Bull. Natl. Mus. Nat. Sci., Ser. B, 45(2), pp. 63–70, May 22, 2019

Lectotypiﬁcation of Usnea confusa (Parmeliaceae, Ascomycota)
Yoshihito Ohmura1, * and Philippe Clerc2
1

Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo Tsukuba, Ibaraki 305–0005, Japan
2
Conservatoire et Jardin botaniques de la Ville de Genève, Geneva, Switzerland* E-mail: ohmura-y@kahaku.go.jp(Received 25 February 2019; accepted 27 March 2019)

Abstract The original material of Usnea confusa Asahina consists of several thalli glued on a
cardboard. In order to avoid any future taxonomic confusion especially presence or absence of“isidiate soredia”, a single specimen with numerous isidioﬁbrils developing on the soralia was chosen as lectotype. The lectotype of U. confusa contains usnic, salazinic, constictic acids and trace
amount of protocetraric acid as the secondary substances. ITS rDNA sequences of Japanese andTaiwanese specimens that have the same morphology and chemistry with the lectotype form two
distinct clades nested within the strongly supported clade representing the core group of Usnea
cornuta (containing U. cornuta Körber s.str.). Our molecular phylogenetic result based only on
ITS rDNA sequences doesnʼt allow to conﬁrm or contradict the conspeciﬁcity of U. confusa with
U. cornuta.
Key words : isidioﬁbrils, ITS rDNA, lectotype, lichenized fungi, phylogeny, soralia, taxonomy.

Introduction
In lichenology, the genus Usnea Adans. is
known as one of the most difﬁcult genera due tothe high morphological variability within species(Clerc, 1998).
Among the more than 350 species of thisgenus widely distributed in the world (Lücking etal., 2017), Usnea cornuta Körb. s. lat. is a good
example showing such taxonomical difﬁculty todelimit the species. The diagnostic features of U.
cornuta s. lat. deﬁned by Gerlach et al. (2019)[as “U. cornuta aggregate” including morphospecies of U. brasiliensis (Zahlbr.) Motyka, U. cornuta and U. dasaea Stirt.] are the following characters: thallus erect-shrubby to subpendulous
with more or less inﬂated branches constricted attheir point of attachment; soralia minute withvarious shapes and ontogeny, ±covered withisidiomorphs; the ratio of cortex, medulla andaxis (CMA) of main branches is of the cornutatype (Truong et al., 2011) or of the brasiliensis-

type (Gerlach et al., 2017), the density of medullary hyphae varies from dense to lax; the many
chemotypes designated by main substances suchas: salazinic, constictic, stictic, norstictic, protocetraric, psoromic, galbinic, thamnolic or lobaric
acids; and the worldwide distribution occurringin Europe, Asia, North and South America, Australia, and Africa. After the ﬁnding that thisgroup is polyphyletic by Truong et al. (2013),
Gerlach et al. (2019) investigated species boundaries in this group and found at least ninestrongly supported distinct lineages. Although U.
cornuta s. lat. should be carefully revised basedon morphological and chemical features in further researches, U. cornuta s. str. can be characterized by the minute and numerous soralia thatare even with the cortex and conﬂuent, a cornuta-type CMA, constictic, stictic, salazinic or
norstictic acid chemotype. This group corresponds to “lineage 5” in the phylogenetic tree
(Gerlach et al., 2019).
To understand such complicated and cosmopo-

