Hikobial4:55-702003 MOlecularphylo窪enyOftheGrimmiales(Musci)basedOnchlOroplast mcLsequencesI HIRoMITsuBoTA,YuMIAGENo,BELENEsTEBANEz,ＴＯＭＩoYAMAGucHIANDHIRoNoRIDEGucHI TsuBoTA，Ｈ､，AGENo，Ｙ､，EsTEBANEz，Ｂ，YAMAGucHI，Ｔ＆DEGucHI，Ｈ、2003． MolecularphylogenyoftheGrimmiales(Musci)basedonchloroplastr6cLsequences・ Hikobial4:55-70． FamilialcircumscriptionsoftheGrimmialesandtheirallies，includingthe Orimmiaceae，thePtychomitriaceaeandtheSeligeriaceae，havebeenrepeatedly discussedHerewepresentphylogeneticinferencesontheGrimmiaceaeandtheir ロ alliesbasedonmaximumlikelihoodanalysesofchloroplast76cLgenesequences・ Oursmdyshowsthat（１）theGrimmiaceae(s」at.)withtheinclusionofthegenera CtJ叩〕ﾉﾉosje/伽ａｎｄＰ〃c〃o〃か伽lismonophyletic;(2)Ｇ〃んo〃"伽hasaclose affinitytMrcjoaoftheDicranales;(3)theSeligeriaceaefblmadistinctcladesisterto theGrimmiaceae（s・lat)；（４）theGrimmiales（s」at.）withtheinclusionofthe SeligeriaceaearemonophyleticwhentheDrummondiaceae,theScouleriaceaeandthe Wardiaceaeareomitted;ａｎｄ(5)theDrummondiaceae-Scouleriaceaecladeappearsat thebasalpositionofthehaplolepidousmossgroupDicranidae，adjacenttothe Bryoxiphiaceae．SincethemonophylyoftheGrimmiales(s」at.)includingmembers oftheSeligeriaceaeiswellsupported,weproposeherethattheordercanbetreatedin awidesense,andincludesfamilieswhichhaveinｃｏｍｍｏｎｔｈｅＧ伽〃α/Mjgel･ja typeperistome、 H1、伽乃"Ｍα,Ｔｂ〃ｏ】'bwmg"cﾙﾉ＆ﾉﾌﾞﾉｧo"MDegz`chj,ＤＥｐｑ"ｍｅ"'ｑ/Ｄｊｏ/ogjcaノ 比je"Ｃｅ，Ｇｍａ"α/ｅ比/ioo／ｑ／Sbje"Ｃｅ,〃ｉｍｓｈｊ"ｚａＵ７ｍﾉeﾊﾉﾉﾘﾉ,ＫｔＪｇα"!(〕ﾉαｍα／-3-/， HjgZzsﾙﾉｰﾊjrosh〃α-sﾊﾉ,HIrosh〃α7398526,ノヒＷｎ ＹｊｌｍＭｇｅ"０，Depα"ｍｅ"／ｑ/Ｄｉｏ/OgjcaノＳｂ/e"Ｃｅ，ＦｔＪＭｌｌ〕ノｑ／Ｓｂｊｅ"Ｃｅ，脇、s/i〃α 肋加e'Fjlﾜﾉ,KtJgzJmJi〕′α"zα/-3-/,ノリｉｇａＷ－ｈ"ｏｓｈｊ"Ｔａ－ｓｈｊ,HIroSﾙ〃α739-8526,.ﾉﾋJpα〃 Be/6〃ＥＳ肋α"ez，Depα"α"Te"'０火Ｂｊｏ/oｇｊａ（Bor伽αZﾉ，Ｆａｃ"ﾉﾉα`ノ火Ｃｉｅ"ｃｊａｓ， 肋ⅣＣＭ/､１Ｍ伽"o"zα火MIC/rj仏280ｲ,α"/o6/α"ＣＯ,Mzc/ｒｊ仏Slpaj〃 TheorderGrimmialesisoneofthebasalmem- Orimmiaceae（asGrinunieae）andPtychomitria- bersofthehaplolepidousmosses，ａcconmodat- ceae（asPtychomitrieae）intheGrimmiales（as ingthetwofamiliesGrimmiaceaeandPtychomi- Grimmiaceae)inthefirsteditionof"Dienatiirli- triaceae・TheorderSeligeriales，accommodating chenPHanzenfamilien，，、Subsequently,Brotherｕｓ 品 thefamilySeligeriaceae，isalsooneofthebasal (1924-1925）transferredthePtychomitriaceaeto orders・Circumscriptionandtaxonomicposition thelsobryales、Nyholm（1954-1960)placedthe ofthefamiliesGrimmiaceae，Ptychomitriaceae， PtychomitriaceaeintheEubryales，inwhichare Seligeriaceaeandtheirallieshavebeenrepeat- includedthefamiliesprimarilywithadiplolepi- edlydiscussedAfterthefirstdescriptionofthe