全 文 ：A Preliminary Analysis of Phylogenetic Relationships in Cymbidium
(Orchidaceae)Based on nrITS Sequence Data
ZHANG Ming_Yong1 , SUN Cai_Yun1 , HAO Gang1 , YE Xiu_Lin1＊ , LIANG Cheng_Ye1 , ZHU Guang_Hua2
(1.South China Insti tute of Botany , The Chinese Academy of Sciences , Guangzhou 510650 , China;
2.Missouri Botanical Garden , St.Louis , MO 63166_0299 , USA)
Abstract:　The infrageneric classification currently in use for Cymbidium is based on gross morphology , with
emphasis on the number of pollinia and state of fusion between lip and column.The sequences of nrDNA re-
gions of 27 species and 3 cultivars of Cymbidium and 3 outgroup species(Eulophia graminea , Geodorum den-
siflorum , Amitostigma pinguiculum)were analyzed using PCR amplification and direct DNA sequencing.The
phylogenetic trees generated from maximum parsimony analysis , however , show that the existing division among
three subgenera(subgen.Cymbidium , subgen.Cyperorchis and subgen.Jensoa)should be evaluated with
more data.Subgenus Cyperorchis was not a monophyletic group , with the unexpected nesting of C.dayanum(subgen.Cymbidium)within it;subgenus Jensoa also appeared paraphyletic , with C.lancifolium being the
sister group to the remainder of the genus;species of subgen.Cymbidium appeared polyphyletic , being split
into several clades and intermixed with the main subgen.Cyperorchis and subgen.Jensoa clades , respective-
ly.However , because of the insufficiency of informative characters of ITS sequences , some of the clades
identified , especially the major lineages of Cymbidium , received relatively low support;sectional delimitations
were also not clear within each subgenus.Further study is needed for achieving a robust phylogeny of Cymbidi-
um.
Key words:　Cymbidium ;Orchidaceae;phylogeny;ITS
　　Cymbidium , the most important orchids in com-
merce , consists of c.48 species[ 1 ,2] .It is widely dis-
tributed in southeast Asia , from northwest India to Japan ,
and south to Australia , with the species diversity center in
N E India , S W China , Indo_China and Malesia.Cym-
bidium species were among the earliest to be cultivated ,
being primarily grown for their attractive and fragrant
flowers.With a few large flower species as parents , some
commercially important hybrids have been produced over a
hundred years.
In Dressler s[ 3] framework of Orchidaceae , Cymbid-
ium is placed in Cymbidieae of Vandioideae , which con-
tains all of the sympodial vandoid orchids mostly with two
pollinia.The generic delimitation of Cymbidium has been
controversial , largely due to the different emphasis of the
character variation of pollinia and lip (Table 1).
Schlechter s[ 4] complete revision of this group of plants is
the basis of the modern infrageneric classification of Cym-
bidium although two genera , Cymbidium and Cyper-
orchis , were recognized in his system.Significantly he
emphasized the fusion of the base of lip and the base of
column as the distinguishing feature of Cyperorchis , rather
than emphasizing the pollinarium and pollinium shapes as
did Blume
[ 5-7] , Reichenbach[ 8] and Hooker[ 9] .
Schlechter
[ 4]
also proposed several sections for both gen-
era , most of which are still being used now somewhat in
their original form.Hunt[ 10] reduced Cyperorchis to within
Cymbidium and maintained Schlechter s sectional divi-
sions.Seth and Cribb[ 11] started to use subgenus concept
and three subgenera were proposed mainly based on the
number of pollinia and state of fusion between lip and col-
umn:subgenus Cymbidium with two pollinia and free lip ,
subgenus Cyperorchis with two pollinia and fusion of the
lip and column_base , and subgenus Jensoa with four
pollinia and free lip.This system was slightly modified by
Du Puy and Cribb[ 1] .
In the present study a phylogenetic analysis of Cym-
bidium has been undertaken based on the sequences of the
internal transcribed spacer region(ITS)of nuclear riboso-
mal DNA.ITS sequences have been widely used to infer
phylogenetic relationships among closely related genera
and species[ 12] .The phylogenetic relationships formulated
were evaluated against the existing supraspecific classifi-
cation proposed by Du Puy and Cribb[ 1] .
