Functional Ecology. 2019;00:1–11.  | 1wileyonlinelibrary.com/journal/fec

Received:13April2019  |  Accepted:3July2019DOI: 10.1111/1365-2435.13408

R E S E A R C H A R T I C L E

Distinct telomere differences within a reproductively bimodal common lizard population

Darryl McLennan1,2  | Hans Recknagel1  | Kathryn R. Elmer1  | Pat Monaghan1

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsuse,distributionandreproductioninanymedium,providedtheoriginalworkisproperlycited.©2019TheAuthors.Functional EcologypublishedbyJohnWiley&SonsLtdonbehalfofBritishEcologicalSociety

DarrylMcLennanandHansRecknagelcontributedequallytothiswork.

1InstituteofBiodiversity,AnimalHealthandComparativeMedicine,UniversityofGlasgow,Glasgow,UK2DepartmentofFishEcologyandEvolution,EAWAG,Kastanienbaum,Switzerland

CorrespondenceDarrylMcLennanEmail:darrylmclennan@outlook.com

Funding informationNaturalEnvironmentResearchCouncil,Grant/AwardNumber:NE/N003942/1;H2020EuropeanResearchCouncil,Grant/Award Number: 322784

HandlingEditor:FrédéricAngelier

Abstract1. Differentstrategiesofreproductivemode,eitheroviparity(egg‐laying)orvivipar-ity(live‐bearing),willbeassociatedwitharangeofotherlife‐historydifferencesthatareexpectedtoaffectpatternsofageingandlongevity.Itisusuallydifficulttocomparetheeffectsofalternativereproductivemodesbecauseofevolution-aryandecologicaldivergence.However,theveryrareexemplarsofreproductivebimodality,inwhichdifferentmodesexistwithinasinglespecies,offeranoppor-tunityforrobustandcontrolledcomparisons.

2. Onetraitofinterestthatcouldbeassociatedwithlifehistory,ageingandlongev-ityisthelengthofthetelomeres,whichformprotectivecapsatthechromosomeendsandaregenerallyconsideredagoodindicatorofcellularhealth.Theshorten-ingofthesetelomereshasbeenlinkedtostressfulconditions;therefore,itispos-siblethatdifferingreproductivecostswillinfluencepatternsoftelomereloss.Thisisimportantbecauseanumberofstudieshavelinkedashortertelomerelengthtoreduced survival.

3. Here,wehavestudiedmaternalandoffspringtelomeredynamicsinthecommonlizard(Zootoca vivipara).Ourstudyhasfocusedonapopulationwhereoviparousand viviparous individuals co‐occur in the same habitat and occasionally inter-breedtoformadmixedindividuals.

4. Whileviviparityconfersmanyadvantagesforoffspring, itmightalso incursub-stantial costs for themother, for example requiremore energy. Therefore,wepredictedthatviviparousmotherswouldhaverelativelyshortertelomeresthanoviparousmothers,withadmixedmothershavingintermediatetelomerelengths.Thereisthoughttobeaheritablecomponenttotelomerelength;therefore,wealsohypothesizedthatoffspringwouldfollowthesamepatternasthemothers.

5. Contrarytoourpredictions,theviviparousmothersandoffspringhadthelongesttelomeres,andtheoviparousmothersandoffspringhadtheshortesttelomeres.Thedifferingtelomerelengthsmayhaveevolvedasaneffectofthelife‐historydivergencebetweenthereproductivemodes,forexampleduetothe increased

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1  | INTRODUC TION

Differingreproductivestrategiescanco‐occurwithinaspecies,dueto both environmental and genetic differences among individuals,aswellastheinteractionbetweenthetwo(Taborsky&Brockmann,2010).Thisphenotypicvariationcanexistonacontinuousspectrumorfallintodiscontinuouslife‐historymodes(Partridge,MacManes,Knapp,&Neff,2016;Tsubaki,Hooper,&Siva‐Jothy,1997),andstrat-egies canbeplasticor fixedwithin an individual's lifetime (Bailey,Gray,&Zuk,2010;Baum,Laughton,Armstrong,&Metcalfe,2004;Bronikowski&Arnold,1999;Meunieretal.,2012;Zamudio&Chan,2008).

Astrikingexampleof fixed,discontinuousreproductivemodesiswhenaspeciesisbimodalforbothoviparity(i.e.egg‐laying)andviviparity(i.e.givingbirthtoliveyoung).Viviparityisevolutionarilyderivedfromoviparityandconfersmanybenefitstooffspring,par-ticularlyamorestableandprotecteddevelopmentalenvironmentinthemother's reproductive tract (Shine, 1995).However, viviparitycanalsorestrictamother'sreproductiveoutputandmayreducethenumberofclutchesproducedwithinagivenyear(Blackburn,1999;Recknagel&Elmer,2019;Sites,Reeder,&Wiens,2011;Wourms&Lombardi,1992).Mostvertebratespeciesexhibitonlyoneofthesereproductivemodes;however,inasmallnumberofspecies,bothre-productivemodesoccur(Murphy&Thompson,2011).Thedifferingmaternalcostsassociatedwitheachreproductivemodehavebeenlittle studied, in part because so few species exhibit bothmodes.However,ithasbeensuggestedthatviviparitymayincursubstantialcostsforthemother.Firstly, theprolongedgestationalperiodandpresumed largerclutchmass inviviparous individuals (Horváthováetal.,2013;Qualls&Shine,1998;Roitbergetal.,2013)couldcarryagreatermetaboliccostforthemother.Secondly,viviparityhasbeenlinkedtotheevolutionoflargerbodysizestocounteractthespaceconstraint in utero (Qualls & Shine, 1995) and there can be costsassociatedwithrapidgrowthand/oralargerbodysize(Metcalfe&Monaghan,2003).

