Skeletal histology of Bothriolepis canadensis (Placodermi...

Skeletal Histology of Bothriolepis canadensis(Placodermi, Antiarchi) and Evolution of the Skeletonat the Origin of Jawed Vertebrates

Jason P. Downs1,2* and Philip C.J. Donoghue3

1Department of Geology and Geophysics, Yale University, New Haven, Connecticut 065112Academy of Natural Sciences of Philadelphia, Philadelphia, PA 191033Department of Earth Sciences, University of Bristol, Bristol BS8 1RJ, UK

ABSTRACT We used light microscopy and scanningelectron microscopy to compile a complete histologicaldescription of the dermal skeleton of the antiarch placo-derm, Bothriolepis canadensis. Placodermi is most oftencited as the sister group of crown group Gnathostomata,but some recent authors propose that placodermsinstead represent a paraphyly of forms leading to thecrown. In either phylogenetic scenario, comparativeanalysis of placoderm and gnathostome histological dataallows us to address the primitive condition of both thegnathostome skeleton and the jawed vertebrate skeleton.The results of this work support the interpretation thatthe external skeleton of Bothriolepis canadensis is com-prised exclusively of cellular dermal bone tissue. Theunique stratification of the antiarch thoracic skeletonthat has led to controversial interpretations in the pastis explained by the nature of the articulations betweenadjacent elements. Skeletal features long thought to begnathostome innovations are instead discovered to arisealong the gnathostome stem. These innovations includesecondary osteons, the systematic reconstruction of theskeleton in response to growth, and unfused, overlap-ping joints that enable marginal growth while maximiz-ing the area of the articulation surface. The extensiveevidence for spheritic mineralization agrees with amodel of the skeleton as one capable of a high growthrate and active remodeling. Dermal skeletal develop-ment in both placoderms and osteichthyans is primarilyskeletogenetic with only a minor odontogenetic contribu-tion in some taxa. This demonstrates the problem inher-ent with assuming a broad application for those hypoth-eses of dermal skeletal evolution that are based on achondrichthyan model. Our results highlight the impor-tance of anatomical and ontogenetic context in the inter-pretation of fossil tissues. J. Morphol. 270:1364–1380,2009. � 2009 Wiley-Liss, Inc.

KEY WORDS: vertebrate; placoderm; histology; dermalskeleton; skeletogenesis; bone

INTRODUCTION

Chondrichthyans are generally perceived to bethe most basal clade of vertebrates with a mineral-ized skeleton and, thus, they have been influentialin attempts to uncover the nature of the primitivevertebrate skeleton (Donoghue, 2002). This has

been especially true in debate over the primacy ofbone versus cartilage, and the developmental evo-lution of the dermal skeleton. Though they may beamong the oldest extant lineages of skeletonizingvertebrates, it has long been recognized that chon-drichthyans are far removed, both temporally andphylogenetically, from those extinct vertebrates inwhich a mineralized skeleton first evolved and inwhich skeletal developmental systems were estab-lished (Heintz, 1929; Romer, 1942). This recogni-tion stems from the discovery that the mineralizedskeleton first arose in extinct jawless relatives ofthe jawed vertebrates, to whom they are relatedby degree (Janvier, 1981). The study of theseextinct stem gnathostomes (see Fig. 1 for meaningof taxonomic concepts) has revealed that peculiar-ities, such as the chimeric embryological composi-tion of the vertebrate skeleton, betray its piece-meal evolutionary assembly over a protracted epi-sode of early vertebrate phylogeny (Donoghue andSansom, 2002; Donoghue et al., 2006). Thus, thevertebrate skeleton may be considered more appro-priately and accurately as a series of distinct skel-etal systems characterized by their distinct devel-opmental and evolutionary origins: the dermalskeleton, neurocranium, splanchnocranium (viscer-ocranium), appendicular and axial skeletons(Donoghue and Sansom, 2002).

Considerable effort has been expended in under-standing the early evolutionary assembly of thevertebrate skeleton, but we remain no closer to

Contract grant sponsors: National Science Foundation, GeologicalSociety of America, Paleontological Society, Yale Institute for Bio-spheric Sciences (Field Ecology and ECOSAVE divisions).

*Correspondence to: Jason P. Downs, Academy of Natural Scien-ces of Philadelphia, 1900 Benjamin Franklin Parkway, Philadel-phia, PA 19103. E-mail: [email protected]

Received 18 December 2008; Revised 21 April 2009;Accepted 24 April 2009

Published online 16 June 2009 inWiley InterScience (www.interscience.wiley.com)DOI: 10.1002/jmor.10765

JOURNAL OF MORPHOLOGY 270:1364–1380 (2009)

� 2009 WILEY-LISS, INC.

understanding the condition from which the skele-tons of chondrichthyans and osteichthyansdeparted. In large part, this occurs because so lit-tle is known concerning the nature of the skeletonin the most basal jawed vertebrates, the placo-derms. Placodermi is an extinct clade or grade ofjawed vertebrates that first appears in the mid Si-lurian (Wenlock, circa 428 Ma) and goes on todominate the Devonian (417-354 Ma) vertebratefossil record (Carr, 1995). Though the systematicposition and interrelationships of the groupremain points of contention, recent analyses placePlacodermi as either the sister group of crownGnathostomata (Fig. 1A; Young, 1986; Goujet andYoung, 1995; Janvier, 1996) or a paraphyleticgrade leading to the crown (Fig. 1B; Johanson,2002; Brazeau, 2009). In either phylogenetic con-text, placoderms are integral to understanding thecondition of the skeleton at the origin of jawed ver-tebrates and immediately before the emergence ofchondrichthyans and osteichthyans.

There is no good explanation for the dearth ofinformation on the composition of the skeleton inplacoderms. Placoderms are well represented inthe fossil record, with over 700 recognized species(Carr, 1995). However, it is perhaps because placo-derms are so richly represented in the fossil recordand possess such complex skeletons that palaeon-tologists have not resorted to skeletal histology, anapproach that has historically been associatedwith the most desperate attempts to classify fossilvertebrates known only from scraps of bone.

The aim of this study has been to remedy thissituation, to completely characterize the skeletalhistology of a placoderm, providing a benchmarkfor future studies that will seek to establish the di-versity of skeletal histology within the group. Ourstudy focuses on the antiarch, Bothriolepis cana-densis (Whiteaves, 1880). This is not because it isrepresentative of placoderms (though they may berepresentative of the very earliest jawed verte-brates; Johanson, 2002; Brazeau, 2009) butbecause Bothriolepis has proven previously to bethe best source of histological data for placoderms.There is an abundance of specimens representingall stages of ontogeny, many of which are fullyarticulated and preserve histological microstruc-ture with great fidelity. Thus, B. canadensis repre-sents the best possible candidate for establishingskeletal composition in placoderms.

Previous Research Into the SkeletalHistology of Antiarch Placoderms

The difficulty in obtaining ontogenetic data forfossil taxa continues to generate controversial tis-sue identifications and developmental interpreta-tions for Antiarchi. Past interpretations of theclade’s skeletal microstructure are marked by alack of anatomical and developmental context.

