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.
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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