Two New Corticolous Buellioid Species from South Korea.

Several buellioid specimens were collected from South Korea during field surveys and two new species are described based on morphology, chemistry, and molecular phylogeny. Buellia boseongensis sp. nov. is similar to B. polyspora but differs in having a UV + orange thallus and cryptolecanorine apothecia. Sculptolumina coreana sp. nov., resembles S. japonica, but differs in having a smooth entire continuous thallus, which reacts K-, a narrower excipulum, thicker epihymenium, narrower subhymenium, and in containing secondary metabolites other than flavo-obscurin and myeloconone. A key to the buellioid lichens reported from Korea is also presented.

Introduction

Buellia De Not., typified by B. disciformis (Fr.) Mudd is a cosmopolitan lichen genus including a large number of crustose lichen species with green photobiont, black lecideine to biatorine apothecia with blackish disc, dark hypothecia and Bacidia-type asci with brown to black, 1- or pluriseptate ascospores [1]. Studies of this group have been conducted since it was firstly described by De Notaris [2], with many species being discovered, several suggestions were also proposed on the homogeneity and the segregation of the buellioid lichens. Scheidegger [3] and Marbach [4] separated 13 genera based on morphological-anatomical characters of mainly corticolous taxa, i.e., ascomata, ascus, spore anatomy, spore wall ornamentation, and conidiophore type. Buellia in the strict sense is characterized by Callispora-type ascospores, bacilliform or weakly clavate conidia and a hymenium with oil droplets [4-6]. However, many species with pluriseptate spores do not form a monophyletic group and remain to be placed in segregate genera, thus they remain classified in the Buellia in the broad sense [1,7-9].

Poelt [10] and Hafellner et al. [11] included the family Buelliaceae in the Physciaceae, and Buellia has long been treated as a well-studied genus of the Physciaceae by investigators worldwide [1,4,7,8,12-14], however, molecular genetic data indicates that the Buellia group is not supported as a monophyletic genus [7,15,16]. Rambold et al. [17] proposed two groups based on the ascus types in Physciaceae, i.e., Buellia group with Bacidia-type ascus and Physcia group with Lecanora-type ascus. Wedin et al. [18,19] reconstructed the phylogenetic tree of Caliciaceae and Physciaceae, and showed that these two families formed a well-supported monophyletic group, but prototunicate, mazaedia-forming representatives of Caliciaceae were derived from within Physciaceae, and fell into the “Buellia-group” clade, so buellioid lichens actually belong to Caliciaceae rather than the Physciaceae [20].

Many substance classes have been reported for buellioid species, including orcinol depsides, β-orcinol depsidones, xanthones, and anthraquinones [7]. However, some of them (e.g., xanthones and anthraquinones) are difficult to distinguish with routine thin-layer chromatography (TLC); therefore, high-performance liquid chromatography (HPLC) was applied to identify the substances [4,21,22], Kalb and Elix [23] investigated the chemistry of 47 buellioid species and confirmed several type species, which gave a great significance for the following chemical and taxonomic studies of buellioid lichens.

Since the first two species have been reported under the genera Amandinea and Hafellia by Park [24], now 25 buellioid taxa belonging to the following four genera Amandinea, Buellia, Hafellia, and Sculptolumina have been recorded in South Korea [25-31]. Most of them are saxicolous. During our field trips from 2013 to 2016, several lignicolous and coniferous buellioid specimens were collected. In this study, morphological, chemical, and molecular analyses were conducted to confirm their systematical position.

Material and methods

Material and morphological studies

All the specimens in this study were collected from South Korea and deposited in Sunchon National University, Korean Lichen Research Institute (KoLRI). Specimens morphology, and spot test were examined under a dissecting microscope (Nikon SMZ 745 T, Tokyo, Japan), and the anatomical characters were recorded under Olympus BX 50 microscope (Olympus, Tokyo, Japan) and photos under Carl Zeiss MicroImaging with Axio Cam ERc 5 s imaging system (Carl Zeiss MicroImaging, Göttingen, Germany). All measurements based on the sections from the thalli and apothecia were made in the water or KOH solution (5–10%). The ranges of ascospore dimensions were calculated from 30 to 50 ascospore size measurements of a single apothecium per specimen.

Chemical studies

Amyloid reactions were tested with Lugol’s reagent (IKI). UV test was performed under the UV Chamber (CE07 21470). Secondary metabolites were studied by spot tests and TLC in solvent A, B, B’, and C as described by Elix [32] and Orange et al. [33].

High-performance liquid chromatography (HPLC)

Samples preparation. Dried thallus and apothecia (ca. 20–30 mg) were scraped from the substrate with a blade under a dissecting microscope, then transferred into 2 mL tubes for 2 h ultrasonic extraction in acetone twice, and after having dissolved with 300 μL acetone, the solution samples were filtered through 0.4 µm syringe membrane before injection to HPLC. Each species was analyzed with three different specimens and two replicates.