Goodrich (1909) provided a diagrammatic figureof the gross structure of the dermal skeleton ofBothriolepis canadensis which he described ascomposed of true bone. He noted that, althoughthe surface is ornamented by tubercles, there is noevidence that they were formed from fused den-ticles. Goodrich recognized a vascular middle layerand a typically lamellated basal layer and his fig-ure (p. 206) shows clear evidence of planar discon-tinuity in the middle vascular layer.

Heintz (1929) provided some of the earliest pub-lished microscopic images of the antiarch externalskeleton. The description, limited to a single sec-tion through an unidentified ‘‘trunk armor plate’’(Pl. XXIV: Heintz, 1929), demonstrates the particu-lar stratified nature of the external skeleton inAsterolepis sp. Heintz (1929) designated four dif-ferent zones of tissue without sharp distinctionsamong them, a superficial and a basal compact, la-mellar bone tissue and two unique middle cancel-lous bone tissues. The superficial and basal cancel-lous tissues were respectively designated theMaschen-schicht (mesh layer) and the Kanal-schicht (channel layer).

Though he did not publish microscopic images,Erik Stensio did observe thin sections to interpretthe skeletal tissues in Bothriolepis canadensis andRemigolepis sp. and to describe the internal fea-tures of those skeletons. Stensio (p. 111: 1931)noted the presence of a ‘‘perichondral layer oflime-bearing tissue ... intermediate between truebone and calcified cartilage’’ in an unidentified ele-

Fig. 1. Evolutionary relationships among the principalgroups of stem- and crown-gnathostomes, applied followingHennig (1981) and Jefferies (1979), effecting an explanation ofthe concepts of gnathostome stem and crown, and how theyrelate to the concept of a clade of jawed vertebrates. Trees differonly in terms of the relations of placoderms and acanthodiansand the topology of the tree otherwise follows Donoghue et al.(2000) and Donoghue and Smith (2001). (A) The traditionalview of placoderm monophyly as promulgated by Goujet andYoung (1995, 2004), Young (1986), and Janvier (1996). (B) Thehypothesis of placoderm paraphyly as promulgated by Brazeau(2009) and Johanson (2002). Icons of representative fishes afterJanvier (1996).

THE SKELETON OF Bothriolepis canadensis 1365

Journal of Morphology

ment of the pectoral limb endoskeleton. Thoughseemingly not based on observations, Stensio(1931) argued that B. canadensis is the only anti-arch species with a mineralized component to theendoskeleton. His description of the mineralizedtissue matches that of ‘‘globular calcified carti-lage,’’ an observation first noted by Ørvig (p. 410:1951), who used topological relationships to fur-ther propose that this zone of calcified cartilage isthe homologue of the ‘‘subperichondral calcifiedlayer’’ observed in other placoderm species, Plour-dosteus canadensis and Coccosteus decipiens.

Walter Gross’s (1931) monograph on Asterolepisornata, makes reference to three stratified layersin the antiarch external skeleton: the superficialTuberkelschicht (tubercle layer), the Spongiosa,and the basal Grundlamellenschicht (basic lamel-lar layer). In unspecified elements of the thoracicskeleton, deep to the Tuberkelschicht, Gross (p. 11:1931) identifies a second cancellous zone without adefinitive association with either the Tuberkel-schicht or the Spongiosa. This is likely a referenceto the Maschen-schicht of Heintz (1929).

In a vertical cross section of the anterior mediandorsal element of Asterolepis ornata, Gross (1935)identifies an acellular, lamellar tissue zone withinthe Spongiosa that divides the cancellous tissuezone into basal and superficial components. Nofurther mention was made of a distinction betweenthe two stratified components of Spongiosa.

Ørvig (1968) recognized that Bothriolepis cana-densis, by possessing two stratified zones of cancel-lous tissue, demonstrates an unusual histologicalorganization. Clear evidence was presented forspheritic mineralization within the basal cancel-lous tissue and the author properly noted thatsuch a mode of mineralization is atypical of bone.To match his observations to the traditional diag-nosis for a dermal bone organ, Ørvig (p. 377: 1968)tentatively designated the peculiar tissue ‘‘globularbone.’’

Burrow (2005) proposed that the spheriticallymineralized, lower middle cancellous tissue in theexternal skeleton of Bothriolepis canadensis repre-sents bone trabeculae that separated large spheresof calcified cartilage. The mineralized spherites ofthis tissue were interpreted as the lacunae ofosteoblasts from the bone trabeculae and chondro-blasts of the cartilage. Burrow interpreted this tis-sue as the precursor to prismatic calcified carti-lage, proposing that the bone trabeculae separat-ing the spaces of the lower middle cancelloustissue in B. canadensis are lost in Chondrichthyesand are replaced by the interlocking prisms thatare characteristic of prismatic calcified cartilage.

This brief but comprehensive review demon-strates that to date, attempts to characterize theskeletal histology of antiarchs have been cursoryand often contradictory. Furthermore, the sug-gested presence of cartilage in the external skele-

ton (Burrow, 2005) perhaps unwittingly challengesthe dogma that there is a fundamental dichotomybetween the dermal skeleton and endoskeleton,both in terms of their development and evolution(Patterson, 1977; Smith and Hall, 1990; Donoghueand Sansom, 2002). There have been isolatedaccounts of developmental integration of the endo-and dermal skeleton, although it is perhaps nota-ble that they are invariably based on fossil mate-rial where development cannot be observed. Forinstance, Scheyer (2007) described a cartilaginousbone from the integument of extinct placodonts (agroup of sauropterygian reptiles). It might beanticipated that such phenomena should be mani-fest early in the evolutionary establishment ofskeletal developmental programs, but there is noevidence of this among extinct jawless vertebrates(Smith and Hall, 1990; Donoghue and Sansom,2002). However, placoderms are the first verte-brate clade in which all vertebrate skeletal sys-tems are manifest (Donoghue and Sansom, 2002)and so it is possible that during this integral epi-sode of vertebrate skeletal evolution, some of thedistinctions that we observe in living vertebratesdid not yet hold.

Thus, it is integral that we obtain a more com-prehensive understanding of the composition ofthe mineralized skeleton of Bothriolepis canaden-sis and this article provides an histological analy-sis and interpretation of its skeletal biology.

MATERIALS AND METHODS

The present study is based on a growth series of 13 near-com-plete specimens of Bothriolepis canadensis from the Frasnian-age exposures of the Escuminac Formation at the Miguashafield site in Quebec, Canada. The Escuminac Formation out-crops exclusively in the Baie des Chaleurs area, on the south-western shore of the Gaspe Peninsula. The sedimentology andstratigraphy of the formation indicate deposition of terrigenousdetritus in a marginal estuarine environment (Hesse and Sawh,1992; Prichonnet et al., 1996). Each specimen was preserved inthree dimensions within a calcium carbonate concretion thatwas collected as float and therefore without specific strati-graphic context.

