sustainability Article Diversity and Distribution Patterns of Endolichenic Fungi in Jeju Island, South Korea Seung-Yoon Oh 1,2 , Ji Ho Yang 1, Jung-Jae Woo 1,3, Soon-Ok Oh 3 and Jae-Seoun Hur 1,* 1 Korean Lichen Research Institute, Sunchon National University, 255 Jungang-Ro, Suncheon 57922, Korea; [email protected] (S.-Y.O.); [email protected] (J.H.Y.); [email protected] (J.-J.W.) 2 Department of Biology and Chemistry, Changwon National University, 20 Changwondaehak-ro, Changwon 51140, Korea 3 Division of Forest Biodiversity, Korea National Arboretum, 415 Gwangneungsumok-ro, Pocheon 11186, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-61-750-3383 Received: 24 March 2020; Accepted: 1 May 2020; Published: 6 May 2020 Abstract: Lichens are symbiotic organisms containing diverse microorganisms. Endolichenic fungi (ELF) are one of the inhabitants living in lichen thalli, and have potential ecological and industrial applications due to their various secondary metabolites. As the function of endophytic fungi on the plant ecology and ecosystem sustainability, ELF may have an influence on the lichen diversity and the ecosystem, functioning similarly to the influence of endophytic fungi on plant ecology and ecosystem sustainability, which suggests the importance of understanding the diversity and community pattern of ELF. In this study, we investigated the diversity and the factors influencing the community structure of ELF in Jeju Island, South Korea by analyzing 619 fungal isolates from 79 lichen samples in Jeju Island. A total of 112 ELF species was identified and the most common species belonged to Xylariales in Sordariomycetes. The richness and community structure of ELF were significantly influenced by the host taxonomy, together with the photobiont types and environmental factors. Our results suggest that various lichen species in more diverse environments need to be analyzed to expand our knowledge of the diversity and ecology of ELF. Keywords: algae; cyanobacteria; Daldinia; host specificity; lichen; oreum; photobiont; sordariomycetes; xylariales 1. Introduction Lichens are symbiotic organisms in which the mycobiont (lichen-forming fungi) and the photobiont (green algae and/or cyanobacteria) live together in a mutualistic relationship: the mycobiont protects the photobiont against external environmental stress by forming a thallus, and the photobiont provides photosynthetic carbon as a reward [1,2]. Lichens play ecologically important roles in ecosystems as food and habitats for animals, as well as participants in nutrient cycling and soil formation [3,4]. In addition, they produce numerous secondary metabolites that are industrially or pharmaceutically effective compounds such as antibiotics, anti-tumor agents, and antioxidants [5]. Recent studies have shown that diverse microorganisms exist within lichen thalli, and they can influence the physiology of host lichen in a similar manner to the influence of endophytes on the host plant [6–8]. Endolichenic fungi (ELF) are non-mycobiont fungal species living in the lichen thallus [9]. They are distinct from the lichenicolous fungi in terms of symptomless characteristics. As for the relationship between ELF and lichens, it is unclear whether they have any kind of intimate association [8]. Given that the endophytic fungi influence the plant physiology and increase a tolerance against environmental stress (e.g., high temperature, drought, or pathogens) [10–12], it is suggested that ELF promote the biological function Sustainability 2020, 12, 3769; doi:10.3390/su12093769 www.mdpi.com/journal/sustainability Sustainability 2020, 12, 3769 2 of 19 of lichens in the ecosystem [8]. Therefore, the biodiversity and distribution pattern of ELF is important not only for understanding the ecology and physiology of lichens, but also for the maintenance of ecosystem sustainability in the era of global climate change. The ELF diversity has been studied in various biomes from tropical areas to polar regions using culture-dependent [13–20] or independent approaches [19,21–25]. ELF are phylogenetically diverse, covering all lineages of Ascomycota and a minority of Basidiomycota and Mucoromycota [9,14]. The evolution of ELF is not well understood, but it is thought to be polyphyletic, and ELF have been suggested as the origin of endophytism, which would explain the similar phylogenetic range [9]. The ELF diversity is different from the co-existing endophytic diversity, which indicates that ELF are a distinctive ecological group differing from endophytic fungi [14,26]. However, the ELF diversity has been studied mainly in Europe [13,27,28], North America [9,14], and Southern Asia [17,29,30], while the ELF in Eastern Asia are largely unexplored [31]. Therefore, it is expected that exploring the ELF diversity in Eastern Asia can expand the knowledge of fungal diversity associated with lichens. Several abiotic and biotic factors drive the pattern of fungal diversity and community structures [32–34]. It is well known that endophytic fungal communities are influenced by the climate [15,35], geographic position of the region [36], and host taxonomy [37,38]. The factors influencing the ELF community structures have rarely been investigated, but a few studies have suggested that geographic characteristics and the host taxonomy are important factors shaping the ELF community structure [9,14,15,27,28]. The ELF diversity was high in tropical or subtropical regions, probably due to favorable climatic conditions [9,15]. The lichen host is one of the factors that determine the ELF communities [14,15,27,28]. However, other studies showed no effect of the host taxonomy [39], which suggests that the pattern of ELF diversity and factors governing the ELF community may vary depending on the geographical region and spatial scales. In this study, we investigated the ELF diversity and community structure in Jeju Island (South Korea). Jeju Island is composed of warm lowlands and subalpine (Mt. Halla, 1947 m above sea level) biomes with deciduous-evergreen broadleaved and coniferous forests [40], as well as many reported lichens [41–44]. The oreums, a special structure of parasitic volcanoes, are distributed from the lowland to the mountain area [45,46] and are covered by forests with a high diversity of animals [47,48], plants [49,50], and microorganisms [51,52]. Although study of lichen flora in oruems is limited, several studies have revealed novel records of lichen species from oreums [53,54]. However, the diversity of ELF is largely unexplored in South Korea as well as in oreums. We collected different species of lichens from several oreums in Jeju Island, and investigated the diversity of ELF by culture isolation. Moreover, we analyzed the pattern of the diversity and community structure of ELF to understand the effect of various factors (host taxonomy, photobiont type, and ecoregions) on the relationship between ELF and lichens. 2. Materials and Methods 2.1. Lichen Sample Collection and ELF Isolation Lichen specimens were collected from sampling sites at 29 oreums covering the whole island from the lowland near the coast to the high mountain area in Mt. Halla, from April to September 2017 (Figure1; Table S1 (Supplementary Materials)). We focused on the foliose lichens, except for Stereocaulon (fruticose), because the wide area of the thallus is helpful to acquire sufficiently large thalli and to avoid contamination. Healthy and fresh thalli of lichens on rock, soil, and trees were collected (Table A1) and transferred to the laboratory in paper bags. Sustainability 2020, 12, 3769 3 of 19 Sustainability 2020, 12, x FOR PEER REVIEW 3 of 19 FigureFigure 1. Map 1. ofMap the of sampling the sampling sites on sites Jeju on Island. Jeju Island. Ecoregions Ecoregions classified classified using elevationusing elevation and bioclimatic and variablesbioclimatic are presented variables inare di presentedfferent colors. in different colors. The lichenThe lichen thalli thalli were were subjected subjected to to sterilization. sterilization. Be Beforefore sterilization, sterilization, the the litter litter and and debris debris attached attached on the thalli were removed using a needle of a syringe, and the thalli were washed in running tap on the thalli were removed using a needle of a syringe, and the thalli were washed in running tap water. The surface of the thalli was sterilized by a modified method used in the previous study [14]: water. The surface of the thalli was sterilized by a modified method used in the previous study [14]: 95% ethanol for 30 s, 0.5% NaOCl for 2 min, 70% ethanol for 30 s, and rinsed three times with sterile 95% ethanoldistilled forwater. 30 s,The 0.5% thalli NaOCl were dried for 2 min,on sterilized 70% ethanol paper forand 30cut s, into and 1 rinsedcm2 pieces three using times sterilized with sterile 2 distilledscissors. water. For Theeach thallispecimen, were a driedtotal of on 20 sterilizedfragments were paper placed and cuton four into plates 1 cm of pieces90 mm usingPetri dishes sterilized scissors.containing For each a potato specimen, dextrose a total agar of(PDA; 20 fragments Difco Labo wereratories, placed Detroit, on fourMI, USA), plates and of 90incubated mm Petri at 25 dishes containing°C for aup potato to 12 weeks. dextrose The agar plates (PDA; were Difco checked Laboratories, every day, Detroit,and fungal MI, isolates USA), were and incubatedtransferred at to 25 ◦C for upPDA to 12medium weeks. for The pure plates culture. were Additionally, checked every ELF strains day, and previously fungal isolatesisolated from were Jeju transferred Island using to PDA mediumthe same forpure procedure culture. and Additionally,deposited in the ELF Korean strains Lichen previously Research Institute isolated (K fromoRLI) Jeju were Island included using in the samethe procedure analysis andto increase deposited the volume in the Korean of the datase Lichent. To Research recover Institute the strains (KoRLI) from KoRLI, wereincluded they were in the cultured on PDA medium at 25 °C. analysis to increase the volume of the dataset. To recover the strains from KoRLI, they were cultured on PDA2.2.