Bull. Natl. Mus. Nat. Sci., Ser. B, 43(3), pp. 79–86, August 22, 2017

Pogonia subalpina (Orchidaceae): a new species from Japan
Tomohisa Yukawa* and Yumi Yamashita
Tsukuba Botanical Garden, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki 305–0005, Japan*E-mail: yukawa@kahaku.go.jp(Received 11 May 2017; accepted 28 June 2017)

Abstract Pogonia subalpina T. Yukawa & Y. Yamashita (Orchidaceae) is newly described frommarshy, subalpine grasslands of central and northern parts in Honshu, Japan. This species is similar to Pogonia japonica Rchb. f. and P. minor (Makino) Makino, but can be distinguished in
ﬂower by its short hairy crests on the disk of the labellum mid-lobe, by its glabrous abaxial surfaceof the labellum, and by its single, broad, purplish pink band along the mid-vein of the petals.
Nucleotide sequence divergences among the three entities in the nuclear ribosomal DNA ITS andthe plastid trnK 5ʼ intron regions warrant independent species status of the new entity.
Key words : Japan, new species, Orchidaceae, Pogonia, taxonomy.

Introduction
The genus Pogonia Juss. is a small orchid
genus in which four species, i.e., P. japonica
Rchb. f., P. minor (Makino) Makino, P. ophioglossoides (L.) Ker Gawl., and P. yunnanensis
Finet, are currently accepted (Pridgeon et al.,2003). P. ophioglossoides is widespread throughout eastern North America and the rest are distributed in continental East Asia and Japan. This
genus thus represents a signiﬁcant case of theeastern North American-Asian biogeographic
disjunction (Chen, 1983). Cameron and Chase(1999) conducted a phylogenetic analysis of P.
japonica, P. minor and P. ophioglossoides andalso calibrated divergence time among themusing the molecular clock hypothesis. They demonstrated that the disjunction between P. ophioglossoides in eastern North America with P.
japonica and P. minor in eastern Asia is best
explained by speciation following a northwestward migration via Bering land bridges in the
Tertiary.
In Japan, distribution of two species, P. japon-

ica and P. minor has been generally recognised(Yukawa, 2015). However, another entity that
does not match morphologically with either ofthem was tentatively designated a Japanese name
“Miyama-tokiso” (Takahashi, 1987). In this study
we evaluate the taxonomic status of thisneglected entity.
Materials and Methods
Observation and measurement of morphological characters was based on living plants, dried
herbarium specimens, and spirit-preserved specimens listed in the section “Other specimens
examined”.
Materials used in the DNA analysis are shownin Table 1. DNA was extracted from fresh ordried leaves. Nucleotide sequences were determined by amplifying the internal transcribedspacer (ITS) regions of the 18S–26S nuclear
ribosomal (nr) DNA and a region of the plastid
genome encompassing ca.600 base pairs of theinitial 5ʼ region of trnK 5ʼ intron via the polymerase chain reaction (PCR) from a total DNA

80

Tomohisa Yukawa and Yumi Yamashita
Table 1.

Materials used for DNA sequencing. All voucher specimens are deposited at TNS

Species

Locality

Pogonia subalpina (new entity)
Pogonia japonica
Pogonia minor
Table 2.

VoucherJapan, Honshu. Miyagi Pref.
Japan, Honshu. Niigata Pref.
Japan, Honshu. Nagano Pref.
Japan, Honshu. Kyoto Pref.
Japan, Hokkaido
Japan, Honshu. Niigata Pref.
Japan, Honshu. Nagano Pref.

Y. Mikanagi s. n.
C. Tsutsumi, T. Kuhara & M. Sato CT1079
T. Yukawa 16-14
J. Nagasawa s. n.
T. Asanuma 2267
C. Tsutsumi, T. Kuhara & M. Sato s. n.
T. Hashimoto s. n.

