Sexual coevolution of spermatophore envelopes and female genital traits in butterflies: Evidence of male coercion?

0.57 MB·English

by  Sanchez, Victor

Sexual coevolution of spermatophore
envelopes and female genital traits in
butterflies: Evidence of male coercion?
V(cid:237)ctorSÆnchez1 andCarlosCordero2
1PosgradoenCienciasBiol(cid:243)gicas,InstitutodeEcolog(cid:237)a,UniversidadNacionalAut(cid:243)nomadeMØxico,MØxico,
DistritoFederal,Mexico
2DepartamentodeEcolog(cid:237)aEvolutiva,InstitutodeEcolog(cid:237)a,UniversidadNacionalAut(cid:243)nomadeMØxico,
DistritoFederal,Mexico
ABSTRACT
Signaaresclerotizedstructureslocatedontheinnerwallofthecorpusbursaoffe-
maleLepidopterawhosemainfunctionistearingopenspermatophores.Thesexu-
allyantagonisticcoevolution(SAC)hypothesisproposesthatthethicknessofsper-
matophoreenvelopeshasdriventheevolutionofthefemalessigna;thisideaisbased
inthefactthatinmanylepidopteransfemalesexualreceptivityisatleastpartially
controlledbythevolumeofejaculateremaininginthecorpusbursa.Accordingto
theSAChypothesis,malesevolvedthickspermatophoreenvelopestodelaythepost-
matingrecoveryoffemalesexualreceptivitythusreducingspermcompetition;in
response,femalesevolvedsignaforbreakingspermatophoreenvelopesfaster,gain-
ingaccesstotheresourcescontainedinthemandreducingtheirintermatingin-
tervals;theevolutionofsigna,inturn,favoredtheevolutionofeventhickersper-
matophoreenvelopes,andsoon.WetestedtwopredictionsoftheSAChypothe-
siswithcomparativedataonthethicknessofspermatophoreenvelopesofeleven
speciesofHeliconiinaebutterflies.Thefirstpredictionisthatthespermatophore
envelopesofpolyandrousspecieswithsignawillbethickerthanthoseofmonan-
drousspecieswithoutsigna.Inagreementwiththisprediction,wefoundthatthe
spermatophoreenvelopesofapolyandrousHeliconiusspecieswithsignaarethicker
Submitted 27October2013 thanthoseoftwomonandrousHeliconiusspecieswithoutsigna.Thesecondpre-
Accepted 29December2013
dictionisthatinsomespecieswithsignamalescouldenforcemonandryinfemales
Published 30January2014
byevolving‘‘verythick’’spermatophoreenvelopes,inthesespecieswepredictthat
Correspondingauthor
theirspermatophoreenvelopeswillbethickerthanthoseoftheircloserpolyandrous
CarlosCordero,
[email protected] relativeswithsigna.Inagreementwiththisprediction,wefoundthatintwooutof
Academiceditor threecomparisons,spermatophoreenvelopesofmonandrousspecieswithsigna
DezeneHuber havethickerspermatophoreenvelopesthantheircloserpolyandrousrelativeswith
AdditionalInformationand signa.Thus,ourresultssupporttheideathatselectivepressuresarisingfromsexu-
Declarationscanbefoundon allyantagonisticinteractionshavebeenimportantintheevolutionofspermatophore
page10
envelopes,femalesignaandfemalematingpatterns.
DOI10.7717/peerj.247
Copyright
Subjects AnimalBehavior,Entomology,EvolutionaryStudies,Zoology
2014SÆnchezetal.
