Arthonia ulleungdoensis, a New Lichenized Fungus from Ulleung Island, South Korea

Arthonia ulleungdoensis Lee & Hur is described as a new lichen species from South Korea. The new species is distinguishable from Arthonia ruana A. Massal. by its large, rounded and non-punctiform apothecia, taller apothecial section, asci with fewer spores, and larger and permanently colorless spores. Molecular analyses employing mitochondrial small subunit (mtSSU) and RNA polymerase subunit II (RPB2) sequences strongly support Arthonia ulleungdoensis as a distinct species in the genus Arthonia. Overall, 22 Arthonia species are currently recorded in South Korea. A surrogate key is provided to assist in the identification of all 10 taxa of Arthonia/Arthothelium with muriform spores in Northeast Asia.


Introduction
The genus Arthonia is one of the least explored species in lichen taxonomy although the genus is comprised of about five hundred species worldwide [1]. One of the main reasons for this challenging genus is its 'paraphyletic origin'. The relationship between Arthonia and related genera is unclear, and many Arthonia species have synonyms for Arthothelium or other genera. Molecular phylogeny also showed that Arthonia species are arranged over genera and even families [2]. Another reason for Arthonia is the 'unstable structure' of the species. Many species in Arthonia are highly pioneering. They disperse rapidly but become sterile with a deformed structure after spore discharge and dispersal in a short term. Therefore, although Arthonia species are encountered in the field, they are useless for analysis in many cases by the old, unfilled and barren ascomata with no ascus and spore. The third reason for the demanding genus is a 'poor description' of previous references as old descriptions are insufficient to explain specific characteristics of microlichens in anatomy, such as genus Arthonia particularly. The deficient depiction in the past is due mainly to the poor quality of microscopes, and still discourages a comparison with other species/specimens. Although the genus Arthonia received much less attention in Asia compared to Europe and America, several Asian countries have actively studied Arthonia. In particular, in Asia, India has explored the genus the most since 2000 and dynamic studies such as local detection, new species/records discovery, diversity assessment and ethnolichenological survey for Arthonia were reported [3][4][5][6]. Turkey disclosed new species and records and reported other Arthonia species which were detected locally [7][8][9]. South Korea intently studied Arthonia since 2013 and many new species and records, including lichenized and lichenicolous fungi, were discovered [10][11][12]. For other countries, some new species/records were announced from Japan and three new records of foliicolous Arthonia species were reported recently from China [13][14][15].
This study aimed to describe a new lichenized fungus in the genus Arthonia. A concentrated field study was achieved in Ulleung Island and Pohang, which are an eastern island and seashore region

Morphological and Chemical Analyses
Hand-cut sections were prepared with a razor blade and examined under a stereomicroscope (Nikon SMZ645; Nikon, Tokyo, Japan) and a compound microscope (Nikon Eclipse E200; Nikon, Tokyo, Japan) and imaged using a software program (AxioVison Release 4.8.2; Carl Zeiss, Jena, Germany ) and an Axiocam ERc 5s camera (Carl Zeiss, Jena, Germany) mounted on a Zeiss scope A1 microscope (Carl Zeiss, Jena, Germany). The ascospores were investigated at 1000× magnification in water. The length and width of the ascospores were measured and the range of spore sizes was shown with averages, standard deviation, and number of measured spores. Thin-layer chromatography (TLC) was performed using solvent systems A and C according to standard methods [16].

Isolation, DNA Extraction, Amplification, and Sequencing
Hand-cut sections of ascomata or thallus from all collected specimens were prepared for DNA isolation and DNA was extracted in line with the manufacturer's instructions (Macherey-Nagel, Düren, Germany). Two-way PCR amplification for the mitochondrial small subunit (mtSSU) and RNA polymerase subunit II (RPB2) genes was achieved using Bioneer's AccuPower PCR Premix (Bioneer, Daejeon, Korea) in 20-μL tubes and primers, mrSSU1 and mrSSU3R [17] and fRPB2-7cF and fRPB2-11aR [18], respectively. The PCR thermal cycling parameters used were previously described by Ekman [19]. Sequencing was accomplished by the genomic research company GenoTech (Daejeon, Korea).

Morphological and Chemical Analyses
Hand-cut sections were prepared with a razor blade and examined under a stereomicroscope (Nikon SMZ645; Nikon, Tokyo, Japan) and a compound microscope (Nikon Eclipse E200; Nikon, Tokyo, Japan) and imaged using a software program (AxioVison Release 4.8.2; Carl Zeiss, Jena, Germany) and an Axiocam ERc 5s camera (Carl Zeiss, Jena, Germany) mounted on a Zeiss scope A1 microscope (Carl Zeiss, Jena, Germany). The ascospores were investigated at 1000× magnification in water. The length and width of the ascospores were measured and the range of spore sizes was shown with averages, standard deviation, and number of measured spores. Thin-layer chromatography (TLC) was performed using solvent systems A and C according to standard methods [16].