dousperistome:Bryaceae,Mniaceae,Aulacomnia- GrimmiaceaeasanindependentfamilybyArnott ceae，Meesiaceae，Timniaceae，Catoscopiaceae， (1825)，manyworkersvariouslyproposedtheir Bartramiaceae，Orthotrichaceae，andHedwigia- systemsontheGrimmiaceaeandtheirallies ceaeNoguchi（1954)discussedtherelationship (Tableｌ）Brotheruｓ（1901-1902）placedthe betweenPltW/io〃/伽加，Ｇ７加伽ａａｎｄＲａｃｏ〃ノー rj"mbasedonleafstrucmres，especiallyoma- lContributionfmmtheLaboratoryofPlantTaxonomy mentationofleafcells,ａｎｄｐｌａｃｅｄＰひc/io〃〃i"m andEcology,HiroshimaUniversity､ＮＳｅｒ・No.５２６． intheGrimmiaceae・Noguchi（1974)suggested 5６ ＨｉｋｏｂｉａＶｏｌｌ４,No.1，２００３ thatRaco伽かｊ"misanintennediategenusbe- PCRamplificationsofsegmentsfbr『bcLgene， tweｅｎＰｔＷｈｏｍかj"ｍａｎｄＧｒ加伽ａｏｎｔｈｅｂａｓｉｓ anddirectsequenceanalyseswerecarriedoutus- ｏｆｔｈｅｌｅａｆｃｅｌｌｓａｎｄｅｘothecialcelldevelopment、 ingstandardconditionsaccordingtothemanufac- Deguchi(1979)treatedP〃c〃o加伽"ｍａｓａｍｅｍ‐ turer'sinstructionsReactionswereperfbnnedin beroftheGrimmiaceaeemphasizingthemorpho- athennalcycler（ThermalCycler9600,Perkin logicalandanatomicalaffinityofcalyptra，leaf Elmer)withPCRamplificationkitsandsynthetic cellsandsetatwistingVitt(1984),andBuckand primers(TsubotaeM/200lb)．DirectDNAse‐ Goffinet（2000）treatedthePtychomitriaceaeas quenceanalysisofthePCRproductswasper- anindependentfamilyoftheGrimmiaceaelnre- lbnnedusingABIorAmershamkitsaccordingto centyears，severalmolecularphylogeneticworks themanufacturer1sinstructions・Thesequences relevanttothephylogenyofhaplolepidousmoss- wereelectrophoresedonautomatedsequencers eshavebeencarriedout(LaFarge＆coworkers (ABI373AandABIPmSM310),andanalyzed 2000,2002;GofTmete/α/､2001),showingthe onthedata-analysisSystems、Thesequencesob- monophylyoftheGrimmialesandSeligeriales， tainedinthepresentstudyweresubmittedtothe ａｌｔｈｏｕｇｈｔｈｅｎｕｍｂｅｒｓｏｆＯＴＵｓｆｂｒｔｈeseorders ＤＤＢＪ/ＥＭＢＬ/GenBanklnternationalNucleotide seemtobenotenoughtoclarifyanddiscusstheir SequenceDatabaseCollaborationDetailsand phylogeneticpositionswithinthesystem・ fUrtherinfbnnationfbrDNAextractingprotocols， Theohjectivesofthisstudywere:（１）tocon‐ amplificationprimers，PCRprotocolsandthose structaphylogenetictreefbrtheGrimmiaceae， ofsequencingreactionsaredescribedpreviously theSeligeriaceaeandtheiralliesbasedonmaxi- inTsubotaandcoWorkers(1999,2000,2001a,ｂ） mumlikelihoodcriteria；（２）ｔｏｔｅｓｔｔｈｅｍｏｎｏ‐ Ｓｂ"'cesα"`Ｍ"αかze`、"α phylyofthefamiliesandclarifytheftlmilialand