Biology of turtles.pdf

Biology of Turtles

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Biology of Turtles

Edited by

Jeanette WynekenFlorida Atlantic University

Boca Raton, FL, U.S.A.

Matthew H. GodfreyNorth Carolina Wildlife Resources Commission

Beaufort, NC, U.S.A.

Vincent BelsMusum of National D Histoire Naturelle

Paris, France

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Cover photos by Stephen L. Barten, D.V.M., Ann C. Burke, and Jeanette Wyneken.

CRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 334872742

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Library of Congress CataloginginPublication Data

Wyneken, Jeanette, 1956Biology of turtles / Jeanette Wyneken, Matthew H. Godfrey, Vincent Bels.

p. cm.Includes bibliographical references and index.ISBN 9780849333392 (alk. paper)1. Turtles. I. Godfrey, Matthew H. II. Bels, V. L. (Vincent L.) III. Title.

QL666.C5W96 2007597.92dc22 2007024320

Visit the Taylor & Francis Web site athttp://www.taylorandfrancis.comand the CRC Press Web site athttp://www.crcpress.com

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Contents

Preface............................................................................................................................................viiAbout the Editors............................................................................................................................ixContributors....................................................................................................................................xi

Chapter 1 HowtheTurtleGetsItsShell.............................................................................................................1Scott F. Gilbert, Judith A. Cebra-Thomas, and Ann C. Burke

Chapter 2 ComparativeOntogeneticandPhylogeneticAspectsofChelonianChondro-OsseousGrowthandSkeletochronology....................................................................................................... 17Melissa L. Snover and Anders G.J. Rhodin

Chapter 3 EvolutionandStructureoftheTurtleShell..................................................................................... 45Peter C.H. Pritchard

Chapter 4 LongBoneAllometryinTortoisesandTurtles...............................................................................85Gustavo A. Llorente, Xavier Ruiz, Adri Casinos, Ignacio Barandalla, and Carles Viladiu

Chapter 5 EvolutionofLocomotioninAquaticTurtles...................................................................................97Sabine Renous, France de Lapparent de Broin, Marion Depecker, John Davenport, and Vincent Bels

Chapter 6 HindlimbFunctioninTurtleLocomotion:LimbMovementsandMuscularActivationacrossTaxa,Environment,andOntogeny................................................................................................ 139Richard W. Blob, Angela R.V. Rivera, and Mark W. Westneat

Chapter 7 CervicalAnatomyandFunctioninTurtles................................................................................... 163Anthony Herrel, Johan Van Damme, and Peter Aerts

Chapter 8 FunctionalEvolutionofFeedingBehaviorinTurtles.................................................................... 187Vincent Bels, Sabine Baussart, John Davenport, Marc Shorten, Ruth M. ORiordan, Sabine Renous, and Julia L. Davenport

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i Contents

Chapter 9 TheStructureofCardiopulmonarySystemsofTurtles:ImplicationsforBehaviorandFunction.................................................................................................................................. 213Jeanette Wyneken

Chapter 10 ReproductiveStructuresandStrategiesofTurtles........................................................................225Jeffrey D. Miller and Stephen A. Dinkelacker

Chapter 11 MixedandUniformBroodSexRatioStrategyinTurtles:TheFacts,theTheory,andTheirConsequences................................................................................................................................. 279Vincent Hulin, Marc Girondot, Matthew H. Godfrey, and Jean-Michel Guillon

Chapter 12 ThePhysiologyandAnatomyofAnoxiaToleranceintheFreshwaterTurtleBrain..................... 301Sarah L. Milton

Chapter 13 TheRelationshipsofTurtleswithinAmniotes.............................................................................. 345Olivier Rieppel

Index .............................................................................................................................................. 355

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ii

PrefaceThefirstbeaststhatweidentifyasturtlesemergedabruptlyintheTriassicabout220millionyearsago.Sincethen,countlessdiverselineagesofturtlesterrestrial,marine,freshwater,andin-betweenhavecomeandgone,yetmanylineagespersistfortensofmillionstomorethan100millionyears.Allbringwiththemthesuiteoftraitsthatareuniquelyturtle:abonyshell,usuallyaretractableneck,lackoftrunkmusculature,andlimbgirdleslocatedinsidetheribcage(insteadoflyingoutsideofit).Thereptilesweidentifyasturtles,ortestudines,orchelonians,havecaptivatedmanynaturalistsandotherscientistsbecauseoftheseandotheruniquetraitsandtheirconsequences.

Some180yearsagowhileinJena,Germany,LudwigHeinrichBojanusdevelopedaninterestintheanatomyoftheEuropeanturtleEmys obicularis.Bydissectingspecimensandillustratingtheir anatomy over the next decade, Bojanus authored one of the most detailed studies on anysinglevertebratespecies,Anatome Testudinis Europaeae(18191821).Thisunparalleledbookonturtleanatomyrepresents the intersectionofhis interestsasanaturalist,comparativeanatomist,veterinarysurgeon,andteacherofanatomicalart.Itwasbecauseofthishistoricalbackdropthatthebiology,particularlytheanatomy,ofturtleswasvisitedagainfittinglyattheSixthInternationalCongressofVertebrateMorphology,whichwasheldinJenain2001.Thesymposiumanditsmanycontributedpapersservedasthefoundationuponwhichthisvolumeisbased.However,thisbookgoesbeyondamorphologicallybasedsymposiuminrecognizingthatthestructuresofturtlesareparticularlyinterestingandbestunderstoodwithinthecontextoftheirformation,theirdiversityoffunctions,theirnovelty,andtheirevolution.Whereasseveralareasofinterest(e.g.,turtlegenetics,sensorysystemsandbehavior,andlifehistoryevolution)arenotincludedasthesefieldscontinuetoprogressrapidlywithoutaclearstablepointwehavebroughttogetherawiderangeofdiscus-sionsonothernovelfeaturesofturtles.

Forthefirsttimeanywhere,wehavearobustdiscussionabouttheorigins,development,anddiversityoftheshellinchaptersbyGilbertetal.(Chapter1)andPritchard(Chapter3).SnoverandRhodin(Chapter2)synthesize the importantworkonbonegrowthandaging,whileLlorenteetal.(Chapter4)bringforwardanunderstandingoflimbbonestrengthinthisuniquegroup.Turtlelocomotionisuniqueinmanywaysbecauseofthepresenceoftheshell.Renousetal.(Chapter5)provideanovelsynthesisofthefieldwhileBlobetal.(Chapter6)presentanexperimentalandfunc-tionalperspectiveonthemotorpatternsusedbyturtlesduringlocomotion.TheretractableneckisexpertlydescribedbyHerreletal.(Chapter7)andgivenfunctionalcontextbeyondthesimpleviewofprotectingthehead.Belsetal.(Chapter8)provideafunctional,anatomical,andbehavioraloverviewoffeedinginherbivorousversuscarnivoroustypesandaquaticversusterrestrialturtlesthathasneverpreviouslybeensummarized.Wyneken(Chapter9)discussescardiopulmonaryanat-omyandfunctionfromfunctionalperspectives.Reproductivestrategiesreceiveathoroughover-viewbyMillerandDinkelacker(Chapter10).Hulinetal.(Chapter11)challengethereadertothinkrigorouslyintheiranalysisoftheconsequencesofenvironmentalsexdeterminationandturtlesexratios,andMilton(Chapter12)remindsusthatatleastsometurtles(particularlytheaquaticspe-ciesthathibernateunderwater)haveanumberofveryuniquestructuralandfunctionaladaptationsfortoleratinglowoxygenlevels.Rieppel(Chapter13)examineswhytheoriginofturtlesandtheirrelationshipstootheramniotesissuchagreatchallenge.Despitemorethanacenturyofintensivestudy,ourunderstandingofthephylogeneticoriginsofturtlesremainsindefinite.Thischapterpar-ticularlychallengesustolookbothinsideandoutsidetheshellaswebuildanunderstandingoftheevolutionofturtles.Thischallengeextendsacrossallfieldsrepresentedinthisvolumeandthoseyettocome.

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iii Preface

Theeditorsthankthemanyexternalrefereeswhorespondedtoourrequestforarigorousreviewofeachchapter.WearegratefulforthecollegialenvironmentinJena,Germany,andattheSixthInternationalCongressforVertebrateMorphology,whichstimulatedtheideasandtopicsforthissynthesis.Particularly,weacknowledgeMatthiasStarckforhisencouragementandsupportofthesymposiumTurtles:FromStructurestoStrategiesofLife,andtothelatePeterLutzforaper-spectiveonthehistoryofthescienceandthemanynovelintegrativeapproachesthathaveledtounderstandingthebiologyofturtles.JohnSulzyckiandDavidFauselatCRCPress/Taylor&Fran-cisprovidedessentialguidance,superbadvice,andgoodhumorasthisbookprogressedataturtlespace.

Jeanette WynekenBoca Raton, Florida, USA

Matthew H. GodfreyBeaufort, North Carolina, USA

Vincent BelsParis, France

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ix

AbouttheEditorsJeanette Wyneken, Ph.D.,isanassociateprofessorofbiologicalsciencesatFloridaAtlanticUni-versityinBocaRaton.SheearnedherB.A.fromIllinoisWesleyanUniversityandlater,herPh.D.inbiologyfromtheUniversityofIllinoisin1988.Shewasaresearchassociatefrom1988to1989attheUniversityofIllinoisbeforetakingaresearchpositionatFloridaAtlanticUniversityin1990.Shelaterassumedapositionasassistantprofessor,thenassociateprofessorofbiologicalsciencesatFloridaAtlanticUniversity.Dr.Wynekenisacomparativeandfunctionalanatomistandaconserva-tionbiologist.Herstudiesarediverseanddealwithgrowth,energetics,migratorybehavior,devel-opment,includingenvironmentalsexdetermination,andmedicalimagingofreptiles.Inadditiontoteachingvertebrateanatomyanddevelopment,shedevelopedandtaughttheBiologyofSeaTurtlescourseofferedatHarborBranchOceanographicInstituteinFortPierce,Florida,andworkedwithSelinaHeppellandLarryCrowdertodevelopasimilarconservation-basedseaturtlebiologycourseattheDukeUniversityMarineLaboratoryinBeaufort,NorthCarolina.SheistheformerpresidentoftheAnnualSeaTurtleSymposium(nowTheInternationalSeaTurtleSociety),theconvenerofthe7thInternationalCongressforVertebrateMorphology,andhasorganizedseveralsymposiaonvariousaspectsofthebiologyofturtles.

