A New Lichen-Forming Fungus, Aspicilia humida, from a Forested Wetland in South Korea, with a Taxonomic Key for Aspicilioid Species of Korea.

Aspicilia humida Lee is described as a new lichen-forming fungus from a wetland forest, South Korea. The new species is distinguishable from Aspicilia aquatica (Fr.) Körb., the most similar species, by the absence of prothallus, black disk without green color in water, olive-brown epihymenium, shorter hymenium, hymenium I + yellowish blue-green, wider paraphysial tips without a vivid pigment, smaller asci, smaller ascospores, and the presence of stictic acid. Molecular analyses employing internal transcribed spacer (ITS) and mitochondrial small subunit (mtSSU) sequences strongly support A. humida as a distinct species in the A. cinerea group. A surrogate key is provided to assist in the identification of all 28 aspicilioid species of Korea.

Introduction

As the Aspicilia was comprising more than 300 species, the large genus has been classified into several infrageneric groups and some of them were finally splitted into some new genera or resurrected as old genera [1-6]. The genus Megaspora was newly introduced by the characteristics of large ascospores with thick walls and anastomosing paraphyses [7,8]. The genus Lobothallia was raised up from the A. radiosa group [9]. The old genus Circinaria (previously the A. contorta/calcarea group) was reintroduced by the characteristics of diverse thalli (crustose, foliose to subfruiticose), broad-ellipsoid to globose ascospores which are shown generally less than eight per ascus, and particularly the presence of aspicilin [3,4]. The old genus Sagedia was reintroduced based on the molecular analysis [3]. The genus Teuvoa was newly introduced by the characteristics of the lack of lobate, radiating thalli, generally absence of algal layer underlining hypothecium, the absence of secondary metabolites, and the substrate preference to barks or woods but not rocks, in comparing with Lobothallia [10]. The genus Oxneriaria (previously the A. mashiginensis group) was newly introduced by defining the characteristics of radiating thalli with wrinkled or lobate periphery, small ascospores, presence of substictic acid, and the habitat preference to polar and alpine areas [5]. The old genus Aspiciliella was reintroduced by the characteristics of consistently ellipsoid ascospores, small conidia, and the presence of norstictic acid in all species [11]. The infrageneric groups are further categorized into seven groups in Aspicilia s. str. [6]. The Aspicilia (200 spp.) is still considered the main genus of the family Megasporaceae (243 spp.) [12].

Hue, a French lichenologist, first described the aspicilioid lichens of Korea including 15 species (Aspicilia adamanticola Hue, A. asteria Hue, A. chinnampoana Hue, A. dimorphodes Hue, A. exserta Hue, A. fauriana Hue, A. geographica Hue, A. leucera Hue, A. microsporeta Hue, A. stellata Hue, A. stenospora Hue, A. tofacea Hue, A. tumens Hue, A. umbrinella Hue, and A. vulcanica Hue) [13]. After a century, Kondratyuk discovered A. contorta ssp. hoffmanniana S. Ekman & Fröberg ex R. Sant. (syn. Circinaria hoffmanniana (S. Ekman & Fröberg ex R. Sant.) A. Nordin) in 2013 [14], and Aptroot and Moon recorded A. cinerea (L.) Körb., A. grisea Arnold, Circinaria caesiocinerea (Nyl. ex Malbr.) A. Nordin, Savić & Tibell, and C. leprosescens (Sandst.) A. Nordin, Savić & Tibell in 2014 [15]. Kondratyuk focused on the aspicilioid lichens of Korea in 2016 and eight species were introduced from Korea (A. pseudoabbasiana S.Y. Kondr., Lőkös & Hur, A. pseudovulcanica S.Y. Kondr., Lőkös & Hur, A. subepiglypta S.Y. Kondr., Lőkös & Hur, A. subgeographica S.Y. Kondr., Lőkös & Hur, A. subgoettweigensis S.Y. Kondr., Lőkös & Hur, A. submamillata S.Y. Kondr., Lőkös & Hur [16], A. geumodoensis S.Y. Kondr., Lőkös & Hur (syn. Rimularia geumodoensis (S.Y. Kondr., Lőkös & Hur) S.Y. Kondr., Lőkös & Hur and R. badioatra (Kremp.) Hertel & Rambold) [17]. Particularly, Paukov reclassified both A. dimorphodes and A. fauriana to A. intermutans (Nyl.) Arnold, and both A. geographica and A. microsporeta to Lecanora oreinoides (Körb.) Hertel & Rambold in 2017 [18]. Kondratyuk detected Rimularia gibbosa (Ach.) Coppins, Hertel & Rambold [19] and Yakovchenko introduced two Rimularia species such as R. badioatra and R. limborina Nyl. in 2018 [20]. Overall 27 species of the aspicilioid lichens were recorded in Korea.