Dataset--9076cLgenesequencesfbr86moss genericrelationshipsWepresentberephylog- specieswereexaminedfbrtheanalyses(Table２， eneticinferencesfbrthefamiliespreviouslytreat- withＤＤＢＪ/EMBL/GenBankAccessionNos.） edintheGrimmialesandSeligeriales，basedon The76cLgenesequencesofthegrinmialeanand themaximum-likelihood(ＭＬ)analysesof90se- seligerialeanmossesweremanuallyalignedwith quencesofthechloroplastribulｏｓｅｌ,5-bisphos- ７１mosssequencesregisteredintheDNAdata- phatecarboxylase/oxygenaselargesubunit(『bcL)， base、Outgrouptaxawereselectedbasedonthe togetherwithrecentinfbnnationonthemolecular phylogenetichypothesispropｏｓｅｄｂｙＬａＦａｒｇｅｅ／ phylogenyofgrimmialeanmosses，anddiscuss α/､(2000)andTsubotaetal.(ｉｎｍｓ.,andtheab‐ theimplicationsofourfindingsontheGrimmia- stractoftheMOSymposium2003） ceaeandtheirrelatedfamilies． 』ﾉﾉg""?e"j-Manualalignmentwascarriedout ibrthedatasetagainstacompleteγbcLsequence Materialsandlnethods ofLe"co6M/"zsca6r"ｍ(ABO29388)withanedi- torprogramandouroriginalprogram，Sclean Phylogeneticanalysesbasedonther6cLgene 287（Tsubotal998-2003，inourhttpsites,see sequenceswereperfbnnedfbllowingthemethods AppendixB)．Undetenninedsites,gaps,andre‐ ofTsubotaandcoworkers（1999,2001a,2002)． gionsnotclearlyalignablefiDrallsequenceswere ② Themethodconsistsoftwosteps:（１）detennin- excludedfiomtheanalyses・Amongsequences ingsequencedata(DNAextractionPCRamplifl- aligned，identicalsequencesarerepresentedbya cationandDNAsequencing，orDNAdatabase singlesequence,andanunalignablesequencewas homologysearch);and(2)dataanalyses， omittedftomthedatasets． ＤＭ欧伽ajo",ＰＣＲ。〃/抗cα/ｊｏ〃α"`/Ｄ/V,４ A"αｌｂＭＭｍ"oqb Se9"e"cmg Treeswereconstructedbythefbllowingthree TotalDNAiswereextractedfi･omfiPeshsamples methods:theneighborjoining(NJ;Saitou＆Nei ordriedherbariumspecimensbymodificationsof l987)，themaximum-parsimony（ＭＰ；Fitch thｅＣＴＡＢｍethod(Murray＆Thompsonl98u l971),andthemaximum-likelihood(ＭＬ;Felsen- Arikawa＆Higuchil999;TsubotaeM2000)or steinl981)．Thetreesobtainedbythethree phenol-chlorofbrmmethod(TsubotaeM/1999)． methodswereappraisedbythelog-likelihood HTsuBoTA,Ｙ・ＡGENｏ,BEsTEBANEz,ＴＹＡＭＡＧｕｃＨＩＡＮＤＨ・DEGucHI 5７ measurewithNucMLinMOLPHY23b3(Ada- MJx〃"川ﾉﾉﾋcﾉﾉﾉjooaα"α加応一MLtreeswere chi＆Hasegawal996,seeAppendixB） constructedwithNucMＬｉｎＭＯＬＰＨＹａｎｄｆａｓｔ－ Ｄｊｓｊα"Ｃｅα"α11〕入,jJ-ANJtreewasconstructed ＤＮＡｍｌ1.2.2(Olsenetα/､1994)．