Functional Ecology. 2017;1–15. wileyonlinelibrary.com/journal/fec  | 1© 2017 The Authors. Functional Ecology © 2017 British Ecological Society

Received:3January2017  |  Accepted:23August2017DOI: 10.1111/1365-2435.12984

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

Environmental conditions shape the chemical signal design of lizards

Simon Baeckens1,2  | José Martín3 | Roberto García-Roa3 | Panayiotis Pafilis4 |  Katleen Huyghe1 | Raoul Van Damme1

1LaboratoryofFunctionalMorphology,DepartmentofBiology,UniversityofAntwerp,Wilrijk,Belgium2DepartmentofOrganismicandEvolutionaryBiology,HarvardUniversity,Cambridge,MA,USA3DepartmentofEvolutionaryEcology,MuseoNacionaldeCienciasNaturales,CSIC,Madrid,Spain4DepartmentofZoologyandMarineBiology,SchoolofBiology,NationalandKapodistrianUniversityofAthens,Ilissia,Greece

CorrespondenceSimonBaeckensEmail:simon.baeckens@uantwerp.be

Funding informationSpanishMinisteriodeEconomíayCompetitividad,Grant/AwardNumber: BES-2012-054387;UniversityofAntwerp

HandlingEditor:KevinMcGraw

Abstract1. Thesignalsthatanimalsusetocommunicateoftendifferconsiderablyamongspe-cies.Partofthisvariationinsignaldesignmayderivefromdifferentialnaturalselec-tion on signal efficacy; the ability of the signal to travel efficiently through theenvironmentandattractthereceiver’sattention.Forthevisualandacousticmo-dalities,theeffectofthephysicalenvironmentonsignalefficacyisawell-studiedselectiveforce.Still,verylittleisknownonitsimpactonchemicalsignals.

2. Here,wetookabroad,phylogeneticcomparativeapproachtotestforarelation-shipbetweenanimals’signalchemistryandpropertiesoftheirnaturalenvironment.OurstudyfocusedonlizardsfromtheLacertidaefamily.

3. Wesampled64speciesacrossthreecontinentsanddeterminedthelipophiliccom-position of their glandular signalling secretions using gas chromatography–massspectrometry.Foreachspecies,anarrayofenvironmentalvariablesofhightempo-ralandspatialresolutionwasobtainedfromclimatedatabases.

4. Speciesvariedconsiderably in theoverall richness (numberofconstituents)of theirsecretions, aswell as in the relative contribution of themajor chemical compoundclasses.Signalrichnessandtherelativecontributionoftherespectivecompoundsex-hibitedlittleevidenceofphylogeneticrelatedness,suggestingthatchemicalsignalsmaychangeveryrapidly.Neitherinsularitynorsubstrateuseaffectedchemicalsignalcom-position,however,wefoundastrongstatisticalrelationshipbetweenthechemistryofthelizards’secretionsandaspectsofthethermalandhydricenvironmenttheyinhabit.

5. Speciesfrom‘xeric’milieuscontainedhighproportionsofstablefattyacidestersandhighmolecularweightalcoholsintheirglandularsecretions,whichlikelyincreasethepersistenceofsecretionscent-marks.Incontrast,speciesinhabiting‘mesic’environ-mentsproducedsecretionsofahighchemicalrichnesscomprisinghighlevelsofal-dehydesand lowmolecularweightalcohols.Thischemicalmixprobablycreatesavolatile-richsignalthatcanbeusedforlong-distanceairbornecommunication.

6. Wearguethattheobservedvariationinsignaldesignresultsfromdifferentialnaturalselection,optimizingsignalefficacyundercontrastingenvironmentalconditions.

K E Y W O R D S

adaptiveevolution,chemicalcommunication,epidermalglandsecretions,Lacertidae,macroevolution,pheromones,phylogeneticcomparativemethods,signalefficacy

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1  | INTRODUCTION

Thevastarrayofsignalsusedinanimalcommunicationisacontinu-oussourceofaweandahottopicinbehaviouralresearch(e.g.HavenWiley,2015;Laidre&Johnstone,2013;Lichtenberg,Zivin,Hrncir,&Nieh,2014;Tibbetts,Mullen,&Dale,2017;tosayafew).Whilesig-naldiversitycanarisethroughgeneticdrift(e.g.Campbelletal.,2010;Picqetal.2016),naturalandsexualselectionseemmorelikelycausesofdifferentiation.Onefeaturecontributingtoasignal’ssurvivalorre-productivevalueisitsefficacy:itsabilitytotravelthroughthemediumandtoattractthereceiver’sattention.Naturalselectionisexpectedtomouldsignaldesigninsuchawayastomaximizetheefficacyofin-formationtransmissionanddetectability(Endler,1992;Wyatt,2010).Sinceefficacydependsonthephysicalpropertiesoftheenvironment,whichmayvarygreatlyinspaceandtime,differentialselectioncanbeexpectedtoresultinconsiderablevariationinsignaldesign.Thislinkbetweenthedesign (or ‘morphology’)ofasignaland itsefficacy (or‘performance’)couldalsobedubbedasthe‘ecomorphology’ofasignal(cf.Arnold,1983;Endler,2000).

Fortheauditoryandvisualsignallingsystem,thereisgrowingem-pirical evidence that signal efficacy largely depends on the climaticconditions and habitat characteristics under which they operate,leading to differential selection along environmental gradients. Forinstance,habitatdensityisknowntoinfluencethecallofbowerbirds(Ptilonorhynchus violaceus),withbirdssingingat lowerfrequencies indense habitats and at higher frequencies in open habitats (Nicholls&Goldizen,2006).Highvocalizationfrequenciesareeasilyabsorbedindensehabitat, so theuseof low frequencycalls in suchanenvi-ronmentenables thebirds tomaximize thedistance their songsarecarried(Nicholls&Goldizen,2006).Ng,Landeen,Logsdon,andGlor(2013) reporteda strong relationshipbetween thephenotypeofananolelizard’sdewlap(i.e.sexualsignallingornament)anditsenviron-ment.Anolis distichuspopulationsinhumidhabitatshavelarge,orangedewlaps,whilepopulationsindryerhabitatshavesmaller,yellowdew-laps.Itisgenerallythoughtthatthedifferentdewlapdesignsincreasevisualdetectabilityintherespectivexericandmesichabitatsinwhichtheyoperate(Driessensetal.,2017;Ngetal.,2013).

Themajorityofresearchontheevolutionofanimalsignalshasfo-cusedonvisualandauditorycommunication,whilethechemicalcom-municationsystemhasreceivedfarlessattention(Searcy&Nowicki,2005; Stevens, 2013; but see e.g. delBarco-Trillo & Drea, 2014;Steiger, Schmitt, & Schaefer, 2011;Weber, Mitko, Eltz, & Ramirez,2016).Thisisrathersurprisingsincechemicalsignalsformasubstan-tial component of communication acrossmany taxa, and can differsubstantially, even among closely related species (Müller-Schwarze,2006;Müller-Schwarze&Silverstein,1980;Wyatt,2014).Theoreticaland(limited)empiricalworksuggeststhatenvironmentalfactorswillaffectthephysico-chemicalproperties(andconsequentlytheefficacy)ofchemicalsignals—justastheyinfluencetheefficacyofauditoryandvisualsignals.

Firstly,temperatureandhumiditymayinfluencethepersistenceof chemical signals, with increasing evaporation rates of chemi-cals inhotandhumidenvironments (Alberts,1992a).Forexample,

the half-life of acetate, a pheromone in many insects (e.g. Durak& Kalender, 2009; Lacey,Millar, Moreira, & Hanks, 2009;Vinson,1972),decreasestwo- tofourfoldwhenair temperaturerisesfrom20 to 30°C (McDonough, Brown, & Aller, 1989). Evidently, tem-perature has the largest functional impact on scent-mark signalscompared to other chemical signals, as scent-marks are only ef-fectivewhen theyaredetectable for longperiodsof time (Alberts,1992a; Apps, Weldon, & Kramer, 2015). To illustrate, high tem-peraturesacceleratescent-markdecay inants,andhence limit thetrail-following behaviour of these insects (Van Oudenhove, Billoir,Boulay,Bernstein,&Cerdá,2011;VanOudenhove,Boulay, Lenoir,Bernstein,&Cerdá,2012).Similarly,high temperatureshavedetri-mentaleffectson thepersistenceanddetectabilityof scent-marksinthelizardIberolacerta cyreni(Martín&López,2013b).Tongue-flickassays showed that the levelof chemosensory investigationof liz-ardswaslowertowardsscent-markskeptinwarmratherthanincoldconditions, probably due to the rapid signal fade-out in thewarmsetting(Martín&López,2013b).Highlevelsofhumiditycanincreaseevaporationandoxidationratesofchemicalmixtures,resultinginac-celeratedratesofsignalfade-out(Alberts,1992a;Appsetal.,2015;Müller-Schwarze, 2006; Regnier & Goodwin, 1977). Experimentswith chemical compounds found in the scent-marks ofMongoliangerbils(Meriones unguiculatus)demonstrateverylittlesignallossoverseveraldaysat0%relativehumidity,butat100%relativehumidity,over60%ofthesignalevaporateswithin2hr(Regnier&Goodwin,1977). Secondly, precipitation may wash away scent-marks. Thefrequencyofrainfall,forinstance,hasbeenarguedtoinfluencethepredator–prey interactions among thewolf spidersPardosa milvina and Hogna helluo,aswaterdegradesmanyof thespiders’chemicalcuesdepositedatnight(Wilder,DeVito,Persons,&Rypstra,2005).Thirdly,highlevelsofsolar—andUVradiation,whichiscloselylinkedwith altitude (Blumthaler,Ambach,&Ellinger, 1997),may increasechemicaldegradationrates.Lastly,extremeaircurrentscanreducetheactive spatial rangeof chemical signalsbecauseofhigh turbu-lentdiffusivities,althoughmoderatelevelsofwindmayfacilitatethetransportof airbornevolatile chemicals and can increasea signal’srange (Alberts, 1992a; Bossert&Wilson, 1963; Elkinton&Cardé,1984).Darwinian selectionmay favour thosechemical compoundsormixturesofcompoundsinchemicalsignalsthatenableanimalstocopewith such harsh signalling environments.On the other hand,asmanychemicalcompoundsareenergy-consumingtoproduceordifficulttoobtain(Clark,DeBano,&Moore,1997;García-Roa,Sáiz,Gómara,López,&Martín,2017;Kopena,Martín,López,&Herczeg,2011;Rantala,Kortet,Kotiaho,Vainikka,&Suhonen,2003;Rundle,Chenoweth,Doughty,&Blows,2005),animalsareunlikelytoinvestincostlysignallingcompoundswhenthebenefitiseconomicallylow.

