Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 DOI 10.1600/036364411X553289 © Copyright2011 bythe American SocietyofPlantTaxonomists Systematic Botany introgressed intoathird. Arnold etal.(1990) investigatedthe tifiable hybrids, genesfrom atleasttwo specieshadbeen found that,inpopulationsdevoid ofmorphologicallyiden- Using allozymeand AFLP markers,respectively, each study Wyatt 1990 ) and species havebeenreported for studies are basedonthegrassesandgraincrops. involving three ormore speciesandthatthemajorityofthese ple hybrids,or3)isfocusedonancienthybridizationevents local floras,2)reports ontheexperimentalproduction oftri- is basedonmorphologyandpresented inthecontextof to theseauthors,mostoftheliterature ontriplehybrids1) natural hybridsbetweenthree (ormore) species. According using moleculartoolstoreveal theoriginofrecently formed In contrast,theliterature containsfewexamplesofstudies that mostofthoseare focusedontheoriginofallotetraploids. erature onplanthybridizationisbasedbinaryhybridsand Liston andKadereit 1995 ; Cronn etal.2003 ). into polyploidlineages( Wendel 1989 ; et al.2003 Dorado etal.1992 ; ; Mason-Gamer 2004 ships involvingmultipleunexpectedgenomedonors( ), andcpDNA introgression Cronn ization events( Soltis and1993 , 1999 ), complexrelation- many haveformedmultipletimesfrom independenthybrid- genetic analysesofallopolyploidplanttaxahaveshownthat (reviewed in Arnold 1997 ; Rieseberg 1997 ). Molecularphylo- clarify thephylogeneticaffinities ofseveralpolyploidspecies the biparentally inherited nucleargenomehavebeenusedto from boththeuniparentally inheritedchloroplast genomeand allozyme data.More recently, DNA sequencedata obtained Early studiesfocusedonmorphological,cytogenetic,and Soltis and Soltis the originsandevolutionaryhistoryofpolyploidplanttaxa. 1997; Rieseberg 1997; 2000 ). Severallinesofevidencehavebeenusedtoelucidate Arnold 1981; ( Grant tributed greatly tothediversification andspeciationofplants

1 Current address: DepartmentofBiologicalSciences,GrandRapidsCommunityCollege,Rapids,Michigan49503U.S. A. Recent naturally occurringhybridsbetweenthree ofmore Kaplan andFehrer (2007) pointoutthatthemajorityoflit- con- have polyploidization and hybridization Interspecific and codedindelsindicate by allaccessions.TheuniquepatternofcpDNA indelswasalsorecovered in the nucleargenomeof hexaploid nature ofthisspecies,butalsowithchloroplast capture ofcpDNA from anunexpectedsource through introgression. together, thesedatareveal bothasingleoriginandcomplexpatternofreticulation that isconsistentnotonlywiththehyp incongruent topologies. Analysis ofclonednrDNA sequencedataindicatedthat allowing ustopositivelylinktheETSandITShomoeologuetypes.Phylogeneticanalysesofnuclearchloroplast datasets accessions indicatedthepresence ofmultiplehomoeologuetypes.Thesewere separatedbymolecularcloningofthe3055bp3 range, and25otherspeciesof loci ( whether thespeciesisofsingleorpolytopicorigin.To studytheevolutionaryhistoryof hypothesized combinationsofparental specieshavebeensuggestedbutnonephylogeneticallytested. Additionally, it Keywords— Abstract— accD-psaI (2011), 36(1):pp.209–226 Department ofBiologicalSciences,Western MichiganUniversity, Kalamazoo,Michigan49008U.S. A. Ncer n Clrpat N Sget Cmlx ige rgn o the for Origin Single Complex a Suggest DNA Chloroplast and Nuclear Threatened Allopolyploid Solidagohoughtonii , , pN nrgeso rN eiuae vlto evolution, reticulate nrDNA, introgression , cpDNA, psbA-trnH Quercus S. houghtonii. , , L.( Dodd and Afzal-Rafii 2004 ). trnL-trnF S. gigantea Solidago Torrey & A. Grayexisafederallythreatened polyploidplantspecieslikelyofhybridorigin.Several Aesculus , , Analysis ofcpDNA sequencedatarevealed thepresence ofmultipleinsertions/deletions thatare shared rps16-trnQ isthematernalgenomedonor. However, wedidnot recover a includingallsympatricspecies.Polymorphismswithinthedirect nrDNA sequencesofall Reticulate EvolutionandIntrogression

1 Author forcorrespondence ([email protected]) L.( dePamphilis and aea . Laureto J. Pamela ), andtheITS3 omnctn Eio: nrw Hipp Andrew Editor: Communicating Solidago houghtonii ′ ETS regions forfouraccessionsof 1 Solidago houghtonii n od . Barkman J. Todd and 209 origin of occurring hexaploid ence ofthree genomesin apersistent,butsterile,naturally the ITSregion andcpDNA sequencedatato reveal thepres- reported tobehexaploid. Kaplan andFehrer (2007) used of thesecaseswere the naturally occurringtriplehybrids three sympatrictaxaasparental donors.However, innone nation ofgeneticmarkersindicatingtheinvolvementall allozyme dataconfirmedthat characteristics ofthree sympatric Louisiana thatdisplayedmorphologicalandchromosomal which varyfrom ashort,stoutcaudex producing clumpsof dent characters: (1)thestructure oftheunderground parts (1991) dividedthegenusintogroups basedonthree indepen- topic speciesandploidynumbers). Gleason andCronquist loids andpolyploids(See Semple andCook2006 forpoly- several specieshavemultiple cytotypesincludingbothdip- hybrids, butalsobecausemany speciesare polytopic, and ther complicated,notonlyby theoccurrence ofinterspecific cies identificationdifficult. Cleardelineationofspeciesisfur- morphological complexitywithinthegenusoftenmakesspe- by theirnumerous, smallheadsofyellowflorets. However, 2008 ), mostspeciesare easilyrecognized as“goldenrods” logical synapomorphiesthatdefine in South America. Although there are nodistinctmorpho- species thatare nativetoEurasia,eightinMexico,andfour ( Semple andCook2006 ). Additionally, there are sixtoten est numberoftheseoccurringineasternNorth America includes about100North American species,withthe great- to theformationandstabilizationofhybridtaxa.Thegenus istics identifiedby Ellstrand et al. (1996) asadvantageous ing breeding system,and clonalreproduction, allcharacter- ization “hotspot”family( Ellstrand etal.1996 ). (Asteraceae: Astereae: Solidagininae),amemberofhybrid- tile hexaploid, gate theoriginandevolutionofnaturallyoccurringfer- Solidago S. gigantea S. riddellii,ptarmicoides Iris nelsonii L.ischaracterizedbyaperennial habit,outcross- . . PhylogeneticanalysisofthecpDNA sequencedata S. houghtonii (Asteraceae)Involving Solidago houghtonii Rand.,astabilizedhybridfrom southern Potamogeton S. gigantea S. houghtonii wesequencedfournoncodingcpDNA , and S. ohioensis I. nelsonii nrDNA sequencetype.Taken L.clone.Here weinvesti- Torrey & A. Grayex , whichspanitsgeographic Iris Solidago L.species.Theuseof havecontributedto possessedacombi- ′ ETS –ITSregion, othesized allo- ( Schilling etal. S. houghtonii is unclear revealed

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 occurring hybrids between arisen byamphidiploidy (chromosome doubling) ofnaturally tested. Morton (1979) proposed that inclusion of to bemostcloselyrelated to 2003 ; Beck etal.2004 ; Schilling etal.2008 bers ofthesubtribeSolidagininae( ) found Zhang 1996 ; Urbatsch etal. Solidago not beenacomprehensive molecularphylogeneticstudyof have chosentousethatnamethroughout. To date,there has origin of Cook 2006 ). other populationsare hexaploid(2 octoploid (2 population isuniquebecausetheplantsare reported tobe Michigan’s LowerPeninsula( Fig. 1 ). TheMichigandisjunct lations; oneinGeneseeCounty, New York andtheotherin shoreline distribution,there are twodisjunctinlandpopu- are known( USFWS 1997 ; COSEWIC 2005 Canada ( ). Inadditiontoits Fig. 1 ) where approximately 70isolatedpopulations Michigan andHuron inMichigan,U.S. A. andOntario, endemic, occurringmainlyonthenorthernshores ofLakes 2005). ( COSEWIC in New York ( Young 1996 ), andas“specialconcern”inCanada threatened” inMichigan( MDNR 1991 ), as“stateendangered” Act of1973( USFWS 1988 ). Additionally, itislistedas“state been awarded protection through theEndangered Species 1999 ), isfederallylistedasathreatened speciesandhas through naturalhybridization ( Morton 1979 ; Semple etal. et al.2004 ). Solidago listed inthe (Torrey & A. GrayexGray)Nesom. Becausethespeciesis boid taxacitedinNesom’srevision as our speciesofinterest, 1 SSEAI OAY [Volume 36 within corymboid taxa,recognized by Nesom (1993) as Ptarmicoidei ognized section sented ataxonomictreatment of Oligoneuron those of of naturalhybridization”betweentaxa and larger achenes. Additionally, Nesomcitesa“paucity topped) inflorescence, punctateleaves,broader phyllaries, SYSTEMATICfrom theothermembersof BOTANY includes sixspeciesthatare distinguishedmorphologically Rydberg. According tothisview, thegenus treated within Nesom alsosegregated twotaxathathadpreviously been tions andsection recognized twosections,section Nesom (1993) presented ataxonomicoverviewinwhichhe divided intodiffering numbersofsectionsandsubsections. form. Basedprimarilyonmorphology, thegenushasbeen described aseitherpaniculiform,racemiform,orcorymbi- but mosthaveaterminalinflorescence thatcanbroadly be rescence type.Inafewspeciestheinflorescence is axillary, from basallydisposedtochieflycauline;and(3)theinflo- stems; (2)theshapeanddistributionofleaveswhichvary stems toamore slenderandelongaterhizome with scattered 210 There havebeenseveral hypothesesregarding thehybrid Solidagohoughtonii Solidago ; however, recent molecularstudiesinvolvingmem- awaitsadefinitivemolecular-based phylogeny( Beck Solidago houghtonii Solidago (House)SempleandGandi.Theyplacedthesix . Mostrecently, Semple andCook(2006) havepre- n Federal Register Oligoneuron =8x72)(J.Pringle pers.comm.)whereas all section Solidago Solidago asfurthersupportforthesegregation of Unilaterales Solidago houghtonii , apolyploidspecieslikelyderived Solidago houghtonii, Ptarmicoidei : : withelevensubsectionsandsection within Oligoneuron S. ohioensis , noneofwhich have beenformally (SW 98) s as (USFWS1988) Solidago Solidago G.Donwithsevensubsections. Solidago Solidago Solidago . Thisissignificantbecause n bytheircorymboid(flat- Riddelland Smalland , whichmaysupportthe isanUpperGreat Lakes =6x54)( Semple and S. houghtonii Oligoneuron houghtonii . Circumscription of L.withfoursubsec- isoneofthecorym- in whichtheyrec- Oligoneuron S. houghtonii S. ptarmicoides Oreochrysum Oligoneuron mighthave Oligoneuron Oligoneuron and we ,

(2 houghtonii COSEWIC (2005) . Boxesindicatefourhypothesizedentitiesfor approximate locationofknownpopulationsbasedon USFWS (1997) and the hybridwith et al.(1999) proposed that of independentoriginfrom theGreat Lakesplants. Semple Sheviak (1981) , furtherproposed thattheNew York plantswere (Torrey & A. Gray)B.Boivin.He, aswell Mitchell and S.ptarmicoides regard toparentage of cies. However, thestatisticalresults were inconclusivewith individuals from across itsgeographicrangewere onespe- of themorphologicalcharacterssuggestedthat parental taxa.Themultivariateanalysisofvariationforeach tion between the eastsideofBruce Peninsula),resulted from hybridiza- plants thatoccuraround the ManitoulinIslandregion andon Figure modifiedfrom Guire andVoss (1963) . study are indicatedbyarrows andreferenced bytheiraccessionnumber. × whose substitution rateandmodeofmolecular evolutionis Rosselló 2007 ). Recently, theETSregion ofnrDNA,amarker sion, andtheoriginofpolyploid species( Nieto Felinerand plant groups andforthestudyofhybridization,introgres- the inference ofphylogenyinalarge anddiversenumberof coides 22 morphologicalcharactersof test thesehypothesesusingelectron microscopy toevaluate for thesehypotheses. Edwards-Wilson (1999) attemptedto ation inchromosome numberandmorphologyasevidence (2 plants), derivedfrom hybridizationbetween and resulted from hybridizationbetween “entity 3”(theoctoploidpopulationdisjunctinMichigan), (2 Michigan, istheresult ofhybridization between clatural typeandlocatedaround theStraitsofMackinacin “entity 1”(true geographic locations.Pringle’shypothesesare asfollows: ing different putativeparents andcorresponding todistinct ties, eachresulting from separatehybridizationeventsinvolv- that S.riddellii ing morphologicalsimilaritiesinleafstructure between

n S. uliginosa n n Fig. The biparentally inherited ITSregion hasproven usefulfor =18);entity2: =18)and =18)and S. uliginosa S. houghtonii andeither 1 . . ( USFWS 1997 ). Entity1: Geographic distributionof and (2 (2 n n S. ptarmicoides S. uliginosa =18).Collectionlocalities forsamplesincludedinthis =18) S. riddellii S. houghtonii Nutt.(2 S. ptarmicoides S. riddellii isactuallycomprisedoffourseparateenti- S. ptarmicoides S. houghtonii ×

S. uliginosa S. houghtonii n (2 =36);and“entity4”(theNew York (2 or (2 n (2 . Pringle( USFWS 1997 ) proposed n S. riddellii =18);“entity2”(theCanadian (2 =18)( Fig. 1 ). Pringlecitesvari- n S. uliginosa n =18) S. ptarmicoides ), represented bythenomen- n =18)and =36);entity4: , ratherthan S. houghtonii Solidago houghtonii , suggestingthat ×

S. ohioensis Frankwasinvolvedin S. ptarmicoides were involved. S. ohioensis (2 S. ptarmicoides n andallputative =18) S. ohioensis (2 S. ptarmicoides S. ptarmicoides n S. houghtonii . Dotsindicate =18);entity3: × (2 (2 S. ptarmi-

