Bull. Natn . Sci . Mus., Tokyo, Ser. B, 28(4), pp. 129- 139, December 20, 2002

Molecular Phylogeny and Character Evolution ofCymbidium (Orchidaceae)
Tomohisa Yukawa 1, Kazumitsu Miyoshi 2 and Jun Yokoyama 3
1

Tsukuba Botanical Ga rden, National Science Museum ,1- 1, Amakubo 4, Tsukuba , 305- 0005 Japan
E-mail : yukawa@ kahaku .go.jp
2
Department of Biological Production, Akita Prefectural University,
Shimoshinj o-nakano, Akita 0 I 0- 01 95 Japan
E-mail: mi .orchids@akita-pu. ac.jp
3
Department of Ecology & Evo lutionary Biology, Tohoku University,
Aramaki , Aoba-ku, Sendai 980- 8578 Japan
E-mail: j yokoyam@mail.cc.tohoku.ac.jp

Abstract

Molecular phylogenetic analyses using two data sets, deri ved from DNA sequences of

matK, the maturase-encoding gene located in an intron of the chloroplast gene trnK, and ITS (inte rnal transcribed spacer) region of the 18S- 26S nuclear ribosomal DNA, were performed to examine rel ationships among 37 taxa in Cy mbidium . Although each data set did not provide conclusive evidence in itself, the data sets combining the two region s yi elded the followin g insights : (I)Cymbidium subgenus Cyperorchis and section Pachy rhizanthe, often treated as independent genera, were nested within the remainin g me mbers of Cymbidium. (2) All the three subgenera of Cymbidium, namely, Cymbidium , Cyperorchis, a nd Jensoa, turned to be para-/polyphyletic. (3) Among
sections of Cymbidium, Austrocymbidium , Cyperorchis, lridorchis, and Maxillarianthe did notshow monophyly. (4) At specific leve l, o ur results indicated the pol yphyl y o f Cymbidium bicolor,although bootstrap supports for the clades including C. bicolor were not robu st.
Key words: Cymbidium , ITS, matK, mol ecular, Orchidaceae, phyloge ny

Introduction
The genus Cymbidium Sw. comprises about 50
species. Geographic distribution extends fromthe northwestern Himalaya to Japan and south
through Indochina and Malesia to northern andeastern Australia. This group exhibits distinctive
ecologica l diversification and occurs as terrestrial
and epiphytic life forms. Moreover, one section,
Pachy rhizanthe Schltr. lacks foliage leaves andhas a strongly mycoparasitic existence.
Swartz ( 1799) established the genus Cy mbidium with a broad generic delimitation. Circumscription of the genus has been variously definedby later workers: Lindley ( 1833), Blume ( 1848,
1849, 1858), Reichenbach (1852 , 1864), Hooker( 1890), Schlechter ( 1924), Hunt ( 1970), Seth &Cribb (1984) , and Du Puy & Cribb (1988). At

generic level , there have been inconsistencies on
the delimitation. For example, Blume ( 1848,
1849, 1858), Reichenbach ( 1852), Hooker( 1890), and Schlechter ( 1924) treated Cyperorchis as an independent genus. Another example is Pachyrhizanthe. As mentioned above, thisgroup shows very distinctive vegetative morphology and nutritional habit, and Nakai ( 1931) thus
elevated Pachy rhizanthe to generic rank .
The first major revision of Cymbidium was undertaken by Schlechter ( 1924) where he proposed sectional limits within the genus . His
framework has been modified by Hunt ( 1970),
and Seth & Cribb ( 1984). So far, the most extensive infrageneric classification of Cy mbidium isthat of Du Puy & Cribb ( 1988), because theirsystem was based on the most recent information
on morphological and anatomical characters.

130

Tomohisa Yukawa et al.

