ORIGINAL ARTICLE

Hybridization drives speciation in Gagea (Liliaceae)

Angela Peterson Æ Doerte Harpke Æ Lorenzo Peruzzi ÆIgor G. Levichev Æ J.-M. Tison Æ Jens Peterson

Received: 7 May 2008 / Accepted: 1 September 2008

� Springer-Verlag 2009

Abstract Hybridization seems to play an important role

in speciation of Gagea Salisb., a genus which is charac-

terised by polyploid taxa lines and in which diploids

(2n = 24) appear only to be common in basal sections.

Hybrid detection was applied utilising direct and cloning

nrDNA ITS data (ITS1, 5.8S rDNA, ITS2) combined with

neighbour and ribotype networks and discussed in con-

nection with previously published cpDNA, morphological

and karyological data of the authors. We have evidence of

the hybrid origin of taxa within the section Gagea (G.

pomeranica, G. megapolitana) and the monophyletic clade

of sections Didymobulbos and Fistulosae (G. microfistul-

osa, G. polidorii, G. cf. bohemica). Morphologically and

karyologically differentiated Gagea megapolitana and G.

pomeranica, adapted to synanthropic habitats, represent

both hybrids of G. pratensis 9 G. lutea. Gagea micro-

fistulosa represents a hybrid of G. villosa 9 G. fragifera;

Gagea polidorii could represent the reverse hybrid.

G. glacialis is also closely related to the latter complex.

Keywords Concerted evolution � Gagea Salisb. �Hybridization � ITS-region � Morphology � Networks �Polyploidization � Speciation

Introduction

Hybridization and polyploidy have been shown to be

amongst the most important evolutionary mechanisms in

plants (reviewed in Arnold 1997; Wissemann 2007;

Rieseberg and Willis 2007; Paun et al. 2007), well

illustrated in the, e.g. Poaceae genus Spartina (Ainouche

et al. 2004). Recently, several macro molecular studies

(reviewed in Hegarty and Hiscock 2005) demonstrated that

hybridization can promote adaptive evolution and specia-

tion. Hybrids often possess odd ploidy levels which

generally cause low fertility or complete sterility (Linder

and Rieseberg 2004). If the hybrids are partially fertile, the

backcrossing to one of the parental species (introgression)

can occur (reviewed in Hegarty and Hiscock 2005; Linder

and Rieseberg 2004).

The parental origin and evolutionary history of hybrids

and allopolyploids can be determined through the com-

bined use of biparental nuclear and maternal organellar

phylogenies (Soltis et al. 2004a; Vriesendorp and Bakker

2005), in particular detected by the incongruence between

plant chloroplast and nuclear DNA trees (e.g. Okuyama

et al. 2005; McBreen and Lockhart 2006). Depending on

inheritance patterns, organelle DNAs can permit the

identification of the direction of hybridization (McDade

1992; Schwarzbach and Rieseberg 2002; Albarouki and

Peterson 2007). In F1 hybrids and alloploids, or also in

A. Peterson (&) � D. Harpke

Biozentrum, Martin-Luther-University of Halle-Wittenberg,

Weinbergweg 22, 06120 Halle (Saale), Germany

e-mail: angela.peterson@biozentrum.uni-halle.de

L. Peruzzi

Department of Biology, Unit of General and Systematic Botany,

University of Pisa, via L. Ghini 5, 56126 Pisa, Italy

I. G. Levichev

Komarov Botanical Institute of the Russian Academy

of Sciences, Prof Popov Str 2, 197376 Saint Petersburg, Russia

J.-M. Tison

14, Promenade des Baldaquins, L’Isle d’Abeau, France

J. Peterson

State Office for Environmental Protection of Saxony-Anhalt,

Reideburger Str. 47, 06116 Halle (Saale), Germany

123

Plant Syst Evol

DOI 10.1007/s00606-008-0102-3

probable relatively young hybrid species when concerted

evolution is not fast enough or even fails to homogenise

the nrDNA repeat units, the rDNA repeats of both parents

are expected to co-occur (e.g. Booy et al. 2000; Peterson

et al. 2004; Saito et al. 2007). However, in some allopo-

lyploids only one parental rDNA repeat type was found

(e.g. Alvarez and Wendel 2003; Volkov et al. 2007) and

this is attributed to concerted evolution (reviewed in Elder

and Turner 1995; and Eickbush and Eickbush 2007). If a

hybrid fixes ITS sequences from one parent via gene

conversion, but has inherited cpDNA from the other

parent, it will have discordant positions on the ITS and

cpDNA phylogenies (e.g. Sang et al. 1997). Very little is

known about how concerted evolution maintains and

eliminates sequence variation after inter-specific hybrid-

ization (Okuyama et al. 2005; Volkov et al. 2007).

Generally, the ITS-region represents a useful marker for

phylogenetic intrageneric reconstruction of the Liliaceae

s.s. (e.g. Fritillaria L.: Ronsted et al. 2005; Lilium L.:

Nishikawa et al. 1999; Ronsted et al. 2005; Ikinci et al.

2006; Erythronium L.: Allen et al. 2003; Gagea: Peterson

et al. 2004, 2008; Peruzzi et al. 2008a, b) and in inves-

tigation of hybrids within these genera (e.g. Tulipa L.:

Booy et al. 2000; Gagea: Peterson et al. 2004).

The genus Gagea Salisb. (Liliaceae s.s.) comprises at

least 275 species, currently divided by Levichev into

sections (Levichev 2008a; in Peterson et al. 2008). The

distribution of Gagea is restricted to the temperate and

sub-tropical regions of Eurasia and does not cover areas

with either tropical climate or permafrost (Levichev

1999a). The greatest richness in species is reported for

Western Tien Shan and Pamir-Alai, representing the

modern centres of species diversity (Levichev 1999a).

The species appear to be typically insect-pollinated plants

which offer nectar and/or pollen as a reward (Gargano

et al. 2007). Generally, sexual reproduction is influenced

by their early flowering and presumably by factors such

as ploidy levels which can reduce sexual efficiency and

favour vegetative propagation (Gargano et al. 2007). The

important role of hybridization and polyploidy in the

speciation within the genus Gagea was becoming

evident in several morphological (e.g. Levichev 1990,

1999b, 2001, 2005, 2006a; Tison 1998; Henker 2005) and

karyological investigations (e.g. Peruzzi 2003, 2008a;

Peruzzi and Aquaro 2005). The basic chromosome num-

ber for Gagea species is x = 12. In Gagea, polyploid

lineages (Henker 2005) can be found, ranging from

triploid to undecaploid with 2n = 132, whereby several

species were found to have different chromosome

numbers (Henker 2005; Peruzzi 2003, 2008a; Table 1).

Putative Gagea hybrid taxa were detected by incon-

gruent cpDNA and nrDNA gene trees (Peterson et al. 2004,

2008; Peruzzi et al. 2008b) and in direct sequencing

procedures indicated by polymorphic nucleotide sites,

sometimes also unexpected (described in this study for G.

cf. bohemica). Evidence from hybridization and polyplo-

idisation could be connected with taxonomic problems in

the genus Gagea and the description of a large number of

species and a nomenclature which is overloaded with

synonyms (Levichev 1999a). It is noteworthy that over 670

specific and intra-specific combinations have been pub-

lished. Generally, the different sections of Gagea are

characterised by a combination of morphological charac-

ters (see Levichev in Peterson et al. 2008) which could be

explained by parallel evolution but also by hybridization

processes.

This paper demonstrates how molecular approaches

can help to understand the past and recent reticulate

history of putative hybrid taxa within two Gagea clades

[G. section Gagea and G. sections Didymobulbos (K.

Koch) Boiss. and Fistulosae (Pascher) Davlian.; Peterson

et al. 2008]. In both clades, several polyploids were

recognised (summarised in Peruzzi 2008a; Table 1); some

of these are expected to be alloploids according to mor-

phological studies; e.g., Gagea megapolitana Henker

(Henker 2005) and G. pomeranica Ruthe (Henker 2005)

of G. section Gagea, G. microfistulosa Levichev (Levichev

2008b), G. polidorii J.-M. Tison (Tison 2004) of G.

section Fistulosae, and G. luberonensis J.-M. Tison

(Tison 1998) of G. sect. Didymobulbos. ITS (ITS1,

5.8SrDNA, ITS2) direct and cloning data of three Gagea

hybrid complexes (G. pratensis (Pers.) Dumort–G. lutea

(L.) Ker Gawl.; Gagea villosa (Bieb.) Sweet–G. fragifera

(Vill.) Ehr. Bayer and G. Lopez (synonym of G. liotardii

(Sternberg) Schult. and Schult. f.; see Table 1); and

Gagea bohemica (Zauschn.) Schult. et Schult. f.) includ-

ing putative hybrid taxa, their parental species and related

taxa were investigated utilising phylogenetic networks

which have become an important tool in molecular

investigations and in the detection of reticulate events

such as hybridization (Huson and Kloepper 2005; Huson

and Bryant 2006; McBreen and Lockhart 2006; Grimm

and Denk 2008). Generally, such detailed molecular

studies on natural hybridogenous taxa of the monocots are

rare (e.g. Booy et al. 2000; Mahelka et al. 2007; Suarez-

Santiago et al. 2007). Our results show that hybrid

detection is often possible in Gagea via maternally and

biparentally inherited molecular markers, but that it also

sometimes has its limitation due to recombination and

concerted evolution of the investigated nrDNA region. In

addition, our comprehensive ITS cloning data provide

insights into the recombination processes of both internal

transcribed spacers which are connected with the con-

certed evolution of this region in the investigated genus.

