全 文 :书广 西 植 物 Guihaia Aug.2015,35(4):447-463 http://journal.gxzw.gxib.cn
DOI:10.11931/guihaia.gxzw201506015
唐光大,张国强,洪文君,等.基于ITS和matK序列的兰科沼兰族分子系统研究及二新种[J].广西植物,2015,35(4):447-463
Tang GD,Zhang GQ,Hong WJ,et al.Phylogenetic analysis of Malaxideae(Orchidaceae:Epidendroideae):two new species based on the combined
nrDNA ITS and chloroplast matK sequences[J].Guihaia,2015,35(4):447-463
Phylogenetic analysis of Malaxideae(Orchidaceae:
Epidendroideae):two new species based on the combined
nrDNA ITS and chloroplast matK sequences
TANG Guang-Da1,2,ZHANG Guo-Qiang2,HONG Wen-Jun1,
LIU Zhong-Jian1,2,ZHUANG Xue-Ying1*
(1.College of Forestry and Landscape Architecture,South China Agricultural University,Guangzhou 510642,China;
2.Shenzhen Key Laboratory for Orchid Conservation and Utilization,The National Orchid Conservation Center
of China and The Orchid Conservation and Research Center of Shenzhen,Shenzhen 518114,China)
Abstract:Malaxideae is a larger cosmopolitan orchid tribe consisting of nearly 2 000species and is distributed worldwide,
excluding the polar and desert regions.It is abundant in the tropical regions,particularly in Australia,and the tropical A-
sia,Southeast Asia,the tropical Americas and Africa.Molecular and morphological analyses have been performed to es-
tablish the phylogenetic relationships within this tribe.However,the subtribe and intergeneric relationships remain un-
clear,and the genus definition of this tribe is considerably controversial.The maximum parsimony,maximum likelihood
and Bayesian inference analyses were performed based on the combined sequences of the nuclear ITS and chloroplast
matK genes of 133taxa(10other alied species as outgroups),which represent nearly al of the major genera of the Mal-
axideae.These results suggested that Malaxideae could be divided into three clades,the epiphytic Oberoninae,the terres-
trial Liparidinae,and a terrestrial Crepidiumclade.The Oberoninae and Liparidinae could be divided into six genera and
five genera respectively,and Crepidiumclade camposes of four genera.The subtribe Ypsilorchidinae should be attributed
to Oberoninae.Disticholiparisshould be removed because it shared the same type with Stichorkis.The phylogenetic re-
lationship of Crepidiumand Dieniais monophyletic,although they have diferent lip structures.In addition,two new
species found in Southwest China and northern Vietnam,Platystyliparis malipoensis G.D.Tang,X.Y.Zhuang &Z.
J.Liu and Cestichis pingtaoi G.D.Tang,X.Y.Zhuang &Z.J.Liu,were described based on the molecular analysis
and morphological observation.
Key words:Malaxideae;nrDNA ITS;matK;phylogeny;Paraphyletic
CLC number:Q943.2 Document code:A Article ID:1000-3142(2015)04-0447-17
基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
唐光大1,2,张国强2,洪文君1,刘仲健1,2,庄雪影1*
(1.华南农业大学 林学与风景园林学院,广州510642;2.深圳市兰科植物保护与利用重点实验室,
国家兰科植物保护中心,深圳市兰科植物保护研究中心,广东 深圳518114)
① 收稿日期:2015-06-13 修回日期:2015-07-06
基金项目:国家林业发展计划项目(2011-100);广东省自然科学基金(2014A030310465);深圳市科技计划项目 (JC201005310689A)。
作者简介:唐光大(1982-),男,贵州普安人,博士,讲师,研究方向为植物系统与进化,(E-mail)gdtang@scau.edu.cn。
*通讯作者:庄雪影,博士,教授,研究方向为植物系统与进化,(E-mail)xyzhuang@scau.edu.cn。
摘 要:沼兰族是兰科植物的大族之一,约2 000种,除了极地和沙漠地区,全球均有分布。该族植物主要分
布在热带地区,尤其在东南亚、热带美洲、非洲以及澳大利亚等地区种类非常丰富。目前,已有关于该族植物
形态和分子系统的研究,但有关该族亚族和属间的系统关系尚不清楚,属的界定争议也较大。该文基于核基
因片段ITS和叶绿体基因片段matK序列,采用最大简约法、最大似然法贝叶斯推理分析法,对现有沼兰族主
要属的123种植物和10个外类群植物进行了分子系统学研究。结果表明:沼兰族主要分为3个亚族分支,包
括附生的鸢尾兰亚族(Oberoninae)、地生的羊耳蒜亚族(Liparidinae)和沼兰亚族分支(Crepidiumclade)。鸢
尾兰亚族包括6个属、羊耳蒜亚族分支包括5个属、沼兰亚族分支包括4个属;丫瓣兰亚族(Ypsilorchidinae)
应归并为鸢尾兰亚族;Disticholiparis属与Stichorkis属的模式标本相同,应并入Stichorkis属;沼兰属(Crep-
idium)和无耳沼兰属(Dienia)的唇瓣结构差异较大,但二者均为单系类群。此外,在收集野外实验材料过程
中,发现了2种产自中国西南部和越南北部的沼兰族新种,分别命名为麻栗坡羊耳蒜(Platystyliparis mali-
poensis G.D.Tang,X.Y.Zhuang &Z.J.Liu)和秉滔羊耳蒜(Cestichis pingtaoi G.D.Tang,X.Y.