Following the methodology developed by Jeppsson et al.(1999), the individual external skeletal elements were isolatedfrom the concretions using repeated baths of 7% acetic acid buf-fered with calcium acetate to a pH of 3.6. The pH was chosen tomaximize dissolution of the calcium carbonate in the concretionwhile preventing the dissolution of the fossil material.

All material to be sectioned was first embedded in StruersSerifix polyester resin. Where possible, transverse sections werecut through skeletal elements of the external head skeleton(premedian, lateral, postmarginal, paranuchal-marginal, postpi-neal, nuchal), thoracic skeleton (anterior median dorsal, ante-rior dorsal lateral, mixilateral, posterior median dorsal, anteriorventral lateral, posterior ventral lateral) and pectoral append-age (dorsal central 1, ventral central 1). The samples were cutin two using a diamond wafering blade mounted on a bench-topBuehler ISOMET low speed saw. Cut surfaces were thencleaned in water using a ultrasonic cleaner and impregnatedwith Buehler EPO-THIN resin. The specimen surfaces weremanually ground using grit sizes ranging from P1200 to P4000and polished using 1.0 and 0.1 lm deagglomerated alpha alu-

1366 J.P. DOWNS AND P.C.J. DONOGHUE


mina powder and finally 0.04 lm colloidal silica solution. Eachspecimen was affixed to a petrographic slide using the EPO-THIN resin as an adhesive. A Ward’s Natural Science IngramThin-Section Cut-Off Saw was used to remove all but the lastmillimeter of the specimen from the slide. The slide was thenground to a thickness of �300 lm using a Buehler ECOMET4 grinder/polisher. Finally, each thin section was ground to �50lm and polished using the manual grinding/polishing methoddescribed earlier.For each element, we prepared two serial sections for the pur-

poses of comparative imaging using transmitted light opticalmicroscopy and scanning electron microscopy (backscatter elec-tron and secondary electron imaging). The scanning electronimages were generated using a JEOL JXA-8600 electronmicroprobe and a Philips Field Emission Gun EnvironmentalScanning Electron Microscope. Following the methodology ofSundstrom (1968), before study, the scanning electron sampleswere etched for 1 h in a bath of 0.5 weight percent solution ofchromium (III) sulfate [Cr2(SO4)3�nH2O] brought to pH 3.5with the addition of sodium hydroxide (NaOH). Immediatelybefore imaging, a carbon coat was applied to each sample usinga Cressington ‘‘208carbon’’ Carbon Coater.

RESULTS

Ordinarily, we would provide a direct interpreta-tion of skeletal histology; however, given that it isthe interpretations of these tissues that has beenthe source of contradiction and controversy wehave chosen instead to provide a neutral descrip-tion of the histological tissues, followed by aninterpretation of this evidence. The followingdescription is conducted according to the followingthree a priori assumptions: 1) the material repre-sents elements of a vertebrate skeleton, 2) thegross morphology of the elements may be used todetermine anatomical direction and to identify theelements of the head and thoracic skeletons, and3) osteocytes, canaliculi, and vascular canals arerecognizable by their distinctive morphology.

Histological Description

All of the tissues of the Bothriolepis canadensisexternal skeleton are cellular. The elements of theexternal skeleton are characterized by a distincthorizontal zonation or stratification. In elements ofthe thoracic skeleton, at least three zones of tissueare discernible: a superficial lamellar tissue with acompact component and a cancellous component; abasal, compact lamellar tissue; and a middle zoneof woven-fiber cancellous tissue between them(Fig. 2A). The boundary plane between the superfi-cial and middle cancellous tissues forms the zoneof overlap between articulating elements of thethoracic skeleton. In other words, in each of theoverlapping joints of the external thoracic skele-ton, the superficial tissue zone of one elementoverlaps with the basal (woven-fiber 1 lamellar)tissue complex of another. In the external headskeleton and proximal pectoral appendage, wheresuch overlapping joints do not occur, compact su-perficial and basal lamellar tissue zones bound adeep zone of woven-fiber, cancellous tissue. In this

section, we separately describe the microstructuralanatomy of the external thoracic, head, and pecto-ral appendage skeletons. Each description remarkson the features common to all observed elementsof the respective skeletal component.

External thoracic skeleton. The most superfi-cial tissue of the external thoracic skeletonincludes the tuberculated ornament and is com-posed of a cellular, lamellar tissue without evi-dence for pulp cavities. This zone of tissue is com-posed of approximately parallel superimposed lam-inae and varies little in depth within a singleelement. Individual lamellae average �10 lm inthickness. Lacunae lie within and between thelamellae and are often round or star-shaped with aslight tendency toward a flattened, elongate shapein the most superficial lamellae (Fig. 2B). Relativeto those of the basal lamellar tissue, the shapes ofthe lacunae are far more variable. Primary proc-esses, or canaliculi, issue from the lacunae roughlyparallel to the tissue lamellae and many additionalramifying processes form a dense networkthroughout the tissue. The degree to which theprimary processes are parallel with the layers oftissue growth is another measure that increaseswith proximity to the superficial surface. The tis-sue’s primary vascular canals are aligned in theplane of the skeletal plates and feature simplereticulating canals (Fig. 2C). Open spaces in thetissue frequently truncate lamellae. In manyinstances, these spaces are lined by concentriclamellae of cellular tissue (Fig. 2D). The lacunaeenclosed in the concentric lamellae are spindle-shaped with long axes parallel to the lamellae.The frequency with which these open spacesappear is highest in the deepest part of this tissue.This cancellous component of the superficial lamel-lar tissue zone corresponds with the Maschen-schicht of Heintz (1929). A sharp division sepa-rates this tissue zone and the middle cancelloustissue deep to it. The boundary itself is planar andis continuous with the plane of overlap at the mar-gins of thoracic skeletal plates (Fig. 2E). Burrow(2005) described thin but distinct bone lamellaewithin this boundary; we find no evidence in ourown material or in any of the material presentedby Burrow (2005). Between plate margins, theboundary is discontinuous due to interruptionsimposed by open spaces that extend between thetwo cancellous layers. These spaces, too, are linedby concentric lamellae of cellular tissue. Becauseof the abundance of these open spaces, in certainpositions, the boundary between the superficialand the underlying tissue zones is not obvious andthe entire region between the superficial and basallamellar tissues takes on the appearance of a sin-gle, gradational cancellous tissue zone. Of finalnote, the superficial tissue exhibits mineralizedspherites. Within this tissue zone, they are mostcommonly observed surrounding the smaller cav-



Figure 2.

ities that reside just beneath the most superficiallamination. Spherites are abundant throughoutthe external skeleton of Bothriolepis canadensisand, while present in this superficial tissue, aremost abundant in the underlying middle cancel-lous zone (Fig. 2F).

