Molecular Phylogeny and Chromosomal Evolution of Japanese Schoenoplectus (Cyperaceae), Based on ITS and ETS 1f Sequences

2005·1.3 MB·English

by  Yano, Okihito

TThhee JJaapapnaesneSeosciee tySociety ffoorPrl anPtlant SSyystsetmaetmicastics
ISSN1346-7565 ActaPhytotax.Geobot.56(2)1:g3-195(200S)

Molecular Phylogeny Chromosomal Evolution
and of

JapaneseSchoenopleetu(sCyperaceaeB)as,ed
on
ITS ETS lfSequences
and
OKIHITO YANO TAKUJI HOSHINO
and
DepartmentofBiosphere-GeosSpyhsetreSemcience,GraduateSehootofinformatOiktcus;,amaUhivensity
qf
SbienceR,idai-cho1-1,Okayama-shi,Okayama 700-OO05,mpan.

ITS and ETS 1f sequence data were used to estimate the phylogeny of 13 Japanes eSchoenqptectus
species, and karyomorphological observations were made on 14 species ofthis genus .IXvo major clades
werc idcntifie din the Japanese Schoenoplectu msoleeular phylogeneti ctree :(1 )one includin gall
species of section Actaeogeton, and (2 )the another comprising the two se¢tions MLilacogeto nand
Sbhoenoplecti Pchgy.logenetic amalysis, including three publishe sdpecies of section Sbhoenoplectu ssu,p-
porte da monophyly of the two majer clades. These molecular phylogenet idcata also support thc intra-
generi crelationships and morphological characters of genus SZrhoenoplect uass ,define dby Smith &
Hayasaka (2001 T)h.e section Actaeogeto cnlade and the sections MdlacQgeto nand Sbhoenopiec ctlaudse
showed thc same chrernosomal evolution; each clade had both high and low chromosomc numbers. The
high chromosome numbers may arise by polyploid byecause chromosome sizes were almost equal in
both .Therefor ech,romosomal evolution in the genus 5bhoenoplect umasy be caused more by polyploidy
more than aneuploidy. In our study, the putativ neatural hybrids S, × trapezoideus and S. × uzenensis
were fbund .The chromosome number ofS × ttmpezoicleu swas 2n = 43 and S. × usenensis was 2n =
58. These two hybrid shad an intermediate ehromosome number ofboth putativ pearents,
Key words: chrornosomal evolution, Cyperaceae d,jffu sceentrorneric chromosome, ITS and ETS 1fphy-
logenMSchoenopiectus

The genusSchoenqplectu(sRchbP.a)llaisdistrib-(ixycai:yumS,t]hoenoplect"Sst,]mpus,7}"ichophorum