Keywords Sexualcoevolution,Sexualselection,SexualConflict,Femalegenitalia,Spermatophore,Signa,
Distributedunder Lepidoptera,Heliconiinae,Sexualcoevolution
Creative-CommonsCC-BY3.0
OPENACCESS
HowtocitethisarticleSÆnchezetal.(2014),Sexualcoevolutionofspermatophoreenvelopesandfemalegenitaltraitsinbutterflies:
Evidenceofmalecoercion?.PeerJ2:e247;DOI10.7717/peerj.247
INTRODUCTION
Duringsexualinteractionsmalesandfemalesexertselectionpressuresontheopposite
sexthatcanproducereciprocaladaptationsinaprocessknownassexualcoevolution
(Parker,1979;Eberhard,1985,1996;Holland&Rice,1998).Thereisincreasingevidence
thatsexualcoevolutionisresponsiblefortheevolutionofmanystructuralandfunctional
aspectsofanimalgenitalia(Eberhard,1985,1996,2010;Hosken&Stockley,2004;Arnqvist
&Rowe,2005;Minder,Hosken&Ward,2005;Brennanetal.,2007;SÆnchez,
HernÆndez-Baæos&Cordero,2011;Breed,Leigh&Speight,2013;Burns,Hedin&Shultz,
2013;Yassin&Orgogozo,2013).Forexample,inspeciesinwhichfemalesincreasetheir
fitnessbymatingwithmultiplemates,malescouldevolvegenitalstructuresfordamaging
femalegenitaliaifthisdamagedecreasesfemalematingrates;thesestructures,inturn,
couldselectforprotectivegenitalstructuresinfemales.IntheDrosophilamelanogaster
speciessubgroupevidenceindicatesthatfemaleshavecoevolvedgenitalstructuresthat
protectthemfromdamagebymalegenitalstructures(Yassin&Orgogozo,2013).Inother
species,alsoexhibitingadaptivepolyandry,femalescouldevolvegenitaltraitsthatallow
themtodiscriminateamongmalesofdifferentqualityduringcopulation;thesetraits
couldselectforelaboratemaleintromittentgenitaliaforinternalstimulationofthe
females(Eberhard,1985).Evidencesuggeststhattheextremelycomplexvaginal
morphologyofwaterfowlspeciescoevolvedwiththelongandcomplexmalephallusasa
crypticchoicemechanism(Brennanetal.,2007).
IntheparticularcaseofLepidoptera,inapreviouspaperwepresentedcomparative
evidencesupportingthehypothesisthatthesclerotizedstructurescalledsigna,presentin
theinnergenitaliaoffemalesfrommanyspecies,areaproductofantagonistic
coevolutionwithmales(SÆnchez,HernÆndez-Baæos&Cordero,2011).Thesignaare
locatedontheinnerwallofthefemale’scorpusbursa(cid:22)thebag-likereceptaclewhere
malesdepositaspermatophoreduringcopulation(cid:22)andareusedforbreakingthe
spermatophoreenvelopeandgainaccesstoitscontents(Hinton,1964;Galicia,SÆnchez&
Cordero,2008;Lincango,FernÆndez&Baixeras,2013).Oursexuallyantagonistic
coevolution(SAC)hypothesisproposesthat,sinceinmanypolyandrousLepidopterathe
lengthoftimeafemaleremainssexuallyunreceptiveaftermatingisdirectlyrelatedtothe
amountofejaculateremaininginhercorpusbursa(Sugawara,1979;Drummond,1984;
Wiklund,2003;Wedell,2005),spermcompetitionselectsformalesthattransfer
spermatophoreswiththickenvelopesthattakemoretimetobebrokenandthusdelay
femalerematingbeyondheroptimumtime(Drummond,1984;Cordero,2005;Fig.1).
Thickspermatophoreenvelopes,inturn,selectforsignathatallowfemalesfaster
breakingoftheenvelopes,thusreducingintermatingintervals(Cordero,2005;Fig.1).
