Two new Rinodina lichens from South Korea, with an updated key to the species of Rinodina in the far eastern Asia.

Rinodinasalicis Lee & Hur and Rinodinazeorina Lee & Hur are described as new lichen-forming fungi from forested wetlands or a humid forest in South Korea. Rinodinasalicis is distinguishable from Rinodinaexcrescens Vain., the most similar species, by its olive-gray thallus with smaller areoles without having blastidia, contiguous apothecia, non-pruinose discs, paler disc color, wider ascospores in the Pachysporaria-type II, and the absence of secondary metabolites. Rinodinazeorina differs from Rinodinahypobadia Sheard by areolate and brownish thallus, non-pruinose apothecia, colorless and wider parathecium, narrower paraphyses with non-pigmented and unswollen tips, longer and narrower ascospores with angular to globose lumina, and the absence of pannarin. Molecular analyses employing internal transcribed spacer (ITS) sequences strongly support the two new species to be unique in the genus Rinodina. An updated key is provided to assist in the identification of all 63 taxa in Rinodina of the far eastern Asia. Beeyoung Gun Lee, Jae-Seoun Hur.

Introduction

, the largest genus in the family , comprises about three hundred species worldwide (Sheard et al. 2017; Wijayawardene et al. 2020). Several infrageneric groups have been studied since Malme (1902) introduced the ascospore-type concept for the groups in (Poelt 1965; Grube and Arup 2001). Although the classification based on different ascospore types has been coarsely accepted, the variety of ascospores does not always correspond to the infrageneric classification. As the pattern of ascospore ontogeny is considered more important than the spore type itself, it is understood that the ascospore types should be respected in developmental stages of a spore (Giralt 1994; Grube and Arup 2001; Sheard 2010; Resl et al. 2016).

The has been studied in Europe (Mayrhofer and Poelt 1979; Giralt et al. 1995; Giralt 2001; Mayrhofer and Moberg 2002), North America (Sheard and Mayrhofer 2002; Sheard 2004, 2010, 2018; Sheard et al. 2011, 2012; Lendemer et al. 2012, 2019; Morse and Sheard 2020), islands of South America (Bungartz et al. 2016), Australia to New Zealand (Mayrhofer 1983, 1984b; Kaschik 2006; Elix 2011; Elix et al. 2020), Asia to Russian Far East (Mayrhofer 1984a; Galanina et al. 2011; Lendemer et al. 2012; Sheard et al. 2017; Galanina et al. 2018; Galanina and Ezhkin 2019; Zheng and Ren 2020; Galanina et al. 2021; Kumar et al. 2021), and South Africa (Matzer and Mayrhofer 1996; Mayrhofer et al. 2014). Molecular works have been accomplished over the continents (Grube and Arup 2001; Wedin et al. 2002; Nadyeina et al. 2010; Resl et al. 2016).

Sheard et al. (2017) achieved the first and comprehensive study on the genus of the far eastern Asia (Korea, Japan, and Russian Far East). Several studies announced further more species in the genus, such as Sheard, H. Magn., (Körb.) Sheard, S.Y. Kondr., Lőkös & Hur and (Tuck.) Herre from South Korea (Kondratyuk et al. 2016, 2017; Yakovchenko 2018; Kondratyuk et al. 2020) and (Nyl.) Müll. Arg., H. Magn., (Ach.) Malme, and H. Mayrhofer & Poelt from the Kuril Islands and the Magadan region, Russian Far East (Galanina and Ezhkin 2019; Galanina et al. 2021). Among them, was discovered as a new species and other eight species were reported as new records to the far eastern Asia. The species of in the far eastern Asia are mainly corticolous and the main genera of the substrate trees are , , , and (Fig. 1) (Lendemer et al. 2012; Sheard et al. 2012; Joshi et al. 2013; Kondratyuk et al. 2013, 2016, 2017, 2020; Aptroot and Moon 2014; Sheard et al. 2017; Yakovchenko et al. 2018; Galanina and Ezhkin 2019; Gananina et al. 2021). Those main substrates vigorously grow in a humid forest, a valley or a wetland, and particularly the genera and often inhabit the water. Inhabiting those tree barks, diverse species are easily detected in shaded forests and forested wetlands in which are one of the representative lichens of the ecosystems.

Figure 1.

Substrates of species in the far eastern Asia. species of the far eastern Asia occur mainly on bark, and the genera , , , and are the main substrates for corticolous species of the far eastern Asia.

This study describes two new lichen-forming fungi in the genus . Field surveys for the lichen biodiversity in the forested wetlands of South Korea were carried out during the summer of 2020, and a couple of specimens of were collected from barks of and , the most common genera of the substrates for corticolous species in the far eastern Asia, in a humid forest and a forested wetland on mountains (Fig. 2). 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 new species, and , and this discovery contributes to the taxonomy with overall 63 taxa in the genus of the far eastern Asia. The type specimens are deposited in the herbarium of the Baekdudaegan National Arboretum (KBA, the herbarium acronym in the Index Herbariorum), South Korea.

Figure 2.

Specific collection sites for two new species A habitat/landscape for B habitat/landscape for C location for (a black star); locations for (two black diamonds).

Materials and methods

Morphological and chemical analyses

Hand 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, Tokyo, Japan) and a DS-Fi3 camera (Nikon, Tokyo, Japan) mounted on a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan). 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 (Orange et al. 2001).

Isolation, DNA extraction, amplification, and sequencing

Hand-cut sections of ten to twenty ascomata per collected specimen 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) RNA genes were achieved using Bioneer’s AccuPower PCR Premix (Bioneer, Daejeon, Korea) in 20-μl tubes with 16 μl of distilled water, 2 μl of DNA extracts and 2 μl of the primers ITS5 and ITS4 (White et al. 1990). 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). 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 genomic research company Macrogen (Seoul, Korea).

