Tail Autotomy and Subsequent Regeneration Alter the Mechanics Of

Home , Autotomy, Christinus marmoratus

*Author ([email protected]) for correspondence NW, Calgary, ABT2N1N4,Canada. Riverside,California, 900University Avenue, Riverside, CA92521,USA. © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3891-3897doi:10.1242/jeb.110916 Received 10July 2014; Accepted10September2014 2 1 range oftaxa(Flemingetal.,2007),includingcrabs(Bennett,1973), functional challengesimposedbysucheventualities. more fullyunderstandhowanimalsovercometheparticular of theeffects ofrapidweight lossonlocomotionisnecessaryto length oftimeassociatedwithweightlossorgain.Anassessment variation inlocomotorstrategieslikelyrelatestodifferences inthe locomotor performancewhengravid.However, amajorsourceof 1980; Shine,2003)andbirds(Leeetal.,1996),suffer areductionin taxa, includingsalamanders(Finkleretal.,2003),lizards(Shine, changes inlocomotorbehavior. Forexample,organisms ofseveral these wayscanresultinavarietyofphysiologicaleffects, including pregnancy andevenlimbortailloss.Losingand/orgainingmassin throughout theirlifetimeasaresultoffamine,obesity, illness, Many animalsencounterepisodesofsubstantialweightchange Locomotion, Centerofmass,Kinematics,Groundreactionforce KEY WORDS:Tail autotomy, Regeneration,Biomechanics, (GRFs) intheleopardgecko by measuringmorphology, 3Dkinematicsandgroundreactionforces associated withtaillossandlong-termregenerationforthefirsttime explored, weinvestigatedchangesinlocomotormechanics general effects ofautotomyonlocomotorperformancearecommonly capable ofbothautotomizingandregeneratingtheirtail.Althoughthe and theresponsestomoregradualweightgain,particularlyinlizards measuring theeffects ofrapidweightchangeonlocomotorbehavior response toapredatorstimulus,providesaneffective modelfor reasons. Autotomy, thevoluntarysheddingofanappendagein Animals canundergosignificantweightchangeforavarietyof Kevin Jagnandan of locomotioninlizards Tail autotomyandsubsequentregenerationalterthemechanics RESEARCH ARTICLE morphology, kinematicsandforce. demonstrates thecausalrelationshipsbetweenvariationsin results suggestanactivelocomotorresponsetotaillossthat recovered topre-autotomyvaluesasthetailregenerated.These locomotor speed.HindlimbkinematicsandGRFsfollowingautotomy weight-specific propulsiveforce,withoutacorrespondingchangein autotomy. ChangesinhindlimbGRFsresultedanincrease hindlimb jointanglessignifyamoresprawledposturefollowing changes inbodyorforelimbkinematicswereevident,decreases only partiallyrecoveredafterfullregenerationofthetail.Althoughno resulted ina13%anteriorshiftthecenterofmass(CoM),which INTRODUCTION ABSTRACT Department of Biological Sciences, University of Calgary, BiologicalSciences, 2500University University Drive of of Department Evolution, Ecology, of andOrganismalDepartment Biology, University of Autotomy isapredator-escape strategydocumented forabroad 1, *, AnthonyP. Russell Eublepharis macularius 2 and Timothy E.Higham . Tail autotomy radical change inmorphologywithmeasured performance mass (CoM)associatedwith tail autotomyiscrucialtolinkthe locomotion changewiththeloss ofweightandshiftincenter of howforceproductionand limbjointmechanicsduring (Higham etal.,2013;McElroy andBergmann, 2013).Knowledge locomotion associatedwithtail autotomyarelesswellunderstood (sprint speed,jumpingandstability), thebiomechanicalchangesin between morphology(tailedversustail-less)andperformance or both. because itgeneratesfrictionwhilebeingdraggedalongtheground, impede fasterrunningbecauseofitsrelativelygreatweight or does notassistinlocomotiontheseanimals,butmayactually ( run morethantwiceasfastwithoutthetail[marbledgeckos ( difference insprintspeed following autotomy[velvetgeckos by thepresenceoftail.Somelizards,however, exhibitno (Punzo, 1982),suggestingthatlocomotorperformanceisenhanced (Punzo, 1982)andgreaterearlesslizards( (Ballinger etal.,1979),desertfringe-toedlizards( been reportedforsix-linedracerunners( For example,adecreaseinsprintspeedfollowingautotomyhas dependent upontheroleoftailinlocomotion(Vitt etal.,1977). (McElroy andBergmann, 2013)andarethushypothesizedtobe have beenfoundtobehighlyvariableamonglizardspecies during jumpingafterautotomy. Effects oftaillossonrunningspeed et al.(Gillisal.,2009)demonstratedadecreaseinin-airstability also foundthatautotomyincreasesthelikelihoodoffalling.Gillis used torecoverfromslipswhileascendingaverticalsurface;they showed thatthetailfunctionsasanemergency fifthlegandcanbe number oflocomotorbehaviors.Jusufietal.(Jusufial.,2008) and Fleming,2009;McElroyBergmann, 2013). behavior andperformance(Vitt etal.,1977;Daniels,1983;Bateman Daniels, 1983)andthusitslossmaymarkedlyaffect locomotor for normalpatternsoflocomotionandbalance(Etheridge,1967; (Smyth, 1974;Vitt etal.,1977).Additionally, thetailcanbeessential Avery, 1970;Congdonetal.,1974),sexual displayandreproduction species canplayasignificantroleinenergy storage(Bustard,1967; subsequent tothepredator–preyinteraction.Thetailsofmanylizard autotomy inlizardscanhavesignificanteffects ontheanimal with apredator(Vitt etal.,1977;DialandFitzpatrick,1983),tail mechanism forincreasingthelikelihoodofsurvivinganencounter response toapredatorystimulus.Althoughthisisaneffective lizards, thisvoluntarysheddingofanappendagegenerallyoccursin and Collins,1918;Layne,1972).Mostcommonlyexaminedin 1985; BatemanandFleming,2009)evensomerodents(Sumner (Congdon etal.,1974;Daniels,1983;Arnold,1984;CooperandVitt, Wilkie, 1980),salamanders(Wake andDresner, 1967),lizards spiders (Parry, 1957;Amaya et al.,2001),echinoderms(Emsonand Oedura lesueurii)(KelehearandWebb, 2006)],whereasotherscan Christinus marmoratus Although suchstudieshaverevealednotablerelationships It isclearfromrecentworkthatthelizardtailimportantina 1 ) (Daniels,1983)],suggestingthatthetail Cnemidophorus sexlineatus) Cophosaurus texanus) Uma notata) 3891