Dr. Wyneken serves on the editorial boards of two professional journals in her field and isactiveinthepeerreviewprocessforanumberofotherjournals.Sheisamemberofseveralprofes-sionalorganizationsincludingtheAAAS,AssociationofIchthyologistsandHerpetologists,Societyfor theStudyofAmphibiansandReptiles,HerpetologistsLeague,AssociationofReptilianandAmphibianVeterinarians,theInternationalSeaTurtleSociety,SigmaXi,theSocietyofIntegra-tiveandComparativeBiology,and theIUCNMarineTurtleSpecialistGroup.Dr.Wynekenhasauthoredmorethan30peer-reviewedpapers,fourbookchapters,onebook(The Anatomy of Sea Turtles),andco-editedThe Biology of Sea Turtles, Volume 2.

Matthew H. Godfrey, Ph.D., is a biologist with the North Carolina Wildlife Resources Com-missionandanadjunctassistantprofessorattheNicholasSchoolfortheEnvironmentandEarthStudies at Duke University, North Carolina. Dr. Godfrey received a B.A. (1991) in history andphilosophyfromtheUniversityofToronto.HewentontoreceiveaM.Sc.(1994)andPh.D.(1997)inzoologyfromtheUniversityofTorontoin1997.Between1997and2002,Dr.GodfreyworkedasaresearchfellowatProjetoTAMAR-IBAMAinBrazilandasaresearcher/lectureratUniversitParis,France.Hisresearchtopicsincludebehavioralandevolutionaryecologyofreptiles,withafocusontemperature-dependentsexualdifferentiation.Dr.Godfreyisalsointerestedinthecon-servationofprotectedspeciesanditslinkstosocialjustice.Hehasauthoredorco-authoredmorethan40researchpapersandfivechaptersineditedbooks.Dr.Godfreyiscurrentlyco-editoroftheMarine Turtle Newsletterandservesontheeditorialboardoftwootherscientificjournals.HeisalsoamemberoftheIUCNMarineTurtleSpecialistGroupandservesonthreenationalandtwostatescientificadvisorycommitteesconcernedwithprotectedspeciesmanagementandconservation.

Vincent Bels, Ph.D.,isprofessorattheMusumNationaldHistoireNaturelle(Paris,France).HeobtainedhisB.A.andhisdoctoratdEtatfromtheUniversityofLige(Belgium)in1989.Dr.BelsbeganhisworkinethologyattheUniversityofLigeandcultivatedhisinterestincomparativeandfunctionalmorphologyofvertebrates.HeisnowassociatedirectoroftheresearchteamUMR7179Mcanismes adaptatifs: des organismes aux communauts (CNRSMNHN Univ. Paris 6 College de France) at the Department Ecologie et Gestion de la Biodiversit at the MusumNationaldHistoireNaturelle. InadditiontoteachingvertebratefunctionalmorphologyattheUni-

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x AbouttheEditors

versityofMons (Belgium)and theMusum(mastersdegreeprogramanddoctoral school),Dr.Belss studies concern a large variety of lower vertebrates from a comparative, functional, andevolutionarypointofview.Hismainworkfocusesonfeedingbehaviorinsquamatesandturtles.Hehasstudiedlocomotorbehaviorinanumberoflowervertebratesincludingfishes,crocodiles,andmarineturtles.Hehasalsoinvestigatedbehavioralandfunctionalmechanismsofbehaviorsinvolvedincommunicationinsquamates.Heisactiveinthepeer-reviewprocessforanumberofjournals.Dr.Belshasauthoredmorethan50peer-reviewedpapers,fivebookchapters,andeditedorco-editedthreebooksonthefunctionalandevolutionarybiologyofvertebrates.

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xi

Contributors

Peter AertsDepartmentofBiologyUniversityofAntwerpAntwerp,Belgium

Ignacio BarandallaDepartmentofAnimalBiologyUniversityofBarcelonaBarcelona,Spain

Sabine BaussartDpartementEcologieetGestiondela

BiodiversitMusumNationaldHistoireNaturelleParis,France

Vincent BelsDpartementEcologieetGestiondela


Richard W. BlobDepartmentofBiologicalSciencesClemsonUniversityClemson,SouthCarolina,USA

Ann C. BurkeBiologyDepartmentWesleyanUniversityMiddletown,Connecticut,USA

Adri CasinosDepartmentofAnimalBiologyUniversityofBarcelonaBarcelona,Spain

Judith A. Cebra-ThomasBiologyDepartmentMillersvilleUniversityMillersville,Pennsylvania,USA

John DavenportDepartmentofZoology,EcologyandPlant

ScienceEnvironmentalResearchInstituteUniversityCollegeCorkCork,Ireland

Julia L. DavenportDepartmentofZoology,EcologyandPlant


Marion DepeckerDpartementEcologieetGestiondela


Stephen A. DinkelackerDepartmentofBiologyUniversityofCentralArkansasConway,Arkansas,USA

Scott F. GilbertBiologyDepartmentSwarthmoreCollegeSwarthmore,Pennsylvania,USA

Marc GirondotLaboratoiredesReptilesetAmphibiensMusumNationaldHistoireNaturelleParis,France

Matthew H. GodfreyNorthCarolinaWildlifeResources

CommissionBeaufort,NorthCarolina,USA

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xii Contributors

Jean-Michel GuillonLaboratoireEcologie,Systmatiqueet

EvolutionFacultdesSciencesdOrsayUniversitParisSudOrsay,France

Anthony HerrelDepartmentofBiologyUniversityofAntwerpAntwerp,Belgium

Vincent HulinLaboratoireEcologie,Systmatiqueet

EvolutionFacultdesSciencesdOrsayUniversitParisSudOrsay,France

France de Lapparent de BroinDpartementHistoiredelaTerre,

PalobiodiversitMusumNationaldHistoireNaturelleParis,France

Gustavo A. LlorenteDepartmentofAnimalBiologyUniversityofBarcelonaBarcelona,Spain

Jeffrey D. MillerDepartmentofBiologyUniversityofCentralArkansasConway,Arkansas,USA

Sarah L. MiltonDepartmentofBiologicalSciencesFloridaAtlanticUniversityBocaRaton,Florida,USA

Ruth M. ORiordanDepartmentofZoology,EcologyandPlant


Peter C.H. PritchardChelonianResearchInstituteOviedo,Florida,USA

Sabine RenousDpartementEcologieetGestiondela


Anders G.J. RhodinChelonianResearchFoundationLunenburg,Massachusetts,USA

Olivier RieppelDepartmentofGeologyFieldMuseumofNaturalHistoryChicago,Illinois,USA

Angela R.V. RiveraDepartmentofBiologicalSciencesClemsonUniversityClemson,SouthCarolina,USA

Xavier RuizDepartmentofAnimalBiologyUniversityofBarcelonaBarcelona,Spain

Marc ShortenDepartmentofZoology,EcologyandPlant


Melissa L. SnoverNationalOceanicandAtmospheric

Administration(NOAA)NationalMarineFisheriesServicePacificIslandsFisheriesScienceCenterHonolulu,Hawaii,USA

Johan Van DammeDepartmentofBiologyUniversityofAntwerpAntwerp,Belgium

Carles ViladiuDepartmentofAnimalBiologyUniversityofBarcelonaBarcelona,Spain

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Contributors xiii

Mark W. WestneatDepartmentofZoologyFieldMuseumofNaturalHistoryChicago,Illinois,USA

Jeanette WynekenDepartmentofBiologicalSciencesFloridaAtlanticUniversityBocaRaton,Florida,USA

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1 HowtheTurtleGetsItsShellScott F. Gilbert, Judith A. Cebra-Thomas, and Ann C. Burke

Ifitwerentforthe250speciesofturtleslivingtodaytheseanimalsencasedinmobilehomescouldeasilybeviewedasbizarreevolutionaryexperimentsthatwereordainedtofailure.

Richard Ellis (2003)

Contents

1.1 TheNatureoftheTurtleShell................................................................................................11.1.1 IntroductiontotheTurtleShell....................................................................................11.1.2 AnatomyoftheTurtleShell.........................................................................................2

1.2 TheFormationoftheCarapacialBones:HeterotopyandParacrineFactors.........................21.2.1 TheDermalBonesoftheCarapace.............................................................................21.2.2 FormationoftheCarapace..........................................................................................4

1.2.2.1 TheCarapacialRidgeandtheEntryoftheRibsintotheDermis.................41.2.2.2 CostalBones:TheOssificationoftheCarapace............................................51.2.2.3 TheNuchalandPeripheralBonesoftheCarapace.......................................6

1.3 TheFormationofthePlastronBones:HeterochronyandNeuralCrestCells........................81.3.1 DermalBonesofthePlastron......................................................................................81.3.2 OssificationofthePlastron..........................................................................................8

1.3.2.1 DevelopmentofthePlastronBones...............................................................81.3.3 RolesofNeuralCrestCellsinPlastronandNuchalBoneDevelopment.................. 10

1.4 EvolutionaryImplications..................................................................................................... 12Acknowledgments............................................................................................................................ 13References........................................................................................................................................ 13

. thenatureoftheturtleshell

1.1.1 IntroductIontotheturtleShell

TheturtleshellisaremarkableevolutionarynoveltythatdefinestheorderChelonia.Theturtleshellisfoundinthreegeneralformsbasedonthenatureanddegreeofossification:hardshells,softshells,andleatherbacks.Thissectionwillconcentratealmosttotallyonthebonycomponentofthoseshellsof thehardbackturtlesof theEmysandChelydaefamilies.Thisshell iscomposedof twomainparts,thedorsalcarapaceandtheventralplastron,connectedalongthemidflanksbylateralbridges.Altogether,theshellcontainsover50dermalbonesthatarehomologoustonootherboneinanyothervertebrateorder.Moreover,thepresenceofthisbonycasinghasnecessitatedextensivemodi-ficationsofthetetrapodbodyplan(Zangerl,1969).Whereasdermalossificationitselfisaprimitivecharacterforvertebrates(Smith&Hall,1993),theturtleshellrepresentsanextremedevelopmentofthedermalskeletonamongtetrapods.

Theshellclearlyhasadaptivevalueforturtlesasphysicalprotection,butitalsoservesphysi-ologicalfunctionsindifferentspeciesasasiteofhematopoiesis,areservoirforwater,fat,orwastes,

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BiologyofTurtles

andabufferforpH.Theembryonicdevelopmentoftheshellinvolvesadramatichypertrophyofthedermisinthedorsalbodywallandaresultantrearrangementofthetypicalrelationshipbetweenthepectoralgirdleandtheaxialskeleton.Thus,turtlesaretheonlyvertebrateswhoselimbsarefounddeeptotheribs.Theparaxialandlimb-girdlemusculaturetheneckandskullarealsogreatlymodified.Aswedetailhere, thekey innovation for thecheloniansappears tobe thecarapacialridge,abulgeofectodermandmesodermthatinfluencesthegrowthoftheribs(Burke,1989a).Theribsareenvelopedwithinthedorsaldermis,resultingintheirlateraldisplacementasthedermisrapidlyexpands.Thusinsteadofextendingventrallyandenclosingthethoraciccavity,theturtleribsbecomeintegratedintothecarapacialdermis.Theneuralarchesofthevertebraealsofusewiththemidlineofthecarapace.Astheanonymousauthor(1676)ofthelettertotheRoyalSocietyofLondonwrotein1676:

TheAnatomieofaTortoise,showingthatwhatweretheRibsinotherAnimalsoneupperShellisintheTortoise,andthattothatupperShellarefirmlyfastenedthespinalVertebrae,sothattheAnimalcannotgooutofitsHome,asSnailsdo.

