A new lichenized fungus, Lecanora baekdudaeganensis, from South Korea, with a taxonomic key for Korean Lecanora species

Abstract Lecanora baekdudaeganensis Lee & Hur is described as a new lichenized fungus from Baekdudaegan Mountains, South Korea. The new species is classified into the Lecanora subfusca group – allophana type and distinguishable from Lecanora imshaugii Brodo by a darker thallus, brownish disc, K–insoluble granules on the surface of the epihymenium, shorter hypothecium, and the presence of oil droplets in the apothecial section. Molecular analyses employing internal transcribed spacer (ITS) and mitochondrial small subunit (mtSSU) sequences strongly support Lecanora baekdudaeganensis as a distinct species in the genus Lecanora. A surrogate key is provided to assist in the identification of all 52 taxa in the genus Lecanora of Korea.


Introduction
The Baekdudaegan Mountains are the main mountain range stretching across the entire Korean Peninsula. The mountains stretch 1,400 km in length from North Korea to South Korea and encompass protected areas of approximately 2,750 km 2 (Korea Forest Service 2019). The Baekdudaegan Mountains, as the main mountain system for whole mountainous areas comprising 70 percent of Korea, are almost totally covered with forest and support a productive ecosystem for specialists as well as generalists,

Morphological and chemical analyses
Hand-cut sections were prepared with a razor blade under a stereomicroscope (Olympus optical SZ51; Olympus, Tokyo, Japan), examined under a compound microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan) and imaged using a software program (AxioVision Release 4.8.2; Carl Zeiss, Jena, Germany) and an Axiocam ERc 5s camera (Carl Zeiss, Jena, Germany) mounted on a Zeiss Axioscope 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 average, standard deviation, and number of measured spores. Thin-layer chromatography (TLC) was performed using solvent systems A and C according to standard methods (Orange et al. 2001).

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 with a NucleoSpin Plant II Kit in line with the manufacturer's instructions (Macherey-Nagel, Düren, Germany). PCR amplification for the internal transcribed spacer region (ITS1-5.8S-ITS2 rDNA) and the mitochondrial small subunit genes was achieved using Bioneer's AccuPower PCR Premix (Bioneer, Daejeon, Korea) in 20-μL tubes and primers ITS5 and ITS4 (White et al. 1990), and mrSSU1 and mrSSU3R (Zoller et al. 1999), respectively. The PCR thermal cycling parameters used were 95 °C (15 sec), followed by 35 cycles of 95 °C (45 sec), 54 °C (45 sec), and 72 °C (1 min), and a final extension at 72 °C (7 min) based on Ekman (2001). DNA sequences were generated by the genomic research company GenoTech (Daejeon, Korea).