uted worldwide, mainly in marsh and wetland habi- and PPkbsteria )by the fo11owin gmorphological
tats ,and include sabout 77 species (Smit h& characters: embryo fbrm, stolon presence ,tuber
Hayasaka 2002, Smith 2002). Strhoenoplec thuass presenc eand form, ligul peresence ,culm branching,
been recently accepted as a distin gcetnus, one ofthe lea fpositio nand structure, spikelet arrangement, flo-
segregated genera from the polymorphic and poly- ral scale indument, and culm anatomy (Smit &h
phyletic genus Sbiizp usesnsu iat o(Goetghe b&eurHayasaka 2001 2002).
,
Simpson 1991, Bruhl I995, Goetghebeur 1998, Smith & Hayasaka (2001 r)ecognized fbur
Hayasaka & Ohashi 2000, Smith & Hyasaka 200l sections, Actaeageto nA4,blacageton ,St:hoenqpiectiAy
,
2002). Genus Sbhoenoplect ucasn be distinguishedand St{pin iw,ithin the genus Schoenoplectus.
from closely related genera (ActinoscirpuRse,cently ,Pignott i& Mariotti (2004 i)dentifi tewdo
Amphiseirpus B,lysmus, Bolboschoenus, lsolepis,groups in the genus S℃hoenqplectu sb,ased on the
NII-Electronic Library Service
TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
L84 APG Xlo1 ,56
micromorphology of the frui tsurface and pollen estimated intragener ireclationships (e. gC.a,rex:
size: (1 )species ofsection St:hoenoplectu sa,nd (2) Starr et aL 1999, Hendrichs et al. 2004a, b;
species ofthe two sections Actaeogeton and Shrpini. EleochaFis: Roalson & Friar 2000, Yane et al,
According to the molecular phylogeneti cstud- 2004). Recently, new sequences, the nrDNA non-
ies of Muasya et aL (199 82,000, 2001) ,genus coding fragments from the external transcribed
Schoenqplectus forms a clade with the genera spacers 1 (ETS ID sequences, were reported to
Actinosctcpus or Bolboschoenus. However, there estimate lower-lev reellationships in sedges (Sta ertr
has been no molecular phylogenet isctudy of the al, 2003), The ETS lf region within intergenic
intragener ireclationships of the genus Schoeno- spacer (IGS i)s variable and infbmiati vmoere than
plectus. ITS region (Sta ert ral, 2003) .Furthermore, nuclear
The chromosome numbers of the genus ITS and ETS 1f sequences were used successfu11y
Sbhoenopiect huasve been reported by Hicks (1928),by Roalson & Friar (200 4an)d Star ret al, (200 4on)
Tanaka (193 8],948) ,Otzen (1962 S)c,huyle r(1969,the genera Ctire axnd Uitcinia .Chromosomal evo-
1972, 1976), Sanyal & Sharma (1972) R,ath & lution based on molecular data of the genus Eleo-
Patnaik (1974 )He,iser (1979 )I,.wasa k&i Ueki charis was also reported by Yano et al. (2004 T)h.e
(1979 )L6,ye (1981 )K,ozhevnikov et aL (1986),goal of the present study was to clarify the intra-
Subramanian (1988 B)i,r et al. (199 1H)os,hino et generic relationships ef Japanes eSchoenoplectus
al. (1993 )S,mith (2002 )a,nd Maeda & Uchino using nuclear ribosomal intern atlranscribe dspacer
(2004 )T.he lowest chromosome number of the (ITS a)nd external transcribed spacer 1 (ET Slf)
genus is 2n-10 (Schuyl 1e9r69) ,and the highest sequence data ,and to identify the relationship
number is 2n=:128 (Hic k1s928) .Cytologica lwotks between chromosomal evolution and the molecular
on Japanese Schoenoplectus were carried out by phylogeny.
Tanaka (193 81,948) ,Iwasak i& Ueki (1979 )a,nd
Maeda & Uchino (2004 a)nd, they observed 2n-38, Materials and Methods
40,42,72,74 76in Intraspecific
and eight species.
2n=38,40 42 inS tacustrisPlantSamples
aneuploids were and
("lan a1k9a38) .The mixoploids were reported as Thirtee nspecies of the genus Schoenqplect twe{rse
2n=32-39 fbr S. mucronatus, 2n=37-44 for S, tri- sampled fbr the phylogenetic analyses (Tab l1e).
angrdatus and 2n==68-76 fbr S. gemmijle (rMaed &a Samples of livin gplant swere collected from difl
Uchino 2004) ,However, there has been no study on feren ltoca litie sin Japan ,and drie dspecimens were
the relationship between chromosomal evolution selected fror nthe Herbarium sofOkayama Univer-
and phylogeny in the genus Schoenoplectus. sity ef Scienc e(oKAy an)d [Ibhok uUniversi t(yTus).
DNA sequences are comrnonly used in phylo- Three sections, Actaeogeton, Malacogeton, and
geneti canalyses and have proven usefu1 in the sub- Schoenoplectus, of the genus Sthoenoptectus, as
family Mapanioideae (Simpso ent ai. 2003). In define dby Smith & Hayasaka (200 1we)re studied.
Cyperaceae, plasti drbcL gene, ndhF, rps16, and Sectio nSupin icould not be included in this study
trnL spacers sequence data have been used to esti- due to the unavailability ofplant material. [I btest for
mated higher-le vpehlylogenet ireclationships, such monophyly of the genus Sbhoenqplectus ,we chose
as interfami liyn,tertri boar ls,uprageneric (PlunketBtulbostylis barbata, Fimbristylis ovata, and
et al 1995, Muasya et aL 1998 ,2000, 2001, Yen & 7hichophoru malpinum as outgroups,
Olmstead 2000, Roalson et at. 2001, Simpson et al,
2003) .The nrlTS sequence data have been used to DMI extraction, PCR amptipcation and segueneing
NII-Electronic Library Service
TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
August 200S YANO & HOSHINOi Molecula prhylogeny efSchoenoptecttts (Cyperaceae) 185
rlbtal
DNA was extracted from O.1 g fresh-frozen including those ofthe 13 Japanese Sbhoenoplectus
material or O.03 g drie dspecimens, using a Nucleon species and three outgroups, were used fbr the phy-
Phytopure plant and funga lDNA extractien kit logenet iacnalyses, The ITS data matrices ofthree
(Amersh aImnc. )A.mplificati ooftnhe nrDNA inter-North American St]hoenoptec tfursom Roalson &
nal transcribe sdpacers (IT Sre)gion and the nrDNA Fria r(2000 we)re also analyzed,
extemal transcribed spacers 1 (ETS 1b region were Sequences were aligned using Clusta lW
performed by polymerase chain reaction (PCR) (Thomps eot nal. 1994) and then adjusted manual-
with faq polymeras e(TaKaR lanc.) R.eact ions were ly as necessary. Phylogenet iacnalyses were per-
perfbrme dusing a DNA thermal cycler (GeneAmpfbrmed fbllowing the modified methods ofTsubota
PCR System 2400, Perkin Elmer Inc, )fbr the ITS et aL (200 22,003) ,Oguri et ai. (200 3an)d, Yano et
region, followi ntghe protoco olfHsiao et aL (1994),aL (2004 G)a.ps were treated as rnissing data and
and fbr the ETS 1 f region, fbllowin tghe protoco olf not used as characters. Phylogeneti ctrees were
Starr et al. (2003 [)[.he entire ITS-region was ampli- censtructed using the methods ofmaximum-parsi-
f i e dw it h pr i'mers ITSL ( 5tmTCG[EIAACAAGG- mony (MP) (Fit c19h71) and maximum-1ikelihood