HPLC analysis. HPLC was carried out under a Shimadzu liquid chromatography (Prominence Modular HPLC LC-20A; Shimadzu, Kyoto, Japan). The chromatographic conditions were as follows: column, YMC-Pack ODS-A 150 × 4.6 mm I.D., S-5 µm, 12 nm (YMC Co., Ltd., Kyoto, Japan); solvent system, MeOH: H2O: Phosphoric acid = 80:20:1; flow rate, 1.0 mL/min; column temperature, 40 °C; detector, SPD-M20A, range 180–700 nm; the retention times and UV-spectra of detected peaks were compared with those of known data [34,35].

Molecular study and phylogenetic analysis

Total genomic DNA was extracted from freshly collected and frozen herbarium specimens using the NucleoSpin Plant II Kit (Clontech Laboratories, Mountain View, CA) following the manufacturer’s instructions. Primers pairs ITS1F [36] and ITS4 [37] were used to amplify the internal transcribed spacer (ITS) region. Protocols of PCR amplification followed Liu et al. [38]. Sequencing was accomplished by the genomic companies GenoTech (Daejeon, Korea) and Macrogen (Daejeon, Korea).

Newly generated ITS sequences were aligned with related buellioid lichens species from GenBank (Table 1). All raw sequences were assembled and edited using SeqMan (DNAstar packages) and BioEdit version 7.09 [39], then automatically aligned with MUSCLE version 3.6 [40]. Ambiguous regions were identified and excluded using Gblocks [41].

Table 1.

Information on ITS sequences used in this study, newly generated parts is in boldface.

Taxon	Accession no.	Collection number	Location
Amandinea lignicola	JX878521	Toensberg 36426 (BG)	USA, Washington, Kitsap Co., Bainbridge Island
A. efflorescens	AY143409	Kalb 27734 (GZU)	Australia, Northern Territory, Darwin
A. punctata 1	MF398994	S. Y. Kondratyuk 163344 (KoLRI)	South Korea, Jeollanam-do
A. punctata 2	AF224353	Nordin5346 (UPS)	Sweden, Uppsala
A. punctata 3	HQ650627	Unknown	Unknown
A. trassii 1	MF399002	S. Y. Kondratyuk 151358 (KoLRI)	South Korea, Gyeongsangbuk-do
A. trassii 2	MF399003	S. Y. Kondratyuk 150827 (KoLRI)	South Korea, Gangwon-do
Buellia aethalea 1	AF540496	Per Johansson 5 (UPS)	Sweden, Narke, Gotlunda Island Valen
B. aethalea 2	AY143410	Hafellner 39069 (GZU)	Italy, Udine
B. chujana 1	KT733597	L. Lőkös 140835-1 (KoLRI)	South Korea, Jeju-si, Mt. Dondae
B. boseongensis 1	MF398999	S. Y. Kondratyuk & L. Lőkös 150409 (KoLRI)	South Korea, Jeollanam-do
B. boseongensis 2	MF398998	S. Y. Kondratyuk & L. Lőkös 150408 (KoLRI)	South Korea, Jeollanam-do
B. boseongensis 3	MF399000	Kondratyuk S.Y. 163435 (KoLRI)	South Korea, Jeollanam-do
B. boseongensis 4	MF399001	D. Liu 163552 (KoLRI)	South Korea, Jeollanam-do, Boseong-gun
B. chujana 1	KT733597	L. Lőkös 140835-1 (KoLRI)	South Korea, Jeju-si, Mt. Dondae
B. chujana 2	KT733598	L. Lőkös 140835 (KoLRI)	South Korea, Jeju-si, Mt. Dondae
B. dijiana	AF250788	Unknown	Unknown
B. disciformis 1	FR799139	Unknown	United Kingdom
B. disciformis 2	AF540498	A. Nordin 4429 (UPS)	Sweden, Uppland, Radmanso Riddersholm
B. epigaea	AF250785	Unknown	Unknown
B. erubescens	AF250786	Unknown	Unknown
B. frigida	AF276066	IMI 384687 (CABI Bioscience)	Eastern Antarctica, Princess Elizabeth Land
B. halonia 1	KT733595	S. Y. Kondratyuk & L. Lőkös 140808 (KoLRI)	South Korea, Jeju-si, Mt. Dondae
B. halonia 2	KT733596	Y. Joshi & J. -U. So 140768 (KoLRI)	South Korea, Jeju-si, Mt. Dondae
B. lauricassiae	AB971692	Watanuki: L01179 (CBM)	Japan, Miyazaki Pref., Kushima
B. mamillana 1	KT733599	140792-1 (KoLRI)	South Korea, Jeju-si, Mt. Dondae
B. mamillana 2	KT733600	Josef P. Halda 141100 (KoLRI)	South Korea, Jeju-si, port of Yecho-ri
B. mamillana 3	MF398995	S. Y. Kondratyuk & L. Lőkös 161370 (KoLRI)	South Korea, Gyeongsangbuk-do
B. ocellata	AF540502	A. Nordin 4284 (UPS)	Faroe Islands, Skovoy
B. petermannii	AF250779	Unknown	Unknown
B. russa	DQ534454	J. S. Hur ANT050942 (KoLRI)	Antarctica, King George Island
B. stellulata	MF398996	120219 (KoLRI)	South Korea, Jeollanam-do
B. subdisciformis	AF352323	13750 (BBC)	Spain, Cap de Creus, Catalonia
Dermatiscum fallax	KX512921	Brusse 4944 (S)	Unknown
Dermatiscum thunbergii	AF540507	H. Sipman 19.908 (B)	South Africa, Transvaal Prov.
Dimelaena radiata 1	KX512923	Nash III 41396 (S)	Unknown
Dimelaena radiata 2	JQ301693	C. Scheidegger 140 (DUKE)	Spain, Almeria, Cabo de Gata
Dirinaria applanata	MF398997	120062 (KoLRI)	South Korea, Jeollanam-do
Pyxine sorediata	AY498683	J. B. Chen & G. R. Hu 21941 (HMAS)	Unknown
Sculptolumina coreana 1	MF399007	S. Y. Kondratyuk 150824 (KoLRI)	South Korea, Chungcheongbuk-do
S. coreana 2	MF399005	S. Y. Kondratyuk, L. Lőkös & C. H. Park 130766 (KoLRI)	South Korea, Jeollanam-do
S. coreana 3	MF399006	S. Y. Kondratyuk 150822 (KoLRI)	South Korea, Chungcheongbuk-do
S. coreana 4	MF399004	S. Y. Kondratyuk 110994 (KoLRI)	South Korea, Jeollanam-do
S. coreana 5	MF399008	D. Liu 163541	South Korea, Jeollanam-do
Tetramelas confusus	DQ201954	Galloway 0239 (UPS)	New Zealand
T. geophila	AF540499	A. Nordin 4429 (UPS)	Sweden, Torne Lappmark, Jukkasjarvi
T. insignis	DQ198358	A. Nordin 5664 (UPS)	Sweden
T. papillatus	AF250790	Unknown	Unknown
T. phaeophysciae 1	DQ201951	A. Nordin 5663 (UPS)	Norway
T. phaeophysciae 2	DQ198359	A. Nordin 4922 (UPS)	Iceland
T. phaeophysciae 3	KX512939	A. Nordin 6896 (UPS)	Unknown
T. pulverulentus	KX512940	A. Nordin 6368 (UPS)	Unknown

