Botanical Studies (2006) 47: 191-197.

*

Corresponding author: E-mail: lwp@hunnu.edu.cn; Tel:

86-731-8871052; Fax: 86-731-8883310.

Natural hybridization between Aster ageratoides var.

scaberulus and Kalimeris indica (Asteraceae): evidence

from morphology, karyotype, and ITS sequences

Wei-Ping LI

*

College of Life Sciences, Hunan Normal University, Changsha 410081, P. R. China

(Received April 14, 2004; Accepted November 4, 2005)

ABSTRACT.

A new natural hybrid from the Hengshan Mountain in Hunan Province, China, was found.

Comparisons of morphology, karyotype, and ITS sequences among the hybrid and its parental species show

that the hybrid represents F

1

progeny from hybridization between Aster ageratoides var. scaberulus and Ka-

limeris indica. Based on morphological and karyotypical observations of the hybrid, this study suggests that

natural hybridization between Aster and Kalimeris could have lead to the shortening of the pappus and a

special karyotype (LS-type) and makes the delimiting of Aster and Kalimeris difficult. This study also indi-

cates that karyotype investigations may be particularly useful in resolving some of the taxonomic confusion

between Aster and Kalimeris.

Keywords: Aster; Kalimeris; Karyotype; Natural hybridization; Pappus.

INTRODUCTION

Natural hybridization is a frequent phenomenon in

plants and plays an important role in plant evolution,

leading in at least some cases to formation of new spe-

cies. In addition, hybridization often generates consider-

able taxonomic confusion by facilitating introgression of

plant traits. Therefore, confirmation of the hybrid nature

and parentage is important in studying plant evolution,

and may help to resolve certain taxonomic problems.

Unfortunately, many hybrids described in the botanical

literature (e.g. Hsieh and Wang, 1989; Chang, 1991; Sun

and Huang, 1994; Zhang et al., 1995; Zhang and Zhao,

1995; Qian and Sun, 1998; Ren, 1996; Zhou and Wu,

2001; Zhang et al., 2003) were determined based only on

their morphological intermediacy between their putative

parents. However, morphological intermediacy may result

from continuous variation and the high plasticity of many

traits (Xu, 1998; Craft et al., 2002). In recent years, mo-

lecular data have played an increasing role in the detection

and verification of putative hybrids (Smith et al., 1996;

Morrell and Rieseberg, 1998; Milne, 1999; Durand, 2002;

Denda and Yokota,

2003

).

Tara (1972, 1977, 1978a, 1978b, 1979) first described

intergeneric hybridization between Aster and Kalimeris

(Asteraceae, Astereae, Asterinae). Based on cytological

analyses, Tara suggested that hybridization had occurred

between Aster ovatus (Franch. et Sav.) Soejima et Mot.

Ito (A. ageratoides subsp. ovatus (Franch. et Sav.) Ka-

tam.) and Kalimeris incisa (Fisch.) DC., and that in fact

A. ovatus was an amphidiploid that originated following

intergeneric hybridization between Aster and Kalimeris.

However, many subsequent authors have overlooked the

morphological characters of hybrids and the potential

impact of intergeneric hybridization on the complex and

uncertain relationship between these two genera. This

study provides the first report of hybridization between

Aster ageratoides var. scaberulus (Miq) Ling and Kalim-

eris indica (L.) Sch.-Bip. with verification of the parent-

age. This study demonstrates the use of karyotyping and

of molecular sequencing as a means to detecting hybrids

and discusses the implication of these results for the phy-

logeny of Aster and Kalimeris.

MATERIALS AND METHODS

Study site and materials

Plant materials for this study were collected from

Hengshan Mountain, located in the middle-subtropical

monsoon zone in central Hunan Province, in China, at

112¢X34¡¦ to 112¢X44¡¦E and 27¢X10¡¦ to 27¢X20¡¦N. The putative

hybrid plants were found at a sunny deforested site near a

subtropical coniferous forest at 300 m alt. These hybrids

appeared to have leaves and inflorescences intermedi-

ate between two sympatric taxa, Aster ageratoides var.

PLANT BIOSYSTEMATICS

192

Botanical Studies, Vol. 47, 2006

scaberulus and Kalimeris indica, which were common

at the site. Aster ageratoides var. laticorymbus (Vant.)