TTTCCGTAGGTG-3 ' ;Hsiao et aL 1994) and (ML) (Felsens 1t9e81i)n for each region. MP trees
ITS4 (5'-TCCTCCGCTTATTGAI/MGC-3 '; were constructed using PAUPRat (Sik e&s Lewis
wnite et al, 1990) .ETS 1fsequence wsere amplified 200 1) ,which is a tool to implement the Parsimony
us mtg primer sETS 1f(5 t-CTGTGGCGTCGCAIi- Ratchet searches (Nixo 1n999) with PAUP", over
GAGTTG-3' ; Star ret al, 2003) and 18S-R (5'- IIAUP' 4.0blO (SwofTb 2r00d3), and DNARARS in
AGACAAGCATMGAC[I)XCTGGCAGG-3' ; StarT PHYLIP 3.573 c(Felsens 1t9e80i-n2001) .ML trees
et al. 2003). PCR product swcre electrophoresed on were constructcd by NucML in MOLPHY vcrsion
1.4% agarose gels to confirm a single product ,and 2,3b3 (Adac h&i Hasegawa 1996) ,fastDNAml ver-
purifie dusing a Qiaqui cPkCR purificati oKint sion 1.2. 2(Ols eetn al, 1994) ,DNAML in PHYLIP
(Qiag eInnc.). 3.573c, and PUZZLE version 5.0 (Strimm e&r
Cycle sequencing reactions were performed Haeseler 1996) .For ML analyses, the HKY85
using the purifie dPCR product sand a BigDye model (Hasegaw eat aL 1985) was used as the esti-
Terminator Vl,1 Cycle Sequencing Kit (Appliedmate model. A transitionftransversion (TsfT pva)ra-
Biosystems Inc,) .For ITS sequencing, one ofthe meter of2.39!1 ,91 (ITS!E TISD was used based on
amplification primers ,ITSL and ITS4, or one of the calculations of PUZZLE.
the interna lsequencing primers ITS2C (5'- A tree comparison with the ML criteria by
GCTACGTTCTTCATCGMCG-3' i Yano et al. NucML was canied out to evaluate thc topology of

2004) and ITS3C (5 ''GCMCGPffGAAGAACG- trccs obtained from the two methods (ML and MP),
TA G C - 3 ' ; Yano et at. 2004) was used, and for and the program CONSEL O,1f(Shimodair a&
ETS lfsequencin gE,TS lfand 18S-R was used. Hasegawa 2001) was also used to calculate thep-
The products werc resolved by capillary clec- values of confidence fbr the bifurcati ncagndidate
trophoresi susing an ABI PRISM 31O automated topologies using test procedures ,Thc ITS tree,
DNA sequencer. Sequences were assembled and includin gthe publishe dITS data of 3 species in
edited using Sequencing Analysis version 3.0 the genus Sthoenoplectu sw,as alse constructed
(Perk Eilnmer Inc,). using the similar method.
The obtained highes tlog-likelih oIoTdS tree
Data analyses and ETS 1ftree were applied by [[btaIM Lin MOL-
The data matrices ofthe ITS and ETS 1fsequences,PHY to evaluate the resulting trees .Using NucML,
NII-Electronic Library Service
TThheJea paJnaespeSaoncieestye Society ffoorr PPllanatnt SSyystsetmaetmicastics
186 APG Vbl.S6

TABLE 1. Tbxa used in this study and the classificatio nofthe genus Sbhoenopleetu saccording to Smith & Hayasaka (2001).
Genbank acccssion numbers listc fdor LTS and ETS 1 fdata .Nloucher sare deposite din oKAy and Tus.
Taxon Localit yand Vk,ucher Chromosome Accessionnumber
nuniber; 2n Cn)Previousreport(2nI)TS ETS If
Schoenoplectus
Sect .Actaeogeton
S. gemmtferC. Sato ,T,Maeda & Uchino 74 (Maed a&
Japan ,Shizuoka Pref., Harnamatsu; Kitamura l9554 (OKAY) 7676 Uchino 2004) AB206257 AB206271
Japan ,Shizueka Pre£, Hamamatsu; Kitamura 19557 (OKAY)
S. hondoensis (Ohwi S)ojak
japan ,Niigata Pref, Ylizawa; Sasak iiP6i8 (OKAY) 38 AB206258 AB206272
S .hotaru (iOhwi H)olub 44 (Iwasa &ki
Japan ,Shizuoka Pref. H,amakita; Ift)shi neot at. i3744 (OKAY) 424242 Ueki 1979)
Japan ,Okayamu PreE, Maniwa; Fleiru t&a Ikedo J5907 (OKAY)
Japan ,Okayama PTe£, Maniwa; filein uett aaL l5425 (OKAY) (2Iii)4242
Japan ,Hiroshima Pref, Fukuyama; Hoshino 160i7 (OKPLY)
Japan ,Okayama Pref, Okayama; Sata et aL J6039. 16040
(OKAY)