64

Yoshihito Ohmura and Philippe Clerc

lite species, examinations of the related typespecimens that were collected from various
localities in the world and the molecular data of
the local populations are prerequisite. However,in many cases, the original material of Usnea
species is heterogeneous due to the high morphological similarity of closely related species. If wewant to progress towards a sound taxonomy of
the genus, it is thus very important to lectotypify
such species represented by such heterogenous
type material.
Usnea confusa Asahina was originallydescribed from Japan (Asahina, 1956) and it iscurrently considered as a synonym of U. cornuta(Clerc, 2004). The original material of this species consists of 10 specimens glued on a cardboard (Fig. 1A). To avoid future taxonomic confusion, a single thallus is here lectotypiﬁed.
This paper further aims at providing ITS rDNA
sequences of Japanese and Taiwanese collections
corresponding morphologically, anatomically andchemically to the lectotype of U. confusa andintegrating them in the general phylogeny of thegroup published by Gerlach et al. (2019).
Materials and Methods
This study is based on the examinations ofherbarium specimens housed in the Departmentof Botany, National Museum of Nature and Science (TNS), Tsukuba, Japan.
Morphological observations for identiﬁcation
were made using a dissecting microscope and abright ﬁeld microscope. The ratios of thickness ofthe cortex, medulla, and axis for the branch were
measured following the method of Clerc (1984,1987). Cross sections of thallus were cut by handwith a razor blade, and observed after mounting
in GAW (glycerin : ethanol : water, 1 : 1 : 1).
Lichen substances were examined using thinlayer chromatography (TLC) (Culberson andJohnson, 1982). Solvent B system (hexane :methyl tert-butyl ether : formic acid, 140 : 72 : 18)
was used for all TLC analyses.
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
of each primer (10 pmol/µL) and 5 µL EmeraldAmp PCR Master Mix (TaKaRa Bio Inc.). PCR
ampliﬁcation of the ITS rDNA region (including
ITS1, 5.8S rDNA 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. In some cases,
when no PCR product was appeared by ITS1F/
LR1 primer pair, USITS1-F as the 5′ primer and
USITS2-R as the 3′ primer (Ohmura, 2008) wereused for the PCR. PCR cycling conditions were
94°C (3 min), followed by 11 cycles of 95°C (30sec), 62°C to 52°C (30 sec) with annealing temperatures lowered by 1°C between cycles, and72°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., 2017) using the default settings.
After removing sites with gaps, missing data andambiguous data, the resulting alignment of 483
sites was used for the molecular phylogenetic
analyses.
The maximum likelihood (ML) (Felsenstein,1981) and neighbor-joining (NJ) (Saitou and Nei,
1987) analyses with the best nucleotide substitution model were performed. Kimura 2-parameter(Kimura, 1980) plus gamma distribution(K2P+G) was selected for the model. The bootstrap values (Felsenstein, 1985) with 1,000 replicates for ML and NJ were shown on the branches
only when both are ≥ 50% simultaneously. All
calculations were conducteed in MEGA 7.0.18(Kumar et al., 2016).
The sample data for molecular analyses andtheir DDBJ/EMBL/GenBank accession numbers
for the obtained ITS rDNA sequences are shownin Table 1.

Lectotypiﬁcation of Usnea confusa

65

Fig. 1. Lectotype of Usnea confusa Asahina. A. Thallus “C” on the sheet was designated as the lectotype. B.
Thallus. C. Base of the thallus. D. Cortex, medulla and axis in a slightly inﬂated branch. E. Isidioﬁbrils on
soralia. F. Merrillii-type cortex. Scales: C=1 mm, D, E=0.5 mm, F=50 μm.

66

Yoshihito Ohmura and Philippe Clerc
Table 1.

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

Usnea brasiliensis
Usnea cornuta s.str.

Usnea rubrotincta
Usnea sp. [=U. cornuta s. lat.
sense Gerlach et al. (2019)]

Voucher of
Y. Ohmura(YO)* or OTU
name

Accession no. for
ITS rDNA sequencebras-13BR
corn-19BR
corn-32US
corn-113BR
corn-171BR
corn-176BR
corn-198BR
YO7376YO8494A
YO8506B
YO10392B