Zhuang &Z.J.Liu)。
关键词:沼兰族;核基因ITS;matK;系统学;并系类群
Malaxideae is one of the oldest orchid tribes and
encompasses nearly 2 000species that are native to
both temperate and tropical areas worldwide(Chen et
al.,2009).Lindley(1826)first classified and revised
21genera into Malaxideae.Six of these genera re-
mained in this tribe,including Malaxis,Microstylis
(attributed to Crepidiumnow),Liparis,Dienia,Em-
pusa,and Pedilea,but other 15genera were attributed
to other tribes.Lindley(1830)redefined this tribe and
emphasized that the most important features were:
polen cohering in masses of a firm waxy texture,with-
out any of the celular substance,and remaining under
the form of a distinct gland lying upon the stigma.
Pfitzer(1887)redefined this tribe again as“plant with
leaves duplicative,stem gracile or incrassate leaved
pseudobulb;leaf lamina except for Cestichis and
Oberonia,with continent basal sheath;petals and la-
belum more conspicuous than external sepals,column
apodal or forming a short spur with the labelum,an-
ther erect or incumbent,polinia 4,waxy,glabrous;
dispersed”.Schlechter(1911)revised the Liparidinae
(Malaxideae)of New Guinea and left Malaxis,Micro-
stylis,Orestias,Liparis,Cestichis,Oberoniaand Hipp-
eophyllumin this subtribe.Dressler(1981)empha-
sized that the main characters of Malaxideae were not
only the naked polinia,but a column lacking a foot at
the gynostemium base,and stated that polinia lacking
a distinct caudicle were also equal characters for distin-
guishing this tribe.Szlachetko(1995)recognized 16
genera in the subtribe Malaxidinae,seven were newly
described by him.He divided Malaxideae into two
subtribes of terrestrial Malaxidinae and Oberoninae,
but the latter was named by Averyanov(1991).Szla-
chetko and Margońska(2002)provided detailed draw-
ings of the column structure of representatives of 15
genera.Pridgeon et al.(2005)stated 14genera in the
tribe of Malaxideae.However,the description of Mal-
axideae was quite augmented,especialy in terms of leaf
structure,floral arrangement within the inflorescences,
tepal form and ovary.
Malaxideae is also one of the few groups of or-
chids that contain large numbers of both obligate ter-
restrial and epiphytic species(Atwood,1986).There
are obvious diferences between terrestrial and epiphyt-
ic taxa in vegetative organs characters.An example is
that the terrestrial genera,such as Crepidium and
Diteilis,have significantly plicate leaves,but the epi-
phytic genera are conduplicate except for Oberoniaand
a fewLiparis species.However,their flower morphol-
ogies are rather homogenous,which are typicaly very
smal,born on a terminal inflorescence,containing a
single,terminal,incumbent anther,usualy with four
naked polinia(Cameron,2005),only a few species
have two polinia(Li et al.,2013).Many species of
Malaxideae are polinated by fungus gnats,and only a
few are polinated by ants and ichneumon flies(Tang
et al.unpublished data).
In recent years,there are considerable continuing
controversy about the size of genera within the Mal-
axideae due to their wide distribution and diverse mor-
844 广 西 植 物 35卷
phology,and the main debates focus on the generic def-
inition of this tribe.In the strictest and most conserva-
tive senses,some scholars considered that the tribe
should be divided into at least three genera:Oberonia,
Liparis,and Malaxis,but stil many genera were es-
tablished or separated(Cameron,2005).For example,
Dressler(1993)recorded that Malaxideae consisted of
six genera:Hippeophyllum,Liparis,Malaxis,Ores-
tias,Oberonia and Risleya,but now Risleyain Cola-
bieae(Xiang et al.,2014).Szlachetko(1995)sugges-
ted there were more than 18genera in this tribe.Fur-
thermore,Margoń-ska and Szlachetko described some
new genera such as Alatiliparis(Margońska et al.,
2001),Disticholiparis(Margońska et al.,2004),Sei-
denforchis (Margońska,2006),Platystyliparis
(Margońska,2007),and Crossoliparis (Margońska,
2009).Margońska et al.(2012)described that 25
genera belonged to this tribe and divided them into
three subtribes:Oberoninae(containing two genera),
Malaxidinae(containing 12genera),and Liparidinae
(containing 11genera).
Although some molecular phylogenetic studies
have examined the phylogenetic and evolutionary rela-
tionships of Malaxideae(Cameron,2005;Margońska et
al.,2012),some results indicated that Liparis and
Malaxis were polyphyletic aliances.Cameron(2005)
described and identified four habit-leaf-morphology
syndromes as indicative of phylogenetic relationships in
Malaxideae,such as epiphytes with several elongate
conduplicate leaves(Liparis)and with unifacial,later-
aly compressed,equitant leaves(Oberonia),terrestrial
with two rounded conduplicate leaves(Malaxis)and
terrestrial with plicate leaves(Crepidium).However,
the generic definition of Malaxideae remains unclear.
