TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics ISSN 1364-7565 ActaPhytotax.Geebot.55 (1)1i9-28(2004) Geographic Distribution GeneticVariation Vulnerable and of a ConiferSpeciesP,iceakcu7amae(Pinaceae) TOSHIO KATSUKIi*, SUGAYA2, KEIKO KITAMURA3, TOSHIKAZU TAKEUCHI2,TAKMAASSHAIKI KATSUTA2 and HIROSHI YOSHIMARU3 i7ltma Fbres tScienc eGarden, Pbi?sir aynd 1;bre sPtroduc' tRsesearch institu tleb,klL ,1o93-0843, ,Lrpan; 2Department oj'Iib Srbeisentce f,iticu totfyRegien Eanlvironmen tStience ,7bkyo Lbeiversi otfy'Agricu l7tbktytore, 156-8502 ,.hrpan ;jDepartment offore Gsetnetics F,brestt )an?d FbrestProduct Rsesearch institut eJ,baraki 305- 8687,Japan Picea kq),ama eis a species which is distribut secadrcely and i sthreatened to extinction, though the details are not well knewn. We invcstigat tehde geographic adlistribut iaondn geneti cvariation ofthis species. Seven habita tosvcr the Yatsugatak meountains and Akaish imountains were confirmed in thi sstudy, and i twas estimated that in these areas combined there were les sthan 1,OOO livin mgature trees .Genetic divers iwittyhin and among 4 population wsas analyzed using allozymes. The mean genetic variation wLthin populations was in the range ofgeneral long-liv etdrce species. However, genetic variation in the Karamatsu-saw apopulation at Yatsugatake mountains showed a rather low value. It i sconceivable that thi spopulatio hnas experienced a bottlene cefkfect. The genetic differentia taniioonng 4 populatie nwsas found to be at an intennedia tlceve lbetween continuously distribut iconnigfers and discQntinuously dis- tributin gones. in thi sstudy, tLR shirasawae" was suggested to be mere]y a morphological variation with- in a populatio onfR kayamae, and was treated as R kayainae in our results. Key words: Akaishi mountains, allozymc, geneticdiversit yh,abita tP,ieea ko.Famae ,Picea shiTu- Yatsugatake sa}vae, mountains Picea koyamae Shiras .(Yatsugatake- tin6hireport on Red Data PIant s(Faij &o nPage 1999, Japanese) is a tall conifer tree species which is dis- Environmental Agency ofJapan 2000). tributcd in Honshu Islan dj,apan .In 191 1 i twas dis- The gcnus Picea is one ofthe most important covered in the Yatsugatake mountains area tree greups in Japancse flora to come out of the (Shirasa w&a Koyama 1913), Since then, R koj,a- Last Glacia Alge, and fbssi lclsassified int oR koya- mae has been believed to be distribut eondly on mae or "R shirasawae Hayashi "have been discov- Yatsugatake mountains (Faij o1n990, Yarnazaki ered in very wide area on Honshu Island (Minaki 1995). Currentl yt,here are les sthan 500 living 1987). In addition, more than 10 genus Picea mature trees in the Yatsugatake mountains area, species, inc]uding now-extinct ones, have bccn and is liste das "threatened wjth extinction" in a reported bascd on foss irlecords in Honshu CSuzuki "CQrresponding author: Toshio Katsuki; Phone/ 0426-61-1121.Fax:0426-6 ] -S261 E-mail :hatsuki(IUt7)ri.qfi}:,go,ip NII-Electronic Library Service TThhee JJaapapnaesneese SSooocieityety ffoorr PPllanatnt SSyystsetmaetmioastics 20 APG Vol. )⊃ 雪 2 薹 2 ・ 晝 三( Z。、 看 【象咽」雪G。 葱量壽.、 『壽宥. 覧爲蠹 鴾耄吻.マ着。α。ぎD Z8。。壽−、D 『自身、亀『量邑超、≦口筐、。鴛雪 莠尋邑 舞⊆謇 豸。島。D 袰2動 2旦。尾っ 堂国鼕 首玄目 豸o呂じ。 ン自墓2o毎じ。 的爵く〉 量卜。 ( 篝 演o 2致 . 己 〔 δ(自. 超窘 「岩脚鷺=o 顕幅岩 (まひ三焉鐔N邸.ま色驀舞N§璽弓コ萋)慧耄=K。弓§。 7§、・ミ、ミq ヨ爵ミ。箆ミ・導窪劬・ 三゜冒誌篭§§「へおζ§耄り. 翌嬬←=冨 箆Σ(も量も゜」ご謎ミ 。..且三善Z【5。 三倉箏Z【冖〔Q。 」ヂ湯§鳴莓尾8。鳴」罨§・、δ冖曁遵ミ自益・うo.ゴ。ミミ。 =[鷲≧箕[冨£O巽篝壽・配δ。り。。旦石耄Z・。。 ヨδ(N塑留邑、丶ミ8口ミ. 气 气 气 へ へ 叫 气 勹 烏 ( 〔 ( )象ひ一 ・ 疂呉 蠹σ・ 覧コし(Oま【)弖」三゜「コN冒邸り壽旨。。.、(Nひ蛮)刷緝胡ゴN お罷9(頑コ顰夢冒§气愚話§電. 受§宅藁曽電8 藷鵡三巴 ミ毳遷震・邸」ひN』恙旨〉署ミミ。,.. 【7冖のトづN畳口.藷略三゜)賢耄慧気(看§藝ミq。。, 聲昌山口易 Σ(うもし』)ミミ浅軋耋日。..