Itislikelythatsuchadivergenceinreproductivemodewillalsobeassociatedwithotherlife‐historydifferences,whichcouldinturnaffectpatternsofageingand longevity.Onetrait thatcouldbeofinterestinthiscontextistelomerelength.Telomerescaptheendsofeukaryoticchromosomesandplayanimportantroleinchromosomeprotection(Blackburn,1991;Monaghan,2010).Thesetelomerecapsshortenwith each round of cell division because of the ‘end rep-licationproblem’ (Levy,Allsopp,Futcher,Greider,&Harley,1992).Certainspeciesarecapableoftelomereelongation,mostlyviatheexpressionoftheenzymetelomerase(Gomes,Shay,&Wright,2010;

Tianetal.,2018).However,intheabsenceofelongationmechanisms,telomeresmayshortentosuchanextentthatthecentralcodingre-gionofthechromosomebecomesvulnerable.Assuch,arelativelyshorttelomerelengthisconsideredtobeanindicatorofpoorcellu-larandbiologicalstate,andanumberofstudieshavelinkedashortertelomerelengthand/orafasterrateoftelomereattritiontoreducedsurvivaland/orlongevity(Boonekamp,Mulder,Salomons,Dijkstra,&Verhulst,2014;Debes,Visse,Panda,Ilmonen,&Vasemagi,2016;Dupoué et al., 2017; Salmón, Nilsson, Nord, Bensch, & Isaksson,2016;Wilbournetal.,2018).

Inadditiontocelldivision,theacceleratederosionoftelomereshas been linked to environmental stressors, potentially via oxida-tivestresspathways(Monaghan&Ozanne,2018;Reichert&Stier,2017,but seealsoBoonekamp,Bauch,Mulder,&Verhulst,2017).Recentstudieshave linked telomeredynamics tovariousstressfulconditions,bothinvitroandinvivo(Barnes,Fouquerel,&Opresko,2018; Cram, Monaghan, Gillespie, & Clutton‐Brock, 2017; Debesetal.,2016;Monaghan,2014;Olssonetal.,2018).Moreover,are-centstudyonAustralianpainteddragons(Ctenophorus pictus)foundthat telomere dynamics differed between individuals with differ-entreproductiveandlife‐historytactics (Rollingsetal.,2017). It isthereforepossiblethatdifferingreproductivecostsassociatedwithoviparityorviviparitywillalsoinfluencepatternsoftelomereloss.

Inadditiontoaffectingmaternaltelomerelength,differencesbetween oviparous and viviparous life‐history strategies couldalso affect the telomere lengthof theoffspring that arise fromthese reproductive modes. A number of studies have reporteda heritable component to telomere length (Bouwhuis, Verhulst,Bauch,&Vedder,2018;Dugdale&Richardson,2018).Moreover,offspringtelomerelengthcouldalsobesubjectedtomaternalef-fects,forexampleviadifferencesinembryonicprovisioningand/or embryonic environment (McLennan et al., 2018a; Noguera,Metcalfe, Reichert, & Monaghan, 2016), as well as potentialmaternal effects on oocyte telomere length (Keefe, Kumar, &Kalmbach, 2015). Further, since oviparous offspring interactwith the environment at an earlier life stage, it is possible thatthetelomeresof theseoffspringcouldthenbedifferentiallyaf-fected by environmental factors, for example via temperatureandgrowtheffectsonearly lifedevelopment (McLennanet al.,2018b;Monaghan&Ozanne,2018;Vedder,Verhulst,Zuidersma,&Bouwhuis,2018).

The common lizard (Zootoca vivipara) is one of only a few ex-tantvertebratespeciesinwhichbothviviparityandoviparityoccur(Surget‐Grobaetal.,2006).Inthisstudy,weexaminematernalandoffspring telomere dynamics in a wild common lizard population

growth rate that viviparous individuals may undergo to reach a similar size atreproduction.

K E Y W O R D S

life‐historyvariation,oviparity,physiologicalstate,squamate,viviparity

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in which different evolutionary lineages with either an oviparousor viviparous reproductivemode coexistwithin the same habitat.Reproductivemodeinthisspeciesisknowntobegeneticallydeter-minedandfixedbetweenlineages(Arrayago,Bea,&Heulin,1996;Recknagel&Elmer,2019;Recknagel,Kamenos,&Elmer,2018).Theoviparousmotherslaycalcifiedeggs~33daysaftercopulation(i.e.oviposition)withameanthicknessof40µm,whicharethenincu-bated by themother for ~28 days (Arrayago et al., 1996; Heulin,1990; Lindtke,Mayer,&Böhme,2010).Offspring fromviviparousmothersareretainedinuteroforthedurationofembryonicdevel-opment (~57 days between copulation and birth; Arrayago et al.,1996)andarefullydevelopedatbirth(i.e.parturition).Eachvivipa-rousoffspringisbornindividuallysurroundedbyathinmembrane,fromwhichtheythen‘hatchout’ofimmediatelyorwithinafewdaysafter parturition (Recknagel & Elmer, 2019). Because this popula-tionoccursinauniquecontactzonebetweenthetworeproductivemodes,admixture isalsopossible (Lindtkeetal.,2010;Recknagel,2018). Admixed offspring are created when interbreeding occursbetween the two reproductive modes. These offspring may befirst‐generationhybridsor result fromthebackcrossingofhybridswith oviparous or viviparous individuals. Offspring from admixedindividualsare laid inthinnerand lesscalcifiedeggs,butata laterdevelopmentalstage,comparedwithoviparousoffspring.However,embryomortalityisalsomuchhigherintheadmixedoffspring,withestimatesofaround40%(Lindtkeetal.,2010).

