Arthonia dokdoensis and Rufoplaca toktoana - Two New Taxa from Dokdo Islands (South Korea).

Arthonia dokdoensis sp. nov., a lichenicolous fungus from the subcosmopolitan Arthonia molendoi complex growing on crustose thalli of species of the genus Orientophila (subfamily Xanthorioideae, Teloschistaceae), as well as the lichen species Rufoplaca toktoana sp. nov. (subfamily Caloplacoideae, Teloschistaceae) similar to Rufoplaca kaernefeltiana, both from Dokdo Islands, Republic of Korea, are described, illustrated, and compared with closely related taxa. In the phylogenetic tree of the Arthoniaceae based on 12S mtSSU and RPB2 gene sequences, the phylogenetic position of the A. dokdoensis and the relationship with the A. molendoi group are illustrated, while the position of the newly described R. toktoana is confirmed by phylogenetic tree based on ITS nrDNA data.

Introduction

During a recent investigation of lichen-forming and lichenicolous* fungi (*lichenicolous, i.e., growing or inhabiting on lichen thalli [or thalli of lichen-forming fungi]) of Dokdo Islands, two members of the genera Arthonia (Ach.) (Arthoniaceae) and Rufoplaca Arup, Søchting & Frödén (subfamily Caloplacoideae of the Teloschistaceae) were newly found.

Within our study from combined phylogenetic analysis of the Arthoniaceae based on mtSSU and RPB2 sequences, the Arthonia molendoi group was positioned in the monophyletic branch. Several arthonioid species were already known to grow on foliose or fruticose members of the Teloschistaceae, namely Arthonia sytnikii S. Y. Kondr., Arthonia anyutae S. Y. Kondr. et Alstrup and Arthonia descruens var. nana Grube et Hafellner, as well as A. molendoi (Heufl. ex Frauenf.) R. Sant. All of them infect the thallus, and some of them also develop fruiting bodies on the apothecia (hymenia) of their hosts. Selected characters of taxa mentioned above have been recently summarized by Fleischhacker et al. [1], who described a new taxon, Arthonia parietinaria Hafellner et A. Fleischhacker, a member of the A. molendoi group, and compared with Arthonia anjutae, A. sytnikii, as well as Arthonia epiphyscia, and A. molendoi. It was concluded that the status of taxa mentioned at the species level had been confirmed by the taxonomic position of hosts in separate genera of the Teloschistaceae, which recently have been proved by three-gene phylogeny (see [2]). Thus A. parietinaria has hosts of the genus Xanthoria Th. Fr. (as it is correctly stressed by Fleischhacker et al. [1]), while lichenicolous fungi previously recorded on Massjukiella polycarpa (Hoffm.) S.Y. Kondr., Fedorenko, S. Stenroos, Kärnefelt, Elix, Hur & A. Thell and/or Oxneria huculica S.Y. Kondr. are highly likely to belong to different taxa. A. anjutae was confirmed as a species of the genus Teloschistes Norm. (the subfamily Teloschistoideae of the Teloschistaceae), A. as a species of the genus Jackelixia S. Y. Kondr., Fedorenko, S. Stenroos, Kärnefelt & A. Thell, and A. molendoi as a species of the genus Rusavskia S. Y. Kondr. & Kärnefelt (both latter genera, i.e., Jackelixia and Rusavskia of the subfamily Xanthorioideae of the Teloschistaceae). The further taxon from the A. molendoi complex, which was confirmed as a species of the genus Orientophila Arup, Søchting & Frödén (subfamily Xanthorioideae, Teloschistaceae), is segregated in this article.

The genus Rufoplaca Arup, Søchting & Frödén (subfamily Caloplacoideae, Teloschistaceae) was introduced in 2013 for six species and two additional species have subsequently been described [3-5]. Additionally, to eight species of the genus Rufoplaca, hitherto known from various regions of the Northern Hemisphere, was added one new species found among Dokdo lichens.

The aim of this article was to present legal descriptions of these two taxa of the genera Arthonia and Rufoplaca.

Materials and methods

Taxon sampling

More than 230 lichen specimens were collected in 17 localities of Dokdo Islands, the Republic of Korea in September 2017. The Dokdo specimens, as well as previous collections included in comparative studies and kept in the KoLRI and other herbaria (BP, KW-L, LE, LWG, and VBI), were examined using standard microscopic techniques and hand-sectioned under a dissecting microscope (Nikon SMZ 645; Nikon, Tokyo, Japan). Anatomical descriptions were based on observations of these preparations under a microscope (Nikon Eclipse E200; Nikon, Tokyo, Japan, and Zeiss Scope, A1; Carl Zeiss, Oberkochen, Germany) with digital camera AxioCam ERc 5s. A section of apothecia was tested with water and with potassium (K) and iodine-potassium iodide (IKI) (10% aqueous potassium iodide) for identification [4,5]. Well-preserved and freshly collected lichen specimens lacking any visible symptoms of fungal infection were selected for DNA isolation. Although not possible for all taxa, an attempt was made to achieve a sample series with at least two specimens of each newly sequenced species, preferably from different localities, to ensure species determination by avoiding contamination through unintentional sampling of a wrong individual or sequencing errors.

A total of 11 new sequences (6 nrITS, 2 mtSSU, and 3 RPB2 sequences) from 8 specimens belonging to 3 species in Dokdo and Ulleung-do islands (Table 1), as well as approximately 150 sequences from GenBank (27 ITS nr DNA, 71 mtSSU, and 78 RPB2), were used for phylogenetic analysis.

Table 1.

