Incipient speciation in Drosophila melanogaster involves chemical signals.

1.2 MB·English

by  Grillet, Micheline

Incipient speciation in Drosophila
melanogaster involves chemical signals
SUBJECTAREAS:
MichelineGrillet1,2,ClaudeEveraerts1,BenjaminHouot1,3,MichaelG.Ritchie4,MatthewCobb2
ANIMALBEHAVIOUR
&Jean-FrançoisFerveur1
SPECIATION
EVOLUTIONARYBIOLOGY
1CentredesSciencesduGouˆtetdel’Alimentation,UMR6265CNRS,UMR1324INRA,Universite´deBourgogne,AgrosupDijon,6,
MOLECULAREVOLUTION BdGabriel,21000Dijon,France,2FacultyofLifeSciences,UniversityofManchester,OxfordRoad,ManchesterM139PT,UK,
3Presentaddress:DepartmentofBiology,WVU,LifeScienceBuilding,53CampusDrive,Morgantown,WV26506,USA,4School
ofBiology,UniversityofStAndrews,NorthHaugh,StAndrews,Fife,KY169TF,UK.
Received
4July2011
ThesensoryandgeneticbasesofincipientspeciationbetweenstrainsofDrosophilamelanogasterfrom
Accepted
Zimbabweandthosefromelsewhereareunknown.Westudiedmatingbehaviourbetweeneightstrains–six
15December2011 fromZimbabwe,togetherwithtwocosmopolitanstrains.TheZimbabwestrainsshowedsignificantsexual
isolationwhenpairedwithcosmopolitanmales,duetoZimbabwefemalesdiscriminatingagainstthese
Published
males.Ourresultsshowthatflies’cuticularhydrocarbons(CHs)wereinvolvedinthissexualisolation,but
19January2012
thatvisualandacousticsignalswerenot.ThematingfrequencyofZimbabwefemaleswashighly
significantlynegativelycorrelatedwiththemale’srelativeamountof7-tricosene(%7-T),whilethematingof
cosmopolitanfemaleswaspositivelycorrelatedwith%7-T.Variationintranscriptionlevelsoftwo
Correspondenceand hydrocarbon-determininggenes,desat1anddesat2,didnotcorrelatewiththeobservedmatingpatterns.
Ourstudyrepresentsastepforwardinourunderstandingofthesensoryprocessesinvolvedinthisclassic
requestsformaterials
caseofincipientspeciation.
shouldbeaddressedto
J.F.F.(jean-francois.
ferveur@u-bourgogne. Sensorysignalsareusedbyanimalstoassessthesex,speciesandreproductivestatusoftheirpotentialmate1.
fr) Theintraspecificdivergenceofsuchsignalsbetweenpopulationscanincreasetheirsexualisolationand
ultimately lead to distinct species2. Although Drosophila melanogaster has a global distribution, several
strains found in some geographic areas show non-random mating indicating partial sexual isolation3–5. The
strongest documented case of intraspecific sexual isolation in this species is found between strains from
Zimbabweandthosefromothergeographicareas(Cosmopolitanstrains).Inparticular,Zimbabwefemalesrarely
mate with Cosmopolitan males whereas Cosmopolitan females show a high mating frequency with both
ZimbabweandCosmopolitanmales6,7.
Several D. melanogaster natural strains vary in their cuticular hydrocarbons (CHs), some of which play a
pheromonalrole8.Mostfemalesproducehighlevelsof7,11-dienes(CHswithtwodoublebondsoncarbons7and
11),whereasthefemalesfromseveralWesternAfricanstrains(suchastheTaistrainfromIvoryCoast9,10)and
fromtheCaribbean11producehighamountsof5,9-dienesandlowamountsof7,11-dienes.However,malesof
Tai-likestrainsstillproducehighlevelsof7-monoenes(withasingledesaturationonC7)andnoorverylowlevels
of5-monoenes9,12.Thesecompounds can actaspheromones: male-predominant 7-tricosene (7-T)stimulates
femalematingbutinhibitsmalecourtship,while7,11-dienesenhancemalepreference13–16.Othercompounds
mayalsobeinvolved:ithasbeensuggestedthat5-tricosene(5-T)inhibitsmalecourtshipinitiation,whiletherole
of5,9-dienesisunclear–thesecompoundswereinitiallythoughttodecreasefemaleattractivityasshownby
matinglevels,butthiswasnotconfirmed10,17.
