American Journal of Plant Sciences, 2011, 2, 781-789
doi :1 0.4236/ aj ps.2011 .26093 Publ i s hed Online December 2011 (http://www.SciRP.org/journal/ajps)
781
Phylogeography of Asparagus schoberioides Kunth
(Asparagaceae) in Japan
Tatsuya Fukuda1*, In-Ja Song2, Hokuto Nakayama3, Takuro Ito4, Akira Kanno5, Hiroshi Hayakawa6,
Yukio Minamiya6, Jun Yokoyama7
1Faculty of Agriculture, Kochi University, Nankoku, Japan; 2Subtropical Horticulture Research Institute, Jeju National University,
Jeju, Ko rea; 3Graduate School of Science, University of Tokyo, Tokyo, Japan; 4Institute for Advanced Biosciences, Keio Un iversity,
Tsuruoka, Japan; 5Graduate School of Life Sciences, Tohoku University, Sendai, Japan; 6United Graduate School of Agricultural
Sciences, Ehime University, Monobe, Japan; 7Department of Biology, Faculity of Science, Yamagata University, Yamagata, Ja-
pan.
Received April 8th, 2011; revised May 3rd, 2011; accepted May 24th, 2011.
ABSTRACT
To describe the phylogeographic structures of Asparagus schoberioides Kunth (Asparagaceae) in Japan, we investi-
gated its nucleotide sequence variations with respect to its geographic distribution pattern. Sequencing of the internal
transcribed spacer (ITS) 1 region in 29 samples of A. schoberioides revealed 20 polymorphic nucleotide sites. As a re-
sult, the 29 samples of A. schoberioides fell in to 15 distinct haplotypes and phylogenetic analyses revealed these haplo-
types fell into two major clad es, Clade 1 and Clade 2. The haplotypes of Clade 1 were distributed chiefly along the Pa-
cific Ocean side of Japan, while those of Clade 2 occurred mainly along the Japan Sea side. This result suggests that A.
schoberioides has migrated via two routes in Japan.
Keywords: Asparagus schoberioides, Internal Transcribed Spacer (ITS), Phyloge ny, Phylogeography
1. Introduction
The genetic and geographic structures of natural plant
populations are a consequence of ecological factors and
historical events. Consequently, the amount of genetic
variation within a species depends on its history and life
strategy. Comparison of nucleotide sequences are the
best way to analyze the history of plant populations, and
phylogeographic studies employing nuclear DNA data
have been used to test ever more sophisticated historical
models [1-3]. In particular, nucleotide divergences of the
nuclear DNA (nrDNA) showed relative higher values
than those of chloroplast DNA (cpDNA) [4], although
recombination of nrDNA is biased the concepts of ho-
mology [5].
Asparagus schoberioides Kunth is a perennial herb
bearing small flowers with yellowish green perianth in a
raceme-like inflorescence and reddish colored berries. This
species ranged from Far East Russia, northern China,
Korea, Sakhalin, Japan and Taiwan [6,7]. In Japan, its
distribution extends from the northern-most part of Hok-
kaido down to Shikoku and Kyushu [6]. A. schoberioides
is one of the most closely related species to the garden
asparagus, A. officinalis L. [8], which is the most eco-
nomically important species in this genus. While the gar-
den asparagus is not nati ve to Jap an, it is cultivated t here
and has escaped from the crop fields [6]. Recently,
Ochiai et al. [9] and Ito et al. [10] reported successfully
generating of interspecific hybrids between A. schobe-
rioides and the garden asparagus. A natural hybrid be-
tween A. schoberioides and the garden asparagus has not
been found to date, but the possibility of genetic intro-
gression from crop to wild relatives suggests the escape
of the garden asparagus from the crop fields may pose a
substantial ecological risk for the environment and its
biodiversity [2,11-16].
In Japan, molecular approaches have been used to ana-
lyze the intraspecific genetic variation of a number of
different plant species (e.g., Abies mariesii [17]; Aucuba
chinensis and A. japonica [18]; Fagus crenata [19-21];
Purimula cunefolia [22]; Quercus serrata and its allied
species [23]; Stachyurus praecox [24], Alpinia japonica,
Arachnioides sporadosora, A. aristata, Daphne kiusiana,
Elaeocarpus sylvestris var. ellip ticus, Prunus zippeliana
[25]). Most of these studies were conducted based on the
chloroplast DNA (cpDNA) sequence. Similarly, in our

Phylogeography of Asparagus schoberioides Kunth (Asparagaceae) in Japan
782
previous study, we used primers designed by Taberlet et
al. [26] and Nishizawa and Watano [27] to sequence
approximately 3000 bp of 16 regions in the cpDNA of
some Asparagus species [8]. However, we detected little
variation in these regions a nd concluded that the cpDN A
is not suitable for phylogenetic analyses of this species.
