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Extensive chromosomal variation in a recently formed natural allopolyploid species, Tragopogon miscellus (Asteraceae)
http://www.pnas.org/content/109/4/1176 ( Publisher's URL )
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Title: Extensive chromosomal variation in a recently formed natural allopolyploid species, Tragopogon miscellus (Asteraceae)
Series Title: www.pnas.org/cgi/doi/10.1073/pnas.1112041109
Physical Description: Journal Article
Creator: Chester, Michael
Gallagher, Joseph P.
Symonds, V. Vaughan
Cruz da Silva, Ana Veruska
Mavrodiev, Evgeny V.
Leitch, Andrew R.
Soltis, Pamela S.
Soltis, Douglas E.
Publisher: National Academy of Sciences
Place of Publication: Washington, DC
Publication Date: January 24, 2012
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Abstract: Polyploidy, or whole genome duplication, has played a major role in the evolution of many eukaryotic lineages. Although the prevalence of polyploidy in plants is well documented, the molecular and cytological consequences are understood largely from newly formed polyploids (neopolyploids) that have been grown experimentally. Classical cytological and molecular cytogenetic studies both have shown that experimental neoallopolyploids often have meiotic irregularities, producing chromosomally variable gametes and progeny; however, little is known about the extent or duration of chromosomal variation in natural neoallopolyploid populations. We report the results of a molecular cytogenetic study on natural populations of a neoallopolyploid, Tragopogon miscellus, which formed multiple times in the past 80 y. Using genomic and fluorescence in situ hybridization, we uncovered massive and repeated patterns of chromosomal variation in all populations. No population was fixed for a particular karyotype; 76% of the individuals showed intergenomic translocations, and 69% were aneuploid for one or more chromosomes. Importantly, 85% of plants exhibiting aneuploidy still had the expected chromosome number, mostly through reciprocal monosomy- trisomy of homeologous chromosomes (1:3 copies) or nullisomy- tetrasomy (0:4 copies). The extensive chromosomal variation still present after ca. 40 generations in this biennial species suggests that substantial and prolonged chromosomal instability might be common in natural populations after whole genome duplication. A protracted period of genome instability in neoallopolyploids may increase opportunities for alterations to genome structure, losses of coding and noncoding DNA, and changes in gene expression.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Michael Chester.
Publication Status: Published
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Source Institution: University of Florida Institutional Repository
Holding Location: University of Florida
Rights Management: All rights reserved by the submitter.
System ID: IR00001290:00001

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Extensivechromosomalvariationinarecentlyformed naturalallopolyploidspecies, Tragopogon miscellus (Asteraceae) MichaelChester a ,JosephP.Gallagher a ,V.VaughanSymonds b ,AnaVeruskaCruzdaSilva c,d ,EvgenyV.Mavrodiev d AndrewR.Leitch e ,PamelaS.Soltis d ,andDouglasE.Soltis a,1 a DepartmentofBiology,UniversityofFlorida,Gainesville,FL32611; b InstituteofMolecularBiosciences,MasseyUniversity,PalmerstonNorth,4442, NewZealand; c EmbrapaTabuleirosCosteiros,CEP49025-040,Aracaju-SE,Brazil; d FloridaMuseumofNaturalHistory,UniversityofFlorida,Gainesville, FL32611;and e SchoolofBiologicalandChemicalSciences,QueenMaryUniversityofLondon,LondonE14NS,UnitedKingdom EditedbyJamesA.Birchler,UniversityofMissouri,Columbia,MO,andapprovedDecember6,2011(receivedforreviewJuly22,2011) Polyploidy,orwholegenomeduplication,hasplayedamajorrole intheevolutionofmanyeukaryoticlineages.Althoughthe prevalenceofpolyploidyinplantsiswelldocumented,themolecularandcytologicalconsequencesareunderstoodlargelyfrom newlyformedpolyploids(neopolyploids)thathavebeengrown experimentally.Classicalcytologicalandmolecularcytogenetic studiesbothhaveshownthatexperimentalneoallopolyploids oftenhavemeioticirregularities,producingchromosomallyvariablegametesandprogeny;however,littleisknownaboutthe extentordurationofchromosomalvariationinnaturalneoallopolyploidpopulations.Wereporttheresultsofamolecular cytogeneticstudyonnaturalpopulationsofaneoallopolyploid, Tragopogonmiscellus ,whichformedmultipletimesinthepast 80y.Usinggenomicand uorescenceinsituhybridization,we uncoveredmassiveandrepeatedpatternsofchromosomalvariationinallpopulations.Nopopulationwas xedforaparticular