A lichen is a symbiotic organism by association of a lichen-forming fungus and an alga and/or a cyanobacterium (Hawksworth, 1988). And the organism is regarded as a consortium of above members and lichenicolous fungi, endolichenic fungi, and other bacteria in a wide sense. Lichen taxonomy is based on the lichen-forming fungi and lichens are classified in the kingdom fungi. The number of lichen species is approximately 19,000 recorded (Lücking et al., 2017). The family Arthoniaceae is the second largest group of the lichen-forming fungi, and this study focuses on a species of the main genus Arthonia in the family.

The genus Bryostigma (syn. Arthonia) was first introduced in 1979 just with a single species B. leucodontis Poelt & Döbbeler (Poelt and Döbbeler, 1979). The authors characterized the genus by the hyphae reaction to red or blue in iodine, the undifferentiated excipulum, the Arthonia-type asci with or without amyloid ring structures, the small ascomata, and importantly the substrate preference to bryophytes (Poelt and Döbbeler, 1979) which is assumingly the key characteristic to name the genus.

However, Bryostigma have been challenged for natural classification. In specific, the genus with the type species B. leucodontis was reclassified into Arthonia s. lat. mainly by its similarity with the specimens of A. apatetica (Coppins, 1989). Both species share the important characteristics of paraphysoids, i.e., brown-walled paraphysoidal tips and periclinally extended paraphysoids at epihymenium, which are the taxonomic key distinguishing from a closely related species, A. patellulata Fée, representing non- or faintly pigmented paraphysoidal tips and erect paraphysoids at epihymenium (Coppins, 1989). The type species was moved into Arthonia as A. muscigena Th. Fr. (Santesson, 1993), and was classified into the Bryostigma clade of Arthonia based on molecular phylogeny (Frisch et al., 2014). The type species of Bryostigma is named B. muscigenum (Th. Fr.) Frisch & G. Thor at present (Frisch et al., 2014). Since Frisch et al. (2014), several new species were discovered in the Bryostigma clade, such as A. parietinaria Hafellner & Fleischhacker, A. lobariellae Etayo, A. toensbergii Holien & Frisch, A. dokdoensis S.Y. Kondr., Lőkös, B.G. Lee, J.J. Woo & Hur, and B. huriellae S.Y. Kondr. & Hur (Fleischhacker et al., 2016; Etayo, 2017; Frisch and Holien, 2018; Kondratyuk et al., 2019, 2020a). The old species A. glebosa is classified in the Bryostigma clade as well (Halıcı and Kahraman, 2021). However, the taxonomy of B. apotheciorum (A. Massal.) S. Y. Kondr. et J.-S. Hur (syn. A. apotheciorum [A. Massal.] Almq.) may not be convincing (Kondratyuk et al., 2020b) as the species was classified far from the Bryostigma clade in molecular phylogeny (Frisch et al., 2014; Lee and Hur, 2016). Overall 18 species are recorded in the Bryostigma clade at present as other possible species, A. fuscopurpurea (Tul.) R. Sant., A. patellulata, and lichenicolous species, are not yet included due mainly to lack of molecular results.

This study describes a lichen-forming fungus in the Bryostigma clade of Arthonia, new to northeastern Asia. Field surveys for the lichen biodiversity in the southern region of the Mts. Baekdudaegan and the forested wetlands of Korea were carried out during the spring and summer of 2021, and overall 25 specimens of Arthonia were collected from the barks of the broad-leaved deciduous trees (Fig. 1). The specimens were comprehensively analyzed in ecology, morphology, chemistry and molecular phylogeny and corresponded to A. apatetica. Arthonia apatetica was previously reported from Europe (Wirth, 1995), North America (Brodo, 1995), the northwest Siberia (Sedelnikova and Taran, 2000), and Mongolia (Hauck and Javkhlan, 2006). We report the specimens as a new record to northeastern Asia. The specimens are deposited in the herbarium of the Baekdudaegan National Arboretum (KBA), South Korea.

Fig. 1. Collection sites for the newly recorded Arthonia apatetica (black star marks).
The brackets indicate substrates, i.e., Ac, Acer tataricum subsp. ginnala; Al, Alnus incana subsp. hirsuta; Ca, Carpinus tschonoskii; Ce, Celtis sinensis; Di, Diospyros kaki.

Morphological and chemical analyses

Hand-cut sections were prepared with a razor blade under a stereomicroscope (Olympus optical SZ51; Olympus), examined under a compound microscope (Nikon Eclipse E400; Nikon) and imaged using a software program (NIS-Elements D; Nikon) and a DS-Fi3 camera (Nikon) mounted on a Nikon Eclipse Ni-U microscope (Nikon). The ascospores were investigated at 1000× magnification in water. The length and width of the ascospores were measured and the range of spore sizes was shown with average, standard deviation, and number of measured spores. Thin-layer chromatography (TLC) was performed using the solvent system C according to standard methods (Orange et al., 2001).