InD.melanogaster,theproductionofCHswithafirstdesaturationeitheronC7oronC5dependsonthe
activity of desat1 and desat2, respectively. These are two closely linked genes that code for carbon-specific
desaturaseenzymes17–19.Thedesat2geneisnotexpressedinCosmopolitanstrains,thusexplainingtheabsence
of5,9-dienesinCosmopolitanfemales20.Themolecularstructureofthedesat2geneisapparentlyrelatedtothe
degreeofsexualisolationinZimbabwefemales21,butnothingisknownofthesensorysignal(s)involvedinthis
case of incipient speciation22,23. The desat1 gene has a pleiotropic effect on both the production of cuticular
pheromonesandtheirdiscrimination24,25.Thesefindingsraisethetantalizingpossibilitythatdesat1anddesat2
arealsoinvolvedintheperceptionofC7-andC5-desaturatedhydrocarbonpheromones.
Aftereliminatingvisualandauditorysignalsassourcesofsensorydiscriminationbetweenstrains,weexplored
the relationship between the sexual isolation of Zimbabwe and Cosmopolitan strains and (i) their cuticular
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pheromonesand(ii)thetranscriptlevelsofdesat1anddesat2.After USAandDijon(Dij)fromFrance)inall64possiblecombinations
investigating the mating patterns between six Zimbabwe and two andwemeasuredtheirmatingfrequenciesforonehour.Thisrevealed
CosmopolitanstrainsandtheirCHprofiles,wechoseoneCosmo- significantassortativematingpatterns(Figure1):ALandDijfemales
politan and one Zimbabwean strain to represent each group and tendedtoshowhigherlevelsofmatingwithALandDijmalesthan
exploredtheroleofCHsindepth.Weshowedthepartialinvolve- with Z2–Z6 males, while Z2-6 females mated more frequently with
ment of CHs in this case of sexual isolation by perfuming experi- Z2-6malesthanwithALandDijmales(Figure1A).Toquantifythe
mentsandexploredthegeneticbasisofthecuticularhydrocarbons degree of sexual isolation, we calculated the joint isolation index
variationbetweenthesetwostrainsbycomparingthelevelofdesat1 (I )foreachofthe64crossesbetweentheeighttypesoffly26.Over-
psi
anddesat2transcripts. all,theindexwashighlysignificant(TotalI 50.17,G 5561.5,
psi 49df
p,0.0001;TableS1).
Results Surprisingly, Z1 flies behaved like AL and Dij flies (I 50.04;
psi
Zimbabwefemalestendnottocopulatewithcosmopolitanmales. t 50.64, p50.52; Figure 1B). These flies come from the
2755df
WepairedmalesandfemalesfromsixstrainsfromZimbabwe(Z1-6) Zimbabwecapital(Harare)andshowacuticularhydrocarbonprofile
and two cosmopolitan strains(Arkansas-Louisiana, (AL) from the closetothatofALandDijflies(seebelow).Cosmopolitan-typeflies
Figure1|(A)MatingfrequencyofsinglepairsofmaleandfemalefliesfromAL,DijandZ1–Z6strains,shownasapercentage. Coupleswereobserved
for60minutes.Eachboxrepresentsacrossbetweenthegenotypeofthefemale(rows)andofthemale(columns).Therelativesurfaceofthesquarefilledis
proportionaltothematingfrequency.n534–156.(B)Dendrogramofahierarchicalclusteranalysis(unweightedpair-groupaveragelinkagemethod)
oftheeightcosmopolitan-typeandZimbabweanstrains,comparingtheirmatingfrequencies.BranchlengthisproportionaltothepairwiseI (see
psi
MaterialsandMethods).Theeightstrainsaredividedinthreesignificantclusters(p50.05):thefirstone(‘‘Cosmopolitan-type’’)includetheAL,Dijand
Z1flies,thesecond(Zimbabwean)includetheZ3toZ6flies,whereastheZ2fliesdifferfromthesetwogroups.