Generally, the important role of hybridization and poly-

ploidy in speciation of Gagea is discussed.

A. Peterson et al.

123

Ta

ble

1In

ves

tig

ated

tax

ao

fG

ag

ease

ctio

ns

(G.

sect

.)G

ag

ea,

Did

ymo

bu

lbo

san

dF

istu

losa

ew

ith

ori

gin

,v

ou

cher

nu

mb

er,

EM

BL

acce

ssio

nn

um

ber

s,k

ary

oty

pes

of

the

inv

esti

gat

edre

gio

ns

and

des

crib

edch

rom

oso

me

nu

mb

ers

Tax

aC

od

eO

rig

inV

ou

cher

ITS

reg

ion

Ch

rom

oso

me

nu

mb

ers

(2n

)a

EM

BL

acce

ssio

nn

um

ber

Sit

esD

escr

ibed

wo

rld

wid

e

G.

sect

.D

idym

ob

ulb

os

(K.

Ko

ch)

Bo

iss.

G.

bo

hem

ica

(Zau

sch

n.)

Sch

ult

.et

Sch

ult

.f.

D-S

/AG

erm

any

:S

axo

ny

-An

hal

t0

95

84

9(H

AL

)A

J42

75

47

24

,3

6,

48

,6

0,

72

D-R

/BG

erm

any

:R

hin

elan

d-P

alat

inat

e0

95

85

6(H

AL

)A

J43

71

99

CZ

Cze

chR

epu

bli

k:

Mo

rav

ia0

95

84

2(H

AL

)A

J42

75

49

60

GR

Gre

ece:

Cre

te1

01

64

5(H

AL

)A

M2

65

52

8

RU

SR

uss

ia:

Dag

esta

n1

01

78

7(H

AL

)A

M1

62

67

2

I-A

BR

Ital

y:

Ab

ruzz

o1

49

25

(CL

U)

AM

28

72

71

48

G.

cf.

bo

hem

ica

I-C

AL

I-C

AL

cf.

bo

h1

–9

Ital

y:

Cal

abri

a1

26

88

(CL

U)

AM

93

19

40

clo

nes

:

AM

93

19

41

-

AM

93

19

49

48

G.

chry

san

tha

Sch

ult

.&

Sch

ult

.f.

I-S

ICIt

aly

:S

icil

y9

23

7(C

LU

)A

M2

87

28

43

63

6

G.

du

bia

A.

Ter

racc

.I-

AB

RIt

aly

:A

bru

zzo

20

58

0(A

PP

)A

M4

22

46

24

82

4,

48

I-S

AR

Ital

y:

Sar

din

ia1

04

33

1(H

AL

)A

M9

03

04

9

I-S

ICIt

aly

:S

icil

y1

04

33

2(H

AL

)A

M9

03

04

84

8

G.

foli

osa

(J.

Pre

slet

C.

Pre

sl)

Sch

ult

.et

Sch

ult

.f.

I-S

AR

Ital

y:

Sar

deg

na

34

69

7(Z

)A

M1

62

67

63

6

I-S

ICIt

aly

:S

icil

y1

26

91

(CL

U)

AM

40

93

46

36

G.

gra

na

tell

ii(P

arl.

)P

arl.

FF

ran

ce1

01

83

7(H

AL

)A

M4

09

33

32

42

4,

36

,4

8

I-A

BR

Ital

y:

Ab

ruzz

o1

49

24

(CL

U)

AM

40

93

42

48

I-C

AL

Ital

y:

Cal

abri

a9

26

6(C

LU

)A

M4

09

34

43

6

I-S

ICIt

aly

:S

icil

y9

26

7(C

LU

)A

M4

09

34

33

6

G.

hel

dre

ich

ii(A

.Ter

racc

.)

Gro

ssh

.

UA

Uk

rain

e:C

rim

ea1

01

80

9(H

AL

)A

M2

65

53

4

G.

loja

con

oi

Per

uzz

i(=

G.

lon

gif

oli

aL

oja

c.n

on

Gan

d)

I-C

AL

Ital

y:

Cal

abri

a9

25

6(C

LU

)A

M2

87

27

23

63

6

I-S

ICIt

aly

:S

icil

y9

25

3(C

LU

)A

M4

22

46

43

6

G.

lub

ero

nen

sis

J.-M

.

Tis

on

I-A

BR

Ital

y:

Ab

ruzz

o1

49

26

(CL

U)

AM

42

24

67

36

36

I-L

AT

Ital

y:

Lat

ium

20

58

1(C

LU

)A

M4

22

46

33

6

G.

ma

uri

tan

ica

Du

rieu

I-A

PU

LIt

aly

:A

pu

lia

12

69

7(C

LU

)A

M9

03

04

63

63

6

G.

ped

un

cula

ris

(J.

&C

.

Pre

sl)

Pas

cher

I-A

PU

LIt

aly

:A

pu

lia

18

16

3(C

LU

)A

M9

03

05

03

63

6

G.

sicu

laL

oja

c.I-

CA

LIt

aly

:C

alab

ria

92

54

(CL

U)

AM

40

93

40

36

24

,3

6

I-S

ICIt

aly

:S

icil

y9

25

8(C

LU

)A

M4

09

34

12

4

G.

ten

era

Pas

cher

KS

Ky

rgy

zsta

n1

01

84

3(H

AL

)A

M4

22

46

03

63

6

Hybridization in Gagea

123

Ta

ble

1co

nti

nu

ed

Tax

aC

od

eO

rig

inV

ou

cher

ITS

reg

ion

Ch

rom

oso

me

nu

mb

ers

(2n)a

EM

BL

acce

ssio

nn

um

ber

Sit

esD

escr

ibed

wo

rld

wid

e

G.

vill

osa

(Bie

b.)

Sw

eet

D-S

/AG

erm

any

:S

axo

ny

-An

hal

t-

AJ4

27

54

54

8,

60

D-B

Ger

man

y:

Bra

nd

enb

urg

-A

J42

75

45

MD

Mo

ldo

va

10

18

07

(HA

L)

AM

18

04

53

I-A

BR

Ital

y:

Ab

ruzz

o1

49

28

(CL

U)

AM

28

72

83

48

I-C

AL

Ital

y:

Cal

abri

a1

52

19

(CL

U)

AM

40

93

45

48

G.

sect

.F

istu

losa

e(P

asch

er)

Dav

lian

G.

gla

cia

lis

K.

Ko

chT

R

gla

c

1–

7

Tu

rkey

:R

ize

dis

tric

t1

01

64

7(H

AL

)A

M2

65

53

5cl

on

es:

AM

93

15

54

-

AM

93

15

60

48

G.

fra

gif

era

(Vil

l.)

Eh

r.

Bay

er&

G.

Lo

pez

=G

.li

ota

rdii

(Ste

rnb

erg

)

Sch

ult

.&

Sch

ult

.f.

(=G

.fi

stu

losa

(Ram

on

dex

DC

)K

erG

awl.

)

UA

Uk

rain

eC

rim

ea1

01

79

7(H

AL

)A

M2

65

53

24

8,

60

,8

4

KZ

Kaz

akh

stan

10

17

96

(HA

L)

AM

18

04

55

BG

Bu

lgar

ia0

70

40

7(H

AL

)A

M1

62

67

7

I-C

AL

Ital

y:

Cal

abri

a1

26

93

(CL

U)

AM

42

24

68

84

I-S

ICIt

aly

:S

icil

y1

26

92

(CL

U)

AM

28

72

85

84

G.

mic

rofi

stu

losa

Lev

ich

ev

UA

mic

ro

1-1

0

Uk

rain

e:C

rim

ea1

01

72

8(H

AL

)A

M4

09

33

2cl

on

es:

AM

93

15

69

-

AM

93

15

78

G.

po

lid

ori

iJ.

-M.

