Bull. Natl. Mus. Nat. Sci., Ser. B, 40(4), pp. 153–161, November 22, 2014

Molecular and Morphological Data Conﬁrm the Occurrence of
Two Varieties of Helwingia japonica subsp.
japonica (Helwingiaceae) in Kochi Prefecture, Japan
Hana Umemoto1,2*, Koh Nakamura3, Ayako Maeda4,
Masatsugu Yokota5 and Goro Kokubugata1,2*
1

Graduate School of Agriculture, Ibaraki University,
Ami, Ibaraki 300–0393, Japan2
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
3
Biodiversity Research Center, Academia Sinica, Nangang, Taipei 115, Taiwan
4Makino Botanical Garden, Kochi, Kochi 781–8125, Japan
5
Laboratory of Ecology and Systematics, Faculty of Science,
University of the Ryukyus, Nishihara, Okinawa 903–0213, Japan* E-mail: humemoto@kahaku.go.jp; gkokubu@kahaku.go.jp(Received 15 August 2014; accepted 24 September 2014)

Abstract Twenty-six plants of Helwingia japonica subsp. japonica, including 23 plants from
Kochi Prefecture and three plants from other areas of Japan with other subspecies of this species,
were taxonomically compared using molecular and morphological data. The molecular phylogenetic analyses placed the 23 Kochi plants in two major clades. Studentʼs t-test revealed a signiﬁcant difference in the number of leaf lateral vines between the Kochi plants of the two clades.
Based on taxonomy, the two groups were respectively identiﬁed as H. japonica subsp. japonica
var. japonica and var. parvifolia. The present study further conﬁrmed that two varieties are distributed in Kochi, Japan, and suggests that gene exchange between the two varieties sympatrically distributed may be hindered by certain isolation mechanism.
Key words : Helwingia, ITS, Kochi Prefecture, leaf lateral vine.

Introduction
The genus Helwingia Willd., morphologicallycharacterized by epiphyllous inﬂorescences, iscomposed of four species in East Asia (Hara andKurosawa, 1975; Xiang and Boufford, 2005).
This genus was traditionally a member of thefamily Cornaceae (Noshiro, 1999), but recent
molecular phylogenetic studies separated as amonotypic family Helwingiaceae (APGIII,
2009), which is thought to be closely related toPhyllonoma (Phyllonomaceae), having epiphyllous inﬂorescences and distributing in Mexico
and Bolivia (Mori and Kallunki, 1977), by Tank

and Donoghue (2010), and Soltis et al. (2011).
In Japan, one species Helwingia japonica(Thunb.) F.Dietr. with two subspecies is found:
H. japonica subsp. liukiuensis (Hatus.) H.Hara etKuros., is characterized by oblong–lanceolate toovate leaves of 5–12 cm long and endemic to the
Ryukyu Archipelago of Japan (Hara and Kurosawa, 1975); and H. japonica subsp. japonica ischaracterized by obovate to elliptic leaves of
4–15 cm long and widely distributed from Japan
to Himalayas (Hara and Kurosawa, 1975;
Noshiro, 1999; Fig. 1). In the later subspecies,
two varieties are reorganized in Japan: H. japonica subsp. japonica var. japonica and var. parvi-

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Hana Umemoto et al.

Fig. 1. Habitat of Helwingia japonica subsp. japonica in Kochi. A. Sympatric population with well-branched(arrow) and poorly branched (arrowhead) plants of H. japonica subsp. japonica. Bar＝15 cm. B. Plants thatare well branched. Bar＝1 cm. C. Plants that are poorly branched. Bar＝2 cm.

folia Makino studied herein (Hara and Kurosawa, 1975; Noshiro, 1999).
The two varieties are distinguished based onleaf and stem morphologies: H. japonica subsp.
japonica var. japonica has more than four pairsof leaf lateral vines and poorly branched habits,
whereas H. japonica subsp. japonica var. parvi-

folia has less than four pairs of leaf lateral vines
and well branched habits (Noshiro, 1999; Kobayashi, 2009) (Fig. 1). Furthermore, H. japonica
subsp. japonica var. japonica has larger leaves
than H. japonica subsp. japonica var. parvifolia(Yamanaka, 1996). In Japan, H. japonica subsp.
japonica var. japonica is widely occurred from

Two Varieties of Helwingia japonica in Kochi

155

Fig. 2. Map showing the 14 collection localities of 32 individuals of Helwingia japonica from Japan and Taiwan.
Abbreviations refer to localities are listed in Table 1.