This study aimed to describe a new lichen-forming fungus in the genus Aspicilia. One of the field surveys for the lichen biodiversity in the forested wetlands of South Korea was carried out in a wetland forest of a high mountain, Gangwon Province in 2020, and two specimens of aspicilioid lichens were collected (Figure 1). The specimens were comprehensively analyzed in ecology, morphology, chemistry and molecular phylogeny and did not correspond to any previously known species. We describe them as a new species, Aspicilia humida, and this discovery contributes to the taxonomy with overall 28 taxa in the genus Aspicilia of Korea. The specimens are deposited in the herbarium of the Baekdudaegan National Arboretum (KBA, the herbarium acronym in the Index Herbariorum), South Korea.

Figure 1.

Specific collection site for the new species Aspicilia humida (black star mark).

Materials and methods

Morphological and chemical analyses

Specimen sections were prepared manually with a razor blade under a stereomicroscope (Olympus optical SZ51; Olympus, Tokyo, Japan), scrutinized under a compound microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan) and pictured 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 examined 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 (SD), length-to-width ratio, and the number of measured spores. Thin-layer chromatography (TLC) was performed using solvent systems A and C according to standard methods [21].

Isolation, DNA extraction, amplification, and sequencing

Hand-cut sections of 10–20 ascomata with thallus from the 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, Düren, Germany). PCR amplifications for the internal transcribed spacer region (ITS1-5.8S-ITS2 rDNA), the mitochondrial small subunit, and the nuclear large subunit ribosomal RNA genes was achieved using the primers ITS5 and ITS4 [22], mrSSU1 and mrSSU3R [23], and LR0R and LR5 [24], 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 [25]. The annealing temperature was occasionally altered by ±1 degree in order to get a better result. PCR purification and DNA sequencing were accomplished by the Macrogen (Seoul, Korea).

Phylogenetic analyses

All ITS and mtSSU sequences (Table 1) were aligned and edited manually using ClustalW in Bioedit V7.2.6.1 [26]. All missing and ambiguously aligned data and parsimony-uninformative positions were removed and only parsimony-informative regions were finally analyzed in MEGA X [27]. The final alignment comprised 1163 (ITS) and 1058 (mtSSU) columns. In them, variable regions were 171 (ITS) and 120 (mtSSU). Finally, the phylogenetically informative regions were 444 (ITS) and 271 (mtSSU). Phylogenetic trees with bootstrap values were obtained in RAxML GUI 2.0 beta [28] using the maximum likelihood method with a rapid bootstrap with 1000 bootstrap replications and GTR GAMMA for the substitution matrix. The posterior probabilities were obtained in BEAST 2.6.4 [29] using the GTR 123141 (ITS) and the GTR 121323 (mtSSU) models, as the appropriate models of nucleotide substitution produced by the Bayesian model averaging methods with bModelTest [30], 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 1000 log parameters. Then, a consensus tree was constructed in TreeAnnotator 2.6.4 [29] with no discard of burnin, 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 [31] and edited in Microsoft Paint. The bootstrapping and Bayesian analyses were repeated three times for the result consistency and no significant differences were shown for the tree shapes and branch values. The phylogenetic trees and DNA sequence alignments are deposited in TreeBASE under the study ID 28153. Overall analyses in the materials and methods were accomplished based on Lee and Hur [32].