Tis

on

I-C

AL

po

li

1–

8

Ital

y:

Cal

abri

a1

27

00

(CL

U)

AM

90

30

53

clo

nes

:

AM

93

15

61

-

AM

93

15

68

72

72

G.

sect

.G

ag

ea

G.

aip

etri

ensi

sL

evic

hev

UA

Uk

rain

e:C

rim

ea1

01

80

1(H

AL

)A

M0

87

95

52

42

4

G.

art

emcz

uki

iK

rasn

ov

aU

AU

kra

ine:

Cri

mea

10

14

81

(HA

L)

AM

40

93

31

24

24

G.

cap

usi

iA

.T

erra

cc.

UZ

Uzb

ekis

tan

10

18

39

(HA

L)

AM

42

24

55

24

G.

eru

bes

cen

s(B

esse

r)

Bes

ser

UA

Uk

rain

e:n

ear

Ch

ark

ow

10

38

61

(HA

L)

AM

49

39

53

24

24

G.

hel

ena

eG

ross

h.

RU

SR

uss

ia:

Dag

esta

n1

01

79

8(H

AL

)A

M2

65

53

12

42

4

G.

lute

a(L

.)K

erG

awl.

D-S

/AG

erm

any

:S

axo

ny

-An

hal

t0

95

84

1(H

AL

)A

J48

85

69

72

12

,2

4,

48

,7

2

RU

SR

uss

ia:

Cau

casu

s1

01

80

0(H

AL

)A

M2

65

53

07

2

I-C

AL

Ital

y:

Cal

abri

a1

26

96

(CL

U)

AM

28

72

82

72

G.

meg

ap

oli

tan

aH

enk

erD

-MV

meg

a

1-9

Ger

man

y:

Mec

kle

nb

urg

-Wes

tern

Po

mer

ania

10

16

44

(HA

L)

AM

93

15

53

clo

nes

:

AM

93

15

44

-

AM

93

15

52

72

,8

47

2,

84

G.

na

kaia

na

Kit

ag.

RU

SR

uss

ia:

Kh

abar

ov

skT

erri

tory

10

17

99

(HA

L)

AM

18

04

54

48

24

,4

8

A. Peterson et al.

123

Ta

ble

1co

nti

nu

ed

Tax

aC

od

eO

rig

inV

ou

cher

ITS

reg

ion

Ch

rom

oso

me

nu

mb

ers

(2n)a

EM

BL

acce

ssio

nn

um

ber

Sit

esD

escr

ibed

wo

rld

wid

e

G.

pa

czo

skii

(Zap

al.)

Gro

ssh

.

I-V

EN

Ital

y:

Fri

uli

-Ven

ezia

10

43

39

(HA

L)

AM

90

30

51

48

G.

po

do

lica

Sch

ult

.&

Sch

ult

.f.

UA

Uk

rain

e1

01

83

2(H

AL

)A

M4

09

33

42

4

G.

po

mer

an

ica

Ru

the

D-S

/A

po

m

1.1

,1

.2,

2.1

,2

.2

Ger

man

y:

Sax

on

y-A

nh

alt

09

58

42

(HA

L)

AJ4

27

54

3cl

on

es:

AM

93

15

37

-

AM

93

15

40

60

60

,7

2

D-M

V

po

m

3.1

–3

.3

Ger

man

y:

Mec

kle

nb

urg

-

Wes

tern

Po

mer

ania

09

58

46

(HA

L)

AJ4

29

19

3cl

on

es:

AM

93

15

41

-

AM

93

15

43

60

,7

2

G.

pra

ten

sis

(Per

s.)

Du

mo

rt.

D-S

/A(1

)G

erm

any

:S

axo

ny

-An

hal

t0

95

84

7(H

AL

)A

J42

75

42

60

24

,3

6,

48

,6

0,

72

D-S

/A(2

)G

erm

any

:S

axo

ny

-An

hal

t–

AJ4

37

20

1

D-S

/A(3

)G

erm

any

:S

axo

ny

-An

hal

t0

95

84

8(H

AL

)A

J43

72

03

D-B

Ger

man

y:

Bra

nd

enb

urg

–A

J43

72

02

60

,7

2

I-C

AL

Ital

y:

Cal

abri

a1

27

03

(CL

U)

AM

90

30

47

60

G.

pu

sill

a(F

.W.

Sch

mid

t)

Sw

eet

RU

SR

uss

ia:

cult

ivat

ed1

01

83

1(H

AL

)A

M4

22

45

32

4,

48

,6

0

I-V

EN

Ital

y:

Fri

uli

-Ven

ezia

10

43

38

(HA

L)

AM

90

30

52

24

G.

rub

icu

nd

aM

ein

sh.

emen

d.

Lev

ich

ev

ES

TE

sto

nia

10

38

55

(HA

L)

AM

49

39

54

48

G.

shm

ako

via

na

Lev

ich

evR

US

Ru

ssia

:A

ltai

10

18

30

(HA

L)

AM

42

24

54

G.

terr

acc

ian

oa

na

Pas

cher

MG

LM

on

go

lia:

Bo

gd

-Ul

Mo

un

tain

s0

70

42

6(H

AL

)A

M2

87

27

92

4

RU

SR

uss

ia1

03

85

3(H

AL

)A

M4

93

95

5

G.

tiso

nia

na

Per

uzz

i&

al.

I-T

US

Ital

y:

Tu

scan

y1

49

20

(CL

U)

AM

42

24

66

24

24

G.

tra

nsv

ersa

lis

Ste

ven

UA

Uk

rain

e:C

rim

ea1

01

78

3(H

AL

)A

M1

62

67

12

4

Pu

tati

ve

hy

bri

dta

xa

are

inb

old

aF

or

refe

ren

ces

see

Per

uzz

i(2

00

3,

20

08

a)

Hybridization in Gagea

123

Materials and methods

Taxon sampling

This study includes the taxa (Table 1) of three Gagea

sections (G. sect.): 18 taxa of G. sect. Gagea, fourteen taxa

of G. sect. Didymobulbos (‘‘Didymobolbos’’ according to

Boissier 1882; see also Levichev 2006a, in Peterson et al.

2008) and four taxa of G. sect. Fistulosae; including the

putative hybrid taxa (Gagea megapolitana, G. pomeranica,

G. luberonensis, G. polidorii, G. microfistulosa, G. cf.

bohemica) and also their putative parental species and

related taxa.

DNA isolation, amplification, cloning and sequencing

of the ITS-region

Plant material (10 mg) was frozen in liquid nitrogen and

used for DNA isolation with the DNeasy Plant Mini Kit

(Qiagen) following the manufacturer’s protocol. The con-

centration of DNA was determined by spectrophotometry.

The ITS-region (ITS1, 5.8S rDNA, ITS2) was amplified

using the primers ITS4 and ITS5 (White et al. 1990). PCR

was performed with 50 ng genomic DNA in 20 ll reac-

tions (Ready To GoTM PCR Beads, Amersham Bioscience)

in a GeneAmp PCR System 9700 (Perkin Elmer) with a

primer concentration of 50 lM. Amplification was per-

formed for 3 min at 96�C and for 30 cycles of 30 s at 95�C,

45 s at 56�C and 30 s at 72�C and a final extension for

5 min at 72�C. PCR products were purified after gel sep-

aration on 1.8% agarose gels using the Mini Elute Gel

Extraction Kit (Qiagen) following the manufacturer’s

protocol, eluted in water.

The ITS-regions of Gagea megapolitana, G. microfi-

stulosa, G. polidorii, G. cf. bohemica and G. glacialis K.

Koch were cloned into the pDrive Vector following the

manufacturer’s protocol (Qiagen PCR Cloningplus Kit).

Ligation-reaction mixtures were placed at 4�C for 4 h

instead of 30 min to increase the number of recombinants.

Individual white colonies were used to inoculate 5 ml

liquid LB Amp media and incubated overnight at 37�C

with shaking (225 rpm). Plasmid DNA was isolated fol-

lowing the manufacturer’s protocol (Eppendorf Fast

Plasmid Mini Kit) and re-suspended in 50 ll of water. A

2 ll sample of the isolated plasmid was used to determine

the DNA concentration by spectrophotometry (Genequant,

Pharmacia).

The double-stranded plasmid DNA from each individual

(ten clones per individual) was sequenced following the

cycle sequencing procedure (BigDyeTM Terminator v1.1

Cycle Sequencing Ready Reaction Kit, Applied Biosys-

tems) in a volume of 20 ll containing 700 ng DNA and

5 lM primer (Vector primer M13 and T7, Qiagen). The

cycling parameters were 25 cycles for 10 s at 96�C for

denaturation and 4 min at 60�C for primer annealing and

extension. The cleaning of sequencing products by ethanol

precipitation was followed by the separation and analysis

of sequencing products on an automated analyser (ABI

310, Applied Biosystems). All sequences were deposited in

the EMBL database (Table 1).