Hokkaido to Kyushu, whereas H. japonica
subsp. japonica var. parvifolia is comparatively
narrowly distributed from southwestern Honshu
to Kyushu (Hara and Kurosawa, 1975). According to Kobayashi (2009), both varieties are distributed in Kochi Prefecture of Shikoku (Fig. 2).
In our botanical expedition in Kochi, we foundpopulations with two types of H. japonica subsp.
japonica plants with well-branched and poorlybranched stems (Fig. 1). Although the branchingof stems is a key character to separate the twovarieties, this character varies in different growthstages and habitats and thereby other salient morphological character should be examined for reliable identiﬁcation of these plants. In this study,
we performed molecular phylogenetic analyses
using the internal transcribed spacer (ITS) regionof nuclear ribosomal DNA and performed a mor-

phological comparison of leaf lateral vine number to conﬁrm the sympatric occurrence of thetwo varieties of H. japonica subsp. japonica var.
japonica and var. parvifolia in Kochi.
Materials and Methods
Plant materials
Information of plant materials for the present
molecular phylogenetic analyses and morphological comparisons are provided in Table 1. For themolecular phylogenetic analyses, we collected 26
individuals of Helwingia japonica subsp. japonica from Honshu (two plants), Kochi of Shikoku(23 plants), and Kyushu (one plant), as well as
three individuals of H. japonica subsp. liukiuensis from the Ryukyu Archipelago of Japan (Haraand Kurosawa, 1975). We also included threeHana Umemoto et al.

156

Table 1. Plant materials of Helwingia japonica investigated in the present study and their ITS accession numbers
and number of leaf lateral vine
Species
INGROUP
H. japonica
subsp. japonica

Collection locality (altitude)

Japan: Honshu, Tochigi, Mt.
Amamaki (350 m)
Japan: Honshu, Ibaraki, Mt.
Gozen (70 m)
Japan: Shikoku, Kochi, Behukyo(546 m)Japan: Shikoku, Kochi, Tosakuwae (128 m)

Japan: Shikoku, Kochi, Kawanaji (213 m)

subsp. liukiuensis

subsp. taiwaniana

OUTGROUP
Phyllonoma
ruscifolia

Japan: Shikoku, Kochi, Oohana(111 m)Japan: Shikoku, Kochi, Yusunose (66 m)
Japan: Kyushu, Kumamoto, Kikuchi
Japan: Ryukyus, Amami Island,
Asato (380 m)
Japan: Ryukyus, TokunoshimaIsland, Mt. Inokawa (540 m)
Japan:
Ryukyus,Okinawa
Island, Mt. Yae (225 m)
Taiwan: Kaohsiung, Taoyuan
Hsiang (2451 m)
Taiwan: Nantou, Chitou (1119 m)
Taiwan: Nantou, Meifan (2361 m)* Referring to Fig. 1.
** aThe present study; bManen et al. (2002).

Abbreviation*

Voucher
no.

H1

ITS

Mean of leaf
lateral vein inthree leaves(±SD）

Accession
no.**

Type

GK10511

AB981678a

A

—

H2

GK14915

AB981679a

A

—

S1

HU39

AB981680a

F

3.00±0.00

S2

HU13

AB981681a

B

4.83±0.37

S2
S2
S2
S2
S2
S2
S2
S2
S2
S3

HU14
HU15
HU16
HU17
HU18
HU19
HU20
HU21
HU22
HU1

AB981682a
AB981683a
AB981684a
AB981685a
AB981686a
AB981687a
AB981688a
AB981689a
AB981690a
AB981691a

