Bull. Natl. Mus. Nat. Sci., Ser. B, 34(2), pp. 89–94, June 23, 2008

Taxonomic Status of Lipairs japonica and L. makinoana (Orchidaceae):
A Preliminary Report
Chie Tsutsumi and Tomohisa Yukawa
Department of Botany, National Museum of Nature and Science, Amakubo 4–1–1, Tsukuba 305–0005, Japan
E-mail: tsutsumi@kahaku.go.jp

Abstract To clarify the taxonomic status of Liparis japonica and L. makinoana, we investigatedmolecular differences among three dubious entities, which have been assigned to L. japonica or L.
makinoana. The three entities were distinct in nucleotide substitutions of the nuclear ribosomal
ITS region. A morphological comparison of the three entities and the original descriptions of thetwo species indicates that the type of L. japonica is distinct from the plants currently called “L.
japonica” and is identical with Malaxis monophyllos. Further, the entity widely called “L. makinoana” does not agree with the protologue of L. makinoana.
Key words : Japan, Liparis japonica, Liparis makinoana, molecular phylogeny, taxonomy.

Introduction
Liparis Rich. (Orchidaceae), consisting of over
400 species (Cribb and Govaerts, 2005), is widely distributed in tropical and temperate regions of
the world. It is characterized by one or more relatively soft, fleshy, green leaves, which sheathe afleshy pseudobulb or corm. A terminal inflorescence has small, resupinate flowers with a longarching column and four pollinia without viscidia. Section Liparis, one of its 19 sections, isdefined by the undeveloped pseudobulb at anthesis and two, subfleshy, non-ribbed leaves borne in
the apical part of the pseudobulb (Garay andGonzalez, 1999). In Japan, nine of 15 described
Liparis species are assigned to sect. Liparis; L.
auriculata Blume ex Miq., L. fujisanensis F.
Maek. ex Konta et S. Matsumoto, L. hostaefolia(Koidz.) Koidz. ex Nakai, L. japonica (Miq.)
Maxim., L. krameri Franch. et Sav., L. kumokiri
F. Maek., L. makinoana Schltr., L. purpureovittata Tsutsumi, T. Yukawa et M. Kato, and L. truncata F. Maek. ex T. Hashim. Our previous molecular phylogenetic study using these species except L. hostaefolia revealed that sect. Liparis inJapan is separated into three clades; L.
auriculata, the Krameri clade including L.

krameri and L. truncata, and a clade comprising
the other species. The last clade is subdivided
into two groups: one comprises L. purpureovittata (labeled as L. sp. [24, 27] in Tsutsumi et al.,2007) and the Kumokiri clade (L. fujisanensis, L.
sp. [treated as L. koreana in Tsutsumi et al.,2007] and L. kumokiri), and the other is theMakinoana clade consisting of L. japonica and L.
makinoana (Tsutsumi et al., 2007).
Our molecular and morphological studies discovered a new and a putatively new species in
the Kumokiri clade and related species. Oneis L. purpureovittata, which is vernacularly recognized as “Azumi-kumokiri”, “Chikumajigabachi”, “Fugaku-kumokiri” and “Nanbukumokiri” (Tsutsumi et al., 2008). The other putatively new species is L. sp., which has been
misidentified as L. makinoana var. koreana
Nakai or L. koreana (Nakai) Nakai ex W. T. Lee(Tsutsumi et al., unpubl. data). Previous insufficient recognition of the group is supposed to becaused by the lack of important diagnostic characters in herbarium specimens, such as the morphology of the anther cap and the three-dimensional structure of the perianth lobes. Anotherreason is insufficient descriptions of these taxa,
in which such diagnostic characters were mostly90