The ITS matrix was analyzed by Maximum likelihood optimality criterion (ML) and Bayesian Inferences (BI) with Dirinaria applanata and Pyxine sorediata as outgroups. ML inferences were performed using RAxML version 7.2.6 [42] with the GTR model, and supported bootstrap values >70% were estimated from the consensus tree built with 2000 trees obtained from nonparametric bootstrapping pseudoreplicates. GTR + I + G were selected best-fitted substitution models based on the Akaike information criterion using jModelTest version 3.7 [43]. BI analyses were performed with MrBayes version 3.1.2 [44] using four chains and run for 1 million generations. Trees were sampled every 1000th generations. Phylogenetic trees were summarized with the first 25% of tree discarded, then the remaining trees were used to generate a majority-rule consensus tree with posterior probabilities (PP), clades of ≥0.95 were considered as significantly supported.

Results

A total of 31 taxa including 51 sequences were aligned into the data set for this study, including 15 new ITS and 36 published sequences of buellioid lichen from GenBank. A total of 449 unambiguous characters were reserved after Gblocks. The phylogenetic tree (Figure 1) depicted based on the ML analysis with bootstrap value and Bayesian PP. Moreover, The ML and the BI trees (Supplementary 1) show different topologies, but accords in the terminal branches.

Figure 1.

Phylogenetic relationships of species of buellioid lichens inferred from ITS sequences under a criterion of maximum likelihood. Nodes supported by ML bootstrap values >70% and Bayesian posterior probabilities >0.95 are identified by thickened branches.

The phylogenetic tree infers that the genus Buellia is polyphyletic; however, Buellia boseongensis and Sculptolumina coreana each form monophyletic groups with strong support, respectively. B. boseongensis is close to Amandinea efflorescens and A. trassil, while S. coreana clade drops out of Buellia. The relationships in Buellia s. lat. are not still full resolved, B. disciformis; the type species of the genus Buellia forms a single clade (Buellia sensu stricto). Additionally, the genus Tetramelas, characterized by large spore and the presence of 6-O-methylarthelin [45], is monophyletic which also agrees with former researches [13,20,46-48], and evidence is also given in this study to separate Tetramelas typified by T. geophila from Buellia.

