Bull. Natl. Mus. Nat. Sci., Ser. B, 46(1), pp. 1–7, February 21, 2020

New Localities of Ropalospora phaeoplaca in Japan and
Far East of Russia with ITS nrDNA Sequences
Yoshihito Ohmura
Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan
E-mail: ohmura-y@kahaku.go.jp(Received 6 December 2019; accepted 25 December 2019)

Abstract Six localities from Honshu in Japan and one from Primorye Territory in Far East of
Russia are newly added for the distribution of Ropalospora phaeoplaca. Although this species isknown from main land of China, Taiwan, Korea and Far East of Russia, only the type locality was
known in Japan before the present report. The ITS nrDNA region was sequenced from the freshmaterials using in this study, and the maximum likelihood phylogenetic tree is also presented. The
tree shows that the samples of R. phaeoplaca formed a monophyletic clade that is a sister to theclade of R. viridis, and they are sister to R. lugubris clade. Although R. chlorantha is considered to
be closely related to R. phaeoplaca, the ITS nrDNA sequence could not be obtained in this study.
Key words : Ascomycota, distribution, eastern Asia, lichenized fungi, phylogeny, Ropalosporaceae.

Introduction
Ropalospora phaeoplaca (Zahlbr.) S.Ekman
belongs to the family Ropalosporaceae (Ascomycota). It was originally described as a species ofBacidia (Zahlbruckner, 1927), and transferred
into Ropalospora by Ekman (1996). This species
is characterized by the following features: thallus
corticolous, crustose, grayish-white to grayish
green, smooth to verrucose areolate, esorediate;
prothallus black or absent; apothecia lecideine,sessile, black, up to 1 mm; disk black, epruinose;
exciple brown externally, inside pale brown tohyaline, with K-soluble crystals; epihymenium
brown; hymenium hyaline, inspersed; hypothecium hyaline; asci clavate, 8–16 spored, Fuscidea-type; paraphysis simple, slightly branched at
the top; ascospores hyaline, acicular with one
attenuate end, straight or curved, 4–8 septate,30–47 × 2–3 μm; pycnidia not seen (Zahlbruck© 2020 National Museum of Nature and Sciencener, 1927; Ekman, 1996; Hu et al., 2013), andchemistry is K–, C–, KC–, P– (thallus andmedulla) and presence of unidentiﬁed substancedetected by thin layer chromatography (TLC) (Rfclass 5–6, UV254 nm＋visible, with solvent C) andmicrocrystal test (MCT) (crystal in GE) (Hu etal., 2013).
Although this species is known from main
land of China, Taiwan, Korea and Far East of
Russia, only the type locality was known inJapan before the present report (Zahlbruckner,
1927; Ekman, 1996; Aptroot and Sparrius, 2003;
Hu et al., 2013; Skirina, 2015, 2017). This paper
contributes to the distribution of R. phaeoplaca
especially for Japan and also one additional
locality for Russia, and the ITS nrDNA region
was also sequenced from the fresh materials.
Materials and Methods
This study is based on the herbarium specimens housed in the herbarium of National

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Yoshihito Ohmura

Museum of Nature and Science (TNS), Tsukuba,
Japan.
Morphological observations were made using
a dissecting microscope (SZX16, Olympus) anda differential interference contrast microscope(BX53, Olympus). Sections of apothecia wereprepared by hand cutting with a razor blade andmounted in water.
Chemical substances were detected by meansof TLC with solvent B′ (hexane : methyl tert.butyl ether : formic acid, 140 : 72 : 18) (Culberson and Kristinsson, 1970; Culberson and Johnson, 1982). MCT was performed using GE(acetic acid : glycerol ＝ 1 : 3) and oT (o-toluidine : glycerol : ethanol ＝1 : 2 : 2) (Asahina,
1936–1940). The amyloidity of ascus was examined using 1% Lugol's iodine solution.
DNA extraction followed a modiﬁed CTAB
protocol (Hosaka, 2009).
For DNA ampliﬁcation, 10 μl of PCR mix contained 1 μl genomic DNA extraction, 0.25 μl ofeach primer (10 pmol/μl) and 5 μl EmeraldAmp
PCR Master Mix (TaKaRa Bio Inc.). PCR ampliﬁcation of the ITS nrDNA region (including
ITS1, 5.8S nrDNA and ITS2) was performed
using the primer set of ITS1F (Gardes and Bruns,1993) as the 5′ primer and LR1 (Vilgalys and
Hester, 1990) as the 3′ primer. PCR cycling conditions were 94°C (3 min), followed by 11 cycles
Table 1.