More comprehensive studies are needed to explore the
exact evolutionary relationships of Malaxideae at the
generic level.Malaxideae should be re-classified,espe-
cialy given the aggregated study results,combined
with the molecular phylogenetics and their habits,veg-
etation,and morphologies.
In this study,the new sequences of nuclear ITS
and chloroplast matK from 35species located in China
were combined with the sequences quoted from Gen-
Bank,and assessed for the phylogenetic relationships a-
mong Malaxideae,in combination with the plant hab-
its,pseudobulbs and leaves.In addition,we provide a
supported phylogenetic resolution for Malaxideae and
comprehensive results regarding this tribe.
1 Materials and Methods
1.1Materials
A total of 121species of Malaxideae were ana-
lyzed,consisting of main genera of Malaxideae,inclu-
ding Liparis,Oberonia,Malaxis,Crepidium,Dienia,
Empusa,Oberonioides,Stichorkis,Cestichis,and Platy-
styliparis.Ten diferent alied species,Acanthephipp-
ium mantinianum,Ancistrochilus rothschildianus,Bul-
bophyllum odoratissimum,Calanthe argenteostriata,
C.sinica,Collabium simplex,Dendrobium crumena-
tum,Listera smalli,Phaius tancarvilleae,and Rid-
leyella paniculata,were selected as outgroups accord-
ing to previous studies(Van den Berg et al.2005).
There are 35sequences of species were generated new
for this study from Yunnan,Guangdong etc,and 98se-
quences of species were downloaded from existing se-
quences in GenBank.
1.2Amplification and sequencing
Total DNA was extracted from fresh material or
silica gel-dried plant tissue using a Multisource Ge-
nomic DNA Miniprep Kit(Axygen Biosciences)ac-
cording to the manufacturer’s instructions.The ampli-
fication reaction included total DNA,primers,Ex-Taq
bufer and Ex-Taq DNA polymerase(Takara Bio).
The polymerase chain reaction(PCR)profile consisted
of an initial 5min pre-melting stage at 95℃,folowed
by 30cycles of 30sat 95℃ (denaturation),30sat 50
℃to 55 ℃ (annealing temperature was determined
based on the primer requirement),1min to 3min at 72
℃ (extension time was determined based on the length
of the target DNA region)and a final 10min extension
at 72℃.
Amplification of the ITS region was performed u-
sing the primer pairs ITS A and ITS B(Mike et al.,
1999).For matK sequences,amplification was per-
formed using the primer matK-731F (Liu et al.,
9445期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
2011).Table 1contains the detailed information.
The PCR products were run on 1.5% agarose
gels
Table 1 Primers used in this study
Primer Sequence(5′to 3′) Origin
ITS A GGAAGGAGAAGTCGTAACAAGG Mike et al.
,
1999
ITS B CTTTTCCTCCGCTTATTGATATG Mike et al.
,
1999
matK-731F AAGAAAAGATTCTTTTGGTTCC Liu et al.
,
2011
to assess the quality of the amplified DNA.The gels
with the target products were excised purified using
DNA Gel Extraction Kits(Axygen Biosciences)and
then sequenced by Life Technologies Corporation.
1.3Sequence editing and assembling
Both forward and reverse sequences,as wel as
electropherograms,were edited and assembled using
DNASTAR (http://www.dnastar.com/).DNA se-
quences were aligned to the muscle model and manual-
ly adjusted using MEGA5.05(Tamura et al.,2011).
The aligned sequences are available from the corre-
sponding authors upon request.
1.4Data analyses
The datasets of a nuclear ITS,plastid DNAmatK
and their combination were used for the subtribe and
genus level analysis.Insertions,deletions and some un-
available sequences were treated as missing.Phyloge-
netic analyses were performed using Bayesian inference
(BI),maximum parsimony(MP)and maximum likeli-
hood analyses.The best-fit model for each dataset was
selected using Modeltest 3.7under the Akaike Infor-
mation Criterion(Posada et al.,1998).The homoge-
neities between nrDNA ITS data,and matK dataset
were tested using the incongruence length diference
(ILD)test(Farris et al.,1995),as implemented in
PAUP* version 4.0b10 (Swoford,2002).TBR
branch was swapping and keeping no more than 100
trees per random additional replicate.As previously
described by Cunningham (1997),a significance level
of P=0.01was adopted for this test.
MP analyses were performed using the PAUP*
version 4.0b10(Swoford,2002).Al characters were e-
qualy weighed and unordered.The settings included
1 000random additional sequences and a heuristic search
with tree bisection-reconnection branch swapping.Table
2list the tree length(TL),consistency index(CI)and
retention index(RI).ML analysis was performed using
RA×ML with 100bootstrap replicates and settings as
described in(Stamatakis et al.,2008).BI analysis was
performed using MrBayes 3.1.2 (Ronquist et al.,
2003).The folowing settings were applied:sampling
frequency=1 000,temp=0.1,burn-in=10 000and
number of Markov chain Monte Carlo generations=40
000 000.The first 10 000trees were discarded as burn-
in to ensure that the chains reached stationarity.A ma-
jority-rule consensus tree was constructed on these trees
sampled after generation 40 000 000.
