Bull. Natl. Mus. Nat. Sci., Ser. B, 42(3), pp. 95–102, August 22, 2016

New Records of Liparis purpureovittata (Orchidaceae)
and Identiﬁcation of its Mycorrhizal Fungi
Chie Tsutsumi1,*, Ayako Maeda2, Yumi Yamashita1,3, Takahide Kurosawa3,
Akitomo Uchida4, Taiga Kuhara5 and Tomohisa Yukawa1
1

Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan
2
Kochi Prefectural Makino Botanical Garden, Godaisan 4200–6, Kochi 781–8125, Japan
3
Faculty of Symbiotic Systems Science, Fukushima University,
Kanayagawa 1, Fukushima 960–1296, Japan
4
Shiretoko Museum, 49–2 Hon-machi, Shari, Hokkaido 099–4113, Japan
5
Niigata Prefectural Botanical Garden, Kanazu 186, Akiha-ku, Niigata 956–0845, Japan* E-mail: tsutsumi@kahaku.go.jp(Received 12 May 2016; accepted 22 June 2016)

Abstract We revise the distribution area of Liparis purpureovittata with new records from western Japan (Ehime Pref. and Kochi Pref.) and central to northern Japan. In molecular analyses of
mycorrhizal fungi of L. purpureovittata, Tulasnella (Tulasnellaceae), which is among predominant
mycobiont groups of the Orchidaceae including the other Liparis species, was detected from theplants, suggesting its status of the fungal parter.
Key words : Japan, Liparis, mycorrhiza, orchid, Tulasnella.

Introduction
Liparis Rich. is a cosmopolitan orchid genus
consisting of about 320 species, including epiphytes and terrestrial plants (Pridgeon et al.,2005). In Japan, 17 species of Liparis are recognized (Yukawa, 2015). Liparis purpureovittata
Tsutsumi, T.Yukawa & M.Kato was recently
described as a new species (Tsutsumi et al.,2008). The species is morphologically and phylogenetically close to L. fujisanensis F.Maek. ex
Konta & S.Matsumoto, L. kumokiri F.Maek. andL. koreojaponica Tsutsumi, T.Yukawa, N.S.Lee,
C.S.Lee & M.Kato. It can be distinguished fromthe congeners by the shapes of labellum and column and the ﬂower color (Tsutsumi et al., 2008).
Liparis purpureovittata was recorded in several areas in Hokkaido, Niigata, Nagano, andGunma Prefectures in central to northern Japan(Tsutsumi et al., 2008). Subsequently, the species

was also reported from Toyama Pref., central
Japan (Toyama Prefecture, 2012). Recently, we
found the species from Ehime Pref. and Kochi
Pref. in Shikoku, western Japan. Furthermore,
the species was also observed in several localities from central to northern Japan. In this report,
we revise the distribution area of L. purpureovittata.
Mycorrhizal symbiosis is a key issue to determine habitats or geographical distributions oforchid plants (Rasmussen, 1995; Batty et al.,
2001; Ogura-Tsujita and Yukawa, 2008; Barrett
et al., 2010; Roche et al., 2010). Most mycorrhizal fungi in the Orchidaceae belong to narrow
ranges of taxa in Basidiomycota, such as Ceratobasidiaceae, Tulasnellaceae, and Sebacinaceae,
whereas Glomeromycota, forming arbuscular
mycorrhizae that are common in most land
plants, have not been recorded from the Orchidaceae (Yukawa et al., 2009). In several Liparis