Diagnostic characters among Pogonia subalpina, P. japonica and P. minor

CharacterP. subalpina (new entity)

P. japonica

P.minorFlowering shoot height (cm)
Leaf width (mm)Flower posture
Sepal openness
Purplish pink band along petal mid-vein
Petal margins
Labellum length (mm)Labellum abaxial surface
Labellum midlobe outline
Labellum midlobe hairy crest length
Rostellum

9–16
10–20
lateralpatentpresent
nearly entire
15–18
glabrous
ligulateless than 0.8 mm
developed

10–35
7–12
lateralpatentabsenterose-dentate
15–22
puberulent
elliptic-obovate
2–3 mmdeveloped

8–20
4–12
upright
semi-closed
absententire
10–11.5
puberulent
ligulate
1 mm
absentextract. Experimental methods follow thosedescribed in Topik et al. (2005) and Yukawa etal. (2005) in which the primer information of thesequenced regions is also shown. Voucher specimens were deposited at TNS.
The ITS and trnK 5ʼ intron data sets werealigned using MUSCLE implemented in MEGA
7.0 (Kumar et al., 2016) and modiﬁed manually
to minimize the number of gaps. All insertions/deletions were coded as missing data in the phylogenetic analysis of ITS. Cleistesiopsis bifaria(Fernald) Pansarin, C. divaricata (L.) Pansarin &F. Barras and Isotria verticillata (Muhl. ex
Willd.) Raf. were chosen as outgroup taxa on thebasis of the results of previous phylogenetic
analyses of Pogonia and allied genera (Cameron
and Chase, 1999; Cameron, 2009). The evolutionary history was inferred by using a modelbased Bayesian approach and the Maximum
Likelihood method. A Bayesian search was performed using MeBayes 3.2 (Ronquist et al.,2012) and using the program Kakusan 4 (Tanabe,2011) to select the ʻbest-ﬁtʼ model of evolution
under the Akaike information criterion. Two separate runs were carried out using the GTR＋I＋Γ

model in each analysis. The Markov chains wererun for 1 million generations and the ﬁrst 10
thousand runs were burned in. Maximum likelihood (ML) analysis was conducted using MEGA
7.0 based on the Tamura-Nei model (Tamura andNei, 1993). The initial tree(s) for the heuristic
search were obtained automatically by applying
Neighbor-Joining and BioNJ algorithms to amatrix of pairwise distances estimated using theMaximum Composite Likelihood approach, andthen selecting the topology with superior log
likelihood value. Bootstrap analysis (Felsenstein,1985) with 1000 replicates was used for ML
analysis to assess the relative strength of support
for all branches. The ML trees were visualizedwith MEGA 7.0.
Results and Discussion
Morphological characters
Characters discriminating the new entity fromthe closely related Pogonia japonica and P.
minor are shown in Table 2. Among these, themost notable diagnostic character of this entity is
its shorter hairy crests of the labellum mid-lobe

A New Orchid Species from Japan

81

Table 3. Variable nucleotide sites and insertions/deletions found in nrITS and trnK 5ʼ intron sequence alignment
among Pogonia subalpina, P. japonica and P. minor
Genetic region
nrITS

trnK 5ʼ intron

Site no.

P. subalpina(new entity)

P. japonica

P. minor

18
210
289
350
547
209
214-18228232
235-41
242-47248-53254-58584
587-90

T
G
G
G
T
A
TAACA
C
T
--TACATA--TACAT
A
TATG

T
A
T
T
G
—
--T
C
ATTTTTATACATA
TTTTTA
TACAT
T
ATCA

C
G
G
G
G
—
--T
C
--------T
ATCA

Fig. 1. Phylogenetic relationships of Pogonia inferred from nrITS sequences. The tree is rooted by outgroup taxa
Cleistesiopsis bifaria, C. divaricata and Isotria verticillata. The tree was calculated using the maximum likelihood method and the topology was the same as the Bayesian inference analysis. Numbers at the nodes indicate
Bayesian posterior probabilities (PP) and bootstrap percentages (BS) shown as PP/BS.