Species of the Arthoniaceae and the Caloplacoideae of the Teloschistaceae included in the phylogenetic analyses and the GenBank accession numbers of the sequences. New sequences generated are indicated in bold.

Species name	Voucher specimen/reference	ITS	12S mt SSU	RPB2
1. Alyxoria ochrocheila	Ertz et al. [6]		EU704073	EU704035
2. Alyxoria ochrocheila	Ertz et al. [6]		EU704072	EU704037
3. Alyxoria ochrocheila	Ertz et al. [6]		EU704071
4. Alyxoria varia	Frisch et al. [7]		KJ851006	KJ851147
5. Alyxoria varia	Frisch et al. [7]		KF707642	KF707664
6. Alyxoria varia	Miadlikowska (unpubl.)		KT232221
7. Alyxoria varia	Ertz et al. [6]		EU704075	EU704039
8. Alyxoria varia	Schoch et al. [8]		FJ772243
9. Arthonia apatetica	Frisch et al. [7]		KJ850994	KJ851126
10. Arthonia apotheciorum	Frisch et al. [7]		KJ850970	KJ851148
11. Arthonia biatoricola	Frisch et al. [7]		KJ850990	KJ851149
12. Arthonia calcarea	Ertz et al. [6]		EU704065	EU704028
13. Arthonia calcarea	Ertz et al. [6]			EU704029
14. Arthonia calcarea	Frisch et al. [7]		KJ850974
15. Arthonia didyma	Ertz et al. [6]		EU704047
16. Arthonia didyma	Frisch et al. [7]		KJ851106
17. Arthonia dokdoensis	SK L06, South Korea, Dokdo Islands, 07.09.2017 J. J.Woo 171029 (KoLRI 045310)	171029	171029	171029
18. Arthonia dokdoensis	SK L05, South Korea, Dokdo Islands, 07.09.2017 J. J.Woo 171032 (KoLRI 045313)			171032
19. Arthonia dokdoensis	SK L04, South Korea, Dokdo Islands, 07.09.2017 J. J.Woo 171034 (KoLRI 045315)		171034	171034
20. Arthonia granitophila	Frisch et al. [7]		KJ850981	KJ851107
21. Arthonia incarnata	Frisch et al. [9]		KY983975	KY983983
22. Arthonia incarnata	Frisch et al. [9]		KY983976	KY983984
23. Arthonia lapidicola	Frisch et al. [7]		KJ850997	KJ851119
24. Arthonia lobariellae	Frisch et al. [7] as Arthonia sp. “lobariicola”		KJ851001	KJ851127
25. Arthonia lobariellae	Frisch et al. [7] as Arthonia sp. “lobariicola”		KJ851002	KJ851128
26. Arthonia molendoi	Frisch et al. [7]		KJ851000	KJ851117
27. Arthonia neglectula	Frisch et al. [7]		KJ850989	KJ851118
28. Arthonia peltigerina	Frisch et al. [7]		KJ850998	KJ851122
29. Arthonia phaeophysciae	Frisch et al. [7]			KJ851112
30. Arthonia physcidiicola	Frisch et al. [7]		KF707646	KF707657
31. Arthonia radiata	Ertz and Tehler [10]			HQ454651
32. Arthonia radiata	Tehler and Irestedt [11]			EF081373
33. Arthonia radiata	Ertz et al. [6]		EU704048	EU704011
34. Arthonia radiata	Frisch et al. [7]			KJ851109
35. Arthonia stereocaulina	Frisch et al. [7]		KJ850999
36. Arthonia subfuscicola	Frisch et al. [7]		KJ850971	KJ851111
37. Arthonia subfuscicola	Frisch et al. [7]		KJ850972
38. Brigantiaea ferruginea	SK 780; Kondratyuk et al. [12]	KF264623	KF264685
39. Brigantiaea ferruginea	SK 779; Kondratyuk et al. [12]	KF264622	KF264684
40. Bryostigma muscigenum	Frisch et al. [7]		KJ850991	KJ851124
41. Caloplaca areolata	SK 714; Kondratyuk et al. [13]
42. Caloplaca cerina	FNM 185; Fedorenko et al. [14]	EU681284	EU680863
43. Caloplaca stillicidiorum	Gaya et al. [15]	KT291510		KT291649
44. Oxneria huculica	FNM 199; Fedorenko et al. [14]		EU680931
45. Oxneria huculica	Gaya et al. [16]	JQ301687		JQ301776
46. Oxneria ulophyllodes	FNM 198; Fedorenko et al. [14]	EU691342	EU680930
47. Oxneria ulophyllodes	Gaya et al. [16]			JQ301779
48. Rufoplaca arenaria	Arup et al. [3]	KC179455
49. Rufoplaca arenaria	Halici et al. [17]	KF007908
50. Rufoplaca arenaria	Vondrák and Malicek [18]	KT934385
51. Rufoplaca kaernefeltiana	South Korea, Ulleung-do Island, Dodong Port, 11.07.2016 Kondratyuk S. Y. & L. Lőkös 162024 (KoLRI 040262)	162024
52. Rufoplaca kaernefeltiana	South Korea, Ulleung-do Island, Dodong Port, 11.07.2016 Kondratyuk S. Y. & L. Lőkös 162044 (KoLRI 040282)	162044
53. Rufoplaca oxfordensis	Arup et al. [3]	KC179456
54. Rufoplaca scotoplaca	Gaya et al. [16]	JQ301618	JQ301497	JQ301756
55. Rufoplaca scotoplaca	Arup et al. [3]	KC179457	KC179573
56. Rufoplaca sp. 43	Arup et al. [3]	KC170458	KC179574
57. Rufoplaca sp. JV 14429	JV 14429, Vondrák et al. [19]	KU926986
58. Rufoplaca sp. JV 18681	Vondrák et al. (unpubl.)	MG954203
59. Rufoplaca sp. D 17245	Vondrák et al. (unpubl.)	MG954209
60. Rufoplaca subpallida	Arup et al. [3]	KC179459
61. Rufoplaca toktoana	SK L03, South Korea, Dokdo Islands, 07.09.2017 J.-J.Woo 171044 (KoLRI 045325)	SK L03
62. Rufoplaca toktoana	SK L07, South Korea, Dokdo Islands, 07.09.2017 J.-J.Woo 171045 (KoLRI 045326)	SK L07
63. Rufoplaca toktoana	SK L08, South Korea, Dokdo Islands, 07.09.2017 J.-J.Woo 171045 (KoLRI 045326)	SK L08
64. Rufoplaca toktoana	162040, South Korea, Ulleung-do Island, Dodong Port, 11.07.2016 Kondratyuk S. Y. & L. Lőkös 162040 (KoLRI 040278)	162040
65. Rufoplaca tristiuscula	Arup et al. [3]	KC179460	KC179575
66. Xanthoria coomae	Arup et al. [3]	NR_120253
67. Xanthoria mediterranea	Fedorenko et al. [14]	EU681297
68. Xanthoria monofoliosa	SK 3191, Fedorenko et al. [20]	JN984136
69. Xanthoria parietina	FNM 177; Fedorenko et al. [14]	EU681297