However, satisfying conclusions have not been
affirmed for the infrageneric relationships andcircumscriptions of several species in Cy mbi-

dium.
Recently, DNA sequences of matK, the maturase-encoding gene located in an intron of thechloroplast gene trnK, and ITS (internal transcribed spacer region) of the 18S- 26S nuclear ribosomal DNA have proven to be powerful tools
for phylogenetic reconstruction within angiosperm families and genera (e.g. , Baldwin,
1992 ; Johnson and Soltis, 1994; Steele and Yilgalys, 1994). In this study we compared andcombined DNA sequences of matK and ITS toestablish more concrete relationships in Cymbidi-um.
Material and Methods
In this study we selected 37 representative taxa
from all the subgenera and sections in Cy mbidium. Results of matK and ITS analyses for tribe
Cymbidieae have indicated that the sister groupof Cy mbidium becomes the clade comprising
Acriopsis, Crammatophyllum, and Th ecostele(Yukawa, unpublished). Consequently, these genera were chosen as outgroup of this study. Materials used in the phylogenetic analyses are shownin Table 1. Voucher specimens are deposited at
TNS .
Experimental methods were described in
Yukawa et al. ( 1993, 1996). Sequences were determined by amplifying the matK (including
parts of trnK introns) and ITS regions (including
5.8S rDNA and parts of 18S and 26S rDNA) via
the polymerase chain reaction (PCR) from a total
DNA extract. All of the primers were from
Yukawa et al. ( 1999) for matK and Douzery et al.
( 1999) for ITS . DNA sequences were aligned
manually. Gaps were treated as missing characters. The aligned data file is available from thefirst author upon request. Parsimony and distanceanalyses were conducted with PAUP* version
4.0b6 (Swofford, 200 I). The huristic option wasused to perform Fitch parsimony analyses (Fitch,
1971 ). Distance trees were obtained using theneighbor-joining (NJ) method (Saitou & Nei ,1987) with a Kimura two-parameter correction(Kimura, 1980). To assess the relative robustness
for branches, the bootstrap method (Felsenstein ,1985) was used with 1000 replicates.

Results

Phylogenetic analyses
Figure 1 shows a strict consensus of 28 ,800
most parsimonious (MP) trees derived from thematK sequences. The tree had a consistency
index (Cl) of 0.7726 (0.6115 excluding uninformative characters) and a retention index (RI) of
0.8033. The NJ topology with more than 50%
boots trap support was identical (Fig . 1). A strict
consensus of 325 MP trees on the basis of ITS
data set is shown in Fig. 2. Cl was 0.6848(0.5606 excluding uninformative characters) and
RI was 0.7790. In this data set the NJ topology
with more than 50% bootstrap support was identical (Fig. 2). On the other hand, analyses ofmatK sequences provided results substantiallyconcordant with those achieved via analyses of
ITS sequences except for positions of Cy mbidium erythrostylum and Cy mbidium sanderae. In
such cases, analyses of combined data sets excluding problematic taxa provide more resolution
and internal support for relationships than do theindividual data sets (e.g. , Olmstead and Sweere,
1995). We thus conducted a combined analysis of
the information derived from matK and ITS sequences. In this analysis C. erythrostylum and C.
sanderae were excluded. The length of aligned
sequence was 2829 base pairs. Figure 3 shows astrict consensus of 168 MP trees from the combined data set. Cl was 0.7283 (0 .5931 excluding
uninformative characters) and RI was 0.7788. In
this data set the clades revealed by NJ analyses
with more than 50% bootstrap support was also
identical (Fig. 3).
In the combined analysis, the following insights were provided (Fig. 3): (I) Cy mbidium
subgenus Cyperorchis and section Pachy rhizanthe, often treated as independent genera, were
nested within the remaining members of Cy mbid-

Molecular Phylogeny of Cymbidium
Table 1.

131

Taxa of Cymbidium and outgroups used for sequencing.