Taxonomic treatment

Buellia boseongensis D. Liu, S.Y. Kondr. & J.-S. Hur, sp. nov

MycoBank No.: MB 821884

Similar to B. polyspora (Tuck.) Marbach but differs in thallus UV + orange and 16-spored apothecia.

Type: South Korea, Jeollanam-do, Boseong-gun, Boseong-eup, Nokcha-ro 775#, Green Tea Garden, 34°43′2′′N, 127°4′46′′E, 240–266 m, on Cryptomeria sp., 23 Jul. 2016, D. Liu 163552 (holotype). Accession number: MF399001 (ITS) (Figure 2).

Figure 2.

B. boseongensis (holotype, KoLRI 041797). (A) apothecia and thallus; (B) algal cells (Trebouxia) in the thallus; (C) vertical section of apothecia; (D) paraphyses with a swollen cap, simple or sparsely branched; (E) asci with 16 spore; (F) spore with big cell or small guttula; (G) spores with numerous small guttula (Scale bar: A = 0.3 mm, B = 20 μm, C = 100 μm, D = 20 μm, E = 20 μm, F = 20 μm, and G = 15 μm).

Thallus crustose, granular, continuous, light grey or yellow to light yellow-green, 1–3 (–8 cm) across. Soredia and isidia absent. Margin usually delimitated. Prothallus absent, hypothallus present, grey to silvery. Cortex and medulla not well developed, medulla ca. 80 µm thick when visible, I–. Photobiont algal cells round, trebouxioid (6–)10–15 µm diam.

Apothecia numerous, common, immersed to sessile (0.1–)0.2–0.56(–0.6) mm diam., disc black, epruinose, plane to convex or rarely weakly concave, cryptolecanorin. Prople exciple 20–40 µm thick, hyaline inner zones, and dark brown or blackish out zones, paraplectenchymatous or pseudoplectenchymatous with cell lumina 6–9(–11) × 3–5(–7) µm. Hymenium 60–70(–80) µm thick, hyaline, not inspersed with oil droplets, amyloid. Epihymenium indistinct, usually with abundant spores, K–. Hypothecium to 30–70(–80) µm thick, brownish or olive-blackish. Asci Basidia-type, clavate, 57–62 × 10–16 µm, 16-spored. Paraphyses 1–2 µm wide, simple to sparsely branched, swollen toward the tips, apex forming a brown to black caps (2–)2.5–3(–6.6) µm. Ascospores Buellia-type, 1-septate, ellipsoid, constricted, end attenuated, hyaline when immature, turning olive brownish to deep brownish when mature, usually one cell is wider than the other, outer wall smooth, septum and wall distinctly thickened, non-amyloid (9–)10–13.8(–15) × 4–6(–6.5) µm in water and (9–)10–16(–18) × (4–)4.5–6(–8) µm in K. Pycnidia rare, conidia fusiform to ellipsoid, 4.3 –5.0 × 2.5–2.9 µm.

Chemistry: Thallus K − or pale yellow, C–, P–, UV + orange, spot after K then UV + bright yellow; 2,7-dichloro-3-O-methylnorlichexanthone (major), thiophanic acid (major), 2,5,7-trichloro-3-O-methylnorlichexanthone (major), norlichexanthone (minor), gyrophoric acid (minor), 4,5-dichloronorlichexanthone (minor), 4,5-dichloro-3-O-methylnorlichexanthone (minor), 2,7-dichlorolichexanthone (minor) by TLC and HPLC.

Etymology: The epithet refers to the location Boseong-gun, South Korea, where the type specimen was collected.

Habitat: This species always grows on bark of various coniferous trees (Cryptomeria, Metasequoia glyptostroboides, Chamaecyparis obtusa, etc.), together Biatora pseudosambuci, Dirinaria, Fellhanera, Fuscidea, Lecanora, Lepraria, and Ochrolechia.

Remarks: Buellia boseongensis is characterized by crustose, light grey or yellow to light yellow-green, granular thallus, without soredia and isidia, numerous apothecia, cryptolecanorine apothecia, 16-spored asci, and containing UV + orange xanthones.

Buellia boseongensis is similar to Amandinea errata Marbach but differs in having much thicker thallus (ca. 180–200 µm vs. 25–50 µm thick), and lacking a well-developed cortical layer and K– (vs. cortical layer K + reddish. B. boseongensis is most similar to A. melaxanthella (Nyl.) Marbach, they share the same class substance xanthones, but the major compounds of B. boseongensis is 2,7-dichloro-3-O-methylnorlichexanthone, thiophanic acid, and 2,5,7-trichloro-3-O-methylnorlichexanthone rather than arthothelin and thuringone, in addition, ascospores of B. boseongensis are wider ((9–)10–13.8(–15) × 4–6(–6.5) µm vs. 9–12(13) × 4 − 5.5 µm), and a hypothallus is present; Buellia boseongensisresembles Amandinea trassii and A. efflorescens, but it can be distinguished by having polyspored asci; Buellia boseongensis resembles B. polyspora, which was known from North America, and corticolous, but it easily distinguished by its UV + orange thallus and in having 16-spored asci. Buellia boseongensis can be distinguished from Amandinea punctata (Hoffm.) Coppins & Scheid. in having better developed and much thicker thallus, much larger apothecia, distinctly subconvex to convex apothecium disc, as well as a hyaline true exciple in lateral portion, 16-spored asci, and much narrower ascospores; Buellia boseongensis differs from B. lauricassiae and B. numerosa in UV + orange; Buellia boseongensis is similar with B. rhizocarpica and B. carballaliana, but the latter two species are 8-spored. The organism grows under the same conditions as Sculptolumina coreana, but differs in having Buellia-type (vs. Mischoblastia-type) ascospores and “polyspored” (i.e., 16-spored asci instead of 8-spored).