OurpreviouscomparativeanalysissupportedthepredictionfromtheSAChypothesis
thatsignatendtobepresentmainlyinpolyandrousspecies,andsuggestedthatpolyandry
andsignaareplesiomorphicintheLepidoptera(SÆnchez,HernÆndez-Baæos&Cordero,
2011).TheSAChypothesisalsopredictsthatwhenmonandryisselectedforinfemales,
theresultingdisappearanceofspermcompetitionfavorstheevolutionofthinner
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 2/12
Figure1 Sexuallyantagonisticcoevolutionhypothesisoftheevolutionofspermatophoreenvelope
thicknessandsignainLepidoptera.SchematicrepresentationoftheSexuallyAntagonisticCoevolution
hypothesisforthecoevolutionofspermatophoreenvelopesandsignainLepidoptera.Arrowsrepresent
selectivepressures.
spermatophoreenvelopes(becausetheyarelessexpensivetoproduce)and,in
consequence,thelossofsignainfemales.Ourpreviousstudyalsofoundsupportforthis
prediction,becauseinseveralgroups(includingthepupalmatingHeliconiusspecies)the
evolutionofmonandrywasaccompaniedbythelossofsigna(SÆnchez,HernÆndez-Baæos
&Cordero,2011).However,insomecasesmonandrycouldbeimposedbymaleson
females(i.e.,couldbemaladaptiveforfemales)byevolvingeventhickerspermatophore
envelopesinresponsetotheevolutionofsigna(ananalogouseffecthasbeenproposedfor
Heliconiusantiaphrodisiacs;Estradaetal.,2011).Inthiscase,theSAChypothesispredicts
theevolutionofthickerspermatophoreenvelopesinmonandrousspecieswithsignathan
inpolyandrousspecies.
PredictionsoftheSAChypothesisontherelationshipbetweenthicknessofthe
spermatophoreenvelopeandpresenceofsignainspeciesdifferinginfemalemating
patternshavenotbeentested.Inthisrespect,theonlyrelevantobservationsweareaware
ofarethosereportedbyMatsumotoandSuzukiinapaperonmatingplugsinsixgenera
ofPapilionidae(Matsumoto&Suzuki,1995).Wehavediscussedthesedataindetailin
previouspublications(Cordero,2005;SÆnchez,HernÆndez-Baæos&Cordero,2011).
Briefly,MatsumotoandSuzuki’sresultssupportpredictionsoftheSAChypothesis:
monandrousgeneraarecharacterizedbyanabsenceofthickspermatophoreenvelopes
(‘‘capsule’’intheirterminology)andalackofsignum;moderatelypolyandrousspecies
havea‘‘relativelythick’’spermatophoreenvelopeanda‘‘small’’signum;whereasmore
polyandrousgenerahavea‘‘thick’’spermatophoreenvelopeandawelldevelopedsignum
(Matsumoto&Suzuki,1995).TheagreementofMatsumotoandSuzuki’sdatawiththe
SAChypothesisispersuasive,butstudiesspecificallydesignedtotestthepredicted
relationshipbetweenthethicknessofspermatophoreenvelopesandsignaarenecessary.
Inthisreport,weusedataonthethicknessofspermatophoreenvelopesofelevenspecies
ofbutterfliesvaryinginpresenceofsignaandinfemalematingpattern(Fig.2A)totest
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 3/12
twopredictionsoftheSAChypothesis.First,wetestedthepredictionthatspermatophore
envelopesofpolyandrousspecieswithsignaarethickerthanthoseofmonandrousspecies
withoutsigna(T1!T2inFig.1).Second,wetestedthepredictionthatspermatophore
envelopesofmonandrousspecieswithsignahavethickerspermatophoreenvelopesthan
theircloserpolyandrousrelativeswithsigna;asexplainedabove,therationalebehindthis
predictionisthatinmonandrousspecieswithsignamonandryisenforcedbymalesvia
the(co)evolutionof‘‘verythick’’spermatophoreenvelopes(T2!T4inFig.1).