S. riddellii n n (2 Solidago =18); =18) n , cit- , =18)

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 hexaploid variants whichcorresponded tothethree parental taxaofthe of thenrDNA ITSregion revealed thepresence ofthree ITS and Kaplan andFehrer (2007) found thatdirect sequencing tion withcpDNA,revealed thepaternalsource ofthe hybrid, following theprotocol of Chase andHills(1991) . Intotal, 26 obtained from thestem,justbelowinflorescence, andsilicagel-dried chased seedorplants. Foreachindividualsampled, asingleleafwas from fieldcollectionsofnaturalpopulationsandinafewcases from pur- likely progenitors? (2)Did (1) Is gin of (GAA), and intergenic spacers, ing 5.8Sgene,the3 gesting asingleandrecent originforthisspecies. bursa-pastoris cpDNA sequencesfrom 20accessionsofthetetraploid origin forthespecies.Likewise, Slotte etal.(2006) foundthat one ofitshypothesizedparental donorssuggestingasingle for thepolyploid Guggisberg etal.(2006) foundasinglechloroplast haplotype lotypes inheritedfrom different maternalsources. Incontrast, hybrid specieswasshowntohavemultiplechloroplast hap- huronensis Aegilopstriuncialis anomalus (Chambers) Chambers( Wallace andJansen1995 ), ( origins fortheallopolyploid used cpDNA restriction sitedatatoshow two independent single versuspolytopichybridorigins. Soltis and(1989) revealing boththematernal parent ofahybridspeciesandalso nally inherited( Corriveau andColeman1988 ), itisusefulfor gin for polymorphic ITSsiteswhichsupportedanallopolyploidori- et al.(2006) foundcompleteadditivityatseveraldiagnostic 211 Glycinetomentella homoeologous locitodetectandcharacterizehybridityinthe allopolyploid taxa. Rauscher etal.(2002 ; 2004 successfully beenusedtoreveal thehybridoriginofseveral ) usednrDNA polyploid origins.Despitethesecautions,theITSregion has ing polyploidformationcanlimititsutilityforthestudyof 2004 ). Inparticular, theprocess ofITShomogenizationfollow- struction (reviewed in Álvarez andWendel 2003 ; Small etal. level processes thatmayaffect itsutilityforphylogeny recon- LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII tioned againstitsuse,citingseveralmolecularandorganism- marker ofchoiceforoveradecade,severalauthorshavecau- 2003 ; Urbatsch etal.2003 ). WhiletheITSregion hasbeenthe data (i.e. Markos andBaldwin2001 ; Andreasen andBaldwin logeny reconstruction, ofteninconjunctionwithITSsequence similar tothatoftheITS,hasincreased inpopularityforphy- 2011] hybridization eventoris Stebbinsoserisheterocarpa including to infermultipleoriginsforseveralotherpolyploidspecies the twodifferent parents. Chloroplast DNA hasalsobeenused ulations hadincorporatedthechloroplast genomeofeach T. dubius In thisstudy, weusetheITS1andITS2 spacersandinterven- Since inmostangiospermsthechloroplast genomeismater- ao Sampling— Taxon S. houghtonii Solidago houghtonii Centaurium bianoris S.F. Blake( Schwarzbach andRieseberg 2002 ), Scop. (Nutt.)Lindl.( Wallace 2003 ) where ineachcase the Senecio cambrensis Potamogeton rps16-trnQ (L.)Medikus lackedintraspecificvariation,sug- ×

T. pratensis Hayataallopolyploidcomplex, Guggisberg Centaurium bianoris aeil ad Methods and Materials ofhybridoriginandifso,whoare itsmost L.( Vanichanon etal.2003 ) and Samples forDNA analysiswere primarilyobtained ′ endoftheETSspacer, andfourcpDNA accD-psaI

× to investigatetheputativehybridori- . Here weaskthefollowingquestions:

(Nutt.)Chambersand torssanderi S. houghtonii (Sennen)Sennenand,inconjunc- L.)where different polyploidpop- Rosser ( Ashton and Abbott 1992 ), S. houghtonii , , Tragopogon miscellus psbA-trnH (Tiselius) Dörfler. thatformedacladewith ofpolytopicorigin? originateinasingle , , trnL (UAA) S. decipiens Platanthera Helianthus Ownbey Capsella Solidago -trnF

tribe (Astereae: Solidagininae)as were chosenasoutgroups becausetheybelongtothesametribeandsub- (Pallas exPursh)G.L.Nesom,and of and likelyderived,were alsoincludedinoursampling.Multiplesamples Ptarmicoidei the phylogeneticbreadth ofthegenus. All membersof genus In addition,atleastonespeciesfrom eachsectionandsubsectionofthe was designedtoincludeallspeciesof Michigan University(WMU)Hanesherbariumunlessotherwisenoted. for eachspeciesexaminedinthisstudyare depositedintheWestern mission oftheBergen SwampPreservation Society. Vouchered specimens and privatelandowners.TheNew York collectionswere madewithper- collected withpermissionfrom theOntarioMinistryofNaturalResources and FederalFishWildlife permitTE074805–0.Canadianmaterialwas of NaturalResources endangered speciespermits#00–1133 and#00–1257 material of data andcollectionlocalitiesare presented in Appendix 1.Michiganplant 1994 ; Goertzen etal.2003 ; Urbatsch etal.2003 netic studieshaveshownthemtobecloselyrelated to ; Beck etal.2004 ). Voucher not shown).Therefore, fouraccessions of additional accessions of ing thepresence ofITStypesslightlydifferent length. Sequencingof directions were clean uptoapointandthenbecameunreadable, indicat- hypothesized parental species, of severalpolymorphicpositions. We alsoobservedthatforoneofour 5 nal primers(SOL-448F5 1990 ). The were alsoobtainedusing the internalprimersITS2and3( White etal. plete forward andreverse reads through theentire ITSregion, sequences sequenced usingtheirrespective PCRprimers( Table 1 ). To ensure com- As expected,direct sequencesof sequences were inspectedforpolymorphicsitesand/or“dirty” sequence. cant differences inlengthwere observed. Electropherograms ofthedirect were alignedtotheoneusedininitialBLASTsearch andnosignifi- to otherITSsequencesfrom the Asteraceae. All additionalITSsequences search inGenBankwhich indicated significantalignmentwithhighscores ITS sequencesobtainedfrom (2007) forobtainingreliable ITSsequencedata. First,usingoneofour fore, wefollowedtheguidelines provided by Nieto FelinerandRosselló against theuseofparalogoussequencesinphylogeneticanalysis,there- (50 mmol),2.0 water, 5.0 Amplifications were performedin50 taxa were sampled. nate hypothesesofasingleorpolytopicorigin were obtainedfrom GenBank(GB)(Appendix1).To investigatethealter- tive variationwasobserved.DataforadditionalITSand3 our phylogeneticanalysesasnointraspecificphylogeneticallyinforma- accessions forsomemarkers;however, wereport onlythoseincludedin For themajorityofspeciesincludedinourstudywesequencedmultiple S. riddellii, 1 cm cies corresponding toeachofPringle’sfourentities( Fig. 1 ). S.houghtonii tocols. TheITS,3 kitsandpro- Coulter CEQ2000XL geneticanalyzerandthemanufacturer’s strands ofallpurifiedPCRproducts were sequenced usingaBeckman- Beverly, protocols. Both Massachusetts)followingthemanufacturer’s AMPure magneticbeadPCRpurificationsystem(AgencourtBioscience, QIAquick PCRPurificationkit(Qiagen,Inc.,Valencia, California)or Hamburg, Germany). All PCRproducts were purifiedusingeitherthe mastercycler programmable thermalcycler(Eppendorf ScientificInc., cycling conditions.Thereaction mixtures were cycledonanEppendorf and 1 μ accD-psaI of thenrDNA ETS,andfournoncodingcpDNA intergenic spacers(IGS), entire ITS,includingITS1,ITS2,andtheintervening5.8Sgene,3 pers. comm.). Doyle and(1987) withtheadditionof1%PEG8000(JoelMcNeal, ′ l ofPlatinum -TCATAGAATGGGTACCTCGA) thatwedesignedforthisstudy. S. houghtonii To investigatetheparentage of Cnimto o IS Homology— ITS of Confirmation Ttl N Extraction— DNA Total PR mlfcto ad Sequencing— and Amplification PCR 2 ofdriedleaftissuefollowingthe2 μ Solidago l oftemplateDNA diluted1:10.See Table 1 forPCRprimersand , , μ and psbA-trnH l oftheprovided 10 accD-psaI Solidago houghtonii ( Semple and Gandhi2004 ), ofwhich were collectedfrom across thegeographicrangeofspe- sensu Semple andCook(2006) were includedtorepresent ® μ , ofallproposed parental taxa( Taq polymerase(Invitrogen Corp.,Carlsbad,California), S. uliginosa l totaldNTPs(10mmol),1.0 ′ ETS, spacerwassequencedusing Euthamia graminifolia , , trnL-trnF trnL-trnF S. ptarmicoides ′ -AAATCGGAAATCTGACCC andnewPSAI-R ), aswell Total DNA wasextractedfrom approximately Solidago houghtonii wascollectedunderMichiganDepartment × , , enzymebuffer, 2.5 , , S. ptarmicoides rps16-trnQ psbA-trnH Solidago S. houghtonii Solidago houghtonii Heterotheca villosa μ Numerous authorshavecautioned Solidago l volumescontaining35.4 produced thesameresults (data S. gigantea S. houghtonii For eachindividualsampled,the (L.)Nutt., andbecauserecent phyloge- × , and , were amplifiedusingPCR. CTAB extractionmethodof μ l (10 thatare sympatric withit. S. ptarmicoides,ohioensis, , sequencereads inboth , weperformedaBLAST S. houghtonii indicatedthepresence rps16-trnQ S. houghtonii Aiton, were collected. μ μ Solidago M) ofeachprimer, 0.1 l magnesiumchloride (O02 HOU008, (HOU002, Ericameria nauseosus (Pursh)Shinners, , taxonsampling Solidago Solidago ′ ETS sequences seii inter- specific regions were isamember , samplesof (Bremer μ section l sterile ′ end

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 xeso 1 Extension neln 1 Annealing ia ylsn=3 0n=3 5n=3 n=34 n=34 n=35 n=34 n=40 1 n=34 Denaturation Final cycles xeso 1 Extension intergenic spacers.Primernamesfollowtheoriginalpublication. neln 1 Annealing nenlFPie ITS3 Internal FPrimer 1 SSEAI OAY [Volume 36 585 inITS2.Theremaining speciesof (see below). HOU015, andHOU060)oneof SYSTEMATIC BOTANY F Primer 212 Final Extension nta yl n=1 n=1 n=1 n=1 n=1 3 n=1 ITS2 Denaturation Initial cycle Internal RPrimer Baldwin 2001 ; rmr26SE R Primer in lengthforall 2002 ). We investigatedtheconserved5.8Sregion andfoundittobe164bp sequence data( Buckler etal.1997 ; Hershkovitz etal.1999 used todeterminethepresence ofnonfunctionalpseudogeneswithinITS ; Hughes etal. indels across sequencesofthenormallyconserved5.8Sgenehavebeen phisms andhad“clean”sequence. the 5.8Sregion. The length of160–170bp. Additionally, wefoundlittlesequencedivergence in tor offunctionalityforalldirectly amplifiedandclonedITSsequences. the Astereae. Therefore, weconsiderthelackof5.8Svariationasanindica- position from thepublishedconsensussequence( Goertzen etal.2003 ) for rate binarycharacters. We treated indelswithidentical5 and nrDNA datasetswere codedandaddedtothe datamatricesassepa- intra andinterspecific level.Therefore, indelsinboththealignedcpDNA indels canbeusefulforinferringphylogenetic relationships bothatthe Freudenstein andChase2001 ; Ingvarsson etal.2003 ) haveshownthat 10768 and10767,respectively). The aligneddatamatricesare availablefrom TreeBASE (studynumbers and analyzedtogethersincethey are typicallyuniparentally inherited. data sets( and Markos1998 ; Markos andBaldwin2001 ). Likewise,thecpDNA IGS together becausetheyare partofthesametranscriptionalunit( Baldwin were inserted.The3 deletions (indels)occurred, manualadjustmentswere necessaryandgaps forward formuchoftheir lengths,butintheregions where insertionsand deposited inGenBank(Appendix1).Sequencealignmentswere straight- et al.1997 ) andfurtheradjustedbyeye.Completesequenceshavebeen Sequence alignmentswere madeusingCLUSTAL_X v1.8 ( Thompson Sequencher ver. 4.01(GeneCodesCorporation,Inc., Ann Arbor, Michigan). sequence wasusedinphylogeneticanalyses(seeresults). ers. Clonesofidenticalorsimilartypewere grouped andtheirconsensus sequenced asdescribedaboveusingtherespective 3 reaction, thePCRproducts from 20–30positivecolonieswere cleanedand elongation at72°C,witha30minfinalextension72°C.Foreachcloning of 1mindenaturationat94°C,20secannealing55°C,and3 cells andinitialdenaturationfor10minat94°C,followedby35cycles ITS region. Temperature andcycling conditions consistedoflysingthe for the3 18S-R1 ( Markos andBaldwin 2001 and Urbatsch etal.2003 Colonies were thenscreened respectively) forinsertsbyPCRusing primers Ast-8 and further grown onLBagarstreak platescontaining100 Approximately 70coloniesfrom eachcloningreaction were selectedand tively linkthe3 protocol. Bycloningthisentire region wewereufacturer’s abletoposi- 3,055 bp,usingaTOPO TA cloningkit(Invitrogen) followingtheman- the entire 3 for allaccessionsof phic nucleotidepositionsindirect sequencesofpurifiedPCRproducts Table Pairwise comparisonsofbasepairdifferences andthepresence of Coig f h 3 the of Cloning Several authors(e.g. Golenberg etal. 1993 ; Vijverberg etal.1999 ; Pyoeei Analyses— Phylogenetic Region ′ ETS region andprimers17SE26SE( Sun etal.1994 ) forthe 1 accD-psaI . . ′ ETS –18SgeneITS15.8SITS2region, atotalof Solidago gigantea PCR andsequencingprimersconditionsforthe3 d Urbatsch etal.2003 ; ′ ETS sequencewiththeITSfrom thesameclone. Solidago , , Solidago Solidago houghtonii psbA-trnH ′ ′ ETS-ITS Region— ETS andITSdatasetswere combinedandanalyzed accessionsandclones,wellwithintheexpected 5 17SE ′ ′ ′ ′ ′ ′ ′ 20 @5° 1 @55°C @9° 1 @94°C 20 @5° 1 @55°C @72°C @9° 3 @94°C 5.8Ssequencesdiffered atonlyonenucleotide For eachmarker, contigswere assembledin ″ ″″ (GIG004)hadapolymorphismatposition ITS @7° 1 @72°C 2, b 2, b , , 1, 2,a 1, 2,a @7° 1 @72°C trnL-trnF 18S-ETS Ast-1 18S-R1 S. ptarmicoides e Baldwin andMarkos1998 ; , andalso Solidago Due tothepresence ofpolymor- , and 7 Ast-8 rps16-trnQ ′ ′ ′ ′ ′ ′ ′ @7° 1 @72°C @5° 1 @55°C @9° 1 @94°C @7° 1 @72°C @5° 1 @55°C @72°C @9° 3 @94°C showednoITSpolymor- S. ptarmicoides 3 ′ ETS (PTR011) were cloned 1, 2,c 1, 2,c 1, 2,d ′ 1, 2,e ETS andITSprim-