Phylogenetic analyses

All ITS sequences (Table 1) were aligned and edited manually using ClustalW in Bioedit V7.2.6.1 (Hall 1999). All missing and ambiguously aligned data and parsimony-uninformative positions were removed and only parsimony-informative regions were finally analyzed in MEGA X (Stecher et al. 2020). The final alignment comprised 974 bp in which 167 variable regions were detected. The phylogenetically informative regions were 523. Phylogenetic trees with bootstrap values were obtained in RAxML GUI 2.0 beta (Edler et al. 2019) 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 (Bouckaert et al. 2019) using the GTR 121343 model, 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 1000 log parameters. Then, a consensus tree was constructed in TreeAnnotator 2.6.4 (Bouckaert et al. 2019) 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 (Rambaut 2014) and edited in Microsoft Paint. The bootstrapping and posterior probability 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 28192. Overall analyses in the materials and methods were accomplished based on Lee and Hur (2020).

Table 1.

Species list and DNA sequence information employed for phylogenetic analysis.

No.	Species	ID (ITS)	Voucher
1	Amandinealignicola	JX878521	Tønsberg 36426 (BG)
2	Amandineapunctata	HQ650627	AFTOL-ID 1306
3	Buelliabadia	MG250192	TS1767 (LCU)
4	Buelliaboseongensis	MF399000	KoLRI 041680
5	Buellianumerosa	LC153799	CBM:Watanuki:L01034
6	Rinodinaafghanica	MT260860	500103 (XJU-L)
7	Rinodinaalba	GU553290	GZU 000272655
8	Rinodinaalbana	GU553297	GZU 000272651
9	Rinodinaanomala	MN587028	Sipman 62934
10	Rinodinaarchaea	DQ849292	H. Mayrhofer 15752 (GZU)
11	Rinodinaatrocinerea	AF540544	H. Mayrhofer 13.740 & U. Arup (GZU)
12	Rinodinabalanina	KY266842	O-L-195705
13	Rinodinabischoffii	DQ849291	M. Lambauer 0031 (GZU)
14	Rinodinacacaotina	DQ849295	H. Mayrhofer 10770 (HO)
15	Rinodinacalcarea	GU553292	GZU 000272654
16	Rinodinacana	MN587029	Sipman 63008
17	Rinodinacapensis	DQ849296	W. Obermayer 09230 (GZU)
18	Rinodinaconfragosa	DQ849297	W. Obermayer 09091 (GZU)
19	Rinodinaconfragosula	DQ849298	M. Lambauer 0044 (GZU)
20	Rinodinadegeliana	KX015681	Tønsberg 42631
21	Rinodinadestituta	KT695382	BIOUG24047-H02
22	Rinodinadisjuncta	MK812529	TRH-L-15387
23	Rinodinaefflorescens	KX015683	Malicek 5462
24	Rinodinaexigua	GU553294	GZU 000272652
25	Rinodinagallowayi	DQ849299	M. Lambauer 0125 (GZU)
26	Rinodinagennarii	AJ544187	B44435
27	Rinodinaglauca	GU553295	GZU 000272662
28	Rinodinaherteliana	DQ849300	M. Lambauer 0177 (GZU)
29	Rinodinaimmersa	DQ849301	M. Lambauer 0129 (GZU)
30	Rinodinainterpolata	AF250809	M263
31	Rinodinajamesii	DQ849303	H. Mayrhofer 10810 (GZU)
32	Rinodinalecanorina	AF540545	H. Mayrhofer 13.120 (GZU)
33	Rinodinalepida	AY143413	Trinkaus 137
34	Rinodinaluridata	DQ849304	H. Mayrhofer 12122 (GZU)
35	Rinodinaluridescens	AJ544183	B42835
36	Rinodinametaboliza	MT260864	20080224 (XJU-L)
37	Rinodinamilvina	GU553299	KW 63379
38	Rinodinamniaroea	KX015689	Spribille 20101 (GZU)
39	Rinodinamniaroea	KX015691	V. Wagner, 15.07.06/1 (GZU)
40	Rinodinamniaroea	KX015692	Spribille 20391 (GZU)
41	Rinodinamoziana	DQ849307	H. Mayrhofer 6729 (GZU)
42	Rinodinamozianavar.moziana	DQ849305	M. Lambauer 0214 (GZU)
43	Rinodinanimisii	AJ544184	B42685
44	Rinodinaobnascens	AJ544185	B42477
45	Rinodinaoleae	DQ849308	M. Lambauer 0178 (GZU)
46	Rinodinaoleae	GU553301	GZU 000272565
47	Rinodinaolivaceobrunnea	AF540547	J. Romeike 2.090300 (GOET)
48	Rinodinaorculata	DQ849309	H. Mayrhofer 15754 (GZU)
49	Rinodinaorientalis	MW832807	BDNA-L-0000284
50	Rinodinaorientalis	MW832808	BDNA-L-0000653
51	Rinodinaorientalis	MW832809	BDNA-L-0000774
52	Rinodinaoxydata	DQ849313	H. Mayrhofer 11406 (GZU)
53	Rinodinaplana	AF250812	E34
54	Rinodinapyrina	AF540549	P. Bilovitz & H. Mayrhofer 483 (GZU)
55	Rinodinaramboldii	DQ849315	G. Rambold 5094 (M)
56	Rinodinareagens	DQ849316	M. Lambauer 0218 (GZU)
57	Rinodinaroboris	MK811851	O-L-206765
58	Rinodinaroscida	DQ849317	S. Kholod plot515 (GZU)
59	Rinodinasalicis	MW832810	BDNA-L-0000558
60	Rinodinasalicis	MW832811	BDNA-L-0000560
61	Rinodinaseptentrionalis	GU553303	GZU 000272561
62	Rinodinasheardii	MK778639	J. Malicek 10238
63	Rinodinasheardii	MK778640	J. Vondrak 15298 (PRA)
64	Rinodinasophodes	AF540550	P. Bilovitz 968 (GZU)
65	Rinodinateichophila	GU553305	GZU 000272659
66	Rinodinatrevisanii	KX015684	de Bruyn s.n. 2011 (GZU)
67	Rinodinatunicata	AF540551	H. Mayrhofer 13.749 & R. Ertl (GZU)
68	Rinodinaturfacea	AF224362	Moberg 10422
69	Rinodinavezdae	DQ849318	H. Mayrhofer 15757 (GZU)
70	Rinodinazeorina	MW832812	BDNA-L-0000642
71	Rinodinazeorina	MW832813	BDNA-L-0000646
72	Rinodinazeorina	MW832814	BDNA-L-0000650
73	Rinodinazeorina	MW832815	BDNA-L-0000651
74	Rinodinazeorina	MW832816	BDNA-L-0000668
75	Rinodinazeorina	MW832817	BDNA-L-0000933
76	Rinodinazwackhiana	AF540552	H. Mayrhofer 13.848 (GZU)
77	Rinodinellacontroversa	AF250814	M281
78	Rinodinelladubyanoides	AF250815	E29
	Overall	78