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96

species such as L. japonica (Miq.) Maxim., L.
kumokiri, L. liliifolia (L.) A.Rich ex Lindl., andL. loeselii (L.) Rich., which are closely related to
L. purpureovittata (Tsutsumi et al., 2007), Tulasnella (Tulasnellaceae) has been found withmolecular techniques (McCormick et al., 2004;
Illyés et al., 2005; Shimura et al., 2009; Ding etal., 2014). To clarify mycorrhizal partners of L.
purpureovittata, molecular identiﬁcation of thefungi was performed.
Materials and Methods
Leaves of Liparis purpureovittata collectedfrom Saijo-shi, Ehime Pref. (Y. Hagino et al.
FOS-009187, FOS-009189) were used formolecular identiﬁcation of plants. DNA from theleaf samples was extracted using a QIAGEN
DNeasy Mini Kit (QIAGEN, Valencia, CA) following the manufacturerʼs instruction. The internal transcribed spacer (ITS) regions with the
5.8S region of nuclear ribosomal DNA were
examined as described by Tsutsumi et al. (2007).
The obtained sequencing patterns were comparedto those of the sample of L. purpureovittata
examined by Tsutsumi et al. (2007).
For fungal identiﬁcation, fungal hyphae wereisolated from ﬁve corms of Liparis purpureovittata collected from Saijo-shi, Ehime Pref. The
corms were thoroughly washed in running water.
After the outer tissues of the corms were
trimmed, the remaining inner tissues were sliced
into pieces using clean razor blades. Each piece
was washed three times in sterilized distilledwater and crushed using a sterilized glass rod in
a Petri dish, into which about 15 mL of corn meal
agar (CMA; Nissui Pharmaceutical Co., Tokyo)
containing 150 ppm streptomycin and 50 ppm tetracycline were added. A few days after incubation at approximately 25°C in the dark, fungal
hyphae growing from pelotons (hyphal coils)
were transferred to new CMA plates for puriﬁcation. The isolates were transferred and cultured
on potato dextrose agar slants (Nissui Pharmaceutical Co.) at approximately 25°C in the dark.
The hyphae were directly used for polymerase

chain reaction (PCR) ampliﬁcation without DNA
extraction. The internal transcribed spacer (ITS)
regions with the 5.8S region of nuclear ribosomal
DNA were used for fungal identiﬁcation. A pairof primers ITS1-F and ITS4 (White et al., 1990;
Gardens and Bruns, 1993) was used. PCR was
performed using a Perkin-Elmer 9700DNA thermal cycler (Applied Biosystems, Foster, CA)with Ex Taq DNA polymerase (TaKaRa Bio,
Tokyo) and Ampdirect Plus (Shimadzu, Kyoto);
the reaction conditions were as follows: 30 denaturation, annealing, and elongation cycles for 30 s
at 94°C, 30 s at 50°C, and 90 s at 72°C, respectively, with a ﬁnal elongation step for 7 min at
72°C. The PCR products were puriﬁed using
illustra ExoProStar (GE Healthcare, Buckinghamshire) following the manufacturerʼs instructions. Sequences were analyzed using ABI3130xl
or ABI 3500xl (Applied Biosystems) and assembled using Seqman II (DNAstar Lasergene, WI).
The sequences analyzed in this study were registered in Genbank (LC158688–LC158689 forTulasnella, LC158690 for Exophiala).
The sequences were submitted to BLAST
searches (Altshul et al., 1997) against the NCBI
sequence database (Genbank) to detect closely
matched sequences. We assigned the genus orfamily names to our samples based on the registered sequences with ＞96% ITS similarity. The
BLAST searches revealed that Tulasnella are
candidates of mycorrhizal partner of L. purpureovittata (see Results and Discussion). Therefore we performed molecular phylogenetic analyses using our samples identiﬁed as Tulasnella,
30 registered sequences with high Max scores by
the BLAST searches, and ITS sequences analyzed by Girlanda et al. (2011). Girlanda et al.
(2011) comprehensively analyzed a close relative
group of Tulasnella to our samples. All assembled sequences were aligned using the Clustal X
program (Thompson et al., 1997) and thenaligned manually. The alignment was easily performed, whereas the nuclear ribosomal operon of
the Tulasnellaceae is known to evolve exceedingly rapidly (Taylor et al., 2002; Binder et al.,2005; Moncalvo et al., 2006). For outgroups,

