Bull. Natl. Mus. Nat. Sci., Ser. B, 41(3), pp. 99–105, August 21, 2015

Hybrid of Osmunda japonica and O. lancea on Mt. Tenjo,
Kozu Island, Izu Islands, Japan
Chie Tsutsumi1,*, Yumiko Hirayama1, Kaoru Yamamoto2, Hidetoshi Kato3,
Noriaki Murakami3, Hirokazu Tsukaya4 and Yoko Yatabe-Kakugawa3
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Yokosuka City Museum, Yokosuka 238–0016, Japan3
Department of Biological Science, Tokyo Metropolitan University, Hachioji, Tokyo 192–0397, Japan
4
Department of Biological Sciences, Graduate School of Science, The University of Tokyo,
Hongo, Tokyo 113–0032, Japan* E-mail: tsutsumi@kahaku.go.jp(Received 14 May 2015; accepted 24 June 2015)

Abstract We found an Osmunda lancea-like plant on Mt. Tenjo, the Kozu Island, the Izu Islands,
and identiﬁed it as Osmunda×intermedia, a hybrid between O. japonica and O. lancea, based on
the morphology and molecular phylogeny. It is the ﬁrst record of O.×intermedia from Izu Islands,
and its habitat differs from the ordinary habitat of O.×intermedia. Morphology suggests that theindividual is a later-generation hybrid.
Key words : Izu Islands, Kozu Island, Osmunda×intermedia.

Introduction
Osmunda lancea Thunb. is a rheophyte and is
adapted to the ecologically specialized river-banksubject to periodical ﬂooding with swift turbulence after heavy rains. It is characterized by thenarrow-lanceolate pinnules resistant to fast current (Imaichi and Kato, 1992, 1993, 1997). Thisrheophilous species is endemic to Japan and distinguished from O. japonica Thunb., mainly by
the shape of pinnule. Osmunda japonica has
oblong pinnules with truncate bases and inhabitssemi-open roadside slopes/excavations and secondary forests in eastern Asia. The pinnule of O.
lancea is lanceolate and has a cuneate base. Phylogenetically, O. japonica and O. lancea formeda monophyletic clade and are quite close (Yatabe
et al., 1999; Metzgar et al., 2008; Tsutsumi et al.,2011). A previous study of the genetic population
structure revealed that O. japonica and O. lancea
are genetically differentiated in nuclear genes

despite shared polymorphism in chloroplast
genes, suggesting recent introgression between
O. japonica and O. lancea (Yatabe et al., 2009).
A natural semifertile hybrid between the two
species is known as O.×intermedia (Honda)
Sugim. with morphological intermediacy in its
pinnule shape. The distribution almost overlapsthat of O. lancea (Kurata and Nakaike, 1990). It
occurs locally abundantly on and above stream
banks but does not invade forest edges where O.
japonica grows (Shimura, 1964). The analyses of
the genetic population structure of those species
inferred that later-generation hybrids are formed,
based on the ﬁndings of putative F2 hybrids in
O.×intermedia (Yatabe et al., 2009). Osmunda×
intermedia has a considerable variation, which
may be caused by phenotype segregation of F2or later progeny, multiple origins, and/or backcrosses (Kato, 2009).
The Izu Islands consist of about 100 Islands
and are ca. 650 km long in the Paciﬁc Ocean off-

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Chie Tsutsumi et al.

shore the Izu peninsula in Japan. Osmunda
japonica is common on the inhabited islands,
although there is no record of rheophilous O.
lancea from the Islands (Kurata and Nakaike,1990). Recently we found an O. lancea-like plantwith narrow-lanceolate pinnules around the topof Mt. Tenjo on the Kozu Island, an inhabitedisland of the Izu Islands locating about 55 kmapart from the nearest Izu Peninsula. To reveal
the taxonomic identity of the plant, we per-formed morphological and molecular phylogenetic analyses using several nuclear markers.
Materials and Methods
Living plant materials of an Osmunda lancealike plant (KL1) and two O. japonica individuals(KJ1 and KJ2) were collected from the Kozu
Island (Fig. 1, Table 1).
In a morphological analysis, ﬁve pinnules ofFig. 1. Localities of Osmunda lancea-like plant (green square) and O. japonica (red squares) at Mt. Tenjo in the
Kozu island. Sample IDs are identical to those of Table 1. The basemap is the 1 : 25,000 topographic map(Kozu island) published by Geospatial Information Authority of Japan. Scale bar＝300 m.

