Standing geographic variation in eclosion time and the ...nosil-lab.group.shef.ac.uk/wp-content/uploads/2019/01/Doellman_et_al-2019-Ecology_and...Shakir‐Botteri, & McPheron, 2005;

Ecology and Evolution 20199393ndash409 emsp|emsp393wwwecolevolorg

Received19July2018emsp |emsp Revised30October2018emsp |emsp Accepted1November2018DOI101002ece34758

O R I G I N A L R E S E A R C H

Standing geographic variation in eclosion time and the genomics of host race formation in Rhagoletis pomonella fruit flies

Meredith M Doellman1 emsp|emspScott P Egan123emsp|emspGregory J Ragland145emsp|emsp Peter J Meyers1emsp|emspGlen R Hood16emsp|emspThomas H Q Powell17emsp|emspPeter Lazorchak18emsp|emsp Daniel A Hahn9emsp|emspStewart H Berlocher10emsp|emspPatrik Nosil11emsp|emspJeffrey L Feder124

1DepartmentofBiologicalSciencesUniversityofNotreDameNotreDameIndiana2AdvancedDiagnosticsandTherapeuticsInitiativeUniversityofNotreDameNotreDameIndiana3DepartmentofBiosciencesRiceUniversityHoustonTexas4EnvironmentalChangeInitiativeUniversityofNotreDameNotreDameIndiana5DepartmentofIntegrativeBiologyUniversityofColoradondashDenverDenverColorado6DepartmentofBiologicalSciencesWayneStateUniversityDetroitMichigan7DepartmentofBiologicalSciencesStateUniversityofNewYorkndashBinghamtonBinghamtonNewYork8DepartmentofComputerScienceJohnsHopkinsUniversityBaltimoreMaryland9DepartmentofEntomologyandNematologyUniversityofFloridaGainesvilleFlorida10DepartmentofEntomologyUniversityofIllinoisatUrbana‐ChampaignUrbanaIllinois11DepartmentofAnimalandPlantSciencesUniversityofSheffieldSheffieldUK

ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttributionLicensewhichpermitsusedistributionandreproductioninanymediumprovidedtheoriginalworkisproperlycitedcopy2018TheAuthorsEcology and EvolutionpublishedbyJohnWileyampSonsLtd

CorrespondenceMeredithMDoellmanDepartmentofBiologicalSciencesUniversityofNotreDameNotreDameINEmailmdoellmandedu

Funding informationDivisionofEnvironmentalBiologyGrantAwardNumberDEB‐1638951DEB‐1638997andDEB‐1639005NationalInstituteofFoodandAgricultureGrantAwardNumberNIFA2015‐67013‐23289DivisionofInformationandIntelligentSystemsGrantAwardNumberIIS‐1560363EuropeanResearchCouncilGrantAwardNumberNatHisGenR129639DivisionofIntegrativeOrganismalSystemsGrantAwardNumberIOS‐1257298andIOS‐1700773InternationalAtomicEnergyAgencyGrantAwardNumberCRPinDormancyManagementtoEnableMass‐rearing

AbstractTaxaharboringhighlevelsofstandingvariationmaybemorelikelytoadapttorapidenvironmental shifts and experience ecological speciation Here we characterizegeographic and host‐related differentiation for 10241 single nucleotide polymor‐phismsinRhagoletis pomonella fruitfliestoinferwhetherstandinggeneticvariationinadulteclosiontimeintheancestralhawthorn(Crataegusspp)‐infestinghostraceasopposedtonewmutationscontributedsubstantiallytoitsrecentshifttoearlierfruitingapple (Malus domestica)Allelefrequencydifferencesassociatedwithearlyvslateeclosiontimewithineachhostraceweresignificantlyrelatedtogeographicgeneticvariationandhostracedifferentiationacrossfoursitesarrayedfromnorthtosouthalonga430‐kmtransectwherethehostracesco‐occurinsympatryintheMidwestUnitedStatesHostfruitingphenologyisclinalwithbothappleandhaw‐thorntreesfruitingearlier intheNorthand later intheSouthThusweexpectedallelesassociatedwithearliereclosiontobeathigherfrequenciesinnorthernpopu‐lationsThispatternwasobservedinthehawthornraceacrossallfourpopulationshoweverallelefrequencypatternsintheappleraceweremorecomplexDespitethegenerally earlier eclosion timing of apple flies and corresponding apple fruiting

394emsp |emsp emspensp DOELLMAN Et AL

1emsp |emspINTRODUC TION

The rawmaterial for novel adaptation can come fromnewmuta‐tions or standing variation (Barrett amp Schluter 2008) Adaptationis expected to occurmore rapidlywhen based on standing varia‐tionbecausetheprocess isnot limitedbywaittimefornewben‐eficial mutations to arise and standing variants are likely to havebeenfilteredfornegativepleiotropicfitnesseffectsordetrimentalepistatic interactions (Barrett amp Schluter 2008 Yeaman 2015)Therefore taxaharboringhigh levelsofstandinggeneticvariationmaybemore likely toadapt to rapidenvironmental shiftsandex‐perienceecological speciation than those requiringnew favorablemutationstorespondIndeedmyriadexamplesareaccumulatingofrapidadaptiveevolutionfueledbystandingvariationincludingcoatcolorintheoldfieldmousePeromyscus polionotus(SteinerWeberampHoekstra2007)reduceddefensivearmorinthethreespinestick‐lebackfishGasterosteus aculeatus(Colosimoetal2005)mimicryinHeliconius butterflies (TheHeliconiusGenomeConsortium2012)and beak morphology in Darwinrsquos Finches (Lamichhaney et al2015)Standingvariationmaybeespeciallyrelevantincaseswhereadaptation involvespolygenic traits and themajorityof favorablealleleshaveonlyminoreffectsonfitnessandgenomicincompatibil‐ity(BarrettampSchluter2008HermissonampPennings2005SchluterampConte2009)Largestoresofstandinggeneticvariationobservedinsomepopulationsmaybeaproductofcomplexevolutionaryhis‐toriesincludingpastgeneflowcoupledwithecologicalselectiontomaintainhighlevelsofgeneticvariationresultingfromtrackinglocalregionalor temporaldifferences inenvironmentalorbioticcondi‐tions(BerglandToblerGonzaacutelezSchmidtampPetrov2016BernerampSalzburger 2015Brawandet al 2014 FederBerlocher et al2003GompertFordyceForisterShapiroampNice2006Jefferyetal 2017 Jones et al 2012 Lamichhaney et al 2015 Loh et al2013PeaseHaakHahnampMoyle2016RoestiGavriletsHendrySalzburger amp Berner 2014 The Heliconius Genome Consortium2012)

Reservoirs of standing genetic variation are often maintainedacrossgeographicclinesgradientsofphenotypicorgeneticchange

inpopulationsacross space that canprovidewindows intounder‐standing adaptive evolution and speciation (Endler 1977 Huxley1938) Geographic clines can be either primary due to selectionacross a landscape or secondary as a result of population subdi‐visionfollowedbyhybridizationandintrogressionof loci Ineithercaseanalysisof thedifferential spatialdistributionofphenotypesand loci can help identify traits and genes involved in adaptationthatmaycontributetoreproductiveisolation(BartonampGale1993Barton amp Hewitt 1981 1985 Gompert Mandeville amp Buerkle2017HarrisonampLarson2016JigginsampMallet2000KruukBairdGale amp Barton 1999Mallet et al 1990 Payseur 2010 Slatkin1973 SzymuraampBarton19861991)However clines canbe in‐dicativeofmorethanjustpastprogresstowardspeciationbutalsobe natural experiments in the speciation process itselfwith geneflow providing an input of new material facilitating divergence(Abbott et al 2013 Arnold 1997 Endler 1977 Gompert et al2014HarrisonampLarson2016Hewitt1988Mallet2007)Inthisregard hybridization can createnovel genotypes thatmay rapidlyleadtodifferentiationfromnearbyparentaltaxawhenhybridsoc‐cupyunderusednichesorenvironmentsviapolyploidorhomoploidmechanisms(Gompertetal2006KangSchartlWalterampMeyer2013Lamichhaneyetal2018Mavaacuterezetal2006RiesebergampWillis2007SalzburgerBaricampSturmbauer2002SchwarzMattaShakir‐BotteriampMcPheron2005YakimowskiampRieseberg2014)Inadditionhybridizationneednothaveanimmediateeffectongen‐eratingnewtaxa(AbbottBartonampGood2016)butcanalsocreateandmaintainextensivestandingvariationenablingdivergenceatalatertimewhenpopulationsexperiencenewecologicalopportuni‐ties(BernerampSalzburger2015)

Rhagoletis pomonella (DipteraTephritidae)amodelforpopula‐tion divergence in sympatry provides an avenue for investigatingtheroleofstandingclinalvariationinrapidadaptationandspecia‐tioninresponsetonovelecologicalopportunity(BerlocherampFeder2002) Ancestral R pomonella infested the fruits of native NorthAmericanhawthorns(Crataegusspp)andshifted(lt170yearsago)todomesticatedapple(Malus domestica)aftertheplantwasintroducedby European settlers ~400years ago (Bush 1966 Walsh 1867)

phenologyallelesonchromosomes2and3associatedwithearlieremergencewereparadoxicallyatlowerfrequencyintheapplethanhawthornhostraceacrossallfoursympatricsitesHoweverlocionchromosome1didshowhigherfrequenciesofearlyeclosion‐associatedallelesintheapplethanhawthornhostraceatthetwosouthernsitespotentiallyaccountingfortheirearliereclosionphenotypeThusalthoughex‐tensiveclinalgeneticvariationintheancestralhawthornraceexistsandcontributedtothehostshifttoapplefurtherstudyisneededtoresolvedetailsofhowthisstand‐ingvariationwasselected togenerateearliereclosingapple flypopulations in theNorth

K E Y W O R D S

clinalvariationeclosiontimeecologicalspeciationhostracesstandingvariation

emspensp emsp | emsp395DOELLMAN Et AL

Previousstudieshave impliedthathawthorn‐infestingpopulationsofR pomonellapossesslargestoresofphenotypicandgeneticvari‐ationnotablyforlifehistorytimingwhichmayhavefacilitatedthehostshifttoapple(Federetal2005FederBerlocheretal2003)Inparticulardifferences in thetimingofadulteclosionhavebeenshown tobe an importanthost‐relatedecological adaptation con‐tributing to partial allochronic premating isolation between appleandhawthornflies(Federetal1994FederHuntampBush1993)Fruitonapplevarietiesfavorableforlarvalsurvivorshipripensabout3ndash4weeks earlier than those of downy hawthorn (C mollis) theprimaryhostofR pomonella in theMidwesternandNortheasternUnitedStates(Federetal1994)Fliesmustsynchronizebreakageofpupaldiapauseandadulteclosionwiththeavailabilityofripehostfruit formating andoviposition This is critical becauseRhagoletis isunivoltineadultstakeaweektoreachsexualmaturityandfliesliveforamaximumof1monthinnature(DeanampChapman1973)

Asaresultappleflieshaveevolvedtoecloseanaverageof10daysearlierthanhawthornfliesasmeasuredinfieldcapturestudiesre‐ducinghostraceoverlapto~80(Federetal19931994)Thispar‐tialallochronicisolationincombinationwithhostfidelity(eghostfruitodorpreference)reducesgeneflowbetweenthehostracesto~4ndash6pergeneration(Federetal19931994)

Eclosion time appears to have a complex evolutionary historyassociatedwithstandingclinalvariation intheancestralhawthornrace (Feder amp Bush 1989 Feder Berlocher et al 2003 FederChilcoteampBush 1990Michel et al 2010Michel Rull AlujaampFeder 2007) It has been hypothesized that part of the variationoriginatedintheEjeVolcaacutenicoTransMexicano(EVTM)~15millionyearsagoduringaperiodofisolationfromotherMexicanandNorthAmericanpopulations (seeFigure1Micheletal2007Xieetal2007)ItthenspreadthroughtheSierraMadreOrientalMountains(SMO)ofMexicoand intoNorthAmericaover thepast~1million