Table 1.

Species list and DNA sequence information employed for phylogenetic analysis.

No.	Species	ID (ITS)	ID (mtSSU)	Voucher
1	Aspicilia abbasiana	KM609324		WDLC-20111154
2	Aspicilia aschabadensis	GU289916		Borisova1934 (LE)
3	Aspicilia berntii	EU502747		Nordin 6392 (UPS)
4	Aspicilia bicensis	KU341407		Anderson 16123 (NY)
5	Aspicilia blastidiata	KX129963		AGP20111009-01
6	Aspicilia brucei	JF703111		UPS:Owe-Larsson 9147
7	Aspicilia calcitrapa	JF703113		MARSSJ:Roux 24309
8	Aspicilia cinerea	EU057899	HM060695	Hermansson 13275 (UPS)
9	Aspicilia cinerea	HQ406799	HM060696	Nordin 5542 (UPS)
10	Aspicilia cinerea	HQ650637	DQ986890	AFTOL-ID 647
11	Aspicilia coronata	EU057901		Lattman 13.V.2006 (UPS)
12	Aspicilia desertorum	HQ406802	HM060689	Owe-Larsson 9814 (UPS)
13	Aspicilia dudinensis	EU057906	HM060710	Nordin 6036 (UPS)
14	Aspicilia dudinensis	MN906265		McCune 36017 (OSC)
15	Aspicilia dudinensis		HM060719	UPS:Nordin 5971
16	Aspicilia epiglypta	MH248866	MH248867	Nordin 7037 (UPS)
17	Aspicilia fluviatilis	HQ259264		UPS:Nordin 6188
18	Aspicilia granulosa	HQ259265		UPS:Nordin 6174
19	Aspicilia humida	MW832805	MW832823	BDNA-L-0000703
20	Aspicilia humida	MW832806	MW832824	BDNA-L-0000711
21	Aspicilia indissimilis	EU057909	HM060708	Nordin 5943 (UPS)
22	Aspicilia laevata	EU057910	HM060692	Tibell 23659 (UPS)
23	Aspicilia prestensis	JF703122		UPS:Roux 24883a
24	Aspicilia pseudoabbasiana	KY249599		KoLRI 018579
25	Aspicilia pseudovulcanica	KY249601		KoLRI 012338
26	Aspicilia pseudovulcanica	KY249602		KoLRI 012420
27	Aspicilia pseudovulcanica	KY249603		KoLRI 023829
28	Aspicilia subdepressa	JF703123		MARSSJ:Roux 24653
29	Aspicilia subepiglypta	KY249607		KoLRI 012535_2
30	Aspicilia subfarinosa	MN989235		Sipman & Raus 63720
31	Aspicilia subgeographica	KY249611		KoLRI 018704
32	Aspicilia subgoettweigensis	KY249616		KoLRI 007090
33	Aspicilia submamillata	KY249620		KoLRI 011146
34	Aspicilia subradians	HQ259267		UPS:Nordin 5984
35	Aspicilia verrucigera	EU057939		Tibell 22669 (UPS)
36	Aspiciliella intermutans	MH248863	MH248869	Roux 25790 (CR)
37	Circinaria affinis	HQ389194		Abbas 20081364 (H)
38	Circinaria caesiocinerea	HQ650636	DQ986892	AFTOL-ID 653
39	Circinaria calcarea	EU057898		Nordin 5888 (UPS)
40	Circinaria calcarea		AY853310	Wedin 6500 (UPS)
41	Circinaria cerebroides	JQ797534		Ringel 5138 (H)
42	Circinaria contorta	HQ650638	DQ986876	AFTOL-ID 1358
43	Circinaria cupreogrisea	EU057903		Nordin 6046 (UPS)
44	Circinaria esculenta	HQ406803		Owe-Larsson 9824 (UPS)
45	Circinaria esculenta	JQ797511	JQ797485	Owe-Larsson 9796 (UPS)
46	Circinaria fruticulosa	HQ389199		Vondrak 5188 (GBFS)
47	Circinaria fruticulosa		KC020253	ASPFRU 2361
48	Circinaria gibbosa	EU057908	HM060702	Nordin 5878 (UPS)
49	Circinaria gyrosa	JQ797528	JQ797487	Sohrabi 10401 A
50	Circinaria hispida	HQ171233		Sohrabi 15099
51	Circinaria jussuffii	JQ797518		Esnault 2033 (GZU)
52	Circinaria lacunosa	JQ797517		Abbas 940003 (H)
53	Circinaria leprosescens	EU057911	HM060711	Nordin 5906 (UPS)
54	Circinaria sphaerothallina	JQ797545		Sohrabi 9369
55	Lobothallia alphoplaca	JQ797516	JQ797480	Sohrabi 3677
56	Lobothallia alphoplaca	KT456207	KT456211	SK A20
57	Lobothallia melanaspis	JF825524		Owe-Larsson 8943a (UPS)
58	Lobothallia melanaspis		HM060688	UPS:Nordin 6622
59	Lobothallia praeradiosa	MK347501	MK348229	UFU L-1264
60	Lobothallia recedens	HQ406807		Nordin 6035 (UPS)
61	Lobothallia recedens		HM060724	UPS:Nordin 6582
62	Oxneriaria dendroplaca	HQ259259	HM060706	UPS:Nordin 5952
63	Oxneriaria mashiginensis	EU057912	HM060694	Nordin 5790 (UPS)
64	Oxneriaria permutata	EU057918	HM060709	Nordin 6027 (UPS)
65	Oxneriaria rivulicola	EU057922	HM060715	Nordin 5957 (UPS)
66	Oxneriaria supertegens	EU057936	HM060704	Owe-Larsson 9002 (UPS)
67	Oxneriaria verruculosa	EU057940	HM060703	Owe-Larsson 9007 (UPS)
68	Oxneriaria virginea	HQ259270		UPS:Nordin 6017a
69	Sagedia mastrucata	EU057913	HM060698	Nordin 5708 (UPS)
70	Sagedia nunatakkorum	KT630250		Malicek-228
71	Sagedia simoensis	EU057926	HM060701	Owe-Larsson 9000 (UPS)
72	Sagedia zonata	EU057946	HM060700	Owe-Larsson 8942 (UPS)
73	Teuvoa tibetica	GU289915		Obermayer 04386
	Overall	68	34