The Journal of Experimental Biology 3892 down theposteriorendoftrunkandthatremovalthisweight running onalevelsurface?We hypothesizethatalarge tailweighs the associatedshiftinCoMaffect locomotormechanicswhile CoM toitsoriginalposition.(2)Howdoesremovalofthetailand shift theCoManteriorlyandregenerationoftailwillreturn the removalofsubstantialcaudalweightasaresultautotomywill loss andregenerationaffect thelizard’s CoM?We hypothesizethat locomotor mechanics,weaddressedthreequestions:(1)howdotail Higham andRussell,2012;Lynn et al.,2013). Russell, 2010;McLeanandVickaryous, 2011; Delormeetal.,2012; inducing autotomyinthisspecieshasbeennoted(Highamand established studysystemforautotomyandtherelativeeaseof locomotor mechanics.Additionally, leopardgeckosarean species iswell-suitedforexaminingtheeffects ofautotomyon a greaterchangeinlocomotion(McElroyandBergmann, 2013),this Russell, 2012).Becauselosingarelativelylarge tailislikelytoelicit of theanimal’s mass(HighamandRussell,2010;Higham for approximatelyone-thirdthelengthofbodyandone-fourth one ofthelargest (relativetobodysize)amonggeckos,accounting macularius autotomy andregenerationintheleopardgecko rapid lossandgradualrecoveryofweightassociatedwithtail 2002). autotomy arerare(DownesandShine,2001;ChappleSwain, locomotor changesassociatedwithregeneratingthetailfollowing differences. Furthermore,studiesconsideringthetimecourseof RESEARCH ARTICLE D B A Tail mass To examinetheeffects ofautotomyandregenerationon We investigatedhowlocomotormechanicsareaffected bythe V snout–ventlength groundreactionforce centerofmass SVL GRF CoM Pre-autotomy abbreviations List of Tail length (mm) (% body mass)

0.15 0.30 Post-autotomy 100 50 0 0 Pre-autotomy Blyth 1854.Thetailofthisdesert-dwellingspeciesis *

rgnlRegenerated Original **

Ps-uooyPost-autotomy Post-autotomy 2

4 6 *

Time (weeks)

