molecular phylogeny of phalaenopsis blume (orchidaceae) on the … · 2017. 8. 29. · molecular...

ORIGINAL ARTICLE

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae)on the basis of plastid and nuclear DNA

C. C. Tsai • Y. C. Chiang • S. C. Huang •

C. H. Chen • C. H. Chou

Received: 29 July 2009 / Accepted: 8 June 2010 / Published online: 4 July 2010

� Springer-Verlag 2010

Abstract The internal transcribed spacers (ITS) of

nuclear ribosomal DNA (nrDNA) and plastid DNA,

including the trnL intron, the trnL-F spacer, and the atpB-

rbcL spacer, from most of the living species in the genus

Phalaenopsis were sequenced. The monophyly of the

genus described by Christenson (Christenson EA (2001)

Phalaenopsis. Timber Press, Portland, p 330), that Doritis

and Kingidium are synonyms of Phalaenopsis, was sup-

ported by these molecular data. Within the genus, subgenus

Polychilos was monophyletic, and the species were divided

into two subclades. The subgenus Phalaenopsis was shown

to be non-monophyletic, because the sections Esmeralda

and Deliciosae appeared separated the from sections

Phalaenopsis and Stauroglottis. Meanwhile, subgenera

Aphyllae and Parishianae were also shown to be

non-monophyletic on the basis of the molecular data.

Furthermore, the monotypic species of subgenus Probos-

cidioides, P. lowii, formed a clade with subgenus Aphyllae.

In accordance with geographical distribution, the historical

geography of Southeast Asia due to the periodic glacial

epochs, and molecular phylogeny, two evolutionary trends

of Phalaenopsis from the original center in South China to

the Philippines, Indonesia, and Malaysia were suggested.

First, using Indochina and some older parts of the Philip-

pines (e.g., Mindoro and Palawan) as stepping stones,

Phalaenopsis species dispersed from South China to the

Philippines, where the sections Phalaenopsis and Stauro-

glottis of subgenus Phalaenopsis developed. Second, using

the Malay Peninsula as a stepping stone, Phalaenopsis

species dispersed from South China to Indonesia and

Malaysia, where the subgenus Polychilos developed.

Keywords Phalaenopsis � Phylogeny � Biogeography �Plastid DNA

Introduction

The genus Phalaenopsis Blume (Orchidaceae) comprises

approximately 66 species worldwide according to the

latest classification of Christenson (2001), who divided

this genus into five subgenera, namely Proboscidioides,

Aphyllae, Parishianae, Polychilos, and Phalaenopsis.

This classification was mainly based on plant size and

floral morphology (callus, lip structure, and pollinium

number). Subgenus Polychilos was subdivided into

four sections (Polychilos, Fuscatae, Amboinenses, and

Zebrinae), and subgenus Phalaenopsis was subdivided

into four sections (Phalaenopsis, Deliciosae, Esmeralda,

and Stauroglottis).

C. C. Tsai and Y. C. Chiang contributed equally to this work.

C. C. Tsai

Kaohsiung District Agricultural Improvement Station,

Pingtung 900, Taiwan

C. C. Tsai � Y. C. Chiang

National Pingtung University of Science and Technology,

Pingtung 912, Taiwan

S. C. Huang

National Plant Genetic Resources Center,

Agricultural Research Institute, Taichung 413, Taiwan

C. H. Chen

Department of Botany, National Museum of Natural Science,

Taichung 404, Taiwan

C. H. Chou (&)

Research Center for Biodiversity, Graduate Institute of Ecology

and Evolutionary Biology, China Medical University,

Taichung 404, Taiwan

e-mail: [email protected]

123

Plant Syst Evol (2010) 288:77–98

DOI 10.1007/s00606-010-0314-1

Species of Phalaenopsis are found throughout tropical

Asia and the larger islands of the Pacific Ocean, and ranges

from Sri Lanka and southern India in the west, to Papua

New Guinea in the east, to the Yunnan Province (southern

China) and Taiwan in the north, and to northern Australia

in the south (Christenson 2001). Different subgenera of

Phalaenopsis have distinct geographic distributions. Sub-

genera Aphyllae, Parishianae, and Proboscidioides are

distributed in southern China and India, extending to

northern Vietnam, Myanmar, and Thailand, respectively.

Subgenus Polychilos has a few species distributed as far

west as northeastern India, but it is primarily centered in

Indonesia and the Philippines (Christenson 2001). Subge-

nus Phalaenopsis is centered in the Philippines with two

species extending into Taiwan (P. aphrodite subsp. for-

mosana and P. equestris) and one wide-ranging species

(P. amabilis) found from the Philippines and Indonesia to

northern Australia (Christenson 2001).

Sweet (1980) divided Phalaenopsis into eight sections

(Table 1). Shim (1982), however, disagreed with Sweet’s

concept (1980) and treated the sections Proboscidioides,

Aphyllae, Parishianae, Polychilos, Zebrinae, Fuscatae,

and Amboinenses as the genus Polychilos, leaving a nar-

rowly defined Phalaenopsis. Christenson (2001) basically

agreed with Sweet’s treatment and raised five subgenera. In

addition, he treated the traditional genera Kingidium and

Doritis as synonyms of Phalaenopsis and split Kingidium

into different parts of Phalaenopsis, placing some species

(i.e., P. braceana, P. minus, and P. taenialis) into the

subgenus Aphyllae and some (i.e., P. chibae and P. del-

iciosa) into the section Deliciosae of subgenus Phalaen-

opsis (Table 1). This generic-level treatment was supported

by several lines of molecular evidence (Padolina et al.

2005; Yukawa et al. 2005; Tsai et al. 2006). Furthermore,

two genera of subtribe Aeridinae, Nothodoritis and Lesliea,

were nested within Phalaenposis on the basis of molecular

data (Topik et al. 2005; Yukawa et al. 2005). Yukawa et al.

(2005) split Phalaenopsis into two genera, Phalaenopsis

and Doritis, in accordance with a narrow genus concept.

According to the systematics of Phalaenopsis described by

Christenson (2001), on the basis of several pieces of

molecular evidence (Padolina et al. 2005; Yukawa et al.

2005; Tsai et al. 2006), the subgenus Aphyllae was not

monophyletic, forming a clade with P. lowii of subgenus

Proboscidioides and the sections Esmeralda and Delicio-

sae of subgenus Phalaenopsis. Therefore, on the basis of

molecular data, the subgenus Phalaenopsis was not

monophyletic. Within subgenus Phalaenopsis, section

Phalaenopsis was shown to be monophyletic on the basis

of nrITS data (Yukawa et al. 2005; Tsai et al. 2006) but not

on the basis of the plastid matK, atpH-F, and trnD-E

spacers (Padolina et al. 2005) and the plastid matK and

trnK introns (Yukawa et al. 2005). The subgenus

Polychilos was shown to be polyphyletic according to

nrITS data (Yukawa et al. 2005; Tsai et al. 2006) but not on

the basis of plastid DNA (Padolina et al. 2005; Yukawa

et al. 2005). Within subgenus Polychilos, only section

Polychilos was shown to be monophyletic on the basis of

internal transcribed spacers (ITS) data (Yukawa et al. 2005;

Tsai et al. 2006), and section Fuscatae was shown to be

monophyletic according to plastid DNA (Padolina et al.

2005; Yukawa et al. 2005).

The objective of this study was to further elucidate the

phylogeny of Phalaenopsis with DNA sequence data,

including the trnL intron, the trnL-F and atpB-rbcL spac-

ers, and nrITS data. Nearly all Phalaenopsis species were

sampled to evaluate in detail the treatments of Christenson

(2001) on the generic, subgeneric, and sectional levels

using both nuclear and plastid DNA. Furthermore, the

biogeographic and evolutionary trends of Phalaenopsis are

discussed in the light of the molecular data, geographical

distribution, and the historical geology of Southeast Asia.

Materials and methods

The taxonomy and nomenclature of the Phalaenopsis

species in this study followed those of Sweet (1980) and

Christenson (2001). The sampled plants included 54 taxa of

Phalaenopsis and plants of seven related genera having

different degrees of hybridization with Phalaenopsis,

including Aerides, Ascocentrum, Neofinetia, Renanthera,

Rhynchostylis, Vanda, and Vandopsis (Sweet 1980). In

addition, Paraphalaenopsis, once treated as Phalaenopsis

(Sweet 1980), and five other genera of subtribe Aeridinae,

Amesiella, Gastrochilus, Haraella, Thrixspermum, and

Tuberolabium, were selected as outgroup species

(Table 2). Leaf materials were collected from living plants

cultivated in the Kaohsiung District Agricultural

Improvement Station (KDAIS) in Taiwan; their voucher

specimens were deposited at the herbarium of the National

Museum of Natural Science, Taiwan (TNM). Flower

photographs of the species sampled for this study are

available from the first author ([email protected]).

Using the cetyltrimethylammonium bromide (CTAB)

method (Doyle and Doyle 1987), total DNA was extracted

from fresh leaves. Ethanol-precipitated DNA was dissolved

in TE (Tris–EDTA) buffer and stored at -20�C. Qiagen

(Hilden, Germany) columns were used to clean DNA

samples that were difficult to amplify.