Japan ,Okayama Pref, Sohjy aK;dtayama l 7521 (OKAY) AB180720 AB206273
Japan, Miyagi Pref,, Watari; Hayasaha & lano 18997 (OKAY) 4444
Japan, HokkaidQ Prefi T,ornakomai; Hoshino et aL i926P AB206259 AB206274
(OKAY)

S. juncoide (sRoxb .Pa)lla 74 (lwasa &ki
Japan, Okayama Pref. S,objya; Iloshin oet aL 17539 (OKAY) Ueki 1979) AB206260 AB206275
Jupan, Yhmaguchi Pre£, Minai; Kdita 18ISi (OKAY) 747474
Japan, Okinawa Pret, Kunigarni; lokot a19387 (OKAY)
Japan, Gunma Prefi A,gatsurna ;Hbshino et al, 19519 (OKAY)
S, kemarovii (Roshe vS,oj)ak
Japan, Yamanashi Pre£, Minami-tsuru; Kdtsayama & AB206261 AB206276
lano IS213 (OKAY)
Japan, Hokkaido Prcfi T,omakomai} Hbshino et aL l9279 38(19Li)
(OKAY)

S .iineola i(uFsranc &h .Sav. T). Koyama 42, ca. 60
Japan, Okayarna Pref, Sohjya; Katctyama 1 7573 (OKAY) 7474 (fana k1a948,
Japan, Yamanashi Pref, Minami-tsuru ;Hoshino et aL I 7864 Kezhevnikov
(OKAY) et al. 1986)
Japan, Yamanashi Pref, Minami-tsuru; Kdtgayama & 74 (37rD AB206262 AB206277
lhno 18216 (OKAY)
S. m"cronatus (L, P)alla 38 (Maed a&
Japan, Okayama Pref, Okayama; Fletrut aet al. 16013, 38 Uchino 2e04)
16014(OKAY)
Japan, Okayama Pref, T,amano; IIirahar a& lhno 18136 38 AB206263 AB206278
(OKAY)
S .multisetus Hayasaka & C. Sato
Japan K,umameto Pref, Kumamoto; Sato s, n. (TUS) AB206264 AB206279
Japan, Okayarna Pref, Okayama; Hirahara et al. J966i 70
(OKAY)

NII-Electronic Library Service
TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
August200S YdLNO & HOSHINO/ Molecular phylogeny efSbhoenoptectzas (Cyperaceae) 187
TABLE1.Continued.

Taxon Localit yand Vbucher Chromosome Accessien number
number; 2n (n)Mevious r(e2pno)rt ITS ETS If
Schoenoplectus
Sect .Actaeogeton
S. triangulatus (Roxb ,So).iak 42 (Tana k1a948,
Japan, Okayama Pre£, Okayama; Fltrut aet al. J5708 (OKAY) 42 Macda & Uchino
2004)
Japan, Hiroshima Prefi F,ukuyama; Hoshino 1 7567 (OKAY) AB206265 AB206280
Japan. Shizuoka Pref, Hikisa ;Kitamura f9548 (OKAY) 4242
Japan, Shizuoka Pre£, Hamamatsu; Kitamura iY556 (OKAY)
S. wallichii (Nes sT), Koyama 72 (Iwasa &ki
JapanK.agawaPref,NakatadoH;inahara 18332(OKAY) Ueki1979) AB206266 AB2062Sl
et al,
S, × trtupezoideus (Koidz .H)ayasaka & H. Ohashi 43
Japan ,Miyagi Pref, Sendai ;Hdyasaka 24S3 (TUS)
S, × uzenensis (T .Koyarna) Hayasaka & H. Ohashi 58
japan ,Fukushima Prefi S,oma; Hayasaka 2309 CTUS)
Sect.Malaeegeton

S. nmponicus (Makin oSo)jfik 76 (knaka
Japan ,Hokkaido Pref. T,omakomai; Hoshino et aL 74 1948) AB206267 AB2062S2
i9265-19267(OKAY)
Sect.Schoenoplectus