than those of the other species. A broad, purplish
pink band along the mid-vein of the petals is
another unique feature of this entity. The heightof the ﬂowering shoot overlaps among the threetaxa, but the new entity is shorter than the others
in most individuals. Flower size of this entity is
generally intermediate between the two others.
Besides, P. ophioglossoides, a North Americancongener, has an entire labellum and P. yunna-

nensis from southwestern part of China does notdevelop hairs on the labellum.
Macromolecular characters
In both ITS and trnK 5ʼ intron regions,sequences were identical for three samples of the
new entity collected at different sites. In ITS, the
new entity was well demarcated from Pogonia
japonica and P. minor by four and two substitu-

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Tomohisa Yukawa and Yumi Yamashita

tions, respectively. In ca. 600 base pairs of theinitial 5ʼ region of trnK 5ʼ intron, the new entity
was discriminated from both P. japonica and P.
minor by seven substitutions as well as four
insertions/deletions (Table 3). Accumulations ofsynapomorphic nucleotide changes in rather stable genetic regions, lack of polymorphic sites in
the nuclear genome, and divergence among thethree taxa in the plastid genome did not indicate
a hybrid origin of the new entity between P.
japonica and P. minor. Phylogenetic analysis of
ITS sequences further supported independent status of the new entity, although the order of cladogenesis was not resolved in the current data set(Fig. 1).
Taking account of both morphological andmacromolecular evidences, we hereby describe
the new entity as a separate species.
Taxonomic Treatment
Pogonia subalpina T. Yukawa & Y. Yamashita,
sp. nov.
TYPE: JAPAN, Honshu, Nagano Pref.,Hakuba-mura, Goryu, alt. 1600 m, 10 July 2016,
T. Yukawa 16-14 (TNS - holotype).
This species is similar to Pogonia japonica
Rchb. f. and P. minor (Makino) Makino, but isseparable by its short hairy crests on the disk of
the labellum mid-lobe, by its glabrous abaxial
surface of the labellum, and by its single, broad,
purplish pink band along the mid-vein of the petals.
Plant terrestrial, 9–16 cm tall. Rhizome cylindric, slender. Roots 4–6, fascicled, ﬁbrous, cylindric, pubescent, up to 9.5 cm long. Stem erect,
2-ridged, with several basal sheaths, green, dark
purple-red at base, 6–11 cm long. Leaf 1, patentsuberect, lanceolate to oblanceolate, acute or
obtuse, ﬂeshy, coriaceous, base contracted andamplexicaul, green, lucid, 24–70 mm long,
10–20 mm wide. Floral bract positioned
3.5–6 cm above leaf, suberect, lanceolate, apex
obtuse, ﬂeshy, coriaceous, persistent, green,
lucid, 11–40 mm long, 2–9 mm wide. Flower sol-

itary, terminal. Pedicellate ovary clavate, 6-sulcate, purplish green-green, 10–22 mm long, 2 mm
in diameter. Dorsal sepal linear-oblanceolate,subacute-obtuse, entire, patent, pale pink,
17–20 mm long, 4–4.5 mm wide. Lateral sepals
linear-oblanceolate, subacute-obtuse, entire,slightely oblique, patent, pale pink, 17–19.5 mm
long, 4 mm wide. Petals oblanceolate, obtuse,nearly entire, clasping, pale pink with a broad,
purplish pink band along mid-vein, 16–19 mm
long, 6–7 mm wide. Labellum weakly recurved,
3-lobed above middle, disk with a wide, longitudinal ridge extending from base and becoming
continuous with crests on mid-lobe, adaxial surface sparsely puberulent, with a pair of globularridges at base, pale to dark pink with cream yellow to cream ridge and hairs, abaxial surface glabrous, 15–18 mm long, 6–6.5 mm wide when ﬂattened; lateral lobes oblique triangular, erect,
apical margin irregularly incised, 2 mm long;
mid-lobe, narrowly triangular-ligulate, obtuse,
with hairy crests of 0.8 mm or less in length,
margin irregularly incised-entire, 4.5–6 mm long,
2–3 mm wide. Column clavate, erect, adaxiallypapillose, 11 mm long, 2.5 mm in diameter; clinandrium erose at margin; operculum rectangular,
2 mm long, 2.5 mm wide; pollinia 2, granular-farinaceous, without accessory organs; rostellum
short, broad, protruding above stigma. Figs. 2, 3,
4.
Japanese name: Miyama-tokiso.
Flowering period: June to August.
Distribution: JAPAN: Honshu (Tohoku andChubu districts).
Ecology: The plants usually inhabit open,
marshy, subalpine grasslands and wet cliffs from1400 to 1750 m a.s.l., as shown in Fig. 4. Exceptionally, a few populations establish below thesubalpine zone around the border of Fukushima
and Niigata Prefectures where the habitats arecharacterized by tuff deposits. Pogonia japonica
always grows in wet bogs in warm-temperate to
subarctic habitats dominated by Sphagnum
mosses, and P. minor unexceptionally grows in
mesic grasslands in warm- to cool-temperateregions. Evidently, these three species occupy