Buellia boseongensis was previously misidentified in South Korea and recorded as Amandinea melaxanthella [49,31] and Buellia polyspora [28,50], and all the specimens cited under A. melaxanthella and B. polyspora by them are now referred to B. boseongensis.

Additional specimens examined: Jeollanam-do: Boseong-gun, Boseong-eup, Nokcha-ro 775#, Green Tea Garden, 34°43′2 ′′N, 127°4′46′′E, 240–260 m, on Cryptomeria sp., October 31 2016, D. Liu 163526, 163527, 163529, 163530, 163531, 163533, 163534, 163535, 163536, 163537 163538, 163539, and 163540; Jangheung County, Mt. Cheongwan, 34°32′56.1′′N, 126°56′11.1′′E, alt. 120 m, on Alnus bark, 07 Oct. 2005, L. Lökös 050632; Sunchon-si: 34°58′00.4′′N, 127°28′32.9′′E, alt. 115 m, on Alnus bark, October 8 2005, L. Lökös 050672; Sunchon National University: 34°58′10′′N, 127°28′40′′E, 40 m, on Chamaecyparis obtusa, October 30 2016, D. Liu 163546, 163548, 163549, 163550, and 163551; October 2 2016, S. Y. Kondratyuk 163553; Humanitarian faculty, 34°57′59.3′′N, 127°28′44.8′′E, 70 m, on bark, October 5 2005, L. Lőkös 050625; 34°58′10.8′′N, 127°28′36.7′′E, on bark of pine tree, October 4 2011, S. Y. Kondratyuk 110994; along river, on bark of Metasequoia glyptostroboides, October 2 2016, S. Y. Kondratyuk 163344, 163345, 163348, 163349, and 163352. Odong-do Island, along the tourist path, 34°44′37.75′′N, 127°45′50.57′′E, 25 m, on bark of Camellia japonica, Machilus thunbergii, Pinus thunbergii, Quercus serrata, July 28 2013, S. Y. Kondratyuk, L. Lőkös & C. H. Park 130733; Yeosu-si: Hwayang-myeon, Yongju-ri, Najin elementary school yard, 34°42′30.00′′N, 127°36′44.46′′E, 15 m, on bark of Cedrus deodara, Cerasus, Pinus densiflora, July 28 2013, S. Y. Kondratyuk, L. Lőkös & C. H. Park 130760; Nam-myeon, Geumoh-do Island, Simjang-ri, Simpo coast, 34°29′33.2′′N, 127°46′13.7′′E, 49 m, on bark, April 27 2012, U. Jayalal, J. S. Park & J. A. Ryu 120464; Jeju-do, Cheju Island, Seogwipo-si, Gangjeong-dong, Yeongtto waterfall, 33°16′01.7′′N, 126°29′49.00′′E, 210 m, on bark, June 19 2014, Y. Joshi & J. U So 140576.

Sculptolumina coreana D. Liu, S. Y. Kondr. & J.-S. Hur, sp. nov

MycoBank No.: MB 821888

Similar to Sculptolumina japonica, but differs in having a smooth, continuous K–thallus, a narrower excipulum, thicker epihymenium, narrower subhymenium, and in containing secondary metabolites other than flavo-obscurins and myeloconone.

Type: South Korea, Jeollanam-do, Boseong-gun, Boseong-eup, Nokcha-ro 775#, Green Tea Garden, 34°43′2′′N, 127°4′46′′E, 240–260 m, on Cryptomeria sp., October 31 2016, D. Liu 163541. Accession number: MF399008 (ITS) (Figure 3).

Figure 3.

S. coreana (A–F, holotype, KoLRI 041786, in water, G, KoLRI 033898, in K). (A) apothecia; (B) thallus; (C) vertical section of apothecia; (D) elongate filiform conidia; (E & G) mature spore; (F) immature spore (Scale bars: A = 0.5 mm, B = 20 μm, C = 100 μm, D = 20 μm, and E–G = 10 μm).