The middle cancellous tissue is less compactthan the superficial cancellous tissue. Round orstar-shaped lacunae within the woven-fiber tissueappear randomly distributed. The globular appear-ance of the woven-fibered tissue results from adense aggregation of mineral spherites. They aresurrounded by a fibrous network that is revealedthrough crossed nicols (Fig. 3A). The mineralspherites average between 10 and 20 lm in diame-ter, smaller than those previously reported fromthe internal skeleton of the Plourdosteus canaden-sis pectoral appendage (Ørvig, 1951). The most su-perficial spherites are themselves comprised ofspherical structures at multiple size scales; thesmallest microspherites have a diameter on theorder of 100 nm (Fig. 3B). These cluster into indi-vidual larger scale units (Fig. 3C) that themselvesmay cluster into even larger scale units (Fig. 3D).Even those units that we here refer to as‘‘spherites’’ cluster together along the woven-fiberframework of this tissue zone. Concentric darkand light banding of the spherites, associated withcompositional differences, is observable in back-scatter electron images (Fig. 3C). In contrast tothe more superficial spherites, many of the deeperspherites are hollow (Fig. 3D). The orientation,size, and shape of the microspherites in theBothriolepis canadensis external skeleton areinconsistent with our understanding of spheriticmineralization in vertebrates.

The middle tissue zone is not entirely withoutlamination. As observed in the superficial tissuezone, there are open spaces that are lined withconcentric lamellar tissue. However, this is a farless common phenomenon in this middle tissuezone. In the majority of instances, the open spacesare lined by dense aggregations of spherites withno evidence at all for tissue lamellae.

Bands of mineralized tissue run along the lateraledges of each element and are approximately paral-lel in orientation to the lateral edge of the element.Within these bands of tissue, dark striations run

parallel to the superficial and basal surfaces of theelement (Fig. 3E). Similar dark striations run in asuperficial to basal direction in lenses of lamellartissue that appear superficial to the lamellae of thesuperficial tissue zone (Fig. 3F).

The lamellae in the compact basal lamellar tis-sue zone average 20 lm in thickness. Abundantlacunae lie within and between the lamellae. Themajority of the lacunae are spindle-shaped withprimary processes that issue parallel to the lamel-lae and additional processes that issue largely per-pendicular to the lamellae (Fig. 3G). As in the su-perficial lamellar tissue, the degree to which thesefactors are developed increases with proximity tothe surface of the element, (the basal surface, inthis instance). In the basalmost lamellae, however,lacunae are extremely rare. Through crossed nic-ols, these most basal lamellae share an interfer-ence color with one another but one that is distinctfrom the single or multiple interference colors dis-played by the overlying lamellae (Fig. 3H).

External head skeleton. Although the relativedepths of the tissue zones are unlike those of theexternal thoracic skeleton, the elements of thehead skeleton demonstrate a similar stratification.The superficial tissue is lamellar with open spacescrosscutting the lamellae and with concentric la-mellar tissue in those spaces. The middle cancel-lous tissue zone exhibits a woven matrix and min-eralized spherites. A compact lamellar tissue formsthe basal tissue zone. Unlike the condition in theexternal thoracic skeleton, there is no sharpboundary plane between the superficial and middletissue zones (Fig. 3I).

In the head skeleton, cross-cutting open spacesof the superficial lamellar tissue exhibit concentriclamellae (Fig. 4A) and, in some instances, mineral-ized spherites. Deep to the surface of the element,the superficial tissue zone exhibits waves of lamel-lae that exhibit the size and appearance of superfi-cial tubercles (Fig. 4B). Between these ‘‘tubercles’’are zones of spheritically-mineralized tissue densewith round osteocytes and canaliculi issuing in alldirection. Waves of lamellar tissue lie superficial tothese internal ‘‘tubercles’’ and the zones of spher-ites between them. These lamellae form the tuber-culated ornament of the element. Like the phe-nomenon described for the external thoracic skele-

Fig. 2. MHNM 02-616, Bothriolepis canadensis, mixilateral in thin section, superficial surface always toward top of frame; (A)LM image, full depth of element illustrates the division into superficial compact and cancellous lamellar tissue zones, middle woven-fibered cancellous tissue zone, and basal compact lamellar tissue zone. The sharp boundary plane between superficial and middlezones is prominent; scale bar 5 0.40 mm; (B) LM image, superficial compact lamellar tissue zone with lacunae and network of canal-iculi; scale bar 5 0.07 mm; (C) LM image, vascular canals of the superficial tissue zone. Primary canals are surrounded by simplereticulating canals; scale bar 5 0.18 mm; (D) LM image, superficial tissue zone shows division into compact and cancellous compo-nents. Open spaces with concentric bands of mineralized tissue are secondary osteons; scale bar 5 0.40 mm; (E) SEI image, bound-ary between superficial and middle tissue zones; scale bar 5 0.08 mm; (F) SEI image, middle woven-fibered tissue showing extensivespheritic mineralization; scale bar 5 0.10 mm. bas, basal lamellar tissue zone; bp, boundary plane between superficial and middletissue zones; lac, lacuna; mid, middle cancellous tissue zone; sph, mineral spherite; sup, superficial lamellar tissue zone.



Figure 3.

ton, in positions where lamellar tissue lies superfi-cial to internal ‘‘tubercles,’’ the superficial tissueoften exhibits dark striations that run perpendicu-lar to the lamellation. Also similar to the conditionin the external thoracic skeleton, the lateral mar-gins exhibit banding of the tissue nearly parallelto the lateral edge of the element and dark stria-tions generally parallel to the element’s basal sur-face (Fig. 4C). The margins of the elements of theexternal head skeleton do not feature the overlapzones that characterize the external thoracic skele-ton. Accordingly, the external head skeleton doesnot exhibit a clearly defined stratification into thesuperficial and basal units that make up the over-lap zones in the thoracic skeleton.

The external head skeleton exhibits an extensivearray of open spaces interrupting not only thelamellae of the superficial tissue zone but alsothe woven matrix of the middle cancellous zone;the postmarginal of MHNM 02-616 even exhibitsthese open spaces in the basal lamellar tissue.These open spaces sometimes feature concentriclamellae within them and sometimes feature denseaggregations of mineralized spherites (Fig. 4D).The degree of interruption imposed by these openspaces makes it nearly impossible to distinguish aboundary between the superficial lamellar tissueand the middle cancellous tissue. However, even inpositions where we might expect to observe theboundary, the transition between the tissue zonesis not sharply defined as it is in the thoracic skele-ton. As a final note, compared to the conditionobserved in the thoracic skeleton, relative to theoverall depth of the element, the basal lamellartissue in the head skeleton is especially shallow.

External pectoral appendage skeleton. Theproximal elements of the pectoral appendage skele-ton (dorsal central 1 and ventral central 1) exhibita superficial lamellar tissue zone, a middle woven-fiber cancellous tissue zone, and a basal lamellartissue zone. The lamellar tissues, like the woven-fiber tissue, exhibit a dense presence of mineral-ized spherites. Like the conditions described in thehead skeleton and thoracic skeleton, the lamellaeof the superficial zone and the woven matrix of thecancellous zone exhibit cross-cutting open spaces.

