge of varia-tion.

HISTORICAL OVERVIEW

The known history of the order Cheloniabegins abruptly in the Triassic with the fos-sil Proganochelys. This animal has the highlyderived trunk morphology by which we rec-ognize the order. Both pre- and post-Dar-winian morphologists have sought affinitiesor ancestors for the Chelonia among variousfossil groups, including the plesiosaurs(Owen, 1849; Baur, 1887), the placodonts(Jaekel, 1902; Broom, 1924), the pariesaurs(Gregory, 1946), and even the labyrintho-dont Gerrothorax (Vallen, 1942).

Because of the extreme morphologicalleap between turtles and other tetrapods,much of the conjecture on turtle ancestryhas resorted to the construction of hypo-thetical taxa. The hypothetical ancestor isoften constructed in light of assumptionsabout the selective advantage of the shell asa protective adaptation. Cope (1871) sug-gested that the leatherback, Dermochelys, isthe lone survivor of the earliest turtles, the"Atheca," and that all other turtles, the"Thecophora," are more recent offshootswhose excessive ossification can beexplained by the rigors of the terrestrialenvironment. This distinction influencedideas of turtle relationships for many years(Gafthey, 1984).

Deraniyagala (1930), again using Der-mochelys as representative of the primitive

NON-CHELONIAN CHELONIAN

j^rTTgj Girdle elements

m ^ Ribs

FIG. 1. Schematic diagram illustrating the differentrelationships between the axial elements and the pec-toral girdle in chelonian and non-chelonian tetrapods.Reprinted with permission from Burke (19896).

condition, proposed the taxon "Saurotes-tudinata." He imagined the carapace andplastron to have evolved from the habit ofhunching the shoulders around the head androlling up "armadillo fashion" for protec-tion. This habit, he felt, led to the migrationof the scapula over and around the anteriorribs into the chelonian position.

Eunotosaurus africanus Seeley from themiddle Permian of South Africa was pre-sented by D. M. S. Watson (1914) as a"missing link" between the Chelonia andmore primitive cotylosaurs. This animal isknown from only five specimens with littlewell-preserved cranial material. The singleheaded thoracic ribs are expanded alongtheir shafts into broad plates that make con-tact with one another. Watson consideredthis character of the ribs to be intermediatebetween normal tetrapods and the carapaceof turtles, and that Eunotosaurus closelyapproaches a hypothetical chelonian ances-tor he called "Archichelone." He proposedthat the pectoral girdle had migrated pos-teriorly in "Archichelone," under an alreadywell-developed shell. Many authors adoptedEunotosaurus as a representative of the che-lonian line (Ruckes, 1929; Deraniyagala,1930; Gregory, 1946). No dermal bones areassociated with the axial skeleton, however,and the pectoral girdle takes the standardtetrapod position, external to the ribs. InRomer's words, "the inclusion of Eunoto-saurus in the Chelonia does violence to anydefinition of that group" (1956, p. 517).Eunotosaurus has now been placed on itsown as an order incertae sedis within thesubclass Anapsida (Carroll, 1988).

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618 ANN CAMPBELL BURKE

Proganochelys has a full carapace andplastron but its cranial anatomy links it toEucaptorhinus, a Permian captorhino-morph (Gaffney and McKenna, 1979; Gaff-ney and Meeker, 1983). The captorhinidshave been identified as the sister group ofturtles, indicating the chelonian formevolved from a tetrapod with typical (non-chelonian) postcranial anatomy. No inter-mediate morphologies are represented in thefossil record between Proganochelys and theCaptorhinomorpha. It is, in fact, very dif-ficult to imagine any functional intermedi-ates, and the evolutionary history of theturtle body plan has remained relativelyobscure.

Having settled on an ancestor, hypothet-ical or fossil, or a sister group, few authorshave been willing to tackle what I see as themost crucial and interesting aspect of che-lonian evolution—the aberrant positioningof the ribs and pectoral girdle. Because oftheir highly derived trunk morphology, Car-roll (1988) comments that turtles might wellbe placed in their own subclass of the Amni-ota.

