American Journal of Plant Sciences, 2011, 2, 521-526
doi:10.4236/ajps.2011.24061 Published Online October 2011 (http://www.SciRP.org/journal/ajps)
521
Interspecific Hybridization between Arisaema
sikokianum and A. serratum (Araceae) Confirmed
through Nuclear and Chloroplast DNA
Comparisons
Hiroshi Hayakawa1,2, Hidenori Hamachi3, Kanako Matsuyama3, Yuko Muramatsu3, Yukio Minamiya1,
Katsura Ito1, Jun Yokoyama4, Tatsuya Fukuda1*
1Faculty of Agriculture, Kochi University, Monobe, Nankoku 783-8502, Japan; 2The United Graduate School of Agricultural Sci-
ences, Ehime University, Monobe, Nankoku 783-8502, Japan; 3The Graduate School of Integrated Arts and Sciences, Kochi Univer-
sity, Monobe, Nankoku 783-8502, Japan; 4Faculty of Science, Yamagata University, 1-4-12 Kojirakawa-machi, Yamagata 990-8560,
Japan.
Received January 22nd, 2011; revised March 24th, 2011; accepted April 1st, 2011.
ABSTRACT
A morphologically intermediate plant between Arisaema sikokianum Franch. et Sav. and A. serratum (Thunb.) Schott
has been newly found in Kochi Prefecture, Shikoku, Japan. The putative hybrid has the intermediate morphological
characteristics of the parental species. Molecular analysis using PCR-RFLP of internal transcribed spacer (ITS) in
nuclear DNA (nrDNA) indicates that the putative hybrid has a combined pattern of the two putative parent species.
Moreover, the sequence result of chloroplast DNA (cpDNA) of the putative hybrid was identical to that of A. si-
kokianum. These results suggest that the putative hybrid is a hybrid between A. sikokianum and A. serratum and that it
was formed by interactive gene excha nging via pollens from A. serratum to A. sikokianum. It is the first record o f a hy-
brid between A. sikokianum and A. serratum.
Keywords: Araceae, Arisaema, A. serratum, A. sikokianum, Chloroplast DNA, Interspecific Hybrid, Molecular Analysis,
Nuclear DNA
1. Introduction
The genus Arisaema Martius (Araceae), which has a
large, often colored and conspicuous bract (spathe), and
subtending and enveloping bisexual or unisexual spadix
with numerous small flowers, comprises approximately
40 - 85 species in Japan [1,2]. Species of Arisaema in the
section Pedatisecta Schott ex Engler have a slender ap-
pendage at the base and are mostly distributed in Japan
[2]. Section Pedatisecta is included in 35 - 80 species in
Japan [2], and presents many taxonomic difficulties
caused by the concentration of closely related species
with few morphological differences [3].
Sixteen patterns of putative natural hybrids have been
reported in Arisaema sect. Pedatisecta (e.g., [4]). Of
them, A. sikokianum Franch. et Sav. and A. tosaense Ma-
kino make the hybrids [5,6]. Moreover, A. ehimense J.
Murata et Ohno is of hybrid origin between A. serratum
(Thunb.) Schott and A. tosaense based on allozyme
analysis [7]. Therefore, it is possible that hybrid speci-
ation or reticulate evolution or both has occurred among
A. tosaense, A. sikokianum and A. serratum. However,
the hybridization between A. sikokianum and A. serratum
was unknown until now.