DNA sequences for the new species Aspicilia humida (in bold) were generated in this study. All others were obtained from GenBank. The species names are followed by GenBank accession numbers and voucher information. ITS, internal transcribed spacer; mtSSU, mitochondrial small subunit; Voucher, voucher information.

Results and discussion

Two independent phylogenetic trees for the genus Aspicilia and related genera were produced from 98 sequences (66 for ITS, and 32 for mtSSU) from GenBank and four new sequences (each two for ITS and mtSSU) from the new species (Table 1). The new species was positioned in the A. cinerea group in both trees. The ITS tree describes that the new species is located in a clade with A. subfarinosa (J. Steiner) Şenkard. & Sohrabi and Circinaria hispida (Mereschk.) A. Nordin, Savić & Tibell, represented by a bootstrap value of 98 and a posterior probability of 0.7 (not shown) for the branch. Other species, such as A. abbasiana S.Y. Kondr., Lőkös, Ismayil & S.Y. Guo, A. blastidiata Paukov, A. Nordin & Tibell, A. cinerea, A. dudinensis (H. Magn.) Oxner, A. pseudoabbasiana, A. pseudovulcanica, A. subepiglypta, A. subdepressa Arnold, A. subgeographica, A. subgoettweigensis, and A. submamillata, are closely located to the new species in the A. cinerea group, represented just by a bootstrap value of 79 for the branch (Figure 2). The mtSSU tree shows that the new species is solely located in the A. cinerea group. Closely positioned species to the new species are A. cinerea, A. dudinensis, A. indissimilis (H. Magn.) Räsänen, and A. laevata (Ach.) Arnold, represented by a bootstrap value of 95 and a posterior probability of 1 for the branch (Figure 3). The phylogenetic analyses did not designate any species identical to the new species in the genus Aspicilia.