Currently, the literatureonchemical signalling isheavilybiasedtowardsinsects;othergroupshavereceivedfarlessattention.Aliter-aturesearchbySymondsandElgarin2008revealedthat79%ofthestudiesonpheromonediversityhave focussedon insects (38%onLepidoptera),comparedto14%onvertebrates (1%onsquamates).Within vertebrates, lizards seem to be a promising clade to studythe evolution of chemical signalling (e.g. Baeckens, Van Damme,

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Cooper,2017;García-Roa,Jara,Baeckens,etal.,2017;García-Roa,Jara, López,Martín, & Pincheira-Donoso, 2017;Mangiacotti etal.,2016;Martín& López, 2014;Mason&Parker, 2010;Pruett etal.,2016).Males ofmost lizard species are equippedwith a series ofepidermal glands located in the dermis of the inner thighs,whichsecrete waxy substances through pore-bearing scales (Figure1),or ‘epidermal (femoral) pores’ (Mayerl, Baeckens, & Van Damme,2015).The lipophiliccompoundswithintheepidermalglandsecre-tionsaregenerallyconsideredtobetheleadingsourceofchemicalsignals involved in lizardcommunication,andmediatebehaviour ina variety of contexts (reviewed byMartín & López, 2014;Mayerletal.,2015;butseeAlberts,Phillips,&Werner,1993),suchaster-ritorydemarcationandassessment(Aragón,López,&Martín,2001;Font,Barbosa,Sampedro,&Carazo,2012;Leu,Jackson,Roddick,&Bull, 2016;Martín&López,2012;Martins,Ord,Slaven,Wright,&Housworth,2006),male rival assessment (Carazo,Font,&Desfilis,2007;Hews,Date,Hara,&Castellano,2011;Khannoonetal.,2011;López&Martín,2002),femalechoice(Carazo,Font,&Desfilis,2011;Gabirot, López, & Martín, 2013; Kopena, López, & Martín, 2014;Kopenaetal.,2011;Martín&López,2013a,2015), assessmentoffemalereproductivestatus(Cooper&Pèrez-Mellado,2002;Thomas,2011), individual recognition (Alberts, 1992b; Alberts & Werner,1993;Gabirot,Castilla,López,&Martín,2010a,2010b),sexidenti-fication(Cooper&Steele,1997;Cooper&Trauth,1992;Khannoon,Breithaupt, El-Gendy, & Hardege, 2010) and species recognition(Barbosa, Font, Desfilis, & Carretero, 2006; Gabirot etal., 2010a;Labra, 2011). Lizards passivelymark or deposit these gland secre-tions into the environmentwhilemoving through their habitat, ortheyexhibit activemarkingbehaviour to leave scent-markson thesubstrateoftheirchoice(deVilliers,Flemming,Mouton,&Le,2015;Mason&Parker,2010).Tobeeffective,ascent-markshouldpersistforaslongaspossibleintheabsenceofthesignaller,andshouldbereadilydetectabletoothers(Alberts,1992a;Hughes,Kelley,Banks,& Grether, 2012). Since the longevity and detectibly of chemicalsare strongly environment-dependent (Alberts, 1992a; Apps etal.,2015),lizardspeciesinhabitingdissimilarenvironmentsareexpectedtovary inparticularcharacteristicsof their secretion (e.g.chemical

composition) in order to optimize the functionality of their signalsin their environment. The glandular secretions of lizard speciesDipsosaurus dorsalis and Crotaphytus bicinctores, for instance, con-tainlargeamountsoffattyacidsofhighmolecularweight,whicharethoughttoprotecttheirscent-marksfromrapidevaporation(Alberts,1992b;Martín,Ortega,&López,2013).Highamountsofsqualeneas found inZootoca vivipara, and high amounts ofα-tocopherol inLacerta schreiberi and Lacerta viridis might protect other lipophiliccompounds in the secretions from oxidation inwet environments,therefore, increasing scent-mark longevity (Gabirot etal., 2008;Kopena etal., 2011, 2014). Cholesterol is usually the most abun-dantcomponentinlizardglandsecretions,andsaidtofunctionasanunreactiveapolar‘matrix’thatholdsandprotectsotherlipidsinthescent-marksfromfading(Escobar,Escobar,Labra,&Niemeyer,2003;Escobar,Labra,&Niemeyer,2001;Martín&López,2014).

Most studies on the ecological factors influencing chemical sig-nalevolutionconcentrateononeortwospecies(e.g.Martín&López,2014; Martín, Ortega, & López, 2015; Rouault, Marican, Wicker-Thomas,&Jallon,2004;Rundleetal.,2005),andcannotprovidethebroad-scaleevolutionaryinsightthatcanbegainedfrommultispeciescomparativestudies(seeGaramszegi,Eens,Erritzøe,&Møller,2005;andOrd&Martins,2006forexcellentexamplesonauditoryandvi-sualsignalling).Here,weexaminetheroleoftheenvironmentontheevolutionof the chemical signalling signaturesof lizards.We takeabroad phylogenetic comparative approach to test for co-evolutionbetweenthe lipophilicchemicalcompositionoftheglandsecretions(or‘chemicalsignaldesign’)oflizardsofthefamilyLacertidae,andtheenvironment(climaticconditionsandhabitatcharacteristics)theyin-habit.Wehypothesize thatspecies fromareas inwhich theprevail-ing environmental conditions severely decrease signal longevity byanincreaseinthelossofsignalcompoundsthroughevaporation(e.g.hot,humid,windy,highlevelsofradiation),willcarrylargeproportionsofstableandheavyweightchemicalsintheirglandularsecretionstocounterrapidsignalfade-out.Lacertidlizards(Figure2)constituteanexcellentmodelforthisparticularstudy,becausespeciesofthiscladearedistributedoverawidegeographicalarea(Arnold,1989),andvaryconsiderablyintheirmicro-andmacrohabitatuse(Baeckens,Edwards,Huyghe,&VanDamme,2015).Allspecies(exceptone)carryepider-mal glands (although theydiffer in the number of glands,Baeckensetal.,2015),andmanyareknowntousechemicalsignallinginseveralcontexts(Martín&López,2014;Mayerletal.,2015).

2  | MATERIALS AND METHODS

2.1 | Study area and species

Between 2003 and 2016, we collected epidermal gland secretionsfrom64lizard(sub)speciesofthefamilyLacertidae(Squamata:Sauria)throughoutEurope,Africa,andAsia.Ourdatasetcovershalfofalllac-ertidgeneraandc.20%ofalllacertidspecies,encompassingspeciesbelonging toboth subfamilies (GallotiinaeandLacertinae), andbothmajor tribes within Lacertinae, i.e. Eremiadini and Lacertini (Uetz,2017).Wecoveredawidearrayofhabitatsandclimateregions;from

F IGURE  1 Photographofthecloacalregionofamalelacertidlizard(Lacerta agilis).Notethenumerousepidermalporeswithprotrudingglandularsecretion.PicturefromMayerletal.(2015)(withpermission)

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theMediterraneanmaquisoverthealpinemeadowsinthePyreneesMountains,tothesandyIsraelidunesandtheKalahariDesertofSouthAfrica. Intotal,wecaptured627 lizardsbyhandornoose in11dif-ferentcountries,at60differentlocations(Figure3).Onaverage,wecaught10individualsperspecies(range1–35).Sinceepidermalglandsdevelopattheonsetofsexualmaturity,andtheiractivityisgreatestduringthereproductiveperiod (Cole,1966;Smith,1946),weexclu-sivelysampledadultlizardsduringthematingseason(i.e.spring–earlysummer;Arnold&Ovenden,2007;Carretero,2007;Pianka,Huey,&Lawlor,1979).Weonlycollectedsecretionfrommales,sincetheepi-dermalglandsofmostfemalelacertidsarevestigialand(justasjuve-nilemales)secretioncollectionissimplynotpossibleinmostspecies(Martín&López,2014;Mayerletal.,2015).Aftersecretioncollection,alllizardswerereleasedatexactlythesiteofcapture.Inaddition,sev-enteenHolaspis guentherilizardswereobtainedthroughthepettrade(FantasiaReptiles,Belgium, licenseHK51101419).MaleH. guentheri werewild-caughtinTanzaniabythetrader’spersonnelc.2weekspriortopurchase,andtheirepidermalglandsecretionswerecollectedinthelaboratoryatthemomentofarrival,attheUniversityofAntwerp.Ofthe64speciessampled,generaldescriptionsofthechemicalcomposi-tionoftheglandsecretionsof16specieshavealreadybeenpublished

byJ.Martín’sresearchgroup(MuseoNacionaldeCienciasNaturales,Madrid,Spain;TableS2).

2.2 | Collection and extraction of secretions

Immediatelyafterthelizardswerecapturedinthefield,wecollectedepidermalglandsecretionbygentlypressingaroundthefemoralporeswhilewearing freshnitrile gloves.We attempted to extract secre-tionsfromallglandsfrombothhindlimbs,providingroughlybetween2and6mgofsecretionsperindividual.Theextractionprocedureisharmless,andthelizardsareabletorapidlyproducemoresecretionafterwards(e.g.Baeckens,Huyghe,Palme,VanDamme,2017).WeimmediatelyplacedthecollectedsecretionsinglassvialswithglassinsertssealedwithTeflon-linedlids.Inordertoobtainblankcontrolvials(onaveragetwoperlocality),wefollowedthesameprocedurewithoutcollectingsecretion,inordertoexcludecontaminantsfromthehandlingprocedureortheenvironment,andtoexaminepotentialimpuritiesinthesolventoranalyticalprocedure.Subsequently,vialswerestoredat−20°Cuntilfurtheranalyses,andfornotlongerthan5months.Theidentificationofeachchemicalcompoundandestima-tionof its relative abundance (as percentage)was estimatedusing

F IGURE  2 Photographsofasubsetoflacertidlizardspeciesusedinthisstudy.Fromthelefttoptotherightbottom:Acanthodactylus beershebensis,Lacerta bilineata*,Dalmatolacerta oxycephala,Podarcis melisellensis*,Tropidosaura gularis,Podarcis siculus,Heliobolus lugubris,Algyroides nigropunctatus*,Lacerta media.Withpermision,picturestakenbyAnneliesJacobsaremarkedwithanasterix(*).TheothersaretakenbySimonBaeckens

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gas chromatography–mass spectrometry (GC–MS). Here, we usedexactly thesamemethodologyandprotocolasdescribed inearlierstudies (cf.Martín&López,2006,2014;Martín,Moreira,&López,2007;Martín,Ortega,etal.,2013).DetailsonthechemicalanalysescanbefoundinAppendixS1insupportinginformation.

For further analyses,weassignedeachchemical compound toamain chemical class, and calculated the relative proportion of eachclass.Themainchemicalclassesusedherearealcohols,aldehydes,car-boxylicorfattyacids,ethylormethylesters,offattyacids,furanones,ketones,steroids,terpenoids,tocopherolsandwaxyesters(i.e.anesterofafattyacidandafattyalcohol).Additionally,wecalculatedthepro-portionsofcholesterol,becausethissteroidisthemaincompoundinthesecretionsofmanylizards(Weldon,Flachsbarth,&Schulz,2008).Sincethemolecularweightofalcoholsandfattyacidsareknowntovarygreatlyamong lizardssecretions (Martín&López,2011;Mayerletal.,2015),wesub-categorizedalcoholsandfattyacidsinalowmo-lecularweight class (compoundswith chains of 16 carbons or less)andahighmolecularweightclass(compoundswithover16carbons).Wealsoincludedthenumberofdifferentlipophiliccompounds(bothidentified and unidentified compounds but that could be character-izedwithinaspeciesbytheirspecificretentiontimesandcharacteristicmassspectra)establishedinaspecies’secretion,orso-called‘chemicalrichness’,asanadditionalchemicalvariableintheanalyses.

Within a lizard species, there is still measurable inter-individualvariation in relativeproportionsofcompounds inglandsecretions—although, small and subtle (Martín & López, 2015). In spite of thisinter-individual variation, themain chemical profile of each species(i.e.presence/absenceofmajorcompoundsandrelativeimportanceofeachcompound)isalwaysmaintained.Essentially,andforthepurposeofthisstudy,naturalvariationwithineachspeciesshouldnotaffectinterspecificcomparisons.

