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First Report of the Lichen Ochrolechia Akagiensis (Ochrolechiaceae, Ascomycota) in Korea

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First Report of the Lichen Ochrolechia Akagiensis (Ochrolechiaceae, Ascomycota) in Korea The Korean Journal of Mycology www.kjmycology.or.kr RESEARCH ARTICLE First report of the lichen Ochrolechia akagiensis (Ochrolechiaceae, Ascomycota) in Korea 1 1 1 2 3 2,* Jung Shin Park , Soon-Ok Oh , Jeong-Jae Woo , Dong Liu , Sook-Young Park , Jae-Seoun Hur 1 Korea National Arboretum, Pocheon 11186, Korea 2 Korean Lichen Research Institute, Sunchon National University, Suncheon 57922, Korea 3 Department of Plant Medicine, Sunchon National University, Suncheon 57922, Korea *Corresponding author: [email protected] ABSTRACT The genus Ochrolechia is a widespread, lichen genus in Korea. Despite being common, little is known about the species diversity and geographical distribution of Ochrolechia. In this study, we detailed the identification procedure of the genus Ochrolechia in a Korean collection and provided the description of each species. Using 104 specimens collected from 2003 to 2017, we identified four species of the genus Ochrolechia via morphological and/or molecular phylogenetic analysis: O. parellula, O. trochophora, O. yasudae and O. akagiensis. Among them, O. akagiensis had not been previously reported in Korea. Moreover, the species identified as O. frigida and O. tartarea in past studies were corrected as O. yasudae and O. parellula, respectively, based on morphological and/or molecular evidence. Phylogenetic analysis using the internal transcribed spacer regions including 5.8S rRNA gene showed that the four species separated clearly, indicating that the morphological identification corresponds to the phylogenetic identification. We provide a taxonomic key for the four species of the genus Ochrolechia. OPEN ACCESS Keywords: ITS region, lichen, new record, Ochrolechia akagiensis pISSN : 0253-651X eISSN : 2383-5249 Kor. J. Mycol. 2019 June, 47(2): 95-104 https://doi.org/10.4489/KJM.20190012 INTRODUCTION Jung Shin Park The genus Ochrolechia is comprised of a common crustose lichen which can be found easily on rocks and/ https://orcid.org/0000-0001-5450-7358 or bark which are exposed to sufficient sunlight in mountainous areas. The genusOchrolechia is distributed Received: May 7, 2019 Revised: June 14, 2019 worldwide ranging from the polar to tropical regions, with a total of 60 species reported worldwide [1-8]. Accepted: June 16, 2019 © 2019 THE KOREAN SOCIETY OF MYCOLOGY. The typical morphology of the genus is a grayish thallus with circular, disc-shaped, and pink apothecia [8]. Regarding the morphology, apothecia and ascospores of the genus Ochrolechia resemble those of the This is an Open Access article distributed under the terms of genus Pertusaria, with a thin layer of asci and ellipsoid ascospores [9]. In chemistry, however, the genus the Creative Commons Attribution Non-Commercial Ochrolechia is known to produce a depside, gyrophoric acid, as a secondary metabolite [9]. License (http: //creativecommons.org/licenses/by- nc/4.0/) which permits unrestricted non-commercial Ochrolechia spp. are identified by morphology, anatomy and chemistry [10]. Based on morphology, use, distribution, and reproduction in any medium, Verseghy [11] divided the genus into four groups: namely tatareae, parellae, harmandii, and upsaliensis. provided the original work is properly cited. The Korean Journal of Mycology 2019 Vol.47 95 Park et al. Later, Brodo renamed the groups as tartarea, parella, africana, and upsaliensis [3]. More recently, phylogenetic studies have been applied in this genus using the ribosomal internal transcribed spacer (ITS) regions. Wei [12] has reported that the genus Ochrolechia is present in the same clade as the genus Circinaria with very low bootstrap support values and is a sister-clade to the genera Varicellaria and Lepra, which are more closely related to one another. In Korea, Moon [13, 14] reported three Ochrolechia species in Mt. Seorak: O. trochophora, O. parellula, and O. yasudae. In addition, O. tartarea and O. frigida were also reported by Zhang [15] and Kondratyuk [16] through a survey of the coastal region of Korea, respectively. Based on these studies, most of the Ochrolechia specimens collected in Korea belong to the tartarea-group, which meet the following criteria: gyrophoric acid in the cortex, disc presence or absence in the margin, epruinose apothecia, and rarely having variolaric acid [10]. In this study, combining newly collected specimens with existing specimens in Korea, we identified all species belonging to the genus Ochrolechia at species level by a morphological, chemical and molecular examination. We also provide a taxonomic key and descriptions in detail. MATERIALS AND METHODS Collection and Morphological characteristics. The