F I G U R E 1 emspMapofthefourpairedsympatriccollectionsitesforappleandhawthornfliesintheMidwesternUnitedStatesAlsogivenaretherangesoftheappleraceandnativehawthorn‐infestingpopulationsofRhagoletis pomonellaintheUnitedStatesandMexico(seeSupportingInformationTableS2fornumericaldesignationsofpopulationsandsiteinformation)Notethatthe430‐kmtransectthroughtheMidwesternUnitedStatesencompassesmuchofthelatitudinalrangeofoverlapoftheappleandhawthornhostracesintheregionTheprimaryhawthornhostofR pomonellaintheNortheasternandMidwesternUnitedStatesisCrataegus mollisHowevermovingsouthfromUrbanaappleisnotinfestedandC mollisbecomesrarealthoughavarietyofthespeciesC mollis texanaexistsinthestateofTexasUnitedStates(site5)OtherhawthornspecieswithvaryingfruitingtimesaretheprimaryhostsoftheflyinthesouthernUnitedStatesandMexico(seeFigure2aandSupportingInformationTableS2Lyons‐SobaskiampBerlocher2009Rulletal2006)DNAsequencingdataimplythathawthorn‐infestingR pomonellapopulationsfromtheEjeVolcaacutenicoTransMexicano(EVTM)andthoseintheSierraMadreOrientalMountainsofMexico(SMO)andUnitedStateshaveundergonecyclesofallopatryfollowedbysecondarycontactandgeneflowoverthepast~15My(Federetal2005FederBerlocheretal2003Micheletal2007Xieetal2007)contributingtothecreationandmaintenanceofgeographicgeneticvariationineclosiontimeinthefly

0 200 400 600 800km

N

Grant MI (1)Fennville MI (2)Dowagiac MI (3)

Urbana IL (4)

5

67

8

91011 12

13 Chiapas MX

EVTM MX SMO MX

Texas USA

Midwest USA

Apple and Hawthorn racesHawthorn-infesting populations only


yearsviaepisodesofsecondarycontactandintrogressionbetweenhawthornflypopulationsinMexicoandtheUnitedStates(Federetal2005FederBerlocheretal2003Micheletal2007Xieetal2007)SubsequentlyselectionrelatedtolatitudinalaltitudinalandspeciesdifferencesinhawthornfruitingtimeintheSMOandUnitedStates likelymaintained this adaptive variation in eclosion timing Consistentwiththisscenariomanyspeciesofhawthornsripenlaterin the yearwithdecreasing latitude reflected in laterdatesof flycollection(Figure2aLyons‐SobaskiampBerlocher2009RullAlujaFederampBerlocher2006Xieetal2007)Asaresulteclosiontimevariesgeographicallywithhawthornfliesfromfurthersouthrequir‐ingmoretimetoeclosethanthosefromfurthernorthbothinnatureandincontrolledrearingexperiments(Figures2band3aDambroskiampFeder2007Hoodetal2015Lyons‐SobaskiampBerlocher2009Xieetal2007)Thisstandinggeographicvariationineclosiontim‐ing of hawthorn flies is thought to have contributed to the adap‐tiveradiationoftheR pomonellasiblingspeciesgroupbyallowingthese short‐lived univoltine flies to attack novel host plantswithdifferingfruitingtimesincludingtheformationofanumberofracesandpotentiallyspeciesondifferenthawthornsinsouthernlatitudes(PowellChaLinnampFeder2012PowellForbesHoodampFeder2014)andmostrecentlytheappleraceintheEasternUnitedStates(FederChilcoteampBush1988)

Previous studies from our group provided evidence for a linkbetweenstandingclinalvariationandhostraceformationbuthadinsufficientgenomicresolutionto inferthegeneticarchitectureofeither the underlying eclosion time phenotypes or host race dif‐ferentiation (FederampBush1989Federetal19902005FederBerlocheretal2003Micheletal20072010Xieetal20082007)Raglandetal (2017)recentlylaidagenomicfoundationforunderstanding the architecture of eclosion timing and its associa‐tionwithhost racedifferentiation inR pomonella (seeSupportingInformationAppendixS1 foradditionaldetails) Inagenome‐wideassociation study (GWAS) of adult eclosion time in hawthorn andapple flies collected from a field site in Fennville Michigan (MI)UnitedStatesRaglandetal(2017)comparedsinglenucleotidepoly‐morphism (SNP) allele frequencydifferencesbetween theearliest(le3)andlatest(ge97)eclosingquantilesoffliesTheyfoundthatSNPs displaying significant allele frequency differences betweenearly‐and late‐eclosingflies inbothhostraceswereconcentratedonthreeofthefivemajorchromosomes(numbers1ndash3)constitutingtheR pomonellagenome(SupportingInformationTableS1)Withinchromosomes 1ndash3 SNPs displaying high levels of linkage disequi‐librium(LD)withoneanotherpresumablyduetochromosomalin‐versions(FederRoetheleFilchakNiedbalskiampRomero‐Severson2003b) showed the greatest frequency responses (Table S1 seeSupporting Information Appendix S1 for further discussion of in‐versionsandtheirorigins)Howeveraproportionof lowLDSNPson chromosomes 1ndash3 also displayed significant responses aboverandom expectation in the GWAS (Supporting Information TableS1) These lowLDSNPspresumably represent loci inmore freelyrecombiningcolinearregionsofchromosomesalsocontributingtoeclosiontiming

Thehighlypolygenicnatureof eclosion timedemonstratedbyRagland et al (2017) provides additional evidence supporting thehypothesis that standinggeneticvariation rather thannewmuta‐tions likely fueledR pomonellarsquos shift to the earlier fruiting applehostHoweverastrongercasewouldbemadeifallelefrequenciesforSNPs responding in theeclosion timeGWASalso showed sig‐nificant and predictable geographic and host‐related variation innatureHerewe investigategenome‐widepatternsofdifferentia‐tion inRhagoletis pomonella (DipteraTephritidae) for10241SNPsbycomparingresultsfromtheadulteclosionGWAS(Raglandetal2017)withapopulationsurveyoffoursitesdistributedfromnorthto south along a 430‐km transect across theMidwestern UnitedStates where populations of hawthorn and apple flies co‐occurinsympatry (Figure1)Wedetermine thedegree towhich the re‐sponsesofeclosion‐associatedSNPsfromtheGWASpredictgeo‐graphicallelefrequencydifferenceswithinthehostracesacrosstheMidwestandlocalhost‐relateddifferencesbetweenappleandhaw‐thornflypopulationsatthefoursympatricsites

In this studywe test four specific predictions of the standingvariation hypothesis regarding how genetic variation for eclosiontimeshouldbeassociatedwithgeographicandhost‐relatedgeneticandphenotypicdifferentiationFirst loci associatedwitheclosiontime inRaglandet al (2017) should show significant relationshipswith geographic variation in the population survey Second haw‐thorn fly populations at the more northern sites (Grant MI andFennvilleMI)shouldpossesshigherfrequenciesofallelesassociatedwithearlieradulteclosiontimeintheGWASthanthemoresouthernsites(DowagiacMIandUrbanaIllinois[IL])totrackthegenerallyearlier fruitingtimeofhawthorns furtherNorth (Figure2a)Thirdthe apple race shouldmirror the geographic pattern exhibited bythehawthornrace(iehigherfrequenciesofallelesassociatedwithearliereclosioninnorthernthansouthernpopulations)Fourthpop‐ulationsofapplefliesatbothnorthernandsouthernsitesshouldpossesshigherfrequenciesofallelesrelatedtoearliereclosionthantheir paired sympatric hawthornpopulationTogether predictionsthreeandfourreflectthatapplefliesphenotypicallyecloseearlierthan hawthorn flies at sympatric sites but that both races ecloseearlierathigherlatitudes(Figure3a)

2emsp |emspMATERIAL S AND METHODS

21emsp|emspGeographic survey

Fliesgenotyped in thegeographicsurveywerecollected fromna‐tureaseggsorearlyinstarlarvaeinfestingappleordownyhawthornfruit at four sympatric field sites across the Midwestern UnitedStateswherehosttreesandtheflyracesco‐occurwithin1kmofeachother (Figure1)Collectionsweremade fromGrantMI (latlong=4335Nminus859Wyear collected=1989n=54adulthaw‐thorn and n=48 adult apple flies genotyped equally divided bysex) FennvilleMI (426N minus8615W 2008n=96 hawthorn andn=93appleflies)DowagiacMI(4188Wminus8623N2006n=32fliesofeachrace)andUrbanaIL(4008Wminus8819N2000n=48


hawthorn andn=38 apple flies) Field‐collected flieswere rearedto adulthood for genotyping using standardRhagoletis husbandrymethods(seeFederChilcoteampBush1989)

22emsp|emspGenotyping by sequencing

Methods for genotyping by sequencing (GBS) of individually bar‐codeddouble‐digestrestrictionsite‐associatedDNAlibrariesofflies(ddRAD‐seq) denovoassemblyof contigs SNPcalling andallelefrequency estimation for field‐collected samples at the four sitessurveyed inthecurrentstudywereperformedas inRaglandetal(2017)Weused custom scripts and theGenomeAnalysis Toolkit(GATK version 25ndash2 DePristo et al 2011) to identify 10241

variablesitespassingqualityfiltersthatweregenotypedintheeclo‐sionstudyandatall fourpairedfieldsitesAverageSNPcoverageperindividualwas62XintheeclosiontimeGWASand33Xinthegeographicsurveyofsympatricsites

23emsp|emspMeasuring linkage disequilibrium

ToassessgenomestructureBurrowrsquoscompositemeasuresof link‐age disequilibrium (∆) standardized to r values between minus1 and 1wereestimatedfollowingWeir(1979)betweenpairsofSNPswithinhawthornandappleflysamplesatthefourcollectingsitesAnalysisofLDwasrestrictedtoasubsetof4244ofthe10241totalSNPsgenotyped thatwerepreviously assigned tooneof the fivemajorchromosomesinthegenome(Eganetal2015Raglandetal2017)TofurthertakeLDandgenomestructureintoaccountwealsosepa‐rately analyzed three different classes of linked SNPs categorizedasdisplayinghighintermediateorlowlevelsofLDwithinchromo‐somesasdefinedinRaglandetal(2017seeSupportingInformationAppendixS1fordetails)ThesehighLDclustersoflinkedSNPspre‐sumablyrepresentinversionswitheightdifferentgroupsidentifiedonchromosome2andoneeachontheremainingfourchromosomes

F I G U R E 2 emspGeographicvariationinhostfruitingphenologyandflyeclosiontimealongalatitudinaltransectof18differentRhagoletis pomonellapopulationsanalyzedat13sitesfromGrantMIUnitedStatestothehighlandsofChiapasMexico(MX)(DataarefromDambroskiampFeder2007Hoodetal2015Lyons‐SobaskiampBerlocher2009Xieetal2007seeFigure1foramapofthe13sampledsitesandSupportingInformationTableS2forpopulationdesignationsandadditionalinformationSiteswiththesamenumberbutdifferentlettersrepresentfliessampledfromdifferenthostsatthesite)(a)Hostfruitingtimeasindicatedbythecollectingdateofflypopulationsatsitesplottedagainstlatitude(b)meantimetoeclosionmeasuredastheaveragenumberofdaysfollowingpostwinterwarmingofflypopulationsdeterminedfromcontrolledlaboratoryrearingstudiesplottedagainstlatitudeand(c)meaneclosiontimeofflypopulationsplottedagainsthostfruitingtimeatsitesGrantMI(site1)andUrbanaIL(site4)representtheapproximatenorthernmostandsouthernmostendsrespectivelywhereapple(greencircles)andCrataegus mollis(redtriangles)hostracesgeographicallyoverlapintheMidwesternUnitedStatesNotethatfliesfromsite5infestC mollis v texana inTexaswhichisalsodepictedwitharedtrianglewhileotherhawthorn‐infestingflypopulationsareshowninblacktrianglesGeographicandhost‐relateddifferentiationinhostfruitingtimeandflyeclosionintheMidwesternUnitedStatesissubsumedwithinalargerpatternofvariationacrossNorthAmericaThereisageneraltrendforbothhostfruittoripenandfliestoecloselaterintheyearwithdecreasinglatitudeasevidencedbysignificantnegativecorrelations(rvaluesaregivenconsideringthehawthorndataalone=randforallhostsincludingapples=rall)TherelationshipiscomplicatedhoweverbyR pomonellaattackingdifferenthawthornspeciesmovingsouthwardfromtheMidwest(seeSupportingInformationTableS2)thatfruitatvaryingtimesduringtheseason(Lyons‐SobaskiampBerlocher2009Rulletal2006Xieetal2007)aswellasaltitudinaleffectsinMexico

(a)

(b)

(c)

(N)

rr

dfpp

pp

pp

df

rr

rr

dfdf

dfdf

(N)