10 C Tail volume (mm3) 10,000

12 5000 4 weeks

0 14 Eublepharis

16 rgnlRegenerated Original

18 Post-autotomy Tail diameter Tail length 22 weeks

*** (repeated measuresANOVA, F P (original, 7.8×10 22% bodymass;regenerated,15%mass)andvolume regenerated tailsweresignificantlysmallerinbothmass(original, 1).Paired and shapefromtheoriginals(Fig. addition totheabsenceofvertebrae,regeneratedtailsdiffered insize growth oftheanimalsasidefromregenerationtail.In weight, end ofthestudy(paired the tail)ofeachindividualdidnotdiffer significantlyatthestartand 22 weeks. Thesnout-ventlength(SVL)andbodyweight(without t=–1.396, d.f.=6,P P hoc autotomy ceasedshowingsignificantchangesafter18 Body weight,taillengthanddiameterofleopardgeckosafter permanent solution. manifestation ofthe‘post-autotomy’ modeoflocomotionasa autotomy’ stateovertimeorthelizardmayadoptsome hypothesize thatthesemetricsmayeitherreturntothe‘pre- a shiftinkinematicsandGRFsimmediatelyfollowingautotomy, we change continuallyandultimatelyreturntotheoriginalstate?After regenerated, dojointkinematicsandpropulsivehindlimbGRFs decrease proportionallywiththelossofmass.(3)Astailis propulsive hindlimbgroundreactionforces(GRFs)areexpectedto changes inforelimbandhindlimbjointkinematics.Additionally, an increaseinhipheightfollowingautotomy, resultinginpassive will resultintherisingofposteriorend.Therefore,weanticipate ANOVA, 131% oftheoriginalmaximumdiameter(repeatedmeasures originals (mass,t RESULTS 22 =0.010). Regeneratedtailsgrewtoonly61%oftheoriginallength =0.432; taillength, *

comparison ofweeks18and20:bodyweight,

0 12 24

t Tail diameter (mm) =–2.238, d.f.=6,P The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110916 F 2,5 ANOVA, P autotomy andfullyregeneratedtails(repeatedmeasures in bothtaillengthanddiameterbetweenpre-autotomy, post- and regeneration.Asteriskindicatessignificantdifferences diameter (blue)measuredbiweeklythroughoutautotomy (paired regenerated tails.Asteriskindicatessignificantdifferences leopard gecko.(B,C)Meanmassandvolumeoforiginal macularius regeneration intheleopardgecko(Eublepharis Fig. =394.159, =6.457, d.f.=6,P

3 .Changesintailmorphologywithautotomy and 1. =0.212), justifyingthecessationofstudyafter mm t -test, P t 3 t <0.05). Errorbarsrepresents.e.m. =–2.303, d.f.=6,P -tests: SVL,t ; regenerated,5.8×10 ). (A) Stagesoftaillossandre-growthinthe <0.05). (D)Meantaillength(red)and P =0.067), indicatingthattherewasno <0.001). Thesechangesintail 2,5 =585.764, =0.001; volume, =0.473, d.f.=6,P =0.061; taildiameter, P <0.001), butattained t -tests indicatedthat 3 mm t =–0.842, d.f.=6, t =3.742, d.f.=6, =0.653; body 3

) thanthe weeks (post

The Journal of Experimental Biology Hindlimb 0.23–2.4 suggesting thattheCoMwasnotrestoredtoitsoriginalposition. original andthefullyregeneratedtail( indicated asignificantdifference inthelocationofCoMwith maximum anglesoffemurdepression( 3).Immediatelyfollowingautotomy, significantdecreasesin (Fig. were unaffected, changesinhindlimbjointangleswereprominent 1).However, althoughforelimbjointkinematics regeneration (Table hindlimbs werenotsignificantlyimpactedbyautotomyand the body, stridelength,stancetimeanddutyfactorsofthefore- P RESEARCH ARTICLE measures ANOVA) ofchangesineachvariableisalso given.Significantresultsareindicated inboldtype. Means +residuals (±s.e.m.)foreachvariableareshown forpre-autotomy, post-autotomyand22 Variable ANOVA, 60.5±1.3% SVL withafullyregeneratedtail(repeatedmeasures 52.7±1.6% SVL withnotailandfinally, aposteriorshiftto CoM fromameanof65.9±1.6%SVL withtheoriginaltailto morphology resultedinasignificantanteriorshiftlocationofthe P significantly different fromoneanother(repeatedmeasuresANOVA, regenerated tails. Fig. Forelimb Body pitch(deg) Table significant effect onspeed(repeatedmeasuresANOVA, =0.017). =0.147). Maximumhipheight,maximumshoulderpitchof Max. ankleangle(deg) Max. femurretraction(deg) Max. femurdepression(deg) Hip height(SVL) Duty factor Stance time(s) Stride length(SVL) Max. kneeangle(deg) Max. elbowangle(deg) Max. humerusretraction(deg) Max. humerusdepression(deg) Shoulder height(SVL) Duty factor Stance time(s) Stride length(SVL) In runningtrials,geckosranatspeedsrangingfrom

.LocationofCoMinlizardswithoriginal,autotomizedandfully 2.

1. Summaryofkinematicvariablesbeforeandafterautotomyintheleopardgecko 0 SVL F 2,5 20 40 60 80 100 =10.174, s − 1 Error barsrepresents.e.m.Allthreepointsare , andautotomyregenerationdidnothavea % SVL P 007 Fg 2).A posthoc =0.017) (Fig.