karyotype;76%oftheindividualsshowedintergenomictranslocations,and69%wereaneuploidforoneormorechromosomes. Importantly,85%ofplantsexhibitinganeuploidystillhadthe expectedchromosomenumber,mostlythroughreciprocalmonosomy-trisomyofhomeologouschromosomes(1:3copies)ornullisomy-tetrasomy(0:4copies).Theextensivechromosomalvariation stillpresentafter ca. 40generationsinthisbiennialspeciessuggeststhatsubstantialandprolongedchromosomalinstability mightbecommoninnaturalpopulationsafterwholegenome duplication.Aprotractedperiodofgenomeinstabilityinneoallopolyploidsmayincreaseopportunitiesforalterationstogenome structure,lossesofcodingandnoncodingDNA,andchangesin geneexpression. compensatedaneuploid | segmentalallopolyploid P olyploidyhasplayedamajorroleintheevolutionofmany extanteukaryoticlineages(1).Inplants,genomicandtranscriptomicdataassociatesomeancientpolyploidyeventswith majorradiations,includingtheemergenceofseedplantsand angiosperms(2)andsomelargecladeswithinangiosperms(3). Thephylogeneticpositioningoftheseancientpolyploidyevents suggeststhattheymighthaveledtokeyphenotypicinnovations oranincreasedtolerancetoextremeenvironmentalchanges(1, 2,4,5).Withingenera,polyploidyhascoincidedwithanestimated15%ofangiospermspeciationevents(6).However,over recenttimescales,polyploidsdiversifymoreslowlythantheir diploidrelativesandaremorelikelytogoextinct,perhapsduein parttotheunstablenatureofneopolyploids(7). Classicalcytologicalstudieshaveshownthatmanynewly formedexperimentalautopolyploidsandallopolyploidsproduce chromosomallyvariablegametesandprogeny(8 – 19).Oneimportantconsequenceforallopolyploidsisthattheymightnot behavestrictlyas “ constantspecies-hybrids ” (20);thatis,disomic inheritanceateachparentallocusmaybeupset,andgenetic heterozygositybetweentheparentalspeciesmaynotremain xed afterwholegenomeduplication(18).Insomecases,theregularity ofmeiosiswasfoundtoincreaserapidlyinexperimentalneoallopolyploidsthatwereinitiallychromosomallyunstable(21, 22);forexample,afterjust veselfedgenerations, Nicotiana neoallotetraploidsdisplayedbivalentpairingand > 99%stainable pollen(22).Verylittleisknownabouttheextentordurationof chromosomalvariabilityinnaturalneoallopolyploidpopulations. Standardchromosomecountscanidentifynumericalaneuploidy, inwhichthereisachangeinthetotalchromosomenumber; however,aneuploidyismuchhardertodetectinapolyploidin whichchromosomenumbersvarywithinparentalsubgenomes butthetotalchromosomenumberremainsunchanged.Such chromosomesubstitutionshavebeenshownforasubsetof chromosomesinafewexperimentalneoallopolyploids(21,22). Onlyonerecentmolecularcytogeneticstudy,onthesynthetic allotetraploid Brassicanapus ,wasabletodetectaneuploidy acrossallchromosomes,includingwhereitresultedinsubstitutions,intheearlygenerationspostpolyploidization(23).Standardmeiotic(24)andmitotickaryotypicanalyses(25),aswellas thedistributionofcentromericandtelomericmarkers(26),have suggestedthattherecentlyformedallopolyploids Tragopogon miscellus and Tragopogonmirus arechromosomallystable. However,preliminaryworkusinggenomicand uorescentinsitu hybridization(GISHandFISH,respectively)revealedaneuploidyandtranslocationsinafewindividualsofbothallotetraploids(27). Herewepresenttheresultsofanin-depthmolecularcytogeneticsurveyofanaturallyoccurringneoallotetraploid, T.miscellus. Thisspeciesis 80yold( ca. 40generationsforthis biennial),havingformedrepeatedlyinNorthAmericaafterthe introductionofitsdiploid(2 n =12)progenitors, Tragopogon dubius and Tragopogonpratensis ,fromEuropeintheearly1900s (24,28).Asnotedearlier,preliminaryGISH/FISHanalyses revealeddeviationsfromtheexpectedadditivekaryotypeintwo ofthethree T.miscellus individualsanalyzed(27),butgiventhis smallsamplesize,theextentofthisphenomenoninnature remainedunclear.Thus,animportantaimofthepresentstudy wastoexaminevariationwithinandbetweenpopulationsacross agreaterpartofitsrange.Usingupdatedinsitumethodology, Authorcontributions:M.C.,A.R.L.,P.S.S.,andD.E.S.designedresearch;M.C.,J.P.G.,V.V.S., A.V.C.d.S.,E.V.M.,P.S.S.,andD.E.S.performedresearch;M.C.,J.P.G.,andV.V.S.analyzed data;andM.C.,V.V.S.,A.R.L.,P.S.S.,andD.E.S.wrotethepaper. Theauthorsdeclarenocon ictofinterest. ThisarticleisaPNASDirectSubmission. FreelyavailableonlinethroughthePNASopenaccessoption. Datadeposition:ThesequencesreportedinthispaperhavebeendepositedintheGenBankdatabase(accessionnos. JN227618 and JN227619 ). 1 Towhomcorrespondenceshouldbesent.E-mail:dsoltis@botany.u .edu. Thisarticlecontainssupportinginformationonlineat www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1112041109/-/DCSupplemental 1176 – 1181 | PNAS | January24,2012 | vol.109 | no.4www.pnas.org/cgi/doi/10.1073/pnas.1112041109