Arisaema sikokianum has a purple upward spathe, a
white capitate appendage and leaves with 3 - 5 leaflets
(Figure 1(a), Table 1), while Arisaema serratum has a
green cylindrical appendage and leaves with 7 - 17 leaflets
(Figure 1(b), Table 1). Although the spathe of A. serra-
tum varies widely in various areas of Japan [2,3], our ob-
servation is that almost all A. serratum in Kochi Prefecture
show a green spathe and appendage (Figure 1(b)). In Ko-
chi Prefecture of Shikoku, A. sikokianum and A. serratum
are found in sympatry. The flowering phenology of the
two species overlaps (Table 1). The chromosome num-

Interspecific Hybridization between Arisaema sikokianum and A. serratum (Araceae) Confirmed through Nuclear
522
and Chloroplast DNA Comparisons
Figure 1. Arisaema species examined in this study. (a) A.
sikokianum; (b) A. serratum; (c) putative hybrid in 2007; (d)
putative hybrid in 2006; (e) putative hybrid in 2010; (f)
newly formed putative hybrids in 2010.
ber of A. sikokianu m and A. serratum is 2 n = 28 in both
species [8]. Therefore, it is possible to generate a hybrid
between A. sikokianum and A. serratum.
Molecular approaches can reveal the processes of past
event such as hybridization and reticulate evolution [9].
Nuclear markers provide information to estimate the pu-
tative parents in hybrids [10,11]. The polymorphisms of
the internal transcribed spacer (ITS) region in nuclear
DNA (nrDNA) are good tools for clarifying the relation-
ship of closely related taxa in many plant groups [12-14],
and can provide evidence of hybridization. In fact, inter-
specific hybrids are most commonly identified by the
heterogeneity of nrDNA [5,15,16]. Moreover, chloroplast
DNA (cpDNA) data can indicate maternal and paternal
parents of hybrids, because cpDNA normally inherit
from maternal parent [17]. In this study, we found a pu-
tative natural hybrid of A. sikokianum and A. serratum in
Kochi Prefecture from the viewpoint of its morphological
characteristics (Figure 1(d), Figure 2). To clarify the
maternal and paternal parents of the putative hybrid, we
conducted molecular analysis using nrDNA and cpDNA
sequences. Our finding suggests that hybridization may
occur between A. sikokianum and A. serratum.
2. Materials and Methods
A morphologically intermediate plant was found at one
locality in Kochi Prefecture (Figure 2, Table 2). The
intermediate hybrid was growing in situ with Arisaema
sikokianum, A. serratum and A. tosaense. A voucher
specimen is deposited in the Herbarium of the Makino
Botanical Garden, Kochi (MBK).
For the molecular analysis, total DNA was isolated
from 200 - 300 mg of leaves with a Plant Genomic DNA
Mini Kit (VIOGENE, Sunnyvale, CA, USA), according
to the manufacturer’s protocol. We amplified the internal
transcribed spacer (ITS) region from nrDNA and the trnL
intron from cpDNA with primers designed by Taberlet et
al. [18] and White et al. [19], respectively. Isolated DNA
was amplified by PCR in a 50 µl reaction solution con-
taining 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 (Ta Ka Ra) and
0.5 µM of each primer. We used the following thermal
cycle profile for amplification: 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. A preliminary sequence
of the ITS region was taken from the parent species to
confirm whether PCR-RFLP can effectively demonstrate
Table 1. Morphological characteristics of samples used in this study.
Trait A. sikokianum Putative Hybrid A. serratum
Pseudostem
Pseudostem Length Short Short-Long Long
Pseudostem Color Green Purplish Dark Brown Purplish Dark Brown
Leaf Characteristics
Leaflets 3 to 5 9 7 to 17
Width of Leaflets Wide Narrow-Wide Narrow
Petiole Length Long Short-Long Short
Reproductive Characteristics
Spathe Tip Direction Upward Middle Downward
Spathe Color Purple Purple Green
Appendage Shape Capitate Capitate-Cylindrical Cylindrical
Appendage Color White White Green
Flowering Phenology April to May Early April Late April to June

Interspecific Hybridization between Arisaema sikokianum and A. serratum (Araceae) Confirmed through Nuclear 523
and Chloroplast DNA Comparisons
Figure 2. Sampling locality of the putative hybrid of Ari-
saema sikokianum and A. serratum. Black squar es indicate
A. sikokianum. Black triangles indicate A. serratum. Open
circle indicates putative hybr id.
hybridization (Figure 3). After amplification, PCR pro-
ducts of the ITS region as well as the trnL intron were
subjected to electrophoresis in 1% low-melting-tem-
perature 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, Foster City, CA, USA) and ABI Prism 3100
Genetic Analyzer (Applied BioSystems, Foster City, CA,
USA) according to the manufacturer’s instructions. For
sequencing, we used the same primers as those used for
amplification.