MATERIALS AND METHODS
WecollectedfemalesfromelevenspeciesofthesubfamilyHeliconiinae(Nymphalidae)
(Luis-Mart(cid:237)nez,Llorente-Bousquets&Vargas-FernÆndez,2003;Table1);specimenswere
capturedunderascientificcollectorpermitgrantedtothesecondauthorbytheMexican
Secretar(cid:237)adeMedioAmbienteyRecursosNaturales(FAUT-0237).Thesespecieswere
selectedtotestthepredictionsmentionedintheintroductiononthebasisoffindings
fromourpreviousresearch(SÆnchez,HernÆndez-Baæos&Cordero,2011).Information
abouttheabsence/presenceofsignawasobtainedfromBrown(1981)andconfirmed
upondissection.Formostspecies,weusedpublisheddataaboutfemalematingpattern
estimatedfromspermatophorecountsinfieldcollectedfemales(Heliconiusspp.(Ehrlich
&Ehrlich,1978;Waltersetal.,2012);Eueidesspp.,Dryadulaphaetusa,Dryasiulia,
PhilaethriadiatonicaandDionejuno:(Ehrlich&Ehrlich,1978);Agraulisvanillae
(Drummond,1984);Dionemoneta:datafromfemalescollectedbyVSinthePedregalde
SanAngelecologicalpreserve,locatedinthemaincampusoftheUniversidadNacional
Aut(cid:243)nomadeMØxicoinsouthernMexicoCity,thesefemalesweredifferentfromthose
usedformeasuringthicknessofspermatophoreenvelopes).Mostfemaleswerecollected
indifferentlocationsinthestateofVeracruz,Mexico.Femaleswerenetted,euthanized,
andtheirabdomenspreservedinvialswith70%ethanoluntildissection.
Inthelaboratory,thecorpusbursaeweredissectedunderadissectionmicroscope
(OlympusSZH10)andonlycorpusbursaecontainingcompletespermatophoreswerecut
intransversalsectionsthatallowedusmeasuringthethicknessofspermatophore
envelopes.(Severalfemalesprovidednodatabecausetheydidnotcontain
spermatophoresorbecausethespermatophorestheycontainedwerepartiallyor
completelydigested.)Toobtaincrosssectionsofspermatophoreenvelopes,thecorpus
bursaecontainingintactspermatophoreswereprocessedinthefollowingsequence:(1)
theywereleftinBouinfixativesolutionfor24h;(2)theyweredehydratedin
progressivelyhigherconcentrationsofalcohol(from50%to100%,leavingthecorpus
⃝
bursae1hineachconcentration);(3)theywereleftina1:1mixtureofParaplastR tissue
⃝ ◦
embeddingmediaandHistoChoiceR clearingagentfor24hinanovenat60 C;(4)they
⃝ ◦
wereleftinParaplastR tissueembeddingmediafor24hinanovenat60 C;(5)blocksof
⃝
ParaplastR containingonecorpusbursawereelaborated;(6)thewholecorpusbursae
containingintactspermatophoreswerecuttransversallyin20(cid:22)mthicksectionswithan
advancedprecisionrotarymicrotome(MD00030);(7)thesectionswereplacedinglass
slides,stainedwithmethyleneblue,andpermanentpreparationsmadeusingCytoseal
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 4/12
Table1Descriptivestatisticsofspermatophoreenvelopethickness(mm)ofeachspermatophoremea-
sured.Eachrowcorrespondstoonespermatophoreofthespeciesindicatedinthefirstcolumn(totalsample:
11speciesand43spermatophores).ns:totalnumberofmeasurementsmadeinsectionsofeachindividual
spermatophore(inalmostallcasestherewerefourmeasurementspersection).Specieswithanasteriskare
polyandrous,alltheothersaremonandrous.
Species/Specimen ns Mean SD Median Min.(cid:21)Max.
