Botanical Studies (2006) 47: 191-197. PLANT BIOSYSTEMATICS Natural hybridization between Aster ageratoides var. scaberulus and Kalimeris indica (Asteraceae): evidence from morphology, karyotype, and ITS sequences Wei-PingLI* College of Life Sciences, Hunan Normal University, Changsha 410081, P. R. China (ReceivedApril14,2004;AcceptedNovember4,2005) ABSTRACT. AnewnaturalhybridfromtheHengshanMountaininHunanProvince,China,wasfound. Comparisonsofmorphology,karyotype,andITSsequencesamongthehybridanditsparentalspeciesshow thatthehybridrepresentsF1progenyfromhybridizationbetween Aster ageratoidesvar.scaberulus and Ka- limeris indica.Basedonmorphologicalandkaryotypicalobservationsofthehybrid,thisstudysuggeststhat naturalhybridizationbetweenAster and Kalimeriscouldhaveleadtotheshorteningofthepappusanda specialkaryotype(LS-type)andmakesthedelimitingofAsterand Kalimeris difficult. This study also indi- catesthatkaryotypeinvestigationsmaybeparticularlyusefulinresolvingsomeofthetaxonomicconfusion betweenAster and Kalimeris. Keywords:Aster;Kalimeris;Karyotype;Naturalhybridization;Pappus. INTRODUCTION analyses,Tarasuggestedthathybridizationhadoccurred betweenAster ovatus (Franch.etSav.)SoejimaetMot. Naturalhybridizationisafrequentphenomenonin Ito(A. ageratoidessubsp.ovatus (Franch.etSav.)Ka- plantsandplaysanimportantroleinplantevolution, tam.)andKalimeris incisa(Fisch.)DC.,andthatinfact leadinginatleastsomecasestoformationofnewspe- A. ovatuswasanamphidiploidthatoriginatedfollowing cies.Inaddition,hybridizationoftengeneratesconsider- intergenerichybridizationbetweenAsterandKalimeris. abletaxonomicconfusionbyfacilitatingintrogressionof However,manysubsequentauthorshaveoverlookedthe plant traits. Therefore, confirmation of the hybrid nature morphologicalcharactersofhybridsandthepotential andparentageisimportantinstudyingplantevolution, impactofintergenerichybridizationonthecomplexand andmayhelptoresolvecertaintaxonomicproblems. uncertainrelationshipbetweenthesetwogenera.This Unfortunately,manyhybridsdescribedinthebotanical studyprovidesthefirstreportofhybridizationbetween literature(e.g.HsiehandWang,1989;Chang,1991;Sun Aster ageratoidesvar.scaberulus (Miq)LingandKalim- andHuang,1994;Zhangetal.,1995;ZhangandZhao, eris indica (L.) Sch.-Bip. with verification of the parent- 1995;QianandSun,1998;Ren,1996;ZhouandWu, age.Thisstudydemonstratestheuseofkaryotypingand 2001;Zhangetal.,2003)weredeterminedbasedonlyon ofmolecularsequencingasameanstodetectinghybrids theirmorphologicalintermediacybetweentheirputative anddiscussestheimplicationoftheseresultsforthephy- parents.However,morphologicalintermediacymayresult logenyofAsterandKalimeris. fromcontinuousvariationandthehighplasticityofmany traits(Xu,1998;Craftetal.,2002).Inrecentyears,mo- MATERIALS AND METHODS leculardatahaveplayedanincreasingroleinthedetection andverificationofputativehybrids(Smithetal.,1996; Study site and materials MorrellandRieseberg,1998;Milne,1999;Durand,2002; Plantmaterialsforthisstudywerecollectedfrom DendaandYokota,2003). HengshanMountain,locatedinthemiddle-subtropical Tara (1972, 1977, 1978a, 1978b, 1979) first described monsoonzoneincentralHunanProvince,inChina,at intergenerichybridizationbetweenAsterandKalimeris 112°34’to112°44’Eand27°10’to27°20’N.Theputative (Asteraceae,Astereae,Asterinae).Basedoncytological hybridplantswerefoundatasunnydeforestedsiteneara subtropicalconiferousforestat300malt. Thesehybrids *Correspondingauthor:E-mail:[email protected];Tel: appearedtohaveleavesandinflorescencesintermedi- 86-731-8871052;Fax:86-731-8883310. atebetweentwosympatrictaxa,Aster ageratoidesvar. 