Hand.-Mazz. also occurs at Hengshan Mountain, but the

population was found to be a hexaploid (Li, unpublished)

and was spatially distant from the putative hybrid, sug-

gesting that it could not be one of the parents of the puta-

tive hybrid. It is thus excluded from this study.

Twelve putative hybrid individuals were collected and

prepared as specimens for morphological analyses, and

rhizomes of three individuals were transplanted to Hunan

Normal University for cytological and molecular investi-

gation. Thirty plants each of A. ageratoides var. scaberulus

and K. indica that grew near the putative hybrids were

prepared as specimens for morphological analyses, and

five each were transplanted for cytological and molecular

investigation. All specimens were identified according to

Ling and Chen (1985) and were deposited as herbarium

vouchers at Hunan Normal University (HNNU).

Methods

After preliminary observation, 16 morphologi-

cal characters¡Xincluding features of branches, middle

leaves, phyllaries, florets, and achenes¡Xwere found

to differ between the two putative parental species. All

field collections were scored for these 16 characters. For

quantitative features, such as size of middle leaves and

number of florets, the measurements are presented as a

range of variation.

Actively growing root tips for chromosome

observations were cut from living plants, pretreated with

0.1% colchicine at 8-12¢XC for 4 h, fixed in Carnoy I

(glacial acetic acid: 95% ethanol=1:3) at room temperature

for 12 h, then macerated in 1 mol/L hydrochloric acid

at 60¢XC for 8 min, stained in 5% NH

4

Fe(SO

4

)

2

, at room

temperature for 3 h, stained in 0.75% hematoxylin for

3 h, washed in distilled water for 30 min, and finally

depigmented and squashed in 45% acetic acid.

Four samples for ITS analysis, one each from A. ag-

eratoides var. scaberulus and K. indica, and two, respec-

tively, from putative hybrid individuals were obtained

from plants cultivated at Hunan Normal University. Total

genomic DNA was isolated from fresh leaf tissue using

the CTAB method of Roger and Bendish (1988). The

two internal transcribed spacers (ITS-1, ITS-2) and 5.8S

nrDNA were amplified using the primers "ITS1" (5 ¡¦ AGT

CGT AAC AAG GTT TCC GTA G 3¡¦) and "ITS4" (5¡¦

TCC TCC GCT TAT TGA TAT GC 3¡¦). The amplification

reaction was performed in 50 £gL volumes using 10-20 ng

of genomic DNA, 5 £gl MgCl

2

(25 mmol/L), 4 £gl dNTP (2.5

mmol/L), 1 £gl (6.25 pmol) of each primer, and 0.25 £gl (5

U/£gl) Taq DNA polymerase. The following temperature

and time profile was used in the amplification of the Poly-

merase Chain Reaction (PCR): an initial denaturation step

of 94oC for 5 min; 36 cycles of 94oC for 1 min, 57oC for 1

min, and 72oC for 2 min, and a final extension of 10 min

at 72oC. The amplified double stranded DNA fragments

were purified and then directly sequenced on an ABI 377

automated sequencer (PE Applied Biosystems, Inc.). Prim-

ers were the same as those of the initial PCR. For each

sample, both forward and reverse sequences of ITS1, 5.8S

and ITS2 were obtained. Each base position was examined

for agreement between complementary strands.

RESULTS

Morphology

A morphological comparison among the three taxa is

summarized in Table 1. The two putative parents differ

significantly in all 16 characters. By contrast, the pre-

sumed hybrids have intermediate character states in eight

characters, including distribution of stomata, number of

florets, size of achenes, and especially length of pappus.

The hybrids resemble A. ageratoides var. scaberulus, in

five characters, such as lack of stomata on upper leaves,

shape of inner phyllaries, width of middle phyllaries, and

lack of glands on phyllaries and ovaries, and resemble K.

indica in the other three, including number of branches

above, size of middle leaves, and color of ray florets.