141.80±3.57 65.77±7.50 160.49±3.57 59.73±5.60 0.15±0.00 0.73±0.00 135.65±3.01 54.61±5.98 29.02±8.62 0.75±0.01 0.43±0.02 0.11±0.00 Pre-autotomy 0.70±0.00 3.72±0.61 0.70±0.03 0.33±0.04 t t =3.306, d.f.=6,P =6.534, d.f.=6,P Regenerated Autotomized Original comparison F 2,5 =0.016), =0.001), =2.877, 129.92±2.55 21.75±4.96 140.78±3.89 133.04±2.60 42.00±3.88 0.15±0.00 0.72±0.00 63.90±4.93 45.00±15.63 0.72±0.01 0.41±0.02 0.12±0.00 otattm 22 Post-autotomy 0.70±0.00 2.44±0.77 0.70±0.02 0.28±0.02 P autotomy stateatweekeight( regeneration ( ANOVA, showed nosignificantchangeoverthe11 trials(repeatedmeasures and regenerationofthetail,weight-specificpeakverticalGRF After correctingforthechangesinmassassociatedwithautotomy ( returning totheoriginalvalueat10weeksfollowingautotomy angle showedamoregradualincreaseasregenerationproceeded, d.f.=6, autotomy state’ 2 the extentoffemoraldepressionandretractionrecoveredto‘pre- increased throughouttailregeneration. ( femur retraction( juvenile animalsandalthoughontogeneticdifferences intail Additionally, suchstudieshavegenerallybeenconductedon 1978; Lynn etal.,2013)andhavenot previouslybeenquantified. disparities insizeandshapeareonlybrieflymentioned(Whimster, (Goss, 1969;McLeanandVickaryous, 2011; Delormeetal.,2012), the structureofregeneratedtaildiffers fromthatoftheoriginal original andregeneratedtails.Althoughitisfrequentlynotedthat gross morphology, anatomy, histologyanddevelopmentbetween relating totheeffects ofautotomyhasfocusedondifferences in rapidly regenerateabulkytail(Lynn etal.,2013).Previouswork macularius, whichhastheabilitytoautotomizeandrelatively body weightandrelativelocationoftheCoM.Forthisweused biomechanical ramificationsoftheassociatedalterationboth attacks. Althoughsurvivalislikelyenhanced,wepresentseveral Tail autotomyisaremarkablestrategyforescapingpredatory irrespective ofthelossmass( still suggestasignificantdecreaseinverticalGRFafterautotomy, hoc peak propulsiveGRFsignificantlyincreasedafterautotomy(post until returningtothepre-autotomystateafter12 ( DISCUSSION t t post hoc =1.080, d.f.=6,P =0.125) (Fig. =3.807, d.f.=6,P Peak verticalGRFsignificantlydecreasedfollowingautotomy test, P =0.083; retraction,t F test, t =–4.473, d.f.=6,P 2,5 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110916

132.66±4.81 160.70±4.40 138.56±3.17 38.36±6.18 67.28±17.20 61.23±6.63 0.15±0.00 0.72±0.00 53.97±3.51 0.73±0.01 0.43±0.02 0.12±0.00 0.70±0.00 2.78±0.80 0.73±0.02 0.35±0.02 weeks post-autotomy. Statisticalsignificance (repeated =4.419, post hoc 4C).

t weeks post-autotomy =6.739, d.f.=6,P weeks followingautotomy(depression, Eublepharis macularius =0.322). t =0.009) wereobserved,whichthennotably =5.872, d.f.=6,P P=0.078) (Fig. 4B).However, posthoc test, =2.294, d.f.=6, t =0.445, d.f.=6,P =0.004) andreturnedtothepre- t =3.207, d.f.=6,P post hoctest, =0.001) andgraduallyincreased =0.001) andkneeflexion Post hoc P 12.244 7.449 1.667 19.465 2.881 16.984 3.044 0.263 4.475 1.984 0.294 1.374 0.682 0.867 0.613 3.344 F =0.062), whereasknee -ratio 062 Fg 4A). =0.672) (Fig. t tests suggestthat =0.018). Relative =–1.782, d.f.=6,

weeks of t=2.078, 0.279 0.147 0.137 0.779 0.077 0.232 0.757 0.334 0.547 0.475 0.578 0.120 0.012 0.004 0.032 0.006 P 3893 tests E.