MORPHOGENESIS OF THE CARAPACE

A good deal of detailed descriptive workhas been done on turtle embryology,especially in the 19th century (e.g., Agassiz,1857; Rathke, 1848; see review by Ewert,1985), but little that investigated the unusualrelationship between the ribs and the pec-toral girdle. An important exception is thework of Ruckes (1929), who examined thestages of carapace formation in embryos ofa wide range of turtles. His observations ofhistological material and embryo whole-mounts led him to conclude that two relatedphenomena are responsible for the posi-tioning of the ribs relative to the girdles.First, an exaggerated lateral growth of thedorsal dermis greatly exceeds dorso-ventralgrowth and causes the apparent flatteningof the body. Second, and most important,the rib primordia are "ensnared" within thisspecialized dermis. This later phenomenon,Ruckes felt, was responsible for the "deflec-tion" of the ribs resulting in their positiondorsal to the limb girdles. He also disagreedwith Watson's idea about the migration ofthe pectoral girdle. Based on his observa-

tions the girdles form in situ at the levelof the eighth cervical vertebra and do notmigrate posteriorly along the axis from theposition in which they first appear.

In addition to Ruckes' work, turtles havebeen the subject of other developmentalstudies (see Ewert, 1985, for review), someof which have provided more detail on theregionalization of somitic derivatives alongthe axis. For example, Vasse (1973, 1974,1977) studied early limb development inembryos of Emys orbicularis and Testudograeca. His studies localized the forelimbbud at the level of somites 6-13 in boththese species. Yntema worked extensivelywith turtle embryos and performed a widevariety of experiments on Chelydra serpen-tina and other species. He created a seriesof normal stages for Chelydra (1968) andpublished a detailed protocol for collectingand working with their eggs (1964). Mostpertinent to the subject of this paper werehis somite extirpations (Yntema, 1970) thatconfirmed the somitic origin of the ribs anddermal carapace in Chelydra, as well as thesomite level of their origin.

I have reinvestigated the problem of car-apace formation and scapular position usingtechniques of immunohistochemistry as wellas classical surgical perturbations. Thesetechniques have identified one develop-mental mechanism of carapace outgrowthand brought the problem of the scapula intosharper focus.

Normal developmentThe species used in this study was Chel-

ydra serpentina, the common snappingturtle. This species is hypothesized to be themost primitive living member of the sub-order Cryptodira (Gaffney and Meylan,1988). A single species study is admittedlytypological. The chelonian body plan, how-ever, defined by the relationship betweenthe axial and appendicular elements, isessentially uniform in all turtles. This uni-formity legitimizes generalization for theorder regardless of the variation in the extentof ossification, size and shape of the shell.Approximately 200 eggs, representing 17different clutches from at least 14 differentChelydra females, were used in variousaspects of this study. Observations were also

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FIG. 2. a) Mid-trunk cross section of an Yntema (1968) stage 15 Chelydra serpentina embryo showing thecarapacial ridge (arrow). Bar = 300 /im. b) Higher magnification of the carapacial ridge. Bar = 28 iim. Reprintedwith permission from Burke (19896).

made on histological material of Chrysemyspicta and Trionyx sp.

For wider comparative purposes I use thechick embryo as a representative non-che-lonian species. Obviously, this choice is forpractical rather than phylogenetic reasons.The chick has been the experimental ver-tebrate of choice for decades and a great dealof developmental data are available in theliterature. The interpretive problems thatarise as a result of the phylogenetic distancebetween birds and turtles is discussed in thefinal section of this paper.

The Chelydra embryo at Yntema's (1968)stage 14 resembles the chick embryo atHamburger and Hamilton's (1951) stage 24;it is a typical tetrapod embryo at the "pha-ryngula" stage of development (Ballard,1981). There is nothing about its morphol-ogy that would reveal its identity as a turtle.The first sign of departure from typical tet-

rapod development is the appearance of aridge along the flank dorsal to the limbs. Insubsequent stages this ridge extends ante-riorly and posteriorly to become continuousover the base of the tail and cervical regionto form the complete margin of the cara-pace. A cross-sectional view of this cara-pacial ridge reveals that it is formed by athickening in the ectoderm underlain by acondensation of mesenchymal mesoderm(Fig. 2). This morphology is typical of areasof epithelial-mesenchymal interactions.