S.tabernaemontani (C.C.Grnel.)Palla 40,42,44

Japan ,Okayama Pref, Bizen; Katayama 1 7660 (OKAY) (Otze 1n962, AB206268 AB206283
Japan ,Kagoshima Pre£, Nase; kusaka 18968 (OKAY) 42 Scbuyler1976,
Hoshino et al.
1993,Smith

2002.)
S.triqueter(L.P)alla 40,41,42,

Japan O,kayarn aPre£, Maniwa; Fintru t&a ikeda i31 72 (OKAY) 42 44 (Tanaka
1948,Otzen
Japan, Okayama Pref. M,aniwa; thtrut aet al. 15415 (OKAY) 1962, Bir et AB206269AB2e6284
aL 1991,
Hoshine et al.
1993, Smith

2002.)
Outgroups
Butbostylis
B. barbata Kunth
Japan, Okayama Pref, Okayama; Hbshino et aL 13779 (OKAY) AB180718 AB206285
Flmbristylis
E ovata (Burm £.) Kem
Japan, Kanagawa Prefi M,iura; lhno 182i8 (OKAY) AB180719 AB2062g6
7)'ichophorum
T aipinum (L )Pers.
Japan, Hekkaido Pref. ,Nakagawa; Sato 13260 (OKAY) AB206270 AB2062g7
NII-Electronic Library Service
TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
18g APG Vbl. 56
loca lbootstrap probabilit i(eLsBPs 9;/a ()Adac h&i (1 :3) for over 6 hr at -20 OC. The anthers were
Hasegawa 1996) were applied to each best topolo- stained in 1% aceto-orcein and then squashed.
gy･
Results
Cliromosomeobservations
Fourteen species ofthe genus SZrhoenoplec tcouls- Seguence analyses
lecte dfrom Japan were used for karyomorphologi-The ITS sequences ofthe 16 species, includin tghe
cal observations. Vbucher specimens are liste idn three outgroups, ranged in lengt hfrom 375 to 462
Tabl e1 , Somati cchromosomes ofthe genus Schoeno- bp. Aligned sequences were 514 bp, ofwhich 342
plectu swere observed in the meristematic cells ef sites were used fbr the phylogenet iacnalyses. The
root tips or shoots. The root tips or shoots were data matrix contained 273 variable sites, ofwhich
inO.O02M 8-hydroxyquinolfionre5hr 188 infbrmative.
pretreate d at were potentiall pyhylogeneticall y
16 OC or fbr 1 hr at 23 OC and 15 hr at 4 OC. They [[he ETS 1fsequences ofthe 16 species, includ-
were fixe din acetic alcohol (1 :3) fbr over 16 hr at ing the three outgroups, ranged in ]engt hfrom 551
-20
eC or for 1.5 hr at 23 OC, stained by Feulgen's to 65 1 bp. A!igned sequences were 736 bp, ofwhich
nuclear reaction, macerated in a mixture of 2% 5l4 sites were used fbr the phylogenet iacnalyses.
pectinase and 2% cellulase for 1 hr at 37 OC, The data matrix contained 395 variable sites, of
restained in l% aceto-orcein, and squashed, Meiotic which 279 were potentiall pyhylogenetical] yinfbr-
in
chromosemes were atso observed pollen mother matlve,
cells (PMCs) .Spikele twesre fixe din acetic alcohol In phylogenetic study using nrlTSfETS
Section
Actaeogeton
38

]Schoenoplectus
- Malaeogeton

-
O.1substitutionsfsite
FtG.1, Total ML tre eofthe ITS and ETS 1fsequenc iens Japanes eSichoenoptect t(tHsKY85 model; ln L=-4738.1 by NucML). Local
bootstra pprobabilit j(eLsBP ;%) more than 50% arc shown above branche s(ITSIE T1SD. The intragener iclcassification by Smith
& Hayasaka (2001 i)s shown on the right T.he numbers after each taxon are chremosome numbcrs (2n )an,d * showcd previously
reported.
NII-Electronic Library Service
TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
August 2005 YANO & HOSHINO: Molecular phylogeny of Schoenopleetu s(Cyperaceae) 189
section
9 S S wallichii 72"
ss Sjuneoides 74
8 S S lineolat u7s
S 2 S m"ttisetus 70
S l S gemmifler 76
7 9 S triang"lat u4s2 Actaeogeton
84 S mucronatus 3S
Schoenoplectus
)
-Malacogeton