Thallus crustose, continuous to dispersed, from very thin to somewhat slightly warty or with undulating surface, granular to subsquamulose, not being leprose and farinose, dark grey, or green to dark green. Soredia, isidia, hypothallus, and prothallus absent. Photobiont green alga (Trebouxia), algae cells round, 4 − 10.2 µm.

Apothecia pseudolecanorine, numerous, common, sessile attached, 0.2–0.67 mm diam., disc black, epruinose, plane to convex, or rarely weakly concave; disc with distinct permanent (40(–80) µm wide) margin. Proper exciple conspicuous, 22–60 µm wide, trachyspora-type, outer zone greenish black to brown-black or carbonaceous, K–; inner zone colorless to pale brown, K–. Hymenium (90–)100–120(–140) µm thick, amyloid, inspersed with oil droplets, 4(–5) µm in diam. Epihymenium (15–)20–25 µm thick, dull yellow brownish or dull brownish to dark brown. Hypothecium (20–)30–50(–60) µm thick, dark brownish to black, ±inspersed with oil droplets. Asci Bacidia-type, clavate, 8-spore, 80–90 × 20–22(–24) µm, often with only 1–2 well-developed ascospores. Paraphyses 1.5–2.2 µm wide, with apical cells 2.5–4.5µm wide, forming a brownish to black cap. Ascospores Mischoblastia-type, hyaline to light grey, greenish grey when young, and dark brown to black when over mature, ascospore wall smooth (15–)17–22(–25) × (8–)9–11(–12) µm. Pycnidia rare, irregular. Conidia filiform, straight, or slightly curved to waved (8–)15–22.5 × 1 µm.

Chemistry: Thallus K–, C–, P–; contains two major dark grey unknown pigments, rhizocarpic acid (minor), and another unknown pulvinic acid derivatives (minor) by TLC and HPLC. Spectral information of unknown compounds as follow: major 1: spot color dark grey, ultraviolet spectrum λmax 225.40, 259.45, 320.33, 356.23; major 2: spot color grey, ultraviolet spectrum λmax 283.94, 322.14, 355.76; minor unknown pulvinic acid derivative: spot color pale grey, ultraviolet spectrum λmax 285.72, 369.54.

Etymology: The species epithet refers to the name of the country, South Korea, where the type specimen was collected.

Habitat: This species always grows on bark of various coniferous trees (Cryptomeria, Metasequoia glyptostroboides, Chamaecyparis obtusa, etc.), adjacent lichens included Lecanora, Lepraria, Ochrolechia, and Physcia.

Remarks: Sculptolumina coreana is characterized by crustose, greenish, granulose to minutely squamulose thallus, numerous pseudolecanorine sessile apothecia, 0.2–0.67 mm diam., with black, epruinose, plane to convex or rarely weakly concave disc; hymenium hyaline, inspersed with numerous oil droplets, Bacidia-type asci with eight Mischoblastia-type spores. Additionally, measurements of ascospore size in water and K differ, being much smaller in water as (15–)17–22(–25) × 9–11(–12) µm vs. in K (21–)24–35(–34) × (12–)13–15(–17) µm (Figure 3(E,G)).

Moon [51] recorded Buellia disculiformis (Nyl.) Zahlbr. from South Korea, but this species was subsequently synonymized with S. japonica [4]. Sculptolumina coreana is similar to S. japonica, but differs in having a smooth, continuous (rather than being leprose-granulose) which is colored grey or grey greenish to dark greenish thallus a thicker epihymenium (20–25 µm vs. 4–8 µm), narrower hypothecium (20–)30–50(–60) µm vs. 80 − 120 µm thick), in having smooth walled (vs. rugulate) ascospores, as well as in lacking K + purple orange pigment and soredia (S. japonica is more or less diffusely sorediate in places). Furthermore, flavo-obscurins and myeloconone, the compounds in S. japonica [4,23,52,53] were not detected by TLC and HPLC analysis. Sculptolumina coreana was previously misidentified as S. japonica in South Korea [30,49,54]. Re-examination relevant specimens indicated that all refer to S. coreana.

Sculptolumina coreana is similar to S. serotina (Malme) Marbach in that it has very similar ascospores (15–)17–22(–25) × 9–11(–12) µm vs. (16–)18–22(–27) × (8–)9–12(–14) µm after Marbach [4], but differs in having funnel-shaped cell lumina in ascospores (vs. lumina rounded). Moreover, the latter contains lobaric acid [55].

Sculptolumina coreana is easily distinguished from S. conradiae H. Mayrhofer, Giralt, van den Boom & Elix by the Mischoblastia-type ascospores and its unique secondary chemistry, while S. conradiae has 1–3-septate ascospore similar to the Conradii-type ascospores and no secondary metabolites [55].