The amplification procedures were as follows: 50-ll

reactions containing 40 mM Tricine–KOH (pH 8.7),

15 mM KOAc, 3.5 mM Mg(OAc)2, 3.75 lg/ml BSA,

0.005% Tween 20, 0.005% Nonidet-P40, four dNTPs

(0.2 mM each), primers (0.5 lM each), 2.5 U Advantage 2

DNA polymerase (Clontech, CA, USA), 10 ng genomic

78 C. C. Tsai et al.

123

Table 1 Comparison of the systematics of the genus Phalaenopsis between Sweet (1980) and Christenson (2001)

Sweet (1980) Christenson (2001)

Genus Phalaenopsis Genus Phalaenopsis

Section Proboscidioides Subgenus Proboscidioides

Phalaenopsis lowii Phalaenopsis lowii

Section Aphyllae Subgenus Aphyllae

Phalaenopsis wilsonii Phalaenopsis wilsonii

Phalaenopsis stobartiana (syn. Phalaenopsis hainanensis) Phalaenopsis stobartiana

Phalaenopsis hainanensis

Phalaenopsis honghenensis (Liu 1988)a

Phalaenopsis taenialis (syn. Kingidium taenialis) (Christenson 1986)b

Phalaenopsis braceana (syn. Kingidium braceana) (Christenson 1986)b

Phalaenopsis minus (syn. Kingidium minus) (Seidenfaden 1988a)b

Section Parishianae Subgenus Parishianae

Phalaenopsis mysorensis Move to the Section Deliciosae

Phalaenopsis appendiculata Phalaenopsis appendiculata

Phalaenopsis gibbosa Phalaenopsis gibbosa

Phalaenopsis parishii Phalaenopsis parishii

Phalaenopsis lobii Phalaenopsis lobii

Section Polychilos Subgenus Polychilos

Section Polychilos

Phalaenopsis mannii Phalaenopsis mannii

Phalaenopsis cornu-cervi

Phalaenopsis lamelligera

Phalaenopsis cornu-cervi

Phalaenopsis borneensis (Garay et al. 1995)a

Phalaenopsis pantherina Phalaenopsis pantherina

Section Fuscatae Section Fuscatae

Phalaenopsis cochlearis Phalaenopsis cochlearis

Phalaenopsis viridis Phalaenopsis viridis

Phalaenopsis fuscata Phalaenopsis fuscata

Phalaenopsis kunstleri Phalaenopsis kunstleri

Section Amboinenses Section Amboinenses

Phalaenopsis micholitzii Phalaenopsis micholitzii

Phalaenopsis gigantea Phalaenopsis gigantea

Phalaenopsis javanica Phalaenopsis javanica

Phalaenopsis amboinensis Phalaenopsis amboinensis

Phalaenopsis robinsonii Phalaenopsis robinsonii

Phalaenopsis venosa (Shim and Fowlie 1983)a

Phalaenopsis doweryensis (Garay et al. 1995)a

Section Zebrinae

Subsection Lueddemanninae

Phalaenopsis pulchra Phalaenopsis pulchra

Phalaenopsis reichenbachiana Phalaenopsis reichenbachiana

Phalaenopsis fasciata Phalaenopsis fasciata

Phalaenopsis fimbriata Phalaenopsis fimbriata

Phalaenopsis hieroglyphica Phalaenopsis hieroglyphica

Phalaenopsis lueddemanniana Phalaenopsis lueddemanniana

Phalaenopsis violacea Phalaenopsis violacea

Phalaenopsis 9 gersenii Phalaenopsis 9 gersenii

(syn. Phalaenopsis 9 singuliflora) Phalaenopsis 9 singuliflora

Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on plastid and nuclear DNA 79

123

DNA, and 50-ll mineral oil. The amplification reactions

were completed in a dry-block with two-step thermal

cycles (Biometra, Goettingen, Germany). The trnL intron

and trnL-F spacer primers were those of Taberlet et al.

(1991). For the atpB-rbcL spacer, two primers were

designed from conserved regions at the 30 end of the atpB

gene and the 50 end of the rbcL gene from GenBank

sequences, i.e., 50 CATCTAGGATTTACATATAC 30 and

50 GTCAATTTGTAATCTTTAAC 30, respectively. The

PCR conditions for the trnL intron were: incubation at

94�C for 3 min, followed by 10 cycles of denaturation at

94�C for 30 s, annealing at 68�C for 10 s, and extension

at 72�C for 45 s, followed by 30 cycles of denaturation at

94�C for 30 s, annealing at 66�C for 10 s, extension at

Table 1 continued

Sweet (1980) Christenson (2001)

Phalaenopsis bastianii (Gruss and Rollke 1991)a

Phalaenopsis floresensis (Fowlie 1993)a

Phalaenopsis bellina (Christenson and Whitten 1995)b

Subsection Zebrinae Section Zebrinae

Phalaenopsis speciosa Phalaenopsis speciosa

Phalaenopsis tetraspis Phalaenopsis tetraspis

Phalaenopsis corningiana Phalaenopsis corningiana

Phalaenopsis sumatrana Phalaenopsis sumatrana

Phalaenopsis inscriptiosinensis (Fowlie 1983)a

Subsection Hirsutae

Phalaenopsis pallens Phalaenopsis pallens

Phalaenopsis mariae Phalaenopsis mariae

Subsection Glabrae

Phalaenopsis modesta Phalaenopsis modesta

Phalaenopsis maculata Phalaenopsis maculata

Subgenus Phalaenopsis

Section Phalaenopsis Section Phalaenopsis

Phalaenopsis amabilis Phalaenopsis amabilis

Phalaenopsis aphrodite Phalaenopsis aphrodite

Phalaenopsis sanderiana Phalaenopsis sanderiana

Phalaenopsis schilleriana Phalaenopsis schilleriana

Phalaenopsis stuartiana Phalaenopsis stuartiana

Phalaenopsis 9 amphitrite Phalaenopsis 9 amphitrite

Phalaenopsis 9 intermedia Phalaenopsis 9 intermedia

Phalaenopsis 9 leucorrhoda Phalaenopsis 9 leucorrhoda

Phalaenopsis 9 veitchiana Phalaenopsis 9 veitchiana

Phalaenopsis philippinensis (Tharp et al. 1987)a

Section Stauroglottis Section Stauroglottis

Phalaenopsis equestris Phalaenopsis equestris

Phalaenopsis celebensis Phalaenopsis celebensis

Phalaenopsis lindenii Phalaenopsis lindenii

Section Deliciosae

Phalaenopsis chibae (Yukawa 1996)a

Phalaenopsis deliciosa (Siegerist 1989)a

Genus Doritis Section Esmeralda

Doritis pulcherrima Phalaenopsis pulcherrima

Doritis regnieriana Phalaenopsis regnieriana

Doritis buyssoniana Phalaenopsis buyssoniana

a New speciesb Revised species


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34

52

AY

38

94

16

Ph

ala

eno

psi

sb

elli

na

(Rch

b.f

.)E

.A

.C

hri

st.

Mal

aysi

a(M

alay

Pen

insu

la)

and

Eas

t

Mal

aysi

a(S

araw

ak)

KD

AIS

KC

-67

/C.

C.

Tsa

i1

59

6A

Y2

65

74

6A

F5

33

46

7A

Y3

89

43

3

Ph

ala

eno

psi

sd

ow

erye

nsi

sG

aray

&E

.A

.C

hri

st.

Eas

tM

alay

sia,

Sab

ah,

wit

ho

ut

ap

reci

se

loca

lity

KD

AIS

KC

-13

8/n

ov

ou

cher

AY

26

57

53

AF

53

34

85

AY

38

93

95

Ph

ala

eno

psi

sfa

scia

taR

chb

.f.

En

dem

icto

the

Ph

ilip

pin

es(L

uzo

n,

Bo

ho

l,an

dM

ind

anao

)

KD

AIS

KC

-18

9/C

.C

.T

sai

17

79

AY

26

57

55

AF

53

34

90

AY

38

94

01

Ph

ala

eno

psi

sfi

mb

ria

taJ.

J.S

m.

Ind

on

esia

(Jav

a,S

araw

ak,

and

Su

mat

ra)

KD

AIS

KC

-62

/C.

C.

Tsa

i1

73

2A

Y2

65

75

6A

F5

33

46

5A

Y3

89

43

1

Ph

ala

eno

psi

sfl

ore

sen

sis

Fo

wli

eE

nd

emic

toth

eis

lan

do

fF

lore

sK

DA

ISK

C-5

4/C

.C

.T

sai

16

10

AY

26

57

97

AF

53

34

62

AY

38

94

28

Ph

ala

eno

psi

sg

iga

nte

aJ.

J.S

m.

En

dem

icto

Sab

ahin

Esa

tM

alay

sia

and

adja

cen

tK

alim

anta

nT

imu

r,fr

om

sea

lev

elto

40

0m

inel

evat

ion

KD

AIS

KC

-13

1/C

.C

.T

sai

15

76

AY

26

57

59

AF

53

34

84

AY

38

93

94

Ph

ala

eno

psi

sh

iero

gly

ph

ica

(Rch

b.f

.)S

wee

tE

nd

emic

toth

eP

hil

ipp

ines

KD

AIS

KC

-33

/C.

C.

Tsa

i1

64

4A

Y2

65

76

0A

F5

33

45

1A

Y3

89

44

3

Ph

ala

eno

psi

sja

van

ica

J.J.

Sm

.E

nd

emic

toIn

do

nes

ia(J

ava)

KD

AIS

KC

-38

/C.

C.

Tsa

i1

39

1A

Y2

65

76

3A

F5

33

45

5A

Y3

89

42

4

Ph

ala

eno

psi

slu

edd

ema

nn

ian

aR

chb

.f.

En

dem

icto

the

Ph

ilip

pin

esK

DA

ISK

C-8

/C.

C.

Tsa

i1

16

2A

Y2

65

76

8A

F5

33

44

4A

Y3

89

43

6

Ph

ala

eno

psi

sm

acu

lata

Rch

b.f

.M

alay

sia

(Pah

ang

),E

ast

Mal

aysi

a(S

abah

and

Sar

awak

),an

dIn

do

nes

ia

(Kal

iman

tan

Tim

ur)

KD

AIS

KC

-49

/C.

C.