Karyotype

The metaphase chromosomes and the karyotype of the

three taxa are shown in Figure 1. All three taxa are tetra-

ploid with 2n=4x=36. However, there are significant dif-

ferences in karyotype among them. All 36 chromosomes

in A. ageratoides var. scaberulus are L-type (large chro-

mosomes), with the length varying from 2.51-4.35 £gm

while all 36 chromosomes of K. indica are S-type (short

chromosomes), ranging in length from 1.4-1.93 £gm. The

longest chromosome pair of K. indica, 1.93 in length, is

shorter than the shortest pair of A. ageratoides var. scab-

erulus. The putative hybrid is intermediate in karyotype,

with 18 L-type chromosomes (2.49-4.1 £gm) and 18 S-type

chromosomes (1.35-1.85 £gm).

ITS sequences

The boundaries of the ITS region (ITS1, 5.8S, and

ITS2) were determined by comparison to published ITS

sequences of Aster amellus L. and Kalimeris integrifolia

(L.) Less (Noyes and Rieseberg, 1999). The length of

the ITS sequence of all the materials studied is 630 bp.

The sequences of A. geratoides var. scaberulus and K.

indica differ from one another at 13 sites (2%; Table 2).

At each of these 13 sites the sequence spectrum of the

putative hybrid individuals shows two overlapped bands

with the peak about half as high as those of neighboring

sites, indicating that two different base pairs are at each

of these sites that correspond with those of A. geratoides

var. scaberulus and K. indica, respectively (Table 2),

exhibiting a combination of the ITS sequences of the

two putative parent species. The forward and reverse

sequences agree with each other for complementary

strands. The ITS sequences from the two hybrids

were identical. Therefore, the overlapped bands, as a

polymorphism, can be attributed to the fact that there are

LI ¡X Natural hybridization between

A. ageratoides

var.

scaberulus

and

K. indica

193

two kinds of ITS sequences in each hybrid plant instead of

to a test error.

DISCUSSION

Confirmation of the hybrid

Sympatry of parents is one of preconditions for

hybridization. The putative hybrid, A. ageratoides var.

scaberulus, and K. indica co-occurred at a sunny deforest-

ed site of the Hengshan Mountain with no other Kalimeris

or Aster species nearby. Their spatial

distribution indicates

the possibility that the putative hybrid is derived from

A. ageratoides var. scaberulus and K. indica. A hybrid is

usually morphologically intermediate between its parents

(Stebbins, 1950), and may also share some features with

them (Rieseberg and Ellstrand, 1993). According to Table

1, the putative hybrid had eight intermediate characters be-

tween A. ageratoides var. scaberulus and K. indica, three

similar to K. indica, and five similar to A. ageratoides var.

scaberulus, supporting the notion that the putative hybrid

was the product of A. ageratoides var. scaberulus and K.

indica.

Even though both Aster ageratoides var. scaberulus

and K. indica at this locality are tetrapolid with 2n=4x=36,

their karyotypes are fortunately quite different (Figure 1),

which makes it easy to test the hybrid cytologically. Since

the karyotype of the two taxa was investigated simultane-

ously with the same method and in the same reagents,

length of the chromosomes was comparable. The shortest

chromosome pair (L-type) of A. ageratoides var. scaberu-

lus was found to be longer than the longest of K. indica

(S-type), and the putative hybrid had 18 chromosomes

each of L- and S-Type, both resembling in size and

morphology the presumed parental species.

Tara (1972, 1978a, 1978b, 1979) studied intergeneric

hybridization between Aster and Kalimeris based on

karyotypical comparisons among hybrids and their parents

as well as observations of meiosis. Tara reported that there

were several hybrid types, such as 2n=72=18 L-chromo-

somes + 54 S-chromosomes, 2n=72=9 L-chromosomes

+ 63 S-chromosomes, 2n=81=27 L-chromosomes + 54

S-chromosomes, and 2n=108=18 L-chromosomes + 90

S-chromosomes, which resulted from the natural hy-

bridization of A. ovatus and K. incisa and the backcross

Table 1. A morphological comparison among Aster ageratoides var. scaberulus, Kalimeris indica and their putative hybrid.