InMLanaly- withNuｃＭＬａｎｄＮＪｄｉｓｔｉｎＭＯＬＰＨＹｕｓｉｎｇｔｈｅ ses，thebest-confbnnedtransition/transversion ＨＫY85model（Hasegawae/αＬ1985）fbrthe (Ts/ＴＶ）parameterwasestimatedbyNucML distanceestimations accordingtocalculationswiththeHKY85model PZJ芯加o'1Ｗ"αOA,js-MPtreeswereconstructed employingthe“-topt，,optionparameteras2､093 byPAUPRat(Sikes＆Lewis2001),whichisa (as4185intheNucMLnotationalsystem)．ML tooltoimplementtheParsimonyRatchetsearches treeswereconstructedwiththelocalrearrange- (Nixonl999)withPAUP*,overPAUP40blO mentsearchffomtｈｅＮＪｔｒｅｅｂｙＮｕｃＭＬａｎｄ (Swoffbrd2002)．hanalysesbyPAUPRat,MP ＮＪｄｉｓｔＩｎａｎａｌｙｓｅｓｂｙｆｔｌｓｔＤＮＡｍｌ，MLtrees treesweresearchedusingtheParsimonyRatchet weresearchedwiththeglobalrearrangements searchstrategyintwenty200iterationruns． optionsTheTs/Tｖｐａｒａｍｅｔｅｒｗａｓｔｈｅｎｕｓｅｄａｓ Table１．ＣｌassificationsoftheOrimmiaceae,PtychomitriaceaeandSeligeriaceae,orpartsoftheftlmily， andpossiblyrelatedgenera・Thetableshowsthedistributionoffamiliesandgenerathathavebeencon- sideredtobelongingtotheGrimmialesandSeligerialesatonetimeoranotherandincludedinthepresent analysis． Ｂｍｃｈ＆Schimper Schimper(1876）Limpricht(1885-1889）Brothems('901-1902）Fleischer(1915-1923） (1837-1846） Seligeriaceae Seligeriaceae Seligeriaceae Dicranaceae 、icranales 6MgUJ7a Seligerieae Stﾀﾉﾙg［?rm Seligerieae Seligeriaceae Bﾉﾉ"c/ｍ Sｾﾉ'9℃rm Bﾉﾉ"ぬａ 比｣I/gcr/α StWgw、 ＣｂｍＰﾘﾉﾉbsje"zｲ耐 Blindieae β〃7c/7ａ βﾉﾉ"cﾒﾉα B"、ﾉｶﾞﾋJ Campylosteliaceae OIthotrichaceae Brachyodonteae Bmcﾉ(〕ﾉﾋﾞﾉb""""１ Grimmiaceae Grimmiales P〃Ｃ〃ｏｍｊｗ""l Cmγ収ｿﾉbsだ"""７ ｃｍ"ﾉﾌﾘﾉﾉＯＳ雌ﾉﾉ""l Ptychomitrieae Grimmiaceae Gblp"o"""７２ｨ"’ G卯/､'''/かｊ"ｍ Sbo"ん'･ｍ ＣＤＳＣｊ"odb〃 Grimmiaceae Grimmiaceae ＰｌＤﾉcho'"ｊ"ｍｍ Ｃｂｓｃｊ"ocjb〃 Grimmieae Cinclidonteae ＥＵ《gOlMo""""'"７ Ｓと〃jSj減""’ Grimmiaceae Gr/"ｍｍＪ C/"cﾉﾉﾋﾉＷ"ｓ ｃｔJ"ﾘﾘﾉﾉnFje"""１ ０r/〃〃ね Sb〃ｊｓｊ城""ｊ Ｒａｃｏｍｊかｊ"m （Sbo"ん'ね） Scoulerieae Ｒ力αＣＯ"""７""Z Ｇ'･〃”ｊａ Ptychomitrieae Ｇｒｉｍｍｉｅａｅ SboMIerm Ｒａｃｏ"""７"'〃 CoFcj"ocﾉb〃 Ｓｂ"1F'jdh/"l Grimmieae Isobryales Ｐ〃c〃ｏｍｊ"7"m CQFc"Todb〃 Cosc/"ＯＬ/b〃 Erpodiaceae GOlpﾉｍｍｊ"７""’ Ｇﾉﾌﾞ"1"ｊｍ Ｇ'．ｊ"ｌ"Jliq EJipoa7""' Ｒａｃｏ〃"'７""l Grj"'""α 随"/"rje"b「 Ptychomitrieae Ｓｃ"ｌＷｊ的Ⅲ"ノ Orthotrichaceae Bmc伽花ﾙﾉｨ"’ Ｒｈａｃｏ"ﾉﾉ〃/""Ｉ G〃んo"'ｊ"７""， （PI(〕ﾉc力o""か/""7） Ｃ、"Pﾘﾉﾉbsr/e"Ⅲ"】 （GOlp/io"""72J"'） （Ch1”γﾉbs'e""腕） Dixon(1924） Brothems(1924-1925）Jones(1933)inGrout，NyhoIm('954-1960）Churchill(1981） ＆GrouMl936） Dicranaceae Dicranales Bryales Dicranales Grimmiaceae Seligerieae Seligeriaceae Seligeriaceae Seligeriaceae Grimmioideae Sb/jgU7m「 Sc/jg〃ね Scﾉigcrm Bﾉﾉ'7., GJ7"】"】ね「 Dicraneae β肋めα B""｡、 sｾﾉﾉgヅノα Ｓどんな'枕""７ B""ぬａ Ｂ'pcﾉiyzjb""z'"’ Coscinodontoideae Grimmiales