The Importance of Ultraviolet and Near-Infrared Sensitivity for Visual

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The importance of ultraviolet and near-infrared sensitivity for visual discrimination in two species of lacertid lizards Melissa Martin, Jean-François Le Galliard, Sandrine Meylan, Ellis R. Loew

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Melissa Martin, Jean-François Le Galliard, Sandrine Meylan, Ellis R. Loew. The importance of ultraviolet and near-infrared sensitivity for visual discrimination in two species of lacertid lizards. Journal of Experimental Biology, Cambridge University Press, 2015, 218 (3), pp.458-465. 10.1242/jeb.115923. hal-02638791

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Distributed under a Creative Commons Attribution - NonCommercial| 4.0 International License Paris, France. (MWS class),short-wavelengthsensitive(SWSclass)and very- wavelength sensitive(LWS class),middle-wavelengthsensitive Biodiversité, MuséumNational d’Histoire Naturelle, 91800 Brunoy, France. France. *Author ([email protected]) for correspondence University,Medicine, Cornell Ithaca,NY14853,USA. 458 Received 27October 2014;Accepted8December2014 1 by thewavelengthofpeakabsorbance, details, seeYokoyama, 2000).Photopigmentsareusuallyspecified transmembrane opsinproteinassociatedwithachromophore (for outer segmentsofretinalphotoreceptorcells,andcomposed ofa by visualpigmentmoleculesembeddedinthemembranesof the (see BradburyandVehrencamp, 2011). Photosensitivityisconferred photopic andcolourvisionareservedbyconephotoreceptor cells environment anddistinguishdifferent visualtargets. Invertebrates, considerable interesttocomprehendhowanimalsperceivetheir Vehrencamp, 2011; LandandNilson,2012).Thus,itisof optimised totheecologicalnicheofeachspecies(Bradburyand predator avoidance,andvisualcapabilitiesareexpectedtobe Vision isakeysenseinvolvedintaskssuchasmating,foragingand vivipara Vitamin A1/A2-basedpigments,Coneabundance, KEY WORDS:Colourvision,Chromaticresolution,UVsensitivity, system andvisualsignalsmightco-evolve. chromatic resolutionincommonlizards,indicatingthatthevisual coloration among and nearinfraredimprovesdiscriminationofsmallvariationsinthroat terrestrial vertebrates.We foundthatspectralsensitivityintheUV spectral sensitivityintothenearinfrared,whichisararefeaturein lizards possessmixedvitaminA1andA2photopigments,extending have purevitaminA1-basedphotopigments,whereascommon abundant in cones, therelativeabundanceofUVSsinglecones(potentiallymore cones. Thetwospeciesdiffer inthespectralsensitivityofLWS one spectralclassofdoubleconesandfourclassessingle lizard contrasting habitats:thewalllizard characterise thespectralsensitivityoftwoLacertidspeciesfrom sensitivity oftheirretinalphotoreceptorsremainsunknown.Here,we systems ofLacertidaehaverarelybeenstudiedandthespectral that isinvolvedinintraspecificcommunication.However, visual Male andfemaleLacertidlizardsoftendisplayconspicuouscoloration Mélissa Martin visual discriminationintwospeciesoflacertidlizards The importanceofultravioletandnear-infraredsensitivityfor RESEARCH ARTICLE © 2015.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2015)218,458-465doi:10.1242/jeb.115923 Pierre-lès-Nemours, France.Pierre-lès-Nemours, UMS 3194,CEREEP–EcotronIleDeFrance, Supérieure, 77140St- ÉcoleNormale INTRODUCTION ABSTRACT CNRS UMR7618,iEESParis, etMarieCurie, 75005Paris, Université Pierre Zootoca vivipara 2 CNRS UMR 7179, Département d’EcologieetdeGestionla CNRS UMR7179,Département , Podarcismuralis 5 Z. vivipara)andthecolorationofoildroplets.Wall lizards Department of Biomedical Sciences, College of Veterinary BiomedicalSciences, Collegeof of Department Z. vivipara.Thus,retinalspecialisationsoptimise 1,2, *, Jean-FrançoisLeGalliard . Bothspeciespossessapure-coneretinawith 4 ESPE deParis-Université SorbonneParis IV, 75016 Podarcis muralis λ max , andincludelong- Zootoca and thecommon 1,3 , SandrineMeylan 3 CNRS and, moresurprisingly, and have foundachromophoremixtureinlizardssuchaschameleons 1984; Crescitelli,1972;KnowlesandDarntnall,1977).Somestudies temperature, seasonorlifestage(Beatty, 1966;Beatty, 1975;Beatty, and A2-basedvisualpigmentswithage,hormonalstate,light, species presentindividualplasticityintherelativeproportionofA1- Bowmaker, 1989).Ithasbeenshownthatsomeamphibianandfish based pigment(rhodopsins)(Hárosi,1994;Whitmoreand absorption peakshiftedtowardlongerwavelengthsthantheA1- opsin protein,A2-basedpigments(porphyropsins)showan terrestrial amphibians(reviewedbyBridges,1972).Forthesame usually associatedwithfreshwaterspeciesortheaquaticphaseof terrestrial vertebratesandmarinespecies,whereasvitaminA2is 2008). Vitamin A1iscommonlyencounteredintheeyesof used, eitherthealdehydeofvitaminA1orA2(Bowmaker, the aminoacidsequenceofopsinproteinandchromophore prerequisite forunderstandingtheevolutionofvisualcapabilities. spectral propertiesoftheretinainvariousspeciesisthereforea from thephotoreceptors(Bowmaker, 2008).Characterisationofthe perceptual mechanismscapableofanalysingandinterpretingsignals differing intheirspectralsensitivityaswelltheneuraland Colour visionrequiresthepresenceofatleasttwovisualpigments short-wavelength sensitive(VS/UVSclass)(Kelberetal.,2003). (300–400 including onephotoreceptorsensitive tolightintheUV range three orfourspectralclassesof photoreceptors (tri-ortetrachromats) photoreceptor type(Bradburyand Vehrencamp, 2011). therefore, partofthenoisesurrounding theresponseofagiven indirect evaluationoftheabundance ofthedifferent conetypes and, 2005)]. Thisspecificityisparticularlyinterestingbecauseitallows examples inbirds,seeHartandVorobyev (HartandVorobyev, humans (e.g.Fleishmanetal.,2011; Loewetal.,2002)[for with specificoildroplettypes,basedonitsapparentcolour to have demonstratedthateachphotoreceptortypecanbeassociated Wilts, 2014;Vorobyev, 2003).Previousstudiesinbirdsandlizards and reducingtheoverlapofspectrallyadjacentcones(Stavenga and restricting therangeofwavelengthsthatentersoutersegment Oil dropletsarebelievedtoimprovehuediscrimination by the sensitivitypeaksofphotoreceptorstolongerwavelengths. pass filterforthephotonsenteringoutersegment,which shifts content andhighconcentrationofcarotenoidpigmentsactasa long- the doublecones(reviewedbyBowmaker, 2008).Theirlipid of theinnersegmentcones,exceptforaccessorymember is thepresenceofpigmentedoildropletslocatedindistalregion based visualpigmentinthevertebrateretinaispoorlyunderstood. al., 2002).TheadaptivesignificanceofvitaminA1-versusA2- retina containingonlyA2pigments(Provencioetal.,1992;Loew Spectral absorptionofthevisualpigmentsisdeterminedbyboth The majorityofdiurnallizards are knowntopossessnorodsand A commonfeatureoftheretina most diurnalreptilesandbirds Podarcis sicula nm) (reviewed by PérezideLanuzaandFont, 2014) 1,4 and EllisR.Loew (Bowmaker etal.,2005;Provencio1992) Anolis carolinensis 5 possesses apure-cone