Features

Aster ageratoides var. scaberulus The putative hybrid Kalimeris indica

Branches above

None or rarely 1-3

Many (>5)

Many (>5)

Size of middle leaves (Length ¡Ñ

Width) (cm)

6-15¡Ñ2-6

6-9¡Ñ2-3.5

6-9.5¡Ñ2-4

Stomata on the upper surface of

middle leaves

None

None

Present, but rare

Size of leave on branches

(Length ¡Ñ Width) (cm)

3-8¡Ñ1.4-3

2-4¡Ñ0.5-1.5

1-2.5¡Ñ0.5-1

Length of peduncles (cm)

0.3-2.1

0.3-9

1.2-10

No. of layers of phyllaries

4-5

3-4

2-3

Shape of inner phyllaries

Linear oblanceolate

Linear oblanceolate Ovate in the upper 2/3, and

constricted and lanceolate in

the lower 1/3

Glands on middle phyllaries None

None

Present

Width of middle phyllaries 0.8-1

0.85-1

1.3-1.6

No. of florets per head

24-40

54-72

100-130

No. of ray florets per head

12-20

19-22

25-35

No. of disc florets per head

12-20

34-51

90-100

Coloure of ray florets

White

Blue-violet

Blue-violet

Glands on ovary

None

None

Present

Length of pappus (mm)

4.75-5.5

0.5-2.25

0.1-0.2

Size of achenes (Length ¡Ñ

Width) (mm)

2-2.5¡Ñ0.65-0.95

1.95-2.25¡Ñ0.9-1

1.75-2.1¡Ñ0.95-1.15

194

Botanical Studies, Vol. 47, 2006

Figure 1. Photomicrographs of the metaphase chromosomes

and the karyotype of Aster ageratoides var. scaberulus, the puta-

tive hybrid, and Kalimeris indica (Scale bar=2

£gm)

. A, D: Aster

ageratoides var. scaberulus; B, E: the putative hybrid; C, F: Ka-

limeris indica.

A

B C

D

E

F

Table 2. ITS comparison among Aster ageratoides var. scaberulus, Kalimeris indica and their putative hybrid.

Positions 43 73 74 117 136 142 179 236 419 488 492 555 583

AST

5

¡¦

.....A.....T

A.............T.............T.............A.............A.............G.............G.............G.............T.............G.............G.............3

¡¦

HYB

5

¡¦

...A & C...T & C A & G.....T & C.....T & A......A & G......A & T......G & T.......G & A......G & C......T & C......G & T......G & T......3

¡¦

KAL

5

¡¦

.....C.....C

G¡K¡K¡KC¡K¡K¡K..A¡K¡K¡K..G¡K¡K¡K..T¡K¡K¡K..T¡K¡K¡K..A¡K¡K¡K..C¡K¡K¡K..C¡K¡K¡K..T¡K¡K¡K..T¡K...3

¡¦

AST: Aster ageratoides var. scaberulus; HYB: The putative hybrid; KAL: Kalimeris indica.

between the F

1

and K. incisa. Moreover, the hybrid chro-

mosome behavior at meiosis confirmed the origin of the

two types of chromosomes. On the other hand, A. ovatus

was verified to be an amphiploid (2n=4x=36=18 L-chro-

mosomes + 18 S-chromosomes), with 18 L-chromosomes

and 18 S-chromosomes derived from Aster and Kalimeris,

respectively (Tara, 1977). Since L-chromosomes are one

of the important traits of Aster and S-chromosomes are a

specific character of Kalimeris (Huziwara, 1957a, b, 1958;

Chen et al., 1992a, b; Soejima and Peng, 1998; Li, 2002,

2003; Li and Zhang, 2004), karyotypical investigation is

useful in testing intergeneric hybridization between Aster

and Kalimeris.

The sequence of the nrDNA of the hybrid should

possess ITS sequences of both parental species, and as

expected, the results (Table 2) showed this to be the case.

Generally, it is expected that concerted evolution in hy-

brids should result in a homogeneous nrDNA with seg-

ments from one or both parents. The fact that concerted

evolution in the hybrid studied had not begun indicates

that this is a recent F1 hybrid.