F
G
F
B
F
C
C
C
B
C

3.33±0.47
3.17±0.37
3.33±0.47
5.33±0.47
4.00±0.00
4.50±0.50
5.00±0.58
5.00±0.58
4.67±0.47
5.50±0.50

S3
S3
S3
S3
S3
S3
S3
S3
S3
S4

HU2
HU3
HU5
HU6
HU7
HU8
HU9
HU10
HU11
HU63

AB981692a
AB981693a
AB981694a
AB981695a
AB981696a
AB981697a
AB981698a
AB981699a
AB981700a
AB981701a

F
F
F
F
F
D
F
B
B
F

3.00±0.00
3.67±0.47
3.33±0.47
4.00±0.00
4.00±0.00
4.33±0.47
3.50±0.50
5.00±0.58
4.50±0.50
3.33±0.47

S5

HU48

AB981702a

F

3.33±0.47

K1

GK17572

AB981703a

E

—

R1

GK11666

AB981704a

H

—

R2

GK9543

AB981705a

H

—

R3

GK10747

AB981706a

H

—

T1

GK10871

AB981707a

I

—

T2
T3

GK14991
GK15097

AB981708a
AB981709a

J
I

—
—

AJ492650b—

Two Varieties of Helwingia japonica in Kochi

individuals of H. japonica subsp. taiwaniana
Y.-P.Yang et H.-Y.Liu, which is endemic to Taiwan, and characterized by oblong–lanceolate toelliptic leaves of 5–12 cm long (Hara and Kurosawa, 1975). In total, 32 plant samples of H.
japonica from Japan and Taiwan were investigated in the molecular analyses. Voucher specimens for the present molecular analyses were
deposited in the herbarium of the National
Museum of Nature and Science (TNS). The ITS
sequence of Phyllonoma ruscifolia Willd. ex
Schult. (Manen et al., 2002) was downloaded
from GenBank (http://getentry.ddbj.nig.ac.jp/
getentry/na/AJ492650), and was used as an outgroup following previous studies (Tank andDonoghue, 2010; Soltis et al., 2011).
DNA extraction, PCR, and sequencing
Genomic DNA was extracted from silica geldried leaves using the DNeasy Plant Mini Kit(Qiagen, Valencia, CA, USA) following the manufacturerʼs protocol. The genomic DNA samples
were deposited in the Molecular Biodiversity
Research Center of the National Museum of
Nature and Science. The ITS region was ampliﬁed using polymerase chain reaction (PCR) with
an iCycler (Bio-Rad, Hercules, CA, USA). Forward primer AB101 (5′-ACG AAT TCA AGG
TCC GGT GAA TGT TTC G-3′) and reverse
primer AB102 (5′-TAG AAT TCC CCG GTTCGC TCG CCG TTA C-3′) were used for PCR(Douzery et al., 1999). Ampliﬁcations were performed using the TaKaRa Sapphire Amp Fast
Master Mix (TaKaRa, Otsu, Japan) with Sapphire Amp (TaKaRa). The PCR proﬁle was 35
cycles of 5 s at 94°C, 5 s at 50°C, and 5 s at 72°C
after an initial denaturing for 3 min at 94°C. PCR
products were checked by electrophoresis before
puriﬁcation using illustra ExoProStar (GEHealthcare, Tokyo, Japan). Cycle sequencing
reaction was performed with the BigDye™ Terminator Cycle Sequencing Kit ver. 3.1 (Applied
Biosystems, Foster City, CA, USA). Primers
used for sequencing were AB101, AB102, ITS2N(5′-TCGCTGCGTTCTTCATC-3′), and ITS3N(5′-GATGAAGAACGCAGCGA-3′) (Douzery et