Osmunda×intermedia in Kozu Island
Table 1.

101

Osmunda individuals found on the Kozu Island. Asterisks (*) show samples used for molecular analyses

Species

Sample ID

LocalityO. lancea like plantO. japonica

KL1*
KJ1*
KJ2*
KJ3
KJ4
KJ5
KJ6
KJ7
KJ8

Japan, Tokyo, Kodzu Isl., Mt. Tenjo (N34°12′59″, E139°09′16″, alt. ca. 500 m)
Japan, Tokyo, Kodzu Isl., Mt. Chichibu (N34°11′59″, E139°08′39″, alt. ca. 170 m)
Japan, Tokyo, Kodzu Isl., Mt. Chichibu (N34°11′59″, E139°08′39″, alt. ca. 170 m)
Japan, Tokyo, Kodzu Isl., Mt. Tenjo (N34°13′09″, E139°09′11″, alt. ca. 500 m)
Japan, Tokyo, Kodzu Isl., Mt. Tenjo (N34°13′10″, E139°09′11″, alt. ca. 500 m)
Japan, Tokyo, Kodzu Isl., Mt. Tenjo (N34°12′51″, E139°09′13″, alt. ca. 500 m)
Japan, Tokyo, Kodzu Isl., Mt. Tenjo (N34°12′50″, E139°09′09″, alt. ca. 500 m)
Japan, Tokyo, Kodzu Isl., Mt. Tenjo (N34°12′50″, E139°09′09″, alt. ca. 500 m)
Japan, Tokyo, Kodzu Isl., Mt. Tenjo (N34°12′51 ″, E139°09′08″, alt. ca. 500 m)

Table 2.

Primers, GenBank accession numbers and total lengths of nuclear EST markers analyzed in this study

Maker

Accession
No.

Total
length(bp)

EST_L141

FS993744

434

EST_L258

FS993861

317

EST_L261

FS993864

320

Primer namePrimer sequence

References

EST_L141F
EST_L141R
EST_L258F
EST_L258R
EST_L261F
EST_L261R

CATTCGTGAGGGTTCAAGGT
ACTGTCCCCTGATTTGATGG
TCATGGCGACTGTGAAGAAGCGCCCTTTGGATTTACGATA
ACGTGGTGGCTTCTGGTTACGCAGCACCAAAACTCCTAGC

Yatabe-Kakugawa et al. 2013
Yatabe-Kakugawa et al. 2013
Yatabe et al. 2009
Yatabe et al. 2009
Yatabe-Kakugawa et al. 2013
Yatabe-Kakugawa et al. 2013

the O. lancea-like plant (KL1) were picked out
randomly in order to measure morphologicalcharacters as indicators of stenophylly using thesame method as Yatabe et al. (2009). The number
of ultimate veinlets of the basal basiscopic vein
was counted, and the angle of pinnule-base wasmeasured.
In a molecular phylogenetic analysis, DNA
was extracted from silica-gel dried material using
a QIAGEN DNeasy mini kit (Qiagen, Valencia
California), following the manufacturerʼs instruction. Three nuclear DNA markers (EST_L141,
EST_L258, EST_L261), which were selectedfrom an expressed sequence tag library developed by Yatabe et al. (2009), and the chloroplastrbcL (ribulose-1,5-bisphosphate carboxylase/
oxygenase large subunit) were analyzed. Primers
for ampliﬁcation and sequencing and detailed
information on the three nuclear markers areshown in Table 2. We used PHASE 2.1.1 toreconstruct probable pairs of haplotypes for eachof the sequences with heterozygous sites (Stephens and Donnelly, 2003). We constructed a
haplotype network using statistical parsimony
methods in TCS v1.2.1 (Clement et al., 2000)

using samples from the Kozu Island, along witheight samples of O. japonica and three of O. lancea analyzed in Tsutsumi et al. (2011) (Appendix
1).
Results
We found only one Osmunda lancea-like individual, which inhabits in a wind beaten areaaround the top of Mt. Tenjo on the Kozu Island(Figs. 1 and 2, Table 1). It has narrow-lanceolate
pinnules, and the base of the pinnules is cuneate,similar to that of O. lancea (Fig. 2). No fertile
leaf was found. Several individuals of O. japonica were found around there (Figs. 1 and 2, Table1), which are moderately small and inhabit under
forests or in crevices of rocks.
The averages of the veinlet numbers of thebasiscopic basal vein and the pinnule-base angles
were plotted for the O. lancea-like individual,
together with samples analyzed in Yatabe et al.
(2009) (Fig. 3). The two values of the O. lancealike individual fell in the range of those in populations of O. japonica and O. lancea. It was,however, an outlier in the putative F1 hybrids of

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Chie Tsutsumi et al.

