Botanical Studies (2010) 51: 217-222. GENETIC DIVERSITY Genetic diversity of Balanophora fungosa and its conservation in Taiwan Shu-ChuanHSIAO*,Wei-TingHUANG,andMaw-SunLIN Department of Life Sciences, National Chung-Hsing University, 250 Kuo-Kuang Road, Taichung 40227, Taiwan (ReceivedOctober11,2007;AcceptedFebruary26,2010) ABSTRACT. Balanophora fungosa is a rare holoparasitic flowering plant in Taiwan, where it is restricted totheHengchunPeninsulainsouthernmostTaiwan,andOrchidIsland(LanyuinChinese),asmallvolcanic islandoffthesoutheasterncoastofTaiwan.Plantsfromthesetwoareasappearintwodifferentgroups basedonthecoloroftheinflorescence,i.e.,thoseofHengchunareyellow,buttheyarepinkishorangeto redonOrchidI.Thisstudyusedaninter-simplesequencerepeat(ISSR)molecularmarkerapproachandthe unweightedpairgroupmethodwitharithmeticmean(UPGMA)analysistoevaluategeneticvariationsamong populationsof B. fungosa.Theresultsshowedthatthetwogeographicalgroupsrepresentthesamespecies as indicated by a high Dice similarity value of 0.78. Populations from the two areas formed two well-defined clusters,asdidpopulationswithineacharea.Theresultsoftheanalysisofmolecularvariance(AMOVA) showedthatthecomponentsofvariationbetweengroups(31.35%),amongpopulationswithingroups (13.74%),andwithinpopulations(54.91%)weresignificant(p<0.001),indicatingthatvariationsamong individualswithinpopulationscontributedmosttothetotalgeneticvariance.Thepopulationsofthetwoareas werealsodifferentiatedwithgeneticdistancesrangingfrom0.44~0.53forpairedcomparisons.Therefore, werecommendthatprotectedareasbesetasideinbothareasfor B. fungosa,thatis,inKentingParkon theHengchunPeninsulaandonOrchidIsland,toallowthepopulationtoexpandnaturallywithouthuman disturbance. Keywords: Balanophora fungosa;Conservation;Populationvariation. Abbreviations:AMOVA,analysisofmolecularvariance;ISSR,inter-simplesequencerepeat;UPGMA, unweightedpairgroupmethodwitharithmeticmean. INTRODUCTION populationismorewidelydistributedwithmanysmall patchesbecauseitgrowsinaprotectedresearchsiteat Balanophora fungosa J. R. & G. Forst. is a thepark.However,veryfewpatcheswerelocatedinthe holoparasiticplantwhichgrowsonrootsofvarioushost latterareaduetotheeffectsofagricultureandtourism.On plants.ThisspeciesisfoundinIndia,EastMalaysia, OrchidI.,despiteless-intensivehumandisturbance,the Taiwan,thePacificIslands,andnortheasternAustralia plant’sdistributionislimitedtoafewisolatedpatches. (Hansen,1972;HuangandHuang,1996).InTaiwan,the TheB. fungosaplantiscomposedofanunderground distributionofthisspeciesisrestrictedtotheHengchun tuberwhichattachestohostroots,andmonoecious PeninsulainsouthernmostTaiwanandalsoonOrchid inflorescences which are only visible during the flowering Island(LanyuinChinese),asmallvolcanicisland90 season.Theinflorescenceisyellowtopinkish-orange, kmoffthesoutheasterncoastofTaiwan.Forsubsequent withpistillateflowerslocatedontheupperpartofthe analyses,populationsofB. fungosainTaiwanarereferred inflorescenceandstaminateflowersonthelowerpart toasgroupsintheHengchunareaandonOrchidI., (Figure1).ThepopulationsofHengchunaredistinguished respectively. from those on Orchid I. by the color of the inflorescence: Duetoitsrarity,B. fungosawasdesignatedavulnerable plantsintheHengchunareahaveyellowinflorescences plantinTaiwanbasedontheIUCN(1994)(LuandChiou, while those onOrchidI.arepinkish-orangetored. 