Here,wehavemeasured telomere length in viviparous, ovipa-rous andadmixed common lizards, ofbothmothers and theiroff-spring.Wepredictedthatviviparousmothers(withthepresumptionthattheyincurahigherreproductiveburden)wouldhaverelativelyshorter telomeres than oviparous mothers, while the telomerelengthsofadmixedmotherswouldbe intermediatebetween theiroviparousandviviparousconspecifics.Lastly,sincethereisknowntobeaheritablecomponenttotelomerelength,wepredictedthatthepatternsoftelomerelengthintheoffspringwouldfollowthoseofthemothers.

2  | MATERIAL S AND METHODS

2.1 | Field study

ThefieldaspectofthisstudywasconductedintheGailtalvalleyoftheCarinthianAlps,Austria(N46.60°,E13.14°),andunderpermitnumber HE3‐NS‐959/2013 (012/2016). This is currently the onlyknown locationwherebothviviparousandoviparouscommon liz-ardsco‐occurinhighdensitiesandoccasionallyinterbreed(Cornettietal.,2015;Lindtkeetal.,2010;Recknagel,2018).Thestudysitecoveredanareaofapproximately0.3km2andanaltitudinalrangeof1,380–1,580m.Wild female lizardswerecaughtbetweenMayandJuly2016;femalescanbedistinguishedfrommalesbytheab-senceofahemipenalbulgeatthebaseofthetail.Whetherornotafemalehadrecentlymatedwasidentifiedbythepresenceofabitingmarkonthefemale'sbellyorflank(Lindtkeetal.,2010).Immediatelyaftercapture,alllizardswereweighed(tothenearest0.001g)and

snout–ventlength(SVL)andtaillength(TL)weremeasured(tothenearest0.01mm).Tailautotomy(self‐amputationofthetailasade-fencefrompredatorsandconspecifics) isacommonoccurrenceinthis species.Tail autotomyhaspreviouslybeen linked to telomeredynamics(Olsson,Pauliny,Wapstra,&Blomqvist,2010);therefore,forthisstudy,wedecidedtoincludeonlyfemalesthathadanon‐au-totomizedtail.

Females thathad recentlymated (andwere therefore likely tobepregnant)weremovedtonearbyholding facilitiesso that theirreproductivemodecouldbeassessed.Allfemaleswereindividuallyhoused inplastic terraria (56×39×28cm) thatwerecoveredbynettingonthetopandononeside,toallowsufficientairflow.Theterrariawerehousedwithintents,sothateachlizardwasexposedtonatural temperaturevariation.Eachterrariumcontainedsandsub-strate,suitableshelter(e.g.piecesofdriedwood),moisturizedmossandabowlofwater. Lizardswere fedad libitumwithmealworms(Tenebrio molitor)andcrickets(Gryllus assimilis).

Eachfemalewascheckeddailyforthepresenceofoffspring.Onthe sameday that a femalehadundergoneoviposition (to shelledeggs)orparturition(toliveyoung),thefemalewasweighed(tothenearest0.001g)andthenumberofoffspringwithinaclutch(here-afterclutchsize,CZ),relativeclutchmass(RCM:clutchmass,includ-ingeggshell,amnioticfluidsandyolk,dividedbyfemalemassafteroviposition/parturition)andrelativeoffspringmass(ROM:summedmass of the offspring after hatching, excluding eggshell, amnioticfluidsandyolk,dividedbyfemalemassafteroviposition/parturition)was measured. After oviposition/parturition, a tail clip was takenfromeachfemaleforsubsequentsequencingandtelomereanalysis.Whileabsolutetelomerelengthsmaydifferamongtissues,studieshavefoundstrongcorrelationsintelomerelengthbetweendifferenttissues in birds (Reichert, Criscuolo,Verinaud, Zahn,&Massemin,2013)andlizards(Rollingsetal.,2019).Moreover,astudyonbrowntroutSalmo truttabyDebesetal. (2016) foundtrends intelomeredynamicstobesimilaramongtissues,includinghighlyregenerativetissues suchas fin.Therefore,wewere confident inusing the tailclips for a non‐invasivemeasurement of telomere length. Shortlyaftersampling,thefemaleswerereturnedandreleasedbacktothesamelocationatwhichtheyhadbeencaught.Unhatchedoffspringwereincubatedat24°CinanExoTerraIncubator.Onthedayofbirthorhatching,each individualwasweighed (to thenearest0.001g),SVLwasmeasured(tothenearest0.01mm),andatailclipwastakenforsubsequenttelomereanalysis.Theoffspringwerethenreturnedand releasedback to thesame locationatwhich themothershadbeencaught.Intotal,wehadcompletetailsamplesfor69mothers(23oviparous,33viviparousand13admixedindividuals).Offspringthatdidnothatchwereexcluded.Intotal,wehadoffspringtailclipsamplesfrom57ofthemothers(20oviparous,29viviparousand8admixedindividuals).