The internal transcribed spacer (ITS) region is a power-
ful tool for resolving historical relationships among po-
pulations of widespread plants [28]. In fact, many re-
searches with various species using ITS region had been
reported in recent years [29-32]. For example, Yoko-
yama et al. [33] analyzed the ITS region of Mitchella
undulata Sieblod et Zucc. in Yakushima Island, they
detected intraspecific variations that suggested this popu-
lation had undergone rapid morphological modification.
Thus, analysis of this region may permit plant relation-
ships to be evaluated down to the intraspecific level.
The aims of this study were to describe the phylo-
geographic structures of A. schoberioides especially in
Japan on the basis of its ITS region and to analyze the
distribution of t hese genetic variations relative to the di s-
tribution of A. schoberioides.
2. Materials and Methods
2.1. Plant Materials
Twenty-nine samples of Asparagus schoberioides were
examined in this study (Table 1). A. kiusianus Makino
and A. officina lis were selected as outgroups on the basis
of phylogenetic analyses of the genus Asparagus [8].
Vouchers for all species sa mpled in this study have been
deposited in the Herbarium, Graduate School of Science,
Tohoku University (TUS) and the Herbarium of Tsumura
Laboratory (THS).
2.2. DNA Extraction, Amplification, and
Sequencing
Total DNAs were isolated from 200 - 300 mg of phyllo-
clades with a Plant Genomic DNA Mini Kit (VIOGENE,
Sunnyvale, USA) used according to the manufacturers’
protocols. The isolated DNA was resuspended in TE and
stored at –20˚C until use.
For phylogenetic analysis, we amplified the ITS1 re-
gion with primers designed by White et al. [34]. Dou-
ble-stranded DNA was amplified by incubation at 94˚C
for 2 min followed by 40 cycles of incubation at 94˚C for
1.5 min, 48˚C for 2 min, and 72˚C for 3 min, wit h a fi nal
extension at 72˚C for 15 min. After the amplification,
reaction mixtures were subjected to electrophoresis in
1% low-melting-temperature agarose gels to purify of the
amplified products. We sequenced the purified PCR pro-
ducts using a DYEnamic ET-terminator Cycle Sequenc-
ing Kit (Amersham Pharmacia) and a Model 373A auto-
mated sequencer (Applied BioSystems) according to the
manufacturers’ instructions. For sequencing, we used the
same primers as those used for amplification.
2.3. Data Analysis
ITS region sequences were aligned with the CLUSTAL
X program [35]. Phylogenetic analysis and a test of clade
support were conducted using the PAUP* program (ver-
sion 4.0b10; [36]). Maximum parsimony analyses were
carried out via a heuristic search with TBR branch swap-
ping and MULPERS optio n. Multiple islands of the most
parsimonious trees [37] were identified using the heuris-
tic option with 100 random sequence additions. To esti-
mate confidence levels of monophyletic groups, the
bootstrap method with 1000 replications were employed
[38].
3. Results
We determined the ITS1 region sequence in 29 samples
of Asparagus schoberioides and two outgroup species,
namely A. kiusianus and A. officinalis. The ITS1 region
of all A. schoberioides plants was 249 bp in length.
Variable sites in this region are shown in Table 2. Fif-
teen ITS1 haplotypes were obtained from A. schoberi-
oides, and the sequence of each haplotype has been de-
posited in the DDBJ/EMBL/GenBank international DNA
data bank (Table 1). There are no indels among the ITS
sequences of A. schoberioides and its allied species.
When we used the ITS1 sequence data set in phyloge-
netic analyses, we obtained 78 most parsimonious trees
of the 28 steps with a consistency index (CI) of 0.71 and
a retention index (RI) of 0.92. One of the most parsimo-
nious trees is shown in Figure 1.
In our phylogenetic tree, all A. schoberioides samples
formed the monophyletic group and two clades (Clade 1
and Clade 2 hereafter) in A. schoberioides were recog-
nized. The relationship between the clades and the lo-
calities of the individuals is indicated in Table 1. The
correspondence between haplotypes and samples is
shown in Figure 2. The geographic distribution of the
samples is shown in Figure 3. Clade 1 consists of the
following fourteen samples: China, South Korea, Hok-
kaido1, Hokkaido3, Hokkaido4, Hokkaido5, Hokkaido6,
Miyagi1, Miyagi2, Saitama, Yamanashi, Nagano1, Na-
gano2 and Shizuoka. Clade 2 contains the following fif-
teen samples: Russia, Hokkaido2, Iwate, Niigata1, Nii
gata2, Niigata3, Nagano3, Kyoto1, Kyoto2, Kyoto3,
Hyogo, Shimane, Yamaguchi, Fukuoka and Nagasaki.