24emsp|emspTests for allele frequency differences

Probabilities of single locus genotypes and allele frequencies forSNPswerecalculatedfollowingMcKennaetal(2010)Testsforsig‐nificantallelefrequencydifferencesforSNPswereperformedusinga nonparametricMonteCarloapproachbetweensamplepopulationsWeestablishedanulldistributionofallelefrequencydifferencesateachSNPforagivenpairofsamples(early‐vslate‐eclosingfliesor

fliesfromGrantvsUrbanaconstitutingthe latitudinalextremesofthesample)bygenerating10000pairsofrandomizedsamplesApairofrandomizedsamplesconsistedofNandMindividuals(representedbytheirgenotypeprobabilities)drawnwithreplacementfromthepoolofthetwosamplesofrespectivesizesNandMAnobservedabsolutedifferencethatexceededtheupper95thquantileofthenullMonteCarlodistributionofabsolutedifferenceswastakenassignificantlydifferentthanexpectedTodeterminewhetheroverallpercentagesof SNPs associated with eclosion time and geographic divergenceweresignificantlygreaterthanexpectedbychancethetotalpercent‐ageofSNPsshowingsignificantdifferences ineachsimulationwascalculatedandthedistributionforall10000runscomparedtotheobservedpercentagestoassesswhetherthesewereintheupper5ofsimulatedpercentagesByusingwholeflygenotypeprobabilitiestogeneratethenulldistributionLDrelationshipsamonglociandthustheeffectsofinversionsandgeneticcorrelationsbetweenSNPswereaccountedforbymirroringtheirassociationsintherandomizedsam‐plesRaglandetal(2017)foundthatallelefrequencydifferencesbe‐tweenearly‐andlate‐eclosingapplevshawthornfliesfromFennvillewere significantly correlated genome wide in the GWAS (r=054plt00001 for all10241SNPsgenotyped)We thereforeused themeanofthefrequencydifferenceaveragedbetweentheappleandhawthornracesforthealleleassociatedwithlatereclosiontimeforallanalysesinvolvingeclosiontimeexceptwherenoted

25emsp|emspTests for genetic associations of eclosion time with geographic and host‐related differentiation

Weperformedlinearregressionanalyses(RCoreampTeam2017)toassessthedegreetowhichallelefrequencydifferencesforSNPsbe‐tween theearliestand latestquantilesof flies in theeclosion timeGWAS were related to (a) allele frequency differences within theappleandthehawthornhostracesbetweentheGrantandUrbanasites(ieameasureoftheextentofgeographicvariationforfliesbe‐tweenthetwomostdistantsiteswesampledessentiallyencompass‐ingthelatitudinalrangeofoverlapofthehostracesintheMidwestseeFigure1)and(b)allelefrequencydifferencesbetweentheappleandhawthornracesatthefoursympatricsites(ielocalhost‐related

F I G U R E 3 emsp (a)Meantimetoeclosionmeasuredastheaveragenumberofdaysfollowingpostwinterwarmingforappleandhawthornfliesincontrolledrearingexperiments(datafromHoodetal2015seealsoSupportingInformationTableS2foradditionalsiteinformation)andbndashd)meanstandardizedallelefrequenciesforSNPvariantssignificantlyassociatedwithlatereclosiontimeintheGWASofRaglandetal(2017)atthefourlatitudinallyarrayedstudysitessurveyedacrosstheMidwesternUnitedStatesPopulationshavinghighermeanstandardizedallelefrequenciesarethereforepredictedtobegeneticallyinclinedtoecloselaterthanpopulationshavinglowermeanstandardizedallelefrequenciesPanel(b)chromosome2and3SNPstogether(c)chromosome1SNPs(d)chromosome1ndash3SNPstogetherseeMaterialsandMethodsfordetailsconcerningthecalculationofmeanstandardizedallelefrequenciesforSNPsn=numberofsignificantSNPsonchromosome(s)contributingtomeanstandardizedallelefrequencies

Mea

n ec

losi

on ti

me

40 41 42 43 4455

60

65

70

75

80 UrbanaDowagiac

FennvilleGrant

(a) Eclosion time

HawthornApple

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(b) Chromosomes 2amp3

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(c) Chromosome 1

Latitude

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(d) Chromosomes 1ndash3


divergence)SignificancelevelsweredeterminedbyMonteCarlosim‐ulations inwhich correlation coefficientswere calculated betweentworandomsamplesofwholeflygenotypeprobabilitiestakenwithreplacementfromtheappropriatepooleddatasetsoffliesasaboveAbsolutevaluesofobservedcorrelationcoefficientsthatexceededtheupper5ofabsolute rvalues in thesimulateddistributionsof10000replicatesweretakenassignificantlydifferentthanexpectedAgainbyusingwholeflygenotypeprobabilitiestogeneratenulldis‐tributionsthenonindependenceofSNPsdisplayinghighLDwithoneanotherwasfactoredintotheanalysestestingforsignificanceTovis‐ualizehowoverallpatternsofSNPallelefrequenciessignificantlyas‐sociatedwitheclosiontimechangedgeographicallyandbetweentheraceswecalculatedmeanstandardizedallelefrequenciesforappleandhawthorn flypopulationsat all sitesForeclosion time‐relatedSNPs(Raglandetal2017)weusedthealleleatahigherfrequencyinlate‐eclosingfliesasthereferencesettheaverageallelefrequencyforeachraceacrossallsitestozeroandcalculatedthemeandevia‐tioninallelefrequencyforeachpopulation

3emsp |emspRESULTS

31emsp|emspLinkage disequilibrium

Patterns of composite LD between SNPs were similar withinand across sites and between the host races Mean pairwise LD

values for SNPs to all loci mapping to the same chromosomewere strongly correlated between populations from the ends ofthe transect at Grant and Urbana (r=083 plt00001 4243df Figure 4) Intrachromosomal mean pairwise LD values were

F I G U R E 4 emspRelationshipbetweenlinkagedisequilibrium(LD)estimatesmadeatGrantMIvsUrbanaILValuesaremeanLDforagivenSNPtoallotherSNPsmappingtothesamechromosomeintheappleandhawthornracesSNPsbelongingtodifferentLDclassesattheGrantsiteasdesignatedinRaglandetal(2017)aredepictedindifferentcolors(red=highLDclassofSNPsblue=intermediateLDblack=lowLD)

r p df

TA B L E 1 emspPercentagesofSNPsdisplayingsignificantgeographicallelefrequencydifferencesbetweenGrantMIandUrbanaILfortheapplerace(uppertable)andhawthornrace(lowertable)

chr 1 chr 2 chr 3 chr 4 chr 5 chr 1ndash5

Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

102(006) 389(011) 431(013) 291(009) 93(006) 242(009)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

72(006) 566(014) 861(024) 786(017) 67(005) 332(011)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

116(006) 386(010) 352(010) 403(010) 115(006) 247(008)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

102(006) 149(007) 149(007) 128(006) 81(006) 118(006)

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

544(014) 504(011) 477(011) 120(006) 154(006) 370(010)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

920(025) 822(016) 839(020) 48(005) 171(007) 583(015)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

455(011) 471(011) 421(009) 120(006) 142(006) 348(009)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

156(005) 207(006) 207(007) 149(006) 158(006) 170(006)

NotesResultsaregivenforallmappedSNPs(MapSNPs)andforhighintermediate(Int)andlowLDclassesofSNPsforeachchromosomeconsideredseparatelyaswellasforallchromosomestogether(chr1ndash5)AlsogiveninparenthesesarethemeanSNPallelefrequencydifferencesbetweenGrantMIandUrbanaILforappleandhawthornflypopulationsforeachclassofSNPconsideredple005 ple001 ple0001 ple00001SignificantpercentagesabovenullexpectationforaclassofSNPsasdeterminedbyMonteCarlosimulationsareinboldn=SNPsgenotypedintheclass


also highly correlated between the host races at Grant (r=090plt00001 4243 df) and Urbana (r=079 plt00001 4243 df)Likelydueinlargeparttosharedinversionsandongoinggeneflow(Feder et al 1994 Feder Roethele et al 2003b) these similarpatterns of LD imply that genome structure is concordant withinandbetweenthehostracesacrosstheMidwestandallowforourbroader application of the high intermediate and low LD SNPsclassescategorizedatGrant

32emsp|emspPatterns of geographic variation within and between the host races

Both hawthorn and apple host races displayed substantial geo‐graphic variation in SNP allele frequencies across sites Of the10241SNPsgenotypedinthestudy2871(28)showedsignifi‐cant allele frequencydifferencesbetweenhawthorn fly popula‐tions fromUrbana (south) vsGrant (north)while2102 (205)differed for apple flies (plt00001 and 0001 respectively) Ofthese SNPs 952 displayed significant geographic variation inboth races Thus allele frequency differences between GrantandUrbanaweresignificantlycorrelatedbetweenthehostraces(r=049plt000110240df )

Twomajor trends ingeographicvariationwereapparentFirstgeographic divergence varied among chromosomes being greater

for SNPs on chromosomes 1ndash3 than on chromosomes 4 and 5(Table 1) In an exception to this pattern chromosome 1 showedless geographic variation in the apple vs hawthorn race (Table1)Converselychromosome4displayedincreaseddifferentiationintheapplevshawthornraceforhighandintermediateLDlociSecondgeographicvariationwashigherforSNPsshowingelevatedlevelsofLD (Table 1) Thepercentagesof SNPsdisplaying significant allelefrequencydifferencesbetweenhawthornflypopulationsfromGrantandUrbanaincreasedfromthelow(17n=845)tointermediate(348n=2368)andtohighLD(583n=1031)classesof loci(G‐heterogeneity test=3499 plt00001 2 df) although low LDSNPsonchromosomes1ndash3stillshowedsignificantgeographicvaria‐tion(Table1)TogetherthesetwotrendssupportthefirstpredictionofthestandingvariationhypothesisHighLDlocionchromosomes1ndash3 displayed both the strongest associations with eclosion time(Raglandetal2017)andthehighestlevelsofgeographicdivergence(Table1)

33emsp|emspEclosion time and geographic variation

Allele frequency differences between the early‐ and late‐eclosingquantilesoffliesintheGWASofRaglandetal(2017)werehighlypositively related to their latitudinal frequency differences fromnorthtosouthbetweenGrantandUrbana(Table2)Forall10241

TA B L E 2 emspCorrelationcoefficients(r)ofallelefrequencydifferencesforSNPsbetweentheearly‐andlate‐eclosingquantilesoffliesintheeclosiontimeGWAS(Raglandetal2017)vsgeographicallelefrequencydifferenceswithinthehostracesbetweenGrantMIandUrbanaILforapplefly(uppertable)andhawthornflypopulations(lowertable)


Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018

NotesResultsaregivenforallmappedSNPs(MapSNPs)andforhighintermediate(Int)andlowLDclassesofSNPsconsideredseparatelyforeachchromosomeaswellasforallchromosomestogether(chr1ndash5)SignificantrelationshipsasdeterminedbyMonteCarlosimulationsareinboldn=ofSNPsgenotypedintheclassple005ple001ple0001ple00001


SNPs the genome‐wide correlation between the eclosion timeGWAS and geographic differentiation in the hawthorn race wasr=060 (plt00001 Figure 5a) The relationship was not as pro‐nouncedbutstillsignificantfortheapplerace(r=035plt00001Figure5b)

For thehawthorn race thegenetic relationshipbetweeneclo‐sion time and geographic SNP variation was significant for chro‐mosomes1ndash3(r=078plt00001n=2620SNPs)butnotfortheremainderofthegenome(chromosomes4and5r=009p=0396n=1624SNPsTable2)Thegeneticcorrelationbetweeneclosion

time and geographic variationwas also high among the eight dif‐ferentgroupsofhighLDSNPsonchromosome2thatpresumablyrepresent smaller inversions (r=088p=000397df Figure6a)Consistentwiththesecondpredictionofthestandingvariationhy‐pothesistheallelethatincreasedinfrequencyinthehawthornracetowardthenorthernmostGrantvssouthernmostUrbanasitewasgenerallytheallelefoundinhigherfrequencyintheearly‐eclosingquantileoffliesintheGWASSpecificallythisrelationshipwasob‐servedfor783ofthe2196SNPsshowingasignificantassociationwitheclosiontimeand901forthesubsetof1376oftheseSNPs

F I G U R E 5 emspRelationshipsofthegeneticresponsesofSNPsintheeclosiontimeGWAS(differencesinallelefrequenciesforSNPsbetweenearly‐andlate‐eclosingquantilesofflies)vsgeographicdivergence(differencesinallelefrequenciesforSNPsbetweenGrantandUrbanasites)for(a)allSNPsinthehawthornrace(b)allSNPsintheapplerace(c)chromosome1SNPsintheapplerace(d)thehighandintermediateclassesofSNPsonchromosome4intheapplerace(e)chromosome1SNPsinthehawthornraceand(f)chromosomes23and5andthelowLDclassoflocionchromosome4intheappleracen=numberofSNPsincomparisonArrowsinpanelbdesignatethedichotomouspatternsofgeographicvariationdisplayedbytheappleraceThelowerarrowrepresentsSNPsshowinglimitedgeneticassociationbetweeneclosiontimeandgeographicvariationwhiletheupperarrowdenotesSNPsdisplayingamoremarkedrelationship

ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race

ndash06ndash04 ndash02 00 02 04 06

r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)


mappingtochromosomes1ndash3 (plt00001 inbothcasesasdeter‐minedbyMonteCarlosimulations)