Table 2 Parsimony statistics from phylogenetic
analyses of the various datasets
Data Taxa Alignedlength
Information
site TL CI RI
ITS 121 847 475(56.1%) 2 946 0.377 8 0.804 5
matK 120 1 405 308(21.9%) 1 030 0.621 4 0.864 1
Combined 139 2 252 783(34.8%) 4 107 0.436 4 0.815 1
2 Results and Analysis
ITS analysis supported that Malaxideae could be
divided into three clades,including two terrestrial
clades and one epiphytic clade(PP=100%,BPMP=
94%,BPML=99%,Fig.1).The first terrestrial clade
included five genera:Malaxis(containing 10taxa),
Liparis(12taxa),Oberonioides(2taxa),and two
unnamed genera(Clade B,C;Fig.1).The second ter-
restrial clade included five genera:Crepidium(15tax-
a),Dienia(4taxa),Diteilis(7taxa),Empusa(4tax-
a)and an unnamed genus(Clade D;Fig.1).Among
these clades,Litsea auriculata was attributed to Mal-
axis,L.maingayi and L.purpureoviridis are sisters
to Oberonioides.The third epiphytic clade consisted of
six genera:Oberonia (15taxa),Platystyliparis (7
taxa),Cestichis(12taxa),Stichorkis(5taxa),and
two unnamed genera(Clade E,F;Fig.1).Oberonia
was monophyletic in this tree and a sister of Lipariss.
l.(Fig.1).
054 广 西 植 物 35卷
The results of the BI,MP and ML analysis of matK
also showed that Malaxideae could be divided into three
clades(PP=100%,BPMP=96%,BPML=97%,Fig.
2).However,the posterior probabilities of the matK
Fig.1 Phylogram obtained from Bayesian inference analysis of the nrDNA ITS data of Malaxideae Numbers at the nodes are
bootstrap percentages and Bayesian posterior probabilities,respectively.“-”indicates that the values of PP or BPMPor BPMPare lower than 50%in
the three analysis.PP is the left value at the nodes;BPMPis the middle value,and BPMLis the right value.
tree were lower than those of the ITS tree at the genus
level.This sequence might be not optimal for the per-
formance of molecular phylogenetics of Malaxideae be-
low the subtribe and genus.These results have also
1545期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
Fig.2 Phylogram obtained from Bayesian inference analysis of the chloroplast matK data of Malaxideae Numbers at the nodes
are bootstrap percentages and Bayesian posterior probabilities,respectively.“-”indicates that the values of PP or BPMPor BPMPare lower than 50%.
BI is the left value at the nodes;BPMPis the middle,and BPMLis the right.
been described in a previous study(Cameron,2005).
The datasets for the ITS and matK sequences
were combined into a single dataset.The consensus
trees supported the division of Malaxideae into three
254 广 西 植 物 35卷
Fig.3 Phylogram obtained from Bayesian inference analysis of the combined data of the nrDNA ITS and chloroplast matK
of Malaxideae Numbers at the nodes are bootstrap percentages and Bayesian posterior probabilities,respectively.“-”indicates that the values of
PP or BPMPor BPMPare lower than 50%.BI is the left value at the nodes;BPMPis the middle value,and BPMLis the right value.
main clades,similar to the ITS and matK trees,but the
posterior probabilities and bootstrap percentages were
higher than these two sequences(PP=100%,BPMP=
99%,BPML=100%;Fig.3).The first terrestrial clade
3545期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
Fig.4 Living plants of Platystyliparis malipoensis and P.assamica A-B.P.malipoensis
A.Flowering plant;B.Flower,front view C-D.P.assamica C.Flowering plant;D.Flower,front view.
was strongly supported by the polyphyletic groups(PP
=98%,BPMP=100%,BPML=100%;Fig.3),consis-
ting of Malaxis,Liparis,Oberonioides,and three un-
named genus(Clade A,B,C;Fig.3).Surprisingly,
their morphological characters were similar,with the
same short pseudobulbs and conduplicate leaves.This
terrestrial clade should be named Liparidinae because it
contains L.loeseli (L.)Rich.,the type species of Li-
paris Rich.The second terrestrial clade,including Cre-
pidium,Dienia,Diteilis,Empusa,and an unnamed
genera(Clade D;Fig.3),was also strongly supported
based on the polyphyletic groups(PP,BPMP,BPML=
100%;Fig.3).The species in this clade are united by
a longer pseudobulb and plicate leaves.Although some
subtribes were named in Malaxideae,such as Malaxidi-
nae(Malaxeae)by Bentham (1883),the type species
of Malaxis(M.spicata)is among the Liparidinae
subtribe.Thus,we introduced the new name Crepidi-
nae for this subtribe tentatively according to the classi-
fied history of Malaxideae (Rasmussen,1979;
454 广 西 植 物 35卷
Fig.5 Platystyliparis malipoensis G.D.Tang,X.Y.Zhuang et Z.J.Liu 1.Flowering plant;2.Flower,side view;3.Flower,
front view;4.Dorsal sepal;5.Lateral sepal;6.Petal;7.Lip;8.Polinia and anther cap(Drawn by X.Y.Ma from type Z.J.Liu 4416).
Margońska et al.,2012).This subtribe consists of
mainly terrestrial plicate leaf species.The third epi-
phytic clade Oberoninae consists of Stichorkis,Platy-
styliparis,Cestichis,Oberoniaand two unnamed genera
(Clade E,F;PP=99%,BPMP=99%,BPML=100%;
Fig.3).