Epithelial-mesenchymal interactions arevery common inductive interactions in ver-tebrate development. A great deal of researchhas addressed the mechanisms of theseinductions (see papers in Sawyer and Fal-lon, 1983). The data generated by thesestudies provide a wealth of information onthe temporal and spatial distribution of var-ious molecules during the development of

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various structures (cf. Edelman, 1986).These data can then be used as charactersto identify epithelial-mesenchymal inter-actions in other systems.

I have shown that the distribution of two"morphogenetic molecules," fibronectin andthe neural cell adhesion molecule (N-CAM),in the carapacial ridge is similar to the dis-tribution of these molecules in the equiva-lent stages of feather formation and earlylimb outgrowth, both well known sites ofepithelial-mesenchymal interactions (Burke,19896). Based on the histological mor-phology and this molecular data, I haveidentified the carapacial ridge as the site ofan epithelial-mesenchymal interaction.Further, I have hypothesized that this inter-action in the early body wall influences riboutgrowth and positioning, and is the causalfactor in what Ruckes called the "ensnare-ment" of the ribs. This mechanism is seenas analogous to the interactions that initiateearly limb bud outgrowth and morphogen-esis of many integumental features (Burke,1989a, b).

EXPERIMENTAL MANIPULATIONS

The hypothesis that the carapacial ridgeis the site of an epithelial-mesenchymalinteraction invites experimental investiga-tion. The certain identification of an induc-tive interaction requires experimental ver-ification to prove its causal role in themorphogenesis of a structure.

In order to further investigate thishypothesis, a series of experiments weredesigned to determine the inductive role ofthe carapacial ridge and the migratorybehavior of somitic cell populations duringbody wall formation. Working from ananalogy with other structures initiatedby epithelial-mesenchymal interactions,especially the limb, two surgical perturba-tions were used to test the causal role of thecarapacial ridge in rib positioning. The sur-geries were performed in ovo, the eggs hav-ing been windowed within 12 hours of ovi-position (see Yntema, 1964, and Burke,19896). The embryos were stained with 0.2%neutral red to enhance visualization ofstructure, and manipulated with sharpenedtungsten needles.

Testing the inductive role of thecarapacial ridge (CR)

In the first set of experiments, the ecto-derm and immediately subjacent meso-derm in the area of the incipient carpacialridge (CR) were surgically removed onembryos from stage 13 through stage 16 (Fig.3a). These stages bracket the period betweenthe first appearance of the CR and its matu-rity. No attempt was made to completelyseparate the ectoderm from the mesoderm.These experiments test the effect of removalof the CR on the formation of the dermalcarapace and the placement of the ribs withinit.

The results from these surgeries demon-strate a remarkable degree of regenerationand regulation on the part of the embryo.However, several trends can be noted.Embryos perturbed at stage 14 showed ahigher percentage (93%) of effects to the car-apace than embryos perturbed at earlier(36%) or later stages (26%). Embryos thatfail to regenerate show definite gaps in thedermal margin of the carapace. When thegaps correspond to levels of normal ribplacement, the ribs are deflected and crowdwith a neighboring rib into the next avail-able marginal scute (Fig. 4).

The source of embryonic cells that pro-duce the ribs of tetrapods has been exten-sively investigated in the chick. Sweeney andWatterson (1969), using tantalum foil bar-riers to block the migration of somitic cellsinto the lateral plate mesoderm, demon-strated that the vertebral and sternal ribs ofchicks are somitic in origin. The quail-chickchimera method of heterospecific trans-plantations has verified these conclusionsand showed that the lateral and ventral der-mis, in which the distal ends of the ribsdifferentiate, is formed exclusively from lat-eral plate mesoderm (Gumpel-Pinot, 1984).Yntema's (1970) somite-removal experi-ments on embryos of Chelydra demonstratethat chelonian ribs and dorsal dermis alsoarise from somitic mesoderm.

I placed barriers between the somitic andlateral plate mesoderm in Chelydra embryoswith 6 to 19 somites. Tantalum foil, 0.005mm in thickness, was cut into rectangularpieces measuring approximately 0.5 by 1.0

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a.