Theweaker relationshipbetweeneclosion timeandgeographicSNP variation in the apple race reflected differences among chro‐mosomesandLDclassesFortheappleraceFigure5bsuggeststhatSNPscanbedividedintotwocategories(a)locidisplayingthesamepositiverelationshipbetweeneclosionandgeographicvariationseeninthehawthornrace(upperarrowinFigure5b)and(b) locithatdonot (lower arrow) This second category in apple flies was largelycomprisedofSNPsmapping tochromosome1andto thehighandintermediateLDclasseson chromosome4 (Table1) Therewasnosignificant relationship between eclosion time and geographic SNPvariationforapplefliesforchromosome1(r=007p=0615n=949SNPsFigure5c)orforthehighandintermediateLDclassesofSNPsonchromosome4 (r=012p=0478n=201SNPsFigure5d)al‐thoughsucharelationshipclearlywasseenatleastforchromosome1inthehawthornrace(r=082p=lt0001n=949SNPsFigure5e)Withoutchromosome1andthehighandintermediateLDSNPsonchromosome 4 the dichotomy seen for apple flies disappeared(Figure5f)andthecorrelationcoefficientbetweeneclosiontimeandgeographicSNPvariationincreasedintheappleracefromr=035to065(plt00001n=3094SNPs)Moreoverlikethehawthornracetherewasa strong relationship in theapple racebetweeneclosiontimeandgeographicvariationfortheeightgroupsofhighLDSNPsonchromosome2(r=096plt000017dfFigure6b)AlsosimilartohawthornfliesthealleleinhigherfrequencyintheappleraceattheGrantcomparedtotheUrbanasitewasthealleleathigherfre‐quencyintheearly‐eclosingquantileoffliesintheGWASconsistentwithpredictionthreeofthestandingvariationhypothesisEvenwithchromosomes1and4included697ofthe2196SNPsshowingasignificantassociationwitheclosion timechanged frequency in theexpecteddirectionintheappleracetowardearlyeclosionallelesatGrantwhilethisrelationshipwasobservedfor887oftheseSNPs(n=1672)mappingtochromosomes2ndash3(plt00001inbothcases)

34emsp|emspHost race eclosion and geographic differentiation across the genome

Genomic variation associated with eclosion time was also sig‐nificantly related to host race divergence at the Grant FennvilleDowagiacandUrbanasitesHoweverthisrelationshipvariedacrosschromosomesandwasdependentuponthepatternofgeographic

F I G U R E 6 emspRelationshipsofthemeangeneticresponsesofSNPsbelongingtotheeightdifferenthighLDgroupsonchromosome2intheeclosiontimeGWAS(ietheiraverageallelefrequencydifferencesbetweenearly‐andlate‐eclosingquantilesofflies)vs(a)theirmeanfrequencydifferencesinthehawthornracebetweenGrantandUrbana(b)theirmeanfrequencydifferencesintheappleracebetweenGrantandUrbanaand(c)theirmeanfrequencydifferencesbetweentheappleandhawthornracesatFennvilleMINumbers(1ndash8)infiguresdesignatetheeightdifferentgroupsofhighLDlocipresumablyrepresentingeightdifferentrelativelysmall‐sizedinversionsresidingonchromosome2

(a)

(b)

(c)


variation displayed by each LD class (Table 3) In particular hostracedivergenceatsympatricsiteswassignificantlyassociatedwitheclosiontimeforchromosomes1ndash3(Table3)Incontrastnosignifi‐cantrelationshipwasdetectedforchromosomes4or5atanysite(Table3)Wethereforefocusonchromosomes1ndash3below

Patternsofhost‐relatedallele frequencydivergenceweresimilarbetweenchromosomes2and3butdifferedforchromosome1Whenconsidered together chromosomes 2 and 3 displayed a consistentpattern of divergence inmean eclosion‐standardized allele frequen‐cies between apple and hawthorn host races across sympatric sites(Figure3bseeSection2fordetailsofeclosionstandardizationcalcu‐lations)Moreoverinaccordwiththethirdpredictionofthestandingvariationhypothesismeanstandardizedallelefrequenciesforchromo‐somes2and3showedaparalleldecreasewithincreasinglatitudeforbothhostraceswithsouthernpopulationsofbothappleandhawthornfliespossessinghigherfrequenciesofallelesassociatedwithlatereclo‐sionintheGWAS(Figure3b)Howeverwhileeclosiontimewassig‐nificantlyrelatedtohostracedivergenceateachofthefoursympatricsitesforchromosomes2and3(r=minus036minus055minus043andminus038forGrantFennvilleDowagiacandUrbanarespectivelyplt00001inallcasesn=1671SNPs)thesigns(directions)oftherelationshipswereallnegativeThispatternextendedtotheeighthighLDgroupsofSNPs

onchromosome2aswell(Figure6c)Thusincontrasttothefourthpredictionofthestandingvariationhypothesisappleflypopulationstended topossesshigher frequenciesofallelesassociatedwith lateradulteclosionatallfoursites(Figure3b)eventhoughapplefliesecloseonaverageearlierthansympatrichawthornflies(Figure3a)

Thedifferentpatterndisplayedbychromosome1maypartiallycounteractthisdeviationfrompredictionfour(Figure3c)ForSNPsonchromosome1geographicvariationwaslesspronouncedintheapple than hawthorn race (Table 1) andwas not significantly cor‐relatedwith eclosion time for apple flies (Figure5c) as itwas forhawthornflies (Figure5e)ThiswastrueeventhoughasignificantpercentageofSNPsonchromosome1wereassociatedwitheclo‐siontime(414=393949SNPsplt00001)andtheassociations(allele frequencydifferencebetweenearly‐and late‐eclosing flies)weresignificantandpositivelycorrelatedbetweenappleandhaw‐thorn flies (r=088plt00001n=949SNPs)As a consequenceof the reducedgeographicvariation in theapple racemeanstan‐dardizedallelefrequencyclinesforchromosome1crossedbetweenthehostraceswithappleflypopulationsatUrbanaandDowagiactendingtohavetheexpectedlowerfrequenciesofallelesassociatedwith later adult eclosionwhile the reversewas trueatGrant andFennville(Figure3c)Asaresultcorrelationsbetweeneclosiontime

TA B L E 3 emspCorrelationcoefficients(r)ofallelefrequencydifferencesforSNPsbetweenearly‐andlate‐eclosingquantilesoffliesintheeclosiontimeGWAS(Raglandetal2017)vshostraceallelefrequencydifferencesbetweenappleandhawthornflieswherepopulationsco‐occuratfieldsitesinGrantMIFennvilleMIDowagiacMIandUrbanaIL


GrantMI

MapSNPs minus064 minus016 minus046 008 002 minus042

HighLD minus008 minus052 minus009 minus014 007 minus063

IntLD minus051 minus008 minus040 020 004 minus029

LowLD minus025 005 minus004 minus003 003 minus006

FennvilleMI

MapSNPs minus075 minus080 minus014 018 minus016 minus056


IntLD minus066 minus075 minus007 015 minus017 minus050

LowLD minus028 minus047 minus014 012 001 minus012

DowagiacMI

MapSNPs 045 minus029 minus048 007 minus002 minus001

HighLD 010 minus033 minus015 028 minus012 001

IntLD 042 minus033 minus036 009 minus001 minus004

LowLD 023 minus018 minus023 001 minus001 minus003

UrbanaIL

MapSNPs 071 minus006 minus052 minus009 011 018


IntLD 062 003 minus047 minus011 013 013

LowLD 025 003 minus010 004 007 003

NotesResultsaregivenforallmappedSNPs(MapSNPs)andforhighintermediate(Int)andlowLDclassesofSNPsforeachchromosomeconsideredseparatelyaswellasforallchromosomestogether(chr1ndash5)SignificantrelationshipspositiveinsignarehighlightedinboldandnegativerelationshipsareinitalicsSeeTables1and2forofSNPsgenotypedineachclassple001ple001ple0001ple00001


andhost‐relateddivergenceweresignificantandpositiveinsignatUrbanaandDowagiacinlinewithpredictionfourwhiletheyweresignificantandnegativeinsignatGrantandFennville(Table3)Whenchromosomes1ndash3wereconsideredcollectivelytheeffectsofchro‐mosome1 outweighed those of chromosomes2 and3 such thatstandardizedallele frequenciescorrectlypredictedthatapple fliesshouldecloseearlier thanhawthornfliesatUrbanaandDowagiac(Figure 3d) Moreover mean standardized allele frequencies pre‐dictedthattheappleflypopulationatDowagiacwoulddeviatefromtheothersitesandecloserelativelylate(Figure3d)asobservedinnature(Figure3a)Neverthelessmeanstandardizedallelefrequen‐ciesforchromosomes1ndash3stillpredictedthatapplefliesatGrantandFennvillewouldecloselaterthanhawthornflies(Figure3d)deviat‐ingfromtheobservedphenotypicpattern (Figure3a)andrunningcountertopredictionfourofthestandingvariationhypothesis

4emsp |emspDISCUSSION

41emsp|emspStanding variation and rapid adaptive evolution

Ourresultssupporttheimportanceofstandingvariationintherecentformationof the apple‐infestinghost raceofR pomonella a primeexampleofecologicaldivergencewithgeneflow(BerlocherampFeder2002)Thepatternsofeclosion‐associatedgenomicdivergenceob‐served in the population survey of theMidwest largely concurredwiththepredictionsof thestandingvariationhypothesiswithonemajorexceptionFirstgeographicandhost‐relateddivergencewasmostpronouncedforchromosomes1ndash3particularlyforthehighLDlocireflectingthepatternofeclosion‐associatedvariationreportedbyRaglandetal(2017)Secondclinalvariationwithintheancestralhawthornhostracetrackedgeographicvariationinhawthornfruitingtime(Figure3a)HawthornflypopulationsatthemorenorthernGrantandFennvillesitespossessedhigherfrequenciesofallelesassociatedwithearlyadulteclosionprovidingapotentialseedfortheshifttotheearlierfruitingappleThirdtheappleracegenerallymirroredthegeographicpatternexhibitedbythehawthornracealthoughinthisregardtherewasanimportantdifferencewediscussfurtherbelowFinallychromosome1lociassociatedwithearlyeclosionwerefoundathigherfrequenciesinapplevshawthornfliesatthetwosouthernsites (DowagiacandUrbana) accounting for theearliereclosionofapplefliesHoweverimportantquestionsregardingtheearliereclo‐sionofapplefliesatthenorthernGrantandFennvillesitesremainasapplefliesatthesesitestendedtohavehigherfrequenciesoflateeclosionallelescomparedtosympatrichawthornflies

PreviousworkhasdemonstratedthateclosiontimeisassociatedwithmanySNPsofmodesteffectspreadthroughoutchromosomes1ndash3(Raglandetal2017)whichisinconsistentwitharecentmuta‐tionaloriginforthegeneticbasisoftheshifttoapplesTheassocia‐tionsofSNPalleleswitheclosiontimeandtheirgeneticarchitectureasimpliedbypatternsofLDaresimilarbetweentheracesInpar‐ticularthesamesetsofallelesinsimilarlinkagephaseonchromo‐somes1ndash3appeartoaffecteclosiontimeinaconcordantmannerboth locallyandgloballyacross theMidwestwithinandbetween

thehostracesTheseobservationscombinedwiththerecenttimeframeofdivergence(~400yearssincetheintroductionofapplestoNorth America) argue strongly against multiple independent ori‐ginsofnewmutationsunderlying thegeneticsofeclosion time Itis highly unlikely that the same variants have arisen and divergedbetweenhostracesinasimilarmannerrepeatedlyacrosssitesandonsucharapidtimescale

Futurestudiesareneededtoidentifythespecificgenesaffectingeclosion time inR pomonella test for signatures of selection anddiscern their demographic histories In this regard Ragland EganFederBerlocherandHahn(2011)andMeyersetal(2016)showedmarkeddifferencesbetweenhostracesingeneexpressionpatternspriortodiapauseterminationandsuggestedgenesintheWntandTORsignalingpathwaysascandidatelociWhole‐genomeDNAse‐quencingisalsoneededexaminetheappleraceforsoftvshardse‐lectivesweepsaroundcandidateloci(HermissonampPennings2005)theformerpredictedunderthestandingvariationhypothesiswhilethe latter isexpected ifnewmutationsenabledtheshift toappleIn addition previous analysis of cDNA loci implied that the puta‐tiveinversionsonchromosomes1ndash3containingeclosionlociareold(gt15MyaFederBerlocheretal2003Federetal2005)ThusasdiscussedearlierifsecondarycontactandgeneflowwithMexicanflies fromtheEVTMgenerated thestandingvariation in thehaw‐thornracethenwepredictthatgenomesequencingstudieswillre‐vealthatmanyallelesaffectingeclosionwilldateto~15Mya

