Bull. Natl. Mus. Nat. Sci., Ser. B, 38(1), pp. 11–17, February 22, 2012

The Phylogeny of Japanese Enkianthus species (Ericaceae)
Chie Tsutsumi* and Yumiko Hirayama
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan* E-mail: tsutsumi@kahaku.go.jp(Received 16 November 2011; accepted 28 December 2011)

Abstract The genus Enkianthus consists of 12–17 species, most of which are distributed in
China and Japan. We performed molecular phylogenetic analyses for Enkianthus species of Japan,
using nuclear ITS, chloroplast matK with trnK intron, and trnS-trnG spacer regions. The result
suggested that Enkianthus was separated into two groups; i.e., sect. Enkianthus with umbellate
inﬂorescences and a clade of sect. Andromedina, sect. Enkiantella and sect. Meisteria with racemose inﬂorescences. It also inferred that the ancestral Enkianthus seems likely to be shrubs withtubular or urceolate corollas and 4–5-apertured pollen, whereas the more derived species may possess campanulate corollas and 3-apertured pollen.
Kew words : Enkianthus, ITS, matK, molecular phylogeny, trnSG.

Introduction
The genus Enkianthus Lour. (Ericaceae) is agenus of shrubs or small trees and often used as
ornamental plants in the gardens. The genus consists of 12–17 species (Anderberg, 1994; Kron etal., 2002; Fang and Stevens, 2005). All of thespecies are distributed from the Himalayas toJapan (Fang and Stevens, 2005) and most ofthem are in China and Japan. Recent molecularphylogenetic studies showed that Enkianthus issister to all other representatives of Ericaceae s.l.
including Empetraceae, Epacridaceae, Monotropaceae and Pyrolaceae (Kron and Chase, 1993;
Kron, 1996; Morton et al., 1996; Anderberg etal., 2002; Kron et al., 2002). A cladistic analysis
using morphological, anatomical, embryologicaland cytological characters suggested monophylyof the genus (Anderberg, 1994). The species ofEnkianthus are distinct from the other Ericaceae
in several characters, such as embryological features and morphologies of anther, pollen, seed
and hair (reviewed by Kron et al., 2002).
The species of Enkianthus differ in leaf tex-ture, inﬂorescences structure, corolla shape, andmorphologies of anther, pollen and seed (Anderberg, 1994; Kron et al., 2002). Infrageneric relationships of Enkianthus were studied by Anderberg (1994), who proposed a classiﬁcation
comprising four sections. Sect. Enkianthus has
more or less coriaceous leaves, tubular or urceolate ﬂowers, umbellate inﬂorescences, 4–5-apertured pollen, glabrous pedicels and smooth
anthers (except for E. serotinus Chun et Fangwith papillose anthers). Sect. Andromedina
shares several features with sect. Enkianthus but
is distinct in racemose inﬂorescences and nonwinged seeds. Sect. Meisteria is characterized byracemose inﬂorescences, 3-apertured pollen,
campanulate corollas, and a papillose corolla surface, and sect. Enkiantella by racemose inﬂorescences (except for E. pauciﬂorus Wils. with solitary ﬂowers), 3-apertured pollen, campanulate
corollas, conspicuously winged and lamellate
seeds, and anthers without a prolonged connective. Anderberg (1994) also suggested that
sect. Enkianthus is sister to sect. Andromedina,then they are sister to sect. Meisteria, and sect.