Fig. 2. Osmunda lancea-like plant and O. japonica at the top of Mt. Tenjo, Kozu Island. A–C. O. lancea-likeplant. A. Habitat. B. Leaf. C. Base of pinnule. D–F. O. japonica.

O.×intermedia, which were identiﬁed by
NewHybrids analysis using ten nuclear DNA
markers in Yatabe et al. (2009).

The genotypes of O. lancea-like plant for thethree examined markers (EST_L141, EST_L258,
EST_L261) revealed that the O. lancea-like plantOsmunda×intermedia in Kozu Island

103

Fig. 3. Morphological characters of pinnules withdata of Kozu island O. lancea-like plant (solid
triangle) added on Fig. 3 of Yatabe et al. (2009).
Osmunda japonica (open circles), O.×intermedia(crosses, identiﬁed as F1 hybrids by NewHybrids
analysis based on ten nuclear DNA markers), O.
lancea (solid diamonds), and possible later-generation hybrids (solid square, identiﬁed as F2
hybrids or backcross hybrids by NewHybrids
analysis).

has two distinct alleles in all markers (Fig. 4);
one is identical to a typical type of alleles in O.
lancea, and the other is coincide with a typicaltype of alleles in O. japonica. There is no difference in the sequences of the chloroplast rbcL
gene of the O. lancea-like plant from those of O.
japonica and O. lancea (data not shown).
Discussion
Results of the morphological and molecularphylogenetic analyses suggested that the
Osmunda lancea-like plant is O.×intermedia, ahybrid between O. japonica and O. lancea. It is
the ﬁrst record of O.×intermedia from the Izu
Islands. It has untypical morphology among
O.×intermedia plants; the angle of pinnule baseis intermediate between those of O. japonica andO.×intermedia, although the number of veinlets
in most basal basiscopic vein is similar to thoseof O.×intermedia (Fig. 3). It suggested that theKozu Island plant is likely an F2 or later-genera-

Fig. 4. Haplotype networks using three nuclear
markers. Doubled circles indicate the haplotypes found in O. lancea-like plant. Size of eachcircle indicates the number of haplotypes(alleles) observed in all the examined individuals. White and black circle show haplotype
derived from O. japonica and O. lancea,
respectively. Sample information is shown inAppendix 1. J1–J8, O. japonica; L1–L3, O. lancea; KJ1–KJ2, O. japonica from the Kozu
island; KL1, O. lancea-like plant from the Kozu
island. Different alphabets following sample
name indicate distinct alleles.

tion hybrid. The genetically putative F2 hybrids
or backcross individuals have extreme morphology in O.×intermedia, which is likely caused bysegregation of genes for morphological characters (Yatabe et al., 2009).
Osmunda japonica occurs in eastern Asia
extending west to the western Himalaya andsouth to northern Vietnam (Kato, 2007). In
Japan, it is widely distributed from northern
Hokkaido to the Ryukyu Islands (Kurata and104

Chie Tsutsumi et al.

Nakaike, 1990). Rheophilous O. lancea isendemic to Japan and has moderately narrow distribution area, from southern Hokkaido toMiyazaki Pref., southern Kyushu, and not distributed in the Ryukyu Islands (Kurata andNakaike, 1990). The distribution of O.×
intermedia almost overlaps that of O. lancea(Kurata and Nakaike, 1990). In the Izu Islands,
however, O. japonica (one parent of O.×
intermedia) is common on inhabited islands
while O. lancea, another parent, is not recorded
to occur (Kurata and Nakaike, 1990). It suggeststhat the Kozu Island O.×intermedia is considered not to be derived from outcrosses between
the two extant neighboring parent plants. The Izu
Archipelago is a chain of volcanic islands without any previous connection to the mainland. The
oceanic islands were probably formed within thepast million years (Kaneoka et al., 1970). The
Kozu Island is composed of at least 16 rhyolitic
monogenetic volcanos and it has been inferredthat the eruption of rhyolites began at least several tens of thousands years ago and was
repeated intermittently (Isshiki, 1982). The latest
and large eruption was at Mt. Tenjo in 838 AD
according to Shoku Nihon Koki, a Japanese old
historical literature, published in 869 AD (Isshiki
et al., 1965). One possible cause of the existenceis that the establishment of O.×intermedia on
the Kozu Island may be involved with a longdispersal of spores of O.×intermedia, which is
known as to be semifertile and has moderate
spore germination rates (4–11%: Shimura, 1964).
Another, less like possibility is that O.×
intermedia was formed in situ by crossing of O.
japonica and O. lancea, which became extinct orwhose spores were dispersed recently.
Osmunda×intermedia usually grows in theupper rheophytic zone. However, the Kozu
Island plant is found on the top of mountain. Its
extreme morphology of narrow-lanceolate pinnules may happen to be appropriate to such awind beaten area around the top of the mountain.