However this does not mean that variants of large effect(Colosimoetal2005HopkinsampRausher20112012Lamichhaneyetal2015Steineretal2007YuanSagawaYoungChristensenampBradshaw2013)andof recentmutationalorigin (eg industrialmelanism in thepepperedmothvanrsquotHof2016)donotalsoplayimportantroles inecologicaladaptationandspeciationThiscouldevenbethecaseforRhagoletiswithrespecttolociaffectingotherselected traits such as host plant choice (Dambroski et al 2005)FutureworkisneededtoestablishthegenomicarchitectureofhostodorpreferenceinR pomonellaandinfertheroleofnewmutationsvsstandingvariationinitscontributiontohostracedivergence

42emsp|emspEclosion time and genomic divergence between the host races

Notably our results differed fromprediction four of the standingvariationhypothesisOnbalanceapplefliesdidnothavehigherfre‐quenciesof allelesassociatedwithearliereclosion than sympatrichawthornfliesAlthoughwefoundsubstantialstandinggeographicgeneticvariationforeclosiontimeonchromosomes1ndash3inthehaw‐thornracewhichwassignificantlycorrelatedwithhost‐relateddi‐vergenceatallsympatricsitesthedirectionoftheallelefrequencydifferencesbetweentheracesgenerallydifferedfromexpectationFrequenciesofchromosome2and3SNPschangedinparallelacrossallsitesinbothappleandhawthornhostracesbutshowedapara‐doxicalpatternwithineachsympatricsitewhereapplefliestendedtopossesshigherfrequenciesofallelesassociatedwithlatereclosionthanhawthornflies(Table3andFigure3d)Howeverclinalpatterns


forchromosome1SNPsdeviatedfrompredictionthreeshowingasteepclineamonghawthornflypopulationsbutremainingrelativelyflat among apple fly populations Therefore chromosome1 SNPsactuallyfitthepredictionfourpatternatDowagiacandUrbanawithapple flies harboring higher frequencies of alleles associatedwithearliereclosionalthoughtheystillshowedthereversedpatternatGrantandFennville

Onepossiblereasonforthisdiscrepancymaybethatallelesaf‐fectingoraffectedbyeclosiontimeareindifferentlinkagephasesbetween the host races at Grant and Fennville vs the twomoresouthernsitesHoweverpatternsofLDbetweenSNPsweresimi‐laracrossgeographicsitesandbetweenthehostracesatsympatricsites (Figure 4) Moreover SNPs on chromosomes 1ndash3 were sig‐nificantlycorrelated in theireffectsoneclosiontimebetweenthehost races (r=074plt00001n=2620SNPs) Thus the earliereclosionphenotypeofapplefliescannotbeeasilyaccountedforbygeographic or host‐associated variation in linkage phase betweengenesaffectingeclosiontimeandthechromosome1ndash3allelesgeno‐typedinthecurrentstudyAdditionaleclosiontimestudiesinvolvingwhole‐genomesequencingatFennvilleandtheothersitesarestillneededhowevertocompletelydiscountthescenariothatvariablelinkagerelationshipsbetweenthehostracesareresponsiblefortheearliereclosiontimeofappleflies

Another importantconsideration isthatwemaynothavegen‐otyped all relevant eclosion‐associated SNPs including key lociresponsible for the earlier eclosion of apple vs hawthorn flies atGrant andFennvilleHowever a relatively high percentage of thephenotypic variation in eclosion time (63)was explained by the10241 SNPswe genotyped (Ragland et al 2017) In addition al‐thoughmanySNPs (n=213) displayed significant allele frequencydifferencesbetweenearly‐andlate‐eclosingfliesonlyintheappleraceGWAS(Raglandetal2017)theselocishowednosignificantgeneticrelationshipbetweeneclosiontimeandgeographicvariationin the apple race (r=0004 p=0972) orwith host‐related diver‐genceatanysite(r=002005010and001forGrantFennvilleDowagiac and Urbana respectively pgt050 in all cases) ThusSNPswitheclosiontimeeffectsuniquetoapplefliescannotreadilyexplainwhyapple fliesecloseearlier thanhawthornfliesatGrantand Fennville Nevertheless the effects of loci not genotyped inthestudywhichareinlinkageequilibrium(lowLD)withthe10241SNPswescoredmayremainundetectedandthusapossiblecauseofearlierappleflyeclosionTheseunresolvedlocicouldconceivablybeoflargeeffectandofrecentmutationaloriginHowevertheymayalsohaverelativelysmalleffectsizesandrepresentolderstandingvariationwhichincombinationwithresidenceinfreelyrecombininggenomicregionscouldfurtheraccountfortheirabsencefromthecurrent study Additional whole‐genome sequencing is needed toresolvetheissueofundetectedloci

Finally gene‐by‐environment or nonadditive epistatic inter‐actionsmayalsoplay a role indeterminingeclosion time (FilchakRoetheleampFeder2000)Intheformercasedifferencesinthepre‐oroverwinteringperiodexperiencedbythehostracesatthemorenorthernGrantandFennville sitesmay result inallelesassociated

with later diapause termination in the laboratory having differenteffectsinnaturecausingapplefliestoecloseearlier(Filchaketal2000) In insectsmost latitudinal clines associatedwith diapausevary in response to photoperiod (Hut Paolucci Dor Kyriacou ampDaan2013) includingvoltinismclines in theEuropeancornborerthat contribute to temporal isolation between pheromone strains(Levy KozakampDopman 2018 Levy KozakWadsworth CoatesampDopman2015)Photoperiodalsoaffectsdiapause inRhagoletis (Prokopy1968)Thusitispossiblethatlongerphotoperiodsexpe‐riencedbylarvalapplefliesorbytheirmothersmaycausethemtoecloseearlierthanhawthornfliesinamannernotpredictedbytheirgenotypesHowevertemperatureandthelengthoftheprewinterperiodfollowingpupariationhavebeenshowntoexertalargerin‐fluence than photoperiod on eclosion phenotype in R pomonella (FilchakRoetheleampFeder2001)MoreoverDambroskiandFeder(2007)controlledforphotoperiodandverifiedthatthereisageneticbasisforappleflieseclosingearlierthanhawthornfliesincludingattheGrantsiteWithrespecttoepistasisitispossiblethatinterac‐tionsbetweenSNPsonchromosomes1ndash3mightcauseapplefliestoecloseearlierthanhawthornfliesElevatedLDbetweentheselocimightbeexpectedifstrongepistaticinteractionsbetweenchromo‐somesinfluencedthetraitHoweverconsideringthehostracesto‐getherwedidnotfindstrongLDbetweeneclosion‐associatedSNPsresidingondifferentchromosomes(datanotshown)Furtherstudyisneededtoresolvethegeneticbasisfortheearliereclosionofappleflies at theGrant andFennville sites althoughwe consider unde‐tectedlociasperhapsthemostlikelyexplanationatthecurrenttime

It also remains to be determinedwhy apple flies atDowagiaceclose relatively late in controlled laboratory rearing experiments(Figure 3a) and as predicted genetically (Figure 3d) yet in naturetheyremaindivergedfromhawthornfliesforchromosome1ndash3al‐leles (Figure 3bc) Apples collected atDowagiac fruited relativelylateintheseasonandatamoresimilartimetohawthorns(seesite3inFigure2ac)whichmaycontributetothegreaterphenotypicsim‐ilarityineclosiontimebetweenDowagiacappleandhawthornfliesrelativetoothersites(Figure3a)ThusallochronicisolationshouldbereducedatDowagiacbutapplefliesstillmaintainsignificantallelefrequencydifferencesforSNPsassociatedwitheclosiontimethere(Figure3bc)Additionalfield‐basedhostfidelitystudiesandGWASforother traits suchashostodorpreference areneeded to fullycharacterizedifferentiationbetweenhostracesatthissite

43emsp|emspGeographic variation not explained by eclosion time

Our results also raise questions concerningwhy genomic regionsother than thoseharboring eclosion variation (chromosomes1ndash3)displaysignificantgeographicvariationForexample thehighandintermediateLDclassesofSNPsonchromosome4showsignificantgeographicvariationintheapplerace(Table1)butarenotassociatedwitheclosiontime(Table2)ThesameistrueinthehawthornraceforthelowLDclassofSNPsonchromosomes4and5(Tables1and2)Theseresultssuggestthatotherfactorsinadditiontoeclosion


time impose differential selection across theMidwest In this re‐gard previous studies imply that the length of the prewinter pe‐riodalsodifferentiallyselectsontheintensityatwhichfliesinitiallyenter pupal diapause (Egan et al 2015 Feder RoetheleWlazloampBerlocher 1997) The prewinter period is longer for apple thanhawthornfliesandalsovarieswithlatitude(lengtheningfromnorthtosouth)resultinginbothgeographicandhost‐relateddifferentialselection on initial diapause intensity (Dambroski amp Feder 2007)Moreover initialdiapauseintensitymaybeunderseparategeneticcontrolfromeclosiontimeatleastinhawthornflies(Raglandetal2017)Thusselectiononotheraspectsofthediapausephenotypemightaccountforthesignificantgeographicvariationnotexplainedbyeclosiontime

5emsp |emspCONCLUSION

Inconclusionwehaveshownthatselectiononeclosiontimevari‐ation significantly sculpts geographic and host‐related genomicdifferentiation in the apple and hawthorn races of R pomonella Diapause lifehistoryadaptation in therecently formedappleraceappears tohavebeenextracted in significantpart fromstandinggeneticvariation in theancestralhawthornraceThusour resultshighlight the potential inherent in geographic clines to contributetotheoriginsofnewbiodiversitywhennewresourceopportunitiesbecomeavailableHoweverdetailsconcerninghowthisprocesshasresultedinappleflieseclosingearlierthanhawthornfliesparticu‐larlyat themorenorthernGrantandFennville sites remain toberesolvedRegardlessinRhagoletispolygenicstandingvariationpar‐ticularlyinputativeinvertedregionsappearstobeduetoahistoryofgeographic isolationandsecondarycontact (Federetal2005FederBerlocheretal2003)HowgeneralthisisforothermodelsystemsofecologicalspeciationremainstobedeterminedSimilarlywhether selection haswidespread genomic effects on divergenceinotherorganisms as itdoes inRhagoletis orhas simplergeneticunderpinnings and consequences for facilitating speciation is anopenquestionNeverthelessitisclearthatintraspecificclinalvari‐ationinlifehistorytimingisextensiveinotherorganismsincludingplants (BlackmanMichaelsampRieseberg2011Chenet al 2012Kawkakamietal2011KellerLevsenOlsonampTiffin2012LowryampWillis2010)vertebrates(Johnsenetal2007OrsquoMalleyFordampHard2010)and insects (AdrionHahnampCooper2015BradfordampRoff1995BradshawQuebodeauxampHolzapfel2003KyriacouPeixotoSandrelliCostaampTauber2008LankinenTyukmaevaampHoikkala 2013 Lehmann Lyytinen Piiroinenamp Lindstroumlm2015Levy et al 2015 Masaki 1978 Paolucci Zande amp Beukeboom2013Roff1980Wanget al2012)Howeverwhetherandhowthisintraspecificclinalvariationistransformedintointerspecificdi‐vergence are lesswell documented Itwould seem that divergentselectiononstandingvariationinlifehistorytimingcouldbeaprimeaxisforcontributingtotheadaptationoforganismstonewhabitatsand environments (review by Taylor amp Friesen 2017) potentiallygeneratingreproductiveisolationandawealthofnewbiodiversity

ACKNOWLEDG MENTS

WethankMMGloverLAMillerJJSmithandCTaitfortheirassistanceandCSmadjaandtwoanonymousreviewersforusefulcommentsforimprovingthemanuscriptThisworkwassupportedbytheUnivNotreDameGenomicsandBioinformaticsCoreFacilitythe Univ of Notre Dame Advanced Diagnostics amp TherapeuticsInitiative(SPEJLF)theUnivofNotreDameEnvironmentalChangeInitiative (GJR SPE JLF) the Florida Agricultural ExperimentStation (DAH) a Food and Agriculture AssociationInternationalAtomicEnergyAgencyCoordinatedResearchProjectinDormancyManagement to EnableMass‐rearing (DAH) a Rice Univ HuxleyFacultyFellowship(SPE)NationalScienceFoundationgrantstoGJRSPEDAH and JLF (DEB‐1638951DEB‐1638997DEB‐1639005andIOS‐1700773andIOS‐1257298)andaUSDeptofAgriculturegrant to JLF (NIFA 2015‐67013‐23289) PN was supported by aEuropeanResearchCouncilStarterGrant (NatHisGenR129639)PLwasfundedbyNationalScienceFoundationResearchExperienceforUndergraduates(IIS‐1560363)

CONFLIC TS OF INTERE S T

Allauthorsconfirmthattheyhavenoconflictsofinterest

AUTHOR CONTRIBUTIONS

MMDSPEGJRPJMGRHTHQPDAHSHBPN and JFLde‐velopedtheconceptualframeworkandexperimentaldesignSPEPJMGRHandTHQPcollectedthedataMMDGJRPLandJLFperformed analyses and all authors contributed to manuscriptdrafting