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havepreviouslybeendescribedinAfricanurbancentres,presumably intenseovipositorextrusion(generallyconsideredasarejectionres-
duetohumantransport27,soonthebasisoftheCHdataweclassified ponse28)inresponsetoALmalesthantoZ6males(X 57.615,p5
2
Z1aspartoftheCosmopolitangroup.Asexpectedinstrainsshowing 0.022).Thecumulativematingcurvesforallfourcrossesinvolving
incipientspeciation,thedistributionofmatingincrossesinvolving these two strains revealed significant differences: over the first 10
ZimbabwestrainsZ2–Z6andthosegroupedas‘Cosmopolitan’(AL, minutes,ALxALpairsshowedasignificantlyhighermatinglevel
DijandZ1)revealedastrongasymmetricsexualisolation(I 50.35; (23%)thanZ6xZ6orALxZ6pairs(10%),whereasZ6xALpairs
psi
t 54.16, p50.0001, Figure 1B. All IA values for crosses be- alreadyshowedasignificantlylowermatinglevel(Figure2B).This
2755df psi
tween‘Cosmopolitan’andZimbabwefliesweresignificant(p50.05; indicatesthatthefactor(s)thatleadZ6femalesnottomatewithAL
TableS1),duetotherelativeabsenceofmatingbetweenZimbabwe malesaredetectedfromtheveryearliestmomentsoftheencounter
femalesandCosmopolitanmales. betweenmaleandfemale.
Weinvestigatedthesensoryeffectsresponsible fortheobserved
matingpatternsbystudyingthecourtshipbehaviouroftheZ6and The nature of the stimulus provided by the male. We first
AL strains (crosses are always given as female x male). We chose investigated whether the Z6 females were discriminating against a
thesestrainsbecause they showedthemostextreme differences in visualsignalprovidedbytheALmales,bypaintingthefemales’eyes.
matingpatternandincuticularhydrocarbonprofile(seebelow),and Thisunusualprocedurewasrequiredinordertomakethefemales
canbetakenasexemplarsofthiscaseofincipientspeciation.First,we blindwhileenablingthemalestobehavenormally.Ithadnoeffecton
exploredwhetherthetendencytopositiveassortativematinginZ6 the courtship intensity of sighted males compared to crosses with
femaleswasduetoalteredmalebehaviour.Z6femalesinducedsig- sighted females, indicating that the males were not discriminating
nificantlyhigherlevelsofcourtshipinALmalesthandidALfemales againstthefemalesbecauseofthepaintontheireyes,andtheZ6xAL
(Figure2A),indicatingthatZ6femaleswerehighlyattractivetoAL crossstillinducedthehighestcourtshipindex(FigureS1).However,
males.Thisinturnsuggests thatthetendency towardsisolation is sight-deprivedZ6femalesstillshowedvirtuallynomatingwithAL
due to female discrimination by Z6 females of a sensory stimulus males,whereasmatingfrequenciesintheotherthreecrosseswereall
providedbytheALmales,notduetoalteredmalebehaviours.This .40%(FigureS1).Asimilarmatingpatternwasobtainedunderred
wasconfirmedbythefactthatZ6femalesmorefrequentlyshowed lightwhere bothpartners arevirtuallyblind: 48%ALand30%Z6
Figure2|(A)Mean(6SE)courtshipindicesinallfourcrossesbetweenALandZ6flies. Courtshipwasdefinedasthetimespentbyamaleshowing
wingsvibration,femalegenitalialicking,circlingandcopulationattempt51.Pairsofflieswereobservedfor10minutes.n548–54.Lettersindicate
significantdifferences.(B)CumulativematingcurvesinallfourcrossesbetweenALandZ6flies.Thetotalobservationperiodlastedfor60min.
n596–156.Lettersindicatesignificantdifferences.
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malesmatedwithALfemales,comparedto2%and41%,respectively AL or Z6 males were used as donors, to produce desat1*AL* and
with Z6 females. We conclude that male visual signals are not desat1*Z6*malesrespectively.Theperfumingprocedurealteredthe
responsibleforthediscriminationshownbyZ6females. CHprofileofdesat1malestoaprofilebetweenthoseofdesat1males
WenextstudiedwhethertheZ6femaleswereusingmalecourt- andthedonor(Figure6B).Perfumingdesat1maleswitheitherZ6or
shipsongtodiscriminateagainsttheALmales.Recordingsofcourt- ALmalessignificantlyincreasedtheirsuccesswithALfemales,with
shipsongfromALandZ6malesrevealedslightdifferencesbetween no difference between the treatments (Figure 6A). Strikingly, AL
the two strains (Figure 3A). However, ablation of male wings and females showed higher mating frequencies than Z6 females in
playbackofsyntheticsongmimickingthesedifferencesshowedthat all three crosses. We conclude that AL females are relatively
thisdifferenceinsongplayednoroleinmatediscriminationbyZ6 indifferenttotheprecisecuticularcocktailcarriedbyamalefly,as
females.Songwasimportanttomatingsuccess,becauseablationof long as it contains some ofthe relevant compounds. However, Z6
malewingsabolishedmatinginallbuttheALxALcross(mating5 females showed a significant increase in mating only when paired
20%),butmatingwasrestoredwhenwinglessmaleflieswereaccom- withdesat1*Z6*males,butnotwithdesat1*AL*males.Thisshows
panied by playback of song typical of either their own strain or a that Z6 females are positively stimulated by the CH profile of Z6
heterotypicmale’ssong(Figure3B).Strikingly,.65%ofALmales males,andsuggeststhatCHmaleprofileandfemalepreferencesare
mated with AL females when accompanied by playback, with no involved in this case of incipient speciation. However, the levels
significantdifferencebetweenthetwoplaybacktypes,suggestingthat of mating shown in the Z6 x desat1*Z6* cross (Figure 6A) were
AL females are highly stimulated by any type of D. melanogaster substantially lower than those in the unmanipulated homotypic
courtship song. The persistence of very low mating of both kinds cross (Z6 x Z6 – Figure 3C), indicating that the pheromonal
of female with Z6 wingless males accompanied by song playback profileoftheperfumedmaleswasstillnotoptimalforZ6females.