-
O.1substitutionstsite
=
FiG .2. The bcst NueML tree by ITS data includin tghe 3 publishe dspecies of the genus Schoenqplect" (sHKY85 model; crfB 5.31S;
ln L= r 1817.16± 73,67) .Local bootstra pprobabiliti (eLsBP; %) more than 50% are shown above branches .The intragene¢ri
classifications by Smith & Hayasaka (200 1ar)e shown on the right T.he nurnbers after cach taxon are chromosome numbers (2n),
and ' showed prcyiousl yrcported. GenBank accession numbers are also to the righ otfeach taxon name.
sequence data ,it is necessary to consider the pres- detaile tdopologie swere searched for through the
ence of ITSIETS intraspecif ipcolymorphisms p,ar- obtained trees using log-likelihoo dmeasure
alogous copies of ITS!ETS in the genome, and (NucM Land CONSEL O. 1D. The highes tlog-1ike-
pseudogene copies of the nrDNA repeat (Roals &on lihood tree for ITS and ETS 1fwas obtained, as in
Friar 2004). However, Roalson & Friar (2004)Fig. 1. Phylogeneti canalyses ofthe Japanese species
reported that paralogou asnd pseudogene copies of revealed two major clades in the genus Schoeno-
ITSfETS are not having a major infiuenc eon the plectus (Fi g1.) :(1 )all sampled species of section
topology of the ITSI ETS phylogeny in Cdrex. In Actaeogeton (100 %LBP value), and (2 )sections
this study, ITSfETS sequences were not cloned, A/latacogeto nand Sbhoenoplectus (10019 s9up%-
because polymorphic sequences which show paral- port in [[bt aMlL tree).
ogous copies ofthe nrDNA were not fbund. Aligned sequences of the ITS region fbr 19
species, including 3 North American and 13
Pitylogeneti canalyses Japanes eSthoenoplectus species and 3 outgroups,
For phylogeneti acnalyses, a total of nine (ITS) were 514 bp, ofwhich 332 sites were used for the
and ten (ETS 1f) nen-overlapping topologie swere phylogenetic analyses. The data matrix contained
obtained from the MP and ML methods. More 277 variable sites, of which 170 were potentially
NII-Electronic Library Service
The JJaapapnaesneese Society ffoorr　 PPllanatnt　 SSyystsetmaetmloastics
APG Vol．56
ア
　 ． 響 機 轢 　 レ 　　　
．
磯叢　 。煽 　蔵 舞　　　
　 掩 濕 ．コづ鋤 　　　
　　　 蔚  講先門b 、纛。ξ　．パみ 鵡 諺績演， 　ホ　ダド　　　
， 　 慧 灘 慰 　　
　A 　 マ母幽ド　ゐ 　の　り　　 　　・：1　 B ．紬愚 ・ ・卿．、もぬ 鱒 》’　　だ　　
蕣 　
“
　 −、輾 婆モ、
　 讐
ズ　　　　げ　　　　　　　　ら　 ’ll 訴野。−、瀦灘爭 鸚　繕魂1
藩
鑽 　 　　 ．欄．。麗黔 轟 囁 鉱 ド
響
　　　 蠣 欝
驫

響　をオサ鍬 　　　　　　　　蠣覊

　むきほ　　　　ぎ　　　　 乱，讌 翁謙瓠
　　　　　　ロ　　　　　ニずし難 熱回 i3tttt
　 　 。
　 磯 ・
　 蕚
タ 聴
　 、
　　　 L ．内印♂
　　へ濾