MF669812
MF669861
JQ837300
MF669835
MF669869
MF669847
MF669872
LC479119
LC479120
LC479121
LC479122

YO10417
YO10991
YO10300
corn6-28BR
corn6-83BR
corn6-90BR
corn6-170BR

LC479123
LC479124
LC479125
MF669826
MF669865
MF669866
MF669868

Specimens (YO)*
which have theidentical sequence

Reference

—
—
—
—
—
—
—

Gerlach et al. (2019)
Gerlach et al. (2019)
Truong et al. (2013)
Gerlach et al. (2019)
Gerlach et al. (2019)
Gerlach et al. (2019)
Gerlach et al. (2019)
This study
YO8494B, YO8494C This study—
This study
YO6218, YO6977,
This study
YO7270, YO7276,
YO7333, YO8509,
YO8850, YO8855B,
YO8688B
YO8855A
This study—
This study—
This study—
Gerlach et al. (2019)—
Gerlach et al. (2019)—
Gerlach et al. (2019)—
Gerlach et al. (2019)*The detail collection data for voucher specimens of YO are shown in the paragraph of specimens used in the phylogenetic analyses in the main text except YO10300 (Usnea rubrotincta Stirt.) which was collected in Japan, Shikoku,
Prov. Iyo (Pref. Ehime): Furuiwaya, Kumakogen-cho, Kamiukena-gun (N33°40′, E132°58′), on trunk of dead tree, elevation about 500 m, 11 November 2013 (TNS).

Results and Discussion
Lectotypiﬁcation of Usnea confusa
Usnea confusa Asahina, Lich. Jpn. 3: 97,
1956. Lectotype (designated here): JAPAN, Honshu, Prov. Suruga, Subashiri-guchi, Mt. Fuji, 7
July 1952, Y. Asahina 5277, thallus “C” as shownin Fig. 1A, (TNS!). % C/M/A = 4.6/38.2/14.5.
Chemistry: usnic, salazinic, constictic and protocetraric (trace) acids.
Although Ohmura (2001) already selected thelectotype of U. confusa as being the material collected by Y. Asahina with collection number
5277 (TNS), further studies showed that thismaterial was in fact heterogeneous. Among theten thalli glued on the cardboard marked as“TYPUS” and prepared by Y. Asahina (Fig. 1A),
two of them correspond to U. hondoensis Asahina that has uninﬂated branches with punctiform
soralia and the chemistry of usnic, norstictic andsalazinic acids (thalli “F” and “H” in Fig. 1A,
annotated by Y. Ohmura as “U. pangiana” in1997). The eight remaining thalli belong to thesame species corresponding to the description of
U. confusa, and they have the same chemistry,i.e. usnic, salazinic, constictic and protocetraric(trace) acids. However, the amount of isidioﬁbrils on soralia in each thallus varies very much.
To avoid any confusion in future taxonomic
treatment, we selected one thallus bearing abundant isidioﬁbrils (thallus “C” in Fig. 1) according
to the mention “soredia isidiosa” in the protologue (Asahina, 1956).
Isidioﬁbrils
The term of isidioﬁbril was deﬁned by Truong
et al. (2011), as being the secondary development of an axis as soon as a certain size isreached during the growth of an isidiomorph.
This was already reported in U. hakonensis Asa-

Lectotypiﬁcation of Usnea confusa

hina, an Asian taxon (Ohmura, 2001), and it ishere further observed in U. confusa (or U. cornuta s.str.). Morphologically, isidioﬁbrils may be
confused with isidiomorphs but they usually aretaller and slender, with sometimes a sinuose
irregular form. Isidioﬁbrils are thus an older
stage of isidiomorphs and more studies are necessary to access whether this character might beused to delimit species in the taxonomy of thegenus Usnea. The amount of isidioﬁbrils varies
depending on the developmental stage of thallus
and on some environmental parameters of thehabitat. Then the presence of isidioﬁbrils would
be more the result of the older age of a thallus orof environmental changes than of the genetic
makeup of the species.
Molecular analyses
A total of 18 sequences of ITS rDNA (491 bp)
for the Japanese and Taiwanese specimens of U.
cornuta morph with usnic, salazinic, constictic
and protocetraric (trace) acids was obtained inthis study, and six haplotypes were recognizedamong them. Based on the molecular phylogenetic analyses, two distinct clades (“confusa 1”
and “confusa 2”) (bootstrap values for ML/NJ =85/85 and 88/91 respectively) were formed
within the strongly supported clade of U. cornuta
s. str. (bootstrap values: 96/98) (Fig. 2). The
topology of the tree is not in conﬂict with the one
shown in Gerlach et al. (2019). Thus, the tree in
Fig. 2 clearly shows that U. confusa belongs to
the core group of U. cornuta (Gerlach et al.
2019: lineage 5). On the base of morphological,
anatomical and chemical studies, Clerc (2004)
considered U. confusa being a synonym of U.
cornuta. At ﬁrst glance, our results might support
this synonymy. However, our study neither reallyconﬁrms or contradicts this afﬁrmation (Fig. 2).
Internal relationships of this group are not well
supported and Gerlach et al. (2019) emphasized
the need of ﬁne-scale studies with a larger sampling over the entire distribution range of the lineage. Within each clade of “confusa 1” and “confusa 2”, the differences among the sequences of
haplotypes are only one residue (homology is