A New Orchid Species from Japan83

Fig. 2. Pogonia subalpina T. Yukawa & Y. Yamashita. A. Habit. B. Flower, side view; C. Lateral sepal; D. Petal;
E. Dorsal sepal; F. Labellum; G. Column and labellum, side view; H. Column; I. Operculum. Drawn from T.
Yukawa 16-14 (holotype, TNS) by K. Hamasaki. Scale bars ＝ 1 cm (A, B), 5 mm (C-G), 3 mm (H), or 1 mm(I).

different niches and are therefore ecologicallyisolated from each other.
Other specimens examined: JAPAN, Honshu:
Akita Pref. Senboku-shi, Tazawako-machi, July

1992, S. Mitsuhashi s.n. (TNS8504799). Iwate
Pref. Ichinoseki-shi, Mt. Kurikomayama, 2 Aug.
1935, Chyoji Suzuki 101638 (TNS). Miyagi Pref.
Katta-gun, Shichikashuku-machi, alt. 1550 m, 14

84

Tomohisa Yukawa and Yumi Yamashita

Fig. 3. Flower close-ups of three Pogonia species in Japan (photo courtesy of Toshiyuki Yamashita). A. P. subalpina T. Yukawa & Y. Yamashita; B. P. japonica Rchb.f.; C. P. minor (Makino) Makino.

Fig. 4. Pogonia subalpina T. Yukawa & Y. Yamashita in the habitats. A. Flowering plant at the type locality(photo courtesy of Hayato Tsuboi); B. Population in a snowbed, subalpine grassland, Yamagata Prefecture(photo courtesy of Toshiyuki Yamashita); C. Population in a marshy, subalpine grassland, Niigata Prefecture(photo courtesy of Taiga Kuhara).