1.1.2 AnAtomyoftheturtleShell

Thecharacterandhomologyofthebonyelementsoftheturtleshellhavealonghistoryofcontro-versy.Theshelliscomprisedoftheendochondralaxialelementsofthetrunkoverlaidbyamosaicof dermal bones and an outer epidermal layer made of keratinous scales (also called scutes orshields).All turtlespossess10 trunkvertebraeassociatedwith thecarapace.Eachvertebrapos-sessesasingle-headedribthatoftensharesanarticulationwiththenextanteriorvertebra.Thefirstandtenthribsarediminutiveandnormallyextendashortdistancebeforemakingcontactwiththesecondandninthribs,respectively.Thetenthribisoftenindistinguishableinbothembryosandadults,butthepresenceofalargetenthribinembryosisanormalvariation.Thethoracicribsenterthedermisoftheshellashortdistancefromtheirarticulationwiththevertebrae,andtheyextendlaterallywithinthecarapacialdermis,terminatingattheperiphery(reviewedbyZangerl,1969).

Inthedermallayeroftheshell,therearegenerally59bones:thecarapacehas38pairedand12or13unpairedbones(sometimesthesuprepygealboneisdividedandsometimesitisnot).Theplastroncontainsoneunpairedandeightpairedbones.Withtheexceptionofafewkeytaxa,theonlyrealvariationsinthisgeneralschemeoccurasindividualvariationsaroundtheneckandtailwheretheaxialskeletonisnotcloselyjoinedtothecarapace.Theshapesandrelativesizesofthebonesdeterminethegeneralformoftheshellindifferentgenera.

Theshellsepidermallayergenerallyconsistsof38scutesinthecarapaceand16intheplastron.However,thiscanvarydependingontheshapeoftheshell(domed,hinged,flapped,andsoon;seeChapter3).Theshieldandbonepatternsarenotinregister;eachshieldcoversaparticularareaofthebonymosaic.Thepatternofthesulcithatformbetweenneighboringscutesandthesuturesthatformbetweenneighboringbonesformtwominimallyoverlappingpatterns.Theepidermalshieldpatterndevelopslongbeforetheshellbonesbegintoossify,andtheunderlyingdermismayplayamajorroleintheformationoftheepidermalscutes,similartotheinfluenceofsomiticdermisoffeatherpatternsinthechick(Yntema,1970;Cherepanov,1989;Alibardi&Thompson,1999a,b).

. theformationoftheCarapaCialBones: heterotopyandparaCrinefaCtors

1.2.1 thedermAlBoneSofthecArApAce

The unpaired midline dermal bones of the carapace, called neurals, are fused with the neuralspinesofthe10thoracicvertebrae(Figure1.1).Thecostalbonesextendfromtheneuralstowardtheperiphery.Thereareeightpairsandeachisintimatelyassociatedwitharib(Figure1.1E).Gen-erally,thereisaone-to-onecorrespondencebetweenthevertebralspinesandtheneuralbones,and

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HowtheTurtleGetsItsShell

betweentheribsandthecostalbonesofthecarapace.Thisrelationshipdoesnotholdintheanteriorandposteriorendsoftheshell,wherethevertebralcentraareshortenedandhavelittleornocontactwiththeshell.Thefirstcostalboneoverliesribsoneandtwo,andtheeighthoverliesribsnineandten(variantshaveninepairsofcostalbones).Thepygalandsuprapygalbonesformtherearofthecarapace.Theseboneshavenocontactwithvertebraandribsbutprojectoverthesacrumandpel-vis.Theperipheralbonesformtheedgeofthecarapace.Therearegenerally11pairsofperipheralbones;beforemakingcontactwiththecostals,theyformasocketaroundthedistaltipofribstwothroughnine.Thenuchalboneformstheanteriormarginofthecarapace,whichoverhangsbutisnotattachedtotheposteriorcervicalvertebra.Thisboneextendslaterallyaroundthemarginsofthecarapacetothelevelofthesecondrib.Itisoverlaidbythefirstthreeperipheralboneslaterallyandcontactsthefirstcostalsandneuralboneposteriorly.Eachofthecarapacialbonesisconnected

peripheralneural

nuchal

costal

pygialsuprapygial

11223344556

67

7 8

8

figure. Developmentof thecarapace. (A).Entryofcartilaginous ribprecursor (arrow) intocarapa-cialridgeofTrachemysembryoaroundstage16.ThefollowingshowboneformationinTrachemys scripta,stainedwithAlcianblue(cartilage)andalizarinred(bone).(B)1.2-cmembryoshowingcartilaginousribsformingtheoutlineoftheshell.(C).Ventralviewof3.1-cmcarapace,showingintramembranousossificationofthenuchalboneandaroundandintheanteriorribs.(D)Lateralviewofthesamecarapace,showingregionofribchondrogenicgrowth(blue,arrow)andtransitionzone(white)betweencartilageandbone(red).(E)Dorsalviewof118-day(CL=3.1cm)hatchlingcarapaceshowingexpandednuchalboneregion,thefusionof theanteriorcostalossificationcenters,and theperipheralboneossificationcenters thatstartanteriorly.Thepigmentationoftheepidermalscutescanbeseen.(F)Dorsalviewof185-day(CL=4.5cm)hatchlingcarapaceshowingfusionofmarginalossificationregionsanteriorly,aswellasthepygalossificationcenterposteriorly.Thecostalossificationcentershavecreatedbonyarmordorsally(thebluestainingisbeneaththecarapace).(G)Predominantpatternoftheadultcarapacialbones.(ModifiedfromGilbertetal.,2001;Gmodi-fiedfromZangerl,1969.)

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BiologyofTurtles

bysuturestoitsneighbors.Thedistaledgeofeachcostalisattachedbysuturetotheperipheralbones.Thiscontactoftendoesnotoccuruntillaterstagesofpost-hatchinggrowth,leavingopenaperipheralringoffontanelsthatsurroundthedistaltipsoftheribs.

Sectionsacrossthecarapacesofadult turtlesshowathree-layeredarrangementofthebone.Thecentralportionoftheboneisaspongylayercontainingsphericalcavities.Oneithersideofthespongylayerarelayersofmorecompactlamellarbone.Thiscompactboneisthoughttoformbeneath the inner and outer periosteal membranes. The shapes and relative sizes of these bonyregionsdetermine thegeneral formof theshell indifferentgenera(Yntema,1970;Ewert,1985;Cherepanov,1997).

1.2.2 formAtIonofthecArApAce

...theCarapacialridgeandtheentryoftheribsintothedermis

Theformationofthecarapaceinvolvesseveralsteps.Thefirstconcernstheentryoftheribprecur-sorcells intothedermis.Theturtleeggis laidat themid-gastrulastage.Turtlegastrulationhasnotbeenstudiedindetailforalmosteightdecadesandpresentsaninterestingcontrasttothewell-studiedaviansystem(seereview;Gilland&Burke,2004).Laterstagesofnerulationandsomiteformationaresimilartothoseprocessesinthechick(Ewert,1985;Pasteels,1937,1957).ThefirstsignthattheorganismistobecomeaturtleratherthansomeothertetrapodoccursatYntemastage14/Greenbaumstage15(Yntema,1968stagesareforChelydra;Greenbaum,2002stagesareforTrachemys.Stage14/15isapproximatelyequivalenttoHamburgerHamiltonchickstage24).Atthisstagearethefirstsignsofridgesonthelateralsurfacesoftheembryo,dorsaltothelimbbuds(Ruckes,1929).Atfirst,theseridgesareseenbetweenthetwolimbbuds,andonlylaterdotheridgesextendanteriorlyandposteriorly.Thisstructurehasbeennamedthecarapacialridge(CR)(Burke,1989b,1989c,1991),andthepairedcarapacialridgeswilleventuallyformtheouteredgeofthecarapace.TheCRisformedbyathickeningoftheectodermandisunderlaidbyacondensedsomite-derivedmesenchyme(Yntema,1970;Burke,1989b,1989c;Nagashimaetal.,2005).

Ruckes(1929)observationsofturtleembryosdescribedtwoimportantfeaturesofturtleshelldevelopment.First,thereisanacceleratedlateralgrowthofthedorsaldermisofthetrunkcomparedtogrowthinthedorso-ventralplane.Second,thereisanapparentensnarementofthegrowingribsbythedermis.Theinvolvementoftheribswiththecarapacialdermisresultsintheirgrowthinapredominantlylateraldirection(Figure1.1A).Thelimbgirdlesdevelopintypicaltetrapodfash-ionbutbecauseofthegrowthtrajectoryoftheribs,thepectoralgirdlebecomesventralanddeeptotheaxialelements.Yntema(1970)performedaseriesofsomiteextirpationexperimentsonsnappingturtles,confirmingasomiticoriginfortheribsanddermisofthecarapace.Post-oticsomitepairs12through21areinvolvedinformingthecarapaceinChelydra.

In1989,BurkeproposedthatthethickenedectodermandcondensedmesenchymeoftheCRistypicalofsitesofepithelial-mesenchymalinteractions.Thedistributionsofthecelladhesionpro-teinsfibronectinandN-CAMintheCRaresimilartotheirlocationsinotherinductivesitessuchastheearlylimbbudorfeatherprimordia.Burke(1991)testedthecausalrelationshipbetweentheCRandthegrowthtrajectoryoftheribs.Inthefirstsetofexperiments,sheremovedtheCRbytungstenneedlesfromonesideofstage1throughstage16embryos.Theseextirpationsincludedbothectodermalandmesenchymalcomponents.InthosecaseswheretheCRdidnotregenerate,thegrowthtrajectoryoftheribwasdeflectedtowardaneighboringregionthatdidhaveaCR.Inasec-ondsetofexperiments,sheplacedtantalumbarriersbetweenthesomiteandthepresumptiveCR.ThesurvivingembryosshoweddisruptionssuchthatwheretheCRwasinterrupted,entireregionsofthedermalcarapaceweremissing.Theribsassociatedwiththesemissingregionsinterdigitatedwiththosebonesoftheplastron.BurkeconcludedthatthenormaldevelopmentoftheribsappearstobedirectedbytheCR.IntheabsenceoftheCR,theseribsprojectventrallyintothelateralplatemesodermliketheribsofnon-Chelonianvertebrates.