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weobtainedcompletekaryotypesof68individualsgrownfrom eld-collectedseedfromsixnaturalpopulations(Fig.1),an additionalsamplecomprisingasetof10siblings( Materialsand Methods ),andeight eld-collectedplants. ResultsandDiscussion GISHandFISHKaryotypingof T.miscellus GISHkaryotypesreadily distinguishedchromosomesderivedfromthediploidprogenitors, T.pratensis and T.dubius (referredtoasP-andD-subgenomes, respectively),whichtogetherconstitutethe T.miscellus genome (Fig.2).GISHsignalsatseveralpositions,includingsomeknown tocompriserepetitiveDNA(Fig.2),permittedidenti cationof allchromosomeswithineachsubgenome(Fig.2).TheeffectivenessofGISHindiscriminatingDNAoftheprogenitorswas con rmedusingapairofdispersedrepeats,eachofwhichis highlyrepresentedinonlyonediploidparent(Fig.3).These repeatsandadditionalFISHprobescon rmedchromosome designationsbasedonGISH. ExtensiveKaryotypicVariationIsPresentinAllAnalyzedPopulations. Fromthesixpopulationsstudied(Spokane-2,Veradale,Post Falls,Coeurd ’ Alene,Oakesdale,andPullman),only31%of T.miscellus plants( n =18)hadadditive(euploid)karyotypes (Fig.4and Figs.S1 – S5 ),whichcontaintheexpectedtwocopies ofeachparentalchromosome(e.g.,Fig.2).Theremaining69% ofplantshadoneormoreaneuploidchromosomes(whichwere notpresentintwocopies);10%wereeither2 n =23( n =2)or 2 n =25( n =4),and59%were2 n =24( n =34).Thesampleof 10siblingplantsfromSpokane-1wasalsohighlyvariable( Fig.S6 and TableS1 )andshowedasimilarfrequencyofeuploids( n = 2)andaneuploidsthatwereeither2 n =25( n =1)or2 n =24 ( n =7)(Table1).Suchextensivevariationinchromosomalcopy numberisunprecedentedwithinnaturalpopulations,buthas beenreportedforsyntheticallotetraploid B.napus (25)and,to alesserextent,forsyntheticallohexaploidwheat(29). AneuploidyFrequentlyResultsinChromosomeSubstitutions. Plants fromallsevennaturalpopulationsof T.miscellus werepooled foranalysesofaneuploidy.Ofthe48aneuploidplantsanalyzed, 41(34fromthesixsamplednaturalpopulationsplus7ofthe10 siblingplantsfromSpokane-1)represent “ compensatedaneuploids ” (22)becausetheyhadthe “ euploid ” number,2 n =24 (Fig.4).Intheseplants,thenumbersofchromosomesdeviating aboveandbelowtwocopiesareequal,giving24chromosomes. Thisisthe rstreportofsuchextensivecompensatedaneuploidy innature,althoughithasbeenfoundinexperimentalneoallopolyploids(23,29),andsuchchromosomesubstitutionshave beenusedextensivelyincerealbreeding(30 – 32).Compensated aneuploidyinnaturalpopulationsof T.miscellus provides apowerfulmechanismin uencingtheinheritanceand xationof allelesinearlyallopolyploidevolution. Forallbutoneofthe41compensatedaneuploidplants,aneuploidywasreciprocalbetweenputativehomeologs(e.g.,A Du andA Pr ),occurringaseithermonosomy(onecopy)forone homeologandtrisomy(threecopies)fortheotherhomeolog,or, lessoften,nullisomy(zerocopies)andtetrasomy(fourcopies) (Fig.4).Thus,thesumofeachhomeologouschromosomegroup (A – F)wasconsistentlyfourin40ofthe41plants.Thesingle Fig.1. Mapsshowing T.miscellus collectionsitesinrelationtothenorthwesternUnitedStates.( Upper )ThePaci cOcean(gray)andstatesofWashington(WA),Oregon(OR),Idaho(ID),andMontana(MT)areindicated; arectangleshowstheareaintheenlargedmap.( Lower )Locationsofcollectionsites,withmajorroadsshowningray.ThegreaterSpokanearea (shadedgray)includesthecityofSpokane(collections2729and2730),and thetownsofVeradale(2731),PostFalls(2736)andCoeurd ’ Alene(2738).Two additionalcollectionsitesareindicated:Oakesdale(2872)andPullman(2785/ 2875-B).TheWashington – Idahostatelineisindicatedbythedottedline. (Scalebar:10mi.) Fig.2. Mitotickaryotypeofa T.miscellus plantshowinganadditivechromosomecomplement.Metaphasechromosomes(fromplant2875 – 1-1)were rstsubjectedtoFISH(toprow)usingprobesfor35SrDNA(green),acentromericrepeat(TPRMBO;red),andasubtelomericrepeat(TGP7;yellow). ThesamespreadwasthenreprobedwithtotalgenomicDNA(GISH;middle row)of T.dubius (green)and T.pratensis (red);chromosomeswerecounterstainedwithDAPI(gray).Thelowerrowshowsthesamechromosomes withonlyDAPIstaining(blue).Eachchromosomeispresentintwocopies (disomic).Examplesofchromosomesthatarehomologsandhomeologsare indicated.(Scalebar:5 m.) Fig.3. Mitotickaryotypeofanadditive T.miscellus plantprobedwith dispersedrepetitiveDNA.Metaphasechromosomes(fromplant2875-B-5) were rstsubjectedtoFISH(upperrow)usingamixtureoftwoprobesfor DNArepeatsabundantinonlyoneofthediploidparentalgenomes, T.pratensis (pra001;red)and T.dubius (dub005;green).Inaddition,asubtelomericrepeat(TGP7;yellow)presentinbothsubgenomeswasincluded, andDNAwascounterstainedwithDAPI(gray;visiblewheretheprobesignal islessintense).Thesamechromosomespreadwasthenreprobedwithtotal genomicDNA(lowerrow)of T.dubius (green)and T.pratensis (red).(Scale bar:5 m.) Chesteretal. PNAS | January24,2012 | vol.109 | no.4 | 1177 EVOLUTION