indicatesthatthewingsofthesemalesareimportanteitherforthe Itispossiblethatthecombinationofthetwoprofiles(desat1males
stimulationordetectionoffemales,orboth.Thesensorymodalities are rich in linear saturated alkanes, and Z6 males are rich in 5-T)
involvedareunknownbutwepresumethemtobeprimarilychem- induced simultaneous antagonistic effects — repulsion and attrac-
ical.Lastly,noZ6femalesmatedwithALmales,irrespectiveofthe tion — on the behaviour of Z6 females. The partial effect of per-
typeofplayback,suggestingZ6femaleswerenotusingmalesongto fumingonmatingfrequency(20%insteadof45%)mayalsobedue
discriminate against AL males. To test this hypothesis, we ablated totheabsenceoralterationofotherunknownpheromonalornon-
female aristae, thereby deafening the females29–31. This procedure pheromonal signals in manipulated males. Z6 females were also
significantly reduced mating by AL and Z6 females, but only in crossed with AL and Z6 males perfumed with either donor male
homotypiccrosses(Figure3C).ThisindicatesthatALfemaleswere (Figure S2A). Z6 males showed higher mating frequency than AL
equallystimulatedbytheacousticand/orthechemicalsignalspro- males,andtheperfumeofALmalesinducedaslightlyhighermating
ducedbymalewingswhereasZ6femalesreliedmoreonthechemical responsethanthatseenwithZ6maleperfume.Thealteredfemale
than on acoustic signals when paired with homotypic males. We responsemaybecausedby(i)thecomplexcombinationofthetwo
concludethatanothersensorysignal,probablychemical,isrespons- CHprofiles (Figure S2B), (ii) the altered distribution of CHtopo-
ibleforthetendencytoisolationintheZ6xALcross. graphy on the male cuticle35, and/or (iii) altered male behaviour
Variation in cuticular hydrocarbons (CHs) has previously been producedbytheperfumingprocedure14,46.
implicated in this assortative mating5–7, but no decisive evidence To identify which part of the male cuticular profile might be
has been found. We found significant and complex CH variation involvedinthiscaseofincipientspeciation,wecalculatedcorrelation
between these strains, which generally paralleled the differences coefficientsbetweenthemeancuticularcompositionofeachstrain,
betweenthetwogroupsofstrainssuggestedbythematingpatterns anditsmatingperformance.ThematingfrequenciesofZ2-6females
(Z2-6 vs Z1, AL and Dij) (Table S2). The most extreme CH dif- werehighlysignificantlynegativelycorrelatedwiththeproportionof
ferences were observed between Z6 and AL flies (Figure 4). Z6 7-Tonthecuticleofthemalestheywerepairedwith(r520.458,
females produced more C5-desaturated CHs (5-tricosene (5-T), d.f.538,p50.003).Forthethreecosmopolitan-typefemalesthe
5-pentacosene (5-P) and 5-heptacosene, 5, 9-hepta- and nona- situationwasthecompleteopposite:therewasasignificantpositive
cosadienes), whereas AL females produced more of the equivalent correlationbetweenmatingfrequencyandtheproportionof7-Ton
C7-desaturated CHs (7-tricosene (7-T), 7-pentacosene and 7- themale(r50.429,d.f.522,p50.036).Nosignificantcorrelations
heptacosene,7,11-hepta-andnona-cosadienes).Similarly,Z6males werefoundbetweenmatingfrequencyandmale%5-T,5-T:7-Tratio
produced significantly more 5-T than 7-T, while the opposite was or C5:C7 ratio for either group of females (data not shown). This
trueinALmales.Multivariateanalysis(Figure5)showedvariability suggests that Zimbabwe females discriminate against males witha
within Zimbabwe males – Z2, Z3 and Z4 showed an intermediate hydrocarbon bouquet containing a high proportion of 7-T, while
positionbetweenZ5andZ6maleswithhighlevelsof5-T15-P1 cosmopolitan-type females are stimulated by 7-T-rich males. We
n-heptacosaneandZ1,ALandDijmales(withhighlevelsof7-T1 concludethatthedifferencesincuticularhydrocarbonsplayarole
n-tricosane). Even though Z2–Z6 males showed inter-strain vari- intheeffect.However,thisisnotthefullstory:other,asyetunknown,
ationforthepercentageof5-T,theyallshowedasignificantlyhigher signalsmayalsocontributetothesensoryprofilethatstimulatesZ
percentage of 5-T than Z1, AL and Dij males. Virtually all these femalesmost.