且ε℃Z口。沼。 且琶咢Z口。ω。 毛♂萱し喫『壽き9面き§魯。。.葛髦鴇蝿慝気ΦN.きミミ ヨ=琶鴦曇嘱气・冂詫屮§§§賄h将冫きミ§ミ。、、且唇℃Z霧沼。 髪三2書受邑ミ。。§・ミ. , 粤 ( で[ 旨軋辺=踴衷∪【紐萄β。』鳴§。口。8・ヨ差。,.ε象一言魯謡 居三δ【鵞』届耄〔一タ)鬟×りuミ寒、, て耄§ド。§ 藷幡三゜三、ミ耄慧篷鼠の邸」聖、弼耄曇ミ嵩9)。. 』唱山彗o。。三発(2彪゜)毳耄団籌防し“』耄妻ミぎ。・., も眉函冒 Σ(葦弓゜巴ミ§麓日り靴..母慧Zぴ 毳隔§へ、富髱Z2考3∀鬟 ぴ§逆×」°§§い物§へ≧鼕・、,.、言§三°」夕唇x。Q−。2D 圭のセミ灘§魯.Σ2曽、程遠葱×。。き、§ミQ.。 暑嵒鰐庸鎚ミ誤§§§」霎き蓬ミ.ρ名ε日ミ 墅o.務 畧蛍躍図邑§。8§. 9量三鷺Σ憙爵雪=当髭・・日・・・・:。.. 蔘=婁零当8。・騒。ヒリ島。」の・.3量のお琶=ξ耄。。ω8。 蠶・ω§:・。g 霧ηモ帽莞ε℃=8崙詈。。』。〉。目り. 『 『 『 『 气 气 曙 曙 軋 配 凝 激 卜 一 NNI工I工-EElleoetcrotniroonic LLiibrbarryary Service TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics June2004 KATSUKI et al : Genetic variations ofPicea kovamae 21 1991, LePage 2001) ,though the detail osf their cally distinguishe dfrom R kqyamae by longe rcone phylogenet ireclationships are unknown. length (8-1 2cm), longe rleaflcng t(h13-18 mm) There are several problems with the classifi- and branchle tcoslor (orange-b irn pouwbnescence) cation of Picea koyamae and it ssibling species, (Hayas h1i960a), However, Shimizu (1992 p)ointed Areund the Japan Sea, six or sevcn species classified out that there was not a differen coteher than the into Sect .Picea have been reported by Fac]'on fbrm and size of cone between those two taxa. In (1990 S)h,imizu (199 2an)d Yamazaki (1995 ()[Ilabalddeition, Kobayash iet al. (200 0re)ported a total of l, Fig .1) .Some botanis thsave pointe dout that R about 2500 bases of nucleotide sequences in fbur kqyamae, R koraiensis ,and R obovata are very regiens of chloroplast DNA fb rone R koyamae similar (Wils o1n916, Wright 1955) ,In panicular,individu aanld one C`R shirasaivae" individua alnd, there was a case that R koraiens iwsas included thcir sequences were identic awlith each other with R koyamae as onc species (Fu et aL 197g), though they showed some differenc efrom the though no detaile edxamination had been made. sequences of other fbur Japanese Sect. Pieea species. This might support the classification of Shimizu(1992). E12o" Elso` In this study, we confirmed the present geo- Nso graphica dlistribut ioofPnicea koyamae and ana- lyzed their genetic variation using allozymes (enzym epolymorphisms )without distinguishing `・R shirasa}vae" as a separate species according to the classfication by Shimizu Ci989 ,1992). Materials Methods and Plant materials N30" Based on descriptio nisn past reports (Hayashi l960a,Yamazaki 1965, Katsuki & Seido 1999), FTG 1 . Distributi oofn Sect .Ptcea specics around the Japan Sea we considered Picea kqyamae to be geographicalEy T(h1c9 s9pe0ci)es. names are fb]lo-・c dby descriptio onfFarion distributed in the Yatsugatake mountains and Akaish imountains areas, Thcn, we confirmed their current habita tbsy means of fiel sdurvey and direct On the other hand ,"Picea shipusawae" (Hirne-examination to identif ythe species. matsuhada in Japanese h)as been sometimes treated After this survey, we studied the genetic vari- as an independen stpecies (Hayas h1i960b, Yama- ation in allozymes of the four populations (Fuki- zaki 1995) .Howeve4 many botanis thasve classified sawa and Karamatsu-saw apopulations in Yhtsuga- it as a variety ofR kayamae (Shimiz 1u9g9) or R take mountains; Ohdaira and Tenshi-iwa popula- aleoquiana (Faci 1o9n90) ,"Pieea, shiTusawae" was tions in Akaishi mountains) from which we had discovere idn the same geographical area in which obtained a sucacient number of samples of Picea R koyamae was discovere dan,d the discoverie sof kayamae individual s(Fi g2)., both were published in the same report (Shirasaw a For allozyme analysis, we collected young & Koyama 1913) ,`LPicea. shirasarvae" rescmbles R branche sofabout 