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2 n =24plantthatdidnotfollowthispattern(Coeurd ’ Alene plant2738 – 2-1)wasmonosomicfortheEchromosomeof T.dubius origin(E Du )andtrisomicfortheFchromosomeof T.pratensis origin(F Pr )(Table1).Thisresultedinatotalofthree groupEchromosomesand vegroupFchromosomes. Ofthe40plantsof T.miscellus with2 n =24andaneuploidy betweenhomeologouschromosomes,29hadonlyonecaseof mono-trisomyornulli-tetrasomy(i.e.,asinglemonosomicor disomicsubstitution,respectively).Theremainingplantsshowed multiplesubstitutionsinvolvingtwo( n= 9),three( n= 1),or fourchromosomegroups( n =1)(Fig.4),butinallcasesthe totalnumberofchromosomecopieswasfourforeachofthe sixgroups. Aneuploidyisexpectedtoresultinloweredmetabolicef ciencyduetodisturbancesinthenormalproteinstoichiometry foraffecteddosage-sensitivegenes(33 – 36).Onepossibleexplanationfortherepeatedoccurrenceofhomeologoussubstitutions isthatthetetrasomicdosagemightbemaintainedforgenes presentonbothhomeologs(37).Thisinferenceofhomeologyin T.miscellus isunderscoredforchromosomegroupsB – E,which werefoundinnulli-tetrasomiccombinationsin T.miscellus .In otherallopolyploids,substantialsyntenyisrequiredforcompensationofcompletechromosomeloss(nullisomy)bychromosomesfromanotherprogenitororrelatedspecies(23,32,38 – 40).ThedatathatwereportsuggestthatchromosomesA – Fof T.dubius and T.pratensis origincorrespondtosixgroupsof largelyhomologous(homeologous)chromosomes.Thecompensatorypatternmayresultfrommispairingatmeiosis(e.g.,in multivalents),leadingtothemissegregationofhomeologous chromosomes(41 – 43). AneuploidyDoesNotShowaConsistentParentalBiasin T.miscellus Acombinedanalysisoftheaneuploidydatafromthesixnatural populationsdidnotdemonstrateasigni cantparentalbiasin termsofeitherchromosomegains(trisomyortetrasomy; P = 0.50,one-sampleproportionstest)orchromosomelosses(monosomyornullisomy; P =0.20,one-sampleproportionstest)(Fig. 5).Opposingtrendswereapparentamongpopulations,however. OnlyplantsfromCoeurd ’ Aleneshowedatrendtowardagainof P-subgenomechromosomesandalossofD-subgenomechromosomes.ThispopulationwasalsounusualinthatonlyEand Fchromosomeswereaneuploid( TableS1 and Fig.S4 ).When Fig.4. Mitotickaryotypesof10 T.miscellus individualsfromOakesdale,WA.GISHwascarriedoutwithtotalgenomicDNAprobesof T.dubius (green)and T.pratensis (red).Arrowsindicatethepositionsoftranslocationbreakpoints.Diamondsymbolsarebelowaneuploidchromosomes(i.e.,thosethatarenot disomic).(Scalebar:5 m.) Table1.Summaryof T.miscellus karyotypes PopulationEuploid Compensated aneuploid Numerical aneuploid Spokane-1(sibs)271 Spokane-2550 Veradale252 PostFalls162 Coeurd ’ Alene460 Oakesdale271 Pullman451 Pullman-B*620 *Pullman-BisasampleofeightadultplantscollectedfromPullman,WA. 1178 | www.pnas.org/cgi/doi/10.1073/pnas.1112041109 Chesteretal.

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theother vepopulations(Spokane-2,Veradale,PostFalls, Oakesdale,andPullman)wereanalyzedtogether,D-subgenome chromosomesweregainedmoreoften( P= 0.05,one-sample proportionstest),andP-subgenomechromosomeswerelost moreoften( P =0.01,one-sampleproportionstest).Thisbias appearsirrespectiveofparentage;plantsinPullmanhave T.dubius asthematernalparent,whereasallothershave T.pratensis asthematernalparent(44). Thebiasagainstchromosomesof T.pratensis origincontrasts withgenomicinvestigationsusingassaysbasedonsinglenucleotidepolymorphisms,whichhaveshownthatgenecopiesof T.dubius originarelostmoreoftenthanthoseof T.pratensis origin(44 – 48).Thus,chromosomegainsandlossesalonecannot explainthegenecopynumberbias,pointingtoothermechanisms. Small-scalenonreciprocalexchangesordeletions,arisingvia translocationsand/orhomeologousrecombinationasobservedin otherallopolyploids(49 – 51),alsomaybeoccurringin T.miscellus IntergenomicTranslocationsOccurPredominantlyBetweenHomeologous Chromosomes. Rearrangementsbetweensubgenomeswerecommonin T.miscellus ,detectedonatleastonechromosomein 76%oftheindividuals(44of58),notincludingthe10sibling progenyfromSpokane-1.ThegroupAchromosomesshowedthe highestfrequencyofapparenthomeologousexchangeswithat least vedifferenttranslocationbreakpointsinindividualsfrom sixpopulations( TableS1 ).Similarly,translocationsonthelong armsofB Du andB Pr wereobservedinplantsfromallseven populations( TableS1 ),suggestingthatthisregionmighthave apropensitytowardintergenomicrecombination. Despitetheextensivekaryotypicvariationseenoverall,some populationsexhibiteddistinctivekaryotypicsignatures.Eight ofthe10plantsfromSpokane-2sharedwhatappeartobethe samehomozygousreciprocaltranslocationsonA Du /A Pr ( Fig.S1 ) OnlyindividualsfromPostFallshadanapparentnonreciprocal translocationontheshortarmofD Du ( Fig.S3 ).Oncarefulvisual inspectionofintergenomictranslocations,allappearedtobeeither reciprocalornonreciprocalexchangesbetweenhomeologous chromosomes. T.miscellus PopulationsRepresentIndependentOrigins. Microsatellitedataobtainedforpopulationssampledinthisstudy, alongwithpreviousstudies(28,52),indicatethateachofthe populationssampledrepresentsanindependentpolyploidorigin ( Fig.S7 ).Microsatellitedatasuggestthatplantsofseparateoriginco-occurinSpokane-2andPostFalls.Inafewcases,genotypicandkaryotypicdatasuggestthatcrossingmighthave occurredbetweenplantsofdifferentpolyploidorigins(e.g., Spokane-2:2730 – 8-3and2730 – 10-1; Figs.S1 and S7 ),butthis appearstobeverylimited. PlantsShowingAneuploidyandRearrangementsPersistinNatural Populations. Thechromosomalvariationdescribedabovewas presentinplantsderivedfrom eld-collectedseed;thus,the extenttowhichthistypeofvariationpersistsamongadults growinginnaturalpopulationsisunknown.Toaddressthisissue, individualrosetteswerecollectedfromPullman,WA.Ofeight individualsanalyzed,twowerecompensatedaneuploids,showingasinglecaseofmonosomy-trisomybetweenhomeologous chromosomes,andsixwereeuploid( Fig.S8 ); veoftheplants alsoshowedtranslocations.Thus,bothaneuploidyandtranslocationswerealsoobservedinplantsfromnaturalpopulations. Ofnote,twooftheseeightplantsappearedtobefullyadditiveof theprogenitorgenomes,showingneitheraneuploidynortranslocations,whereasonlythreefullyadditiveplantswerefound amongthe68plantsgrownfromseed.This ndingsuggeststhat innaturalpopulationstheremaybeselectionagainstkaryotypes withmoreextremeaneuploidyandrearrangements;thishypothesismeritsfurthertesting. T.miscellus RemainsChromosomallyVariable. Unlikethediploid progenitorsof T.miscellus T.dubius and T.pratensis ,bothofwhich havestablekaryotypes(24,25),noneofthe ca. 40-generation-old populationsof T.miscellus appearedtobechromosomallyuniform.No T.miscellus populationwas xedforasinglekaryotype, andfewplantshadakaryotypethatwascompletelyadditiveof thetwoparents.Furthermore,thenumberofaneuploidsibling progenygeneratedbyoneplant(theSpokane-1maternalplant) wascomparabletothepercentageofaneuploidsobservedacross allofthepopulationsexaminedhere. Theextensiveaneuploidy(69%)observedacross T.miscellus populationsissimilartowhathasbeenreportedforothersyntheticorspontaneousneoallopolyploids,mostofwhichareof youngeragethan T.miscellus .FISHrevealedthepresenceof asimilarpercentageofaneuploids(71%)inthe fthgeneration (S 5 )ofsynthetic B.napus ;thisvaluecontinuedtorise,reaching 95%bytheS 10 generation(23).Classicalcytologicalstudies revealedthatinthe rstgenerationofaspontaneous Crepis allotetraploid(21),78%oftheplantswereaneuploid(comprising 49%numericalaneuploidsand29%compensatedaneuploids) (21),andinthe rstgenerationofaninduced Cyrtanthus allotetraploid(22),61%oftheplantswereaneuploid(comprising 57%numericaland4%compensatedaneuploids).Onlyafew chromosomalsubstitutionsin Crepis and Cyrtanthus neoallopolyploidsweredetectable,becausenotallparentalchromosomes weremorphologicallydistinct;thus,thesevaluesarelikelyunderestimates(18).SyntheticS 2 breadwheat( Triticumaestivum )lines exhibited0 – 50%aneuploidy,withvariationattributabletoprogenitorbackground(29).Instabilityinwheatneoallopolyploids probablycouldbeincreasedfurtherintheabsenceof Ph1 ,which preventshomeologouspairing(29,53 – 55). Selectionforincreasedfertilityshouldstabilizethegenome andreducetheextentofaneuploidyovertime(56).However,if acompensatedaneuploidplanthaslittlereductionin tness(i.e., homeologouschromosomessubstituteforeachother),andifthe productionofaneuploidsisongoing,thentheinterplaybetween generationofandselectionagainstaneuploidsisunclearand dif culttopredict.Likewise,ifnewchromosomalcombinations Fig.5. Stackedbarchartshowingthenumberofchromosomelossesand gainsfromGISHkaryotypesofseed-grownplants.Onthe y -axisarethe numbersofaneuploidchromosomesobservedinthe48plantsgrownfrom seed,whichwerenotchromosomallyadditiveoftheparents.Eachbaron the x -axisrepresentsoneofthesixhomeologouschromosomegroups,A – F, of T.pratensis (magenta)and T.dubius origin(green).Casesofchromosome loss(eithermonosomyornullisomy)andgain(eithertrisomyortetrasomy) areshownbelowandabovetheorigin,respectively.Theseverityofthe aneuploidyisindicatedbycolorintensity,withmonosomyortrisomyshown bylightercolorsandnullisomyortetrasomyindicatedbydarkercolors. Chesteretal. PNAS | January24,2012 | vol.109 | no.4 | 1179 EVOLUTION