12Chie Tsutsumi and Yumiko Hirayama

Enkiantella is ﬁrstly diverged group in the genus
based on morphology. Therefore, the ancestral
Enkianthus seems to have deciduous leaves, racemose inﬂorescences, campanulate corollas, andwinged seeds, whereas the more derived species
likely possess coriaceous leaves, umbellate inﬂorescences, ﬂowers with tubular or urceolate
corollas, and non-winged seeds (Anderberg,
1994). The classiﬁcation was supported by a pollen morphological study (Golam Sarwar and
Takahashi, 2007). The molecular phylogeny of
the genus, however, remains to be examined.
In Japan, six species and two varieties of Enkianthus are recognized (Yamazaki, 1993).
According to the section concept sensu Anderberg (1994), Enkianthus perulatus (Miq.)
C.K.Schneid. is assigned to sect. Enkianthus, E.
nudipes (Honda) Ueno and E. subsessilis (Miq.)
Makino are assigned to sect. Andromedina,
E. campanulatus (Miq.) G.Nicholson, and E.
sikokianus (Palib.) Ohwi and E. cernuus (Siebold
et Zucc.) Makino belong to sect. Meisteria.
Enkianthus perulatus is distributed in Japan andTaiwan, although plants in Taiwan are sometimes
treated as a distinct species, E. taiwanianus Ying.
The other ﬁve species are endemic to Japan. In
this study, the molecular phylogeny of the six
Japanese Enkianthus species was analyzed using
nuclear ITS regions and two chloroplast DNA
regions, matK with trnK intron and trnS-trnG
spacer. Enkianthus quinqueﬂorus Lour. distributed in China to Vietnam, and Clethra species,
one of sister candidates to Enkianthus, were also
analyzed in this study.
Materials and Methods
Leaf samples of seven Enkianthus species
were collected in the ﬁeld or from cultivatedplants in the Tsukuba Botanical Garden, National
Museum of Nature and Science (Table 1). Clethra barbinervis Siebold et Zucc. was also analyzed (Table 1). DNA was extracted from freshor silica-gel-dried material using a QUIAGEN
DNeasy Mini Kit (QUIAGEN, Valencia, CA)following the manufacturerʼs instruction.

The internal transcribed spacer regions of 18S26S nuclear ribosomal DNA (ITS), and two chloroplast regions, matK (the maturase-encodinggene) with trnK intron, and trnS-trnG spacer,
were analyzed. Primers for ampliﬁcation were
17SE (Sun et al., 1994) and ʻ26SEʼ (Topik et al.,2005, modiﬁed 26SE in Sun et al., 1994) in ITS
region, and trnK-3914F and trnK-2R (Johnson
and Soltis, 1994) in matK with internal primers
MK-F1 (Koi et al., 2008), matK-E728F (5′-ATAGATCTCAGCAACATGAC-3′), matK-E1166F(5′-CATCCATCTGGAAATCTTGG-3′), matKE780R (5′-GTGGATATATGAAGTCATGTTGCTG-3′), and matK-E1391R (5′-TTGGCACTATGGTATCGAACT-3′), and trnS (GCU) andtrnG (UCC) in trnS-trnG spacer (Hamilton,
1999). PCR for ITS region was performed usinga DNA thermal cycler (Perkin-Elmer 9700,
Applied Biosystems, Foster, CA) with LA Taq
DNA polymerase (TaKaRa Bio, Tokyo, Japan) in
35 denaturation, annealing, and elongation cycles(30 sec at 95°C, 30 sec at 55°C and 90 sec at72°C) with a ﬁnal elongation step (5 min at
72°C). PCR for matK with trnK intron and trnStrnG spacer regions was conducted with Ex Taq
DNA polymerase (TaKaRa Bio, Tokyo, Japan) in
35 denaturation, annealing, and elongation cycles(30 sec at 95°C, 30 sec at 55°C and 90 sec at72°C) with a ﬁnal elongation step (7 min at
72°C). The PCR products were puriﬁed with
ExoSAP-IT (USB corporation, Cleveland, OH)following the manufacturerʼs instruction.
Sequencing was conducted using an ABI PRISM
3130xl Genetic Analyzer (Applied Biosystems).
The sequences obtained were assembled using
Seqman II (Dnastar, Madison, WI). The assembled sequences were aligned by Clustal X program (Thompson et al., 1997) and then aligned
manually. In addition to the sequences analyzed,
several registered sequences of Enkianthus andCyrilla racemiﬂora L. (Cyrillaceae) in GenBank
were added for phylogenetic analyses.
Phylogenetic analyses were performed by
Bayesian analyses and maximum parsimony(MP). Bayesian analyses were performed by
MrBayes 3.1.2 (Huelsenbeck and Ronquist,

Phylogeny of Enkianthus

13

Table 1. Species used in molecular phylogenetic analysis, sources and GenBank accession no. of ITS, matK with
trnK intron and trnS-trnG spacer regions.
SpeciesSect. Meisteria
E. campanulatus (Miq.)
G.Nicholson
E. campanulatus(Miq.)*1
E. campanulatus(Miq.)*2
E. sikokianus (Palib.) Ohwi
E. cernuus (Siebold etZucc.) Makino f. cernuus
Sect. Andromedina
E. nudipes (Honda) Ueno
E. subsessilis (Miq.) Makino
Sect. Enkianthus
E. perulatus (Miq.)
C.K.Schneid.
E. quinqueﬂorus Lour.