3 Discussion
3.1Systematic status of Malaxideae
Malaxideae have been dificult to place within
Epidendroideae because they lack typical appendages
on their polinia,although they appear to have smal
viscidia in most cases,there are stil some exceptional
5545期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
Fig.6 Living plants of Cestichis pingtaoi and C.mannii A,C,D.C.Pingtaoi A.Flowering plant;C.Inflorescence;
D.Flower,front view B,E,F.C.mannii B.Flowering plant;E.Inflorescence;F.Flower,front view.
and unique species(Li et al.,2013;Su et al.,2014).
Column morphology,particularly the structure of the
polinia and associated appendages,has always been
given pre-eminence in orchid classification.Therefore,
proposals as to the relationships within this tribe have
been limited.Dressler(1993)hypothesized that al-
though Dendrobieae appear to be secondarily naked,
Malaxideae may be primitively naked.In the morpho-
logical analyses about Malaxideae,two species of Li-
paris were among a large number of Epidendroideae on
apolytomy(Freudenstein et al.,1999),whereas an a-
nalysis by Cameronet al.(1999)on rbcL data revealed
that Liparis and Malaxis were sisters to Dendrobieae
but without bootstrap support greater than 50%.The
addition of plastid psaB or matK sequences to the rbcL
dataset did not achieve a greater resolution for the po-
sition of Malaxideae(Cameron,2004;Freudenstein et
al.,2004).Van den Berg et al.(2005)placed Den-
654 广 西 植 物 35卷
Fig.7 Cestichis pingtaoi G.D.Tang,X.Y.Zhuang et Z.J.Liu 1.Flowering plant;2.Flower,front view;3.Flower,side view;
4.Dorsal sepal;5.Lateral sepal;6.Petal;7.Lip;8.Polinia and Anther cap(Drawn by X.Y.Ma from type Z.J.Liu 5814).
drobieae and Malaxideae as two successive sister clades
to the rest of Epidendroideae.These results revealed
that Malaxideae was a monophyletic clade and closely
related to the tribe Dendrobinae,although the wel-
supported consensus trees can not be acquired.
In Malaxideae tribe,the basic taxonomic problem
7545期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
is the lack of unequivocal criteria for distinguishing the
genera.Many genera suggested in previous studies ap-
pear artificial or exhibit polymorphism (Margońska et
al.,2012).Even the location,access to type speci-
mens,and original references are unavailable because of
their dispersal,age and state of preservation(Szlachet-
ko et al.,2008).Most taxonomists agree that the tra-
ditional classification of Malaxideae divided into three
main genera is unsatisfactory and unnatural,but no
clear strategy has been ofered to organize the tribe
previously(Cameron,2005).In this study,an updated
phylogeny of Malaxideae is proposed with a more sam-
pling of taxa and combined chloroplast and nuclear data
than previous molecular phylogenies.And the results
suggest that the tribe should be supported with three
main subtribes,rather than two (Averyanov,1991;
Szlachetko,1995).
3.2Molecular data analysis of Malaxideae
In the Malaxideae tribe,two main genera of Li-
paris and Malaxis were polyphyletic.Many new gene-
ra are established in recent years,but these genera are
controversial.In this study,we analyzed 11established
genera and 121species of Malaxideae,and these taxa
included most of the representative genera in this
tribe,although the sequences from species within some
genera were not examined,such as Allatiliparis,Hip-
peophyllum,Seidenforchis and Crossoliparis.The
phylogenetic analyses of these 121species were per-
formed using Bayesian inference(BI),maximum parsi-
mony(MP)and maximum likelihood(ML)for each
dataset.The BI,MP and ML trees of each dataset had
higher similar topological structures(Figs.1,2,3),in-
dicating that the analysis had the high congruence and
that the experimental data were reliable.The difer-
ences between the BI,MP and ML trees were the val-
ues of the bootstrap percentages or posterior probabili-
ties in some nodes.
This study revealed that the sequence of ITS was
better than matK in resolving the intergeneric and in-
terspecific relationships within Malaxideae.The ITS
and combined data of two sequences produced approxi-
mately equivalent tree topologies(Figs.1,3).The
consensus tree of matK was weakly supported for the
genus-level classification of Malaxideae(Fig.2),which
may be due to insuficient variations in the sequence of
matK(Table 2).Many studies have demonstrated that
at lower taxonomic levels(tribes and below),the ITS
nuclear ribosomal DNA spacer can yield better results
than protein-coding genes(Marcin et al.,2010).Con-
versely,at the family level,plastid protein-coding genes
have become the primary focuses,including rbcL
(Chase et al.,1994,Freudenstein et al.,2004)and
matK(Freudenstein et al.,2004).This study focused
on lower taxonomic levels(tribes and below).Thus,it
was reasonable that the consensus trees of matK pro-
duced weakly supported results.However,when the
ITS and matK dataset were combined to analyze these
relationships,the best tree topology was obtained and
was consistent with the delimitation based on their
morphological characters.Using this combined data-
set,most of the clades were better delimited and more
strongly supported.In the present study,the matK
fragments were stil useful in resolving the relation-
ships below subtribe and the generic rank when com-
bined with ITS sequences.
