Taiwania 68(1): 101‒105, 2023
DOI: 10.6165/tai.2023.68.101

NOTE

Gyalectidium setiferum (Gomphillaceae, Ascomycota), a foliicolous lichen,
new to East Asia and its molecular phylogenetic position
Kento MIYAZAWA1,*, Yoshihito OHMURA2,3, Izumi OKANE4
1. Degree Programs in Life and Earth Sciences, Graduate School of Science and Technology, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki, 305-8577, Japan. 2. Department of Botany, National Museum of Nature and Science, 4-1-1 Amakubo, Tsukuba,
305-0005, Japan. 3. School of Integrative and Global Majors, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8577,
Japan. 4. Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, 305-8572, Japan.
*Corresponding author’s email: miyazawa.kento.ss@alumni.tsukuba.ac.jp(Manuscript received 31 August 2022; Accepted 29 November 2022; Online published 25 February 2023)
ABSTRACT: Gyalectidium setiferum Vězda & Sérus., a foliicolous lichen characterized by having some vertically oriented whitishtranslucent cilia surrounding a swollen greenish diahyphal mass, is reported as new to East Asia. It was collected from two localities
on the lowlands of central Honshu in Japan where it grew on leaves of Aucuba japonica and Maesa japonica. The description with
illustration based on the Japanese material is given. Gyalectidium setiferum has been included in this genus due to its morphologicalfeatures even though no fertile specimen has been found so far. The monophyly of this species and its position in this genus were
newly confirmed by the molecular phylogenetic analysis based on mtSSU and nrLSU.
KEY WORDS: Conidia, conidiomata, diahyphae, distribution, hyphophore, lichenized fungi, mtSSU, nrLSU, photobiont.

INTRODUCTION

species and its taxonomic position in the genus.

The genus Gyalectidium (Gomphillaceae, lichenizedAscomycota) consists of 45 accepted species that mainly
grow on living leaves, very rarely on barks or rocks(Ferraro et al., 2001; Herrera-Campos and Lücking, 2003;
Safranek and Lücking, 2005; Lücking et al., 2007;
Lücking, 2008; Suto and Ohtani, 2018). This genus ischaracterized by a smooth to coarsely verrucose thallus
with a cartilaginous corticiform layer; immersed toerumpent, zeorine, rounded apothecia; squamiform
hyphophoral conidiomata with a scale protecting an
adnate, applanate, diahyphal mass (the scale is sometimes
reduced or disintegrated into a cluster of erect cilia thatare difficult to distinguish from setae); and branchedmoniliform diahyphae forming sausage-like shaped
segments (Lücking, 2008).
During the course of studies for the Japanesefoliicolous lichenized mycota, G. setiferum Vězda &Sérus. was confirmed to occur in Japan, which is the firstreport from East Asia. This taxon is considered to belong
to the genus Gyalectidium due to the presence of reduced
hyphophores with diahyphal mass which is produced at
the base and the diahyphal mass composed of sausagelike shaped cells (Sérusiaux, 1993; Ferraro et al., 2001).
However, no apothecia of G. setiferum have been foundso far (Sérusiaux, 1993; Ferraro et al., 2001; van den
Boom, 2021), so a molecular phylogenetic analysis wasneeded to confirm the position of this species in this genus.
The purpose of this study was to describe themorphological and chemical features of G. setiferum basedon the Japanese material as well as performing a molecularphylogenetic analysis to confirm the monophyly of the

MATERIALS AND METHODSAll voucher specimens are housed in the herbarium of
National Museum of Nature and Science (TNS), Tsukuba,
Japan.
Morphology and chemistry
Morphological observations and photography were
performed using a dissecting microscope (M165C; Leica,
Wetzlar, Germany) with a digital camera (Flexacam C3;
Leica) and a differential interference contrast microscope(BX53; Olympus, Tokyo, Japan) with a digital camera(EOS Kiss X9i; Canon, Tokyo, Japan). Anatomical
examinations were performed using hand-cut sections
mounted in GAW (glycerin: ethanol: water = 1: 1: 1)
solution (Asahina, 1936). The digital image of Fig. 1A
was prepared using CombineZP image stacking software(developed by Alan Hadley).
Calcium oxalate crystals were identified under the
microscope by adding 25% H2SO4 following Thor et al.
(2000). Color spot tests for K, C, KC, and Pd were
performed according to Orange et al. (2001).
Secondary substances were examined using a highperformance thin layer chromatography (HPTLC)
following Schumm and Elix (2015). Solvent systems B′(n-hexane: methyl tert-butyl ether: formic acid, 140: 72:
18) (Culberson and Johnson, 1982) was used for HPTLC.
The spot color was checked under 254 and 366 nm
wavelength of UV and visible light, before and after
spraying with 10% sulfuric acid on the HPTLC plate andcharring at 90°C for 20 minutes.
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Vol. 68, No. 1