2.3 | Environmental data

Foreachspecies,wecollatedanarrayofenvironmentalvariablesfromclimatedatabases,usingthegeographicalcoordinatesofthespecies’catchinglocality(TableS1).DataweredownloadedfromWorldClim(Hijmans, Cameron, Parra, Jones, & Jarvis, 2005) and ERA-Interim(Deeetal.,2011).FromtheglobalWorldClimdatabase,whichpro-vides recent long-termaverageclimaticconditions (monthly;1950–2000)onaspatialresolutionof±1km2,weextracteddataonmeanairtemperature(taken±2mabovethesurface;Tair),meanprecipitation,andaltitude.Inaddition,wecalculatedasinglemeasurefor‘aridity’,theQindex:

whereP is the average annual precipitation (mm),Tmax is the high-estmonthlymean temperature, andTmin the lowestmonthlymeantemperature. Arid environments are characterized by a lower Q,whereas mesic environments have a higher Q (Oufiero, Gartner,Adolph, & Garland, 2011; Tieleman, Williams, & Bloomer, 2003).TheERA-Interimdatabase is the latest global atmospheric reanaly-sis produced by the European Centre for Medium-RangeWeatherForecasts (ECMWF), and provides complete datasets of multiplevariables at high spatial (±17km2) and temporal resolution (6-hr;1979-present).Weextracted informationonrelativehumidity,windspeed, proportion cloud cover, downwardUV radiation at the sur-face level (RUV), totalsurfacesolarradiation (or ‘irradiance incident’;Rsolar), soil temperature (temperaturemeasured between 0 to 7cmin the soil; Tsoil), and ground surface temperature (air temperaturec. 5cm above the surface; Tsurface).We restricted ourselves to thetime and period that lizards are thought to be active (and scent-marking) and, therefore, exclusively included environmental datameasured at mid-day (12:00h), and from March until Septemberfor European/Mediterranean lacertid species (Arnold, Arribas, &Carranza, 2007; Carretero, 2006; Perry etal., 1990), October untilMarchforSouthAfricanlacertids(Huey&Pianka,1977,1981;Piankaetal.,1979),andannualdataforspeciesaroundtheequator(i.e.only H. guentheriinourdataset).Basedondailyinformation,wecalculatedspecies’ means for every environmental variable across its activityseasonduringtheperiod1979–2015.

Additionally,we (1) documentedwhether specieswere sampledonthemainlandoronanisland,and(2)assignedeachspeciestooneoffoursubstrateclassesbasedondatafromBaeckensetal. (2015):sandy,rocky,vegetationorgeneralist.

Q=P

(

Tmax+Tmin

) (

Tmax−Tmin

) ×1000,

F IGURE  3 GeographicalmapofEurope,AfricaandwesternAsia,showingthesamplelocalitiesofthe64lacertidlizardspeciesunderstudy

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2.4 | Phylogenetic analyses

Oneofthemostrecent(comprehensive)phylogeneticreconstructionofthefamilyLacertidae,whichisbasedonbothmitochondrialandnu-cleargeneregions,comprises162speciesandcoversalllacertidgen-era (seeBaeckensetal.,2015),but,alas,doesnot includeallspeciessampledinthecurrentstudy.Inordertoobtainasuitablephylogenetictreeforourphylogeneticcomparativetests,were-rananidenticalphy-logeneticanalysisasBaeckensetal.(2015),butincludedanadditional12(sub)speciestofitourdataset.DetailsonthephylogeneticanalysescanbefoundinAppendixS2.Overall,theresultedtreeofthelacertidfamilycorroboratesmanypreviously reported inter-and intragenericrelationships(Arnoldetal.,2007;Baeckensetal.,2015;Edwardsetal.,2013;Fu,2000;Kapli,Poulakakis,Lymberakis,&Mylonas,2011;Mayer&Pavlicev,2007;Mendes,Harris,Carranza,&Salvi,2016;Pavlicev&Mayer,2009;Pyron,Burbrink,&Wiens,2013).Hence, thisphyloge-netictreewasusedinallfurtherphylogeneticcomparativetests.

2.5 | Statistical analyses

Weperformedallanalysesusingthe ‘ape’, ‘geiger’, ‘phylocurve’and‘phytools’packagesinr,version3.3.1(Adams,2014;Goolsby,2016;Harmon,Weir, Brock,Glor,&Challenger, 2008; Paradis, Claude,&Strimmer,2004;RCoreTeam,2013;Revell,2012,2013).Probabilities(p)lowerthan.05wereconsideredstatisticallysignificant.

Thephylogeneticsignalfortheproportionsofeachchemicalclass,and for the complete multivariate chemical matrix, was calculatedusingPagel’sλandBlomberg’sK(function‘phylosignal’and‘K.mult’).Phylogeneticsignalisthetendencyofrelatedspeciestoresembleoneanotherduetotheircommonancestry,andBlomberg’sKandPagel’sλaretwoquantitativemeasuresofthispattern(Blomberg,Garland,&Ives,2003;Pagel,1999).Kvaluesthatareapproximatelyequalto1matchtheexpectedtraitevolutionundertheBrownianmotion(BM),andindicateanapparentphylogeneticsignal;Kvaluesfarunder1andclosertozeroindicatelittleornophylogeneticsignalassociatedwithrandomtraitevolutionorconvergence;Kvaluesgreaterthan1sug-geststrongersimilaritiesamongcloselyrelatedspeciesthanexpectedunderBMand,therefore,indicatesasubstantialdegreeoftraitcon-servatism(Blombergetal.,2003).Pagel’sλisascalingparameterthatranges from zero to 1. Lambdavalues of zero indicate no phyloge-neticsignal,whereasvaluesof1indicateastrongphylogeneticsignal,matchingtraitevolution,expectedunderBM(Pagel,1999).

To investigate the relationshipbetweenenvironment and chemi-calcomposition,weusedaphylogeneticcanonicalcorrelationanaly-sis(pCCA,function‘phyl.cca’).Thismultivariatemethodenablesustocalculateandanalysethecorrelationbetweencharactersetswhileac-countingforthenon-independenceofspeciesduetophylogeny(Revell&Harrison,2008).Thismethodallowsusto identifythe linearfunc-tionsofeachsetofvariablesthathavemaximumcorrelationwithothersuchsets (Miles&Ricklefs,1984).ThedetailsofthismethodcanbefoundinRevellandHarrison(2008),andaneasilyaccessibleexampleofitsapplicationinHarrison,Revell,andLosos(2015).Priortoanalyses,wetransformedallvariablestoconformtothestatisticalexpectations

oftheanalysis.Percentageswerearcsinesquare-roottransformed,andcount-data(i.e.chemicalrichness)square-roottransformed.

We used phylogenetic MANOVAs (function ‘aov.phylo’) to testwhethersubstrateuseandinsularityaffectsspecies’secretioncompo-sition.WeimplementedthecompletechemicalmatrixasmultivariatecharactersetintheMANOVAanalyses.

3  | RESULTS

3.1 | Chemical composition and richness

The most abundant chemical class found in the glandular secre-tions of the 64 lacertid species under study were steroids, with amean (±SE) proportion of 64.2±2.5% (of TIC) ranging from 19.6%in Acanthodactylus erythrurusto96.3%inDalmatolacerta oxycephala. Alcohols(8.2±1.9%),fattyacids(8.1±1.3%),waxyesters(7.9±1.5%)andtocopherols(6.9±1.5%)werepresentinintermediateconcentra-tions.Aldehydes(1.6±0.3%),terpenoids(1.4±0.3%),estersoffattyacids (0.7±0.2%), furanones (0.5±0.1%) and ketones (0.3±0.1%)were the five chemical classeswith the lowest averageproportion.Descriptive statistics on the relative abundance of chemicals areshownforeachspeciesinTableS3,andvisualizedinFigure4.

Thechemical richnessof thesecretionwasonaverage (±SE)50(±2),andrangedfrom14different lipophiliccompoundsinOphisops elegans and 18 in Z. vivipara,to98chemicalsinTropidosaura gularis and 103 in Gallotia galloti galloti(Figure5).

3.2 | Phylogenetic signal

Theoverallcompositionoftheepidermalglandsecretioninlacertidliz-ardsexhibitedarelativelyweak,albeitsignificant,phylogeneticsignal(Blomberg’smultivariateK=0.45,p<.001).Thecontributionof theindividualchemicalclassesalsocarriedrelativelyweakphylogeneticsignals(TableS3).Theproportionoffattyacidesters,furanonesandterpenoids,however,showedhighPagel’sλvalues(all0.99,p<.001),butintermediateKvalues(K<0.6,p>.05).Asthesoleexception,al-coholsexposedahighλvalue(λ=0.99,p<.001)andaK value over one (K=1.33,p=.001),which implies thatneighbouring lizardspe-ciestendtoresembleeachothermore—withregardtotheproportionofalcoholsintheirsecretion—thanexpectedunderBrownianmotionofevolution.

3.3 | Environmental effects

Aphylogenetic canonical correlationanalysis revealeda significantrelationshipbetween theenvironmental and chemicalmatrices, in-dicating that climatic conditions are affecting the overall chemicalcomposition of lizard epidermal gland secretion (canonical axis 1;R=0.86,χ2=233.58,p=.002).Onlythefirstcanonicalaxisbetweenenvironmentandchemicalsprovedsignificant;thesecondaxiswasnot(canonicalaxis2;R=0.76,χ2=167.36,p=.157).Wethereforefocused on canonical variable 1 (CV1). The canonical loadings (onCV1)fortheenvironmentalandchemicalvariablesaregiveninTable

     |  7Functional EcologyBAECKENS Et Al.

S4.Temperature (Tair,Tsurface and Tsoil)andaridityallhadhighposi-tive loadings, while altitude, precipitation, relative humidity, radia-tion(Rsolar and RUV)andwindspeedcorrelatednegativelywithCV1.Cloud cover proved to have a low impact. Loadings onCV1werehighlypositivefortherelativecontributionofhighmolecularweightalcoholsandfattyacidesters,while lowmolecularweightalcohols,aldehydes and chemical richness showed high negative loadings.Cholesterol,fattyacids,furanones,ketones,steroids,terpenoids,to-copherolandwaxyestershadalowerimpact.Figure6visualizestherelationshipbetweentheenvironmentandthechemicalcompositionusingthespecies’canonicalscores(forCV1)inascatterplot(seealsoAppendixS3).

PhylogeneticMANOVAsdidnotdetect any significant effectofinsularity(F16,47=0.160,p=.904)orsubstrateuse(F64,174.53<0.001,p=.321)onspecies’secretioncomposition.