GenBank accession no.

SourceCultivated in Tsukuba Botanical Garden;
Tsutsumi s.n.
from Genbank
from Genbank

ITS

matK

trnSG

AB697666

AB697674

AB697682

AF091940

U61344

GU176714

AF133752

Cultivated in Tsukuba Botanical Garden(Origin; Kochi, Japan); Tsutsumi s.n.
Cultivated in Tsukuba Botanical Garden;
Tsutsumi s.n.
Shinshiro, Aichi, Japan; Tsutsumi
Ho3-20100514
Cultivated in Tsukuba Botanical Garden(Origin; Tochigi, Japan); Tsutsumi s.n.
Cultivated in Tsukuba Botanical Garden;
Tsutsumi s.n.
Cultivated in Tsukuba Botanical Garden(Origin; Hong Kong); Tsutsumi s.n.
from Genbank

E. quinqueﬂorus Lour.*Sect. Enkiantella
E. chinensis Franch.*from Genbank
Clethraceae
Clethra barbinervis Siebold et Kagoshima Pref. Japan; Tagane & Fuse
Zucc.
(TNS763118)
Cyrillaceae
Cyrilla racemiﬂora L.*from Genbank

AB697667

AB697675

AB697683

AB697668

AB697676

AB697684

AB697669

AB697677

AB697685

AB697670

AB697678

AB697686

AB697671

AB697679

AB697687

AB697672

AB697680

AB697688

FJ378572

FJ357264

FJ378571

FJ357263

AB697673

AB697681

AF396236

AJ429282

AB697689* Asterisks show the data derived from registered sequence in Genbank.

2001; Ronquist and Huelsenbeck, 2003).
MrModeltest 2.0 (Nylander, 2004) was used todetermine the nucleotide substitution model for
Bayesian analysis. Bayesian searches were conducted by mcmc with four chains over one million generations for each of ITS, matK with trnK
intron, trnS-trnG spacer regions, and over two
million generations for combined data, sampling
every 100 generations. Trees obtained before stationary generations were discarded as burn-in
trees and the rest of trees were used to calculate
posterior probabilities. Maximum parsimony(MP) inference was conducted with PAUP*
4.0b10 (Swofford, 2002). The bases that could
not been identiﬁed were treated as unknown (N),and gaps were treated as missing data. Bootstrap
values were calculated from 1000 pseudo-replicates, each with 100 random additions replicates

involving TBR branch swapping. Clethra
barbinervis and/or Cyrilla racemiﬂora were usedas outgroups (Anderberg et al., 2002; Soltis etal., 2011).
Homogeneity among the different datasets wascalculated with the ILD test (Farris et al., 1995;
Johnson and Soltis, 1998) as implemented in
PAUP. The ILD tests were performed between
two of the three datasets. The test was conductedby heuristic search with 100 replicates, using 10
random additions with parsimony-uninformative
characters excluded. All ILD-tests for different
partitions tested indicated that p-values werehigher than 0.05 (the lowest p-value was 0.09
between ITS and matK with trnK intron), suggesting that the datasets were not incongruent tobe combined.

14

Chie Tsutsumi and Yumiko Hirayama

Fig. 1. Consensus tree by Bayesian analysis based on nuclear ITS sequence dataset. Figures below branches indicate
posterior probabilities (p≧0.8) calculated by Bayesian analysis and those above branches indicate maximum parsimony bootstrap values (＞50).

Results
The molecular phylogenetic trees deduced
from each of the nuclear ITS region, the matKwith trnK intron sequences, and the trnS-trnG
spacer region showed that all the species of Enkianthus examined formed a monophyletic group(Figs. 1–3). The results showed that each of thethree sections (sect. Enkianthus, sect. Andromedina, sect. Meisteria) formed a clade in all thetrees except sect. Meisteria in the ITS tree. Supports were high of the sect. Andromedina in all
the trees, of the sect. Meisteria in the trees ofmatK with trnK intron and the trnS-trnG spacer(including E. chinensis in the trnS-trnG spacertree), and of the sect. Enkianthus in the matKtree. In the trees deduced from the two chloroplast regions, sect. Enkianthus was ﬁrstly
diverged from the other species of Enkianthus,
and sect. Meisteria was sister to sect. Andromedina, although the infrageneric relationships
remained unsolved in the ITS tree. The molecu-