Tsa

i1

77

4A

Y2

65

79

8A

F5

33

46

0A

Y3

89

42

6

Ph

ala

eno

psi

sm

ari

ae

Bu

rb.

exW

arn

.&

B.

S.

Wm

s.

En

dem

icto

the

Ph

ilip

pin

esan

dIn

do

nes

ia

(Kal

iman

tan

and

Bo

rneo

)

KD

AIS

KC

-30

/C.

C.

Tsa

i1

03

0A

Y2

65

77

0A

F5

33

44

9A

Y3

89

41

4

Ph

ala

eno

psi

sm

ich

oli

tzii

Ro

lfe

Ph

ilip

pin

es(M

ind

anao

)K

DA

ISK

C-8

5/C

.C

.T

sai

16

74

AY

26

57

71

AF

53

34

71

AY

38

94

38

Ph

ala

eno

psi

sm

od

esta

J.J.

Sm

.E

nd

emic

toth

eis

lan

do

fB

orn

eoin

Eas

t

Mal

aysi

a(S

abah

)an

dIn

do

nes

ia

(Kal

iman

tan

)

KD

AIS

KC

-15

9/C

.C

.T

sai

17

56

AY

26

57

93

AF

53

34

88

AY

38

93

98

Ph

ala

eno

psi

sp

all

ens

(Lin

dl.

)R

chb

.f.

En

dem

icto

the

Ph

ilip

pin

esK

DA

ISK

C-1

17

/C.

C.

Tsa

i1

11

7A

Y2

65

77

3A

F5

33

47

9A

Y3

89

38

9

Ph

ala

eno

psi

sp

ulc

hra

(Rch

b.f

.)S

wee

tE

nd

emic

toth

eP

hil

ipp

ines

(Lu

zon

and

Ley

te)

KD

AIS

KC

-17

/C.

C.

Tsa

i1

28

1A

Y2

65

77

8A

F5

33

49

4A

Y3

89

39

9

Ph

ala

eno

psi

sre

ich

enb

ach

ian

aR

chb

.f.

&

San

der

En

dem

icto

the

Ph

ilip

pin

esK

DA

ISK

C-2

35

/C.

C.

Tsa

i1

23

5A

Y2

65

77

9A

Y2

66

12

0A

Y3

89

41

0

Ph

ala

eno

psi

sve

no

saS

him

&F

ow

lie

En

dem

icto

Ind

on

esia

(Su

law

esi)

KD

AIS

KC

-14

/C.

C.

Tsa

i1

01

4A

Y2

65

78

5A

F5

33

49

3A

Y3

89

40

6

Ph

ala

eno

psi

svi

ola

cea

Wit

teIn

do

nes

ia(S

um

atra

)an

dM

alay

sia

(Mal

ay

Pen

insu

la)

KD

AIS

KC

-15

2/C

.C

.T

sai

15

98

AY

26

57

96

AF

53

34

87

AY

38

93

97

Sec

tio

nZ

ebri

na

eP

fitz

.

Ph

ala

eno

psi

sco

rnin

gia

na

Rch

b.f

.B

orn

eo(S

araw

akan

del

sew

her

eo

nth

e

isla

nd

)

KD

AIS

KC

-29

/C.

C.

Tsa

i1

77

6A

Y2

65

75

0A

F5

33

44

8A

Y3

89

41

3

Ph

ala

eno

psi

sin

scri

pti

osi

nen

sis

Fo

wli

eE

nd

emic

toIn

do

nes

ia(S

um

atra

)K

DA

ISK

C-4

8/n

ov

ou

cher

AY

26

57

61

AF

53

34

59

AY

38

94

23


123

Ta

ble

2co

nti

nu

ed

Tax

aan

dsy

stem

atic

clas

sifi

cati

on

aG

eog

rap

hic

ald

istr

ibu

tio

nS

ou

rceb

/vo

uch

erc

Gen

Ban

kac

cess

ion

no

.

trnL

intr

on

trnL

-Fsp

acer

atp

B-r

bcL

spac

er

Ph

ala

eno

psi

ssu

ma

tra

na

Ko

rth

.&

Rch

b.f

.W

ides

pre

adfr

om

My

anm

ar,

Th

aila

nd

,

Vie

tnam

,to

Ind

on

esia

(Jav

aan

d

Su

mat

ra),

Mal

aysi

a(P

erak

and

Joh

ore

),

Eas

tM

alay

sia

(Sab

ah),

and

the

Ph

ilip

pin

es(P

alau

an)

KD

AIS

KC

-32

/C.

C.

Tsa

i1

65

0A

Y2

65

78

3A

F5

33

45

0A

Y3

89

41

5

Ph

ala

eno

psi

ste

tra

spis

Rch

b.f

.In

dia

(An

dam

anan

dN

ico

bar

Isla

nd

s)an

d

Ind

on

esia

(Su

mat

ra)

KD

AIS

KC

-40

/C.

C.

Tsa

i1

69

3A

Y2

65

78

4A

F5

33

45

6A

Y3

89

41

9

Su

bg

enu

sP

ha

laen

op

sis

Sec

tio

nP

ha

laen

op

sis

Ben

th.

Ph

ala

eno

psi

sa

ma

bil

is(L

.)B

lum

eW

ides

pre

adfr

om

Su

mat

raan

dJa

va

toth

e

sou

ther

nP

hil

ipp

ines

,an

dea

stto

New

Gu

inea

and

Qu

een

slan

d,

Au

stra

lia

KD

AIS

KC

-96

/C.

C.

Tsa

i1

93

AY

26

57

42

AF

53

34

72

AY

38

94

40

Ph

ala

eno

psi

sa

ph

rod

ite

Rch

b.f

.N

ort

her

nP

hil

ipp

ines

and

sou

thea

ster

n

Tai

wan

KD

AIS

KC

-99

/C.

C.

Tsa

i1

42

0A

Y2

65

74

4A

F5

33

47

3A

Y3

89

44

1

Ph

ala

eno

psi

sp

hil

ipp

inen

sis

Go

lam

coex

Fo

wli

e

&T

ang

En

dem

icto

the

Ph

ilip

pin

esK

DA

ISK

C-2

6/C

.C

.T

sai

13

33

AY

26

57

76

AF

53

34

46

AY

38

94

11

Ph

ala

eno

psi

ssa

nd

eria

na

Rch

b.f

.E

nd

emic

toth

eP

hil

ipp

ines

KD

AIS

KC

-35

/C.

C.

Tsa

i1

52

6A

Y2

65

78

0A

F5

33

45

3A

Y3

89

41

7

Ph

ala

eno

psi

ssc

hil

leri

an

aR

chb

.f.

En

dem

icto

the

Ph

ilip

pin

esK

DA

ISK

C-4

/C.

C.

Tsa

i1

00

4A

Y2

65

78

1A

F5

33

44

3A

Y3

89

42

5

Ph

ala

eno

psi

sst

ua

rtia

na

Rch

b.f

.E

nd

emic

toth

eis

lan

do

fM

ind

anao

inth

e

sou

ther

nP

hil

ipp

ines

KD

AIS

KC

-2/C

.C

.T

sai

14

19

AY

26

57

82

AF

53

34

92

AY

38

94

03

Sec

tio

nD

elic

iosa

eE

.A

.C

hri

st.

Ph

ala

eno

psi

sch

iba

eY

uk

awa

En

dem

icto

Vie

tnam

KD

AIS

KC

-27

/C.

C.

Tsa

i1

79

2A

Y2

65

80

0A

F5

33

44

7A

Y3

89

41

2

Ph

ala

eno

psi

sd

elic

iosa

Rch

b.f

.W

ides

pre

adfr

om

Sri

Lan

ka

and

Ind

iato

the

Ph

ilip

pin

esan

dS

ula

wes

i

KD

AIS

KC

-73

/C.

C.

Tsa

i1

66

4A

Y2

65

75

2A

F5

33

46

8A

Y3

89

43

4

Sec

tio

nE

smer

ald

aR

chb

.f.

Ph

ala

eno

psi

sp

ulc

her

rim

a(L

ind

l.)

J.J.

Sm

.W

ides

pre

adfr

om

no

rth

east

Ind

iaan

d

sou

ther

nC

hin

ath

rou

gh

ou

tIn

do

chin

ato

Mal

aysi

a(M

alay

Pen

insu

la),

Ind

on

esia

(Su

mat

ra),

and

Eas

tM

alay

sia

(Sab

ah)

KD

AIS

KC

-20

/C.

C.

Tsa

i1

02

0A

Y2

65

77

7A

F5

33

49

5A

Y3

89

40

4

Sec

tio

nS

tau

rog

lott

is(S

cha

uer

)B

enth

.

Ph

ala

eno

psi

sce

leb

ensi

sS

wee

tE

nd

emic

toIn

do

nes

ia(S

ula

wes

i)K

DA

ISK

C-6

4/C

.C

.T

sai

16

71

AY

26

57

99

AF

53

34

66

AY

38

94

32

Ph

ala

eno

psi

seq

ues

tris

(Sch

auer

)R

chb

.f.

Ph

ilip

pin

esan

dT

aiw

anK

DA

ISK

C-5

9/C

.C

.T

sai

16

89

AY

26

57

54

AF

53

34

64

AY

38

94

30

Ph

ala

eno

psi

sli

nd

enii

Lo

her

En

dem

icto

the

Ph

ilip

pin

esK

DA

ISK

C-1

18

/C.

C.