67

99.8%). In contrast, the difference between thesequences of the clades “confusa 1” and “confusa
2” is 7 to 11 residues (homology is 97.8 to
98.6%). No morphological and chemical difference were found between the specimens representing these two clades. So far, other haplotypesof U. cornuta s. str. were not detected from Japanand Taiwan. It is then possible that the eastern
Asian population of U. cornuta are on the way of
speciation. However probably due to the use forthe phylogenetic analyses of ITS rDNA
sequences only, the relationships among the U.
cornuta core group are not well resolved. The
use of multigene analyses or of high-throughput
sequencing technology will be necessary to
improve the deep relationships inside the U. cornuta group to settle this case.
The ITS rDNA region is considered as a good
DNA barcoding marker to recognize fungal taxa
including lichenized fungi (Begerow et al. 2010;
Orock et al. 2012). However, our study agreewith Badotti et al. (2018) who stated that the useof this marker only might in some cases not be
sufﬁcient to separate fungal species, especiallywhen the speciation is a recent event and processes like incomplete lineage sorting and reticulation are at work, obscuring species boundaries
and relationships (Naciri and Linder, 2015).
Specimens used in the phylogenetic analyses(all vouchers in TNS). JAPAN. Hokkaido. Prov.
Kushiro: Aikappu, Akkeshi-cho, Akkeshi-gun(N43°01′09″, E144°50′20″), on bark of Cerasus
sargentii, elevation about 80 m, 28 May 2012, Y.
Ohmura et al. 8850; Tobai, Akkeshi-cho,
Akkeshi-gun (N42°59′54″, E144°53′31″), on
twig of Abies sachalinensis, elevation about 80
m, 28 May 2012, Y. Ohmura et al. 8855A,
8855B. Honshu. Prov. Musashi (Tokyo Metropolis): Inamura-iwa, Nippara, Okutama-machi,
Nishitama-gun (N35°50′28″, E139°02′00″), on
trunk of Juniperus rigida, elevation about 800 m,
24 September 2015, Y. Ohmura 10991. Prov. Kii(Pref. Wakayama): Fukuchiin Temple, Mt. Koya,
Koya-cho, Ito-gun (N34°12′, E135°35′), on barkof pine tree, elevation 820 m, 2 March 2012, Y.
Ohmura 8494 & A. Frisch; Kiyotaka-Inari68

Yoshihito Ohmura and Philippe Clerc

Fig. 2. Molecular phylogenetic tree of Usnea cornuta and the related taxa based on ITS rDNA 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 ≥ 80% simultaneously. The OTU names indicated the vouchers of Y. Ohmura(YO) and those used in Gerlach et al. (2019) (see Table 1). Jp and Tw indicating after “:” of YO vouchers are
Japan and Taiwan as the collection locality. Clade numbers or names in the double quotations are identicalwith those in Gerlach et al. (2019). The topology of this tree with statistically supported branches is not conﬂicted with that in Gerlach et al. (2019).