4  | DISCUSSION

Natural selection favours signals thatwork effectively (Boughman,2002;Endler, 1992,1993). For the auditory andvisual communica-tionsystems, there isgrowingevidence that theefficacyof signalsisstronglydependentontheenvironmentalconditionsunderwhich

signalsoperate,leadingtodifferentialselectionalongenvironmentalgradients(Alberts,1992a;Fleishman,1988,1992;Morton,1975;Ngetal.,2013;Nicholls&Goldizen,2006).Thisstudysoughttotesttheideathatthephysicalpropertiesoftheenvironmentdrivetheevolu-tion of chemical signals by comparing the chemistry of 64 speciesoflizardsinhabitingawiderangeofhabitats.Ouranalysesrevealedsubstantialvariationinthechemicalrichnessandtherelativepropor-tionsofthecomponentsoftheglandularsecretions.Shared-ancestryfailed to explain among-species patterns of variation, although therelative proportion of alcohols proved to be a highly phylogeneticconservativetraitwithinthechemicalsignallingsignatureoflacertidlizards.Most interestingly, our findings revealed a strong relation-ship between the environmental conditions species live in and thechemicalcompositionoftheirepidermalglandsecretions.Ontheonehand,lizardslivingin‘xeric’environments,characterizedbyhightem-peraturesandaridconditions,hadlargeproportionsofhighmolecularweightalcoholsandfattyacidestersintheirglandsecretions.Ontheotherhand,inlizardsinhabitingmore“mesic”environmentswithhighlevelsofprecipitation,humidity,UV-andsolar-radiation,andwind,wefoundglandsecretionsofahighchemicalrichness,withlargepro-portionsofaldehydesandlowmolecularweightalcohols.Insularityorsubstrateusedidnotaffectchemicalsignalcomposition.Thisstudyprovidesthefirstevidenceofastrongrelationshipbetweenchemical

F IGURE  4 A‘heatmap’-visualizationofthevariationintherelativeabundanceofthemainchemicalclasses(range0%–72%)establishedintheepidermalglandsecretionof64lacertidspecies.Steroidsareexcluded,astheywouldskewthecolorationintherepresentationduetotheirhighproportioninlacertidsecretion

High %Low %

8  |    Functional Ecology BAECKENS Et Al.

signaldesignandprevailingenvironmentalconditionsacrossdifferentanimalspecies,whichmayresultfromselectiononsignalefficacy.

4.1 | Diversity in secretion composition

The lipophilic compounds of epidermal gland secretion of lacertidsserve importantfunctions in inter-and intraspecificcommunication(reviewedbyMartín&López,2014;Mayerletal.,2015).Inall64lac-ertidspeciesexamined,steroidswerethemostcommoncompoundsintheglandularsecretion,buttherelativeproportionsofallthecom-pounds,andthechemical richness,varieddramaticallyevenamongclosely related species. Our analyses were unable to describe theinterspecificdisparityinchemicalsignaldesigntophylogeneticrelat-edness; thephylogeneticsignal forchemical richness, thecompletechemicalcomposition,andthemajorchemicalclassesseparately(ex-ceptalcohols)wereallrelativelylow.Similarfindingsarereportedforother signallingmodalities,with low phylogenetic signal in e.g. theadvertisementcallsofAfricanclawedfrogs(Tobias,Evans,&Kelley,2011)andthedisplaybehaviourofCyclurarockiguanas(Martins&Lamont, 1998). Similar tomany behavioural traits (Blomberg etal.,2003), the chemical signal design in lacertids seems highly evolu-tionarymalleableandappearstohavechangedrapidlyoverasmall

evolutionary time-scale. Interestingly, and in contrast to all otherchemicalclasses,therelativeproportionofalcoholsappearedhighlyphylogenetically conservative within the Lacertidae (Blomberg’sK>1).While thereasonor functional significanceof theconserva-tivecharacterofalcoholsinlizardsecretionisuncertain,behaviouralstudies linkthefunctionalbiologyofalcoholswithterritoriality.Forexample,inrocklizards,Iberolacerta monticola,malesrespondaggres-sivelytowardscottontipsimpregnatedwiththealcoholshexadecanolandoctadecanol,andmaleswithhighlevelsofthesealcoholsintheirsecretion have a high dominance status (Martín etal., 2007).Malespiny-footed lizards (A. erythrurus and Acanthodactylus boskianus)respondwith aggressivebehaviour towards the alcoholshexacosa-nolandtetracosanol,bothabundantinthesecretionoftheselizards(Khannoonetal.,2011).Secretionsthatmarkterritoriesmaynotonlytargetconspecifics,butalsomembersofcompetingspecies.Inlacer-tids,pairsofcloselyrelatedspeciesoftenliveinsympatryandfeedonsimilarprey(Arnold,1987,1989),whichiswhylizardswouldbenefitfromkeepingbothhetero-andconspecificsoutoftheirterritory.Theconservative character of alcohols promotes alcohols as a suitablesignal candidate involving interspecific communication. Evidently,moreresearchisnecessarytofullyunderstandthesignallingfunctionofalcohols.

F IGURE  5 Ancestralcharacterestimationofchemicalsignalrichnessalongthebranchesandnodesofthetreefor64lacertidlizardspecies.Theillustrationsucceedsinvisualizingthelargevariationinchemicalrichnessscatteredamong(evencloselyrelated)species.IllustrationmadeinR(functioncontMapusingtype=‘fan’,inpackagephytools;Revell,2013)

Acan beershebensis

Acan boskianus

Acan eagyptius

Acan erythrurus

Acan opheodurus

Acan schreiberi

Acan sc

utellatus

Algy m

archi

Algy moreoticus

Algy nigropunctatus

Dalmat oxycephala

Gall galloti galloti

Gall galloti palmae

Gall simonyi

Gall stehlini H

elio

lugu

bris

Hellen graeca

Hol

a gu

enth

eri

Iber aranica

Iber aurelioi

Iber bonnali

Iber cyreni Iber galani

Iber martinezricai

Iber monticola cantabrica

Iber monticola monticola

Lac agilis

Lac bilineata

Lac media Lac schreiberi

Lac trilineata

Lac viridis M

er s

quam

ulos

us

Mes

guttu

lata

Mes

oliv

ieri

Nuc

taen

iola

ta

Oph

eleg

ans

Ped

io li

neoo

cella

ta

Phoen kulzeri Phoen laevis

Pod bocagei

Pod ca

rbone

lli

Pod

cret

ensi

s

Pod erhardii Pod gaigeae

Pod

guad

arra

mae

Pod lilfordi

Pod

liole

pis

Pod melisellensis

Pod milensis

Pod

mur

alis

Pod

pel

opon

nesi

acus

Pod pityusensis

Pod siculus

Pod tauricus

Pod

vauc

heri Pod

vire

scen

s

Psamm algirus

Psamm hispanicus

Teira perspicillata

Timon lepidus

Timon tangitanus

Tro

p gu

laris

Zoot vivipara

Chemical signal richness

Low High

     |  9Functional EcologyBAECKENS Et Al.

4.2 | Chemical signal efficacy

Whilephylogenywasunabletopredicttheobservedpatternsofvari-ationinthechemicalsignallingsignatureoflizards,environmentalfac-tors succeeded. Earlier theoretical and experimental work (such asAlberts,1992a;Martínetal.,2015;Regnier&Goodwin,1977)sug-gests that theobserved covariationmay reflect differential adapta-tion of signal chemistry to climatic conditions, serving transmissionefficacy.

‘Xeric’milieus,typifiedbydryconditionswithhightemperatures,canformaharshchemicalsignallingenvironmentforanimalsbecausethesehightemperaturesincreasetheevaporationanddiffusionratesofchemicals,thusaffectingthedurabilityofchemicalsignals(Alberts,1992a;Müller-Schwarze2006;Regnier&Goodwin,1977).Speciesin-habitingsuchxericenvironmentsarethereforeexpectedtocompen-sate for these external factors that reduce chemical signal efficacy.Onewayofcopingwithahostilesignallingenvironmentisbyalteringthechemicalcompositionofscent-marks; investing inchemicalsen-compassingheavymoleculeswith lowvapourpressureswillcounterrapid evaporating rates and hence increase chemical signal durabil-ity(Appsetal.,2015).Forexample,aninterpopulationalstudyonthelacertid lizard Podarcis guadarramae revealed small, but significant,differencesinthechemicalsignalcompositionoftwopopulationsin-habitingenvironmentswithdisparateclimaticconditions(Martínetal.,2015).Moreover, chemosensory tests showed that chemical signalshadalowerefficacywhentemperatureanddrynessincreases,butthatthesenegativeeffectsweremorepronouncedforsignalsfromlizardsinhabitingnaturallycolderandmorehumidenvironmentsthansignalsfrom lizards living inwarm and dry conditions (Martín etal., 2015).This suggests that abiotic conditions can causea selectivepressureonthedesignofchemicalsignalsinordertomaximizesignalefficacy.Our findings strongly support this hypothesis on a broad species-wide level,with lizard species living in xericmilieus exhibiting large

proportionsofhighmolecularweightalcoholsandfattyacidestersintheirglandsecretions.Theheavyweightfeaturesandthelowvapourpressureoflong-chainedC>16alcoholsandstablelong-chainedfattyacidesters (Benzianeetal.,2011;Saxena,Patel,&Joshipura,2013)probably allow epidermal gland secretion to persist for longer peri-odsoftimeintheenvironment.Whilescent-marksobviouslybenefitfromthesephysico-chemicalproperties, theslowemissionprobablyproduceslowgasphaseconcentrations,hencedecreasingsignalde-tection by airborne components (Apps etal., 2015). The glandularsecretionsof lizards fromxeric environments, therefore,most likelyserve a scent-marking function that can be detected by substrate-borne chemical sampling.Whether lizards from xeric milieus reallyexhibitmore ‘tongue-touches’ than ‘air-flicks’ (terminology followingAlberts, 1989;Carazo etal., 2007), andwhether this potential shiftinchemosensorybehaviourhas implicationsontheevolutionofthevomeronasal-lingual system of these lizards, is uncertain. Among-speciescomparisonsof tongueandvomeronasalorganmorphology,usinge.g.micro-CTimaging(asinBaeckens,Herrel,etal.2017),wouldbehighlyinformative.

‘Mesic’environments,heredefinedbyrelativehumidconditionswithhighlevelsofprecipitation,radiationandwind,mayalsocompli-catechemicalcommunication,althoughthechallengesdifferslightlyfromthosefacedinxericclimates.Ourresultsshowedthatlizardspe-cieslivinginsuchmesicmilieusexhibitlargeproportionsofaldehydes,lowmolecularweightalcoholsandahighchemical richness intheirsecretions.Lowmolecularweightalcoholsandaldehydesevaporaterapidly, and aldehydes are alsovery susceptible to oxidation (Appsetal., 2015; Falbe, Bahrmann, Lipps, &Mayer, 2000; Kohlpaintneretal.,2013)Whilethevolatilenatureofthesechemicalsmakesthem,apriori,unsuitablecandidatesforascent-markingfunction,theharshsignallingconditionsofmesicenvironmentswouldonlyacceleratetheemissionofthechemicals,makingthemevenlesssuitable.Intuitively,thesefindingsarenotinaccordancewithourpredictionsonsignalling

F IGURE  6 Scatterplotshowingthesignificantrelationshipbetweentheenvironmentalandchemicalmatrices,indicatingthatclimaticconditionsaffecttheoverallchemicalcompositionoftheepidermalglandsecretionsoflacertidlizards.Eachdotrepresentsaspecies’canonicalscore(forCV1)obtainedfromthephylogeneticcanonicalcorrelationanalysisbasedonthemultivariateenvironmentalandchemicalcharactersets

–0.5 0.0 0.5

–0.5

0.0

0.5

Environmental matrix

Che

mic

al m

atri

x

‘Mesic’ ‘Xeric’

% Fatty acid esters

% Heavy-weight alcohols

% Aldehydes

% Low-weight alcohols

Chemical signal richness

10  |    Functional Ecology BAECKENS Et Al.

efficacy. However, the competing requirements for efficient scent-marksignals (persistenceby lowvolatility)and long-distancesignals(detectabilitybyhighvolatility)maytriggerashiftinchemicalsignal-lingtacticinmesicenvironments.Itisplausiblethatlizardsfrommesicenvironmentscompensatetheprevailingsignallingchallengesbyuti-lizingtheirsecretionsforlong-distanceairbornecommunication(‘highdetectability-tactic’), rather than substrate-bound scent-marking(‘highdurability-tactic’).Indeed,ameta-analysisbyAppsetal.(2015)in searchof relationshipsbetween signal compoundproperties andsignal function in terrestrial vertebrates found that aldehydes areoverall less common in amniotes’ scent-marks, but more commonas long-distance ‘odours’. Besides, aldehydes are highly odoriferous(many fragrances are aldehydes;Gounaris, 2010; Indradas,Hansen,Palmer,&Womack,2014;Kohlpaintneretal.,2013).Theirstrongar-omatic character, togetherwith themoderate air currents inmesicenvironments,mayincreasetheactivespaceoftheairbornechemi-calsandfacilitatesignaldetectionbyhetero-orconspecificsfromalongdistanceaway(Martín&López,2014).Theneedtoshiftbetweenchemicalsignallingtactics(‘highdetectability’vs.‘highdurability’tac-tic) could also be influenced by population density. Scent-markingin low-density populations is only effectivewhen depositingmanyscent-marksovera largerange,which is,byhookorbycrook,verycostly (Moorcroft&Lewis,2006). Lizards frommesicenvironmentshousinglow-densitypopulationswouldthereforebenefitfromoper-atingalong-distancesignallingtacticoverscent-marksignalling.Alas,wedonotpossessreliabledataonpopulationdensitiesofthespeciessampledinthisstudy.