3.3Subtribe-level analyses
Although some subtribes have been previously
described by botanists,they were confusing and did not
have consistent morphological characteristics.Bentham
(1881)divided“Epidendreae”into nine subtribes,in-
cluding“Microstyleae”(Microstylidinae)which com-
bined with the already existing“Liparideae”(Liparidi-
nae Lindl.ex Miq.).However,his description of“Mi-
crostyleae”was very short,“anther erect or decumbent
often persistent”,and did not list any of its genera.
Soon thereafter,Bentham and Hooker(1883)changed
the name“Microstyleae”to“Malaxieae”(Malaxidi-
nae)and separately described“Liparieae”(Liparidinae
Lindl.ex Miq.).Both their subtribes contained plants
with a pseudobulbous shoot and terminal inflores-
cences.In addition,the typical characters of“Malaxe-
ae”were defined as having one to three leaves in the
shoots,smal often minute flowers,and anthers that are
erect or decumbent often persistent.They placed Mal-
axis and Microstylis in the subtribe.In contrast,“Li-
parieae”was characterized as plants with “1-many
854 广 西 植 物 35卷
leaves or aphylus shoot;4rarely 8,subequal,gathered
or often free,inappendiculata polinia.”“Liparieae”
embraced these genera of Liparis(the type-genus),
Oberonia,Gastroglotis,Microstylis,Chrysoglossum,
Orysicera,and Dendrochilum.Oberoninae was desig-
nated by Averyanov(1991),it contains only two gene-
ra,Oberonia (the type-genus)and Hippeophyllum.
Many common characteristics exist in these two gene-
ra,such as a slender shoot,duplicate fleshy,lateraly
compressed leaves,erect gynostemium column.Thus,
the latter was attributed to Oberonia (Chen et al.,
2009).However,the phylogenetic relationship of
Oberonia was very close with some species of Liparis
and formed a main subtribe clade.Therefore,this sub-
tribe should contain more genera in addition to Obero-
nia.In addition,Liu et al.(2008)proposed a subtribe
Ypsilorchidinae based on a single Liparis species‘Li-
paris fissipetala’.They recorded“2granular-waxy
polinia each with a somewhat elastic caudicle and
deeply bilobed petals”,but the results of the molecular
phylogenetic analysis did not support that the subtribe
Ypsilorchidinae was a single subtribe(Figs.1,2,3).
Our results suggest that the tribe Malaxideae
should be divided into three subtribes.The type genus
(the type is Liparis loeseli)of Liparidinae and the
type genus(the type is Malaxis spicata)of Malaxidi-
nae were attributed to one subtribe in this phylogenetic
analyses.Liparidinae Lindley et al.(1855),has pre-
cedence over Malaxidinae Benth etal.(1883).Thus,
we propose that Malaxidinae should be attributed to
the Liparidinae.Moreover,a new subtribe should be
established according to the results of the molecular
phylogenetic analyses,which we named Crepidinae
tentatively.Nearly al of the datasets supported the di-
vision of Liparis into 11genera and Malaxis divided
into five genera in this study.Oberonia is a single
monophyletic genus as the type genus of subtribe of
Oberoninae.
3.3.1Subtribe Liparidinae Lindl.ex Miq. Liparidi-
nae was established by Lindley et al.(1855),accord-
ing to the characteristics of column part erect,basaly
elongated,and this subtribe contained the genera Li-
paris(the type-genus),Oberonia,Gastroglotis,Micro-
stylis,Chrysoglossum,Orysicera,and Dendrochilum.
However,Gastroglotisis a synonym of Dienia,which
was established by Lindley(1824).Microstylis is a
synonym of Malaxis,and Chrysoglossumis attributed
to Collabiumby Schlechter(1911).In addition,no
other information is acquired on Orysicera.Dendro-
chilumis replaced in the tribe Coelogyninae(Pridgeon
et al.,2005).Recently,11genera were included in this
subtribe,including Stichorkis,Liparis,Orestias,Cros-
soglossa,Kornasia,Lisowskia,Oberonioides,Alatili-
paris,Disticholiparis,Platystyliparis and Crossoli-
paris(Margońska et al.,2012).However,these gene-
ra clades contained terrestrial and epiphytic habits taxa
and lacked consistent morphological characteristics.
According to this molecular analysis,this subtribe
consists of six genera.Malaxis were polygenetic and
divided into Malaxiss.s.,Oberonioides,Liparis s.s.,
an unnamed clade containing M.monophyllos,and two
Liparis species(Figs.1,3).These genera share the
same terrestrial vegetative habits,mainly growing in
grasslands,distributing over al of the European,Asian
and American temperate grasslands,except for
Oberonioides,which grows on the unique Danxia rocks
located in south China.In addition,their similar con-
duplicate leaves wither in winter,reserving their short
and fleshy pseudobulbs to overcome the cold winter.A
high level of diferentiation appears in their floral mor-
phology,such as inflorescence,lips and column,and the
length of the ovary in this subtribe.Our phylogenetic
results suggest that the subtribe-level classification of
Malaxideae should employ the characteristics of their
habits,pseudobulbs and conduplicate leaves.Thus,we
suggested that nearly al of the grassland species in the
tribe Malaxideae should be included in the subtribe Li-
paridinae.