specimens were collected and deposited in the Korea Lichen Research Institute (KoLRI). The morphological and anatomical characteristics of the specimens were observed using a dissecting microscope (Nikon SMZ645; Nikon, Tokyo, Japan) and a compound microscope (Olympus BX 50, Olympus, Tokyo, Japan). Zeiss Scope, A1 compound microscope (Carl Zeiss, Oberkochen, Germany) was used to take the microphotographs. Sections of the fruiting bodies were mounted in distilled water to measure the ascomata structure and spore size. A spot test was carried out on the thallus cortex, apothecial disc and apothecial margin, and the color reaction was observed under a compound microscope. Secondary compounds were determined by thin layer chromatography (TLC) in solvent system C (toluene: acetic acid = 85: 15) [17]. DNA extraction and amplification of ITS regions. For molecular analysis, fresh lichen materials were ground with a mini bead-beater-16 (3450 RPM, 115V, 10A, Biospec products) and the grounded lichen materials were applied to extract DNA using a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). The extracted DNA was used to amplify the internal transcribed spacer (ITS) regions. Polymerase chain reaction (PCR) was performed using an AccuPower® PCR Premix (Bioneer, Daejeon, Korea) with initial denaturation for 5 min at 94 C, 30 cycles of 1 min ° denaturation at 94 C, 1 min annealing at 55 C, 1 min extension at 72 C, followed by a final extension ° ° ° for 5 min at 72 C. The PCR products were confirmed by gel electrophoresis purified using an AccuPrep® ° PCR purification kit (Bioneer, Daejeon, Korea) and bi-directionally sequenced on both strands with the The Korean Journal of Mycology 2019 Vol.47 96 Lichen genus Ochrolechia in Korea same primers used for PCR amplification. The ITS regions (ITS1-5.8S-ITS2) were amplified with ITS1F (5'- CTTGGTCATTTACAGGAAGTAA-3') [18] and ITS4 (5'- ATTTGAGCTCTTCCCGCTTCA-3') [19]. The sequence alignments were obtained using BIOEDIT 7.0.9 [20], in addition with the reference sequences downloaded from GenBank. The sequences were initially aligned using Clustal W ver. 1.83 [21]. Phylogenetic analysis. Eight representative specimens were used for phylogenetic analysis. Trapelia coarctata (KR017098) was selected as an outgroup. Evolutionary history was inferred using the maximum likelihood (ML) method based on the general time reversible model [22]. The tree was drawn to scale, with branch lengths measured as the number of substitutions per site. A total of 26 nucleotide sequences were used in phylogenetic analysis. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7 [23]. One thousand bootstrap replications were tested for the reliability of the inferred tree. RESULTS AND DISCUSSION Morphological characteristics and dominant species in Korea. During 2003-2017, a total of 104 specimens were collected. Applying the classification keys used in China and North America, we identified the specimens by morphological and chemical analysis [3, 8]. Based on thin layer chromatography (TLC) analysis, all 104 specimens produced gyrophoric acid as a secondary metabolite, which is a characteristic of the genus Ochrolechia. Four species were identified from our specimens: O. trochophora, O. parellula, O. yasudae, and O. akagiensis (Fig 1). Among the four species, O. trochophora (40 specimens, 38.5%) and O. parellula (39 specimens, 37.5%) were the predominant species in Korea, followed by O. akagiensis (23 specimens, 22.1%) and O. yasudae (2 specimens, 1.9%). With the exception of O. akagiensis, the other three species identified, equating to 81 specimens (77.9%), have been reported in Korea [13-16]. Interestingly, all O. trochophora specimens were collected from mountainous regions, whereas all O. parellula were collected in coastal areas. In China and Poland, O. trochophora has been collected only from various trees in mountainous areas, but not from stones [8, 24]. In addition, O. parellula has been reported only in Japan and Korea until now [13]. In previous reports, these species have been found at altitudes below 500 m and existed in a saxicolous habit on acidic rocks [13]. Our findings are consistent with previous studies [13, 14]. This result implies that O. trochophora is likely to have adapted to higher altitudes, whereas O. parellula preferentially adapted to coastal areas in Korea. In this study, we report the presence of O. akagiensis in Korea for the first time. O. akagiensis forms round granule-shaped isidia ranging from 0.2 to 0.3 mm in diameter, only on trees (Fig. 1A), and is very similar to O. yasudae in morphological characteristics. Unlike O. akagiensis, O. yasudae forms cylinder- shaped isidia ranging from 0.1 to 0.3 mm in diameter on rocks [8]. In a previous study, Moon [13] reported that O. yasudae are present on rocks with mosses or on tree bark. In this study, however,
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