Tsa

i1

57

4A

Y2

65

76

6A

F5

33

48

0A

Y3

89

39

0

Ou

tgro

up

s

Aer

ides

mu

ltifl

ora

Ro

xb

ury

Wid

esp

read

fro

mN

epal

toIn

do

chin

aK

DA

ISV

an-1

0/n

ov

ou

cher

DQ

19

49

83

DQ

19

49

98

DQ

19

50

15

Am

esie

lla

ph

ilip

pin

ensi

s(A

mes

)G

ara

yE

nd

emic

toth

eP

hil

ipp

ines

KD

AIS

KC

-46

/no

vo

uch

erD

Q1

94

98

4D

Q1

94

99

9D

Q1

95

01

6


123

Ta

ble

2co

nti

nu

ed

Tax

aan

dsy

stem

atic

clas

sifi

cati

on

aG

eog

rap

hic

ald

istr

ibu

tio

nS

ou

rceb

/vo

uch

erc

Gen

Ban

kac

cess

ion

no

.

trnL

intr

on

trnL

-Fsp

acer

atp

B-r

bcL

spac

er

Asc

oce

ntr

um

am

pu

lla

ceu

m(L

ind

l.)

Sch

ltr.

En

dem

icto

Nep

alan

d

Th

aila

nd

KD

AIS

Van

-24

/no

vo

uch

erD

Q1

94

98

5D

Q1

95

00

0D

Q1

95

01

7(c

lon

e1

)

DQ

19

50

18

(clo

ne

2)

DQ

19

50

19

(clo

ne

3)

Ga

stro

chil

us

jap

on

icu

s(M

akin

o)

Sch

ltr.

En

dem

icto

Tai

wan

and

Jap

anK

DA

ISK

C-6

9/n

ov

ou

cher

AY

26

57

94

AY

26

61

22

DQ

19

50

20

Ha

rael

lare

tro

call

a(H

ayat

a)K

ud

oE

nd

emic

toT

aiw

anK

DA

ISK

C-7

0/n

ov

ou

cher

DQ

19

49

86

DQ

19

50

01

(clo

ne

1)

DQ

19

50

02

(clo

ne

2)

DQ

19

50

03

(clo

ne

3)

DQ

19

50

21

(clo

ne

1)

DQ

19

50

22

(clo

ne

2)

DQ

19

50

23

(clo

ne

3)

DQ

19

50

24

(clo

ne

4)

DQ

19

50

25

(clo

ne

5)

Neo

fin

etia

falc

ata

(Th

un

b.)

H.H

.H

uF

rom

Jap

anan

dK

ore

ato

the

Ry

uk

yu

Is.

(Jp

n.)

KD

AIS

Aer

-36

/no

vo

uch

erD

Q1

94

98

7D

Q1

95

00

4D

Q1

95

02

6

Pa

rap

ha

laen

op

sis

lab

uke

nsi

s(P

.S.

Sh

im)

A.

Lam

b&

C.L

.C

han

.

En

dem

icto

Bo

rneo

KD

AIS

KC

-12

1/n

ov

ou

cher

AY

26

57

89

AF

53

34

81

AY

38

93

91

Ren

an

ther

am

atu

tin

a(B

lum

e)L

ind

l.W

ides

pre

adfr

om

Th

aila

nd

,

Su

mat

ra,

Jav

aan

dM

alay

sia

KD

AIS

Van

-33

/no

vo

uch

erD

Q1

94

98

8D

Q1

95

00

5D

Q1

95

02

7(c

lon

e1

)

DQ

19

50

28

(clo

ne

2)

DQ

19

50

29

(clo

ne

3)

DQ

19

50

30

(clo

ne

4)

DQ

19

50

31

(clo

ne

5)

Rh

ynch

ost

ylis

gig

an

tea

(Lin

dl.

)R

idle

yIn

do

chin

ese

pen

insu

laK

DA

ISK

C-4

5/n

ov

ou

cher

DQ

19

49

89

DQ

19

50

06

DQ

19

50

32

(clo

ne

1)

DQ

19

50

33

(clo

ne

2)

DQ

19

50

34

(clo

ne

3)

Th

rixs

per

mu

mfo

rmo

san

um

(Hay

ata)

Sch

ltr.

En

dem

icto

Tai

wan

KD

AIS

KC

-78

/no

vo

uch

erD

Q1

94

99

0(c

lon

e1

)

DQ

19

49

91

(clo

ne

2)

DQ

19

49

92

(clo

ne

3)

DQ

19

50

07

(clo

ne

1)

DQ

19

50

08

(clo

ne

2)

DQ

19

50

09

(clo

ne

3)

DQ

19

50

10

(clo

ne

4)

DQ

19

50

35

Tu

ber

ola

biu

mko

toen

seY

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123

72�C for 45 s, with a final extension for 5 min at 72�C. The

PCR conditions for the trnL-F spacer were: incubation at

94�C for 3 min, followed by 10 cycles of denaturation at

94�C for 30 s, annealing at 68�C for 30 s, and extension

at 72�C for 30 s, followed by 30 cycles of denaturation at

94�C for 30 s, annealing at 66�C for 30 s, extension at

72�C for 30 s, with a final extension for 5 min at 72�C. The

PCR reaction conditions for the atpB-rbcL spacer were:

incubation at 94�C for 3 min, followed by 10 cycles of

denaturation at 94�C for 45 s, annealing at 54�C for 30 s,

and extension at 72�C for 1 min, followed by 30 cycles of

denaturation at 94�C for 45 s, annealing at 52�C for 30 s,

extension at 72�C for 1 min, with a final extension for

5 min at 72�C. The PCR products were stained with

0.5 lg/ml ethidium bromide, detected by agarose gel

electrophoresis (1.0%, w/v in TBE), and photographed

under UV light. The PCR products were purified using

Glassmilk (BIO 101, CA, USA).

The amplicons were sequenced in both directions using

the BigDye� Terminator v3.1 Cycle Sequencing Kit

(Applied Biosystems, CA, USA). The sequencing primers

were the same as those used for PCR. The sequencing was

performed on an Applied Biosystems 3730 automated

sequencer. Each sample was sequenced two or three times

for confirmation. Cloning was used to clarify the intra-

individual variation (additive peaks) found in some sam-

ples. The recovered PCR products were ligated into a

pGEM-T Easy vector (Promega, WI, USA), and the

resulting recombinants were transformed into Escherichia

coli. Five to seven clones for each individual were ran-

domly selected, and the plasmid DNA was purified with

Qiagen spin mini-prep kits. The plasmid DNA was

sequenced with vector-specific primers (SP6 and T7). In

addition, the nuclear ITS sequences of Phalanopsis and

outgroup species studied were obtained from Tsai et al.

(2006).

The sequence alignment was determined using the

Clustal W multiple alignment program in BioEdit ver.

7.0.5 (Hall 1999). The alignment was subsequently

checked visually. Maximum parsimony (MP) analyses

(Fitch 1971) were performed using code modified from the

close-neighbor-interchange (CNI) algorithm (Rzhetsky and

Nei 1992) in MEGA ver. 4 (Tamura et al. 2007). The strict

consensus of the most parsimonious trees was constructed

using MEGA ver. 4 (Tamura et al. 2007), and bootstrap

consensus values were calculated using 1,000 replicates

(Felsenstein 1985; Hillis and Bull 1993). The phylogenetic

relationships among the haplotypes were also evaluated

using Bayesian inference and MrBayes ver. 3.1.2 (Ronquist

and Huelsenbeck 2003). The general time-reversible GTR

model with the gamma-distributed rate variation across

sites and a proportion of invariable sites was determined

to be the most suitable model by jModelTest ver. 0.1.1

(Posada 2008). This model was used for the subsequent

nucleotide analyses, coded as the MrBayes likelihood set-

tings hLset nst = 6 rates = invgammai. Two parallel runs

and four Markov Chain Monte Carlo (MCMC) chains were

run for 10,000,000 generations, and a tree was sampled

every 1,000 generations after a burn-in period of 3,000,000

generations, after which the standard deviation of the split

frequencies was below 0.01 as suggested in the manual.

The aligned data matrix and tree files are available from the

first author ([email protected]).

Results

Sequence alignment and characteristics

All PCR products were directly sequenced except for those

of the trnL intron of Thrixspermum formosanum, the trnL-F

spacers of Haraella retrocalla and T. formosanum, and the

atpB-rbcL spacers of Ascocentrum ampullaceum, Haraella

retrocalla, Renanthera matutina, Rhynchostylis gigantea,

and Vandopsis lissochiloides. Five separate clones were

randomly selected for sequencing within an individual

(Table 2). In addition, Phalaenopsis lowii showed PCR

products of different lengths for both the plastid trnL intron

and atpB-rbcL spacer, and P. gibbosa for the atpB-rbcL

spacer. These PCR products were separately cloned. By

comparing the long and short forms of the trnL intron and

atpB-rbcL spacer in P. lowii, it was shown that the short

forms had long deletions of 139 bp (data not shown) and

244 bp plus nine nucleotide substitutions, respectively

(Fig. 1). For P. gibbosa, the short form had a long deletion

of 158 bp, four short deletions, and 16 nucleotide substi-

tutions (Fig. 2). In this study, the molecular phylogeny of

Phalaenopsis was clarified using the long form of the

plastid DNA fragment. The GenBank accession numbers of

those plastid DNA sequences from the 54 species of

Phalaenopsis plus the 13 outgroup species are shown in

Table 2. Four sequences (the trnL introns from Aerides

multiflora, Amesiella philippinensis, and Tuberolabium

kotoense were 283, 353, and 365 bp, respectively, and the

atpB-rbcL spacer from Gastrochilus japonicus was

486 bp) were extremely short compared with the others

and were therefore excluded from further analyses.