Table 1. Vouchers and their GenBank accession numbers. New sequences obtained in this study are in bold.
Taxa
Aderkomyces sp. (sterile)
Asterothyrium longisporum
Asterothyrium sp. (as Calenia
monospora)
Calenia lobulata
Calenia monospora
Gyalectidium catenulatum
Gyalectidium aff. denticulatum
Gyalectidium filicinum
Gyalectidium imperfectum
Gyalectidium aff. imperfectum
Gyalectidium setiferum

Gyalectidium sp.
Gyalectidium sp.
Tricharia lancicarpa
Tricharia vainioi
Tricharia sp. (sterile)

Voucher
Brazil; A.B. Xavier-Leite 2004 (ISE)
Costa Rica; R. Lücking s.n. (F)Costa Rica; R. Lücking s.n. (F)

mtSSU
MZ827178
AY341363
AY341365

nrLSU
MZ851543
AY341349
AY341351

Reference
Xavier-Leite et al., 2022
Lücking et al., 2004
Lücking et al., 2004

Costa Rica; R. Lücking s.n. (B)Cuba; R. Lücking et al. 41885c (B, HAJB)
Costa Rica; R. Lücking 16032b
Brazil; A.B. Xavier-Leite et al. 1245 (ISE)
Brazil; A.B. Xavier-Leite 1951 (ISE)
Brazil; A.B. Xavier-Leite 1970 (ISE)
Brazil; A.B. Xavier-Leite 1973 (ISE)
Guatemala; R. Lücking 4353 (B)Costa Rica; R. Lücking 55 (B)Japan; K. Miyazawa 1017 (TNS)Japan; K. Miyazawa 1018 (TNS)Japan; K. Miyazawa 1023 (TNS)
Brazil; A.B. Xavier-Leite 1986 (ISE)
Brazil; M. Cáceres & R. Lücking 143 (B, URM)
Brazil; A.B. Xavier-Leite 1564 (ISE)
Brazil; A.B. Xavier-Leite et al. 1402b (ISE)
Brazil; A.B. Xavier-Leite 1950 (ISE)

MZ827242
MZ827264
KF833335
MZ827272
MZ827174
MZ827171
MZ827170
MZ827236
MZ827257
LC726432
LC726433
LC726434
MZ827180
MZ827253
MZ827275
MZ827228
MZ827173

MZ851715
MZ851728
KF833323
MZ851626
MZ851514
MZ851521
MZ851522
MZ851731
MZ851712
LC726435
LC726436
LC726437
MZ851529
MZ851699
MZ851491
MZ851658
MZ851513

Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
unpublishedXavier-Leite et al., 2022
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
This study
This study
This study
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022
Xavier-Leite et al., 2022

Fig. 1. Gyalectidium setiferum collected from Japan (K.
Miyazawa 1017, TNS). A: Thallus with vertically oriented cilia
surrounding the swollen diahyphal masses (each arrow indicatesswollen diahyphal mass). B: Moniliform hyphae with sausage-likeshaped segments in a diaphyphal mass with photobiont cells. C:
Photobiont cells in the thallus with calcium oxalate crystals(arrow). Scale bars: A = 0.5 mm, B & C = 10 μm.

102

DNA extraction, PCR amplification and sequencing
DNA extraction for PCR was performed following amodified method of Izumitsu et al. (2012) (see also
Miyazawa et al., 2022). The partial sequences of the small
subunit of the mitochondrial ribosomal RNA gene
(mtSSU) and the large subunit of the nuclear ribosomal
RNA gene (nrLSU) were amplified using the primer sets
mrSSU1 and mrSSU3R (Zoller et al., 1999) for mtSSU,
LIC24R (Miadlikowska and Lutzoni, 2000) and LR7(Vilgalys and Hester, 1990) for nrLSU, according to thefollowing protocol. PCR was performed in a 15 μL
reaction solution containing 2 μL DNA template, 7.5 μL
GenRED PCR Mix Plus (Nippon Gene, Tokyo, Japan),
1.5 μL each primer (2 pmol/μL), and 2.5 μL distilledwater. The PCR conditions followed the method of Wanget al. (2020) for mtSSU and a modified method of Frischet al. (2014) (45 cycles to 35 cycles) for nrLSU, using a
TaKaRa PCR Thermal Cycler Dice® Touch (TaKaRa,
Tokyo, Japan). The PCR products were checked byelectrophoresis on a 1.5% agarose gel stained with Midori
Green Direct DNA Stain (Nippon Genetics, Tokyo, Japan)
and visualized using WSE-5200 Printgraph 2 M (ATTO
Corporation, Tokyo, Japan).
The PCR products were purified using an ExoSAPIT™ PCR Product Cleanup Reagent (Thermo Fisher,
Tokyo, Japan). 13 μL of PCR products with 2 μL of 4
times diluted ExoSAP-IT™ were incubated at 37°C for
15 minutes, then 80°C for 15 minutes.
Sequences were obtained by a DNA sequencing
service (Eurofins Genomics, Tokyo, Japan). The taxon
name and GenBank accession numbers for the obtained
sequences are shown in Table 1.