Ourresultsalsoshowthatlizardspeciesfrommesicenvironmentsproducedglandsecretionsofahigherchemicalrichness(i.e.numberof different lipophilic chemical compounds) than species inhabitingxericenvironments. It isknown thatmerelyone individual chemicalcompoundcanholdvaluableinformation(e.g.cholesta-5,7-dien-3-olin I. cyreni lizards, López & Martín, 2005a; Martín & López, 2006; (Z)-7-dodecen-1-ylacetateinElephas maximuselephants,Rasmussen,Lee,Zhang,Roelofs,&Daves,1997),oracombinationoftwospecificcompounds(e.g.oleicacidandergosterolinI. cyrenilizards;López&Martín,2012),oracombinationofcompoundsinapreciseratio(e.g.67:33blendoftrans-11andcis-11-tetradecenylacetates intheoakleaf roller Archips semiferanus,Miller,Baker,Carde,&Roelofs,1976).Therefore, animals emitting rich chemical signals probably have thepotential to convey more elaborate (multiple-message hypothesis)or strongermessages (redundantmessage hypothesis) than animalsproducingsignalsofalowchemicalrichness(Hebets&Papaj,2005;Møller&Pomiankowski,1993;Steigeretal.,2011;Symonds&Elgar,2008).Thefunctionalsignificanceofthedifferenceinchemicalrich-nessbetweenlizardsfrommesicorxericenvironmentscouldbelinkedto theeffectofhumidityon thechemoreceptiveorgansof animals.Mammalianchemoreception,forinstance,ishighlymodulatedbyrela-tivehumidityduetosensoryimpairmentfromthedrying-outofnasalmucosa in lowhumidconditions (Laska,Rother,Schmidt,&Schmid,1986;VanderWall,1998).Optimalfunctioningofthechemoreceptiveorgansinhumidconditionsmayenableanimalstodetectandprocess

more(and)differentairbornechemicals.Whetherthis istruefor liz-ards,however,isstilluncertain.

Although plausible, many of the above-mentioned explanationsstillremainspeculative.Whiletheprevailingenvironmentalconditionsstronglypredicttheobservedvariationinthechemicalsignaldesignin lizards, it still remains tobe testedwhether thisvariation is trulyadaptiveintermsofmaximalsignalefficacy.

Altering the chemical composition or the relative proportionsof the compounds in the chemical signal isonewayof copingwithsuboptimalsignallingenvironments,butotherwaysarealsopossible.Increasing the total quantity of the deposited glandular secretions,for example, has been shown to increase the signal detectability inthe lacertid lizardsPodarcis muralis (Baeckens,Huyghe,etal.,2017),Specificsiteselectionforsecretiondepositmaybeanotherwayofin-creasingsignallingefficacy.LizardsofthespeciesI. cyrenideposittheirexcrementsatnon-randomsites(preferringhighpositionedrocks)tofacilitatechemicalorevenvisualdetectionbyothers(López,Aragón,&Martín,1998).Lizardsmightalsoselectsitesofmoresuitablemi-croclimaticconditions(undervegetation,intheshade,etc.)dissimilartothatofthesuboptimalmacroclimate.Forexample,thevolatile-richsecretionofspiny-tailedlizards(Uromastyx aegyptia microlepsis)doesnotseemtobeadaptedtotheextremearidconditionsinthedesert,but theymight be useful under themicroclimatic conditions insideburrowswheretheselizardsspendlongperiodsoftime(Martínetal.,2016).Ifenvironmentalconditionsbecometoohostileorthecostsofproducingchemicalstoocostly, lizardsmighteventrade inchemicalsignalsforothersignallingmodalities:the‘between-channelcompen-sationhypothesis’,asproposedbyBaeckensetal.(2015)(seealsoFox&Shipman,2003;Plath,Parzefall,Körner,&Schlupp,2004).

4.3 | Insularity and substrate use

Sixteen of the 64 species included in this studywere sampled onislands.Ourfindingsshowthatinsularitydoesnotaffectthechemi-calsignaldesigninlacertidlizards.However,lizardspeciesonislandstendtodiffernotablyfromcloselyrelatedspeciesonthemainlandin a number of morphological, behavioural, ecological, physiologi-calandlife-historycharacteristics(Losos&Ricklefs,2009;Vervust,2011).Furthermore,Martín,López,Garrido,Pérez-Cembranos,andPérez-Mellado(2013)observedinter-islandvariationinthechemicalcompositionofthesecretionofthelacertidPodarcis lilfordionthreeislands in theBalearics.However,withall islandssharingallmajorcompounds,whichaccountedfor97.7%ofthetotalchemicalcom-position,theseamong-islanddifferencesturnedouttobeveryminor(Martín, López, etal., 2013).Whilst theoverall chemical profile oflacertidswasunaffectedbyinsularityinouranalyses,island-lifecantriggersubtlechangesinlacertidsecretionthatmaybebiologicallysignificant (Runemark, Gabirot, & Svensson, 2011). Clearly, large-scaleislandcomparisonsarenecessary—preferablyonapopulationallevel—inordertodeterminetheadaptivesignificanceofsmallvaria-tioninthechemicalcompositionoflizards’secretiononislands.

     |  11Functional EcologyBAECKENS Et Al.

AcomparativestudybyBaeckensetal.(2015)encompassing162lacertid species showed that shrub-climbing species tend to havefewersecretionglandsthanspeciesinhabitingothersubstrates,infer-ringthatshrub-climbingspeciesinvestlessinandrelylessonchemicalsignalling.Thepresentstudywasunabletofindanyeffectofsubstrateuseonthechemicalsignalcompositionoflacertidsecretion.However,thisresultmaybeascribedtothelownumberofshrub-climbingspe-ciesinourdataset(nineoutof64species;14%).

In summary, by taking a multispecies comparative approach,wetestedwhethertheenvironmentisresponsiblefortheremark-ableandubiquitouschemicalsignaldiversityinlizards,detectedinthis study. Using by far the largest comparative chemical datasetamassedto-datetoexaminethisquestion,ourstudyhasprovidedstrongevidenceforasignificantrelationshipbetweenchemicalsig-naldesignandprevailingenvironmentalconditions,whichmayre-sultfromdifferentialselectiononsignallingefficacy.Inaddition,wedemonstratedthatlizardsprovideapromisingmodelsystemforthestudy ofmacroevolutionary patterns of chemical signalling evolu-tion.Futureexperimentalstudiesonthebiologicalfunctionalityofthedifferentchemicalcompoundstoserveastruechemicalsignalsorpheromonesarehighlyencouraged.

ACKNOWLEDGEMENTS

Wewerefortunateenoughtobeabletocountonsomanypeoplefor assistance and guidance in the field: ShaiMeiri and hiswon-derful team, Chris Broeckhoven, Genevieve Diedericks, le FrasMouton,JanScholliers,JorritMertens,CharlotteVanMoorleghem,andmanymore.We thank JosieMeaney for linguistic advice, ‘ElVentorillo’MNCNFieldStationforuseoftheirfacilities,AnneliesJacobsforprovidinglacertidpictures,andfouranonymousreview-ersforsignificantlyimprovingdraftsofthismanuscript.ThisworkwasconductedunderpermitsforCroatia(UP/I-612-07/14-48/111&UP/I-612-07/14-48/33);TheNetherlands (FF/74A/2015/009);Israel(2014/40323),SouthAfrica(SA)FreeStateProvince(S54C-515022511060), SA Eastern Cape Province (CRO 45/15CR &46/15CR),SAWesternCapeProvince(0056-AAA041-00093),SANorthernCapeProvince(FAUNA229/2015&230/2015),andSALimpopoProvince(0092-MKT001-00004),andinaccordancewithUniversityofAntwerp(Belgium)animalwelfarestandardsandpro-tocols(ECD2014-32).Capturesoflizardsandsamplingprocedureswere performed under different licenses for the EnvironmentalAgenciesofthedifferentRegionalGovernmentsofSpainwhereliz-ardswerestudied.AllGreekspecieswerecollectedinaccordancewiththeHellenicNationalLegislation(PresidentialDecree67/81).Financial support to JM and RGRwas provided by the Spanish’sMinisterio de Economía y Competitividad projects MICIIN-CGL2011-24150/BOS and MINECO CGL2014-53523-P. RGR isindebtedtotheSpanishMinisteriodeEconomíayCompetitividad(FPIpredoctoralgrantnumberBES-2012-054387).ThisworkwaspartofSB’sdoctoralthesisattheUniversityofAntwerp,andmadepossiblethroughfinancialsupportfromtheUniversityofAntwerp.

CONFLICT OF INTEREST

Theauthorsdeclarethattheyhavenoconflictofinterest.

AUTHORS’ CONTRIBUTIONS

S.B.,andR.V.D.conceivedthestudy;allauthorscollecteddata;S.B.,M.J.,R.G.R.,andR.V.D.analysedthedata;allauthorsdiscussedthefindings;S.B.,M.J.,andR.V.D.wrotethefirstdraftofthemanuscript;S.B.designedthefiguresandrevisedthemanuscript.

DATA ACCESSIBILITY

Data deposited in the Dryad Digital Repository: https://doi.org/10.5061/dryad.340g8(Baeckens,Martín,etal.,2017).

ORCID

Simon Baeckens http://orcid.org/0000-0003-2189-9548

REFERENCES

Adams, D. C. (2014). A generalized K statistic for estimating phyloge-neticsignalfromshapeandotherhigh-dimensionalmultivariatedata.Systematic Biology,63,685–697.

Alberts, A. (1989). Ultraviolet visual sensitivity in desert iguanas:Implicationsforpheromonedetection.Animal Behavior,38,129–137.

Alberts,A.C.(1992a).Constraintsonthedesignofchemicalcommunica-tionsystemsinterrestrialvertebrates.American Naturalist,139,62–89.

Alberts, A. C. (1992b). Pheromonal self-recognition in Desert iguanas.Copeia,1,229–232.

Alberts,A.C.,Phillips,J.,&Werner,D.(1993).Sourcesofintraspecificvari-ability in the protein-composition of lizard femoral gland secretions.Copeia,1993,775–781.

Alberts,A.C.,&Werner,D.(1993).Chemicalrecognitionofunfamiliarcon-specificsbygreen iguanas:Functional significanceofdifferent signalcomponents.Animal Behavior,46,197–199.

Apps,P.J.,Weldon,P.J.,&Kramer,M.(2015).Chemicalsignalsinterrestrialvertebrates:Search fordesign features.Natural Products Reports,32,1131–1153.

