First report of the lichen Ochrolechia akagiensis (Ochrolechiaceae, Ascomycota) in Korea

INTRODUCTION

The genus Ochrolechia is comprised of a common crustose lichen which can be found easily on rocks and/or bark which are exposed to sufficient sunlight in mountainous areas. The genus Ochrolechia is distributed worldwide ranging from the polar to tropical regions, with a total of 60 species reported worldwide [1-8].

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 genus Pertusaria, with a thin layer of asci and ellipsoid ascospores [9]. In chemistry, however, the genus Ochrolechia is known to produce a depside, gyrophoric acid, as a secondar ymetabolite [9].

Ochrolechia spp. are identified by morphology, anatomy and chemistry [10]. Based on morphology, Verseghy [11] divided the genus into four groups: namely tatareae, parellae, harmandii, and upsaliensis. Later, Brodo renamed the groups ast artarea, 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 them argin, 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 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 ClustalW 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 anlaysis. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7 [23]. One thousand bootstrap replications were tested fo rthe 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 cylindershaped 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, we found that O. yasudae existed only on rocks and was rarely distributed in Korea. In addition, the specimens formerly identified as O. yasudae have been re-identified as O. akagiensis on account of the presence of granular isidia. Because these two species are very similar in the morphology, the probability of misidentification is very high. Molecular biological evidences should help clearly distinguish the two species.

Phylogenetic analysis by maximum likelihood method based on ITS rDNA sequences

We generated ITS region sequences from two representative specimens each of the four Ochrolechia species (O. akagiensis, O. parellula, O. trochophora, and O. yasudae). The length of the ITS1, 5.8S, and ITS2 regions ranges from 432 bp to 498 bp. The alignment of the ITS included 500 unambiguously aligned nucleotide positions, 301 of which were conserved and 187 were variable. All the generated ITS region sequences were deposited in GenBank.

Using the generated sequences, phylogenetic analysis was performed using the maximum likelihood (ML) method. The ML tree is shown in Figure 3. The clustering of O. parellula with O. trochophora is wellsupported by a higher bootstrap value (Fig. 3). O. yasudae and O. akagiensis formed a monophyletic group and was a sister clade to the clade composed of O. parellula and O. trochophora with high bootstrap values.

In the morphological observation, O. yasudae and O. akagiensis are quite similar because both species have isidia. In this phylogenetic analysis, two species were clustered and placed in the same clade (Fig. 3). In a previous study, these two species were divided by the substrate [8], but phylogenetic analysis revealed clade division corresponding to the diference in the isidia type than that of the substrates.

In a previous study, specimen KU933682 was identified as O. tartarea, following morphological and chemical identification [15]. In this study, however, phylogenetic analysis showed that putative O. parerulla specimens, including specimen KU933682, is clearly clustered in the clade ofO . parellula rather than with O. tartarea [15].

Taxonomy

1) Ochrolechia akagiensis Yasuda & Vain., Bot. Mag., Tokyo 35: 54 (1921) (Figs. 1A, 2A). Morphology: Thallus gray, thick, continuous, isidiate, granular, isidia covers thallus, 0.2~0.5 mm tall, 0.1~0.2 mm wide. Apothecia abundant or indistinct, round, sessile, 0.8~1.5 mm diam., apothecial discpinkish, concave, without pruina; margin concolorous with thallus, prominent, smooth becoming rugose to verrucose; well-developed, epihymenium brownish, 40~44.5 μm, hymenium hyaline, 294~338 μ m, hypothecium hyaline, 75.6~100 μm, algal layer mainly confined to margin, with spotty algal below hypothecium. Ascospores, simple, hyaline, ellipsoid. 50~77 × 28~36 μm.

Chemistry: Thallus and isidia: K-, C+ red, P-, UV-; apothecial disc and margin C+ red; gyrophoric acid.

Note: Ochrolechia akagiensis is confused with O. yasudae, which differs by having cylindrical isidia 0.1~0.3 mm in diam, and grows directly on rocks or moss-covered rocks, while O. akagiensis has granular

isidia and grows on bark to moss-covered bark [3]. O. frigida is also similar to O. akagiensis, but differs by having a yellowish apothecial disc and granular thallus with spine-like extensions.