of 95°C (30 s), 62°C to 52°C (30 s) with annealing temperatures lowered by 1°C between cycles,
and 72°C (1 min), followed by 30 cycles at 52°C
annealing temperature and a ﬁnal extension at
72°C (7 min). Sequencing was done on an ABI
Prism 3130x genetic analyzer (Applied Biosystems) using the BigDye Terminator ver. 3.1
Cycle Sequencing Kit according to the manufacturerʼs instructions.
The sequences were aligned in MAFFT ver. 7(Katoh et al., 2019) using the default settings. A
sequence of Stereocaulon apocalypticum Nyl.
was used as an out group because BLAST search(Altschul et al., 1997) showed high score ofhomology. After removing sites with gaps, missing data and ambiguous data, the resulting alignment of 410 sites was used for the molecularphylogenetic analyses.
The maximum likelihood (ML) (Felsenstein,1981) and neighbor-joining (NJ) (Saitou and Nei,
1987) analyses with the best nucleotide substitution model were performed. Tamura 3-parameter(T92) (Tamura, 1992) plus gamma distribution(＋G) was selected for the model. The bootstrap
values (Felsenstein, 1985) were calculated from
1,000 replicates for ML and NJ. All calculations
were conducteed in MEGA 7.0.18 (Kumar et al.,2016).
The sample data for molecular analyses and

DDBJ/EMBL/Genbank accession numbers of samples used for molecular analyses in this study
Taxon

Ropalospora lugubris

R. phaeoplaca

R. viridis

Voucher(Herbarium Code)*

Accession no. for
ITS rDNA sequence

Reference

Timdal 11170 (O)O-L-173455 (O)BGL-98792 (BG)
O-L-168439 (O)HK51464 (TNS)HK50726 (TNS)YO6297 (TNS)
YO8913 (TNS)YO10900 (TNS)YO11100 (TNS)JL24387 (TNS)
M. Kukwa & A. Łubek
17266 (KTC, UGDA)

MG926020
MK812610
MK812617
MK812670
LC516199
LC516205
LC516201
LC516202
LC516203
LC516204
LC516200
MN387183

Kistenich et al. (2018)
Marthinsen et al. (2019)Marthinsen et al. (2019)Marthinsen et al. (2019)
This study
This study
This study
This study
This study
This study
This study
Singh et al. (2019)*The detail collection data for voucher specimens of HK (＝ H. Kashiwadani), JL (＝J. Lendemer) and YO (＝Y.
Ohmura) are shown in the paragraph of specimens examined except Stereocaulon apocalypticum which was collected inJapan, Honshu, Prov. Kai (Yamanashi Pref.): Sensui Pass, Minami-Alps-city (N35°44′44″, E138°14′03″), on rock, elevation 2260 m, 3 September 2012, Y. Ohmura 9268 (TNS) (GenBank accession no. for ITS rDNA sequence: LC516206).

Ropalospora phaeoplaca

their DDBJ/EMBL/GenBank accession numbers
for the obtained ITS nrDNA sequences areshown in Table 1.

3

Results and Discussion
Ropalospora phaeoplaca (Zahlbr.) S.Ekman,
Op. Bot. 127: 128, 1996.
≡Bacidia phaeoplaca Zahlbr., Bot. Mag., Tokyo

Fig. 1. Ropalospora phaeoplaca. A. Habitat (Y. Ohmura 11100, TNS). B. Thallus with apothecia (fresh material)(Y. Ohmura 11100, TNS). C. Thallus with apothecia (old specimens) (isotype in TNS). D. K-soluble crystals
in proper exciple observed using a differential interference contrast microscope (around arrows) (isotype in
TNS). E. Ascospores (H. Kashiwadani 51829, TNS). F. Asci with spores (H. Kashiwadani 51829, TNS).
Scales: B ＝ 5 mm, C ＝ 1 mm, D ＝ 20 μm, E ＝ 10 μm, F ＝ 20 μm.