1.Heliconiusismenius* 227 0.034 0.010 0.03 0.01(cid:21)0.06
2.H.ismenius* 157 0.035 0.012 0.03 0.01(cid:21)1.00
3.H.ismenius* 315 0.033 0.009 0.03 0.01(cid:21)0.07
4.H.ismenius* 106 0.034 0.010 0.03 0.01(cid:21)0.06
5.H.ismenius* 154 0.037 0.010 0.04 0.02(cid:21)0.06
1.Heliconiushortense 188 0.028 0.009 0.03 0.01(cid:21)0.05
2.H.hortense 127 0.027 0.009 0.03 0.01(cid:21)0.06
3.H.hortense 179 0.028 0.008 0.03 0.01(cid:21)0.05
4.H.hortense 78 0.028 0.009 0.03 0.01(cid:21)0.04
5.H.hortense 163 0.031 0.007 0.03 0.02(cid:21)0.05
1.Heliconiuscharithonia 187 0.021 0.007 0.02 0.01(cid:21)0.04
2.H.charithonia 55 0.024 0.005 0.02 0.02(cid:21)0.04
1.Eueidesaliphera* 123 0.039 0.009 0.04 0.02(cid:21)0.06
2.E.aliphera* 89 0.032 0.009 0.03 0.01(cid:21)0.06
3.E.aliphera* 102 0.036 0.008 0.04 0.02(cid:21)0.05
4.E.aliphera* 83 0.036 0.008 0.04 0.01(cid:21)0.05
1.Eueidesisabella 136 0.047 0.015 0.05 0.02(cid:21)0.09
2.E.isabella 147 0.049 0.014 0.05 0.02(cid:21)0.09
3.E.isabella 232 0.060 0.018 0.06 0.02(cid:21)0.12
4.E.isabella 147 0.054 0.012 0.05 0.03(cid:21)0.10
5.E.isabella 93 0.052 0.015 0.05 0.03(cid:21)0.09
6.E.isabella 209 0.052 0.015 0.05 0.03(cid:21)0.12
7.E.isabella 248 0.069 0.027 0.06 0.03(cid:21)0.16
1.Dryadulaphaetusa* 285 0.048 0.013 0.05 0.02(cid:21)0.08
2.D.phaetusa* 221 0.045 0.011 0.05 0.02(cid:21)0.10
3.D.phaetusa* 238 0.042 0.012 0.04 0.01(cid:21)0.08
4.D.phaetusa* 413 0.054 0.013 0.05 0.03(cid:21)0.09
5.D.phaetusa* 280 0.045 0.016 0.04 0.02(cid:21)0.11
1.Dryasiulia* 236 0.047 0.014 0.05 0.01(cid:21)0.09
2.D.iulia* 195 0.033 0.012 0.03 0.01(cid:21)0.08
3.D.iulia* 120 0.043 0.020 0.04 0.01(cid:21)0.09
1.Philaethriadiatonica 272 0.069 0.018 0.07 0.03(cid:21)0.12
2.P.diatonica 333 0.070 0.018 0.07 0.03(cid:21)0.12
3.P.diatonica 316 0.063 0.022 0.06 0.02(cid:21)0.21
1.Agraulisvanillae* 248 0.047 0.011 0.05 0.02(cid:21)0.08
2.A.vanillae* 154 0.054 0.011 0.05 0.03(cid:21)0.09
3.A.vanillae* 184 0.053 0.018 0.05 0.02(cid:21)0.14
1.Dionemoneta 226 0.032 0.008 0.03 0.01(cid:21)0.06
2.D.moneta 140 0.034 0.011 0.03 0.01(cid:21)0.08
3.D.moneta 219 0.037 0.013 0.04 0.01(cid:21)0.10
1.Dionejuno 134 0.053 0.013 0.05 0.03(cid:21)0.09
2.D.juno 210 0.048 0.018 0.04 0.02(cid:21)0.10
3.D.juno 151 0.049 0.011 0.05 0.03(cid:21)0.10
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 5/12
Figure2 Comparativetestsofthesexuallyantagonisticcoevolutionhypothesis(SAC)oftheevolu-
tionofspermatophoreenvelopethicknessinbutterflies.(A)Phylogeneticrelationshipsbetweenthe
elevenbutterflyspeciesincludedinthecomparisons(thisfigureispartofthephylogeneticsupertree
usedinthecomparativestudyofSÆnchez,HernÆndez-Baæos&Cordero(2011).(B)Comparisonofsper-
matophoreenvelopethicknessbetweenonepolyandrousspecieswithsignaandtwomonandrousspecies
withoutsigna.AspredictedbytheSAC,thepolyandrousspecieswithsignahasthickerenvelopesthan
themonandrousspecieswithoutsigna.(C-E)Threecomparisonsofspermatophoreenvelopethickness
betweenpolyandrousspecieswithsignaandmonandrousspecieswithsigna.AspredictedbytheSAC,
incomparisonsAandBthemonandrousspecieswithsignahasthickerenvelopesthanpolyandrous
specieswithsigna;thispatternwasnotobservedincaseC.