Heterogeneous sequences were found in six outgroup

species as described above, and the first clone sequenced

from each of these samples was selected for the phyloge-

netic analyses because the individual sequences had high

nucleotide sequence identity to each other within a sample

(data not shown). The three plastid DNA regions were

combined after removal of 95 bp within the trnL intron,

because of an unalignable TA-rich region caused by dif-

ferences in the P8 stem loop region, as described by


123

Kocyan et al. (2008). The resulting alignment was 2,107 bp

in length, of which 527 sites were variable (the trnL intron,

trnL-F spacer, and atpB-rbcL spacer accounted for 166,

107, and 254 variable sites, respectively) and 193 were

potentially parsimony-informative sites (the trnL intron,

trnL-F spacer, and atpB-rbcL spacer accounted for 66, 35,

and 92, respectively). The sequence alignment of the

combined nuclear and plastid DNA data matrix was

2,886 bp, of which 963 were variable and 498 were

potentially parsimony-informative sites.

Phylogenetic reconstruction

The Bayesian inference (BI) tree derived from the nrITS

data is shown in Fig. 3. Using the plastid DNA, the MP

analysis yielded 425 equally most-parsimonious trees, each

of length (L) = 868 steps, with a consistency index (CI) of

0.71 and a retention index (RI) of 0.76. The strict con-

sensus tree is shown in Fig. 4. The BI tree is generally

congruent with the MP strict consensus tree (Fig. 5). On

the basis of the combined data matrix, the MP analysis

yielded 13 equally most-parsimonious trees, each of length

(L) = 1,913 steps, with a CI of 0.64 and an RI of 0.78. The

MP strict consensus tree is shown in Fig. 6 and is generally

congruent with the BI tree (Fig. 7).

The monophyly of Phalaenopsis was supported by the

nrITS, plastid DNA, and combined DNA data sets (84, 99,

and 99% bootstraps in the MP tree, 100, 100, and 100% in

the BI tree; Figs. 3, 4, 5, 6, 7). Within Phalaenopsis, the

monophyly of subgenus Polychilos was supported by the

5 15 25 35 45 55 65P. lowii-normal type TACAACATAT ATTACTGTCA AGAGAGGGGA CCGGGTCCTA TATTCTTTCT TTTTCTTTCT ATATTAGATAP. lowii-variant type .........G .......... .......... .......... .......... .......... ..--------

75 85 95 105 115 125 135P. lowii-normal type TTTCTATTTA CTATTTATTA TCTTGACTTT AAAATTTTTA TAATTGAATT TTATTCATTC TAGAATTTCTP. lowii-variant type ---------- ---------- ---------- ---------- ---------- ---------- ----------

145 155 165 175 185 195 205P. lowii-normal type TAATTCGAAT TTAGAATTCT ATTTCTATTC AATTTAATAT TTATCTATTT GAATTGAATT CTATTTAAACP. lowii-variant type ---------- ---------- ---------- ---------- ---------- ---------- ----------

215 225 235 245 255 265 275P. lowii-normal type TAGATTTATG AATTGAAATG AAATCGAAAT TTTTCATTTT ATTTGATGTT TTTTTCTCTT TATTTTTATAP. lowii-variant type ---------- ---------- ---------- ---------- ---------- ---------- ----------

285 295 305 315 325 335 345P. lowii-normal type TTCTTATTTA TTTATTTTTT TTCTATTTAT ATTCTATATC ATATTCATTA TTTATAAAAA ATATTAAGAAP. lowii-variant type ---------- ---------- ------.C.. .......... .......... ....G..... ..........

355 365 375 385 395 405 415P. lowii-normal type GATTAGAAAT TCCATTAAGA ATAGAAATTT TCAAGAAGAT TGGGTTGCGC CATATATATC AAAGAGTCTAP. lowii-variant type ...G...... .......... .......... .......... .......... .......... ..........

425 435 445 455 465 475 485P. lowii-normal type AAATAATGAT GTATTTGGTG AATCAAATAA ATGGTCCAAT AACGAACCCT TTTCAAAATT TCATTATTCAP. lowii-variant type .......... .......... .......... .......... .......... .......... ..........

495 505 515 525 535 545 555P. lowii-normal type TTAGTTGATA ATATTAATTT AGAGTTTAGT TGAATCTTTT TTGAATTGTA AAGATTTTTG TCAAAGGTTTP. lowii-variant type .......... ....G..... CT........ .......... .........C ..T....... ..........

565 575 585 595 605 615 625P. lowii-normal type CATTCACGCT TAATTCATAT CGAGTAGACC TTGTTGTTGT GAGAATTCTT AATTCATGAG TGTAGGGAGGP. lowii-variant type .......... .......... .......... .......... .......... .......... ..........

635 645 655P. lowii-normal type GACTTATGTC ACCACAAACA GAAACTAAAG CAP. lowii-variant type .......... .......... .......... ..

Fig. 1 The sequence alignment of the different forms of atpB-rbcL spacers of plastid DNA from a P. lowii individual


123

plastid DNA and the combined data (55 and 72% boot-

straps in the MP tree, 100 and 100% in the BI tree). Within

subgenus Polychilos, section Fuscatae was monophyletic

on the basis of the plastid DNA, combined data, and the

nrITS, excluding one clone of ITS in P. viridis (91, 88 and

95% bootstrap in the MP tree, 100, 100 and 100% in the BI

tree; Figs. 3, 4, 5, 6, 7). Section Polychilos was mono-

phyletic according to the nrITS (75% bootstrap in the MP

tree; Tsai et al. 2006) but not in the trees based on the

plastid DNA and combined DNA data, because of the

position of P. mannii (Figs. 4, 5, 6, 7). Species of both

sections Amboinenses and Zebrinae were not monophyletic

5 15 25 35 45 55

P. gibbosa-normal type TACAACATAT ATTACTGTCA AGAGAGGGGA CCGGGTCCTA TATTCTTTCT TTTTATTTCTP. gibbosa-variant type .........G .......... .......... .......... .......... ....C.....

65 75 85 95 105 115P. gibbosa-normal type ATATTAGATA TTTCTATTTA CTATTTATTA TCTTTAATAT CTTTATTTTA TAATTGAAATP. gibbosa-variant type .......... .......... .......... .......... .......--- ----------

125 135 145 155 165 175P. gibbosa-normal type TTCTTCATTC TAGAATTTCT GAATTCTAAT TTAGAATTCT ATTTCTATTC AATTTAATATP. gibbosa-variant type ---------- ---------- ---------- ---------- ---------- ----------

185 195 205 215 225 235P. gibbosa-normal type TTATCTATTT GAATTGAATT CTATTTAAAC TAGATTTCTG AATTGAAATG AAATCGAAATP. gibbosa-variant type ---------- ---------- ---------- ---------- ---------- ----------

245 255 265 275 285 295P. gibbosa-normal type TTTTCATTTT CTTTGATGTT TTTTTCTCTT TATTTTGATA TTCTTATTTC TTTATTTTTTP. gibbosa-variant type ---------- ---------- -----..... .C........ .......... ...T....C-

305 315 325 335 345 355P. gibbosa-normal type TATATATTCA TATTCTATTA TCATATTCAT TATTTCATTA TTTATAAAAA AATATTAAGAP. gibbosa-variant type ----...... ........-. .......... ...----... .C-----... ..........

365 375 385 395 405 415P. gibbosa-normal type AGATGATAAA TTCCATTAGG AATAGAAATT TTCAAGAAGA TTGGGTTGCG CCATATATATP. gibbosa-variant type ......G... .......... .......... .......... .......... ..........

425 435 445 455 465 475P. gibbosa-normal type CAAAGAATAT AAAATAATGA TGTATTTGGT GAATCAAAGA AATGGTCCAA TAACGAACCCP. gibbosa-variant type ......G..G .......... .......... .......... .......... ..........

485 495 505 515 525 535P. gibbosa-normal type TTTTCAAAAT TTCATTATTC ATTAGTTGAT AATATTAATT TCTAGTTTAG TTGAATCTTTP. gibbosa-variant type .......... .......... .......... .....G.... .......... ..........

545 555 565 575 585 595P. gibbosa-normal_type TTTGAATTGT AAATATTTTT GTCAAAGGTT TCATTCACGC TTAATTCATA TCGAGTAGACP. gibbosa-variant_type .......... C......... .......... .......... .......... ..........

605 615 625 635 645 655P. gibbosa-normal_type CTTGTTGTTG TGAGAATTCT TAATTCATGA GTTGTAGGGA GGGACTTATG TCACCACAAAP. gibbosa-variant_type .......... .......... .......... .......... .......... ..........

665 675P. gibbosa-normal_type CAGAAACCTA AAGCAP. gibbosa-variant_type .......TC. .GCA.

Fig. 2 The sequence alignment of the different forms of atpB-rbcL spacers of plastid DNA from a P. gibbosa individual


123

in all the analyses (Figs. 3, 4, 5, 6, 7). Parts of section

Amboinenses, namely P. bastianii, P. fasciata, P. hiero-

glyphica, P. lueddemanniana, P. pallens, P. pulchra, and

P. reichenbachiana, formed a clade according to the nrITS,

plastid DNA, and combined data (99, 76% excluding

P. reichenbachiana, and 99% bootstrap in the MP tree,

100, 93, and 100% in the BI tree). Phalaenopsis mariae

was sister to the aforementioned group (Figs. 3, 4, 5, 6, 7).

Furthermore, parts of section Amboinenses, namely

P. doweryensis, P. gigantea, and P. maculata, formed a

strongly supported clade with section Fuscatae in all the

phylogenetic trees (Figs. 3, 4, 5, 6, 7).