Molecular phylogeny

Isolation, DNA extraction, amplification, and sequencing: hand-cut sections of ascomata or thallus from all collected specimens were prepared for DNA isolation and DNA was extracted with a NucleoSpin Plant II Kit in line with the manufacturer’s instructions (Macherey-Nagel). PCR amplification for the mitochondrial small subunit (mtSSU), the nuclear large subunit ribosomal RNA (LSU), and the RNA polymerase subunit II (RPB2) genes was achieved using Bioneer’s AccuPower PCR Premix (Bioneer) in 20-μl tubes and primers mrSSU1 and mrSSU3R (Zoller et al., 1999), and LR0R and LR5 (Rehner and Samuels, 1994), and fRPB2-7cF and fRPB2-11aR (Liu et al., 1999), respectively. The PCR thermal cycling parameters used were 95°C (15 sec), followed by 35 cycles of 95°C (45 sec), 54°C (45 sec), and 72°C (1 min), and a final extension at 72°C (7 min) based on Ekman (2001). DNA sequences were generated by the genomic research company GenoTech.

Phylogenetic analyses: all mtSSU, LSU and RPB2 sequences were aligned and edited manually using ClustalW in Bioedit V7.2.6.1 (Hall, 1999). All missing and ambiguously aligned data and phylogenetically uninformative positions were removed and phylogenetically informative regions were finally analyzed in MEGA X (Stecher et al., 2020). The final alignment comprised 1347 (mtSSU), 2260 (LSU), and 2065 (RPB2) columns. In them, variable regions were 94 (mtSSU), 353 (LSU), and 32 (RPB2). Finally, the phylogenetically informative regions were 637 (mtSSU), 1113 (LSU), and 472 (RPB2). A concatenation was carried out for combining all mtSSU, LSU and RPB2 gene loci. They were manually combined for the informative regions. Two problematic sequences were removed when conflicting results occurred in the internal branches with the bootstrap values ≥ 70% and the posterior probabilities ≥ 95% in the concatenated tree. Phylogenetic trees with bootstrap values were obtained in RAxML GUI 2.0 beta (Edler et al., 2021) using the maximum likelihood method with a rapid bootstrap with 1,000 bootstrap replications and GTR GAMMA (TVM + G4 for both mtSSU and the concatenation of mtSSU, LSU and RPB2) for the substitution matrix. The posterior probabilities were obtained in BEAST 2.6.4 (Bouckaert et al., 2019) using the GTR 123421 (mtSSU) and the GTR 123121 (concatenation) models, as the appropriate model of nucleotide substitution produced by the Bayesian model averaging methods with bModelTest (Bouckaert and Drummond, 2017), empirical base frequencies, gamma for the site heterogeneity model, four categories for gamma, and a 10,000,000 Markov chain Monte Carlo chain length with a 10,000-echo state screening and 1,000 log parameters. Then, a consensus tree was constructed in TreeAnnotator 2.6.4 (Bouckaert et al., 2019) with the first 25% discard as a burn-in, no posterior probability limit, a maximum clade credibility tree for the target tree type, and median node heights. All trees were displayed in FigTree 1.4.2 (Rambaut, 2014) and edited in Microsoft Paint. Most sequences employed for the analyses are based on Frisch et al. (2014). Overall analyses in the materials and methods were accomplished based on Lee and Hur (2020).

Taxonomy

Arthonia apatetica (Massal.) Th. Fr., Botaniska Notiser 56 (1866) (Fig. 2).

Fig. 2. Arthonia apatetica (KBA-L-0001570 for [A, B, D, & F–K]; KBA-L-0002050 for [C and D]) in morphology.
(A–E) Habitus and apothecia. (F) Adnate apothecia with chlorococcoid algae. (G) Interascal hyphae anastomosing with coarsely periclinally arranged paraphysoids at epihymenium. Paraphysoidal tips not pigmented but swollen a little. (H) Hymenium representing blue in iodine. (I) Asci clavate to widely clavate with 8-spores per ascus. (J–L) Ascospores consistently 1-septate with constriction at septum. Bars: (A), 2 mm; (B and C), 500 μm; (D and E), 200 μm; (F), 50 μm; (G–I), 20 μm; (J–L), 10 μm.

Thallus corticolous, crustose, smooth, olive-green, not developed or indistinct as getting old and mostly being disappeared; photobiont chlorococcoid, cells globose to subglobose, 5–20 μm diam. Prothallus absent. Apothecia simple, generally rounded, somewhat convex, dark brown to black, more brownish in water, not pruinose, 0.1–0.4 mm diam.; epihymenium brown to slightly grayish brown, 10–15 μm high; hymenium hyaline, 30–40 μm high; hypothecium pale brown, 30–40 μm high. Crystals or oil droplets not present. Interascal hyphae paraphysoidal, anastomosing, irregular but rather periclinally arranged at epihymenium, tips slightly swollen, not pigmented, branched, 1.5–2 μm thick, brown epihymenium not clearly pigmented by paraphysoidal tips. Asci 8-spored, clavate to narrowly clavate, 24–35 × 14–20 μm (n = 10). Ascospores constantly 1-septate, constricted at septum, upper cell wider and the lower longer, occasionally tales present on one or both ends, 11.5–17 × 4–6 μm (mean = 13.6 × 5.2 μm; SD = 1.1(L), 0.5(W); L/W ratio 2.0–3.4, ratio mean = 2.6, ratio SD = 0.3; n = 50), without epispore.

Chemistry: Epihymenium K+ greenish gray to blackish, hymenium I+ red-brown or blue, UV–. No lichen substance was detected by TLC.

Ecology and distribution: The species occurs on barks of diverse deciduous trees (e.g., Acer, Alnus, Carpinus, Celtis, or Diospyros). This species is distributed in Europe, North America, the northwest Russia, Mongolia, and Korea.