Thus, in our phylogenetic analysis, the samples from
Hokkaido and Nagano extend into the two lineages that
form Clades 1 and 2 (Figures 1, 3). Apart from these
samples, Clade 1 consists of samples from the Pacific

Phylogeography of Asparagus schoberioides Kunth (Asparagaceae ) in Japan 783
Table 1. List of taxa, sources, haplotypes and accession numbers of plant materials.
Taxon Sample name Locality Collector HaplotypeAcc. no.
Asparagus
scho berioides Ku nth Russia RUSSIA: Sakhalin, Dovie-alexandrowsk Kudo 25406 K AB196766
China CHINA: Liaoning, Dalian, Lushun Suzuki s.n . D AB196739
SouthKorea SOUTH KOREA: Junranam, Gwangyang, OkryongIm 21864 E AB196767
Hokkaido1 JAPAN: Hokkaido, Kamikawa, Toma Deguchi 8326 A AB196744
Hokkaido2 JAPAN: Hokkaido, Oshima, Matsumae Kudo 25410 F AB196741
Hokkaido3 JAPAN: Hokkaido, Iburi, Tomakomai Kudo 25407 A AB196745
Hokkaido4 JAPAN: Hokkaido, Okushiri Isl. Mt. Kamui Kudo 25411 B AB196743
Hokkaido5 JAPAN: Hokkaido, Abashiri, Abashiri Kudo 25409 A AB196742
Hokkaido6 JAPAN: Hokkaido, Hidaka, Samani Yamaji s.n. A AB196760
Iwate JAPAN: Iwate, Mt. Hayachine Endo s.n. M AB196747
Miyagi 1 JAPAN: Miyagi, Tome, Towa Sugaya & Soma 4203 A AB196752
Miyagi2 JAPAN: Miyagi, Shiroishi, Mt. Ohagi Suzuki 366 A AB196751
Saitama JAPAN: Saitama, Chichibu Yamaji s.n. A AB196759
Yamanashi JAPAN: Yamanashi, Hokuto Yamaji s.n. A AB196765
Niigata1 JAPAN: Niigata , Nakakubiki, Kasu ga Iwano 1177 H AB19675 6
Niigata2 JAPAN: Niigata, Nishikubiki, Nou Iwano 5257 N AB196758
Niigata3 JAPAN: Niigata, Nishikanbara, Maze Takeuchi s.n. I AB196757
Nagano1 JAPAN: Nagano, Minamisaku, Minamimaki Takahashi 21353 A AB196755
Nagano2 JAPAN: Nagano, Chiisagata, Aoki Hisauchi s.n . C AB196754
Nagano3 JAPAN: Nagano, Kitasaku, Karuizawa Kimura s.n. L AB196753
Shizuoka JAPAN: Shizuoka, Fujinomiya, Nehara Konta et al. 771 A AB196 7 62
Kyoto1 JAPAN: Kyoto, Takeno, Amino Tsugaru & Miyah ara 26674 I AB196748
Kyoto2 JAPAN: Kyoto, Kuma no, Kumiha ma Tsugaru & Takahashi 27970 H AB19674 9
Kyoto3 JAPAN: Kyoto, Maizuru, Kan mu r i Isl. Tsugaru & Takahashi 30054 I AB19 6750
Hyogo JAPAN: Hyogo, Kobe, Fukiai Miyake 3869 G AB196746
Shimane JAPAN: Shimane, Yatsuk a, Shi mane Miki s.n. I AB196761
Yamaguchi JAPAN: Yamaguch i, Toyoura, Toyoura Imada 4414 I AB196764
Fukuoka JAPAN: Fukuoka, Munakata Watanabe s.n. J AB196740
Nagasaki JAPAN: Naga s aki, Tsushima Isl., Shimoa gata Ohash i & Soma 7454 I AB196763
Outgroup
A. kiusianus Makino JAPAN: Fukuoka, Munakata Watanabe 93 AB196738
A. offic inalis L. JAPAN: Mi yagi, Furuka wa (cult. ) Komatsu 041118 AB195716

Phylogeography of Asparagus schoberioides Kunth (Asparagaceae) in Japan
784
Table 2. Apomorphic characters of the ITS1 sequences obtained from Asparagus schoberioides and two outgroups.