Species

VoucherSubgenus Cymbidium
Section Cymbidium

Cy mbidium a/oifolium (L.) Sw.
C. bicolor Lindl. subsp. obtusum Du Puy & P J. Cribb
C. bicolor Lindl. subsp. pubescens (Lindl.) Du Puy & P J. Cribb
C.finlaysonianum Lindl.
C. rectum Rid!.
Section Hymantophyllum Schltr.
C. dayanum Rchb. f.
Section Borneense Du Puy & P. J. Cribb
C. borneense J. J. Wood
Section Austrocymbidium Schltr.
C. cana/iculatum R. Br.
C. chloranthum Lindl.
C. hartinahianum J. B. Comber & Nasution
C. madidum Lindl.
Section Floribundum Seth & P J. Cribb
C..floribundum Lindl.
C. suavissimum Sander ex C. Curtis
Section Bigibbarium Schltr.
C. devonianum Paxton
Subgenus Cyperorchis (Biume) Seth & P J. Cribb
Section lridorchis (Biume) P. Hunt
C. e1y thraeum Lindl.
C. hookerianum Rchb. f.
C. insigne Rolfe
C. iridioides D. Don
C. lowianum (Rchb. f.) Rchb. f.
C. sanderae (Ro1fe) Du Puy & P J. Cribb
C. tracyanum L. Castle
Section Eburnea Seth & P. J. Cribb
C. eburneum Lindl.
C. roseum J. J. Sm.
Section Annamaea (Schltr.) P Hunt
C. e1y throstylum Rolfe
Section Cyperorchis (Biume) P. Hunt
C. elegans Lindl.
C. cochleare Lindl.
Section Parishiella (Schltr.) P Hunt
C. tigrinum Parish ex Hook.
Subgenus Jensoa (Raf.) Seth & P. J. Cribb
Section Jensoa (Raf.) Schltr.
C. ensifolium (L.) Sw.
C. kanran Makino
C. sinense (Jackson in Andr.) Willd.
Section Maxil/arianthe Schltr.
C. aliciae Quisumb.
C.faberi Rolfe
C. goeringii (Rchb. f.) Rchb. f.
C. tortisepalum (Fukuyama) Y. S. Wu & S. C. Chen
Section Geocymbidium Schltr.
C. lancifolium Hook.
Section Pachyrhizanthe Schltr.
C. aberrans (Finet) Sch1tr.
C. macrorhizon Lindl.
Acriopsis liliifo/ia (Koen.) Ormerod
Grammatophyllum scriptum (L.) Blume
G. 5peciosum Blume
G. stapeliiflorum (Teijsm . & Binn.) J. J. Sm.
Th ecostele alata (Roxb.) Par. & Rchb. f.

TBGII8253*
TBGI24328
TBGII8509
TBGI22826
Leiden Bot. Gard. 913298
TBGI27899
Suzuki s.n.
TBGI35991
TBGI27438
Cibodas Bot. Gard. s.n.
TBG119076
TBG127357
TBG 128817
TBGII9073

TBGI35399
TBG 133783
TBG135526
TBG 118251
TBG54943
Hiroshima Bot. Gard. 5138
TBGI24406
Suzuki s.n.
TBGI33724
TBGI26650
Karasawa s.n.
Ohba s.n.
TBGI33781

TBGII8255
TBGI33782
lwagawa s.n.
TBGII9071
TBGI33593
TBGII5919
TBGI33595
TBG56111
Yagame s.n.
Yagame s.n.
Kyoto Bot. Gard. s.n.
TBGII8874
Shinjukugyoen s.n.
TBGI24396
TBGII8540*TBG series indicate accession numbers in living collection database at Tsukuba Botanical Garden.

132

Tomohisa Yukawa et al.