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Yoshihito Ohmura

41: 333, 1927.
Type: JAPAN. Honshu. Prov. Uzen (Yamagata
Pref.): Takayu, 24 June 1904, U. Faurie 5882
(Holotype in W; Isotype in TNS!, KYO).
(Fig. 1)The detail description and illustration of R.
phaeoplaca are provided in Hu et al. (2013).
This species is considered to be closely related to
R. chlorantha (Tuck.) S.Ekman but differs inhaving the proper exciple inspersed with minute
K-soluble crystals and in usually eight (occasionally ≤16) spores per ascus (Ekman, 1996).
The materials from Japan and Russia used inthis study coincide with the protologue and thefeatures mentioned above as well as with morphology and chemistry of the isotype (TNS). The
size of these ascospores was 24–37 × 2.8–
3.5 μm (n ＝ 13) with 3–7 septa. Although it was
shorter length and slightly wider width than theformer reports (30–47 × 2–3 μm) (Zahlbruckner,
1927; Hu et al., 2013), the observed shorter ascospores could be immature because they were
only 3 septa (usually 4–7 septa). So that, thisstudy treats the shorter size as variation. Amountof the K-soluble crystals in the proper exciple
vary very much from few to abundant in thematerials of this study. For example, those in isotype are less amount (Fig. 1D) and abundant
crystals are like the illustration shown in Hu etal. (2013).
An unidentiﬁed substance was detected by
TLC as reported in Hu et al. (2013), but it was
not crystalized by MCT using GE and oT in thisstudy. The TLC sport was visible with UV254 nm
and yellow color after 110°C heating for 10 min
with 10% H2SO4 spray. The Rf class is 7 in solvent B′ (slightly higher than atranorin and perlatolic acid) and Rf class 6 in solvent C (sameheight with perlatolic acid) (Fig. 2). It would be
difﬁcult to distinguish from perlatolic acidbecause only slight difference was seen in solvent B' and the same position in solvent C. But
the unidentiﬁed substance was detected from all
samples of R. phaeoplaca used in this study,
which would have an important taxonomic value
for the species.

Fig. 2. Diagnostic TLC spots of lichen substances
detected in Ropalospora phaeoplaca in Solvents B' and C. Control (C) using Stereocaulon
japonicum which contains atranorin (At) for Rf
class 7, norstictic acid (Nor) for Rf class 4, stictic acid (St) for Rf class 2, and constictic acid
(ConSt) for Rf class 1. Per ＝ perlatolic acid asa reference substance which is common in R.
chlorantha. Unid ＝ unidentiﬁed substancedetected from R. phaeoplaca.

A total of ﬁve sequences of ITS nrDNA for
Japanese and Russian specimens of R. phaeoplaca, one for R. lugubris (Sommerf.) Poelt collected in Japan, and one for R. viridis (Tønsberg)
Tønsberg collected in U.S.A. (Lendemer: Lich.
East. N. Amer. Exs. 420) were obtained in thisstudy. The genus Ropalospora includes sevenaccepted species: i.e., R. chirisanensis S.Y.
Kondr. et al., R. chlorantha, R. hibernica (P.
James & Poelt) Tønsberg, R. lugubris, R. phaeoplaca, R. rossii Øvstedal, and R. viridis (see Hu
et al., 2013; Kondratyuk et al., 2016). Amongthem, the ITS nrDNA sequences of R. phaeoplaca and R. viridis were ﬁrst registered into
GenBank in this study. In order to conﬁrm theindependency of R. phaeoplaca, molecular phylogenetic analyses based on these ITS nrDNA
sequences are performed. As the result, samplesof R. phaeoplaca formed a monophyletic cladewith high support value (ML/NJ ＝ 100/100)(Fig. 3). The clade of R. phaeoplaca forms a sister clade (99/98) with R. viridis that is sorediateRopalospora phaeoplaca