lar phylogenetic positions of Enkianthus chinensis in sect. Enkiantella differed in trees; in the
ITS tree it formed a clade with the species ofsect. Andromedina, and it was nested in the cladeof sect. Meisteria in the trnS-trnG spacer tree.
Deduced phylogenies based on the combined
datasets of the nuclear ITS and two chloroplast
regions also showed that the genus Enkianthus
was monophyletic with high supports (Fig. 4).
The tree indicated that sect. Enkianthus was
ﬁrstly separated from the remaining species ofEnkianthus, and sect. Meisteria including E. chinensis was sister to the clade comprising the species of sect. Andromedina. Those relationships
were supported by high credibilities.
Discussion
The present results showed that the phylogenetic relationships of mainly Japanese Enkianthus species by the nuclear ITS and two chloroplast regions. The infrageneric classiﬁcation wasPhylogeny of Enkianthus

15

Fig. 2. Consensus tree by Bayesian analysis based on sequence dataset of chloroplast matK with trnK intron. Figures
below branches indicate posterior probabilities (p≧0.8) calculated by Bayesian analysis and those above branches
indicate maximum parsimony bootstrap values (＞50).

Fig. 3. Consensus tree by Bayesian analysis based on chloroplast trnS-trnG spacer sequence dataset. Figures below
branches indicate posterior probabilities (p≧0.8) calculated by Bayesian analysis and those above branches indicate
maximum parsimony bootstrap values (＞50).

16

Chie Tsutsumi and Yumiko Hirayama

Fig. 4. Consensus tree by Bayesian analysis based on combined sequence datasets of ITS, matK with trnK intron and
trnS-trnG spacer. Figures below branches indicate posterior probabilities (p≧0.8) calculated by Bayesian analysis
and those above branches indicate maximum parsimony bootstrap values (＞50).

generally consistent with Anderberg (1994),
except for sect. Enkiantella, whose samples were
not well analyzed in this study (Figs. 1–4). The
previous cladistic analysis based on the morphological characters suggested that sect. Enkianthus
was sister to sect. Andromedina, then they weresister to sect. Meisteria, and sect. Enkiantella
was ﬁrstly diverged group from the remaining
species (Anderberg, 1994). The present resultswere, however, incongruent to the topology
derived from the previous cladistic analysis. Our
tree deduced from combined datasets showedthat sect. Enkianthus with umbellate inﬂorescences was separated from the other sections
with racemose inﬂorescences (Fig. 4). The tree
also indicated that sect. Andromedina was sisterto sect. Meisteria including E. chinensis with
3-apertured pollen, hairy anthers, and campanulate corollas, although sect. Andromedina sharesa number of character states with the species ofsect. Enkianthus. Therefore, the ancestral Enkianthus, like sect. Enkianthus, seems to be shrubswith tubular or urceolate corollas and 4–5-apertured pollen, while campanulate corollas and
3-apertured pollen seem likely to be apomorphic.
Anderberg (1994) noted the similarities of E.
nudipes and E. subsessilis, both of which differ
mainly in the indumentum of the calyx and thepedicel, while Ueno (1950) suggested that they
are distinct by mount of crystals in the leaf mesophylls. The molecular differences between thetwo species were three substitutions in ITS
regions, seven substitutions, one insertion andthree variations of poly-(N) in matK with trnK
intron, and two insertions in trnS-trnG spacer. It
suggests that the two species seem likely to begenetically differentiated. Further, the taxonomic
treatments of E. campanulatus and E. sikokianus
varied by scientists. Anderberg (1994) treated it
as one species with variations. Yamazaki (1993)
opined that E. sikokianus is a distinct species
from E. campanulatus by the long racemes andshort pedicels with many ﬂowers, and that E.
campanulatus consists of three varieties, var.

Phylogeny of Enkianthus

campanulatus, var. palibinii and var. longilobus,
which differed from each other in corolla colorand sizes of corolla and lobes. The molecular differences between E. campanulatus and E. sikokianus were not identical in the three regions
examined; two substitutions in ITS region, four
substitutions in matK with trnK intron, and one
insertion in trnS-trnG spacer. It needs further
analysis to clarify the cause of these.
Acknowledgments
We thank A. Ebihara, K. Fuse, T. Muramatsu
and S. Tagane for providing the materials and/or
help during the ﬁeld trips. We also gratefully
thank A. Uehara for technical assistance and M.
Kato for useful comments on the manuscript.
This study was supported by Grants-in-Aid for
Scientiﬁc Research from the Japan Society for
the Promotion of Science.
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