compounds have previously been suggested to play a pheromonal Asaninitialexplorationofthegeneticbasesofthisvariation,we
roleinD.melanogastercourtship10,13–15. usedRT-PCRtomeasurethelevelsofthedesat2transcriptandoffive
This combined profile of male and female CHs in Zimbabwe desat1transcripts17–19,24,25inALandZ1flies,andinZ6andTai(Ivory
strainsisunliketheprofilesofeithercosmopolitanorotherAfrican Coast)flies(Figure7;dataarepresentedasexpressionrelativetoAL
strains;althoughtheC5:C7ratiofoundinZ2–Z6femalesissimilar flies).Taiflieswereincludedbecause–theypossessawell-studied
tothatfoundinCentralAfricanstrains,thehighlevelsof5-Tshown CHprofilethatisintermediatebetweenthoseofZ6andALflies.The
by Z2–Z6 males are unique4,9,10,20. This unique combination may cuticle ofTai males is mainly covered with7-monoenes (as in AL
be a local adaptation to environmental parameters such as latitude males)whileTaifemalesmainlycarry5,9-dienes(asinZ6females).
and/ortemperature22,32,33. desat1 and desat2 genes are both involved in the desaturation of
fatty acids, a key step in the cuticular hydrocarbon biosynthesis
Theroleofmalehydrocarbonsinisolation.Toinvestigatewhether pathway36,37. Inconclusive findings have linked desat2 to this case
male CHs are used by females in selecting a mate, we perfumed of incipient speciation21, while desat1 is involved in the detection
desat1 mutantmales, which lackany of theCHs described here34.. of cuticular hydrocarbons as well as their production24,25. desat2
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Figure3|(A)CompositionofthecourtshipsongofALandZ6males.n510.(B)Isolationisnotduetoauditorysignalsfrommales.Matingfrequency
inallfourcrossesbetweenALandZ6flieswasobservedfor60minutes.Maleshadtheirwingsclippedoffandwereeitherallowedtomateintheabsenceof
anysong(Ø)orwereaccompaniedbyasyntheticsongcorrespondingtoALorZ6males.n55–56.(C)Isolationisnotduetosignalsdetectedbythe
femalearistae.MatingfrequencyinallfourcrossesbetweenALandZ6flieswasobservedfor60minutes.Femaleswereeitherintact(‘Control’),orhad
theiraristaeremoved(‘Aristae-’).n528–49.Lettersindicatesignificantdifferences.
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Figure4|Zimbabweandcosmopolitanflieshavequalitativelydifferentcuticularhydrocarbonprofiles. Typicalgaschromatographytracesareshown.
