Extensive Electrophoretic Variation in the Apogamous Fern Species, Dryopteris nipponensis (Dryopteridaceae)

10 Pages·2003·0.97 MB·English

by  Ishikawa, Hiroshi

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ISSN13647565 Acta Phytotax ,Gcobot .54 (1) 5:9-68 (2003)
ExtensiyeElectrophoretViacriationinthe Apogamous FernSpecies,

Dryopteris (Dryopteridaceae)
nipponensis

HIROSHIISHIKAWAi, MOTOMI IT02, YASUYUKI WATAN03
and SIRO KURITA3
iL de a nd Bi or es ou rc e S ci e n ce , G raduaie School ofScien acnde 7lechnoto gCrhliba Liniversit yi,-33 1'ayoi-cho,
Jnage-ku ,Chiba 263-8522, lapan, `-tatlti-Disciptinail･' Sciences G.raduate School qf'Art asnd Seiences ,C,Fniversi4,
qf'7bkyo, 3-8-1 komaha, Mqguro-ku, 7bft.v o153-8902 ,Jtipa n3,Dqpartnient qfBiology Flacut 4o?fStien cChei,ba
LrniverTi A1.-･3;3 )tiyoi-c hJo},iage-ku ,Chiba 263-8522, JLcpan
To determine geneti cvariation in the apegamous fern species Di:yny)ter insilrponensis, allozyme
analysis was canied out. Among thc 196 individua olfsD, nmponensis examined, 45 electrophorctically
distinguisha bvalreiants were detecte dT,his is the !argest number of electrophoretic variants fbund in
one apogameus fem species so fbr examined. Only one variant was characterized by a unique allele. All
ofthe other variants were distinguish beyd the combinations ofthe ljmitc dnumber ef alleles that were
shared by some variants. The evidence strongly suggests that this apogamous species is ofrccurrent ori-
gin .The possibi lofi thyomoeQlogous chromosome pairing was also evaluated. Theoretical lgeyn,otypes
oftwelve of45 variants can be generate dfrom those ofother variants via homoeologous chromosome
pairing.
Key words: allozyme, apegamous species, clonal diversit, yDnyopteris nilzponensis, homoeologo uchsro-
mosomepalrlng
Apogamy in fern sis a fbrm ofasexual reproduction (Tlakam iety aaL 2001), and so on, while Di7opteris
in which unreduced spores are forrne adnd the resul- yakusilvico Kluarata (Darna eetd aiL 1990) and D.
tant gametophyte sproduce sporophytes without Jemota (A .Braun) Druce (Schnel &l eHorlderegger
fertilizat iIon ng.encral ,individual sproduced by 1994) showed no gencti vcariation. Those obserNJa-
apogamy are clones ef the parent .It is therefore tions suggest that many apogamous species arc not
expected that only minor variation will be secn in ef single origin, but of recurrent origin. In addi-
apogamous species. Hewever, numerous apoga- tion, there may be other mechanisms by which
mous species show great morphological variation, geneti cvariation in apogamous species arises, such
and often fbrm species complexes that are taxo- as homoeologous chromosome pairin (gKlekowski
nomically problernatica lA.llozyme studies also 1973 ,Klekowski & Hickok 1974, Chapman et aL
have shown genetic variation in Asplenium t{nilat- 1979, Masuyama 1984).
erale Lam. sensu lato (Watan o& Iwatsuk i1988), The Dip,opter ei}ysthrosora group (Miyamoto
Pteris cretica L. (Suzuk i& Iwatsuki 1990), & Nakamura 1988, Dryopteridacea ec)ontains a
Dsyopteris bissetia n(aBakc rC.) Chr. (Li net al. number ofapogamous specics. Among about twen-
1995), Diptazium doederlein i(iLuers sMa.k)ino ty taxa ofthis complex in Japan, only three spccies
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(D .caudipinna Nakai, D. koidzumiana Tagawa, Materials and Methods
and D. kinkiens iKsoidz. ex Tagawa) are sexual,
while all of the other taxa are known to be apoga- Dryopteris nipponensis bear ssome resemblances to
mous (Iwats u1k9i92, Takamiya 1996) .Most of D. eiythrosora, D. caudipinna, D. koideumiana,
thos eapogamous taxa are triploids .Morphological- and D. .fbrmosana. The fbllowin gcharacters have