Figure 2.

Phylogenetic relationship among available species in the genus Aspicilia based on a maximum likelihood analysis of the dataset of ITS sequences. The tree was rooted with six Lobothallia and Teuvoa sequences. Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Aspicilia humida is presented in bold, and all species names are followed by the Genbank accession numbers. Reference Table 1 provides the species related to the specific GenBank accession numbers and voucher information.

Figure 3.

Phylogenetic relationships among available species in the genus Aspicilia based on a maximum likelihood analysis of the dataset of the mitochondrial small subunit (mtSSU) sequences. The tree was rooted with five Lobothallia sequences. Maximum-likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Aspicilia humida is presented in bold, and all species names are followed by the GenBank accession numbers. Reference Table 1 provides the species related to the specific GenBank accession numbers and voucher information.

Taxonomy

No: MB839181

Figure 4

Figure 4.

Aspicilia humida (BDNA-L-0000703, holotype for A–K; BDNA-L-0000711, paratype for L & M) in morphology. (A–D): Habitus and apothecia emerging single to several per an areole; (E): Adnate apothecia without constriction at the base in section; (F): Epihymenium in olive-brown pigment; (G–J): Clavate asci with eight spores; (K): Ellipsoid or globose ascospores with no septation; (L): Immersed pycnidia; (M): Thread-like pycnoconidia. Bars: A–D 1 mm; E 200 μm; F 50 μm; G–K 10 μm; L 100 μm; M 10 μm.

Diagnosis

Aspicilia humida differs from A. aquatica by the absence of prothallus (vs. thick gray prothallus), black disk without green color in water (vs. black disk with translucent green when wet), olive-brown epihymenium (vs. olive-green epihymenium), shorter hymenium (50–60 μm vs. 150–170 μm), hymenium I + yellowish blue-green (vs. hymenium I + blue or turning dark red-brown), wider paraphysial tips without vivid pigment (4.5–6 μm wide vs. blackened tips in 2–5 μm wide), smaller asci (64–72 × 17–27 μm vs. 80–140 × 25–35 μm), smaller ascospores (10.5–23 × 6–13.5 μm vs. 20–35 × 13–20 μm), and the presence of stictic acid (vs. no substance).

Type

South Korea, Gangwon Province, Pyeongchang-gun, Daegwallyeong-myeon, Hoenggye-ri, a forest wetland, 37°46'0.02"N, 128°42'19.58"E, 1,047 m alt., on siliceous rock, 03 June 2020, B. G. Lee & H. J. Lee 2020-000503, with Diplotomma alboatrum (Hoffm.) Flot. and Endocarpon maritimum Y. Joshi & Hur (holotype: BDNA-L-0000703!; GenBank MW832805 for ITS, MW832823 for mtSSU, and MW832826 for LSU); same locality, on siliceous rock, 03 June 2020, B. G. Lee & H. J. Lee 2020-000511, (paratype: BDNA-L-0000711; GenBank MW832806 for ITS, MW832824 for mtSSU, MW832827 for LSU).

Thallus saxicolous, crustose, mainly areolate and partially rimose, pale gray to white, margin determinate, not pruinose, 175–300 μm thick; cortex hyaline, 25–30 μm thick; medulla 25–30 μm thick; photobiont coccoid, algal layer 35–50 μm thick, cells globose to subglobose, 5–15 μm. Small crystals in cortex, medulla and between algal cells, not dissolving in K. Prothallus inconspicuous.