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Miyazawa et al.: Gyalectidium setiferum, new to East Asia

Fig. 2. Maximum likelihood tree of selected taxa in Gomphillaceae showing the phylogenetic position of Gyalectidium setiferum
collected from Japan (in bold) within the Gyalectidium clade. Calenia monospora-Asterothyrium group is used as an out group. NJ(first) and ML (second) support values are presented for each node. Branches highly supported (≥70) by both analyses are indicatedwith bold black lines, and a branch supported by only one of the analyses with a bold grey line.

Molecular phylogenetic analyses
The newly obtained mtSSU and nrLSU sequences of
Gyalectidium setiferum from the Japanese material werealigned with registered sequences of selected taxa in
GenBank (Table 1) in MAFFT ver. 7 (Katoh et al., 2019)using the default settings. For the outgroup, the sequencesof Calenia monospora (MZ827264, MZ851728),
Asterothyrium sp. (AY341365, AY341351) and A.
longisporum (AY341363, AY341349) from GenBank
were used to be consistent with the phylogenetic tree
generated by Xavier-Leite et al. (2022). Each data set(mtSSU and nrLSU) was separately aligned. Afterremoving sites with gaps, missing data and ambiguous data,
the data were concatenated. The final alignment of 897
sites was used for the molecular phylogenetic analyses.
The neighbor-joining method (NJ) and maximum
likelihood method (ML) analyses were performed by
Tamura 3-parameter model (Tamura, 1992) plus gamma
distribution which was selected as the best fitting model. The
bootstrap values (≥70%) with 1,000 replicates for NJ and
ML were placed on each branch. A branch with highbootstrap values (≥70%) by both analyses was indicatedwith bold black line, and a branch supported by only one of
the analyses was shown with bold grey line. All calculations
were conducted in MEGA Ⅹ (Kumar et al., 2018).

RESULTS AND DISCUSSION
Molecular analysis
Among the aligned sites of each locus within theJapanese material, there are ten variable sites and one gap
site in 739 sites of mtSSU (the percent identity among
three specimens: 98.5–100%) and nine variable sites in
501 sites of nrLSU (98.2–99.2%). The topologies ofphylogenetic trees constructed by both methods are
consistent. The ML phylogenetic tree is shown in Fig. 2.
The monophyly of G. setiferum and the its position in thegenus Gyalectidium were confirmed with high support
values (NJ/ML = 100/96, 100/99 respectively).
There was no conflict between the topology of our
phylogenetic tree including G. setiferum and that of
Xavier-Leite et al. (2022). However, the relationshipbetween G. setiferum and other Gyalectidium species was
unclear due to the low bootstrap values (Fig. 2). Further
phylogenetic analysis with additional DNA informationof other loci of more related species would clarify thephylogenetic position of G. setiferum within the genus.