Phylogenetic analyses
All ITS and mtSSU sequences were aligned and edited manually using ClustalW in Bioedit V7.2.6.1 (Hall 1999). All missing and ambiguously aligned data and parsimony-uninformative positions were removed and only parsimony-informative regions were finally analyzed in MEGA X (Stecher et al. 2020). The final alignment comprised 564 (ITS) and 1032 (mtSSU) columns. In them, variable regions were 51 (ITS) and 100 (mtSSU). Finally, the phylogenetically informative regions were 359 (ITS) and 464 (mtSSU). Phylogenetic trees with bootstrap values were obtained in RAxML GUI 2.0 beta (Edler et al. 2019) 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 (Drummond et al. 2012) using the HKY (Hasegawa, Kishino and Yano) model, as the appropriate model of nucleotide substitution based on the Bayesian Information Criterion (BIC) (Schwarz 1978) as evaluated by bModelTest (Bouchaert and Drummond 2017), empirical base frequencies, gamma for the site heterogeneity model, four categories for gamma, and a 10,000,000 Markov chain Monte Carlo chain length with a 10,000-echo state screening and 1000 log parameters. Then, a consensus tree was constructed in TreeAnnotator 1.8.0 (Drummond and Rambaut 2007) with a burn-in of 5000, 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 (Rambaut 2014) and edited in Microsoft Paint. The bootstrapping and Bayesian analyses were repeated three times for the result consistency and no significant differences were shown for the tree shapes and branch values. The phylogenetic trees and DNA sequence alignments are deposited in TreeBASE under the study ID 25859. Phylogenetic relationships among comparable species related mainly with the Lecanora subfusca group based on a maximum likelihood analysis of the nuclear ribosomal ITS1-5.8S-ITS2 region. The tree was rooted with several sequences in the genus Protoparmeliopsis. Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Lecanora baekdudaeganensis is presented in bold, and all species names are followed by GenBank accession numbers. A dash indicates branches with posterior probabilities <95%. The Lecanora subfusca group is marked with a black diamond (♦). Reference Table 1 provides the GenBank accession numbers for the included species and voucher information. . Phylogenetic relationships among comparable species related mainly with the Lecanora subfusca group based on a maximum likelihood analysis of the mitochondrial small subunit (mtSSU) sequences. The tree was rooted with several sequences in the genus Protoparmeliopsis. Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Lecanora baekdudaeganensis is presented in bold, and all species names are followed by GenBank accession numbers. A dash indicates branches with posterior probabilities <95%. The Lecanora subfusca group is marked with a black diamond (♦). Reference Table 1 provides the GenBank accession numbers for the included species and voucher information DNA sequences for the new species Lecanora baekdudaeganensis (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. ITS, internal transcribed spacer; mtSSU, mitochondrial small subunit; Voucher, voucher information. Description. Thallus corticolous, crustose, without lobes, continuous or cracked, rimose to areolate or verruculose, usually rounded or irregular, bluish gray in the beginning (margin) and olivish-or pale brownish-gray when mature (center), not pruinose, 30-70 mm diam., 100-170 μm thick; cortex hyaline to pale yellow or pale brown, Apothecia abundant, rounded, smaller and scattered around the margin and larger and aggregated in the center, constricted at the base, 0.2-1.6 mm diam. Disc flat to slightly concave, not pruinose, brown to dark brown from the beginning, 270-430 μm thick; margin persistent, prominent, generally entire or slightly flexuous, some a little crenulate when old, concolorous to thallus. Amphithecium well-developed, with numerous small crystals in both algal-containing and cortical parts (allophana-type) not dissolving in K, 60-100 μm thick laterally, 110-130 μm thick basally; amphithecial cortex distinct, 7-12 μm thick. Parathecium hyaline, indistinct in water, 15-25 μm thick in I. Epihymenium pale yellowish brown to pale brown, with small granules on the surface not dissolving in K, pigment slightly paler in K but not diluted, without oil droplets, 5-15 μm high. Hymenium hyaline, 50-75 μm high. Subhymenium hyaline, 20-40 μm high. Hypothecium hyaline, coarsely prosoplectenchymatous (periclinal) in the lower and marginal parts and prosoplectenchymatous (irregular) in the upper and central parts, 15-25 μm high. Oil droplets present in hypothecium, subhymenium and the base of hymenium. Hypothecial base not extending or a little extending to the substrate and always closed by medulla of amphithecium. Paraphyses septate, anastomosing, 1-2.5 μm wide, simple or sparsely branched at tips but not, or only slightly, swollen, 2.5-4 μm. Asci clavate, 8-spored, 41-51 × 13-20 μm (n = 10). Ascospores simple, often biguttulate in the beginning then having an oval-shaped oil drop by assembly of guttules when mature, narrowly or widely ellipsoid, or eye-shaped, 10-18.5 × 4.5-9.5 μm (mean = 15.2 × 6.5; SD = 1.58 (L), 1.10 (W); n = 128), wall ca 0.5 μm thick when exist. Pycnidia only once detected, pale brown at tip, ovoid, 315 × 330 μm, without conidia as old.
Distribution and ecology. The species occurs on the bark of Quercus mongolica and Q. dentata which are the most dominant tree species on the mountain. This species is currently known from four different sites on the mountain.
Etymology. The species epithet indicates the lichen's geography, namely the main mountains called Baekdudaegan stretching from north to south in the entire Korean Peninsula.
Notes. The new species is classified to the Lecanora subfusca group -allophana type, representing the main characteristics of a crustose thallus without lobes containing atranorin as a major constituent, small calcium oxalate crystals in both algalcontaining and cortical parts of the amphithecium, and trebouxioid photobionts in the thalline margin, dark brown discs, and colorless ellipsoid simple spores in the range of 10-20 × 6-9 μm (Brodo 1984;Miyawaki 1988;Lumbsch 1995;Lumbsch et al. 2003). The new species is compared with Lecanora chionocarpa Hue, L. horiza, L. imshaugii, L. japonica Müll. Arg., and L. megalocheila (Hue) H. Miyaw., as those species are in the L. subfusca group with only small crystals in the amphithecium (allophana or campestris type) which is defined by the main characteristics such as K+ yellow thallus reaction (containing atranorin), small calcium oxalate crystals in algal-containing and/or cortical parts of amphithecium, and ascospores in the size of 10-20 × 6-9 μm (Hue 1915;Brodo 1984;Miyawaki 1988;Smith et al. 2009). The new species is most similar to L. imshaugii by a continuous, rimose, verruculose or areolate thallus, the absence of soredia, the absence of a prothallus, apothecia size, and ascospore size (Brodo 1984). However, Lecanora baekdudaeganensis differs from L. imshaugii by a darker thallus (bluish, olivish, or pale brownish gray vs. greenish or yellowish gray), brownish disc (vs. reddish brown disc), K-insoluble granules on the surface of epihymenium (vs. absence of granules), a shorter hypothecium (15-25 μm vs. 50-75 μm), and the presence of oil droplets in the apothecial section (Brodo 1984).
The new species differs from L. horiza by a darker thallus (bluish, olivish or pale brownish gray vs. yellowish white to whitish gray), and the crystals in amphithecium not dissolving in K (vs. crystals dissolving in K) (Smith et al. 2009).