ly ,interrnedi aftoerms can be identjfie dbetween been used to djstingui sD.h nipponensis (Iwatuski
various combinations oftwo taxa, With regard to the 1992) ,The basiscop ipcinnule nsext to the rachis on
origin of apogamous fern species, hybrid origin the lowes tpinnae are shorter or slightly longe rthan
has been documented through hybridizatj eoxpner- the adjacent pinnules .The basiscopi cpinnules next
iments and through studies ofwild plant s(Manton to the rachis on the lowes tpinnae are parted .The
1950, Walker 1962, Suzuki & Iwatsuki 1990, young frond sand indusi aare not reddish.
Darnaed iet aL 1990), as well as autopo[yploid ori- Sampres ofD?:yopteris nipponensis were col-
gin (Gasto nl9y88, Gastony & Windham 1989) .It lecte dfrom the localiti geisven in Table I, Ap-
seems that complex speciation events involving proximate]y 200 stocks were planted in the field
hybridizatio nand the acquisition of apogamous at the Facult yof Science ,Chiba University ,and
reproduction have occurred in the Dp:yopter eirs:y- used for allozyme analysis. Xk)ucher specimens are
throsora group. Dr:yopteri nsippenensis Koidz., one deposite din the herbariur nef the Natural History
taxon of this group, is also a triploid apogamous Museum and Institut eC,hiba, Japan (cBM).
species (Kuri 1t9a66, Hirabayash i1967, Ishikawa Fresh leave swere taken from livin gplants
unpublished), It occurs mainly on Honshu, Shikoku, and analyzed electrophoretically, One hundred mg
and Kyushyu in Japan ,and rarely in China and of lea fmaterial was ground in 1 ml of extraction
South Korea (Oh 1978, Kurata & Nakaike 1985, bufll ecormposed ofO,1 M Tris-HCI pH7.5, 1O mM
lwatsuki 1992, Flora of Zhejiang Editori aClom- MgC12, O.5 % Sodium mctabisulfite, O.5 % Sodium
mittee 1993) .Drlyopteri nsipponensis shows exten- ascorbate, 8 % PVP (SIGMA ,St .Louis ,MO, USA),
sivc merphological variability and is treated as a and O,5 9, 62-mercaptoethanol .The resulting slurTy
variety ofD. eiythivsora (D,C,Eat oKunn)tze by was centrifuged at 1OOOO rpm fbr 1O min, and the
some authors (Ohw i1957 ,Kurata & Nakaike l985, supernatant was used fbr electrophoresis.
Nakaike 1992) ,or as a distinc stpecies by others (Ito Enzyme electrophoresis was conducted for the
1939,Tagawa 1959, Iwatsuki 1992).Mitsuta & fo11owing
seven enzymes: asparate aminotrans-
Nagamasu (1985 )reported that D. nipponensis feras c(AP(I'fG OleTu)ci,ne aminopeptidase (LAP),
exhibits intermedia cthearacters between D. koideu- isocitr adteehydrogenas e(IDH )m,alate dehydro-
miana and D. formosa n(aH.Chri Cs.tC)hr .and is genase (MDH) ,6-phsphoglucona dteehydrogenase
possibl oyfhybrid origin. (6PGD) ,phosphoglucose isomeras e(PGI )a,nd
In this study, 188 individua o]fsD, nipponen- triosephosphat eisomerase (TPI )AM, and LAP
sis were collected from 19 localit iceovsering the were examined by the polyacrylamid egel system of
distribut iaroena of the species in Japan, and eight Shiraishi (1988 )I.DH, MDH, and 6PGD were
from one locali tiyn South Korea. Allozyme analy- resolved on 1 1 .5% starch gel and the histidi cniterate
sis was carricd out to determine the geneti cvariation buHbr system ofCardy et al. (1981 P)G.I and TPI
in this apogamous species, Below we djscu stshe were electrophosed on 1 1.5% starch gel and buffer
causes of the polymorphism we observed, system 8 of Solti set al. (1983 S)t.aining s¢hed-
ules fo11owe dthe methods of Shirais h(i198 8f)br
AAI' and LAP, and the methods of Solti set al.
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TABLE1. Localities and voucher specirnens ofDo,opteris niru)onensis used in thi sstudy
SymbolLocality No. ot'
plantsLioucher
ABCDEFGHIJKALMsNaohPiQ-RsmTura,NiigataPref. 2111 HI95091201,HI95091208