Remarks: Arthonia apatetica is characterized by small, black apothecia with 1-septate ascospores, absence of crystals, pale brown hypothecium, periclinally arranged paraphysoids at epihymenium, and chlorococcoid photobiont among corticolous species. Arthonia patellulata differs from A. apatetica by erect paraphysoids at epihymenium and darker hypothecium. Although B. muscigenum share the characteristic, i.e., the periclinally arranged paraphysoids at epihymenium, the former differs from the latter by narrower ascospores (2.5–4 μm) and darker hypothecium (Smith et al., 2009).

The Korean specimens are similar to those of Europe and North America with a little difference (Table 1). The Korean specimens are supposed to be classified into A. apatetica in morphology and molecular phylogeny and we regard that the difference in morphology is an infraspecific variation.

Table 1 Infraspecific comparison of Arthonia apatetica among different continents

Specimens	Korea	Europe	North America
Hymenium height (μm)	30–40	40–60	40–50
Hymenium color	Hyaline	Hyaline to pale red	Pale brown
Hymenium in iodine	Red-brown	Red	Red
Hypothecium color	Pale brown	Hyaline to straw-colored	Pale brown
Paraphysoidal tip pigmentation	Not pigmented	Distinct dark brown	–
Paraphysoidal tip thickness (μm)	1.5–2	3–4	c. 1 (but variable)
Ascospores (μm)	11.5–17 × 4–6	12–15 × 4.5–5	10–13 × 4–5
Reference	Present study	Smith et al. (2009)	Nash et al. (2007)

The morphological characteristics are referenced from the previous literatures. All information on A. apatetica of Korea is produced from collected specimens in this study.

Specimen examined: SOUTH KOREA, South Jeolla Province, Jinan, Jucheon-myeon, a forested wetland, 35°59’45.4”N, 127°21’58.2”E, 680 m alt., on bark of Acer tataricum subsp. ginnala, 10 May 2021, B.G. Lee & D.Y. Kim 2021-000098, with Lecanora sp., Phaeophyscia rubropulchra (KBA-L-0001570; GenBank ON116972 for mtSSU, ON115808 for LSU, and ON260924 for RPB2); SOUTH KOREA, South Jeolla Province, Gwangju, Gwangsan-gu, Dodeuk-dong, a forested wetland (Jang Valley), 35°08’55.1”N, 126°42’42.9”E, 60 m alt., on bark of Diospyros kaki, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000165, with Graphis scripta, Phaeophyscia rubropulchra, Porina hirsuta (KBA-L-0001637; ON116973 for mtSSU, ON115809 for LSU); same locality, on bark of Alnus incana subsp. hirsuta, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000182, with Hypotrachyna pseudosinuosa, Traponora varians (KBA-L-0001654); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000194, with Hypotrachyna pseudosinuosa, Phaeophyscia rubropulchra, Porina hirsuta (KBA-L-0001666); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000197, with Hypotrachyna pseudosinuosa, Phaeophyscia rubropulchra (KBA-L-0001669); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000199, with Hypotrachyna pseudosinuosa, Phaeophyscia rubropulchra (KBA-L-0001671); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000200, with Hypotrachyna pseudosinuosa, Porina hirsuta (KBA-L-0001672); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000201, with Phaeophyscia rubropulchra (KBA-L-0001673); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000202, with Phaeophyscia rubropulchra (KBA-L-0001674); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000203, with Phaeophyscia rubropulchra, Porina hirsuta (KBA-L-0001675); same locality, on bark of Celtis sinensis, 11 May 2021, B.G. Lee & D.Y. Kim 2021-000205, with Graphis scripta, Hypotrachyna pseudosinuosa, Phaeophyscia rubropulchra, Porina hirsuta (KBA-L-0001677); SOUTH KOREA, North Jeolla Province, Jangsu, Mt. Youngchi, 35°38’35.5”N, 127°36’59.7”E, 905 m alt., on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000565, with Bacidia ekmaniana, Lecidella euphorea (KBA-L-0002037); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000568, with Lecidella euphorea, Phaeophyscia adiastola (KBA-L-0002040); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000570, with Anisomeridium polypori, Lecidella euphorea (KBA-L-0002042); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000571, with Anisomeridium polypori, Pertusaria sp. (KBA-L-0002043); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000572, with Caloplaca flavorubescens, Lecanora hafelliana, Lecidella euphorea (KBA-L-0002044); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000573, with Bacidia ekmaniana, Lecanora aff. perflexuosa, Lecidella euphorea, Porina hirsuta (KBA-L-0002045); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000574, with Rinodina orientalis (KBA-L-0002046); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000575, with Graphis scripta, Rinodina orientalis (KBA-L-0002047); same locality, on bark of Carpinus tschonoskii, 08 Jun 2021, B.G. Lee & H.J. Lee 2021-000576, with Pyrenula laevigata, Rinodina orientalis, Scoliciosporum chlorococcum (KBA-L-0002048); SOUTH KOREA, North Jeolla Province, Jangsu, Mt. Jangan, 35°38’35.0”N, 127°36’57.8”E, 925 m alt., on bark of Carpinus tschonoskii, 09 Jun 2021, B.G. Lee & H.J. Lee 2021-000774, with Lecidella euphorea, Micarea aff. elachista (KBA-L-0002046); same locality, on bark of Carpinus tschonoskii, 09 Jun 2021, B.G. Lee & H.J. Lee 2021-000775, with Lecanora sp., Lecidella euphorea (KBA-L-0002047); same locality, on bark of Carpinus tschonoskii, 09 Jun 2021, B.G. Lee & H.J. Lee 2021-000776, with Rinodina orientalis (KBA-L-0002048; ON115810 for LSU, ON260925 for RPB2); same locality, on bark of Carpinus tschonoskii, 09 Jun 2021, B.G. Lee & H.J. Lee 2021-000778, with Caloplaca gordejevii, Lecanora hafelliana, Rinodina orientalis (KBA-L-0002050; ON260926 for RPB2); SOUTH KOREA, North Jeolla Province, Jangsu, Mt. Jangan, 35°38’36.1”N, 127°36’53.9”E, 950 m alt., on bark of Carpinus tschonoskii, 09 Jun 2021, B.G. Lee & H.J. Lee 2021-000788, with Lecanora sp., Lecidella euphorea, Myelochroa xantholepis, Pertusaria multipuncta, Rinodina archaea, Rinodina orientalis (KBA-L-0002060).