DNA sequences which were generated in this study, i.e., two new species such as and , and another compared species, , are presented in bold. All others were obtained from GenBank. The species names are followed by GenBank accession numbers and voucher information. ITS, internal transcribed spacer; Voucher, voucher information.

Results and discussion

An independent phylogenetic tree for the genus and related genera was produced from 67 sequences from GenBank and 11 newly generated sequences for the two new species and related species (Table 1). The two new species were positioned in the genus in the ITS tree. The ITS tree describes that , a new species, is coming alone in a single clade. Several species such as (Ach.) Körb., (Sommerf.) Arnold, (Hepp) A. Massal., (Körb.) H. Mayrhofer, Scheid. & Sheard, Vain., (A. Massal.) A. Massal., M. Steiner & Poelt, (Kremp.) Körb., (Hepp ex Arnold) Arnold, (Körb.) J. Steiner, H. Mayrhofer & Poelt, (A. Massal.) H. Mayrhofer & Poelt, and (Hepp) H. Mayrhofer & Poelt, are situated close to the new species; this particular clade lacks statistical support (bootstrap value of 58 and a posterior probability of 0.75). , the other new species, was located in a clade with Tønsberg, represented by a bootstrap value of 89 and a posterior probability of 0.88 (not shown) for the branch (Fig. 3).

Figure 3.

Phylogenetic relationships among available species in the genus based on a maximum likelihood analysis of the dataset of ITS sequences. The tree was rooted with the sequences of the genera and . Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown as fatty lines. Two new species, and are presented in bold as their DNA sequences were produced from this study. All species names are followed by the Genbank accession numbers.

Taxonomy

B.G. Lee & J.-S. Hur sp. nov.

1B918156-AB08-58A8-890C-E6E52C49DFCB

MB839186

Fig. 4

Figure 4.

(BDNA-L-0000558, holotype) in morphology A–D habitus and apothecia. Thallus olive-gray composed of tiny areoles and non-pruinose apothecia E well-developed amphithecium and algal layer extending to the base F asci clavate with eight spores G ascospores simple in the beginning and developed 1-septate, -type II, rarely -type at mature. Scale bars: 1 mm (A–D); 200 μm (E); 10 μm (F, G).

Diagnosis.

differs from by olive-gray thallus with smaller areoles without blastidia, contiguous apothecia, the absence of pruina on disc, paler disc color, wider ascospores in the -type, and the absence of secondary metabolites.

Type.

South Korea, Gangwon Province, Gangneung, Seongsan-myeon, Eoheul-ri, a forested wetland, , 212 m alt., on bark of Andersson, 02 June 2020, B.G.Lee & H.J.Lee 2020-000358 (holotype: BDNA-L-0000558; GenBank MW832810 for ITS); same locality, on bark of , 02 June 2020, B.G.Lee & H.J.Lee 2020-000360, with (Tomin) Oxner, sp., and sp. (: BDNA-L-0000560; GenBank MW832811 for ITS).

Thallus corticolous, crustose, minutely bullate, some developing to conglomerate and continuous, rarely lobulated, thin, grayish-green to olive green, margin indeterminate, vegetative propagules absent, areoles 0.1–0.2 mm diam., 85–90 μm thick; cortex hyaline, 10 μm thick, cortical cells 5–9 μm diam.; medulla 60–65 μm thick, intermixed with algal cells, without crystals (PL–); photobiont coccoid, cells globose, 5–15 μm. Prothallus absent.

Apothecia abundant, rounded, often contiguous, emerging on the surface of thallus and sessile when mature, constricted at the base, 0.2–1.3 mm diam. Disc flat, not pruinose, pale brown or dark brown from early stages, 220–260 μm thick; margin persistent, prominent, generally entire or somewhat flexuous, a little crenulate, thalline margin concolorous to thallus but proper margin near disc distinctly pale brown. Amphithecium well-developed, with small crystals in both cortical layer and the algal-containing medulla, crystals extending to the base, not dissolving in K, 60–70 μm wide laterally, algal layers continuous to the base or solitary, algal cells 5–15 μm diam., cortical layer hyaline, 10–20 μm thick. Parathecium hyaline but light brown at periphery, 45–50 μm wide laterally and 70–80 μm wide at periphery. Epihymenium brown, not granular, pigment slightly paler in K but not diluted, 5–10 μm high. Hymenium hyaline, 70–90 μm high. Hypothecium generally hyaline, with pale yellow pigment, prosoplectenchymatous (irregular), 70–80 μm high. Oil droplets are present mainly in hypothecium and a little in hymenium. Paraphyses septate, anastomosing, 1–1.5 μm wide, simple or branched at tips, tips swollen, pigmented, epihymenium pigmented by paraphysial tips, 4.5–7.5 μm wide. Asci clavate, 8-spored, 68–90 × 20–25 μm (n = 5). Ascospores ellipsoid, 1-septate, -type II, rarely -type, Type A development, hyaline when young and light brown to brown in mature, 14–24 × 8–13.5 μm (mean = 18.2 × 10.5 μm; SD = 2.12(L), 1.19(W); L/W ratio 1.2–2.4, ratio mean = 1.7, ratio SD = 0.2; n = 105). Pycnidia not detected.