Araka",a-choN,iigataPretl HT95091209

Shibata,NiigataPref, HI9509121S

Yahiko-mura,Niigata Pref, HI95091319

Nishiyumu-cho, Niigata Pref 12927114HI95091320
Suzu ,Ishikawa Prcfi HI95093001, HI950930IO, I[195093013 .HI95093014, HI95093017
Anamizu-ch oI.shikaw ?are£ HI95093030,HI95093034,HI95093035,HI9S093049,HI95093051
Kashima-ch oI,shikaw RPre£ HL95092901, HI950929e4, Hl95092907. HI95092908 H,I95092909
Oamishirasato-cho,ChibaPref HI94040209,HI94040227,HI94040229,HI9404023O
Ichinomiya-cho,ChibaPrcf. ]342 HI94061807
iluttsuC,hibaPreC HI94030639,HI94030641,Hl94030644

Amatsukominate-cho,ChibaPrefi HI94022701,HI94041209, H194041211, HI94041212,HI94e41213

Oshima-cho, Tokyo Pref. 121629 HI94060120
Eigeriji-c hSoh,iga Pre£ IIt94080302,HI940g0303
Owase, Mie Prctl HI95040504, H195040511, HI95040512, H1950405]7, HI95040521
Nishinoshima-cho,Shimane Prcf, HI950402LJ,HI95040212

Izuhara-choN,agasakiPref, HI95033OOS,HI95033008,H19S033O]3,HI95033e3G,HI95033034

Tarumizu,KagoshimaPrefi 827 H195032701,HI95032704.HI9503280],H19S032802,HI95032806
Kamiyaku-cho,KagoshimaPref H194062504,HI94062513,HI94062519H,I94062606H,194062612