2.2 | DNA extraction

ThetailclipsamplesweretransferredtotheUniversityofGlasgow,UK.DNAwasextractedfromallsamplesusingtheMacherey‐Nagel

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DNA NucleoSpin Tissue Extraction Kit, following the manufac-turer'sprotocol.Foreachofthemothers,across‐sectionalsampleofthetail(approximately3mmwide)wasincubatedin180µllysisbuffer+20µlofproteinaseKsolution(20mg/ml)at56°Covernight.Sampleswerethencentrifugedtoseparatebonefragmentsfromthetissuelysate,withthelysatebeingusedinthesubsequentDNAex-traction.Fortheoffspring,allsiblingswithinaclutchwerepooled(min1offspring,max10offspring)sothattherewasoneaveragedoffspringDNAsamplepermother.Again,across‐sectionalsampleoftail (approximately1mmwide)wastakenfromeachofthesib-lingsandthepooledtissuewasthenprocessedinthesamewayasforthemothers.DNAconcentrationandpuritywasmeasuredspec-trophotometricallyusingaNanoDrop8000,whichconfirmed thatallsamplesmettherecommendedA260/280ratioandhadaDNAconcentration>20ng/µl.

2.3 | Identifying maternal reproductive mode by ddRADSeq

Oviparous and viviparous females are easily distinguishable byphenotype:oviparousoffspringarelaidincalcifiedshellsandthenrequire ~28days of incubation prior to hatching,while viviparousoffspringarebornfullydeveloped,butinathinmembranethattheythen‘hatchout’ofwithinafewhourstodaysafterbirth(Arrayagoetal.,1996;Lindtkeetal.,2010).However,becauseourstudypopula-tionoccurs inauniquecontactzone,therewerealsofemalesthatlaidadmixedoffspringthatexhibitedintermediatephenotypes,suchaspartiallycalcifiedshellsandfewerdaysofincubationcomparedtooviparousclutches.Therefore,weusedadouble‐digestrestrictionsite‐associatedsequencing(ddRADSeq)approachtogeneticallydis-tinguishbetweenthereproductivemodesandadmixedindividuals.Todoso,wefollowedtheddRADSeqlibrarypreparationprotocolofRecknageletal.(2018);seeAppendixS1fordetails.

Femaleswereassignedamembershipvalue(Q)toestablishfromwhichlineageofwhichreproductivemodetheyderived(Recknageletal.,2018).Femalesthathadastrongsignatureofoviparousge-nomic background (Q value ≤0.01) were assigned as oviparous,whilefemaleswithastrongviviparousgenomicbackground(Q value ≥0.99) were assigned as viviparous. Females that had a genomicbackgroundofadmixture(Qvalue>0.01and<0.99)wereassignedasadmixedindividuals.Weassignedareproductivemodeto67outof69of the femalesbasedon theirgenotypes.Twoof the lizardswerenotincludedintheddRADSeqlibrary;however,theirnumberofincubationdayswasclearlyintheviviparousandoviparousranges(2and35, respectively); therefore,wewereconfident inassigningtheirreproductivemodebasedonphenotypealone.

2.4 | Telomere analysis

Common lizard telomere length has been previously measuredusingtheTeloTAGGGTelomereLengthAssay(Dupouéetal.,2017),confirming that common lizard telomeresarealsomadeupof therepetitivesequenceTTAGGG.For this study, telomere lengthwas

measured in all samples using the quantitative PCR method de-scribedbyCawthon(2002).TheuniversalTel1bandTel2bprimersdesignedbyCawthon(2002)andmodifiedbyEpeletal.(2004)wereused for amplification of the telomere repeats. The recombina-tionactivatinggene1(RAG‐1)waschosenasthesingle‐copygene,and the Z. vivipara RAG‐1 sequence (GenBank accession number:KY762205.1)wasusedtodesignprimers.ThefollowingforwardandreverseRAG‐1primerssuccessfullyamplifiedasingleamplicon,asdeterminedbymeltcurveanalysis,andweresubsequentlyusedintheanalysis:

LizRAG1‐F5′‐GCCAACTGCAACAAGATACAC‐3′andLizRAG1‐R5′‐GATATGCTCACAGACCTGACAA‐3′.

A full outlineof theqPCRprotocol is provided in theAppendix S1.The samples (69mothers and 57 offspring) were randomly distrib-utedacrosssixsetsofPCRplates.qPCRdatawereanalysedusingtheqBASEsoftwareforWindows(Hellemans,Mortier,Paepe,Speleman,&Vandesompele,2007),asdescribedinMcLennanetal. (2016).Foreachsample, theqBASE softwareproducedacalibratednormalizedrelativequantity(CNRQ).ThisissimilartotheT/SratiodescribedbyCawthon(2002)butwithgreatercontroloftheqPCRefficiencyandinter‐platevariation(seeAppendixS1forfurtherdetails).Threepointsfromthestandardcurve(5,10and20ng/well)wereusedasinter‐runcalibratorsduringqBASEanalysis, tohelp correct for inter‐runvari-ation.Theremainingthreepointsofthestandardcurve(40,2.5and1.25ng/well)wereusedtocalculateaninter‐assaycoefficientofvari-abilityoftheCNRQs(whichwas8.22).Theaverageintra‐platevaria-tionoftheCtvalueswas0.94forthetelomereassayand0.33fortheRAG‐1assay,respectively.Theaverageinter‐platevariationoftheCtvalueswas1.44forthetelomereassayand0.34fortheRAG‐1assay,respectively.TheefficienciesofthetelomereandRAG‐1assaysrangedfrom95.6% to 108.5% and 91.7% to 97.9%, respectively, andwerethereforewithintheacceptablerange(85%–115%).Theaveragequan-tificationcycle(Ct)forthetelomereandRAG‐1assayswas12.04and22.87,respectively.WefoundnosignificantdifferenceintheRAG‐1Ctvaluesamongthethreereproductivemodes(oviparous,viviparousandadmixed)(GLMF2,66=1.08,p=.35),butahighlysignificantdifferenceamongthetelomereCtvalues(GLMF2,66=19.08,p<.001).