Chemistry.

Thallus K–, KC–, C–, Pd–. Hymenium I+ purple-blue. UV–. No lichen substance was detected by TLC.

Distribution and ecology.

The species occurs on the bark of . The species is currently known from the type collections.

Etymology.

The species epithet indicates the lichen’s substrate preference, namely the substrate tree .

Notes.

The new species is similar to and Sheard & Lendemer in having bullate thallus. However, the new species differs from by olive-gray thallus with smaller areoles without having blastidia, contiguous apothecia, the absence of pruina on disc, paler disc color, ascospore type, larger ascospore, and the absence of secondary metabolites (Sheard 1966; Sheard et al. 2012).

The new species is closer to in having small bullate areoles without having blastidia. However, the new species differs from the latter by olive-gray thallus, contiguous and larger apothecia, proper margin with pale brown color, crystals present in both cortex and medulla in amphithecium, larger ascospores, K– reaction on thallus, and the absence of lichen substance (Sheard et al. 2012, 2017).

The new species is comparable to Vain. as the latter represents thallus with minute areoles. However, the new species differs from the latter by thallus color, slightly smaller areoles without blastidia, abundance of apothecia without pruina, -type II ascospores, K– reaction on thallus, and the absence of lichen substance (Giralt et al. 1994; Galanina et al. 2011). Reference Table 2 provides the key characteristics distinguishing from the compared species above.

Table 2.

Comparison of with closely-related species.

Species	Rinodinasalicis	Rinodinabullata	Rinodinaexcrescens	Rinodinagranulans
Thallus growth form	bullate without blastidia	bullate without blastidia	bullate with blastidia	bullate with blastidia, forming leprose crust
Areoles (mm in diam.)	0.1–0.2	0.1–0.15(–0.2)	up to c. 1.98	(0.1–)0.2–0.3(–0.5)
Thallus color	olive-gray	light gray	gray	gray to gray-brown
Apothecia (mm in diam.)	0.2–1.3	0.3–0.6	up to c. 1.26	up to 0.3
Apothecia contiguation	often contiguous	not contiguous	not contiguous	not contiguous
Apothecia abundance	abundant	abundant	abundant	very rare
Pruina	absent on disc	–	often present on disc	often present on disc
Disc color	pale to dark brown	brown	brown to black	reddish brown
Proper margin	pale brown	indistinct	–	indistinct
Crystals in amphithecium	present in medulla and cortex	present in cortex	–	present
Ascospore type	Pachysporaria-type II	Pachysporaria-type II	Physcia-type	Physcia-type to Milvina-type
Ascospores (μm)	14–24 × 8–13.5	14.5–16.5 × 8–9	17.5–19.5 × 8.5–9.5	18–25 × 10–14
Spot test	thallus K–	thallus K+ yellow	thallus K–	thallus K+ faint yellow
Substance	absent	atranorin	pannarin, (rarely zeorin)	pannarin
Reference	BDNA-L-0000558 (holotype), BDNA-L-0000560 (paratype)	Sheard et al. 2012, 2017	Sheard 1966; Sheard et al. 2012, 2017	Giralt et al. 1994; Galanina et al. 2011

The morphological and chemical characteristics of 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-0000558 and BDNA-L-0000560) in this study.

B.G. Lee & J.-S. Hur sp. nov.

C2162038-9969-5520-BB2F-DE0B93647AE6

MB839187

Fig. 5

Figure 5.

(BDNA-L-0000933, holotype for A–G; BDNA-L-0000668 for H–K) in morphology A–C habitus and apothecia on bark of . Thallus brownish and areolate and non-pruinose apothecia D well-developed amphithecium and pigmented hypothecium E epihymenium with brown pigment which extending to the cortical layer of amphithecium. Parathecium light brown at periphery F hypothecium with light (olive-)brown pigment G ascospores 1-septate, -type but lumina angular to globose H habitus and apothecia on bark of . Thallus more grayish I apothecial section representing well-developed amphithecium and pigmented hypothecium J asci clavate with eight spores K ascospores 1-septate, -type but lumina angular to globose. Scale bars: 1 mm (A–C); 200 μm (D); 50 μm (E, F); 10 μm (G); 1 mm (H); 200 μm (I); 10 μm (J, K).

differs from by areolate, brownish thallus, apothecia without pruina, hyaline and wider parathecium, narrower paraphyses with hyaline and unswollen tips, longer and narrower ascospores with just angular to globose lumina, and the absence of pannarin.

South Korea, North Gyeongsang Province, Bonghwa-gun, Seokpo-myeon, Mt. Cheongok, , 1,104 m alt., on bark of , 16 June 2020, B.G. Lee & H.J. Lee 2020-000733, with sp., (Flörke) Kremp., (Turner) Nyl., and sp. (: BDNA-L-0000933; GenBank MW832817 for ITS).

Thallus corticolous, crustose, areolate, rimose to continuous, thin, light gray to light brownish gray, margin indeterminate or determinate with prothallus, vegetative propagules absent, 160–250 mm diam., 80–170 μm thick, areoles 0.1–0.5 mm diam.; cortex brown, 5–8 μm thick, with epinecral layer, hyaline, 3–7 μm thick; medulla 35–40 μm thick, intermixed with algal cells, without crystals (PL–); photobiont coccoid, cells globose, 5–9 μm. Prothallus absent or brownish black when present.