Isolation, DNA Extraction, Amplification, and Sequencing
Hand-cut sections of ascomata or thallus from all collected specimens were prepared for DNA isolation and DNA was extracted in line with the manufacturer's instructions (Macherey-Nagel, Düren, Germany). Two-way PCR amplification for the mitochondrial small subunit (mtSSU) and RNA polymerase subunit II (RPB2) genes was achieved using Bioneer's AccuPower PCR Premix (Bioneer, Daejeon, Korea) in 20-µL tubes and primers, mrSSU1 and mrSSU3R [17] and fRPB2-7cF and fRPB2-11aR [18], respectively. The PCR thermal cycling parameters used were previously described by Ekman [19]. Sequencing was accomplished by the genomic research company GenoTech (Daejeon, Korea).

Phylogenetic Analysis
All mtSSU and RPB2 sequences were aligned and edited manually using ClustalW in Bioedit (V7.2.5; Carlsbad, CA). The bootstrap values were obtained in RAxML GUI 1.5 beta (Heidelberg, Germany) [20] using the maximum likelihood method with a rapid bootstrap with 1000 bootstrap replications and GTR GAMMA for the substitution matrix. The posterior probabilities were obtained in BEAUti 1.8.0 and BEAST 1.8.0 [21] using the HKY (Hasegawa, Kishino and Yano) method for the substitution model, empirical base frequencies, gamma for the site heterogeneity model, four categories for gamma, and a 1,000,000 Markov chain Monte Carlo chain length with a 10,000-echo state screening and 200 log parameters. Then, the best tree was constructed in TreeAnnotator 1.8.0 [22] with a burn-in of 100, no posterior probability limit, a maximum clade credibility tree for the target tree type, and median node heights. All trees were displayed in FigTree 1.4.2 [23] and edited in Microsoft Paint.

Results and Discussion
A concatenated tree was produced from two different loci, which involved overall 70 sequences (35 sequences for each mtSSU and RPB2) ( Table 1). This concatenation employing mtSSU and RPB2 sequences allows a more comprehensive classification on diverse and comparable Arthonia/Arthothelium taxa to show the position of the new species in molecular phylogeny. The integrated tree shows that the new species is classified in the genus Arthonia ( Figure 2). Arthonia ulleungdoensis is positioned in a highly supported clade with Arthonia didyma Körb., Arthonia granitophila Tr.Fr., Arthonia physcidiicola Frisch & G. Thor, and Arthonia ruana, represented by a bootstrap value of 100 and a posterior probability of 95 for the branch. The new species is separately located in the group without any closely positioned taxa. All Arthothelium taxa including Arthothelium norvegicum Coppins & Tønsberg, Arthothelium spectabile A. Massal., and Arthothelium sp. are situated quite far from the new species in the tree although those taxa are similar to the new species mainly by the spore morphology (i.e., muriform spores). Arthonia ulleungdoensis exhibits important characteristics of the genus Arthonia, particularly A. sect. Arthonia, such as maculate ascomata, olive-brown ascomatal pigments, persistently colorless spores, and photophilous [24], although the epihymenium is brownish without a distinct olive color for the new species. Regarding the above analyses and characteristics for the new species, Arthonia ulleungdoensis is identified as a unique species in both morphology and molecular phylogeny, being positioned in the genus Arthonia. DNA sequences for the new species and Arthonia ruana (in bold) were generated in this study. All others were obtained from GenBank. The species names are followed by GenBank accession numbers and voucher information. mtSSU, mitochondrial small subunit; RPB2, RNA polymerase subunit II; Voucher, voucher information.  Table 1 provides the species related to the specific GenBank accession numbers and voucher information.  Thallus corticolous, crustose, hypophloedal, white to gray, without bleaching; epinecral layer 15-18 µm; medulla indistinct; photobiont trentepohlioid, forming a distinct algal layer, parallel to the substratum, between the epinecral layer and the brown bark layer or under apothecia, often in top layer of the brown bark, 45-54 µm thick, cells irregular, globose to angular, single or in chains, 4.5-6 × 6-9 µm, looking somewhat shrunken and supposed to be a little larger originally. Apothecia rounded, erumpent, black, epruinose, with or without epinecral bark layer on the surface of apothecia, 0.79-2.63 × 0.72-1.97 mm (length x = 1.56, SD = 0.73, width x = 1.32, SD = 0.51, n = 6); bark layer hyaline, 10-15 µm; apothecial section 100-130 µm thick; epihymenium brown to dark brown, 15-20 µm high; hymenium hyaline to light brown, 50-60 µm high, oil droplets near base of hymenium or in hypothecium; hypothecium brown to dirty brown, 25-35 µm high; paraphysoids septate, anticlinally Microorganisms 2019, 7, 0205 6 of 9 arranged, 1-1.5 µm wide, somewhat branched at tips; tips swollen and pigmented, 1.5-2 µm wide. Asci wide to narrow clavate, 2-, 4-or 6-spored, 32-49 × 20-31 µm (n = 5); ascospores hyaline, muriform, 4-to 8-transverse and 0-to 3-longitudinal septa, without a gelatinous sheath, old spores with dark septum, 20.5-31 × 9-13 µm (length x = 25.9, SD = 2.81, width x = 11.4, SD = 2.60, n = 28). Pycnidia not detected.
Distribution and ecology: This species occur on the bark of Acer takesimense. It is currently known by a specimen in the type collection (Ulleung Island, South Korea).