New Records of Liparis purpureovittata

mycorrhizal fungi from Dendrobium crumenatum (AJ313438) and Vanda ʻMiss Joaquimʼ
(AJ313443) were used in the ITS analysis basedon the result of phylogenetic analysis in Girlanda
et al. (2011).
Phylogenetic analyses were performed using
Bayesian analysis. Ambiguous bases and gaps
were treated as unknown (N) and missing data,
respectively. MrModeltest 2.0 was used to determine nucleotide substitution models (Nylander,2004). GTR ＋ I ＋ G models were selected.
Bayesian searches were conducted using Markov
Chain Monte Carlo with two independent sets offour chains; each was run for 10 million generations with sampling every 100 generations byusing MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and Huelsenbeck, 2003).
The program Tracer (Rambaut and Drummond,2009) was used to check the runs that hadreached stationarity; the effective sample size ofall the parameters was high (＞200). The ﬁrst 2.5
million generations were discarded as burn-in
periods and the remaining trees were used to calculate posterior probabilities.
Results and Discussion
New localities
Liparis purpureovittata was found in cooltemperate mixed conifer and deciduous forests atthe border between Saijo-shi, Ehime Pref. andAgawa-gun, Kochi Pref. (Y. Hagino et al. FOS009187, FOS-009189, ca. 1670 m alt., 2 Jul.
2015 [MBK0272829, MBK0272831]; Figs. 1, 2.
T. Ishijo and M. Hyodo 17922, 17923, ca. 1700 m
alt., 29 Jun. 2015. Saijo-shi, Ehime Pref. [TNS]).
Identiﬁcation was conﬁrmed by molecular analyses; the ITS sequences of the three individuals
collected by Hagino et al. were identical to thosepreviously examined by Tsutsumi et al. (2007),
except one ambiguous nucleotide site (data notshown). It is the ﬁrst record of the species fromwestern Japan. The habitats were characterized
by leaf litter and mosses along roadsides or at thesides of open spaces. There were more than 30
individuals, about ﬁve of which were ﬂowering97

at 2 July 2015. The large individuals with aboutnine ﬂowers were observed in partially shaded
places under the shrubs, and the small individuals with 3–6 ﬂowers and juveniles were in lightplaces. Some individuals had no purple tints in
the green ﬂowers. In this area, several individuals of the orchid Malaxis monophyllos (L.) Sw.
occurred together.
We also recorded L. purpureovittata fromother localities in central and northern Japan(Fig. 2); Shari-gun, Hokkaido, 280 m alt., 21 Jul.
2013 (A. Uchida s.n. [TNS]), Rebun Island, Hokkaido (S. Miyamoto pers. comm., determined byphotographs), Yuza-machi, Yamagata Pref.,1120 m alt. (K. Sawa and S. Kawakami, pers.
comm., determined by photographs), Yama-gun,
Fukushima Pref., 1385 m alt., 9 Jul. 2013 (Y.
Yamashita 264 [FKSE 69074]), and Ohno-gun,
Gifu Pref. (H. Nakayama pers. comm., determined by photographs). In the Shari-gun plants,
green ﬂowers without purple tints were also
observed. Those ﬂowers are morphologicallysimilar except in the ﬂower color.
In Shari-gun, Hokkaido, three individuals
were observed in the semi-open and partiallyshaded places. In Yuza-machi, Yamagata Pref.,almost 10 individuals were found on the damp
ground with Nephrophyllidium crista-galli (Menzies ex Hook.) Gilg. In Yama-gun, Fukushima
Pref., 30 individuals occurred along a trail in themountain grassland. Based on those results, wefound out that L. purpureovittata is widely distributed in light and partially shaded places inmountain grasslands, in damp grounds or alongroadsides in open places of the cool temperatezones from western to northern Japan.
Mycorrhizal partner
We analyzed the ITS regions of 13 isolates
randomly selected from 30 fungal isolates
obtained from L. purpureovittata. BLAST
searches for the ITS regions of the samples
showed that four of the 13 isolates had highafﬁnities with Tulasnella (Tulasnellaceae, Basidiomycota) and a single isolate was closely related
to Exophiala (Herpotrichiellaceae, Ascomycota).

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98

Fig. 1. Liparis purpureovittata from new locality, at border of Saijo-shi, Ehime Pref. and Agawa-gun, Kochi
Pref. A, habitat. B, ﬂower with a purple dot at center of lip. C, green ﬂowers with no purple dot.

New Records of Liparis purpureovittata

99

Fig. 2. Distribution map of L. purpureovittata. Solid circles, new localities determined by voucher specimens;
solid triangles, new localities determined by photographs; open circles, localities recorded in Tsutsumi et al.
(2008).