111111 1 1 1 111
3 3 3 4 4 9 023 556 6 7 7 789
sample 5 9 2 5 8 6 8 1 195 454 6 1 2 330
Russia C A A T T GACCCCATC T C T GAG
China . G C C C . . G. .. . . T . T A .G .
South Korea . G C C C . . G. .. . . . . T A .G .
Hokkaido1 . G C C C . . G. .. . . . . . A .G .
Hokkaido2 . G . . C A. G. .. C. . . . A .. .
Hokkaido3 . G C C C . . G. .. . . . . . A .G .
Hokkaido4 . G C C . . . G. .. . . T . . A .G .
Hokkaido5 . G C C C . . G. .. . . Y . . A .G .
Hokkaido6 . G C C C . . G. .. . . . . . A .G .
Iwate . . . . . . . G. .. C. . . . A .. .
Miyagi1 . G C C C . . G. .. . . . . . A .G .
Miyagi2 . G C C C . . G. .. . . . . . A .G .
Saitama . G C C C . . G. .. . . . . . A .G .
Yamanashi . G C C C . . G. .. . . . . . A .G .
Niigata1 . . C . . . . G. .. C. . . . . .. .
Niigata2 . . . . . . . G. .. C. . . . . .. .
Niigata3 . . . . . . . G. .. C. . . . A .. T
Nagano1 . G C C C . . G. .. . . . . . A .G .
Nagano2 . G C C C . . G. T. . . . . . A . G.
Nagano3 . . . . . . . G. .. . . . . . . .. .
Shizuoka . G C C C . . G. .. . . . . . A .G .
Kyoto1 . . . . . . . G. .. C. . . . . .. .
Kyoto2 . . C . . . . G. .. C. . . . . .. .
Kyoto3 . . . . . . . G. .. C. . . . . .. .
Hyogo . G C . . . . G. .. . . . . . . .. .
Shimane . . . . . . . G. .. C. . . . . .. .
Yamaguchi . . . . . . . G. .. C. . . . . .. .
Fukuoka . . . . . A. G. .. C. . . . . .. .
Nagasaki . . . . . . . G. .. C. . . . . .. .
A. kiusianus . G C . C ACG. .ACC. C . A TG.
A. officina lis T G C . C . C GT .. CC . . . A TG.
Numbers indicat e the position from the first nucleotide of 5’ region of ITS sequence rem oving the primer region.

Phylogeography of Asparagus schoberioides Kunth (Asparagaceae ) in Japan785
Figure 1. One of the 78 most parsimonious trees of 29 sam-
ples of Asparagus schoberioides and outgroups. The num-
bers above the branches indicate the synapomorphic char-
acters; the bootstrap values are indicated below branches
(only those more than 50% are indicated on the tree). Ar-
rowheads indicate branches that do not appear in the strict
consensus tree.
Oceanic side of northern and central Honshu in Japan,
while Clade 2 is comprised of samples from the Japan
Sea side of Honshu (Figures 1, 3). Moreover, some of
the samples from the neighboring locations in Japan fall
into different phylogenetic position within the clade (Fig-
ures 1).
4. Discussion
The phylogenetic analyses in this study indicate that Ja-
panese Asparagus schoberioides could not be detected
the haplotype of the garden asparagus, suggesting that
there are no genetic introgressions from the crops or the
escaped individuals of the crop fields into wild relatives.
Moreover, our results suggest that the current distribu-
tion in A. schoberioides has employed two routes of ex-
pansion. One is along the Japan Sea side while t he other
is along the Pacific Oceanic side. This is consistent with
the geographical features of Japan, which has a lofty
backbone range that basically runs along the axis of Hon-
shu and divides the island into two areas, namely, the
Figure 2. Schematic of the correspondence between haplo-
types and samples in Figure 1.
Pacific Oceanic and the Japan Sea sides. The range affect
the climate of both sides and may have blocked or li-
mited the migration of plants to the opposite sides. This
is supported by studies of plant intraspecies variation
between the two sides. For example, the leaf of Euptelea
polyandra Siebold et Zucc. growing on the Japan Sea
side is broader than that of the same species growing on
Pacific Oceanic side [39]. Yonekura and Ohashi [40] re-
ported similar observations with regard to Bistorta tenui-
caulis (Bisset et Moore) Nakai. Moreover, Fujii et al. [20]
and Okura and Harada [21] found that a phylogenetic
tree constructed on the basis of the cpDNA variability of
Fagus crenata was roughly divided into two clades that
were composed of populations growing on the Pacific
Oceanic side and the Japan Sea side, respectively. Thus,
the expansion of A. schoberioides may similarly have
been limited by the mountain range, leading to two dif-
ferent lineages on the Pacific Oceanic and the Japan Sea
sides.