Molecular phylogeny

Two phylogenetic trees (mtSSU, and the concatenation of all mtSSU, LSU and RPB2) for the genus Arthonia and related genera were produced from overall 184 sequences (72 for mtSSU, 55 for LSU, and 57 for RPB2) of GenBank and eight new sequences (two for mtSSU, and each three for LSU and RPB2) from the new record (Table 2). The sequences of the new record were positioned in the Bryostigma clade in both the mtSSU and the concatenation trees (Figs. 3 and 4). Both trees describe that the sequences of the new record are located in the clade and between the tips of A. apatetica, supported by the bootstrap values of 98 (mtSSU) and 100 (concatenation), and the posterior probabilities of 1.0 (both) for the branches.

Table 2 Species list and DNA sequence information employed for phylogenetic analysis

No.	Species	mtSSU	LSU	RPB2	Voucher
1	Arthonia apatetica	KJ850992	KJ851045	KJ851125	UPS:Svensson 2017 (Europe)
2	Arthonia apatetica	KJ850993	KJ851056		UPS:Hermansson 17868b (Europe)
3	Arthonia apatetica	KJ850994	KJ851050	KJ851126	UPS:Svensson 1939 (Europe)
4	Arthonia apatetica	ON116972	ON115808	ON260924	KBA-L-0001570
5	Arthonia apatetica	ON116973	ON115809		KBA-L-0001654
6	Arthonia apatetica		ON115810	ON260925	KBA-L-0002048
7	Arthonia apatetica			ON260926	KBA-L-0002050
8	Arthonia apotheciorum	KJ850970		KJ851148	UPS:Frisch 11/Se23
9	Arthonia biatoricola	KJ850990		KJ851149	UPS:Thor 24350
10	Arthonia caesia	FJ469671	FJ469668	FJ469670	AFTOL-ID 775
11	Arthonia calcarea	EU704065		EU704029	Ertz 7540
12	Arthonia didyma	EU704047	EU704083	EU704010	Ertz 7587
13	Arthonia didyma		KJ851081	KJ851106	UPS:Frisch 11/Se41
14	Arthonia dispersa	AY571383	AY571381		UPSC 2583
15	Arthonia eos	KJ850987	KJ851053	KJ851134	UPS:Thor 26000
16	Arthonia glebosa	MZ502372			JR 0.154
17	Arthonia granithophila	KJ850981	KJ851049	KJ851107	UPS:Frisch 10/Se74
18	Arthonia graphidicola	KJ850980	KJ851034		UPS:Frisch 10/Jp102
19	Arthonia ilicina	KJ850982	KJ851069		UPS:McCune 31067
20	Arthonia incarnata	KY983974		KY983982	Frisch 13/Jp215
21	Arthonia incarnata	KY983976		KY983984	Thor 26035
22	Arthonia lapidicola	KJ850997	KJ851070	KJ851119	UPS:Frisch 11/Se47
23	Arthonia lobariellae	MT153920	MT153975		A.F.25324
24	Arthonia mediella	KJ851014	KJ851032	KJ851133	UPS:Frisch 11/Se22
25	Arthonia mediella	KJ851015	KJ851031	KJ851132	UPS:Thor 12/8
26	Arthonia molendoi	KJ851000	KJ851051	KJ851117	UPS:Frisch 11/Se36
27	Arthonia molendoi	MH177777	MH177781	MH177770	Frisch 17/No10
28	Arthonia neglectula	KJ850989	KJ851037	KJ851118	UPS:Frisch 10/Se91
29	Arthonia parietinaria	MH177778	MH177782	MH177771	Frisch 17/No25
30	Arthonia peltigerina	KJ850998		KJ851122	UPS:Westberg Frisch 11/Se46
31	Arthonia phaeophysciae	MN955428	MN963753		KT180627002
32	Arthonia physcidiicola	KF707646		KF707657	UPS:Frisch 11/Ug318
33	Arthonia picea	MG201836	MG201847		Frisch 13Jp107
34	Arthonia picea	MG201837	MG201848		Frisch 13Jp124
35	Arthonia pinastri	MN842780	MN842779		MFLU 17-3497
36	Arthonia punctiformis	KJ850973	KJ851044	KJ851113	UPS:Thor 26158
37	Arthonia aff. punctiformis	KJ850975	KJ851043		UPS:Thor 24702
38	Arthonia radiata	EU704048		EU704011	Ertz s.n.
39	Arthonia radiata	KJ850969		KJ851109	UPS:Frisch 11/Se25
40	Arthonia ruana	MG495133	MG495139		KoLRI 044321
41	Arthonia ruana	MG495134	MG495140		KoLRI 044324
42	Arthonia ruana	MG495135	MG495142	MG589416	KoLRI 044331
43	Arthonia ruana	MG495136		MG589417	KoLRI 044332
44	Arthonia ruana		MG495141	MG589415	KoLRI 044325
45	Arthonia rubrocincta	GU327684			Nelsen 4010
46	Arthonia sanguinaria	MG201838	MG201846		Frisch13Jp59
47	Arthonia spadicea		KJ851029	KJ851115	UPS:Frisch 11/Se31