The Journal of Experimental Biology 3894 locomotion in regenerating areplicaofthe original mightnotbeessentialfor hypothesize thatthetaildoesnot returntoitsoriginalshapebecause the costsofregeneratingalarger tailmayoutweighthebenefits. We regeneration isnotafforded suchahighpriorityinadults and that are notsmallerinweight(Lynn etal.,2013),suggestingthattail Interestingly, theregeneratedtailsofjuvenilessamespecies on anewshape,whichisshorterandwiderthantheoriginal(Fig. smaller inbothweightandvolumethantheoriginaltails take mature adultfemaleleopardgeckos,fullyregeneratedtails are 1974; Vitt etal.,1977;Lynn etal.,2013).Here,weshowthatin suggested toinfluenceregenerationratesingeckos(Congdon et al., regeneration areunknown,growthandmaturityhave been comparisons, autotomy trials(repeatedmeasuresANOVA and values thatarenotsignificantlydifferent fromvaluesobtainedinthepre- from sevenindividuals.Errorbarsrepresents.e.m.Opencirclesindicate depression, (B)femurretractionand(C)kneeanglearemeans+residuals measured throughoutautotomyandregeneration. Fig. RESEARCH ARTICLE

.Meansofmaximumhindlimbjoint anglesduringstancephase 3.

Pre-autotomy

P Post-autotomy Max. knee angle (deg) Max. femur retraction (deg) Max. femur depression (deg) 125 150 175 >0.05). 25 50 75 40 80 E. macularius. 0 B A C 2

4 In

6 Time (weeks) Aspidoscelis sexlineatus

post hoc 14

16 Values for(A)femur

18 tests formultiple

22 , thetailis

1). shorter andweighs lessthantheoriginal,CoM neverreturnedto the CoManteriorly. Additionally, becausetheregeneratedtailis the autotomizableportionof tailresultedinasignificantshiftof regeneration werenotsurprising. Asexpected,completeremovalof locomotion. et al.,2008),whichmayalsobe linkedtothefunctionoftailin tails inotherlizardspecies(Vitt et al.,1977;Daniels,1983;Medger been foundwhencomparingthesizesoforiginalandregenerated 2013), forwhichtailshapemaybelesscritical.Mixedresults have hypothesized tobedevotedprimarilyfatstorage(Lynn etal., (Ballinger etal.,1979).Incontrast,leopardgeckotails are suggested tofunctionasacounter-balance mechanism whilerunning in thepre-autotomytrials(repeatedmeasuresANOVA and circles indicatevaluesthatarenotsignificantlydifferent fromvaluesobtained residuals toremovetheeffect ofspeed.Errorbarsrepresents.e.m.Open specific peakpropulsiveGRF. Values forpropulsiveforcesaremeans+ (A) PeakverticalGRF. (B)Weight-specific peakverticalGRF. (C)Weight- Fig. multiple comparisons, The changesobservedinCoM associatedwithautotomyand

.MeansofpeakGRFsfromthepropulsive halfofstancephase. 4.

Pre-autotomy

Rel. peak propulsive GRF Rel. peak vertical GRF Peak vertical GRF (N)

The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110916 0.05 0.15

Post-autotomy 0.06 0.12 0.18 0.1 0.2 0.2 0.4 0.6 0 0 0 A C B P >0.05). 2

6 Time (weeks)

18 post hoc

22 tests for

The Journal of Experimental Biology RESEARCH ARTICLE tail, likethatof the leopardgecko,isproposed topositiontheCoM the musclesresponsibleforbody support.Additionally, along heavy posture oftheanimalandresulting changeintheleverarmsof the hindlimbs,whichmaybedirectly relatedtothemoresprawled immediately afterlosingthetail bygeneratinglessverticalforcein geckos maybeovercompensating forthelossofcaudalmass force afterautotomy(Fig. were stillfoundtoproducesignificantlylessweight-specificvertical when peakverticalGRFwascorrectedforweight,thehindlimbs the tailregenerated(Fig. the animalagainstforceofgravityanditgraduallyincreased as autotomized becauselessforceisneededtoliftthereducedmass of expected, peakverticalGRFdecreasedafterthetail was thought tohavesignificanteffects onhindlimbGRFproduction.As number ofstridesnecessaryfortheanimaltobegin‘recovering’. following autotomywouldlikelyrevealtheamountoftime or of hindlimbkinematicsoverthedays,orevenhoursandminutes response inhindlimbkinematics.Additionally, frequentobservations mass throughoutregenerationisnotlinear, resultinginanon-linear until weeks6–8(Fig. length, whereastaildiameterdoesnotbegintosignificantlyincrease stages oftailregenerationarerepresentedmostlybyincreasesin uniform changesintailformasregenerationproceeds.Theinitial depression, retractionandkneeanglesislikelyrelatedtothenon- return topre-autotomyconditionsobservedamongfemoral those obtainedwiththeoriginaltailbyweek10.Thedifferential after autotomy)andvaluesarestatisticallyindistinguishablefrom begins toreturnthepre-autotomystatebynexttrial(2weeks its tail.Interestingly, eachofthesehindlimbkinematicparameters to thechangeinweightdistributionduringitsfirststepsafterlosing (Rewcastle, 1981;FosterandHigham,2012)astheanimaladjusts means ofenhancingstabilityandbalancebyloweringtheCoM not surprising.We hypothesizethatthissprawledposturemaybea loss wouldoccurhere.Thus,thechangesinhindlimbkinematicsare 2006), itislikelythatalocomotorresponsetoautotomyandweight generators oflocomotorthrust(RussellandBels,2001;Chenetal., al., 2011). Becausethehindlimbsareperceivedtobeprimary indicative oftheanimaladoptingamoresprawledposture(Fulleret (Fig. by thedecreasesinfemoraldepression,retractionandkneeangles able toimmediatelyadjusttheshiftinweight.Thisissupported anterior shiftinCoMwasnotupheld,suggestingthattheanimalis Our predictionthatgeckobodypitchwouldbeaffected bythe kinematic adjustmentsofthelimbsand/orbody(Gillisetal.,2013). take toquicklycompensateforsuchreducedstabilityisdeploy reduction instabilityduringlocomotion.Oneapproachanimalscan propulsive system(thehindlimbs)followingautotomy, weexpecta the case. of neuromuscularfunctionareneededtodeterminewhetherthisis Lind etal.,2010;Kuo2011). However, long-termrecordings animals canadjusttoincreasesinbodyweight(Irschicketal.,2003; permanently ‘adapt’ tothisalteredmorphology, muchlikeother neuromuscular controloflocomotion.Possibly, theselizards that long-termcompensationmayoccurintheunderlying The factthattheCoMneverreturnedtoitsoriginallocationsuggests that changesinCoMareproportionallyrelatedtothesizeoftail. mathematical modelsofCoM(Gillisetal.,2013),whichsuggest was fullyregenerated.Thesefindingsprovideempiricalsupportfor about halfofitsinitialantero-posteriordisplacementoncethetail its originalposition.Instead,theCoMlocationrecoveredtoonly Furthermore, thechangesweobservedinlocationofCoM are Because theCoMislocatedinadifferent positionrelativetothe 3) observedimmediatelyfollowingautotomy, allofwhichare 1D). Thus,itislikelythattheincreaseintail