PCR-RFLP (restriction fragment length polymorphism)
analysis for the ITS regions of 18 - 26 S nuclear ribo-
somal DNA were conducted to clarify the hybrid nature
of the putative hybrid after checking the sequencing re-
sults and alignments. We used 2 Mse I sites (TTAA) as
an autapomorphic character of nrDNA (Figure 3). The
ITS region of A. sikokianum has one Mse I site in ITS 1,
while A. serratum has two sites (ITS 1 and ITS 2) di-
gested by the restriction enzyme. After the designation of
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 there was one putative hybrid in the locality. As for
the morphological characteristics, the putative hybrid has a
purple spathe and white appendage (Figures 1(c), (e), (f)),
which is similar to that of Arisaema sikokianum, but the
leaves have nine leaflets, which is similar to that of A.
serratum (Figures 1(c), (d), (f); Table 1). Additionally,
two clones of the putative hybrid were newly formed from
a corm of the original putative hybrid in cultivation (Fig-
ure 1(f)). Although A. sikokianum can not grow lateral
buds on its corm [20], A. serratum can grow lateral buds
and become new clones [3,21]. Therefore, the clones gen-
erated by germinated lateral buds in the hybrid may inherit
the traits of A. serratum.
In this study, the hybrid of Arisaema sikokianum and A.
serratum showed an intermediate appendage compared
with the parents in 2007 (Figure 1(c)). The shape of the
appendage of the hybrid in this study transformed from
intermediate (in 2007) to cylindrical (for 2008-2010) un-
der a cultivated condition (Figures 1(c), (e)). Moreover,
the pseudostem length also showed the same phenomenon
as the appendage: the hybrid was short in 2006 (Figure
1(d)), which is similar to A. sikokianum, but it became
long for 2007 to 2010, which is similar to A. serratum. It is
curious that the shape of the appendage and the pseu-
dostem length transformed in different years. In the future
to understand this transformation, an analysis of the genes
involved in the appendage and the pseudostem is needed.
In the Arisaema sect. Pedatisecta, there are some nu-
cleotide polymorphisms in the ITS sequences, e.g., A.
angustatum, A. ringens, A. serratum. and A. sikokianum
(Accession numbers: AF291914, AF274295, EF017383,
and AB513178, respectively). In this study, the length of
the ITS sequence of nrDNA is 648 bp in Arisaema ser-
Table 2. Locality where samples were collected.
No. Species Locality Collector Collected Date
1 Arisaema sikokianum Kochi Pref. Aki City, Ochial, Suginokuma RiverH. Hayakawa 2009-7-4
2 A. sikokianum Kochi Pref. Nankoku City, Nareai, Nebiki PassH. Hayakawa 2009-5-1
3 Putative Hybrid Kochi Pref. Aki City, Doi H. Hamachi 2006-5-25
4 A. serratum Kochi Pref. Aki City, Ochial, Suginokuma RiverH. Hayakawa 2009-7-4
5 A. serratum Kochi Pref. Nankoku City, Nareai, Nebiki PassH. Hayakawa 2009-6-8
S: Arisaema sikokianum type. Accession numbers: AB513178 (ITS) and AB513176 (trnL intron). E: A. serratum type. Accession numbers: AB605025 (ITS)
and AB605026 (trnL intron).