48	Arthonia spadicea		KJ851071	KJ851116	UPS:Thor 26200
49	Arthonia stereocaulina	KJ850999	KJ851062		UPS:Westberg Frisch 11/Se48
50	Arthonia subfuscicola	KJ850972		KJ851111	UPS:Frisch 11/Se15
51	Arthonia subfuscicola			KJ851110	UPS:Thor 44866
52	Arthonia subfuscicola	KJ850971			UPS:Thor 11/1
53	Arthonia thoriana	MG207685			Sanderson 2174
54	Arthonia thoriana	MG207687			Sanderson 2176
55	Arthonia thoriana	MG207686			Sanderson 2175
56	Arthonia toensbergii	MH177775	MH177779		Frisch N4-2-Pa4-1
57	Arthonia toensbergii	MH177776	MH177780		Frisch N1-2-Pa6-1
58	Arthonia ulleungdoensis	MG495138	MG495144	MG589418	KoLRI 044339
59	Arthonia sp.	EU704049	EU704084	EU704012	Ertz 7775
60	Arthonia sp.	EU704050		EU704013	Ertz 9090
61	Arthonia sp.	KJ850985	KJ851064	KJ851129	UPS:Frisch 11/Ug212
62	Arthonia sp.	KJ850986	KJ851068	KJ851131	UPS:Frisch 11/Ug218
63	Arthonia sp.	KJ850995	KJ851046	KJ851120	UPS:Svensson 2148
64	Arthonia sp.	KJ850996		KJ851121	UPS:Svensson 2295
65	Arthonia sp. ‘lobariicola’	KJ851002	KJ851036	KJ851128	UPS:Frisch 10/Jp124
66	Arthonia sp.	KJ851019	KJ851082	KJ851123	UPS:Svensson 2324
67	Arthonia sp.	KJ851025	KJ851039	KJ851100	UPS:Frisch 11/Ug41
68	Arthothelium galapagoense		HQ454515	HQ454657	Ertz 11654
69	Arthothelium galapagoense		HQ454516	HQ454658	Ertz 11790
70	Arthothelium norvegicum	KJ851003	KJ851038	KJ851114	UPS:McCune 31061
71	Arthothelium orbilliferum	KY983977			TRH-L-15449
72	Arthothelium spectabile	KP870144		KP870160	TNS:Frisch 12Jp179a
73	Arthothelium spectabile	MN955429		MN947670	Frisch 12Jp164
74	Bryostigma dokdoense	MH510022		MH510019	SK L06
75	Bryostigma dokdoense	MH510023		MH510021	SK L04
76	Bryostigma muscigenum	KJ850991	KJ851052	KJ851124	UPS:Thor 26206
77	Opegrapha lithyrga	EU704068	EU704096	EU704032	Ertz 8784
78	Opegrapha niveoatra	EU704070	EU704098	EU704034	Ertz 7529
79	Opegrapha vermicellifera	EU704077	EU704105	EU704041	Ertz 7562
80	Opegrapha vulgata	EU704080	EU704108	EU704044	Ertz 7564
81	Opegrapha zonata	EU704081	EU704109	EU704045	Vigneron 104
	Overall	72	55	57

DNA sequences which were generated for the new record A. apatetica in this study, are presented in bold. All others were obtained from GenBank. The species names are followed by GenBank accession numbers and voucher information. mtSSU, mitochondrial small subunit; LSU, nuclear large subunit ribosomal RNA; RPB2, RNA polymerase subunit II; Voucher, voucher information.

Fig. 3. Phylogenetic relationships among available species in the genus Arthonia based on a maximum likelihood analysis of the dataset of mtSSU sequences.
The tree was rooted with Opegrapha sequences based on Lee and Hur (2016). Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The sequences of newly recorded Arthonia apatetica specimens are presented in bold, and all species names are followed by the GenBank accession numbers. Table 2 provides the species related to the specific GenBank accession numbers and voucher information.

Fig. 4. Phylogenetic relationships among available species in the genus Arthonia based on a maximum likelihood analysis of the dataset of the concatenated dataset of all mitochondrial small subunit (mtSSU), nuclear large subunit ribosomal RNA (LSU), and RNA polymerase subunit II (RPB2) sequences.
The tree was rooted with Opegrapha sequences based on Lee and Hur (2016). Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The sequences of newly recorded Arthonia apatetica specimens are presented in bold, and all species names are followed by the voucher information. Table 2 provides the species related to the specific GenBank accession numbers and voucher information.