NtC

FIG. 3. Three surgical manipulations discussed in the text: a) Tissue removed in carapacial ridge extirpations,b) Placement of tantalum foil barriers. Foil thickness = 0.005 mm. c) Extirpation of somitic mesoderm. Drawingsare not to scale.

mm, and folded along the long axis to forman "L." One flange of the "L" was insertedinto an incision made lateral to the somites,and the other lay medially over the ecto-derm (Fig. 3b). These experiments have twopurposes. First, placing a physical barrier atthe border between somitic and lateral plateshould interfere with or inhibit the forma-tion of the carapacial ridge and provideadditional data on the relationship betweenthe ridge and the ribs. Secondly, barrierswill evaluate the extent of migration of som-itic cells into the lateral plate, and the roleof these somitic cells in ventral body wallformation.

Barrier placement is highly disruptive todevelopment and survivorship is low (38%).Of the survivors, slightly more than halfrejected the barrier and are normal or showonly superficial scute disruptions. The restof the embryos (44%) retained the barrierand show disruption of the body wall. Inthese cases, the carapace margin is discon-tinuous and entire quadrants of the dermalcarapace are absent. The ribs associated with

these gaps can be seen to interdigitate withthe bones of the plastron, which are of der-mal origin. Even in the most extreme casesof carapace disruption, all of the bones ofthe plastron are present.

These results show that development ofthe carapacial ridge can be inhibited result-ing in severely disrupted axial morphology.Further, they show that while somitic cellsare necessary for the normal developmentof the carapace, the plastron has some degreeof developmental independence (Burke,1987). The interdigitation of the ribs withthe plastral elements indicates that the ribshave entered the lateral plate as they do inavian embryos.

The following conclusions have beendrawn from the results of these two exper-imental perturbations: (1) The carpacialridge is the site of an epithelial-mesenchy-mal interaction that is responsible for theoutgrowth of the carapacial dermis. (2) Thenormal placement of the ribs is dependenton the integrity of the carapacial ridge (Burke1989c). This system is likely analogous to

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FIG. 4. Left rear quadrant of the carapace of a spec-men in which the carapacial ridge was removed atYntema's (1968) stage 14. Marginal scute 7 is reducedand 8 is missing; m = marginal.

the role of the apical ectodermal ridge innormal limb development (cf. Saunders,1948).

Homology of the scapulaA developmental segregation parallels the

functional distinction between the axial andappendicular skeletons of tetrapods. Theaxial elements arise from somitic meso-derm, the appendicular elements from lat-eral plate mesoderm. Recent data generatedby experimental embryologists, however,have shown that in at least one group ofhigher tetrapods the scapula does not con-form to this neat developmental segregationof functional skeletal systems. Chevallier(1977), using quail-chick chimeras, foundthe chick scapula to be formed at least inpart by somitic cells. Grafting blocks of quailsomites into the same level of a chick hostresulted in quail cells in the scapular carti-lage in segmental fashion, more anteriorsomites contributing to more proximal

FIG. 5. a) Illustration showing anterior view of a spec-imen in which the somitic mesoderm at the level ofsomites 8-12 was removed from the right side of theembryo at Yntema's (1968) stage 6. The head has beenremoved for clarity, the neck hangs ventrally. b) Pho-tograph of scapula and cervical vertebrae of the spec-imen drawn in (a); sc = scapula; cv = cervical vertebrae.

regions of the scapula. Therefore this seriesof somites (15—24) contributes both to theaxial and appendicular skeletons. The samemethods demonstrate that the rest of thelimb skeleton, the coracoids, the entire pel-vis as well as the distal limb bones, all arisefrom lateral plate mesoderm.

The cervical vertebrae in primitive tet-rapods are equipped with ribs. As axial ele-ments (the serial homologues of thoracicribs) they arise from somitic mesoderm.Cervical ribs of amniotes are reduced orabsent, presumably reflecting specializa-tions for mobility. The fossil Proganochelyshas quite well-developed cervical ribs foran amniote though they are small relativeto the ribs associated with the carapace. Inall living turtles cervical ribs are present onlyas occasional, atavistic rudiments (Wil-

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TURTLE DEVELOPMENT AND EVOLUTION

a. b .