Subgenus

100
97

72
51
98
92
90
84

100
100

93
93

100
99

C.aloifolium
Cymbidium
C.finlaysonianum
CymbidiumC.bicolor subsp.pubescens Cymbidium
C.bicolor subsp.obtusum
CymbidiumC.rectum
CymbidiumC.aliciae
JensoaC.borneense
Cymbidium
C.canaliculatum
CymbidiumC.madidum
Cymbidium
C.dayanum
Cymbidium
C.erythrostylum
Cyperorchis
C.cochleare
Cyperorchis
C.elegans
Cyperorchis
C.sanderae
Cyperorchis
C.insigne
Cyperorchis
C.erythraeum
Cyperorchis
C.iridioides
Cyperorchis
C.lowianum
Cyperorchis
C.hookerianum
Cyperorchis
C.eburneum
Cyperorchis
C.roseum
Cyperorchis
C.tracyanum
Cyperorchis
C.tigrinum
Cyperorchis
C.devonianum
Cymbidium
C.hartinahianum
CymbidiumC.chloranthum
Cymbidium
C.floribundum
Cymbidium
C.suavissimum
Cymbidium
C.goeringii
JensoaC.faberiJensoaC.tortisepalum
JensoaC.ensifolium
JensoaC.kanranJensoaC.sinense
JensoaC.lancifolium
JensoaC.macrorhizon
JensoaC.aberrans
JensoaGrammatophyllum speciosum
Grammatophyllum scriptum
Grammatophyllum stapeliaeflorum
Thecostele alata
Acriopsis lilifolia

SectionCymbidium
Cymbidium
Cymbidium
Cymbidium
Cymbidium
Maxillarianthe
Borneense
Austrocymbidium
Austrocymbidium
Himantophyllum
Annamaea
Cyperorchis
Cyperorchis
lridorchis
lridorchis
lridorchis
lridorchis
lridorchis
lridorchis
Eburnea
Eburnea
lridorchis
Parishiella
Bigibbarium
Austrocymbidium
Austrocymbidium
Floribundum
Floribundum
Maxillarianthe
Maxillarianthe
Maxillarianthe
JensoaJensoaJensoaGeocymbidium
Pachyrhizanthe
Pachyrhizanthe

Fig. I. Strict consensus of 28,800 most-parsimonious Fitch trees based upon matK sequences: length =4 75, consistency index =0. 7726 (0.611 5 excluding uninformative characters), retention index of 0.8033. Numbers
above internodes indicate bootstrap values from I ,000 replicates of Fitch parsimony analysis (maxtree limitof 200 per rep licate). Numbers be low intern odes indicate bootstra p va lues from I ,000 replicates of ne ighborjoining distance analysis.

Mo lec ular Phy logeny of Cymbidium

Subgenus

66
52

100
100

86
89

17
16

39
59

58
52

100
100

C.aloifolium
Cymbidium
C.finlaysonianum
CymbidiumC.bicolor subsp.pubescens Cymbidium
C.bicolor subsp.obtusum
CymbidiumC.rectum
Cymbidium
C.canaliculatum
CymbidiumC.madidum
CymbidiumC.aliciae
JensoaC.borneense
Cymbidium
C.dayanum
Cymbidium
C.cochleare
Cyperorchis
C.elegans
Cyperorchis
C.insigne
Cyperorchis
C.erythraeum
Cyperorchis
C.eburneum
Cyperorchis
C.roseum
Cyperorchis
C.sanderae
Cyperorchis
C.tracyanum
Cyperorchis
C.erythrostylum
Cyperorchis
C.iridioides
Cyperorchis
C.hookerianum
Cyperorchis
C.lowianum
Cyperorchis
C.tigrinum
Cyperorchis
C.devonianum
Cymbidium
C.goeringii
JensoaC.tortisepalum
JensoaC.faberiJensoaC.ensifolium
JensoaC.kanranJensoaC.sinense
JensoaC.lancifolium
JensoaC.macrorhizon
JensoaC.aberrans
JensoaC.hartinahianum
CymbidiumC.chloranthum
Cymbidium
C.floribundum
Cymbidium
C.suavissimum
Cymbidium
Grammatophyllum speciosum
Grammatophyllum scriptum
Grammatophyllum stapeliaeflorum
Thecostele alata
Acriopsis lilifolia

133

SectionCymbidium
Cymbidium
Cymbidium
Cymbidium
Cymbidium
Austrocymbidium
Austrocymbidium
Maxillarianthe
Borneense
Himantophyllum
Cyperorchis
Cyperorchis
lridorchis
lridorchis
Eburnea
Eburnea
lridorchis
lridorchis
Annamaea
lridorchis
lridorchis
lridorchis
Parishiella
Bigibbarium
Maxillarianthe
Maxillarianthe
Maxillarianthe
JensoaJensoaJensoaGeocymbidium
Pachyrhizanthe
Pachyrhizanthe
Austrocymbidium
Austrocymbidium
Floribundum
Floribundum