Cheju Island, South Korea 8 HI9S042201, HI95042308, HI95042319, HI9504232 5H,I9504233I
(1983 )for the other enzymes. Q, R, S, and T) was quantifie dby Simpson' sdiver-
When multiple banding zenes were observed, sity index D (Piel o19u69),
presumed loci wcre numbered sequentially with
the most anodally migrating locu sdesignate ads 1, D-1-Z{[ni(ni-1)]f[N(N-1)])
Allozymes at individu alloci were lettere dalpha-
beticall yw,ith the faste smtigrating allozyme des- where ni is the number of individua ]ofs variant i,
ignated as a. The genotypes were interpret beadsed and N is the number ofcollected individua lTsh.e
on the known subunit structurc of the enzymes, other localitie swere excluded in this analysis
and on the number of isozymes typical fbr each because the numbers of individual scollected were
enzyme (Solt eits al, 1983, Weeden & Wendel too small.
1989) .As fbr the locu sflgi- 2th,e gene dosage was
estimated based on the relative intensi toyf the Results
stained bands .Genotypes of triploi dplants were
coded by three letter ss,uch as aab and hcc, The Among the seven enzymes examined, the band paF
genotypes ofthe other loc iwcre coded only by the terns of MDH were too complex to be interpreted.
combination of alleles, such as alb ,and b!c ,because AM and TPI showed no geneti cvariatien, Finally,
we were not abl¢ to clearly identi ftyhe gene dosage we recorded the genotypcs at fbur loci L,ap, (ff'gd-
for those loci, 2, ldh ,and Rgi- 2Th,e bands at I]lgi -a7nd 6Pgd-1
Clonal diversi tiyn 9 localiti e(sF ,G, H, L, O, were not well reselved, and not scored,
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At the locus Lop, six alleles were detected, locali tarye shown in fable 3 and Fig .2.
and one to three alleles were fbund in each indi-
vidual. Twclve differe ngtenotypes were distin- Discussion
guishe dby the combinations ofallcles. At the loci
bl]gd- 2l,dh ,and Rgi-2 t,hree, two, and four alleles In this studM 45 electropheretically distinguishable
were detecte da,nd three, two, and fifte edni fferent variants were detectcd ,In previous studies on the
gcnotypes were observed rcspectively, geneti cvariation in apogamous ferns ,14 variants in
Representat ibvaend pattern sofPGI-2 are shown in Dryopteri bsissetia n(aLi ent aL 1995) ,six triploid
Fig. 1 , Finally ,by integratin tghe genotype sat fbur and fiv ediploi vdariants in Pteris cretica (Suzu &ki
loci 4,5 electrophoretically distinguishab lvcariants Iwatsuk i1990) ,and one tetraploid and four triploid
(clone swe)re recognizcd in 196 individua losf variants in Diptazium doederlein (irilakam eit yala,
DoJopteris nijzponensis, They were narned variant 1 2001) have been reported. In the asexually-repro-
to variant 45. The nurnber of individua lansd the ducing gametophytic population sof Vittaria
genotype ofeach variant are shown in Table 2, The appatachiana Farrar & Mickel, ten variants were
a]lele Ltu)-a was observed only in variant 1. All recognized (Farr a1 9r90). The number ofvariants in
alleles cxccpt Lap-a were shared by more than two Dryopteris nimponensis is far large rthan in thosc
varlants, specles.
The number of individua lnsum,ber of vari- Among the 45 variants, 23 variants (51%)
ants, and Simpson' sdiversi tiyndex (D) in each were solitary ,and 33 variants (739 iwGer)e fbund
1 2 3 4
･ilt
il..'/･y,
a
ptiti't".,,,,,veY-//llg,, a
b b b b
c c c
c
.,tiXptt':'E'

fu･''

'W..,,,-,e?-,f.'

.. r,,/i..t,.',,ll.,1'1
'/i'//'af'lll/il,ee
" t
d･ d
d
d
/gtt g e l tend
acc bcd hcc bcld ccld bcc bcld
bcd bcc bed bcd boo ccd acd
}･'IC1. iR.epresentative band patteni osl'PGI-2. The position osfhomedimcrs are shown by the lette rons thc lef tside ofthe photographs,
Homodimer dd is locate dout ef the gel in photograph 4, The interprct cgdenotyp easre shewn below bands.
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TABLE2.Interprete dgenotype esf45 variants ofDiyopteris n4eponensis at fburlocj
Vbriant No. ofplants No. ofplants Lap**6Pgd-2"" Idh** Pgi-2
fromlocality

1234567891011122l19311141516171821T9*230I2,12223242526272829a3lc0b3bX1cb/cb/cbldbbl/dbcdaeb/dceb-/*eb*/*ebb/aleabca/aceabalrbleabca!abeabtafaceabaalaXfaabcaalacbfabb/ab/accfaabab/acfacbac/adfabcaccaccab/adcdcclcedcaiceccciccdcb/dedca/aebcc/cedea/aebacdbbdbcdabdbbdbcdbcdbddbcdcccaababdbccbedcdddddaababc
IK, 14L,IT

IRISIQIS2SIIISISIJILILIS140,3R2SlL,
l.1211l111117231962
alcalcbtcbtcbicbtealealca!cblcblcalcbXcat'ca/ca!cal'calca,rcblcblc
2QIL9G4L,