2.5 | Statistical analysis

WeusedaPearsoncorrelationcoefficientmatrixtoassesspotentialcollinearitybetweenallcovariates(withacut‐offcoefficientof0.8).Mass and lengthwere highly collinear for bothmothers (Pearsonr=.91,p<.001)andoffspring(Pearsonr=.91,p<.001);therefore,onlymasswasused forsubsequentanalyses.Allvariablesused intheanalysesareshowninTable1.

All statistical analyseswere conducted using r version 3.5.0 software. Intotal,weraneightstatisticalmodels.Firstly,werangeneral linear models (GLMs) to assess whether maternal mass(model 1), offspring mass (model 2), clutch size (model 3) andrelative clutchmass RCM (model 4) and relative offspringmass

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ROM (model 5) varied between the female reproductive mode.Secondly,weconductedalinearmixedmodel(LME)usingthelme4andlmerTestfunctions(Bates,Maechler,Bolker,&Walker,2015;Kuznetsova, Brockhoff, & Christensen, 2014) to assesswhetherRTL (relative telomere length) differed between life stage andreproductivemode,which includedfamily IDasarandomfactorto control for non‐independencebetweenmother andoffspring(model 6). Estimates of marginal (fixed effects) and conditional(fixedeffects+randomeffects)R2valuesofthemixedmodelwerecalculatedusingtheMuMinpackage.Finally,welookedatfactorsaffecting variation in RTL at each life stage by conducting twoseparateGLMs.Firstly,maternalRTLwasassessed inrelationtoreproductivemode,maternalmass,clutchsizeandRCM(model7).Thismodelwasthensimplifiedbybackwardselimination,startingwiththemostinsignificanttermandcontinuingwithinsignificantmaineffectsuntilthemodelcontainedonlysignificantterms;pro-vidingthatthisresultedinareductionoftheAICscore.Secondly,meanoffspringRTLwasassessedinrelationtomaternalRTL,re-productivemode,offspringmass,clutchsize,RCMandtheinter-actionmaternal RTLwith reproductivemode to assesswhetherthe relationship betweenmaternal RTL andmean offspring RTLdiffered between the reproductivemodes (model 8). As before,model 8 was then simplified by backwards elimination, startingwith insignificant interactions and continuing with insignificantmain effects until the model contained only significant terms.Variableswereonlyremovedfromthemodel ifthisresultedinareductionoftheAkaikeinformationcriterion(AIC)score.

3  | RESULTS

3.1 | Life‐history variation

Wedidnotfindasignificantdifferenceinsomaticmassamongthere-productivemodesatthematernalstage,measuredafteroviposition/parturition(Table2.1andFigure1a);however,therewasasignificantdifference at the offspring stage, measured on the day of birth or

hatching,withoviparousoffspringbeingtheheaviest,viviparousoff-springbeingthelightestandadmixedoffspringbeingtheintermediate(GLMF2,54=30.85,p< .001;Table2.2andFigure1b).Thenumberofoffspringwithinaclutchalsodifferedsignificantlyamongthe re-productive modes, with viviparous mothers producing the smallestclutchesandoviparousmothersproducingthelargestclutches(GLMF2,66=3.99,p= .023;Table2.3andFigure2c).RCM(relativeclutchmass, including eggshell, amniotic fluids, etc.) did not significantlydiffer among themodes, suggesting that therewas similar resourceinvestmentamongthemothers(Table2.4).However,wedidfindasig-nificantdifferenceinROM(relativeoffspringmass,excludingeggshell,amnioticfluids,etc.)amongthereproductivemodes,withROMbeinghighestintheoviparousfamiliesandlowestintheviviparousfamilies(GLMF2,54=33.15,p<.001;Table2.5andFigure2b).Thissuggeststhatwhiletherewasasimilarinvestmentofmaternalresourcesamongthereproductivemodes(intermsofembryo,eggshell,yolkandamni-oticfluidproduction),theoviparousfemaleshadahighernetgainperclutch,supportedbythelargerclutchsizeandoffspringmass.