157al., 1999). The Sanger sequencing products werethen puriﬁed using ethanol precipitation. Automated sequencing was performed on a 3130xl
Genetic Analyzer (Applied Biosystems). The
electropherograms were analyzed using ATGC
ver. 4.01 (Genetyx Co., Tokyo, Japan). Sequence
data from this study were deposited in the DDBJ
database (http://www.ddbj.nig.ac.jp/; Table 1).
Phylogenetic analyses
DNA sequences were aligned using the program ClustalW 1.8 (Thompson et al., 1994), andthen manually adjusted. Phylogenetic analyses
were conducted based on a Bayesian approach
using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) and a maximum parsimony (MP)criterion using PAUP* version 4.0b10 (Swofford,
2002). The hierarchical likelihood ratio test
(hLRT) implemented in MrModeltest 2.2 (Nylander, 2004) was used to estimate the appropriate
evolutionary model of nucleotide substitutions.
Based on the model selected, two separate runsof Metropolis coupled Markov chain Monte
Carlo (MCMCMC) analyses were performed,
each with a random starting tree and four chains(one cold and three heated). The MCMCMC
length was 2 million generations, and the chain
was sampled every 100th generation from thecold chain. The ﬁrst 5000 sample trees (25% of
the total 20,000 sample trees) were discarded asburn-in after checking that the mean standard
deviation of split frequencies (ASDSF) reached astationary state at ＜0.01 thereafter. As a guide toconvergence, the potential scale reduction factors(PSRFs) were close to 1.0 for all parameters inan output table. A 50% majority consensus treeof the output tree ﬁle from MrBayes was visualized using TreeView (Page, 1996).
In MP phylogenetic analysis, characters weretreated as unordered, and character transformations were equally weighted. The branch collapse
option was set to collapse at a minimum lengthof zero. A heuristic parsimony search was performed with 200 replicates of random additionsof sequences with ACCTRAN character optimization, tree bisection–reconnection (TBR) branch

Hana Umemoto et al.

158

swapping, and MULTREES and STEEPESTDESCENT options on. Statistical support foreach clade was assessed using bootstrap analysis(Felsenstein, 1985). A thousand replicates of heuristic searches, with the TBR branch swapping
switched on and MULTREES options off, were
performed to calculate bootstrap values (BS).
Comparison of leaf lateral vine number
In the morphological comparison of leaf lateral vines, 23 plants collected from Kochi werestudied on herbarium specimens (Table 1). Leaf
lateral vine number was examined using threeleaves per individual to calculate the mean for
the individual. After molecular phylogenetic
analyses, if 23 plants collected from Kochi are
divided into multiple clades, equality of the meanof leaf lateral vine were statistically evaluatedusing Studentʼs t-test between (among) the clades
after checking the equality of variances using theF-test with PRISM ver. 4 (GraphPad Software,
La Jolla, CA, USA).
Results
Phylogenetic distinction based on ITS
After alignment of ITS sequences from 33
operational taxonomic units (OTUs; 32 ingroups
and an outgroup member), a matrix of 790 bp
was obtained. Ten ITS types (A–J) were recognized in the ingroup of H. japonica from Japanand Taiwan (Table 1).
The model of HKY＋G was selected for the
ITS data. The 50% majority rule consensus treeof all post-burn-in trees is depicted in Fig. 3 withmean branch lengths and posterior probabilities(PPs). Because the topology of the MP strict consensus tree (CI＝0.989; RI＝0.987; RC＝0.976,
not shown) was compatible with that of theBayesian tree, the bootstrap percentage (BS;
1,000 replicates) was plotted on the Bayesiantree (Fig. 3).
The Bayesian and MP analysis (Fig. 3)
showed two major clades in the ingroup (Clades
I and II). Clade I consisted of 13 plants of H.
japonica subsp. japonica from Kochi of Shikoku(ITS types F and G), three plants of H. japonica
subsp. liukiuensis (ITS type H), and three plantsof H. japonica subsp. taiwaniana (ITS types I
and J); and clade II consisted of 1 plant of H.
japonica subsp. japonica from Honshu (ITS typeA), Kochi of Shikoku (ITS types B, C and D)
and Kyushu (ITS type E) of Japan. Therefore, thepresent molecular analyses divided 23 plants of
H. japonica subsp. japonica from Kochi into two
major clades.
Comparison of the leaf vine number
In 23 plants in Kochi of Japan, the means ofleaf lateral vines number of the plants of H.
japonica subsp. japonica placed in Clade I (ITS
types F and G) ranged from 3.00 to 4.00, while
those of plants placed in Clade II (ITS types B, C
and D) ranged from 4.33 to 5.33 (Table 1, Fig.
4). The F-test showed no signiﬁcant difference inthe variance in the leaf lateral vine number
between the Kochi plants in Clade I and Clade II,
and thus, Studentʼs t-test was applied to evaluate
equality of the mean in the leaf lateral vine numbers. Studentʼs t-test statistically revealed a signiﬁcant difference in the mean in leaf lateral vine
number between the Kochi plants placed in
Clades I and II (p＜0.005).
Discussion
Sympatric occurrence of two varieties of Helwingia japonica in Kochi
As brieﬂy mentioned in the introduction,
Noshiro (1999) noted that H. japonica subsp.
japonica var. japonica and var. parvifolia weredistinguishable based on the number of lateralFig. 3. The Bayesian 50% majority rule consensus tree of Helwingia japonica based on nrITS sequences. The
topology of the maximum parsimony strict consensus tree was compatible with the Bayesian tree. Alphabets
in parentheses indicate nrITS types. Numerals above branches indicate Bayesian posterior probabilities(upper) and bootstrap percentages in the maximum parsimony analysis (lower; ＜−50%).