CHICK SOMITE LEVELS TURTLE SOMITE LEVELS

623

WING&SHOULDER

MUSCLES

SOMITE DERIVATIVES SOMITE DERIVATIVES

CERVICALVERTEBRAE

I THORACICRIBS

o•

CARAPACE

VERTEBRALBODIES

FIG. 6. a) Schematic fate map of the skeletal somitic derivatives in the chick compiled from the literature, b)Schematic fate map of the somitic derivatives in Chelydra serpentina.

liams, 1959). This latter case is also the sit-uation seen in birds.

A fate map compiled from the literatureon chick development shows that the cer-vical-thoracic transition lies at somites 18-19 (Fig. 6; see Gumpel-Pinot, 1984, forreview of mapping). The first trunk somitein chicks is somite 5, and chicks have 14cervical vertebrae. According to Chevallier(1977), the scapula arises from a series ofsomites that overlap the cervical-thoracictransition. Somites 19-24, contributing cellsto the middle and distal shaft of the scapularblade, also provide cells that form thoracicvertebrae and ribs. This requires a degreeof cell segregation and skeletal pattern for-mation not usually expected of trunksomites.

Given the unusual position of the scapulain turtles, the embryonic origin of the scap-ula vis-d-vis the thoracic ribs is critical to

understanding the evolution of cheloniananatomy. To this end, I performed somiteremoval experiments on embryos of Chel-ydra serpentina. The levels chosen for extir-pation were based on the results of Yntema(1970). His experiments demonstrated thatthe thoracic vertebrae and ribs arise fromsomites 13-21, and the dermal carapacefrom somites 12-21. He reported no dam-age to the scapula or cervical vertebrae, butremoved no somites anterior to somite 12.Yntema's data place the cervical-thoracictransition at somites 12-13. I removedsomites 8-12 and 12-16 in an attempt tobracket the cervical-thoracic boundary (Fig.3c).

The number of embryos that survived thesurgery without total regeneration are toofew to determine with certainty the effectsof somite removal (57.7% survivorship, 34%of these with skeletal effects). The only spec-

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imens showing disruption of the scapula arethose perturbed at somite levels 8-12. Theextent of the effect varies from minor mal-formations of the shaft to total absence ofa recognizable scapula. In 5 of 7 cases, thedisruption is limited to the dorsal scapularshaft; the acromion and glenoid are rela-tively normal. Scapular defects are alwaysassociated with malformations of the car-tilagenous arches of cervical vertebrae 4-7,and in three specimens the distal forking ofthe scapula makes contact with the mal-formed arches of cervicals 5-7 (Fig. 5a, b).Three embryos have cervical disruptionsand normal scapulae. In every case withscapular and cervical disruption, the ante-rior thoracic vertebrae and ribs are com-pletely normal. The coracoid is also normal,though in two cases the distal limb showsabnormalities. The contralateral limb showsadditional digits in one specimen (illus-trated in Fig. 5a).

This mixture of results does not allow aninterpretation free from the effect of artifact,but a working model can be proposed thatwill be further tested over future nesting sea-sons. In the majority of cases (71%) the limbskeleton, including the coracoid, is normal,indicating that the lateral plate was unaf-fected by the surgery. The defects in thecervical vertebrae indicate successful per-turbation of the somitic mesoderm. Thecorrelation of scapular and cervical defects,and the close association of scapular andcervical cartilages in the affected specimens(Fig. 5), allow the tentative conclusion thatthe chelonian scapula is formed in part bysomitic cells from the levels of somites8-12 (Fig. 6b). The somitic contributionappears to be limited to the suprascapularcartilage and the dorsal extent of the scap-ular shaft.

It is interesting to note that the "scapular"somites (8-12) form cervical vertebrae 4-7.In normal development the scapula differ-entiates in situ at the level of the eighthcervical. This indicates a posterior migra-tion of scapular cells along the axis fromtheir point of origin to their site of differ-entiation. This ontogenetic migration ech-oes the phylogenetic migration of the scap-ula proposed by Watson (1914) that wasundetected by Ruckes (1929) in his analysisof static developmental stages.