Molecular data

Genomic DNA was isolated from lichen specimens using the CTAB extraction protocol [21]. For some tiny crustose species (e.g., Arthonia or Rufoplaca), contaminations with co-occurring fungi are frequent when using standard DNA isolation protocols on large parts of the lichen thalli. To avoid such contamination, hand-made sections of the hymenium or the thallus were used for direct polymerase chain reaction (PCR) [22]. Any pigmented or crystal-encrusted portions were removed with a razor blade. In addition, the lichen material was sometimes washed with acetone or a 1% KOH solution, and then rinsed with water to remove remnants of pigments.

The material was then added to a tube containing the PCR reaction mixture and amplified directly. Amplification reactions were prepared for a 50 µL final volume containing 5 µL 10× DreamTaq Buffer (Fermentas, Waltham, MA), 1.25 µL of each of the 20 µM primers, 5 µL of 2.5 mg mL−1 bovine serum albumin (Fermentas #B14), 4 mL of each of the 2.5 mM dNTPs (Fermentas), 1.25 U DreamTaq DNA polymerase (Fermentas) and 1 µL of template genomic DNA or tiny fragments of lichen material.

The nuclear ribosomal RNA gene region including the internal transcribed spacers 1 and 2 and the 5.8S subunit (ITS) was amplified using the primers ITS1F [23] and ITS4 [24], the 28S LSU using the primer LR5 [25], and the 12S mtSSU using the primers mtSSU1-mtSSU3R and mtSSU2R [14,20]. Methods of DNA extractions, data on primers, and phylogenetic analysis are provided in our previous article [26].

A fragment of about 1 kb of the RPB2 protein-coding gene was amplified using primers fRPB2-7cF and fRPB2-11aR [27]. The yield of the PCRs was verified by running the products on a 1% agarose gel using ethidium bromide. The amplicons were sequenced by Macrogen® using the amplification primers. Two additional primers, RPB2-2488F and RPB2-2492R [28], were used for sequencing RPB2. See also Park et al. [29] and Kondratyuk et al. [26] for extractions, amplifications, and sequencing procedures.

Sequence fragments were assembled with Sequencher version 4.6 (Gene Codes Corporation, Ann Arbor, MI). Sequences were subjected to MEGABLAST searches to verify their closest relatives and to detect potential contaminations.

Phylogenetic analyses

The NucITS, mtSSU, and RPB2 sequences for taxa listed in Table 1 were aligned manually using MacClade version 4.05 (Sunderland, MA) [30].

A conflict was assumed to be significant if two different relationships (one being monophyletic and the other being non-monophyletic) for the same set of taxa were both supported with bootstrap values ≥70% [31].

The mtSSU and RPB2 datasets were concatenated. The combined two-locus dataset consisted of 180 terminals and 1777 unambiguously aligned sites: 856 for the mtSSU and 921 for RPB2. Bayesian inference, maximum likelihood, and parsimony were used to estimate the phylogeny of Arthoniaceae based on a concatenated sequence matrix of the two loci. For the Bayesian and the maximum likelihood analyses, the best fit model for the two loci, as well as for the codon positions in the RPB2 gene, were calculated by applying the Akaike Information Criterion [32] and the program MrModeltest version 2.2 (Uppsala, Sweden) [33] in conjunction with PAUP* [34]. The prior selection of substitution models supported the GTR + I+Γ model for both the two individual loci, as well as for each codon position in RPB2. In the Bayesian analysis, the dataset was analyzed in four partitions, mtSSU and by codon positions for RPB2. Posterior probabilities of trees and parameters in the substitution models were approximated with MCMC and Metropolis coupling using the program MrBayes version 3.2.1 (Uppsala, Sweden) [35]. In parsimony analysis, the concatenated dataset was analyzed using the same settings as those used for testing the topological incongruence. The phylogenetic tree of the Arthoniaceae obtained from parsimony analysis based on the concatenated mtSSU and RPB2 sequences as the most illustrative one was included in the article (Figure 1).

