Introgressive Hybrids of <i>Arisaema sikokianum</i> and <i>A. tosaense</i> (Araceae) Confirmed through Nuclear and Chloroplast DNA Comparisons

doi:10.4236/ajps.2011.23034

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American Journal of Plant Sciences, 2011, 2, 303-307

doi:10.4236/ajps.2011.23034 Published Online September 2011 (http://www.SciRP.org/journal/ajps)

303

Introgressive Hybrids of Arisaema sikokianum and

A. tosaense (Araceae) Confirmed through Nuclear

and Chloroplast DNA Comparisons

Hiroshi Hayakawa1,2, Hidenori Hamachi1, Kanako Matsuyama3, Yuko Muramatsu3, Yukio Minamiya1,

Katsura Ito1, Jun Yokoyama4, Tatsuya Fukuda1*

1Faculty of Agriculture, Kochi University, Nankoku, Japan; 2United Graduate School of Agricultural Sciences, Ehime University,

Nankoku, Japan; 3The Graduate School of Integrated Arts and Sciences, Kochi University, Nankoku, Japan; 4Faculty of Science,

Yamagata University, Yamagata, Japan.

Email: *[email protected]

Received January 22nd, 2011; revised March 24th, 2011; accepted April 1st, 2011.

ABSTRACT

Morphologically putative introgressive hybrids of Arisaema sikokianum Franch. et Sav. and A. tosaense Makino were

newly found in Kochi and Tokush ima Prefectures in Japan. All the individ uals have the same morph ological char acter-

istics as A. tosaense excluding a purple spathe. Molecular analysis using PCR-RFLP of internal transcribed spacer

(ITS) in nuclear DNA (nrDNA) indicates that these putative introgressive hybrids have the same pa ttern as A. tosaense.

Moreover, the sequences of chloroplast DNA (cpDNA) of the putative introgressive hybrids were identical to A. si-

kokianum from Kochi Prefecture and A. tosaense from Tokushima Prefecture. The results suggest that the plants are

introgressive hybrids of A. sikokian um and A. tosaense and that they ha ve highly exchanged genes with A. tosaense.

Keywords: Araceae, Arisaema, A. sikokianum, A. tosaense, Chloroplast Capture, Introgression, ITS, Molecular

Analysis, PCR-RFLP, Trn

1. Introduction

Studies of natural hybridization and introgression and

their genetic composition can shed new light on issues

concerning reproductive barriers and fitness of them and

give important insights into evolutionary processes and

the adaptation of species [1].

The genus Arisaema Martius (Araceae), which has a

large, often colored and conspicuous bract (spathe), sub-

tending and enveloping a bisexual or unisexual spadix

with numerous small flowers, comprises 40 - 85 species

in Japan [2,3]. Species of Arisaema in the section Pistil-

lata Nakai have a slender appendage at the base and are

mostly distributed in Japan [3]. Section Pistillata is in-

cluded in 35 - 80 species in Japan [3], and presents many

taxonomic difficulties caused by the concentration of

closely related species with few morphological differ-

ences [4].

Sixteen patterns of putative natural hybrids among the

species in section Pistillata have been reported in Ari-

saema (e.g., [5]). Of them, the hybrids between Arisaema

sikokianum Franch. et Sav. and Arisaema tosaense Maki-

no grow only in Kochi Prefecture in Japan [2,6]. How-

ever, Hayakawa et al. [7] newly found hybrids of these

two species in Tokushima Prefecture and, using molecu-

lar analysis, revealed that the hybrids occur bidirection-

ally and that they have exchanged genes with the two

parental species. A. sikokianum has a purple upward sp a-

the, a white capitate appendage and leaves with 3 to 5

leaflets (Figure 1(a), See detail Table 1). A. tosa en se has

a characteristic green spathe extending approx. 30 - 45

cm before it bends downward to the ground and has

leaves with 7 to 11 leaflets (Figure 1(b), Table 1 ). These

species have the same chromosome numbers (2n = 28)

[8,9]. Sympatry of the two species extends over a wide

area in Shikoku, and the hybrids of them share various

morphological characters [7,8]. Therefore, it is possible

that the parental species could make the hybrid progenies

and introduce gene(s) in each other through chloroplast

captures, backcrossing and recombination, but introgres-

sive hybrids including near isogenic lines (NILs) and

recombinant lines (RLs) have not been made so far.

To clarify chloroplast captures of the introgressive hy-

brids of Arisaema sikokianum and A. tosaense, we con-

Introgressive Hybrids of Arisaema sikokianum and A. tosaense (Araceae)

304 Confirmed through Nuclear and Chloroplast DNA Comparisons

ducted molecular analysis using nuclear DNA (nrDNA)

and chloroplast DNA (cpDNA) sequences. Our findings

regarding the introgressive hybrids suggest that chloro-

plast captures and gene(s) flow may occur in A. si-

kokianum and A. tosaense.

