Sexual dimorphism and population differentiation in the Chilean Neotropical moth Macaria mirthae (Lepidoptera, Geometridae): a wing geometric morphometric example

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Hugo

A. Benítez

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_Héctor

_A.

_Vargas

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a_Universidad_de_Tarapacá,_Facultad_de_Ciencias_{Agronómicas,}_Departamento_de_Recursos_Ambientales,_Arica,_Chile b_Cambridge_University,_Museum_of_Zoology,_Cambridge,_United_Kingdom

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Articlehistory: Received16April2017 Accepted20June2017 Availableonline5July2017 AssociateEditor:LiviaPinheiro Keywords: Wingshape Sexualdimorphism Geometridae Azapavalley Chacavalley

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Sexualshapedimorphismisthedifferentiationofmaleandfemaleorganismsbasedontheirshape variation;thisdefinitionwasproposedfortheuseofgeometricmorphometricsanalysiswherethe geometric features of the shape are analyzed without the influence of the size. Macaria mirthae (Lepidoptera:Geometridae)isamoththatinhabitsdifferentvalleysinthenorthofChileprincipally associatedtoAcaciamacracanthaandlatelyLeucaenaleucocephalabothtreesoftheFabaceaefamily. TheSexualdimorphismwasanalyzedinthisspeciesinordertocorroboratestudiesontheuseofwing asasexualdifferentiationtrait,andspecificinfluenceoflocalitieswasalsoevaluated.Aclearshape variationwasfoundwherethemalewingsaremorecontractedcomparedtofemalewings.Aclimate influenceisalsosuggestedthatcoulddifferentiatethewingshapefromtheindividualsthatinhabittwo differentvalleysintheneotropicalregionofthenorthofChile.Thisresearchsupportspreviousstudies identifyingaclearSexualshapedimorphisminthewing,asaselectedtrait,suggestingthatoviposition andmalecompetitionofthisgroupofmothsisreflectedintheirwings.Thesedifferencesraisethe questionwhetherSexualshapedimorphismcanbemodulatedbynaturalselection.

Introduction

Inthelastcenturies,comparativebiologistshavebeenfocused instudiesofmorphologicalvariationwherethemostusedmethod isthecomparisonofphenotypicvariation.Oneimportantsourceof variationisthesexualdimorphismwithitscorresponding classiﬁ-cationandunderstanding(Andersson,1994).

Sexualdifferencesinmorphologicalcharactersareacommon phenomenonin many animaltaxa, and theirmostconspicuous aspectisbodysize(Andersson,1994;Fairbairn,1997;Tederand Tammaru,2005;StillwellandDavidowitz,2010).Nevertheless,size variationisnotenoughtoidentifyorcharacterizethesexual dimor-phism(sexualsizedimorphism(SSD))(Benitezetal.,2013).

InLepidoptera,Benítezetal.(2011)identiﬁedthrough geomet-ricmorphometricsapproacha pattern ofsexualdimorphismin externalmorphologyusingtheshapeoftheorganism(sexualshape dimorphism,SShD).Theabove-mentionedauthorsfoundthatthis toolwasusefultodifferentiatemalesandfemalesofageometrid

∗ Correspondingauthor.

E-mail:hbenitez@uta.cl(H.A.Benítez).

moth(Synneuriasp.)usingonlythewings(thisanalysisusesonly theshape,excludingthesize).

Additionally,anotherfactorthatcontributestothe morphologi-calvariationinorganismsisthevariationontheclimaticcondition, wheresamespeciesmodifytheirmorphologyforabetter adap-tation totheenvironment (adaptation of thephenotypeto the environment).

TheAtacamaDesertisthemostariddesertoftheworld(Clarke, 2006).InthenorthernmostpartofChile,itiscrossedbyseveral narrow,sub-parallel,transversevalleysextendingfromthe west-ernslopesoftheAndesMountainRangetothePaciﬁcOcean.These valleysarerecognizedtobeamongthemostimportantplacesfor thebiodiversityoftheAtacamaDesert(LuebertandPliscoff,2006; Estadesetal.,2007).However,contiguousvalleysareseparated bya widerareaofabsolutedesertmainlydevoidofvegetation, whichcouldworkaseffectivebarriersforlowdispersalof orga-nismsinhabitingdifferentvalleys.Itisexpectedthat,depending onthedispersalcapacities,thiseffectcouldbeparticularlyserious forsomespecializedphytophagousinsectsassociatedwithplants restrictedtothebottomofthevalleys.

Studiesonmorphological variationduetoclimaticcondition aremany;nevertheless,therearefewthatcombineLepidoptera http://dx.doi.org/10.1016/j.rbe.2017.06.003

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Fig.1.GraphicalschemeofthelocationofthetwoValleysinAtacamaDesertinthe northofChile.

andgeometricmorphometrics.Sanzanaetal.(2013)usethewing shapeofAucacoctei(Lepidoptera: Nymphalidae)toidentifythe wingshapeadaptationintheirdistributionalcline,suggestingthe presenceofecologicalrulesregardingthemorphology(Converse BergmanRule).Benitezetal.(2013)studiedtheisolatedeffectof islandonthemorphologyoftenebrionidbeetlesandtheyfound thatindividualsofthesamespeciescanvarytheirgeometricbody shaperespondingtodifferentclimaticconditions.