Apothecia abundant, generally rounded but subangular or even irregular when several apothecia contiguous or coalescent, emerging single to several per an areole, adnate when mature, not constricted at the base, 0.2–1.7 mm diam. Disk flat or somewhat concave, smooth or slightly rugose, not pruinose, black from the beginning and partially paler when old, 100–130 μm thick; lecanorine, thalline margin present and same color to thallus or slightly darker, proper margin indistinct. Amphithecium well-developed, with small crystals in both cortical layer and medulla, crystals extending to the base, not dissolving in K, 90–100 μm wide laterally, 50–60 μm wide at periphery. Parathecium inconspicuous, hyaline but olive-brown at periphery, 10–15 μm wide laterally, 15–25 μm wide at periphery, disappearing to the base. Epihymenium olive green to brown, smooth and not granular, brown pigment dissolving in K, 10–15 μm high. Hymenium hyaline, 50–60 μm high, I + yellowish blue-green. Hypothecium hyaline, 25–50 μm high. Oil droplets present mainly in hypothecium and also along paraphyses in hymenium. Paraphyses septate, anastomosing, 2–2.5 μm wide, simple or branched at tips, tip cells somewhat bead-like (moniliform), bead-like formation clearer in staining, swollen but not pigmented, 4.5–6 μm wide. Asci clavate, 8-spored, 64–72 × 17–27 μm (n = 5). Ascospores constantly simple, ellipsoid or somewhat globose, 10.5–23 × 6–13.5 μm (mean = 17.3 × 9.8 μm; SD = 2.6 (L), 1.5 (W); L/W ratio 1.2–2.7, ratio mean = 1.8, ratio SD = 0.3; n = 106). Pycnidia immersed, ostiolar region slightly projected, rounded, black, 250–275 × 200–230 μm. Pycnoconidia thread-like, straight, slightly curved or v-shaped, 5.5–28 × 0.5–1.0 μm (mean = 15.1 × 0.7 μm; SD = 3.7 (L), 0.1 (W), n = 110)

Chemistry

Thallus K–, KC–, C–, Pd–. Medulla K + yellow, I–. UV + gray to dull white. Stictic acid was detected by TLC.

Distribution and ecology

The species occurs on a siliceous rock nearby a stream in an open wetland forest of a high mountain. The species is currently known from the type collections.

Etymology

The species epithet indicates the lichen’s geography, namely a humid wetland.

Notes

The new species is similar to A. aquatica, A. vulcanica and A. pseudovulcanica in having white to gray thallus with negative reaction in K among saxicolous species. However, the new species differs from A. aquatica by the absence of prothallus, black disk without green color in water, olive-brown epihymenium, shorter hymenium, hymenium I + yellowish blue-green, wider paraphysial tips without vivid pigment, smaller asci, smaller ascospores, and the presence of stictic acid [33,34].

The new species is different from A. vulcanica by apothecia emerging one to several per single areole, larger apothecia, narrower paraphyses, shorter and wider asci, and the substrate preference to siliceous rock [13,16].

The new species is distinguished from A. pseudovulcanica by thicker, larger apothecia without pruina, shorter hymenium, smaller ascospores [16]. Reference Table 2 provides the key characteristics distinguishing A. humida from the compared species above.

Table 2.

Comparison of Aspicilia humida with closely-related species.