Apothecia abundant, rounded, erumpent in the beginning and sessile when mature, constricted at the base, 0.2–0.6 mm diam. Disc flat, not pruinose but epinecral debris shown in water, black to dark brown from early stages, 150–200 μm thick; margin persistent, prominent, generally entire or a little crenulate, concolorous to thallus. Amphithecium well-developed, with small crystals in the algal-containing medulla and particularly near the base, dissolving in K, 70–90 μm wide laterally, algal cells evenly distributed from periphery to base, 10–15 μm diam., cortical layer brownish, cortical cells granular, 2–3 μm diam., with epinecral layer, up to 5 μm thick. Parathecium hyaline but light brown at periphery, 5–10 μm wide laterally and 20–50 μm wide at periphery. Epihymenium red-brown, small granules not dissolving in K, 8–10 μm high. Hymenium hyaline, 90–95 μm high. Hypothecium brown with olive pigment in upper part, prosoplectenchymatous (irregular), 60–65 μm high. Oil droplets present a little in hypothecium. Paraphyses septate, anastomosing, 0.5–1 μm wide, simple or branched at tips, tips generally not swollen or little swollen, not pigmented, epihymenium pigmented by small granules, not by paraphysial tips, up to 1.5 μm wide. Asci clavate, 8-spored, 60–75 × 15–21 μm (n = 3). Ascospores ellipsoid, 1-septate, -type but lumina angular to globose, Type B development not detected, septum inflated a little or not, without a torus, hyaline when young and generally brown or dark brown in mature, 11–20 × 5–8.5 μm (mean = 15.4 × 7.1 μm; SD = 1.77(L), 0.70(W); L/W ratio 1.5–3.4, ratio mean = 2.2, ratio SD = 0.3; n = 105). Pycnidia raised, asymmetric, 175–225 μm wide. Pycnoconidia bacilliform, 3–4 × 0.5 μm.

Thallus K–, KC–, C–, Pd–. Hymenium I+ blue. UV–. Zeorin was detected by TLC.

The species occurs on the bark of , Rupr., and Rupr. & Maxim. The species is currently known from a humid forest and a forested wetland of two mountainous sites.

The species epithet indicates that the lichen’s substance, zeorin, is a major compound.

The new species is similar to , , and sp. A in having a pigmented hypothecium. However, the new species differs from by areolate, brownish thallus, apothecia without pruina, hyaline and wider parathecium, narrower paraphyses with hyaline and unswollen tips, longer and narrower ascospores with just angular to globose lumina, and the absence of pannarin (Sheard et al. 2017).

The new species differs from by the absence of vegetative propagules, and -type ascospores in smaller size (Sheard et al. 2017).

The new species differs from sp. A by wider parathecium, narrower paraphyses with swollen tips, smaller ascospores -type, and the absence of pannarin (Sheard et al. 2017).

The new species can be compared with and in having erumpent apothecia, small ascospores(<21 μm long) with swollen septum among corticolous species. However, the new species differs from by crystals present in the amphithecium, wider parathecium, narrower paraphyses without swollen tips, pigmented hypothecium, and longer and narrower ascospores (Tønsberg 1992; Sheard et al. 2017).

The new species is distinguished from by narrower ascospores, and pigmented hypothecium (vs. hyaline hypothecium) (Sheard et al. 2017). Reference Table 3 provides the key characteristics distinguishing from the compared species above.

Table 3.

Comparison of with closely-related species.

Species	Rinodinazeorina	Rinodinahypobadia	Rinodinamanshurica	Rinodinasheardii	Rinodinaaff.oleae	Rinodina sp. A
Thallus growth from	areolate, rimose to continuous	rimose, not areolate	rimose, rimose-areolate	±areolate to ±continuous, sorediate	continuous, rimose-areolate	continuous to areolate
Thallus color	light gray to light brownish gray	light to dark gray	gray-brown	yellow, yellow-brown, or pale brown or greenish	(dark gray to olive-green)	dark gray to gray-brown
Pruina	absent, but epinecral debris shown in water	slightly pruinose	absent	absent	(absent)	–
Parathecium color	hyaline and light brown at periphery	red-brown	–	red-brown to brown	(hyaline to brownish)	–
Parathecium at periphery (μm)	20–50	10–20	c. 20	c. 30	(up to 30)	c. 25
Paraphyses (μm)	up to 1.5	2–2.5	2.0	2.0	(1–2)	3.0
Paraphysial tips	not or little swollen, not pigmented	3–4 μm, lightly pigmented	c. 3 μm, light pigmented	c. 3 μm	–	c. 4.5 μm, pigmented
Hypothecium color	brown with olive pigment	reddish or chestnut brown	hyaline	dilute brown to red-brown	hyaline	light brown
Crystals in amphithecium	present in medulla	present in both cortex and medulla	absent	present	–	present in medulla
Ascospore type	Dirinaria-type with angular-globose lumina	Dirinaria-type with Physcia- or Physconia-like lumina	Dirinaria-type, with Physcia-like lumina	Pachysporaria-type I	Dirinaria-type with Physcia-like lumina	Pachysporaria-type I
Ascospores (μm)	11–20 × 5–8.5	12.5–18.5 × 6.5–10	14–16.5 × 7.5–8.5	16–35 × 8–17	15.5–19 × 6.5–9.5	22–28.5 × 10.5–15.5
Pycnidia	175–225	up to 300	–	–	–	–
Pycnoconidia (μm)	3–4 × 0.5	3.5 × 1.0	–	–	(4–5 × 1)	–
Substance	zeorin	pannarin, zeorin	absent	zeorin	(absent)	pannarin, zeorin
Reference	BDNA-L-0000933 (holotype), BDNA-L-0000642, BDNA-L-0000646, BDNA-L-0000650, BDNA-L-0000651, BDNA-L-0000668	Sheard et al. 2017	Sheard et al. 2017	Tønsberg 1992; Sheard et al. 2017	Joshi et al. 2013; Smith et al. 2009; Sheard et al. 2017	Sheard et al. 2017

The morphological and chemical characteristics of several species close to the new species are referenced from the previous literature. All information on the new species is produced mainly from the type specimen (BDNA-L-0000933) in comparing with additional specimens (BDNA-L-0000642, BDNA-L-0000646, BDNA-L-0000650, BDNA-L-0000651, BDNA-L-0000668) in this study . The brackets in the column of are referenced from as some information of is not mentioned in the reference.