The Journal of Experimental Biology throat andabellycolorationrangingfromyellowtodark red habitats dominatedbyagreenbackground.Malesbearwhitish common lizardiscommonlyfoundinmoistandgrassy open communication (Martin,2013;Vacher andGeniez,2010).The patches thatreflectUV and usevisualsignalsforintraspecific inhabit contrastinghabitats,displaybright,non-nuptialcolour the studyofvisualsystemslacertidsbecausetwospecies lizard belong tofiveclasses: C1,C2,G,Y, O,plusadispersedpigment (seeListofabbreviationsand Table 2). W snl)147C 5±3C1 G C2 Y DP 456±23 497±19 367±9 584 558 3 3 2 1 1 C1 O C2 DP G 487±14 437 358±8 544±4 624±27 614±17 20 1 4 2 5 6 LWS (accessorymemberofdouble) LWS (principalmemberofdouble) LWS (single),formA2 LWS (single),formA1 MWS (single) SWS (single) UVS (single) Number ofcountedcells,spectralsensitivity (mean Pigment class 2011). male contestandfemalematechoice(Bajeretal.,2010;Bajer indicate thatlacertidscanuseUV signalsofconspecificstosettle (Pérez ideLanuzaandFont,2014).Inaddition,behaviouraltests visual sensitivitytoUV fromretinalstructureandmoleculardata communication. Recentworkinlacertidsprovidedevidencefor and Parker, 2010),visualsignalsarealsoinvolvedinintraspecific navigation andcommunicationinthisfamilyoflizards(seeMason al., 2013).Eventhougholfactionplaysamajorroleforforaging, sexes, includingintheUV range(e.g.Fontetal.,2009;Martin lacertid speciesdisplaycolouredornamentsthatdiffer between most ofthediurnalcommonEuropeanlizardspecies.Several on theLacertidaefamilyofLacertibaeniagroup,whichincludes unknown (seesupplementarymaterialTable sensitivity ofseveralentirelizardinfraordersremainsessentially species belongtoalimitednumberoffamiliesand,date,spectral of pigmentshasbeeninvestigatedinseverallizardspecies,butthese Lanuza andFont,2014).Overthepastdecades,spectralabsorbance pigments (Bowmakeretal.,2005;Loew2002;Pérezide droplets arealwaysassociatedwithcellscontainingUVSandSWS MWS andLWS pigments,andoneortwotypesofcolourlessoil yellow (tothehumaneye)colouredoildropletsarepairedwith spectral classesofoildroplets.Onetothreetypesgreenand/or (supplementary materialTable distinction between absorptionprofilesofLWS singleconesfittedbya vitaminA1orA2template,the RESEARCH ARTICLE Table The commonlizard yellowoildroplet Y orangeoildroplet VS/UVS very-short-wavelength UV ultraviolet sensitive microspectrophotometry SWS short-wavelength O MWS medium-wavelength greenoildroplet sensitive MSP LWS long-wavelength colourlesstype2oildroplet sensitive JND just-noticeable colourlesstype1oildroplet G DPsensitive dispersed C2 difference C1 pigment List of abbreviations List of 1. Characteristicsofvisualpigmentsfoundinconescommon andwalllizards Podarcis muralis Zootoca vivipara Laurenti 1768areinterestingcandidatesfor