Key to the species of Arthonia in northeastern Asia (54 taxa)

An updated taxonomic key is introduced for the species of Arthonia in northeastern Asia (Korea, Japan, and China) (Table 3). Based on the key of Lee and Hur (2016), eight species were additionally reported from Korea and Japan, i.e., A. almquistii Vain., A. apatetica, A. dokdoensis S.Y. Kondr., Lőkös, B.G. Lee, J.J. Woo & Hur, A. incarnata Kullh. ex Almq., A. lopingensis Zahlbr., A. picea Vain., A. sanguinaria Frisch & Y. Ohmura, and A. ulleungdoensis B.G. Lee & Hur (Zhurbenko et al., 2015; Zhurbenko et al., 2015, 2018; Kondratyuk et al., 2019; Lee and Hur, 2019). Although previously recorded in China, A. lopingensis was newly discovered in Japan with specific information (Frisch et al., 2018).

Table 3 Key to the species of Arthonia in Northeast Asia (Korea, Japan and China)

1.	Lichenicolous	2
–	Not lichenicolous	7
2.	Hypothecium dark brown or blackish brown; epihymenium dark brown, on Rinodina species	A. rinodinicola
–	Hypothecium hyaline to pale brown; epihymenium pale olive-brown or yellow-orange	3
3.	Epihymenium yellow to orange; on Graphis species	A. lopingensis
–	Epihymenium pale olive-brown to brown	4
4.	On Phaeophyscia species; asci 35–45 × 16–20 μm	A. phaeophysciae
–	On Rhizoplaca species; asci 28–33 × 14–17 μm	A. clemens
–	On various species of Porpidiaceae and Agyriaceae, or Amygdalaria pelobotryon, esp. Trapelia species in NE Asia; asci 28–38 × 17–20 μm	A. almquistii
–	On Caloplaca, Orientophila, or Xanthoria species	5
5.	Ascospores 3-septate, 12–17 × 4.5–6 μm; on Xanthoria species, but usually on bark	A. varia
–	Ascospores 1-septate, 8–13 × 4–6 μm	6
6.	Ascospores 1-septate, 10–13 × 4–5 μm; on Orientophila species	A. dokdoensis
–	Ascospores 1-septate, 8–11 × 4–6 μm; on Caloplaca or Xanthoria species	A. molendoi
7.	Foliicolous, saxicolous or terricolous	8
–	Corticolous	16
8.	Foliicolous	9
–	Saxicolous or terricolous	13
9.	Ascospores grayish brown when mature	10
–	Ascospores colorless when mature	12
10.	Thallus K+ purple; ascospores 1-septate; usually on bark	A. vinosa
–	Thallus K−; ascospores 2-septate	11
11.	Apothecia blackish brown to black, usually rounded to oval, naked or with a very thin, indistinct, marginal pruina; ascospores 11–19 (–24) × 4–6 (–9) μm, 3–3.5 times as long as broad	A. trilocularis
–	Apothecia blackish brown with a purplish tinge, irregularly angular, with a thin but distinct marginal pruina which sometimes spreads over the apothecial surface; ascospores 13–19 × 2.5–4.5 μm, 3–5 times as long as broad	A. fuscocyanea
12.	Apothecia light to dark brown, non-pruinose; ascospores consistently 2-septate, 11–16 × 3–5 μm	A. accolens
–	Apothecia dark greenish gray but with a whitish pruina which gives them a conspicuous bluish tinge; ascospores variably 2- to 4-septate, 11–25 (–29) × 3.5–6.5 (–8) μm	A. cyanea
13.	Terricolous; thick thallus; dark brown hypothecium; ascospores single to 1-septate, hyaline	A. glebosa
–	Saxicolous	14
14.	Thallus foliose, thick	A. rufidula
–	Thallus crustose, thin	15
15.	Thallus rimose-cracked, thin; apothecia slit, lirellate, simple, branched or stellate, 0.6–1.2 (–2) × (0.1–) 0.15–0.3 mm; ascospores 3-septate, 16–20 (–24) × 4–5 (–6.5) μm; especially on limestone	A. calcarea
–	Thallus partly immersed to irregularly scurfy granularverrucose, thin; apothecia globose, simple, convex, dark brown to black, not branched, (0.1–) 0.2–0.4 (–0.5) mm; ascospores 1-septate, 11–15 (–18) × 4–7 μm; on limestone or basalt	A. lapidicola
16.	Apothecia brownish black or black	17
–	Apothecia colored, not just brownish or blackish	45
17.	Ascospores 1-septate	18
–	Ascospores 2-septate or multi-septate	22
18.	Apothecia curved or flexuose, often branching	A. excipienda
–	Apothecia rounded, not branching	19
19.	Thallus white to pale fawn, K+ purple	A. vinosa
–	Thallus green to dark gray, K−	20
20.	Apothecia 0.1–0.4 mm; thallus olive-green	A. apatetica
–	Apothecia 0.2–1.5 mm; thallus dark gray to black-gray	21
21.	Apothecia 0.75–1 mm; ascospores 20–23 × 7–9 μm	A. henoniana
–	Apothecia 0.2–1.5 mm; ascospores 7–11 × 3–4 μm	A. spadicea
22.	Ascospores 2-septate, sometimes 1-septate, 11–12 × 4.5–5 μm; apothecia sub-angular or irregularly stellate; thallus whitish macular	A. biseptella
–	Ascospores 3-septate or more	23
23.	Ascospores 3-septate	24
–	Ascospores more than 3-septate	30
24.	Thallus bleached as whitish or grayish macular	25
–	Thallus not bleaching	27
25.	Apothecia stellate, few radiate; ascospores bigger, 17–19 × 6–7 μm	A. astropica
–	Apothecia linear, round or thinly stellate; ascospores smaller, 12–17 × 4.5–6 μm	26
26.	Apothecia linear, round to irregular, flat, not branching; ascospores 12–17 × (3–) 4.5–6 μm; thallus light gray; on bark or sometimes on Xanthoria parietina	A. varia
–	Apothecia divided or thinly stellate, erumpent, dendritically branching; ascospores 15–17 × 5–6 μm; thallus white macular; on bark only	A. varia var. stenograpella
27.	Locules unequal, apical cell enlarged; thallus cinnamon-gray, shiny	A. schoepfiae
–	Locules equally divided; thallus whitish or grayish	28
28.	Photobiont present, chlorococooid; apothecia rounded to subangular; ascospores 7.4–18 × 3.8–5.1 μm; I+ blue on apothecial section	A. zelkovae
–	Photobiont absent; apothecia rounded to irregularly branched	29
29.	Apothecia brown, distinctly pruinose; I+ blue turning red on apothecial section; hypothecium hyaline; paraphysoidal tips not thickened, without distinct pigment caps	A. glaucella
–	Apothecia black, usually epruinose; I+ persistently blue on apothecial section; hypothecium tinged light brownish; paraphysoidal tip cells slightly thickened, with apical pigment caps	A. pinastri
