Geographic Distribution and Genetic Variation of a Vulnerable Conifer Species, Picea koyamae (Pinaceae)

2004·0.92 MB·English

by  Katsuki, Toshio

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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
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一
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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
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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.
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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
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(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
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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.
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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.
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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.
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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,,
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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-

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NationalForestAgency,40individuals kaya- Study ofJapanese Conifers. Norin Shuppan, [Ibkyo.
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