Representative specimen: Korea, Gyeongsangnam-do, Hadong-gun, Mt. Jiri, 35˚19'06.79"N, 127˚39'05.22"E, alt. 1346 m, on bark, Y. Joshi, X. Y. Wang, J.-S. Hur 091390 (KoLRI-011307); Gangwon-do, Gangneung-si, Mt. Seokbyeong, 37˚34'40.66"N, 128˚51'36.11"E, alt. 807 m, on bark, J. S. Park, 170089 (KoLRI-044213).

2) Ochrolechia parellula (Müll. Arg.) Zahlbr., Cat. Lich. Univers. 5: 693 (1928) (Figs. 1B, 2B).

Morphology: Thallus gray, thick, 1.2~1.5 mm, continuous, cracked, verrucose, verrucae hemispheric, crowded, 0.1~0.3 mm in diam., prothallus indistinct to zonated. Apothecia abundant, scattered, sessile, round, 0.5~2.5 mm; disc white, pink to yellowish-pink, flat to concave, apothecial margin smooth, thick, concolorous with thallus, prominent above the disc level; epihymenium pale brown, 12.5~15 μm thick; hymenium hyaline, 270~290 μm tall; hypothecium pale brown to pale yellow, 40~70 μm thick, amphithecium fairly lax, algal layer continuous in margin, with spotty to continuous below the hypothecium. Ascospores eight per ascus, simple, hyaline, ellipsoid, 40~52.5 × 20~22.5 μm.

Chemistry: Thallus K-, C+ red, P-, UV-; apothecia disc K-, C+ red; apothecial margin cortex C+ red, medulla of thallus and margins K-, C-, P-; gyrophoric acid.

Note: O. parellula is a saxicolous species and grows on rock. In Moon􏌿s description, this species showed a C positive reaction, without soredia or isidia and apothecia with subdivided disc and grows on exposed rock, such as granite [25]. This species is similar to O. trochophora in the early stage, but the former has an increasingly thick thallus with a rimose-areolate form. Moreover, O. trochophora has a verrucose apothecial margin when well-developed, while O. parellula has a smooth margin. This species has been reported in India and Japan [25, 26]. O. parellula specimens have been reported as O. tartarea because of the verruculose thallus [15], but this was corrected through a re-examination.

Representative specimen: Korea, Jeollanam-do, Wando-gun, Cheongsan Island, 34˚09'01.87"N, 126˚52'08.21"E, alt. 2 m, on a rock, X. Y. Wang, J. A. Ryu, 110733 (KoLRI-013758; Gyeongsangbukdo, Ulleung-gun, Ulleung Island, 37˚28'59.09"N, 130˚54'40.07"E, alt. 20 m, on rock, J. S. Park, 162244 (KoLRI-040482).

3) Ochrolechia trochophora (Vain.) Oshio, J. Sci. Hiroshima Univ., Ser. B, Div. 2, 12: 145 (1968) (Figs. 1C, 2C).

Morphology: Thallus gray to pale gray, thin when young, becoming verrucose to verruculose, thick, with dispersed pustulate areole, thick, prothallus indistinct. Apothecia round to subround, 1.1~1.3 mm in diam., apothecial disc pinkish, concave becoming flat, occasionally developing radiation bands of sterile tissue. Apothecial margin smooth becoming verrucose, epihymenium brownish, 25~37.5 μm, hymenium hyaline, 232~268 μm, hypothecium pale brownish 70~103 μm, amphithecium medulla lax, algal layer absent or spotty, bellow on hypothecium. Ascospores eight per ascus, simple, hyaline, ovoid to ellipsoid, 38.3~62.2.7 × 21~22.5 μm.

Chemistry: Thallus K-, C+ red, P-, Medulla K-, C-, P-, UV-; apothecial disc C+ red; apothecial margin cortex C+ red; apothecial margin medulla C-; gyrophoric acid.