20IH,

ISIPIT2A,

l12913
IE,16F,1OH

ILIR,
2SIP4S2LIG,ILIB,

142241
IC,ID,13F,

17G,2L,1M,3Q,2T

323334353637383191410042114324413445414l51I2TIRIK, c/'ecleci'ecfec/fbc,lf/cclfbcilfccbtf,c'/fccabfafiada'lecdfedlf abdaccbcdbddbbdbcdcddabcabdbddbddaababcabc
5L, IQ,2R, IS

2SIT3L4SIS
atca/cb/cb,icblcbalac/abtacaaala'lcbbataca
IK, 3L3L,
2S

ISININ2Q
* Symbol oflocality is shown in Tablc 1. 2T indicate 2s plant fsrorn localit Ty,
** Combinations ofalleles are shown. See text.
*** No data,
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toc.B
N=1

[31]tocA
.
N=2
toe.c
[24]
N=1

[31]

Loc.D
N=1

[31]

Loc.E
N=1
[24]
ltt
t･r
'7 Loc.1
v "-- N=4
[2,8]
r Loc.J

N=1
'?'S')(..\ [11]Loc.L

)P`j"
N=42

i..fi'l:>?'" [2,12,13,17,18,

lv ?N>>s (20,25,29,30,31
'
34,37,40,41]
S;"te' Loc.M tro.ee
/ b N=1 Loc.K
[31]
N=3
[2,34,4(1
toc.N

N=2

"i' [43,4tl]

f' LO;i;Rs toc.o
L.}
toc.Q N=16
･-
N=9 [3,15,26,33,3 4][15,2C"
[5,17,31,34,4 5 ] D=O.86 D=O.23
D=O.86 ;oa----toc.s
N=27[4,6,7,9,10,14,16,21,26,28,34,35,38,39,41,42]
Loe.T (Chei Lu, S.Korea) D=O.95
N=8

[1,2,23,31,32,36]
ur
Dn.93
hn
FiG ,2. Wiriant snu,mber ofplants, and diversi tiyndex in each locali tSyy,rnbel soflocalities are shown in Tlabl 1e. N: Number ofplants,
D: Simpson's diversi tiyndex ,V2riant sfound in each locality are shown in brackets.
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TABLE3. Number ofplallts, nurnberofvariants and clonal diyersi tiyn each locality
Loea}ityNo. ofplants No. ofval'iallts Vlariant Diversityindex
A*BCDEFGHIJ2KL 11111232213l4 243131312424,3119, **
1
11l29214
O,5]O.51O.18
30,31