3.2 | Telomere length variation

TherewasasignificantdifferenceinRTLbetweenmothersandoff-spring.Mothershadrelativelylongertelomeresthantheiroffspringwithineachofthethreereproductivemodes(LMMF1,55.03=32.14,p< .001;Table3andFigure3).Telomere lengthalsodifferedsig-nificantly between the reproductivemodes. The viviparousmodehad the longest telomeres, the oviparous mode had the shortesttelomeres,andtheadmixed individualshadrelatively intermediatetelomeres(LMMF2,59.13=72.55,p<.001;Table3andFigure3).Thiswastrueforbothmothers(GLMF2,66=37.98,p<.001;Table2.7andFigure3)andoffspring(GLMF2,54=63.12,p<.001;Table2.8andFigure3).Whenfocusingonlyonthemothers,maternalmass,clutchsizeandRCMwerenotsignificantlyassociatedwithmaternalRTL.For the offspring,maternal RTL, offspringmass, clutch size, RCMandtheinteractionmaternalRTLXreproductivemodewerenotsig-nificantlyassociatedwiththerelativemeantelomerelength.

TA B L E 1  Summaryofallthevariablesusedinstatisticalanalyses

Variable name Variable description

Lifestage Lifestageatwhichasamplewastaken(i.e.whetherfrommotheroroffspring).Factor

Reproductivemode Reproductivephenotypeofeachmother,basedontheddRADSeqanalysis.Offspringwereassignedthesamemodeastheirmother.Factor

MaternalRTL Relativetelomerelengthofeachmother,measuredattheindividuallevel.Covariate

MeanoffspringRTL Relativeoffspringtelomerelength,measuredasanaverageforeachmotheracrossalloffspring.Covariate

Maternalmass Somaticmassofeachmotherafterovipositionorparturition(tothenearest0.001g).Covariate

Offspringmass Somaticmassofoffspringatthetimeofhatching,measuredasanaverageforeachmother(tothenearest0.001g).Covariate

RCM Relativeclutchmass:clutchmassdividedbyfemalemassafterovipositionorparturition.Covariate

ROM Relativeoffspringmass:sumoftheoffspringmassafterhatchingdividedbyfemalemassafteroviposition/parturition.Covariate

Clutchsize Numberofoffspringbornwithinagivenclutch.Covariate

Note: SeeSection22foranoutlineofeachmodel.

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4  | DISCUSSION

This study has shown that different reproductivemodes of thecommon lizard significantly differ in telomere length. The vi-viparousmodehad the longest telomeres (forbothmothersandoffspring), the oviparous mode had the shortest telomeres (forbothmothers and offspring), while the admixed individuals hadrelatively intermediate telomeres (again, for both mothers andoffspring).Sincethesignificantdifferenceintelomerelengthwasalreadyevidentattheoffspringstage,itsuggeststhattherepro-ductive modes have evolved differing telomere dynamics overtime.However, it iscurrentlyunclearwhetherand/orhowthesedifferences in telomere length have co‐evolved with other life‐history traits.We also found thatmothers had relatively longer

telomeresthantheiroffspringamongthereproductiveecotypes.However,wecurrentlydonotknowwhetherthisisduetophysi-ological processes (e.g. telomere elongation mechanisms duringdevelopment;Gomesetal.,2010)ordue to the selectivedisap-pearanceofindividualsbornwithrelativelysmallertelomeres(e.g.Salmón,Nilsson,Watson,Bensch,&Isaksson,2017).

Weinitiallyhypothesizedthattheviviparousmotherswouldhaveahigher reproductive investment (Lindtkeetal.,2010)becauseoftheprolongedpregnancyandpresumedlargerclutchmassinvivipa-rousspecies(Horváthováetal.,2013;Qualls&Shine,1998;Roitbergetal.,2013).However,therelativeclutchmass(asaproxyforrepro-ductive investment)was similarbetween the reproductivemodes.Anotherpotentialcosttoviviparity is thatviviparousfemalesmaybelessabletohuntandacquireresourcesduringpregnancy,whichcouldresultinanetlossofenergy.However,thiswasnotpossible

F I G U R E 1  Therelationshipbetweenmaternalreproductivemodeandthesomaticmassof(a)mothersand(b)offspring.Dataplottedasindividuals+mean.Somaticmassdidnotdiffersignificantlybetweenreproductivemodesatthematernalstage;however,therewasasignificantdifferenceattheoffspringstage,measuredonthedayofbirthorhatching(p<.05;seeTable2)

TA B L E 2  SummaryofthefinalGLMscorrespondingtomodels1,2,3,4,5,7and8

# Explanatory variable Estimate SE t p

1 Maternalmass — — — — —

2 Offspringmass Intercept 0.204 0.010 19.67 <.001

Repro. mode—oviparous 0.044 0.012 3.57 <.001

Repro. mode—viviparous −0.023 0.012 −1.97 .054

3 Clutchsize Intercept 7.154 0.553 12.94 <.001

Repro. mode—oviparous 0.411 0.691 0.60 .554

Repro. mode—viviparous −1.063 0.653 −1.63 .108

4 RCM — — — — —

5 ROM Intercept 0.414 0.034 12.11 <.001

Repro. mode—oviparous 0.086 0.040 2.13 .037

Repro. mode—viviparous −0.140 0.039 −3.62 <.001

7 MaternalRTL Intercept 0.043 0.033 1.28 .204

Repro. mode—oviparous −0.166 0.041 −3.98 <.001

Repro. mode—viviparous 0.118 0.039 3.01 <.001

8 MeanoffspringRTL Intercept −0.077 0.030 −2.52 .014

Repro. mode—oviparous −0.135 0.036 −3.73 <.001

Repro. mode—viviparous 0.146 0.034 4.24 <.001

Note: SeeSection22forfulldefinitionsofthemaineffectsandinteractionsinitiallyincludedineachmodel.SeeSection33foranalysisofvarianceteststatistics.