2. Materials and Methods

Morphologically, the putative introgressive hybrids of

Arisaema sikokianum and A. tosaense were found at two

localities in Kochi and Tokushima Prefectures (Figure 2,

Table 2). Although hybrids of A. sikokianum and A. to-

saense were found with both parental species [7,8], all

the putative introgressive hybrids were found with A.

tosaense (Figure 2(I) and (II)). Voucher specimens of

the introgressive hybrids are deposited in the herbarium

of the Makino Botanical Garden, Kochi (MBK).

Figure 1. Species of Arisaema examined in this study. (a) A. sikokianum; (b) A. tosaense; (c)-(f) putative introgressive hybrids

along Koishikawa River; (e) introgressive hybrid with spathe tip raised to show appendage (same plant shown in (d)).

Table 1. Morphological characteristics of samples used in this study.

Arisaema Putative Introgressive Hybrid Arisaema

Trait sikokianum I II tosaense

Leaf Characteristics

Leaflets 3 to 5 9 to 15 over 7 7 to 11

Reproductive Characteristics

Spathe Tip Long Very Long Very Long Very Long

Spathe Tip Direction Upward Drooping Drooping Drooping

Spathe Color Purple Purple Purple Green

Appendage Shape Capitate Cylindrical Cylindrical Cylindrical

Appendage Color White Green Green Green

Flowering Phenology April to May Early June Early June Late May to June

Introgressive Hybrids of Arisaema sikokianum and A. tosaense (Araceae) 305

Confirmed through Nuclear and Chloroplast DNA Comparisons

Figure 2. Sampling localities of putative introgressive hybrids of Arisaema sikokianum and A. tosaense. Blacked squires, tri-

angles and circles indicate A. sikokianum, A. tosaense and putative introgressive hybrids, respectively. (I) Kochi Pref., Aki-

gun Umaji-mura, Yanase, along Koishikaw a River ; (II) Tokushima P re f., Kaifu-gun Kaiyo-cho, Ogawa, Kirikoshi Pass.

Table 2. Locality where samples were collected.

No. Species Locality Collector Date

1 Arisaema sikokianum Tokushima Pref. Naka-Gun, Naka-Cho, Senbondani Valley HH, MuY, MiY 2009-7-2

2 Kochi Pref. Nankoku City, Nareai, Nebiki Pass HH 2009-5-1

3 Putative Introgressive Hybrid I Kochi Pref. Aki- G un, Umaji-Mura, Yanase, Koishikawa Valley HH, FT 2010-6-4

4 Kochi Pref. Aki-Gun, Umaji-Mura, Yanase, Koishikawa Valley HH, FT 2010-6-4

5 II Tokushima Pref. Kaifu-gun, Kaiyo-Cho, Ogawa, Kirikoshi PassHH, FT 201 0 -6-4

6 Tokushim a Pref. Kaifu-gun, K ai yo-Cho, Ogawa, Kirikoshi Pa s sHH, FT 2010-6-4

7 A. tosaense Tokushima Pref. Naka-Gun, Naka-Cho, Senbondani Valley HH, MuY, MiY 2009-7-2

8 Kochi Pref. Nankoku City, Nareai, Nebiki Pass HH 2009-6-8

HH: Hayakawa Hiroshi; MuY: Muramatsu Yuk o; MiY: Minamiya Yukio; FT: Fukuda Tatsuya.

For the molecular analysis, total DNA was isolated

from 200 - 300 mg of leaves using a Plant Genomic

DNA Mini Kit (VIOGENE, Sunnyvale, USA), according

to the manufacturer’s protocol. We amplified the internal

transcribed sp acer (I TS) region fro m nrDNA and the trnL

intron from cpDNA with primers designed by White et al.

[10] and Taberlet et al. [11], respectively. The isolated

DNA was amplified by PCR in a 50 µl reaction solution

containing approximately 50 ng total DNA, 10 mM

Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.2 mM

of each dNTP, 1.25 units Taq DNA polymerase (TaKaRa)

and 0.5 µM of each primer. We used the following ther-

mal cycle profile for amplification by the PCR Thermal

Cycler Dice (TaKaRa): 1 min at 94˚C, 2 min at 48˚C, and

2 min at 72˚C for 45 cycles, followed by 15 min of final

extension at 72˚C. After amplification, the PCR products

of the ITS region as well as the trnL intron were sub-

jected to electrophoresis in 1% low-melting-temperature

agarose gels to remove by-products and purify amplified

products. We sequenced the purified PCR products using

a BigDye Terminator ver. 3.1 (Applied BioSystems) and

ABI Prism 3100 Genetic Analyzer (Applied BioSystems)

according to the manufacturer’s instructions. For se-

quencing, we used the same primers as those used for

amplification.

For the ITS region, we carried out PCR-RFLP (restric-

Introgressive Hybrids of Arisaema sikokianum and A. tosaense (Araceae)

306 Confirmed through Nuclear and Chloroplast DNA Comparisons

tion fragment length polymorphism) analysis, because an

autapomorphic character of the nrDNA is the restriction

site Mse I (TTAA) [7]. After designating the restriction

sites, the amplified products were digested by Mse I at

37˚C for more than an hour. The digested DNAs were

separated on 1.5% agarose gel and the size of each band

was determined.