μ ′ newPSAI-R g/mL ampicillin. and3 ) were combined

, wecloned ′ terminias 7 SOL-448F f thispaper; 1 ′ ′ ′ ′ ′ ′ ′ PCRPrimer; 20 @5° 1 @55°C @9° 1 @94°C 20 @5° 1 @55°C @9° 3 @94°C @72°C ″ ″ accD-psaI @7° 45 @72°C @7° 45 @72°C ′ ETS andITSregions andcpDNA 1, 2,f

g 1, 2,f atntl 20 2001; Saltonstall sequences. Ofthe 3,349charactersinthecpDNA datamatrix, of nucleotide variability foreachofthefour cpDNA IGS test ( Shimodaira andHasegawa1999 ). constrained analysisusingtheconservative Shimodaira-Hasegawa(S-H) ologue typeswere freely estimated,wascompared tothescore from the from anunconstrainedanalysis, where relationships oftheclonedhomoe- straint toML searches usingthecombinednrDNA dataset.TheML score S.houghtonii straint topologythatforced theclonednrDNA homoeologuetypesof Using MacClade4( Maddison and2001 ), wedesignedacon- by comparingalternativetopologiesasin Barkman andSimpson(2002) . that was appliedtoproduce the network. indel structure. The95%probability limitofparsimoniousconnections data, butthree binarycharacters were addedtothedatamatrixreflect all othersitesandincludedintheanalysis.Gapswere treated asmissing positions were scored asidenticaltotheaccessionthatwasmostsimilarat lems withtheTCSanalysis.Following Rauscher etal. (2004) theambiguous otide positionsindicatingthepresence ofanybase(N)thatcausedprob- S.ohioensis in theTCS1.21program ( Clement etal.2000 ). set usingtheparsimonymethodof Templeton etal.(1992) asimplemented MulTrees optionselected. one randomadditionsequenceandTBRbranchswappingwiththe 3.06 ( Posada andCrandall1998 ). A heuristicsearch wasperformedwith ( Rodríguez etal.1990 ), aschosenfrom likelihoodratiotestsinModeltest performed onthenrDNA datasetusingthebest-fitmodelGTR+GI licate withtwotrees savedineachsearch replicate. MulTrees on,andonerandomadditionsearch replicate perjackknife rep- pseudoreplicates, 37%characterdeletion,TBRbranchswappingwith Freudenstein etal.(2004) , PAUP* wassettoemulateJacresampling, 2,000 was assessedintermsofjackknifesupport(JS)( Farris etal.1996 ). As in combined toconstruct strictconsensustrees. Therobustness ofclades each datasetusingPAUP* andthemultiplemostparsimonioustrees were sequences. Tree statisticsandmeasures ofhomoplasywere calculated for cpDNA datasetwasanalyzedasabovebutwith10randomaddition and theMulTrees optionselected.Duetocomputationalconstraints,the addition sequences,TBR(tree bisectionreconnection) branchswapping, independently. ThenrDNA datasetwasanalyzedwith100randomtaxon search algorithmwere conductedonthecpDNA andnrDNA datasets, ( Swofford 1998 ). Maximumparsimony(MP)analysesusingtheheuristic the nrDNA data,respectively. data matrixcellsscored asmissingwas1.36%ofthecpDNA and3.46%of coding methodof Simmons andOchoterena (2000) . Theportionofthe been derivedindependentlyofoneanother. Followingthesimpleindel- homologous andthosethatdiffered intheir5 2 PSBA SequencingPrimer; hools DA Data— DNA Chloroplast Nuclear rDNA PhylogeneticTest ofHybridization— An unrooted haplotype networkwasconstructed forthenrDNA data A maximumlikelihood(ML)analysis ( Felsenstein 1981 ) wasalso All phylogeneticanalyseswere performedusingPAUP* 4.0b4a Solidago houghtonii 3 TRNH(GUG) sequencesobtainedfrom GenBankhadIUPAC codednucle- ′ ′ ′ ′ ′ @5° 1 @50°C @9° 1 @94°C @5° 1 @50°C @9° 3 @94°C @72°C ″ ″ tobemonophyletic(nonhybrid origin).We appliedthiscon- @7° 1 @72°C @7° 1 @72°C psbA-trnH 1, 2,g h aelt t l 1991 al. et Taberlet trnF(GAA)-f

a isofhybridorigin wasphylogeneticallytested 1, 2,g u e a. 94; 1994; al. et Sun accD-psaI

Results Overall, there wasalowlevel 7 trnL(UAA)3-e , , ′ ′ ′ ′ ′ ′ ′ 20 @5° 1 @55°C @9° 1 @94°C 20 @5° 1 @55°C @9° 3 @94°C @72°C psbA-trnH ″ ″ @7° 1 @72°C @7° 1 @72°C trnL-trnF b White etal.1990 ; ′ and/or3 , ,

1, 2,h trnL-trnF 1, 2,h Solidago rigida TRNQ

′ terminiashaving , and h hypothesis The c aks and Markos 7 RPS16F ′ ′ ′ ′ ′ ′ ′ 20 @50°C @94°C 20 @50°C @94°C @72°C rps16-trnQ rps16-trnQ ″ ″ L.and @72°C @72°C 1, 2,g 1, 2,g

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 tonii mean cpDNA divergence between allaccessionsof coides, Here variation inthenumberofrepeats inan“AT” microsatellite. nucleotide position632( Fig. 2D ). Thisindelistheresult of of 26indels,whichthere wasasingleinformativeindelat sampled species. for theNew York accession(GIG005)( Fig. 2C ) butin noother of including a15bpdeletionthatwaspresent inallaccessions have removed theseindelsfrom Fig. 2B . species (datanotshown).Foreaseofdatapresentation we These insertionswere notpresent inotheraccessionsofthese of S.uliginosa was shared by ( Fig. 2B ). Thisdatasetalsoincludesa95bpinsertionwhich sion), S.canadensis,nemoralis,nitida,riddellii, except fortheNew York accession(GIG005)andalsowith length variationsare shared withallaccessionsof six “AT” repeats andonlyoneofthe“C five bpdeletionofa“C in thenumberofrepeats inan“AT” microsatellite, and2)a variation isprimarilyattributedtotwofactors:1) York (GIG005), share thisinsertion( Fig. 2A ). U. S. A. andOntario,Canada,butnottheaccessionfrom New accessions of rather with not withoneoftheproposed parents aswehadexpected,but or ploidylevel.To oursurprise,thisinsertionwasalsoshared, sions of revealed aunique29bpinsertion thatisshared byallacces- section and/orsubsection.Sequencealignmentofthisdataset cies orshared byspeciesthought tobeinthesametaxonomic indels, thevastmajorityofwhichare uniquetoasinglespe- informative. (7.4%) were variable,andofthese87(2.45%)were parsimony- bined cpDNA datamatrixcontained3,552characters,263 taxa. Followingtheadditionofcodedindels,com- these, 95(47%)occurred betweentheoutgroup and ingroup our cpDNA alignmentwere codedforpresence/absence. Of 213 inclusion inthedatamatrix. A totalof203lengthmutationsin forward. Thisallowedustoconfidentlycodetheindelsfor nucleotide substitutions,alignmentwasrelatively straight- otide substitutions;however, becauseoursequenceshadfew number ofindelsflankedbyregions withnumerous nucle- Sequence alignmentcanbedifficult whenthere is alarge primarily toinsertionsordeletionsofshort(1–30bp)repeats. 869/999 bp( 212/290 ( LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII the fourcpDNA regions were 763/939bp( minimum/maximum sequencelengthsobtainedforeachof was highvariabilityinsequencelengthacross thetaxa.The to therelatively lowrateofnucleotidesubstitution,there of these,44(1.31%)are parsimony-informative.Incontrast 121 (3.61%)are variableinatleastoneoftheaccessions,and 2011] The analysisofpairwisedifferences indicated thatthe Lastly, the In the Te The Te The S. rigida S. houghtonii andaccessionsof S. houghtonii and S. juncea trnL-trnF accD-psaI S. houghtonii psbA-trnH psbA-trnH and S. speciosa hasa101bpinsertionwhichisidenticaltothat Solidago gigantea rps16-trnQ rps16-trnQ S. gigantea , , , aswellallaccessionsof S. simplex datasetcontained16indels.Sequencelength S. rigida sequencealignmentincludedatotalof132 S. nemoralis , , S. gigantea sequencealignment wefound29indels, IGS),407/514bp( havethesamenumberof“AT” repeats. , regardless oftheirgeographiclocation S. gigantea ATAT” repeat. sequencealignmentincludedatotal IGS).Lengthvariationisattributed and from diverselocalitiesinMichigan, butwithsixadditionalnucleotides. andnoothersampledspecies. All , , (exceptfor theNew York acces- S. ohioensis S. simplex from Michiganand Ontario Solidago houghtonii ATAT” repeats. These . Inthesameregion, , , trnL-trnF S. patula S. gigantea and accD-psaI IGS),and , , S. gigantea S. velutina S. ptarmi- S. hough- except IGS), has ,

at position484inthe3 at positions698inITS2and55the3 ITS1 (positions138–142ofourITSalignment),aguanine(G) Solidagoptarmicoides ITS2 andacytosine(C)atpositions55484inthe3 142 ofourITSalignment),athymine(T)atposition698 in 2). (Table micoides types ITS divergent for heterozygous fering length.Cloningof ITS1, indicatingthepossibilityofmultipleITS1typesdif- 698 (G/T)intheITS2sequence,andbecameunreadable in 55 (C/G)and484(C/T)inthe3 S.ptarmicoides cies lackedevidenceofsinglesitepolymorphisms.However, vation ofsequenceelectropherograms revealed thatmostspe- similar lackofsequencevariationfor cies sampledinthisstudy. Schilling etal.(2008) observeda data displayedalackofdivergence amongthe26 Ptarmicoidei a cladecomprisedofallaccessions clade (JS=98).Theanalysisalsostrongly supports(JS=90) members ofsection ernmost MichiganaccessionandGIG004Ontario,Canada) ferences betweentwo was quitelow(0.044%)andthatthere were nocharacter dif- tionships were weaklysupported (JS Triplinerviae ported clade(JS=70)and (subsection and three accessionsof four accessionsof ( Table 2 ). Subsequently, cloningofPCRproducts from the three ofthesepositionswere foundinallfouraccessions morphic amongallthe 3 gle sitepolymorphismsintheelectropherograms ofaligned the polyploid four accessions(HOU002,HOU008,HOU015,HOU060) of tion York). Bruce Peninsula,Ontario,Canada,andHOU015from New ern tipofMichigan’slowerpeninsula,HOU008from the HOU008, 30from accessionHOU015,and 29from accession regions; 30 clonesfrom accessionHOU002, 22from accession A totalof111 cloneswere sequencedforthe3 hypothesized entities( USFWS 1997 ; Fig. 1 ), wasperformed. supported clade(JS=87). (GIG005) didnotdiffer from other and ITSsequencesfrom theNew York accessionof two nucleotidesubstitutions.However, thenrDNA 3 bined cpDNA sequencedata,havingsixuniqueindelsand considerably from otheraccessionsof of section Solidago sis strongly supports(JS=100)themonophylyofgenus all speciesrelationships are notwellresolved, theanaly- (400 steps,CI=0.885,andRI0.720). Although theover- sets ( Fig. 3 ) resulted in143,316equallyparsimonioustrees correctly identifiedthisaccession. nuclear databelow).Therefore, weare confidentthatwehave ′ ETS andITSsequencedata.Intotal,12siteswere poly- Following direct sequencingofPCRproducts from our Ncer N Data— DNA Nuclear The MP analysisofthefourcombinedcpDNA IGSdata Junceae Solidago gigantea . . -Type 1hasathree bpdeletioninITS1(positions140– Solidago bicolor Solidago (Rydberg) G.L.Nesom)and ) andthree accessionsof (Torrey & A. Gray)G.L.Nesom). All otherrela- Multiradiatae haddetectablepolymorphismsatpositions Solidago houghtonii subsection S. houghtonii -Type 2hasafivebasepairdeletionin Ptarmicoidei ′ ETS. S. gigantea S. houghtonii L.and In general, our3 (GIG005)from New York differed Semple)formamoderatelysup- S. houghtonii Solidago missouriensis S. ptarmicoides S. ohioensis Squarrosae S. speciosa , representing Pringle’s four , formastrongly supported accessions(GIG003north- , weobservedseveralsin- ′ ETS sequence,atposition S. gigantea (HOU002from thenorth- and < ′ Solidago S. gigantea ETS, andathymine(T) 70). accessions, butonly S. gigantea ′ ETS andITSsequence S. houghtonii Nutt.,bothmembers A. Grayformawell revealed thatitwas S. multiradiata S. ptarmicoides, accessions(see . Careful obser- Nutt.(subsec- ′ Solidago ptar- ETS andITS Solidago (subsection initscom- S. gigantea (section Aiton ′ both spe- ETS. ′ ETS

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 condition in all accessionsof tonii accessions of S.uliginosa 1 SSEAI OAY [Volume 36 SYSTEMATIC BOTANY 214 Fig. , three accessionsof 2 . . (seetext)which were removed in thisfigure butnotfrom thedatamatrix.C. A 15bpdeletion from thecpDNA Informative cpDNA indels. A. A 29bpinsertionfrom thecpDNA S. houghtonii S. gigantea S. houghtonii . B.Microsatellite lengthvariationandafivebpdeletion from thecpDNA S. gigantea and and three accessions of S. gigantea , andfiveadditionalspecies(seetext).Backslashes (//)represent large insertionspresent in aswellotherspecies (seetext). S. gigantea . D. Microsatellite lengthvariationfrom thecpDNA accD-psaI IGSthatisshared byallaccessionsof trnL-trnF IGSthatisshared byallaccessionsof rps16-trnQ psbA-trnH IGS showing theidentical Solidago houghtonii S. rigida,simplex, IGSthatisshared by S. hough- andsix and Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 S.gigantea 215 LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII 2011] Fig. 3 . . formsamonophyletic cladewithallaccessionsof Strict consensusof143,316 trees from themaximum parsimonyanalysisof combined cpDNA datasets. A keyresult ofthis anal S. houghtonii . Numbersabove thebranchesrepresent jackknifesupport. ysis isthat Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 niyTaxon andCloneTypes Entity several cloned sequencescontainedsingleton changesthat likely ofmeiotic origin.InadditiontoPCR recombinants, evidence ofonethehypothesized parental taxaand is also sion HOU060wasincludedin ouranalysisasitalsoshowed Likewise, thesinglerecombinant clonerecovered from acces- tion. Therefore, thisclone typewasincludedinouranalysis. parental taxaandmaybetheresult ofmeioticrecombina- This clonetypeshowedevidence ofoneourhypothesized HOU015, fouridenticalrecombinant cloneswere recovered. believed tobetheresult ofPCRrecombination. Foraccession as theyoccurred almostexclusivelyinsingleclonesandwere We excludedmostputativerecombinants from ouranalysis and accessionHOU060hadonerecombinant sequence(3%). from accessionHOU008,10(32%)from accessionHOU015 (50%). An additional15recombinants (68%)were identified From accessionHOU002weidentified15recombinants especially prone toPCRrecombination ( Kovarik etal.2005 ). et al.2002 ), andthemulticopy ETSandITSmarkersseem produce PCRrecombinants ( Bradley andHillis1997 ; Cronn templates (e.g.allopolyploids)are cloned,theyfrequently processes. WhenPCRproducts from heterogeneous DNA tion (PCRmediatedormeiotic),acombinationofthese variable sequenceswere the result of few were completelyidentical. Itappeared thatmanyofthe sequences from eachaccession, weobservedthatrelatively HOU060. Intheprocess of independentlyaligningthecloned [Volume 36 SYSTEMATIC BOTANY additivity between perfect additivityofpolymorphicsitesbetween (accession HOU002)showsperfectadditivityofpolymorphicsitesbetween 216 viduals representing eachofthefourgeographicentitiesare shownfollowedbythenumberofclonesrecovered perconsensusty bases are shadedand O05# (4) (7) T (4) T G T G C G T T T T A C G G A G A G A C A C G C G C G C C C C C C C A C A A A A C A C G C T G T G T C HOU015-#4 HOU015-#3 HOU015-#2 4 HOU015-#1 4 4 4 4 O00# (5) T G T T A G A C G C C C A G C G T T HOU060-#6 HOU060-#5 3 3 O00# (4) (6) T (4) T (2) G T (4) G (1) T C G T T G T T (12) T G (1) T C A A (2) T C T A T T T G G T A T G G A C A C A G A A T G A C T A T G C T A G G A C C A G A C A C G G A C C G G C C A G T G C A C G C C A C C C C C T A G C C C A G C C A C G A C A C A T C C A T A C C A C G C C C A T G C C A T A G T C T A G T A C C G A T G C A T HOU060-#4 C C G HOU060-#3 T T G HOU060-#2 3 T G HOU060-#1 3 T HOU008-#3 3 C HOU008-#2 3 HOU008-#1 2 HOU002-#4 2 HOU002-#3 2 HOU002-#2 1 HOU002-#1 1 1 1 4 3 2 1 Table