The other eight isolates were assigned to members of Clonostachys rogersoniana, Myrothecium
gramineum, M. roridum, Paraphaeosphaeria
neglecta, Peyronellaea glomerata, Pleosporalessp., Tolypocladium sp., and Trichoderma polysporum. They were excluded from the candidatesof mycorrhizal fungi, because they were suggested to be members of Hypocreales andPleosporales, which have been regarded as either
endophytic fungi or contaminants (Dearnaley etal., 2012; Oliveira et al., 2014).
The four isolates were obtained from four of
the ﬁve corms of L. purpureovittata, suggestingthat Tulasnella are commonly associated withthis species. Three of the four isolates of Tulasnella had 100% identical sequence and the fourth
was 99.2% identical to the other three in the ITS
region. Considering that Tulasnella has beenfound in other Liparis species (McCormick etal., 2004; Illyés et al., 2005; Shimura et al.,2009; Ding et al., 2014), Tulasnella are likely
represent of the main fungal partner of L. purpureovittata. Exophiala was detected only oncefrom a single sample in this study; it is unlikelythat Exophiala is the main mycorrhizal partner of
L. purpureovittata. Exophiala has previously
been identiﬁed as possible orchid endophytes(Stark et al., 2009), but their ecological role inorchids is unclear (Pecoraro et al., 2013).
Preliminary molecular phylogenetic analyses
using several sequences of Tulasnellaceae registered in Genbank suggested that the four isolatesof Tulasnella were included in the “A1” clade inGirlanda et al. (2011) (data not shown). The ITS
phylogenetic tree using the four Tulasnella isolates detected in this study, closest matchingsequences of them revealed by BLAST searches,and representative sequences in “A1” clade used
by Girlanda et al. (2011), revealed that the fungi
isolated from L. purpureovittata was Tulasnellacalospora or relatives (Fig. 3). The tree also
showed that the fungi of L. purpureovittata wereclose to those of several terrestrial orchids, Anacamptis laxiﬂora (Lam.) R.M.Bateman, Pridgeon& M.W.Chase, Chloraea bletioides Lindl., Dactylorhiza incarnata (L.) Soó, Evotella rubiginosa

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Chie Tsutsumi et al.

Fig. 3. Consensus tree of Tulasnella A1 clade by Bayesian analysis based on ITS sequences (522 bp). Mycorrhizal fungi isolated from Liparis purpureovittata are shown by bold font. Other materials than the fungi from L.
purpureovittata were shown by the names of fungi, or host plants, followed by Genbank accession numbers,
or sample ID of Girlanda et al. (2011). Figures above branches indicate posterior probabilities (p ＞ 0.9) byBayesian method. Outgroups, fungi from Dendrobium crumenatum (AJ313438) and Vanda ʻMiss Joaquimʼ
(AJ313443), were determined based on the results of Girlanda et al. (2011).

New Records of Liparis purpureovittata(Sond. ex Bolus) Kurzweil & H.P.Linder, Goodyera pubescens R.Br., Gymnadenia conopsea(L.) R.Br., Liparis japonica, Ophrys fuciﬂora(F.W.Schmidt) Moench, Serapias parviﬂora
Parl., and Serapias vomeracea (Burm.f.) Briq
(Fig. 3). These orchids usually live in light andsometimes wet places in temperate grasslands inthe northern hemispheres, Chile, and South
Africa. Therefore, the mycorrhizal partner of L.
purpureovittata and its relatives are possiblycommon in terrestrial orchids distributed in lightgrasslands in worldwide temperate zones. The
four isolates were also close to those from otherdeciduous terrestrial Liparis; L. japonica, L.
kumokiri, and L. loeselii, suggesting that these
fungi may be predominant mycorrhizal partnersof the deciduous terrestrial Liparis.
Acknowledgments
We thank Y. Hagino, S. Hagino, H. Hongo,
M. Hyodo, T. Ishijo, S. Kawakami, M. Matsui,
S. Mitsuhashi, S. Miyamoto, T. Nagai, H.
Nakayama, K. Oikawa, K. Sakamoto, M. Sato,
K. Sawa, T. Sekijyo, H. Takeuchi, and K. Watanabe for providing the samples or helping us during our ﬁeld trips. We are also grateful to K.
Suzuki, Y. Yamada, M. Nakajima, and K. Abe forcultivating the living plants and fungi and to P.
Abe for teaching us the techniques of fungal isolation.
References
Altshul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J.
and Zhang, Z. 1997. Gapped Blast and PSI-BlAST: a
new generation of protein database search programs.
Nucleic Acids Research 25: 3389–3402.
Barrett, C. F., Freudenstein, J. V., Taylor, D. L. and Kõljalg, U. 2010. Rangewide analysis of fungal associations in the fully myco-heterotrophic Corallorhiza striata complex (Orchidaceae) reveals extreme speciﬁcity
on ectomycorrhizal Tomentella (Thelephoraceae)
across North America. American Journal of Botany 97:
628–643.
Batty, A. L., Dixon, K. W., Brundrett, M. and Sivasithamparam, K. 2001. Constraints to symbiotic germinationof terrestrial orchid seed in a Mediterranean bushland.