The monophyly of subgenus Phalaenopsis was not

supported in all the analyses (Figs. 3, 4, 5, 6, 7). Within

subgenus Phalaenopsis, section Phalaenopsis was mono-

phyletic, but section Stauroglottis was not, on the basis of

the nrITS and combined DNA data (99 and 99% bootstraps

in the MP tree, 100 and 100% in the BI tree; Figs. 3, 6 and

7). Section Deliciosae did not form a clade based on the

nrITS data (Fig. 3) but formed a clade in plastid DNA

analyses (Figs. 4, 5). Phalaenopsis pulcherrima (section

Esmeralda) formed a clade with the members of subgenera

Parishianae, Aphyllae, and Proboscidioides on the basis of

the nrITS and combined data (81 and 70% bootstraps in the

MP tree, 100 and 93% in the BI tree; Figs. 3, 6 and 7).

Subgenus Parishianae appeared monophyletic in the BI

tree derived from plastid DNA only (Fig. 5). Subgenus

Aphyllae was not monophyletic because P. lowii (subgenus

Proboscidioides) was nested within subgenus Aphyllae in

all the obtained phylogenetic trees (Figs. 3, 4, 5, 6, 7).

Discussion

Heterogeneous plastid DNA within individuals of two

Phalaenopsis species

The short forms of DNA fragments in both the trnL intron

and the atpB-rbcL spacer had also indels based on the

sequence alignment. There are at least two explanations for

different copies of plastid DNA within an individual:

1 these different copies are all retained in the plastid

genome because of the occasional biparental inheri-

tance of plastid genomes (Second et al. 1989; Liu et al.

2004; Matsushima et al. 2008); or

2 the short form of the plastid DNA might be retained in

the nuclear or mitochondrial genome through horizon-

tal gene transfer (Ellis 1982; Timmis and Scott 1983;

Cheung and Scott 1989; Ayliffe and Timmis 1992;

Ayliffe et al. 1998).

Horizontal transfer can occur in large fragments

([30 kb) coming from the organellar genome into the

nuclear genome (Yuan et al. 2002; Huang et al. 2005). In

this study, the sequence of the short form of DNA was

different from that of the other species of Phalaenopsis.

Thus, we suggest that these short forms were not retained

through biparental inheritance, but were retained through

horizontal gene transfer. In addition, the mutation rate in

nuclear DNA is higher than that in plastid DNA in plants

(Wolfe et al. 1987). This could explain the high mutation

rate for the short form of plastid DNA in the nuclear

genome in both P. lowii and P. gibbosa.

The phylogeny of the genus Phalaenopsis

Phalaenopsis, as described by Christenson (2001), was

monophyletic on the basis of the nrITS data (Tsai et al.

2006, Fig. 3), plastid trnL intron, trnL-F and atpB-rbcL

spacers (Figs. 4, 5), and combined data (Figs. 6, 7). These

results are consistent with those from previous phyloge-

netic analyses based on other molecular evidence, includ-

ing the plastid matK, atpH-F, and trnD-E (Padolina et al.

2005) and the plastid matK and trnK introns and the nrITS

(Yukawa et al. 2005). Excluding the monotypic subgenus

Proboscidioides, only subgenus Polychilos was monophy-

letic in the plastid DNA and combined data analyses,

and the same patterns have been shown in other studies

(Padolina et al. 2005; Yukawa et al. 2005). On the basis of

the phylogenetic tree from the combined data, three major

clades are shown within genus Phalaenopsis. The first

clade, which includes species of subgenus Polychilos, was

also found in previous studies (Padolina et al. 2005;

Yukawa et al. 2005), as was the second clade, including

subgenus Phalaenopsis sections Phalaenopsis and Stauro-

glottis (Kao 2001; Padolina et al. 2005; Yukawa et al.

2005). The third clade, comprising subgenera Parishianae,

Aphyllae, and Proboscidioides and sections Deliciosae and

Esmeralda of subgenus Phalaenopsis, was also retrieved in

other molecular phylogenetic studies (Padolina et al. 2005;

Yukawa et al. 2005; Carlsward et al. 2006). The species

included in this latter clade also have similar morphology

(four pollinia, Sweet 1980; Seidenfaden 1988a) and dis-

tributions (mainly distributed in South China and Indo-

china; Christenson 2001).

Two monotypic genera, Nothodoritis and Lesliea, were

nested within Phalaenopsis on the basis of the mole-

cular phylogeny of subtribe Aeridinae (Topik et al. 2005).

Yukawa et al. (2005) re-evaluated Phalaenopsis at the

Fig. 3 The Bayesian inference (BI) tree resulting from analysis of 53

Phalaenopsis and 13 outgroup ITS sequences. Posterior probabili-

ties [50% are shown above each branch for the BI tree and below

each branch for the MP tree. A filled circle indicates that the species

was traditionally treated as the genus Doritis. A filled square indicates

that the species was traditionally treated as the genus Kingidium.

Redrawn from Tsai et al. 2006

c


123

P. corningiana

P. sumatrana

P. tetraspis

P. zebrina

Section Zebrinae

Section ZebrinaeP. inscriptiosinensis

Section AmboinensesP. modesta

P. fimbriata

P. floresensis

P. javanica

Section Amboinenses

P. amboinensis

P. venosa

P. bellina

P. violacea

Section Amboinenses

P. bastianii

P. fasciata

P. hieroglyphica

P. reichenbachiana

P. pulchra

P. luddemanniana

P. pallens

P. mariae

Section Amboinenses

P. borneensis

P. cornu-cervi

P. lamelligera

P. pantherina

Section Polychilos

Section PolychilosP. mannii

Section AmboinensesP. micholitzii

Subgenus Polychilos

Section AmboinensesP. maculata

Section FuscataeP. viridis-clone 3

Section AmboinensesP. doweryensis

P. viridis-clone 2

P. viridis-clone 1

P. kunstleri

P. cochlearis

Section Fuscatae

Subgenus Polychilos

Section StauroglottisP. celebensis

P. lindenii

P. equestrisSection Stauroglottis

P. stuartiana

P. schilleriana

P. philippinensis

P. aphrodite

P. sanderiana

P. amabilis

Section Phalaenopsis


Section Esmeralda ----------------- Subgenus PhalaenopsisP. pulcherrima

Section Deliciosae ------------------ Subgenus PhalaenopsisP. chibae

--------------------------------------------- Subgenus AphyllaeP. minus

--------------------------------------------- Subgenus ProboscidioidesP. lowii

P. honghenensis

P. wilsonii

P. braceana

Subgenus Aphyllae

--------------------------------------------- Subgenus ParishianaeP. appendiculata

P. parishii

P. lobbii

P. gibbosa

Subgenus Parishianae

Section Deliciosae ------------------ Subgenus PhalaenopsisP. deliciosa

Amesiella philippinensis

Tuberolabium kotoense

Ascocentrum ampullaceum

Vanda coerulescens

Neofinetia falcata

Paraphalaenopsis labukensis

Rhynchostylis gigantea

Haraella retrocalla

Gastrochilus japonicus

Renanthera matutina

Aerides multiflora

Vandopsis lissochiloides

Thrixspernum formosanum

100

100100

10089

96

100100

72

76

9799100

58

100

10091

100

59100

88100

82

76

10078

94

100

9953

80

10080

100

75

100

98

100

100

98

97100

84

83

100

62

95 99

100 99100

100

100

9956

9092

71

51

9299

99

9983

99

9975

59

7264

91

9583

92

54

67

86

99

94

99

8384

5052

9581

99

99

90

63


123

generic level. The results suggested that Nothodoritis and

Lesliea belong in Phalaenopsis, and that there are two main

genera, Phalaenopsis (including subgenera Polychilos and

Phalaenopsis sections Phalaenopsis and Stauroglottis; the

two-pollinium clade) and Doritis (including subgenera

Parishianae, Aphyllae, Proboscidioides, and Phalaenopsis

P. hieroglyphica P. pallens P. lueddemanniana P. bastianii P. pulchra P. fasciata P. mariae

Section Amboinenses

Section Polychilos P. mannii P. reichenbachiana P. amboinensis P. venosa

Section Amboinenses

P. floresensis P. bellina P. violacea

Section Amboinenses

Section Amboinenses P. micholitzii P. cornu-cervi P. lamelligera P. borneensis P. patherina

Section Polychilos

Section Zebrinae P. inscriptiosinensisSection Amboinenses P. modestaSection Amboinenses P. fimbriataSection Zebrinae P. tetraspisSection Zebrinae P. corningianaSection Amboinenses P. javanicaSection Zebrinae P. sumatranaSection Amboinenses P. maculata

P. doweryensis P. gigantea Section Amboinenses

P. cochlearis P. fuscata P. kunstleri P. viridis

Section Fuscatae

Subgenus Polychilos

Section Esmeralda ---- Subgenus Phalaenopsis P. pulcherrima P. equestris P. lindenii P. celebensis

Section Stauroglottis

P. schilleriana P. philippinensis P. stuartiana


P. amabilis P. aphrodite P. sanderiana



------------------------------ Subgenus Aphyllae P. wilsonii------------------------------ Subgenus Proboscidioides P. lowii------------------------------ Subgenus Aphyllae P. braceana------------------------------ Subgenus Aphyllae P. honghenensis------------------------------ Subgenus Aphyllae P. minus

P. chibae P. deliciosa Section Deliciosae ---- Subgenus Phalaenopsis

P. appendiculata P. gibbosa P. lobbii P. parishii


Thrixspermum formosanum-1 Renanthera matutina Rhynchostylis gigantea-1 Paraphalaenopsis labukensis Ascocentrum ampullaceum-1 Neofinetia falcata Vanda coerulescens Haraella retrocalla-1 Vandopsis lissochiloides-1