Theaimofthisstudywastoevaluatethewingsexualshape dimorphismandthewingmorphologicalvariationontwo contigu-ouscoastalvalleysoftheAtacamaDesertintheNeotropicalmoth Macariamirthae.

Materialsandmethods

Studysites

Thestudywasundertakenin twocoastalvalleysofthe Ata-cama Desert in northern Chile: the Azapa Valley (18◦3112S, 70◦1041W)andtheChacaValley(18◦4856S,70◦0907W),both belongingtotheAricaProvince(Fig.1).Thisareaischaracterized byatropicalhyperdeserticbioclimatewithmeanannual precip-itationabout0mm(LuebertandPliscoff,2006).Asaresultofthe summerrainsthatoccurinthehighplateauandthewesternslopes oftheAndes,thetwovalleysharboursemiperennialstreamswhich enablethepresenceofnativevegetation.

Samplingandrearing

Larvae(onlylast instar)of M.mirthaewerecollectedin Jan-uary2013onA.macracanthaintheAzapaandChacavalleys.AsM. mirthaeisabletouseadditionalFabaceaehostplants(Vargasand Mundaca,2014)inthetwostudysites,andsincewing morphol-ogycanbeaffectedbydifferentlarvalhostinthismoth(Benítez etal.,2015),surveysforlarvaewererestrictedtoisolatedtreesof A.macracanthacollectedbycanningtrees.Thecollectedlarvaewere placedintoindividualplasticvialswithtowelpaperonthebottom. Thevialswerekeptinthelaboratoryatroomtemperatureandwere cleaneddaily.FreshleavesofA.macracanthawereaddeddailyuntil

Fig.2.Benítezetal.(2015),representationofthe13morphologicallandmarks identiﬁedintheforewingsofMacariamirthae.

thelarvaeﬁnishedfeeding.Pupationoccurredbetweenthetowel paperpiecesatthebottomofthevial.Theadultsobtainedfrom thepupaewerekilledwithethylacetate24hoursafteremergence. Wingswereremovedfromthebodyoftheadultsandthen com-pressedbetweentwomicroscopeslidesforthesubsequentimage capture.

Geometricmorphometricsanalysis

Following the procedure of Benítez et al. (2015), from 230 individualsrightandleftforewingswerephotographedwitha dig-italcameraMicropublisher3.3RTV-QImaging(Q-imaging,Canada) attachedto a stereoscopic microscopeOlympus SZ61. Thirteen landmarksweredigitized,usingTpsDig2.17software(Rohlf,2013). Onallwings(bothleftandright)upperwingsidelandmarkswere identiﬁedaccordingtoexternalanatomyandwingsveinpattern (Fig.2)(Benítezetal.,2015).

TheshapeinformationwasextractedusingaProcustesﬁt(Rohlf andSlice, 1990;Drydenand Mardia,1998)and theexploratory shapevariationbetweensexeswasanalyzedusingprincipal com-ponentsanalysis(PCA).Inordertoevaluatethelocationeffecton themorphology,adiscriminantanalysiswasperformedusingthe covariancematrixofshape.APCAofthepoolbysexcovariance matrixwasperformedtoevaluateiflocalitieseffectwasnot con-fusedwiththeanalyzedsexualdimorphism.Finally,amultivariate regressionofsizeonshapewasperformedinordertoanalyzeifthe sizehasaninﬂuenceonthesexualdimorphism(Monteiro,1999). Allthemorphometricsanalysiswereperformedusingthesoftware MorphoJ1.06d(Klingenberg,2011).

Results

The geometrical attributes of the wing shape were visible usingthePCA,theaccumulatedvariationofshapein theﬁrst3 principalcomponentsreached61.4%(PC1=34.12%;PC2=16.20%; PC3=11.08%).Thescatterplotofvariation(simulationofthe mor-phospaceinaPCA)showsacleardifferentiationbetweenmales (blackdots)andfemales(greydots)(Fig.3).Thereisanoticeable SShDbythecontractionofthetipandradialveinsofthewingin maleswerethemeanvectorofmovementonthelandmarksisto theleftdirection.Onthecontrary,femalewingshapeexpandsthe tipsectionofthewingbutcontractsthelandmarks1,2,3,5ofthe wings.ThePCAtoidentifyvariationbetweenlocalitieswasnotable toﬁnddifferencesfromthepoolbysexcovariancematrix; how-ever,thediscriminantanalysisshowsanoticeabledifferentiation

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Fig.3. PCAanalysisofthesexualshapedimorphismofMacariamirthae:theﬁgureshowstheﬁrsttwoorthogonalPCcomponents’axesthatrepresenttheshapespace dimensions,alsoadecompositionofshapevariationbetweensexes.*Eachpointrepresentsadifferentshape.