192 Botanical Studies, Vol. 47, 2006 scaberulus andKalimerisindica,whichwerecommon automatedsequencer(PEAppliedBiosystems,Inc.).Prim- atthesite.Aster ageratoidesvar.laticorymbus(Vant.) erswerethesameasthoseoftheinitialPCR.Foreach Hand.-Mazz.alsooccursatHengshanMountain,butthe sample,bothforwardandreversesequencesofITS1,5.8S populationwasfoundtobeahexaploid(Li,unpublished) andITS2wereobtained.Eachbasepositionwasexamined andwasspatiallydistantfromtheputativehybrid,sug- foragreementbetweencomplementarystrands. gestingthatitcouldnotbeoneoftheparentsoftheputa- tivehybrid.Itisthusexcludedfromthisstudy. RESULTS Twelveputativehybridindividualswerecollectedand preparedasspecimensformorphological analyses,and Morphology rhizomesofthreeindividualsweretransplantedtoHunan Amorphologicalcomparisonamongthethreetaxais NormalUniversityforcytologicalandmolecularinvesti- summarizedinTable1.Thetwoputativeparentsdiffer gation.ThirtyplantseachofA. ageratoidesvar.scaberulus significantlyinall16characters.Bycontrast,thepre- andK. indicathatgrewneartheputativehybridswere sumedhybridshaveintermediatecharacterstatesineight preparedasspecimensformorphological analyses,and characters,includingdistributionofstomata,numberof five each were transplanted for cytological and molecular florets, size of achenes, and especially length of pappus. investigation. All specimens were identified according to ThehybridsresembleA. ageratoidesvar. scaberulus,in LingandChen(1985)andweredepositedasherbarium five characters, such as lack of stomata on upper leaves, vouchersatHunanNormalUniversity(HNNU). shapeofinnerphyllaries,widthofmiddlephyllaries,and lackofglandsonphyllariesandovaries,andresembleK. Methods indicaintheotherthree,includingnumberofbranches After preliminary observation, 16 morphologi- above, size of middle leaves, and color of ray florets. calcharacters—includingfeaturesofbranches,middle leaves,phyllaries,florets,andachenes—werefound Karyotype todifferbetweenthetwoputativeparentalspecies.All Themetaphasechromosomesandthekaryotypeofthe field collections were scored for these 16 characters. For threetaxaareshowninFigure1.Allthreetaxaaretetra- quantitativefeatures,suchassizeofmiddleleavesand ploid with 2n=4x=36. However, there are significant dif- numberofflorets,themeasurementsarepresentedasa ferencesinkaryotypeamongthem.All36chromosomes rangeofvariation. inA. ageratoidesvar.scaberulusareL-type(largechro- Actively growing root tips for chromosome mosomes), with the length varying from 2.51-4.35 μm observationswerecutfromlivingplants,pretreatedwith whileall36chromosomesofK. indicaareS-type(short 0.1%colchicineat8-12°Cfor4h,fixedinCarnoyI chromosomes), ranging in length from 1.4-1.93 μm. The (glacial acetic acid: 95% ethanol=1:3) at room temperature longestchromosomepairofK. indica,1.93inlength,is