96

72

99

80

5964

88

60

7599

71

51

91

71

96

85

76

91

8892

99

88

55

9971

56

99

60

Fig. 4 The strict consensus tree of the most parsimonious trees

resulting from analysis of the combined plastid DNA (including the

plastid trnL intron, trnL-F spacer, and atpB-rbcL spacer) from 54

Phalaenopsis and nine outgroup species. Bootstrap values [50% are

shown above each branch. A filled circle indicates that the species

was traditionally treated as the genus Doritis. A filled square indicates

that the species was traditionally treated as the genus Kingidium


123

P. amboinensis

P. venosaSection Amboinenses

P. bastianii

P. fasciata

P. hieroglyphica

P. lueddemanniana

P. pallens

P. pulchra

P. reichenbachiana

P. mariae

Section Amboinenses

P. floresensis

P. bellina

P. violacea

Section Amboinenses

Section Amboinenses P. micholitzii

P. cornu-cervi

P. borneensis

P. lamelligera

P. patherina

Section Polychilos

Section Zebrinae P. corningiana

Section Amboinenses P. javanica

Section Zebrinae P. sumatrana

Section Zebrinae P. tetraspis

Section Amboinenses P. fimbriata

Section Zebrinae P. inscriptiosinensis

Section Polychilos P. mannii

Section Amboinenses P. modesta

Section Amboinenses P. maculata

P. doweryensis

P. giganteaSection Amboinenses

P. cochlearis

P. fuscata

P. viridis

P. kunstleri

Section Fuscatae

Subgenus Polychilos

Section Esmeralda --- Subgenus Phalaenopsis P. pulcherrima

------------------------------ Subgenus Aphyllae P. wilsonii

------------------------------ Subgenus Proboscidioides P. lowii

------------------------------ Subgenus Aphyllae P. braceana

------------------------------ Subgenus Aphyllae P. honghenensis

------------------------------ Subgenus Aphyllae P. minus

P. celebensis

P. equestris

P. lindenii

Section Stauroglottis

P. stuartiana

P. schilleriana

P. philippinensis


P. sanderiana

P. aphrodite

P. amabilis



P. deliciosa

P. chibaeSection Deliciosae --- Subgenus Phalaenopsis

P. parishii

P. lobbii

P. gibbosa

P. appendiculata


Vandopsis lissochiloides-1

Haraella retrocalla-1

Renanthera matutina

Rhynchostylis gigantea-1


Neofinetia falcata

Vanda coerulescens

Ascocentrum ampullaceum-1

Thrixspermum formosanum-1

7995

100

86

100

93

74

98100

100

96

100

10095

100

9670

100

59

100

100

100

77100

99

55

93

86

100

70

98

10095

98

100

100

50

100 100

10099

Fig. 5 The Bayesian inference tree resulting from analysis of the

combined plastid DNA (including the plastid trnL intron, trnL-F

spacer, and atpB-rbcL spacer) from 54 Phalaenopsis and nine

outgroup species. Posterior probabilities [50% are shown above each

branch. A filled circle indicates that the species was traditionally

treated as the genus Doritis. A filled square indicates that the species

was traditionally treated as the genus Kingidium


123

P. reichenbachiana P. hieroglyphica P. pulchra P. fasciata P. pallens P. bastianii P. luddemanniana P. mariae

Section Amboinenses

P. violacea P. bellina P. venosa P. amboinensis

Section Amboinenses

P. javanica P. floresensis P. fimbriata

Section Amboinenses

Section Zebrinae P. inscriptiosinensisSection Amboinenses P. modesta

P. tetraspis P. sumatrana P. corningiana

Section Zebrinae

Section Polychilos P. manniiSection Amboinenses P. micholitzii

P. borneensis P. cornu-cervi P. lamelligera P. pantherina

Section Polychilos

Section Amboinenses P. maculataSection Amboinenses P. doweryensis

P. cochlearis P. kunstleri P. viridis-1 P. viridis-2 P. viridis-3

Section Fuscatae

Subgenus Polychilos

Section Stauroglottis P. celebensis P. equestris P. lindenii Section Stauroglottis

P. amabilis P. sanderiana P. aphrodite P. philippinensis P. schilleriana P. stuartiana



Section Esmeralda --- Subgenus Phalaenopsis P. pulcherrima-------------------------- P. lobbii

P. parishii P. gibbosa


Section Deliciosae --- Subgenus Phalaenopsis P. deliciosa------------------------------- Subgenus Parishianae P. appendiculataSection Deliciosae --- Subgenus Phalaenopsis P. chibae------------------------------- Subgenus Aphyllae P. minus------------------------------- Subgenus Proboscidioides P. lowii---------------------- P. wilsonii

P. braceana P. honghenensis

Subgenus Aphyllae

Thrixspernum formosanum Rhynchostylis gigantea Renanthera matutina Vandopsis lissochiloides Haraella retrocalla Paraphalaenopsis labukensis Neofinetia falcata Ascocentrum ampullaceum Vanda coerulescens

87

99

99

79

99

99

99

86

99

99

5299

98

55

8765

8599

99

51

94

9761

87

9997

88

98

99

6698

50

99

99

72

62

99

54

58

51

63

70

99

99

96

97

99

Fig. 6 The strict consensus tree of the most parsimonious trees

resulting from analysis of the combined data matrix (the nuclear

ribosomal ITS, plastid trnL intron, trnL-F spacer, and atpB-rbcL

spacer) from 52 Phalaenopsis and nine outgroup species. Bootstrap

values [50% are shown above each branch. A filled circle indicates

that the species was traditionally treated as the genus Doritis. A filledsquare indicates that the species was traditionally treated as the genus

Kingidium


123

P. bastianii

P. fasciata

P. hieroglyphica

P. reichenbachiana

P. pulchra

P. luddemanniana

P. pallens

P. mariae

Section Amboinenses

Section Polychilos P. mannii

P. violacea

P. bellina

P. venosa

P. amboinensis

Section Amboinenses

P. javanica

P. floresensis

P. fimbriata

Section Amboinenses

Section Zebrinae P. inscriptiosinensis

Section Amboinenses P. modesta

P. tetraspis

P. sumatrana

P. corningiana

Section Zebrinae

Section Amboinenses P. micholitzii

P. borneensis

P. cornu-cervi

P. lamelligera

P. pantherina

Section Polychilos

Section Amboinenses P. maculata

Section Amboinenses P. doweryensis

P. cochlearis

P. kunstleri

P. viridis-1

P. viridis-2

P. viridis-3

Section Fuscatae

Subgenus Polychilos

Section Stauroglottis P. celebensis

P. equestris

P. lindeniiSection Stauroglottis

P. amabilis

P. sanderiana

P. aphrodite

P. philippinensis

P. schilleriana

P. stuartiana



Section Esmeralda --- Subgenus Phalaenopsis P. pulcherrima

P. lobbii

P. parishii

P. gibbosa


------------------------------ Subgenus Parishianae P. appendiculata

Section Deliciosae --- Subgenus Phalaenopsis P. deliciosa

Section Deliciosae --- Subgenus Phalaenopsis P. chibae

------------------------------ Subgenus Aphyllae P. minus

------------------------------ Subgenus Proboscidioides P. lowii

--------------------- P. wilsonii

P. braceana

P. honghenensis

Subgenus Aphyllae

Vandopsis lissochiloides

Haraella retrocalla

Ascocentrum ampullaceum

Vanda coerulescens

Neofinetia falcata


Renanthera matutina

Rhynchostylis gigantea

Thrixspernum formosanum

100

100

100

9490100

75

88

89

100

100

100

100

62100

88

76

99100

100

65

100

100

100100

100

100

100

100

100100

56

58

9798

100

58

99

93

100

100100

10099

100

96

100 100

99

99100

100

100

Fig. 7 The Bayesian inference tree resulting from analysis of the

combined data matrix (the nuclear ribosomal ITS, plastid trnL intron,

trnL-F spacer, and atpB-rbcL spacer) from 52 Phalaenopsis and nine

outgroup species. Posterior probabilities [50% are shown above each

branch. A filled circle indicates that the species was traditionally

treated as the genus Doritis. A filled square indicates that the species

was traditionally treated as the genus Kingidium


123

sections Emeralda and Deliciosae plus genera Nothodoritis

and Lesliea; the four-pollinia clade). This result was only

moderately supported by the nrITS sequences (Yukawa

et al. 2005, Tsai et al. 2006) but not supported by plastid

data from the matK, atpH-F, and trnD-E spacers (Padolina

et al. 2005), matK and trnK introns (Yukawa et al. 2005),

or by the trnL intron, and trnL-F and atpB-rbcL spacers of

this study (Figs. 4, 5). This supports clear incongruences

between plastid DNA and nrITS data in the lineage of

subgenus Phalaenopsis, sections Phalaenopsis and Stau-

roglottis. Because plastid DNA is maternally inherited and

nrITS is biparentally inherited, the inconsistency might be

derived from an ancient hybridization event or maybe

because of the paralogous ITS sequences.

Intrageneric relationships within Phalaenopsis

Subgenus Polychilos

The plastid DNA and combined data support the mono-

phyly of subgenus Polychilos with weak or moderate

support. However, this group cannot be separated from

sections Phalaenopsis and Stauroglottis of subgenus Pha-

laenopsis on the basis of the nrITS data (Yukawa et al.

2005; Tsai et al. 2006). Furthermore, within subgenus

Polychilos, only section Fuscatae was shown to be

monophyletic on the basis of the plastid DNA and com-

bined data. Section Polychilos appeared polyphyletic based

on the position of P. mannii in the plastid DNA and

combined data phylogenies, but formed a clade with

moderate support in the MP tree derived from the nrITS

data (Tsai et al. 2006). Those molecular data are in

agreement with morphological data, which show that spe-

cies in section Polychilos have a fleshy, flattened rachis,

with the exception of P. mannii which has a fleshy, roun-

ded rachis (Christenson 2001). In addition, one species of

the section Amboinenses, P. micholitzii, grouped with

section Polychilos. These results are consistent with other

molecular phylogenetic results (Goh et al. 2005; Yukawa

et al. 2005).