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arisethatareselectivelyadvantageous,thencompensatedaneuploidymaybemaintained.Therefore,chromosomalvariation, suchasthatobservedin T.miscellus ,perhapsmightrepresent additional variation ratherthan instability SegmentalAllopolyploidBehavior. Stebbins(57)coinedtheterm “ segmentalallopolyploid ” todescribepolyploidsthatdonot exhibitstrictbivalentformationacrossallchromosomesordisomicinheritanceatallloci.Atatimewhenpolyploidswere classi edasautopolyploidsorallopolyploidsonthebasisof chromosomepairingbehavior(quadrivalentvs.bivalentformation,respectively),Stebbinsconsideredtheparentsofsegmental allopolyploidstooccupyanintermediatelevelofchromosomal divergencebetweenthoseofautopolyploidsandallopolyploids (57).Heproposedthatresidualhomologybetweenhomeologous chromosomesisprimarilyresponsibleforinconsistentbivalent formationandnondisomicinheritance.Meioticirregularities mayrendersegmentalallopolyploidsunstable(58,59),withincreasinglyrearrangedkaryotypesandshiftstopolysomicinheritance(59).Stebbins ’ sde nitionwasbasedonaconceptof structuralhomologybetweenprogenitorchromosomes,butits applicationwasbasedonpatternsofinheritanceand/orchromosomebehavior,withanunderlyingassumptionofadditivityof theparentalgenomes.However,thesamepatternmightarise throughcompensatedaneuploidywithpairingbetweenhomologsthatare,forexample,trisomicortetrasomic.Basedonthe extensivecompensatedaneuploidyobservedin T.miscellus ,itis likelythatdeviationsfromstrictbivalentpairinganddisomic inheritanceinothersegmentalallopolyploidsmayalsore ect postallopolyploidizationprocessesratherthanpartialchromosomalhomologyoftheparentalgenomes. ConsequencesforEstablishmentandEvolutionofYoungAllopolyploids. Classicalcytologicalstudiesofsyntheticandspontaneousneoallopolyploidsindicatethatsubstantialchromosomalinstability oftenfollowswholegenomeduplication.Recentmolecularcytogeneticstudiesofsynthetic B.napus (25)anddataprovided hereforthenaturalallotetraploid T.miscellus alsoshowcytologicalvariability,butrevealthatmuchofthevariationinvolves chromosomesubstitutionsandrearrangementsbetweenhomeologousregions.Thesetypesofchangeswouldhavebeendif cultorimpossibletodetectusingclassicalapproaches.The consequencesofsuchchromosomalinstabilitymaybeconsiderable,possiblyincludinggenelossandalterationsingene expression,bothofwhichhavebeendetectedinnaturalpopulationsof T.miscellus (44 – 47)andinsyntheticlinesof B.napus (49,60 – 62).Evenallohexaploidwheat( T.aestivum ),whichis ca. 10,000yold(63),stillexhibitssomechromosomalinstability; aneuploidscompose 1%ofintervarietalpopulations(64)and 2 – 3%ofcultivatedlines(29). Chromosomalvariation,suchasnonreciprocalintergenomic translocationsandcompensatinganeuploidy,willyieldsegregatinggeneticvariation.Giventhatallindividualsresultingfrom asinglepolyploidizationeventaregeneticallyhighlysimilar orpotentiallyevengeneticallyuniform(followingamodelof spontaneoushybriddoubling),thesechromosomalmechanisms wouldsupplygeneticvariationinadvanceofpointmutationsin thenewallopolyploid.Availabledatasuggestthatnaturalpopulationsofneoallopolyploidsmayundergoaprolongedperiodof aneuploidyandrearrangementsbeforegenomicstabilization, increasingopportunitiesforalterationsingenomestructure, lossesofcodingandnoncodingDNA,changesingeneexpression,segregatinggeneticvariationandthepossibilityofgenetic andphenotypicnovelty. MaterialsandMethods SeedCollections. Seedswerecollectedfrom60plantsfromsixpopulations, plus10siblingprogeny(sharingatleastthematernalparent)collectedfrom asingleplantfromaseventhpopulation(Spokane;2729).Detailsareprovidedin SIMaterialsandMethods .Vouchersforeachofthepopulations weredepositedattheUniversityofFloridaHerbarium. PlantCollectionsfromtheField. Asampleof22plantswascollectedfrom Pullman,WAonApril14,2011,asplantsemergedinthespring(justbefore bolting).Theseplantshadoverwinteredasbasalrosettes.Theywereshipped totheUniversityofFlorida,planted,andgrowninthegreenhouse,after whichrootswereobtainedforanalysis. ProgenitorDNARepeatIdenti cation/Isolation. Repetitivesequenceswere identi edfromgenomic454sequencesusinganapproachsimilartothat describedpreviously(65).Detailsareprovidedin SIMaterialsandMethods ChromosomePreparation. The nal2cmofgrowingrootswereharvestedand pretreatedinanaqueoussolutionof2mM8-hydroxyquinoline(SigmaAldrich)for4.5hat4C.Metaphasechromosomespreadswerepreparedas describedpreviously(66).Detailsareprovidedin SIMaterialsandMethods GISHandFISH. ProbesofgenomicDNAforGISHandrepetitiveDNAforFISH were uorescentlylabeledandthenappliedtochromosomespreadsas describedbyBirchleretal.