Analysis of the ITS-region

For the hybrid taxa Gagea cf. bohemica, the second puta-

tive parental ITS sequence was generated (G. spec.)

according to the direct and cloning ITS data of G. cf.

bohemica and G. bohemica (Table 3).

Neighbour networks (NN) were constructed for the

direct sequencing results of species of G. sect. Gagea (18

taxa), and of G. sect. Didymobulbos (14 taxa) and G. sect.

Fistulosae (4 taxa) using the neighbour network method of

SplitsTree version 4.6 (Huson and Bryant 2006) based on

uncorrected p-distances.

Ribotype networks were constructed for three hybrid

complexes (G. pratensis-G. lutea, G. villosa–G. fragifera,

G. bohemica). The cloning data of a previous study

(Peterson et al. 2004; Table 1) of three individuals of G.

pomeranica (G. pom1-3) were also included in the ribotype

network of G. pratensis–G. lutea. Ribotype networks were

applied using the statistical parsimony algorithm of TCS

version 1.21 (Clement et al. 2000). Gaps were treated as

fifth character state and identical ITS alleles were com-

bined into one ribotype.

Generally, all investigated ITS-regions are putatively

functional. Their functionality was checked through their

ability to build up the proper 5.8S secondary structure

(Schnare et al. 1996) using the RNA structure mask option

of BioEdit version 7.0.9 (Hall 1999) and the presence of

eukaryotic conserved 5.8S motifs (Harpke and Peterson

2008).

Results

Section Gagea

The section Gagea was found to be divided into three

major clusters (Fig. 1). The first cluster (I) contained dip-

loid species (except G. rubicunda; Table 1). The other two

clusters (II and III) included both diploids and polyploids.

G. pratensis (cluster III) and G. lutea (cluster II) were

found in separated clusters. G. pomeranica and G. mega-

politana shared a position close to the centre of the graph

(see Fig. 1). The ITS direct sequencing and cloning data

provide evidence that both parental ITS types from G.

pratensis and G. lutea could be found in G. megapolitana.

A. Peterson et al.

123

The cloned ITS-regions had a length of 618 or 619 bp, with

a variability of up to 2%. In total, 16 variable positions in

ITS1 and six variable positions in ITS2 were detected.

TCS calculated a 95% parsimony connection limit and

resulted in a network of 30 ITS ribotypes for G. pratensis

and G. lutea and their putative hybrids (Fig. 2). Sixteen

ribotypes were not found in the analysed individuals and

occur as missing intermediates. Gagea megapolitana rib-

otypes were found to be identical to G. lutea (mega 2),

were related to the parental species (mega 1, 6–8 to G.

pratensis and mega 3 and 4 to G. lutea), and were related to

intermediates (mega 5 and 9). G. pomeranica ribotypes

were found to be identical to the parental species (pom 1.1,

2.1, 3.1 to G. pratensis and pom 1.2, 2.2 to G. lutea) and

occurred as intermediates (pom 3.2) or were related to

intermediates (pom 3.3).

Sections Didymobulbos and Fistulosae

The Neighbour networks analyses of the G. sections Didy-

mobulbos and Fistulosae resulted in two clusters (I and II;

Fig. 3). Cluster I mainly contained polyploids (2n = 36,

48, 60, 72, 84) whereas in cluster II diploids to tetraploids

were found (2n = 24, 36, 48). In cluster I, Gagea

bohemica I-ABR falls in one lineage with G. luberonensis

I-ABR and I-LAT. Gagea villosa grouped together with G.

microfistulosa, G. glacialis, G. fragifera and G. polidorii.

Gagea microfistulosa and G. polidorii were located on the

edge between G. villosa and G. glacialis and G. fragifera.

Gagea cf. bohemica I-CAL was located between cluster I

and II.

According to the cloning data of G. microfistulosa, both

parental ITS types (G. villosa, G. fragifera) were recog-

nised in addition to chimeric ITS types (Table 2). The

cloned ITS types ranged from 615 to 617 bp with a vari-

ability of up to 3%. In G. polidorii, ITS clones ranging

from 615 to 617 bp and in G. glacialis from 616 to 617 bp

were recognised, both having a low intra-specific vari-

ability (up to 1%).

TCS calculated a 95% parsimony connection limit and

resulted in a network of 55 ITS ribotypes (Fig. 4) for the G.

villosa–G. fragifera hybrid complex. Twenty-eight ribo-

types were not found in the analysed individuals and occur

as missing intermediates in the network. Gagea microfi-

stulosa ribotypes were found as intermediates or were

related to intermediates of G. villosa and G. fragifera

(micro 1–6) and were related (micro 7–9) or identical

(micro 10) to the ribotype glac 1 of G. glacialis. Gagea

Fig. 1 NeighbourNet splits

graph of ITS sequences of G.sect. Gagea. Edge lengths are

proportional to the uncorrected

p-distances. Taxon name is

accompanied by voucher

information (see Table 1). The

position of direct sequencing

results of putative hybrids are

indicated with dots. Clusters are

labelled (I–III)

Hybridization in Gagea

123

polidorii ribotypes were found to be identical (poli 6) or

related (poli 1–5, 8) to fra I-CAL, UA ribotype of G.

fragifera and as an intermediate of fra I-CAL, UA and the

G. glacialis ribotype glac 1 (poli 7). Ribotypes of G. gla-

cialis were separated from G. fragifera by at least three

intermediates and were not directly related to G. villosa.

After direct sequencing, in the voucher Gagea cf.

bohemica (cf. boh) multiple peaks were recognised on 14

sites in ITS1 and on 19 sites in ITS2. According to the

cloning data (Table 3), generally two ITS types were found

and recombination between parental spacers was recogni-

sed. The ITS clones ranged from 615 to 617 bp, with a

variability of up to 7%. For the cloning results of Gagea cf.

bohemica I-CAL, G. bohemica I-ABR, and the generated

parental sequence, TCS calculated an ITS network of 65

ribotypes (Fig. 5). Fifty-five ribotypes were not found in

the analysed individuals and occur as missing intermedi-

ates in the network. Gagea bohemica I-CAL ribotypes

were found to be identical (cf. boh 1) or related (cf. boh 2,

3) to the putative parental species G. bohemica I-ABR. One

ribotype was connected to an intermediate (cf. boh 4) and

the remaining ribotypes (cf. boh 5–8) were found to be

related to the generated parental sequence.

Discussion

Gagea pomeranica and G. megapolitana—hybrid taxa

share the same parents

Gagea megapolitana and G. pomeranica (G. sect. Gagea)

both are morphologically intermediate between G. pratensis

Fig. 2 Ribotype network for

ITS ribotypes of G. pratensis(prat), G. lutea (lutea) and their

putative hybrid taxa G.megapolitana D-MV (mega1-9)

and G. pomeranica D-S/A

(pom1.1-1.2, 2.1-2.2) and D-

MV (pom 3.1–3.3). Parental

species accompanied by

voucher information (Table 1)

are boxed and indicated in bold.

Empty dots refer to missing

intermediates not found among

the analysed sequences

Fig. 3 NeighbourNet splits

graph of ITS sequences of G.sections Didymobulbos and

Fistulosae. Edge lengths are

proportional to the uncorrected

p-distances. Taxon name is

accompanied by voucher

information (Table 1). The

generated putative second

parental species (G. spec.) of G.cf. bohemica I-CAL is marked

with an asterisk. The positions

of direct sequencing results of

putative hybrids are indicated

with dots. Clusters are labelled

(I, II)

A. Peterson et al.

123

and G. lutea (Henker 2005) concerning the colour of

the leaves and the habitus, and features of bulbils (for G.

pomeranica see also Peterson et al. 2004). Both are charac-

terised by the presence of a single vegetative bulbil (Peterson

et al. 2004).

Interestingly, we found that the parental species are

identical for both in the same direction: Gagea praten-

sis 9 G. lutea (see also cpDNA and ITS data, Peterson et al.

2004, 2008). Hybrid speciation has the potential to occur

repeatedly at different times and in different geographical

areas and this may also result in morphological differences,

leading to the different naming of offspring of the same

hybridising taxa (Hegarty and Hiscock 2005) as it is true for

both taxa. Different evolutionary histories could be caused

by recombination, genetic drift, selection or the inheritance

of different alleles of parental loci. Whereas in the stabilised

G. pomeranica 3 (Peterson et al. 2004) most ITS clones

(80%) corresponded to G. pratensis, in G. megapolitana

most clones (66.7%) corresponded to G. lutea.

Both parental species occur with different chromosome

numbers from 2n = 24 up to 2n = 72, but only Gagea

pratensis displays odd stages such as 2n = 36 and 2n = 60

connected with a loss of ability for generative reproduction.