1996). Our field observations also showed that population Therefore,itwouldbereasonabletoassumethatB. declinescouldbeamajorthreattoB. fungosainboth fungosapopulationsinTaiwancanbedividedintotwo areas.InHengchun,onlytwopopulationsofB. fungosa distinctgroupsbothmorphologicallyandgeographically. werefoundatKentingParkandatKuanshan.Theformer GeneticvariationsamongpopulationsofB. fungosa havenotbeenanalyzedbefore.Inthisstudy,inter-simple *Correspondingauthor:E-mail:[email protected];Tel: sequencerepeat(ISSR)molecularmarkerswereappliedto 886-4-22840416;Fax:886-4-22874740. estimategeneticvariationswithinthespeciesinTaiwan. 218 Botanical Studies, Vol. 51, 2010 Figure 1.Balanophora fungosa in Taiwan. A, Plants of the Hengchun area of southern Taiwan; B, plants of Orchid I. Bars = 1 cm. The ISSR technique has been widely used to study Genomic DNA was extracted using the modified CTAB populationvariationinplantswithoutbackgroundgenetic methodforplantscontaininghighpolysaccharideand informationbecauseofitshighnumberofpolymorphisms polyphenolcomponents(Porebskietal.,1997).Intotal, andthehighreproducibilityofitsbands(Culleyand 125ISSRprimers(OperonTechnologies,USA)were Wolfe,2001;Werneretal.,2003;Jianetal.,2004). initiallyusedtoscreenforpolymorphisms,andsevenof The aim of this study was to investigate genetic thesewerechosenfortheirdistinctbandpatternsandare variationsamongindividualsofB. fungosaofpopulations listed in Table 2. Extracted DNA solutions were quantified fromHengchunandOrchidI.inTaiwan.Furthermore,we underaspectrophotometeratawavelengthof260nm, proposesuitableconservationmeasuresforthespecies and were diluted to 25 ng/μl as the working solution for basedontheseresults. polymerasechainreaction(PCR)amplification.Each 25 μl of the PCR solution contained 175 μM dNTPs, 0.35 μM ISSR primer, 1 U Taq DNA polymerase MATERIAL AND METHODS (ProtechTechnologyEnterprise,Taiwan),and25ng PlantsofB. fungosawerecollectedfromtheHengchun oftemplateDNA.Amplificationwasperformedina areaandonOrchidI.Duetotherarityofthespecies, DNAthermocycler480(PerkinElmer,USA)underthe onlytwopopulationsweresampledineachofthetwo followingthermo-cycleconditions:5minat94°C;then regions(Table1).IntheHengchunarea,onepopulation 38cyclesof1minat94°C,30sofannealingat50°C,and wassampledatKentingandanotheratKuanshan.The a 2-min extension at 72°C; followed by a final extension habitatofbothpopulationsconsistsmostlyofuplifted stepof10minat72°C.Theamplifiedproductswere coralreefcoveredbyathinsoillayer,andtheseparasitic electrophoreticallyseparatedon2.5%agarosegelsin plantsweredistributedinpatchesonhostroots.To 0.5 × TBE buffer, and the band patterns were visualized by distinguishspecimensfromdifferenthostplants,we ethidiumbromidestaining. dividedthepopulationintoafewpatches,eachofwhich ThebandpatternsofallDNAsampleswerescoredas containedindividualsfromthesamehostplant.Oneto present(1)orabsent(0),andallweakandambiguousones fourindividualswerecollectedfromasinglepatch,and wereexcluded.Thedatawereusedtoobtainasimilarity