S1). Therearealsothreetofive Jacquin 1789andthewall 23 N Z. vivipara

λ S1). Here,wefocused max ± s.d.)andassociatedoildroplettypes forthedifferent conetypes. Becausewecouldmakeaclear 617±23 λ max } based pigmentsarea10:90proportionin profile ofLWS pigments,weestimatedthatvitaminA1-andA2- depending onthetestedinnersegment.Basedabsorption rhodopsin (vitaminA1)oraporphyropsinA2)template, visual pigmentsfrom Figs S1andS2forrepresentativeexamples).Absorptionprofilesof (seesupplementarymaterial 1 of bothspeciesareprovidedinTable signals. of alternativevisualsystemsagainstnaturallyoccurring visual systemstructureinlizardspeciesbytestingforoptimisation exercise helpedustogainfurtherinsightintotheevolutionof cone densityandchromophoretypeaffect chromaticresolution.This capabilities ofthecommonlizardinordertoinvestigatehowUV than in suggest thatUV conesmightbetwicemoreabundantin presented anA1/A2-basedchromophoremixtureandourdata those ofdiurnallizardsstudiedsofar. Nevertheless, eye. Inbothlacertidspecies,wefoundvisualcharacteristicscloseto of thedifferent oildroplettypes,basedontheircolourforhuman photomicrographs, wealsoaimedtoevaluatetherelativeabundance droplets inZ.vivipara determine thespectralabsorbanceofvisualpigmentsandoil displays (PérezideLanuza,2012;Martin,2013). spots thattheyexhibitbypresentingtheirflankandpush-up males alsohavebright,UV–bluemarginal ventralscalescalledblue yellow andorange)(Galeottietal.,2010;Sacchi2007) Adults ofbothsexesexhibitthreeventralcolourmorphs(white, open habitatsdominatedbyagrey, highlyreflectivebackground. The walllizardinhabitsstonewallsandnaturalrockoutcropsin conspecifics sightduringagonisticinteractions(Martinetal.,2013). UV range,especiallyonthethroatofmaleswhichisexposedto 1987; Vercken etal.,2007).Theventralornamentalsoreflectsinthe interspersed withblackspots,andfemalesareduller(Bauwens, as UVS,SWS,MWSandLWS. Thedetailsofpigment distinct single-coneclasseswereidentifiedandcharacterised a dispersedpigmentinitsinnersegment.Ineachspecies,four principal memberwithanoildropletandaccessory oil dropletintheirinnersegmentanddoubleconesconsistingofa possessed apure-coneretina,whichcontainedsingleconeswithan species (PérezideLanuzaandFont,2014).Thetwostudy range 350–750 analyses ofthecornearevealednosignificantabsorptionin We didnotmeasurespectralpropertiesofocularfluidbutourMSP template. In Spectral characteristics of lacertid lizards lacertid characteristicsof Spectral RESULTS In thisstudy, weusedmicrospectrophotometry (MSP)to G orO Oil droplet P. muralis.We thususedphysiologicaldatatomodelvisual The JournalofExperimentalBiology(2015)doi:10.1242/jeb.115923 Z. vivipara nm asinarecentanalysisofeightlacertidlizard λ max of eachLWS pigmentformisreported.Oildroplets P. muralis , pigmentabsorptionswerebestfittedbya P. muralis –– –– 11 N and were bestfittedbyavitaminA1 P. muralis.Fromretinal 562±17 λ max Z. vivipara.However, this Z. vivipara λ Z. vivipara max Y orG Oil droplet values 459

The Journal of Experimental Biology muralis. cones aretwiceasabundantin Thus, theMSP dataandoildropletcountsbothsuggestthatUV of colourlessoildropletsinZ.vivipara Counting ofphotoreceptorsfromretinaphotographsrevealed19% and SWSconescannotthereforebeestimatedfromphotographs. oil dropletswereindistinguishableforahumanviewer, andUVS 1.Inthesameway, colourless pigment classesareprovidedinTable LWS cones. Data ontheassociationbetweenoildropletclassesand which impededanyestimateoftherelativeabundanceMWSand second onewasassociatedwithbothMWSandLWS pigmenttypes, droplets wasexclusivelyassociatedwithLWS pigments,butthe representative examples).Inbothspecies,onetypeofcolouredoil yellow oildroplets(seesupplementarymaterialFig. thus a‘+’ indicatestheirpresenceinthecellsofretina. nmisnotmeasureableand images (asapercentage)werereportedforeachoildroplettype.Cut-off oftheC2dropletsovermeasurementrange340–750 i.1). Fig. 2; abundant thantheothertypeofcolouredoildroplets(Table colourless oildroplet,andgreendropletswereonaverageless Both speciespossessedgreenoildropletsandtwotypesof elw()–––540464(53–69) – – 470±4 27(22–42) + 460±11 5 500±8 2 – 7 55 – – 29(13–63) – 52(15–71) – 503±10 485±11 + 538±6 406±9 – 24 2 28 4 9 Dispersed pigment(DP) Colourless, type2(C2) Colourless, type1(C1) Yellow (Y) Green (G) Orange (O) 460 RESEARCH ARTICLE vivipara forbothspecies.Thespectralsensitivitiesof 2 illustrated inFig. Vorobyev, 2005) forvisualpigmentsandoildroplets,is calculated basedonHartandVorobyev’s templates(Hartand The relativespectralsensitivityofeachsingleconeclass was chromophore type.However, itissafetoassumethatifthe template matchingtoMSP dataisnotthebestwaytoassess conspecific colourdiscrimination.Itshouldbeemphasisedthat modelling inordertotesttheimportanceofthisparameter retina regions;thatiswhywealsouseda50:50proportionin estimate doesnottakeintoaccountpotentialvariationofdifferent Number andspectralfeatures( Oil dropletclass Table al forestimatesof 2 Table one typeofdispersedinnersegmentpigmentineachspecies(see cell thatmatters. chromophores areusedbecauseitisthespectralsensitivityof component. Forecologicalstudies,itislessimportantwhich a measuredpigmentisgreaterthan580 Quantitative modelling of visual performances of lacertids of visualperformances Quantitative modellingof estimate based onMSP dataforthemodelling exercise cone abundancecannotbeprecisely estimated,weusedarough mixture observedintheLWS visualpigment. Giventhattherelative wavelengths thantheretinaof retina ofZ.vivipara that oftheothercones,especially in the oildroplet,relativesensitivity ofMWSconeswaslessthan cones overlappedinbothspecies.Becauseofthefilteringeffect of overlap. Bycontrast,therangeofsensitivityMWSand LWS and 480 MSP allowedustoidentifyfourspectralclassesofoildropletand 2. Characteristicsofoildropletsinretinalsamplescommonandwalllizards Z. vivipara nm, wherethesensitivityofUVSandSWSconeshadlittle and P. muralis had orangeoildropletswhereas displayed awiderrangeofsensitivity inlong were closeinthespectralrangebetween300 λ mid λ mid of oildropletsanddispersedpigment). P. muralis,owingtothechromophore ± s.d.,thewavelengthatwhichabsorbanceis50%)ofoildropletsmeasuredbyMSP andabundancebasedonretina N Z. vivipara Z. vivipara Z. vivipara.Inaddition,the

nm, itmostlikelyhasanA2 but only9%inP. muralis. compared with λ mid P. muralis

S3 for } λ max had of Z. P. 19 (15–25) % (Range) a modearound5just-noticeabledistance(JND)and<1%ofthe empirical model)wascharacterisedbyafattailskewedtotheright, 10/90 andconeratiosof1:2:5:9,hereafterreferredtoasthe our MSP estimates(modelwithanA1/A2chromophoremixtureof zone). Thesampledistributionofthroatorbellycolourdistancesfor the bellyonother(3486pair-wise comparisonsforeachbody pairs ofmalesamongspectrafromthethroatononehandand quantified theCartesiandistanceincolourspaceforallpossible common lizardsdescribedinMartinetal.(Martinal.,2013),we (model 1:2:6:6,resultsnotpresentedhere). abundances forMWSandLWS conesbasedonoildropletcounts model outputswerealmostidenticalwhenweassumedanequal (UVS:SWS:MWS:LWS, 1:2:5:9).However, itshouldbenotedthat Fig. muralis. vivipara droplet intheretina of coloured oildroplets inbothspeciesandtheabundance ofcolourlessoil droplets arevisible.Notethepresence oftwoclearlydistinguishabletypes Using thespectraldataofventralcoloration84adultmale