for12h,thenmaceratedin1mol/Lhydrochloricacid shorterthantheshortestpairofA. ageratoidesvar.scab- at60°Cfor8min,stainedin5%NH4Fe(SO4)2,atroom erulus.Theputativehybridisintermediateinkaryotype, temperaturefor3h,stainedin0.75%hematoxylinfor with 18 L-type chromosomes (2.49-4.1 μm) and 18 S-type 3h,washedindistilledwaterfor30min,andfinally chromosomes (1.35-1.85 μm). depigmentedandsquashedin45%aceticacid. FoursamplesforITSanalysis,oneeachfromA. ag- ITS sequences eratoidesvar.scaberulus andK. indica,andtwo,respec- TheboundariesoftheITSregion(ITS1,5.8S,and tively,fromputativehybridindividuals wereobtained ITS2)weredeterminedbycomparisontopublishedITS fromplantscultivatedatHunanNormalUniversity.Total sequencesofAster amellusL.andKalimeris integrifolia genomicDNAwasisolatedfromfreshleaftissueusing (L.)Less(NoyesandRieseberg,1999).Thelengthof theCTABmethodofRogerandBendish(1988).The theITSsequenceofallthematerialsstudiedis630bp. twointernaltranscribedspacers(ITS-1,ITS-2)and5.8S ThesequencesofA. geratoidesvar.scaberulusandK. nrDNA were amplified using the primers “ITS1” (5´AGT indicadifferfromoneanotherat13sites(2%;Table2). CGTAACAAGGTTTCCGTAG3´)and“ITS4”(5´ Ateachofthese13sitesthesequencespectrumofthe TCCTCCGCTTATTGATATGC3´). The amplification putativehybridindividualsshowstwooverlappedbands reaction was performed in 50 μL volumes using 10-20 ng withthepeakabouthalfashighasthoseofneighboring of genomic DNA, 5 μl MgCl2 (25 mmol/L), 4 μl dNTP (2.5 sites,indicatingthattwodifferentbasepairsareateach mmol/L), 1 μl (6.25 pmol) of each primer, and 0.25 μl (5 ofthesesitesthatcorrespondwiththoseofA. geratoides U/μl) Taq DNA polymerase. The following temperature var.scaberulusandK. indica,respectively(Table2), and time profile was used in the amplification of the Poly- exhibitingacombinationoftheITSsequencesofthe meraseChainReaction(PCR):aninitialdenaturationstep twoputativeparentspecies.Theforwardandreverse of94ºCfor5min;36cyclesof94ºCfor1min,57ºCfor1 sequencesagreewitheachotherforcomplementary min, and 72ºC for 2 min, and a final extension of 10 min strands.The ITS sequences from the two hybrids at72ºC.TheamplifieddoublestrandedDNAfragments wereidentical. Therefore,theoverlappedbands,asa were purified and then directly sequenced on an ABI 377 polymorphism,canbeattributedtothefactthatthereare LI — Natural hybridization between A. ageratoides var. scaberulus and K. indica 193 Table 1.AmorphologicalcomparisonamongAster ageratoidesvar.scaberulus,Kalimeris indicaandtheirputativehybrid. Features Aster ageratoidesvar.scaberulus Theputativehybrid Kalimeris indica Branchesabove Noneorrarely1-3 Many(>5) Many(>5) Sizeofmiddleleaves(Length× 6-15×2-6 6-9×2-3.5 6-9.5×2-4 Width)(cm) Stomataontheuppersurfaceof None None Present,butrare middleleaves Sizeofleaveonbranches 3-8×1.4-3 2-4×0.5-1.5 1-2.5×0.5-1 (Length×Width)(cm) Lengthofpeduncles(cm) 0.3-2.1 0.3-9 1.2-10 No.oflayersofphyllaries 4-5 3-4 2-3 Shapeofinnerphyllaries Linearoblanceolate Linearoblanceolate Ovateintheupper2/3,and constrictedandlanceolatein thelower1/3 Glandsonmiddlephyllaries None None Present Widthofmiddlephyllaries 0.8-1 0.85-1 1.3-1.6 No. of florets per head 24-40 54-72 100-130 No. of ray florets