anytwopatcheswereatleast2mapart.OnOrchidI., matrixbasedontheDiceformula(SAB=2NAB/(2NAB+ twopopulationswerefoundinnaturalthickets,butthe NA+NB)onNTSYS-pcvers.2.0(Rohlf,1993),whereNA populationsizesweresmall;oneconsistedoffewerthan isthenumberofbandspresentinsampleAbutabsentfrom fiveindividuals.Informationonallthesamplesislisted sample B, NB is the number of bands present in sample B inTable1.Sampledplantswerebroughttothelaboratory butabsentfromsampleA,andNABisthenumberofbands forfurthercleaning,andfreshbractsandslicesofthe presentinbothsamples(Dice,1945).Thissimilarity inflorescencestalkwerepreservedinliquidnitrogen matrix(S)wasusedtoconstructaUPGMAdendrogram, immediatelyafterbeingcutofffromtheplantforlater whichwasfurtherconvertedintoadistancematrix(D)for DNAextraction. analysisofmolecularvariance(AMOVA),inwhichD= HSIAO et al. — Genetic diversity of Balanophora fungosa 219 Table 1.SamplinginformationofBalanophora fungosa. Area Populationno. Patchno. Individualno.a Hostb Totalno.ofindividuals Hengchun 1 1 111,112,113 ▲ 18 (Kenting) 2 121,122,123,124 ▲ 3 131,132,133 ▲ 4 141,142,143,144 ▲ 5 151,152,153,154 ▲ 2 1 211,212,213,214 10 (Kuanshan) 2 221,222,223,224 3 231,232 OrchidI. 3 1 311 ? 10 (Hsiaotienchih1) 2 321 ? 3 331,332 ? 4 341,342,343 ? 5 351,352,353 ? 4 1 411,412 ? 2 (Hsiaotienchih2) aIndividualswerelabeledbytheorderoftheirpopulationandpatchwithathree-numbercode;e.g.,individual123wasthethird plant(3)collectedinpatch2(2)oftheKentingpopulationintheHengchunarea(1). b▲, Host was Diospyros philippensis;,Macaranga tanarius;?,hostunknown. n (1-S), where n is the total number of bands (Excoffier et Allsamplesof B. fungosaformedtwowell-defined al.,1992).Thedistancematrixwasusedtocalculate(1) clusters,withaDicesimilarityof0.78(Figure2).This thevariancecomponentsforvariationsbetweenregions, resultfitsthegeographicaldifference(Hengchunand amongpopulations,andwithinpopulations;and(2) OrchidI.),andfurtherindicatesthatmorphological geneticdistancesbetweenpairsofpopulations,withthe differences in inflorescence colors, that is, yellow in the significance levels based on 9999 permutations. Hengchunareaandpinkish-orangeonOrchidI.,couldbe oneofthecharacteristicsinvolvedingeneticvariations RESULTS AND DISCUSSION betweenthetwoareas.Althoughtheresultisinagreement with Hansen’s (1972) opinion that coloration of B. Cluster analysis fungosashouldnotbeusedfordelimitingthespecies, ForB. fungosasamples,250bandsweregenerated thischaracteristicissodiscretethatitisworthfurther fromthesevenISSRprimers,and75.2%ofthebands studiesonaglobalscaletoinvestigateanycorrelationof werepolymorphic(Table2).Thebandingpatternswere colorationwithoverallgeneticvariation.Forexample, usedtogenerateaDicesimilaritymatrixwhichwasthen sinceplantsofB. fungosafromOrchidI.andtheRyukyu visualizedbyUPGMAclustering. Islands(ofsouthernJapan)arebothpinkish-orange (Hatusima,1971;Hansen,1972;KawakitaandKato, Table 2.SevenISSRprimersusedinthisstudy.Numbersofbandsandpolymorphiconesgeneratedfromeachoftheprimersare alsolisted. Primera Sequence(5’to3’) No.ofbands No.ofpolymorphicbands(%) AM5 GTGTGTGTGTGTYR 24 14(58.33) IS44 ACACACACACACACACG 30 19(63.33) IS50 AGAGAGAGAGAGAGAGYC 44 37(84.09) IS51 AGAGAGAGAGAGAGAGYG 44 36(81.82)