These spaces feature concentric lamellae andspheritic mineralization (Fig. 4E). In the dorsalcentral 1, such spaces additionally appear in thebasal lamellar tissue. The margins of these ele-ments do not possess the overlap zones that arecharacteristic of the external thoracic skeleton.Relative to those of the corresponding tissue in thehead and thoracic skeletons, the trabeculae of themiddle cancellous zone are thicker and with adenser aggregation of spherites. In the distal pec-toral appendage, the terminal plate fuses withmembers of the lateral marginal, medial marginal,dorsal central, and ventral central series. Both thedorsal central and the ventral central elements arecomposed entirely of superficial and basal lamellartissues. Only a narrow, dense zone of mineralizedspherites separates the two. Open spaces linedwith concentric lamellae perforate the lamellar tis-sue (Fig. 4F). The only lamellae distinguishable inthe medial and lateral marginal elements arealong the superficial and basal surfaces. The mid-dle of these elements is a mass of mineralizedspherites without any obvious structure. Numer-ous open spaces perforate this mass of spherites.Many of these spaces have a vascular appearance(Fig. 5A). These open spaces exhibit both concen-tric lamellar tissue and mineralized spherites.

In the most distal pectoral appendage, fusionamong the elements means that individual ele-ments are discernible only in section. Stensio(1948) described the element boundaries followingacid preparation of the surface of the distalappendage. In transverse section, the boundariesare clearly observable. The dorsal and ventral cen-tral elements and the medial and lateral margin-als between them feature bands of tissue at theirlateral edges. In the zones of fusion, a mass ofspheritically-mineralized tissue fills in the spacebetween adjacent elements. Lamellae superficialand basal to the zone of fusion are continuousacross the two elements. Between fused elements,open spaces crosscut not only the bands of tissueat the edges of the elements but also the spheritictissue between the elements (Fig. 5B). These openspaces feature aggregations of spherites and con-centric lamellar tissue.

Fig. 3. MHNM 02-616, Bothriolepis canadensis in thin section, superficial surface always toward top of frame; (A) LM image(crossed nicols), mixilateral, crossed nicols reveal the woven-fibered network of the middle tissue zone; scale bar 5 0.14 mm; (B-D)BSE images, mixilateral middle cancellous tissue zone; (B) microspherites; scale bar 5 0.91 lm; (C) mineral spherites, dark and lightbands signify compositional differences; scale bar 5 3.77 lm; (D) mineral spherites, black spots at bottom of frame are hollow spaceswithin spherites; scale bar 5 14.46 lm; (E) LM image, mixilateral, basal lamellar tissue zone, lateral edge shows dark striations par-allel with tissue lamellae; scale bar 5 0.14 mm; (F) LM image, mixilateral, superficial lamellar tissue zone showing dark striationsperpendicular to tissue lamellae; scale bar 5 0.14 mm; (G, H) LM images, mixilateral, basal lamellar tissue zone; (G) lacunae andcanaliculi; scale bar 5 0.04 mm; (H) crossed nicols show that basalmost lamellae share a single interference color and therefore align-ment of their collagen fibers; scale bar 5 0.15 mm; (I) LM image, postmarginal, full depth of element shows the division into superfi-cial compact and cancellous lamellar tissue zones, middle cancellous tissue zone, and basal compact lamellar tissue zone. The externalhead skeleton does not exhibit a sharp plane of division between superficial and middle tissue zones; scale bar 5 0.50 mm. bas, basallamellar tissue zone; hsp, hollow spherite; lac, lacuna; mid, middle cancellous tissue zone; str, dark striation.



Figure 4.

DISCUSSIONTissue Homologies

The bone classification scheme of de Ricqles(1975) and de Ricqles et al. (1991) is based on mor-phology and topology and therefore is readily ap-plicable to bone in the fossil record. The majorityof the tissues comprising the external skeletonBothriolepis canadensis can readily be interpretedas cellular bone tissues; however, the spheriticallymineralized tissue comprising the middle cancel-lous layer requires greater consideration (seebelow).

Inotropic mineralization. The most superfi-cial zone of the external skeleton of Bothriolepiscanadensis, including the tuberculated ornament,is comprised of compact, lamellar, dermal bone tis-sue with simple vascular canals. Several genera-tions of osteons cross-cut earlier fabrics within thetissue and are evidence for multiple cycles of boneresorption and redeposition.

In the external thoracic skeletal elements, thereis a sharp division between the superficial lamellarbone and the middle cancellous bone. This bound-ary is identified as a rest cementing line because itrepresents a discontinuity in bone deposition anddoes not occur at a resorption surface (de Ricqleset al., 1991). This rest line, especially obvious insecondary electron images, is also the plane ofoverlap between articulating elements of the exter-nal thoracic skeleton. The middle tissue zone inthe B. canadensis external skeletal elements iscomprised of spheritically-mineralized primarycancellous bone tissue. This rare example of spher-itic mineralization in a bone tissue makes it partic-ularly difficult to classify, though it otherwise fitsthe diagnosis of spongiosa of dermal origin.Observable in even the smallest specimens ofBothriolepis canadensis (Fig. 5C), this primary tis-sue is identified according to the large open spacesin mineralized tissue, the lack of lamellation, andthe delicate scaffolding of its trabeculae (Fran-cillon-Vieillot et al., 1990).

The basal zone of the Bothriolepis canadensisexternal skeleton is comprised of a compact, lamel-lar, avascular, dermal bone tissue. This tissue isdiagnosed by the combination of a topological posi-tion superficial to the organism and basal withrespect to the element, a distinct lamellation, alack of vasculature, and flattened cell spaces with

long axes parallel to the lamellation. Observedthrough crossed nicols, the most basal lamellae ofthis tissue exhibit a parallel-fiber bone matrix(Fig. 3H). The homogeneity of interference colorsindicates that the mineral crystallites, and there-fore the collagen fibers, in this series of lamellaeare parallel to one another.

All three stratified tissue zones exhibit dark hor-izontal lines that are observable in the opticalmicroscope images. Their topological positions sug-gest that they are attachment fiber spaces thatrun between the body of the skeletal element andzones of new tissue growth. The attachment fiberspaces occur along the lateral margins of theexternal skeletal elements in the bands of tissuethat represent zones of marginal tissue growth.Elements of the head skeleton exhibit multiplegenerations of tubercles in the superficial zone oflamellar bone. The most superficial, latest genera-tions of tubercles also exhibit these striations per-pendicular to the lamellae.