EVOLUTIONARY INFERENCES

The experimental data reviewed abovegive insight into the developmental patternsresponsible for the ontogeny of the turtlebody plan. In conjunction with experimen-tal data generated from non-chelonianembryos, inferences can be made about thedevelopmental changes involved in the evo-lution of the chelonian morphology from anon-chelonian ancestor. Furthermore, thesecomparative data increase our knowledgeof the nature of morphological variation,and the role of development in generatingthis variation.

The primary difference between chelo-nian and non-chelonian body wall forma-tion is the migratory route followed by thepresumptive costal cells of the somiticmesoderm. I have proposed that the epi-thelial-mesenchymal interaction in the bodywall, the carapacial ridge, has a causal con-nection to this migration and therefore toplacement of the ribs (Burke, 19896). Basedon this interpretation of the ontogeny of thechelonian trunk, I further proposed that thisnovel interaction in the body wall wasinstrumental in the evolutionary transitionfrom the typical tetrapod arrangement ofthe trunk to that of the chelonian. I hypoth-esize that the origin of the carapace involvedthe progressive elaboration of a primitiveintegumental interaction in the proto-che-lonian.

The differentiation of the integument isgenerally a late stage phenomenon, occur-ring well after the morphogenesis of theskeleton. The precocious initiation of aninductive interaction would change thedevelopmental context of its influence. Inessence, the inductive effect would act upona smaller embryo and could influence deeplayers of as yet undifferentiated meso-derm—in this case the costal sclerotome.

The detailed mechanisms of epithelial-mesenchymal interactions remain fairlymysterious, but their wide distribution invertebrate development indicates that theyare a primitive phenomenon. Their initialinvariant morphology and subsequent gen-erative versatility indicate they are sourcesof developmental potential and, as such,vehicles for evolutionary novelties (Mad-erson, 1983). A temporal shift of an origi-

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nally integumental interaction may well haverapidly produced the highly modified che-lonian body plan from a non-chelonianancestor, and set the stage for all the sub-sequent modifications in turtle evolution.

This theory is analogous both conceptu-ally and mechanistically to the lateral finfold theory of the evolution of the pairedappendages (Thacher, 1877;Balfour, 1881).This theory, based on observations ofmedian fin development, proposes that anoriginally integumental lateral fin fold waselaborated by the invasion of somitic mus-cle buds that provoked the in situ formationof endochondral skeletal structures. It seemsreasonable to propose that an epithelial-mesenchymal interaction was involved inthe formation of the early integumental finfold, and evolved in concert with theincreasing complexity of the limb.

In a "systems" approach to vertebratemorphology, an implicit assumption per-sists that the functional segregation of axialand appendicular systems is paralleled bysome degree of developmental segregation.This is also implicitly if not explicitlyassumed in the theory of the origin of thepaired appendages mentioned above. Thegirdle elements are seen to arise as exten-sions of the primary radials into the bodywall to anchor the fin (see Goodrich, 1906,and Jarvik, 1965, for discussion). The scap-ula in chicks, however, is known to arisefrom somitic mesoderm, in contrast to therest of the appendicular skeleton. The somiteremoval experiments on turtles outlinedabove indicate that the chelonian scapulaalso has a somitic component. (The somiticnature of the scapula is very interesting con-sidering Owen's [1849] interpretation of itsidentity. He described the scapula as thehomologue of a haemal arch of an arche-typal occipital vertebra, in other words, anaxial element. Thus, to his own satisfaction,he explained the positioning of the scapulain turtles by its axial nature.)

In contrast to the chick embryo, thesomites that contribute to the turtle scapulado not overlap with those that form the tho-racic ribs, but arise from cervical levels only(Fig. 6). The most straightforward expla-nation for this fact requires only that fewersomites are involved in the formation of thescapula in the turtle than in the chick. The

chick and turtle scapulae are thereforedevelopmentally incongruent. While fewwould argue that they are not homologouselements, clearly the difference betweenthem—a matter of somites—reflects theirdivergent phylogenetic history.