A New Orchid Species from Japan

July 2005, Y. Mikanagi s.n. (TNS). Yamagata
Pref., Tsuruoka-shi, Mt. Gassan, July 1888, T.
Nagasawa s.n. (TNS54312). Fukushima Pref.
Minamiaizu-gun, Hinoemata-mura, Mt. Aizukomagatake, 26 Aug. 1934, H. Koidzumi 93962(TNS788253); Minamiaizu-gun, Hinoemata-mura,
Mt. Aizukomagatake, 26 Aug. 1934, H. Koidzumi 93963 (TNS788255). Niigata Pref. Minamiuonuma-gun, Yuzawa-machi, Mt. Naebayama,
alt. 1680 m, 15 July 2006, C. Tsutsumi, T. Kuhara& M. Sato CT1079 (TNS 766274, 8505060);
Uonuma-shi, Mt. Asakusadake, 28 July 1971,
Joju Haginiwa JH003081 (TNS953081); Kitauonuma-gun, Irihirose-mura, Mt. Asakusadake, 10Aug. 1961, Satoshi Saito 818 (FKSE40094);
Kitauonuma-gun, Irihirose-mura, OoshirakawaMt. Sumondake, alt. 1350 m, 7 Aug. 1981, Y.
Kadota s.n. (TNS715904); Kitauonuma-gun,Yunotani-mura, 24-25 July 1974, Satoshi Saito
39571 (FKSE44340); Nakauonuma-gun, Tsunanmachi, Komatsubara bog, Mt. Naebayama, H.
Koidzumi 24215 (TNS788254); Nakauonumagun, Tsunan-machi, Komatsubara bog, Mt. Naebayama, 9 July 1969, Takajirou Kusumi s.n.
(TNS257698); Nakauonuma-gun, Tsunan-machi,
Mt. Naebayama, 12 July 1958, Kengo Soma s.n.
(TNS01064261); Nakauonuma-gun, Tsunanmachi, Mt. Naebayama, 21 July 1930, H. Koidzumi 24126 (TNS788249); Nakauonuma-gun,
Tsunan-machi, Mt. Naebayama, 21 July 1930, H.
Koidzumi 24127 (TNS788260). Nagano Pref.
Kitaazumi-gun, Hakuba-mura, Mt. Happodake,
1923, H. Koidzumi 48755 (TNS788265); Kitaazumi-gun, Hakuba-mura, Mt. Happodake, 1923,
H. Koidzumi 48756 (TNS788248); Kitaazumigun, Hakuba-mura, Mt. Happo, 14 July 1923, H.
Koidzumi 5521 (TNS); Kitaazumi-gun, Hakubamura, Mt. Hakuba-happo-yama, 24 July 1954,
Nobuaki Ookawa s.n. (TNS629153). Gunma
Pref. Tone-gun, Minakami-machi, Mt. Ichinokurayama, 6 Aug. 1934, H. Koidzumi 92009(TNS788256); Tone-gun, Minakami-machi, Mt.
Mantaroyama, 9 Aug. 1934, H. Koidzumi 92225(TNS788263); Tone-gun, Minakami-machi, Mt.
Mantaroyama, 9 Aug. 1934, H. Koidzumi 92226(TNS788271).

85

Photographic records: JAPAN, Honshu: Yamagata Pref. Nishimurayama-gun, Nishikawa-machi,
Mt. Gassan, alt. 1440 m, 31 July 2011, photographed by Toshiyuki Yamashita. Miyagi Pref.
Shiroishi-shi, Mt. Fubosan, alt. 1300 m, 9 July
2011, photographed by Toshiyuki Yamashita.
Fukushima Pref. Minamiaizu-gun, Shimogomachi, Mt. Nagareishiyama, alt. 1755 m, 14 July
2012, photographed by Toshiyuki Yamashita;
Nishiaizu-gun, Nishiaizu-machi, Koboiwa Rock,
alt. 340 m, June 2016, photographed by Toshiyuki Yamashita. Niigata Pref. Gosen-shi, Kotsuradani, alt. ca. 250 m, 1 June 2013, photographedby Naoyuki Shimizu. Gifu Pref., Ohno-gun, Shirakawa-mura, Mt. Sanpoiwadake, alt. 1600 m,
July 2011, photographed by Hiroshi Nakayama.
Acknowledgments
We would like to thank Taiga Kuhara, Yuki
Mikanagi, Shunkei Mitsuhashi, Hiroshi Nakayama, Masaji Sato, Kazuhiro Sawa, Naoyuki
Shimizu, Hayato Tsuboi, Chie Tsutsumi, Mihoko
Watanabe, and Toshiyuki Yamashita for sharing
observations and providing photographs,
Hakuba-goryu Alpine Botanical Garden for permission to collect samples, Stephan Gale for critical reading of the manuscript, and Kumi Hamasaki for preparing the illustrations. We are
grateful to the director of FKSE for allowing us
to examine specimens. This study is partly supported by JSPS KAKENHI Grant Number
JP15H04417 and by a National Museum of
Nature and Science research grant entitled “Integrated analysis of natural history collections forconservation of highly endangered species” for T.
Yukawa.
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