21,242,8112,
1342
34,402,

12,13,17, O.86

1S,20,25,29,

30.31,34,37,

40,413143,

MNoPQRs l21629g27 12225516
4415,

2022, O.23

27S,17,31,34,4S

O.86O.86O.95
3,15,26,33,34
4,6,7,9,10,14,

16,21,26,28,
34,3S,38,39,

4L 421,

'1'
8 6 2,23,31,32,36 O.93

' Symbol of localit iys shown in Tab]e 1 .
"* Net calculated, Scc tcxt.
in only one locali t(yTab l2e), indicatin gthat the 1973, Klekowski & Hickok l974, Chapman et al.
clenes ofD. nmponensis have a highl yrestricte ddis- 1979, Masuyama 1984), and recurrent origin
tribution pattem ,A similar pattern of clone distrib-(Walk e1r962, Watano & Iwatuski 1988 ,Suzuki &
ution is reported fbr many agamospermous and Iwatsuk i1990, Lin et al. 1995).
vegetatively reproducing clonal seed plants In this study, only one uniquc allele was fbund
(Ellst &r aRnoodse 1987) ,Considcrin tghe highly at on ¢ locu sof variant 1 (Lap-a T)h.e rarity Qf
restricted distribut ipoanttern of each variant, 45 unique alleles suggests that the contribution of
may be fewer than the actual number of existing mutation to genetl vcarlatlon ln D. n4nponensis ls not
clones of D. nipponensis. A large rscale sampling signifi¢ant. All alleles except Ltop-a were shared
may reveal a greate nrumber of variants. b v. vmaorreian ttshan two variants. As a result, most of the
The possible causes fbr geneti cvariation in are distinguish beyd the combinations ofa
apegamous species are mutation (Manto 1n950), limited number of alleles, which implies that D.
homoeologous chromosome pairin g(Klekowskinipponensis originated multiple times independently
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TABLE 4, Possibl ecombinations of ancestor-desccndant pairs ether variants by segregation of the llgi- 2geno-
poaf ivrariinan gtisnv ounldverin gtahsseu rlnoedcu s hPgoim-o2eologou cshromosonie types through homoeologous chromesorne pairing
Variants Genotypcs ([Il e4)t, bTlo eexamine segregation ofgenotypes in
16 . 15 bcd ' 'bbd progeny under experimental conditions, spores were
17 . 18* abd - bbd collected from one sporophyte with the genotype
19 - 18 bcd " bbd abc at Pgi-2 (vari a3n1, tlocalj tMy), and gameto-
20 .21 bcd - bdd phytes and offspring sporephytes were prepared.
25 - 24 abd "' aab As a result, fiv eof 284 progenies (25 0gameto-
27 -. 26 bcd - bcc phyte sand 34 sporophytes) showed differe gnetno-
2278 -- 2298 bcdcdd -- cddddd type s(thr aeace, one bbc, and one bcc) from that of
31 30* -' the parent ,which can be explained by segregation
- abc aab
32 - 30 abd ' aab through homoeologous chromosome pairing
31 - 33 abc - acc (Ishik eat wal.a in press).
32 - 35* abd - bdd Although homoeologous chromosome pairing
443044 -.. 344513 baabbcdcd ---' .babaddbdd iDs, onnmepo noefntshies, pos tshieb olre icgaiunsses o foaflgle nveatriicant sva rciaantniootn bien
eNkiriants 18, 30 and 35 can be dcrived from twe anccstral vari- explained only by this mechanisrn, For example,
ants each. variants with the genotype abc at flgi- 2are inca-
pable ofproducing gcnetypes with allelc d, such as
abd, by homoeologous chromosome pairing,
from limited geneti cresources, Consequentl yi,t is almost certain that D. nippo-
The pattern of genetic variation in D. nmpo- nensis is not ofa singlc origin. PresumablM D, nip-
nensis does not preclude the possibili otfhyomoe- ponensis has arisen many times independently,
ologous chromosome pairin gT,here has been no Diversity index (Tab l3e, Fig .2) is high in
electrophoretic evidence fbr homoeologous chro- localit iLe, sQ, R, S, and T (D ranges finyo Om.86 to
mosome pairin ign fems (Gasto &n yGottlie 1b982, e,95) a,nd low in localit iFe, Gs, H, and O (D ranges
Gastony & Darrow 1983). However, there have fi-o rOn. 1 8 to O.5 I ) ,There are only twe variants (24
been few electrophoretic studies that have verified and 31) in 35 individua closllected from six locali-
the possibili toyf such a mechanism, and it is stM a ties A, B, C, D, E, and E It appears that clonal
controyersial topic ,Thc sporogenesis pathway in the diversit yis lower in the northern part ofthe distri-
apogamous species ofDFyopteris fbllow sthe D6pp- butio narea ofD, nipponensis than in other parts,
Manton scheme (Manto n1950, Kurita 1967, This may refiect the histor yofgeographical expan-
Hirabayash 1i974) ,and potentiall yhomoeologous sion of D, nipponensis. Additional analyses with
chromosome pairing can take place (Klekowskistandardized sampling methods should be carried
1973) .For example, variants 30 and 31 have the out.
same genotype at Lcrp ,611gd- 2a,nd Idh ,but differ- An apogamous species may arise from the
ent genotype sat llgi-2 ,aab, and abc, Ifhomoeole- results of hybridizati obnetween two sexual indi-
gous chremosome pairing and the resulting segre- viduals, fo11owe dby the acquisition of apogamous
gatio nin the progeny occur, the genotyp eaab of reproduction through mutation (Darnae edti al.
variant 30 can be generate fdrom the abc ofvariant 1990) ,AltemativelM hybridizat iboentween a sexual
31. Theoretical lgye,notypes of twelve of the 45 individu aanld an apogamous one can produce a
variants of D. nipponensis can be induced from new apogamous hybrid (Walke r1962, Watano &
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Iwatsuki 1988, Suzuki & Iwatsuki 1990) .It has Ellstran dN,. C. & M. L. Roosc. 1987. Patterns of geno-
been pointe dout that D. nimponensis is morpho- typic divers iitn yclonal p]ant specics. Amer. J. Bot.
logical ilnytennediat ebetween D. koideumiana and 74:123-131.
D. formosan aan,d is of pogsibl ehybrid origin FlorZa hoefj Zihaenjgi.aVno glE.d1i.tPotreir aCilodmompihtytteaGey. m1n9o9s3p. eFrlmoarea o.f