30 individua lfrsom each ofthe kqyamae, and has been reported to be morphologi- four populatio nisn June. 2000. Collected branches NII-Electronic Library Service TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics 22 APG M)1,55 were stored at -80℃ until the enzymes were extract- Levels ofgenctic variation were calculated for each ed. of the population asccordjng to a number of para- meters including :the proportio onf polymoxphic Attoayme analysis loci (P )at the 95% criterion, the ayerage number of Procedures for enzyme extraction and polyacry- alleles per locu s(Na) t,he effective number of alle- larrii gdeel electrophoresis fbllowe dthose describedles pcr locus (Ne ;Kimura & Crow 1964), the in Tsumura et al, (1990 a)nd Tsumura & Ohba obscrved heterozygosit y(Ho) a,nd the expected (1993 e)xce,pt that 100 mg of young leaf tissue unbiased heterozygosi tuynder Hardy-Weinberg from each tree and 80 mg ofpolyvinyl-polypyrroli- equilibrium (He ;Nei 1978, Nei & Roychoudhury done were greund to a past ewith a mortar and pes- 1974). tle in 1 ml of extraction buffe r(1O OmM Tris-HCI F-statisti c(sFi s()Wrig h1t951) that represent pH 7.5 ,23.49, 6glycero ]O,.6% tween 80, l.2 mM the deviation of genotypi cproportion fsrom the dithiothrei Ot.o6l m,M NAD, O.5 mM NADR O.59i6 Hardy-Weinber gexpectation were calculated fbr 2-mercaptoethan oanld, O.08% albumin). polymorphic loci in each populatio nS.tatistical Eight deviationfsi'om enzyme systems were employed: analysis Qf such expectations was diaphoras (eDia E,C 1.6.4.3. )t,imiarat ehydratase made using the x2 test (L i& Horvitz 1953). (F} nEC, 4.2. 1.2.) ,glycerel-2-dehydroge (nCas2edh, Gene differentiati wointhin and among popu- EC 1.1.1.29 .g)lu,tamate dehydrogenas (eGcV iE,C lation swere calculated according to the fbllowing 1 .4 .1 ,2.) g,lutamate oxaloacetate transaminase (Got,geneti dciversi tstyatistics (Ne i1973, Nei & Chesser EC 2,6,1,1. )p,hosphoglucomutase (Pgm ,EC 1983) :the total population gene diversi tHyT,; the 2,7.5.1.) ,shikimate dehydrogenase (Shdh ,EC average gene diversit iweitshin populations ,Hs; 1.1.1.25. )a,nd UDP glucos epyrophosphorylase and the relative extent ofgene differentia atmionogn (Uklp pEC, 2.7,7,9, )T.he stain{ng method for blglzp populations ,GsT. is described in Harris & Hopkingon (1976 )an,d Nei's unbiased standard geneti dcistance swere these fbr the other enzyme systems fo11ow the meth- calculated for all populatio pnair s(Ne i1972, Nei & ods set out by Tsumura et aZ. (199 0an)d Tsumura & Roychoudhury 1974) ,and clusters were analyzed Ohba (1993). using the unweighted pair-grou pmethod with arith- metic averages (UPGMA; Sneath & Sokal 1973). S t a t i s ti c a l a nalvsis The above calculations were carried out using / TABLE 2. The eonfirrned population sofPicea.kcij;umcre in this study. Attitudc(rnP)opulation Mountains Habitat Location g sizc Fuki-sawa Nishidake-305ge,Fujimi,Nagano ISOO 30 Yatsugatake Karamatsu-sawa Nishidake-31egfi,Fuiimi,Nagano 1650 100 OhdairaKitazawa-t6ge Nirasaki-66ia Hga,kushu, Ylwnanashi 16001500]45100t<41500<11705<010so Miwa-278fiG H,ase, Nagano Akaishi Mikobuchi Inasato-78ww,Hase,Nagano Koma-sawa lnasato-85g Hga,se. Nagano Tbnshi-iwa Ohkawara-76gOYh,shikaN,agano g Roughry estimated nurnbcr ofmature trees (bigg teharn about 20cm DBH). g? ・Forest compartment assigncd by the Nationa lForest Agency. gru Fores tcompartinent assigned by Yamanashj Prefecture. NII-Electronic Library Service TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics June 2004 KATSUKI et al. : Geneti vcariations ofPicea keu,amae 23 GENESTRUT (Constant ietn ael. 1994) and