2 HOU015-#5 S.ptarmicoides S.ohioensis S.riddellii S.ptarmicoides S. houghtonii S. houghtonii S. houghtonii S. houghtonii S.rigida S.nitida . . ie fr 3 for Sites TGCGACCCGCAGATTGT TGCAACCCGCAAATTGT CTATTGAATAT GCAGACT ACTCT CGT S. ohioensis TGCAACCCGCAGATTGT S. ptarmicoides -O05YSCRACCCGCAGAYYGT T G T G T Y R Y R Y Y Y Y A Y A Y G A G A A G A G C A C A G C G C C G Y G C Y Y Y C C C C A Y A Y R M R M C R C A S Y S Y Y G Y S -HOU015 Y -HOU060 Y -HOU008 -HOU002

′ ETS andITSthatwere polymorphicinfour ye1T T -Type 1 ye2T T -Type 2 and S. ptarmicoides informativebasesare inboldfacetype. T 05026244890122898 55052583334449139 0

C C G 0 G G C C CAAC CAAC CAAC CAAC CAAC CAAC Taq

S. riddellii andpartialadditivitywith err, recombina- errors, C 3 E G 3 T and A 4 S S. ohioensis S. houghtonii C 4

. Entity3(accessionHOU060)andentity4HOU015)showperfect S. riddellii * C C T 4 C C T C =deletion. S. riddellii C C CGC CGC CGC CGC six from accessionHOU060 ( Table 2 ). from accessionHOU008, fivefrom accessionHOU015,and types from clonesof in ouranalysis.We identifiedfourconsensushomoeologue by usingaconsensussequencefrom clonesofsimilartype we attributedto three outgroup taxawere aligned togenerateacombined of homoeologue typesrecovered from clonesoffour accessions ered from clonesof we chosenottoclonetheirPCR products. to increase ourunderstandingoftheorigin since resolution ofasinglesiteinthesetaxawasnotexpected ity ofthepolymorphismsoccurred atuninformativesites,and cause lackofresolution ( Soltis etal.2008 ), butsincethemajor- could increase thenumberofmostparsimonioustrees and ize thatincludingcodedpolymorphismsinourMP analyses we codedthepolymorphismsusingIUPAC coding.We real- polymorphism intheir3 Solidago phisms were observedintheremaining 23sampledspeciesof in theITSandtwo3 S.gigantea S.houghtonii

individualsrepresenting fourhypothesizedgeographicentities.Entity1 In additionto In total,sixtysequences,including tworepeat typesrecov- S. houghtonii C 1 , ,

S. ohioensis . Consensusclonetypesfrom PCRproducts of , but5of the23specieswere found topossessasingle Nucleotide Sites G 1 * *

GIG004hadsingle site polymorphisms;one

, bothofwhichwecloned, ouraccessionof C * 1 * , and

, tensequences obtainedfrom GenBank, and

Taq Solidago ptarmicoides S. ptarmicoides * A * 1 I * * * * Solidago ptarmicoides error. We essentiallyignored

S. houghtonii ′ ETS sequence.Fortheseindividuals * G * 1 T * * * *

′ ETS regions. NoITSpolymor- * A * * * * * 1 S . Entity2(accessionHOU008)shows TTG TTG TTG TTG TTG TTG accessionHOU002, three T 5

andthepolyploid pe. , eighteenconsensus 6 T S. riddellii

S. houghtonii 6 G S. houghtonii

informative Taq T T G T 6 T T G (1) (10) (3) (1) (3) (5) errors

indi- (5)

, Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 sions of weakly supported(JS=60)cladewith 1991 ) as sidered insomeclassifications (e.g. Gleason andCronquist houghtonii Notably, thiscladedoesnotincludeanyclonesof (JS =72),and tion S.juncea this cladethatare notmembers ofthesubsection sion (GIG005).There are three additionalspeciesincludedin all accessionsof subsection clade (JS=92)thatcontainsallmembersofsection monophyletic (JS=100). Also resolved wasawell-supported most relationships within A. Gray). members ofsection eny isthatsection (JS =58)assisters. approximately 150miles apart, were alsoweaklysupported of This isconsistentwith Zhang’s (1996) cpDNA RFLP analysis resolved withothermembersofsection ment of 217 and section cies belongingtobothsection sus from theMP analysiswithrespect totheplacementofspe- (-lnL =3757.7823)thatwere congruent withthestrictconsen- 0.789) isshownin Fig. 4 . TheML analysisresulted intwotrees equally parsimonioustrees (335steps,CI=0.842andRI simony-informative. Thestrictconsensustree from 33,522 1,455 characters,178were variableand92(6.3%)were par- presence/absence ( LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII Simmons andOchoterena 2000 ). Ofthe due toindelsintheoutgroup taxa.Gapswere coded for gene. Alignment required theinsertionof17gaps,mostly 5.8S gene,212from ITS2,and69from the5 from the3 characters, includinggaps;520charactersfrom 3 3 2011] Ptarmicoidei Solidago junct), occupiesapositionwithintheunresolved polytomyof HOU015-#1, representing Pringle’sentity4(New York dis- as the“RiddelliiClade”( Fig. 4 ). Clonedhomoeologuetype ologue typesHOU002-#1andHOU008-#1.We identifythese ported clade(JS=99)withGBaccessionsof tion betweenspecies andclonedhomoeologue typeswithin “Ptarmicoidei Clade” ( Fig. 4 ). Thepaucityof sequencevaria- form amonophyleticclade(JS homoeologues generatedfrom cloningof micoides ported (JS=60)assisterto entity 3(Michigandisjunctoctoploid)( Fig. 1 ), isweaklysup- tonii Canada) perPringle( USFWS 1997 , HOU008-#1 representing Fig. 1 ). representing two ′ ETS andITS1–5.8S-ITS2datamatrixwhichcontained1,455 While thenrDNA analysis( Fig. 4 ) wasunabletoresolve A notableaspectofthecombined Solidagoriddellii The remainder ofthespecieswithin Solidago homoeologuetypeHOU060-#1,representing Pringle’s Junceae S. houghtonii species. , and S. houghtonii Aiton and S. gigantea S. canadensis . Within this “Triplinerviae Clade,” ′ where endofthe18Sgene,252from ITS1,164from the Triplinerviae ( , whichformamoderatelysupportedsubclade Ptarmicoidei S. nitida S. houghtonii S. rigida S. multiradiata S. gigantea (section Ptarmicoidei clonedhomoeologuetypes:HOU002-#1 S. riddellii , bothfrom withinMichiganandlocated Ptarmicoidei S. missouriensis clonetypes(datanotshown). Torrey & A. Gray, var. : : L.),alongwiththeremainder of , andalsowithrespect totheplace- S. canadensis entity1(NorthernMichigan)and S. riddellii Ptarmicoidei Solidago scabra S. houghtonii , includingtheNew York acces- alsodidnotresolve withother from subsection isparaphyletic; Solidago (Zhang’s < Torrey & A. Gray, formsa 50)identifiedhere asthe , itdidresolve , bothmembers ofsubsec- and Solidago L., S. canadensis subsection ) formsastrongly sup- entity2(Ontario, S. houghtonii Corymbosea Ptarmicoidei S. ohioensis S. altissima nrDNA phylog- Solidago ′ S. altissima endofthe28S S. riddellii Solidago hough- S. riddellii S. houghtonii Multiradiatae Triplinerviae Triplinerviae . Two acces- Solidago ′ Torrey & ETS, 123 , , section (Fg 4). (Fig. L.,and homoe- Solidago Solidago S. ptar- isnot , con- , and as . : ,

single haplotypewiththeputativediploid of HOU015, andHOU060)intotenhaplotypes.Eachaccession sion OHI005. our fouraccessionsof the 3 These homoeologueshaveaguanine(G)atposition352in S.houghtonii subclade (JS=61)includes share auniquecytosine(C)atbp618intheITS2.The“D” (JS =61)identifiedasthe“C”subclade.Thesehomoeologues HOU015-#3, andHOU060-#3formamonophyleticclade Solidagohoughtonii #6 whichissistertothe “B” subclade(JS=85)iscomposedofhomoeologueHOU060- which are sistertothe homoeologues, HOU002-#4,HOU060-#4,andHOU015-#4, subclade identifiedas“A”(JS=87)iscomposedofthree haplotype with repeat types. HOU060-#3 wasderivedfrom thishaplotype. phy, auniquecytosine(C)atbp618 inITS2.Homoeologue differed from HOU008-#3, andHOU015-#3formedasecondhaplotypethat S.nitida which severalsubcladeswere resolved. Ouraccession of the “PtarmicoideiClade”resulted inapolytomywithin Clade” isnestedwithinthegroup of species includedinourMP analysis( Fig. 5 ). The“Riddellii the “Triplinerviae Clade”andtheremainder ofthe three groups thatcorrespond tothe“PtarmicoideiClade”, alternative connections.Thenetworkisbroadly dividedinto positions thatprevented TCS from distinguishingbetween network. Theseloopswere causedbyhomoplasticalignment which couldnotbeunambiguouslyresolved, occurred inthe as missingintermediatesinthenetwork.Sixclosedloops, by TCSwere notfoundin theanalyzedindividualsandoccur data matrixpriortotheanalysis).Fifty-sixhaplotypesinferred species andclones(outgroup taxawere removed from the network ofthe57nrDNA sequencesobtainedfrom mony connectionlimitof16stepsandresulted inahaplotype work ( Fig. 5 ). TCS( Clement etal.2000 ) calculateda95%parsi- Solidagohoughtonii the homoeologousrepeat typesrecovered from cloningof nrDNA sequencesofpotentialdiploidprogenitor speciesand that fivehadevidenceofG. 3 ohioensis but noother HOU008-#1 formed asinglehaplotypewith theputative homoplasy exists. mediate leadingto the and HOU060-#5.Thishaplotype formedaclosedloopwith gle haplotypewith was derivedfrom thishaplotype.The HOU060-#4 inthenetworkanalysis.HomoeologueHOU002-#4 logue HOU060-#6wasderivedfrom ′ ETS sequencedatafrom 14additionalaccessionsrevealed The diploid TCS grouped the18nrDNA homoeologuerepeat typesfrom To gainbetterinsightintotherelationships betweenthe Solidagohoughtonii Solidagoohioensis S. houghtonii S. ohioensis ′ ETS whichisshared with issistertotheGBaccessionof appearstobepolymorphicforthispositionbecause Solidago homoeologues,HOU015-#5andHOU060-#5. Solidago houghtonii S. ohioensis Solidago ptarmicoides (OHI005)haplotypeandanunsampledinter- containedahomoeologuetypethatformed Solidago ptarmicoides S. houghtonii , weestimatedaparsimonyhaplotypenet- homoeologuesHOU002-#3,HOU008-#3, (GB) and S. houghtonii speciesincludedinouranalysis. S. ptarmicoides homoeologue typesHOU002-#1and Solidago houghtonii S. ptarmicoides (OHI005)byasinglesynapomor- S. ptarmicoides S. rigida,ohioensis homoeologuesHOU015-#4and S. rigida S. rigida homoeologues,HOU002-#3, homoeologues HOU015-#5 -Type 1formedasingle –Type 1suggestingthat possessed twonrDNA -Type 1repeat type.The S. ptarmicoides Solidago (RIG002) formedasin- S. houghtonii S. nitida and -Type 2repeat type. (HOU002,HOU008, S. ohioensis S. ohioensis species. (GB),andtwo (JS=76).The -ye 2. -Type homoeo- Solidago Solidago Solidago acces- (GB),

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 1 SSEAI OAY [Volume 36 SYSTEMATIC BOTANY 218 analysis isthat of section Fig. 4 . . Ptarmicoidei. Strict consensus of33,522trees from themaximumparsimonyanalysis ofcombinednrDNA and3 S. gigantea Numbersabove thebranchesrepresent jackknifesupport. isnotsisterto S. houghtonii , rather, S. houghtonii homoeologuesare polyphyletic, nestingwith ′ ETS andITSdata sets. A keyresult ofthis S. riddellii andvariousmembers Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 diploid 219 LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII 2011] hexaploid from the S.riddellii HOU015-#1, whichare likely chimericsequencesbetween haplotype composedof meric homoeologuesisonlysixmutationstepsawayfrom the cal (network)approaches toaddress thesegoals. data andmolecular phylogenetic(tree-based) andgenealogi- or polytopicorigin.We usednuclearand cpDNA sequence firmed hybrid,andtodetermine if of S.houghtonii gin fortheintraindividualnrDNA homoeologuetypesin tonii The SHtestsignificantlyrejected monophylyof was obtainedforthemonophyletic(nonhybrid)hypothesis. of 3,767.8forthehybridhypothesis,whilea–lnL of 3,843.1 unconstrained analysisresulted ina–loglikelihood(-lnL) ate ahybridvs.nonhybridstatusfor of S.houghtonii S.houghtonii h “idli Clade”. “Riddellii the inferred byTCS.GraylinesseparatesequencetypesidentifiedinMP analysisasbelongingtothe“Triplinerviae Clade”,the“P Fig. The goalsofthispaperwere toconfirmthehybridorigin The phylogenetictestforhybriditywasdesigned toevalu- S. gigantea Solidago houghtonii homoeologues( 5 . . S. riddellii S. houghtonii S. riddellii Statistical parsimonynetworkofnrDNA sequencesfrom 24 and homoeologuetypeisconnectedto any accession . inthenetworkanalysisofnrDNA sequences. homoeologuetypes.Itisnoteworthythatno S. ohioensis , whilehomoeologuetypesHOU060-#1and haplotype,respectively. Eachofthesechi- anddiploid , toinvestigatetheparentage ofthecon- p =0.002),consistentwithahybridori- Discussion S. ohioensis , are 11 and15mutationstepsaway S. ptarmicoides S. houghtonii (GB), Solidago houghtonii . Linesrepresent singlemutation stepsandsmallcircles are hypotheticalintermediaterepeat types S. rigida isofasingle , and two S. hough- . The . Solidago inference of parentage. ( Cronn etal.2003 ; et al.1999 Mason-Gamer 2004 ), andunexpected introgression ofdivergent sources ) cancomplicatethe genome duplicationduring polyploidization ( Rabinowicz gene amplificationabovethe numberexpectedfrom direct ploidization (reviewed in Soltis andSoltis1999 ; Wendel 2000 ), nonadditivity duetonon-Mendelian changesfollowingpoly- For example,polytopicorigins ( Soltis and1993 , 1999 shown thatgenomeevolutioninpolyploidscanbecomplex. ), specific hybridizationevents.However, recent studieshave phylogenetic patternswhichare usefulforinferringinter- nuclear andcpDNA sequencesfrequently produce different logues from theirprogenitor taxa( Wendel 2000 ). Therefore, that inphylogeneticanalysistheywillbesistertotheortho- will beadditivewiththoseofthediploidprogenitors and from thebiparentally inheritednucleargenomeofthehybrid parent. Incontrast,theexpectationisthatsequencesgenerated will formacladewiththosegeneratedfrom thematernal maternally inheritedchloroplast genomeoftheallopolyploid tion whenusingcpDNA isthatsequencesgeneratedfrom the markers withdiffering modes ofinheritance.Theexpecta- ence ofspeciesrelationships reconstructed from molecular 2000 ). Theseeventscanoftenbedetectedfrom theincongru- ( Soltis and1993 ; Wendel andDoyle1998 ; Soltis and spread andfrequent modeofplantevolutionandspeciation tion resulting inallopolyploids hasbeenshowntobeawide- speciesandhomoeologuesrecovered through molecularcloningofthe Allopolyploid OriginofSolidagohoughtonii— tarmicoidei Clade”and Hybridiza- Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 tence ofancestors withtheirderivatives,multifurcations, and processes that actatthespecieslevel, such asthecoexis- and Crandall2001 ). Networkanalysis takesintoaccount resent reticulate rather thanhierarchical relationships ( Posada ing tree asthegenessampledfrom allopolyploidspeciesrep- in historiesthatmaynotbeadequately modeledbyabifurcat- try inabifurcating diagram.Hybridization,however, results Phylogenetic trees describethishierarchical patternofances- cal structure betweengenessampledfrom different species. traditional methods,suchasMP andML,assumeahierarchi- genetic analysesofclonedhomoeologuetypes. between hypothesizedparental speciesandalsobythephylo- ence ofsequencepolymorphismsthatappeared tobeadditive of hybridorigin The usefulnessoftheITSandETSmarkersforouranalysis allopolyploid genome( Sang etal.1995 ; Whittall etal.2000 vide evidencefordivergent parental rDNA). typeswithinan 2004 ). Therefore, polymorphic nucleotidepositionscanpro- longer inallopolyploidsthandiploidtaxa( Lihová etal. somes sothatdivergent rDNA typesare expectedtopersist proceed more efficiently withinratherthanamongchromo- Feliner etal.2004 ). However, homogenization isbelievedto only oneparental typeis evident ( Wendel etal.1995b ; Nieto the divergent repeat typescanberapidlyhomogenizedsothat ( Wendel etal.1995b ). Eveninallopolyploidsofrecent origin, ies thatare frequently homogenizedbyconcertedevolution the parental taxaconsistof multipletandemlyrepeated cop- lyploid taxa,isthatthehomoeologouslociinheritedfrom contemplating theiruseforphylogeneticanalysisofallopo- of thesemarkers,whichmustbetakenintoaccountwhen taxa contributetothehybridoffspring. An importantfeature sequence data,suchastheITSandETSspacers,allparental logue typesthatnestedwithinbothclades.With nuclearDNA four accessionsof ( Table 2 ). Molecularcloningoftheentire 3 seven informativenucleotidepositionsseparatingthem “Ptarmicoidei Clade”andthe“Riddellii( Fig. 4 ) with Solidago tion (MP) analysisofthenrDNA sequencesindicated could leadtotheformationofwithin-individualrepeats. for thisspeciesalthoughotherprocesses, asdiscussedbelow, variants isconsistentwiththehypothesisofahybridorigin polyphyletic originof instead theyarose from atleastthree different lineages.The vidual clonednrDNA homoeologueswere notmonophyletic; phylogenies, phylogeneticanalysesrevealed thatwithin-indi- members ofsection S.houghtonii subsection placement of ITS lociwithvariousmembersofsection supports thenestingof S.gigantea the placementofallaccessions Phylogenetic analysisofcpDNA strongly supports(JS=90) 2 SSEAI OAY [Volume 36 SYSTEMATIC BOTANY for show evidenceofanincongruent topologicalplacement data ( Fig. 3 ) andourcombinednrDNA sequencedata( Fig. 4 ) tionships reconstructed from ourcombinedcpDNA sequence 220 A secondconsiderationisthatphylogeneticanalysesusing Parentage— of Evidence Nuclear Visual examinationoftheMP trees depictingspeciesrela- S. houghtonii Ptarmicoidei section . Incontrast,phylogeneticanalysisofthenrDNA Triplinerviae hasalwaysbeentaxonomicallyplacedwithother S. gigantea asexpectedforaspeciesofhybridorigin. maynotbemonophyletic.Specieswithin Ptarmicoidei S. houghtonii S. houghtonii Ptarmicoidei withothermembersofsection S. houghtonii (JS=92).Thisisnotsurprising,as S. houghtonii resolved intotwoclades,the resulted inconsensushomoeo- . Inadditiontoincongruent wasindicatedbythepres- S. houghtonii homoeologous3 nrDNA homoeologue aiu parsimony Maximum Ptarmicoidei ′ ETS –ITSregion in inacladewith Solidago ′ ETS and andthe Solidago sec-