MountingMedium.Photographsofthesepreparationsweretakenunderthemicroscope
(OlympusBX-51)withadigitalcamera(OlympusC-5050),andthethicknessof
spermatophoreenvelopesmeasuredinthephotographsofthesectionswiththe
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 6/12
UTHSCSAImageToolforWindowsversion3.00software.Ineachphotographwetraced
animaginarycrosscenteredinthemiddlepointofthesectionandmeasuredthe
thicknessofthespermatophoreenvelopeateachofthefourintersectionpointsbetween
thecrossandthespermatophoresection.Thenumberofspermatophoresusedper
speciesvariedbetween2and7(totalnumberofspermatophoresstudiedD43);thetotal
numberofmeasurementsofenvelopethicknessperspermatophorevariedbetween55
and413(abouthalfofthesamplehadbetween150and250sectionsmeasured),mainly
duetodifferencesinspermatophoresize(Table1).
Thepredictionthatspermatophoreenvelopesofpolyandrousspecieswithsignaare
thickerthanthoseofmonandrousspecieswithoutsignawastestedbycomparingthree
speciesofHeliconius,twobelongingtothemonandrouscladewithoutsigna(H.hortense
andH.charithonia)andtheothertothepolyandrouscladewithsigna(H.ismenius)
(BeltrÆnetal.,2007;Fig.2A).Thepredictionthatspermatophoreenvelopesof
monandrousspecieswithsignaarethickerthanthoseoftheirpolyandrousrelativeswith
signawastestedinthreeindependentcomparisons(Fig.2A):(a)polyandrousEueides
alipheravs.monandrousE.isabella;(b)polyandrousDryadulaphaetusaCDryasiuliavs.
monandrousPhilaethriadiatonica;and(c)polyandrousAgraulisvanillavs.monandrous
DionejunoCD.moneta.
RESULTS AND DISCUSSION
Arethespermatophoreenvelopesofpolyandrousspecieswithsignathickerthan
thoseofmonandrousspecieswithoutsigna?
Thespermatophoreenvelopesofthepolyandrousspecieswithsigna(H.ismenius)were
thickerthanthoseofthemonandrousspecieslackingsigna(H.hortenseandH.
charithonia)(Kruskal-WallisANOVA,H2;12 D 8:33,p D 0:016;Fig.2B).Thisresultisin
agreementwiththeSAChypothesisthatproposesthatpolyandryselectsformalesthat
producethickerspermatophoreenvelopestodelayfemaleremating,andthat,in
response,femalesevolvedsignathatallowedthemtoincreasetherateofspermatophore
digestion,thusincreasingtheirrematingrate(Cordero,2005;SÆnchez,HernÆndez-Baæos
&Cordero,2011).Therewerealsodifferencesinspermatophoreenvelopethickness
betweenthetwomonandrousspecies(Fig.2B).SinceWaltersandcolleaguesfoundthat
inlargesamplesofsomepupalmatingmonandrousHeliconiusspeciesthereisavery
smallproportionofdoublematedfemales(Waltersetal.,2012),itwouldbeinterestingto
studylargesamplesofH.hortenseandH.charithoniatoseeifsomefemalesmatemore
thanonceand,incasetheydo,iftheproportionofmultiplematedfemalesislargerinH.
hortense,aswouldpredicttheSAChypothesis.