1　Materials and Methods
A total of 30 taxa of Cymbidium were sampled(voucher specimens deposited in IBSC).Their distribu-
tion , source , and the GenBank accession numbers are
shown in Table 2.Du Puy and Cribb s[ 1] classification of
Cymbidium was followed for the purpose of this phyloge-
netic analysis.Two species from Tribe Cymbidieae , Eu-
lophia graminea , Geodorum densiflorum , and one from
Tribe Orchideae , Amitostigma pinguiculum , were
Received:2001-08-07　Accepted:2001-10-15
Supported by the National Natural Science Foundation of China(39870086), the Natural Science Foundation of Guangdong Province(960467)and the President
Foundation of The Chinese Academy of S ciences(767).
＊Author for correspondence.E_mail:.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2002 , 44(5):588-592
Table 1　Comparison of supraspecific classifications proposed for Cymbidium (modified from Du Puy and Cribb[ 1])
Blume[5-7] Schlechter[4] Seth &Cribb[11] Du Puy &Cribb[ 1]
Cymbidium Sw. Cymbidium Sw. Cymbidium Cymbidium
subgen.Cymbidium subgen.Cymbidium
　sect.Eucymbidium 　sect.Cymdidium 　sect.Cymbidium
　sect.Borneense
　sect.Himantophyllum 　sect.Himantophyllum 　sect.Himantophyllum
　sect.Austrocymbidium 　sect.Austrocymbidium 　sect.Austrocymbidium
　sect.Floribundum 　sect.Floribundum
　sect.Suavissimum
　sect.Bigibbarium 　sect.Bigibbarium 　sect.Bigibbarium
Cyperorchis Bl. subgen.Cyperorchis subgen.Cyperorchis
Iridorchis Bl. 　sect.Iridorchis 　sect.Iridorchis 　sect.Iridorchis
　sect.Eburnea 　sect.Eburnea
　sect.Annamaea 　sect.Annamaea 　sect.Annamaea
Cyperorchis Bl. 　sect.Eucyperorchis 　sect.Cyperorchis 　sect.Cyperorchis
　sect.Parishiella 　sect.Parishiella 　sect.Parishiella
Cymbidium Sw. subgen.Jensoa subgen.Jensoa
　sect.Jensoa 　sect.Jensoa 　sect.Jensoa
　sect.Maxillarianthe 　sect.Maxi llarianthe 　sect.Maxillarianthe
　sect.Geocymbidium 　sect.Geocymbidium 　sect.Geocymbidium
　sect.Macrorhizon 　sect.Pachyrhizanthe 　sect.Pachyrhizanthe
designated as outgroups.All samples were collected from
wild in China and cultivated in IBSC.　　Total DNA was extracted from fresh leaves , following
the 2 ×CTAB protocol[ 13] .Nuclear ribosomal internal
transcribed spacers(ITS1 , 5.8S , ITS2 region)were am-
plified using the forward primer ITS5
[ 14]
and the reverse
primer CA26[ 15] .Amplified double stranded DNA frag-
ments were purified using QIAquick Gel Extraction Kit(QIAGEN).Automated sequencing was performed using
fluorescent dye_labeled nucleotides on an ABI 377 DNA
Sequencer , using at least two of the four primers of
N18L18
[ 15] , ITS2 , ITS3 and ITS4[ 14] .
The combined sequences of ITS1 , 5.8S and ITS2
were aligned using Clustal X[ 16] , adjusted manually where
necessary.Maximum parsimony analyses were performed
using PAUP 4.0＊[ 17] with all changes weighted equally ,
using HEURISTIC searches with TBR branch swapping
and 100 random addition sequences.Multiple most parsi-
monious trees were summarized as strict consensus tree.
To assess the relative support for clades found in the par-
simony analysis , bootstrap analysis (BS)was conducted
using 1 000 replicates and the same tree search procedure
as described above except with simple taxon addition.