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TAXONOMIC TREATMENT
Gyalectidium setiferum Vězda & Sérus., in Sérusiaux,
Nord. J. Bot. 13: 454 (1993). Type: GEORGIA, Colchis,
Soci, valley of Chosta River, 50 m elev., on leaf of
Laurocerasus officinalis, 1980, Vězda s. n. [holotype: PRA,
not seen; a photo is shown in Ferraro et al. (2001)].
Fig. 1
THALLUS crustose, more or less circular, forming
rounded to irregular patches, up to 4 mm diam. and 20–60
μm thick, with corticiform layer, finely but irregularly
verrucose due to incrustation with calcium oxalate crystals,
greenish grey; prothallus present, membranaceous, palegrey; sterile setae scattered on the thallus that resemble thecilia of hyphophoral conidiomata, tapering towards the tips,
0.2–0.4 mm long and 15–35 μm wide, white translucent.
ASCOMATA not seen. CONIDIOMATA hyphophoral,
laminal, sessile on thallus, their scales divided into 2–5
vertically oriented cilia surrounding the swollen diahyphalmass; cilia, tapering towards the tips, 0.3–0.5 mm long and
15–30 μm wide, often abruptly broadened at the base (upto 90 μm), white translucent; diahyphal mass green, 0.1–
0.15 mm diam., composed of moniliform hyphae, terminal
segments sausage-like shaped, cells 3–8 × 1.5–2.5 μm.
Photobiont Chlorella (s. lat.), green, with a subparietal
chloroplast bearing a conspicuous central pyrenoid,
spherical, 6–11 μm diam. in thallus and spherical toellipsoid, 3–7 × 2–5 μm diam. in diahyphal mass.
Chemistry. C–, K–, KC–, Pd– (thallus). No secondary
substances were detected by HPTLC.
The morphological and chemical features of specimens
collected from Japan are consistent with the protologue(Sérusiaux, 1993) and descriptions provided by Ferraro etal. (2001) and van den Boom (2021). Among the Japanese
specimens, no clear differences in morphology wereobserved, so this study treats the genetic differences (seeabove; Fig. 2) as variations within a single species.
This species is characterized by a greenish grey thallus
with some vertically oriented whitish translucent cilia
surrounding the swollen greenish diahyphal mass (Fig. 1A).
Similar hyphophoral structures are also known in G.
eskuchei Sérus. and G. rosae-emiliae Herrera-Camp. &Lücking (Lücking, 2008) within this genus, but both differ
in lacking a prominent diahyphal mass. The latter also
differs in lacking sterile setae.
A subparietal chloroplast bearing a conspicuous central
pyrenoid in a spherical to ellipsoid cell was observed in thephotobiont cells of G. setiferum from Japan (Fig. 1C).
From the morphology, it can be identified as a species of
Chlorella (s. lat.). However, molecular phylogenetic
analyses of Chlorella (s. lat.) were revealed it as being
polyphyletic and consisting of at least twelve independent
lineages of the Trebouxiophyceae and Chlorophyceae(Huss et al., 1999; Krienitz et al., 2004; Darienko et al.,2010, 2016; Pröschold et al., 2011; Krienitz et al., 2015;
Darienko and Pröschold, 2019). Without DNA data, it
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would be difficult to identify a genus of the photobiont of
G. setiferum. Since photobionts of other Gyalectidum
species belong to Jaagichlorella Reisigl (asHeveochlorella J. Zhang, V.A.R. Huss, X. Sun, K. Chang& D. Pang) (Sanders et al., 2016) which have similar
intracellular structures with a single chloroplast bearing acentral pyrenoid, the photobiont of Japanese G. setiferum
could belong to Jaagicholorella or a related genus within
Chlorella (s. lat.).
The size of photobiont cells in the diahyphal mass aremuch smaller than those in the thallus (Fig. 1B & C). Suchsmall algal cells in the diahyphal mass were also reported
from the type material of G. setiferum (Sérusiaux, 1993).
Gyalectidium setiferum may be confused with otherfoliicolous species having whitish setae or hyphophores on
the thallus (e.g., Aderkomyces spp., Echinoplaca spp. andother Gyalectidium spp.), but these species do not produce
swollen greenish diahyphal masses surrounded byvertically oriented whitish translucent cilia.
This species was known only from western Europe andCaucasus as an obligately foliicolous species, especiallyreported on Buxus and Abies (see Ferraro et al., 2001; vanden Boom, 2021). In Japan, G. setiferum was found on
leaves of Aucuba japonica Thunb. in Ibaraki Prefectureand Maesa japonica (Thunb.) Moritzi & Zoll. in Chiba
Prefecture at ca. 100 m elevation of central Honshu. It ishere reported as new to East Asia, resulting a total of eightspecies of Gyalectidium are recorded at present in thisregion, i.e., G. australe Lücking, G. catenulatum (Cavalc.
& A.A. Silva) L.I. Ferraro, Lücking & Sérus., G.
caucasicum (Elenkin & Woron.) Vězda, G. ciliatum
Lücking, G. Thor & Tat. Matsumoto, G. filicinum Müll.
Arg., G. setiferum Vězda & Sérus., G. shimanense Y. Sutoand G. radiatum Lücking, G. Thor & Tat. Matsumoto(Thor et al., 2000; Aptroot and Sparrius, 2003; Aptroot etal., 2003; this study).
Specimens examined. JAPAN. Honshu. Hitachi Prov. (Ibaraki
Pref.): near Fudotaki Waterfall, Shiio, Makabe-cho, Sakuragawa-city(N36°14′, E140°04′), 120 m elev., on leaf of Aucuba japonica, 5 May 2022,
K. Miyazawa 1017, 1018 pr. p. (in collection Fellhanera bouteillei) (TNS).
Kazusa Prov. (Chiba Pref.): near headstream of Minato River, Toyooka,
Futtsu-city (N35°10′, E139°59′), 130 m elev., on leaf of Maesa japonica,
21 May 2022, K. Miyazawa 1023 (TNS).

ACKNOWLEDGMENTS
We thank Dr. Robert Lücking and an anonymous reviewer for
critical reading of our manuscript and valuable comments; Hiyori
Itagaki for an advice to molecular analysis. This study was partly
supported by JSPS KAKENHI 22J20567 for the first author.

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