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Loredoandcolleagues(2001)werethefirsttoanalyzetheCRwithmolecularprobesandfoundfibroblastgrowthfactor-10(FGF-10)expressioninthemesenchymecondensedbeneaththeTrache-mysCR.Fibroblastgrowthfactorsareparacrinefactorsthatarecriticalinthepatterning,migration,anddifferentiationofnumerouscell types,and theyareespecially important indetermining thefatesofcellsinthefaceandinthelimbs.Vincentandcoworkers(2003)foundtheturtlehomologueoftranscriptionfactormsx1isexpressedinthemesenchymeoftheEmysCR.ThisresultfurtheredthenotionthattheCRwasmadethroughmesenchymal/epithelialinteractionssimilartothosethatgeneratethelimbbud.TheWntsignalingpathwayisusedinseveralembryonicinductionsandcanmediatetheeffectsoffibroblastgrowthfactors(inthelimbbud).ByusingRT-PCR,Kurakuandcolleagues(2005)foundturtleorthologsofSp5andWnttargetsAPDCC-1andLEF-1intheCRmesenchymeandectodermoftheChinesesoftshellturtlePelodiscus.TheyalsofoundCRABP-1expressedintheCRectoderm.However,theydidnotdetecttheexpressionofeitheroftheprevi-ouslyreportedgenes,msx1,orFGF-10intheCRmesenchymeofthisspecies.SpeciesdifferencesmightbeimportantinthesepatternsbecausethecostalbonesofPelodiscusmightformbydifferentmethodsfromthatofthehardshellturtles(Zangerl,1969),andthepatternofFGF-10distributioninthelimbsofPelodiscusdifferedfromtheexpressionpatternseeninthelimbsofTrachemys.

TheFGFfamilyofparacrinefactorsisofteninvolvedinchemotaxis,andinthechicklimb,FGF-10appearstobecriticalindirectingtheendodermalchemotaxisinthelung(Parketal.,1998;Weaveretal.,2000).Cebra-Thomasandcolleagues(2005)demonstratedthatFGF-inducedchemo-taxisplaysanimportantroleincausingtheribprecursorstoentertheCR.Theyculturedeviscer-atedtrunkexplantsofstage15Trachemysembryosventral-sidedownonnucleoporemembranes.Atthisstage,theCRisvisibleandthesclerotomehasbeenspecified.Afterthreedaysinculture,theribshavemigratedintotheCR,andtheridgesarevisiblyraised.However,ifSU5402(aninhibitorofFGFsignaling)isaddedtotheculturemediawhentheexplantsareestablished,theCRdegen-eratesandtheribstravelventrally,liketheribsofnon-Chelonians.Cebra-ThomasandcolleaguesalsoshowthatchickribprecursorcellsareresponsivetoFGF-10,andbeadscontainingFGF-10willredirectchickribgrowthinculture.Thus,theCRappearstobecriticalfordirectingthemigrationofribprecursorcellsintoit.FGFsignalingintheCRappearstobecrucialinthemaintenanceoftheCRandiseitherdirectlyorindirectlyresponsibleforguidingtheribprecursorcellsintotheCR.

Another finding of Cebra-Thomas and colleagues (2005) was that the distal tip of each ribexpressedFGF-8.HighlevelsofFGF-8expressionhavenotbeenreportedinthedistalribsofotherorganisms.Cebra-Thomasandcolleagues speculate thatFGF-8 (in the ribs)andFGF-10 (in theCRmesenchyme)mayestablishapositivefeedbackloopsuchthatthegrowthoftheribbecomescoordinatedwiththegrowthofthecarapace.Suchapositivefeedbackloophasbeenshowntoberesponsibleforthecoordinatedoutgrowthofthechickandmouselimbbuds(Ohuchietal.,1997;Kawakamietal.,2001).

... CostalBones:theossificationoftheCarapace

Theribprecursorcells thatenter into theCRareprechondrocytes (Figure1.1A,B),and the ribsundergonormalendochondralossification,replacingthecartilagewithbonecells(Figure1.1C,D).Cebra-Thomas and colleagues (2005) have proposed that bone morphogenetic proteins (BMP),whicharesecretedbyhypertrophicchondrocytesduringendochondralossification,arecapableofinducingthedermistoossifyaswell.Thus,theyclaimthatcostalboneformationiscausedbytheBMP-dependentossificationofthedermisbytheribs.Theribprecursorcellsenterthedermisoftheshellashortdistancefromtheirorigininthevertebraeandgrowlaterallywithinthecarapacialdermis(Ruckes,1929;Burke1989b,1989c;Gilbertetal.,2001).Whenendochondralossificationtakesplace,theribisconvertedtobone,beginningattheproximalend(Figure1.1E).However,thedistalportionoftheribremainscartilaginousbeyondtheboundarybetweenpleuralandmarginalscutes,andtheydonotmakecontactwiththeperipheralbonesuntillaterinlife.Thereisanante-rior-posteriorpolarity,inthattheanteriorribsbeginossificationearlier.

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BiologyofTurtles

Asendochondralossificationensues,theribsappeartobecometheorganizingcentersforthecostalbones thatmake theplateof thecarapace (Gilbertetal.,2001).Thesecostalbones formaroundtheribsbyintramembranousossification(Burke,1991;Gilbertetal.,2001;Klin,1945).Thus,thecarapaceisacompositeofendochondralaxialskeleton(fromtheribs)plusintramem-branousdermalbone.Thecostalbonesbegintoformastheribsbecomeencasedinathintubeofbone,andtrabeculaeextendbothcaudallyandcraniallyfromthisbonycasing.Later,spiculesformbetweentheribandtheepidermis,formingapatternreminiscentoftheformationofthemandiblearoundMeckelscartilage(Suzuki,1963).Themostintenseareaofcostalboneformationisinitiallylocatedatthesiteswheretheribshadfirstenteredthedermis.

Bone-formingparacrinefactorsaresecretedbythecartilaginousribcellsduringendochondralossification.Inthosevertebratesstudiedthusfar(andtheturtleisnotoneofthem),Indianhedgehoghomolog(Ihh)secretedbytheribsprehypertrophiccartilageinducesBMPsintheperichondrium(Vortkampetal.,1996).Pathiandcolleagues(1999)demonstratedthatinchicklimbs,perichon-drialBMP-2,BMP-4,BMP-5,andBMP-7areinducedbyendogenousandectopicIhh.Similarly,Wuandcolleagues(2001)demonstratedtheinductionofBMP-2/BMP-4byIhhinchickjawtissue.BothIhhandBMPsareknowntoinduceboneformationinsurroundingcompetentcells(Barlow&Francis-West,1997;Ekanayake&Hall,1997),thecompetenceofdermalcellstorespondtoBMPsbyproducingintramembranousbonehasbeendemonstratedinadultdermalandperiostealtissues(Shafritzetal.,1996;Shoreetal.,2006).

Inturtleembryosandhatchlings,thedermalcellsaroundtheribappeartoberespondingtoBMPs.Thiswasshown(Cebra-Thomasetal.,2005)byusinganantibodyagainstphosphorylated(activated)Smad1.(TheSmad1proteinisatranscriptionfactorsubunitthatbecomesphosphory-latedinresponsetoaBMPsbindingtoitscellmembranereceptor.)Whereastheribanditsperi-chondriumremainunstained,therewasintensestainingintheperiosteumandinthecellsadjacenttoit(Figure1.2).Moreover,whencomparedtoalcianandalizarin-stainedadjacentsections(whichstaincartilagematrixandbonematrix,respectively),ahighlevelofstainingwasobservedinthecellsthatwereintheareadestinedtobecomebone.Thus,itappearsthatBMPsignalingfromtheribduringendochondralossificationisabletoinduceintramembranousossificationinthedermalcellssurroundingthem.Moreover,asthecellsossifytheyappeartotransmittheBMPsignaltothecellssurroundingthem,therebycontinuingacascadethroughwhichBMPwouldbeproducedbythedermalcellsastheyossify.

Althoughtheribsbegintoossifyin ovo,thedermalbonesofthecarapacedevelopprimarilyafterhatching.The ratesofosteogenesis, andperhaps to somedegree thepattern, is influencedbyenvironmentalconditions(Ewert,1985).Sizeandagearebothimportantparametersforbonepattern.Turtlesofthesameagecanbeatdevelopmentallydifferentstages,andthereissignificantvariationevenamongturtlesofthesamesize.Hatchingtimeisalsovariable,andembryosandjuve-nilespecimensaredescribedbytheircarapace length(CL)aswellastheiragesincetheeggwaslaid.ItisalsoprobablethatBMPinhibitorsinthedermisregulatetheprogressionofossificationbecausetheossificationfrontslowsdownandendochondralossificationintheribisfinishedlongbeforethefusionofthedermalbonesintoacarapacialplate(Figure1.1F).

Intheformationofthecarapace,oneseesheterotopy(changeinplacementbetweenancestoranddescendent)atseverallevels.Heterotopyofboneformationisobviousinthatthesebonesaredevelopinginthedorsaldermis,whichrepresentsanewsiteofboneformation.Thisheterotopyofboneformationispredicatedontheheterotopyoftheribs,whichhavemigratedintoapartofthebodywheretheydonotusuallygo.ThisribheterotopyisfurtherpredicatedontheheterotopyofFGF-10expression,whichisactivatedinatissuethatdoesnotusuallyexpressthisgene.

...thenuchalandperipheralBonesoftheCarapace

InChelydraandTrachemys,thenuchalboneshowstwodistinctphasesofossification.Werefertothesephasesasprimaryandsecondary,referringtoboththemodesofossificationandtheelements

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themselves(Burke,1989a;Gilbertetal.,2001).Thispatternofprimaryandsecondaryossificationisalsoseenintheplastronandmayhavephylogeneticsignificance.

TheprimaryportionoftheChelydranuchalformsearly(CL=1.4cm,Yntemastage2021),appearingasathinbandofcondensedcellswithinthedermis,continuousacrossthemidlineandextendinglaterallyaroundthemargintothelevelofthethirdmarginal.Thebandisvisibledeepinthedermisbeforethetissuestainswithalizarin,indicatingthatthewell-definedcondensationofcellsformswellbeforethedepositionofcalcium.Itunderliesthemarginal/vertebralsulci,whichisclearlyvisibleatthisstage.Asevidencedbypositivestainingwithalizarin,calciumdepositionstartsbilaterallyatthelevelofthefirstmarginalscuteandspreadsalongthebarsmediallyandlaterally.

