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

Abstract 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.

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, K€ arnefelt, 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, K€ arnefelt & A. Thell, and A. molendoi as a species of the genus Rusavskia S. Y. Kondr. & K€ arnefelt (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 & Fr€ od en (subfamily Xanthorioideae, Teloschistaceae), is segregated in this article.
The genus Rufoplaca Arup, Søchting & Fr€ od en (subfamily Caloplacoideae, Teloschistaceae) was introduced in 2013 for six species and two additional species have subsequently been described [3][4][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.

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 handsectioned 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.

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 mL final volume containing 5 mL 10Â DreamTaq Buffer (Fermentas, Waltham, MA), 1.25 mL of each of the 20 mM primers, 5 mL of 2.5 mg mL À1 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 mL 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 proteincoding 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 V R 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.
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 Ã [34]. The prior selection of substitution models supported the GTR þ IþC 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). 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. 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.
Conidiomata very often observed below ascomata and probably especially numerous at first stages of infection development; conidia bacilliform, 3-4 Â 0.8 mm.
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.
Chemistry: Epihymenium K þ crimson-purple, while brownish color disappearing. Cortical layer of thalline exciple K þ purple only in the uppermost lateral portion. Cortical layer of thallus KÀ.
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.

Discussion
A. dokdoensis is similar to the recently described A. parietinaria in having ascomata distributed over the surface of the host thallus, including apothecial  Table 2).
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 þ 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 D€ obbeler 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 lichenforming 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 oldgrowth 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.
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. Vondr ak et al. [17][18][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.

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.