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9-8-2019 Influence of host plant, geography and strain on genomic differentiation in sympatric populations of nubilalis Brad S. Coates U.S. Department of Agriculture, [email protected]

Genevieve M. Kozak University of Massachusetts ta Dartmouth

Kyung Seok Kim Iowa State University, [email protected]

Jing Sun Iowa State University

Yangzhou Zhang JFiloinl loAcwadem thiys ofand Ag raicddultiturional Scienal wceorks s at: https://lib.dr.iastate.edu/ent_pubs Part of the Behavior and Ethology Commons, Entomology Commons, Genetics Commons, and See next page for additional authors the Population Biology Commons The ompc lete bibliographic information for this item can be found at https://lib.dr.iastate.edu/ ent_pubs/547. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the Entomology at Iowa State University Digital Repository. It has been accepted for inclusion in Entomology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Influence of host plant, geography and pheromone strain on genomic differentiation in sympatric populations of Ostrinia nubilalis

Abstract Patterns of mating for the (Ostrinia nubilalis) depend in part on variation in sex‐pheromone blend. The ar tio of (E)‐11‐ and (Z)‐11‐tetradecenyl acetate (E11‐ and Z11‐14:OAc) in the pheromone blend that females produce and males respond to differs between strains of O. nubilalis. Populations also vary in female oviposition preference for and larval performance on (C4) and non‐maize (C3) host plants. The er lative contributions of sexual and ecological trait variation to the genetic structure of O. nubilalis remains unknown. Host‐plant use (13C/14C ratios) and genetic differentiation were estimated among sympatric E and Z pheromone strain O. nubilalis males collected in sex‐pheromone baited traps at 12 locations in Pennsylvania and New York between 2007 and 2010. Among genotypes at 65 single nucleotide polymorphism marker loci, variance at a position in the pheromone gland fatty acyl‐reductase (pgfar) gene at the locus responsible for determining female pheromone ratio (Pher) explained 64% of the total genetic differentiation between males attracted to different (male response, Resp), providing evidence of sexual inter‐selection at these unlinked loci. Principal coordinate, Bayesian clustering, and distance‐based redundancy analysis (dbRDA) demonstrate that host plant history or geography does not significantly contribute to population variation or differentiation among males. In contrast, these analyses indicate that pheromone response and pgfar‐defined strain contribute significantly to population genetic differentiation. This study suggests that behavioral divergence likely plays a larger role in driving genetic variation compared to host plant‐defined ecological adaptation.

Keywords population genetics, sexual selection, reproductive isolation

Disciplines Behavior and Ethology | Ecology and Evolutionary Biology | Entomology | Genetics | Population Biology

Comments This is a manuscript of an article published as Coates, Brad S., Genevieve M. Kozak, Kyung Seok Kim, Jing Sun, Yangzhou Wang, Shelby J. Fleischer, Erik B. Dopman, and Thomas W. Sappington. "Influence of host plant, geography and pheromone strain on genomic differentiation in sympatric populations of Ostrinia nubilalis." Molecular ecology (2019). doi: 10.1111/mec.15234.

Rights Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The onc tent of this document is not copyrighted.

Authors Brad S. Coates, Genevieve M. Kozak, Kyung Seok Kim, Jing Sun, Yangzhou Zhang, Shelby J. Fleischer, Erik B. Dopman, and Thomas W. Sappington

This article is available at Iowa State University Digital Repository: https://lib.dr.iastate.edu/ent_pubs/547 Accepted Article This by is protectedarticle reserved. Allrights copyright. doi: 10.1111/mec.15234 lead to differences between this version and the throughbeen the copyediting, pagination typesetting, which process, may and proofreading hasThis article been accepted publicationfor andundergone peerfull but review not has 4 3 2 1 Section: Ecological genomics Submission: MolecularEcology Article type : Article Original DR. ERIK DOPMAN (Orcid :ID 0000-0002-8633-5527) DR. YANGZHOU WANG (Orcid : ID 0000-0001-9683-0428) DR. GENEVIEVEM.KOZAK (Orcid ID: 0000-0001-6413-1403) DR. BRAD STEVEN COATES (Orcid ID:0000-0001-8908-1529) Brad S. Coates S.Coates Brad

Shelby J. Fleischer J.Shelby Iowa State University, Department USA ofEntomology,Ames, State IA, University, Iowa State Iowa DepartmentUniversity, ofNatural Resource Ecology ofMassachusetts-Dartmouth,Department Biology, of University IA Ames, Unit, Research Genetics Crop & Corn USDA-ARS, Influence of host plant, andpheromone ofhost geography Influence differentiation in strain ongenomic USA 1* , Genevieve M. Kozak 6 , Erik B. Dopman Dopman B. , Erik sympatric populations of 7 , and Thomas W. Sappington Thomas , and 2, 7 2, , Kyung Seok Kim Kyung SeokKim , Version of Record. Please Version as this cite ofRecord. article Ostrinia nubilalis Ostrinia 3 , Jing Sun Sun , Jing and Management, Ames, IA, and Management, Dartmouth, MA, USA.

, USA.

1

4 , Yangzhou Wang Wang Yangzhou , 5 , Accepted Article [email protected] ORCID 0000-0001-8908-1529 Tel: 515-294-6948 Ames, IA USA Iowa StateUniversity 103 Laboratory Genetics USDA-ARS, Corn Insects Crop Genetics & ResearchUnit Research Geneticist Brad S.Coates, Ph.D. This by is protectedarticle reserved. Allrights copyright. of between strains and Z11-14:OAc) in pheromonethe blendthat females produce and of( insex-pheromoneThe variation ratio blend. Patterns matingof for the European corn borer ( Abstract * 7 6 5

and Z strain pheromone sexual and ecological trait variation to the genetic structure structure tothe genetic trait variation ecological sexual and plants host (C3) non-maize and (C4) maize on performance larval locations in Pennsylvania and New York and NewYork 2007between locationsand 2010.in Pennsylvania acyl-reductase ( posit variancea at loci, marker polymorphism nucleotide single Host-plant Host-plant ( use Corresponding author: Corresponding Tufts ofBiology, University, Department Medford, MA, USA. Park, USA. College PA, University, State Pennsylvania AcademyJilin ofAgricultural 13 pgfar C/ O. nubilalis 14 ) gene at the locus responsible for determining female pheromon female determining for responsible atthe) gene locus C ratios) and genetic differentiation were estimated symp were among differentiation andgenetic C ratios)

O. nubilalis . Populations also vary oviposition fora female in preference Sciences,Changchun, China. males insex-pheromone collected baited traps at12 males E Ostrinia nubilalis Ostrinia )-11- and( of Z )-11-tetradecenyl acetate (E11- )-11-tetradecenyl (E11- acetate O. nubilalis ion in the ion inthe glandfatty pheromone ) moth inparton ) moth depend . The relative contributions of contributions The relative . Among genotypes at 65 65 at genotypes Among males respond to differs remains unknown. unknown. remains e ratio atricE nd Accepted Article mating preferencesmating forsexual areoftenoneof traits the most 2002; Ravigne andresponse attraction phenotypes & (Kirkpatrick matingloci toassortative due assorting between independently (Safran (Panhuis speciation byreducing the frequ to population sexual fra selection However, can also contribute to host plant defenses (Feder to host defenses (Feder plant disrupting random mating (Pashley 1989; Nason among herbivorous insects can increase genetic variance among s Doebeli 2000; Schluter 2001; Schluter & Nosil Conte 2009; 2012) of accumulation genetic changes with localcoincident ecologica 1 Introduction | This by is protectedarticle reserved. Allrights copyright. larger genetic roleindriving v This suggests b genetic study that differentiation. population pheromoneindicate that response and topopulation differentiation or among contribute mal variation history or geograp host that analysis plant (dbRDA) demonstrate loci.unlinked Principal coordinate, clustering,Bayesian and (malepheromones response, ( Keywords: Pher ) explained 64% of the total genetic differentiation between between 64%) ma ofthe differentiation explained totalgenetic Recent models of speciation predict that populations diverge pr thatRecent predict populations ofspeciation diverge models et al et al population genetics, sexual selection, reproductive isolation isolation reproductive selection, sexual populationgenetics, . 2013). linkage ofgenerating Sexual dis capable is selection . 2001), thus arguably functioning as a force independent of na independent asaforce 2001), . functioning thus arguably Resp et al et ariation compared to host plant ency ofmating individu between . 1998; Berlocher & Feder 2002; Drès & Mallet 2002). ), providing evidence of sexual inter-selection at these at inter-selection of sexual evidence ), providing pgfar -defined strain contribute significantly to contribute -defined strain et al . 2002) orselecting foroptimal responses distance-based redundancy ehavioral divergence likely plays a important reproductive isolating isolating reproductive important -defined ecological adaptation. ecological -defined adaptation. gmentation and ultimately gmentation l adaptation & (Diekmann ladaptation es. Incontrast, these analyses between mates with compatible between hynot does significantly als with variant sexual traits traits sexual als variant with . . example,shiftsFor host plant ympatric subpopulationsympatric by Kirkpatrick 2017). Divergent les attracted to different todifferent les attracted imarily due to the equilibrium (LD)