The second phase of nuchal ossification involves the nuchal plate, which begins to form inChelydraembryosofCL=1.8cm.Thenuchalplateformsasalooselatticeworkofbonewithinthecarapacialdermisthatextendsforwardoverthebaseoftheneck.Thepatternofossificationisverysimilartothatseenintheinitialstagesofossificationintheskullroofingbones.Itbeginsincontactwiththeanterior-medialnuchalbarandextendslaterallyalongthebarandposteriorlyintothedermisabovetheneuralspinesofthelasttwocervicalvertebrae.Thisposteriorextensionofsecondarydermalboneformsthemainbodyofthenuchalandliesunderthefirstvertebralscute.Itwilleventuallyformasutureposteriorlywiththefirstneuralbone,whichdevelopsaroundtheneuralspineofthefirstthoracicvertebra.

InspecimensofCL=2.6cm,thenuchalisfullydevelopedandossified.Thelateralbarsoftheprimaryossificationextendtothemidpointofthefourthmarginalscute,tothelevelofcontactwith

figure. Formationofthecostalbonesofthecarapace.Sagittalsectionthroughtheposteriorthreeribsofa156-dayhatchlingTrachemys(aboutamonthafterhatching).Theribsareatdifferentlevelsofmaturity,theanterior(A)beingthemostmature.ThesectionsstainedwithHallstain(Alcianandalizarin)areneartotheslidesstainedwithantibodiestophosphorylatedSMAD1(PS1).NuclearexpressionofphosphorylatedSmad1(brown)isseenintheperiosteumoftheboneandintheimmediatelyadjacentdermalcells.Beloweachlow-power(200)isaphotographtakenat400magnification.

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BiologyofTurtles

thecartilaginousdistaltipofthesecondrib.Itunderliesthesulciseparatingthemarginalsfromthefirstvertebralandcostalscutes.Thelateralextensionsoftheprimarynuchalboneareneverinassociationwiththesecondarynuchalbone,butrathercometobeoverlainbythefirstandsecondperipheralbones.

Theperipheralbonesareformedinananterior-to-posteriormanner.Here,smallcrescentsofboneconcaveoutwardappearinthedermisontheextremeedgeofthecarapaceimmediatelysubjacent to the intermarginalsulci.Thefirstperipheralappearsunder thesulciof thefirst twomarginalscutes.Theossificationsthatproducetheperipheralbonesarealsoseentobegininthelargestofthenewhatchlings.Theperipheralossificationcentersarefirstseenintheanteriorofthecarapaceonday78Trachemys(CL=3.1cm)andastheturtlegrows,moreperipheralossificationcenterscanbeseencaudallyontheshell.Theseossificationcentersformontheouteredgeofthecarapaceandexpandbothlaterallyandinternallyastheygrow.Thepygalboneformsinsequenceasthelastperipheralandisthereforethelastbonetoossify.Itisnotknownwhatinducesthesecen-terstoformwheretheydo.Itispossiblethattheirpositioningiscoordinatedbythemarginalscutes,andthatsonichedgehog,whosegeneisexpressedinthemarginalscuteformingregion(Lewisetal.,2005)alsoinducesthebonetoformthere.

EvidencefromGilbertandCebra-Thomas(Gilbertetal.,2007)suggeststhatthenuchalbonemayformfromneuralcrestcells.Thisisalsoamechanismbeingproposedforplastronbonesandwillbediscussedlater.

. theformationoftheplastronBones: heteroChronyandneuralCrestCells

1.3.1 dermAlBoneSoftheplAStron

Theplastrongenerallyiscomposedofninebones,formedbyintramembranousossification(Fig-ure1.3)(Rathke,1848;Clarketal.,2001).Thepairedepiplastraandthecentral(unpaired)ento-plastronformthethreeanteriorbonesoftheplastron.Thehyoplastraformtheaxillarybuttressesandtheanteriorbridgeregion.Thebridgeextensionsofthesebonesapproachthecarapaceatthelevelofperipheralfiveandribfour.Thebilateralhyoplastrameeteachotherattheventralmidlineandformtheanteriorrimofthecentralumbilicalfontanel.Duringembryonicdevelopment,thisfontanelsurroundstheyolkstalkthatconnectstothegut.Thepairedhypoplastraformtheinguinalbuttresses,theposteriorbridgeregion,andtheposteriorrimofthecentralfontanel.Theyapproachthecarapaceatthelevelofperipheralssixandsevenandribsfiveandsix.Thepairedxiphiplastraformtheposteriorlobeoftheplastron.

1.3.2 oSSIfIcAtIonoftheplAStron

... deelopmentoftheplastronBones

Theplastronbeginstoossifybeforehatching.Intheembryonicturtle(CL=1.0cminTrachemys,CL=2.0cminChelydra),thefutureplastroncanbeidentifiedbynineossificationcentersintheventraldermis.NoAlcianbluestainingisseenpresagingthesesites.InTrachemys,thethreeossifi-cationcenterscorrespondingtothethreeanteriorplastronbonesappeartofusearoundday78(CL=2.2cm).Thetwoepiplastralbonesformasuturewithoneanother,whereastheentoplastronboneformsmoremediallyandprojectscaudally.Asthehatchlingturtlegetslarger,thesixpairedossifi-cationcentersoftheplastrongrowtowardoneanotherandformsutures.Condensedmesenchymeisseeninadvanceofthecalcifiedtissue(Burke,1989a;Gilbertetal.,2001).Thesesitescontainbothalizarinred-stainedbonyspiculesandaregionofcondensedmesenchymethathascoalescedintothestellatearraysthatwilllatershowstainingforbonematrix.Thisisanotherexampleofprimaryossification,asinthenuchal.

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Oneoftheinterestingthingsobservedaboutplastronossificationisthatthebonyspiculescrossthemidline.Themidlinedoesnotappeartoberespectedbythedevelopingspicules.Moreover,astheycrossedthemidlinethespiculesdidnotimmediatelyfuse.Rather,itappearsasiftheossifyingspiculesoneithersideavoidedoneanother,alteringtheircourseofossificationsuchthattheyinter-digitateratherthanrunintoeachother(Figure1.3E).Thisisverylikelyaprerequisiteforcontinuedgrowththroughsutureformation.

AsimilarsituationisseeninChelydra.Theplastralbonesappearwithaslightanterior-pos-teriorbias,theepiplastraandentoplastronfirstandthexiphiplastronlast.TheyareallpresentinspecimensofCL=1.5cm,precededonlybytheappearanceoftheprimarynuchalbar.Likethenuchalbone,theplastralbonesshowtwophasesofdevelopment.Theyfirstappearasslenderbarsofcondensedcellsthatthencalcifyfromtheircentersoutward.

Thecharacterandhomologyofthebonyelementsoftheplastronhasbeenextremelycontrover-sial(Hall,2001;Vickaryous&Hall,2006).In1834,Caruswasperhapsthefirsttosuggestthatthe

entoplastron epiplastron

hyoplastron

hypoplastron

xiphiplastron

figure. Dermalossificationoftheplastron.(A)55-day(CL1.0cm)Trachemysplastronshowingthethreeanteriorossificationcentersandthethreelaterallypairedossificationcenters.Thedarkbluerepresentsgirdlecartilage.(B)78-day(CL=2.2cm)plastronshowingspiculesradiatingfromtheossificationcenters.(C)78-day(CL=2.4cm)plastronshowingfusionoftheanteriorossificationcenters.(D)118-day(CL=3.1cm)plastronshowingepidermalpigmentationandthecrossingofthemidlinebythespicules.Thespiculesdonottouchbutgetoutofeachothersway.(E)185-day(CL=4.5cm)plastronshowingfusionofossificationcentersandtheformationofplastron.Nocartilageprecursorsareseen.Notethat(B)and(C)areboth78-dayincubations.Theholeinthecenteroftheplastronistheumbilicalfontanelthroughwhichthegutattachestotheyolkstalk.(F)Predominantpatternofplastronbones.(ModifiedfromGilbertetal.,2001.)

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0 BiologyofTurtles

carapaceandplastroninvolvedboththeendo-(endochondral)andtheexoskeletal(dermal)bones.Heproposedthattheplastronformedbyoverlyingtheendoskeletalsternumwithdermalossifica-tions.Rathke(1848)arguedthattheplastronbelongedexclusivelytotheexoskeletonandwasinnowayhomologoustothesternum.However,Owen(1849),adheringtohisidealvertebralarchetype,proposedthattheplastralboneswerehomologuesofthethoracicvertebralhemapophyses,andassuchwerepartoftheendoskeleton.MorerecenthistologicalstudiesconfirmedRathkesassessmentthatthebonesoftheplastronallossifyintramembranouslywithoutanycartilaginousprecursorsandbelongtothedermalexoskeleton(Zangerl,1939,1969;Gilbertetal.,2001).Currently,theconsen-susisthattheepiplastraandentoplastronarehomologous,respectively,totheclaviclesandinter-claviclebonesofother reptilian lineages(Zangerl,1969;Cherepanov,1997;Vickaryous&Hall,2006;Parker,1868;Rieppel,1996),whereasthemoreposteriorplastralbonesarehomologoustothegastralia(floatingribsorabdominalribs)ofothertetrapods(Zangerl,1939;Claessens,2004).

1.3.3 roleSofneurAlcreStcellSInplAStronAndnuchAlBonedevelopment

The embryonic origins of the plastral bones are also controversial. The Swarthmore laboratory(Clarketal.,2001;Cebra-Thomasetal.,2007)hasputforththeproposalthattheplastronbonesarederivedfromthetrunkneuralcrestandformmuchthesamewaythatvertebratefacialbonesform.In2001,Clarkandhercolleaguespublishedevidencethattheturtleplastronbonesareexo-skeletalandthattheyformbytheintramembranousossificationofneuralcrestcells.Thisassertionhasarousedspiriteddebate(Pennisi,2004)because trunkneuralcrestcellsarenotsupposedtoformskeletalelements,andcranialneuralcrestcells(whichareskeletogenic)arenotsupposedtomigratemoreposteriorlythanthecollarboneandshoulderbasedonamniotemodelslikethechickandmouse(Hall,2005;Matsuokaetal.,2005).Clarkandcolleagues(2001)showedthattheninedevelopingplastronbonesofthe50-dayTrachemysembryoareformedbycellsthatstainedposi-tivelyforthecellsurfacecarbohydratedeterminantrecognizedbythemonoclonalantibodyHNK-1(Figure1.4C)andforthemembranereceptorproteinPDGFRa.

HNK-1immunoreactivityisthestandardmarkerforneuralcrestcells,andturtleneuralcrestcellsstainedpositivelyandstronglyforHNK-1(Hou,1999;Hou&Takeuchi,1994).However,inthose studies,onlyearly (Yntemastage12)embryoswereexaminedand thepossiblemigrationofneuralcrestcellstotheplastronwasnotaddressed.PDGFRaisamarkerforskeletogenicandodontogenicneuralcrestcells.PDGFRahasbeendetectedonthebone-formingneuralcrestcellsofmiceandfrogsaswellasinteethandotherfirstbranchialarchderivatives.AntibodystainingagainstPDGFRaintheturtleembryoshoweditslocalizationinthemandibularmesenchyme,asexpected,aswellasineachofthedevelopingplastronbones(Clarketal.,2001).