Most G. pomeranica populations (G. pom 1 and 2 from

D-S/A, Table 1) have 2n = 60 (Henker 2005) and are

probably primarily the product of crossing tetraploid G.

pratensis and hexaploid G. lutea. These G. pomeranica

populations are sterile but proliferately effectively through

Table 2 Parsimony informative ITS sites of G. microfistulosa (G. villosa 9 G. fragifera), G. polidorii, and G. glacialis

Positiona 1 1 2 2 2 2 3 5 5 5 5 5 6

5 6 0 1 1 4 8 7 7 7 8 9 0

5 3 3 1 2 2 2 2 3 5 2 2 5

G. fragifera

fra UA,KZb G T A A T A C G A T C C G

fra I-CALb G T A A T A Y G A T C C G

fra I-SICb G T A A T A Y G A T S Y R

G. villosa

vill D-SA,Bb,I-ABR,CALb t t g g c t t a t c g t a

vill MDb t t g g Y t t a t c g t a

G. microfistulosa

micro UAb: AM409332 K Y R A T A t R A Y g t a

micro 1c AM931569 t t g g T t t G A T g t a

micro 2c: AM931570 t t g g T t C G A T C C G

micro 3c: AM931571 t C g g T t t G A T g t a

micro 4c: AM931572 t t g g T t t a t c g t a

micro 5c: AM931573 t t g g T t t G t c g t a

micro 6c and 7c: AM931574-75 t t A g T t t a t c g t a

micro 8c and 9c: AM931576-77 G C A A T A t a t c g t a

micro 10c: AM931578 G C A A T A t G A T g t a

G. polidorii

poli I-Calb:AM903053 G T A A T A Y G A Y S Y R

poli 1-6c: AM931561-66 G T A A T A C G A T C C G

poli 7c: AM931567 G T A A T A C G A T C C a

poli 8c: AM931568 A T A A T A C G A T C C G

G. glacialis

glac TRb: AM265535 G C A A T A C G A T g t a

glac 1-7c: AM931554-60 G C A A T A C G A T g t a

ITS1 ITS2

Nucleotides in big letters corresponding to G. fragifera, in small letters corresponding to G. villosa; letters in italics did not correspond to the

latter two taxaa Position of informative sites are numbered according to the EMBL accession number of AM409332 (G. microfistulosa)b Direct (K = G and T; M = A and C; R = A and G; S = G and C; W = A and T; Y = C and T) ITS data (see also Table 1)c Cloning ITS data

Hybridization in Gagea

123

bulbils. The actual and repeated origin of G. pomeranica in

northern Germany, the centre of distribution of these spe-

cies, appears unlikely because tetraploid G. pratensis

populations were not detected in this area (Henker 2005).

Nevertheless, in rare cases populations of G. pomeranica

are found with 2n = 72, which produce germinable seeds

(Henker 2005). Most G. megapolitana populations are

fertile and hexaploid (2n = 72, also the investigated sam-

ple). Thus G. megapolitana could be the result of crossing

hexaploid G. pratensis 9 hexaploid G. lutea. In northern

Germany, the only known range of Gagea megapolitana,

hexaploid G. lutea populations are frequent and in rare

cases also hexaploid G. pratensis populations occur

(Henker 2005). Backcrosses of both hexaploid hybrid taxa

with their hexaploid parental species appear to be possible,

particularly with the frequent and fertile Gagea lutea.

Both G. pomeranica and G. megapolitana mostly occur

in anthropogenically influenced sites such as churchyards,

parkways, and parks, and occasionally within extensively

used meadows and pastures. These habitats frequently

share both hybrid taxa with sterile pentaploid Gagea

pratensis populations. Gagea lutea is much less frequent

in these habitats. The latter species inhabits almost

exclusively near-natural deciduous forests in which G.

pomeranica and G. megapolitana are only and very rarely

be found (Henker 2005). The possibility of backcrossing of

fertile G. megapolitana and the rare fertile hexaploid G.

pomeranica individuals with G. lutea is therefore restric-

ted. In principle, G. pomeranica and G. megapolitana are

the same biological unit: both possibly originated in the

course of the development of cultivated landscape in

Central Europe. In some respects some well-documented

cases of hybridogenic speciation after introduction of alien

species resemble the speciation pattern described here, e.g.

in Spartina (Ainouche et al. 2004), Tragopogon (Soltis

et al. 2004b), or Senecio (Abbott and Lowe 2004). In our

Gagea example, the driving force behind these speciation

events was not the present day introduction of alien species

but the deforestation and cultivation of Central European

landscapes over the last millennia, which permitted contact

between species of forest edges and open vegetation (G.

pratensis) and highly specialised forest species (G. lutea),

resulting in hybrids which are well adapted to anthropo-

genic habitats.

Hybridization in the clade of G. sect. Didymobulbos

and G. sect Fistulosae

Although the Gagea sections Didymobulbos and Fistulosae

are morphological different from each other (Levichev in

Peterson et al. 2008), according to our molecular data both

were found in the same highly supported clade (Peterson

et al. 2008). Within this clade, the close relationship

(cpDNA and ITS data) of Gagea fragifera (=G. liotardii),

G. glacialis (Peterson et al. 2008), and G. polidorii

(Peruzzi et al. 2008b) was recognised: three taxa which are

also morphologically closely related. Gagea fragifera and

G. polidorii are characterised by fistulose basal leaves,

which are absent in G. villosa. Gagea microfistulosa

(Levichev 2008b), placed within G. sect. Fistulosae based

on morphology (Levichev in Peterson et al. 2008), corre-

sponded according to our cpDNA to G. villosa and was

Fig. 4 Ribotype network for

ITS ribotypes (Table 2) of the

G. fragifera complex, including

the putative hybrid taxon G.microfistulosa UA (micro1–10), G. polidorii I-CAL (poli1–8) and the near related G.glacialis TR (glac1-7). Parental

species accompanied by

voucher information (Table 1)

are boxed and indicated in bold.

Empty dots refer to missing

intermediates not found among

the analysed sequences

A. Peterson et al.

123

Ta

ble

3P

arsi

mo

ny

info

rmat

ive

ITS

site

so

fG

.cf

.b

oh

emic

a(G

.sp

ec.

xG

.b

oh

emic

a)

Po

siti

on

a3

34

5–

77

89

11

–1

12

23

34

44

44

44

44

44

44

55

55

55

58

12

08

68

12

77

00

89

01

23

33

44

68

88

80

77

88

9

12

14

37

58

59

10

16

35

51

25

60

69

03

2

G.

cf.

bo

hem

ica

I-C

AL

b:

AM

93

19

40

KW

RW

–R

RW

KR

Ka

YK

YR

MR

MY

ST

YR

YY

RK

TK

YY

RW

MY

Y

cf.

bo

h1

c:

AM

93

19

41

TT

AT

–A

GA

TA

T–

CT

CG

AA

AT

CT

CG

CC

AT

TG

CC

AT

CT

C

cf.

bo

h2

c:

AM

93

19

42

TT

AT

–A

GA

TA

T–

CT

CG

AA

AT

CT

CG

CC

Ag

TG

CC

AT

CT

C

cf.

bo

h3

c:

AM

93

19

43

TT

AT

–A

GA

TA

T–

CT

CG

AA

AT

CT

CG

CC

Ag

ct

tt

ga

ac

t

cf.

bo

h4

c:

AM

93

19

44

ga

ga

ag

at

gg

ga

Cg

ta

AA

AT

CT

CG

CC

Ag

TG

CC

AT

CT

C

cf.

bo

h5

c:

AM

93

19

45

ga

gT

–A

at

gg

ga

tg

ta

cg

cc

gc

ca

tt

gg

ct

tt

ga

ac

t

cf.

bo

h6

c:

AM

93

19

46

ga

gT

ag

at

gg

ga

tg

ta

cg

cc

gc

ca

tt

gg

ct

tt

ga

ac

t

cf.

bo

h7

c:

AM

93

19

47

ga

ga

–g

at

gg

ga

tg

ta

cg

cc

gc

ca

tt

gg

ct

tt

ga

ac

t

cf.

bo

h8

c:

AM

93

19

48

ga

ga

ag

at

gg

ga

Cg

ta

cg

cc

gc

ca

tt

gg

ct

tt

ga

ac

t

cf.

bo

h9

c:

AM

93

19

49

ga

ga

–g

at

gg

ga

tg

ta

cg

cc

gc

ca

tt

gg

ct

tt

ga

ac

t

G.

bo

hem

ica

I-A

BR

bT

TA

T–

AG

AT

AT

–C

TC

GA

AA

TC

TC

GC

CA

TT

GC

CA

TC

TC

G.

spec

.dg

ag

aa

ga

tg

gg

at

gt

ac

gc

cg

cc

at

tg

gc

tt

tg

aa

ct

ITS

1IT

S2

Nu

cleo

tid

esin

big

lett

ers

corr

esp

on

din

gto

G.