C. Tsutsumi and T. Yukawa

overlooked.
Much confusion exists in interpretations on Liparis japonica and L. makinoana. L. japonicaand L. makinoana have been recorded from eastern Asia; China, Japan, Korea, Russia, and Taiwan for L. japonica, and Japan, Korea and Russia for L. makinoana (Komarov, 1968; Satomi,1982; Chen et al., 1999; Su, 2000; Lee, 2002).
Historically, key taxonomic references on Japanese flora interpreted that the two species are distinguishable in the length of inflorescence andthe size of flower: L. makinoana has a shorter inflorescence and a larger flower than L. japonica(Maekawa, 1971; Ohwi, 1978; Satomi, 1982;

Takahashi, 1985; Hashimoto, 1990; Hashimotoand Kanda, 1991). These diagnostic characters,
however, do not separate the two species satisfactorily. Our preliminary observation on the basisof morphological characters indicated that thisspecies complex may comprise three entities(Fig. 1).
To clarify the taxonomic status of these entities in the species complex, we performed preliminary macromoleular and morphological studies, using three entities assigned to L. makinoanaand L. japonica (Fig. 1). The three entities arecalled Types 1–3 here to avoid further name confusion.

Fig. 1. Types 1–3 in Liparis cultivated at Tsukuba Botanical Garden, National Museum of Nature and Science.
A–B, Type 1 (C. Tsutsumi & H. Nakayama L6); C–D, Type 2 (C. Tsutsumi L7); E–F, Type 3 (C. Tsutsumi L8).
A, C, E, Flowering plant; B, D, F, Flower, front view. Photographs taken by C. Tsutsumi.

Taxonomic status of Liparis91

Table 1.

Nucleotide variations of the three Liparis entities in the nuclear ribosomal ITS region.

EntitySource; voucher

Nuclear ribosomal ITS

Type 1
Type 2Type 3Kanagawa, Japan; C. Tsutsumi and H. Nakayama L6
Unknown; C. Tsutsumi L20
Hokkaido, Japan; C. Tsutsumi, K. Watanabe and H. Hongo L2
Unknown; C. Tsutsumi L7
Unknown; C. Tsutsumi L8

Materials and Methods
Materials were collected in the field or fromplants cultivated at Tsukuba Botanical Garden,
National Museum of Nature and Science (Table1). Vouchers are deposited in the Department ofBotany Herbarium (TNS). Molecular analyses,
procedures of extraction, amplification and sequencing were conducted, following Tsutsumi etal. (2007). Genetic regions examined were internal transcribed spacer regions of the 18S-26S nuclear ribosomal DNA (ITS) and three plastid regions, trnL and its flanking trnL-trnF spacer,
trnS-trnG spacer and part of the maturase-encoding gene (matK ). GenBank accession numbers ofITS, trnL and its flanking trnL-trnF spacer, trnStrnG spacer and partial matK of Type 3 (C. Tsutsumi L8) are AB435655, AB435656, AB435657,AB435658, respectively. The others were shownin Tsutsumi et al. (2007), in which Types 1 and 2
were named as L. makinoana and L. japonica, respectively.
The phylogenetic relationships were deducedby the maximum likelihood (ML) method with
PAUP* 4.0b10 (Swofford, 2002), following Tsutsumi et al. (2007). Modeltest 3.7 (Posada andCrandall, 1998) was used to determine the nucleotide substitution model. Bootstrap values
were calculated with 1000 replicates by the maximum parsimony (MP) method. Bayesian inference of phylogeny was performed using MrBayes
3.1.2 to estimate posteriori support of clades inML tree (Huelsenbeck and Ronquist, 2001) withthe nucleotide model determined by MrModeltest 2.0 (Nylander, 2004). Bayesian searches