The new species is compared further with other species having the substance zeorin, (Tuck.) Tuck., Sheard, Malme, Zahlbr., (Nyl.) Vain., H. Magn., and Sheard & Giralt. However, all of them are different from the new species because those species represent larger ascospores in - to -type for ; sorediate thallus, mostly light brown hypothecium and -type ascospores and the presence of pannarin for ; sorediate thallus, colorless hypothecium, -type ascospores and the presence of pannarin and secalonic acid A for ; colorless hypothecium, larger ascospores in -type and the presence of thiomelin for ; larger spores in -type and the presence of pannarin for ; larger spores in -type for ; sorediate thallus and the presence of pannarin for (Sheard et al. 2012, 2017).

Additional specimens examined.

South Korea, Gangwon Province, Pyeongchang-gun, Daegwallyeong-myeon, Heonggye-ri, a forested wetland, , 1,047 m alt., on bark of , 03 June 2020, B.G. Lee & H.J.Lee 2020-000442, with (Fr.) Mudd, sp., (Nyl.) J. Steiner, (Hue) H. Miyaw., (Ach.) Ach., , and (J. Lowe) Resl & T. Sprib. (BDNA-L-0000642; GenBank MW832812 for ITS); same locality, , 1,047 m alt., on bark of , 03 June 2020, B.G. Lee & H.J.Lee 2020-000446 (BDNA-L-0000646; GenBank MW832813 for ITS); same locality, , 1,047 m alt., on bark of , 03 June 2020, B.G. Lee & H.J.Lee 2020-000450 (BDNA-L-0000650; GenBank MW832814 for ITS); same locality, , 1,047 m alt., on bark of , 03 June 2020, B.G. Lee & H.J.Lee 2020-000451 (BDNA-L-0000651; GenBank MW832815 for ITS); same locality, , 1,047 m alt., on bark of , 03 June 2020, B.G. Lee & H.J.Lee 2020-000468, with (Hoffm.) Coppins & Scheid., Körb., sp., Fr., (Poelt) Kashiw., Bagl., (Ach.) J. Kalb & Kalb (BDNA-L-0000668; GenBank MW832816 for ITS).

Key to the species of from the far eastern Asia (63 taxa)

Eleven more species have been recorded since Sheard et al. (2017), such as , , , , , , , , , and two new species from this study (Kondratyuk et al. 2016, 2017; Yakovchenko et al. 2018; Galanina and Ezhkin 2019; Kondratyuk et al. 2020; Galanina et al. 2021). Particularly, of Aptroot and Moon (2014) was rejected by Sheard et al. (2017), but Galanina et al. (2021) confirmed the species in the far eastern Asia. This key includes all above species except for because the species was just announced for a new record to Korea without any specific description for reference (Kondratyuk et al. 2020). (Ach.) Körb., (Wahlenb.) Th. Fr., and C.W. Dodge & G.E. Baker were reported from Korea and Russian Far East (Kondratyuk et al. 2016; Galanina et al. 2021) as expected to occur (Sheard et al. 2017). All expected species are remained with an asterisk mark(*).

Overall, 63 taxa of are currently recorded or expected to the far eastern Asia (Korea, Japan and Russian Far East).