However,neitherHNK-1norPDGFRastainingarecompletelyspecificforneuralcrestcellsand theirderivatives.TheHNK-1antibodydetectsnotonlycellsof theneuralcrest lineagebutalso stains theneural tube, cerebellarneurons,motorneurons, andcertain leukocytes. Inmice,PDGFRa is detected not only on skeletogenic neural crest cells but also on rib precursors andin the embryonicmesenchymecells contributing tobone, hair,mammarygland, gut, and lung.Thedefinitiveidentificationofneuralcrestcellscanonlybeconfirmedbylineagemapping,Thus,whereastheClarkstudystronglysuggestedneuralcrestinvolvementinplastronformation,itdidnotconclusivelydemonstratethatthesewereneuralcrestcellsand,ifso,whethertheywerefromthetrunkorcranialneuralcrest.

Cebra-Thomasandcolleagues (2007) attempted tofind theoriginof theseplastron-formingHNK-1+cellsandusemoremarkers to identifyneuralcrestcells.Theyfound thatstage17andstage18Trachemysembryos(threeweeksincubation)hadastagingareainthetrunkcarapacialdermiswhere theHNK-1+cells resided (Figure1.4A).Thecells in this regionwerepositivenotonlyforHNK-1immunoreactivitybutalsofortwoadditionalmarkersforneuralcrest:theneuralcrest-specifyingtranscriptionfactorFoxD3andthelow-affinityneurotrophinreceptor,p75.FoxD3stainingofnucleiwasseeninthedorsal-mostportionoftheearlystage17neuraltubeaswellasin

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cellsinthedermisbetweentheneuraltubeandsurfaceectoderm.ThefactthatthesearedorsalcellsstainingwithHNK-1,FoxD3,andp75makesthemexcellentcandidatestobeneuralcrestcells.

Theseneuralcrestcellswould representavery lateemigratingpopulation,and theyappeartocomedirectly from theneural tube (andnot from theneuralplate/epidermalboundary)afterthefirstwaveofneuralcrestemigrationhasalreadyformedthedorsalrootganglia,pharyngealderivatives,melanoblasts,andentericneurons.Afterleavingthedorsalneuraltuberegion,thesecellsresidewithintheformingcarapacialdermisandbystage18,thesecellsformabroadbandinthedorsalportionofthecarapace.Thesecellsconstituteamigratorypopulation,andDiIstainingshowsthemmovinglaterallyandventrally.Inaddition,stage18embryosalsoexhibitHNK-1+cellsmigratingnearthevertebraeandmigratingdownthelateralwallsoftheembryowithinthedermis.TheseHNK-1+andp75+cellscanbeseencondensingintheplastralmesenchymeandformingbone(Figure1.4B).Unlikechickormouseembryos,thebone-formingneuralcrestcells(suchasthoseinthehead)retaintheHNK-1andp75markersevenastheyareformingbone(Clarketal.,2001;Cebra-Thomasetal.,2007).

ThispatternofHNK-1expression isunique to the turtleandsuggests that the lateemigrat-ingturtletrunkneuralcrestcellshavetakenonthecharacteristicsofcranialneuralcrestcells.InadditiontoexpressingPDGFRa,amarkerusuallyassociatedwithcranialneuralcrestcells,theselate-emergingneuralcrestcellsappear tocontribute to thesclerotome-derivedvertebraland ribcartilages.Thus,theturtlevertebraeandribsmayhaveadualoriginthesomiteandtheneuralcrest.AbipartitepatterninthecartilagewouldbeexpectedifthetrunkcrestcellshadthepropertiesofcranialneuralcrestcellsbecauseLeDouarinandTeillet(1974)showedthataviancranialneuralcrestcellscontributedtotrunkcartilagewhentransplantedintothetrunkregion.

figure. Late-emigratingHNK-1+cellsformingtheplastronofTrachemys.(A)Dorsalregionofstage17(three-week)embryoshowingthecarapacialstagingareawhereinHNK-1+cells(brown-redstain)reside.(B)PlastronbonebeingformedbyHNK-1+cellsinastage18embryo.(C)Hyoplastronofa50-dayembryo.Thebonestainswithhemotoxylin,whereastheHNK-1+cellsarered-brown.(A,BafterCebra-Thomasetal.,2007;(C)adaptedfromClarketal.,2001.)

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BiologyofTurtles

GilbertandCebra-Thomassuggestthatthenuchalboneandtheplastronbonesmayformtotallyorpredominantlyfromtrunkneuralcrestcells.Thedevelopingplastronandnuchalbones(butnottheperipheralcarapacialbonesofthesameturtle)stainpositivelyforneuralcrestmarkers.AlthoughHNK-1reactivityisnotspecificforneuralcrestcells(itisalsoseeninsomeneurons,leukocytes,andcartilagecells),theobservationthattheplastronandnuchalbonesdevelopintramembranously(withoutcartilaginousintermediates),expressadditionalneuralcrestmarkers,arenearnoneurons,andareobviouslynotmadeofwhitebloodcellssuggestsaneuralcrestoriginforthem.

Howmighttrunkneuralcrestcellsformbone?Inmostvertebratesstudied,celllabelingstud-iesdemonstratedthatthedermalcranialandfacialbonesofthevertebrateexoskeleton(aswellasthedentineoftheteeth)comefromthecranialregionoftheneuralcrest,whereasthetrunkneuralcrestisunabletoformbone(Smith&Hall,1993;Matsuokaetal.,2005;Hall,2005).Onedistinc-tionbetweencranialandtrunkneuralcrestcellsliesintheexpressionofHoxgenes.Theneuralcrestcellsthatarisefromthefore-andmidbrainproduceMeckelscartilageandthebonesoftheskull,face,andjawdonotexpressHoxgenes.WhenHoxgeneswereexperimentallyexpressedincranialneuralcrestcellsthatwouldnormallygiverisetothecraniofacialskeleton,theresultingchickembryosshowedsevereskeletaldeformities(Creuzetetal.,2002).SmithandHall(1993)pos-tulatedthattheabilitytoformboneswasaprimitivepropertythatcharacterizedearlyvertebrates,andTrainorandcolleagues(2003)sawtheevolutionofjawsasresultinglargelyfromthelossofmandibularHoxgeneexpressionbetweenthelamprey-likeagnathansandthegnathostomes.

RecentevidencehasshownthattrunkneuralcrestcellscangainskeletogenicpotentialiftheirHoxgeneexpressionpattern isdownregulated.McGonnellandGraham(2003)found thatchicktrunkneuralcrestcellsinlong-termcellculturecanproduceosteoblastsandchondrocytes.More-over,Abzhanovandcolleagues(2003)confirmedthisobservationanddemonstratedthatthecul-turedtrunkcrestcellsthathadgainedskeletogenicpotentialhadalsolosttheirHoxgeneexpression.ItispossiblethatthelateemigratingneuralcrestcellsinturtleembryoshavelosttheirHoxexpres-sionpatterns(eitherbyemigratingfromtheneuraltubeatalatedateorbyremaininginthestagingareaforaprolongedperiodoftime)andhavetherebyacquiredtheabilitytoformbone-likecranialneuralcrestcells.

Thecurrentevidencesupportsthecontentionthatthetrunkneuralcrestcellsoftheturtlehavegained(orregained)theabilitytoformaskeleton.Therefore,itispossiblethatthenuchalboneandthebonesoftheplastronareformedbyneuralcrestcellsusingmethodssimilartoformingthecal-vareumandface.Theseconclusionscanbeconfirmedbydetailedlineagemappingoftrunkneuralcrestcellsinturtleembryos.

. evolutionaryimpliCations

Weretherenoturtlesliving,wewouldlookuponthefossilturtlesasthestrangestofallvertebratesanimalswhichhaddevelopedthestrangehabitofconcealingthemselvesinsidetheirribs,forthatisliterallywhatturtlesdo.

Samuel Williston (1914)

TheorderCheloniaemergesabruptlyintheTriassicabout210millionyearsagowiththefossilspe-ciesProganochelys(Gaffney,1990).Thisreptilehadthecharacteristicderivedtrunkmorphologynowassociatedwithturtles,includingbothacarapaceandplastron.Basedoncranialcharacters,turtleshavetraditionallybeenclassifiedasanapsids,withrootsinoneofseveralTriassicformsofparareptiles.Manyoftheseformssportextensivedermalarmorintheformofbonyossiclesthatwereembeddedintheskin.

Anevolutionarymodelwherethecheloniancostalsandotherboneswerederivedfromosteo-dermsthatsecondarilyfusedwiththeribsandvertebraewasthepredominantviewamongpale-ontologists for many years (Klin, 1945; Romer, 1956; Sukhanov, 1964; Carroll, 1988; Laurin& Reisz, 1995; Lee, 1996, 1997a, 1997b). However, among the candidate ancestorsincluding

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captorhinomorphs,pareiasaurs,andprocolophonidsthefossilrecordprovidesnocluestotheori-ginoftheuniquechelonianrearrangementoftheaxialandappendicularskeletons.Carroll(1988)commentsthattheirbizarreanatomymightbesufficienttoplaceturtlesintheirownsubclassoftheReptilia.

Theanapsidstatusofturtleshasbeenchallengedinrecentyears.Inarecentreview,ZardoyaandMeyer(2001)analyzesixalternativecladogramscurrentlybeingusedtorepresenttherelationshipsofturtlestootherreptilesandbirds.Incontrasttothetraditionalpaleontologicviewthatturtlesareanapsids,adifferentviewrelyingonthephysiologicalandmorphometricevidencefromextantturtles,aswellasfromtheirpancreaticpolypeptidesequences,nuclearDNA,andmitochondrialDNAhascausedseveralgroupstoarguethatturtlesaremodifieddiapsidswithinthereptilianclade.PlatzandConlon(1997)andHedgesandPoling(1999)usesequencedatatoproposethatturtlesgroupwithcrocodiliansamongthearchosaurs.FurtherproteinsequencedatafromIwabeandcolleagues(2005)indicatethatturtlesareasistergrouptothearchosaurclade.Rieppel(2001)andRieppelandReisz(1999)alsoassignturtlestothediapsida.Theyproposeanaquaticoriginoftheturtleswhereintheancestorwouldhavealreadyhadaplastron-likegastraliatowhichthenewlymadecarapacecouldattach.Gastraliaarepresentinnumerousordersofreptilesandwouldprob-ablyhavealreadybeenpresentintheancestorsofturtles.Claessens(2004)summarizes,Gastraliamaybeplesiomorphicfortetrapods,butareonlyretainedinextantCrocodyliaandSphenodon,andpossiblyaspartofthechelonianplastron.