Figure 1.

Position of the newly described Arthonia dokdoensis in the phylogenetic tree of the Arthoniaceae obtained from parsimony analysis based on concatenated mtSSU and RPB2 sequences.

Three outgroup species Oxneria ulophyllodes, Oxneria Alfredii, and O. huculica were chosen for the Arthoniaceae tree, but Brigantiaea ferruginea for the Rufoplaca phylogenetic tree. These taxa were used as the rooting taxa in all the analyses. In total, the dataset for the multilocus phylogenetic tree included 141 sequences and ca. Hundred specimens representing approximately 70 species, while the final tree presented in this article included 38 specimens representing 23 species.

Results and discussion

New taxa

(Figure 2)

Figure 2.

Arthonia dokdoensis (holotype), general habit. Scale 2 mm (top, with enlarged section of apothecium, scale 50 µm and enlarged portion of hymenium, scale 10 µm) and 500 µm (bottom, with enlarged ascospores, scale 10 µm).

MycoBank No.: MB 831133.

This species is similar to A. parietinaria but differs in causing much smaller infection spots, and in having smaller ascomata, a lower mean number of ascomata per infection spot, more common conidiomata, and bacilliform conidia.

Type: Republic of Korea, Gyeongsangbuk-do, Ulleung-gun, Dokdo-ri, Seodo (= Western) Island, on rocks, growing on thalli of Orientophila yokjidoensis, growing together with Polyozosia aff. dispersa and Orientophila dodongensis. Lat.: 37° 14′ 27″ N, Long.: 131° 51′ 54″ E, Alt.: 100 m a.s.l. Coll:. Woo, J. J. (171028), 07.09.2017 (KoLRI 045309 sub Polyozosia – holotype); the same locality, growing on thalli of O. yokjidoensis, growing together with Physciella aff. melanchra and Lecanora sp. Coll.: Woo, J. J. (171029), 07.09.2017 (KoLRI 045310 sub O. yokjidoensis – holotype); the same locality, growing on thalli of O. yokjidoensis, growing together with Polyozosia aff. dispersa. Coll.: Woo, J. J. (171030), 08.09.2017 (KoLRI 045311 sub Polyozosia – isotype); the same locality, growing on thalli of O. yokjidoensis, growing together with Polyozosia aff. dispersa, Physciella aff. melanchra and Diplotomma alboatra. Coll.: Woo, J. J. (171032), 08.09.2017 (KoLRI 045313 sub Polyozosia – isotype); the same locality, growing on thalli of O. yokjidoensis, growing together with Polyozosia aff. dispersa, Physciella aff. melanchra, Buellia ulleungdoensis and Lecanora sp. Coll.: Woo, J. J. (171034), 08.09.2017 (KoLRI 045315 sub Polyozosia – isotype); the same locality, growing on thalli of O. yokjidoensis, growing together with Polyozosia aff. dispersa, Physciella aff. melanchra, Diploicia canescens, and B. ulleungdoensis. Coll.: Woo, J. J. (171036), 08.09.2017 (KoLRI 045317 sub D. canescens – isotype).

Morphology: Lichenicolous fungus forming very indistinct infection spots (from very indistinct to more or less recognizable to 0.5–1 mm across) in the central areolate portion of lobate lichen O. yokjidoensis, where the peripheral zone of host thalli to 1.5–2.5 mm wide with radially orientated lobes usually not damaged; infection spots often include very small, punctiform, scattered, and distant ascomata or conidiomata.

Ascomata (0.08–)0.1–0.13(–0.14) mm wide, single, round and more or less regular, scattered and distant, and rather inconspicuous, or aggregated in very irregular shape aggregations with 5–10 apothecia together in spots to 0.4–0.6 mm diam./across, often covering one side/portion of the host thalline areole, and better seen. In section, epihymenium olivaceous brown to blackish brown, 4.6–8 µm thick; hymenium 32–40 µm high, in the middle and lower portions more or less hyaline; interascal hyphae branched and anastomosing ca. 2 µm wide; subhymenium (48–)56–64(–80) µm thick, hyaline or light brown; asci clavate, 8-spored; ascospores hyaline (0–)1-septate; lower cell slightly attenuated, 9.6–12.8 × 4–4.8 µm (42 measurements).

Conidiomata very often observed below ascomata and probably especially numerous at first stages of infection development; conidia bacilliform, 3–4 × 0.8 µm.

Ascomatal gel I + red; KI + blue; asci with KI + blue ring-structure.

Ecology: The species grows in the crustose central portion of thalli of lobate lichens O. yokjidoensis and O. dodongensis growing on siliceous rock.

Etymology: It is named after the type locality, namely Dokdo Islands, Republic of Korea, Eastern Asia.

Distribution: It is so far known only from the type collection in Dokdo Islands, Republic of Korea, Eastern Asia, where it is rather abundant.

Taxonomic notes: The lichenicolous fungus A. dokdoensis usually damages the central portion to 0.5–1 mm across, while it hosts thalli to 7–8 mm across with peripheral zone to 1.5–2.5 mm wide with radially orientated lobes, which are usually not damaged by lichenicolous fungi.

Very small ascomata (ca. 80–120 µm in diam. at first) are rather scattered and distant, and are very barely noticeable. Lichenicolous fungus is usually better distinguished when aggregated ascomata form irregular, often confluent aggregations to 0.4–0.5 mm wide. At this stage, ascomata of lichenicolous fungus often entirely cover the areoles of the central portion of host thalli. The A. dokdoensis infection on thalli of O. yokjidoensis can be most easy to be recognized at the latest stage.