Aragón,P.,López,P.,&Martín,J. (2001).Effectsofconspecificchemicalcues on settlement and retreat-site selection ofmale lizards Lacerta monticola. Journal of Herpetology,35,684–686.

Arnold,S.(1983).Morphology,performanceandfitness.American Zoologist,23,347–361.

Arnold,E.N.(1987).ResourcepartitionamonglacertidlizardsinsouthernEurope.Journal of Zoology,1,739–782.

Arnold, E. N. (1989). Towards a phylogeny and biogeography of theLacertidae: Relationships within an Old-World family of lizards de-rivedfrommorphology.Bulletin of the British Museum Natural History, Zoology,55,209–257.

Arnold,E.N.,Arribas,Ó.,&Carranza,S.(2007).SystematicsofthePalaearcticand Oriental lizard tribe Lacertini (Squamata: Lacertidae: Lacertinae),withdescriptionsofeightnewgenera.Zootaxa,1430,1–86.

Arnold,N. E., &Ovenden,D.W. (2002).A field guide to the reptiles and amphibians of Britain and Europe,2nded..London,UK:HarperCollinsPublishers.

Baeckens,S.,Edwards,S.,Huyghe,K.,&VanDamme,R.(2015).Chemicalsignalling in lizards: An interspecific comparison of femoral porenumbers in Lacertidae.Biological Journal of the Linnean Society,114, 44–57.

12  |    Functional Ecology BAECKENS Et Al.

Baeckens, S., Herrel, A., Broeckhoven, C., Vasilopoulou-Kampitsi, M.,Huyghe,K.,Goyens, J.,&VanDamme,R. (2017). Evolutionarymor-phologyofthelizardchemosensorysystem.Science Report,7,10141.

Baeckens, S.,Huyghe,K., Palme,R.,&VanDamme,R. (2017).Chemicalcommunication in the lacertid lizardPodarcis muralis: The functionalsignificanceoftestosterone.Acta Zoology,98,94–103.

Baeckens,S.,Martín,J.,Garcia-Roa,R.,Panayiotis,P.,Huyghe,K.,&VanDamme, R. (2017). Data from: Environmental conditions shape thechemical signaldesignof lizards.Dryad Digital Repository, https://doi.org/10.5061/dryad.340g8

Baeckens,S.,VanDamme,R.,&Cooper,W.E.(2017).Howphylogenyandforagingecologydrivethelevelofchemosensoryexplorationinlizardsandsnakes.Journal of Evolutionary Biology,30,627–640.

Barbosa, D., Font, E., Desfilis, E., & Carretero,M.A. (2006). Chemicallymediated species recognition in closely relatedPodarciswall lizards.Journal of Chemical Ecology,32,1587–1598.

delBarco-Trillo,J.,&Drea,C.M.(2014).Socioecologicalandphylogeneticpatterns in the chemical signals of strepsirrhine primates. Animal Behavior,97,249–253.

Benziane,M.,Khimeche,K.,Mokbel, I., Sawaya,T.,Dahmani,A.,&Jose,J. (2011). Experimental vapor pressures of five saturated fatty acidethyl ester (FAEE) components of biodiesel. Journal of Chemical and Engineering Data,56,4736–4740.

Blomberg,S.P.,Garland,T.,&Ives,A.R. (2003).Testingforphylogeneticsignalincomparativedata:Behavioraltraitsaremorelabile.Evolution,57,717–45.

Blumthaler, M., Ambach,W., & Ellinger, R. (1997). Increase in solar UVradiationwith altitude. Journal of Photochemistry and Photobiology. B, Biology,39,130–134.

Bossert,W.H.,&Wilson,E.O.(1963).Theanalysisofolfactorycommuni-cationamonganimals.Journal of Theoretical Biology,5,443–469.

Boughman,J.W.(2002).Howsensorydrivecanpromotespeciation.Trends in Ecology & Evolution,17,571–577.

Campbell,P.,Pasch,B.,Pino,J.L.,Crino,O.L.,Phillips,M.,&Phelps,S.M.(2010).Geographicvariationinthesongsofneotropicalsingingmice:Testingtherelativeimportanceofdriftandlocaladaptation.Evolution,64,1955–1972.

Carretero,M.A. (2007). Reproductive cycles inMediterranean lacertids:Plasticity and constraints. Mainland and Insular Lacertid Lizards: A Mediterranean Perspective,8,1–22.

Carazo,P.,Font,E.,&Desfilis,E.(2007).Chemosensoryassessmentofrivalcompetitiveabilityandscent-markfunctioninalizard,Podarcis hispan-ica. Animal Behavior,74,895–902.

Carazo,P.,Font,E.,&Desfilis,E.(2011).Theroleofscentmarksinfemalechoiceofterritoriesandrefugesinalizard(Podarcis hispanica).Journal of Comparative Psychology,125,362–365.

Carretero,M.A. (2006). Reproductive cycles inMediterranean lacertids:Plasticityandconstraints.In:C.Corti,P.LoCascio,&M.Biaggini(Eds.),Mainland insular lacertid lizards: A Mediterannean perspect (pp. 1–22).Florence,Italy,FirenzeUniversityPress.

Clark,D.,DeBano,S.,&Moore,A. (1997).The influenceofenvironmen-tal quality on sexual selection in Nauphoeta cinerea (Dictyoptera:Blaberidae).Behavioral Ecology,8,46–53.

Cole, C. (1966). Femoral glands in lizards: A review. Herpetologica, 22,199–205.

Cooper,W.E.,&Pèrez-Mellado,V.(2002).Pheromonaldiscriminationsofsex,reproductivecondition,andspeciesbythelacertidlizardPodarcis hispanica. Journal of Experimental Zoology,292,523–527.

Cooper,W.E.,&Steele,L.J.(1997).Pheromonaldiscriminationofsexbymale and female leopard geckos (Eublepharis macularius). Journal of Chemical Ecology,23,2967–2977.

Cooper,W.E.,&Trauth,S.E.(1992).Discriminationofconspecificmaleandfemalecloacalchemicalstimulibymalesandpossessionofaprobablepheromoneglandbyfemalesinacordylidlizard,Gerrhosaurus nigrolin-eatus. Herpetologica,48,229–236.

de Villiers, A., Flemming, A., Mouton, P., & Le, F.N. (2015). Generationglandsofcordylidlizards:Mechanismofsecretiontransfertotheenvi-ronment.Amphibia- Reptilia,36,351–360.

Dee,D.P.,Uppala,S.M.,Simmons,A.J.,Berrisford,P.,Poli,P.,Kobayashi,S.,&Vitart,F(2011).TheERA-Interimreanalysis:Configurationandper-formanceofthedataassimilationsystem.Quarterly Journal of the Royal Meteorological Society,137,553–597.

Driessens,T.,Baeckens,S.,Balzarolo,M.,Vanhooydonck,B.,Huyghe,K.,&VanDamme,R.(2017).Climate-relatedenvironmentalvariationinavisualsignallingdevice:ThemaleandfemaledewlapinAnolis sagreiliz-ards.Journal of Evolutionary Biology,https://doi.org/10.1111/jeb.13144

Durak,D.,&Kalender,Y.(2009).FinestructureandchemicalanalysisofthemetathoracicscentglandsecretioninGraphosoma lineatum(Linnaeus,1758) (Heteroptera, Pentatomidae). Comptes Rendus Biology, 332,34–42.

Edwards,S.,Branch,W.R.,Vanhooydonck,B.,Herrel,A.,Measey,G.J.,&Tolley,K.A. (2013).Taxonomicadjustmentsinthesystematicsofthesouthern African lacertid lizards (Sauria: Lacertidae). Zootaxa, 3669,101–114.

Elkinton,J.S.,&Cardé,R.T. (1984).Effectofwildandlaboratory-rearedfemale gypsy moths (Lepidoptera: Lymantriidae) on the capture ofmales in pheromone-baited traps. Environmental Entomology, 13, 1377–1385.

Endler,J.A.(1992).Signals,signalconditions,andthedirectionofevolu-tion.American Naturalist,139,125–153.

Endler,J.A.(1993).Somegeneralcommentsontheevolutionanddesignofanimalcommunicationsystems.Philosophical Transactions of the Royal Society of London. Series B, Biological sciences,340,215–225.

Endler,J.A. (2000).Evolutionary implicationsofthe interactionbetweenanimalsignalsandtheenvironment.InY.Espmark,T.Amundsen,&G.Rosenqvist (Eds.),Animal signals: Signalling and signal design in animal communication(pp.11–46).Devon,UK:TapirAcademicPress.

Escobar,C.M.,Escobar,C.A.,Labra,A.,&Niemeyer,H.M.(2003).ChemicalcompositionofprecloacalsecretionsoftwoLiolaemusfabianipopula-tions:Aretheydifferent?Journal of Chemical Ecology,29,629–368.

Escobar, C.A., Labra,A., &Niemeyer,H.M. (2001). Chemical composi-tionofprecloacal secretionsofLiolaemus lizards. Journal of Chemical Ecology,27,1677–1690.

Falbe,J.,Bahrmann,H.,Lipps,W.,&Mayer,D. (2000).Alcohols, aliphatic. Weinheim,Germany:Wiley-VCH.

Fleishman,L.J. (1988).Sensoryandenvironmental influencesondisplayform in Anolis auratus,agrassanolefromPanama.Behavioral Ecology and Sociobiology,22,309–316.

Fleishman,L.J.(1992).Theinfluenceofthesensorysystemandtheenvi-ronmentonmotionpatternsinthevisualdisplaysofanolinelizardsandothervertebrates.American Naturalist,139,S36–S61.

Font, E., Barbosa, D., Sampedro, C., & Carazo, P. (2012). Social behav-ior, chemical communication, and adult neurogenesis: Studies ofscentmark function inPodarciswall lizards.General and Comparative Endocrinology,177,9–17.

Fox,S.F.,&Shipman,P.A.(2003).Socialbehaviorathighandloweleva-tions.EnvironmentalreleaseandphylogeneticeffectsinLiolaemus.InS.Fox,J.McCoy,&T.Baird(Eds.),Lizard social behavior(pp.310–355).Baltimore,MD:JohnsHopkinsUniversityPress.

Fu,J.Z.(2000).TowardthephylogenyofthefamilyLacertidae-Why4708base pairs ofmtDNA sequences cannot draw the picture.Biological Journal of the Linnean Society,71,203–217.

Gabirot,M.,Castilla,A.M.,López,P.,&Martín,J.(2010a).Chemosensoryspecies recognition may reduce the frequency of hybridization be-tweennativeand introduced lizards.Canadian Journal of Zoology,88,73–80.

Gabirot, M., Castilla, A. M., López, P., &Martín, J. (2010b). Differencesin chemical signals may explain species recognition between an is-land lizard,Podarcis atrata, and relatedmainland lizards,P. hispanica. Biochemical Systematics and Ecology,38,521–528.

     |  13Functional EcologyBAECKENS Et Al.

Gabirot,M.,López,P.,&Martín,J. (2013).Femalematechoicebasedonpheromone contentmay inhibit reproductive isolation between dis-tinctpopulationsofIberianwalllizards.Current Zoology,59,210–220.

Gabirot,M.,López,P.,Martín,J.,deFraipont,M.,Heulin,B.,Sinervo,B.,&Clobert,J.(2008).Chemicalcompositionoffemoralsecretionsofovip-arousandviviparous typesofmalecommon lizardsLacertavivipara.Biochemical Systematics and Ecology,36,539–544.