The taxonomic relationship between Kalimeris

and Aster

Kalimeris, native to East Asia, is a difficult taxon. This

is reflected mainly in its complicated relationship with

Aster. Since the name Kalimeris was first published as a

subgenus of Aster by Cassini in 1822, the species group

has changed many times in its taxonomic position, being

treated as part of Asteromoea Blume, a genus (Calimeris

Ness), a section of Aster L. (sect. Calimeris Hoffmann or

sect. Asteromoea Makino), a section of Boltonia (sect. As-

teromoea Benth. et Hook. F.), or the genus Kalimeris Cass

(see Ling and Chen, 1985; Nesom, 1994, 2000; Ito and

Soejima, 1995; Gu and Hoch, 1997). Most Asian botanists

consider the types of Kalimeris and Asteromoea to be con-

generic (Gu and Hoch, 1997), so Asteromoea is regarded

as a synonym of Kalimeris. Although Boltonia shares

several floral and achene characters with Kalimeris, the

long-discussed possibility of a close relationship between

the New World Boltonia and Old World Kalimeris was re-

jected by Ling and Chen (1985) and Gu and Hoch (1997).

However, the relationship between Kalimeris and Aster

is still controversial. Some botanists treated Kalimeris as

LI ¡X Natural hybridization between

A. ageratoides

var.

scaberulus

and

K. indica

195

a genus (Ling and Chen, 1985; Nesom, 2000), and some

maintained that it should be a section (sect. Callimeris) of

Aster (Ito and Soejima, 1995). Still others thought that its

taxonomic position was pending (Gu and Hoch, 1997).

The primary morphological character that differentiates

Aster from Kalimeris is length of pappus. The pappus of

Kalimeris is less than 2 mm long while that of Aster usu-

ally exceeds 3 mm (Ling and Chen, 1985; Gu and Hoch,

1997). Over-dependence upon the generic diagnostic

character is what has caused taxonomic confusion. Some

species were classified into Kalimeris based on their short

pappus, but other evidence suggested different taxonomic

positions.

Ling and Chen (1985) thought that some species with

a short pappus such as Aster prorerus Hemsl., blur dif-

ferences between Aster and Kalimeris. Aster prorerus

(Kalimeris prorerus (Hemsl.) S. Y. Hu) is controversial

in taxonomic position (Hu, 1967; Ling and Chen, 1985).

Aster giraldii Diels was placed in Aster sect. Calimeris

Ness by Diels in 1905 (see Ling and Chen, 1985). Aster

prorerus and A. giraldii, as well as A. smithianus Hand.-

Mazz., A. dolichopodus Ling, A. hunanensis Hand.-Mazz.,

and A. menelii Levl., were thought to resemble Kalimeris

because all of them have a short pappus (Ling and Chen,

1985). According to the description of Ling and Chen

(1985), these six species are also similar to Kalimeris in

other features. For instance, their heads are terminal and

solitary on leafy peduncles with few to numerous heads

forming a loosely corymboid inflorescence. Wang et al.

(1999) reported that A. procerus and A. smithianus share

the same characters of pollen grains with Kalimeris. Of

course, these six species also possess features of Aster,

hence their placement in Aster (Ling and Chen, 1985).

Based on the fact that these species are similar to Aster in

some characters and to Kalimeris in others, it is necessary

to provide karyotypical and molecular data to determine

their taxonomic positions.

By contrast with pappus, karyotype is more reliable in

discriminating between Aster and Kalimeris. As shown

here, the karyotype is one character that can verify that a

putative hybrid is a true hybrid. Aster ovatus possesses a

mixed LS karyotype (2n=18 long chromosomes + 18 short

chromosomes) and appears to be derived from hybrid-

ization between Aster and Kalimeris (Tara, 1977). Aster

miquelianus Hara was treated as a species of Kalimeris

(namely K. miquelianus) because it has short pappus

britles (Kitamura, 1937; Ling and Chen, 1985). However,

Ito and Soejima (1995) and Gu and Hoch (1997) excluded

it from Kalimeris and left it as part of Aster, a m ov e

which is supported strongly by cytological evidence (Gu,

1989). Gu (1989) reported that 18 chromosomes of A.

miquelianus (2n=2x=18) are much longer than those of

Kalimeris and like those of Aster. Therefore, karyotypi-

cal studies may be particularly useful in clarifying the

taxonomic confusion produced by the six species such as

A. procerus, for which cytological study has not been re-

ported.

Acknowledgments. I thank two anonymous reviewers

for very valuable comments and important improvements

on the manuscript. Support for this research was provided

by grants from the National Natural Science Foundation

of China to Li Weiping (Grant No. 30470131, 39899400).

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