Fig. 2. Strict consensus of 325 most-parsimonious Fitch trees based upon ITS sequences: length=368, consistency index=0.6848 (0.5606 exc luding uninformative characters), retention index of0.7790. Numbers above
intern odes indicate bootstrap va lues from I ,000 replicates of Fitch parsimony analysis (maxtree limit of 200
per replicate). Numbers below intern odes indicate boots trap values from I ,000 replicates of neighbor-joining
distance analysis.

134

Tomohisa Yukawa er al.

Subgenus
C.aloifolium
Cymbidium
C.finlaysonianum
CymbidiumC.bicolor subsp.obtusum
CymbidiumC.bicolor subsp.pubescens Cymbidium
C.rectum
CymbidiumC.aliciae
Jensoa
100
79
100
72
C.borneense
Cymbidium
C.canaliculatum
Cymbidium
100
100
C.madidum
Cymbidium
C.dayanum
Cymbidium
C.cochleare
Cyperorchis
C.elegans
Cyperorchis
C.insigne
Cyperorchis
C.erythraeum
Cyperorchis
C.iridioides
Cyperorchis
C.lowianum
Cyperorchis
C.hookerianum
Cyperorchis
60
94
C.eburneum
Cyperorchis
100
100
C.roseum
Cyperorchis
C.tracyanum
Cyperorchis
C.tigrinum
Cyperorchis
Cymbidium59r--------------- C.devonianum
81
C.hartinahianum
CymbidiumC.chloranthum
Cymbidium
C.floribundum
Cymbidium
C.suavissimum
Cymbidium
C.goeringii
Jensoa82
C.tortisepalum
Jensoa65
C.faberiJensoaC.ensifolium
JensoaC.kanranJensoaC.sinense
JensoaC.lancifolium
JensoaC.macrorhizon
JensoaC.aberrans
JensoaGrammatophyllum speciosum
Grammatophyllum scriptum
Grammatophyllum stapeliaeflorum
100
Thecostele alata
100
Acriopsis lilifolia

SectionCymbidium
Cymbidium
Cymbidium
Cymbidium
Cymbidium
Maxillarianthe
Borneense
Austrocymbidium
Austrocymbidium
Himantophyllum
Cyperorchis
Cyperorchis
lridorchis
lridorchis
lridorchis
lridorchis
lridorchis
Eburnea
Eburnea
lridorchis
Parishiella
Bigibbarium
Austrocymbidium
Austrocymbidium
Floribundum
Floribundum
Maxillarianthe
Maxillarianthe
Maxillarianthe
JensoaJensoaJensoaGeocymbidium
Pachyrhizanthe
Pachyrhizanthe

Fig. 3. Strict consensus of 168 most-parsimon ious Fitch trees based upon matK and ITS sequences:length= 828, consistency index =0. 7283 (0.5731 excludi ng uninformative characters), retention index of
0. 7788. Numbers above internodes indicate bootstrap va lues from I ,000 replicates of Fitch parsimony analysis (maxtree limit of I ,000 per replicate) . Numbers be low internodes indicate bootstrap values from 1,000
replicates of neighbor-joining distance analysis.

Molecular Phyloge ny of Cymbidium

ium . (2) All the three subgenera of Cymbidium,
namely, Cymbidium, Cyperorchis , and Jensoa,
turned to be para-/polyphyletic. (3) Among sections of Cymbidium, Austrocymbidium , Cyperorchis, lridorchis, and Maxillarianthe did notshow monophyly. (4) At specific level , our results
indicated the polyphyly of Cymbidium bicolor,although bootstrap supports for the clades including C. bicolor were not robust.
Discussion