Shrine, Mt. Koya, Koya-cho, Ito-gun (N34°13′,
E135°36′), on wooden pole, elevation 820 m, 3
March 2012, Y. Ohmura 8509 & A. Frisch; Okunoin, Mt. Koya, Koya-cho, Ito-gun (N34°13′,
E135°36′), on bark of Cryptomeria japonica,
elevation 820 m, 3 March 2012, YO8506B. TAIWAN. Miaoli Co.: Xuejian Recreation Area,

Taian Township (N24°25′36″, E121°00′53″), on
trunk of Prunus sp., elevation 1886 m, 8 October
2013, Y. Ohmura 10392B; the same locality(N24°26′44″, E121°01′32″), on bark of Pinus taiwanensis, elevation 2150 m, 8 October 2013, Y.
Ohmura 10417. Taichung Co.: between 0 km and6.8 km point of mountain trail, en route fromLectotypiﬁcation of Usnea confusa

Shiyuan Yakou to Mt. Nanhu (N24°23′14″,
E121°21′29″), on bark of broad-leaf deciduous
tree, elevation 1977 m, 30 September 2010, Y.
Ohmura 7333; the same locality, on bark of
Pinus sp., Y. Ohmura 7276; the same locality(N24°23′29″, E121°21′09″), on bark of Betulasp., elevation 1948 m, 30 September 2010, Y.
Ohmura 7270; the same locality (N24°22′30″,
E121°21′08″), on bark of Chamaecyparis obtusa,
elevation 2249 m, 30 September 2010, Y. Ohmura 7376. Nanto Co.: Keitau, 9 November
1968, U. Mizushima s.n. (TNS-L-22389); Renluen (N23°42′27″, E120°55′08″), on bark of evergreen tree, elevation 1699 m, 30 August 2008, Y.
Ohmura 6218; en route from Sunghsueh Hostel
to Chengkung No. 2, 3 Cabin, Mt. Chilai, Taroko
National Park (N24°07′01″, E121°18′58″), on
bark of Tsuga sp., elevation about 2900 m, 29
September 2009, Y. Ohmura 6977. Chiayi Co.:
Mt. Alishan, Alishan Township (N23°31′,
E120°48′), on bark of Chamaecyparis formosensis, elevation 2340 m, 4 October 2011, Y. Ohmura 8688B.
Acknowledgments
We thank Dr. Alice da Cruz Lima Gerlach,
Universida de Federal do Rio Grande do Sul(Brazil), for providing raw chromatogram data of
Usnea species used in their publication; andanonymous reviewers, for critical reading of thismanuscript.
References
Asahina, Y. 1956. Lichens of Japan, vol. 3, genus Usnea.
Research Institute for Natural Resources Shinjuku,
Tokyo.
Badotti, F., Fonseca, P. L. C., Tomé, L. M. R., Nunes, D.
T. and Góes-Neto, A. 2018. ITS and secondary biomarkers in fungi: review on the evolution of their use
based on scientiﬁc publications. Brazilian Journal of
Botany 41: 471–479.
Begerow, D., Nilsson, H., Unterseher, M. and Maier, W.
2010. Current state and perspectives of fungal DNA
barcoding and rapid identiﬁcation. Applications in
Microbiology and Biotechnology 97: 99–108.
Clerc, P. 1984. Contribution à la revision de la systém