Sometimes numerous brown hyphae with rounded cells to 4–4.8 µm wide are also observed in host thalli damaged by A. dokdoensis. On the other hand, they probably belong to another lichenicolous fungus.

Additional specimens examined: Republic of Korea, Gyeongsangbuk-do, Ulleung-gun, Dokdo-ri, Seodo (= Western) Island, on rock, growing on thalli of O. yokjidoensis growing together with Buellia halonia, and Lecanora sp. Lat.: 37° 14′ 29.04″ N, Long.: 131° 51′ 51.4″ E, Alt.: 20–25m a.s.l. Coll.: Park, J. S. (170860), 07.09.2017 (KoLRI 045141 sub B. halonia); Dongdo Island, on rock, growing on thalli of O. yokjidoensis, growing together with O. dodongensis, Polyozosia aff. Dispersa, and Physciella aff. melanchra. Lat.: 37° 14′ 21.61″ N, Long.131° 52′ 5.71″ E, Alt.: 12 m a.s.l. Coll.: Oh, S. O. (171086), 08.09.2017 (KoLRI 045367 sub Polyozosia aff. dispersa); Seodo Island, near the top level of the trail, on rock, growing on thalli of O. yokjidoensis growing together with Myriolecis aff. dispersa, B. ulleungdoensis and Physciella aff. melanchra. Lat.: 37° 14′ 30.02″ N, Long.: 131° 51′ 50.44″ E, Alt.: 25 m a.s.l. Coll.: Lee, B. G. (170928), 07.09.2017 (KoLRI 045209 sub O. yokjidoensis); Seodo Island, on rocks, growing on thalli of O. yokjidoensis, growing together with Rufoplaca toktoana. Lat.: 37° 14′ 27″ N, Long.: 131° 51′ 54″ E, Alt.: 100 m a.s.l. Coll.: Woo, J. J. (171044), 07.09.2017 (KoLRI 045325 sub R. toktoana).

(Figure 3)

Figure 3.

Rufoplaca toktoana (holotype), general habit with enlarged section of apothecium (top, scale 25 µm) and with enlarged ascospores (bottom, scale 25 µm). Scale 500 µm.

MycoBank No.: MB 825110.

Similar to Rufoplaca kaernefeltiana but different in having well distinct and much larger thallus, thinner and K − cortical layer of thallus, larger and biatorine-like apothecia, lower hymenium and narrower paraphysis tips and shorter and narrower ascospores, and mainly hardly visible and narrower ascospore septum.

Type: Republic of Korea, Gyeongsangbuk-do, Ulleung-gun, Dokdo-ri, Seodo Island, on rocks, growing together with O. yokjidoensis damaged by A. dokdoensis, Lecanora sp. and Physciella sp. Lat.: 37° 14′ 27″ N, Long.: 131° 51′ 54″ E, Alt.: 100 m a.s.l. Coll: Woo, J. J. (171044), 07.09.2017 (KoLRI 045325 – holotype of R. toktoana); the same locality, growing together with O. yokjidoensis, Coll: Woo, J. J. (171045), 07.09.2017 (KoLRI 045326 – isotype of R. toktoana).

Morphology: Thallus rather thick, areolate to continuous, whitish gray, or grayish-white; apothecia seem to be biatorine, orange, or somewhat reddish-orange. Thalline areoles (0.5–)1.5–2.5 mm across. Thallus in section to (64–)80–160(–280) µm thick, cortical layer 8–11.2(–12.8)[-24] µm thick, very thin, paraplectenchymatous, cell lumina rounded to 4.8 µm in diam.; algal zone filling in the whole thallus below cortical layer to 80(–96) µm thick; algal cells trebouxioid, 16–19.2(–22.4) µm in diam.

Apothecia to 0.9–1 mm in diam. and 0.2–0.27 mm thick in section, seem to be biatorine, while lecanorine or zeorine in section; 1–3(–5) per areole; in section thalline exciple to 80–96(–112) µm thick, cortical layer not distinct or very thin, to 8–16 µm thick, better seen on underside, paraplectenchymatous; true exciple (56–)80–96(–112)[-144] µm wide in the uppermost lateral portion, more or less scleroplectenchymatous, hyphal lumina to 1.6 µm, and to 16–32(–48) µm thick in lower lateral and to 16 µm thick in the basal portion, more or less Blastenia-type in the latter two portions or scleroplectenchymatous; hymenium 64–72 µm high, epihymenium 8–11.2 µm thick, dark brown; paraphysis tips more or less brownish, richly branched to 2.4–4 µm in diam. in K brownish color disappearing; subhymenium (48–)80–96 µm thick, hyaline, without oil; asci 8-spored, but usually only simple ascospore seen (in K too); ascospores narrowly ellipsoid, mainly simple observed, sometimes becoming slightly darker or slightly brownish (9.6–)11.2–12.8(–14.4)×3–4.8 µm in water (45 measurements) and 11.8–13.4(–14.4)×3.6–3.9(–4.5) µm in K (37 measurements); septum very rarely observed, usually seen only at sides of equatorial portions in water to (0.5–)1–2.4 µm wide in water and better seen in K (0.5–)1.5–2(–2.5) µm thick. Conidiomata and conidia not observed.

Chemistry: Epihymenium K + crimson-purple, while brownish color disappearing. Cortical layer of thalline exciple K + purple only in the uppermost lateral portion. Cortical layer of thallus K−.

Ecology: It grows on siliceous rock in the supralitoral zone.