30.	Ascospores up to 5-septate	31
–	Ascospores 5-septate or more	40
31.	Apothecia rounded to substellate; ascospores less than 20 μm long	32
–	Apothecia mostly rounded or orbicular to angular, sometimes linear to stellate; ascospores over 20 μm long at maximum	35
32.	Apothecia rounded; thallus yellowish gray; Ascospores 11–15 × 5–6 μm; on Rhus vernix	A. vernicis
–	Apothecia various, rounded, lirellate or stellate; thallus whitish gray to greenish gray	33
33.	Heavily blue-gray or whitish pruinose on apothecia; ascospore end cell noticeably enlarged	A. cinereopruinosa
–	Epruinose or almost not pruinose on apothecia; ascospore cells equal and end cell not enlarged	34
34.	Ascospores 3- to 4-septate, 15–20 × 4.5–6 μm; I+ blue on hymenium	A. radiata
–	Ascospores 4- to 5-septate, 14–16 × 4–6 μm; I+ purplish red on hymenium	A. taediosula
35.	Locules unequal, end cells or center cells enlarged; spores larger, 16–32 × 6–12 μm	36
–	Locules equal, certain cells not enlarged; spores smaller, 13–23 × 4–7 μm	39
36.	Ascospores usually 5-septate; apothecia rounded, angular or sometimes elongated	37
–	Ascospores usually 3- or 4-septate; apothecia irregularly rounded to elongated or stellate	38
37.	Ascospores usually 5-septate, sometimes 4- or 6-septate, one or both spore end cells enlarged, 22–32 × (6–) 9–12 μm; apothecia round, somethimes elongated up to 1.2 mm, flat to convex, black with yellow tinge, 0.3–0.7 (–1.5) mm diam.; thallus smooth to rough and chinky, greenish gray to white	A. complanata
–	Ascospores consistently 5-septate, two intermediate cells greater than else, 20–26 × 6.5–8 μm; apothecia orbicular or angular, not radiant, flat, black, 0.15–0.25 mm diam.; thallus smooth, gray-white macular formed	A. gregantula
38.	Apothecia irregularly rounded or elongated, 0.1–0.6 mm diam.; ascospores 3- or 4-(5-)septate, (16–)18–23 × 7–9 μm; pycnidia 60–80 μm, pycnoconidia 7–9 × 1 μm	A. ilicinella
–	Apothecia linear, or more usually irregularly branched to stellate, up to 2 mm diam.; ascospores (2-) 3- or 4-septate, (13–) 16–22 (–24) × (5–) 6–7 (–9) μm; pycnidia 40–60 μm; pycnoconidia 4.5–5.5 × 0.5–1 μm	A. stellaris
39.	Ascospores usually 3-septate (sometimes up to 5-septate), 13–23 × 4–7 μm; I+ dark vinose-red on hymenium	A. punctiformis
–	Ascospores mostly 4-septate, 15.5–21.3 × 4.5–6.3 μm; I+ blue on epihymenium and hymenium, and I+ vinose-red on ascus and spores	A. coreana
40.	Prothallus with black thin line; apothecia lirellate to sublinear	41
–	Prothallus indistinct or sometimes with brown line; apothecia rounded to substellate	42
41.	Ascospores 5- to 6-septate, two center locules distinctly larger, 21–24 × 6–7 μm; asci 8-spored; thallus slightly olivaceous, shiny	A. leioplacella
–	Ascospores 4- to 8-septate, end cell enlarged, 28–37 × 8–12 μm; asci 4- to 6-spored; thallus milky-white macular	A. linearis
42.	Hypothecium indistinct or ± hyaline; epihymenium blackish; ascospores 5- to 6-septate, (13–) 18–23 × 5–8 μm; apothecia 0.01–0.3 (up to 1–1.5) mm	A. reniformis
–	Hypothecium brownish; epihymenium brownish; ascospores over 6-septate at the maximum	43
43.	Ascospores 5- to 7-septate, 18–24 × 6–8 μm; apothecia 0.5–0.6 mm	A. pertabescens
–	Ascospores 4 to 8 transeversely septate and 0 to 3 longitudinally septate, muriform; apothecia 0.7–2.6 mm	44
44.	Apothecia irregular to stellate, punctiform; apothecial section 70–95 μm thick; hymenium 35–50 μm thick; asci 8-spored; ascospores brown when mature, 15–26 × 7–10.5 μm	A. ruana
–	Apothecia rounded, non-punctiform; apothecial section 100–130 μm thick; hymenium 50–60 μm thick; asci 2- to 6-spored; ascospores colorless with dark septum when mature, 21–31 × 9–13 μm	A. ulleungdoensis
45.	Ascospores 9- to 13-septate, over 40 μm in length	A. ochropallens
–	Ascospores 3- to 7-septate, less than 40 μm in length	46
46.	Apothecia pruinose or epruinose, violet-brown or brown with purple-red pigment	47
–	Ascospores epruinose, cinnamon or pale yellow	50
47.	Apothecia epruinose, dark red to dark brown-red	48
–	Ascospores pruinose, violet-brown to cinnabar red	49
48.	Apothecia dark red; ascospores 4–5-septate, colorless with pale brown wall when mature, 17–19.5 × 6–7.5 μm; confluentic acid or ±psoromic acid present	A. sanguinaria
–	Apothecia dark brown-red; ascospores 4–6-septate, brown when mature, 27–32.5 × 10–11.5 μm; no substance above	A. picea
49.	Apothecia violet-brown with grayish white pruina at margin; ascospores usually 5-septate, 21–24 (–28) × 7–8 μm	A. adspersa
–	Apothecia brown to blackish with cinnabar-red pruina at margin and white pruina in center; ascospres 3- to 5-septate (up to 7-septate), 20–33 × 5.5–11 μm	A. cinnabarina (Coniocarpon cinnabarinum)
50.	Apothecia cinnamon or somewhat red-brown; anthraquinone (parietin) or depsidone(± psoromic acid, ± norstictic acid) present	A. lopingensis
–	Apothecia pale straw-yellow; xanthone (arthothelin) or lichexanthone present or no substance	51
51.	Pycnidia among apothecia, I+ purplish red on hymenium	A. perpallens (A. antillarum f. spermogonifera)
–	Pycnidia unknown	52
52.	Apothecia adnate and convex; hymenium I+ vinose-red or orange-red; UV– or UV+ white on thallus	53
–	Apothecia innate and almost flat; hymenium I+ blue; UV+ orange to red on apothecia	54
53.	Prothallus pale yellow when present; asci 20–40 × 13–18 μm; ascospores generally 3-septate; xanthone present	A. superpallens
–	Prothallus black when present; asci 40–50 × 14–20 μm; ascospores 2-septate; no substance	A. incarnata
54.	UV+ orange on apothecia (indicative of lichexanthone); apothecia smaller, 0.4–0.5 mm	A. antillarum
–	UV+ red on apothecia (indicative of xanthone); apothecia larger and less branched, 0.3–0.7 mm	A. parantillarum