tural selection tural selection Accepted Article the two strains preferentially respond (Glover the two strainspreferentially al (voltinism)generations year aswell per pheromone assex commu nubilalis expanded south andwesttothe1972; Sh Rocky Mountains (Chiang 1917; Smith 1920; McLaine 1922; Caffrey & Worthley 1927). Popul Massachusetts, Amsterdam,New Yor introductions North broomcorninto America onimported from Eur nubilalis & Brindley 1970; Kochansky blends of99:1 or3:97 ( (Maanisolation &Seehausen maintained isestablished and 2011; mechanisms unique insights intothe both provide inunison may speciat waysduring various interact naturalin selection might of debate (Servedio selection & Boughma alone a matter still is 2011; Lackey &Boughman 2017), a This by is protectedarticle reserved. Allrights copyright. (L are found in nature(Klun& Maini 1979; Roelofs al specificity contributes to partial reproductive isolation (Lieb (Dopman 2011; Lassance 2016; Kozak and ecological mechanisms studying theof forma diversification and traits, this inecological ), spec vary sexual and populations (Svensson sympatric in barriers . 1985; Glover . 2007; Dopman ö fstdt Genetic loci contributing to lociGenetic contributing Populations of the European corn borer, populations in the northeastern United intheStates vary numb northeastern United the populations in et al is a polyphagous lepidopteran native toEurope andwestern native Asia lepidopteran is apolyphagous . . with pheromone 1989), female blend andmale responserespecti et al et al et . 1991; Sorenson . 2010), hybrid females producing a 65:35 blend of E )-11- and( et al . 1975; Roelofs O. nubilalis Z lthough whether divergent prefe divergent whether lthough )-11-tetradecenyl acetate (11-14:OAc), respectively, (Klun)-11-tetradecenyl (11-14:OAc), respectively, acetate k, andSt.Thomas, Ontario bet et al et al . 1992). produceSpecifically,E- females andZ-strain . 2017; Wang et al sexual isolation are inherited independently inherited are isolation sexual et al Ostrinia nubilalis et al et al . 2006; Dopman . 1987). Although pheromone blend blend pheromone Although 1987). . . 1985; DuRant . 1972), towhich corresponding of males et al et herr & PélozueloRoelofs 1975; . 2017; Coates ies has asamodelfor emerged ies ion, andopportunitiesion, tostudy n). Furthermore, sexual and n). Furthermore, tion of incipienttion species of by which reproductive Butlin et al (Hübner) (: nication systems (Roelofs systems nication ween 1909 and 1914 (Vinal ations subsequently ations owers 1993). Current ope occurred near Boston, et al rences arisethrough sexual . 2010; Grace & Shaw er of reproductive er of . 1995). et al E . Multiple inadvertent Multiple. inadvertent 11- to et al et . 2012). vely controlled controlled vely . 2018). Z 11-14:OAc Ostrinia O. et et Accepted Article This by is protectedarticle reserved. Allrights copyright. by autosomal (SNP) (SNP) fixed between Pheromone strains andhybrids can bedistinguished byasingle biosynthesis components ofisomeric female pheromone of blends fixed amino acid changes between corresponds tothe pheromone gland fatty acylreductase gene ( indicates history of C3- or C4 photosynthetic-pathways hostfor insectscorrelatedis directly with source food (DeNiro & Epste males fromtraps baited with the E-strain pheromone lurefedon nubilalis et al within insectpopulations (Ehrlich Raven 1964).Although & among sympatric of (sexual divergencecontribution traits selection), insexual al na basedon markeramong vary mating assortative this autosomal larvae (Ponsard (Ponsard larvae over maize (Tate et al degree of local specialization oncultivatedplant host a maize 200 monocot and host dicot plants 1928;(Hodgson Lewis and autosomal loci donot(Dopman show E- strong individuals between differentiation and Z-strain chromosome (Dopman addition to andfunction (Koutr development neuronal involvedin putatively . 2013a). .2017). The proportion relative ofcarbon (δ stable isotope .2010). choiceLaboratory tests suggest pre neonates that may Host-plant interactions have long been recognized as long th factors recognized have been Host-plant interactions strains fed mostly on C4 plants (predominantly maize), but on mostly a h strains C4but plants maize), fed (predominantly Resp Resp Pher , a major , for QTL voltinism differences loca between strainsis is located is region Z-chromosomewithin ofthe a that encodesa et al et al et O. nubilalis and sex-linked andsex-linked . 2004; Malausa Malausa 2004; . pgfar-e . 2006), but survivorship is highest on maize (Calcagno et al . 2004). Several loci the on chromosome sex ( and and populations remains populations relatively unknown. remains pgfar-z Resp Resp pgfar-e et al et et al loci (Glover (Glover loci alleles.Estimates ofhybridization and positive . 2005; Dopman 2011; Lassance . 2005). Analysis of δ and pgfar-z et al alleles . 1990; Dopman Dopman 1990; . et al O. nubilalis O. to genome-wide to substructure

cause differences the in nd other plant species(O’Rourke 13 in 1978;in Boutton . 1975), evidence supports some plants ingested by plants ingested 13 while other sex chromosomal while sex other pgfar C demonstrated that bothC demonstrated nucleotide polymorphism polymorphism nucleotide non-maize hosts non-maize larvae in as C) integrated into developing intodeveloping C) integrated oumpa (Lassance (Lassance turalpopulations (Coates fer alternate weedy hosts weedy alternate fer ; Lassance ;Lassance at impact the diversity the diversity at impact larvae can feed on over feed onover can larvae igher proportionigher of et al et al et al Tpi , . 2004). et al . 2011). Thus, the . 2016). In et al OR1 ted Z- the ted on suite of of suite genes et al et al . 2007; Fisher and . 2013). O. nubilalis . 2010), and . 1999), and Pher OR3

O. ) et as as Accepted Article This by is protectedarticle reserved. Allrights copyright. North America (O'Rourke associations among associations among tov & contribute and volatilesMason (Udayagiri 1995),and may (Savinelli byplant phenology influenced oviposition can be E11- and Z11-14:OAc were initially tobeE- andZ-strai assumed Malusa Z-strain Z-strain France systemin pheromone were Malausa 2005; 2014, which were accompanied by significant levels of genetic d for maize (C4) andthe E-system mothsC3 hostfor (Bonteplants associations were demonstrated among usingthe Z-pheromon Based on anonymous microsatellite marker data, data, Based marker microsatellite onanonymous pheromone strains inNorth Americaremains unknown. withidifferentiation isassociated preference withany genetic C3 (O’Rourke host respectively C4 and plants, but America whichless andspatial pronounced show significant, of these studies notdirectly European are transferrable E-to percentage of of percentage sampled the (15 ofmarkers 194: Levy States,thealthough MidwestUnited a north-south t in transect when comparing relationship isolation-by-distance a significant (Liebherr & Roelofs 1975; Glover flow, influence possibleto gene pheromone of differ the strain crop landand fragmented Appalachianincluding the Mountains mi et al fromin sites differentiated genetically were and Pennsylvania . 2011). Although authors the s et al Genetic drift also drift Genetic contribute todifferentiation may among O. nubilalis . 2008). In France, et al (Frolov . 2007). However, subsequent studies concluded2007). studies . subsequent However, that Ostrinia et al et al species (Bontemps Europe within . 2010). . for Nevertheless, host plant attraction Ostrinia . 2007; Malausa et al O. scapulalis uggested landscapeuggested wit features . 1991) could notbe ruled out. A separate study predicted populations using the alternative blendspheromone of the alternative using populations et al , and a different species compared a different to sympatric , and et al O. nubilalis . . 2010). towhichThe degree host plant . 2008; Calcagno et al . 2015). et al from two locations from inNewYork and Z-strain and Z-strain n or between n between or ences among geographic regions Ohio westward Coloradoto (Kim et al his pattern was driven small driven was by a his pattern . 1988; Spanger & Calvin 2000) only populations along Z-strain ifferentiation (Bethenod ifferentiation n mps ariation in host ariation in plant ly-dependent associations with ly-dependent O. nubilalis O. nubilalis ght serve as a partial barrier barrier ght serveasapartial . 2004; Bethenod hin northeastern states, hin states, northeastern et al et al e communication system e communication system Ostrinia . 2004; Bourguet . 2010). Thus, results O. nubilalis O. O. nubilalis O. . Strong populations. populations. O. nubilalis using the E- usingthe et al inNorth

et al et . 2005; et al . . Accepted Article instructions (Qiagen,Hilden, Germany). DNA concentrationin r the DNeasy accordi Qiagen using Blood kit Extraction Tissue and 20 lined plate PCR 96-well a ml of wells 0.2 placed individual in Ames, Iowa, where thorax tissue and all were dissected4 wings shippedSamples were on dryUSDA-ARS, ice tothe Corn Insects& adult ofthe(i.e. strain bivoltine overwintering offspring gen (12 sitestotal; Table Fig.1; 1; Table S1).Malescaptur were collection York atNew traps Scentry cloth and at Pennsylvania Incorporated, Adair, OK) were us This by is protectedarticle reserved. Allrights copyright. variation and gene flow andgene di variation among theand sex genetic contributions short-term drift, ofecology, ( traits relevant ecologically sexually and available regarding driftselection, andgenetic as 2.1 | Collection and phenotypic differentiation phenotypic and Collection | 2.1 and Methods 2 Materials | nubilalis strain, differences,host plant and geographic distance onpopu Using anonymous markers,genomic i SNP the estimates this study broader understanding of tospeciation acro their contributions Wagner 2011; onlypotent coupledwhen with ecological Doorn factors (van ° C until used isotopeanalysis.for carbon Totalgenomic DNA wa Pheromone lures with ablendof97:3 or1:99 synthetic E11- and Of the handful ofstudiesthat address roles the relative ofec