Two Varieties of Helwingia japonica in Kochi

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Hana Umemoto et al.

Fig. 4. Comparison of leaf lateral vines between
Kochi plants of Helwingia japonica subsp.
japonica placed in two clades using the boxand-whisker plots of the vein numbers.

leaf vines; the former has 4–6 pairs of leaf lateralveins, whereas the latter has 2–4 pairs. Following
the taxonomic treatment of Noshiro (1999),
plants with leaf lateral vein means of 3.00 to 4.00
placed in Clade I are identiﬁed as H. japonica
subsp. japonica var. parvifolia (Fig. 1: B), andplants with leaf lateral vein means of 4.33 to 5.33
placed in Clade II are identiﬁed as H. japonica
subsp. japonica var. japonica (Fig. 1: C). The
present molecular and morphological results elucidated the occurrence of H. japonica subsp.
japonica var. japonica and var. parvifolia in
Kochi.
In taxonomy, however, we found that H.
japonica subsp. japonica followed by the previous taxonomic concepts (e.g., Hara and Kurosawa, 1975; Noshiro, 1999) was not a monotypictaxon at subspecies level; and that leaf size andnumber of leaf lateral vine have parallel-evolvedin H. japonica. Therefore, it is necessary to comprehensively reconsider these intraspeciﬁc lankof H. japonica based on comparisons using not
only leaf morphologies but also ﬂoral morphologies.

Possible hindrance of hybridization between thetwo varieties
To examine the hybridity of vascular plants,
ITS region is often examined: a hybrid isexpected to have heterozygotic nucleotide sites
showing polymorphisms from putative parental
species in ITS region (e.g., Saito et al., 2007;
Kokubugata et al., 2011). In the present study, 18
segregation sites were detected between plants
identiﬁed as H. japonica subsp. japonica var.
japonica and var. parvifolia, from two localities
Tosa-kuwae and Kawanaji in Kochi, where thetwo varieties occurred sympatrically within anarea of ca. 4 m2 (Fig. 1: A), but additive of nucleotide polymorphisms were not found. This result
suggested that no hybrid were present in two
localities of Kochi, and implied that certain isolation mechanisms might exist to prevent geneexchange between the two varieties in the sympatric population.
Acknowledgments
We thank C.-I Peng (HAST) and H. Yamashita
for providing valuable information on the habitatof Helwingia plants. This study was funded by aGrant-in-Aid for Scientiﬁc Research (B) (JSPS
KAKENHI Grant Number 25290085) and supported in part by the Mitsui & Co., Ltd. Environment Fund (No. RC10-C097).
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