DIS- tribution of mature trees shows a peak at 30-40cm, PAN (Ot a1993) computer software. The age ofa big tree (DBH = 50.0cm; H =: 22.3m) damagcd in 2001 by typhoon was estimated to be Results 103 years, In the Akaishi mountains area, Ohdaira is a Presentgeographicaldistrihution localit ythat has been confirmed recently as a habi- In our survey, a total of about 220 mature trees of tat ofPicea kayamae and P mczximowiczii (Katsuki Picea kqyamae at 7 habitat swere conimed (Fi g2., & Seido 1999) ,and about 30 individua olfsR kq),a- Tbble 2). mae with a heigh tover l.3m were observed in a In the Yatsugatak meountains area, Fuki-sawa small area (14ha )T.he number of mature trees was thought to be the type localit yof the Picea seems to be about 10, Kitazawa-t6ge ,Mikobuchi, koyamae collected by Mr. Koyama in 191 1 (Wilsonand Koma-sawa are the localiti eresported by 1916), but this fores tsuffbred heavy damage by Hayashi (1960 aH)ow.ever, the nurnbers of samples the Isewan typhoon in 1959 .At present ,over l,OOO ofR kcl}・'a mian ethose habita twesre insurncie fnbtr young trees and about 30 mature trees (bigg ethran the present allozyme analysis, since the number of about 20cm DBH) with cones can be observed in mature trees in each of those localiti easppeared Fuki-sawa. Karamatsu-sawa, where there are about to be less than 10, Tenshi-iwa was reported by 100 mature trees ofR koyamae, is the mest well Yamazaki (196 5to) be the southern limi tlocalit oyf known habita t(Hayas 1h9i60a) T,he Nationa Florest "R shirasavvae". Though the detai lofsthis popuia- Agency registered i tas a gene resource preservatien tion were not reported in the previous paper, our fores itn 1987. In this populatio nth,e diameter dis- fiel dsurvey c]arified that there are more than 50 mature trees ofR koyamae in this locality. E180" OO' E18e" 30i 1 / Geneti cvariation in 4population sat two mottn- tatnoetsareas The sampled individua flosr allozyme analysis were N36"oo・ mainty maturc trees in Fuki-sawa K,aramatsu-sawa and Tenshi-iwa and the mixture of mature trees and young trees in Ohdaira. Ten putativ leoci were detecte odn eight enzyme systems: two loc ieach for Dia and Got and one locus fbr the others. At the 95% criterion, four loc i(Dia-2 G,2d, Ilgm, and Shdh) were polymorphic ;one locu s(Dia-1 w)as classified as monomorphic with very littl vearia- tion ;and the other fiv eloc i(Fb eGidh,, Got-l ,Got-2, N35' 30i and Cigpp) showed no geneti vcariation at all ('lable 3) ,[Ilab 4le shows a summary ofthe degre eofgenet- ic variation, It can be seen that three populations FIG, 2, Reported distributi oannd confirmed habitat sof Picea (Fuki-sa wOah,daira and Tenshi-iwa) showed ke.vamae .Hatched area, geegraphi cdailstributi doenscribed approximately the same degree of geneti cvaria- i1n9 6H5a,y aXs hmia rk1,96 0haa, biKtaat tsiun kwi hi&ch S eisadmop le1s99 9 ",aenrde Ycoalmleaczteadk,i tion (}[fe=:O.185 T-hOe, K1a9ra6m)at,su-sawa pop- Circle ,observed habitat. ulation appcared to hold slightly lower variation NII-Electronic Library Service TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics 24 APG Vbl.55 (He=O.13 6t)han these three population st,hough feare dthat small populations will disappear in the the difTeren cwaes not statistically significant, future. The estimated values ef Fis fbr polymorphic With regard to genet¢i variation within the 3 loc iare summarized in fable 5. A significant FIs populatio n(sFuki-sa wOahd,air aand Tenshi-iw aof) value is seen only for the flgm locu sin the Ohdaira Picea koyamae, shown in [Ibbl 4e, estimated values populatio not;her Fis values were not