ing toeliminatethe the evolutionaryprocess of concertedevolutionmaybeact- enization ( Soltis etal.2008 ) ofnrDNA repeat typesthrough study isunknown. Additionally, differential partialhomog- genomic rDNA copiesforanyofthespeciesincludedinour on itscopynumber( Rauscher etal.2002 ), andthenumberof nrDNA repeat typewithinapolyploidgenome isdependent such asPCRselectionordrift. Alternatively, thedetectionofa be duetounpredictable eventsduringPCRamplification, lii and HOU015(entity4)eachyieldedfourchimeric were recovered from each. Accession HOU060(entity3) that ologues andtwoaccessionsof there were nosequencedifferences betweenthesetwohomoe- related to represented byHOU002-#1andHOU008-#1,was closely analysis indictedthatoneclassofclonedhomoeologuetypes, 2001 ). adjacent haplotypesinthenetwork( Posada andCrandall collapsed intohaplotypesandsinglemutationstepsseparate logical relationships are reflected aslikesequencesthatare simony networkapproach. Instatisticalparsimony, genea- we additionallyanalyzedthesequenceswithastatisticalpar- phylogenetic informationcontainedinournrDNA dataset 2001 ). BecauseMP analysismaynotcompletelyreveal the information containedinadataset( Posada andCrandall reticulations, thereby incorporatingmore ofthephylogenetic HOU008 (entity2)althoughseveral entity; twofrom accession HOU002(entity1)andonefrom ing of tributor tothegenomeof tonii from of theallohexaploid S.ptarmicoides et al.1999 ) andasindicatedbybothMP andnetworkanalysis, Pringle ( USFWS 1997 foreachentity),andSemple( Semple network analysis.Thus,ashypothesizedby Morton (1979) , homoeologue typesclonedfrom ploid genomeof tive paucityof only forentity1,tobeaprogenitor species.However, arela- by Semple etal.(1999) and byPringle( USFWS 1997 ), albeit to amplifythe missinghomoeologueandinstead amplified repeat specific andexclusionprimers;however, thesefailed sion, asitwasinallotheraccessions, weattemptedtodesign Assuming that homoeologue couldbeamplified withrepeat-specific primers. morphisms) forasingleITS homoeologue,thesecondITS versal primersyieldedclean sequence (noobservablepoly- Rauscher etal.(2004) showedthatintetraploidswhere uni- resolved withthe homoeologue wasrecovered from HOU060(entity3)that Type 1repeat intheMP andnetworkanalysis. An additional 3, and4respectively) thatresolved withthe tonii Homoeologues were recovered from thegenomesof types were identifiedfrom found tobecloselyrelated to repeats. Thepaucityof Prnae y oiao riddellii— Solidago by Parentage Solidagoptarmicoides ptarmicoides— Solidago by Parentage . Itisnotsurprisingthat accessionsHOU002,HOU060,andHOU015 (entities1, S. riddellii S. houghtonii S. houghtonii S. riddellii isoneprogenitor oftheallohexaploid isasecondparental contributortothegenome S. ptarmicoides S. riddellii S. houghtonii S. ptarmicoides accessionHOU008(entity2-Canada). individuals representing eachgeographic . Statisticalparsimonyanalysisindicated S. houghtonii S. riddellii homoeologueswere notrecovered S. houghtonii repeats were recovered from clon- S. riddellii S. ptarmicoides S. ptarmicoides . wouldbepresent inthisacces- Solidago riddellii homoeologuefrom thepoly- S. riddellii -Type 2repeat intheMP and . -like homoeologuesmay Solidago houghtonii S. riddellii asithasbeenproposed aiu parsimony Maximum , providing evidence accessionPTR011. A secondclassof . Two rDNA repeat isalikelycon- S. ptarmicoides recombinants S. hough- S. hough- S. riddel- was - Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 with psbA-trnH tion inthe and 25other of thecombinedcpDNA sequencedatafrom source oftheallopolyploid serves asasuitablemarkerforinvestigatingthematernal and Coleman1988 ; Reboud andZeyl1994 chloroplast genomeisusuallymaternallyinherited( Corriveau ) andtherefore it seemunlikelythattheywere acquired from thisspecies. S.rigida logues isunclearbasedontheseanalysesalone,thefactthat S.ohioensis formed acommonhaplotype,with and HOU060-#5,whichwasphylogeneticallyrelated to,and S.gigantea relationship between and rosatellite lengthvariationthatwasshared by and ologue typewasrecovered from our datasetorisnowextinct. A finalclassofclonedhomoe- cate thatthere isadifferent parental taxonthatismissingin cate parentage by While thepositionofthisgroup ofhomoeologuesmayindi- appear derivedfrom thehaplotype formedbyOHI005( Fig. 5 ). HOU015-#2, andHOU060-#2are monophyletic( Fig. 4 ) and network. ClonedhomoeologuesHOU002-#2,HOU008-#2, of four geographicalentitiesare identicaltotherepeat type HOU008-#2, HOU015-#2,andHOU060-#2from eachofthe network analysisindicatedthathomoeologuesHOU002-#2, age for sequences, MP analysiswas unabletodirectly indicateparent- these homoeologuesclearlyrepresent Ptarmicoidei-type formed apolytomy(multifurcation) ( Fig. 4 ). Thus,although with anyotherclonetypesinourMP analysis;insteadthey HOU060-#2, were notresolved withanyextantspeciesor 221 Alternatively, silenced orlostinindividualsfrom thisgeographicregion. perhaps the concerted evolutionmayhaveeliminated LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII S.ptarmicoides ber, there are severalotherpossibleexplanationsforwhythe the 2011] the data from these markersdonotrefute thedataobtainedfrom inferred from the of S.ohioensis ologue typeswere recovered from although morphologysuggestsotherwise. in theparentage ofindividualsfrom thisgeographic region ploidy levelindividualsand clones isrequired torecover allhomoeologuesfrom higher Small etal.(2004) indicatedthatscreening aminimumof40 clones foraccessionHOU008,aseeminglylarge number, but lution. With regard toincompletesampling,wescreened 22 of clones,orevolutionaryprocesses suchasconcertedevo- problems (See Rauscher etal.2002 ), toincompletesampling tonii Clrpat vdne f Parentage— of Evidence Chloroplast Prnae y oiao ohioensis— Solidago by Parentage S. houghtonii S. ohioensis psaI-accD S. ohioensis accessionHOU008.Theserangefrom PCRassociated rps16-trnQ S. gigantea S. gigantea S. houghtonii ismorphologicallydistinctfrom IGS. isthesource of (GB)( Figs. 4 , 5 ). Whiletheoriginofthesehomoeo- asathird parent. Theclonedhomoeologuetypes psaI-accD and S. ptarmicoides Solidago homoeologuewasnotrecovered from (OHI005),formingasinglehaplotypeinthe S. ptarmicoides homoeologue.Inadditiontolowcopynum- markersrevealed afivebpdeletionandmic- , aswellother andnoother Solidago houghtonii , HOU002-#2,HOU008-#2,HOU015-#2, and psbA-trnH trnL-trnF S. ohioensis from thesehomoeologues.However, the IGSandaunique15bpdeletioninthe speciesrevealed aunique29bpinser- S. houghtonii S. houghtonii and markers.Furthermore, there isno homoeologouslocushasbeen S. houghtonii Solidago maynothavebeeninvolved Solidago houghtonii , thesesequencescouldindi- rps16-trnQ shared theseuniqueindels Solidago and S. houghtonii S. houghtonii he adtoa homoe- additional Three species,suggestingthat S. rigida cpDNA.The n h Atrca, the Asteraceae, the In S. gigantea ishexaploid.Biased S. houghtonii species.Whilethe markersalone,the S. ptarmicoides (RIG002),and , HOU015-#5 , thatsuggest S. houghtonii S. houghtonii . Alignment . cannotbe trnL-trnF S. hough- makes , or ,

Lakela, washighlydivergent from theremaining foursec- found thatonesection, of subsectionsthegenus lyzed cpDNA variationacross fivesections and themajority without nuclearDNA introgression. Soltis etal.(1991) ana- more oftenthannuclear geneflow, andfrequently occurs Furthermore, theyindicate thatcytoplasmicgeneflowoccurs S.houghtonii sive examplesthat,likethepresence of and foundtheliterature to containahighnumberofconclu- ture through hybridizationandintrogression occursinplants Soltis (1991) examinedtheextenttowhichchloroplast cap- genitor of tonii from The absenceofanyevidence resembling 10–15 clonesperaccession.We failedtoisolateanyclones we hadinitiallyclonedjusttheITSregion andsequenced of clones,22–30peraccessionfortheentire 3 shared morphologicalcharacters betweenthem. the genomeof ITS primersandwere unabletoamplify tonii S.gigantea also berepresented inthenucleargenome.Theexistence of tation thatthematernalsource oftheallopolyploidwill S.ohioensis that progenitor wouldhavehadtoserve asthematernal ancestor of tonii that anowextinctintermediate tetraploidancestorof tive section occurred. Itispossiblethatintrogression intoone oftheputa- mation of donors of tion intersectional hybridizationbecause ization iscommon.Ourresults provide anotherexampleof gression, indicatingthat,in S.houghtonii data supportthepresence ofthree potential parents of expected fororganisms ofallohexaploidorigin.OurnrDNA S.houghtonii 2) capture of 1) maternalparentage of These datacanbeexplainedbytwoalternativescenarios: S.gigantea H.hallii of section authors proposed thatthedistinctivechloroplast genotype closest relatives were notfrom withinthissection.These when morphologicalandallozymedatahadindicatedtheir & A. Grayresolved withmembersofsection Rosendahl, Butters,&Lakela,and ysis indicatedthatthree species, tions. Thisissignificantinlightofourresults astheiranal- HOU008, andHOU015). All accessionsof GIG003 andGIG004) cpDNA sequencedivergence between The alternative,that It remains unclearatwhatpointinthehistoryoffor- The presence ofthree separategenomiccontributionsis arose asaresult ofchloroplast capture byaparent orpro- isimprobable becausewedesigned captured the Solidago S. houghtonii , , H. nivalis S. houghtonii S. houghtonii sequenceswithinthenucleargenomeof S. houghtonii formawellsupportedcladeintheMP analysis. , butnot Rhodoheuchera S. houghtonii S. gigantea . Ptarmicoidei subsection , were largely unexpectedbytheinvestigators. S. gigantea involving S. houghtonii , and . Inadditiontosequencingalarge number S. gigantea S. gigantea H. parvifolia belongtosection , maybemore probable. Rieseberg and cpDNA introgression from from anyaccessionof cpDNA byoneoftheparental taxaof Solidago gigantea parents of Rhodoheuchera Triplinerviae captured thecpDNA of wascaptured by populationsof S. houghtonii S. ptarmicoides isconsistent withanabsenceof S. houghtonii Heuchera Heuchera cpDNA. Regardless ofwhich . Thisconflictswiththeexpec- S. gigantea viahybridizationandintro- H. hallii H. parvifolia S. houghtonii andthenuclear genome S. gigantea L.(Saxifragaceae)and , intersectional hybrid- Rosendahl,Butters,& S. gigantea (accessionsHOU002, S. gigantea cpDNA in S. gigantea Ptarmicoidei by -like nrDNA within A. Gray, , , S. gigantea ′ S. houghtonii S. riddellii, ETS –ITSregion, NuttexTorrey belongstosec- S. gigantea Rhodoheuchera S. houghtonii occurred or (accessions S. gigantea cpDNA in sequences S. gigantea H. nivalis S. hough- . S. hough- S. hough- -specific and and or, . ,