DATA ACCE SSIBILIT Y

DatafromtheeclosionGWASwerepreviouslydepositedinDRYAD(httpsdoiorg105061dryadkn568)Allnewsequencedatagen‐eratedforthisstudyaredepositedinDRYAD(httpsdoiorgdryadk42t7g2)

ORCID

Meredith M Doellman httpsorcidorg0000‐0002‐0903‐6590

R E FE R E N C E S

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AbbottRJBartonNHampGoodJM(2016)Genomicsofhybrid‐ization and its evolutionary consequencesMolecular Ecology 252325ndash2332httpsdoiorg101111mec13685

AdrionJRHahnMWampCooperBS(2015)RevisitingclassicclinesinDrosophila melanogasterintheageofgenomicsTrends in Genetics31434ndash444httpsdoiorg101016jtig201505006


ArnoldML (1997)Natural hybridization and evolutionNewYorkNYOxfordUniversityPress

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BartonNHampHewittGM(1981)Achromosomalclineinthegrass‐hopper Podisma pedestris Evolution 35 1008ndash1018 httpsdoiorg101111j1558‐56461981tb04966x

Barton N H amp Hewitt G M (1985) Analysis of hybrid zonesAnnual Review of Ecology and Systematics16 113ndash148 httpsdoiorg101146annureves16110185000553

BerglandAOToblerRGonzaacutelezJSchmidtPampPetrovD(2016)Secondarycontactandlocaladaptationcontributetogenome‐widepatterns of clinal variation in Drosophila melanogaster Molecular Ecology251157ndash1174httpsdoiorg101111mec13455

Berlocher S H amp Feder J L (2002) Sympatric speciation in phy‐tophagous insects Moving beyond controversy Annual Review of Entomology 47 773ndash815 httpsdoiorg101146annurevento47091201145312

BernerDampSalzburgerW(2015)Thegenomicsoforganismaldiver‐sification illuminatedbyadaptive radiationsTrends in Genetics31491ndash499httpsdoiorg101016jtig201507002

BlackmanBKMichaelsSDampRiesebergLH (2011)ConnectingthesuntofloweringinsunfloweradaptationMolecular Ecology203503ndash3512httpsdoiorg101111j1365‐294X201105166x

BradfordM JampRoffDA (1995)Genetic and phenotypic sourcesof life history variation along a cline in voltinism in the cricketAllonemobius socius Oecologia 103 319ndash326 httpsdoiorg101007BF00328620

BradshawWEQuebodeauxMCampHolzapfelCM(2003)Circadianrhythmicity and photoperiodism in the pitcher‐plant mosquitoAdaptiveresponsetothephoticenvironmentorcorrelatedresponseto the seasonal environment American Naturalist 161 735ndash748httpsdoiorg101086374344

BrawandDWagnerC E Li Y IMalinskyM Keller I Fan ShellipDiPalmaF(2014)ThegenomicsubstrateforadaptiveradiationinAfricancichlidfishNature513375ndash381httpsdoiorg101038nature13726

Bush G (1966) The taxonomy cytology and evolution of the genusRhagoletisinNorthAmerica(DipteraTephritidae)Bulletin of the Museum of Comparative ZoologyHarvardUnivCambridgeMA134431ndash562

ChenJKaumlllmanTMaXGyllenstrandNZainaGMorganteMhellip LascouxM (2012)Disentangling the roles of history and localselection in shaping clinal variation of allele frequencies and geneexpression inNorwayspruce (Picea abies)Genetics191865ndash881httpsdoiorg101534genetics112140749

ColosimoPFHosemannKEBalabhadraSVillarrealGDicksonM Grimwood J hellip Kingsley DM (2005)Widespread parallelevolution in sticklebacks by repeated fixation of Ectodysplasinalleles Science 307 1928ndash1933 httpsdoiorg101126science1107239

DambroskiHRampFederJL (2007)Hostplantandlatitude‐relateddiapause variation inRhagoletis pomonella A test formultifacetedlife history adaptation on different stages of diapause develop‐ment Journal of Evolutionary Biology 20 2101ndash2112 httpsdoiorg101111j1420‐9101200701435x

DambroskiHLinnCBerlocherSForbesARoelofsWampFederJ(2005)ThegeneticbasisforfruitodordiscriminationinRhagoletisflies and its significance for sympatric host shifts Evolution 591953ndash1964httpsdoiorg101111j0014‐38202005tb01065x

DeanRWampChapmanP J (1973)Bionomics of the apple maggot in eastern New YorkIthacaNYCornellUniversity

DePristoM A Banks E Poplin R Garimella K VMaguire J RHartlChellipDalyMJ (2011)Aframeworkforvariationdiscoveryandgenotypingusingnext‐generationDNAsequencingdataNature Genetics43491ndash498httpsdoiorg101038ng806

EganSPRaglandGJAssourLPowellTHQHoodGREmrichShellipFederJL(2015)Experimentalevidenceofgenome‐wideimpactofecologicalselectionduringearlystagesofspeciation‐with‐gene‐flowEcology Letters18817ndash825httpsdoiorg101111ele12460

EndlerJA(1977)Geographic variation speciation and clinesPrincetonNJPrincetonUniversityPress

FederJLBerlocherSHRoetheleJBDambroskiHSmithJJPerryWLhellipAlujaM(2003)Allopatricgeneticoriginsforsympat‐richost‐plantshiftsandraceformationinRhagoletis Proceedings of the National Academy of Sciences U S A10010314ndash10319httpsdoiorg101073pnas1730757100

FederJLampBushGL (1989)GenefrequencyclinesforhostracesofRhagoletis pomonellainthemidwesternUSAHeredity (Edinb)63245ndash266httpsdoiorg101038hdy198998

FederJLChilcoteCAampBushGL(1988)GeneticdifferentiationbetweensympatrichostracesoftheapplemaggotflyRhagoletis po‐monella Nature33661ndash64httpsdoiorg101038336061a0

Feder J LChilcoteCAampBushG L (1989) Inheritance and link‐age relationships of allozymes in the apple maggot fly Journal of Heredity 80 277ndash283 httpsdoiorg101093oxfordjournalsjhereda110854

FederJLChilcoteCAampBushGL (1990)Thegeographicpatternof genetic differentiation between host associated populations ofRhagoletis pomonella(DipteraTephritidae)intheEasternUnitedStatesandCanadaEvolution44570ndash594httpsdoiorg1023072409436

FederJHuntTampBushG(1993)Theeffectsofclimatehost‐plantphe‐nologyandhostfidelityonthegeneticsofappleandhawthorninfestingracesofRhagoletis pomonella Entomologia Experimentalis Et Applicata69117ndash135httpsdoiorg101111j1570‐74581993tb01735x

Feder J L Opp S BWlazlo B Reynolds K GoW amp Spisak S(1994) Host fidelity is an effective premating barrier betweensympatricracesoftheapplemaggotflyProceedings of the National Academy of Sciences U S A917990ndash7994httpsdoiorg101073pnas91177990

FederJLRoetheleJBFilchakKNiedbalskiJampRomero‐SeversonJ(2003b)Evidenceforinversionpolymorphismrelatedtosympat‐richostraceformationintheapplemaggotflyRhagoletis pomonella Genetics163939ndash953

FederJLRoetheleJBWlazloBampBerlocherSH(1997)SelectivemaintenanceofallozymedifferencesamongsympatrichostracesoftheapplemaggotflyProceedings of the National Academy of Sciences U S A9411417ndash11421httpsdoiorg101073pnas942111417

FederJLXieXRullJVelezSForbesALeungBhellipAlujaM(2005)MayrDobzhanskyandBushand thecomplexitiesof sym‐patric speciation inRhagoletis Proceedings of the National Academy of Sciences U S A 102 6573ndash6580 httpsdoiorg101073pnas0502099102

Filchak K E Roethele J B amp Feder J L (2000) Natural selectionandsympatricdivergenceintheapplemaggotRhagoletis pomonella Nature407739ndash742httpsdoiorg10103835037578

FilchakKERoetheleJBampFederJL(2001)Effectsofphotoperiodandlightintensityonthegeneticsofdiapauseintheapplemaggot(DipteraTephritidae)Annals of the Entomological Society of America94902ndash908 httpsdoiorg1016030013‐8746(2001)094[0902EOPALI]20CO2

GompertZFordyceJAForisterMLShapiroAMampNiceCC(2006)HomoploidhybridspeciationinanextremehabitatScience3141923ndash1925httpsdoiorg101126science1135875

GompertZLucasLKBuerkleCAForisterMLFordyceJAampNiceCC(2014)Admixtureandtheorganizationofgeneticdiver‐sity in a butterfly species complex revealed through common and


raregeneticvariantsMolecular Ecology234555ndash4573httpsdoiorg101111mec12811

GompertZMandevilleEGampBuerkleCA(2017)Analysisofpop‐ulation genomic data from hybrid zonesAnnual Review of Ecology Evolution and Systematics 48 207ndash229 httpsdoiorg101146annurev‐ecolsys‐110316‐022652

Harrison R G amp Larson E L (2016) Heterogeneous genome diver‐gence differential introgression and the origin and structureof hybrid zones Molecular Ecology 25 2454ndash2466 httpsdoiorg101111mec13582

HermissonJampPenningsPS(2005)SoftsweepsMolecularpopula‐tiongeneticsofadaptationfromstandinggeneticvariationGenetics1692335ndash2352httpsdoiorg101534genetics104036947

HewittGM (1988)Hybrid zones‐natural laboratories for evolution‐arystudiesTrends in Ecology and Evolution3158ndash167httpsdoiorg1010160169‐5347(88)90033‐X

Hood G R Forbes A A Powell T H Q Egan S P HamerlinckG Smith J J amp Feder J L (2015) Sequential divergence andthemultiplicativeoriginof communitydiversityProceedings of the National Academy of Sciences U S A112E5980ndashE5989httpsdoiorg101073pnas1424717112

HopkinsRampRausherMD(2011)IdentificationoftwogenescausingreinforcementintheTexaswildflowerPhlox drummondii Nature469411ndash414httpsdoiorg101038nature09641

HopkinsRampRausherMD (2012)Pollinator‐mediatedselectiononflower color allele drives reinforcement Science335 1090ndash1092httpsdoiorg101126science1215198

Hut R A Paolucci S Dor R Kyriacou C P amp Daan S (2013)Latitudinal clines An evolutionary view on biological rhythmsProceedings of the Royal Society B‐Biological Sciences28020130433httpsdoiorg101098rspb20130433

HuxleyJ (1938)ClinesAnauxiliarytaxonomicprincipleNature142219ndash220httpsdoiorg101038142219a0

Jeffery N W Stanley R R E Wringe B F Guijarro‐Sabaniel JBourretVBernatchezLhellipBradburyIR(2017)Range‐wideparal‐lelclimate‐associatedgenomicclinesinAtlanticsalmonRoyal Society Open Science4171394httpsdoiorg101098rsos171394

JigginsCDampMalletJ(2000)BimodalhybridzonesandspeciationTrends in Ecology amp Evolution15250ndash255httpsdoiorg101016S0169‐5347(00)01873‐5

JohnsenAFidlerAEKuhnSCarterKLHoffmannABarrIRhellipKempenaersB(2007)AvianClockgenepolymorphismEvidencefor a latitudinal cline in allele frequenciesMolecular Ecology 164867ndash4880httpsdoiorg101111j1365‐294X200703552x

JonesFCGrabherrMGChanYFRussellPMauceliEJohnsonJhellipKingsleyDM (2012) The genomicbasis of adaptive evolu‐tion in threespine sticklebacks Nature 484 55ndash61 httpsdoiorg101038nature10944

KangJHSchartlMWalterRBampMeyerA(2013)Comprehensivephy‐logeneticanalysisofallspeciesofswordtailsandplaties(PiscesGenusXiphophorus) uncovers ahybridoriginof a swordtail fishXiphophorus monticolusanddemonstratesthatthesexuallyselectedswordoriginatedintheancestrallineageofthegenusbutwaslostagainsecondarilyBMC Evolutionary Biology1325httpsdoiorg1011861471‐2148‐13‐25

KawkakamiTMorganTJNippertJBOcheltreeTWKeithRDhakalPampUngererMC(2011)NaturalselectiondrivesclinallifehistorypatternsintheperennialsunflowerspeciesHelianthus maximiliani Molecular Ecology202318ndash2328httpsdoiorg101111j1365‐294X201105105x

KellerSRLevsenNOlsonMSampTiffinP(2012)Localadaptationin the flowering‐timegenenetworkofBalsamPoplarPopulus bal‐samiferaLMolecular Biology and Evolution293143ndash3152httpsdoiorg101093molbevmss121