significant. ofNe (,1.23-1 .a2nd5 )He (O.185-O,1 9i6n )this [Ilab 6l eshows the level osfgenetic differenti-study were not clearly differe nftrom the summa- ation among the 4 populations ,The average GsT rized values (Ne=1,3 2He,-O.173) of 195 long- value over all loc iwas O.067. lived tree species (Hamri c&k Godt 1992) ,In com- Genetic distance samong the 4 populations pariso nwith Japanes econifer species, the genetic w ¢ re between O.OOI (Ohdai vrs.a Tenshi-iwa) and variation ofR koyamae is nearly the same as those O.045 (Fuki-s avsw, aKaramatsu-sawa )([fa b7l)e. of Crxptoineri .afaponica (Ne=1.33 ,He==O.189; Based on standard geneti dcistances t,he dendrogram Tomaru et al. 1994), Chamaee.vparis obtusa by the UPGMA method is shown in Fig .3. The (Ne=1.3 3He,=O,193; Uchida et aL l 997) ,and Pinus bootstra pprobability suggested combining the pumila (Ne=1,3 H8e,-O.225; Tani et al. 1996) ,and Ohdaira and Tenshi-iwa population ass the most greate rthan that ofAbies mariesii (Ne==1,08, closely related at 69%. He-O.054 ;Suyama et at. 1997). On the other hand, variation o'f the Karamatsu-sawa population Discussion (Ne=1, 1He6=,O,136) was rather lower than those of the other populatio nisnvestigat iend this study. In In this study, 7 habitat osf Picea kayamae were this populatio nd,iameter distributi oofn mature confirmed (Tab l2e), but the number of trees in trees shows one peak, and the fores atge was esti- Akaish imountains area ",ere shewn to be underes- mated to be about 1OO years old. An investigation timated, since our survey was not conducted in all conducted in 1911 showed no old growth at that areas ofAkaishi mountains, Furthermore, in some time (Shiras &aw Kaoyama 1913) .From those evi- cases, a stand of R Jtqyamae individua olccsurred on dences ,this populatio mnight have regenerated at a steep slope, and we could not reach such a habitat,oncc, about 1OO years ago. It is conccivable that the Note et al. (199 9rep)orted many R koyamae indi- low variation within this population would have viduals standing on steep slopes or near cliffs on the been caused by a bottlene cefkflect through a limit- other side ofthe Mibu River at Mikobuchi by view- ed number ofmother trees. ing them with binocular bsu,t i tw'as not possibl eto With regard to geneti cdifferentiati oanmong approach thes etree sand identi ftyhei srpecies direct-the 4 populatio nofsPicea kayamae shown in Tttble ly .Table 2 include sonly the number of rnature 6, the estimated GsT value (O,06 7i)n this study trees we could identi fdyirect lbyy touching them. was slightly lower than the mean estimated value Consequently, it is supposed that in the area of the (O.08 4su)mmarized fbr 195 long-live dtree species Akaish imountains there are more than SOO mature (Hamri c&k Godt 1992). GsT values of Japanese trees that can produce cones, This estimation sup- conifers were estimated to be O.034 for O"Jiptomeria porte dinclusi oofnthis species as one "vulnerable to .iaponica ([Ibma ert ual, 1994), O.030 for Chamae- extinction" on the red lis tby the Environmental c)paris obtusa (Uchid eat al 1997), O.170 for Pinus Agency ofJapan (2000 H)o.wever, population size pumila (Tan eit aL 1996) ,and O,144 for Abies and densit yare not sufficient fbr regeneration in mariesii (Suyam aet ai, l997) .The fbrmer two habita twshere mature trees stand alone, and i tis species have wide (ove rall of Japan except NII-Electronic Library Service TThheJea paJnaespeSaoncieestyefo rSPloacntiSyestteym atficosr Plant Systematics June 2004 K A T S U KI e t a L / G e n e t ic v ar ia tio ns of P ic ea k o'vamae 25 TABLE 3,Allelefrequencie