Fig.4.DiscriminantanalysisofthewingshapebetweenthetwoAzapa(greybars)andChacavalley(whitebars)attheAtacamaDesert. betweenthewingshapeofAzapavalleymothsandthewingof

theindividualsfromChacavalleymoths(Fig.4).Thewingshapeof Azapamothsshowsamorevariableshapewithmoredisorganized movementofthevectoratthedifferentsectionsofthewing,in con-trasttothewingsfromtheindividualsofChaca.Theseshape differ-enceswerestatisticallysignificant(p-valuesforpermutationtests (1000permutationruns):<0.0001).Finally,themultivariate regres-sionoftheshapeoncentroidsize(Fig.5)showsthatthesizehasa significantbutsmallinfluence(8.4%)ontheshapedifferentiation.

Discussion

Analysisofgeometricmorphometricscanconﬁrmaclear dif-ferenceinboth:sexualshapedimorphismandlocalities.Thetool

of geometric morphometrics is a good method to analyze the sexualdifferences in thewings ofM. mirthae.Wing shape was welldistinguishedinthetwovalleysintheextremenorthregion ofChile.The Scatterplotof thePCA(Fig.3)showsthat thePC1 differentiatesthegroupofmalesandfemales.Thelandmark dif-ferencesdetectedattheSShDwereprincipallylocatedinthebase oftheradialveins,keytraitstodistinguishdifferentwing mor-photypesbetween sexes.Benítezet al.(2011) explainthat this morphological changeontheveins couldbedue todispersion, migrationandsexualselection.Additionally,inmalesitcouldbe theresultofthenuptialﬂight,territorialityandsexualselection, andinfemales,primarilyduetotheirﬂightbehaviourwhile search-ing for hostplants to lay eggs(Dockx, 2007; Johansson et al., 2009).

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Fig.5.MultivariateregressionofthewingshapeonthewingcentroidsizeofMacariamirthae.Greypointsrepresentfemalewingsandblackpointsrepresentmalewings. Thishypothesiscouldworkforinsectsincasetheirmalesexual

behaviourwasmorepronouncedthaninfemales,conditionthat couldforcesexualselection(Thornhill,1976;SavalliandFox,1998; Fairbairnetal.,2007).Nevertheless,morestudiesareneededonthis topicforM.mirthae.

Theobserveddifferencesinthetwovalleypopulationscouldbe causedbyapossibleisolationofthespecies(30kmofdesert)from thepopulations(Fig.4).Nevertheless,morestudiesareneededin ordertoclarifyifthemodiﬁcationsbetweenvalleyshaveagenetic impactor due tolocal adaptation(Cepeda-Pizarroet al., 2005;

Valdiviaetal.,2011;Benítezetal.,2014).Benítezetal.(2014)found similarshapeadaptationinnon-wingedbeetle(Praocisspinolai). Also,morphologicalplasticitycouldbeobservedininsectsat mul-tipletraits,causedbytheinﬂuenceofdifferenthosts(Mozaffarian etal.,2007;Sotoetal.,2008)orclinallatitudinalandaltitudinal effects(OutomuroandJohansson,2011;Sanzanaetal.,2013).

The multivariate regression of the shape and centroid size (Fig.5)wasusedtotesttheinfluenceofcentroidsizeontheshape. Itiswellknownthatininsectthesizeisoneofthemost impor-tantattributesinsexualdimorphism(Andersson,1994;Fairbairn, 1997;TederandTammaru,2005;StillwellandDavidowitz,2010). However,forspecifictraitsthisruleisnolongereffective,since theresultsshown thatcentroid sizeisnot a significanttraitto differentiatemalesfromfemales.

ForM.mirthaemorphologicalvariationhasbeenfoundatthe wingsduetohostinﬂuence,wheretheindividualsfromthenorth (Azapa)havebeenforcedtochangetheirdietbehaviourfroma uniquehost plant(Acacia macracantha)to include intheir diet amoreabundantplant(Leucaenaleucocephala).Thischangehas affectedthe symmetric pattern of thewings in populations by developmentalinstabilityasaconsequenceofstochasticprocesses andenvironmentalstress(Benítezetal.,2015).

Finally,thisresearchcorroboratesthestudiesofBenítezetal. (2011)identifyinga clearSShD,suggesting that theoviposition behaviourandmalecompetitionofthismothisreflectedintheir wings.Thisstudyalsoconfirmstheefficiencyofthegeometric mor-phometricstoolandraisesthequestionofwhetherSShDmaybe modulatedbynaturalselection.Inaddition,GMtoolsenabledus todetectsubtlemorphological differentiationof isolated popu-lations of a specialized phytophagous. Accordingly, geometric morphometrictoolscanbehelpfultostudythepatternsof sex-ualdimorphismand morphologicaldifferentiationof organisms inhabitingtheextremeenvironmentsoftheAtacamaDesert.

Conﬂictsofinterest

Theauthorsdeclarenoconﬂictsofinterest.

Acknowledgements

TheauthorswouldliketothankstoMsMariaRaquelLazode laVega,fortheproofreadingofthearticleandtotheDr.Wilson Huancatoprovidethefacilitiesonthemicro-labpictures.Financial supportwasobtainedfromprojectDGI-9719-17,fromUniversidad deTarapacá.

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