Whetheroneviewsturtlesasanapsidsordiapsids,thereisadramaticabsenceoftransitionalforms.Thisraisesthepossibility that turtlesarosesaltationally,without intermediatemorpholo-giesthatwouldlinkthemtonon-Chelonianreptiles.ThemodelproposedbyBurke(1989c)setsthetimingandpositionoftheCRasthepivotaleventintheevolutionofthenewbodyplan.Itisasafeassumptionthatepithelial/mesenchymalinteractionsweretheinductivemechanismsfortheforma-tionofdermalarmor inearlyamniotes.Theprecocious initiationofanepithelial/mesenchymalinteractioninthedorsalbodywalloftheearlychelonianembryomayhavebeentheinitialnoveltyintheevolutionofthedermalcarapace.ThemodelproposedbyCebra-Thomas(2005)providesamechanismfortherapidmorphogenesisofthebonyshelloncetheribsarerepositionedintothedermis.

Thedevelopmentoftheturtleisfullofsurprises.Indeed,whatwehavehereisatentativeout-lineofhowtheturtlegetsitsshell,buttherearemanymorequestionstoask.Ifthetrunkneuralcrestcellsformtheplastron,howaretheydirectedthereandwhatcausesthemtobecomebone?Whatcausessometurtlestohaveadome-shapedcarapacewhereasotherturtleshaveaflattenedcarapace?Whatcausesthesexuallydimorphicconcavitiesoftheplastron,andhowdosometurtlesdevelopahingeinthisventralshell?Developmentalbiologyisjustbeginningtojoinpaleontologyandstructuralmorphologyinexploringthisfascinatingstructure,andthisunionmayenableustoseehowevolutionaryinnovationscanrapidlyemergeandtofinallydeterminetheplaceoftheturtleinthehistoryoflife.

aCKnoWledgments

WewishtothankMs.DianeFritzforherassistanceinhelpingpreparethismanuscript.Also,wewishtothanktheNationalScienceFoundationandtheHowardHughesMedicalInstituteforsup-portingmuchoftherecentworkreportedhere.

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2 ComparativeOntogeneticandPhylogeneticAspectsofChelonianChondro-OsseousGrowthandSkeletochronology

Melissa L. Snover and Anders G.J. Rhodin

Contents

2.1 Introduction........................................................................................................................... 172.2 SkeletochronologyinTurtles................................................................................................ 18

2.2.1 Background................................................................................................................ 182.2.1.1 ValidatingAnnualDepositionofLAGs.......................................................202.2.1.2 ResorptionofLAGs.....................................................................................202.2.1.3 SkeletochronologyandGrowthLinesonScutes......................................... 21

2.2.2 ApplicationofSkeletochronologytoTurtles............................................................. 212.2.2.1 FreshwaterTurtles........................................................................................ 212.2.2.2 TerrestrialTurtles......................................................................................... 212.2.2.3 MarineTurtles.............................................................................................. 21

2.3 ComparativeChondro-OsseousDevelopmentinTurtles......................................................222.3.1 ImplicationsforPhylogeny........................................................................................ 322.3.2 ImplicationsforGrowth............................................................................................. 33

References........................................................................................................................................ 39

. introduCtion

Formandfunctionarefundamentalinterdependentstrategiesofalllife.Fromstudiesofskeletaland chondro-osseous structure and development, we can gain insights into phylogenetic differ-encesandtaxonomicclassifications,andwecanalsobetterunderstandhowdifferentspeciesandindividualswithinspeciesgrowtomaturityandrespondtothephysiologicaldemandsof theirparticularlifestrategies.Corticalbandingpatternswithinbonescorrelatetoactivitypatternsoftheindividualaswellasendogenousrhythms,allowingforinferencesnotonlyaboutageandcyclicalgrowthpatternsbutalsopreviousgrowthandcircumstancesthathaveinfluencedgrowth(Suzuki,1963;Enlow,1969;Castanet,2006).Studiesofthesebandingpatternswithincorticalbone(skel-etochronology)havebeenappliedtonumerousspeciesofturtlesandhaveallowedustounderstandpatternsandratesofgrowth.

In addition to skeletochronology, detailed studies of the chondro-osseous development ofappendicular bones have revealed strong similarities among most living chelonians, but with

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BiologyofTurtles

strikingdifferencesforcertainlarge,fast-growingseaturtles(e.g.,theleatherback,Dermochelys)thatseparatesthemfromallotherturtles(Rhodinetal.,1980,1981,1996;Rhodin,1985).

In thischapter,wesummarize theapplicationofskeletochronologyforestimatesofageandgrowthratesinturtles,reviewthetwobasicpatternsofbonegrowththatoccurinturtles,andcor-relatethesepatternsofchondro-osseousdevelopmentwithphylogeny.Finally,wediscusshowthesefactorsinfluenceratesofgrowthtosexualmaturity,highlightinghowtheleatherbackstandsapartfromotherturtles.

. sKeletoChronologyinturtles

2.2.1 BAckground

Skeletochronologyhasbeenusedtoestimateageandgrowthinnumerousspeciesofreptilesandamphibians(Castanet,1994;Smirina,1994).Bonesaregoodrecordingstructures,astheycontainlayersthatformwithapredictableperiodicityandthelayersaredifferentinmorphologyandopti-caldensity,makingthemeasilydiscernable(Klevezal,1996).Inhistologiccross-sectionsofboneareconcentricthinlayersthatstaindarkwithhematoxylin.Alternatingwiththeseconcentricthinlayersarebroadhomogeneouslight-staininglayers(Castanetetal.,1993;Klevezal,1996).Castanetetal.(1977)introducedthetermline of arrested growth(LAG)toidentifythethindarklineschar-acteristicofskeletalgrowthmarks(Figure2.1).

Inbonemorphology,LAGsareinthegeneralclassofcementorcementinglinesandarecom-monthroughoutallvertebratebones.ResorptioncementlinesarefoundaroundHaversiancanalsystems(secondarilyremodeledbonewithvascularingrowth),differentiatingthemfromcorticalbone,andinthelamellarperiostealdepositionofsecondaryendostealbone.Restingcementlines(theclasstowhichLAGsbelong)arefoundinthelayeringpatternofperiostealdepositionofnewcorticalbone(Enlow,1969;Francillon-Vieillotetal.,1990).

Many skeletochronological studies of herpetological species indicate that LAGs are formedasaresultoflowmetabolismandslowedornogrowthassociatedwithseasonalclimaticchanges.Thisislikelytruebutservesonlyasapartialexplanation,consideringthatLAGsalsooccurinthehardstructuresofnonhibernatingmammalianspecies(Klevezal,1996;Castanet2006).Castanetetal.(1993)extendedtheterminologyofLAGstobothpoikilothermsandendothermsasageneraldescriptionofarestingcementlinemarkingperiodicityingrowth.Castanetetal.(1993)alsopro-posedthattheformationofLAGsislikelytobeendogenouswhilestillpotentiallysynchronizedtoenvironmentalconditions.

CyclicalformationofLAGsappearstobeauniversalphenomenoninvertebrates(Castanetetal.,1993;Klevezal,1996;Simmons,1992),andthereisevidenceforendogenouscontrol(Schauble,1972;Castanetetal.,1993;Simmons,1992;Estebanetal.,1999).Boneformationandremodelingratesarehormonallycontrolledandsynchronizedtocircadianpatterns(Simmons,1992).Parathy-roidhormone(PTH),calcitonin,andvitaminsA,C,D,andKhavebeenfoundtoinfluenceratesofboneformationandremodeling(Buchanan&Preece,1991;Narbaitzetal.,1991).Specifically,PTHwhichstimulatesboneresorptionissecretedinresponsetoserumcalciumlevels.

Studies have demonstrated seasonal variability in skeletal growth rates, not just in poikilo-therms(Schauble,1972;Snover&Hohn,2004)butalsoinendothermicmammals(Klevezal,1996;Castanet, 2006).Thesepatternsmaypotentiallybe evolutionarily related to an increased avail-abilityofvitaminsA,C,andD,withtheonsetofspringintemperateclimatesorthewetseasonin tropical climates (Buchanan & Preece, 1991; Simmons, 1992). However, there is substantialevidencethatthespringsurgeingrowthratesisalsounderendogenouscontrol,asanimalsthataremaintainedincaptivityalsodemonstratethispattern.Schauble(1972)amputatedlimbsfromthenewt,Notophthalmus viridescens,atdifferenttimesoftheyearandobservedtheregenerationrates.Shefoundthatregenerationratesweresignificantlyhigherinthespringorearlysummermonths,followedbysummer,latesummer,earlyfall,andwinter,respectively.Astemperature,lightlevels,

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ChelonianChondro-OsseousGrowthandSkeletochronology

andfoodavailabilitywerecontrolled,thesefactorscouldnothaveplayedaroleintheregeneration

rates,suggestingthattheresultsimplytheinfluenceofaninternalbiologicalrhythm,eitherendo-

crineornonendocrineinnature.

AnotherlineofevidenceforseasonalvariabilityinskeletalgrowthratesisSnoverandHohns

(2004)analysisofbone-growthincrementspast the lastcompleteLAGinKempsridleyhumeri

relativetostrandingdate.Theyfoundasignificantandpositiverelationshipbetweentheamount

ofnewbonedepositedafterthelastLAGandtheJuneNovembertimeframe.FromNovemberto

June,therelationshipwasnotsignificantlydifferentfromzero,suggestingthatverylittlenewbone

LAGs

LAGs

1 mm

figure. Cross-sectionsfromhumerioftwoterrapins(Malaclemys terrapin)thathavebeendecalcifiedandstainedwithEhrlichshematoxylin.Arrowshighlightthethin,darklystainedlinesofarrestedgrowth(LAGs),andthelightlystainedregionbetweenLAGsistermedthegrowthzoneandtogetheroneLAGandonezonecompriseagrowthmark.NotehowtheLAGsarebeginningtocompressattheouteredgeofthelowerimage.Theupperimageisfroma15.1-cmstraightcarapacelength(SCL)female,andthelowerisfroma16.5-cmSCLfemale.

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0 BiologyofTurtles

depositionoccursduringthewinterandthatLAGsaredepositedinthespringforKempsridleysalongtheU.S.Atlanticcoast.