sof iO allezyme loc iin 4 populations, Yatsugateke Akaishi Locus-AlleleFuki-sawa Karamatsu-sawa Ohdaira Tbnshi-iwa (31gge) (25) (32). (31)O.97O.03 Dia-Ji g"ah O,97O,03 1.00o.oo 0,97 O.03 Dia-2 ab O.60O,40 O.08O,92 O.30 O,47O.53 O.70 ho a 1,OO 1.00 1.00 1.DOO.45D.S5 G2d ab O,42O,58 O.38O,62 O.36 O.64 Got-1 a 1,OO 1,OO 1.00 1DO Got-2 a l.OO 1.00 1.00 1.00 Pgm ab O.48O.52 O,70O,30 O.69 O.68O,32 O.31 Shdh aha O,27O.731.00 O,18O.82 O.34 O,32O.68 O.66 Lcgge 1.00 1.00 1.00 "Thc locus with -1 in itsdesignatio nrepresents tt form moving clectrophuretically faste rthan tha twith -2, Yl iA}lel ea stands for the a11el emoving faste rthan alle]e h in electrophoresis, eg" sampl cnumber TABLE 4, Genetic },ariatien -,ithin4pepulations. MountainsPepulation P(Yo) NaNeHe He(SE) YlaLtsugatakc Fuki-sawa 404040410,501,4011,.520S1L.1S6OLO2.3211.32O4.132O.196(O,076) Karamatsu-sawa O,136(O,062) Akaishi OhdairaTenshi-iwa O.150O.184O.185(O.072) O.196(O.076) R percentag oefpolyinorphic iec iat a 95% criterion; Na, number ofalleles per loeus ;Ne, effec- tive number ofalleles per locus ;Ho, average obscnred heteroaygesit Hye;. avcrage expected heterozygosity. TABLE 5.Fixatio nindices (FIs w)ithin 4 population sfor polymorphic loci, MountainsPopu]atjen Dia-2-6.i'Gi2id-" Pgni Shdh 'Loii73O.085.O.136O,]03 ifatsugatake Fuki- sawa O.110O.160 -O.196-O.19S Karamatsu-sawa -O.065 Akaishi OhdairaTcnshi-iwa O.042O.044 O.570**O,1O2,9318-O.O17 ** p<o,ot. NII-Electronic Library Service TThhee JJaapapnaesneSeosc-ei etSyociety ffoorPrl anPtlSyastnetma tSicysstematics 26 APG VbL 55 TABLE 6. Levels ofgenetic diffbrcntiat iamoonng 4 populations tinuous distribut ioonn Honshu Island in the Last based on 4 polyrnorphi cIoc iand on all loc iincluding GlacialAge. those that were polymerphic and monomorphic. The dendrogram (Fi g3.) based on standard LocusDia-2HCT;2dflgmSHhsdlt GsT o.4seO.48O1.O3.S436O2.e4,7408o2O31.64O4.7OO0.53O9.S034e.o2ogOe,n06e9tOi. d0ci67stance ssuggests that Ohdair aand Tenshi- iwa inAkaishi populations mountains are very closely related, and tha tthe Karamatsu-sawa popu- latio nis remote from the other three populations. MeantiAII O,449O,184O.418O.172 The two populatio nisn Akaishi mountains might be continuous through some unknown population isn g Mean on 4 polymorphic loci. areas which our fiel dsurvey did not cover, The geneti cremoteness of Kararnatsu-sawa from the Hokkaido) and continuous distributi oanreas, the other populatio ncosuid be explained by the proba- latt etwro species' distributi oarneas are restricted to ble bottlenec ekffect, central and northern Japan and are discontinuous. The Karamatsu-saw paopulatio nis preserved as The value of O.067 in R kqyamae is large rthan a fores rteserve, but it sgenetic variatien is low and those ofthe firs ttwo species and smaller than those cannot represent that of the species Picea koya- of the latt etrwo species. The GsT value ofR koya- mae. Therefbr ef,or the purpose of preservin tghe mae might have been affected by it swide-area cen- genetic variation across the species, not only TABLE 7. Standar dgeneti cdi stanccs for all pair sfrom each of4 popul ations, Fuki-sawa Karamatsu-sawa Ohdaira KaTamatsu-sawa O.045(O,042) OhdairaTenshi-iwa O,O14(O.O12) O.OIO(O.O09) O.O03(O.O04) O.024(O.025)O.OOI(O.OO3) The figure sin parenthese sstand fbr .q.tand earrrodrs ofgcnetie distances. Karamatsu-sawa Fuki-sawa Ohdaira Tenshi-iwa - O.OIO O.O05 O Standar dgenetic distance 'I'he FiG .3, Dendrogram from UPGMA analysis based en pairwis estandard geneti cdistance s, figur ein th cphylogeneti tcree presents bootstra pprobabili tbyased on 1,OOO