Acknowledgments
The authors thank S. Akiyama, M. K. Bhattacharya, A. Ebihara, C. R. Fraser-Jenkins, H. K.
Goswami, M. Kato, S. Kobayashi, G. Kokubugata and T. Minamitani for their help during thetrips or for providing materials. We are also
grateful to M. Kato for useful suggestions. We
also thank Kenya Ishida who guided the practicaltraining of students of Department of Biological
Sciences, University of Tokyo for the help of theﬁeldwork of Osmunda in the Kozu Island.
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Appendix 1. Localities, voucher information and sequence data deposited at DDBJ/GenBank for material of
Osmundaceae used in molecular analyses. *; analyzed in Tsutsumi et al. (2012).
Osmunda×intermedia. Japan: KL1; Tokyo, Kodzu Isl., Mt. Tenjo; K. Yamamoto et al. s.n. (TNS); EST_L141,
LC057166; EST_L258, LC057169; EST_ L261, LC057183.
Osmunda japonica. Japan: KJ1; Tokyo, Kodzu Isl., Mt. Chichibu; N. Murakami et al. s.n. (TNS); EST_L141,
LC057164; EST_L258, LC057167; EST_L261, LC057181. KJ2; Tokyo, Kodzu Isl., Mt. Chichibu; N. Murakami et al.
s.n. (TNS); EST_L141, LC057165; EST_L258, LC057168; EST_L261, LC057182. J1: Cult. in Tsukuba Botanical Garden (originated from Japan); C. Tsutsumi s.n. (TNS); EST_L141, LC057153; EST_L258, AB672830*; EST_L261,
LC057170. J2; Miyazaki Pref., Kobayashi; G. Kokubogata GK9416 (TNS); EST_L141, LC057154; EST_L258,
AB672831*; EST_L261, LC057171. J3; Kagoshima Pref., Amami Island, Mt. Yuwan; M. Kato et al. s.n. (TNS:764134); EST_L141, LC057155; EST_L258, AB672832*; EST_L261, LC057172. China: J4; Fujian, Mt. Wuyi; A. Ebihara s.n. (TNS: 762111); EST_L141, LC057156; EST_L258, AB672833*; EST_L261, LC057173. J5; Yunnan, Gongshan, Chichi; L.-Y. Kuo 9468 (TAIF); EST_L141, LC057157; EST_L258, AB672834*; EST_L261, LC057174. Bhutan:
J6; Sha-ngawang; S. Matsumoto W.G.M.124 (TNS); EST_L141, LC057158; EST_L258, AB672835*; EST_L261,
LC057175. India: J7; Meghalaya State, Shillong Peak ridge, by site of ruined “Peak Lodge”; C. R. Fraser-Jenkins s.n.
(TNS: 740377); EST_L141, LC057159; EST_L258, AB672836*; EST_L261, LC057176. J8; Central Himalaya, from
Fern Garden, Department of Botany, Government Postgraduate College, Pithoragarh, 262502. (Originated from Champawat); C. R. Fraser-Jenkins s.n. (TNS: 777785); EST_L141, LC057160; EST_L258, AB672837*; EST_L261,
LC057177.
Osmunda lancea. Japan: L1; Cult. in Tsukuba Botanical Garden (transplanted from Shizuoka Pref.); C. Tsutsumi s.n.
(TNS); EST_L141, LC057161; EST_L258, AB672838*; EST_L261, LC057178. L2; Kochi Pref.; A. Ebihara et al.,KC2007-1408 (TNS: 766627); EST_L141, LC057162; EST_L258, AB672839*; EST_L261, LC057179. L3; Miyazaki
Pref.; T. Minamitani s.n. (TNS); EST_L141, LC057163; EST_L258, AB672840*; EST_L261, LC057180.