According to all the phylogenetic trees of this study,

section Fuscatae grouped with parts of section Amboinen-

ses, namely P. doweryensis, P. gigantea, and P. maculata.

These results are congruent with the 5S spacer data (Kao

2001), nrITS ? matK-trnK intron data (Yukawa et al.

2005), and the RAPD data of Goh et al. (2005). Morpho-

logically, the aforementioned three species of section

Amboinenses and the species of section Fuscatae all have

striped lips with a longitudinal keel (Christenson 2001). In

addition, we have also observed that the species of this

clade do not have post-pollination chlorophyll in the peri-

anth, as opposed to the remaining species of subgenus

Polychilos described by Christenson (2001). The molecular,

morphological, and physiological data are, therefore, evi-

dence that P. doweryensis, P. gigantea, and P. maculata are

closely related to section Fuscatae.

Sweet (1980) separated section Amboinenses from sec-

tion Zebrinae on the basis of the shape of the perianth and

the lip midlobe. However, Christenson (2001) disagreed,

distinguishing section Zebrinae from section Amboinenses

by the presence of a hooded anther bed. In this study, the

molecular evidence shows that section Amboinenses cannot

be separated from the species of section Zebrinae of both

Sweet’s (1980) and Christenson’s (2001) treatment. Within

section Amboinenses, several species from the Philippines,

including P. bastianii, P. fasciata, P. hieroglyphica,

P. lueddemanniana, P. pallens, P. pulchra, P. reichenba-

chiana, and P. mariae, formed a clade separating them

from the remainder of section Amboinenses. Excluding

P. mariae and one newly described species, P. bastianii

(Gruss and Rollke 1991), this group was treated as one

highly variable species, P. lueddemanniana, in traditional

classifications (Christenson 2001). Therefore, according to

molecular data, species of sections Zebrinae and Amboi-

nenses should not be placed in separate section.


The subgenus was shown to be non-monophyletic because

the sections Esmeralda and Deliciosae appeared separated

from sections Phalaenopsis and Stauroglottis, in agreement

with other molecular evidence (Chen et al. 1995; Kao

2001; Goh et al. 2005; Padolina et al. 2005; Yukawa et al.

2005). These results can partly explain why species of the

sections Deliciosae (i.e., P. chibae and P. deliciosa) and

Esmeralda (i.e., P. pulcherrima) were traditionally treated

as separate genera, Kingidium (Seidenfaden 1988a) and

Doritis (Seidenfaden 1988b), respectively.

In our study, section Phalaenopsis formed a clade with

section Stauroglottis, and this relationship was also sup-

ported by other molecular evidence (Chen et al. 1995; Kao

2001; Goh et al. 2005; Padolina et al. 2005; Yukawa et al.

2005), floral morphology (flowers lack transversely barred

patterns; Christenson 2001), geographic distributions (most

of both sections are distributed in the Philippines; Chris-

tenson 2001; Table 2), cytology (species of both sections

have small chromosome sizes; Shindo and Kamemoto 1963;

Kao et al. 2001), and DNA content (species of both sections

have a low DNA content; Lin et al. 2001). The monophyly of

section Phalaenopsis was supported by nrITS and combined

data in this study and in Yukawa et al. (2005), but not in the

plastid DNA analyses of this study and other plastid DNA

studies (Padolina et al. 2005; Yukawa et al. 2005). According

to the nrITS, this section was divided into two subclades. One

includes P. schilleriana, P. stuartiana, and P. philippinensis,

which have marbling on the upper surface of their leaves, and


123

the other includes P. amabilis, P. aphrodite, and

P. sanderiana, which lack this marbling (Sweet 1980;

Christenson 2001). Section Stauroglottis did not form a

clade in this study or any other molecular studies (Tsai

et al. 2003; Padolina et al. 2005; Yukawa et al. 2005). All

data showed that P. celebensis was separated from

P. equestris and P. lindenii. This result is also consistent

with the geographic distribution of the section, in which

both P. equestris and P. lindenii are distributed in the

Philippines, but P. celebensis is distributed in Sulawesi

(Christenson 2001; Table 2).

Subgenera Parishianae, Aphyllae and Proboscidioides

Subgenus Parishianae was not monophyletic but formed a

clade with section Deliciosae, on the basis of plastid DNA

data in this and previous molecular studies (Padolina et al.

2005; Yukawa et al. 2005; Carlsward et al. 2006). This

subgenus formed a clade with section Deliciosae in the

plastid DNA data, but the aforementioned relationship was

not supported in the nrITS tree (Tsai et al. 2006; Yukawa

et al. 2005). Supporting the plastid data, species of both

section Deliciosae and subgenus Parishianae share a

similar geographical distribution (Indochina) and have

four pollinia (Christenson 2001; Table 2). Subgenus

Aphyllae was not monophyletic on the basis of the plastid

DNA and nrITS data, because the monotypic subgenus

Proboscidioides (P. lowii) was nested within it. With the

exception of P. lowii, on the basis of molecular data

P. minus (syn. K. minus) and P. braceana (syn. K. braceana)

could be placed in the subgenus Aphyllae proposed by

Christenson (2001). Phalaenopsis lowii was traditionally

regarded as unique because of its extremely long beak-like

rostellum and the possession of lateral lobes of the lip in

the form of recurved hooks (Sweet 1980; Christenson

2001). However, P. lowii is embedded within subgenus

Aphyllae in this and previous studies (Goh et al. 2005;

Padolina et al. 2005; Yukawa et al. 2005; Carlsward et al.

2006). These molecular data are in agreement with several

morphological (four pollinia), ecological (deciduous habit),

and geographic distribution (Sweet 1980; Christenson

2001).

Fig. 8 Putative map of Southeast Asia 30 Mya (modified from Hall 1996)


123

Biogeography

According to the evolutionary trend seen in pollinia num-

ber (Holttum 1959; Dressler 1993), the four-pollinium

clade was suggested as the basal group in the genus Pha-

laenopsis. The four-pollinium clade of Phalaenopsis

developed in South China and Indochina and then dis-

persed into Indonesia, Malaysia, and the Philippines.

Thereafter, the two-pollinium clade developed. Within the

two-pollinium clade, there are two major groups, sections

Phalaenopsis and Stauroglottis of subgenus Phalaenopsis,

and subgenus Polycholis. According to current geographi-

cal distribution of Phalaenopsis, sections Phalaenopsis and

Stauroglottis are only distributed in the Philippines with

the exception of the widespread species, P. amabilis and

P. celebensis, which are distributed in Sulawesi, and

P. aphrodite subsp. formosana and P. equestris, which are

distributed in Taiwan. In contrast, subgenus Polychilos is

distributed in Indonesia and Malaysia with the exception of

the P. luddemanniana complex and P. mitcholitzii, which

are distributed in the Philippines. Therefore, the evolu-

tionary trend between subgenus Phalaenopsis sections

Phalaenopsis and Stauroglottis and subgenus Polychilos

seem to be different.

During the Pleistocene (about 0.01–1.8 Mya), when sea

levels were low, the Malay Peninsula, Borneo, Sumatra,

Java, Bali, and various parts of the Philippines would have

been interconnected. The sea level was approximately

120 m below the current level in the last glacial maximum

(Hanebuth et al. 2000) when the Sunda Shelf connected the

Thai–Malay Peninsula and Borneo, forming Sundaland

(0.02 Mya; Sathiamurthy and Voris 2006). This would

have made crossings relatively easy among these regions

(van Oosterzee 1997). Therefore, the Phalaenopsis species

might have dispersed from South China and Indochina to

Indonesia and Malaysia, using the Malay Peninsula as a

stepping stone, from which the subgenus Polychilos

developed. In addition, on the basis of historical geology

most of the Philippine islands are young (\5 Mya), the

exceptions being Palawan, Mindoro, Zamboanga, and parts

of the western Philippines (Aurelio et al. 1991; Quebral

et al. 1994). The older islands of the Philippines, including

Palawan and Mindoro, are on the margin of the Eurasian

Plate and may have begun to slide away from the main

Fig. 9 Evolutionary trends and biogeography of the Phalaenopsis on the basis of molecular data from this study


123

mass in the middle Oligocene (*30 Mya; Fig. 8). There-

fore, the Phalaenopsis species might have dispersed from

South China and Indochina to the Philippines, using some

older lands of the Philippines (e.g., Mindoro and Palawan)

as stepping stones, from which the sections Phalaenopsis

and Stauroglottis developed (Fig. 9).

Until 5–10 Mya, the crust of the older plate, Palawan,

was combined with Borneo (Karig et al. 1986; Stephan

et al. 1986; Hall 1996). This provided an opportunity for

interchange between the Philippines species and Borneo

species, and thereafter, P. amabilis dispersed into Borneo,

Java, New Guinea, and Australia, and species of subgenus

Polychilos dispersed into the Philippines during the glacial

period. Because P. mariae is distributed in both Borneo

and Palawan (Christenson 2001) and belongs to the basal

species of the P. luddemanniana complex, according to the

combined data of the nrITS and plastid DNA, P. mariae

was suggested as a basal species in the P. luddemanniana

complex. This species dispersed from Borneo into the

Philippines, and thereafter, the P. luddemanniana complex

developed. Two Phalaenopsis taxa, P. aphrodite subsp.

formosana and P. equestris, belonging to sections Pha-

laenopsis and Stauroglottis, respectively, are found in

Taiwan. Therefore, these two Taiwanese Phalaenopsis

species must have come separately from the Philippines.

Acknowledgments The authors thank Dr James Beck and

Dr Yi-Shan Chao for their valuable comments and helpful discussion,

which improved the manuscript. This research was supported by

funding from the National Science Council, Executive Yuan, Taiwan.

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