4A). Althoughthisrelationshipvanished

4B). Itisthereforepossible that the gravidity areavailable (ScalesandButler, 2007),thereare likelya Although forcedatadescribing the effects ofmasschangerelated to vertebrates andinvertebrates canoccurforanumberofreasons. selection toacton. compensation asaresultofthe lackofapost-autotomicperiodfor autotomizing speciestodetermine whetherthereisalackof interesting parallelstudywouldbetoremovethetailsofa non- for thoselizardsexhibitingtheabilitytoautotomizetheirtail. An locomotion followingautotomyhasbeenunderselectivepressure something tobeaddressedinfuturework.Itispossible that mechanics andmotionresultinadecreaseescapeability is predator–prey interactions.Whetherthesepermanentlymodified into howlizardsactuatemovementduringpost-autotomic regeneration areavailable.Suchobservationswillprovideinsight related tothechangesinweightassociatedwithautotomy and autotomy. Furthermore,noothersteadylocomotionforcedata Neither ofthesevariableswassignificantlyaffected bytail curvature andstridelength)wererecorded(Medgeretal.,2008). associated withautotomy, butjusttwokinematicvariables(body has incorporatedkinematicdataintoperformancemeasures rapid weightchangeonlocomotormechanics.Onlyoneotherstudy and formabaselineforfutureworkindeterminingtheeffects of (McElroy andReilly, 2009).Ourfindingsprovidesupportforthis variation, whichthendrivesforcevariationinterrestrialrunning location oftheCoMbyaddingmass. something thatisrarely(ifever)doneinstudiesmanipulatethe autotomy allowedustoassessGRFsoveraperiodoftime– locomotion, highlightingtheutilityofthissystem.Finally, tail reduces overallbodymassandmaynotimpacttheenergetic costof apart thedifferential impactsofthesevariables.Tail autotomy and thelocationofCoM,whichwouldmakeitdifficult totease of masslikelyresultsinachangetheenergetic costoflocomotion we founddifferences inkinematicsbetweentheconditions.Addition in agreaterthan10%reductionbodymass.Thismayexplainwhy in mass,especiallybecausetailautotomyleopardgeckosresults 2004). Itisdifficult toequatethisincreaseinmassourdecrease force wasunchangedfromthatofunloadedtrotting(Leeetal., This alterationdidnotimpactkinematicsandthemeanfore–aft was achievedbyadding10%ofbodymassatthepectoralgirdle. trotting indogs.AnanteriorshiftCoM(asoccurredourstudy) manipulated thedistributionofmassonmechanicslevel to beaddedthebody. For example, Leeetal.(Leeal.,2004) of theCoMinothervertebrates,suchasmammals,masswouldneed naturally occurringevent.Inaddition,inordertoalterthelocation shifting thelocationofCoM,especiallygiventhatitisa to supportthishypothesis. from thehindlimbs.However, dataontheforelimbGRFsareneeded significant roleinbrakingtocounteracttheeffect ofincreasedforce 4–6 without anincreaseinspeed,wehypothesizethatduringthefirst Because thehindlimbsareactuallyproducingmorepropulsiveforce propulsive GRFsfollowtheoppositeofexpectedtrend(Fig. interestingly, ourfindings show thatweight-specifichindlimb changes inspeedfollowingautotomyandperhapsmore (Ballinger etal.,1979;Punzo,1982).However, wedidnotfindany propulsive force,whichwould,inturn,reducesprintspeed the CoManteriorly, tailautotomy shoulddecreasehindlimb to thesubstrateduringlocomotion(Snyder, 1949).Thus,byshifting posteriorly sothatthehindlimbscanapplyagreaterpropulsiveforce Rapid weightchangeisa ubiquitous phenomenonacross Morphological variationhasbeenproposedtodrivekinematic Tail autotomyisavaluablewayofassessingtheimpacts weeks followingautotomy, the forelimbsmaybeplayingamore The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110916 3895 4C).