bo

hem

ica

,in

smal

lle

tter

sco

rres

po

nd

ing

tocr

eate

dse

con

dp

aren

tal

seq

uen

ce(G

.sp

ec.)

aP

osi

tio

no

fin

form

ativ

esi

tes

are

nu

mb

ered

acco

rdin

gto

EM

BL

acce

ssio

nn

um

ber

of

AM

28

72

71

(G.

bo

hem

ica

I-A

BR

)b

Dir

ect

(K=

Gan

dT

;M

=A

and

C;

R=

Aan

dG

;S

=G

and

C;

W=

Aan

dT

;Y

=C

and

T)

ITS

dat

a(s

eeal

soT

able

1)

cC

lon

ing

ITS

dat

ad

Gen

erat

edse

qu

ence

(see

‘‘M

ater

ials

and

met

ho

ds’

’)

Hybridization in Gagea

123

found intermediated between G. fragifera and G. villosa in

the ITS trees (Peterson et al. 2008). Our molecular studies

provide evidence of the hybrid origin of Gagea microfi-

stulosa (G. villosa 9 G. fragifera) and lead to the

speculation that G. polidorii could represent the reverse

hybrid (G. fragifera 9 G. villosa). Both Gagea micro-

fistulosa and G. polidorii grow at elevations below alpine

G. fragifera and above G. villosa populations, the latter

occurring in lowlands and low mountains. Gagea micro-

fistulosa (Levichev 2008b) differs from G. fragifera in a

larger habitus but smaller flowers, the normally non-fistular

second basal leaf (the first is fistular) which is always

present at adult stage and the non-fistular peduncle. The

peduncle of G. microfistulosa is not abbreviated in the

immature stage. Gagea microfistulosa is morphologically

closely related to G. polidorii (Levichev 2008a), but differs

from the latter in the gradually acuminate apex of the lower

cauline leaf. Whereas the local endemic G. microfistulosa

could represent a relative young hybrid, G. polidorii

appears to have a longer history; this assumption is sup-

ported not only by its wider range, but also by ITS ribotype

networks and the stage of recombination and conversion of

the investigated ITS-region during the process of concerted

evolution. Although the cpDNA data of G. polidorii mostly

corresponded to G. fragifera we found four multiple ITS2

positions corresponding to both G. fragifera and G. villosa

(see Table 2; see also Peruzzi et al. 2008b). Interestingly,

these positions were also found in one of the G. fragifera

vouchers (I-SIC) which could be explained by introgres-

sion. Although G. polidorii was according to molecular

data found to be more closely related to G. fragifera (see

also Peruzzi et al. 2008b), it is morphologically interme-

diate between G. villosa and G. fragifera (Tison 2004;

Peruzzi 2008b). Gagea polidorii differs from the latter in

its basal leaves with narrower fistula; its second leaf nor-

mally always present at adult stage, becoming larger in old

plants (disappearing in old plants in G. fragifera), its first

cauline leaf not tubulate at apex (a conspicuous feature in

the herbarium), and its spreading fruiting pedicels. Gagea

polidorii differs from G. villosa in its spongy tunics, its

normally pedunculate (and not sessile) head of bulbils at

immature stage, its often smaller second leaf and its fis-

tulose, erect basal leaves which emerge in late winter

(Tison 2004).

An explanation for the relationships observed by the

neighbour network analyses could be that tetraploid G.

villosa and tetraploid G. fragifera are the ancestral species

and that G. microfistulosa, G. polidorii and probably also

G. glacialis represent descendant hybrid species.

According to our molecular data (see also Peterson

et al. 2008) and the ribotype networks represented herein,

G. glacialis can be differentiated from G. fragifera. This

is also true according to morphological studies of living

material of G. glacialis [e.g. from Lazistan and Ararat

mountains (Armenia), Levichev unpubl]. It is character-

ised by its small height (3)-5-8-(12) cm, the occurrence

of one (very seldom 2) flowers; the length of the tepals

\8 mm and the occurrence of one basal leaf with no

lumen (fistulous) in the centre. We do not agree with

Rix (1984) and Zarrei et al. (2007) who considered

G. glacialis to be synonymous with G. fragifera. Mor-

phological data rather suppose a hybridogenous origin of

G. glacialis: it could also have appeared (Levichev un-

publ.) as a result of the hybridization of G. fragifera and

G. tenuissima Miscz., a local endemic of north-eastern

Turkey.

Based on our ribotype network and chromosome

numbers (Table 1), a second hypothesis would be that

G. fragifera, now widely spread in alpine environments in

Europe and Western Asia, could be the result of hybrid-

ization of tetraploid G. villosa and G. glacialis. Again,

introgression of the genetic material of G. villosa into

G. fragifera could result in the development of local

endemic species in Crimea (G. microfistulosa) and the

French Alps, Corsica and Calabria (G. polidorii). The latter

scenario appears to be unlikely due to the range sizes,

particularly the large range of G. fragifera (see below) and

the assumed age of the involved taxa.

Fig. 5 Ribotype network for ITS ribotypes (Table 3) of the G. cf.

bohemica I-CAL hybrid (cf. boh 1–9), including G. bohemica and

putative sequence of its second unknown parental species (G. spec.;see also ‘‘Materials and methods’’). Parental species accompanied by

voucher information (Table 1) are boxed and indicated in bold. Emptydots refer to missing intermediates not found among the analysed

sequences

A. Peterson et al.

123

Gagea bohemica hybrids and their detection

Gagea bohemica displays considerable variability with

regard to its morphology (Peterson et al. 2004; Levichev

2006a), molecular patterns (Peterson et al. 2004) and kar-

yological data (2n = 24, 36, 48, 60, 72; Levichev 2006a;

Peruzzi 2008a). This species is described as a parent of the

putative hybrid taxon G. luberonensis (Tison 1998, 2004)

which is characterised by a morphology intermediate

between G. bohemica and G. granatellii (Parl.) Parl. or

G. dubia A. Terracc. (Tison 1998, 2004; Peruzzi and

Bartolucci 2006). According to the molecular data from

direct sequencing, both investigated Italian G. luberonensis

vouchers (from Abruzzo and Latium) were found to be

identical (ITS-region, psbA-trnH IGS) to G. bohemica (see

also Peruzzi et al. 2008b). Perhaps as a result of concerted

ITS-evolution (reviewed in Elder and Turner 1995 and in

Eickbush and Eickbush 2007; see, e.g. Volkov et al. 2007),

it may not be possible to detect the other hybrid partner of

G. luberonensis.

On the contrary, we found one interesting Gagea cf.

bohemica voucher (I-CAL), which morphologically cor-

respond exactly to G. bohemica, but in consideration of

molecular data was clearly differentiated from the latter.

For this taxon, incongruent cpDNA (AM932484: psbA-

trnH IGS, AM932485: trnL-trnF IGS) and ITS trees were

found (Peruzzi et al. 2007). The ITS direct sequencing and

cloning data show that Gagea bohemica is one of the

parental species, but the other parental taxon could not be

recognised. In the cpDNA tree, the hybrid was found in the

same unsupported clade as G. peduncularis (J. and C.

Presl) Pascher (56% branch support, data not shown), a

species which is morphological very similar to G. bohe-

mica, but distinct through its longer pedicels (Peruzzi

2003). For this reason, the second parent was not included

in our studies: it is perhaps not yet detected or has become

extinct. Further investigations including all known repre-

sentatives of the section Didymobulbos could give a new

insight into this interesting taxon which should be

accompanied by further morphological studies. Two addi-

tional vouchers identified as G. bohemica (from Calabria:

CLU9236, Basilicata: CLU12685) appear to correspond to

G. cf. bohemica I-CAL in consideration of cpDNA data

(Peterson, unpublished). Interestingly, Gagea cf. bohemica

showed the same chromosome number (tetraploid, see also

Peruzzi 2003) as the G. bohemica from Abruzzo, the lat-

ter corresponding molecularly to all other investigated

G. bohemica vouchers (Peterson et al. 2004, 2008).

Accordingly, this peculiar hybrid is only known from the

mountains separating Calabria from Basilicata regions

(Pollino Massif and the Verbicaro-Orsomarso range),

where it appears to exclude the ‘‘pure’’ lineages of

G. bohemica. Generally, hybrids in Gagea may be

overlooked when the parental species are morphologically

similar and morphology of hybrids is overlapping with that

of the parental species (Albarouki and Peterson 2007;

Mahelka et al. 2007).

Hybridization, polyploidy and speciation in the genus

Gagea

We assume the existence of two centres for the origin of

the evolution of Gagea: one in the Eastern Mediterranean

region, the second in the Himalayas. In both regions, basal

lineages represented by diploid species occur: in the first

region the two species of Gagea section Anthericoides

A. Terracc. [G. graeca (L.) Irmisch, G. trinervia (Viv.)

Greuter] and in the second representatives of the Lloydia

clade (Peterson et al. 2008). Between these centres, in the

region of South-West Asia, intensive speciation took place.