101

New Phytologist 152: 511–520.
Binder, M., Hibbett, D. S., Larsson, K. H., Larsson, E.,
Langer, E. and Langer, G. 2005. The phylogenetic distribution of resupinate forms across the major clades ofmushroom-forming fungi (Homobasidiomycetes). Systematics and Biodiversity 3: 113–157.
Dearnaley, J. D. W., Martos, F. and Selosse, M.-A. 2012.
Orchid mycorrhizas: Molecular ecology, physiology,
evolution and conservation aspects. In: Hock, B. (ed.),Fungal Associations, 2nd ed. Springer, Berlin.
Ding, R., Chen, X. H., Zhang, L. J., Yu, X. D., Qu, B.,
Duan, R. and Xu, Y. F. 2014. Identity and speciﬁcity ofrhizoctonia-like fungi from different populations of
Liparis japonica (Orchidaceae) in Northeast China.
Plos ONE 9: e105573.
Gardes, M. and Bruns, T. D. 1993. ITS primers withenhanced speciﬁcity for basidiomycetes—application
to the identiﬁcation of mycorrhizae and rusts. Molecular Ecology 2: 113–118.
Girlanda, M., Segreto, R., Cafasso, D., Liebel, H. T.,
Rodda, M., Ercole, E., Cozzolino, S., Gebauer, G. andPerotto, S. 2011. Photosynthetic Mediterranean
meadow orchids feature partial mycoheterotrophy andspeciﬁc mycorrhizal associations. American Journal of
Botany 98: 1148–1163.
Huelsenbeck, J. P. and Ronquist, F. 2001. MrBayes:
Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755.
Illyés, Z., Rudnóy, S. and Bratek, Z. 2005. Aspects of in
situ, in vitro germination and mycorrhizal partners of
Liparis loeselii. Acta Biologica Szegediensis 49: 137–
139.
McCormick, M. K., Whigham, D. F. and OʼNeill, J. 2004.
Mycorrhizal diversity in photosynthetic terrestrial
orchids. New Phytologist 163: 425–438.
Moncalvo, J. M., Nilsson, R. H., Koster, B., Dunham,
S. M., Bernauer, T., Matheny, P. B., Porter, T. M.,
Margaritescu, S., Weiss, M., Garnica, S., Danell, E.,
Langer, G., Langer, E., Larsson, E. and Larsson K.-H.
2006. The cantharelloid clade: dealing with incongruent gene trees and phylogenetic reconstruction methods. Mycologia 98: 937–948.
Nylander, J. A. A. 2004. Mrmodeltest 2.0. Uppsala, Sweden: Evolutionary Biology Centre.
Oliveira, S. F., Bocayuva, M. F., Veloso, T. G. R.,
Bazzolli, D. M. S., da Silva, C. C., Pereira, O. L. andKasuya, M. C. M. 2014. Endophytic and mycorrhizal
fungi associated with roots of endangered nativeorchids from the Atlantic Forest, Brazil. Mycorrhiza
24: 55–64.
Ogura-Tsujita, Y. and Yukawa, T. 2008. Epipactis helleborine shows strong mycorrhizal preference towards ectomycorrhizal fungi with contrasting geographic distributions in Japan. Mycorrhiza 18: 331–338.
Pecoraro, L., Girlanda, M., Kull, T., Perini, C. and102

Chie Tsutsumi et al.