1	Substratum rock	2
–	Substratum bark, wood, soil, decaying ground vegetation, bone or other lichens	17
2	Thalli with vegetative propagules	3
–	Thalli lacking vegetative propagules	4
3	Thallus effigurate, typically with isidia; when fertile spores belong to the Physconia-type; associated with seabird colonies; northern	R.balanina
–	Thallus not effigurate, vegetative propagules blastidia with budding soredia; spores Pachysporaria-type II; not coastal; southern	R.placynthielloides
4	Always maritime, typically on coastal rocks; spores Dirinaria-type	R.gennarii
–	Generally inland or occasionally maritime; spores belonging to a different type	5
5	Medulla orange, K+ red-violet; spores Pachysporaria-type I, ultimately developing satellite apical lumina	R.cervina
–	Medulla not orange, not K+ red-violet; spores of various types but never developing apical lumina	6
6	Thallus and apothecium margins K+ yellow, atranorin in cortex	7
–	Thallus and apothecium margins K−, atranorin absent	10
7	Spores with angular lumina, walls thickened at septum and apices, Physcia-type; proper exciple hyaline throughout, or if lightly pigmented not aeruginose (N−); thalline margin never pigmented	8
–	Spores with ‘hourglass’-shaped lumina, Mischoblastia-type; proper exciple typically aeruginose at periphery (N+ red under microscope); thalline margin often becoming pigmented	9
8	Apothecia 0.1–0.3 mm diam., hymenium 80–100 μm high, hypothecium 65–135 μm high, asci 75–80 × 16–19 μm, spores 17–27 × 8–13 μm	R.confragosa
–	Apothecia 0.6–1.5 mm diam., hymenium 55–85 μm high, hypothecium 10–55 μm high, asci 45–50 × 13–20 μm, spores 11–16 × 5–9 μm	R.occulta
9	Thallus plane; spores averaging <21 μm in length, rarely swollen at septum	R.oxydata
–	Thallus verrucose; spores averaging >21 μm in length, often swollen at septum when mature	R.moziana (syn. R.destituta)
10	Spores elongately ellipsoid, l/w ratio c. 2.0, Pachysporaria-type	R.cinereovirescens
–	Spores broadly ellipsoid, l/w ratio <2.0, belonging to various types	11
11	Spores >20 μm long at maximum, Teichophila-type, often swollen at septum, more so in KOH	12
–	Spores <20 μm long, never swollen at septum, belonging to another type	13
12	Spores 18.5–25 × 10–12.5 μm	R.tephraspis
–	Spores 20–32 × 11–19 μm	R.teichophila
13	Spores with broad pigmented band around septum, Bischoffii-type	R.bischoffii *
–	Spores lacking a broad pigmented band around septum, belonging to another type	14
14	Spores with Physcia-like lumina when immature, becoming rounded especially at the apices, lateral walls thin	15
–	Spores with rounded lumina from beginning, lateral walls relatively thick	16
15	Thallus thick, dark brown; spores constricted at septum when mature, Milvina-type; secondary metabolites absent	R.milvina
–	Thallus thin, gray to light brown; spores Physconia-type; thalline margin C+ red (under microscope), gyrophoric acid in medulla	R.sicula
16	Apothecial discs pruinose; spores Pachysporaria-type	R.compensata
–	Apothecial discs not pruinose; spores Pachysporaria- to Milvina-like	R.kozukensis
17	On soil, decaying ground vegetation, wood, bone or lichenicolous	18
–	Strictly corticolous or lignicolous	27
18	Spores 1-septate	19
–	Spores 3-septate or submuriform	20
19	Spores Teichophila-type	R.herrei
–	Spores Physcia-type, rarely with apical satellite lumina	21
20	Spores strictly 3-septate, type B development (apical wall thickened prior to septum formation); secondary metabolites absent	R.conradii
–	Spores 3-septate at first, typically becoming submuriform, type A development (apical wall thickening after septum formation); deoxylichesterinic acid present	R.intermedia
21	Strictly lichenicolous, on Aspicilia or Rhizocarpon	R.parasitica
–	Generally not lichenicolous	22
22	Sphaerophorin crystals in medulla (sometimes lichenicolous)	R.turfacea
–	Sphaerophorin lacking in medulla (never lichenicolous)	23
23	Cortex K+ yellow or medulla orange, K+ red	24
–	Cortex reaction absent	25
24	Thallus light gray; K+ yellow, atranorin in cortex	R.mniaroeiza *
–	Thallus a shade of brown; medulla orange, K+ red, skyrin or other anthraquinones present	R.cinnamomea *
25	Spores averaging <23 μm in length	R.olivaceobrunnea
–	Spores averaging >23 μm in length	26
26	Thallus and apothecia not pruinose; apothecial discs becoming convex, thalline margin then excluded; spores averaging 24.5–25.5 μm in length, l/w ratio 2.0–2.2	R.mniaroea
–	Thallus and apothecia typically pruinose; apothecial discs plane or concave, not convex, thalline margin never excluded; spores averaging 30–32 μm in length, l/w ratio 2.2–2.5	R.roscida
27	Vegetative propagules present	28
–	Vegetative propagules absent	37
28	Thallus typically golden yellow	29
–	Thallus a shade of gray or brown	30
29	Thallus with small, dense isidia; very rarely with apothecia; spores Pachysporaria-type I	R.chrysidiata
–	Thallus with marginal, labriform soralia, sometimes becoming pustulate; frequently, but not always, with apothecia; spores Physcia-type	R.xanthophaea
30	Phyllidia present	R.oxneriana
–	Blastidia or soredia present	31
31	Thallus mainly blastidiate, blastidia 35–60 μm diam.	R.colobinoides
–	Thallus generally not blastidiate, but sorediate or sometimes blastidiate	32
32	Blastidia present at margin, no substance, spores Teichophila-type	R.herrei
–	Soredia and/or blastidia present, atranorin or pannarin present, spores in various types	33
33	Thallus light gray; soralia labriform at first, soredia whitish; K+, P+ yellow, cortical atranorin present, pannarin absent	R.subparieta (syn. R.degeliana)
–	Thallus darker gray; soredia never whitish; K−, P+ cinnabar, atranorin absent, pannarin present	34
34	Thallus usually of convex to bullate areoles; blastidia often present, sometimes breaking into soredia; zeorin typically absent, when fertile pannarin also in epihymenium	R.excrescens
–	Thallus never consisting of bullate areoles; soredia always present; zeorin typically present, pannarin never in epihymenium	35
35	Soredia typically yellowish, secalonic acid A present; spores Physcia-type when fertile, averaging <20 μm in length	R.efflorescens
–	Soredia never yellowish, secalonic acid A absent; spores not Physcia-type, averaging >20 μm in length	36
36	Thallus minutely verrucose, verrucae central on areoles, quickly forming raised soralia, later spreading over thallus surface; soredia >40 μm diam.; spores Teichophila-type	R.buckii
–	Thallus with plane areoles, soredia developing marginally on areoles, never raised centrally on verrucae, later spreading over thallus surface; soredia <40 μm diam.; spores Pachysporaria-type I	R.willeyi