(66),withafewminormodi cations.Detailsare providedin SIMaterialsandMethods NuclearMicrosatelliteAnalysis. Twelvemicrosatellitelociforplantsfromall sevensiteswereampli edandanalyzedasdescribedpreviously(52)and detailedin SIMaterialsandMethods StatisticalAnalysis. One-sampleproportiontestswereconductedwithR version2.13.0toidentifyanysigni cantdeviationfromanullexpectationof equalitybetweensubgenomesforchromosomecopynumberincreases(eitherthreeorfourcopies)andfordecreases(eitherzerocopiesoronecopy). ACKNOWLEDGMENTS. WethankChuckCody(WashingtonStateUniversity) forcollectingadult T.miscellus plants,Dr.RobertHarrisforassistinginthe laboratory,Dr.PatriceAlbertforadvisingonchromosomepreparationand insituhybridizationmethodology,andDr.RichardBuggsforcommenting onthemanuscript.ThisworkwassupportedbyNationalScienceFoundation GrantDEB-0922003.Publicationofthisarticlewasfundedinpartbythe UniversityofFloridaOpen-AccessPublishingFund. 1.VandePeerY,MaereS,MeyerA(2009)Theevolutionarysigni canceofancient genomeduplications. NatRevGenet 10:725 – 732. 2.JiaoY,etal.(2011)Ancestralpolyploidyinseedplantsandangiosperms. Nature 473: 97 – 100. 3.SoltisDE,etal.(2009)Polyploidyandangiospermdiversi cation. AmJBot 96:336 – 348. 4.DeBodtS,MaereS,VandePeerY(2005)Genomeduplicationandtheoriginof angiosperms. TrendsEcolEvol 20:591 – 597. 5.FawcettJA,MaereS,VandePeerY(2009)Plantswithdoublegenomesmighthave hadabetterchancetosurvivetheCretaceous-Tertiaryextinctionevent. ProcNatl AcadSciUSA 106:5737 – 5742. 6.WoodTE,etal.(2009)Thefrequencyofpolyploidspeciationinvascularplants. Proc NatlAcadSciUSA 106:13875 – 13879. 7.MayroseI,etal.(2011)Recentlyformedpolyploidplantsdiversifyatlowerrates. Science 333:1257 – 1257. 8.PooleCF(1933)Constantspecieshybrids. AmNat 67:188 – 190. 9.NewtonWCF,PellewC(1929) Primulakewensis anditsderivatives. JGenet 20: 405 – 467. 10.UpcottM(1939)Thenatureoftetraploidyin Primulakewensis JGenet 39: 79 – 100. 11.McNaughtonIH(1973)Synthesisandsterilityof Raphanobrassica Euphytica 22: 70 – 88. 12.TokumasuS(1976)Theincreaseofseedfertilityof Brassicoraphanus throughcytologicalirregularity. Euphytica 25:463 – 470. 13.HowardHW(1938)Thefertilityofamphidiploidsfromthecross Raphanussativus Brassicaoleracea JGenet 36:239 – 273. 14.BuxtonB,DarlingtonC(1931)Behaviourofanewspecies, Digitalismertonensis Nature 127:94 – 94. 15.ShkutinaFM,KhvostovVV(1971)Cytologicalinvestigationof Triticale TheorAppl Genet 41:109 – 119. 16.MarchantCJ(1963)Correctedchromosomenumbersfor Spartina townsendii andits parentspecies. Nature 199:929. 17.GottschalkW(1978)Openproblemsinpolyploidyresearch. Nucleus 21:99 – 112. 18.RamseyJ,SchemskeDW(2002)Neopolyploidyin oweringplants. AnnuRevEcolSyst 33:589 – 639. 1180 | www.pnas.org/cgi/doi/10.1073/pnas.1112041109 Chesteretal.

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19.DavisBM(1943)AnamphidiploidintheF1generationfromthecross Oenothera franciscana Oenotherabiennis ,anditsprogeny. Genetics 28:275 – 285. 20.WingeO(1932)Ontheoriginofconstantspecies-hybrids. SvenBotTidskr 26: 107 – 122. 21.PooleCF(1932)Theinterspeci chybrid, Crepisrubra C.foetida ,andsomeofits derivatives,II:Twoselfedgenerationsfromanamphidiploidhybrid. UnivCalifPubl AgrSci 6:231 – 255. 22.IsingG(1966)Cytogeneticstudiesin Cyrtanthus ,I:Segregationinanallotetraploid. Hereditas 56:27 – 53. 23.XiongZ,GaetaRT,PiresJC(2011)Homoeologousshuf ingandchromosomecompensationmaintaingenomebalanceinresynthesizedallopolyploid Brassicanapus ProcNatlAcadSciUSA 108:7908 – 7913. 24.OwnbeyM(1950)Naturalhybridizationandamphiploidyinthegenus Tragopogon AmJBot 37:487 – 499. 25.OwnbeyM,McCollumG(1954)Thechromosomesof Tragopogon Rhodora 56:7 – 21. 26.PiresJC,etal.(2004)Molecularcytogeneticanalysisofrecentlyevolved Tragopogon (Asteraceae)allopolyploidsrevealakaryotypethatisadditiveofthediploidprogenitors. AmJBot 91:1022 – 1035. 27.LimKY,etal.(2008)Rapidchromosomeevolutioninrecentlyformedpolyploidsin Tragopogon (Asteraceae). PLoSONE 3:e3353. 28.SoltisDE,etal.(2004)Recentandrecurrentpolyploidyin Tragopogon (Asteraceae): Cytogenetic,genomicandgeneticcomparisons. BiolJLinnSocLond 82:485 – 501. 29.MestiriI,etal.(2010)Newlysynthesizedwheatallohexaploidsdisplayprogenitordependentmeioticstabilityandaneuploidybutstructuralgenomicadditivity. New Phytol 186:86 – 101. 30.UnrauJ,PersonC,KuspiraJ(1956)Chromosomesubstitutioninhexaploidwheat. Can JBot 34:629 – 640. 31.LawCN,WorlandAJ(1996)Inter-varietalchromosomesubstitutionlinesinwheat, revisited. Euphytica 89:1 – 10. 32.MillerTE(1984)Thehomoeologousrelationshipbetweenthechromosomesofrye andwheat:Currentstatus. CanJGenetCytol 26:578 – 589. 33.BirchlerJA,BhadraU,BhadraMP,AugerDL(2001)Dosage-dependentgeneregulationinmulticellulareukaryotes:Implicationsfordosagecompensation,aneuploid syndromes,andquantitativetraits. DevBiol 234:275 – 288. 34.GoffSA(2011)Aunifyingtheoryforgeneralmultigenicheterosis:Energyef ciency, proteinmetabolism,andimplicationsformolecularbreeding. NewPhytol 189: 923 – 937. 