2　Results
The length of the entire ITS region in the Cymbidi-
um species surveyed ranges from 646 bp to 661 bp , with
ITS1 ranging from 240 to 245 bp , 5.8 S 162 bp for all
species , and ITS2 ranging from 243 to 255 bp.Of the
681 aligned ITS sequence , 132_bp sites are variable with
25 sites being potentially phylogenetically informative.
There was one INDEL (insertion/deletion)of an 8_base_
pair(bp)deletion in the ITS2 region that was observed in
subgenus Cyperorchis and C.dayanum of subgen.Cym-
bidium , but not in other Cymbidium species(aligned ma-
trix not shown).
The maximum parsimony analysis , treating gaps as
missing data , resulted in 33 most parsimonious trees ,
with a length of 272 steps , CI =0.66(excluding unin-
formative characters), and RI=0.79.These trees essen-
tially differ in the internal composition of the clade com-
prising subgen.Cyperorchis and C.dayanum , and the
clade comprising subgen.Jensoa (without C.lancifoli-
um).The strict consensus tree is presented in Fig.1.
There is consistent but weak support for a sister_group re-
lationship between C.lancifolium and the clade contain-
ing all other Cymbidium species (BS<50).The latter
consists of two clades , one including three species of sub-
gen.Cymbidum sect.Floriundum , with high bootstrap
support(BS =99).The other clade comprises two sub-
clades(both with BS<50);one subclade consists of all
species of subgen.Cyperorchis and C.dayanum of sub-
gen.Cymbidium (BS<50), and the other includes two
species of subgen.Cymbidium (C.aloifolium and C.
bicolor)with 100% bootstrap support , which becomes
the sister to the clade containing all species of subgen.
Jensoa except C.lancifolium (BS<50).Poor resolution
is presented for species relationships of both subgen.
Cyperorchis and subgen.Jensoa.
Treating gaps as a fifth state , the maximum parsimo-
ny analysis generated 33 most parsimonious trees , with a
length of 325 steps , a CI of 0.68(excluding uninforma-
tive characters), and a RI of 0.83.The strict consensus
tree is largely congruent with that of treating gaps as
missing data (Fig.1), except that C.erythraeum be-
comes the sister to the clade comprising C.eburneum
CV , C.eburneum , C.mastersii , and C.insigne.(figure not shown).
ZHANG Ming_Yong et al:Analysis of Phylogenetic Relationships in Cymbidium (Orchidaceae)Based on nrITS Sequence Data 589　
Table 2　Taxa sampled in the molecular study of Cymbidium.Supraspecific classification follows Du Puy and Cribb[ 1]
Taxon Distribution
Source;voucher
specimen
GenBank accession
number
C.aloifolium (L.)Sw. Sri Lanka , Nepal , India , China , Malaysia Yunnan;Sun 98_2 AF284695
C.bicolor Lindl.subsp.obtusum Du
　　Puy et Cribb Sri Lanka , Nepal , Indo_China , Burma , Thailand Yunnan;Sun 98_4 AF284696
C.dayanum Rchb.f. India , China , Japan , Philippines , Thai land , Malaysia Yunnan;Sun 98_5 AF284697
C.floribundum Lindl. China , Taiwan Yunnan;Sun 98_6 AF284698
C.pumilum Rolfe China , Japan Yunnan;Sun 98_7 AF284699
C.suavissimum Sander ex C.Curtis Burma , China Yunnan;Sun 98_8 AF284700
C.tracyanum L.Castle China , Burma , Thailand Yunnan;Sun 99_9 AF284701
C.iridioides D.Don Nepal , India , Burma , China Yunnan;Sun 98_51 AF284702
C.erythraeum Lindl. Nepal , India , Burma , China Yunnan;Sun 99_11 AF284703
C.hookerianum Rchb.f Nepal , India , China Yunnan;Sun 99_12 AF284704
C.wilsonii (Rolfe ex Cook)Rolfe China Yunnan;Sun 98_13 AF284705