Peakswereidentifiedbymassspectrometry.Keyforcompounds:A57,11-TD,B523MeBr19-T,C57-T,D55-T,E5n-C23,F59,13-PD,G57,11-PD,
H55,9-PD,I59-P,J57-P,K55-P,L5n-C25,M59,13-HD,N57,11-HD,O527MeBr,P55,9-HD,Q57-H,R55-H,S5n-C27,T59,13-ND,
U57,11-ND,V529MeBr,W55,9-ND,X5n-C29.CompoundshighlightedincyanandmagentaaredesaturatedonC5andC7,respectively.Detailed
hydrocarboncompositionisshownonFig.S2.
transcriptlevelsweresignificantlyhigherinthebodiesofZ6andTai fliesreactinginoppositewaystotheproportionof7-T,themaincu-
fliesthaninZ1andALflies,withnodifferencebetweenthesexes ticularcomponentofmaleD.melanogasterfliesfromallstrainsexcept
(Figure 7). Of the five desat1 transcripts, RC, RE and RB showed Zimbabwe. Zimbabwe females discriminate against cosmopolitan-
significantinter-strainvariationbetweenmalestrains(Figure7),but likemalesonthebasisoftheproportionof7-TintheirCHbouquet,
noneofthetranscriptswassignificantlydifferentsolelyinZ6males. whilefemalesfromotherstrainsarestimulatedbyhigherproportions
Our data suggest that the desat2 gene is involved in the C5:C7 of this substance. Although we cannot exclude the possibility that
ratiothatcharacterisesfemalesofbothZ6andTaiAfricanstrains. correlatedsignals,forexamplevolatilecompounds,arealsoinvolved
However,thesex-specificdifferencebetweenthetwoAfricanstrains infemalediscrimination,thefactthattransferringALCHstodesat1
—lowlevelsof7-Tandhighlevelsof5-TinZ6malesbutnotinTai males did not significantly increase their mating levels with Z6
males—maybefurtherinfluencedbyeither(i)amoresubtletissue- females, while transfer of the Z6 profile did significantly increase
specificvariationofdesat2expressioninmalesand/or(ii)adiffer- mating (Figure 6), indicates that the % of 7-T as a component of
entialvariationofRAandREtranscriptsbetweenfemales.SinceRA thetotalCHbouquetmaypartiallyaccountfortheobservedpattern
and RE are implicated in hydrocarbon production25, their interac- of isolation in these strains. In male D. melanogaster, 7-T plays an
tionwithdesat2transcriptmayaffectmaleandfemaleCHprofiles inhibitoryroleinmale-malecourtshipandhasbeenshowntoactivate
differently. bittergustatorysensoryneurons38.WehypothesisethatinZimbabwe
femalesthereisasimilareffect,leadingthesefemalestoreject7-Trich
Discussion males.
Wehaveprovidedevidencethatcuticularhydrocarbonsareinvolved Although we found no evidence of quantitative differences in
in this example of incipient speciation between D. melanogaster transcription levels of desat1 between two cosmopolitan lines and
strains from Zimbabwe and from elsewhere in the world. Fur- two lines from Zimbabwe, it is possible that there is a qualitative
thermore,wehaveshownthatthiseffectispartiallyduetofemale differenceintheformsofthisgeneinthesetwokindsofstrain.desat1
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Figure5|Discriminantanalysisoffemale(A)andmale(B)fliesofalleightstrains,projectedontothefirsttwoprincipalcomponents. Probability
ellipsescorrespondto95%probability.Thevariouscomponentsinthecuticularcocktail(A-X)werealsoprojectedontothesecomponents(arrows)to
giveasenseofthedimensionality.Foreachsex,weconductedaforwardstepwisediscriminantanalysis(withanentrythresholdvalueofp50.05anda
removalthresholdvalueofp50.10)usingtheadditive/logratiotransformedproportionoftheCHs)usingtheabsoluteamountoftheuncorrelatedCHs
asquantitativevariablesandthestrainasaqualitativevariable.MagentaarrowsindicateC7compounds,cyanarrowsindicateC5compounds,black
arrowsindicateotherscompounds.Forlegends,seefigure4.
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Figure6|(A)Chemicalsignalsarepartiallyresponsibleforisolation. CumulativematingcurvesforALandZ6femaleswithdesat1malesthatwere
eitherunmanipulated(desat1)orreceivedhydrocarbonsfromALmales(desat1*AL*)orfromZ6males(desat1*Z6*).n548–80.Lettersindicate
significantdifferences.(B)DiscriminantanalysisofmalefliesfromAL,Z6anddesat1strains,andfrommalesthathadpheromonetransfers
(desat1*AL*anddesat1*Z6*),projectedontothefirsttwoprincipalcomponents.Equiprobableellipsescorrespondto95%probability.Thevarious
componentsinthecuticularcocktailwerealsoprojectedontothesecomponents(arrows)togiveasenseofdimensionality.Weconductedaforward
stepwisediscriminantanalysis(withanentrythresholdvalueofp50.05andaremovalthresholdvalueofp50.10)usingtheadditive/logratio
transformedproportionoftheCHs)usingtheabsoluteamountoftheuncorrelatedCHsasquantitativevariablesandthestrainasaqualitativevariable.