(Mitsu&tNaagamasu 1985).SinceD.
nijzponensis Zhejiang Scienc eand Technolog yPublishin Hgousc,
is probably ofrecurrent origin, ifbot hparents wcre Hangchou. (i Cnhinese).
sexual, apogamous reproduction should have Farrar D,. R. 1990. Specie sand evolution in asexually
evolved recurrently, This is nQt very likel yto have reproducing independen tfem gametophytes .Syst.
Bot.15:98-111,
occurred, although it cannot be ruled out absolute-
ly .It seems more reasonable that one of the parents G a s ttornoyp ,hGo.r Jet. i1 ce9v8i8de. nTchee lflour tlhle ad egcatraibvealtli cao oomnpfsl etxh:e ag aemLoe-c-
of D. nipponensis was an apogamous type. The sporous taxa and a revised taxonomy. Amcr. Fern J,
available data show that D. koidet{mian ais a diploid 78: 44.67.
sexual species, and D. formosa nisa a triploid apog- & D, C. Darrow. 1983. Chloroplast iancd cytosolic
amous species ([Iakam i19y9a6) .Because D. nip- isozymes of the hemesporous fern Athyri"n .ifitix-
ponensi sis a triploid ,the origin ofD. nipponensis .femina L. Amer. J .Bot 70: 1409-1415,
can not be explained by simple hybridization e&ro zLy.g oDsi, tyGo titn la iheo bm1,o9s8p2o. rEovuis dfeenmc. e Afmoerr .g eJn. eBtoti .ch 6e9t:-
between the known cytotypes ofD. koideumiana 634-637.

and D. formosa Tnoa c,larify the origin of D. nip- & M. D. Windham. 1989. Species concepts in pteri-
ponensis it is therefbre necessary to carry out an dophytes :The treatmcnt and definiti oofn agamo-
extensive survey of cytotypes in related species, sporous species, Amer. Fern J, 79: 65-77.
includin tghe putativ eancestors, and to investigateHirabayash iH,. 1967. Chrornosome numbers in Japanese
phylogcnct ircelationships among them by using s.p ec1i9es74 . ofCl Dyrtyoopgteeorgirsa (p2h i) SJct.. uJdaipe. sBoont . D4r2y: q4p4t-e4r8i .sof
DNAs,
genetic markers such as allozymes, plasti d Japan .Hara shobo, Tokyo,
and nuclear DNAs, Ishikawa ,H., M. Ito ,Y. Watane & S. Kurita .2003.
Electrophoretic evidence for homoeologous chro-
We wQuld ]ik eto thank Mikio Watanabe (Aic hUniiversity mosome pairin gin the apos,amous fern species
ofEducation) and Ryosuke Sano (Th eInstimt eofPhysical Dr:yopteri snilrponensis' (Dryopterida cJ. ePalean)t.
and Chemical Research) for their teehnical advice, and Res. (i npress).
Koichi Uehara (Chib Unaiversity )for his help in sampling. Ito ,H. 1939. Polypodiaceae-Dryopteridoid ea1.e In:
Nakai, T. & M, Honda, Nova Flora Japonica no,4.
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Received Octoher 28, 2002; accepted Flebniar 2p4), 2003
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