The genus Sculptolumina was originally described by Marbach [4], subsequently Giralt et al. [55] described that long filiform conidia provided an additional diagnostic feature for this genus. Although the type species of Sculptolumina has not been sequenced, we believe that the new species should be placed in Sculptolumina based on following morphological characteristics: crustose thallus, lecideine apothecia with epruinose discs, the hymenium inspersed with oil droplets, Mischoblastia-types ascospores and further filiform conidia. Although S. coreana is similar to several Endohyalina species, but Endohyalina has bacilliform conidia.

Additional specimens examined: Gyeongsangnam-do: Tongyeong-si, Yokgi-myeon, Saryang Island, 34°50′10.6′′N, 128°10′47.6′′E, 73 m, on Pinus sp., March 17 2007, 070015. Incheon Metropolitan City: Ganghwa-gun, Samsan-myeon, Seokmo Island, 37°44.475′N, 126°19.368′E, 16 m, on Quercus sp., September 29 2010, X. Y. Wang et al. 100991. Jeju-do: Seogwipo-si, Donnaeko-ro, Donnaeko, 33°18′04.3′′N, 126°34′53.07′′E, 330 m, on bark, June 19 2014, S. Y. Kondratyuk 140616-2; Wolsan-ro, along the tourist path to Eongtto waterfall, 33°16′03.26′′N, 126°29′52.31′′E, 285 m, on bark, June 19 2014, L. Lőkös 140573. Jeollabuk-do: Namwon-si, Unbong-eup, Sandong-myenon, Mt. Gonam, 35°28′44.9′′N, 127°30′48.6′′E, 621 m, on Pinus, July 18 2015, J. J. Woo & S. -O. Oh 152254. Jeollanam-do: Boseong-gun, Boseong-eup, Nokcha-ro 775#, Green Tea Garden, 34°43′13.45′′N, 127°4′55.60′′E, 274 m, on Cryptomeria sp., May 7 2016, D. Liu 160585; 240–260 m, 34°43′2′′N, 127°4′46′′E, October 31 2016, D. Liu 163524, 163525, 163528, 163542, 163543, 163544, and 163545; Hwasun-gun, Buk-myeon, Mt Baegasan, 35°10′32.4′′N, 127°08′23.1′′E, 320 m, on bark, October 8 2005, L. Lőkös 050669; Gwangyang-si, Taein-dong, 34°56′36.02′′N, 127°44′33.66′′E, 17 m, on bark of Pinus among gardens, June 22 2015, S. Y. Kondratyuk & L. Lőkös 150303; Gwansan-eup, Okdang-ri, Cheongwansan Mts, along the tourist track No. 2, near the temple, 34°32′55.65′′N, 126°55′43.11′′E, 154 m, on Camellia/Diospyros, growing together with Cresponea proximata, June 23 2015, Kondratyuk S. & L. Lőkös 150405, 150407; Jangheung-gun: Gwansan-eup, Okdang-ri, Cheongwansan Mts, along the tourist track No. 2, 34°32′26.63′′N, 126°55′13.67′′E, 456 m, on Quercus, June 23 2015, S. Y. Kondratyuk & L. Lőkös 150330; Mt. Cheongwan, 34°32′50.6′′N, 126°55′43.4′′E, 207 m, on bark, October 7 2005, 050638; Sunchon City, Sunchon National University, Humanitarian Faculty, 34°58′10.8′′N, 127°28′36.7′′E, on bark, October 4 2011, S. Y. Kondratyuk 110995; Songgwang-myeon, Sinpyeong-ri, Mt. Jogye, Songgwang-sa, 34°00′13.7′′N, 127°16′16.3′′E, 210 m, on Quercus bark, October 11 2005, L. Lökös 050699; Yeosu-si: Hwayang-myeon, Yongju-ri, Najin elementary school yard, 34°42′30.00′′N, 127°36′44.46′′E, 15 m, on bark of Cedrus deodara, Cerasus, Pinus densiflora, July 28 2013, S. Y. Kondratyuk & L. Lőkös et al. 130758, 130762, and 130766; along the road at seacoast, 34°39′5.33′′N, 127°34′45.67′′E, 8 m, on bark of Camellia japonica, Celtis, Quercus variabilis, Sorbus amurensis, July 28 2013, S. Y. Kondratyuk et al. 130784, 130762, and 130766; Odong-do Island, along the tourist path, Machilus thunbergii, Pinus thunbergii, 34°44′37.75′′N, 127°45′50.57′′E, 25 m, on bark of Camellia japonica, July 28 2013, S. Y. Kondratyuk et al. 130735.

Discussion

Buellia s. lat. is a heterogeneous and species-rich group where the genetic relationships from molecular phylogeny remain ambiguous and poorly resolved in this study, even though several morphological and phylogenetic investigations have been performed [4,7,19]. However, as a result of this study some segregates matched those in a morphological investigation conducted [4], such as genus Tetramelas. Nevertheless, further study should be continued to clarify the natural relationship in Buellia s. str. clade, which remains highly polyphyletic, Figure 1.