Discrepancy between the genus Bryostigma and the Bryostigma clade

It should be noted that there is a discrepancy between the genus Bryostigma and the Bryostigma clade. Although most species in the Bryostigma clade including recently reported new species (Fleischhacker et al., 2016; Etayo, 2017; Frisch and Holien, 2018; Kondratyuk et al., 2019, 2020a) are classified into the genus Bryostigma (Kondratyuk et al., 2020b), many species in the clade do not correspond to the definition of the genus Bryostigma of Poelt and Döbbeler (1979). Although the authors emphasized bryophytes for the genus substrate when they defined the genus, just one single species, B. muscigenum, is muscicolous yet all other species in the clade are lichenicolous, corticolous, saxicolous or terricolous. In addition, there has been a wrong taxonomy for B. apotheciorum (Kondratyuk et al., 2020b). Arthonia apotheciorum was classified far from the Bryostigma clade (Frisch et al., 2014; Lee and Hur, 2016), and the species should be remained in Arthonia s. lat. Kondratyuk et al. (2020b) confused the species assumingly with A. apatetica for the genus Bryostigma. The definition of the genus Bryostigma should be emended for the Bryostigma clade based on a preliminary definition (Frisch et al., 2014; Frisch and Holien, 2018) or remained for further studies without just combinationing of species in the clade to the genus. Other species possibly belonging to the Bryostigma clade should be considered in the new definition (e.g., A. fuscopurpurea, A. patellulata, and many other lichenicolous species).

티끌별지의 [Arthonia apatetica (A. Massal.) Th. Fr.]가 북동아시아지역 미기록종으로서 한국에서 최초 발견되었다. 너무 작아서 간과되기 쉬운 이 미생물은, 작고 검은 자낭과 속에 1-격벽의 포자를 가지고 있고, 수정결정이 없으며, 옅은 갈색의 자낭하층을 보여주고, 자낭상층의 균사체가 수평으로 정렬되어있고, 클로로코코이드(chlorococcoid) 녹조류를 가진 수피착생형 지의류이다. 분자분석법을 통해 mtSSU, LSU, RPB2 유전자 염기서열을 이용 종합 분석한 결과, 한국에서 채집된 지의류 표본이 티끌별지의(A. apatetica)임을 증명하였다. 이번 연구를 통해 해당 미기록종의 유전정보를 아시아 최초로 확보하였다. 더불어 북동아시아 별지의(Arthonia)속 지의류 총 54분류군에 대한 분류검색표를 새롭게 마련하였다.

This work was supported by a grant from Korean Forest Service Program through the Korea National Arboretum (KNA-202003127AF-00) for the forested wetland conservation of Korea.

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.