69

atique des usnées (Ascomycotina, Usnea) d′Europe. I.
– Usnea ﬂorida (L.) Wigg. emend. Clerc. Cryptogamie
Bryologie et Lichénologie 5: 333–360.
Clerc, P. 1987. Systematics of the Usnea fragilescens
aggregate, and its distribution in Scandinavia. Nordic
Journal of Botany 7: 479–495.
Clerc, P. 1998. Species concepts in the genus Usnea(lichenized Ascomycetes). Lichenologist 30: 321–340.
Clerc, P. 2004. Notes on the genus Usnea Adanson. II.
Bibliotheca Lichenologica 88: 79–90.
Clerc, P. 2016. Notes on the genus Usnea (lichenizedAscomycota, Parmeliaceae) IV. Herzogia 29: 403–411.
Culberson, C. F. and Johnson, A. 1982. Substitution of
methyl tert.-butyl ether for diethyl ether in the standardized thin-layer chromatographic method for lichen
products. Journal of Chromatography 238: 483–487.
Felsenstein, J. 1981. Evolutionary trees from DNA
sequences: A maximum likelihood approach. Journal of
Molecular Evolution 17: 368–376.
Felsenstein, J. 1985. Conﬁdence limits on phylogenies anapproach using the bootstrap. Evolution 39: 783–791.
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.
Gerlach, A. da C. L., Clerc, P. and Borges da Silveira, R.
M. 2017. Taxonomy of the corticolous, shrubby, esorediate, neotropical species of Usnea Adans. (Parmeliaceae) with an emphasis on southern Brazil. Lichenologist 49: 199–238.
Gerlach, A. da C. L., Toprak, Z., Naciri, Y., Caviró, E. A.,
Borges da Silveira, R. M. and Clerc, P. 2018 (2019).
New insights into the Usnea cornuta aggregate (Parmeliaceae, lichenized Ascomycota): Molecular analysis
reveals high genetic diversity correlated with chemistry. Molecular Phylogenetics and Evolution 131: 125–
137.
Hosaka, K. 2009. Phylogeography of the genus Pisolithus
revisited with some additional taxa from New Caledonia and Japan. Bulletin of the National Museum of
Nature and Science, Series B (Botany) 35: 151–167.
Katoh, K., Rozwicki, J. and Yamada, K. D. 2017. MAFFT
online service: Multiple sequence alignment, interactive sequence choice and visualization. Brieﬁngs in
Bioinformatics 2017 Sep 6. doi: 10.1093/bib/bbx108.
[Epub ahead of print].
Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative
studies of nucleotide sequences. Journal of Molecular
Evolution 16: 111–120.
Kumar, S., Stecher, G., and Tamura, K. 2016. MEGA7:
Molecular Evolutionary Genetics Analysis version 7.0
for bigger datasets . Molecular Biology and Evolution
33: 1870–1874.
Lücking, R., Hodkinson, B. P. and Leavitt, S. T. 2017.

70

Yoshihito Ohmura and Philippe Clerc

The 2016 classiﬁcation of lichenized fungi in the Ascomycota and Basidiomycota – Approaching one thousand genera. Bryologist 119: 361–416.
Naciri, Y. and Linder, H. P. 2015. Species delimitation
and relationships: the dance of the seven veils. Taxon
64: 3–16.
Ohmura, Y. 2001. Taxonomic study of the genus Usnea(lichenized Ascomycetes) in Japan and Taiwan. Journalof the Hattori Botanical Laboratory 90: 1–96.
Ohmura, Y. 2008. Taxonomy and molecular phylogeny of
Usnea rubicunda and U. rubrotincta (Parmeliaceae,
lichenized Ascomycotina). Journal of Japanese Botany
83: 347–355.
Orock, E. A., Leavitt, S. D., Fonge, B. A., St. Clair, L. L.
and Lumbsch, H. T. 2012. DNA-based identiﬁcation oflichen-forming fungi: Can publicly available sequence
databases aid in lichen diversity inventories of Mount

Cameroon (West Africa) ? Lichenologist 44: 833–839.
Saitou, N. and Nei, M. 1987. The Neighbor-Joining
method: A new method for reconstructing phylogenetic
trees. Molecular Biology and Evolution 4: 406–425.
Truong, C., Bungartz, F. and Clerc, P. 2011. The lichen
genus Usnea (Parmeliaceae) in the tropical Andes andthe Galapagos: Species with a red-orange cortical or
subcortical pigmentation. Bryologist 114: 477–503.
Truong, C., Divakar, P. K., Yahr, R., Crespo, A. and Clerc,
P. 2013. Testing the use of ITS rDNA and protein-coding genes in the generic and species delimitation of thelichen genus Usnea (Parmeliaceae, Ascomycota).
Molecular Phylogenetics and Evolution 68: 357–372.
Vilgalys, R. and Hester, M. 1990. Rapid genetic identiﬁcation and mapping of enzymatically ampliﬁed ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246.