within the northeastern United States. States. United within the northeastern et al et . 2012; Servedio & Boughman 2017), but inmost systems limited drivers of genomic divergence, ed toseparately oneofa bait stinct subpopulations, subspeci subpopulations, stinct

ed during ed during secondthe flight theof eration) andfrozen -20 at with aluminum foil and stored at - at stored and foil aluminum with Safran Safran many find that findthat many sexualselection is ual traits on patterns of genomic onpatterns traits ual ofgenomic et al pair of Harstack cone wire traps lation genetic structure of lation structure genetic sites between 2007 and2012sites between ss time scales may be gained. gained. be may scales time ss esulting extracts was quantified extractsesulting was quantified from each moth. Wings were ng to to manufacturer ng es, es, and a incipient species, .2009; Mann & Seehausen Crop Genetics Research Unit, nfluence of pheromone nfluence ofpheromone s purified eachfrom thorax ological adaptation, sexual et al . 2013). . By dissecting Z11-14:OAc (Trécé details are ° C. C. O. Accepted Article flight towards E andZblends (Roelofs pheromone the twostrai that of responsemales given pheromone phenotype, of malesinpheromone traps with the baited E- lure orZ-strain homozygous assign to used was assay PCR-RFLP This by is protectedarticle reserved. Allrights copyright. were amplified in fourPCR (W1, multiplexes W3 W2, andW4; Coat 2.2 |Population genetic differentiation μl on spectrophotometera NanoDrop2000 (Thermo Scientific, Wilming were were each from dissected trap collected of male, and ratio the collected pheromone traps ana basedon in isotope carbon stable to respective strains, to respective strains, and >98.7% with correlated E- or Z-pheromone strains (Coates the at marker a of genotype SNP on based converted to δ units relative to Pee Dee Belemnite carbonate(δ Dee toPee toδunits relative converted California Center for St Berkley, mass by continuousflow-isotopic ratio spectrometry c (CF-IRMS) dideoxynucleotides dideoxynucleotides using the (Sequenom; iPLEX-GoldMastermix Ta subsequently SBEused as for assaystemplate incorporated that Plant for Center IA. InAmes, loc University, Genomics, brief, base MassARRAY®assay extension products analyzed onaSequenom R -1 = the ratio of = the ratio with deionized nuclease free nuclease with free and storedwater, deionized at-20 A setof108anonymous biallelic markers wasSNP usedtogenoty Host plant history,Host C3plant or C4 (non-maize) (probable was maize), In addition, strainIn ofpheromone assignment genetic addition, was determi 13 C/ 12 C atomic percentage). percentage). atomic C pgfar-e able Isotope Biogeochemistry ( / pgfar-z heterozygotes toahybrid heterozygotes class. capture Additionally,

pgfar locus. This assay detects alleles that are that locus. detectsalleles This assay pgfar-e et al . 1987). / ° pgfar-e C. C. et al i containing containing i 13C to14C (δ wasused as aproxy for Berkley,The CA). results were 13 . 2013a). Specifically, the allele-specific mass-modified allele-specific mass-modified and lysis. this, For the wings four C = ( = C onducted at the University of the University onducted at ns strongly differ in their in differ ns strongly es ng ng ton, ton, to10ng DE),diluted pgfar-z R et al located at the Iowa State IowaState locatedat the sample et al assigned assigned to each male a priori ned foreach male pe eachmale single by . 2011), and . 1999). Genotypes / 13 / R C) was estimated C) estimated was pgfar-z standard defined SNPs –1)×1000; genotypes genotypes Taq I Accepted Article exact tests implemented in the Arlequin software package (v. (v. package 3. software testsimplementedin exact Arlequin the by querying the the by querying 2015). Linkage groups (LGs) were assigned to physicallocation tocompared known map positions onaconsensus permutation steps using Arlequin 3.2.2 (Excoffier between alldetermined loci, significance and unphased of pairs preceding Fisher’sfilters, testsexact were conducted to predi departures were predicted across populations E- orZ-strain all in≥20%populations. HWE of Exceptions were made expectation missing ≥25% for of individuals, genotypicor proportions show Loci were subsequently excluded f tests for were performed E-, and hybrid Z- This by is protectedarticle reserved. Allrights copyright. Weinberg equilibrium (HWE), with ofobserved departures heteroz (2013b). were fromdetermined rawMassARRAY® Sequenom outputasdescribe and analysis of molecular variance variance (AMOVA) molecularg and analysis for were estimated of 14:OAc lurepheromone (male response)pheromone or by b of genotypes partitioned males position in in position to bombykol production (Moto KAIKOBASE (Shimomura whichmarkers were SNP (Coatesdeveloped heterozygosity ( heterozygosity O. nubilalis Processed SNPSequenom and Locus-by-locus Locus-by-locus O. nubilalis O.

pgfar B. mori H E )across sitessignificance collection tested for (α= 0.05) us (Lassance (Lassance . All BLAST database results were filtered for BLAST werefiltered results . database All genome assembly database assemblywith database genome F ST et al , F IS et al , and . 2009). Additionally,the et al y beencollectedintrap having . 2003; GenBank accession BAC79425) ortholog isthe nearest rom further analyses ifmonomo . 2010), andwas used predictthe relative orthologous F IT estimates were made based on homozygous homozygous on based made were estimates pgfar pgfar PCR-RFLP genotype data assessed data were PCR-RFLPfor Hardy– genotype et al PCR-RFLP-defined partitions within location. PCR-RFLP-defined . 2011) using the BLASTx algorithm at et al et O. nubilalis O. . 2005). Any of pair SNP loci were in LD B. mori O. nubilalis pgfar ct disequilibrium (LD) linkage genetic linkage map (Levy linkage genetic map fatty acyl-reductase involved in in involved acyl-reductase fatty . For locipassed that the 5.2; Excoffier s baited s with baited E11- or Z11- by 10,000 Markov Chain on ed a significant departure ed asignificantfrom departure genotype. genotype. Global enotypes partitioned by enotypes in instanceswhere no HWE rphic, genotypic data were were data rphic, genotypic ygosity ( ygosity Bombyx mori Bombyx contigsequencesfrom E -values -values ≤ 10 d by Coates H et al O ing Markov chain ) expectedfrom chromosomes . 2005). HWE -50 pgfar F . et al -statistics -statistics

. et al . Accepted Article represented intherepresented analyses. Pairwise e This by is protectedarticle reserved. Allrights copyright. 12 Pairwise collectionsites. responsepheromone and differentiation were assessed using Arlequin v. 3.5.2 v. were (Excoffie differentiation Arlequin assessed using theusing BH procedure (Benjamini& Hochberg 1995). All estima each for rate) discovery Significance statisti threshold (false collection site between response,pheromone Principal coordinate analysis the conducted Principal(PCoA) c analysis using coordinate was pairwise also assessed. pheromone response which in the corresponding SNP of65 dataset dataset of64loci (omitting genotypes in the 12 collection sites for each year; note t 12that note the in each collection sites for genotypes year; and again with 64 loci after excluding with and again 64 the excluding loci after made for 52 groups ( Population structure was inferred using a model-based clusteri model-based a using inferred was structure Population by STRUCTURE 2.3.4 (Prichard 2.3 Bayesian | clustering relationshipspopulations. genetic among visualize coordinates accountedthat mostvariationthe for were usedto score Factor analyses. the in notrepresented combinations were individuals with hosthistoryC3 plant in ofthe 13 categories of C3 andC4 ( delimited groups t with by collection sites partitioned further / pgfar-e

F 1), intwoofthe individuals so those (Table collections comb ST s using GENALEXv. (Peakall6.503 2006). & Smouse Pairwise n =1,421 genotypes) using the 65 SNPloci includedthat the n 1,411individuals),= creating pgfar

pgfar F ST genotype, as byplant well host orhistory C4), as across(C3 et al estimates collection estimates were within and between made sites, ) forcomparisonswas used allinstances, except in of . 2000; Falush F ST estimates forthe werealso same 52 made pgfar pgfar locus. locus. The represent the 52 groups three pgfar et al genotype, and host plant history. history. host and plant genotype, . 2003; Hubisz plus sitecollection groups,so those 91 total that note groups; ther c was adjusted for multiple testing testing multiple for adjusted was c here werenohomozygous he twohost history plant r et al et construct a scatter diagram to constructscatter a s along the two thes along two principal tes of tesof subpopulation . 2005). A filtered SNP loci (including (including loci et alet inations not were ng method implemented ng ovariance matrix of . 2009). . STRUCTURE F ST estimates were pgfar pgfar pgfar e wereno locus, pgfar- ) was pgfar