Knowledge of the primitive developmen-tal pattern of the tetrapod scapula is nec-essary in order to determine the evolution-ary polarity of this developmental character.This information would clarify the possibleintermediate morphologies involved in thetransition of the chelonian from the non-chelonian body plan. For example, if onewere to accept that the situation in chicksis primitive, the proto-chelonian scapulawould have been formed from a series ofsomites which, as in the chick, overlap thecervical-thoracic transition. One couldhypothesize that a decoupling of costal andscapular somites may have been one of thedevelopmental modifications leading to thenew position of the scapula in turtles. Thereis no reason to suppose, however, that chicksrepresent a primitive tetrapod pattern ofdevelopment. It is more likely that the sit-uation in chicks is a uniquely derived char-acter of birds related to the evolution offlight. The participation of the somiticmesoderm, with the capacity to behave asa mosaic, should allow for developmentaland therefore evolutionary plasticity in thepectoral girdle.

Unfortunately, no data exist on theembryonic origin of the pectoral girdle inany other vertebrate. With the availabledevelopmental data and the variation ofadult structure seen in phylogeny, predic-tions can be made that can be tested byfurther experimentation on the appropriateembryos. As a current working hypothesis,I would make the following predictions:

1) Primitively in vertebrates the primarypectoral girdle (scapulocoracoid) wasderived from lateral plate mesodermconsistent with its appendicular identityand with the current theories on the ori-gin of the paired appendages in verte-brates.

2) At some point during the modificationof the limbs for terrestrial locomotion,somitic cells were recruited to augmentthe pectoral girdle. Therefore, a somitic

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contribution to the scapula is a derivedcondition among tetrapods, perhaps atthe level of the amniotes.

3) The extent of the somitic contributionhas been shown to vary between avianand chelonian embryos. A variable con-tribution by the somites reflects differentlocomotor adaptations among differentgroups of tetrapods. The overlap of cos-tal and scapular somites (as seen inchicks) is derived among the amniotes.

In order to test these predictions, exper-imental work is now in progress to deter-mine the embryonic origin of the scapulo-coracoid in several species of urodeles andanurans.

CONCLUSIONS

This study is an example of how obser-vations of development lead to hypothesesof both ontogeny and phylogeny. Ontoge-netic hypotheses are accessible to the tech-niques of experimental embryology and theunderstanding of mechanism gained by thesemethods can be used to form predictionsabout variation in phylogeny. That thesepredictions can be tested using classicalmethods of experimental embryology, tes-tifies to the utility of this approach to thestudy of the origin of form in phylogeny.Variation in developmental pattern pro-vides information about intrinsic aspects ofthe evolutionary process. Comparative workis needed in order to fully understand theconstraints which lead to the invariantaspects of form, and the plasticity of mech-anisms which generate novelties. A prob-lematic consequence of the lack of compar-ative developmental data is that one isforced to make inappropriate phylogeneticcomparisons. But, by using the frameworkof independently derived phylogenies,hypotheses can be tested, and gaps in ourknowledge filled, by experimentation on theappropriate embryos.

ACKNOWLEDGMENTS

This work was done at the Museum ofComparative Zoology and is distilled fromparts of a dissertation submitted to HarvardUniversity in partial fulfillment of therequirements for a Ph.D. degree. I would

like to thank P. Alberch in general, and B.K. Hall, P. M. Mabee and J. E. A. Bertramas well as two anonymous reviewers forhelpful comments on the manuscript. Fig-ures 1 and 2 are reprinted from Journal ofMorphology, 1989, 199:363-378, and Ithank Alan R. Liss for permission. L. Me-szoly (MCZ) drew Figures 1 and 3, T. Miyakeadapted Figure 5a from a color plate by K.Wing-Brown. M. Primrose printed the pho-tographs. Financial support for this researchcame from NSF grant BSR-84-07430 (to P.Alberch) and from Sigma Xi (to A.C.B.).Symposium support was provided by aNational Science Foundation grant (BSR-8904370) to J. Hanken and M. H. Wake.

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The Development and Evolution of the Turtle Body Plan

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