KruukLEBBairdSJEGaleKSampBartonNH(1999)Acompar‐isonofmultilocusclinesmaintainedbyenvironmentaladaptationorbyselectionagainsthybridsGenetics1531959ndash1971

KyriacouCPPeixotoAASandrelliFCostaRampTauberE(2008)ClinesinclockgenesFine‐tuningcircadianrhythmstotheenviron‐ment Trends in Genetics 24 124ndash132 httpsdoiorg101016jtig200712003

LamichhaneySBerglundJAlmeacutenMSMaqboolKGrabherrMMartinez‐BarrioAhellipAndersson L (2015)EvolutionofDarwinrsquosfinchesandtheirbeaksrevealedbygenomesequencingNature518371ndash375httpsdoiorg101038nature14181

Lamichhaney S Han FWebster M T Andersson L Grant B RampGrant P R (2018) Rapid hybrid speciation inDarwinrsquos finchesScience359224ndash228httpsdoiorg101126scienceaao4593

LankinenPTyukmaevaVIampHoikkalaA(2013)NorthernDrosophila montana flies show variation both within and between cline pop‐ulations in the critical day length evoking reproductive diapauseJournal of Insect Physiology59 745ndash751 httpsdoiorg101016jjinsphys201305006

LehmannPLyytinenAPiiroinenSampLindstroumlmL(2015)LatitudinaldifferencesindiapauserelatedphotoperiodicresponsesofEuropeanColorado potato beetles (Leptinotarsa decemlineata) Evolutionary Ecology29269ndash282httpsdoiorg101007s10682‐015‐9755‐x

Levy R KozakGampDopman E (2018)Non‐pleiotropic coupling ofdaily and seasonal temporal isolation in theEuropeanCornBorerGenes (Basel)9180httpsdoiorg103390genes9040180

LevyRCKozakGMWadsworthCBCoatesBSampDopmanEB (2015) Explaining the sawtooth Latitudinal periodicity in a cir‐cadiangenecorrelateswithshifts ingenerationnumberJournal of Evolutionary Biology2840ndash53httpsdoiorg101111jeb12562

LohY‐H‐EBezault EMuenzel FM Roberts RB SwoffordRBarluengaMhellipStreelman JT (2013)Originsof sharedgeneticvariationinAfricancichlidsMolecular Biology and Evolution30906ndash917httpsdoiorg101093molbevmss326

LowryDBampWillisJH(2010)Awidespreadchromosomalinversionpolymorphism contributes to a major life‐history transition localadaptation and reproductive isolationPLoS Biology8 e1000500httpsdoiorg101371journalpbio1000500

Lyons‐SobaskiSampBerlocherSH(2009)Lifehistoryphenologydiffer‐encesbetweensouthernandnorthernpopulationsoftheapplemag‐gotflyRhagoletis pomonella Entomologia Experimentalis Et Applicata130149ndash159httpsdoiorg101111j1570‐7458200800805x

Mallet J (2007)HybridspeciationNature446279ndash283httpsdoiorg101038nature05706

MalletJBartonNLamasGSantistebanJMuedasMampEeleyH(1990)Estimatesof selectionandgene flow frommeasuresofclinewidthand linkagedisequilibrium inHeliconiushybridzonesGenetics124921ndash936

Masaki S (1978) Climatic adaptation and species status in the lawnground cricket Oecologia 35 343ndash356 httpsdoiorg101007BF00345141

MavaacuterezJSalazarCABerminghamESalcedoCJigginsCDampLinaresM(2006)SpeciationbyhybridizationinHeliconiusbutter‐fliesNature441868ndash871httpsdoiorg101038nature04738

McKenna A Hanna M Banks E Sivachenko A Cibulskis KKernytskyAhellipDePristoMA(2010)Thegenomeanalysistool‐kit A MapReduce framework for analyzing next‐generation DNAsequencing data Genome Research 20 1297ndash1303 httpsdoiorg101101gr107524110

MeyersPJPowellTHQWaldenKKOSchiefereckeAJFederJLHahnDAhellipRaglandGJ(2016)Divergenceofthediapausetranscriptome in apple maggot flies Winter regulation and post‐winter transcriptional repression Journal of Experimental Biology219(17)2613ndash2622httpsdoiorg101242jeb140566

MichelAPRullJAlujaMampFederJL(2007)Thegeneticstructureof hawthorn‐infestingRhagoletis pomonella populations inMexicoImplicationsforsympatrichostraceformationMolecular Ecology162867ndash2878httpsdoiorg101111j1365‐294X200703263x


MichelAPSimSPowellTHQTaylorMSNosilPampFederJL(2010)Widespreadgenomicdivergenceduringsympatricspe‐ciationProceedings of the National Academy of Sciences U S A1079724ndash9729httpsdoiorg101073pnas1000939107

OrsquoMalley KG FordM J ampHard J J (2010) Clock polymorphisminPacificsalmonEvidenceforvariableselectionalongalatitudinalgradientProceedings of the Royal Society B‐Biological Sciences2773703ndash3714httpsdoiorg101098rspb20100762

PaolucciSvandeZandeLampBeukeboomLW(2013)Adaptivelat‐itudinalclineofphotoperiodicdiapause induction in theparasitoidNasonia vitripennisinEuropeJournal of Evolutionary Biology26705ndash718httpsdoiorg101111jeb12113

PayseurBA(2010)Usingdifferentialintrogressioninhybridzonestoiden‐tifygenomicregionsinvolvedinspeciationMolecular Ecology Resources10806ndash820httpsdoiorg101111j1755‐0998201002883x

Pease J B Haak D C Hahn M W amp Moyle L C (2016)PhylogenomicsrevealsthreesourcesofadaptivevariationduringarapidradiationPLoS Biology14e1002379httpsdoiorg101371journalpbio1002379

PowellTHQChaDHLinnCEJrampFederJL(2012)Onthescent of standing variation for speciation Behavioral evidencefor native sympatric host races of Rhagoletis pomonella (DipteraTephritidae)inthesouthernUnitedStatesEvolution662739ndash2756httpsdoiorg101111j1558‐5646201201625x

PowellTHQForbesAAHoodGRampFederJL(2014)Ecologicaladaptation and reproductive isolation in sympatry Genetic andphenotypicevidence fornativehost racesofRhagoletis pomonella Molecular Ecology23688ndash704httpsdoiorg101111mec12635

ProkopyRJ (1968) Influenceofphotoperiod temperatureandfoodoninitiationofdiapauseintheapplemaggotCanadian Entomologist100318ndash329httpsdoiorg104039Ent100318‐3

RCoreTeam(2017)R A language and environment for statistical comput‐ingViennaAustriaRFoundationforStatisticalComputing

RaglandG JDoellmanMMMeyers P JHoodGR Egan S PPowellTHQhellipFederJL (2017)Atestofgenomicmodularityamonglife‐historyadaptationspromotingspeciationwithgeneflowMolecular Ecology263926ndash3942httpsdoiorg101111mec14178

RaglandG J Egan S P Feder J LBerlocher SHampHahnDA(2011)Developmental trajectories of gene expression reveal can‐didatesfordiapauseterminationAkeylife‐historytransitionintheapplemaggotflyRhagoletis pomonella Journal of Experimental Biology2143948ndash3959httpsdoiorg101242jeb061085

RiesebergLHampWillisJH(2007)PlantspeciationScience317910ndash914httpsdoiorg101126science1137729

RoestiMGavriletsSHendryAPSalzburgerWampBernerD(2014)ThegenomicsignatureofparalleladaptationfromsharedgeneticvariationMolecular Ecology233944ndash3956httpsdoiorg101111mec12720

RoffD(1980)OptimizingdevelopmenttimeinaseasonalenvironmentThe ldquoups and downsrdquo of clinal variation Oecologia 45 202ndash208httpsdoiorg101007BF00346461

RullJAlujaMFederJampBerlocherS (2006)Distributionandhostrangeofhawthorn‐infestingRhagoletis(DipteraTephritidae)inMexicoAnnals of the Entomological Society of America99662ndash672httpsdoiorg1016030013‐8746(2006)99[662DAHROH]20CO2

SalzburgerWBaricSampSturmbauerC (2002)Speciationvia intro‐gressivehybridizationinEastAfricancichlidsMolecular Ecology11619ndash625httpsdoiorg101046j0962‐1083200101438x

SchluterDampConteGL (2009)GeneticsandecologicalspeciationProceedings of the National Academy of Sciences U S A106 9955ndash9962httpsdoiorg101073pnas0901264106

SchwarzDMattaBMShakir‐BotteriNLampMcPheronBA(2005)HostshifttoaninvasiveplanttriggersrapidanimalhybridspeciationNature436546ndash549httpsdoiorg101038nature03800

Slatkin M (1973) Gene flow and selection in a cline Genetics 75733ndash756

SteinerCCWeberJNampHoekstraHE(2007)AdaptivevariationinbeachmiceproducedbytwointeractingpigmentationgenesPLoS Biology5e219httpsdoiorg101371journalpbio0050219

SzymuraJMampBartonNH(1986)Geneticanalysisofahybridzonebetween the fire‐bellied toadsBombina bombina andB variegatanearCracowinSouthernPolandEvolution401141ndash1159httpsdoiorg101111j1558‐56461986tb05740x

SzymuraJMampBartonNH(1991)Thegeneticstructureofthehy‐bridzonebetweenthefire‐belliedtoadsBombina bombinaandB var‐iegataComparisonsbetweentransectsandbetweenlociEvolution45237ndash261httpsdoiorg1023072409660

Taylor R S amp Friesen V L (2017) The role of allochrony in specia‐tion Molecular Ecology 26 3330ndash3342 httpsdoiorg101111mec14126

TheHeliconiusGenomeConsortium (2012) Butterfly genome revealspromiscuous exchange of mimicry adaptations among speciesNature48794ndash98httpsdoiorg101038nature11041

vanrsquotHofAECampagnePRigdenDJYungCJLingleyJQuailMAhellipSaccheri I J (2016)The industrialmelanismmutation inBritishpepperedmothsisatransposableelementNature534102ndash105httpsdoiorg101038nature17951

WalshB(1867)Theapple‐wormandtheapple‐maggotJournal of the American Society for Horticultural Science2338ndash343

WangX‐PYangQ‐SDalinPZhouX‐MLuoZ‐WampLeiC‐L(2012)GeographicvariationinphotoperiodicdiapauseinductionanddiapauseintensityinSericinus montelus(LepidopteraPapilionidae)Insect Science19295ndash302httpsdoiorg101111j1744‐7917201101473x

WeirBS(1979)InferencesaboutlinkagedisequilibriumBiometrics35235ndash254httpsdoiorg1023072529947

Xie X Michel A P Schwarz D Rull J Velez S Forbes A A hellipFeder J L (2008) Radiation and divergence in the Rhagoletis pomonella species complex Inferences from DNA sequencedata Journal of Evolutionary Biology 21 900ndash913 httpsdoiorg101111j1420‐9101200801507x

XieXRullJMichelAPVelezSForbesAALoboNFhellipFederJL(2007)Hawthorn‐infestingpopulationsofRhagoletis pomonella inMexicoandspeciationmodepluralityEvolution611091ndash1105httpsdoiorg101111j1558‐5646200700091x

YakimowskiSBampRiesebergLH(2014)Theroleofhomoploidhy‐bridization inevolutionAcenturyofstudiessynthesizinggeneticsandecologyAmerican Journal of Botany1011247ndash1258httpsdoiorg103732ajb1400201

YeamanS(2015)LocaladaptationbyallelesofsmalleffectAmerican Naturalist186(S1)S74ndashS89httpsdoiorg101086682405

YuanY‐WSagawaJMYoungRCChristensenBJampBradshawHD(2013)GeneticdissectionofamajoranthocyaninQTLcontrib‐uting to pollinator‐mediated reproductive isolation between sisterspeciesofMimulus Genetics194255ndash263httpsdoiorg101534genetics112146852

SUPPORTING INFORMATION

Additional supporting information may be found online in theSupportingInformationsectionattheendofthearticle

How to cite this articleDoellmanMMEganSPRaglandGJetalStandinggeographicvariationineclosiontimeandthegenomicsofhostraceformationinRhagoletis pomonella fruitfliesEcol Evol 20199393ndash409 httpsdoiorg101002ece34758