C.lowianum (Rchb.f)Rchb.f Burma , China , Thailand Yunnan;Sun 99_14 AF284706
C.insigne Rolfe Vietnam , China , Thailand Zhejiang;Ye 15 AF284707
C.wenshanense Y.S.Wu et F.Y.Liu China Yunnan;Sun 98_16 AF284708
C.eburneum Lindl. Nepal , India , Burma , China Yunnan;Sun 98_17 AF284709
C.eburneum Lindl.CV China Hainan;Xin 94_18 AF284710
C.mastersii Griff.ex Lindl. India , Burma , China , Thailand Yunnan;Sun 99_19 AF284711
C.elegans Lindl. Nepal , India , Burma , China Yunnan;Sun 98_21 AF284712
C.tigrinum Parish ex Hook. Burma , India , China Yunnan;Sun 98_22 AF284713
C.ensifolium(L.)Sw. Sri Lanka , India , China , Philippines , Japan Guangdong;Ye 32 AF284716
C.ensifolium(L.)Sw.CV. China Guangdong;Ye 31 AF284717
C.defoliatum Y.S.Wu et S.C.Chen China Yunnan;Sun 99_33 AF284718
C.sinense(Jackson ex Andr.)Willd. India , Burma , Thailand , China , Japan Guangdong;Zhang 98_23 AF284714
C.sinense(Jackson ex Andr.)Willd.CV. China Guangdong;Zhang 98_24 AF284715
C.cyperifolium Wall.ex Lindl. Nepal , India , Burma , Phi lippines , China Yunnan;Sun 99_34 AF284719
C.kanran Makino China , Japan , Korea Guangdong;Ye 37 AF284720
C.faberi Rolfe Nepal , India , China Yunnan;Sun 98_38 AF284721
C.goeringii (Rchb.f.)Rchb.f. Japan , Korea , China , India Yunnan;Sun 99_39 AF284722
C.lancifolium Hook. Nepal , Japan , China , India , Burma , Malaysia , New
Guinea , Ryukyu Islands Yunnan;Sun 99_47 AF284724
C.lianpan Tang et Wang China Yunnan;Sun 99_43 AF284723
Eulophia graminea Lindl. Nepal , India , Burma , China Yunnan;Sun 99_62 AF284726
Geodorum densiflorum (Lam.)Schltr. Sri Lanka , India , China , Philippines , Japan Yunnan;Sun 99_63 AF284727
Amitostigma pinguiculum (Rchb.f.
　　et S.Moore)Schltr. India , Nepal , China
Yunnan;Sun 99_61
(cultivated) AF284725
3　Discussion
Level of variability was low across all accessions of
Cymbidium species in the ITS region studied.The limited
number of parsimony_informative characters resulted in
relatively weak support for some of the clades identified(especially for the major lineages of Cymbidium) and
several conclusions discussed below should be tested with
additional data.
The infrageneric classification currently in use for
Cymbidium is based on gross morphology , with emphasis
on the number of pollinia and the state of fusion between
lip and column.The ITS tree , however , shows that such
subgenus delimitations
[ 1]
should be evaluated with more
data.Subgenus Cyperorchis is not a monophyletic group ,
with the unexpected nesting of C.dayanum (subgen.
Cymbidium)within it.Cymbidium dayanum , represent-
ing sect.Himantophyllum , is placed in subgen.Cym-
bidium with two cleft pollinia and without any fusion be-
tween the lip and base of the column.It is distinctive in
subgen.Cymbidium and superficially resembles some of
the species in subgen.Jensoa , especially in the vegeta-
tive habit and the slender , acute , arching leaves[ 1] .The
systematic position of C.dayanum clearly merits addi-
tional study.
Cymbidium tigrinum , the single , highly distinctive
species representing sect.Parishiella , is sister to the re-
mainder of subgen.Cyperorchis and C.dayanum , al-
though with low bootstrap support(<50%).It is quite
different from other Cymbidium species , especially vege-
tatively , but possesses all the diagnostic characters of
subgen.Cyperorchis.It probably has close affinity with
sect.Iridorchis , to which the pollinarium shape closely
resembles[ 1] .