the

Accepted Article lure), and C3 and C4 host plant history (δ hostlure), plant andC3C4 and (1,421 2 partitionsgenotypes, b 12partitionsgenotypes, response by pheromone location), combi This by is protectedarticle reserved. Allrights copyright. provides likelihood estimates of 999 The ofanydifferences permutations. significance inthe d usingMantel’s Test, determined and si associated probabilities distance. analysesIBD performed were GENALEXusing (Peakall& (Hubisz (Hubisz admixture model of individual ancestry prior andauniform for Pr(X| foreach log valueoftheprobability median estimated clusteri ( genotypes SNP 65 of set the using repeated to -32). Allthe using set of64 runs conducted were SNP genot replications 1 with × 10 STRUCTURE with 2.3.4 running iteratively marker data using Markov a MonteChain Carlo Es (MCMC) method. NC, USA). Additionally, a distance-based redundancy ( distance-based Additionally, analysis NC, a USA). grouped by sample site weremade single-factorvia ANOVA using between genetic distance (based on estimates of of genetic on estimates (based distance andbehavioral relationships 2.4 Ecological | Distruct (Rosenberg using 2004).visualized ancestries ( for distribution K ), the usedtocompute was posteriorprobability of Possible isolation-by-distance (IBD)(Slatkin 1993) was assesse et alet pgfar Q ., 2009) was used ongenotypes partitioned into12 collection s -matrix) using the -defined E-, Z- and hybrid (3 partitions) or responsepheromone K . Optimized values for for values Optimized . 7 iterations preceded by a burn-in of 10 preceded byaburn-in iterations Δ y collection in traps baited wi baited traps in collection y the number ofdistinctthe populat K statistic (Evanno statistic F K ST were derived from the werederived resultingfrom population co- 13 among sample12 locations) on geographic (km) K C estimates for C4 for to-17host andC3 =-8 C estimates host =-22 values from 1 valuesto from 10. Each run consistedof 10 pgfar

included). For all STRUCTURE results,the et al. 2005). Individual K , Pr( th E11- orZ11-14:OAc pheromone ng analysis,ng conditional on istribution of δ ypes ( gnificance determined basedon gnificance determined ions ( dbRDA; Legendre & dbRDA; & Anderson Legendre 5 K ALPHA. The LOCPRIOR option iterations. iterations. Runs an used |X), assuming a uniform uniform a assuming |X), ned across 12 the sites sample SAS 9.2 (SAS Institute, Cary, SASCary, 9.2 (SAS Institute, Smouse, Smouse, 2006) with the timates were obtained by obtained were timates pgfar K ) from unlinked genetic genetic unlinked ) from d by regressing pairwise regressing d by omitted), then omitted), ites (1,421 Q 13 -matrix data were -matrix (2partitions) C estimates estimates C K prior , ln

Accepted Article strain). Foreachpopulation, δ the mean 22 PA total in Nor (Leh), from analysis,was removed resulting were PA collected(Nor), at anddue Northampton, to proximity ( lure),pheromone and2) by male ways:different 1) by pheromone traps response(collected ba in Legendre & Fortin 2010). At each site, males sampling clas were Mantel Tes partial each distinctfactorsthe unlike parsed into to variables for predictor Redundancymultiple allows analysis ( traits This by is protectedarticle reserved. Allrights copyright. z/-z Oksanen 1999; package (Kamvar (Kamvar package as twoindividuals ofallelic number the differences between (a addition, an individual-based analysis inwhich was performed g each other. each predictor (while forother accounting factors) was determi composed of multilocus (http://cc.oulu.fi/~jarioksa/softhelp/vegan/html/capscale.html) dbRDA using was performed the CAPSCALE functionin the VEGAN R (http://cc.oulu.fi/~jarioksa/so inthe VEGANfunction R package for component eigenvectors distances into transformed was (km) pheromone), mean δ mean pheromone), = 0 trait, binary (a pheromone to set were variables predictor factors distance,(geographic pheromone difference, δ and mean Mantel in VEGAN alsowere Tests performed using matric distance verify that the results werenotinfluencedby the d geographic Theand longitude asfactor latitude using wasdbRDA replicated as Z-strain individuals; asZ-strain all pgfar P or pheromone response), and geographic distance onpopulation -valuesusing were corrected multiplethe for Bonferron testing et al et al . 2017) was used theto test roles ofhost relative ( use plant 13 . 2014). The predictor variables in dbRDAthe were pheromone ( C, and the first three geographic distance eigenvectors. Signi three geographic first distanceeigenvectors. the and C, F ST estimates[from of64SNPS the ( dataset pgfar-e/-z pgfar genotype ( genotype 13 were were excluded). Since very E-strainfew individuals C value was calculated, and the matrix of geographic pgfar-e/-e asE-strain individuals and t (Legendre & Legendre 1999; same pheromone,same 1 =different istance transformation. Partialistance transformation. llelic dissimilarity) in thePPOPR in dissimilarity) llelic populations (11 E- populations and11Z- be analyzed simultaneously and and simultaneously analyzed be ned by permutation ( ned bypermutation sified assified E- intwo orZ-strain ited withited E11- or Z11-14:OAc 13 s insteadofthe eigenvectorsto enetic was enetic distance calculated ~5 km) of this of ~5 km) site toLehigh, fthelp/vegan/). Subsequent Subsequent fthelp/vegan/). . The constrained was . matrix constrained The Cdifference) sequentially on each location using the PCNM es andconditioning pairs of pgfar package package excluded)], and the i method. In i In method. genetic structure. structure. genetic C3:C4), sexual sexual C3:C4), ficance of of ficance N 1000).= pgfar pgfar- or Accepted Article with δ with 1,160; 82.0% of total) and C3 ( This by is protectedarticle reserved. Allrights copyright. pheromone response), δ from C3 not between 2009a hostsignificantly differ plants did from further analysesinvolving 3.1 | Collection and phenotypic differentiation phenotypic and Collection | 3.1 Results 3 | dataset). in ofnumber eigenvectors the individual estimates ofδ collected with abimodal showed mode distribution, peaksat-10 data were insubsequent analyses. partitions used the of These male pheromone response, = 0.043; Fig.S1A). The δ at in heterozygosity n e distinctgenotypes assaythree yielded whichgenotyping as were Results (Table baited traps, the of pheromone 1). respectively and Pennsylvania2007 from to2010(Fig.1), / 355; = Table 1; doi:10.5061/dryad.kj34mq9). Assessment oftem pgfar-e 13 A total of 1,421adult male C estimates ranging -18 to-21 wereC estimates between considered ambiguou ; n

= 355), Z-strain ( 13 C between to-17-8 C and-22to between -32were respectively assigned t pgfar 13 13 acrosslocations 2009in vs 2010 indicated a significant diffe C value, and the first four distance eigenvectors (due to the l the to (due eigenvectors distance four first the and value, C C values calculated for 1,415 males from which isotope readings isotope which from males 1,415 for calculated C values pgfar-z n = 18.0%)255; groups. host Thehistory plant individu of five host plant. dataset, hostFor this plant. pgfar O . / nubilalis pgfar-z -defined pheromone strain, andhosthistory plant ;

werecollected at12 trap locations inNewYork with 704with and717collected fromE- andZ- n =711), oraheterozygote class (

the proportionthe of males derived

nd 2010 ( pgfar and-28 (Fig. S1B signed toE-strain ( poral (year-to-year) change -specific PCR-RFLP-specific P = 0.109; s and wereexcluded o the o the C4 ( pgfar-e ).

Fig. S1C). pgfar- The arger arger rence ( rence / pgfar-z n = were als P

; Accepted Article This by is protectedarticle reserved. Allrights copyright. those wereexcluded,markers that 31 were notin HWE. Subseque Of the 108SNP markers, (including 65 accession doi database dataset(Dryad within the final retained 3.2 |Population genetic differentiation Contig06721.1113 was the only SNP marker the on Z-chromosome an assigned toanorthologous on position contig01257.497 LG11on waspreviously positioned ofan positions 5,150,695 to5,153,581 ~or 2.7 Mbp away fromthe estimates indicated five SNP loci, enzyme E2 ( nearest ortholog to across locatedon exons) (Table S2B). S2B). (Table either by betweenstrainsdefined pheromon differ significantly or Z-strain pheromone lure (Table S2A and contig06864.843,differed significantlym of groups between pgfar by locus between pheromone defined strains differentiation contig01453.160, contig05858.945, contig06634.250, and contig06 independence among the 65 filtered loci detected LD only betwee accounted≥ 10% for ofthe total variance genetic in locusboth samples pheromone with trapped different andthe lures, mar SNP from the mapping of ortholog pos ( with the the with nearest P <0.0001; results ofother locus ignored). The B. mori B. mori E -values ≤ 10 ortholog of ortholog gene model gene BGIGMGA010458 ( O. nubilalis O. pgfar B. mori locus accounted 64% for of the t -56 O. nubilalis ; 80.0 to100%identity across exons)on SNPmarker locus contig01257.497 is aubiquitin conjugating chromosome 12 (positions 2,426,074 to2,437,943). The pgfar itions. Specifically, the pred pairs notshown). Secondary

pgfar , contig00078.322, contig05852.999, contig06634.250, ) B. mori . Similarly,markers contig00078.322, contig01257.497,

, BAC78425 (Lassance chromosome 12(Levy pgfar E -values ≤8 10 x ) passed stringency criteria and were otal genetic variation between variation genetic otal O. nubilalis pgfar icted genome position icted genome of the pgfar -by-locus analyses. Tests of analyses. Tests of -by-locus :10.5061/dryad.kj34mq9 e response S2A) (Table confirmation of linkage came confirmation linkage of -39 ales defined by response ales defined toE- n (Table S2B; nt locus-by-locus nt locus-by-locus ker locus contig01257.497 et al ; 84.5 to 100%identity 864.843 showed locus-by- pgfar ortholog. The SNPmarker B. mori genetic map, and also et al d . 2010), is synonymous F andcontig01257.497 ST ST . 2015). estimates not did chromosome 12

significance of significance F ST

or )