1emsp |emspINTRODUC TION

The rawmaterial for novel adaptation can come fromnewmuta‐tions or standing variation (Barrett amp Schluter 2008) Adaptationis expected to occurmore rapidlywhen based on standing varia‐tionbecausetheprocess isnot limitedbywaittimefornewben‐eficial mutations to arise and standing variants are likely to havebeenfilteredfornegativepleiotropicfitnesseffectsordetrimentalepistatic interactions (Barrett amp Schluter 2008 Yeaman 2015)Therefore taxaharboringhigh levelsofstandinggeneticvariationmaybemore likely toadapt to rapidenvironmental shiftsandex‐perienceecological speciation than those requiringnew favorablemutationstorespondIndeedmyriadexamplesareaccumulatingofrapidadaptiveevolutionfueledbystandingvariationincludingcoatcolorintheoldfieldmousePeromyscus polionotus(SteinerWeberampHoekstra2007)reduceddefensivearmorinthethreespinestick‐lebackfishGasterosteus aculeatus(Colosimoetal2005)mimicryinHeliconius butterflies (TheHeliconiusGenomeConsortium2012)and beak morphology in Darwinrsquos Finches (Lamichhaney et al2015)Standingvariationmaybeespeciallyrelevantincaseswhereadaptation involvespolygenic traits and themajorityof favorablealleleshaveonlyminoreffectsonfitnessandgenomicincompatibil‐ity(BarrettampSchluter2008HermissonampPennings2005SchluterampConte2009)Largestoresofstandinggeneticvariationobservedinsomepopulationsmaybeaproductofcomplexevolutionaryhis‐toriesincludingpastgeneflowcoupledwithecologicalselectiontomaintainhighlevelsofgeneticvariationresultingfromtrackinglocalregionalor temporaldifferences inenvironmentalorbioticcondi‐tions(BerglandToblerGonzaacutelezSchmidtampPetrov2016BernerampSalzburger 2015Brawandet al 2014 FederBerlocher et al2003GompertFordyceForisterShapiroampNice2006Jefferyetal 2017 Jones et al 2012 Lamichhaney et al 2015 Loh et al2013PeaseHaakHahnampMoyle2016RoestiGavriletsHendrySalzburger amp Berner 2014 The Heliconius Genome Consortium2012)

Reservoirs of standing genetic variation are often maintainedacrossgeographicclinesgradientsofphenotypicorgeneticchange

inpopulationsacross space that canprovidewindows intounder‐standing adaptive evolution and speciation (Endler 1977 Huxley1938) Geographic clines can be either primary due to selectionacross a landscape or secondary as a result of population subdi‐visionfollowedbyhybridizationandintrogressionof loci Ineithercaseanalysisof thedifferential spatialdistributionofphenotypesand loci can help identify traits and genes involved in adaptationthatmaycontributetoreproductiveisolation(BartonampGale1993Barton amp Hewitt 1981 1985 Gompert Mandeville amp Buerkle2017HarrisonampLarson2016JigginsampMallet2000KruukBairdGale amp Barton 1999Mallet et al 1990 Payseur 2010 Slatkin1973 SzymuraampBarton19861991)However clines canbe in‐dicativeofmorethanjustpastprogresstowardspeciationbutalsobe natural experiments in the speciation process itselfwith geneflow providing an input of new material facilitating divergence(Abbott et al 2013 Arnold 1997 Endler 1977 Gompert et al2014HarrisonampLarson2016Hewitt1988Mallet2007)Inthisregard hybridization can createnovel genotypes thatmay rapidlyleadtodifferentiationfromnearbyparentaltaxawhenhybridsoc‐cupyunderusednichesorenvironmentsviapolyploidorhomoploidmechanisms(Gompertetal2006KangSchartlWalterampMeyer2013Lamichhaneyetal2018Mavaacuterezetal2006RiesebergampWillis2007SalzburgerBaricampSturmbauer2002SchwarzMattaShakir‐BotteriampMcPheron2005YakimowskiampRieseberg2014)Inadditionhybridizationneednothaveanimmediateeffectongen‐eratingnewtaxa(AbbottBartonampGood2016)butcanalsocreateandmaintainextensivestandingvariationenablingdivergenceatalatertimewhenpopulationsexperiencenewecologicalopportuni‐ties(BernerampSalzburger2015)

Rhagoletis pomonella (DipteraTephritidae)amodelforpopula‐tion divergence in sympatry provides an avenue for investigatingtheroleofstandingclinalvariationinrapidadaptationandspecia‐tioninresponsetonovelecologicalopportunity(BerlocherampFeder2002) Ancestral R pomonella infested the fruits of native NorthAmericanhawthorns(Crataegusspp)andshifted(lt170yearsago)todomesticatedapple(Malus domestica)aftertheplantwasintroducedby European settlers ~400years ago (Bush 1966 Walsh 1867)

phenologyallelesonchromosomes2and3associatedwithearlieremergencewereparadoxicallyatlowerfrequencyintheapplethanhawthornhostraceacrossallfoursympatricsitesHoweverlocionchromosome1didshowhigherfrequenciesofearlyeclosion‐associatedallelesintheapplethanhawthornhostraceatthetwosouthernsitespotentiallyaccountingfortheirearliereclosionphenotypeThusalthoughex‐tensiveclinalgeneticvariationintheancestralhawthornraceexistsandcontributedtothehostshifttoapplefurtherstudyisneededtoresolvedetailsofhowthisstand‐ingvariationwasselected togenerateearliereclosingapple flypopulations in theNorth

K E Y W O R D S

clinalvariationeclosiontimeecologicalspeciationhostracesstandingvariation






0 200 400 600 800km

N


Urbana IL (4)

5

67

8

91011 12

13 Chiapas MX

EVTM MX SMO MX

Texas USA

Midwest USA


















(a)

(b)

(c)

(N)

rr

dfpp

pp

pp

df

rr

rr

dfdf

dfdf

(N)








Mea

n ec

losi

on ti

me

40 41 42 43 4455

60

65

70

75

80 UrbanaDowagiac

FennvilleGrant

(a) Eclosion time

HawthornApple

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010


Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(c) Chromosome 1

Latitude

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010




3emsp |emspRESULTS





r p df



Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

102(006) 389(011) 431(013) 291(009) 93(006) 242(009)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

72(006) 566(014) 861(024) 786(017) 67(005) 332(011)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

116(006) 386(010) 352(010) 403(010) 115(006) 247(008)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

102(006) 149(007) 149(007) 128(006) 81(006) 118(006)

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

544(014) 504(011) 477(011) 120(006) 154(006) 370(010)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

920(025) 822(016) 839(020) 48(005) 171(007) 583(015)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

455(011) 471(011) 421(009) 120(006) 142(006) 348(009)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

156(005) 207(006) 207(007) 149(006) 158(006) 170(006)












Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018







ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S






















































































































0 200 400 600 800km

N


Urbana IL (4)

5

67

8

91011 12

13 Chiapas MX

EVTM MX SMO MX

Texas USA

Midwest USA


















(a)

(b)

(c)

(N)

rr

dfpp

pp

pp

df

rr

rr

dfdf

dfdf

(N)








Mea

n ec

losi

on ti

me

40 41 42 43 4455

60

65

70

75

80 UrbanaDowagiac

FennvilleGrant

(a) Eclosion time

HawthornApple

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010


Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(c) Chromosome 1

Latitude

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010




3emsp |emspRESULTS





r p df



Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

102(006) 389(011) 431(013) 291(009) 93(006) 242(009)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

72(006) 566(014) 861(024) 786(017) 67(005) 332(011)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

116(006) 386(010) 352(010) 403(010) 115(006) 247(008)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

102(006) 149(007) 149(007) 128(006) 81(006) 118(006)

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

544(014) 504(011) 477(011) 120(006) 154(006) 370(010)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

920(025) 822(016) 839(020) 48(005) 171(007) 583(015)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

455(011) 471(011) 421(009) 120(006) 142(006) 348(009)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

156(005) 207(006) 207(007) 149(006) 158(006) 170(006)












Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018







ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S

































































































































(a)

(b)

(c)

(N)

rr

dfpp

pp

pp

df

rr

rr

dfdf

dfdf

(N)








Mea

n ec

losi

on ti

me

40 41 42 43 4455

60

65

70

75

80 UrbanaDowagiac

FennvilleGrant

(a) Eclosion time

HawthornApple

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010


Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(c) Chromosome 1

Latitude

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010




3emsp |emspRESULTS





r p df



Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

102(006) 389(011) 431(013) 291(009) 93(006) 242(009)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

72(006) 566(014) 861(024) 786(017) 67(005) 332(011)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

116(006) 386(010) 352(010) 403(010) 115(006) 247(008)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

102(006) 149(007) 149(007) 128(006) 81(006) 118(006)

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

544(014) 504(011) 477(011) 120(006) 154(006) 370(010)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

920(025) 822(016) 839(020) 48(005) 171(007) 583(015)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

455(011) 471(011) 421(009) 120(006) 142(006) 348(009)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

156(005) 207(006) 207(007) 149(006) 158(006) 170(006)












Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018







ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S

























































































































(a)

(b)

(c)

(N)

rr

dfpp

pp

pp

df

rr

rr

dfdf

dfdf

(N)








Mea

n ec

losi

on ti

me

40 41 42 43 4455

60

65

70

75

80 UrbanaDowagiac

FennvilleGrant

(a) Eclosion time

HawthornApple

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010


Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(c) Chromosome 1

Latitude

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010




3emsp |emspRESULTS





r p df



Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

102(006) 389(011) 431(013) 291(009) 93(006) 242(009)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

72(006) 566(014) 861(024) 786(017) 67(005) 332(011)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

116(006) 386(010) 352(010) 403(010) 115(006) 247(008)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

102(006) 149(007) 149(007) 128(006) 81(006) 118(006)

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

544(014) 504(011) 477(011) 120(006) 154(006) 370(010)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

920(025) 822(016) 839(020) 48(005) 171(007) 583(015)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

455(011) 471(011) 421(009) 120(006) 142(006) 348(009)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

156(005) 207(006) 207(007) 149(006) 158(006) 170(006)












Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018







ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S
























































































































Mea

n ec

losi

on ti

me

40 41 42 43 4455

60

65

70

75

80 UrbanaDowagiac

FennvilleGrant

(a) Eclosion time

HawthornApple

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010


Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010

(c) Chromosome 1

Latitude

Mea

n al

lele

freq

uenc

y

40 41 42 43 44ndash015

ndash010

ndash005

000

005

010




3emsp |emspRESULTS





r p df



Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

102(006) 389(011) 431(013) 291(009) 93(006) 242(009)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

72(006) 566(014) 861(024) 786(017) 67(005) 332(011)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

116(006) 386(010) 352(010) 403(010) 115(006) 247(008)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

102(006) 149(007) 149(007) 128(006) 81(006) 118(006)

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

544(014) 504(011) 477(011) 120(006) 154(006) 370(010)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

920(025) 822(016) 839(020) 48(005) 171(007) 583(015)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

455(011) 471(011) 421(009) 120(006) 142(006) 348(009)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

156(005) 207(006) 207(007) 149(006) 158(006) 170(006)












Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018







ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S



















































































































3emsp |emspRESULTS





r p df



Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

102(006) 389(011) 431(013) 291(009) 93(006) 242(009)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

72(006) 566(014) 861(024) 786(017) 67(005) 332(011)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

116(006) 386(010) 352(010) 403(010) 115(006) 247(008)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

102(006) 149(007) 149(007) 128(006) 81(006) 118(006)

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

544(014) 504(011) 477(011) 120(006) 154(006) 370(010)

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

920(025) 822(016) 839(020) 48(005) 171(007) 583(015)

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

455(011) 471(011) 421(009) 120(006) 142(006) 348(009)

LowLD n=128 n=87 n=174 n=235 n=221 n=845

156(005) 207(006) 207(007) 149(006) 158(006) 170(006)












Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018







ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S



























































































































Apple

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

007 072 072 014 002 042

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

049 085 037 002 006 048

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

005 065 063 013 minus001 039

LowLD n=128 n=87 n=174 n=235 n=221 n=845

minus005 043 015 003 minus003 009

Hawthorn

MapSNPs n=949 n=675 n=996 n=436 n=1188 n=4244

082 073 069 004 011 072

HighLD n=263 n=129 n=223 n=42 n=374 n=1031

059 086 041 017 minus003 084

IntLD n=558 n=459 n=599 n=159 n=593 n=2368

075 067 061 minus009 010 063

LowLD n=128 n=87 n=174 n=235 n=221 n=845

037 044 019 010 001 018







ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S






















































































































ndash04

ndash02

0

02

04

06

08

r = 032 P lt 00001 10240 df

Apple race


r p n

r p n

r p n

r p n

r p n

r p n

(a) (b)

(c) (d)

(e) (f)







(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S























































































































(a)

(b)

(c)








GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S
























































































































GrantMI





FennvilleMI





DowagiacMI





UrbanaIL




LowLD 025 003 minus010 004 007 003


























ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S









































































































































ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S






























































































































ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S





















































































































ACKNOWLEDG MENTS








ORCID


R E FE R E N C E S












































































































































































































































































































































Documents

Standing geographic variation in eclosion time and the ...nosil-lab.group.shef.ac.uk/wp-content/uploads/2019/01/Doellman_et_al-2019-Ecology_and...Shakir‐Botteri, & McPheron, 2005;