Spheritic mineralization. Spheritic minerali-zation involves the radial deposition of mineralcrystallites around a mineralization nucleus.Because it does not require the construction of acollagen fibril matrix, it is simple, quick, andenergy efficient relative to inotropic mineralization(Ørvig, 1951, 1968) or the deposition of mineralcrystallites between the individual molecules thatcomprise collagen fibrils (Francillon-Vieillot et al.,1990). Evidence for spheritic mineralization occursin all observed elements of the Bothriolepis cana-densis external skeleton and it is exhibited in eachof the three stratified tissue zones. Spheritic min-eralization of the endoskeletal tissue, cartilage, iswidely documented in the vertebrate endoskeleton.This fact, along with the sharp division betweenthe superficial and basal tissues in the externalthoracic skeleton, has led authors to interpret thebasal tissues in the B. canadensis external skele-ton as a contribution of the endoskeleton (Burrow,2005).

To test this hypothesis, we use evidence of phy-logenetic consistency, morphology, topology andcomparative anatomy. First, the presence of miner-alized endoskeletal tissues, including both calcifiedcartilage and perichondral bone, in placoderms, isconsistent with present understanding of verte-brate phylogeny (Donoghue and Sansom, 2002;

Fig. 4. MNHM 02-616, Bothriolepis canadensis in thin section, superficial surface always toward top of frame; (A) LM image,postmarginal, secondary osteons of the superficial lamellar tissue zone; scale bar 5 0.10 mm; (B) LM image, lateral, subsurfacetubercles of the superficial lamellar tissue zone; scale bar 5 0.14 mm; (C) LM image, lateral, lateral edge of element showing darkstriations; scale bar 5 0.56 mm; (D) LM image, lateral, secondary osteons of the superficial lamellar tissue zone showing evidenceof spheritic mineralization; scale bar 5 0.10 mm; (E) LM image, ventral central 1, full depth illustrates the division into superficialand basal lamellar tissue zones and middle woven-fibered zone. There is extensive evidence for resorption and redeposition of min-eralized tissue; scale bar 5 0.18 mm; (F) LM image, unidentifiable element of the distal dorsal/ventral central series, full depthshows lamellar tissue interrupted by secondary osteons; scale bar 5 0.10 mm. bas, basal lamellar tissue zone; int.t, internal tuber-cle; re.l, resorption line; s.os, secondary osteon; sph, mineral spherite; str, dark striation; sup, superficial lamellar tissue zone.



Fig. 5. Bothriolepis canadensis in thin section, superficial surface toward top of frame except where noted otherwise; (A) LMimage, MHNM 02-616, unidentifiable element of the distal medial/lateral marginal series, dense mass of spherites interrupted bysecondary osteons, superficial surface toward left side of frame; scale bar 5 0.15 mm; (B) LM image, MHNM 02-616, zone of fusionbetween the dorsal/ventral central series and the medial/lateral marginal series, superficial surface toward bottom of frame. Notethe spherites and secondary osteons in the zone of fusion; scale bar 5 0.15 mm; (C) LM image, MHNM 02-2355, juvenile, mixilat-eral, full depth shows superficial and basal lamellar tissue zones and middle woven-fibered tissue zone with mineral spherites;scale bar 5 0.09 mm; (D) LM image, MHNM 02-2617, juvenile, mixilateral, full depth without middle woven-fibered zone, superfi-cial and basal lamellar tissue zones separated by zone of mineral spherites; scale bar 5 0.09 mm; (E) BSE image, MHNM 02-616,anterior median dorsal, bands of tissue growth at element’s lateral edge. Note presence of mineral spherites; scale bar 5 0.06 mm;(F) LM image, MHNM 02-616, mixilateral, full depth, zone of offset shows evidence for repair of the mineralized tissue; scale bar5 0.45 mm; bas, basal lamellar tissue zone; gr.l, marginal growth lines; s.os, secondary osteon; sph, mineral spherites; str, darkstriation; sup, superficial lamellar tissue zone; z.of, zone of offset.



Wang et al., 2005; Donoghue et al., 2006). Mineral-ized endoskeletal tissues have been reported inPetromyzontidae (Bardack and Zangerl, 1971; Lan-gille and Hall, 1993), Astraspida (Ørvig, 1951;Denison, 1967; Sansom et al., 1997), Anaspida(Janvier and Arsenault, 2002), Arandaspida (Gag-nier, 1993), Galeaspida (Janvier, 1984, 1990;Wang, 1991; Wang et al., 2005; Zhu and Janvier,1998), Pituriaspida (Young, 1991), Osteostraci(Stensio, 1927; Denison, 1947, 1951; Wangsjo,1952; Ørvig, 1957a; Gross, 1961) and Placodermi(Ørvig, 1951). The fusion of endoskeletal tissues tothe internal surface of external dermal skeletalelements has previously been reported in the stemgnathostome clades Osteostraci (Stensio, 1927;Denison, 1947, 1951; Wangsjo, 1952; Ørvig, 1957a;Gross, 1961) and Galeaspida (Janvier, 1984; 1990;Wang, 1991; Wang et al., 2005; Zhu and Janvier,1998). The external skeleton of the galeaspid headexhibits a basal zone of calcified cartilage that isseparated from the dermal bone by a hypomineral-ized zone of spherites (Wang et al., 2005). In someosteostracans, the external head skeleton can ex-hibit a basal zone comprised entirely of cellularperichondral bone, or of perichondral bone bound-ing a core that is unmineralized or spheriticallymineralized (Wang et al., 2005).

Second, the basic organization of a middle spher-itically-mineralized tissue and a basal lamellar tis-sue approximates the condition described inosteostracans (Denison, 1947; 1951; Gross, 1961).However, the woven, cancellous tissue of Bothriole-pis canadensis is structurally unlike the compact,densely spheritic tissue described in osteostracans(Ørvig, 1951), galeaspids (Wang et al., 2005) and,indeed, chondrichthyans (Dean and Summers,2006), where spherites are bound by universalmineralization fronts.

Third, the tissue zones hypothesized to be endo-skeletal in the Bothriolepis canadensis are distrib-uted throughout the entire thoracic skeleton. Inthe galeaspid and osteostracan examples, endocra-nial tissues fuse to the basal surface of the dermalskull roof elements only (Janvier, 1984; Wanget al., 2005). In B. canadensis, the endoskeletalinterpretation of these tissues imposes the sugges-tion that the entire thoracic region is encapsulatedby an endoskeletal ‘‘armor.’’ This interpretation isinconsistent with existing knowledge of the topol-ogy of the vertebrate endoskeleton and, indeed,knowledge of the topology of endoskeletal struc-tures in Bothriolepis. Endoskeletal tissues that wewould anticipate to be present in Bothriolepis aremanifestly absent, for instance in the chondrocra-nium (Young, 1984), because they were not miner-alized in the first instance. Finally, the tissuesthat are hypothesized to belong to the endoskele-ton are sandwiched between tissue layers thatare otherwise readily interpreted as superficialand basal lamellar dermal bone; there is certainly

no relationship between the lamellar tissue zonesand the intervening cancellous zone that couldsupport a perichondral interpretation for the lamellartissues.