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 while Midwestpopulationsare tetraploid(2 type. Easternpopulationsof A finalpossibilityinvolvesdifferences associatedwithcyto- may itselfbeanintrogressed individualfrom anotherspecies. S.gigantea (Great Lakesregion only)introgression from unique 29bpinsertionleadingustoreject theideaoflocal We foundthat theseadditionalaccessionsalsocontainedthe (GIG007), Colorado(GIG008),andTexas (GIG009)( Fig. 2A ). ( Fig. 2A –D).Sequencedivergence between match thecpDNA from accessionsofGreat Lakes to findthatthecpDNA of in thehistoryofformation it appearsthat populations havedescended. tonii it wasintrogressed/captured earlyinthehistoryof its presence inallpopulationsstudiedthusfarsuggeststhat be maintainedthrough thevarious haplotype, thenthechloroplast polymorphismwouldneedto for and Zhong2000 ). Ifwehypothesizelineagesortingtoaccount ing ofancestralpolymorphism( Wendel andDoyle1998 ; Sang result notfrom hybridization,butratherfrom lineagesort- tially complicatephylogeneticinterpretations, astheymay cpDNA andthosegeneratedfrom nuclear DNA canpoten- the source ofcpDNA variationwithin ern populationsof al. 1984 ; Semple etal.1999 ). More extensivesamplingofeast- itor speciesifthey were theresult of recent geneduplication. sequence types tobemonophyleticwiththeproposed progen- S.houghtonii analysis. Thiswasnotthecase forsequencesrecovered from divergent sequenceswouldbecloselyrelated inphylogenetic ual sequencevariation.Inthis case,onewouldexpectthatthe ple, recent geneduplicationwouldproduce within-individ- account forwithin-individual sequencevariation.Forexam- origin. However, processes otherthanhybridizationcould etic, leadingustoconcludethat except have tobefollowedbyextinctionofonealleleinallspecies separating [Volume 36 the S.houghtonii Great Lakesaccessionsof the identification. Another possibleexplanationisthatthe its placementinthenrDNA phylogeny, wefeelconfidentin is incorrect, butuponstudyingourvoucherandconsidering Lakes region. Itispossiblethatthespecimenidentification tonii SYSTEMATICalmost anorder ofmagnitudehigherthanwhen BOTANY the chloroplast haplotypeof it isnotclearhow houghtonii formation ofboththehexaploidandoctoploidcytotypes parent foranysubsequenthybridizationeventsleadingtothe 222 ered from cloningof simonious explanationfortheobservedtopologies. of thisscenariowefindchloroplast capture tobeamore par- allele inthesetwospecies.Becauseofthecomplicatednature Alternative ExplanationsforMolecularResults— Topological discrepancies betweentrees generatedfrom The SHtestindicatedthatthenrDNA homoeologuesrecov- S. houghtonii psaI-accD possiblyevenintothehexaploidancestorfrom whichall wascompared toaccessionsof S. houghtonii becausetheseare monophyletic( Fig. 3 ). Although . Alternatively, theNew York accessionof S. houghtonii . Additionally, onewouldnotexpectthevaried . To investigatethisquestionwesequenced IGSforaccessionsof S. gigantea and S. gigantea and S. gigantea S. gigantea Solidago houghtonii S. gigantea from cpDNA wascaptured atsomepoint S. gigantea S. gigantea cpDNA passedinto S. houghtonii S. gigantea S. gigantea wouldbenecessarytoexplain S sharingthesamechloroplast . . gigantea andextinctionoftheother S. houghtonii Solidago S. gigantea were introgressed from from New York didnot S. gigantea S. gigantea are diploid(2 were notmonophyl- from New York was , wewere surprised . Thiswouldthen speciationevents n S. houghtonii S. houghtonii =36)( Semple et from theGreat from Illinois isofhybrid S. houghtonii . S. gigantea S. gigantea Although Although S. hough- S. hough- n =18) into and S. ,

houghtonii S.ohioensis logically similar S.houghtonii ing heads).Infact,thesecharactersare usedtodistinguish pubescence onthepedunclesandlarge capitula(flower- S.houghtonii with aclavatetip,andthetransversesectionedachenesof both to theinflorescence. Inmarked contrast,theinflorescences of inflorescence, are widelyseparatedgivinganopenappearance The floweringheadsof ber (2–)5–50(–100+)tothoseofeither appears tobeintermediateindensityandflower-head num- flowers of large rayflowersof cal for basal leavesare slightlyconduplicate, aconditionthatistypi- micoides S.gigantea nal sources of supports to base,atraitobservedin to thatof tics oftwotheputativeparents; alongsharppointsimilar the marginal leafciliaof of sis of descriptions). Edwards-Wilson’s (1999) microscopic analy- inflorescence (See Semple and Cook2006 formorphological ing 30–450headsand resembles anaturallyoccurringhybridof or lesslinearleaves.More specifically, have acorymbiforminflorescence andabasalrosette ofmore “favors” thatof those oftheGreat Lakespopulations.It has longbeenknown marl fen,where environmental conditions are different from sions of and fouroffivenrDNA homoeologuetypeswith otheracces- or novelcharacters.Themorphologyof showed that89%oflatergenerationhybridsdisplayextreme intermediate morphologicalcharacters.However, theyalso true USFWS 1997 ). Ourdataindicatethatthe New York plantsare S.houghtonii the disjunctNew York populationisadistinctspeciesfrom leaf margins of S.houghtonii at anthesis,andapaniculiforminflorescence. Additionally, predominantly caulineleaves,basalleavesthatare withered two speciesappearmorphologicallydistinct; F than novelones. Rieseberg andEllstrand(1993) showedthat intermediate anddisplayablendofparental charactersrather tion regarding hybridspeciesisthattheyare morphologically tonii indicated thatthevariablesequencesrecovered from ing theprotocol setforthby Nieto FelinerandRosselló(2007) ogs orpseudogenes.Careful assessmentofhomologyfollow- variation couldbeduetotheevolutionofdivergent paral- However, ifthegeneduplicationwasmore ancient,thenthe 1 hybridsare amosaicofbothparental species,displaying Morphological variationhasledmanytopropose that Mrhlgcl upr o Parentage— of Support Morphological S. houghtonii were notlikelypseudogenesordivergent paralogs. S. houghtonii S. riddellii S. houghtonii S. riddellii ( S. houghtonii S. S. ptarmicoides,riddellii, alsoresembles S. ptarmicoides isinvolvedintheparentage of S. ptarmicoides hasglabrous pedunclesandsmallcapitula.

× tobesimilarthose of

generallyhasentire leafmargins whereas the kratkovii from narrow-leaved formsofthemorpho- ( Mitchell andSheviak 1981 ; USFWS 1988 ; S. houghtonii and tobemostlikethatof S. gigantea Solidago , whichalsohashairypeduncles.Lastly, the S. ohioensis astheyshare thesamecpDNA haplotype anditsputativeparents foundthepappus S. ohioensis S. houghtonii B.Boivin)astheybothhaveastrigillose . TheNew York populationexistsina S. ohioensis section S. ptarmicoides , andaconstantlengthfrom leaftip andtheinflorescence of S. houghtonii are serrate. . Morphologydoesnotsuggestthat S. riddellii S. riddellii , withwhichitoftenoccurs,as are densewith Ptarmicoidei are similarinsizetotheray having10–500+headsper and . Therefore, morphology asitslowerstemand S. riddellii S. riddellii displayedcharacteris- , usually1–25(–50)per S. ptarmicoides S. houghtonii S. ohioensis S. houghtonii cmo assump- common A S. ohioensis S. houghtonii whosemembers S. riddellii S. gigantea or . Additionally, . S. houghtonii S. ohioensis aspater- strongly S. hough- × Solidago , robust , clearly S.ptar- asthe hav- has .

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 related tothosefoundin identical ornearlyhomoeologuesthatappeartobe Solidagohoughtonii but appearstoprovide further supportforasingleoriginof progenitor thatgaveriseto of asingleoriginforthespecies,oratleasttetraploid was closelyrelated to species formingawell-supportedmonophyleticcladethat S.houghtonii not straightforward. 223 genomic evolution,theinterpretation ofsequencevariationis involved intheformationofanallohexaploidandsubsequent ent parents. However, giventhecomplexityofprocesses not bemonophyleticandwouldlikelyrelated todiffer- parents. Ifpolytopic,wepredicted thesequences would of thespecieswouldbemonophyleticandrelated tothesame hypothesized entitiesfrom throughout thegeographic range sequences from theindividualscollectedfrom thefour S.houghtonii LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII houghtonii have beenseveralproposals regarding theoriginof tions ( Sultan 2000 ). in individualorganisms dependingonenvironmental condi- that development,physiology, andlifehistorycanbealtered 2011] ies. Thepaucity ofparsimonyinformativecharacters inboth capture through introgression across intersectionalboundar- ing bothhybridizationbetween three speciesandchloroplast the firstreported exampleofarecently formedhybridinvolv- hexaploidy andintrogressive chloroplast capture. Thismaybe lution andspeciationinthe genus be octoploid. to reveal theoriginoffourthgenomeifitisconfirmedto it shares thesameparental lineages,furtherworkisneeded related to they share thesamematernalgenomeandrDNA repeat types S.houghtonii analysis indicatethattheputativelyoctoploidpopulation of is reported tobeoctoploid.BothMP analysisandnetwork range; however, theMichigandisjunctpopulation,entity3, Solidagohoughtonii cytotypes (See Semple andCook2006 forploidynumbers). is ofdifferent parentage. flowers of is believedtobethemostlikelycontributoroflarge ray yet reveal thepresence of cloning additionalaccessionsfrom itsgeographicrangemay genome ofindividualsrepresenting entity2remains unclear, whereas theotherthree entitiesdo. Although the third parental does nothavehomoeologuescloselyrelated to tial polytopichistoryfor shared history( Figs. 4 , 5 ). Themainresult hintingatapoten- sampled parental repeat typesbutstillindicateacommon cases, homoeologues#1,2,3,and5are derivedrelative tothe at leastderivedfrom thehexaploid population. Whileourresults indicatethatthispopulationis nal speciesisthecauseforincreased ploidylevelofthis sess fourgenomes.Perhaps abackcross tooneofthepater- if thispopulationwere octoploiditwouldbeexpectedtopos- Interpretation ofthenrDNA analysesare more complicated Our analysisofcpDNA sequencevariationresulted in Origin— Polytopic vs. Single This study describesacomplexpatternofreticulate evo- Several speciesof S. ohioensis involvingbothsingleandpolytopicorigins.If S. houghtonii iscloselyrelated tothehexaploidpopulations as cpDNA haplotypesfrom across therangeof originatedonce,wepredicted thatisolated becauseeachofthefouraccessionsshare isknowntobehexaploidthroughout its , , Solidago S. ptarmicoides S. gigantea . However, itispossiblethatthisentity S. houghtonii S. riddellii S. ptarmicoides S. houghtonii are knowntopossessmultiple . Theseresults are suggestive As reviewed above,there , and and isthatHOU008(entity2) S. houghtonii Solidago . -like sequences,asit S. riddellii S. ohioensis involvingboth S. ptarmicoides withwhich . However, . . Insome Solidago

Crieu J L ad . . oea 98. Rpd cenn mto t detect to method screening pro- Rapid 1988. computer Coleman. W. a A. and TCS: L. J. 2000 . Corriveau, Crandall. field A. for K. material and ideal Posada, an D. gel: M., Silica Clement, 1991 . Hills. H. H. and W. M. Chase, Bclr E S, . poio ad . . otfr 97. Te vlto o ribo- of evolution The 1997. Holtsford . P. T. and Ippolito, A. S., E. Buckler, 1994. K. Bremer, E. gen- sequences E. DNA and Recombinant Small, 1997. Hillis. L. M. R. D. Baird , and D. I. R. G. Bradley, Calie , J. P. Nesom, L. G. B., J. Beck, of parentage and origin Hybrid 2002 . Simpson. B. B. and J. T. Barkman, Blwn B G ad . aks . 19 hlgntc tlt o te external the of utility Phylogenetic 1998. Markos. S. and G. B. Baldwin, Aho, . . n R J Abt 92. Mlil oiis n gntc diversity genetic and origins Multiple 1992. Abbott. J. R. and A. P. Ashton, variation Allozyme 1990. Bennet. D. B. and Hamrick, L. J. L., M. Arnold, 1997. L. M. Arnold, Thomsen forfieldcollectionsof mission tocollectontheirproperties andMichaelSchuldmanCraig and theMichiganDepartmentofNaturalResources are thankedforper- Ontario MinistryofNaturalResources, theUSFishandWildlife Service, the WhitefishRiverFirstNation,Nature ConservancyofCanada,the houghtonii securing collectionpermits,andhelpfuldiscussionsregarding of NaturalResources forproviding locationinformation,assistancein Natural Features Inventory, andMikeOldhamoftheCanadianMinistry Michigan NaturalFeatures Inventory, Steve Young oftheNew York Western MichiganUniversity. TheauthorsthankMikePenskarofthe sertation submittedbyPJL totheDepartmentofBiologicalSciencesat Ávrz I ad . . edl . 20 iooa IS eune ad ln phy- plant and sequences ITS Ribosomal 2003. Wendel . F. J. and I. Álvarez, relationships, of Reexamination 2003. Baldwin. G. B. and K. Andreasen, Foundation. donation totheFund,byHanesandMichiganBotanical Nongame Wildlife FundontheirMichiganincometaxformorbyadirect is basedhasbeensupportedinpartbycitizenswhocontributedtothe assistance withfieldwork.Fieldandlabworkuponwhichthispaper with labwork,andJamesDoyle,PegHaviland,Tom Laureto for tina ities withinthegenus. ploid beneficial forelucidatingtheoriginsofthisandotherpoly- able lowcopynucleargenesforusein nuclear andchloroplast genomes.Thedevelopmentofvari- species, thesamelevelofdivergence hasnotoccurred inthe within thegenushasallowedrecognition ofover100 These results suggestthatwhilemorphologicaldivergence dently testourquestionsregarding theoriginof the nrDNA andcpDNA datasetslimitedourabilitytoconfi- Acknowledgements. , respectively. We thankTalline MartinsandSeok-HongLimfor help 200 angiospermspecies . potential biparental inheritanceofplastidDNA and results forover gram toestimategenegenealogies . preservation ofleafsamplesforDNA studies . Genetics somal DNA:divergent paraloguesandphylogeneticimplications . Press. Timber 592–593. erated byPCRamplification . monophyletic? (Asteraceae) Taxon Solidagininae subtribe Is 2004. Schilling. 209–220. 27: text usingnuclearandplastidDNA sequencedata . Dendrochilum acuiferum Evolution ITS trees of transcribed spacer(ETS)of18S-26SrDNA:congruence ofETSand Cmoia). (Compositae) . in thenewlyarisenallopolyploidspecies, Heredity in Louisianairises:atestforintrogression andhybrid speciation . Press. University logenetic inference . of Botany Malvaceae) basedonmolecularphylogeneticdata . habital evolution,andphylogeographyofcheckermallows( Solidago . TheBergen SwampPreservation Society, Mr. Clifford Fielding, 3 691–698. 53: 4: 821–832. 145: 5 297–306. 65: 0 436–444. 90: 0 449–463. 10: speciesandfordeterminingphylogeneticaffin- Asteraceae: cladisticsandclassification. Calycadenia Heredity Natural hybridizationandevolution Molecular PhylogeneticsandEvolution This studyrepresents partofadoctoraldis- ieaue Cited Literature 8 25–32. 68: (Orchidaceae) inferred inaphylogeneticcon- (opste (Compositae) . American Journalof Botany Ericameria nauseosus Molecular BiologyandEvolution Molecular Ecology Molecular Phylogeneticsand Senecio cambrensis Solidago Taxon and 5 1443–1458. 75: otad, Oregon : Portland , . Ofr Oxford Oxford : . Systematic Botany 0 215–220. 40: American Journal : 1657–1660. 9: S. houghtonii mayprove Solidago velu- 9 417–434. 29: Sidalcea Solidago Rosser 14: . ;