. Of pgfar Accepted Article but significant ( but significant S3D). Among these, the strains (Tableco-occurred 1) in210 significant were of 27 the byhomozygousidentified at genotypes This by is protectedarticle reserved. Allrights copyright. and Roc; 1)Fig. where pairwise sympatric E- andZ-strains were northernonly detected at colle ( S3C), Table (15.2%; estimates on toeither response E- orZ-strain pheromone lures ( comparisons setofgenotypes was This(Table S3A). same partit for estimates betweenthe significant were collection sites 12 pairwise pairwise pheromone response groups, and were located at a significantly asignificantly located responseat andwere pheromone groups, differentiation between E- and Z-strain locations across and Z-strain males E- between differentiation location) collectedgroups E- Z-pheromone-baited in or traps.T differentiation across locations between E- and Z-strain males in113ofsignificant 132 instances of11(85.6%; 10 sympatric than those that were non-significant (mean latitude (mean latitude than40.54 those non-significant were that Eleven the 12(92%) of pairwise which 125 (86.8%) were significant groupsbetween responded to ranged from -0.09583 to0.05852 and144of 276comparisons (52. comparisons responseof pheromone inalower of resulted number omis (Tablesubpopulations S4A). Conversely, between variation P strain as 6.78% for (Table oftheaccounting total variation S4 F <0.0001), and AMOVA pheromone implicated response as accounti ST 0.0047,= Based onthe dataset genotypes entire of 65 from SNP loci (incl Pairwise Pairwise F ST comparisons were significant between sympatric (within locatio between comparisonssignificant sympatric were P <0.0001; genotypic TableS4B). Interestingly, di significant F F ST ST ST = 0.0085, estimates from 64SNP lociestimates (excluding F ST estimates betweenE-and estimates within Z-strains acrossand wer sites P

0.0001),< and AMOVA implicated and a reduced global estimate of subpopulation differentiation global ofsubpopulation differentiation and areduced estimate F F ST ST estimates were ≤ 0.0087; Table S3C). Five of 12 (42%) comparisons between were sympatric significant (within pgfar marker locus across 21trap locations where n 24groups).= Resulting o pgfar N; C). C). 13 of the 66 (19.7%) pairwise T 6 comparisons (76.1%; Table E-Z comparisons). Global Global comparisons). E-Z was also significant ( was alsosignificant based on based on 4.78 he global estimate of estimate global he ction sites (Oak, Int, Ran,Int, Plan (Oak, sites ction ) between E- and Z-strains and Z-strains E- ) between ioned ioned across samplesitesbased higher latitude (mean (mean 41.79 higher latitude pgfar sion ofthesion =2.81, different lures (Table S3B). S3B). (Table lures different 2%) of were significant, significant pairwise ng for 3.22% ofthe for total ng usive of -defined pheromone pgfar P = 0.039). fferences between fferencesbetween pgfar n) E- and Z- n) E- genotype waslow genotype pgfar F locus from from locus ST estimates estimates F ), ST F =0.0322, ST ST e F ST o N) Accepted Article This by is protectedarticle reserved. Allrights copyright. Fifty-five of 276 (19.9%) pairwise 3.3 | Bayesian 3.3 Bayesian | clustering when to assigned strain)C3(pheromone andlocation or C4host ofgroups inclustering that ac nodiscernable revealed pattern no were groups heterozygotes and Z- E-, for scores factor where contrasted with fromPCoA results the data based 64 l SNP of on clusters the unique along Coordin into individuals heterozygote scores 1,accounted25.1% ofthe for Coordinate along totalvar (PCoA) from all basedon data (including loci 65 SNP genotype as by the tested IBDmodel ( Ransom (Ran), and Rockspring (Roc)]. Ransom estimates Rockspring Overallglobal (Ran),and todifferentincluding assigned hostwith plant history groups, these S3E).of 55 (47.3%) Twenty-six significant comparisonsw andhost theby defined data both SNPgenotype collection site planthistory groups across collection lowall andno sites was predicted anoptimalpredicted of number or subpopulations, represented multilocusby 0.1779; response males of0.7786 compared to 0.0451 among Z-pheromone r inclusion of the datasetwith using 64 based on response loc E-and Z-strain response asthe and excluded (64loci;

An iterative Bayesian Ansimulation iterative approach estimated the optimal There was no significant correlation ofpairwise correlation There was nosignificant Table S4D). Table S4D). pgfar prior (65 loci) resulted proportion inamean of Cluster 1among E-p . Co-ancestries (clusters) between Co-ancestries homogenous wererelatively . Δ K = 36.3) from the = 36.3) genotypic from by datasetpartitioned pheromone

P = 2).0.4280; TheFig. results ofprincipal analys coordinate K =3bothwhen F ST ST estimates were significant between partitions of partitions estimateswere between significant O. nubilalis O. pgfar was included either loci;(65 F SNPgenotypes. STRUCTURE 2.3.4 ST estimates with geographic distance n-significant ( n-significant in three in three (Int), sites [Interlaken counted for for counted pgfar iation, and groupediation, E-, Z- and plant historyplant or (C3 C4) (Table i (Fig. S2A). S2A). Ini (Fig. contrast, ate 1 axis ate 1axis 3A).(Fig. This oci excluding the of t (Fig.distinct 3B). PCoA ere between between ere genotypes plant history groups (Fig. 3C). F ST ) indicated that factor esponse males (Fig. S2A). between hostC3 andC4 number of of number clusters ( pgfar F ST = 0.0004; genotype heromone heromone pgfar Δ groups K locus, = 47.7) P es = = K ), Accepted Article This by is protectedarticle reserved. Allrights copyright. homozygous ofco-ancestries Assessment inassignment ofthese differences differentiated into into clustersdifferentiated corresponding to (including (population (population pheromone response Approximatelygroups. proportionsequal mean STRUCTURE 2.3.4 estimated l 65 for Specifically, results. similar yielded option LOCPRIOR 22). 1, = that tothe responded lure E-strain (0.4305±0.2249; ANO one-way than ofhomo proportion the Table greater S5)wassignificantly 0.226) defined by either with E- (C3: 0.892 ± 0.148; C4: 0.873 ±0.147) orZ-stra C3an toClusterco-ancestries proportion1 assigned between of pgfar informationBayesian for clustering of been setasthe estimates history) (C3 having plant or C4 host Use ofhost Use history plant or (C3 basedC4 designation) onδ and (Fig. S3). ofUse 64loci ( pheromone response pgfar-z/-z ) resulted inan optimum Partitioning data usingPartitioningthe 12collection data as sites pgfar Q pgfar-z/-z -score) of 0.850 ±0.240 and 0.830 ± 0.241 wereassigned toClu genotypes, respectively (remaining data not shown). data genotypes, (remaining respectively ) resulted inanoptimum pgfar -pgfar-e males that respondedtothe Z-strain (mean =0.8972 lure ± 0.0 genotype (Fig. S2B). Bayesian c pgfar K / omitted) in resulted omitted) =3( -e K of3( )

and Z-strain (Z-pheromone response- Δ K Δ O. nubilalis O. = 28.5) with clusters 1 and 2corresponding to E- (E- K = 42.0). There was no difference in = 42.0). There was in mean nothe difference K =3 ( pgfar-e Δ K genotypes genotypes across 64SNPloci (excluding = 569.6) with shared co-ancestries K /- 2( = e lustering of genotypes from65 of genotypes lustering , a priori pgfar-e Δ K = 29.4), = 29.4), and among no structure oci (including the oci (including in (C3: 0.836 ± 0.218; C4: 0.837 ± prior revealed that proportion the of information under the information / zygous zygous d C4 plants for d C4comparisons plants for -z VA: and 13 (Fig. S2B).(Fig. C estimates as C estimates individual co-ancestries F pgfar-z pgfar-e/-e pgfar-z/-z =49.12, ster 1for / pgfar -z P despite δ despite genotypes genotypes < 0.0001, d.f. ), respectively a priori locus), SNP loci loci SNP 512; pgfar-e/e

13

C Accepted Article This by is protectedarticle reserved. Allrights copyright. andbehavioral relationships 3.4 Ecological | ANOVA showed the proportionno significant in ofdifference hom (C3:C4; (C3:C4; 4 | with differences in sexual sexual with (Groot signals differences in In diverging populationsmany Lepidoptera, of ecological factor These indicated analysesdbRDA also pheromone response by or strain pairwise comparisonssignificantly hadhigher amongdifferentiation) populations significantly differed only ( assigned totheassigned δ pgfar-e among group) history plant host however, the effect of host plant history (C3:C4; ofhosthistory the(C3:C4; effect however, plant locations, orwith different C3:C4 le significant showed ratios (Table S6A, 6B; S7).Lastl Table pgfar that tests indicated factors)Mantel partial using distance or = 2, 22). 22). 2, = between groups asdefined 1, 23), or strain defined by pheromone responseor 1, across defined 23), location strain by pheromone η 2

=0.0017) orpheromone response ( Discussion genotype) significantly contributed to genetic togenetic contributed but h genotype) significantly variance, / The overall proportions Analyses of dissimilarity matrices using dbRDA showed that P pgfar-z ≥0.268) or geographic distance (

(E-/Z-strain hybrids) (E-/Z-strain were0.1 13 C bin (-8 to -32) across to-32) populations C( (-8 bin pgfar pgfar y, males responding to differe responding y, males of individualsof with δ homozygotes acrosslocations ( genotype (Fig. 4A, B; Table 2), pgfar-e F ST η / estimates were not significant for historyhost not significant estimates were plant et al 2 pgfar-e

= 0.0034; Table S8). P ≥0.369). Similarly, latitude andlongitude (used as . 2008; Joyce 61, 0.203, and0.151, respective