Interspecific Hybridization between Arisaema sikokianum and A. serratum (Araceae) Confirmed through Nuclear
524
and Chloroplast DNA Comparisons
M
M
M
M
A. sikokian um
A.serratum
A. sikokian um
A. serratum
A. sikokian um
A. serratum
A. sikokian um
A. serratum
A. sikokian um
A. serratum
A. sikokian um
A. serratum
A. sikokian um
A. serratum
A. sikokian um
A. serratum
A. sikokian um
A. serrarum
A. sikokian um
A. serratum
A. sikokian um
A. serratum
Figure 3. Expected restriction sites of Mse I for molecular characteristics of ITS regions by PCR-RFLP. M: restriction site.
ratum (Accession number: AB605025). This sequence is
the same as A. tosaense (Accession number: AB513179).
Therefore, we conducted PCR-RFLP in the nrDNA to
obtain further evidence of the hybrid nature, because the
ITS region of A. sikokianum has one Mse I site, while A.
serratum and A. tosaense have two sites digested by this
restriction enzyme (Figure 3) [5]. The digestion patterns
of all samples of A. sikokianum and A. serratum showed
expected patterns and the putative hybrid showed the
combined pattern of A. sikokianum and A. serratum
(Figure 4). Thus, from the evidence of morphological
and molecular analyses, we confirmed that the putative
hybrid was truly a hybrid of A. sikokianum and A. serra-
tum.
The length of the trnL intron of cpDNA is 467 bp in
Arisaema serratum (Accession number: AB605026).
This region is a good molecular marker for distinguish-
ing Arisaema sikokianum, because the trnL intron has a
17 bp-insertion or deletion (indel) in the sequence of A.
sikokianum (450 bp) (Figure 5) [5]. We therefore deter-
mined the sequences of the trnL intron. In the hybrid, the
sequence result from the cpDNA analysis was identical
to that of A. sikokianum (Table 1). Therefore, the hybrid
Figure 4. PCR-RFLP profile of Arisaema sikokianum, A.
serratum and putative hybrid. Arrowheads indicate ex-
pected fragments of both A. sikokianum and A. serratum. M:
size marker. The cpDNA types correspond to the types in
Table 2 and Figure 5.
transferred pollen from A. serratum to A. sikokianum.
In Shikoku, Arisaema ehimense J. Murata et Ohno is
of hybrid origin between A. serratum and A. tosaense
[7,22]. Additionally, the hybrids between A. sikokianum
and A. tosaense are known [5,6]. Moreover, this study
eveals a hybrid between A. sikokianum and A. serratum. r

Interspecific Hybridization between Arisaema sikokianum and A. serratum (Araceae) Confirmed through Nuclear 525
and Chloroplast DNA Comparisons
A. sikokia nu m
A. serratum
A. sikokia nu m
A. serratum
A. sikokia nu m
A. serratum
A. sikokia nu m
A. serratum
A. sikokia nu m
A. serratum
A. sikokia nu m
A. serratum
A. sikokia nu m
A. serratum
Figure 5. The result of alignment of trnL intron sequences of Arisaema sikokianum and A. serratum.
To our knowledge, it is first report of a hybrid between
this Arisaema pair. These facts indicate that all the com-
bination patterns of hybridization among A. tosaense, A.
sikokianum and A. serratum have been in Shikoku.
Therefore, these facts suggest that hybrid speciation or
reticulated evolution or both might have occurred among
the three species in Shikoku.
4. Acknowledgements
We wish to thank N. Tanaka, the curator of the MBK
herbarium, for allowing us to examine specimens of Ari-
saema, and R. Arakawa, M. Saito, K. Ohga and N. Yo-
koyama for providing much help. I would also like to
thank Dennis Murphy from the United Graduate School
of Agricultural Sciences, Ehime University, for checking
the English in this manuscript. This study was partly sup-
ported by a Grant-in-Aid for Scientific Research from the
Ministry of Education, Science and Culture of Japan (to
TF and JY).
5. Conclusions
In Figure 3 is reported the shifting of the three switching
frequencies and also in this case it is evident.
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