MagentaarrowsindicateC7compounds,cyanarrowsindicateC5compounds,blackarrowsindicateotherscompounds.
simultaneously controls both the production and the detection of genesintermsoftheircombinedeffectsontheintraspecificvariation
someDrosophilasexpheromones24,25.Thisoccursthroughdifferent forproductionandperceptionofpheromones.
promoterregions25thatdriveitsexpressioninoenocytes(abdominal Ourdatahelpansweraconundrumthathasexistedforover15
tissues that control pheromone production)24,39; and in brain neu- years6,23 but they do not fully resolve this case of incipient spe-
ronsinvolvedinpheromoneperception24,25.Zimbabwefemalesmay ciation. The factthat AL females apparently respondsignificantly
carryaspecificformofthisgeneorofitspromoter(s)thatresultin tomanipulationofthemale’scourtshipsong,whereasZ6females
alteredresponses.Wealsoneedtoexplore,bothatthegeneticand did not, suggests that there may be further differences between
tissuelevels,thefunctionalrelationshipbetweendesat1anddesat2 thesestrains,whichmayaddtothepheromonally-drivenisolation
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Figure7|Transcriptionlevelsofdesat1anddesat2donotdirectlycorrelatewithmatingpatterns.RT-PCRwascarriedoutonthetranscriptionlevelsof
desat1anddesat2formaleandfemalefliesfromTai(IvoryCoast),andZ6andZ1;differencesincomparisontoALfliesareshownasstarsabove/below
bars(*:P,0.05;**:P,0.01).SignificantdifferencesintranscriptlevelsratiobetweenZ1,Z6andTaistrains(shownasdifferentlettersabove/belowbars)
weredetectedwiththeRelativeExpressionSoftwareTool(REST,REST-MCSbetasoftwareversion247)wheretheiterationnumberwasfixedat2000.
we have demonstrated here. Our findings indicate the potential until4to6daysold.Allexperimentswereperformedinat2460.5uCand6565%
importance of sensory processes in speciation, and lay the basis humidity.Testswerecompletedoverseveraldaysandtookplace1–4hafterlightson.
Onemalewasaspirated(withoutanaesthesia)underawatchglassusedasan
forafullunderstandingofthisclassiccaseofincipientspeciation.
observationchamber(1.6cm3).After10minutes,avirginfemalewasintroduced.
Eachtestwasperformedfor60minutesinwhitelightandtheoverallfrequencyof
Methods copulatingpairswasmeasuredforeachtreatment.
Courtshipbehaviour.Flieswerepreparedasforthematingbehaviourtests.Asingle
Strainsandflyhusbandry.AllD.melanogasterstrainswereraisedonyeast/cornmeal/
malewasaspirated(withoutanaesthesia)underawatchglassandthevirginfemale
agarmediumandkeptat2460.5uCwith6565%humidityona12512light/dark
wasintroduced10minuteslater.Eachtestwasperformedfor10minutesinwhite
cycle.TheZimbabweandtheALlineswereprovidedbyProfessorJerryCoyne
lightandthetimethatmalespentcourtingwasmeasured.Wethencalculatedthe
(UniversityofChicago,Illinois).TheZimbabwelineswerecollectedin1990eitherin
courtshipindex–thepercentageoftimethemalespentcourtingthefemale.Tomake
theWildlifeReserveofSengwaforZ3,Z5andZ6lines,orinHarare,theZimbabwe
thefemaleblind,wepaintedbothhereyeswithblacknailvarnish(‘MissHelen’
capital,forZ1,Z2andZ4lines.Forthesakeofsimplicity,wemodifiedthenamesofthe
Zimbabwestrainsusedhere.TheoriginalnameswereZ15H23,Z25H18,Z35S6, nu112),usingasmallbrush.Thisprocedurewasperformedonehourbeforethetestto
Z45H42,Z55Z53andZ65S30.ThecosmopolitanlineArkansas-Louisiana(AL) allowthevarnishtodry.Testsperformedinredlightproducedsimilarmating
comesfromtheUnitedStates.TheDijon2000(Dij)linewascollectedbyDr.J-F patterns(seeResults&Discussion).Wethereforeconcludethatpaintingtheeyesdid
FerveurinDijon(France)in2000.Althoughsomeofthesestrainshavebeenkeptin makethefliesblindanddidnotintroduceanyotherconfoundingsensoryvariable.