37	Ascospores 3-septate or submuriform	38
–	Ascospores 1-septate, rarely with satellite apical cells	39
38	Spores strictly 3-septate, type B development (apical wall thickened prior to septum formation); secondary metabolites absent	R.conradii
–	Spores 3-septate at first, becoming submuriform, type A development (apical wall thickening after septum formation); deoxylichesterinic acid present	R.intermedia
39	Thallus brightly pigmented; xanthone present, UV+ orange	40
–	Thallus a shade of gray or brown; xanthone absent, UV−	41
40	Thallus citrine, thiomelin present; spores averaging 31.0–34.5×16.0–17.5 μm, Pachysporaria-type I; not sorediate; subtropical, Tsushima Island, Japan	R.luteonigra
–	Thallus golden yellow, secalonic acid A present; spores averaging 23.5–28.5×2.0–15.0 μm, Physcia-type; frequently sorediate; temperate, widely distributed	R.xanthophaea
41	Thallus K+ yellow or P+ cinnabar, atranorin or pannarin present	42
–	Thallus K−, P−, both atranorin and pannarin absent	49
42	Thallus K+ yellow, atranorin present, pannarin absent	43
–	Thallus P+ cinnabar, pannarin present, atranorin absent	45
43	Spores averaging >33 μm long, Pachysporaria-type I	R.megistospora
–	Spores averaging <33 μm long, Physcia- or Physconia-type	44
44	Spores averaging >26 μm long, strictly Physcia-type; never sorediate; distribution limited to coastal foreshores	R.macrospora
–	Spores averaging <26 μm long, Physcia- to Physconia-type; most frequently sorediate; distribution inland	R.subparieta (syn. R.degeliana)
45	Hypothecium pigmented dark reddish brown; spores Dirinaria-type, (12–)14–16.5(–18)× (6.5–)7.0–8.5(–9.5) μm, lightly pigmented	R.hypobadia
–	Hypothecium never strongly pigmented; spore type otherwise	46
46	Spores averaging <20 μm in length, Physcia-type; thallus becoming bullate, often with minute blastidia	R.excrescens
–	Spores averaging >20 μm in length, not Physcia-type; thallus sometimes verrucate but never bullate or blastidiate	47
47	Thallus persistently plane; epihymenium lacking crystals, P−; spores averaging >29 μm	R.tenuis (syn. R.adirondackii)
–	Thallus becoming verrucate; epihymenium with or without crystals, P+ or P−; spores averaging <29 μm	48
48	Epihymenium typically possessing pannarin crystals, P+ cinnabar; spores lacking apical canals; widely distributed in Japan and adjacent mainland	R.subalbida
–	Epihymenium lacking pannarin crystals, P−; spores with very obvious apical canals; Cheju Island, Korea	Rinodina sp. A
49	Spores 16 per ascus	R.polyspora
–	Spores 4–8 per ascus	50
50	Medulla with sphaerophorin crystals, PL+	51
–	Medulla lacking sphaerophorin crystals, PL−	52
51	Thallus dark gray, typically dark brown; areoles becoming contiguous, plane, 0.40–0.55 mm wide; spores averaging 26.5–27.5 × 13.5–14.5 μm	R.badiexcipula
–	Thallus light gray, sometimes brownish; areoles remaining discrete, convex, 0.20–0.30 mm wide; spores averaging 23.0–25.5 × 11.5–13.5 μm	R.cinereovirens
52	Spores swollen at septum, more so in KOH, type B development (apical wall thickening prior to septum formation), Dirinaria-type	53
–	Spores not swollen at septum, even in KOH, type A development (apical wall thickening after septum formation), various types	59
53	Spores averaging >21 μm long	R.endospora
–	Spores averaging <21 μm long	54
54	Spores lacking wall thickening at maturity (septal and apical thickenings may be present briefly in immature spores)	55
–	Spore lumina Physcia-like, with persistent apical wall thickening	56
55	Thallus gray to ochraceous, rugose, areoles to 0.7 mm wide; apothecia to 0.8 mm in diam., discs plane, never convex; spores averaging 15.5–18.0 × 8.0–8.5 μm, l/w ratio 1.9–2.1	R.mongolica
–	Thallus gray, never ochraceous, continuous to rimose; apothecia to 0.30–0.50 mm in diam., discs often becoming convex; spores averaging 12.5–13.5 × 5.5–6.0 μm, l/w ratio 2.1–2.4	R.pyrina *
56	Apothecia not erumpent; spores averaging 17.5–21.5 × 9–11 μm	R.metaboliza
–	Apothecia erumpent; spores smaller	57
57	Hypothecium pigmented with brown, spores 11–20 × 5–8.5 μm, zeorin present	R.zeorina
–	Hypothecium colorless, spores 15.5–18 × 8–9 μm, no substance	58
58	Spores averaging 15.5–16.0 μm in length	R.manshurica
–	Spores averaging 16.5–18.0 μm in length	R.aff.oleae
59	Spores averaging >22 μm in length	60
–	Spores averaging <22 μm in length	61
60	Margins of apothecia often radially cracked; spores Physcia- to Physconia-type	R.ascociscana (syn. R.akagiensis, R.melancholica)
–	Margins of apothecia not radially cracked; spores Pachysporaria-type I	R.dolichospora
61	Spores Pachysporaria-type II	R.salicis
–	Spores Physcia- or Physconia-type	62
62	Spores Physcia- to Physconia-type, some lumina becoming rounded at apices, at maturity thin-walled	63
–	Spores strictly Physcia-type, apical walls remaining thick	67
63	Thallus dark brown, spores darkly pigmented at maturity, torus prominent; oro-arctic to coastal	64
–	Thallus a shade of gray, sometimes brownish, spores typically pigmented at maturity, torus present but not prominent; boreal	66
64	Thallus inconspicuous; apothecia mostly crowded, typically broadly attached	R.olivaceobrunnea *
–	Thallus of dispersed or contiguous areoles; apothecia mostly dispersed, narrowly or broadly attached	65
65	Ascospores 20–21.5 × 10–11.5 μm, thallus well-developed, flat, scurfy or thick rugose areolate, apothecia broadly attached in the beginning then becoming narrow and even stipitate, discs convex when mature	R.sibirica
–	Ascospores 18.5–19.5 × 8.5–9.0 μm, thallus poorly developed, evanescent, thin or scabrid, sometimes areolate, apothecia broadly attached to thallus, discs typically flat	R.laevigata
66	Thallus thick, rugose, areolate; apothecia crowded, discs persistently plane, thalline margins persistent	R.archaea *
–	Thallus thin, plane, continuous or rimose-areolate; apothecia dispersed, discs becoming convex, often excluding thalline margin	R.trevisanii *
67	Spores averaging >18 μm long, zeorin present	R.subminuta
–	Spores averaging <18 μm long, zeorin absent	68
68	Apothecia erumpent at first, discs often becoming strongly convex; spores with lightly pigmented tori at maturity	R.orientalis
–	Apothecia never erumpent, discs persistently plane; spores with very dark, prominent tori at maturity	69
69	Apothecia crowded, broadly attached; thalli associated with leaf scars or other mesic microhabitats; areoles plane, contiguous, to >0.2 mm in diam.	R.freyi
–	Apothecia mostly scattered, narrowly attached; thalli typically in more xeric microhabitats; areoles convex, scattered, to 0.2 mm in diam.	R.septentrionalis