Today, the South-West Asian region, including North and

East Anatolia, Iraq, Iran, Afghanistan, the Middle Asia

states and the South of Kazakhstan, harbour more than 75%

of the species of the genus; there are representatives of

eleven Gagea sections (Levichev 1999a, 2008a). Some

lineages [e.g. G. sect. Plecostigma (Turcz.) Pascher, G.

sect. Platyspermum Boiss. and G. sect. Stipitatae (Pascher)

Davlian.] maintained a mostly diploid level and adapted to

dry steppe grasslands conditions and colonised dry and

desert areas of South-West and Central Asia and small

regions of Southern Europe and Northern Africa. Others

(e.g. G. sect. Gagea, G. sect. Didymobulbos, G. sect.

Fistulosae) evolved mainly through polyploidisation,

hybridization, and introgression at various degrees and

colonised large areas of temperate Eurasia. Many species

from these sections possess larger distribution ranges, for

example G. lutea (practically throughout Europe, Turkey,

and the North of Iran), and G. bohemica (Central and

Southern Europe). The largest range with a disjunctive

arcto-alpine distribution shows G. fragifera (from Morocco

to Mongolia, from Northern India to the city of Vorkuta

beyond the polar circle; Levichev 2006a), indicating its

pre-glacial age (Levichev 2008a).

Many Gagea species display reduced or non-existent

sexual reproduction along with massive vegetative propa-

gation (Caparelli et al. 2006; Gargano et al. 2007). Only

four Gagea species without vegetative reproduction are

known (Levichev 2006b). This means that in most cases

hybridization can lead to the swift radiation of new lin-

eages, even in the event of completely or partially sterile

progeny (Peruzzi 2008b).

According to Leitch et al. (2007) the evolution of

genome size within Liliaceae appears to be punctuated

rather than gradual. The latter authors hypothesized that

recent diversification has increased the rate of genome size

evolution through time. For Gagea, we demonstrated that

Hybridization in Gagea

123

hybridization and polyploidisation are common; this could

be the causes for the rapid gene evolution with profound

effects on genome size, which, in turn, could drive speci-

ation in the genus. This is in agreement with the finding

(Peterson et al. 2008; Peruzzi et al. 2008a) that the species

of the basal Gagea section Anthericoides are diploid

(2n = 24; IPCN), whereas the other Gagea sections are

characterised by both diploids and polyploids; several

species occur with a variety of ploidy levels. In addition, in

a few taxa from the large sections G. sect. Gagea and G.

sect Stipitatae, chromosome numbers of 2n = 18 were

recognised (e.g. Davlianidze and Levichev 1987). The

occurrence of these low numbers in the morphologically

and molecularly relatively basal section Gagea (Peterson

et al. 2008) and in the morphologically most evolutionary

advanced section Stipitatae (Levichev 2008a) is probably a

result of reduction. It could be hypothesised that Gagea has

a diploid origin and is developing towards polyploid lin-

eages of taxa, probably followed by genome diploidisation,

which was suggested, e.g. for the allopolyploid (2n = 108)

Iris versiciolor (Lim et al. 2007). It must be noted that the

basal diploid Gagea taxa with a basic chromosome number

of x = 12 could also have their origin in polyploids and,

consequently, would not be true diploids. Genome

sequence analyses indicate that many plant species,

including Arabidopsis, maize, rice, and poplar are recent or

ancient diploidised (paleo-) polyploids (Chen et al. 2007)

and provide evidence that angiosperms have undergone

multiple rounds of polyploidisation events during their

evolution (reviewed in Paun et al. 2007). Today in Gagea,

speciation has resulted in a polymorphic, species-rich

genus currently divided into independent sections (Levichev

in Peterson et al. 2008; Levichev 2008a; Peruzzi et al.

2008a), possessing overlapping morphological features

(Levichev in Peterson et al. 2008) which could be caused

by several recombination processes during hybridization

and polyploidisation/diploidisation events.

Conclusion

We were able to demonstrate that hybridization is common

in Gagea, utilising ITS data from direct sequencing and

cloned sequences. Although the ITS-region is generally

concertedly evolved in Gagea, in relatively young hybri-

dogenous taxa the occurrence and recombination of both

parental spacers were recognised. The ITS-region therefore

has the potential to detect hybrid events when concerted

evolution is not sufficiently fast. Our investigation shows

that hybridization took place in both directions at different

places and times. Generally, our results of neighbour joining

and ribotype networks with their high potential of demon-

strating the relation between hybrids and their putative

parental species are in agreement with existing mor-

phological investigations and molecular phylogenetics.

Hybridization and polyploidisation seem to be main forces in

speciation of Gagea. Generally, we surmise that it is essen-

tial to combine all available morphological, karyological,

and molecular investigations for the detection of hybrids.

References

Abbott RJ, Lowe AJ (2004) Origins establishment and evolution

of two new polyploidy species of Senecio in British Isles. Biol

J Linnean Soc 82:467–474

Ainouche ML, Baumel A, Salmon A, Yannic G (2004) Hybridization,

polyploidy and speciation in Spartina (Poaceae). New Phytol

161:165–172

Albarouki E, Peterson A (2007) Molecular and morphological

characterization of Crataegus L. species (Rosaceae) in southern

Syria. Bot J Linnean Soc 153:255–263

Allen GA, Soltis DE, Soltis PS (2003) Phylogeny and biogeography

of Erythronium (Liliaceae) inferred from chloroplast matK and

nuclear rDNA ITS sequences. Syst Bot 28:512–523

Alvarez I, Wendel JF (2003) Ribosomal ITS sequences and plant

phylogenetic inference. Mol Phylogenet Evol 29:417–434

Arnold ML (1997) Natural hybridization and evolution. Oxford

University Press, New York

Boissier E (1882) Gagea. Flora Orientalis. Geneve et Basiliae 5:203–

211

Booy G, Van der Schoot J, Vosman B (2000) Heterogeneity of the

internal transcribed spacer 1 (ITS1) in Tulipa (Liliaceae). Plant

Syst Evol 225:29–41

Caparelli KF, Peruzzi L, Cesca G (2006) A comparative analysis of

embryo-sac development in three closely-related Gagea Salisb.

species (Liliaceae), with some consideration on their reproduc-

tive strategies. Plant Biosyst 140:115–122

Chen ZJ, Ha M, Soltis D (2007) Polyploidy: genome obesity and its

consequences. New Phytol 174:717–720

Clement M, Posada D, Crandall KA (2000) TCS: a computer program

to estimate gene genealogies. Mol Ecol 9:1657–1660

Davlianidze MT, Levichev IG (1987) Chromosome numbers of

species of the genus Gagea from Middle Asia. Bot J (Leningr)

72:1271–1272

Eickbush TH, Eickbush DG (2007) Finely orchestrated movements:

evolution of the ribosomal RNA genes. Genetics 175:477–485

Elder JF Jr, Turner BJ (1995) Concerted evolution of repetitive DNA

sequences in eukaryotes. Q Rev Biol 70:297–320

Gargano D, Peruzzi L, Caparelli KF, Cesca G (2007) Preliminary

observations on the reproductive strategies in five early-flower-

ing species of Gagea Salisb. (Liliaceae). Bocconea 21:349–358

Grimm GW, Denk T (2008) ITS evolution in Platanus (Platanaceae):

homoeologues, pseudogenes and ancient hybridization. Ann Bot

101:403–419

Hall TA (1999) BioEdit: a user-friendly biological sequence align-

ment editor and analysis program for Windows 95/98/NT.

Nucleic Acids Symp Ser 41:95–98

Harpke D, Peterson A (2008) 5.8S motifs for the identification of

pseudogenic ITS regions. Botany 86:300–305

Hegarty MJ, Hiscock SJ (2005) Hybrid speciation in plants: new

insights from molecular studies. New Phytol 165:411–423

Henker H (2005) Die Goldsterne von Mecklenburg-Vorpommern

unter besonderer Berucksichtigung kritischer und neuer Sippen.