Species	Aspicilia humida	Aspicilia aquatica	Aspicilia pseudovulcanica	Aspicilia vulcanica
Thallus growth form	mainly areolate, partially rimose	areolate to rimose	areolate or slightly cracked to continuous	rimose-areolate
Thallus color	pale gray to white	white to gray, often with a yellow or olive tinge	whitish gray to light gray	whitish to white-gray
Thallus thickness (μm)	175–300	200–400	150–200	120–160
Prothallus	inconspicuous	black to gray	–	–
Apothecia per areole	one to several	1 to 2(–4)	–	1 to 2
Apothecia (mm in diam.)	0.2–1.7	0.2–0.5	0.5–0.9	0.3–0.4
Pruina	absent	absent	white pruina on disk	white pruina on disk
Disk color	black without green in water	black with translucent green in water	gray to gray-black	(gray to gray-black)
Epihymenium color	olive green to brown	green to olive	dirty greenish brown	colorless
Hymenium height (μm)	50–60	150–200	110–130	(110–140)
Hymenium in Iodine	yellowish blue-green	blue, or turning dark red-brown	–	–
Paraphyses	2–2.5	1.5–2	–	c. 4
Paraphysial tip width (μm)	4.5–6	2–5	5–6	5–7
Paraphysial tip pigment	inconspicuous	blackened	brownish	colorless
Asci (μm)	64–72 × 17–27	85–140 × 25–35	–	80–90 × 14–12
Ascospores (μm)	10.5–23 × 6–13.5	20–35 × 13–20	18–23 × 10–12	17–20 × 7–10
Substance	stictic acid	no substance	stictic acid	–
Substrate	siliceous rocks nearby streams	siliceous rocks nearby streams	siliceous rocks	volcanic rock
Reference	BDNA-L-0000703 (holotype), BDNA-L-0000711 (paratype)	[33,34]	[16]	[13,16]

The morphological and chemical characteristics for several species close to the new species are referenced from the previous literature. All information on the new species is produced from type specimens (BDNA-L-0000703 and BDNA-L-0000711) in this study. The brackets for Aspicilia vulcanica, i.e. disk color and hymenium height, are assumed from previous literature.

The new species is further compared with A. straminella Hue and A. verrucigera Hue in having grayish, areolate thallus and K + yellow medulla in saxicolous species. However, A. straminella is different from the new species by thicker and straw-gray thallus, smaller apothecia, taller hymenium and wider ascospores [13,16]. Aspicilia verrucigera differs from the new species by thicker and darker thallus occasionally with brown color, smaller apothecia, taller hymenium, wider ascospores and the presence of norstictic acid [16,33].

Key to aspicilioid species of Korea (28 taxa)

Overall 28 species have been recorded for the aspicilioid lichens including the genera Aspicilia, Circinaria, Lecanora, and Rimularia in Korea, except for synonyms. For synonyms after taxonomic revision, A. adamanticola is corresponded to A. cinerea [18], A. contorta ssp. hoffmanniana is reclassified to C. hoffmanniana [35], A. geographica and A. microsporeta are conspecific to L. oreinoides [18], A. dimorphodes and A. fauriana are indistinguishable to A. intermutans [18], and A. geumodoensis is reclassified to R. geumodoensis [18]. This key is revised from Kondratyuk’s work [16] only for Korea territory, and seven more species are included such as A. grisea, A. humida, A. intermutans, C. caesiocinerea, C. hoffmanniana, L. oreinoides, and R. limborina. A. exserta is corrected from A. excerta of Kondratyuk’s work [16].

Paraphysis cells not bead-like, disk umbonate or gyrose..2

Paraphysis cells bead-like (moniliform), disk generally smooth ..5

Thallus C– or C ± pink (not or containing ± gyrophoric acid), hymenium up to 150 μm, ascospores 18–30 × 10–18 μm ..

Thallus C + red (containing gyrophoric acid), hymenium up to 120 μm, ascospores 10–25 × 7–13 μm..3

Thallus lighter, whitish gray, ascospores 20–25 × 9–13 μm....

Thallus darker, pinkish brown to gray brown, ascospores 10–25 × 7–13 μm..4

Thallus pinkish brown to dark gray-brown, hymenium 70–100 μm, apothecia 0.2–0.4 mm diam., disk concave to umbonate, ascospores 10–22 × 7–13 μm....

Thallus pale gray-brown to beige, hymenium up to 75 μm, apothecia 0.5–0.8 mm diam., disk slightly convex, ascospores 16–25 × 10–13 μm....