590　 植物学报　Acta Botanica Sinica　Vol.44　No.5　2002
Fig.1.　Strict consensus trees of the 33 most parsimonious trees from the entire ITS sequences with gaps coded as missing.
Tree length , 272 steps;CI , 0.66;RI , 0.79.Numbers above lines represent bootstrap values in 1 000 replicates.Supraspecific classification
follows Du Puy and Cribb[ 1] .
　　Subgenus Jensoa also appears paraphyletic in the
ITS tree (Fig.1), with C.lancifolium becoming the
sister group to the remainder of the genus , although with
low bootstrap support (<50).Cymbidium lancifolium ,
the single species comprising sect.Geocymbidium , is the
most widespread species in the genus and highly distinc-
tive in vegetative characters.Its habit and flowers indicate
a close relationship with the saprophytic species C.
macrorhizon of sect.Pachyrhizanthe , which lacks leaves
and chlorophyll and is apparent only when in flower[ 1] .It
is speculated from the early divergence of C.lancifolium
that ancestors of Cymbidium species diversified with two
paths , one leading to the main epiphytic or lithophytic
species , the other into the saprophytic ones.Apparently ,
the addition of the saprophytic species of sect.
Pachyrhizanthe will be invaluable to verify this hypothesis
of evolutionarily diversifying paths.
Two species of sect.Cymbidium , C.aloifolium
and C.bicolor , constitute the sister group to subgen.
Jensoa (without C.lancifolim).The splitting feature of
subgen.Cymbidium apparent in the ITS tree suggests that
subgen.Cymbidium is polyphyletic.As implied above ,
the number of pollinia is usually considered to be a con-
servative character and has been employed for the infra-
generic classification of Cymbidium by several au-
thors[ 1 , 4 , 11] .Nevertheless , this criterion may not be
general:the Borneo species C.borneense of sect.
Borneense has four pollinia , but has more other diagnostic
characters and is more reliably to be placed in subgen.
Cymbidium.No matter whether the two or four pollinia
are advanced , state transformation of this character may
have occurred more than once in Cymbidium .
In conclusion , the tree generated from ITS sequences
is only partially congruent with the current taxonomic clas-
sification of the genus.The removal of subgen.Cyper-
orchis and Jensoa from Cymbidium is not justifiable , con-
sidering the fact that species of subgen.Cymbidium are
nested within both subgenera.On the other hand ,
ZHANG Ming_Yong et al:Analysis of Phylogenetic Relationships in Cymbidium (Orchidaceae)Based on nrITS Sequence Data 591　
eventually taxonomic changes may be needed to better re-
flect relationships in Cymbidium.However , because of
the insufficiency of informative characters of ITS se-
quences , this study does not permit unambiguous place-
ment of the major lineages of Cymbidium ;sectional limits
are also not clear within each subgenus.Clearly , more
data , especially more sources of evidence are required be-
fore a new taxonomic delimitation can be formalized for
Cymbidium.
References:
[ 1 ] 　Du Puy D , Cribb P J.The genus Cymbidium.Poregon:
Timber Press , 1988.1-236.[ 2] 　Chen S_C(陈心启).Cymbidium.Chen S_C.Fl Reipubl
Popularis Sin(中国植物志).Beijing:Science Press ,
1999 , 18:191-227.(in Chinese)[ 3 ] 　Dressler R L.The Orchids:Natural History and Classifica-
tion.Cambridge:Harvard University Press , 1981.1 -
332.
[ 4] 　Schlechter R.Die Gattungen Cymbidium Sw.und Cyper-
orchis Bl.Fedde , Repertorium Species Novarum , 1924 ,
20:96-110.
[ 5 ] 　Blume C L.Orchideae.Rumphia , 1848 , 4:38-56.[ 6 ] 　Blume C L.Cyperorchis.Mus Bot Lugduno_batavia , 1849 ,
1:48.[ 7 ] 　Blume C L.Collection des Orchidees les plus remarquables
de larchipel Indien et du Japon.1858 , 1:90-93.[ 8 ] 　Reichenbach H G.Orchidaceae (Cymbidium and Cyper-
orchis).Walpers Ann , 1852 , 3:547-548.