η (E-strain), 2 = 0.0009)= wasweaker than those ofdistance 13 F C values in the range of to -24 inthe -32range (C3C values ST F estimates when classified by =3.42 × 10 et al pgfar-z pheromone strainpheromone (response and vels of mean allelic differentiation; differentiation; mean allelic vels of nt pheromone lures, from different by pheromone strain. E-and Z- strain. pheromone by . 2016), but fewin cases has the but not by host plant (Fig. 4C plant butnotbyhost (Fig. s such plant ashost useco-vary s ( F ozygous male =1.75 × 10 F ost plant not history did / =2.59 × 10 pgfar-z -5 F , ST P estimates (genetic (genetic estimates =1.0000, d.f. =2, 22), (Z-strain), and (Z-strain), -5 ly. Single factor factor ly.Single , -5 P pgfar , = 0.9967, d.f. = P = 0.9981, d.f. genotypes genotypes ). Accepted Article O. scapulalis the between s sympatric Europeto bemarkedly appears different ecological factor impacting as the strongly influenced by variation at the at byvariation influenced strongly individual co-ancestries assigne 2; dbRDA-based Likewise, eigenvectors forvariance in C3:C4 ratios ecotypes or voltinismstrains that in vary and sex-pheromone co earlier population genetics studieswhere lowpairwise to variation amongcollection sites (Table Table2; S7).T S6, Similarly, ourdbRDA that suggest geographic analyses distance planthistory on Frolov This by is protectedarticle reserved. Allrights copyright. pairwise nubilalis presencedifferentiation in the flowrates ofgene of high rema (Alexandre Frolov Thomas 2014; America (O’Rourke occur between differences preference hostin closely plant rela Olsson 2010; et al ofthesebee togenetic factors structure contribution relative suggested high flow gene longover distances (Bourguet

Table S6) and partial Mantel Tests (Table S7) were notsignific S7)were Table MantelS6) Tests (Table and partial . 2017). 2017). . prior et alet et alet Our current study suggests that gene flow among northeastern po flow studyamong that suggests gene Our current remains relatively high at neutral genetic loci, as inferred b as inferred loci, neutral genetic high at relatively remains F ST . 2012). Additionally, current thein study we detected nosi . 2012). Although system pheromone and host plant useco-vary i (Fig. S2B). these results Together, (Fig. host that indicate plant et al . Specifically, estimates collection between sites (Table S3A) Ostrinia nubilalis Ostrinia et al et al . 2013), elucidating the relative contribution of selec contributionvarious of the 2013), relative . elucidating O. nubilalis O. . 2010; Lassance 2016; Kozak . 2003; Pélozuelo et al . 2010) and among Ostrinia population genetic structure 3C inPCoA genetic (Table S4D)or (Fig. population O. nubilalis O. is an emerging isanemerging modelfor speciation dynamics due sympatricto d tosubpopulationsin Bayesian from use the communicationFrance that E-pheromone from et al population inNorth structure America. The scenario in pgfar . 2004; Ostrinia locus, not host plant despite the latter used beings latter the despite locus,host not plant et al

Bethenod species Europein (Bourguet . 2017; Coates F et al. et ST estimates andshallow slopes IBD et al

and by IBDmodeling his his is supportedconclusion by n tested(Prowell 2000b; Kim ins a challenge. achallenge. ins ted . 2005; Pelozuelo clusteringanalysis were did not contribute significantly significantly did notcontribute mmunication (Dopman among collection sites (Table collection sites among ant. Finally, the proportion of of proportion the Finally, ant. ibling species, O. nubilalis O. nubilalis et al y mostly non-significant y mostly non-significant history is notastrong . 2018). Ecological gnificant impact of host pulationsof et al et tive forces to forces to tive n strains inNorth . 2009, 2011; Ostrinia et al O. nubilalis et alet et al

(Fig. 2). . 2004; Gouin . 2000a, . 2007; O.

) et al .

and . Accepted Article could not be tested directly and remain the subject of future w of future subject the couldremain and directly tested be not locus, and therefore atthe determined. variation causal Thus, or involved, markers, withgenetic correlated strongly most mal putatively involved in neurogenesis (Koutroumpa involvedin neurogenesis putatively chromosome-linked phenotype ( phenotype traps withstrain-specific pheromonebaited luresas aproxy fo (Coates research hinders current selection (Groot proposed toexplain the co-evolu remain LD(Kirkpatrick in &Ravigne 2002; Kirkparick 2017). Se This by is protectedarticle reserved. Allrights copyright. including factors for represent accounting significant captured males ( host than or use. Speci differentiation eithergeography plant and Z-strain conclusions fromthe current stu 2007; Frolov are system male response are not physically linked in all species ofLepid linkedspecies inallmale response not physically are S6, S8). Even though genetic loci determining female pheromone when andgeography-based history- eigenv hostto plant compared pgfar conclusion ismost clearly drawn dbRDA,from wherein pheromone genotype are the most significant eigenvectors determining deg genotype arethe eigenvectors most determining significant Our study suggests that pheromon O. nubilalis O. O. scapulalis O. Resp O. nubilalis et al et al ), adirect measure allelicof variation at the pgfar . 2007), whereas Resp (Roelofs . 2016), butlack ofinformation onthe genomicarchitecture of or phenotypehomozygous pheromone response ( genotype) which predominantly feed and develop feedanddevelop mugwort on (Malausa predominantly which may not be transferrable to the sibling species in Europe. tothe speciesinEurope. sibling notbetransferrable may locus (Dopman (Dopman locus et al dy the regarding basisofecolo tion of contributi tionloci genetic of et al . 1987; Löfstedt O. nubilalis . 2018). Although we used capture of male et al e strain o has effect greater a O. nubilalisO. are Z-strain andmostly Thus,are on maize. feed . 2004) . is located ofgenes asuite within et al et al population genetic variance. population This .2016). or The mutation(s) specific gene . 1989; Dopman Resp fically, of strain pheromone optera studied (Groot ng tong mate and attraction potential ofselection impacts ork. r pheromone pheromone r attraction gical diversification betweenE- diversification gical e e response are yet to be veral models have been been have models veral signaland corresponding locus The is lacking. Z- ectors (Table4;Table 2; Fig. response phenotype and ree of variance genetic et alet n O. nubilalis . 2004), these loci O. nubilalis these traits Resp et al genetic et al ) each each ) . 2015), . in in Accepted Article This by is protectedarticle reserved. Allrights copyright. markers (excluding the inZ-strain latitude is with correlated strongly individuals pheromonethat differ in strainand voltinism ecoty 2015) ofasegregating region the Z-chromosomeof which differs phenomenon may bedue (probably toa rearrangement in part an i chromosome compared toonthe autosomes(Levy to high differentia also genetic stain pheromone contribute may pheromone strains. Ecological inseasonalvariation timing(vo evolutio singular necessarily the not sexual aloneis selection timing (Liebherr 1975;circadian & Roelofs Levy assortative (Royer mating 1992; 20 & McNeil Lassance& Löfstedt the ar genome in ofdifferentiation possible that islands other nubilalis greater number of anonymous markers could clarify whether popul the when strains likely Hybridization Z-strains. fl gene and incomplete is mating assortative suggests S1A) Fig. al hybridfemale pheromone andselection pheromone against blends consistent with high c mating ofassortative observed the rates of factor than maintenance ecological the strain and divergence nubilalis contributing to female pheromone signaling and corresponding ma where the strains are also known to differ in voltinism (bivolt response groupscollected location in the mostlysame at northe . 2010). However, observed levels ofhybridization (as evidenc Although we found no evidence that host-plant use contributes no evidencethat Although host-plant use found we concurrentl acting is selection sexual that suggest results Our . charactersThese tohavesignificant appear on impact amore is limited tolimited is pgfar Pher locus genotype was removed analyses. from Future studies using pgfar and locus), we detected significant locus), significant detected we explains why genome-wide struct Resp , and genome isal , regionsloci. It linked tothese physically O. nubilalis O. et al et al . 2018). . 2015; Kozak (Showers 1981). onSNP Based F nary force nary force maintaining e associated withe associated loci promoting ine E- and univoltine E- andunivoltine Z-strain; ine ST ow isoccurring between E- and aused bymaleaused to response ltinism) conjunctionin withltinism) pe (Kozak s between E- andZ-pheromone host plant use. These host plantuse. results are tion on the the on tion rn sites (Int, Oak, Pla,rn sites (Int, andRan) Oak, uring was not detected among uring ed by heterozygosity at at by heterozygosity ed in frequency most among mostamong frequency in 09) in including differences nversion;Wadsworth le response phenotypes in (Linn ation differentiation in differentiation ation y on unlinked loci loci unlinked on y et al inter-strain genetic to genetic structure, structure, genetic to et al et al . 2017). This O. nubilalis . 1997; Dopman . 2017). Voltinism O. nubilalis O. Z- a pgfar et al O. O. . ; O. so et