Buellia is a cosmopolitan lichen genus and contains ca. 453 species worldwide, although several segregations were separated [4,12], the taxonomy of Buellia s. l. is still controversial. Most of species in this group have 8-spored asci, and less than 20 species have polyspored asci [1,4]. B. boseongensis is characterized by 16-spored asci is similar with B. polyspora, A. errata, and A. melaxanthella; however, it is unfortunate that we are not available to get the ITS sequences and have a contrast with each other on the basis of DNA information. The phylogenetic tree infers that B. boseongensis is far from A. punctate, although it is close to A. trassil and A. efflorescens, the morphological difference among them is easy to be distinguished; moreover, the determination of the genus Amandinea need further study.

The genus Sculptolumina was separated from Buellia by Marbach [4] based on following characteristics: the lecideine apothecia, hymenium inspersed oil droplets, Mischoblastia, or Pachysporaria types spores; then Giralt and Clerc [46] expanded the diagnostic character with a long and often curved filiform conidia based on the re-examination of holotype. Giralt et al. [55] described S. conradiae from Guatemala on the basis of spore type, but lacking conidia information; the morphology of new species S. coreana matches the genus characters recorded by Marbach and Giralt, and distinctly differs from those of Buellia. The phylogenetic tree also shows S. coreana forms a separated clade with Buellia (typed by B. disciformis).

Large numbers of chemical compounds have been recorded in buellioid lichens, like xanthones, which may be used as an important character for species identification. However, it is still difficult to confirm the actual chemistry due to the absent of standard controls, but when combined with the HPLC chemical fingerprint chromatography, the characteristics of unknown compounds like UV spectra can be recorded. Previously, Sculptolumina contained three species, all of which differ in either chemistry or spore type. The chemistry of S. coreana differs from all three, although it is not clear what compounds it contains.

1a. Habitat corticolous (growing on bark)	2
1b. Habitat saxicolous (growing on rock)	8
2a. Hymenium with numerous oil droplets; ascus 8-spored	3
2b. Hymenium without numerous oil droplets; ascus 8- to 16-spored	4
3a. Thallus rimose, white to grey yellow, exciple dipersa-type; ascospore Callispora-type	Buellia. disciformis
3b. Thallus granular to subsquamulose, green; exciple trachyspora-type, ascospore Mischoblastia-type	S. coreana
4a. Thallus sorediate, rimose to areolate; ascus 8-spored, ascospores submuriform, 8–12 cells	B. griseovirens
4b. Thallus granular to subsquamulose; ascus 8- or more-spored, ascospores 1-septate	5
5a. Ascus 16- or more-spored	6
5b. Ascus 8-spored	7
6a. Thallus aerolate; ascus (24–)32–48-spored	Amandinea pseudomultispora
6b. Thallus continuous; ascus 16-spored	B. boseongensis
7a. Sorediate	A. trassii
7b. Esorediate	A. punctata
8a. Thallus C + orange or pink	9
8b. Thallus C–	12
9a. Thallus P + yellow or orange	10
9b. Thallus P–	11
10a. Ascospore (11.5) 12.4–15.7 (19) × (6)6.8–8(9) μm	B. halonia
10b. Ascospore (9)10–13 (14) × 5–7 μm	B. chujadoensis
11a. Prothallus usually present, thallus C + pink, ascospore (8)9.1–11.1(14) × (4.5)4.9–6.3(7) μm; medulla amyloid (I + blue)	B. cf. uberior
11b. Prothallus absent, thallus C + orange, ascospore (9)10–13 (–14) × 5–7 μm; medulla non-amyloid (I–)	B. ocellata
12a. Thallus UV + orange	13
12b. Thallus UV –	14
13a. Thallus K + yellow, exciple mamillana-type	B. mamillana
13b. Thallus K–, exciple aethalea-type	B. prospersa
14a. Thallus K–	15
14b. Thallus K + yellow or K + yellow then red	17
15a. Exciple mamillana-type, proper septum distinctly thickened, ascospore 6–8 × 7–9	B. chujana
15b. Exciple aethalea-type, proper septum not thickened	16
16a. Thallus parasitic on other lichen	B. badia
16b. Thallus not parasitic	B. sequax
17a. Thallus pruinose, with a large amount of calcium oxalate (H2SO4+)	18
17b. Thallus epruinose, without calcium oxalate (H2SO4–)	19
18a. Thallus deep brown to olive brown, prothallus absent exciple aethalea-type	B. nashii
18b. Thallus different shades of gray, never brown, prothallus present, exciple dispersa-type	B. maritima
19a. Thallus K + yellow; containing 2’-O-methylperlatolic and ± confluentic acids	B. stellulata
19b. Thallus K + yellow then red; containing norstictic and ± stictic acids	20
20a. Apothecia remaining immersed, not emergent; containing norstictic acid; medulla amyloid (I + blue)	B. aethalea
20b. Apothecia superficial at maturity; containing norstictic or stictic acid, non-amyloid (I–)	B. spuria