3.3.2Subtribe Oberoninae Aver This clade was de-
scribed by Averyanov (1991) and revised by
Margońska et al.(2012)Margońska et al.(2012)re-
vised this subtribe.They considered the morphologi-
cal characteristics of Oberoninae as“a stem-like,slen-
der shoot;duplicate fleshy,lateraly compressed leaves;
a short and massive,erect gynostemium column and
suberect anther”.Most epiphytic genera and taxa
9545期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
formed a single subtribe clade according to this phylo-
genetic analysis(Figs.1,2,3).Thus,we suggested
that Oberoninae should consist of more genera in addi-
tion to Oberonia,such as Cestichis,Platystyliparis,
Stichorkis and two unnamed genera (Figs.1,3).
These genera distribute from Central Africa to French
Polynesia,and from India to New Guinea.The main
similar morphological characteristics of this subtribe
are epiphytic habits,leaf duplicate or compressed unifa-
cial or bifacial features.According to the results of
phylogenetic relationship of Malaxideae,we suggest
that the subtribe Oberoninae should include most of
the epiphytic species in the tribe Malaxideae.
In Oberoninae,Resmussen(1979)established the
genus Stichorkis Thouars according to the drawing of
Malaxis disticha Thours(1809),which was designat-
ed as iconolectotypes. Disticholiparis Marg.et
Szlach.is an objective junior synonym of Stichorkis
due to the identical type species(Thouars 1809,1822;
Margońska et al.,2004).Liuet al.(2008)proposed a
subtribe Ypsilorchidinae on the basis of two granular-
waxy polinia,each with a relatively elastic caudicle and
deep bilobed petals.Only the monotoypic Ypislorchis
was included.Its species,Y.fissipetala was not re-
covered as a distinct lineage in this phylogentic study,
nor do morphological attributes support its distinctness
at the subtribe level(Figs.1,2,3).More detailed
studies are required for this clade.
3.3.3Crepidinae clade In this study,we proposed a
new subtribe Crepidinae tentatively and designated the
genus Crepidiumas its type-genus,because the species
of Crepidiumare more abundant than other genera in
this clade.Although the type species of C.rheedi
Blume was designated as the lectotype species of Crep-
idium(Seidenfaden,1978;Alrich et al.,2011),but it
has attributed to Empusa (Figs.1,2,3).The new
subtribe forms a stable clade on this phylogenetic
trees.The genus Dienia is close with Crepidium
(Figs.1,3).The subtribe Crepidinae consists of five
genera subclades:Crepidium,Diteilis,Dienia,Empusa
and an unnamed genus(Figs.1,3).These genera
share some common morphological characteristics:
they own the same terrestrial vegetative habit,mainly
growing in the deep forests,and distributing in the
tropical and subtropical islands of Southeast Asia.
They are particularly abundant on the islands of Ma-
laysia,Java,Indonesia and New Caledonia.Only one
species of Diteilis nervosa(Liparis nervosa)distributs
from the Pacific islands to Central and South America.
Their longer pseudobulbs and evergreen plicate leaves
enable them to adapt to forest habitats.Two Liparis
taxa of L.laxaand L.chalandei form a monophylum
(PP,BPMPand BPML =100%;Fig.1)as the base
group of this clade,and these two Liparisspecies dis-
tribut on the island of New Caledonia.Thus,more de-
tailed studies should be performed on these species.
According to the main similar morphological character-
istics,we suggest that this clade should include the ter-
restrial and plicate leaf species in the tribe Malaxideae,
because they share samiliar forest habitats and longer
pseudobulbs.
4 Conclusions
In the present study,we investigated the phyloge-
netic relationships of Malaxideae based on combined
molecular evidence.Oberoniais monophyletic,but Li-
paris and Malaxis are not monophyletic.They could
be divided into three major clades,which are the sub-
tribes Liparidinae,Crepidinae(We named here,but
more detailed data and new sequence should be used to
confirm its phylogeny),and Oberoninae.The terres-
trial Liparidinae comprises Malaxis,Liparis and
Oberonioides and three unnamed genera.The newly
established subtribe Crepidinae comprises Crepidium,
Dienia,Diteilis Empusa and an unnamed genus.The
epiphytic Oberoninae comprises six genera,Oberonia,
Stichorkis,Cestichis,Platystyliparis and two unnamed
genera.These unnamed genera should be studied more
carefuly in the future before their phylogenetic rela-
tionships are revised.Finaly,we described a new sub-
tribe and two new species based on molecular and mor-
phological analysis.
5 Taxonomic treatment
064 广 西 植 物 35卷
5.1New subtribe
Crepidinae G.D.Tang,Z.J.Liu et X.Y.
Zhuang,nov.subtribe.New subtribe is distantly relat-
ed to the subtribe Liparidinae,from which it difers by
having longer pseudobulbs,evergreen and plicate leav-
es.It consists of three known genera and one unnamed
genera.They are Crepidium,Diteilis,Empusaand an
unnamed genus.Typus genus:CrepidiumBlume,Bi-
jdr.Fl.Ned.Ind.,8:387,t.63(1825).Lectotype
species:C.rheedi Blume designated by Seidenfaden,
Dansk Bot.Arkiv,33(1):43(1978).
5.2New species
(1)Platystyliparis malipoensis sp.nov.G.D.