Accepted Article This by is protectedarticle reserved. Allrights copyright. This research funded was by the Science National Foundation(DE the Iowa Agriculture and Ec Home (USDA), Agricultural Research Service (ARS) (CRIS Project 5030- This research a was joint contribution United from the States D flow between pherom female selection (maternal ecological distance contributions of use), (host plant factors causal Regardless,und important for are mutations. data these the dev upon future quantifiable voltinism ongenomic variancebetween evolutionary time remains unclear. The relative contributions whether byitselftomai but in Northit issufficient America, betwe differentiation ofgenetic the maintaining observed level thisData from andearlier studiessuggest that sexual selectio were drawn from congruent results fromBayesian clustering, PCo Statesnortheastern United to geographic compared h distance or greaterproportion ofthe amongpopulat geneticdifferentiation both (Maan naturalButli &Seehausen selection 2011; and sexual beamulti isolation may ofreproductive subsequent development ecologicaltraits andsexual may Glover Acknowledgments Acknowledgments evolutionary trajectories. et al In blend study,this female re pheromone wefoundthat and male

. 1991; Calvin & Song 1994). These results suggest that that 1994). interac & Song suggest 1991; CalvinThese . results O. nubilalis subpopulations, which bepropelling may alongdifferent them reinforce one reinforce another to limit elopment of tha markers genetic one blendandmaleresponse) i onomicsExperiment Station, Ame O. nubilalis pheromone strains more may preciselybe ntain the pheromone strains over strainsntain the over pheromone (geographic isolation), and sexual n may function function as acomponent n may gene flow in flow in gene of of male pheromone response and en sympatric pheromone strains en sympatric pheromone erstanding the relative ions of of ions t detect or tightly or linkedto t detect are n ost plant. These ost These conclusions plant. variate processvariate on dependent A, IBDmodeling, and dbRDA. n forming barriers to gene gene to barriers n forming et al et epartmentof Agriculture 22000-018-00D), and22000-018-00D), O. nubilalis . 2012). B-1257251; E.B.D.), E.B.D.), B-1257251; sponse toa contributes tions between certain tions certain between s, IA (Projects, IA 3543), O. nubilalis in the the in , and the Accepted Article Bourguet, D., Bethenod, M. T., Pasteur, N. & Viard, F. (2000b). F. &Viard, N. Pasteur, T., M. Bethenod, D., Bourguet, This by is protectedarticle reserved. Allrights copyright. Bourguet, D., Bethenod M. T., Trouve C., & Viard F. (2000a). Ho Pélo D., Bourguet, A., Bontemps, Pélozuelo B., Frérot, F., Rousset, Y., Thomas, T., Bethenod, M. Sympatric L. speciation i Berlocher,Feder, (2002). S. J. H., & Y. Benjamini, Y.,& Hochberg, Alexandre,Ponsard, H.,S., Bourguet, D., R.,Vitalis, Audiot, References and two anonymous reviewers provided helpful comments on the ma USDA-ARS isan equal opportunity employer and provider. Graham mention of products or servicesdoes not constitute an endorsem of results the reports article This study. this in used assays We thank Natalie Wertish and Dietz forMiranda carryingout por and cooperative agreementbetween USDA-ARS 58-5030-7-066 and Tu corn borer borer corn Sciences maize. insecticidal Sciences Btmaize? insecticidal Europeanborer corn Biological Sciences adult malesat aggregation sites. Europeanborer, corn the evolutionof oviposition. for preferences plant borer, corn European the isol D. Genetic Bourguet, (2005). controversy? beyond moving B(Methodological) Series andpowerful approachto multiple testing. J e69211. plant adaptation two closely relatedin lepidopteran species. (2013).When history repeats itself: exploring the genetic arch , , 267 267 Ostrinia nubilalis (1439), 117-122. (1439),117-122. (1449),1177-1184. 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Accepted Article This by is protectedarticle reserved. Allrights copyright. Tang, K., Fu, D. J., Julien, D., Braun, A., Cantor, C. R., &Kö Effect (2006). M. Friberg, & F., Eroukhmanoff, E.I., Svensson, Spangler, S. M., & Calvin, D. D. (2000). Influence of sweet cor Sorenson, C. E., Kennedy,G. G., Van Duyn, W., Bradley Jr, J. R andno Slatkin, M. bydistanceinequilibrium (1993). Isolation Shimomura, M., Minami, H., Suetsugu, Y., Ohyanagi, H., Satoh, C Servedio, M.R., & Boughman, J.W. (2017) The role of sexual sele Schluter,D. &Conte, G.L. (2009). Geneticsand spec ecological Schluter,an D. (2001).Ecology Savinelli, C. E.,Bacheler, J. S., & Bradley JR, J.R. (1988). S., E. Scordato, J., R. Safran, Smith, H. E. (1920). Broom corn, the probablehost in which Showers, W. (1981). Geographic variation of the respon cage conditions in North Carolina. Carolina. North in conditions cage European cornborer (Lepidoptera: Pyralidae) for field corn and 643-650. reproductive isolation: a research agenda. Contributions naturalof and sexual selectionto the evolution 10020. by mass spectrometry. damselfly. selectionon adaptive population divergencepremating andisola 1235. borer (Lepidoptera: Crambidae) oviposition. nubilalis Geographical pheromonein response variation ofthe European co America. Evolution Genomics (2009). KAIKObase: silkworman integrated genome andd database Sciences of Academy National Evolution speciation. speciation. sciences. Springer, New York. sciences.Springer, New In: Insect Life Insect History Patterns , in North Carolina. , Journal of Economic of Journal Entomology , , 10 47 16 Annual Review of Ecology, Evolution, Systematics and Ecology, Annualof Review Evolution (1), 486. (1), 264-279. (7), 372-380. (7), 372-380. , 60 Proceedings of the National Academy of Sciences Symes, L.B., Rodríguez, R. L., (6), 1242-1253. (6), 1242-1253. Entomologia Experiment d the origin of species. species. d of the origin (R. &Dingle H.,Denno eds), pp. 97-111, Proceedings inlife , 106 (Supplement 1), 9955-9962. (Supplement 1), 9955-9962. Environmental Entomology Environmental , 13 , 425–430. Environmental Entomology Trends inEcology & Evolution ster, H. (1999). Chip-based genotyping alis et Applicata et alis TrendsEcology in & ., & Walgenbach, J. F. (1992). Ovipositional Ovipositional preferencesof the n growth stages onEuropean stages n growth corn Pyrausta nubilalis Pyrausta &Mendelson,T. C.(2013). n-equilibrium populations. ., Antonio,B., ... &Nagaraju, J. ction in local adaptation and inlocalction adaptation se in the European corn borer. s of and natural sexual iation. , 48 rn borer rn , , 85-109. of premating , ata tool.ata mining 17 Proceedings of the 64 cotton under field field under cotton , tion ina (4), 688-690. (4), 688-690. (2), 177-185. 96 Hübner reached , 29 (18), 10016- , Ostrinia (6), 1226- , 28 (11), (11), BMC Accepted Article

This by is protectedarticle reserved. Allrights copyright. Wang, Y., Kim, K. S., Guo, W., Li, Q., Zhang, Y., Wang, Z., & C Wagner, C. E., Harmon, L. J., & Seehausen, O. (2012). Ecologica van Doorn, G. S., Edelaar, P., & Weissing, F. J. (2009). On the a E. Host plant (1995). &Mason, S., constituents C. Udayagiri, Thomas, Y., Bethenod, M.T., Pelozuelo, L., Frérot, B., & Bourg Tate, C. Tate, C. D., Hellmich,R. L., & Lewis, C.L. (2006). E doi:10.5061/dryad.kj34mq9. doi:10.5061/dryad.kj34mq9. High throughput single nucleotide polymorphism fo genotype data accessibility Data samples;EBD advice andcontributed and TWS provided towriting data;SJFexperiments strategy designed andcollected locat for d the of analyzed otherauthors;and YWeach portions KSK, GMK, BSC experiments, designed analyzed and data, wrotethe manuscri Author Contributions Vinal, S.C. (1917). generalist herbivore: European corn borer. 273. Hübner pheromone, I.Sex moth emergence timing, and parasitism. cornEuropean b ofthe strains host-plant two sympatric between Economic Entomology corn preferences neonate for and weeds Crambidae) (Lepidoptera: evolution and pestarthropods.adaptation of of cornregion borer a species in I divergent sympatry: between predict adaptive radiation. together Bulletin I78:Station Experiment Agricultural 147 Massachusetts sexual selection. Applicata , 76 The European corn borer, a recently established pest in Massachusetts pest arecently established European corn borer, The (1), 59-65. Science , 99 , 326 (6), 1987-1993. , 1704-1707. Nature , Entomologia Experimentalis et et Entomologia Experimentalis 487 Molecular Ecology Molecular , 366-370. 366-370. , valuation of of valuation oates, oates, S.(2017).B. Introgression origin of and of bynatural species origin uet, D.(2003). Genetic isolation s oviposition stimulants fora l opportunity and sexual selection selection sexual and opportunity l ion oftraps andcollection of ata; JS conducted laboratory JSconductedlaboratory ata; pt with contributions from r all samples are available at at available all are r samples portions the manuscript. of , Ostrinia nubilalis Ostrinia mplications on the 26 -152. orer, (24), 6892-6907. 6892-6907. (24),

in corn. Evolution Ostrinia nubilalis Ostrinia Journal of of Journal , 57

(2), 261- .