replicates. NII-Electronic Library Service TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics June 2004 KATSUKI et al. / Genetic variations efPieea ktv)amae 27 Karamatsu-sawa but also other population ssuch 85i336-337. as Fuki-sawa or Tenshi-iw aare importan tI,t is Crow, J, F. & M. Kimura, 1970. An Introduction to desirabl tehat care will be taken to preserve the Population Genetic sTheery. Harper & Row, New populatio nisn these other habitat tso,o, Fortunately, En v iYrbornkm.entalAgency Japan2000,Threatened of the Ohdaira populatio nis scheduled to be regis- Wildlife Red DataBook 2nd Xlo1.8. ofJapan. ed. tered as a preserve fdores bty YIimanash Pirefecture, Vascula rPlants .Japan Wildlif eResearch Center, "Picea shirasawae" was include das R koya- Tokyo. (i nJapanese), mae in this study. There was no clear geneti cevi- Faljon ,A. 1990. Pinaceae. Koeltz Scientif iBcooks, dence to suggest the existence of two independent Konigstein. & C.N.Page(eds,L9)99,Conifers,StatusSurvey taxa in our results, as fo11ow sI,fR koyamae and "R shiFzisawae" are indepcndent taxa and had not mated Fu , Sa.nd,W .ConWsaenvga,tiWo.n ACchteinogn P&!aLn.. IFUuC.N,19 7C8a.mbPriicdegae..in: with each other in any of our samples, the Fis cal- Chen, W, & L, Fu (eds .F)l ,Reipub. Popul. Sinicae, culated on the assumption ofa single random mat- 7: 123-1 67 ,(i nChinese). ing populati osnhould have a positi vvaelue accord- Hamrick, J .L. & M. J. W. Godt. I992. Allozyni ediversity in in:Brown,A,H.D.,M. T,CleggA,, ingto "Wahlund's Principle("Crow& Kimura plant species. L.Kahler& B. S.Weir(eds.P)lantPopulation c1l9ea7r0 r)e s.Hulotw eofv esirgn, itfiacbalnte l5y s phoowssit tih vFaeit st ,heesrpeec iwaalsly n ion G6e3 n.eStiincas uB ,erreS, eudni dnger alndan GdeM, nAe.tic Resour ces, pp. 43- the Fuki-sawa and Karamatsu-sawa populations Harris ,H. & D. A. Hopkinson. 1976. Handbook of which were reported as mixed stands ofR kq)?amae Enzyme Electrophores iin sHuman Genetics N.orth- and "R shirasawae" by Shirasawa & Koyama Holland Publishing Company, Amsterdatn, (1913 a)nd Hayashi (1960a )Ac.cording to the Hayashi, YZ 196ea, Taxonomical and Phytogcographical NationalForestAgency,40individuals kaya- Study ofJapanese Conifers. Norin Shuppan, [Ibkyo. ofR (inJapanese). 31 individuals mae and of`tR shirasawae" were 1960b .Note on Japanes etrees and shrubs (4 )B.ull. registered from Karamatsu-saw paopulati oinn 1965. Gov. For .Exp. Stn .125: 71-74. This evidence would suggest that `CR shiiusawae" is Katsuki, T. & K, Seido .1999 .Populatio nosfPicea kcv,a- not an independent taxon, but rathcr, represents a mae and R maximovviczii i' nHakushu-sho ,Yama- morphological variation within R koyamae. nashi Prefecture .Trans. Kanto Jpn. For. Soc. 50/ 69-70. (i nJapanese). WY.e Nciosrdhiiaylalmya t,h aannkd T . KM.a cSdhai,m Ya/d aT s(umFuorrae, Ks. tYarnbdys hFiomruersat, Ki m urc4aa9n,: M7b.2e S &m-a 7iJ3n. t8Fa..i nCerdffw .i1n 9a6 4f. i1n[iIt h peneuombpeurla toifaell neGl.eesne ttihacts ProductsResearchrnstitutfeb)rhelp at sampling and Kobayashi, K,.J.Ydshikawa, & M, Suzuki.2000. DNA e(MoAFxfagoaUm trritFnsieaostucsi rmutouOenole;nsttntoo uc Smrfe(ce o,iCUCirS yhnhhe. iuieanSvblbc h.upeaU i a ) nRtrMD, ieas,evgaip m,i Ssap )o(luifrinzbtKnaugrm y lu;kFesuione (frsatuneTh1 d s eoU u ftctOnho htimToeih venNkc .ekaUen)u ynnt ,,Tisoas.;nv hd .TiU) ansNn, ntDaaiuinditvdmcsni.ot ttsoMoron.fiaelt L e P aii(Ig1dnse6P nel,7 iona.Brntnt.h ida eAAf,rc.nri e cc2 FtaaJ0r ita0oeo fpcim1C)ao P.an nitN,nc heaJeew dapm ains d.p, sd eJlpBcee i, oecHstii e . sEs Jtoo f.oc toLrehfe ,niPBienoc n eLtoa. af ASsx8ote l:cAG6., l7 Ia -DH3ci8e5 iei0:at b1.Arle3gir7ecg-h Forestt Aygency for sarnpling perrn{ssion. Li, C. C. & D. (} .Horvitz. 