Botanischer Rundbrief fur Mecklenburg-Vorpommern 39:5–88

A. Peterson et al.

123

Huson DH, Bryant D (2006) Application of phylogenetic networks in

evolutionary studies. Mol Biol Evol 23:254–267

Huson DH, Kloepper TH (2005) Computing networks from binary

sequences. Bioinformatics 21:159–165

Ikinci N, Oberprieler C, Guner A (2006) On the origin of European

lilies: phylogenetic analysis of Lilium section Liriotypus (Lili-

aceae) using sequences of the nuclear ribosomal transcribed

spacers. Willdenowia 36:647–656

Leitch IJ, Beaulieu JM, Cheung K, Hanson L, Lysak MA, Fay MF

(2007) Punctuated genome size evolution in Liliaceae. J Evol

Biol 20:2296–2308

Levichev IG (1990) On age variation and hybridization of some

representatives of Gagea (Liliaceae). Bot J (Leningr) 75:658–667

Levichev IG (1999a) Phytogeographical analysis of the genus GageaSalisb. (Liliaceae). Komarovia 1:47–59

Levichev IG (1999b) Zur Morphologie in der Gattung Gagea Salisb.

(Liliaceae). I. Die unterirdischen Organe. Flora 194:379–392

Levichev IG (2001) New species of the genus Gagea Salisb.

(Liliaceae) from western districts of Asia. Turczaninowia 4:5–35

Levichev IG (2005) New taxa of the genus Gagea (Liliaceae) in the

flora of the Caucasus. Bot J (St. Petersburg) 90:1580–1592

Levichev IG (2006a) A review of the Gagea (Liliaceae) species in the

flora of Caucasus. Bot J (St. Petersburg) 91:917–951

Levichev IG (2006b) Four new species of the genus Gagea Salisb.

(Liliaceae) from Western Himalayas and the adjoining regions.

Pakistan J Bot 38:47–54

Levichev IG (2008a) System of genus Gagea (Liliaceae) from

positions of the neotenical divergence. Turczaninowia 11 (in

press)

Levichev IG (2008b) New species of genus Gagea Salisb. (Liliaceae)

from the Crimean yaila. Novitates systematicae plantarum

vascularium, Moscow, St. Petersburg. T. 40 (in press)

Lim KY, Matyasek R, Kovarik A, Leitch A (2007) Parental origin and

genome evolution in the allopolyploid Iris versicolor. Ann

Botany 100:219–224

Linder CR, Rieseberg H (2004) Reconstructing patterns of reticulate

evolution in plants. Am J Bot 91:1700–1708

Mahelka V, Fehrer J, Krahulec F, Jarolımova V (2007) Recent natural

hybridization between two allopolyploid wheatgrasses (Elytrigia,

Poaceae): Ecological and evolutionary implications. Ann Botany

100:249–260

McBreen K, Lockhart PJ (2006) Reconstruction reticulate evolution-

ary histories of plants. Trends Plant Sci 11:398–404

McDade LA (1992) Hybrids and phylogenetic systematics II. The

impact of hybrids on cladistic analysis. Evolution 46:1329–1346

Nishikawa T, Okazaki K, Uchino T, Arakawa K, Nagamine T (1999)

A molecular phylogeny of Lilium in the internal transcribed

spacer region of nuclear ribosomal DNA. J Mol Evol 49:238–

249

Okuyama Y, Fujii N, Wakabayashi M, Kawakita A, Ito M, Watanabe

M, Murakami N, Kato M (2005) Nonuniform converted

evolution and chloroplast capture: heterogeneity of observed

introgression patterns in three molecular data partition phylo-

genies of Asian Mitella (Saxifragaceae). Mol Biol Evol 22:285–

296

Paun O, Fay MF, Soltis DE, Chase MW (2007) Genetic and

epigenetic alterations after hybridization and genome doubling.

Taxon 56:649–656

Peruzzi L (2003) Contribution to the cytotaxonomical knowledge of

Gagea Salisb. (Liliaceae) sect. Foliatae A. Terracc. and

synthesis of karyological data. Caryologia 56:115–128

Peruzzi L (2008a) Contribution to the cytotaxonomical knowledge of

the genus Gagea Salisb. (Liliaceae). III. New karyological data

from the C Mediterranean area. Caryologia 61:92–106

Peruzzi L (2008b) Hybridity as a main evolutionary force in the genus

Gagea Salisb. (Liliaceae). Plant Biosyst 142:179–184

Peruzzi L, Aquaro G (2005) Contribution to the cytotaxonomicalknowledge of Gagea Salisb. (Liliaceae). II. Further karyological

studies on Italian populations. Candollea 60:237–253

Peruzzi L, Bartolucci F (2006) Gagea luberonensis J.-M. Tison

(Liliaceae) new for the Italian flora. Webbia 61:1–12

Peruzzi L, Peterson A, Tison J-M, Peterson J (2007) Phylogenetic

Relationships of Gagea Salisb. (Liliaceae) in Italy, inferred from

molecular and morphological matrixes. XII Optima Meeting,

Pisa, Italy, 10–16 September 2007. p. 118

Peruzzi L, Peterson A, Tison J-M, Peterson J (2008a) On the

phylogenetic position and taxonomic value of Gagea trinervia(Viv.) Greuter and G. sect. Anthericoides A. Terracc. (Liliaceae).

Taxon 57:1201–1214

Peruzzi L, Peterson A, Tison J-M, Peterson J (2008b) Phylogenetic

relationships of Gagea Salisb. (Liliaceae) in Italy, inferred from

molecular and morphological data matrixes. Pl Syst Evol

276:219–234. doi:10.1007/s00606-008-0081-4

Peterson A, John H, Koch E, Peterson J (2004) A molecular

phylogeny of the genus Gagea (Liliaceae) in Germany inferred

from non-coding chloroplast and nuclear DNA sequences. Plant

Syst Evol 245:145–162

Peterson A, Levichev IG, Peterson A (2008) Systematics of Gageaand Lloydia (Liliaceae) and infrageneric classification of Gageabased on molecular and morphological data. Mol Phylogenet

Evol 46:446–465

Rieseberg LH, Willis JH (2007) Plant Speciation. Science 317:910–

914

Rix EM (1984) Gagea Salisb. In: Davis PH (ed) Flora of Turkey and

the East Aegean Islands. Edinburgh, pp. 312–327

Ronsted N, Law S, Thornton H, Fay MF, Chase MW (2005)

Molecular phylogenetic evidence for the monophyly of Fritil-laria and Lilium (Liliaceae; Liliales) and the infrageneric

classification of Fritillaria. Mol Phylogenet Evol 35:509–527

Saito Y, Kokubugata G, Moller M (2007) Molecular evidence for a

natural hybrid origin of Doellingeria x sekimotoi (Astaraceae)

using ITS and matK sequences. Int J Plant Sci 168:469–476

Sang T, Crawford DJ, Stuessy TF (1997) Chloroplast DNA phylo-

geny, reticulate evolution, and biogeography of Paeonia(Paeoniaceae). Am J Bot 84:1120–1136

Schnare MN, Damberger SH, Gray MW, Gutell RR (1996) Compre-

hensive comparison of structural characteristics in eukaryotic

cytoplasmic large subunit (23 S-like) ribosomal RNA. J Mol

Biol 256:701–719

Schwarzbach AE, Rieseberg LH (2002) Likely multiple origin of a

diploid hybrid sunflower species. Mol Ecol 11:1703–1715

Soltis DE, Soltis PS, Tate JA (2004a) Advances in the study of

polyploidy since plant speciation. New Phytol 161:173–191

Soltis DE, Soltis PS, Pires JC, Kovarik A, Tate JA, Mavrodiev E

(2004b) Recent and recurrent polyploidy in Tragopogon (Aster-

aceaee). Cytogenic, genomic, and genetic comparisons. Biol J

Linnean Soc 82:485–501

Suarez-Santiago VN, Salinas MJ, Romero-Garcıa AT, Garrido-

Ramos MA, de la Herran R, Ruiz-Rejon C, Ruiz-Rejon M,

Blanca G (2007) Polyploidy, the major speciation mechanism in

Muscari subgenus Botryanthus in the Iberian Peninsula. Taxon

56:1171–1184

Tison JM (1998) Note complementaire sur quelques Gagea francais.

Le Monde des Plantes. 462:7–8

Tison JM (2004) Gagea polidorii J.M. Tison, an unrecognised species

from South western Alps and Apennines. Acta Botanica Gallica

151:319–326

Volkov RA, Komarova NY, Hemleben V (2007) Ribosomal DNA in

plant hybrids: inheritance, rearrangement, expression. Syst

Biodivers 5:261–276

Vriesendorp B, Bakker FT (2005) Reconstruction pattern of reticulate

evolution in angiosperms: what can we do? Taxon 54:593–604

Hybridization in Gagea

123

White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct

sequencing of fungal ribosomal RNA genes for phylogenetics.

In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR

protocols: a guide to methods and applications. Academic Press,

San Diego, pp 315–322

Wissemann V (2007) Plant evolution by means of hybridization. Syst

Biodivers 5:243–253

Zarrei M, Zarre S, Wilkin P, Rix M (2007) Systematic revision of the

genus Gagea Salisb. (Liliaceae) in Iran. Bot J Linnean Soc

154:559–588

A. Peterson et al.

123