     |  7Functional EcologyMCLENNAN Et AL.

toquantifyinthisstudy,sincethepregnantfemaleswerefedadlibi-tum.Ithasalsobeensuggestedthatviviparousspecieshavelongerlifespans,comparedtooviparousspecies,andarethereforeableto

producemoreclutchesovertheyears(Meiri,Brown,&Sibly,2012;Tinkle, Wilbur, & Tilley, 1970). However, it is currently unknownwhether thesamealsoapplies tooviparousandviviparousmodes

F I G U R E 2  Therelationshipbetweenmaternalreproductivemodeand(a)relativeclutchmass(clutchmass,includingeggshell,amnioticfluidsandyolk,dividedbyfemalemassafteroviposition/parturition),(b)relativeoffspringmass(summedmassoftheoffspringafterhatching,excludingeggshell,amnioticfluidsandyolk,dividedbyfemalemassafteroviposition/parturition)and(c)thenumberofoffspringwithinaclutch.Dataplottedasindividuals+mean.RCMdidnotdifferbetweenthereproductivemodes;however,therewasasignificantreproductivemodeeffectonROMandclutchsize(p<.05;seeTable2)

# Explanatory variable Estimate SE df t p

6 RTL Intercept 0.035 0.025 74.78 1.35 .18

Repro. mode—oviparous

−0.155 0.031 62.04 −5.00 <.001

Repro. mode—viviparous

0.127 0.029 62.80 4.32 <.001

Lifestage—offspring −0.094 0.017 55.03 −4.67 <.001

Note: FamilyIDwasincludedasarandomfactortocontrolfornon‐independencebetweenmotherandoffspring.Thefamilylevelvariancewas0.0029(SD0.0542),andtheresidualvariancewas0.0083(SD0.091).SeeSection33foranalysisofvarianceteststatistics.Estimatesofmarginal(fixedeffects)andconditional(fixedeffects+randomeffects)R2valueswere0.62and0.72,respectively.

TA B L E 3  Summaryofthelinearmixed‐effectmodel,correspondingtomodel6

F I G U R E 3  Therelationshipbetweenreproductivemodeandrelativetelomerelength(RTL).Circlescorrespondtomothers,whiletrianglescorrespondtotheoffspring.Dataplottedasindividuals + mean

8  |    Functional Ecology MCLENNAN Et AL.

co‐occurringwithinthesamespecies,andwewereunabletoquan-tify theageandpast reproductivehistoryof themother lizards inthisstudy.

Alsoincontrastwithourhypothesis,theviviparousfemaleshadthelongesttelomeres,whiletheoviparousfemaleshadtheshortesttelomeres. Inmammals, telomere length is generally phylogeneti-callyconserved,althoughexceptionstothisdoexist(Gomesetal.,2011,2010). It isunknowntowhatextent thisapplies to reptiles.Onepossibilityisthatdifferingtelomerelengthsincommonlizardshaveevolvedasaneffectofthelife‐historydivergenceamongthereproductivemodes. It isalsopossible thatwewronglypresumedoviparitytocarryalessermaternalburden.Wedidnotfindasignif-icantdifference inmaternalmassamong the reproductivemodes,and a study by Demarco and Guillette (1992) found viviparity tocarry a minimal metabolic cost. Moreover, within a reproductiveseason,oviparousfemalesmightinvestmoreintheiroffspringcom-pared toviviparous females (Recknagel&Elmer,2019).We foundthatROMwaslargerforoviparousfemalescomparedtoviviparousfemales, indicating that oviparous females provide their offspringwithmorenutrients(i.e.yolk).Whilewecouldnotassessthishere,theshortertimeofpregnancymightalsoallowoviparousfemalestoproducemorethanoneclutchperyear(Heulin,Guillaume,Vogrin,Surget‐Groba,&Tadic, 2000; Lindtke et al., 2010), resulting in anevenlargerreproductiveeffortperseasonforoviparousfemales.

It is worth noting that post‐embryonic telomere repair mech-anisms, such as telomerase expression, have been documented inseverallizardspeciestodate(Alibardi,2015;Ujvarietal.,2017).Inaddition, several studieson fishhave foundhigher telomeraseex-pressioninactivelydividingcells(Peterson,Mok,&Au,2015;Yap,Yeoh,Brenner,&Venkatesh,2005).We found that theviviparousoffspringweresignificantlysmallerathatchingbutwereofequiva-lentsizeatthematernallifestage,perhapsduetoanincreasedrateof growth. Therefore, this possible divergence in telomere lengthbetween the reproductivemodesmay have occurred, in part be-causeofdifferencesingrowthrateanditspossibleassociationwithtelomeraseexpression.Longertelomeresinviviparouscommonliz-ardsmightthereforehaveco‐evolvedwith(a)asmallerreproductiveinvestmentperseasonand(b)anincreaseingrowthrateandpoten-tiallyalsolongevity.