... validatingannualdepositionoflags

Three common methods can be employed to directly validate the annual deposition of skeletalgrowthmarks:thestudyofknown-ageanimals,mark-recapturestudies,andmark-recapturestud-ies that incorporatefluorescentmarking (Castanet,1994).All threeof thesemethodshavebeenappliedtoturtles(Castanet&Cheylan,1979;Klinger&Musick,1992;Colesetal.,2001;Snover&Hohn,2004;Curtin,2006;Snoveretal.,2007b).SnoverandHohn(2004)lookedathumerifromknown-ageKempsridleyseaturtles(Lepidochelys kempii)thathadbeentaggedashatchlingsandreleasedintothewild.Theturtlesfromtheirstudyweresubsequentlyrecoveredasdeadstrand-ingsandallowedforvalidationofannualLAGformationand the recognitionofanannulus,ordiffusemarkratherthanadistinctLAG,thatrepresentedanannualgrowthmark.Curtin(2006)usedbonesfromknown-agedeserttortoises(Gopherus agassizii)frommark-recapturestudiestotestandvalidateback-calculationmethodstoaccountforLAGslosttoresorptioninolderanimals.Snover(2007a)usedhumerifromdeadstrandedloggerheadturtles(Caretta caretta)thathadbeenpreviouslycapturedandtaggedtovalidatethatcarapacelengthcanbeback-calculatedfromthedimensionsofearlierLAGs.CastanetandCheylan(1979)usedfluorescentmarkingtovalidatethatgrowthmarkswereannualinHermannstortoises(Testudo hermanni)andGreektortoises(Testudo graeca).KlingerandMusick(1992) injectedwild loggerheadswithoxytetracyclineandreleasedthem.Bonebiopsiesweretakenfromturtlesrecaptured1to2yearslatertovalidateannualLAGformation.Aturtlefromthatsamestudywasfoundstrandeddead8yearsafterinjectionandpre-sentedadditionalvalidation(Colesetal.,2001).

... resorptionoflags

Asboneincreasesinsizeduringgrowth,itisconstantlyremodeledandreshaped(Enlow,1969).Hardbonetissuescannotgrowthroughinternalexpansion,butrathertheygrowbyappositionalprocesses(onperiosteallyderivedcorticalbone)withthedepositionofnewtissueonthesurfacetogetherwithendostealresorption(Enlow,1969).Thisprocessofresorptionresultsinthelossoftheinnermost(earliest)growthmarksandisaseriouslimitationinestimatingageusingskeletochronol-ogy.Whilenotaseriousissueforshorter-livedamphibiansandreptiles,itisespeciallyproblematicin long-lived turtles, and the problem is noted to be extreme in age-estimate studies of marineturtles(Klinger&Musick,1995;Zugetal.,1995,1997,2002;Parham&Zug,1997;Zug&Glor,1998;Snover&Hohn,2004;Snoveretal.,2007b),resultinginthedevelopmentofseveralmethodsofback-calculationtoestimatethenumberofgrowthmarkslost.

Back-calculation techniques in sea turtles relyon theconcept that the spatialpatternof theLAGsisrepresentativeofthegrowthoftheanimal,andtoconfirmthisassumptionacorrelationmustbeestablishedbetweenbonedimensionsandbodysize (Hutton,1986;Klinger&Musick,1992; Leclair & Laurin, 1996; Snover, 2002; Snover & Hohn, 2004). Using loggerhead turtles,Snover(2007a)demonstratedthattherelationshipbetweencarapacelengthandhumerusdiametercanbeusedtoaccuratelyestimatecarapacelengthatthetimeofearlierLAGdeposition.

Mostback-calculationproceduresappliedtoturtleshavenotbeenvalidatedandmakeassump-tionsaboutearlygrowthrates(Klinger&Musick,1995;Zugetal.,1995,1997,2002;Parham&Zug,1997;Zug&Glor,1998).Curtin(2006)wasabletotestandvalidateback-calculationpro-cedures for the desert tortoise using humeri from known-age animals. She tested two methodspresentedbyParhamandZug(1997),therankingprotocol,andthecorrectionfactormethodsandfoundthatthecorrectionfactormethodprovidedthemostaccurateageestimatesforjuvenilesandsubadults;however,itunderestimatedadultages.Foradulttortoises,therankingprotocolprovidedthemostaccurateestimates.

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ChelonianChondro-OsseousGrowthandSkeletochronology

...skeletochronologyandgrowthlinesonscutes

Formostspeciesoffreshwaterandterrestrialturtles,ageismostcommonlyestimatedfromcountsofgrowthlinesonthescutesofeitherthecarapaceortheplastron(Germano&Bury,1998;Wilsonetal.,2003).Thisisapowerfultechniqueas,unlikeskeletochronologyinturtles,itcanbeappliedto livinganimalsandusedtounderstandtheagestructureofpopulations.However,manystud-iesthatapplythistechniquedonotprovideanyvalidation(Castanet&Cheylan,1979;Wilsonetal.,2003)andinaliteraturereview,Wilsonetal.(2003)foundthatofthestudiesthatdidattemptvalidation,37%wereunabletodoso.Similarly,Berry(2002)foundthateveninjuveniledeserttortoises,agecouldnotbeaccuratelydeterminedthroughscutecountsalone.Hence,itappearsthatwhereascountingscutegrowthlinesmaybeaviablemethodofageestimationinsometurtles(i.e.,Stone&Babb,2005),itisnotaccurateforallturtlesandassumptionsshouldnotbemadethatthemethodisapplicabletoagivenspecieswithoutvalidation.Whilenotstrictlyvalidwhenusedinconjunctionwitheachother,skeletochronologyandscutegrowthlinecountsfromdeadturtlescanserveassupportingevidenceoftheannualnatureofthetwomethods(Castanet&Cheylan,1979;Hart&Snover,unpublisheddata).

Evenwhenscutegrowthlinecountsaccuratelyestimateage,anadvantageofskeletochronologyoverscutegrowthlinecountsappearswitholderadultanimals.Asgrowthslowstonearlyimmea-surableratesinolderanimals,growthlinescannolongerbedifferentiatedonscutes(seeWilsonet al., 2003, for review), hence only minimum ages can be estimated. However, in histologicalpreparationsofbonesLAGscanbegenerallydifferentiatedeveninolderanimalswithnearcessa-tionofgrowth(Snover&Hohn,2004),allowingforestimatesofadultgrowthratesandlongevity(Figure2.1)(Snover,2002;Snover&Hohn,2004;Snoveretal.,2007b).

2.2.2 ApplIcAtIonofSkeletochronologytoturtleS

... freshwaterturtles

Freshwaterturtleswerethefirstturtlestohaveskeletalgrowthmarksrecognizedintheirlongbones.Mattox(1936)notedskeletalgrowthmarksinthelongbonesofpaintedturtles,Chrysemys picta marginata,andfoundacorrelationbetweencountsofthemarksandturtlesize.Peabody(1961)andHammer(1969)documentedperiostealcyclicalringsinsnappingturtles,Chelydra serpentina.Suzuki (1963) and Enlow (1969) found them in the slider, Trachemys scripta. Hart and Snover(unpublisheddata)comparedskeletochronologypreparationsofhumeriwithplastronscutegrowthlinecountstodemonstratethestrongcomparisonofthetwotechniquesinthebrackish-waterdia-mondbackterrapin(Malaclemys terrapin).Countingofgrowthlinesonplastronorcarapacescutesremainstheprimarymeansofestimatingageforfreshwaterturtles.

... terrestrialturtles

Thefirststudytovalidatetheannualnatureofskeletalgrowthmarkswasconductedwithtwospe-ciesoftortoises.CastanetandCheylan(1979)usedfluorescentmarkingtovalidateannualgrowthmarksinHermanns(Testudo hermanni)andGreek(Testudo graeca)tortoises.Recently,skeleto-chronologyhasbeenappliedtodeserttortoises(Gopherus agassizii):Curtin(2006)validatedtheannualnatureoftheLAGsinhumerifromknown-ageanimalsanddevelopedcorrectiontechniquestoestimatethenumberofLAGslosttoresorption.Similartothefreshwaterturtles,growthlinesonscutescontinuetobeaprimarymeansofestimatingageinthisgroupofturtles.

... marineturtles

Ofalloftheturtlegroups,skeletochronologyhasbeenappliedmostfrequentlytomarineturtles.Thescutesoftheplastronandcarapacedonotretaingrowthlineslikethefreshwaterandterrestrial

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BiologyofTurtles

turtles(however,seeTuckeretal.,2001).Hence,skeletochronologyhasbeentheprimarymeansofestimatingageandinferringgrowthratesintheseturtles.

Todate,skeletochronologyhasbeenappliedtofiveofthesevenspeciesofmarineturtles,theloggerhead(Caretta caretta:Zugetal.,1986,1995;Klinger&Musick,1992,1995;Parham&Zug,1997;Colesetal.,2001;Snover,2002;Bjorndaletal.,2003;Snover&Hohn,2004),theleatherback(Dermochelys coriacea:Zug&Parham,1996),theKempsridley(Lepidochelys kempii:Zugetal.,1997;Snover&Hohn,2004;Snoveretal.,2007b),thegreen(Chelonia mydas:Bjorndaletal.,1998;Zug&Glor,1998;Zugetal.,2002),andtheoliveridley(Lepidochelys olivacea:Zugetal.,2006).TheannualdepositionofLAGshasbeenvalidatedforloggerheads(Klinger&Musick,1992;Colesetal.,2001;Snover&Hohn,2004)andKempsridleys(Snover&Hohn,2004).

With the exception of leatherbacks, all of these studies used the humerus bone. Generally,LAGsaremostclearlyvisibleinthelongbones,andthehumerusisidealasitiseasilyremovedfromdeadanimalsandithasmuscleinsertionscarsthatcreatelandmarksthatallowfortheiden-tificationofsectioningsitesthatareconsistent(Snover&Hohn,2004).Humeriofleatherbacksaremorphologicallydifferentfromthehard-shelledturtles,andahighlevelofvascularizationandboneremodelingischaracteristicoftheleatherbackskeleton(Rhodin,1985).Thishighlevelofvascu-larizationmaylimittheusefulnessoflongbonestoskeletochronologystudies.However,Rhodin(1985)documentedtwowidecyclicalgrowthzonesintheperiostealboneofthehumerusofanadultfemaleleatherbackturtlethatsuggestedthepossibilityofgrowthcyclesrelatedtomigrationornest-ingpatterns(Figure10inRhodin,1985).ZugandParham(1996)predictedageatsexualmaturityofleatherbacksbyskeletochronologybasedonLAGsfoundinscleralossicles;skeletochronologyofleatherbackshasalsobeenconductedbyAvensandGoshe(unpublisheddata).However,thepos-sibleannualnatureofthesemarkshasnotbeenvalidated,andtheymayinsteadsimplyrepresentthecyclicalresultofvaryingratesofbonedepositionandgrowthrelatedtofeedingormigrationcyclesinthishigh-metabolismspecies.

. ComparativeChondro-osseousdevelopmentinturtles

Formandfunctionareindeedfundamentalinterdepen

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