thelaboratoryfor20years,ouraimwastostudyincipientspeciationratherthan
whetherthesestrainsrepresentcurrentwild-typevariation.Moreimportantly,the Songrecordingandplayback.4–5dayoldmaleflieswereisolatedateclosionand
performanceofthesestrainswasvirtuallyconstantcomparedtoresultsreportedinthe thenintroducedintotherecordingchambertogetherwithafemalethathaditswings
1990s6,7.Itshouldbenotedthatafterfiveyearsofacclimationinthelaboratory,wild- cutoff,topreventfemalewing-producedsoundsfrombeingrecorded.Malesongwas
typefliesshownoimportantchangesinCH,courtshipormating40.Thedesat1linehas recordedusinganInsectavoxrecorder,linkedtoacomputer.Songwasfilteredbefore
amutationinthedesat1geneanddesat1malesandfemalesshowdrasticallyreduced beingvisualisedonscreenusingtheAudacityprogrammeandanalysedwithSpike2
levelsof7-tricosene(7-T)and7,11-heptacosadiene(7,11-HD),respectively. software.ALandZ6malesproducedslightlydifferentsongs(Figure3A).Basedon
thesedataandamoreextensivesurveyinColegraveetal.41artificialsongswere
Behaviour.Matingbehaviour.AllflieswereisolatedunderlightCO anaesthesia0– synthesisedusingSignal(ElectronicDesign).Eachsongconsistedofanidentical
2
4haftereclosion.Maleflieswereheldindividuallyinfreshglassfoodvialsfor4or5 pulsesongaround300HzwithanIPIofeither35.5ms(representingthesongof
daysbeforetesting.Femaleswerekeptfor4or5daysingroupsoffivetoten.Maleand Cosmopolitanflies)or30.3ms(representingthesongofZimbabweanflies).
femalefliesusedasdonorsforhydrocarbontransferwerekeptinvialsingroupsof20 VariationinIPIfollowedarhythmiccycle42.Fulldetailsofsongsynthesisare
SCIENTIFICREPORTS |2:224|DOI:10.1038/srep00224 9
www.nature.com/scientificreports
describedinRitchieetal.43.Thedaybeforethetest,maleswereanaesthetisedbyCO2 4. Haerty,W.,Jallon,J.M.,Rouault,J.,Bazin,C.&Capy,P.Reproductiveisolationin
andhadbothwingscompletelyremoved.Thematingcell,1.6cmdiameterx1cm naturalpopulationsofDrosophilamelanogasterfromBrazzaville(Congo).
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insulatedchamberat25uC.Thesongwascontinuallybroadcastthroughoutthe Drosophilamelanogasterpopulations.Evolution62,2112–2121(2008).
durationoftheexperiment(30minutes). 6. Wu,C.I.etal.SexualisolationinDrosophilamelanogaster:apossiblecaseof
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andC31hydrocarboninternalmarkers,accordingtostandardprocedures44.Analyses 8. Ferveur,J.F.Cuticularhydrocarbons:theirevolutionandrolesinDrosophila
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detector,withaCP-sil/5CBcapillarycolumn(Varian,25m30.32mmID)anda 9. Jallon,J.M.&Pechine,J.M.AnovelchemicalraceofDrosophilamelanogasterin
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detectortemperatureswere260and280uC,respectively.Thecolumnwasheld Drosophilamelanogastersexpheromone:behaviouraleffects,geneticbasesand
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5.2,Varian). phermonesunderlieincipientsexualisolationbetweenusandCaribbean
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inparallel.Weusedahighlysensitivedetectionmethod(SolidPhaseMicro 15.Marcillac,F.&Ferveur,J.F.Asetoffemalepheromonesaffectsreproduction
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RC,RE,RBandRD)andofthedesat2transcriptin35to45headlessbodiesof5-day-
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reversetranscribedwiththeiScriptcDNASynthesisKit(Biorad).QuantativePCR
19.Dallerac,R.etal.ADelta9desaturasegenewithadifferentsubstratespecificityis
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cycler(MyIQ,Biorad)accordingtotheprocedurerecommendedbythe
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for30sec,TMuCfor30secand72uCfor30sec),precededby3mindenaturation
governingcuticularhydrocarbonvariationandtheirevolutioninDrosophila
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22.Greenberg,A.J.,Moran,J.R.,Coyne,J.A.&Wu,C.I.Ecologicaladaptation
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