Garamszegi,L.Z.,Eens,M.,Erritzøe,J.,&Møller,A.P.(2005).Sexuallysizedimorphic brains and song complexity in passerine birds.Behavioral Ecology,16,335–345.

García-Roa,R.,Jara,M.,Baeckens, S., López,P.,VanDamme,R.,Martín,J., & Pincheira-Donoso, D. (2017),Marcoevolutionary diversificationofglandsforchemicalcommunication insquamatereptiles.Scientific Reports,7,9288.

García-Roa, R., Jara, M., López, P., Martín, J., & Pincheira-Donoso, D.(2017).Heterogeneous tempo andmodeof evolutionarydiversifica-tionofcompoundsinlizardchemicalsignals.Ecology and Evolution,7,1286–1296.

García-Roa,R.,Sáiz,J.,Gómara,B.,López,P.,&Martín,J. (2017).Dietaryconstraintscanprecludetheexpressionofanhonestchemicalsexualsignal.Scientific Reports,7,6073.

Goolsby, E.W. (2016). Likelihood-based parameter estimation for high-dimensional phylogenetic comparativemodels: Overcoming the lim-itationsof“distance-based”methods.Systematic Biology,65,852–870.

Gounaris,Y.(2010).Biotechnologyfortheproductionofessentialoils,fla-voursandvolatileisolates,areview.Flavour and Fragrance Journal,25,367–386.

Harmon,L.J.,Weir,J.T.,Brock,C.D.,Glor,R.E.,&Challenger,W.(2008).GEIGER: Investigating evolutionary radiations. Bioinformatics, 24,129–131.

Harrison,A.S.,Revell,L.J.,&Losos,J.B. (2015).Correlatedevolutionofmicrohabitat,morphology,andbehaviorinWestIndianAnolislizards:Atestofthehabitatmatrixmodel.Behaviour,152,1187–1207.

Haven Wiley, R. (2015). Noise matters: The evolution of communication. Cambridge,MA:HarvardUniversityPress.

Hebets,E.A.,&Papaj,D.R.(2005).Complexsignalfunction:Developingaframeworkoftestablehypothesis.Behavioral Ecology and Sociobiology,57,197–214.

Hews,D.K.,Date,P.,Hara,E.,&Castellano,M.J.(2011).FieldpresentationofmalesecretionsalterssocialdisplayinSceloporus virgatusbutnotS. undulatuslizards.Behavioral Ecology and Sociobiology,65,1403–1410.

Hijmans,R.J.,Cameron,S.E.,Parra,J.L.,Jones,P.G.,&Jarvis,A.(2005).Veryhighresolutioninterpolatedclimatesurfacesforgloballandareas.International Journal of Climatology,25,1965–1978.

Huey,R.B.,&Pianka,E.R.(1977).SeasonalvariationinthermoregulatorybehaviorandbodytemperatureofdiurnalKalaharilizards.Ecology,58,1066–1075.

Huey,R.B.,&Pianka,E.R. (1981). Ecological consequencesof foragingmode. Ecology,62,991–999.

Hughes,N.K.,Kelley,J.L.,Banks,P.B.,&Grether,G.(2012).Dangerousliaisons:Thepredationrisksofreceivingsocialsignals.Ecology Letters,15,1326–39.

Indradas,B.,Hansen,C.,Palmer,M.,&Womack,G.B.(2014).Autoxidationasatriggerfortheslowreleaseofvolatileperfumerychemicals.Flavour and Fragrance of Journal,29,313–323.

Kapli,P.,Poulakakis,N.,Lymberakis,P.,&Mylonas,M.(2011).Are-analysisofthemolecularphylogenyofLacertidaewithcurrentlyavailabledata.Basic and Applied Herpetology,25,97–104.

Khannoon,E.,Breithaupt,T.,El-Gendy,A.,&Hardege,J.D.(2010).Sexualdifferences inbehavioural response to femoralglandpheromonesofAcanthodactylus boskianus. The Herpetological Journal,20,225–229.

Khannoon,E.R.,Flachsbarth,B.,El-Gendy,A.,Mazik,K.,Hardege,J.D.,&Schulz,S.(2011).Newcompounds,sexualdifferences,andage-relatedvariationsinthefemoralglandsecretionsofthelacertidlizardAcanthodactylus boski-anus. Biochemical Systematics and Ecology,39,95–101.

Kohlpaintner, C., Schulte, M., Falbe, J., Lappe, P., Weber, J., & Frey, G.(2013). Aldehydes, aliphatic. In Ullmann’s encyclopedia of industrial chemistry(pp.647–698).Weinheim,Germany:Wiley-VCH.

Kopena,R.,López,P.,&Martín,J.(2014).RelativecontributionofdietarycarotenoidsandvitaminEtovisualandchemicalsexualsignalsofmaleIberian green lizards: An experimental test. Behavioral Ecology and Sociobiology,68,571–581.

Kopena,R.,Martín,J.,López,P.,&Herczeg,G. (2011).VitaminEsupple-mentation increases the attractiveness of males’ scent for femaleEuropeangreenlizards.PLoS ONE,6,e19410.

Labra,A.(2011).ChemicalstimuliandspeciesrecognitioninLiolaemusliz-ards.Journal of Zoology,285,215–221.

Lacey,E.S.,Millar,J.G.,Moreira,J.A.,&Hanks,L.M.(2009).Male-ProducedaggregationpheromonesofthecerambycidbeetlesXylotrechus colonus and Sarosesthes fulminans. Journal of Chemical Ecology,35,733–740.

Laidre,M.E.,&Johnstone,R.A.(2013).Animalsignals.Current Biology,23,R829–R833.

Laska,M.,Rother,G.,Schmidt,U.,&Schmid,U.(1986).DiebeeinflussungdesreichvermogensdurchdieluftfeuchtebeiCarollia perspicillata und Phyllostomus discolor (Chiroptera). Zeitschrift für Säugetierkunde, 51,129–138.

Leu,S.T.,Jackson,G.,Roddick,J.F.,&Bull,C.M.(2016).Lizardmovementtracks:Variation inpath re-usebehaviour isconsistentwitha scent-markingfunction.PeerJ,4,e1844.

Lichtenberg,E.M.,Zivin,J.G.,Hrncir,M.,&Nieh,J.C.(2014).Eavesdroppingselectsforconspicuoussignals.Current Biology,24,R598–R599.

López,P.,Aragón,P.,&Martín,J.(1998).Iberianrocklizards(Lacerta mon-ticola cyreni) assess conspecific information using composite signalsfromfaecalpellets.Ethology,104,809–820.

López,P.,&Martín,J.(2002).Chemicalrivalrecognitiondecreasesaggres-sion levels inmale Iberianwall lizards,Podarcis hispanica. Behavioral Ecology and Sociobiology,51,461–465.

López, P., & Martín, J. (2005a). Female Iberian wall lizards prefer malescents that signal a better cell-mediated immune response. Biology Letters,1,404–406.

López, P., &Martín, J. (2012). Chemosensory exploration ofmale scentbyfemalerock lizardsresult frommultiplechemicalsignalsofmales.Chemical Senses,37,47–54.

Losos,J.B.,&Ricklefs,R.E. (2009).Adaptationanddiversificationonis-lands.Nature,457,830–836.

Mangiacotti,M.,Fumagalli,M.,Scali,S.,Zuffi,M.A.L.,Cagnone,M.,Salvini,R., & Sacchi, R. (2016). Inter- and intra-population variability of theprotein content of femoral gland secretions from a lacertid lizard.Current Zoology,zow113.https://doi.org/10.1093/cz/zow113

Martín,J.,Castilla,A.M.,López,P.,Al-Jaidah,M.,Mohannadi,S.,&Al-Hamaidi,A.(2016).Chemicalsignalsindesertlizards:Arefemoralglandsecretionsof male and female spiny-tailed lizards,Uromastyx aegyptia microlepis adaptedtoaridconditions?Journal of Arid Environments,127,192–198.

Martín, J., & López, P. (2006). Links betweenmale quality,male chemi-calsignals,andfemalematechoice in Iberianrock lizards.Functional Ecology,20,1087–1096.

Martín,J.,&López,P.(2011).PheromonesandreproductioninReptiles.InD.O.Norris,&K.H.Lopez(Eds.),Hormones and reproduction in verte-brates,(Vol.3,pp.141–167).ReptilesSanDiego,CA:AcademicPress.

Martín,J.,&López,P.(2012).Supplementationofmalepheromoneonrocksubstratesattractsfemalerocklizardstotheterritoriesofmales:Afieldexperiment.PLoS ONE,7,e30108.

Martín,J.,&López,P.(2013a).Responsesoffemalerocklizardstomultiplescentmarksofmales:Effectsofmaleage,maledensityandscentover-marking.Behavior Processes,94,109–114.

Martín,J.,&López,P.(2013b).Effectsofglobalwarmingonsensoryecol-ogyofrocklizards:Increasedtemperaturesaltertheefficacyofsexualchemicalsignals.Functional Ecology,27,1332–1340.

Martín,J.,&López,P.(2014).Pheromonesandotherchemicalcommuni-cation inanimals. InJ.L.Rheubert,D.S.Siegel,&S.E.Trauth (Eds.),

14  |    Functional Ecology BAECKENS Et Al.

Reproductive biology and phylogeny of lizards and tuatara (pp. 43–77).BocaRaton,FL:CRCPress.

Martín,J.,&López,P.(2015).Condition-dependentchemosignalsinrepro-ductivebehavioroflizards.Hormones and Behavior,68,14–24.

Martín,J.,López,P.,Garrido,M.,Pérez-Cembranos,A.,&Pérez-Mellado,V. (2013). Inter-islandvariation in femoral secretions of theBaleariclizard,Podarcis lilfordi(Lacertidae).Biochemical Systematics and Ecology,50,121–128.

Martín, J., Moreira, P. L., & López, P. (2007). Status-signalling chemicalbadgesinmaleIberianrocklizards.Functional Ecology,21,568–576.

Martín,J.,Ortega,J.,&López,P. (2013). Lipophilic compounds in femo-ralsecretionsofmalecollaredlizards,Crotaphytus bicinctores(Iguania,Crotaphytidae).Biochemical Systematics and Ecology,47,5–10.

Martín,J.,Ortega,J.,&López,P.(2015).Interpopulationalvariationsinsex-ualchemicalsignalsofIberianwalllizardsmayallowmaximizingsignalefficiencyunderdifferentclimaticconditions.PLoS ONE,10,e0131492.

Martins,E.P.,&Lamont,J.(1998).Estimatingancestralstatesofacommu-nicativedisplay:AcomparativestudyofCyclura rock iguanas.Animal Behavior,55,1685–1706.

Martins,E.P.,Ord,T.J.,Slaven,J.,Wright,J.L.,&Housworth,E.A.(2006).Individual, sexual, seasonal, and temporalvariation in theamountofsagebrushlizardscentmarks.Journal of Chemical Ecology,32,881–893.

Mason,R.T.,&Parker,M.R.(2010).Socialbehaviorandpheromonalcommu-nicationinreptiles.Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology,196,729–749.

Mayer,W.,&Pavlicev,M.(2007).ThephylogenyofthefamilyLacertidae(Reptilia)basedonnuclearDNAsequences:ConvergentadaptationstoaridhabitatswithinthesubfamilyEremiainae.Molecular Phylogenetics and Evolution,44,1155–1163.

Mayerl,C.,Baeckens,S.,&VanDamme,R. (2015).Evolutionandroleofthe follicular epidermal gland system in non-ophidian squamates.Amphibia- Reptilia,36,185–206.