Thus, while the fabric of the spheritically miner-alized tissues grossly resembles spheritically min-eralized cartilage, the topology of these tissuesrequires that they are interpreted as another com-ponent of the dermal skeleton. Reaching this con-clusion does not, however, address the questionsabout antiarch skeletal microstructure that origi-nally motivated the endoskeletal interpretation.These ask first, why the middle and basal tissuezones are separated from the superficial by asharp line of division, and second, why spheriticmineralization was extensively employed in theconstruction of the external skeleton, and third, isthis tissue cartilage or bone? To resolve theseissues, we offer interpretations that are informedby the anatomical and developmental context ofour histological data.

The dividing line between the basal cancellouspart of the superficial lamellar tissue zone and themiddle cancellous zone is the plane of overlapbetween articulating elements. This line of divisionis observed in all the specimens of the growth se-ries. Sections taken through the mixilateral ofMHNM 02-2355 (with an anterior median dorsallength of just 10 mm) demonstrate that, even atthis early stage of ontogeny, the division into su-perficial and basal components is present. Even atcertain positions in the plates (Fig. 5D) that areentirely lacking in cancellous tissue, the elementis divided into a superficial and a basal compactlamellar tissue, separated by a shallow zone ofmineralized spherites. Independent development ofthese two units enables the differential pacing ofmarginal growth in each, a condition that allowsfor overlapping articulations. Each element of theBothriolepis canadensis external skeleton appearsto have a single center of ossification (Graham-Smith, 1978) and growth out from this center ofossification is recorded in the marginal growthlines (Fig. 5E). As the organism grew, the centersof the elements in the thoracic shield migratedaway from one another (Graham-Smith, 1978). B.canadensis and any organism with an externalskeleton composed of large articulating plates,requires a mechanism of growth that does notrequire disarticulation of the component elements.Placoderms represent one of the most basal exam-ples of overlapping or ‘‘scarf ’’ jointing in verte-brates, a type of skeletal articulation that isobserved in many more derived vertebrate clades.These unfused joints enable growth along the ele-ment margins while maximizing the surface areaof the articulation.

The ontogenetic growth of Bothriolepis canaden-sis was extensive, even after the initial develop-ment of the dermal skeleton (Werdelin and Long,



1986). Along with the evidence for resorption andsecondary osteon development, spheritic minerali-zation points to the B. canadensis external skele-ton being a dynamic system at least capable ofrapid growth and reconstruction. Spheritic miner-alization is often observed in secondarily depositedtissues in B. canadensis. In addition to the pres-ence of spherites in resorption spaces, there is atleast one example among our sections of a healedinjury to the external skeleton. In the mixilateralelement of MHNM 02-616, a zone of offset runs allthe way through the element from the superficialto the basal surface (Fig. 5F). This healed fractureexhibits a concentration of spherites along theplane of fracture. Through the action of resorptionand secondary deposition of mineralized tissues,the external skeleton had the ability to continuallystrengthen itself, heal its injuries, and potentiallysustain a high growth rate.

Ultimately, we must consider whether the spher-itically mineralized tissue represents evidence ofcartilage in the dermal skeleton. Although it haslong been accepted that there is a fundamental di-chotomy between the dermal skeleton and theendoskeleton (Patterson, 1977), and that cartilagehas always been exclusive to the endoskeleton(Smith and Hall, 1990), there has been report ofspheritically mineralized cartilage in associationwith the dermal skeleton of extinct placodonts(Scheyer, 2007). However, this interpretation reliesentirely upon the presence of a mode of minerali-zation that transcends tissue type and germ layer(Ørvig, 1951; Donoghue et al., 2006). Ultimately,the distinction between tissue types can be exceed-ingly challenging, sometimes even more so whendealing with the tissues of living organisms wherebatteries of histochemical tests and molecular

markers often fail to clearly delineate one canoni-cal tissue type from another. This has led to sug-gestions that the canonical tissue types are compo-nent elements of a spectrum of intermediates(Hall, 2005; Hall and Witten, 2007; Kawasaki andWeiss, 2008). However, in this discussion, it is im-portant to distinguish between the question ofwhether there are distinct tissues types and theentirely separate question of whether we are ableto distinguish between them. The topology of thetissues provides crucial, albeit inferential insightinto the developmental origins of the tissues inquestion and these indicate that they are dermal.No compelling evidence has ever been presentedfor the presence of primary cartilage in the dermalskeleton (Smith and Hall, 1990).

Comparisons to Other GroupsIs Bothriolepis canadensis representative

of antiarchs? Of placoderms? The skeletal his-tology of the dermal skeleton appears to be gener-ally representative of antiarchs and other placo-derms, at least within the context of what little isknown currently. Like all placoderms, indeed, themajority of stem gnathostomes, Bothriolepis cana-densis possesses a dermal skeleton with a basal com-pact, lamellar bone layer; a spongiose middle bonelayer; and an ornamented superficial layer. WhereB. canadensis and other antiarchs (Gross, 1931;Ivanov et al., 1995) depart from other placoderms,is in the composition of this superficial layer,which is composed solely of compact bone in anti-archs (see Fig. 6). In the majority of other placo-derms, the superficial layer is characterized byodontode-derived ornamental units composed ofsemidentine (a type of dentine identified by the en-

Fig. 6. Block diagram showing the internal structure of the dermal skeleton in Bothriolepiscanadensis.



closure of unipolar odontocytes; Ørvig, 1967) andbone (Ørvig, 1957b; Denison, 1978). Semidentinehas been considered a synapomorphy of Placo-dermi (Denison, 1978) and, under existing schemesof placoderm phylogeny (Goujet and Young, 1995,2004), there have been multiple independent lossesof the tissue throughout the clade (Ørvig, 1957b).If antiarchs are the sister group of all remainingplacoderms plus crown gnathostomes as hasrecently been suggested (Johanson, 2002), the ab-sence of semidentine in antiarchs would be a prim-itive character, and its later acquisition would rep-resent a synapomorphy of the remaining clade ofplacoderms. Although the polarity of placodermphylogeny is open to question, the lack of dentaltissues (dentine, enameloid, bone of attachment) inantiarchs must represent a secondary loss sincedental tissues are manifest in stem gnathostomes(Donoghue and Sansom, 2002).

The independent patterning of the dental andunderlying skeletal tissues comprising the dermalskeleton is not unparalleled. Indeed, although bothare primitive to the dermal skeleton (Donoghueand Sansom, 2002), they exhibit independentgrowth histories and in groups such as the galeas-pids (skeletal only) and chondrichthyans (dentalonly), as well as the antiarchs, the dental andunderlying skeletal tissues are mutually exclusive(Donoghue and Sansom, 2002). This may occurbecause, in the few groups that have been studied,the ectomesenchymal neural crest cells that con-tribute to the dermal skeleton are known to differ-entiate into distinct odontogenic and skeletogeniccell lineages with distinct potential (Smith andHall, 1990; Donoghue and Sansom, 2002; Sire andHuysseune, 2003). The independent patterning ofthese cell lineages facilitates the common or mutu-ally exclusive development of dental or skeletalderivatives.