The Journal of Experimental Biology were sacrificedattheconclusionofexperimentsusing0.05 pinched toinitiateautotomyattheproximal-mostfractureplane.Animals Following pre-autotomymeasurements/trials,thebaseoftailwasgently of thefourthtoe.Jointswereallmarkedonleftforelimbsandhindlimbs. shoulder/hip, elbow/knee,wrist/ankleandthemetapodial-phalangealjoint midline oftheback,centerpectoral/pelvicgirdlesalongmidline, for visualizationinhigh-speedvideos:dorsalmidpointofthebodyalong dots ofwhitenailpolishwereappliedtothefollowingpointsonanimals throughout thedurationofexperiment.Priortolocomotortrials,small 28–33°C. Geckoswerefedadietoflivecrickets pentobarbital (390 along thetrackway ataconstantspeed(wedidnotattempt toelicitmaximal recorded foreach combinationofindividualandtrial astheanimalmoved coordinates fordigitizing.Threeforelimb andthreehindlimbstrideswere calibration objectconstructedofLEGO™ blockswasusedtoproduce3D view (45 cameras wereorientedtoprovidea directlateralviewandanobliquedorsal two PhotronAPX-RScameras(Photron USA,SanDiego,CA,USA).The (A-20110025 andA-20110038). with theUniversityofCalifornia,RiversideAnimalCareandUseProtocols were frozenforlateruse.Allanimalresearchwasconductedinaccordance 22 until week14and4 intervalsthereafter, untilregenerationceasedat 3896 Lizards werefilmedat250 (Langkilde andShine,2006).Strideswerethenrecordedat2 potential effects offatigueor stressassociatedwiththeremovaloftail the firststridesananimalmadewithoutatail,whilealsominimizing movement. Thisensuredthatthepost-autotomytrialwasrepresentative of animal wasallotted20–30 filming. Followingthepre-autotomytrials,autotomywasinducedand each The temperatureoftheexperimentalroomwasmaintainedat~30°C when a levelflattrackway(1.0×0.15 Stride kinematicsandGRFdatawereobtainedfromeachgeckoasit ran on tail) andoriginaltail(reattachedusing2–3dropsofsuperglue). fully regeneratedtail(stillattached),no(afterremovalofthe 1983). ThelocationoftheCoMwasdeterminedforeachindividualwithits measurements wereobtainedviaathread-suspensionmethod(Alexander, in agraduatedcylinder(Vitt etal.,1977). regenerated tails.Volumes ofthetailsweremeasuredbyliquiddisplacement sacrifice inordertocomparetheweightandvolumeoforiginal regenerated tailswereremovedwithascalpelatthepointofautotomyafter 18 tail lengthanddiameterceasedshowingsignificantchangesafter weekly for22 the tail)weremeasuredbeforeandaftertailwasautotomizedbi- the tipoftail)andtaildiameter(obtainedfromwidestpointalong Body mass(includingtailmass),SVL,length(distancefromtheventto individually interraria(50.8 with originaltailswereobtainedfromcommercialsuppliersandhoused Seven adultfemale distribution withoutdetrimentaleffects tothestudyorganisms. lizards allowforprecisemanipulationsofweightchangeandmass rapid weightloss.Autotomyandregenerationstudiesconductedon autotomy islikelyagoodmodelsystemforstudyingtheeffects of physiological changesthatcouldimpactlocomotionisunclear, but that cancontributetolocomotion.Whetherautotomyalsoresultsin number ofotherphysiologicalvariablesassociatedwithgravidity RESEARCH ARTICLE Stride kinematics Experimental set-up andCoM tailmorphology of Measurement Study organisms MATERIALS ANDMETHODS After sacrificingandfreezingthegeckostailsuntilrigid,CoM weeks, yieldingatotalof11 trials. weeks, suggestingthattailregenerationwascompletebyweek22.Fully

deg) andsynchronizedwithanexternal trigger. A pre-measured weeks asthetailregenerated.Measurementsofbodyweight, mg E. macularius ml −

1 frames ). Allsacrificedanimalsandautotomizedtails

min torestinaclothbagwithrestricted

m) coveredwithcorktopreventslipping.