1953. Some meLhods of esti- mating the inbreedin cgoeMcient. A. J. Hurn. Genet. Referenees 5:107-117, Minaki, M. 1987, Fessi lplant softhe Last Glacia lAge Constantin eC,. C., R, P. Hobbs & A. J, Lymbery. 1994, and it sevolutional signifieance, Iden 41(12)i30- 3(5in FOI(I'RAN prograrn fsbr analyzing populatio sntruc- Japanese). ture from mult"ocus genotypic data .J. Hered, Nei, M. I972, Genetic distanc ebetween populations. NII-Electronic Library Service TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics 28 APG Vbl,55 Am. Nat, 106:283-292. ResearchBulletin59:1-41. . 1973. Analysis of gene diversity in subdivided [Ibni ,N., N. Tbmaru, M, Araki & K, Ohba. 1996, Genetic population sP.roc. Natl. Acad. S¢i .USA 70/3321- diversit yand differentiati oinn population sof 3323, Japanes estone pine (Pin uptstmila i)n Japan .Can. J. . 1978, Estimatio nof average heterozygosi tanyd For. Res. 26:1454-1462, genetic distanc ferom a srnal1 number ofindividuals. Ibmaru. N., Y. Tsumura & K. Ohba. 1994. Genetic vari- Genetics89:583-590. difTk]rcntiaitnion ation and population natural popu- & R. K, Chesser .1983. Estimatio nof fixation lation sof Cny)ptorneriajmponi cPaI.ant Sp. Biol. indice asnd gene diversit iAensn., Hum. Genet, 9:191-l99. 47:253-259. Tsumura, Y & K. Ohba. 1993, Geneti cstmcture ofgeo- & A. K. Roychoudhury, 1974, Sampling variances graphica lmarginal population sof CFIzptomeria of hcterozygosity and geneti cdistance. Genetics J'qponic aC.an. J. For. Res. 23:859-863. 76:379-390. , N. Tbmaru, Y/ Suyama, M. Na'iem & K. Ohba. Note, H,, S, Okitsu & A, Momohara. 1999. Habita tof 1990. Laborator ymanual of isozyme analysis. Bull, P i c e a k oivamai Shirasawa and Picea maxtmowiczii Tsukuba Univ. Forest s6:63-95 .Ci nJapanese). Regel ex Mast, in Japan .J. Jpn .For .Sci .81i236-244, Uchida, K., N. Tomaru, C. Tbmaru, C. Yatnamoto & K. (inJapanese), Oliba.1997.Allozyme in variation natural population Ota, T, 1993 .DTSPAN: Geneti cdistanc aend phylogenetic of hinoki ,Chamaeoparis obtusa (Sie bet, Zucc.) analysis. The Pennsylvani aStat eUniversity. Endl. a,nd it scomparison with the plus-tr eseelected Shimiz uT,. 1989, New names ofsome Japane spelant sJ., from artificial stands. Breed. Sci .47:7-14, Phytogcogr. Taxon. 37i 120. Wilsen, E, H, l916, The Conifers and Taxads ofjapan. , 1 992. 1leixonom aynd phytogeograph yofconifers, The University Press, Cambridge. with special reference to some subalpine genera ('2) Wright, J .W. 1955. Specie scrossability in spruce in The genus Picea .Jpn. J .Histor .Bet 9: 3-11. (in relation to distribut iaonnd taxonorny. Fores tSci, Japanese). 1:319-349. Shirasawa, Y. & M, Koyama, 1913. Some new species of Wright, S, 1951. The genetic asltructure of population. Picea and Abies in Japan .Bot. Mag. Tokyo 27: 127- Ann. Eugen, 15:323-354. 131, pl. 2. (i nJapanese), Yamazaki T,. 1965 .Materiai sfor the distribu tofivoasn- Sneath, P. H. A, & R, R, Sokal. 1973, Numerica] cular plants in Japan (41 )Jo.urn. Jpn. Bot. 40:328 .(in Tlaxonomy.W, H.Freeman,SanFrancisco. Japanese), Suyama, Y, Y Tsumura & K. Ohba, 1997. A cline of 1995 .Pinacea ei.n :Iwattsuk Ki.,, T, Yamazaki ,D. E, a]lozyme variation jn Abies mariesii J. Plant Res, Bouffbrd & H. Ohba. (eds .Fl)ora of Japan ,vol. 1 110i219r226. Pteridophyta and Gymnospermae. pp, 266-277, Suzuki, M. 1991. Pieea cone-fossils from Pleistocene Kodansha, [Ibkyo, strata ofNortheast Japan. Saito-Ho-on Kai Museum Received Nbvember i4 ,2003 ; accepted Jtznuat) ,1 7, 2004 NII-Electronic Library Service