Arespermatophoreenvelopesofmonandrousspecieswithsignathickerthanthose
oftheirpolyandrousrelativeswithsigna?
Intwoofthethreegroupscompared,theenvelopesofthespermatophoresreceivedby
monandrousfemaleswithsignawerethickerthanthoseofpolyandrousspecieswith
signa(Eueidesspecies[Fig.2C]:Mann-WhitneyTest,U D0,p D0.006;Dryadula/
Dryas/Philaethria[Fig.2D]:Kruskal-WallisANOVA,H2;11 D6.91,p D0.032).These
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 7/12
resultsagreewithexpectationsfromtheSAChypothesis,thatpredictsperpetual
coevolutionbetweenmaleandfemaletraitsand,therefore,considersthepossibilityof
findinginstancesinwhichtheinterestsofoneofthesexes(malesinthepresentcase)
prevailoverthoseoftheoppositesex(femalesinthepresentcase),aswouldbethe
situationdepictedintime4ofFig.1.However,althoughtheseresultsareconsistentwith
theprediction,theydonotprovethatinE.isabellaandP.diatonicamonandryisimposed
bymalesand,therefore,maladaptiveforfemales.Totestthis,itisnecessarytoshowthat
femalesofthesetwospeciesdonotremateduetothetimetakentobreakanddigestthe
spermatophore,andthatfemalefitnessdecreasedwhentheylosttheabilitytorematedue
totheevolutionofthickerspermatophoreenvelopes.
Ontheotherhand,thethirdcomparison(Fig.2E)doesnotsupporttheprediction:the
thinnerspermatophoreenvelopeswerepresentinoneofthemonandrousspecies(Dione
moneta),whiletheother(D.juno)hadspermatophoreenvelopesasthickasthoseofthe
polyandrousspecies(Agraulisvanillae)(Kruskal-WallisANOVA,H2;9 D6.23,p D
0.044).AhypothesistoexplainthiscaseisthatselectionfavoredmonandryinfemaleD.
moneta,which,inturn,favoredtheevolutionofthinspermatophoreenvelopes.However,
ifthereductioninenvelopethicknessevolvesgradually,thedecreaseinsignasizeand/or
inthesizeofthespinescoveringthesigna(seenextparagraphandFig.3)alsocouldbe
gradual,andthepresenceofsignaandarelativelythinspermatophoreenvelopecouldbe
expectedasatransitoryevolutionarystate.Itisinterestingtonotethat,althoughthinner
whencomparedwithD.junoandA.vanillae,thespermatophoreenvelopesofD.moneta
arethickerthanthoseofthetwomonandrousHeliconiusspecieswithoutsigna(Fig.2B).
Afinalobservationisconsistentwiththehypothesisofantagonisticcoevolution
betweensignatraitsandspermatophoreenvelopes:InthepolyandrousH.ismeniusand
themonandrousEueidesisabella,femaleshavetwosignashapedlikelongandthinplates
coveredwithsmallspines(thisgeneralstructureispresent,withvariants,inmostspecies
includedinthispaper),andpreviousobservationsindicatethatthesesmallspineshelp
breakingopenthespermatophoreenvelope(Galicia,SÆnchez&Cordero,2008).Whenwe
comparedthethicknessofthespermatophoreenvelopeswiththeaveragelengthofthe
spinescoveringthesignawefoundagoodmatchbetweenthesetwomeasures(Fig.3).As
theSAChypothesiswouldpredict,thespinesarelongerinthespecieswiththicker
spermatophoreenvelopes(E.isabella)andinbothspeciestheyareofalengthsimilarto
thethicknessofthespermatophoreenvelopesproducedbymalesofitsownspecies.