Classification
Generic circumscription of Cymbidium bymodern taxonomi sts was supported in this study.
Cyperorchis, sometimes treated as an independent genus (e.g. Blume, 1848, 1849, 1858; Reichenbach , 1852; Hooker, 1890; and Schlechter,
1924) was nested within other clades of Cymbidium. Pachyrhizanthe, another problematic groupwith reduced vegetative morphology and strongly
mycoparasitic nutritional existence, was elevatedto a new genus by Nakai ( 1931 ). The results ofthi s study showed that Pachy rhizanth e makes asister group relationship with section Geocymbidium and that it is reasonable to treat this groupas a section of Cymbidium. Moreover, these two
sections have the following synapomorph ies: (I)underground rhizomes that survive throughout
their life history, (2) whitish perianth lobes withpurpl e striations and markings.
We clarified that all the three subgenera ofCymbidium proposed by Seth & Cribb ( 1984)
represent para-/polyphyly. Subgenus Cymbidium
comprises four clades and these clades become
polyphyletic assemblage. It is apparent that thesubgenus was defined by plesiomorphic characters such as a lip without any fusing structures
and two pollini a. The monophyly of subgenus
Cyperorchis was broken by a nested position ofsubgenus Cymbidium section Himantophyllum.
Transfer of section Himantophyllum to a memberof subgenus Cyperorchis results in the loss of aprominent synapomorphic character of subgenus
Cyperorchis, that is, a fused basal part of the lipwith the column. Subgenus Jensoa will become a135monophyletic group, if Cymbidium aliciae is removed . The four pollinia is a principal synapomorphic character of subgenus Jensoa, but C.
aliciae and Cymbidium borneense, distantly related to subgenus Jensoa , also share this character. Taking the phylogenetic relationships and themorphological characters into consideration, subdivisions of Cymbidium at subgeneric level are
not useful. In other words, we did not find any
stable synapomorphic characters that featureeach major clade clarified in this study.
Our results further indicated that sectional delimitation of Cymbidium also has several problems. Among members of subgenus Cymbidium,
section Austrocymbidium exhibits paraphyly andcomprises the following three clades : (I) Cymbidium canaliculatum and Cymbidium madidum ;(2) Cymbidium hartinahianum ; (3) Cymbidium
chloranthum. The first clade includes the type of
this section (C. canaliculatum) and corresponds
to a revised circumscription of section Austrocymbidium. Since the remaining two clades further form another major clade together with section Floribundum and morphological charactersof the two cl ades are consistent with those of section Floribundum, we extend the limits of thissection to include members of the two clades ,namely, C. hartinahianum and C. chloranthum.
In subgenus Cyperorchis, the monophyly of
the clade including sections Cyperorchis,Eburnea, and lridorchis was supported by highbootstrap values. However, support on several relationships among members of sections Cyperorchis and lridorchis was weak due to few
synapomorphic nucleotide substitutions for thesegroups. It is likely that section lridorchis was established on the basis of plesiomorphic characters such as widely-opened perianth lobes and an
operculum without an obvious backwards-pointing beak, but explicit rel ationships on the basisof more molecular signal s are needed to rearrange the classification for these taxa.
As mentioned above, Cymbidium aliciae broke
the monophyly of subgenus Jensoa, more specifically section Maxillarianthe. Du Puy & Cribb( 1988) placed C. aliciae into subgenus Jensoa

136

Tomohisa Yukawa et al.