McDonough,L.M.,Brown,D.F.,&Aller,W.C.(1989).Insectsexphero-mones–Effectoftemperatureonevaporationratesofacetatesfromrubbersepta.Journal of Chemical Ecology,15,779–790.

Mendes,J.,Harris,J.D.,Carranza,S.,&Salvi,D.(2016).Evaluatingthephy-logeneticsignallimitfrommitogenomes,slowevolvingnucleargenes,and the concatenation approach.New insights into the Lacertini ra-diationusingfastevolvingnucleargenesandspeciestrees.Molecular Phylogenetics and Evolution,100,254–267.

Miles,D.B.,&Ricklefs,R.(1984).Thecorrelationbetweenecologyandmor-phologyindeciduousforestpasserinebirds.Ecology,65,629–1640.

Miller,J.R.,Baker,T.C.,Carde,R.T.,&Roelofs,W.L.(1976).Reinvestigationofoakleafrollersexpheromonecomponentsandthehypothesisthattheyvarywithdiet.Science,192,140–143.

Møller,A.P.,&Pomiankowski,A.(1993).Whyhavebirdsgotmultiplesex-ualornaments?Behavioral Ecology and Sociobiology,32,167–176.

Moorcroft, P., & Lewis, M. (2006). Mechanistic home range analysis. Princeton,NJ:PrincetonUniversityPress.

Morton, E. S. (1975). Ecological sources of selection on avian sounds.American Naturalist,109,17–34.

Müller-Schwarze, D. (2006). Chemical ecology of vertebrates. Cambridge,UK:CambridgeUniversityPress.

Müller-Schwarze, D., & Silverstein, R. M. (1980). Chemical signals: Vertebrates and aquatic invertebrates.NewYork,NY:PlenumPress.

Ng,J.,Landeen,E.L.,Logsdon,R.M.,&Glor,R.E.(2013).Correlationbe-tweenanolislizarddewlapphenotypeandenvironmentalvariationin-dicatesadaptivedivergenceofasignal important tosexualselectionandspeciesrecognition.Evolution,67,573–582.

Nicholls,J.A.,&Goldizen,A.W.(2006).Habitattypeanddensityinfluencevocalsignaldesign insatinbowerbirds.Journal of Animal Ecology,75,549–558.

Ord,T.J.,&Martins,E.P.(2006).Tracingtheoriginsofsignaldiversityinanole lizards: Phylogenetic approaches to inferring the evolution ofcomplexbehaviour.Animal Behavior,71,1411–1429.

Oufiero,C.E.,Gartner,G.E.A.,Adolph,S.C.,&Garland,T.(2011).LatitudinalandclimaticvariationinbodysizeanddorsalscalecountsinSceloporuslizards:Aphylogeneticperspective.Evolution,65,3590–3607.

Pagel,M. (1999). Inferring thehistoricalpatternsofbiologicalevolution.Nature,401,877–884.

Paradis,E.,Claude,J.,&Strimmer,K.(2004).APE:Analysesofphylogenet-icsandevolutioninRlanguage.Bioinformatics,20,289–290.

Pavlicev,M.,&Mayer,W.(2009).FastradiationofthesubfamilyLacertinae(Reptilia: Lacertidae): History or methodical artefact? Molecular Phylogenetics and Evolution,52,727–734.

Perry,G.,Lampl,I.,Lerner,A.,Rothenstein,D.,Shani,E.,&Sivan,N.(1990).Foragingmode in lacertid lizadrs:Variationandcorrelates.Amphibia- Reptilia,11,373–384.

Pianka,E.R.,Huey,R.B.,&Lawlor,L.R.(1979).Nichesegregationindes-ertlizards.InD.J.Horn,D.J.Mitchell,&G.R.Stairs(Eds.),Analysis of ecological systems(pp.67–115).Columbus,OH:OhioStateUniversityPress.

Picq, S., Alda, F., Bermingham, E., & Krahe, R. (2016). Drift-driven evo-lution of electric signals in a Neotropical knifefish. Evolution, 70, 2134–2144.

Plath,M.,Parzefall,J.,Körner,K.E.,&Schlupp,I.(2004).Sexualselectionindarkness?Femalematingpreferencesinsurface-andcave-dwellingAtlantic mollies, Poecilia mexicana (Poeciliidae, Teleostei). Behavioral Ecology and Sociobiology,55,596–601.

Pruett,J.A.,Zúñiga-Vega,J.J.,Campos,S.M.,Soini,H.A.,Novotny,M.V.,Vital-García, C.,…Hews,D.K. (2016). Evolutionary interactions be-tweenvisualandchemicalsignals:Chemosignalscompensatefortheloss of a visual signal inmale Sceloporus lizards. Journal of Chemical Ecology,42,1164–1174.

Pyron,R.,Burbrink,F.,&Wiens,J.(2013).Aphylogenyandrevisedclassifi-cationofSquamata,including4161speciesoflizardsandsnakes.BMC Evolutionary Biology,13,1–53.

RCoreTeam.(2013).R: A language and environment for statistical computing. Vienna,Austria:RFoundationforStatisticalComputing.Retrivedfromhttp://www.R-project.org

Rantala, M. J., Kortet, R., Kotiaho, J. S., Vainikka, A., & Suhonen, J.(2003). Condition dependence of pheromones and immune func-tion in the grain beetle Tenebrio molitor. Functional Ecology, 17, 534–540.

Rasmussen,L.E.,Lee,T.D.,Zhang,A.,Roelofs,W.L.,&Daves,G.D.Jr.(1997).Purification,identification,concentrationandbioactivityof(Z)-7-dodecen-1-ylacetate:SexpheromoneofthefemaleAsianelephant, Elephas maximus. Chemical Senses,22,417–437.

Regnier,F.E.,&Goodwin,M.(1977).Onthechemicalandenvironmentalmodulationofpheromonereleasefromvertebratescentmarks.InD.Müller-Schwarze,&M.M.Mozell(Eds.),Chemical signals in vertebrates (pp.115–149).London:PlenumPress.

Revell,L.J.(2012).phytools:AnRpackageforphylogeneticcomparativebi-ology(andotherthings).Methods in Ecology and Evolution,3,217–223.

Revell,L.J.(2013).Twonewgraphicalmethodsformappingtraitevolutiononphylogenies.Methods in Ecology and Evolution,4,754–759.

Revell, L.J.,&Harrison,A.S. (2008).PCCA:Aprogram forphylogeneticcanonicalcorrelationanalysis.Bioinformatics,24,1018–1020.

Rouault, J. D.,Marican, C.,Wicker-Thomas, C., & Jallon, J.M. (2004).Relations between cuticular hydrocarbon (HC) polymorphism, re-sistance against desiccation and breeding temperature; a modelforHCevolution inD. melanogaster and D. simulans. Genetica,120,195–212.

Rundle, H. D., Chenoweth, S. F., Doughty, P., & Blows, M. W. (2005).Divergentselectionandtheevolutionofsignaltraitsandmatingpref-erences.PLoS Biology,3,1988–1995.

Runemark,A.,Gabirot,M.,&Svensson,E.I.(2011).Populationdivergenceinchemicalsignalsandthepotentialforprematingisolationbetweenislet- and mainland populations of the Skyros wall lizard (Podarcis gaigeae).Journal of Evolutionary Biology,24,795–809.

     |  15Functional EcologyBAECKENS Et Al.

Saxena,P.,Patel,J.C.,&Joshipura,M.H.(2013).Predictionofvaporpres-sureoffattyacidmethylesters.Procedia Eng.,51,403–408.

Searcy,W.A.,&Nowicki,S.(2005).The evolution of animal communication: Reliability and deception in signalling systems.Princeton,NJ:PrincetonUniversityPress.

Smith,H.M.(1946).Handbook of lizards: Lizards of the United States and of Canada.NewYork,NY:CornellUniversityPress.

Steiger,S.,Schmitt,T.,&Schaefer,H.M. (2011).Theoriginanddynamicevolution of chemical information transfer. Proceedings of the Royal Society of London. Series B, Biological Sciences,278,970–979.

Stevens,M.(2013).Sensory ecology, behaviour, and evolution.Oxford,UK:OxfordUniversityPress.

Symonds,M.R.E.,&Elgar,M.A.(2008).Theevolutionofpheromonediver-sity.Trends in Ecology & Evolution,23,220–228.

Thomas,M.L. (2011).Detectionof femalematingstatususingchemicalsignalsandcues.Biological Reviews,86,1–13.

Tibbetts,E.A.,Mullen,S.P.,&Dale,J.(2017).Signalfunctiondrivespheno-typicandgeneticdiversity:Theeffectsofsignallingindividualidentity,qualityorbehavioural strategy.Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences.,372,20160347.

Tieleman,B.I.,Williams,J.B.,&Bloomer,P.(2003).Adaptationofmetabo-lismandevaporativewaterlossalonganariditygradient.Proceedings of the Royal Society B- Biological Sciences,270,207–214.

Tobias,M.,Evans,B.J.,&Kelley,D.B.(2011).EvolutionofadvertisementcallsinAfricanclawedfrogs.Behaviour,148,519–549.

Uetz, P. (ed.) (2017).TheReptileDatabase, http://www.reptile-database.org.AccessedMay6,2017.

VanOudenhove,L.,Billoir,E.,Boulay,R.,Bernstein,C.,&Cerdá,X.(2011).Temperaturelimitstrailfollowingbehaviourthroughpheromonedecayinants.Naturwissenschaften,98,1009–1017.

VanOudenhove,L.,Boulay,R.,Lenoir,A.,Bernstein,C.,&Cerdá,X.(2012).Substrate temperatureconstrains recruitmentand trail followingbe-haviorinAnts.Journal of Chemical Ecology,38,802–809.

VanderWall,S.B.(1998).Foragingsuccessofgranivorousrodents:Effectsofvariationinseedandsoilwateronolfaction.Ecology,79,233–241.

Vervust,B.(2011).Lizards on islands: a model system for studying fast pheno-typic change. (Doctoral thesis).LaboratoryofFunctionalMorphology,UniversityofAntwerp,Belgium.

Vinson,S.B.(1972).CourtshipbehaviorandevidenceforasexpheromoneintheparasitoidCampoletis sonorensis(Hymenoptera:Ichneumonidae).Environmental Entomology,1,409–414.

Weber,M.,Mitko,L.,Eltz,T.,&Ramirez,S.(2016).Macroevolutionofper-fumesignallinginorchidbees.Ecology Letters,19,1314–1323.

Weldon,P.J.,Flachsbarth,B.,&Schulz,S.(2008).Naturalproductsfromtheintegumentofnonavianreptiles.Natural Products Reports,25,738–56.

Wilder,S.M.,DeVito,J.,Persons,M.H.,&Rypstra,A.L.(2005).Theeffectsofmoistureandheatontheefficacyofchemicalcuesusedinpreda-tordetectionbythewolfspiderPardosa milvina (Araneae,Lycosidae).Journal of Arachnology,33,857–861.

Wyatt,T.D.(2010).Pheromonesandsignaturemixtures:Definingspecies-wide signals andvariable cues for identity inboth invertebratesandvertebrates. Journal of Comparative Physiology. A, Neuroethology, Sensory, Neural, and Behavioral Physiology,196,685–700.

Wyatt,T.D.(2014).Pheromones and animal behaviour. Chemical signals and signatures.Cambridge,UK:CambridgeUniversityPress.

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How to cite this article:BaeckensS,MartínJ,García-RoaR,etal.Environmentalconditionsshapethechemicalsignaldesignoflizards.Funct Ecol. 2017;00:1–15. https://doi.org/10.1111/1365-2435.12984

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