138453534672710

A
AA
A
GGGGGCCCCCTAAGGAGGAAG

were conducted by mcmc with four chains overone million generations, sampling every 100 generations. A quarter of trees obtained were discarded as burn-in trees.
Results and Discussion
The molecular phylogenetic analysis showedthat the three entities formed a monophyleticclade, although the phylogenetic relationshipamong the three was unclear (Fig. 2). In the ITS
region of the three entities, Types 1 and 2 are distinguishable by two substitutions, and Type 3 differs from Type 1 by three substitutions and fromType 2 by five substitutions (Table 1). There is nosubstitution among the three types in the plastid
regions. However, two to five substitutions in theITS region among Types 1–3 endorsed independent status of each type.
A preliminary comparison of floral characters
was made for the three entities, Types 1–3. The
result is summarized in Table 2. The three entities are distinct in the combination of floral characters: Type 1 has the largest labellum andblooms earlier than the others. Type 2 is intermediate between Types 1 and 3 in the labellum size.
Type 3 has the longest inflorescence, on which
the flowers are sparser than those of the other twotypes. Type 3 is also characterized by a somewhat
thickened, deep-colored apex of lateral sepal.
The present study revealed that the species
complex currently interpreted as Liparis japonica and L. makinoana includes three entities,
which are distinct by the molecular and the morphological characters. Types 1 and 2 were ap-92

C. Tsutsumi and T. Yukawa

Fig. 2. Maximum Likelihood (ML) tree of Liparis section Liparis (ln4586.53) based on ITS sequences (825bp.), revised by Tsutsumi et al. (2007). Figures above and below branches indicate posterior probabilities(0.9) calculated by Bayesian analysis and bootstrap values (50%) by maximum parsimony analysis, respectively. L. nervosa is chosen as an outgroup.

plied to be L. makinoana and L. japonica, respectively, in the pictures of Satomi (1982) andHashimoto and Kanda (1991), and Type 3 was
identified as L. japonica in the illustration ofIinuma (1913). To clarify this nomenclatural confusion, we need to examine protologues and typespecimens of L. japonica and L. makinoana.
Liparis japonica was originally described asTaxonomic status of Liparis

Microstylis japonica by Miquel (1866) based on
Buerger s.n. (a specimen with fruits collected inJapan). Although the type specimen has not been
located (Ohba et al., 2005; A. Schuiteman, pers.
commun.), morphological characters described in
the protologue suggested that this species issynonymous with Malaxis monophyllos Lindl.
Transfer of this entity to Lparis is inappropriate
and identification of Type 2 or 3 to this namemust be mistaken.
Liparis makinoana was described bySchlechter (1919), where he emphasized the differences from L. liliifolia of North America. Thetype specimen collected from Hokkaido, Japanwas destroyed during the World War II and theduplicates were not located (Yukawa and Ohba,1995). Furthermore, Schlechter (1919) did not
mention any qualitative characters to distinguishthe three entities. However, the size of labellum(12 mm long, 8 mm wide) in the description is
identical to those of Type 2, rather than Type 1
currently interpreted as L. makinoana.
In conclusion, we demonstrated that Type 2,
designated as “Liparis japonica” in most references available in Japan, is likely to represent L.
makinoana, and Types 1 and 3 do not match withany published scientific names.
AcknowledgmentsWe thank M. Goto, H. Hongo, K. Miyoshi, T.
Nagai, H. Nakayama, K. Oikawa, K. Sakamotoand K. Watanabe for their assistance during ourfield trips. We also thank Y. Hirayama for sequencing, E. Isono for translation of German, M.
Nakajima for useful advices, and K. Suzuki forskilful cultivation of the living materials. This
study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (T.Y.) and a research grantfrom the Fujiwara Natural History Foundation (toC.T.).

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93

Table 2. Comparison of the three Liparis entities
in floral characters and flowering season.
Type 1
Flower stalk length
Flower number
Labellum length
Labellum width
Flowering season

Type 2Type 3

10–25 cm 10–30 cm 15–40 cm
4–16
4–30
10–40
14–17 mm 9–12 mm 8–10 mm
11–15 mm 6–8 mm
5–7 mmMay–Jun. Jun.–Jul.
Jun.–Jul.

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