[ 9] 　Hooker J D.Fl British India , 1891 , 6:8-15.[ 10] 　Hunt P F.Notes on Asiatic Orchids 5.Kew Bull , 1970 ,
24:93-94.[ 11] 　Seth C J , Cribb P J.A reassessment of the sectional limits
in the genus Cymbidium Swarta.Arditti J.Orchid Biology ,
Reviews and Perspectives.Ithaca:Cornell University Press ,
1984 , 3:283-322.[ 12] 　Baldwin B G , Saunderson M J , Porter J M , Wojciechowski
M F , Campbell C S , Donoghue M J.The ITS region of nu-
clear ribosomal DNA:a valuable source of evidence on an-
giosperm phylogeny.Ann MO Bot Gard , 1995 , 82:247-
277.[ 13] 　Doyle J J , Doyle J L.A rapid DNA isolation method for
small quantities of fresh tissues.Phytochem Bull , 1987 ,
19:11-15.[ 14] 　White T J , Bruns T , Lee S , Taylor J.Amplification and di-
rect sequencing of fungal ribosomal RNA genes for phyloge-
netics.Innis M , Gelfand D , Sninsky J , White T.PCR Pro-
tocols:A Guide to Methods and Application.San Diego:
Academic Press , 1990.315-322.
[ 15] 　Wen J , Zimmer E A.Phylogeny and biogeography of Panax
L.(the ginseng genus , Araliaceae):inferences from ITS
sequences of nuclear ribosomal DNA.Mol Phylogen Evol ,
1996 , 6:166-177.[ 16] 　Thomson J D, Gibson T J , Plewniak F.The Clustal X win-
dows interface:flexible strategies for multiple sequence
alignment aided by quality analysis tools.Nucl Acids Res ,
1997 , 25:4876-4882.
[ 17] 　Swofford D L.PAUP＊4.0.Phylogenetic Analysis Using
Parsimony(and other Methods).Sunderland:Sinauer As-
sociates , 1998.
基于 nrDNA ITS序列数据的兰属系统发育关系的初步分析
张明永1　孙彩云1　郝　刚1　叶秀 1＊　梁承邺1　朱光华2
(1.中国科学院华南植物研究所 , 广州 510650;2.密苏里植物园 , 圣·路易斯 , MO 63166_0299 , 美国)
摘要:　现存的兰属分类系统是基于宏观形态学性状 、尤其是花粉块的数目以及唇瓣与蕊柱的愈合程度而建立的。
兰属因此而划分为 3个亚属:兰亚属(subgenus Cymbidium), 大花亚属(subgenus Cyperorchis)和建兰亚属 (subgenus
Jensoa)。本文运用 PCR扩增和直接测序的方法分析兰属 (Cymbidium)27 种 、3 个栽培品种以及 3 个外类群的核
DNA ITS 区段序列。通过最简约性分析产生的 ITS 系统发育树表明 ,兰属的 3个亚属均可能为不自然的类群。大花
亚属表现为一复系群 , 兰亚属的冬凤兰(C.dayanum)隐藏于其中;建兰亚属为一并系群 , 它的成员之一兔耳兰
(C.lancifolium)偏离出去而成为兰属一最基部的分支;兰亚属为一复系群 , 它分为几支而分别与另两个亚属组合
在一起。由于兰属 ITS 序列位点变异率较低 ,最简约性分析产生的几支主要分支均得不到 Bootstrap 分析的高度支
持 ,各亚属内组之间的关系也不明确。研究兰属的系统发育关系还需要新的数据。
关键词:　兰属;兰科;系统发育;ITS
中图分类号:Q941+.2;Q949.71+8.43　　　文献标识码:A　　　文章编号:0577-7496(2002)05-0588-05
收稿日期:2001-08-07　接收日期:2001-10-15
基金项目:国家自然科学基金(39870086);广东省自然科学基金(960467);中国科学院院长基金(767)。
＊通讯作者。E_mai l:.
(责任编辑:梁　燕)
592　 植物学报　Acta Botanica Sinica　Vol.44　No.5　2002