25.9 s (mass, 39.4±1.2 − 1 with ashutterspeedof1/2000 ×

32.0 cm (l ad libitum

g; SVL,121.9±2.2 ×

h) maintainedat

week intervals until satiation

ml ofsodium

s using mm) statistical analyseswereperformedusingSYSTAT 13.00.05. equal varianceswerenotviolatedforanyofthevariablesmeasured. All autotomy trialwitheachsuccessivetrial.Assumptionsfornormality and Bonferroni correctionswereusedforpair-wise comparisonsofthepre- variable throughoutautotomyandregeneration, form. A repeated-measuresANOVA wasagainusedtocompareeach statistical analyseswhereasallothervariablesremainedintheiroriginal were obtainedbycorrectingfortheweightofeachindividual. accelerative phaseofeachhindlimbfootfall.Relative(weight-specific)GRFs UK). PeakverticalandpeakpropulsiveGRFswerecalculatedfromthe AcqKnowledge 4.0.0software(BIOPAC Systems,University ofCambridge, and verticalgroundreactionforcesweresampledat5000 Three runswereobtainedperindividualforeachtrial.Fore-aft,medial-lateral one hindlimb(withoutcontactfromanyotherlimbs)wereusedinthisstudy. Only runsinwhichthegeckosteppedcompletelyontoforceplatformwith gecko tomoveovertheforceplatewithonlyonehindlimbmakingcontact. surface (0.10×0.05 for themovinganimalandembeddedincenteroftrackwaywithits Elbridge, NY, USA).Thiswascoveredincorktopreventachangesubstrate Automation, Apex,NC,USA)andacarbon-fibertopplate(DragonPlate, which consistedofaNano176-axisforce/torquesensor(ATI Industrial Ground reactionforceswerequantifiedusingacustom-madeforceplatform, R2012a, TheMathWorks, Natick, MA,USA)inordertoobtain using DLT DV 5customsoftware(Hedrick,2008)forMATLAB (version performance). We digitizedthepoints markedalongthebodyandlimbs IOS-1147043] andstart-up fundstoT.E.H. Financial supportwas providedbytheNationalScienceFoundation [grantnumber findings andrevisingthemanuscript. T.E.H. andA.P.R. wereinvolvedintheconceptionofstudy, interpretation ofthe manuscript. T.E.H. assistedwiththedesign andexecutionoftheexperiments, experiments, interpretationofthefindings anddraftingrevisionofthe K.J. madesubstantialcontributionsto the designandexecutionof The authorsdeclarenocompetingfinancialinterests. also thanktwoanonymousreviewerswhosecommentsimprovedthemanuscript. collection andforprovidinghelpfulfeedbackonthisprojectmanuscript. We We thankallmembersoftheHighamLabforassistancewithanimalcare,data had asignificantrelationship( variables individuallyagainstbodyspeed.Theresidualsofallthat on kinematicsandforcevariableswereremovedbyregressingallofthe individual pertrialwereusedforallstatisticalanalyses.Theeffects ofspeed tails. CoM positionbetweenlizardswithoriginal,autotomizedandregenerated repeated measures.Thelattermethodwasalsousedtocompareshiftsin autotomy, post-autotomy, 2 throughout autotomyandregeneration.Measurementsateachtrial(pre- measures ANOVA wasusedtocomparechangesintaillengthanddiameter were usedtoanalyzedifferences inmassandvolumearepeated- To compareoriginalandfully-regeneratedtailmorphology, paired Hindlimb locomotorforces Funding Author contributions Competing interests Acknowledgements Statistical analyses calculations aremadeavailableelsewhere(FosterandHigham,2012). ankle angles)throughouteachstride.Extensivedetailsofhowthese (humerus/femur depression,humerus/femurretractionandelbow, kneeand duty factor, hipheight,shoulderbodypitchandjointangles throughout thestridedividedbydurationofstride),length, body speed(distancetraveledbythepointatcenterofpectoralgirdle movements, respectively. Thesecoordinateswerethenusedtocalculate coordinates todescribeantero-posterior, medio-lateralanddorso-ventral Averages ofthekinematicandGRFvariablesthreerunsforeach The JournalofExperimentalBiology(2014)doi:10.1242/jeb.110916 m) flushwiththelatter. Thisallowedampleroomforthe

weeks post-autotomy, etc.)wereusedasthe α≤0.10) withspeedwereusedforsubsequent post hoc

tests with Hz using x , y t -tests and z

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