Etymology: It is named after the type locality, namely Dokdo Islands (in Korean Tokto Islands), Republic of Korea, Eastern Asia.

Distribution: The species is so far known from the type collection Dokdo Islands, as well as Ulleung-do Island, both the Republic of Korea, Eastern Asia, where it was rather abundant in some places.

Taxonomic notes: R. toktoana is similar to R. kaernefeltiana S. Y. Kondr., L. Lőkös et J. S. Hur, recently described from Ulleung-do Island, South Korea (Eastern Asia), but differs in having well distinct and much larger thallus and larger thalline areoles or often forming almost continuous thallus (thalline areoles (0.5–)1.5–2.5 mm vs. (0.2–)0.4–0.8 mm across, usually very indistinct, distant, and scattered), in having thinner and K − cortical layer of thallus (8–11(–13) µm vs. 30–40(–50) µm thick, K + purple), in having larger and biatorine-like apothecia (vs. seem to be lecanorine), in having lower hymenium (65–70 µm vs. 70–90 µm high) and narrower paraphysis tips (2.4–4 µm vs. to 5(–6) µm in diam.), and in having shorter and narrower ascospores ((9.5–) 11–13(–14.5)×3–4.8 µm vs. (10–)12–15(–16) × 7–8 µm) and mainly hardly visible and narrower ascospore septum ((0.5–)1–2.4 µm vs. (4–) 5–6(–7) µm wide) [4].

Additional specimens examined: Republic of Korea, Gyeongsangbuk-do, Ulleung-gun, Dokdo-ri, Seodo Island, on rocks, growing together with Lecanora cf. campestris. Lat.: 37° 14′ 27″ N, Long.: 131° 51′ 54″ E, Alt.: 100 m a.s.l. Coll.: Woo, J. J. (171042), 07.09.2017 (KoLRI 045323 sub Lecanora cf. campestris); Republic of Korea, Gyeongsangbuk-do, Ulleung-gun, Dokdo-ri, Seodo Island, on rocks, growing together with Caloplaca dodongensis. Lat.: 37° 14′ 26.66″ N, Long.: 131° 51′ 51.50″ E, Alt.: 20 m a.s.l. Coll.: Lee, B. G. (170909), 07.09.2017 (KoLRI 045190 sub R. toktoana); the same locality, growing together with B. ulleungdoensis, Coll.: Lee, B. G. (170910, 170911), 07.09.2017 (KoLRI 045191, KoLRI 045192 sub R. toktoana); Republic of Korea, Gyeongsangbuk-do, Ulleung-gun,Ulleung-eup, Dodong-ri, Dodong Port, on siliceous rocks. Lat.: 37° 28′ 59.9″ N, Long.: 130° 54′ 40.7″ E, Alt.: 20 m a.s.l. Coll.: Kondratyuk, S. Y., Lőkös, L. (162040), 11.07.2016 (KoLRI 040278).

Discussion

A. dokdoensis is similar to the recently described A. parietinaria in having ascomata distributed over the surface of the host thallus, including apothecial margins and hymenia, but differs in causing much smaller infection spots (to 0.5 mm across, often rather indistinct, vs. to 3–5 mm in diam.), in having smaller ascomata (80–120(–140) µm vs. up to 0.25 mm in diam.), in having lower mean number of ascomata per infection spot (to 5–10 vs. (10–)20–30(–50)), in having more common conidiomata being often aggregated, while ascomata rarely observed (vs. ascomata well developed while conidiomata usually indistinct), and in having bacilliform conidia (vs. ellipsoid; unfortunately measurements on the conidia of A. parietinaria were not provided in the original description)[1] (Table 2).

Table 2.

Comparison of morphological/anatomical characters of Arthonia dokdoensis, A. parietinaria and A. molendoi.

Characters	Arthonia dokdoensis	Arthonia parietinaria	Arthonia molendoi
Infection spots	to 0.5 mm across, often rather indistinct	to 3–5 mm in diam.	to 3–5 mm in across
Mean number of ascomata per infection spot	to 5–10	(10–)20–30(–50)	1–5(−10)
Ascomata (in diam.)	(80–120(–140) µm	up to 0.25 mm	0.1–0.24 mm
Hymenium (µm high)	32–40	30–45	45–50
Subhymenium (µm thick)	64–80	[data not provided in the original description][1]	50–60 µm
Ascospores (µm)	9.6–12.8 × 4–4.8, hyaline	(9-)10–12(−13.5)×(3−)4–5(−6), hyaline with thin hyaline perispore (getting condensed and brownish with age)[1]	11–14 × 5–6.5, slightly pigmented with age
A gelatinous epispore	absent		present
Conidiomata	being often aggregated, more common while ascomata rarely observed	usually indistinct while ascomata well developed	pycnidia of Arthonia molendoi still not observed after Grube [30]
Conidia	bacilliform	ellipsoid; unfortunately measurements on conidia not provided in the original description)[1]	pycnidia of Arthonia molendoi still not observed after Grube [30]
Hosts	Orientophila spp.	Xanthoria parietina	Rusavskia spp.

A. dokdoensis is similar to A. molendoi but differs in causing much smaller infection spots (to 0.5 mm in diam. often rather indistinct vs. to 3–5 mm diam. across), in having higher mean number of ascomata per infection spot (5–10 vs. 1–5(–10) in A. molendoi), in having smaller ascomata (80–120(–140) µm vs. 0.1–0.24 mm in diam.), in having lower hymenium (32–40 vs. 45–50 µm high) and thicker subhymenium (64–80 µm vs. 50–60 µm thick), in having shorter and narrower ascospores (9.6–12.8 × 4–4.8 µm vs. 11–14 × 5–6.5 µm), as well as being hyaline ascospores (not being slightly pigmented with age), and in having bacilliform conidia (pycnidia of A. molendoi still not observed after Grube [36]), as well as in the lack of a gelatinous epispore [36].