section Maxillarianthe as a synonym of Cymbidium cyperifolium Wall. ex Lindl. Our results did
not support this placement and suggested a sistergroup relationship of C. aliciae to Cymbidium
borneense, a sole representative of subgenus
Cymbidium section Borneense. Observationsof morphological and anatomical characters(Yukawa & Stern, 2002; Yukawa, unpublished)of C. aliciae also supported the affinity of these
two species and a distant relationship of section
Maxillarianthe to them . For example, C. aliciae
and C. borneense have hypodermal fiber strands
both in abaxial and adaxial surfaces, but members of section Maxillarianthe lack this characterat least in abaxial surface. Du Puy & Cribb( 1988) established section Borneense, because C.
borneense shows mixed diagnostic characters ofsubgenus Cy mbidium section Cymbidium andsubgenus Jensoa. The present results corroborated the soundness of section Borneense and thetwo species, C. borneense and C. aliciae, constitute the section.
Relationships at species level also may include
problems. Our results indicated the polyphyly of
Cy mbidium bicolor, although bootstrap supports
for the clades including C. bicolor were not robust. On the other hand, a species complex including Cy mbidium ensifolium , Cymbidium kanran , and Cymbidium sinense has low resolution.
Accumulation of more molecular signals is needed to clarify these relationships.
On the other hand, we found discrepant positions for Cymbidium erythrostylum and Cymbidium sanderae between matK and ITS trees. Sincematernal inheritance of plastid DNA was confirmed in Orchidaceae (Corriveau & Coleman,
1988; Sears, 1980), inconsistent phylogenetic positions for these two species in matK trees can becaused by introgressions. Interestingly, collection
records of C. erythrostylum and C. sanderae arevery few, and this fact also may account for theirhybrid origin. Moreover, Du Puy and Cribb( 1988) also indicated a hybrid nature of C.
sanderae. Further studies by use of more markers
both from nuclear and plastid genomes can confirm this hypothesis.

Pollinia number
The most controversial morphological character of Cy mbidium is different numbers of pollinia(two or four) in the genus. Most members of
Cy mbidium have two pollinia, but subgenus Jensoa and subgenus Cymbidium section Borneense
exhibit four pollinia. On the other hand, the sistergroup of Cymbidium (A criopsis , Grammatophyllum, and Th ecostele) and the sister group to thesetaxa (Bromh eadia : Yukawa, unpublished) share
two pollinia. Since the pollinia number of Orchidaceae is usually considered to be stable and has
been treated as a cardinal character among taxonomists, variations in this genus have provokedmuch discussion (e .g. Du Puy & Cribb, 1988).
The results of this study showed that the four
pollinia are an apomorphic character in Cy mbidium and likely evolved twice on the lineages leading to subgenus Jensoa and subgenus Cy mbidium
section Born eense (both in ACCTRAN and
DELTRAN optimizations). Our results also indicated that the conservative nature of pollinia
number is not a rule, but it is a kind of "dogma"
in orchid classification . Actually, variations ofpollinia numbers are found in other small- to
middle-sized monophyletic groups such as Thunia (e .g. Seidenfaden, 1986) and Phalaenopsis(e.g. Christenson, 200 I).
Life form
As mentioned before, Cy mbidium exhibits agreat variation of life forms. In this study we define epiphytes in a broad sense, that is, a group of
species inhabiting trees and/or rocks. In Cy mbidium , many species grow on trees and some ofthem also have potentials to live on rocks . However, accurate figures on habitats of each species
are not known due to the lack of observations insitu. Besides, there are no Cy mbidium species
that grow exclusively on rocks. We thus use abroad concept of epiphyte.
The sister group taxa of Cymbidium (A criopsis, Grammatophyllum , and Thecostele) are
epiphytes and the sister group to these taxa
(Bromheadia : Yukawa, unpublished) comprises
both epiphytic and terrestrial species. The mostMo lecu lar Phy logeny of Cymbidium

137

Life form.---- C.aloifolium
C.finlaysonianum
C.bicolor subsp.obtusum
C.bicolor subsp.pubescens
C.rectumC.aliciae
C.borneense
C.canaliculatum
C.madidum. - - - - - - - - - - C.dayanum
C.cochleare
C.elegans' - - l t - - C.insigne
c____ _ _ C.erythraeum