CONCLUSIONS
Ingeneralterms,mostofthecomparisonspresentedinthispaperareconsistentwiththe
ideathatsexuallyantagonisticselectivepressureshavebeenimportantforcesinthe
evolutionoffemalematingpatterns,signaandspermatophoreenvelopethicknessin
heliconiinaebutterflies(Cordero,2005;SÆnchez,HernÆndez-Baæos&Cordero,2011;
Fig.1):(a)Thespermatophoreenvelopesofapolyandrousspecieswithsignaarethicker
thanthoseoftwomonandrousspecieswithoutsigna(Fig.2B);(b)intwooutofthree
cases,malesfrommonandrousspecieswithsignaproducedthickerspermatophore
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 8/12
Figure3 Thelengthofthespinescoveringthesignacorrelateswithspermatophoreenvelopethick-
ness.(A)Comparisonsbetweenthethicknessofspermatophoreenvelopesandthelengthofthespines
thatcoverthesignaintwospeciesselectedforproducingthickspermatophoreenvelopes,thepolyan-
drousHeliconiusismeniusandthemonandrousEueidesisabella.(B)Sectionofsignumcoveredwith
spinesnexttoasectionofthespermatophoreenvelopefromafemaleH.ismenius.(C)Sectionofsignum
coveredwithspinesnexttoasectionofthespermatophoreenvelopefromafemaleE.isabella.Pho-
tographs(B)and(C)takenfromGalicia,SÆnchez&Cordero(2008)withpermissionfromTheEntomo-
logicalSocietyofAmerica.
envelopesthanrelatedpolyandrousspecieswithsigna(Fig.2C,2D);and(c)intwo
speciesthelengthofthespinescoveringthesignamatchedthethicknessofthe
spermatophoreenvelopesproducedbymalesofitsownspecies(Fig.3).Ontheother
hand,oneofthecomparisonsdidnotfittheprediction(Fig.2E),andfurtherstudiesare
necessarytotestifmonandryisimposedbymalesinE.isabellaandP.diatonica.When
weconsiderthat,atleastinsomespecies,signacouldaccomplishdifferentoradditional
functionstospermatophoretearing(forexample,protectionfromspinesinmale
genitalia;(Galicia,SÆnchez&Cordero,2008;Cordero,2010),itisnotsurprisingthatnot
allvariationinthepresenceofsignaandinspermatophoreenvelopethicknesscouldbe
Sánchezetal.(2014),PeerJ,10.7717/peerj.247 9/12
explainedbytheSAChypothesis.Futurecomparativeandfunctionalstudiesare
necessarytofullyunderstandtheevolutionofthesetraits.
ACKNOWLEDGEMENTS
ThisworkispartofVSdoctoralthesis(PosgradoenCienciasBiol(cid:243)gicas,Universidad
NacionalAut(cid:243)nomadeMØxico).WethankDr.RaœlCuevadelCastilloandDr.Rogelio
Mac(cid:237)asforcommentsandsuggestionstoourresearch;twoanonymousreviewersandDr.
DezeneHuberprovidedcommentsthathelpedustoimprovethemanuscript.Wethank
RaœlMart(cid:237)nezfortechnicalhelp.
ADDITIONAL INFORMATION AND DECLARATIONS
Funding
ThisresearchwassupportedbyPAPIIT/DGAPAIN208413(UNAM),theInstitutode
Ecolog(cid:237)a(UNAM),andthePosgradoenCienciasBiol(cid:243)gicas(UNAM).V(cid:237)ctorSÆnchez
receivedadoctoralscholarshipfromCONACYT.Thefundershadnoroleinstudydesign,
datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.
Grant Disclosures
Thefollowinggrantinformationwasdisclosedbytheauthors:
PAPIIT/DGAPAIN208413(UNAM).
InstitutodeEcolog(cid:237)a(UNAM).
PosgradoenCienciasBiol(cid:243)gicas(UNAM).
Competing Interests
Theauthorshavenocompetinginterests.
Author Contributions
(cid:15) V(cid:237)ctorSÆnchezconceivedanddesignedtheexperiments,performedtheexperiments,
analyzedthedata,wrotethepaper.
(cid:15) CarlosCorderoconceivedanddesignedtheexperiments,analyzedthedata,
contributedreagents/materials/analysistools,wrotethepaper.
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