Unfortunately, a relatively recent full description of A. molendoi published only in Grube [36] is still incomplete. Fleischhacker et al. [1] also provided some data on the diagnostic characters of A. molendoi. Unfortunately, data on some measurements of ascomata, subhymenium, and shape or morphology of conidia are still missing.

Thus far, all species of the A. molendoi aggregation have been recorded from the members of the subfamily Xanthorioideae of the Teloschistaceae (see [2]), i.e., genera Rusavskia (type host of A. molendoi s. str.), Xanthoria (type host of A. parietinaria), Orientophila (type host of A. dokdoensis) and Calogaya. On the other hand, material of A. molendoi previously recorded from members of the genus Calogaya is in urgent need of revision and may belong to another taxon.

The data confirm the conclusion of Grube [36] in that careful studies of the A. molendoi complex are still needed in the future to determine if specimens on different lineages in Caloplaca and Xanthoria belong to the same species.

Arthonia destruens differs from taxa of the A. molendoi aggr. in having permanently brownish ascospores at overmaturity.

Fleischhacker et al. [1] segregated Ar. parietinaria growing on the members of the Xanthoria s. str. from the A. molendoi complex, which was believed to be foliose lichens of the genera Xanthoria and Rusavskia, as well as crustose lichens of the genus Calogaya (the former Caloplaca saxicola group). After segregation of A. parietinaria from the complex mentioned, A. molendoi is confirmed to Rusavskia elegans (type host) and to other species of the genus Rusavskia, as well as of the genus Calogaya.

The suggestion about the worldwide distribution of A. parietinaria and Holarctic is somewhat doubtful given that the host lichen species Xanthoria parietina itself is confirmed only from a few collections outside Europe and the Mediterranean region. Most records of Xanthoria parietina from outside Europe belong to other taxa (Kondratyuk, in prep.) and even to various genera of the Teloschistaceae in current stage (see [2]). Fleischhacker et al. [1] hesitated to use modern generic groups of the Teloschistaceae, but the generic groups Jackelixia, Rusavskia, Massjukiella, and Oxneria, recently received further confirmation [2,37].

Unfortunately, the diagnostic character of the A. molendoi complex, such as fast vine red reaction of hymenium (in contrast to I + blue then red in case of Arthonia destruens or Arthonia incarnata), was not discussed in the description of A. parietinaria [1].

Initially, close relations of the Korean material of A. dokdoensis to Arthonia patellaria (ITS sequences FR799123, FR799124) were found after ITS phylogeny of the members of the Arthoniaceae. On the other hand, only very limited data exists on the ITS nrDNA sequences of the family Arthoniaceae, while phylogeny of this family and the entire Arthoniales is built mainly on nrLSU, mtSSU, and RPB2 sequences. An attempt was made to obtain the three sequences mentioned above. On the other hand, only the mtSSU and RPB2 sequences for the new taxon could be obtained.

A separate mtSSU and RPB2 analysis (not shown) revealed that A. molendoi is positioned in a separate branch from the Arthonia s. str. branch. The same results were obtained in the combined phylogenetic analysis based on concatenated mtSSU and RPB2 sequences (Figure 1).

It was firstly found in this study that the A. molendoi group is positioned within the Bryostigma clade of the combined phylogenetic tree of the Arthoniaceae based on concatenated mtSSU and RPB2 sequences. The Bryostigma clade includes the genus Bryostigma Poelt et Döbbeler with type species Bryostigma muscigenum (Th. Fr.) Frisch et G. Thor, as well as members of the A. molendoi group [38]. We agree with previous authors [9] that there is probably more than one generic group within the Bryostigma clade. Positions of seven of ten taxa of the Bryostigma clade are confirmed by combined mtSSU and RPB2 sequences (Figure 1), as well as by nrLSU phylogeny [1].

A. dokdoensis, together with the following two species, i.e., Arthonia phaeophysciae and A. parietinaria, for which molecular data were provided, are the members of the A. molendoi complex. Unfortunately, only data on nrLSU sequences of A. parietinaria were recorded and illustrated in phylogenetic tree by Fleischhacker et al. [1] from this species of the A. molendoi complex. These data could not be obtained from GenBank within this study. A. epiphyscia, the position of which should be confirmed by molecular data in the future, is highly likely to be a member of the Bryostigma molendoi complex. These taxa, which are characterized by lichenicolous habit, as well as numerous ascomata forming very characteristic aggregations in host thallus, may be segregated in the future in a separate genus. On the other hand, only insufficient data exist on differences of lichenicolous taxa and lichen-forming fungi of the Bryostigma s. l. clade, as well as epibryophilous Bryostigma muscigenum (Th. Fr.) Frisch et G. Thor itself (i.e., the genus Bryostigma s. str.).

These results provide further evidence for the still incomplete understanding of the character evolution in Arthonia s. l. and the relevance of morphological characters used for the delimitation of genera and species groups in Arthoniaceae, as was previously shown, e.g., by the studies of Frisch and Thor [39], Frisch et al. [40,41], and Aptroot et al. [42]. Given the combination of morphological characters presented above, in addition to the isolated position on the phylogenetic tree in Figure 1, A. incarnata appears to be a rather common species in old-growth forests in Japan (collected only once in Korea) [9], and cannot be connected easily with any of the generic names currently accepted in the synonymy of Arthonia [43]. At the current state of knowledge and with a proper revision of Arthonia s. l. still pending, this species should be in Arthonia for the time being instead of describing it for another poorly monotypic genus.