.Light microscopyoftheretinaZootocavivipara 1. and (B)P. muralis Images ofasmallrepresentativepatch oftheretinafrom(A) B A The JournalofExperimentalBiology(2015)doi:10.1242/jeb.115923 Z. vivipara N P. muralis 429±22 4 . Individualphotoreceptors(elongated cells)andoil . λ mid } and Podarcis 9 (6–11) % (Range) Z.

The Journal of Experimental Biology those between1and4 calculated theproportionofcolourdistanceslowerthan1 Lacertid lizards (Lacertidae)suggestedtoprevious researchersthat Natural historydataonthelife style, foragingmodeandanatomyof and totheoutputofmodelwith pureA1pigmentsforbellydata. to theoutputofmodelwith anA1/A2mixtureforthroatdata, patch. Theoutputsofthemodel withpureA2pigmentsweresimilar for thethroatcolourpatchand,toalesserextent,belly in theretinaofcommonlizardenhancedchromaticresolution to avisualsystemwithpureA1pigments,chromophoremixture decreased chromaticresolutionslightly. Furthermore,withrespect abundance ofUV cones(model, 2:1:1:1)relativetootherconetypes variation inthroatandbellycoloration.However, increasingthe the abilityofvisualsystem absence ofsensitivitytoUV light(trichromacy)stronglydecreased respect toavisualsystemwithconetypesinequalratios, the but slightlylessdiscriminationofUV throatpatches. showed thatveryefficient discrimination ofthebellycolourpatches, 3 the subsequentanalyses.ModellingresultspresentedinTable ‘not distinguishable’ and‘poorlydistinguishable’,respectively, in distances lessthan1 Fig. RESEARCH ARTICLE associated oildroplet. normalised to calculated astheproductofabsorbancespectrumvisualpigments Podarcis muralis DISCUSSION oprsn ewe oe upt ihihe Tbe3),with Comparisons betweenmodeloutputshighlighted(Table

2. Relativesensitivityofsingleconesin

Relative sensitivity 0.25 0.75 1.00 0.25 0.75 1.00 0.5 0.5 λ max 0 0 . 0 0 0 0 700 600 500 400 300 0 0 0 0 700 600 500 400 300 and thenormalisedtransmissionspectrumoftheir B A (A) Z.vivipara UVS UVS

JND. Basedontheseobservations,wethen

JND, andassumedthatthesedistancesare SWS Wavelength (nm) and (B)P. muralis SWS MWS Z. vivipara MWS Zootoca vivipara LWS LWS . Relativesensitivitywas to discriminate and

JND and (supplementary materialTable the sameorderofmagnitudeasintraspecificvariation variation inthisstudy. our sampleoflizardswastoosmalltoinvestigateinter-individual et al.,2002).We randomly sampledseveralregionsoftheretina,but and betweendifferent regions oftheretina(Fulleretal.,2003;Loew characteristics ofoildropletsmayindeedvaryamongindividuals of conesandahighersamplesize.Theabundance observation shouldbeconfirmedwithmoreexhaustiveMSP counts by ahighrelativeabundanceinUVScones,althoughthis species. Third,theretinaof whereas thisoildropletisyelloworgreeninotherdiurnallizard the red-shiftedLWS andtheMWSvisualpigmentof so farusejustA1.Second,anorangeoildropletwasassociatedwith A1/A2 chromophoremixturetemplate,whereasmostlizardsstudied First, wefoundthattheLWS absorbancewasbestfittedbyan visual systemof 1994; Loewetal.,2002;Macedonia2009).Interestingly, the et al.,2005;Ellingson1995;Fleishman2011; Loew, diurnal lizardsinvestigatedsofar(Barbouretal.,2002;Bowmaker had visualpropertiesoftheirretinasimilartothoseseeninmost inhabiting contrastinghabitats.However, commonandwalllizards expected todiscoveratypicalvisualfeaturesinourtwostudyspecies Cordylidae (MasonandParker, 2010;Vitt etal.,2009).Thus,we to othergroupsoflizardssuchasIguanidae,Agamidaeor these speciesaremoredependentonolfactionthanvisionrelative density andA1/A2ratio. percentages correspondtotheempiricalmodelwithobservedcone Lower percentagesshowhigherdiscriminatingabilityoftheviewer. Bold densities, andwiththeempiricalconedensitybutdifferent A1/A2ratios. with a10/90proportionofA1/A2LWS photopigmentsbutdifferent cone formodels orpoorlydiscriminable(1–4JND) are notdiscriminable(<1JND) difference. Values arepercentageoftotalthroatorbellycolourcontraststhat Cone densityisexpressedasUVS:SWS:MWS:LWS. JND,just-noticeable 1–4JND <1JND A1/A2 ratio 1–4JND <1JND Cone density Model parameter Fg 3)highlightsthatinterspecificvariationin (Fig. A reviewofthespectraldatacollectedsofarinlizardspecies spectra forthevisualsystemofcommonlizards Table also suggestsignificant variationinthespectral sensitivityofLWS Fuller etal.,2003;Kröger 1999).Nevertheless,availabledata of thevisionphysiologyare strongly conserved(Archer, 1999; species inaccordancewithprevious suggestionsthattheseaspects of thespectralsensitivitythese visualpigmentsamonglizard 3).Thus,thereappearstobelittleevidenceofadaptivetuning (Fig. to thoserecordedinthemajority ofotherdiurnallizardspecies MWS visualpigmentsinourtwomodelspeciesisalsoverysimilar infrared sensitivity infrared near- of andtheimportance sensitivityinlizards Spectral ueA .81.001 4.45 2.64 4.05 0.11 0.06 13.91 0.17 8.38 14.20 14.54 19.77 0.52 21.44 0.20 0.68 1.86 0.72 37.14 1.10 27.63 40.68 Pure A2 3.16 10/90 1.92 50/50 10.70 Pure A1 1:2:5:9 2:1:1:1 1:1:1:1 0:1:1:1 3. Chromaticdiscriminabilitybetweenthroatandbelly The JournalofExperimentalBiology(2015)doi:10.1242/jeb.115923 Z. vivipara Throat contrast .01.800 2.32 0.03 2.32 13.28 0.03 0.60 13.28 0.60 Z. vivipara also presentedsomeatypicalfeatures.