　 ，、 ・バ『 ＾
　　 　 　 鱒 　　 ム ダ 　
灘 　 1鐙鸛

ぎ

　　タ N 　o　　ド織　1 ∴ ・T　．錘漁 蘿噂
13 1
　　　ぐ 蟹軍欝 〜
へ　　　　’
毒ご．：ド無譲 　　　　　 　　　　曽．・s’1き械野げ四説　凝・嶺1r囁諄　，・　〜、　，　・夢芒嫁　ゴt囁1｛彜t 　　彫　　 　’　・響」薪あ／〆．1；・、． 　 鷺選’，　tU　　　，：灘　　　　　1　　覧　　　　　じ　　　　，到
P 　ハ’　　　　 爆歩 Q．、 ∴：塾ザ靴’窮・ ∵ 羅，ぴき1　　’　，　　　 5μm
　 　 　 で　
FIG．3．　Photomicrograph　sof　somatic　metaphase 　chromosomes （A−0）and 　melotlc 　metaphase 　I　chromosomes （P−R）Df　the　genus
　　　＆フhoen〔）pte覦 ts．　A−J，　N一民Sectie　nActaeogeten ．　K，　Section　Malacogeton．　L，　M ，　Sectie　nSehoenoplectt．　tAs，　SchoenoplecnS加ル
　　　　　　　　　d∫Go．，e如∫n．bsmeiulrst（ni2sane・＝召m齠3o8脚）（．　2B頗n，；　S72．0　nl）＝t，　4oH2m，　）aS．　r．Moノuv，nic∫（oi2．i　dnten・s・g3u8et）（．2e　Cηi・＝，（7324n，　）m≡，t41｛2，　eSr）o．．　nN伽at，usθ801α×（2’麗nt5r＝（a32p8nez）＝．o　7Di4d．）e　」Eus，，　3S〔，2　．　hng＝oem4t3a履）r．fu0（ei2，r〔∫n2＝n4×；27，t64t）z4．　e）nK．e　nF，　s，Si8s．　tnii（p2’lρnano＝gn5uitc8aut）u，．　ss・P（，25刀（，　＝2hn7o4＝’）a4，r　2uLi），
　　　（2n＝・42＝21夏エ）．　Q，　s．　komaroレii（2n＝38＝19【1）．　R，5．　lineo’atus （2n＝74＝37H）．
一
NNI工I工-EElleoetcrotnlroonic　 LLlibrbarryary 　Service
TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
August 2005 YANO & HOSHINO: Molecular phy]ogeny ofSchoenoplectus (Cyperaccae) 191
phylogenetical liynformativ eT.he ML tree, obtained two individua flrsom two tocaliti Se. st,riangutatus
from phylogenetic analyses using the 19 species, was 2n=42 in 51 cells of three individua lfsrom
als oshowed two major clades in the genus Sbhoeno- three localiti eansd, S gemmijZi wras 2n=76 in 28
plectus (Fi g2.): (1 )all sampled species of section cells oftwo individua lfrsom two localitie Isn. this
Actaeageton with 1OO% bootstra spupport, and (2) study, mixoploids were not found in thesc three
th erest of5trhoenoplectus with 999' 6bootstra spirp- speciesofSchoenoptectus.
port. In our study, two kinds of putative natural
hybrids S,choenoplect u×s tr[rpezoideus andS ×
C7iromosomeobservations uzenensis, were found .The chromosome number of
Cytologic asltudies were conducted in 14 species. S. × trapezoideus was 2n;43 and S. × uxenensis
Chromosome numbers were observed te be 2n=38 was 2n=58, These chromosome numbcrs were
fbr Sbhoenoptect um"scronatus, 2nX2 fbr S. taber- determine dfbr the firs ttime in this study. The 43
naemontani, 2n=42 fbr S triangtilatus ,2n;42 for S metaphase chromosomes of S. X trapezoideus
trigueter ,2n=44 for S hotani ian,d 2n==7 4for S .iun- ranged from O.7 to 1.3 pm in iength, and 58
([Ilanaka
coides, which confirrn previou sreports metaphase chromosomes efS. × uzenensis ranged
1948, Iwasaki & Ueki 1979, Hoshino et al. 1993, from O.5 to 1.3 ptm in length.
Smith2002,Maeda& Uchino2004)([Ila1b).lThee