Tang,X.Y.Zhuang &Z.J.Liu.(Figs.4,5)
Type:China,Yunnan,Malipo,alt.1 400m,27
Feb.2009,Z.J.Liu 4416(type,NOCC!)
Diagnosis:The new species is similar to Platy-
styliparis assamica,from which it difers by having o-
void pseudobulbs and lip base conspicuous with 2au-
ricular lobes on both sides,apex caved.Column broad
and large.
Description:Epiphytic herbs.Pseudobulbs aggre-
gate,oblong-fusiform,1.5-2.5cm ×4-6mm,api-
caly with 2-3leaves.Leaves eliptic or oblong-obo-
vate,1.2-2.6cm ×5-7mm,apex acute,base con-
tracted into a short stalk,articulated.Scape 5-10cm
long,nearly wingless,near lower half of raceme with 5
-8sterile bracts;raceme 3-6cm long,7-24-flow-
ered;bracts ovate-lanceolate,3-4mm long;pedicel
and ovary 3.5-4.5mm long;flowers yelowish-green
when young,later became orange-yelow;dorsal sepal
narrowly ovate-oblong,5-6×1.5-1.7mm,apex ob-
tuse,margins bent forward;lateral sepals suboblong,5
-6×2-2.2mm,apex obtuse,margins bent forward;
petals narrowly linear,5-6×0.3mm,apex acute;la-
belum ovate-oblong,3.7-4mm long,apex truncate
and mucronate,1.5mm above base conspicuous crisped
and flexuose,looked like 2auricular materials,middle
with a thick calus concave centraly;column erect,3-
3.5mm long,front apicaly with 2wings,relatively
broad,below middle with a pair of wings on both
sides.Polinia 4,2pairs,narrowly ovate.Fl.Decem-
ber-March.
Distribution and habitat:Platystyliparis mali-
poensis is epiphytic,forming scattered colonies on shad-
y and wet areas on the trunks at elevations of 1 200-1
600min evergreen broad-leaved forests of Malipo,
Yunnan Province,China;Ha Giang Vietnam.
Conservation status:The new species has been
found in four populations,each of which has no more
than 50individuals.
Polinators:We observed that this new species
was polinated by the ants,but more detailed research
should be taken in future for deeply understanding this
unique species.
(2)Cestichis pingtaoi sp.nov.G.D.Tang,X.
Y.Zhuang &Z.J.Liu.(Figs.6,7)
Type:China,Yunnan,Malipo,alt.1 400m,14
Nov.2011,Z.J.Liu 5814(type,NOCC!)
Diagnosis:The new species is similar to Cestichis
manni,from which it difers by having a longer pedicel
and ovary,flower large but the number are lesser,la-
belum 3-4mm ligule,long and 2-3mm broad,api-
cal margins entire.
Description:Epiphytic herbs.Pseudobulbs ob-
long-ovoid,ovoid and oblong,1.5-2cm×5-9mm,
nearly flat,apicaly with 1leaves.Leaves narrow linear
and lanceolate,9-17cm×8-18mm,papery,apex a-
cuminate,base contracted into a short stalk,articula-
ted.Inflorescence 21-24cm long,petiole flat,very
narrowly winged,near lower half of raceme with 1-8
sterile bracts;raceme 13-16cm long,10-20flow-
ered;bracts narrow lanceolate,4-5mm long;pedicel
and ovary 1-1.4cm long;flowers light green and
white;sepal narrowly ovate-oblong,4-5×0.7mm;
petals narrowly linear,5-6×0.4mm;labelum oblong
ligule,3-4×2-3mm,nearly 3-lobed,lateral lobes e-
rect,obovoid,obtuse,mid-lobe oblong,2-2.5×1.8-
2mm,base ecalose,apical margins entire;column 1.8
-2mm long,slightly arcuate forward,base dilated and
thick.Polinia 4,2pairs,oblong ovate.Flowering,Oc-
tober-December.
Distribution and habitat:Cestichis pingtaoi is epi-
phytic,forming scattered colonies on the wet areas on
the trunks at elevations of 1 300-1 600min ever-
green broad-leaved forests of Malipo,Yunnan Prov-
1645期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种
ince,China.
Conservation status:The new species has been
found in two populations,each of which has no more
than 30individuals.
Polinators:We observed that this new species
was polinated by the fungus gnats.More detailed re-
search should be undertaken in future.
Acknowledgments We would like to thank
CHEN Xu-Hui,GUO Xi-Bin,CHEN Li-Jun for their
help in the field work,MA Xue-Yong for the drawing
of new species;Philipp Bayer of University of
Queensland,Australia,for helping in the manuscript
preparation.
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此次改版,是顺应广大读者的要求,也是适应时代发展特别是我国科技发展日新月异,新技术新成果不断
涌现的迫切需要而采取的重要举措。
改版后的《广西植物》,将一如既往地以服务读者、办好刊物为中心,继续秉承并更加凸现“支撑生态文明建
设和区域经济社会发展,竭力报道植物科学领域新理论、新发现、新技术和新方法”的办刊宗旨,为促进植物科
学繁荣发展、满足广大读者的精神文化需求,将发挥更加积极的作用。
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《广西植物》编辑部
2015年7月22日
3645期 唐光大等:基于ITS和matK序列的兰科沼兰族分子系统研究及二新种