S1). Theλ was potentiallycharacterised max Belly contrast of UVS,SWSand λ max is smallandof Z. vivipara 461

The Journal of Experimental Biology 462 RESEARCH ARTICLE previously recordedin (Jacobs, 2010;Yokoyama, 2000).Vitamin A2chromophorewas exclusively vitaminA1chromophoresintheirvisualpigments whereas mostdiurnallizardsandterrestrialvertebratesuse (Archer, 1999;Hárosi,1994;WhitmoreandBowmaker, 1989), Z. vivipara was bestattributedtotheexistenceofvitaminA2chromophoresin Table droplets associatedwithsinglecones(supplementarymaterial single conesaswellvariationintheabundanceandtypeofoil was alsoshownbychromatographyin Provencio etal.,1992)andamixtureofA1A2chromophores conspicuous (Bauwens, 1987;Martin,2013;Vercken andClobert, coloration. Infact,theyellow-red bellypatchisstrongly of thetypechromophoreon discriminationofyellow-redbelly this rangeofsensitivity. Ourmodel,however, predictedlittleeffect colours, whereaspureA1-orA2-based pigmentretinacouldnarrow visual discriminationofsmallvariations intherangeofyellow-red chromophore mixtureasobserved in red spotsusedduringsexualdisplays.Inthesamemanner, a ability todetectcolourvariationsinthedifferent yellow, orange and polymorphism inlong-wavelengthconesmayberelatedto the Archer etal.(Archeral.,1987)suggestedthat,inguppies, for intraspecificinteractionsundercertainconditions.Forexample, towards longerwavelengths(Hárosi,1994),whichmaybeoptimal vitamin A1,A2shiftsabsorbanceofthevisualpigment advantage A2-basedvisualpigmentsprovide.Compared with variation ofvisualsensitivityinlizards,itremainstobeseen what sporadically appearedduringtheadaptiveradiationoflizards. that theabilitytosynthesisevitamin-A2-basedvisualpigments 2013). Ourresultsandfindingsinotherlizardspeciesthussuggest vivipara synthesis andincreasedaccumulationofvitaminA2inZ did notattributethisresulttodifferential feedingbuttoapreferential A compoundincommonlizards,where itisstoredintheliver. They al., 2013)recentlyfoundthatvitaminA2wasthedominant electrophysiology (Loewetal.,2002).San-Joseal.(San-Jose A2 intheeyeofZ.vivipara Furcifer pardalis et al.,1992)andtwochameleonspecies,

Although itisclearthatthenatureofchromophoresgenerates Peak sensitivity of photopigments (nm) 350 400 450 500 550 600

S1). Variation inthespectralsensitivityofLWS singlecones , whichisusuallyabsentinmostspecies(San-Joseetal., that extendedspectralsensitivityintothenearinfrared Pure A2 Pure A1 UVS Pure A1/no Lacertibaenia Scinciformata Iguania Gekkota A1/A2 UVS (Bowmaker etal.,2005).Thepresenceofvitamin Anolis carolinensis Type ofphotopigmentcells SWS remains tobeconfirmedby Z. vivipara Podarcis sicula Chamaeleo dilepis (Loew etal.,2002; MWS could easethe (Provencio ootoca and LWS chromophores (supplementarymaterialTable among specieswithachromophoremixtureorpure A2 MWS andLWS photopigmentsduetoA2chromophores.However, adaptation inresponsetothelong-wavelength-shiftedsensitivity of 2003). Itislikelythattheorangecolourofoildroplets is an spectral sensitivityfunctions(StavengaandWilts, 2014;Vorobyev, photoreceptors towardslongerwavelengthsandnarrowtheir Table diurnal lizardspeciesstudiedsofar(supplementarymaterial pigments ofZ.vivipara droplets associatedwiththered-shiftedLWS andMWSvisual 2013). better assessmentofthequalityapotentialmateorrival(Martin, differences betweenthroatcolourofconspecificsand,therefore, near infraredmaythereforeallowbetterdetectionofslight Martin etal.,2013).Possessionofavisualsystemsensitivetothe dominance toothermalesandattractfemales(Martin,2013; expose theirthroat,butnotbelly, tosignalaggressivenessand of conspecifics.Duringbehaviouraldisplays,malecommonlizards be relatedtoanappreciationofthedifferences inthroatcoloration sensitivity innearinfrared(i.e.presenceofA2chromophores)may intraspecific variationofthroatcoloration.Theseresultssuggestthat well asapureA2chromophoresystemindiscriminating system withapureA1chromophoreandperformedequally system basedonachromophoremixtureoutperformedvisual detect inter-individual variations.Bycontrast,wefoundthatavisual 2008) andfine-tunedchromaticresolutionmaynotbenecessaryto with aUV component, andUV vision is thoughttobetuned many lizardspecies,socialsignalling encompassescolourpatches 2002), includinglacertids(Pérez ideLanuzaandFont,2014).In UV visioniscommoninlizards(Fleishmanetal.,2011; Loewet al., chromatic resolution UVScones for lizard of theabundance of importance The which, toourknowledge,hasnotbeenaddresseddate. adaptive significanceofoildropletcolour(i.e.carotenoidpigments) Table droplets isnotparticularlynoticeable(supplementarymaterial variation intransmissionpropertiesofthedifferent typesofoil only onetoshowsuchacharacteristic.Eventhoughinterspecific * * Unexpectedly, MSP analysesalsorevealedatypicalorangeoil