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 Hge, . . C D Bie , n S A Hri 02. Dvret n reticulate and Divergent 2002. Harris. A. S. and Bailey , D. C. E., C. Hughes, DNA Ribosomal 1999. Hahn. J. W. and Zimmer , A. E. A., M. Hershkovitz, Ggibr, . F Beanle ad . aso 06. Alplpod origin Allopolyploid 2006. Mansion. G. and Bretagnolle , F. A., Guggisberg, Lso, . n J W Kdri 95. Clrpat N eiec fr intro- for evidence DNA Chloroplast 1995 . Kadereit . W. J. and A. 2004. Liston, Feliner . Nieto G. and Marhold , K. Aguilar , Fuertes J. J., Lihová, Fednti, . . C vn e Br , . . oda , . . Kores , 1991. J. Cronquist . P. A. and Goldman, A. H. H. Gleason, D. Berg , den van C. V., J. Freudenstein, Gie K E ad . . os . 16 itiuin o dsicie shoreline distinctive of Distributions 1963. Voss . G. E. and E. K. Guire, 1981. V . Grant, 1993. Ma. P. D. and Doebley , J. Durbin, L. M. Clegg, T. M. ITS M., E. 2003. Golenberg, Jansen. K. R. and Gutell, R. R. Cannone, J. J. R., L. Goertzen, mitochondrial of Analysis 2001. Chase. W. M. and V. J. Freudenstein, Flesen J. 18 vltoay re fo DA eune: maximum a sequences: DNA from trees Evolutionary 1981. J. Felsenstein, Kvrk A, . . ie , . . eth K Y Lm A M Sherwood, M. A. Lim, Y. K. Leitch, R. A. Pires , C. J. A., Kovarik, Fri, . . V A Abr , . älrj , . isob ad . . lg 1996. Kluge. G. A. and Lipscomb, D. Källersjö, M. Albert, A. V. S., J. Farris, for procedure isolation DNA rapid 1999. A L. 1987. M. Edwards-Wilson, Doyle. L. J. and J. J. Doyle, Igaso, . . S Rbti , n D R Tyo 03. Mlclr evolu- Molecular 2003. Taylor . R. D. and Ribstein, S. K., P. Ingvarsson, intro- DNA Chloroplast 1992. Arias. M. D. and Rieseberg , H. L. O., Dorado, Kpa, . n J Fhe 07. Mlclr vdne o a aua primary natural a for evidence Molecular 2007. Fehrer . J. and Z. Kaplan, [Volume 36 SYSTEMATIC BOTANY multispe- a in dispersal and Selection 2004. Afzal-Rafii. Z. and S. R. Dodd, of confirmation Electrophoretic 1990. Cryptic Wyatt . 2003. R. Wendel and . W. F. C. J. dePamphilis, and Haselkorn, T. Small, L. R. R., 2002. Wendel . Cronn, F. J. and Grover the , C. Haselkorn, on T. Cedroni , report M. R., status Cronn, and assessment COSEWIC 2005. COSEWIC. 224 Eltad N C, . htu , n L H Reeeg . 19 itiuin of Distribution 1996. Rieseberg . H. L. and Whitkus, R. C., N. Ellstrand, species relationships in Limited. Francis and Taylor London: Gornall. J. R. and tematics andplantevolution sequences andangiospermsystematics . Pp. 268 – 326 in inferred bymolecularmarkers . of theMediterraneanendemic, vus gression andlongdistancedispersalinthe desertannual Journal ofBotany assessed withnuclearandchloroplast DNA sequencedata . Origin ofthedisjuncttetraploid lyploids ofrecent andrecurrent origin . g phy- egy . DNA plastid expanded An 2004. logeny ofOrchidaceae andanalysisofjackknifebranchsupportstrat- Chase. W. M. and Molvray , M. plants intheGreat Lakesregion . Phylogenetics andEvolution Evolution ofanoncodingregion ofthechloroplast genome . Phylogenetics andEvolution for sequencealignmentandphylogenyofthe Asteraceae . secondary structure derivedfrom comparativeanalysis:implications The New York BotanicalGarden. eastern UnitedStatesandadjacentCanada change characters . b-c intron sequencesinOrchidaceae: utilityandcoding oflength- likelihood approach . 99–124. . aysk J Rca D E Sli , n P S Sli 05. Rpd con- Rapid 2005. Soltis . S. P. and Soltis, E. D. certed evolutionofnuclearribosomal DNA intwo Rocca, J. Matyasek, R. Parsimony jackknifingoutperformsneighbor-joining . small quantitiesoffresh leaftissue . tion ofinsertionsanddeletioninthechloroplast genomeof gression insouthernCaliforniasunflowers . Botany triple hybridinplantsrevealed from direct sequencing . MolecularBiologyandEvolution phism . data sources: insightsinto polyploidoriginsandnrDNA polymor- cies oakhybridzone . genetic structure ofabroad hybridzone . interspecific hybridizationin sypiodes repeated genomicrecombination duringspeciationin from polyploidcotton . PCR-mediated recombination inamplificationproducts derived the StatusofEndangered Wildlife inCanada . Ottawa . vi Houghton’s goldenrod . + 17 pp. Sciences USA spontaneous planthybrids . University. Michigan Central Michigan: attempt todiscoveritshybridorigins. (seaee (Asteraceae). American JournalofBotany 9 1213–1222. 99: American JournalofBotany . Evolution Plant speciation 3 5090–5093. 93: 1 1231–1242. 91: Plant Systematicsand Evolution 7 2475–2489. 57: Systematic Botany Journal ofMolecularEvolution Evolution Theoretical andAppliedGenetics Solidago houghtonii . E. e Yr ouba nvriy Press. University Columbia York : New 2. Ed. . Leucaena A taxonomicstudyofSolidagohoughtonii:an , d. P M Hligwrh R M Bateman, M. R. Hollingsworth, M. P. eds. , : 52–64. 2: 216–234. 29: Proceedings oftheNationalAcademy 1 149–157. 91: 0 1737–1740. 20: Aesculus 8 261–269. 58: Systematic Botany Centaurium bianoris Cardamine amporitana The MichiganBotanist 9 1057–1073. 89: (Fabaceae)inferred from multiple Manual ofvascularplantsnorth- Phytochemical Bulletin 6 643–657. 26: M. S.thesis.MountPleasant , (Hippocastanaceae)andthe Genetics . E. e Yr, e York : New York, New 2. Ed. . Evolution inCanada.Committeeon Evolution 6: 931–944. 169: 9: 33–41. 197: 1 368–379. 31: 7 368–376. 17: 4 1295–1317. 44: Tragopogon (Gentianaceae), 6 566–572. 46: 0: 482–489. 104: Gossypium gos- (Brassicaceae) : 99–114 . 2: Molecular sys- Cladistics 9 11 –15. 19: Senecio fla- Molecular Molecular Annals of American allopo- Silene nad1 12: .

Ruce, . . J J Dye ad . . . rw 02. Itra transcribed Internal 2002. Brown . D. H. A. and Doyle, J. J. T., J. Rauscher, Rbnwc, . . E L Ban A D Wle B Bwn ad . Grotewold . E. and Bowen, B. Wolfe , D. A. Braun, L. E. D., trees P. genealogies: Rabinowicz, gene Intraspecific 2001. Crandall. A. K. and D. Posada, Psd, . n K A Cadl 98. MDLET tsig h mdl of model the testing MODELTEST: 1998. Crandall. A. K. and D. Posada, Neo eie, . B Gtérz aea ad . ure Aulr . 20 Fine- 2004. Aguilar . Fuertes J. and Larena , Gutiérrez B. G., know? Feliner, Nieto you devil the Better 2007. Rosselló. A. J. and G. Feliner, Nieto of infrastructure Taxonomic 1993. L. G. Nesom, Mro, . 99. Osrain o Huho’ glerd ( goldenrod Houghton’s on Observations 1979. K. J. Morton, 365, Part 1991. Resources . Natural of Department Michigan (MDNR) 1981. Sheviak. J. C. and S. R. Mitchell, inter- and introgression, evolution, Reticulate 2004. J. R. Mason-Gamer, Shlig E E, . . ek P J Cle ad . . ml 08. Mlclr anal- Molecular 2008. Small . L. R. and Calie, J. P. Beck, B. J. E., E. Schilling, Sml, . . n R E Co 06. 2006 . Cook. E. R. and C. a J. of origins Semple, multiple Likely 2002. Rieseberg . H. L. and E. A. Schwarzbach, Mro, . n B G Blwn . 20 ihrlvl eainhp ad major and relationships Higher-level 2001. Baldwin. of G. Analysis B. and 4: S. Markos, MacClade 2001. Maddison. P. W. and R. D. Maddison, Slosal K. 20 st f rmr fr mlfcto o nnoig regions noncoding of amplification for primers of set A 2001. K. Saltonstall, Sml, . . n K N Gnh 04. 2004 . Gandhi. N. K. and C. J. Semple, Sn, . D J Cafr , n T F Susy . 19 ouetto o reticu- of Documentation 1995. Stuessy . F. T. and Crawford , J. on D. based T., hypotheses Sang, hybridization Testing 2000. Zhong. Y. and T. Sang, Rdíuz F, . . lvr A Mrn ad . . eia . 19 h general The 1990. Medina. R. J. and Marín, A. Oliver , L. J. F., cyto- Rodríguez, of consequences Phylogenetic 1991. Soltis. E. D. and H. L. Rieseberg, Reeeg L H ad . . lsrn 93. Wa cn oeua ad mor- and molecular can What 1993. Ellstrand. C. N. and H. species. L. plant Rieseberg, of origins Hybrid plants. 1997. in H. inheritance L. Rieseberg , Organelle 1994. Zeyl. C. and X. Reboud, and origins Multiple 2004. Brown . D. H. A. and Doyle, J. J. T., J. Rauscher, spacer repeat-specific primersandtheanalysisofhybridizationin in thehigherplants . Maize 1999. grafting intonetworks . DNA substitution . Armeria recombination innuclearribosomalinternaltranscribedspacers scale geographicalstructure, intra-individualpolymorphismand 911 –919. 44: evolutionary studiesinplants . Guidelines forinsightfulutilizationofnrDNA ITSinspecies-level tion . (Asteraceae: Astereae) andobservationsontheirphylogeneticposi- houghtonii to 324.36507.SpecialPlantListdatedNovember14,1991 . Environmental Protection Act, 1994,PA 451.MCL Sections324.36501 Endangered SpeciesProtection oftheNaturalResources and Museum. State York New York : New Albany , 455. No. 25–37. tribal genecapture inanallohexaploidgrass . SystematicBotany internal andexternaltranscribedspacer(ITSETS)sequences . ysis of of theBotanicalResearch InstituteofTexas diploid hybridsunflowerspecies . lineages of Inc. Associates, Sinauer Massachusetts : phylogeny andcharacterevolution . Version 4.03. Sunderland, of chloroplast DNA inthegrasses . A. Gray(Asteraceae: Astereae) . combination toreplace a“rankless”name usedbyTorrey and Press . University Flora ofNorth America EditorialCommittee . New York America : Oxford Sciences USA phy andconcertedevolution . sequences ofnuclearribosomalDNA:Implicationsforbiogeogra- late evolutioninpeonies( incongruent genetrees . Biology stochastic modelofnucleotidesubstitution . plasmic geneflowinplants . in PlantSciences phological markerstellusaboutplanthybridization? Ecology andSystematics 132–140. 72: 987–998. 166: Glycinetomentella nrDNA internaltranscribedspacerhomeologueevolutioninthe Ecology the Glycine tomentella Phytologia Solidaster 4: 485–501. 142: 1 2691–2702. 11: (lmaiaee (Plumbaginaceae). vol. 20, Asteraceae, Part2. Astereae andSenecioneae,eds. ). ). Lessingia The MichiganBotanist R2R3 Myb 2 6813–6817. 92: 5 1–44. 75: 2 213–241. 12: c.v. Lemore, ahybridgoldenrod (Asteraceae) . 6 168–183. 26: (Leguminosae)allopolyploidcomplex . Bioinformatics (Compositae, Astereae) basedonnuclearrDNA Genetics (Leguminosae)polyploidcomplex . genes:sequenceanalysisreveals amplification 8 359–389. 28: Trends inEcology&Evolution Systematic Biology Paeonia Annals ofBotany 5: 427–444. 153: Evolutionary Trends inPlants Solidago Proceedings oftheNational Academyof Sida Molecular PhylogeneticsandEvolution 4 817–818. 14: Rare plantsofNewYork state. 8 31–35. 18: ) usinginternaltranscribedspacer Molecular Ecology Molecular EcologyNotes Solidago 1 755–757. 21: . p 0 6 n in 107–166 Pp. . : 7–18. 2: 9 422–434. 49: sect. 3 189–200. 93: Solidago Systematic Biology Journal ofTheoretical Ptarmicoidei 1 1703– 1715. 11: Annual Reviewof and 6 37–45. 16: Critical Reviews Flora ofNorth : 65–84. 5: Oligoneuron : 76–78. 1: Molecular Heredity Bulletin Genetics Solidago , anew Journal 53:

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 ntd tts ih n Wllf Srie UFS . 18 nagrd and Endangered 1988. (USFWS). Service Wildlife and Fish States United Topo, . . T J Gbo , . lwik F Jamui , n D G. D. and Jeanmougin, F. Plewniak, F. Gibson, J. T. D., J. Thompson, Tmltn A R, . . rnal ad . . ig . 19 caitc analysis cladistic A 1992. Sing. F. C. and Crandall, A. K. R., A. Templeton, for primers Universal 1991. Bouvet. J. and Pautou, G. Gielly , L. P., Taberlet, (*and parsimony using analysis Phylogenetic PAUP*: 1998. L. Phylogenetic D. Swofford, 1994. Hulbert. H. S. and Liang, H. G. Skinner , Z. D. Y., Sun, Sla, . 00. Peoyi patct fr ln dvlpet function development, plant for plasticity Phenotypic 2000. E. S. Sultan, Sli, . . n D E Sli 00. Te oe f eei ad eoi attri- genomic and genetic of role The 2000. Soltis. E. D. and S. P. Soltis, Sli, . . P S Sli , . . ole , n M L Egro 91. Chloroplast 1991. Edgerton. L. M. and Collier , G. T. Soltis, S. P. E., D. Soltis, Sli, . . E V Mvoiv J J Dye J Ruce , n P S Sli 2008. Soltis. S. P. and Rauscher , J. Doyle, J. J. Mavrodiev , V. E. E., D. Soltis, Sli, . . n P S Sli 99. Plpod: eurn frain and formation recurrent Polyploidy: 1999. Soltis. S. P. and nature E. dynamic the D. and Soltis, data Molecular 1993. Soltis. S. P. and E. D. Soltis, Sli, . . n P S Sli 99. Alplpod pcain n in speciation Allopolyploid 1989. Soltis. S. P. and E. Review Johnson D. S. Soltis, S. L. 2004. Wendel . F. J. and Cronn , C. R. L., R. Small, Sot, . A Cpii , . efe , . uk , n M Lsox . 2006. Lascoux. M. and Hurka, H. Neuffer , B. Ceplitis, A. T., Slotte, Smos M P ad . coeea . 20 as s hrces n sequence- in characters as Gaps 2000. Ochoterena . H. and P. M. Simmons, log-like- of comparisons Multiple 1999. Hasegawa. M. and H. Shimodaira, 01 LUEOADBRMN RGNO OIAOHUHOI 225 of goldenrods The 1999. Zhang. LAURETO AND BARKMAN:ORIGINOFSOLIDAGOHOUGHTONII J. J. and Ringius, S. G. C., J. Semple, Chromosome 1984. Morton. G. and Leeder , C. Ringius, S. G. C., J. Semple, 2011] ntd tts ih n Wllf Srie UFS . 19 eoey ln for Plan Recovery 1997. (USFWS). Service Wildlife and Fish States United Wlae R S ad . . asn . 19 N eiec fr utpe origins multiple for evidence DNA 1995. Jansen . K. R. and S. R. Wallace, Wlae L E. 20 oeua eiec fr looyli seito and speciation allopolyploid for evidence Molecular 2003. E. L. Wallace, Vjebr, . T H M Ms ad . ahan . 19 hools DNA Chloroplast 1999 . Bachmann. 2003. K. and Talbert . Mes, E. M. H. L. T. and K., Vijverberg, Sherman, D. J. Blake, K. N. A., Vanichanon, eval- Phylogenetic 2003. Karaman. V. and Roberts, P. R. E., L. Urbatsch, for threatened wildlifeandplants; determinationofthreatened status Hgis . 19 h CUTLX idw itrae feil strate- NucleicAcidsResearch flexible interface: windows gies formultiplesequencealignmentaidedbyqualityanalysistools CLUSTAL_X . The 1997. Higgins. siain. estimation . tion endonucleasemappingandDNA sequencedata.III.Cladogram of thephenotypicassociationswithhaplotypesinferred from restric- Molecular Biology amplification ofthree non-codingregions ofchloroplast DNA . other methods) , version4.0 Sunderland : Sinauer Associates, Inc . of nuclearribosomalDNA . analysis of and lifehistory . of SciencesUSA butes inthesuccessofpolyploids . AmericanJournalofBotany (Saxifragaceae): evidenceforchloroplast transferandparaphyly . DNA variationwithinandamonggeneraofthe lyploids andhybrids . ITS andETSsequence dataandphylogenyreconstruction inallopo- genome evolution . of polyploidy . insights from chloroplast DNA . AustralianSystematicBotany No. 2:Useofnucleargenesforphylogenyreconstruction inplants . sequences . the tetraploid Intrageneric phylogenyof based phylogeneticanalyses . and Evolution lihoods withapplicationstophylogeneticinference . Biology Series Ontario: Brittonia Astereae). II. Additional countswithcommentsoncytogeography . numbers ofgoldenrods, Houghton’s Goldenrod Register Systematics andEvolution of intergeneric allopolyploidsinannual Journal ofPlantSciences recurrent origins in mrc America . and NewZealandafterlong-distance dispersalfrom western North evidence fortheevolutionof Applied Genetics Multiple originsofallopolyploid Journal ofBotany Astereae) basedonETS and ITSnrDNA sequencedata . uation of Minnesota. Solidago houghtonii 3 27134–27137. 53: 6 280–292. 36: Solidago American JournalofBotany Xylothamia, Gundlachia Sorghum American JournalofBotany Genetics 9 1–90. 39: 6 1114 –1116 . 16: C. bursa-pastoris Critical ReviewsinPlantSciences 7 7051–7057. 97: 0: 804–810. 106: Trends inPlantScience 0 634–649. 90: 7 1105 –1109 . 17: L.and andrelated taxausinginternaltranscribedspacers Trends inEcology&Evolution 3: 619–633. 132: Platanthera huronensis 5 4876–4882. 25: Systematic Botany (Houghton’sgoldenrod). FinalRule . 6: 907–916. 164:

9: 253–265. 198: ( Euthamia Solidago houghtonii 8 1091–1112 . 78: Theoretical andAppliedGenetics Capsella 7 145–170. 17: Euthamia basedonchloroplast andnuclearDNA Systematic Biology American JournalofBotany Microseris , andrelated genera(Asteraceae, Nt d 3. 3. Ed. Nutt. Proceedings oftheNationalAcademy Aegilops triuncialis (Brassicaceae)andtheoriginof 6 1448–1463. 86: 3 1714–1724. 93: : 537–542. 5: and 3 7–20. 33: Microseris (rhdca). (Orchidaceae) . (Asteraceae)in Australia A. Gray) . Fort Snelling , Solidago 2 243–273. 12: 9 369–381. 49: University ofWaterloo 4 348–352. 14: (seaee (Asteraceae) . Molecular Biology Heuchera . (Compositae: Theoretical and 6 1119 –1124 . 76: International 9 26–32. 89: Tragopogon American group Federal Plant Plant

: rugosa HQ142471, HQ142542,HQ142506; souriensis HQ142584, HQ142455,HQ142490, HQ142560,HQ142525; juncea HQ142492, HQ142562,HQ142527; ULG003, Michigan:KalamazooCo.,HQ142397,HQ142587,HQ142457, AF477732, AF477668, —,—; HQ142456, HQ142491,HQ142561,HQ142526; rens or plant(collectionlocalitynotavailable). with anasterisk(voucherinformationnotavailable).†=purchased seed for whichpreviously publishedGenBankaccessionswere usedare noted deposited atWMUunlessotherwisenoted.PJL =PamelaJ.Laureto. Taxa and GenBankaccessionnumbers(3 HQ142444, HQ142479, HQ142549,HQ142514; altissima HQ142523; LaSalle Co.,HQ142399,HQ142589, HQ142453,HQ142488,HQ142558, Solidagoulmifolia Co., HQ142398,HQ142588,HQ142452, HQ142487,HQ142557,HQ142522; Solidago HQ142437, HQ142472,HQ142542,HQ142507; Aiton, trnH HQ142473, HQ142543,HQ142508; SIM003, Michigan:LeelanauCo.,HQ142386,HQ142576,HQ142438, Solidagosimplex HQ142540, HQ142505; bicolor HQ142474, HQ142544,HQ142509; petiolaris and GenBankaccessionnumbers(3 followed byvouchernumber, DNA accessionnumber, collectionlocality, section andsubsectionfollowing Semple andCook(2006) . Taxon nameis Yug S M. 19 1996. M. nucle- S. Detecting Young, 2000. Meinke. J. R. and Gisler , S. Liston, A. J., Whittall, direct and Amplification 1990. Taylor . J. and Lee, S. Bruns , T. J., T. White, ribosomal unusual An 1995b. Seelanan. T. and Schnabel, A. F., J. Wendel, into window incongruence: Phylogenetic 1998. Doyle. J. J. and F. J. Wendel, polyploids. in evolution Genome 2000. F . J. Wendel, Solidagopatula Co., HQ142393,HQ142583,HQ142454,HQ142489,HQ142559,HQ142524; patula HQ142477, HQ142547,HQ142512; PJL1171 HQ142441, HQ142476,HQ142546,HQ142511; HQ142545, HQ142510; Michigan: Crawford Co.,HQ142388,HQ142578,HQ142440,HQ142475, Zag J. 19 1996. J. Zhang, World Old contain cottons tetraploid World New 1989. F . J. Wendel, L. var. HQ142548, HQ142513; SPC001, Michigan:Crawford Co.,HQ142580,HQ142443,HQ142478, Solidago Appendix , , , and Sidalcea otide additivityfrom direct sequencesisaSNAP:anexamplefrom California: Diego, San White. T. and Press . Sninsky , Academic J. Gelfand, D. Innis, M. in 315–322 sequencing offungalribosomalRNA genesforphylogenetics . Pp. 298–313. intergenomic introgression . DNA sequencefrom Kluwer . Boston: systematics ofplantsII genome historyandmolecularevolution . Pp. 265 – 296 in 225–249. 42: 4132–4136. sis. related genera(Asteraceae:Astereae) basedonchloroplast DNA RFLPanaly- of Environmental Conservation. New York NaturalHeritageProgram. Latham , New York : Department cytoplasm . PJL1181 Muhl. exWilld. subsp. Atn Aiton, L.†, Millersubsp. caesia PJL1194 , FLX001,Illinois:LaSalleCo.,HQ142392,HQ142582,HQ142442, , , Ph. D.thesis . Waterloo, Ontario,Canada : University ofWaterloo . Atn, Aiton†, subsect. Nutt.†, PJL1175 PJL1206 rps16-trnQ sect. , , Solidago (avca). (Malvaceae). 1 1. Species of , SEMP01,Michigan:Washtenaw Co.,HQ142396,HQ142586, PJL1170 A molecularbiosystematic studyonNorthAmerican Muhl.exWilld.*, EU125376,EU125355, —,—; , MUL002,Colorado:Jefferson Co.,HQ142385,HQ142575, PJL1097 Kunth(A.Gray)var. Muhl.exWilld. var. Proceedings oftheNationalAcademySciencesUSA PCR Protocols: A guidetomethodsandapplications , ALT002, Michigan:KentCo.,HQ142400, HQ142590, Solidago PJL1207 , BCL001,HQ142389,HQ142579,HQ142435,HQ142470, PJL1216 Maritimae New York naturalheritageprogram rare plantstatuslist. , CES005, Michigan:KentCo.,HQ142391,HQ142581, rugosa subsect. ). FornrDNA clonedaccessionstheclonenumber , JUN001,Michigan:Crawford Co.,HQ142394, Solidago Solidago hispida Solidago speciosa Plant Biology , PET001,HQ142387,HQ142577,HQ142439, Gossypium gossypioides , MIS001,HQ142395,HQ142585,HQ142436, , d. D E Sli , . . ots ad . . Doyle. J. J. and Soltis, S. P. Soltis, E. D. eds. , var. subsect. Solidago . . Triplinerviae patula Solidago sempervirens rugosa subsect. Molecular PhylogeneticsandEvolution Solidago Solidago includedinthisstudyare listedby Solidago ′ Solidago Solidago ′ ETS, ITS, ETS, ITS)follows. All vouchersare Multiradiatae , , ulmifolia , , Muhl.exWilld., : 211 –217. 2: PJL1040 Solidago uliginosa PJL0908 gillmanii Nutt.var. — Glomeruliflorae subsect. subsect. . . =sequencenotobtained. subsect. subsect. subsect. , , Solidago altissima Solidago , PAT001, Michigan:Kent accD-psaI PJL1172 , RGS001,Michigan:Kent Solidago canadensis Solidago sempervirens reveals ancient,cryptic, G.S.Nesom, Solidago flexicaulis Plant MolecularBiology . . Thyrsiflorae L.subsp. Solidago multiradiata Squarrosae , ULM001,Illinois: subsect. speciosa Venosae Argutae , , Junceae PJL1119 Nutt., trnL-trnF . . Solidago caesia Solidago mis- Solidago , , L.subsp...... Molecular , HIS002, , Humiles . sempervi- . . PJL0154

PJL1138 PJL1173 Solidago Solidago Solidago Solidago Solidago L.var. , , , eds. , psbA- L.*, L., 86: and and 4:

, . , ,

Copyright (c) American Society for Plant Taxonomists. All rights reserved. Delivered by Ingenta to IP: 192.168.39.151 on: Tue, 28 Sep 2021 22:00:33 HQ142422, HQ142612,cloneHOU015-#3,HQ142423,HQ142613; HQ142530; cloneHOU015-#1,HQ142421,HQ142611; cloneHOU015-#2, HOU015, New York: GeneseeCo.,—,HQ142460,HQ142495,HQ142565, HQ142420, HQ142610; HQ142608; cloneHOU008-#2,HQ142419,HQ142609;HOU008-#3, HQ142494, HQ142564,HQ142529;cloneHOU008-#1,HQ142418, PJL1211 HOU002-#4, HQ142417,HQ142607; HQ142415, HQ142605;cloneHOU002-#3,HQ142416,HQ142606; HQ142528; cloneHOU002-#1,HQ142414,HQ142604;HOU002-#2, Michigan: CheboyganCo.,—,HQ142458,HQ142493,HQ142563, Ptarmicoidei HQ142597, HQ142451,HQ142486,HQ142556,HQ142521; nica HQ142485, HQ142555,HQ142520; PJL1045 Solidago HQ142519; Genesee Co.,HQ142405,HQ142595,HQ142449,HQ142484,HQ142554, HQ142553, HQ142518; Canada, StrawberryIs.,HQ142404,HQ142594,HQ142448,HQ142483, HQ142552, HQ142517; Grand Traverse Co.,HQ142403,HQ142593,HQ142447, HQ142482, HQ142551, HQ142516; GIG002, Michigan:KentCo.,HQ142402,HQ142592,HQ142446,HQ142481, AF477729, AF477665, —,—; HQ142445, HQ142480,HQ142550,HQ142515; 2 SSEAI OAY [Volume 36 SYSTEMATIC BOTANY canadensis 226 (Nutt.)Semple, , HOU008,OntarioCanada:Bruce Peninsula,—,HQ142459, , NEM001,Michigan:KentCo.,HQ142406,HQ142596,HQ142450, subsect. , , PJL1176 Solidago gigantea . . Solidago houghtonii , CAN003,Michigan:KentCo.,HQ142401,HQ142591, Nemorales PJL1185 Solidago gigantea Solidago gigantea Solidago gigantea Solidago houghtonii Aiton*, EU125379,EU125362,—,—; , CAL001,California: Yolo Co.,HQ142407, . . Solidago nemoralis T. &G.ex A. Gray, Solidago velutina Solidago houghtonii io, Aiton, Atn Aiton, Solidago gigantea Atn Aiton, T. &G.ex A. Gray, PJL1178 PJL1180 PJL1179 Aiton subsp. Solidago canadensis D.C.subsp. , GIG005,New York: , GIG003,Michigan: T. &G. ex A. Gray, PJL1102 , GIG004,Ontario Atn Aiton, Solidago , HOU002, , nemoralis PJL1183 PJL1177 califor- sect. L.*, , , , HQ142567, HQ142532; NIT001, Texas: Bastrop Co.,HQ142408,HQ142598,HQ142462,HQ142497, HOU060-#6, HQ142430,HQ142620; HQ142429, HQ142619;cloneHOU060-#5,HQ142431,HQ142621; HQ142617; cloneHOU060-#3,HQ142428,HQ142618;HOU060-#4, clone HOU060-#1,HQ142426,HQ142616;HOU060-#2,HQ142427, Crawford Co.,—,HQ142461,HQ142496,HQ142566,HQ142531; Solidagohoughtonii HOU015-#4, HQ142424,HQ142614;cloneHOU-#5,HQ142425,HQ142615; HQ142533; Delta Co.,HQ142409,HQ142599,HQ142463,HQ142498,HQ142568, —, —; Iosco Co.,—,HQ142464,HQ142499,HQ142569,HQ142534;clone1, —; Q454 HQ142539. HQ142574, Michigan: Crawford Co.,HQ142434,HQ142624,HQ142469,HQ142504, HQ142573, HQ142538; California: MonoCo.,HQ142433,HQ142623,HQ142468,HQ142503, HQ142571, HQ142536; Michigan: KalamazooCo.,HQ142413,HQ142603,HQ142466,HQ142501, HQ142465, HQ142500,HQ142570,HQ142535; Frank, HQ142410, HQ142600;clone2,HQ142411, HQ142601; nauseosa HQ142622, HQ142467,HQ142502,HQ142572,HQ142537; —; EU125371, —,—; EU125387, EU125370,—,—; Solidago ptarmicoides Heterotheca villosa PJL1164 (PallasexPursh)G.L.Nesom&I.Baird, Solidago ohioensis , RID005,Michigan:HillsdaleCo.,HQ142412,HQ142602, Solidago ohioensis T. &G.ex A. Gray, (Pursh)Shinners†, Solidago rigida Euthamia graminifolia (T. &G.)B.Boivin, Solidago nitida Solidago rigida Riddell*,EU125386,EU125369,—, Riddell, L.*, AY292528, AY523851, —, Solidago riddellii Solidago nitida T. &G.*,EU125383,EU125367, L.subsp. PJL1217 PJL1117 (L.)Nutt., PJL1153 PJL1127 Solidago riddellii rigida , HOU060,Michigan: , HET001,HQ142432, , OHI005,Michigan: , PTR011, Michigan: T. &G., Frank*, EU125388, PJL1186 , , PJL1118 PJL1112 Solidago riddellii , THAM01, , , EUTH01, , Ericameria , RIG002, , Frank*, PJL1199 ,