Perotto, S. 2013. Fungi from the roots of the terrestrial
photosynthetic orchid Himantoglossum adriaticum.
Plant Ecology and Evolution 146: 145–152.
Pridgeon, A. M., Cribb, P. J., Chase, M. W. and Rasmussen, F. N. 2005. Genera Orchidacearum: Epidendroideae (Part one). Vol. 4, 453–486. Oxford University
Press, New York.
Rambaut, A. and Drummond, A. J. 2009. Tracer v1.5,
Available at http://beast.bio.ed.ac.uk/Tracer.
Rasmussen, H. N. 1995. Terrestrial orchids from seed to
mycotrophic plant. Cambridge University Press, Cambridge.
Roche, S. A., Carter, R. J., Peakall, R., Smith, L. M.,
Whitehead, M. R. and Linde, C. C. 2010. A narrowgroup of monophyletic Tulasnella (Tulasnellaceae)
symbiont lineages are associated with multiple speciesof Chioglottis (Orchidaceae): Implications for orchid
diversity. American Journal of Botany 97: 1313–1327.
Ronquist, F. and Huelsenbeck, J. P. 2003. MrBayes 3:
Bayesian phylogenetic inference under mixed models.
Bioinformatics 19: 1572–1574.
Shimura, H., Sadamoto, M., Matsuura, M., Kawahara, T.,
Naito, S. and Koda, Y. 2009. Characterizat ion of
mycorrhizal fungi isolated from the threatened Cypripedium macranthos in a northern island of Japan: two
phylogenetically distinct fungi associated with theorchid. Mycorrhiza 19: 525–534.
Stark, C., Babik, W. and Durka, W. 2009. Fungi from theroots of the common terrestrial orchid Gymnadenia
conopsea. Mycological Research 113: 952–959.
Swofford, D. L. 2002. PAUP*, Phylogenetic analysis
using parsimony (* and other methods), Version
4.0b10. Sinauer Associates, Sunderland, MA.
Taylor, D. L., Bruns, T. D., Leake, J. R. and Read, D. J.
2002. Mycorrhizal speciﬁcity and function in myco-

heterotrophic plants. In: van der Heijden, M. G. A. andSanders, R. I. (eds.), The Ecology of Mycorrhizas,
pp. 375–414. Springer, Berlin.
Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin,
F. and Higgins, D. G. 1997. The Clustal X Windows
Interface: ﬂexible strategies for multiple sequencealignment aided by quality analysis tools. Nucleic
Acids Research 25: 4876–4882.
Toyama Prefecture 2012. Red Data Book of Vascular
Plants, http://www.pref.toyama.jp/cms_sec/1709/kj000
13513-010-01.html (in Japanese).
Tsutsumi, C., Yukawa, T., Lee, N. S., Lee C. S. and Kato,
M. 2007. Phylogeny and comparative seed morphology
of epiphytic and terrestrial species of Liparis (Orchidaceae) in Japan. Journal of Plant Research 120: 405–
412.
Tsutsumi, C., Yukawa, T. and Kato, M. 2008. Liparis purpureovittata (Orchidaceae)—a new species from Japan.
Acta Phytotaxonomica et Geobotanica 59: 73–77.
White, T. J., Bruns, T. D., Lee, S. and Taylor, J. W. 1990.
Ampliﬁcation and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M. A.,
Gelfand, D. H., Sninsky, J. J. and White, T. J. (eds.),
PCR Protocols: A Guide to Methods and Application.
Academic Press, San Diego, CA.
Yukawa, T., Ogura-Tsujita, Y., Shefferson, R. P. andYokoyama, J. 2009. Mycorrhizal diversity in Apostasia(Orchidaceae) indicates the origin and evolution oforchid mycorrhiza. American Journal of Botany 96:
1997–2009.
Yukawa, T. 2015. Orchidaceae. In: Ohashi, H., Kadota,Y., Murata, J., Yonekura, K. and Kihara, H. (eds.), Wild
Flowers of Japan. Vol. 1, pp. 178–231, pl. 125–164.
Heibonsha, Tokyo (in Japanese).