S1). Basically, oildropletsshiftthesensitivitypeaksof S1), thisdiscoveryraisestheinterestingquestionof supplementary materialTable separately (reddotandstarwithanasterisk,respectively).See Fig. gecko type(s) ofchromophores].Allspeciesarediurnal,except visual systemspecificities[presence/absenceofUVSconesand point colorreferringtoinfraorderandshape photopigment class,onedatapointcorrespondstoaspecieswith species forwhichMSP dataareavailabletodate. A1- andA2-basedLWS pigments of Teratoscincus scincus The JournalofExperimentalBiology(2015)doi:10.1242/jeb.115923

3. Spectralsensitivity( , Hemidactylusturcicus whereas theyareyelloworgreeninother (blue dotswithblackoutline).The λ , Hemidactylusgarnotii

S1 forrawdata. max ) ofvisualpigmentslizard Z. vivipara

S1), Z.vivipara are reported and For each λ Gecko max is the of

The Journal of Experimental Biology 1999) (supplementarymaterial Table relatively conserved(Archer, 1999;Fuller et al.,2003;Kröger therefore, theancestralvisualsystemofthisgroupappears to be confirm thattherearefewadaptationsindiurnallizards and, chromatic resolution(FleishmanandPersons,2001).These data similar tothatofdiurnallizards,whichischaracterisedby good in ourtwostudymodels,lacertidsalsodisplayavisualsystem (Mason andParker, 2010)andourresults demonstrate that,atleast Chemoreception isknowntobeanimportantsenseinlacertids UVS cones(seePérezideLanuzaandFont,2014)]. which spectralsensitivitydatahadnotbeencollected[except for Lacertidae lizards,awidespreadgroupofSquamatereptiles for Our studyprovidesadditionaldataonthevisualsystems of involving dualvisualsignals. cones existsthatmaximisesdiscriminationofcolourpatches cones gottoohigh.Thus,anoptimalrelativeabundanceofUVS capacity wasslightlynegativewhenrelativeabundanceofUVS signals (Martinetal.,2013),theneteffect ondiscrimination lizards involvesbothstructural(UV)andpigmentary(yellow-red) yellow-red colourrange.Giventhatthethroatcolorationofcommon coloration attheexpenseofcapabilitytodetectvariationin increased thedetectionofsubtleinter-individual variationinUV of UVSconesrelativetothesensitivehumanvisiblelight visual pigmentsimportantforvision.Here,increasingtheabundance traded off againstadecreaseintherelativeabundanceofother increase intherelativeabundanceofonetypevisualpigmentis patches (Kelberetal.,2003;Vorobyev andOsorio,1998).Any oil dropletsaswelllightenvironmentandcontrastsamongcolour sensitivity andrelativeabundanceofpigmentstheirassociated lizards. Inthemodel,chromaticresolutionisconsequenceof density ofUVSconesdecreasedvisualperformancecommon choice andintra-sexualcompetitioninthisspecies(Martin,2013). and UV colorationplaysanimportantroleinsexrecognition,mate especially thatofthethroat,encompassastrikingUV component consistent withourexpectationsbecausebothornaments,but to alesserextent,bellycolourofconspecifics.Thisresultis performance fordetectingsmallvariationsinthethroatcolourand, showed thatthepresenceofUV conesstronglyimprovedvisual same vein,modellingofthespectralsensitivity detect byavisualsystemwhereUVSconesaredominant.Inthe species havesmallvariationsinUV reflectancethatareeasierto Platysaurus broadleyi of conspecificsduringmale–malecompetitioninflatlizards superabundance ofUV conesintheretinaenhancesdiscrimination (Fleishman etal.,2011) previouslysuggestedthatthe chromatic resolution.Usingasimilarapproach,Fleishmanetal. whether theabundanceofUVSconesisimportantforlizard small samplesizeofconesintheMSP analysis,itraisesthequestion muralis. Eventhoughthisdifference couldbeanartefactduetoour suggest thatZ.vivipara sensitive conesinboth (Fleishman etal.,2011; PérezideLanuza,2012).We foundUV- detect smallvariabilityintheUV reflectanceofconspecifics important forsocial interactions. discrimination ofdifferences inthecoloursofconspecifics thatare evolved jointlywithvisual signalsinordertomaximise as conedensity, oildropletcolourandchromophoretypemay have also suggestthatsomedesigncomponents ofvisualsensitivitysuch Squamate reptiles(Vidal andHedges,2009).Nevertheless,our study RESEARCH ARTICLE Colour vision in diurnal lizards Colour visionindiurnal Nevertheless, themodelalsopredictedthatadoublinginrelative , becausethethroatsoflizardsfromthis Z. vivipara might havetwiceasmanyUVScones and