Wedonotknowtheageofthematernallizardsandcanthere-fore not rule out possible age effects. It may be that viviparouscommonlizardsdifferintheirlongevityortheirageatmaturity,al-thoughtherearecurrentlynodatatosuggestthis.Telomereshavebeenfoundtoshorten inhumansandother longer‐livedmammalsand birds (Haussmann et al., 2003), in part because many larger,longer‐livedendothermspeciesappear todown‐regulate telomererepairmechanismsinpost‐embryonicsomatictissues,perhapsasatumoursuppressionmechanism(Gomesetal.,2010).However,thedirectionofthetelomere–agerelationshipislesswellestablishedinectothermicspecies,andstudieshavedetectedtelomeraseexpres-sioninanumberofreptile,amphibianandfishspecies(Gomesetal.,2010;Simide,Angelier,Gaillard,&Stier,2016;Ujvarietal.,2017).Therefore,wemayhave identifiedadifference in telomere length

betweenthereproductivemodes,inpartbecauseageatreproduc-tionmayalsodifferamongthemodes.However,ageandbodysizearethoughttobecorrelatedinthecommonlizard(Richard,Lecomte,Fraipont,&Clobert,2005)andweincludedbodymass(highlycor-relatedwith body size) as a covariate in analyses. In addition, theexactsamepatternwasfoundattheoffspringstage,whenagewassimilaramongthereproductivemodes(i.e.dayssincecopulation).

Aswith the adult females,we also found that the viviparousoffspringhadthe longest telomeres, theoviparousoffspringhadtheshortesttelomeres,andtheadmixedoffspringhadintermedi-atetelomerelengths.Hadthisembryonicdevelopmentoccurredinthewild,wemighthavehypothesizedthattheviviparousoffspringhadlongertelomeresbecauseviviparityisthoughttoconfermorestableembryonicconditions(Vedderetal.,2018).However,alloftheembryosinthisstudydevelopedinstableincubatorconditions.While theremay still be some degree of variation in embryonicconditions(e.g.higherlevelofrespiratoryexchangeinthevivipa-rousmode),itisunlikelythatthiscausedtheobserveddifferencesintelomerelength.Thecomplexityoftelomereheritabilityisstillnot fully understood in lizards. It could be that offspring inherittheir telomere lengthvia the initial telomere lengthof the fertil-izedegg(Dugdale&Richardson,2018),presumingthatviviparousmothersalsohavealongergermlinetelomerelength.Additionally,offspring may also be inheriting the genetic information thatcontrols telomere elongation during embryogenesis (Kalmbach,Robinson,Wang,Liu,&Keefe,2014;Liuetal.,2007;Schaetzleinetal.,2004).Forexample,itisthoughtthattheseembryonictelo-mereelongationprogrammesmayrestoretelomerestoasetlength(Schaetzleinetal.,2004);therefore,itispossiblethatthisgeneticinformationhasalsodivergedbetweenthereproductivemodes.

Westillknowrelativelylittleaboutthedeterminantsofspecies‐specific telomere length ranges, and how theymay have evolvedfromspecies‐specifictrade‐offsbetweenlongandshorttelomeres.Itisoftenhardtodisentanglesuchdifferences,inpartbecausespe-ciesdiffersignificantlyinthetypesofenvironmentthattheyinhabit.However,bystudyingauniquecommonlizardpopulationthatexhib-itsbothoviparousandviviparousreproductioninthesamehabitat,we have identified potential links between life‐history divergenceandtelomerelength,suggestingthatpopulationssuchasthesemayprovefruitfulinfuturestudies.Itwouldalsonowbeinterestingtoexaminewhetherthedifferenceintelomere lengthbetweenovip-arous and viviparous individualsmay affect long‐term patterns ofsenescenceandlongevity.

ACKNOWLEDG EMENTS

WethankA.Adam,M.CapstickandW.Bonerforadviceandhelpwiththelaboratoryanalyses.WewouldalsoliketothankM.Layton,M.Sutherland,H.LeitaoandAlpenferienparkReisachforassistancein the field.AllprocedureswerecarriedoutunderpermitnumberHE3‐NS‐959/2013(012/2016).ThetelomereanalyseswerefundedbyaGlasgowNaturalHistorySocietyBlodwenLloydBinnssmallre-searchgrant,awardedtoD.M.andH.R.Duringtheperiodofdata

     |  9Functional EcologyMCLENNAN Et AL.

collection, analysis andwritingof this paper,D.M.was fundedbyERCAdvancedGrant322784andH.R.byaUniversityofGlasgowLordKelvin‐AdamSmith PhD Studentship and aNERC grantNE/N003942/1.

AUTHOR ' S CONTRIBUTIONS

D.M.,H.R.,K.R.E.andP.M.conceivedoftheideaforthisstudy.H.R.ledthefieldaspectofthestudy.D.M.andH.R.conductedlaboratoryanalyses.D.M.andH.R.leddataanalysisandwritingofthemanu-script, with critical contribution from K.R.E. and P.M. All authorsgavefinalapprovalforpublication.

DATA AVAIL ABILIT Y S TATEMENT

Data are available via the Dryad Digital Repository https://doi.org/10.5061/dryad.712hc6m (McLennan, Recknagel, Elmer, &Monaghan,2019).

ORCID

Darryl McLennan https://orcid.org/0000‐0002‐8566‐9437

Hans Recknagel https://orcid.org/0000‐0003‐2728‐0690

Kathryn R. Elmer https://orcid.org/0000‐0002‐9219‐7001

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SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle.

How to cite this article:McLennanD,RecknagelH,ElmerKR,MonaghanP.Distincttelomeredifferenceswithinareproductivelybimodalcommonlizardpopulation.Funct Ecol. 2019;00:1–11. https://doi.org/10.1111/1365‐2435.13408