With regard to our phylogenetic analyses, Clade 1
showed relatively little mutation accumulation, whereas
many more nucleotide substitutions were observed in
Clade 2 (Figure 1). This discrepancy suggests that Clade
1 had different colonization or migration time from

Phylogeography of Asparagus schoberioides Kunth (Asparagaceae) in Japan
786
Figure 3. Geographical distribution of the A. schoberioides samples belonging to each clade. Alphabets indicate haplotypes of
ITS1 (see Figure 2). ○: Clade 1, ●: Clade 2.
Clade 2. Clade 1 consists of samples from the Pacific
Oceanic side of Japan, South Korea and China. Since
only one sample each from South Korea and China were
employed, the genetic diversity of A. schoberioides in
these regions could not be detected. There are two phy-
togeographic hypotheses of Japanese A. schoberioides
belonging to Clade 1. One is that they had migrated from
the mainland of China or the Korean Peninsula into Ja-
pan, at which point it expanded to Hokkaido and the Pa-
cific Oceanic side. In this study, nucleotide variation of
Clade 1 is very small, suggesting that there may have
experienced extinct events in the western and the Japan
Sea sides of Japan. The other is that they had migrated
from the mainland of China into Hokkaido, and ex-
panded rapidly to the Pacific Oceanic side from Miyagi
to Shizuoka of Honshu. In this case, they had migrated
from the mainland of China into Korean Peninsula inde-
pendently. However, it is not certain which hypothesis to
support from our results.
In contrast to Clade 1, our phylogenetic analyses sug-
gest that a common ancestor of Clade 2 originated from
more ancient times than that of Clade 1. Clade 2 consists
of samples from the Japan Sea side of Japan and Sakha-
lin, and does not include samples from the mainland of
China or the Korean Peninsula, although not enough
samples were collected from the Asian Continent to be
certain of this. Therefore, the relationships between the
Japanese and Asian Continent populations of A. schobe-

Phylogeography of Asparagus schoberioides Kunth (Asparagaceae ) in Japan787
rioides remain unclear at present.
Our phylogenetic analyses also indicate that within
each clade, the phylogenetic relationship and geographic
distribution do not correlate. For example, some neigh-
boring locations in Japan belong to different phyloge-
netic position within the clade. Examples of this are the
three samples from Niigata and the three Kyoto samples
in Clade 2 (Figure 1). This suggests that A. schobe-
rioides populations in Niigata and Kyoto consist of indi-
viduals that have experienced different histories. Fur-
thermore, northern samples such as those from Sakhalin,
Hokkaido and Iwate also appeared in various phyloge-
netic positions in Clade 2 (Figure 1). These results indi-
cate that the migration of A. schoberioides along the Ja-
pan Sea side of Japan has occurred repeatedly.
Thus, our phylogeographic analysis has outlined the
different histories of A. schoberioides between the Pa-
cific Oceanic and the Japan Sea sides in Japan. What has
aided the rapid migration of samples in Clade 1 and the
frequent colonization of samples in Clade? One answer
may lie in the fact that A. schoberioides bears reddish
berries that may be bird-dispersed [41-44]. This may aid
the rapid dispersal of seeds of A. schoberioides through-
out the two routes of Japan.
In general, a morphologically recognized species may
be regarded as a composite of subtly defined cryptic spe-
cies each of which has equal status [45]. In fact, such a
definition takes the systematic approach to an extreme
which would appear to be unworkable in plants. For
example, Yatabe et al. [46] concluded that there are
some cryptic species of Asplenium nidus L. (Asplenia-
ceae) in West Jawa on the basis of sequence variations
using rbcL of cpDNA. In this study, A. schoberioides
had nucleotide differentiation between Clade 1 and Clade
2, suggesting that there are cryptic species with the geo-
graphical features of Japan.
5. Conclusions
There are no genetic introgression between A. schobe-
rioides and A. officinalis (garden asparagus) and two
migr ation r outes o f A. schoberioides exist in Japan. Such
research, when applied to A. schoberioides and to other
Japanese plant taxa, will provide new insights into the
phytogeography of Japanese plants.
6. Acknowledgements
We wish to thank H. Yamaji, M. Kawata, M. Maki, T.
Yamashiro, P.-Y. Yun, T. Ito, H. Ashizawa, Y. Mashiko,
M. Nakada, R. Shinohara, S.-Y. Kim, M. Hirai and H.
To kairin for providing muc h help and a dvice. T his stud y
was partly supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Science and
Culture of Japan and Priority Research Centers Program
through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education Science and
Technology (2010-0029630) and in part by the Sasakawa
Scientific Research Grant from The Japan Science Soci-
ety.
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