S1) givingrisetopresentday P. muralis,butourdataalso Z. vivipara P. Ce5/2011/044). National deRéflexionEthiquesurl’ExpérimentationAnimale,no. French NationalEthicsCommitteeonAnimalExperimentation(Comité and thuspooledalldataforouranalysis.Allprotocolswereapprovedbythe found noobviousdifferences betweenthetwosamplesorsexes, data werenotbiasedbytheuseofanimalsemerging fromhibernation.We using wild-caughtanimals(twoadultindividualsperspecies)toensurethat conditions asinFrance.AllanalyseswererepeatedFranceMay2013 a darkboxand,uponarrival,weremaintainedinthesamehusbandry live food.Afterwards,animalswereshippedtotheUSA byairtransportin a terrariumprovidedwithlightandheatsource,waterdish,shelter Lizards werethenremovedfromthechamberandmaintainedfor1 progressively increasedover48 al., 2005).InFebruary2012,thetemperatureinchamberwas maintained constantat4°Ctomimicnaturalwinteringconditions(Heulinet progressively cooledfrom14to4°Cduringthefirstweekandafterwards were placedinthedarkaclimatechamber. Temperature wasthen damp sandandwetmosses.Afterseveraldaysofaccommodation,terraria capture, eachlizardwasmaintainedinanindividualterrariumlitteredwith in awildpopulationlivingthestonewallsoffieldstation.After natural populations.AdultEuropeanwalllizardswerecapturedbynoosing enclosures locatedinameadowwheretheycanfeedandbehaveas above sealevel,48°17 were carriedoutunderinfraredillumination(>800 Kodak, Rochester, NY, USA).Subsequentpreparationandmeasurements female) attheCEREEP-EcotronIleDeFrancefieldstation(France,60 and fourEuropeanwalllizards( under hypertonicbuffer solutionofCa was isolatedandtheretinascarefullyremovedfrompigmentepithelium 11 orIRLEDs)usingimageconverters.Eyeswerehemisected,thecornea assumed thatA2mustbepresent. Calculated optical density. Foridentification, thevalueofwavelength atwhichthe dispersed pigment absorbancespectrawereplotted directlyinunitsof ±1.0 nm andarereportedheretothenearest wholeinteger. Oildropletand reptiles. However, ifthecalculated noisy datasuchasthatfromthe verysmalloutersegmentsofdiurnal Template fittingaloneisnot the bestdeterminantofA1orA2statusfor standard datausingthemethod ofMacNichol(MacNichol,1986). normalised tothisabsorbancevalueandtemplatefiteitherA1 or A2 differentiated toestablish the peakwavelength.Thespectrumwas a Gaussianfunctionwasfittothetop40datapointsat1 method previouslydescribedbyLoewetal.(Loewal.,2002).Briefly, common lizards[ In September2011, attheendofactivity season,wecapturedfour and theeyesenucleatedunderdimredlight(safelightNo.2,15 animals wereanaesthetisedwithisoflurane,decapitatedsharpshears species, atleastonefemaleperspecies).After2 four commonlizardsandwall(twoindividualsperyearforeach same asthosedescribedbyLoew(Loew, 1994;Loewetal.,2002).We used Microspectrophotometry wasconductedbyE.R.L.andprotocolswerethe using essentiallythesametechniqueasforretina. visual pigment.Cornealabsorbancewasmeasuredfromisolated pieces differ. Post-measurementbleaching wasusedtoconfirmthepresenceof segment foreachcellandthussamplesizesoildropletspigments cones). Insomecases,itwasnotpossibletoscantheinnerand outer or dispersedinnersegmentpigment(theaccessorymembersofthedouble the samecellwasalsoscannedtomeasureabsorbanceofoildroplet accuracy of~1 from 750to350 Absorbance spectrawereobtainedforallclearlyidentifiedoutersegments on thestageofacomputer-controlled single-beamMSP (Loew, 1994). sandwiched betweentwocoverslipsedgedwithsiliconegrease,andplaced pH Study animals MATERIALS ANDMETHODS Spectral absorbance of pigments and oil droplets pigmentsandoildroplets of absorbance Spectral Visual pigment 7.2 supplementedwith6%sucrose.Piecesofretinaweremacerated, The JournalofExperimentalBiology(2015)doi:10.1242/jeb.115923

nm (Loew, 1994).Wheneverpossible,theinnersegmentof

nm, andbackagainfrom350to750 Zootoca λ max ′ N, 2°41′ was determinedbytemplatefittingusingthe ( Lacerta) E). Adultcommonlizardswerecapturedin

h untilitreachedambienttemperature. Podarcis muralis,threemalesandone vivipara, twomalesandfemales] λ max 2+/ was greaterthan580 Mg 2+ λ -free Ringer’s solutionat max

nm, KodaksafelightNo.

values areaccurateto nm, withawavelength

h ofdarkadaptation,

nm intervalsand

nm, itwas

W bulb, week in 463

The Journal of Experimental Biology the normalisedabsorbance spectrumofvisualpigments (outersegment)and 2004). Relativesensitivity ofsingleconeswascalculated astheproductof fraction of0.05assuggestedforamphibians bySiddiqietal.(Siddiqial., that receptornoiseisindependent oflightintensityandusedaWeber are indistinguishable. colours canbediscriminated,whereas valuesbelow1.0indicatethatcolours the opponentdiscriminationmodel, valuesof discriminate foragivenvisualsysteminenvironment.According to in colourspacebetweentwocoloursindicatesthattheseareeasier to in unitsofmultiplesjust-noticeabledifference (JND).A greater‘distance’ 464 RESEARCH ARTICLE receptor type.Thedistanceinthetetrahedralcolourspace and Osorio,1998),whichassumesopponentmechanismsnoisein each calculated followingequation5intheVorobyev andOsoriomodel(Vorobyev distance belowwhichtwostimuliareindistinguishable)canthen be Vorobyev etal.,1998).A threshold distancebetweentwocolours(i.e.the tetrahedral colourspace(Goldsmith,1990;StoddardandPrum, 2008; calculation placesobjectsseenunderincidentlightintoacalculated droplet absorptionandvisualpigmentabsorbance.Foratetrachromat, this the spectralsensitivityofreceptor, includinglens,ocularmediaandoil by eachphotoreceptortypeaccordingtodataonlightenteringtheeyeand but notinreptiles.Inanutshell,themodelcalculatesrelativequantumcatch against behaviouraldiscriminationtestsinsomebirds,mammalsandinsects, et al.,2004;Vorobyev etal.,1998)].Thismodelhasbeensuccessfullytested applications areavailableforotherspecies(seeOsorioetal.,2004;Siddiqi abundance andnoiselevelsinthephotoreceptors[furtherdetails and canbeparameterisedwithdataonreceptorspectralsensitivities, assumes areceptornoise-limitedcolouropponentdiscriminationmechanism of theVorobyev andOsoriomodel(Vorobyev andOsorio, 1998).Thismodel We modelledvisualsignalperceptionbythecommonlizardusingaversion Xenon lightsource(PX-2)covering220–750 Optics Inc.,Dunedin,FL,USA)calibratedbetween200and850 males intheearlysummerusingaspectrophotometer(USB2000;Ocean spectra weremeasuredinthecentreofthroat,chestandbellyfor84 lizards describedinMartinetal.(Martinal.,2013).Briefly, reflectance We usedthereflectancedataofventral colorationofadultmalecommon where theinnersegmentwasattachedtoadroplet. between oildropletclassesandpigmentweredeterminedfromdata ensured thatwecapturedtheaveragepropertyofeye.Associations photoreceptors ontheretina(Newetal.,2012).However, ourprotocol though lizardsmayexhibitheterogeneousspatialdistributionoftheir We didnotattempttoscoreseparately thedifferent regionsoftheretinaeven images. Intotal,wecountedaround200–800oildropletsfromeacharea. each retinawerecapturedandoildropletscountedbyeyefromthese microscope at×40magnification.Severalimagesfromdifferent areasof with agrease-edgedcoverslipandexaminedusinganOlympusBHT lizards afteranaesthesia.Sampleswereplacedindropofbuffer andcovered pieces ofretinafromeachthreecommonlizardsandmalewall In ordertoquantifythedifferent typesofoildroplets,wecollectedtwosmall absorbance ishalfwaybetweentheminimumandmaximumvalues( throat andbellyspectrainthisstudy. coloration werenotsignificantlydifferent (Martinetal.,2013),weusedonly average spectrum.Becausespectralcharacteristicsofchestandbelly measured tworeflectancespectraforeachbodyzoneandcalculatedthe white diffusive standard(WS-1,OceanOpticsInc.).Foreachlizard,we was determinedusingthemethodofLipetz(Lipetz,1984). Oil droplet abundance Oil droplet Quantitative model Body colorationmeasurements the lizard’s skinwasbevelledat45 be visibletolizards(Fleishmanetal.,2011). Theendprobeincontactwith 300–700 nm, whichincludesthebroadestrangeofwavelengthsknownto probe (R400-7-UV/VIS,OceanOpticsInc.).We restrictedouranalysesto approximately 1 No dataonphotoreceptornoiseisavailable forreptiles.Here,weassumed