Accepted Article Significance asdetermined by dbRDA (Table 2)are showneac for and 16 degrees offreedomand 16 ( degrees ( (km) distance lure (3:97 E-:Z11-14:OAc), or lure (trap), homozygous genotypes from the This by is protectedarticle reserved. Allrights copyright. Harstack wire cone trap; of content ratio w from plant determined as host with larval count ofmales from based on (3:97 E-:Z11-14:OAc). Pheromone strainof the same males was d baitedwith eitheran E-lure ateach ( traps location pheromone where 1. Table based on based on contributions to subpopulations (collection sites; 1)basedondbRDATable analy 4 Figure ( markers including the C4 (δ C4 Figure 2 Figure trap.Scentry Figure 1 Figure Table 2. Table genotypes). Range of pgfar polymorphism (SNP) genotype datapartitioned into among variation the greatest 3 Figure fromvalues 1,000estimated iterations performed using MantelT pairwise distance( genetic subpopulations (collection sites; 1). for isolaTable Tests an abbreviations in Table 1 and coordinates Tableabbreviations inTable1and in S1). η 2 ), andassociated 13 locus, and and locus, Ostrinia nubilalis Ostrinia C range of -8 to -17)]. to -8 of range C Samplelocations andacrosswithin yearsn 12 sitesthe in for Distance-based redundancy analysis (dbRDA) of eigenvectors (EV ofeigenvectors redundancy analysis(dbRDA) Distance-based Influence of ecological differentiation of Influence onthe factors genetic Influence of geographic distance on genetic differentiation be Topographic map ofgeographic locations 12 collection for site plot Scatter of factor scores the along Principal major Coordi pgfar A) male response (attraction) to either E-:Z11 lure E-strain to (99:1 (attraction) response male genotype (homozygous pgfar 13 C) Ostrinia nubilalis Ostrinia C/ host plant history host using plant 64SNP markers excluding the 14 P genotype excluded). The sumof squares (Sum ofSq.), -values are shown. are -values C δ (i.e., F pgfar ST E- and Z-pheromone strainsand Z-pheromone are E- E-estimates whenbetween andZ-strain by partitioned pheromon ● = = F locus, ST F ♦ cloth Scentry trap. Ostrinia nubilalis Ostrinia 1,16 13 ) and geographic distance. Associated R Associated distance. geographic and ) B) = C) are indicated within brackets [C3 (δ within brackets [C3 C)indicated are ), proportion of the total variance attributed to each to ), componen theof attributed proportion totalvariance Harstack wire cone trap; wirecone Harstack

pgfar population genetic structure ofpheromone structure population [assigned genetic strain B) pheromone strain using pheromone 64SNP markers the excluding genotype], host planthistory and (C3:C4), geographic pgfar-e pgfar population-derived single population-derived nucleotide / -e and genetic marker, orC3:C4 (host ratio plant). A) pgfar-z ♦ pheromone strain using 65 pheromone strain SNP = sympatric. Males were captured Males sympatric. were ● Harstack wire cone cone trap; wire Harstack = = tion-by-distance model between cloth Scentry trap. trap. Scentry cloth / -z 99:1 E-:Z11-14:OAc) or Z-lure ses loci,excluding (64 ; heterozygous heterozygous ; ing stable carbon isotope ing stable isotope carbon ests. ests. h comparison. comparison. h etermined independently independently etermined between between 2 13 nates (PCs) accounting for for (PCs) nates accounting (goodness and offit) ortheast United ortheast United States tween C range of of C range to -22 -32); s (location pgfar -14:OAc) orZ-strain ) forthe relative F -statistic based 1 on -statistic Ostrinia nubilalis Ostrinia Ostrinia nubilalis Ostrinia locus. pgfar-e ● = cloth cloth ♦ / pgfar -z = = in ). The The ). e P

-

t

Accepted Article

This by is protectedarticle reserved. Allrights copyright. Year Bro10 2 Location ID # of content ratio w from plant determined as host with larval count ofmales from based on (3:97 baitedwith eitheran E-lure ateach ( traps location pheromone where 1. Table Roc10 Roc092 1 Ran101 1 ♦ 0 1 ♦ ♦ Pla10 Pla09 9 ♦ Oak07 8 ♦ Nor10 7 ♦ Leh10 6 ♦ Lan10 Lan09 ♦ Lan08 5 Int07 4 ♦ Dru09 3 ♦ ♦ ♦ Bed09 1 C4 (δ C4

o0 ♦ Roc08 ♦ Ran09 ♦ Por09 13 E Ostrinia nubilalis Ostrinia C range of -8 to -17)]. to -8 of range C -: Sample locations within and across years for Samplelocations andacross n 12 sitesin the within yearsfor Z pgfar 11-14:OAc). Pheromone strain of the 11-14:OAc). same Pheromone of strain was determined males ● ● ● rolw,P 201 PA Brooklawn, lis A 201 200 PA Matilda, Port 200 Plains, PA Plains, PA 200 NY Corners, Oaks 201 Lehigh, PA 201 PA Landisville, 200 PA Landisville, 200 PA Landisville, 200 200 NY Interlaken, PA Drums, 200 Bedminster, PA okpig A 201 PA Rockspring, 200 PA Rockspring, 200 201 PA Rockspring, 200 PA Ransom, PA Ransom, 201 PA Northampton, genotype (homozygous 13 C/ 14 C δ (i.e., E- and Z-pheromone strainsand Z-pheromone are E- ♦ 13 = C) are indicated within brackets [C3 (δ within brackets [C3 C)indicated are Harstack wire cone trap; wirecone Harstack 9 0 0 9 8 0 0 9 0 9 7 0 0 9 8 7 9 9 pgfar-e 48 48 21 lure E- counts Trap 704 717 717 704 48 47 32 44 47 48 28 48 48 3 20 48 48 32 46

/ 48 48 48 lure Z- 48 46 32 22 48 18 11 18 48 48 48 48 45 45 48 -e and

pgfar-z 10 [ 1;; 9] 9] 1;; [ 10 0;;26] [ 26 4] 0; 4[ pgfar-e C3/C4] plant [host strain Pheromone [57;298] [57;298] 355 1;34 [ 35 2] [28; 30 0;24] [ 24 1;35] [ 36 0] 0; 0[ 1;15] [ 16 1;18] [ 19 5;15] [ 20 0;22] [ 22 0] 0; [ 0 9] 0; [ 9 3;26] [ 29 1;14] [ 15 0;18] [ 18 42 [15;;27] sympatric. Males were captured Males sympatric. were ● = = cloth Scentry trap. trap. Scentry cloth / / -e -z 99:1 ] ; heterozygous heterozygous ; ing stable carbon isotope ing stable isotope carbon E 65 [20;45] 65 [20;45] 0;43] 43 [ 47 [11;36] pgfar-z [140;571] [140;571] 711 2;42] 44 [ 1;41] 42 [ 0;33] 33 [ 3;16] 20 [ 48 [13;35] 4;15] 19 [ 5] 3; 8[ 6;10] 16 [ 1;49] 49 [ 8;36] 44 [ 46 [19;27] 46 [16;33] 49 [21;25] 6;40] 46 [ 6;40] 46 [ -: 13 ortheast United ortheast United States Z C range of of C range to -22 -32); 11-14:OAc) or Z-lure / -z independently independently

pgfar-e 21 [ 3;18] 3;18] 21 [ 3;24] 27 [ 5;13] 18 [ pgfar-e [46;309] [46;309] 355 2;15] 17 [ 8;13] 21 [ 7] 0; 7[ 0;10] 10 [ 4;43] 47 [ 0;31] 31 [ 0;12] 12 [ 4;26] 30 [ 1;24] 25 [ 5] 2; 7[ 2;11] 13 [ 4;17] 21 [ 6;23] 29 [ 0;13] 13 [ 4] 2; 6[ / -z

in ). The The ). / -z

Accepted Article and 16 degrees offreedomand 16 ( degrees ( (km) distance lure (3:97 based on contributions to This by is protectedarticle reserved. Allrights copyright. B) A) 2. Table (

η 2 Population by by Population tr lure-baited in (males response pheromone by assigned Strain eiul .081 0.0038010 0.49550 0.8389 Residual 0.0001993 0.48951 0.8711 0.59640 EV3 distance Geographic 0.7151 0.0002069 0.69730 0.0001699 0.6603 EV2 distance Geographic 0.0001569 EV1 distance Geographic C3:C4 Mean 0.00135226 ( strain Pheromone eigenvector Component 0.047 0.36963 0.059 0.027 0.26873 1.0361 0.71528 0.00008757 1.2943 Residual 0.028 0.5864 0.00010939 0.73826 0.00004956 EV3 distance Geographic 0.6105 0.00005160 EV2 distance Geographic EV1 distance Geographic C3:C4 Mean (response) Sq. Sumof strain Pheromone eigenvector Component ), andassociated Distance-based redundancy analysis (dbRDA) of eigenvectors (EV ofeigenvectors redundancy analysis(dbRDA) Distance-based A) E -: male response (attraction) to either lure E-strain to (99:1 (attraction) response male Z 11-14:OAc), or pgfar pgfar Ostrinia nubilalis Ostrinia P pgfar genotype excluded). The sumof squares (Sum ofSq.), genotype genotype -values are shown. are -values

00159 .32 0.00199 4.4362 0.0010539 ) F 1,16 B)

Sum of Sum Sq.of 0.00021459 2.5390 0.03497 0.115 0.115 0.03497 2.5390 0.00021459 ), proportion of totalvariance the attributed toeach componen

pgfar population genetic structure ofpheromone structure population [assigned genetic strain genotype], host plant history and geographic(C3:C4), F F 1,16 1,16

4 0 3 4 P P 8

aps) η 0.035 0.035 0.037 0.028 0.030 η 0.189 0.189 2 2 E -: Z 11-14:OAc) or Z-strain Z-strain or 11-14:OAc) ) forthe relative F -statistic based 1 on -statistic t Accepted Article

Accepted Article