epiphyte
epiphyte
epiphyte
epiphyte
epiphyteterrestrial
terrestrial
epiphyte
epiphyte
epiphyte
epiphyte
epiphyteterrestrial
epiphyte
C.iridioides
epiphyte
epiphyte
C.lowianum'---- C.hookerianum
epiphyte...._______ C.tracyanum
epiphyte
epiphyte
C.eburneum
C.roseum
epiphyte' - - - - - - - - - - - - C.tigrinum
epiphyte. - - - - - - - - - C.devonianum
epiphyte. - - - - - C.hartinahianum
terrestrial.---- C.chloranthum
epiphyte
C.floribundum
epiphyte
epiphyte
C.suavissimum
C.goeringii
terrestrial
C.tortisepalum
terrestrial'---- C.faberi
terrestrial
C.ensifolium
terrestrial'----+-- C.kanranterrestrial
C.sinense
terrestrial.---- C.lancifolium
terrestrial
C.macrorhizon
terrestrial
C.aberrans
terrestrial
Grammatophyllum speciosum
epiphyte
Grammatophyllum scriptum
epiphyte
Grammatophyllum stapeliaeflorum epiphyte
Thecostele alata
epiphyte._______ Acriopsis lilifolia
epiphyte
Fi g. 4. Reconstruction of diversification in life form in Cymbidium under ACCTRAN optimization. T he tree is
one of 168 most-parsimonious Fitch trees based upon matK and ITS sequences. Black bars = change of statesfrom epiphytic to terrestria l habitat ; grey bars= change of states from terrestrial to epiphytic habitat.

138

Tomohisa Yukawa et al.

parsimonious reconstruction of character evolution on life forms indicated changes of character
states at internodes shown by bars (Fig. 4) . The
ancestor of Cymbidium was likely an epiphyte.
Under ACCTRAN optimization, the epiphytic
Cymbidium evolved terrestrial plants three times;
moreover, a sing le reversal from terrestrial to epiphytic condition likely occurred in Cymbidium.
Under DELTRAN optimization, the epiphytic ancestor of Cymbidium evolved into terrestrial
plants four times, that is, (I) in the common ancestor of Cymbidium section Borneense, (2) inthe ancestor of Cymbidium insigne, (3) in the ancestor of Cymbidium hartinahianum, (4) in theancestor of Cymbidium subgenus Jensoa (results
not shown in figures).
Recent phylogenetic analyses clarified that Orchidaceae had a terrestrial ancestry (Neyland and
Urbatsch, 1995 ; Cameron et al., 1999). Therefore, evolution from epiphytic to terrestrial habits
in Cymbidium represents reversal in Orchidaceae
as a whole. These results indicated that thechanges of habitats appeared repeatedly in thelineage of Cymbidium and are not rare evolutionary events in Orchidaceae.

Nutritional habit
Cymbidium section

Pachyrhizanthe Sch ltr.

keeps strong mycotrophy throughout life history
and lacks foliage leaves as well as roots. Albeit
Nakai (1931 )'s suggestion on its separate taxonomic status from Cymbidium, we found that thisgroup belongs to Cymbidium. The present results
also showed that the state, mycotrophy throughout li fe history, evolved only once within a cladeof terrestrial Cymbidium species. All the terrestrial species of Cymbidium have obligate mycotrophy in their juvenile stage of life history.
During this stage, the rhizome only represents
the body and functions as the home of symbiotic
fungi. Undoubtedly, the existence of obligate mycotrophy during juvenile stage in terrestrial
spec ies of Cymbidium acted as preadaptation ofcomp lete mycotrophy throughout life history insection Pachyrhizanthe. Furthermore, the growthhabit of section Pachyrhizanthe only composedof rhizomes and inflorescences represents a caseof heterochrony.
Our molecular data further showed low genetic
divergence between the mycotrophic clade andits autotrophic sister clade, namely, Cymbidium
lancifolium (section Geocymbidium). The number of base differences and sequence divergences
within the ITS region was as follows : Cymbidium
macrorhizon-C. lancifolium: 9, 0.0118; Cymbidium aberrans-C. lancifolium : 9, 0.0 I 06. This factindicated that these apparently drastic shifts ofnutritional habit and morphological characters
were established within relatively short period oftime.

Acknowledgements
would like to thank Dedy Darnaedi , Ed de
Vogel , Genjiro lshida, Fum ihi ro lwagawa, Kohji
Karasawa, Jun ' ichi Nagasawa, and Takahiro
Yagame for providing the plant material , andOsamu M iikeda, Koichi Kita , and Hideaki
Shimizu for technical assistance. This study ispartly supported by a Grant-in-Aid to Scientific
Research from the Japan Society for the Promotion of Science (13640708).

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