Note: Ochrolechia trochophora is a common species in Korea. This species is characterized by having verrucose apothecia margin, the algal layer below the hypothecium is absent or spotty and grows on bark. This species has highly variable apothecial discs and magrins. First, apothecial margin at the immature stage is very smooth, and the disc is flat. After the mature stage, the apothecial margin becomes verrucose and disc concave. In the final stage, the apothecial disc becomes wheel-like or rosulate. According to Kukwa, O. trochophora have two types: 1) O. trochophora s. str. from Europe with weakly developed thallus; 2) O. trochophora s. str. from U.S.A with pustulate areoles on the apothecial margin and over the thallus. Korean O. trochophora has both types. This species resembles O. subpallescens and O. laevigata but the former has a continuous algal layer below the hypothecium while the latter has a thin thallus and apothecial margin almost devoid of the algal layer, and lacking algae below the hypothecium.

Representative specimen examined: Korea, Jeju-do, Jeju-si, Mt. Halla, 33˚24'43.03"N, 126˚32'52.04"E, alt. 713 m, on bark, S.-O. Oh, U. Jayalal, J. S. Park, J.-S. Hur, 120983 (KoLRI-016013); Gangwando, Jeongseon-gun, Gohan-eup, 37˚11'31.26"N, 127˚31'77.1"E, alt. 957 m, on bark, J. S. Park, 163583 (KoLRI-041828); Jeollanam-do, Suncheon-si, Songgwang-myeon, Temple Songgwang, 35˚00'10.07"N, 127˚16'07.00"E, alt. 80 m, on bark, J. S. Park, J.-JW. oo, 164045 (KoLRI-042313).

4) Ochrolechia yasudae Vain., Bot. Mag., Tokyo 32: 155 (1918) (Figs. 1D, 2D).

Morphology: Thallus grows directly on rock or moss-covered rock, ashy-gray, thick, continuous, with a distinct, often zoned border, isidiate, isidia cylindrical, knobby, persistent, isidia covers the whole thallus, 0.2~0.5 mm tall, 0.1~0.2 mm wide.A pothecia not seen.

Chemistry: Thallus and isidia: K-, C+ red, P-; Medulla: K-, C-, P-; UV-; gyrophoric acid.

Note: Ochrolechia yasudae has been reported in East Asia and North America [8]. The main morphological characters include isidia covering the thallus and are coralloid or cylindrical in shape. Ochrolechia frigida is similar to O. yasudae but differs by having a yellowish apothecial disc and granular thallus with spine-like extensions.

Representative specimen examined: Korea, Gangwon-do, Yangyang-gun, Seo-myeon, Mt. Jobong, 37˚56'01.07"N, 128˚33'07.47"E, alt. 980 m, on rock covered by moss, Y. Joshi, X. Y. Wang, J. A. Ryu, J.-S Hur, 090283 (KoLRI-010027); Gangwon-do, Chuncheon-si, Mt. Maebong, 37˚54'92.4"N, 127˚58'48.6"E, alt. 576 m, on rock, J. S. Park, J.-JW. oo, B. G. Lee, 152863 (KoLRI-37172).

Key to the lichen genus Ochrolechia in South Korea

1. Thallus isidiate or with a finger-like extension .................................................................................................... 2

- Thallus lacking isidia and soredia ................................................................................................................... 4

2. Thallus with a spine-like extension ........................................................................................................ O. frigida

- Thallus isidiate, isidia cylindrical or granular ..................................................................................................... 3

3. Thallus isidia cylindrical, knobby; saxicolous ............................................................................................. O. yasudae

- Thallus isidia granular; muscicolous or occasionally corticolous ......................................................................... O. akagiensis

4. Thallus corticolous, apothecial disc epruinose to haze-like pruina present, apothecial margin welldeveloped

to verrucose .............................................................................................................................. O. trochophora

- Thallus saxicolous ................................................................................................................................... 5

5. Thallus white and tartareous (strongly granular), disc yellowish .......................................................................... O. tartarea

- Thallus not tartareous, whitish-gray, continuous, rimose-areolate, verrucose, apothecial disc C+ red ...........

............................................................................................................................................. O. parellula

ACKNOWLEDGEMENTS

This work was supported by a grant from the National Research Foundation of Korea (NRF-2017R1D1A1B04035888) and the Korean Forest Service Program through the Korea National Arboretum (KNA1-1-22, 17-2).