35.BirchlerJA,YaoH,ChudalayandiS(2007)Biologicalconsequencesofdosage-dependentgeneregulatorysystems. BiochimBiophysActa 1769:422 – 428. 36.BridgesCB(1922)Theoriginofvariationsinsexualandsex-limitedcharacters. AmNat 56:51 – 63. 37.KhushGS(1973) CytogeneticsofAneuploids (Academic,NewYork). 38.Linde-LaursenI,Heslop-HarrisonJS,ShepherdKW,TaketaS(1997)Thebarleygenomeanditsrelationshipwiththewheatgenomes:Asurveywithaninternationally agreedrecommendationforbarleychromosomenomenclature. Hereditas 126:1 – 16. 39.SearsER(1944)Cytogeneticstudieswithpolyploidspeciesofwheat,II:Additional chromosomalaberrationsin Triticumvulgare Genetics 29:232 – 246. 40.LongwellJH,SearsER(1963)Nullisomicsintetraploidwheat. AmNat 97:401 – 403. 41.PhillipsLL(1962)Segregationinnewallopolyploidsof Gossypium ,IV:Segregationin NewWorld AsiaticandNewWorld wildAmericanhexaploids. AmJBot 49:51 – 57. 42.PhillipsLL(1964)Segregationinnewallopolyploidsof Gossypium .V.Multivalent formationinNewWorld AsiaticandNewWorld wildAmericanhexaploids. AmJ Bot 51:324 – 329. 43.KostoffD(1935)Studiesonpolyploidplants,X:Ontheso-called “ constancy ” ofthe amphidiploidplants. ComptRendAcadSciURSS 1:653 – 657. 44.TateJA,JoshiP,SoltisKA,SoltisPS,SoltisDE(2009)Ontheroadtodiploidization? Homoeologlossinindependentlyformedpopulationsoftheallopolyploid Tragopogonmiscellus (Asteraceae). BMCPlantBiol 9:80. 45.BuggsRJA,etal.(2009)Genelossandsilencingin Tragopogonmiscellus (Asteraceae): Comparisonofnaturalandsyntheticallotetraploids. Heredity 103:73 – 81. 46.TateJA,etal.(2006)Evolutionandexpressionofhomeologouslociin Tragopogon miscellus (Asteraceae),arecentandreciprocallyformedallopolyploid. Genetics 173: 1599 – 1611. 47.BuggsRJA,etal.(2010)Characterizationofduplicategeneevolutionintherecent naturalallopolyploid Tragopogonmiscellus bynext-generationsequencingandSequenomiPLEXMassARRAYgenotyping. MolEcol 19(Suppl1):132 – 146. 48.BuggsRJA,etal.(2012)Rapid,repeatedandclusteredlossofduplicategenesinallopolyploidplantpopulationsofindependentorigin. CurrBiol 22:1 – 5. 49.LukensLN,etal.(2006)Patternsofsequencelossandcytosinemethylationwithin apopulationofnewlyresynthesized Brassicanapus allopolyploids. PlantPhysiol 140: 336 – 348. 50.SalmonA,FlagelL,YingB,UdallJA,WendelJF(2010)Homoeologousnonreciprocal recombinationinpolyploidcotton. NewPhytol 186:123 – 134. 51.ChantretN,etal.(2005)Molecularbasisofevolutionaryeventsthatshapedthe hardnesslocusindiploidandpolyploidwheatspecies( Triticum and Aegilops ). Plant Cell 17:1033 – 1045. 52.SymondsVV,SoltisPS,SoltisDE(2010)DynamicsofpolyploidformationinTragopogon(Asteraceae):Recurrentformation,gene ow,andpopulationstructure. Evolution 64:1984 – 2003. 53.Snchez-MornE,BenaventeE,OrellanaJ(2001)Analysisofkaryotypicstabilityof homoeologous-pairing( ph )mutantsinallopolyploidwheats. Chromosoma 110: 371 – 377. 54.RileyR,ChapmanV(1958)Geneticcontrolofthecytologicallydiploidbehaviourof hexaploidwheat. Nature 182:713 – 715. 55.SearsE,OkamotoM(1958)Intergenomicchromosomerelationshipsinhexaploid wheat.In XInternationalCongressofGenetics (UniversityofTorontoPress,Toronto, Canada),pp258 – 259. 56.KostoffD(1938)Studiesonpolyploidplants,XXI:Cytogeneticbehaviouroftheallopolyploidhybrids Nicotianaglauca Grah. Nicotianalangsdorf i Weinm.andtheir evolutionarysigni cance. JGenet 37:129 – 209. 57.StebbinsGL,Jr.(1947)Typesofpolyploids:Theirclassi cationandsigni cance. Adv Genet 1:403 – 429. 58.StebbinsGL(1950) VariationandEvolutioninPlants (OxfordUnivPress,London). 59.SybengaJ(1996)Chromosomepairingaf nityandquadrivalentformationinpolyploids:Dosegmentalallopolyploidsexist? Genome 39:1176 – 1184 60.GaetaRT,PiresJC,Iniguez-LuyF,LeonE,OsbornTC(2007)Genomicchangesinresynthesized Brassicanapus andtheireffectongeneexpressionandphenotype. Plant Cell 19:3403 – 3417. 61.SzadkowskiE,etal.(2010)The rstmeiosisofresynthesized Brassicanapus ,agenome blender. NewPhytol 186:102 – 112. 62.SongKM,LuP,TangKL,OsbornTC(1995)Rapidgenomechangeinsyntheticpolyploidsof Brassica anditsimplicationsforpolyploidevolution. ProcNatlAcadSciUSA 92:7719 – 7723. 63.FeldmanM,LevyAA(2005)Allopolyploidy — ashapingforceintheevolutionof wheatgenomes. CytogenetGenomeRes 109:250 – 258. 64.RileyR,KimberG(1961)Aneuploidsandcytogeneticstructureofwheatvarietal populations. Heredity 16:275 – 290. 65.MacasJ,NeumannP,NavrtilovA(2007)RepetitiveDNAinthepea( Pisumsativum L.)genome:Comprehensivecharacterizationusing454sequencingandcomparison tosoybeanand Medicagotruncatula BMCGenomics 8:427. 66.BirchlerJA,AlbertSA,GaoZ(2008)Stabilityofrepeatedsequenceclustersinhybrids ofmaizeasrevealedbyFISH. TropPlantBiol 1:34 – 39. Chesteretal. PNAS | January24,2012 | vol.109 | no.4 | 1181 EVOLUTION