Thallus or medulla K + red or K + yellow turning to red (containing norstictic acid) ....6

Thallus or medulla K– red or K + yellow (containing aspicilin or stictic acid, but not containing norstictic acid) ..16

Thallus with farinose-erose soredia..

Thallus without soredia7

Apothecia rare or solitary per areole when present..8

Apothecia occurring one to several per areole..12

Thallus whitish..9

Thallus grayish to brownish..11

Thallus thin, apothecia 0.2–0.3 mm diam

Thallus thick, apothecia 0.5–1.5 mm diam. ..10

Asci 120 × 24 μm, ascospores 15–20 × 9–10 μm..

Asci 80 × 16 μm, ascospores 17–20 × 7–8 μm.

Apothecia 0.3–0.5 mm diam., ascospores 16–20 × 6–7 μm..

Apothecia 0.4–1.0 mm diam., ascospores 12–19 × 7–11 μm..

Thallus white without grayish or brownish color..13

Thallus white-gray or gray to gray-brown..14

Thallus thin 0.16–0.22 mm thick, apothecia occurring one to two per areole, 0.4–0.8 mm diam. ..

Thallus thick 0.5–0.6 mm thick, apothecia occurring one to several per areole, 0.3–0.5 mm diam. ..

Apothecia 0.2–0.3 mm diam., occurring three to five per areole, conidia 15–20 × 0.7–0.9 μm..

Apothecia 0.4–1.2 mm diam., occurring one to three per areole, conidia 7–16 × 1 μm..15

Apothecia occurring one to two per areole, ascospores 12–22 × 6–13 μm, conidia 11–16 × 1 μm..

Apothecia occurring two to three per areole, ascospores 22–28 × 12–14 μm, conidia 7–11 × 1 μm..

On calcareous or volcanic rocks..17

On siliceous rocks..18

On calcareous rocks, thallus with pruina, apothecia prominent, black, asci 4-spored....

On volcanic rocks, thallus without pruina, apothecia immersed, flesh-colored (beige), asci 8-spored..

Thallus with soredia or isidia, containing aspicilin..

Thallus without soredia or isidia..19

Thallus areolate, thick (0.4–0.6 mm thick) ..20

Thallus areolate to rimose or subsquamulose, thin or thick..23

Thallus with pruina..21

Thallus without pruina..22

Apothecia 0.2–0.5 mm diam., disk blackish with grayish pruina..

Apothecia 0.4–1.0 mm diam., disk gray to brownish with white pruina.

Thallus dark gray to lead-gray, apothecia 0.25–0.3 mm diam., ascospores 17–22 × 8–12 μm, conidia 13–17 × 0.7–0.8 μm..

Thallus light gray to slightly brownish gray, apothecia 0.9–1 mm diam., ascospores 19–22 × 12–16 μm, conidia 3.5–5.5 × 0.7–0.8 μm..

Thallus with pruina..24

Thallus without pruina..26

Thallus thick (up to 0.4 mm thick), white to cream white, or pale yellow, apothecia occurring one to three per areole, ascospores 9–14 × 4–6.5 μm...........

Thallus thin, white, light gray or pale brown, apothecia occurring one to two per areole, ascospores 16–24 × 9–14 μm..25

Apothecia 0.2–0.5 mm diam., thallus white or pale brown......................

Apothecia 0.5–0.9 mm diam., thallus white-gray to light gray.................

Thallus subsquamulose in center, ascospores 14–30 × 7–16 μm, containing aspicilin........

Thallus areolate to rimose only, ascospores 10–23 × 6–14 μm, not containing aspicilin27

Thallus bluish gray, apothecia mostly solitary per areole, rarely two, 0.2–0.5 mm diam., asci 100–132 × 22–26 μm, ascospores 16–20 × 10–14 μm......

Thallus pale gray to white, apothecia occurring one to several per areole, 0.2–1.7 mm diam., asci 64–72 × 17–27 μm, ascospores 10.5–23 × 6–13.5 μm, containing stictic acid..