R. toktoana is also similar to the recently described Rufoplaca ulleungensis S. Y. Kondr., L. Lőkös et J. S. Hur, from Ulleung-do Island, South Korea (Eastern Asia), but differs in having much better developed and often almost continuous thallus (vs. rather indistinct and consisting of distant and scattered areoles), in having larger apothecia (to 0.9–1 mm vs. (0.2–)0.3–0.8 mm diam.), and in having shorter and narrower ascospores ((9.5–) 11–13(–14.5)×3–4.5 µm vs. 14–16(–18) × 4–5.5(–6) µm), while ascospore septum ((0.5–)1–2.4 µm vs. 1.5–2(–2.5) µm wide) is similar [5] (Table 3).

Table 3.

Comparison of morphological/anatomical characters of Rufoplaca toktoana, Rufoplaca kaernefeltiana, and Rufoplaca ulleungdoensis.

Characters	Rufoplaca toktoana	Rufoplaca kaernefeltiana	Rufoplaca ulleungdoensis
Thallus	well distinct and large	usually very indistinct	rather indistinct and consisting of distant and scattered areoles
Thalline areoles mm across	(0.5–)1.5–2.5, well distinct or often forming almost continuous thallus	(0.2–)0.4–0.8, usually very indistinct, distant and scattered	0.3–0.7(–1.3), distant and separate to aggregated in small groups
Cortical layer of thallus (µm thick)	8–11(–13) and K−	30–40(–50), K + purple	7–8 and K–
Apothecia	to 0.9–1 mm, biatorine-like	0.3–0.7 mm, seem to be lecanorine	(0.2–)0.3–0.8 mm, often in groups, biatorine
Hymenium (µm high)	65–70	70–90	70–80
Paraphysis tips (µm in diam.)	2.4–4	to 5(–6)	to 4
Ascospores (µm)	(9.5–) 11–13(–14.5)×3–4.5	(10–)12–15(–16)×7–8	14–16(–18)×4–5.5(–6)
Ascospore septum(µm wide)	(0.5–)1–2.4, mainly hardly visible	(4–) 5–6(–7)	1.5–2(–2.5)

R. toktoana is similar to “Caloplaca” fraudans (Th. Fr.) H. Olivier growing on sea coastal rock, rarely on wood or bones, in Arctic regions of the Holarctic, but differs in having well distinct, thick thallus, in having concave or plane light reddish orange apothecia (vs. convex, dark orange, or rusty red), in having rather thin own margin and concolorous with apothecium disc (vs. to 0.1–0.2 mm wide slightly shiny and lighter of disc, bright orange, or yellow), in having thinner subhymenium (vs. 60–100(–130) µm thick), in having lower hymenium (65–70 µm vs. 85–100 µm high), in having shorter and narrower ascospores ((9.6–) 11–13(–14.5)×3–4.8 µm vs. (10–)12–14(–15)×4–6 µm), and in having slightly narrower (or mainly undeveloped) ascospore septum ((0.5–)1–2.4 µm vs. 2.5–4.5 µm wide) [44].

R. toktoana is similar and can be keyed to Caloplaca erythrocarpa (Pers.) Zwackh growing on limestones, sandstones enriched by calcium in Europe, the Caucasus, Asia (Syria, Israel, Jordania, and Egypt), and North Africa, but differs in having larger apothecia (0.9–1 mm vs. 0.2–0.5(–0.8) mm in diam.), in having biatorine, but lecanorine or zeorine in section apothecia (vs. zeorine), in having superficial (vs. seem to be immersed) apothecia; in having irregular and larger thalline areoles (0.5–1(–1.5) mm vs. 0.2–0.5(–1) mm across), in having 1 apothecium per areole (vs. (1–)2–3 apothecia per areoles in peripheral and 1(–2) per areoles in the center of thallus); in having dull orange or dull reddish orange disc (vs. dark red, dark rusty red, to dark rusty brown), in having weakly developed, seen at sides or on underside thalline margin (vs. own margin lighter of disc, red-orange, thin, and permanent), in having narrower and shorter ascospores ((9.6–) 11–13(–14.5)×3–4.8 µm vs. 12–16(–18)×(5–)7–9(–10) µm, and in having narrower ascospore septum ((0.5–)1–2.4 µm vs. 3–5 µm wide [40].

Twenty-five sequences are available for members of the genus Rufoplaca in GenBank. The main portion of data was provided by Arup et al. [3], while a few specimens were added by J. Vondrák et al. [17-19]. On the other hand, after ITS phylogeny, the Eastern Asian material, i.e., R. toktoana and R. kaernefeltii, for which molecular data are for the first time provided in this article, form a separate branch within the genus Rufoplaca (Figure 4). Both Eastern Asian taxa have the highest level of bootstrap support. Therefore, the material of both taxa mentioned is rather homogenous from the molecular point of view.

Figure 4.

Phylogenetic tree of the genus Rufoplaca based on ITS nrDNA showing position of the new species R. toktoana and the recently described Rufoplaca kaernefeltiana.

Conclusion

The species diversity of the Bryostigma s. lat. clade includes 13 species based on a combined phylogenetic analysis of the Arthoniaceae based on mtSSU and RPB2 gene sequences. Data on ITS nrDNA sequences are provided herein for the first time for A. dokdoensis, R. kaernefeltiana, and R. toktoana.