3. Results and Discussion

From the results of the molecular analysis we determined

that five putative introgressive hybrids occurred at each

locality. The morphological analysis showed that all the

putative introgressive hybrids have a purple spathe (Fig-

ures 1(c)-(f)) which is similar to Arisaema sikokianum,

but have a green cylindrical appendage as an elongate

spathe tip and a large number of leaflets, which is similar

to A. tosaense (Figure 1, Table 1). The putative intro-

gressive hybrids seem to have the same characteristics as

A. tosaense excluding the color of the spathe, and share

the same morphological features.

We conducted PCR-RFLP in nrDNA because the ITS

region of Arisaema sikokianu m has one Mse I site, while

A. tosaense has two sites digested by this restriction en-

zyme [7]. The digestion patterns of all samples of A. si-

kokianum and A. tosaense showed expected patterns and

all the putative introgressive hybrids showed the same

patterns as those of A. tosaense (Figure 3). We therefore

confirmed that the putative introgressive hybrids have an

A. tosaense type of ITS in nrDNA.

The trnL intron region in cpDNA is a good molecular

marker for distinguishing Arisaema sikokianum, because

the trnL intron h as a 17 bp -insertion or deletio n (indel) in

the sequence of A. sikokianum (450 bp) and A. tosaense

(467 bp) [7]. We therefore determined the sequences of

the trnL intron. In the introgressive hybrids, the sequence

results from the cpDNA analysis were identical to the A.

sikokianum from Kochi Prefecture and to A. tosaense

from Tokushima Prefecture (Table 3), and we could not

find any different pattern of cpDNA for the introgressive

hybrids in any of the two localities.

From the results of morphological and molecular

analyses using nrDNA and cpDNA, the introgressive

hybrids from Kochi Prefecture can be determined but no t

from Tokushima Prefecture. The flowering phenology of

the hybrids between Arisaema sikokianum and A. to-

saense was inherited from A. tosaense [7]. Ther efore, the

flowering phenology of hybrid progenies may overlap

with that of A. tosaense and would intr oduce A. tosaense

genes. In this study, although all the putative introgres-

sive hybrids in Tokushima Prefecture had a purple spathe

and had an outward appearance of A. tosaense, we could

not detect any molecular evidence using the ITS region.

Figure 3. PCR-RFLP profile of Arisaema sikokianum, A. to-

saense and putative introgressive hybrids. Arrows indicate

expected fragments of both A. sikokianum and A. tosaense.

M: size marker. The cpDNA types correspond to the types

in Table 3.

Table 3. Genotype of samples used in this study.

Species Locality

nrDNA

(ITS) cpDNA

(trnL intron)

Arisaema sikokianum S S

Putative Introgressive

Hybrid IKoishikawa T S

Putative Introgressive

Hybrid IIKirikoshi pass T T

A. tosaense T T

S: Arisaema sikokianum type. Accession numbers; AB513178 (ITS) and

AB513176 (trnL intron). T: A. tosaense type. Accession numbers; AB513179

(ITS) and AB513177 (trnL intron).

Additional nrDNA sequences such as TPI and PGI may

provide the answers about the putative introgressive hy-

brids in Tokushima Prefecture.

From our results and previous reports [6-8], the hy-

bridization and introgressive hybrids of Arisaema si-

kokianum and A. tosaense seem to be widely distributed

in eastern Shikoku. In some limited areas of Shikoku and

its margin areas, the appearance of A. tosaense is varied,

not only in spathe color but also the shape of the leaflets

(wide to narrow) and the shape of the appendage (cylin-

drical to capitate) ([12] and Hayakawa unpubl.), imply-

ing that various characteristics are generated in intro-

gressive hybrids. More detailed investigations around

eastern Shikoku will be needed because it may reveal

new localities of introgressive hybrids carrying different

characteristics to those in this study. Moreover, in Shi-

zuoka Prefecture, Kakishima et al. [13] indicated that

introgressive gene flow occurs from A. angutatum Fran-

ch et Sav. to A. suwoense Nakai. Those reports, along with

our findings, suggest that endemic species might have

occurred through introgressive h ybrids in some Arisaema

Introgressive Hybrids of Arisaema sikokianum and A. tosaense (Araceae)

Confirmed through Nuclear and Chloroplast DNA Comparisons

307

sect. Pistillata.

4. Acknowledgements

We wish to thank N. Tanaka, curator of the MBK her-

barium, for allowing us to examine specimens of Ari-

saema, and R. Arakawa, A. Hirata, M. Saito, K. Ohga,

and N. Yokoyama for providing additional help. I would

also like to thank Dennis Murphy from the United

Graduate School of Agricultural Sciences, Ehime Uni-

versity, for checking the English in th is manuscript. This

study was partly supported by a Grant-in-Aid for Scien-

tific Research from the Ministry of Education, Science

and Culture of Japan (to T.F. and J.Y.).

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