Whether or not we consider the dermal skeletonof Bothriolepis canadensis to be representative ofplacoderms, its skeleton as a whole cannot be con-sidered representative because, unlike a good dealof other placoderms, elements of its endoskeleton,such as the neurocranium (Young, 1984), were notmineralized. Little is known concerning the micro-structure of this and other endoskeletal elementsin placoderms (Ørvig, 1951).

Perhaps the most significant insight that thisanalysis of Bothriolepis canadensis has revealed isthe extent of resorption and secondary bone depo-sition in its dermal skeleton (see Fig. 6). Boneresorption has been reported in clades that branchmore basally within vertebrate phylogeny, specifi-cally in heterostracans (Gross, 1935; HalsteadTarlo, 1964) and in osteostracans (Denison, 1952).However, in vertebrate phylogeny, placoderms pro-vide the most basal evidence of secondary osteonaldevelopment (Johanson and Smith, 2005), andwhat clearly represents a concerted program of

skeletal tissue remodeling. This insight may high-light dermal skeletal responses to the lifestylechanges imposed by the advent of jaws. Jaws ulti-mately allowed vertebrates to access the ecologicalrole of the active predator and representatives ofPlacodermi offer the most primitive examples ofactive vertebrate predation. Gans and Northcutt(1983) proposed that evolutionary changes in feed-ing habits correlate with evolutionary changes ingrowth rate. We may hypothesize then that thegrowth adaptations observed in the skeleton of B.canadensis are correlated to a shift in growth rateat the origin of jawed vertebrates. Certainly, suchhypotheses will remain tenuous as long as placo-derm systematics remain unresolved and untilreaching a better understanding of the skeletalanatomy in other placoderms and in more basalstem gnathostomes.

Is the placoderm dermal skeleton represen-tative of basal crown gnathostomes? It is clearfrom this description of Bothriolepis canadensisthat the dermal skeleton of placoderms is moredirectly comparable to both the extinct ‘‘ostraco-derms’’ (armored, jawless stem gnathostomes) andosteichthyans, than to chondrichthyans. Indeed,the placoderm and osteichthyan dermal skeletonsare most closely comparable given their divisioninto component cranial dermal plates (Graham-Smith, 1978), in comparison to the fused head cap-sule of the ‘‘ostracoderms.’’ Given existing hypothe-ses on the evolutionary relationships of thesegroups (Janvier, 1996), the dermal skeleton of thelast common ancestor of crown gnathostomeswould have been more comparable to the conditionseen in placoderms and osteichthyans, than to ei-ther stem or crown chondrichthyans. Thus, asRomer recognized of the skeleton in general andthe dermal skeleton in particular, the condition inchondrichthyans is highly derived and should notserve as a model for understanding skeletal evolu-tion (Romer, 1942). It is therefore unfortunate thatchondrichthyans have been so influential in thesynthesis of hypotheses of dermal skeletal develop-mental evolution among vertebrates more gener-ally (Stensio, 1961; Ørvig, 1951, 1968, 1977; Reifand Richter, 2001; Donoghue, 2002; Reif, 1980,1982, 2002; Sire et al., 2009). The problem is thathypotheses such as the odontode regulation theory,and the lepidomorial theory from which it isderived, address only the odontogenically-derivedcomponent of the dermal skeleton, which is char-acteristic of the dermal skeleton of chon-drichthyans, but represents only a relatively minorcomponent of the dermal skeleton of the majorityof early skeletonising vertebrates, and is otherwiseonly rarely manifest in living jawed vertebrates(Donoghue and Sansom, 2002). Future attempts toderive an holistic model for interpreting dermoske-letal developmental evolution must focus on inte-grating the skeletogenically-derived component.



This may be realized through molecular investiga-tion of scale induction and patterning in teleosts(Sire and Akimenko, 2004), Polypterus, Latimeriaand lungfishes.

CONCLUSIONS

In summary, tests of phylogenetic consistency,morphology, topology, and comparative anatomysupport the conclusion that the external skeletonof Bothriolepis canadensis lacks dental tissues andis comprised exclusively of cellular dermal bonetissue. Despite variation in the organization of thebone tissues, the data support the identification ofeach according to the bone tissue classificationscheme of de Ricqles (1975) and de Ricqles et al.(1991). The stratification of the external thoracicskeleton therefore does not suggest fusion betweendermal and endoskeletal systems, but is ratherassociated with the overlapping nature of thearticulations between individual elements. Discov-ery and confirmation of certain skeletal features inB. canadensis, once thought to be gnathostomeinnovations, demonstrate an earlier origin. Withinvertebrate phylogeny, B. canadensis exhibits themost basal examples of secondary osteon develop-ment as well as a systematic program of skeletalremodeling through the resorption and secondarydeposition of mineralized tissue. The presence ofspheritic mineralization in the dermal skeletonand its association with zones of secondary min-eral deposition indicate the potential for rapidgrowth and skeletal remodeling.

Whether placoderms represent the sister groupof crown gnathostomes (Young, 1986; Goujet andYoung, 1995; Janvier, 1996) or a paraphyly of stemgnathostomes (Johanson, 2002; Brazeau, 2009),the skeletal similarities between placoderms andosteichthyans suggest that it is these groups, andnot chondrichthyans, that can best inform theprimitive condition of the crown gnathostomeskeleton.

The anatomical survey of histological microstruc-ture that supports the interpretations of this workis the first for an antiarch placoderm. The evidenthistological similarities between the dermal skele-ton of Bothriolepis canadensis and those ofosteichthyans and basal stem gnathostomes high-light the problem with assuming a broad applica-tion for those hypotheses of dermoskeletal evolu-tion that were based strictly on a chondrichthyanmodel. By addressing only the odontogenetic com-ponent of the dermal skeleton, hypotheses like thelepidomorial and odontode regulation theories donot account for the skeletogenetic component thatcomprises the majority or entirety of the dermalskeleton in most skeletonizing vertebrates.

The controversies that have long surrounded theinterpretation of the antiarch dermal skeletondemonstrate that histological data require anatom-

ical and ontogenetic context if they are to supporttissue interpretations. With this perspective, histo-logical examinations of additional clades of basalvertebrates will generate considerable progress to-ward resolving our understanding of the phylog-eny of vertebrate skeletal tissues.

ACKNOWLEDGMENTS

The acquisition of specimens for this studyrelied on the generosity of Sylvain Desbiens andJohanne Kerr at the Musee d’Histoire naturelle deMiguasha. The authors are indebted to MarilynFox and the laboratories of Jay Ague, Ruth Blake,James Eckert, Shun-ichiro Karato, and Danny Ryefor the guidance and equipment necessary to per-form the histological sample preparations. SimonPowell produced the block model of Bothriolepiscanadensis dermal skeletal histology. The authorsgratefully recognize Edward Daeschler, JacquesGauthier, Richard Lund, John Lundberg, Armandde Ricqles, Mark Sabaj Perez, and H. CatherineSkinner for useful discussions and suggestions.The first author was financially supported by anNSF graduate research fellowship.

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