5

Fig. 3. Molecular phylogenetic tree of Ropalospora phaeoplaca and the related taxa based on ITS nrDNA
sequences. The tree was constructed by ML method, and the reliability of each branch was tested by ML and
NJ methods. The bootstrap values for ML/NJ analyses are shown on the branches only when both are ≥ 50%
simultaneously. Bold branches indicate they are ≥ 50% simultaneously. The OTU names indicated the vouchers of H. Kashiwadani (HK), Y. Ohmura (YO) and J. Lendemer (JL), and the GenBank accession number(italic) (see Table 1). Stereocaulon apocalypticum (YO9268) was used as an outgroup.

and usually sterile. The samples of R. lugubris, asaxicolous species, forms a monophyletic clade
combining Japanese and European samples(96/99). The clade of R. lugubris is a sister to R.
phaeoplaca – R. viridis clade. Although R.
chlorantha is considered to be closely related toR. phaeoplaca (Ekman, 1996), the ITS nrDNA
sequence could not be obtained for the phylogenetic analysis in this study because no freshmaterial was available.
In Japan, the specimens were collected onbarks of broad-leaf deciduous trees such as Alnus
hirsuta, Fagus crenata, Magnolia obovata, andQuercus crispula at elevation between 400 to1160 m (Fig. 1A). In Russia, it was collected onbark of broad-leaf deciduous tree at elevation c.
400 m.

Specimens examined (R. phaeoplaca). JAPAN.
Honshu. Prov. Ugo (Akita Pref.): Akataki,
Higashinaruse-mura, Ogachi-gun, on bark of
Alnus hirsuta, 480 m elev., 20 November 2012,
H. Kashiwadani 50726 (TNS); Genryu Area
(Sangaizawa-Minamisawa Deai) of Kasugegawa in Shirakami Mts, Fujisato-machi, Yamamoto-gun, on bark of decayed wood of Fagus
crenata, c. 400 m elev., 23 October 1999, M.
Inoue 27649 (TNS). Prov. Shimotsuke (Tochigi
Pref.): Nikko Botanical Garden, Nikko-city(N36°45′01″, E139°35′17″), on trunk of Magnolia obovata, 630 m elev., 11 September 2012, Y.
Ohmura 8913 & A. Frisch (TNS). Prov. Kozuke(Gunma Pref.): Akagisawa, Tone-machi,
Numata-city (N36°35′48″, E139°11′22″), on barkof Alnus sp., 930 m elev., 31 May 2016, Y. Ohm-

6

Yoshihito Ohmura

ura 11100 (TNS); the same locality, on bark of
Alnus sp., 930 m elev., 10 June 2016, Y. Ohmura
11112 (TNS). Prov. Kai (Yamanashi Pref.):
Aokigahara, Saiko Fuji-Kawaguchi-cho, Minamitsuru-gun (N32°29′35″, E138°38′60″), on barkof Quercus crispula, c. 950 m elev., 15 April
2017, H. Kashiwadani 51829 (TNS); Nishizawa
Valley,Mitomi-Kawaura,
Yamanashi-city(N35°52′06″, E138°44′53″), on bark of Alnus
hirusta, 1160 m elev., 8 February 2009, Y. Ohmura et al. 6297 (TNS). RUSSIA. Primorsky
Kray: c. 4 km N of Mt. Livadiyskaya(N43°06′19″, E132°41′31″), on bark of broadleaf deciduous tree, c. 400 m elev., 23 September
2013. Y. Ohmura 10900 (TNS).
Other specimens examined (R. lugubris).
JAPAN. Hokkaido. Prov. Tokachi: around summit area of Mt. Haku-un, Kamishihoro-cho,
Kato-gun (N43°15′23″, E143°07′09″), on rocks,
1120–1140 m elev., 6 July 2014, H. Kashiwadani
51464 (TNS).
Exiccata examined (R. viridis). U.S.A. Pennsylvania. Potter County: Susquehannock State
Forest, E side of Ridge Rd. 0.75N of jct w/Hunts
Run Rd. (N41°32′36″, W78°06′16″), 1749 ft.
elev., Larix stand in maple (Acer) forest withsandstone boulders, on Acer, 2 September 2010,
J. C. Lendemer 24387 (J. C. Lendemer: Lichensof Eastern North America Exsiccati 420) (TNS).
Acknowledgments
This study was partly supported by Japan-Russia Research Cooperative Program between JSPS
and RFBR (no. JPJSBP 120194829).
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