chromosome numbers of 2n==38 for S. hondoensis, Discussion
2n-38=19I Ifbr S komarovi ian,d 2n=70 for S mul-
tisetu swere determine dfbr the firs ttime in this Innageneric nelationships and morphological char-
study. New chromosome numbers observed in this actersSmith
study were 2n=42=21T ifor S. hotarui 2,n=74=37u & Hayasaka (2001 r)ecognized four sections
fbr S. Iineolatu s2,n==7 4fbr S. nipponieus, and Uctaeoget oMani,acogeton, Scheenoplectus and
2n-76 for Sl gem,nijZi (rFi g3., Tabl e1) ,IntraspecifiScupini )in the genus Schoenoplectu sIn. their clas-
aneuploids were found to be 2n=42 and 44 in S. sification, the omarnent and color of achenes, and
hotaru iI,isi mjaojor groups were recognized by the the typ eofflora1 scale apices, are used as the major
fbllowin gkaryomorphologic aolbservations: (1) characters that separate sections ActaeQgeto nand
low chromosome numbers, includin sgpecies with Sbepi nfirom the other two sections. in species of sec-
2n=38=:19ii ,2n=42=2Iii and 44, and where somat- tions Actaeageto annd Shrpi ntihe achenes are rugu-
ic chremosomes ranged from O.7 to 1.3 gem in lose and blackis hwhen ripe and the flora lscale
lengt hand meiotic metaphase I ranged from 1 ,8 to apiccs are entire, but in speeies ofsections Addlaco-
2.8 ptm in length a,nd (2 )high chromosome num- geton and Schoenoptectas the achenes are smooth
bers, includin sgpecies with 2n=70, 2n==74=37 ianid and yellow-dar kbrown when ripe and the floral
76, and where somatic chrontosomes ranged from scale apices are emarginate to bifi dor very obscure.
O.5 to 1. 1ym in length ,and meiotic metaphase 1 The veins and apices of fiora lscale apices, rhi-
chromosornes ranged from 1 ,2 to 2.2 gm in length zomes and tubers are importan ctharacters to sepa-
(Fi g3),. rate sections Sehoenoplect uasnd Matacogeton. In
Maeda & Uchino (2004 r)eported a continuous section imlacageton f,lor asclales have 2-10 promi-
series ofmixoploids in root-tip cells: 2n==32-39 for nent veins and are very obscure apices, the rhi-
Schoenoplect umsucronatus, 2n=37-44 fbr S. tri- zomes are weak or soft and tubers are present,
angulatas, and 2n=68-76 fbr S. gemmijbr, In our whereas in section Schoenoplectus the veins are
results, S. mucronattts was 2n=38 in 39 cells of ahscn tand apices are emarginate to deqply bifi drh,i-
NII-Electronic Library Service
TThhee JJaapapnaesneSeosciee tySociety ffoorr PPllanatnt SSyystsetmaetmicastics
192 APG Nbl, 56
zomes are strong and tubers are absent. From the low chromosome numbers (2n== 3482 ,and 44).
ML tree (Fig s1, .2), sections Actaeogeton and Sections imlacageton and &]hoenoplectus also had
Sthoenoplectus are monophyletic with a high sta- both high chromosome numbers (2n=: 6648,, 74
tistica lconfidence (100 9,7f98, and 739t 6LBP). and 78) and low chromosome numbers (2n==42),
Sectio nMdlacogeton i,ncludi nognly one species in Intraspecifi caneuploids were reported in
this study, clearly divide dfrom section SOhoeno- Schoenoplectu lsacustr i(s2n-3 480, and 42), S
plectus .These molecular phylogenetic data sup- tabernaemontani (2n=4 04,2 and 44), and S tri-
port intrageneri crelationships in genus Schoeno- queter (2n=4 041,, 42 and 44) ([Ila n1a9k38a ,1948,
plectusa,s definedby Smith & Hayasaka(200l).Otzen1962,Schuyler1976,Bir 1991,Hoshino
et al.
Moreover ,our study reinforces that the ornament et at. 1993, Smith 2002). In this study, we founded
and color of achenes, the type of flora slcale, rhi- intraspeci afnieucploids in S. hotaru i(2n=4 424,),
zomes and tubers are importan mtorphological char- and in S. gemmijler and S nmponicus (2n-7 47,6)
acters. (Fi g3,, [Ilab 1l)e ,The origin of intraspecif iacneu-
Pignott i& Mariotti (2004 )identifi etdwo ploid sis thought to be by chromosome mutation
groups in genus Schoenoplectu sbased on the (Yan eet al, 2004). Hoshino et al. (200 0r)eported
micromorphology of fi;u siutrface and pollen size: tha taneuploids in Cyperacea eare thought to arise by
(1 )species of section Schoenoplectus, and (2) chromosome fusio nor fragmentation .Practically,
species compnsing sections Actaeogeton and Supini intraspeci afneiucp]oids are common in members of
(sens Sumith & Hayasaka 2001), The cells of the Cyperacea eplant swith difiUs ceentromeric chro-
pericarp in species of section Schoenqplectus are mosomes, In this study, intraspeci faniecuploids
roughly hexagona land isodiametrica la,nd pollen may also arise by chromosome fusio nor fragmen-
grain sin these species are longe rthan 40 ym, but tation, be¢ause genus Schoenoptectus possesses
the cell of the pericarp in species comprising sec- diffus ceentromeric chromosomes.
tions Actaeogete nand Supini are extremely nar- Schoenoplectus × trapezoideus and S. ×
row, longitudina lolblyong, arranged transversely, uzenensis are putativ neatural interspeci hfyibcrids
and polle ngrain sare shorter than 40 ym, Our phy- based on morphological data (Koyam a1958,
logenet itcrees show strong support for monophyly Hayasaka & Ohashi 2000) .Sthoenoplect t×{ bsupe-
of sections Actaeageto nand Sbhoenoptectus (Figs.zoideus is considered the natural hybridizati oorn
1, 2). Therefore m,icromorpholegy of frui sturface introgress ioofnS hotaru iand S, triangulatu sa,nd
and pellen size may be also usefu1 characters for the S, × uzenensis is considered the natural hybridiza-
classification ofthe genus Schoenoplectus. tion or mtrogression of S. triangulatus and S line-
olatus. In our study, S. × trapeioideus showed
Kai:yompe and chromosomal evotution 2n43, and the putativ pearent sof this hybrid were
The genus SZrhoenqplec tcuasn be classified into S hotaru i(2n;4 4an)d S triangulatu (s2n:= 4S2 )×.
two major groups based on karyotyp e(Fi g3.): (1) uzenensis showed 2n=58, and the putativ pearents
species with high chromosome numbers (2n=70,were S. triangulatus (2n== 4an2d) S, iineolatus
74 and 76) ,and (2 )species with low chromosome (2n=74 T)h.ese two hybrid shad intermedia tchero-
numbers (2n=3 84,2 and 44). When these kary- mosome numbers of both putative parents .Our
omorphological data and previou sreported chro- cytological results supported that these two hybrids
mosome numbers are superimposed on the ML tree were arisen by the natural hybridizati oorn intro-
(Fig s1., 2) ,section Actaeogeton had both high gressio nF.urther analysis ofthe meiotic configura-
chromosome numbers (2n-7 072,, 74 and 76) and tion sis needed to detemiin tehe parent osf thes enat-
NII-Electronic Library Service