mm 2 . Reflectancewasmeasuredrelativetoadarkand

deg andthecircularreadingspotwas Δ

S nm anda400 above 1.0JNDindicatethat Δ S μm fibreoptic was calculated

nm, a λ mid ) cones inZ.vivipara of theabundanceSWS,MWSandLWS singleconesrelativetoUVS relative tothosein other singlecones(basedonUVSabundanceobservedin of mostlizards,model1:1:1:1);(3)UVSconestwiceasabundantthe 0:1:1:1); (2)UVSconesequalinabundancetoothersingle(typical proportion. Fourvisualsystemsweretested:(1)noUVScones(model pigments withanA1orA2chromophorewereassumedtobeina10:90 the modeltodetectsmallcolourvariationsforeachpatch.LWS presents reversecolourproperties.We werethusinterestedintheabilityof throat isrichinUV andpoorinyellow-redpigmentationwhereasthebelly conducted separatelybecauseoftheirdifferences ofspectralproperties:the throat orbellycolourpairs.Analysesofanddatawere (empirical estimate)and(4)pureA2-basedLWS pigments. A1- andA2-basedlongwavelength-sensitivepigmentsin10:90proportion vitamin A1-andA2-basedLWS pigmentsin50:50proportion,(3)vitamin assuming theempiricalconedensity:(1)pureA1-basedLWS pigments,(2) spectra fromthe84malecommonlizards.We testedfourconditionsby their proportion,weranthemodelonallpossiblepairsofthroatorbelly ae,K,Mlá,O,Trk .adHrzg G. Herczeg, and J. Török, O., Molnár, K., Bajer, droplet templatesfromthesamereferenceandestimatesof absorbance spectraforeachtypeofvisualpigment.Inaddition,weusedoil assuming atransparentlensandocularmediaintherange350–700 of therelativetransmissionspectrumoildroplets(innersegment) http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.115923/-/DC1 Supplementary materialavailableonlineat French SocietyofEcologytoM.M. the Ministèredelarechercheetl’enseignementsupérieur, andagrantfromthe This researchwassupportedbytheCentreNationaldelaRechercheScientifique, manuscript. and J.-F.L.G. draftedthemanuscript;M.M.,J.-F.L.G., S.M.andE.R.L.revisedthe E.R.L. performedtheexperiments;M.M.describedandanalyseddata; M.M., J.-F.L.G., S.M.andE.R.L.designedconceivedtheresearch;M.M. The authorsdeclarenocompetingorfinancialinterests. discrimination. We thankDorisGomezfor hercriticalhelpwiththequantitativemodellingofvisual Vorobyev, 2005)andestimatesof modelling purposes,weusedHartandVorobyev’s templates(Hartand rhr .N,Ede,J . yho,J .adPrrde J.C. Partridge, and J.N. Lythgoe, J.A., Endler, S.N., Archer, S.N. Archer, ae,K,Mla,O,Trk .adHrzg G. Herczeg, and J. Török, O., Mólnar, K., Bajer, 84 malecommonlizardsandcalculatedthevalueof ran themodelonallpossiblepairsofthroatspectraandbellyfrom the modelwererunusingAvicol version6(Gomez,2006). daylight (D65spectrum)(Wyszecki andStiles,1982)allcalculationsof by thetransmissionspectra.We usedastandardirradiancespectrumfor separately, multipliedby0.5andaddedbeforenormalisingmultiplying mixture, theabsorbancespectraofbothtypespigmentswerecalculated for lizards.IfbothvitaminA1-andA2-basedpigmentswerepresentinthe These templatesweredesignedforbirdsandthereisnoequivalenttemplate MSP datatocalculatenormalisedtransmission spectraoftheoildroplets. References Supplementary material Funding Author contributions Competing interests Acknowledgements 7 determines fightsuccess inmaleEuropeangreenlizards( Ecol. Sociobiol. pigment polymorphismintheguppy Vallerga), pp.25-42.Dordrecht:Kluwer. in theEcologyofVision lizards ( Furthermore, toexploretheimportanceoftwochromophoretypesand To evaluatetheimportanceofrelativeabundanceUVScones,we , 866-868. Lacerta viridis (1999). Visual pigmentsandphotoreception.In The JournalofExperimentalBiology(2015)doi:10.1242/jeb.115923 64, 2007-2014. P. muralis,model2:1:1:1);and(4)empiricalestimates (model 1:2:5:9). ) prefermaleswithhighultravioletthroat reflectance. (ed. S.N.Archer, M.B.A.Djamgoz,E.R.LoewandS. Poecilia reticulata λ max from ourMSP datatofitnormalised (2010). Femaleeuropeangreen (2011). Ultravioletnuptialcolour . Vision Res. Lacerta viridis Adaptative Mechanisms Δ S for eachofthese 27, 1243-1252. λ (1987). Visual mid Z. vivipara ). from our

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