A New Lichenized Fungus, Lendemeriella luteoaurantia , with a Key to the Species of Lendemeriella

: Lendemeriella luteoaurantia B.G. Lee is described as a new lichen species from South Korea. The new species is identiﬁed by smaller, yellow-orange apothecia, larger ascospores with wider septum width, and the absence of Cinereorufa-green pigment and teloschistin, different from the closest species, L. aureopruinosa I.V. Frolov, Vondr á k, Arup, Konoreva, S. Chesnokov, Yakovczenko and Davydov in morphology and chemistry. Molecular phylogeny employing internal transcribed spacer (nuITS), nuclear large subunit ribosomal RNA (nuLSU), and mitochondrial small subunit (mtSSU) sequences strongly supports the new species as nonidentical in the genus Lendemeriella . A preliminary key is provided to assist in the identiﬁcation of all 10 species of Lendemeriella .

Lendemeriella is a genus in the subfamily Caloplacoideae and can be confused with similar genera in the subfamily, i.e., Bryoplaca Søchting, Frödén and Arup, Olegblumia S.Y. Kondr., Lőkös and Hur, Rufoplaca Arup, Søchting and Frödén, and Pyrenodesmia A. Massal. Vondrák et al. mentioned the similarity of Lendemeriella, Bryoplaca, and Parvoplaca Arup, Søchting and Frödén in the aspect of their habitat preference to humid alpine areas [1]. However, Lendemeriella differs from Bryoplaca by substrate preference mainly for rock or bark, biatorine apothecia, and the presence of parietin. Parvoplaca is easily distinguishable from Lendemeriella as the genus inhabits moss, plant debris, or bark with a poorly developed thallus and belongs to the subfamily Xanthorioideae [4]. Kondratyuk et al. compared Olegblumia as the most closely related genus to Lendemeriella in the introduction of Lendemeriella [2]. However, Lendemeriella quite differs from Olegblumia by the crustose thallus without lobes, the presence of the chemosyndrome A, and the narrow distribution limited to arctic-alpine and boreal-montane areas. Frolov et al. compared Lendemeriella with Rufoplaca because they have similar ecology [3]. However, Lendemeriella differs from Rufoplaca by an ascospore septum width of over 3.5 µm and the presence of episamma (the minute granules in epithecium). Pyrenodesmia, previously the Caloplaca xerica group, can be compared with Lendemeriella. The C. xerica group is similar to Rufoplaca, and Pyrenodemia s. lat. previously included Olegblumia, i.e., O. demissa (Flot. ex Körb.) S.Y. Kondr., Lőkös, Jung Kim, A.S. Kondr., S.O. Oh and Hur [3,4]. However, Lendemeriella differs

Morphological and Chemical Analyses
Hand sections were prepared manually with a razor blade under a stereomicroscope (Leica LED2500; Leica, Wetzlar, Germany), scrutinized under a compound microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan), and photographed using a software program (NIS-Elements D; Nikon, Tokyo, Japan) and a DS-Fi3 camera (Nikon, Tokyo, Japan) mounted on a Nikon Eclipse Ni-U microscope (Nikon, Tokyo, Japan). The ascospores were examined 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 (SD), length-to-width ratio, and number of measured spores. Thin-layer chromatography (TLC) was performed using solvent systems A and C according to standard methods [5]. The specimens are used in the herbarium of the Baekdudaegan National Arboretum (KBA), South Korea.

Isolation, DNA Extraction, Amplification, and Sequencing
Hand-cut sections of 10 to 20 ascomata per collected specimen 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 nuclear large subunit ribosomal RNA genes was achieved using Bioneer's AccuPower PCR Premix (Bioneer, Daejeon, Republic of Korea) in 20 µL tubes with 16 µL of distilled water, 2 µL of DNA extracts, and 2 µL of primers ITS5 and ITS4 [6] or LR0R and LR5 [7]. The PCR thermal cycling parameters used were 95 • C (15 s), followed by 35 cycles of 95 • C (45 s), 54 • C (45 s), and 72 • C (1 min), and a final extension at 72 • C (7 min) based on Ekman [8]. The annealing temperature was changed by ±1 degree for a better result. PCR purification and DNA sequencing were carried out by the Macrogen genomic research company (Seoul, Republic of Korea).

Phylogenetic Analyses
All nuITS, nuLSU, and mtSSU sequences were aligned and edited manually using ClustalW in Bioedit V7.7.1 [9]. All missing and ambiguously aligned data and phylogenetically uninformative positions were removed and only phylogenetically informative regions were finally analyzed. The final alignment comprised 1265 (nuITS), 2102 (nuLSU), and 965 (mtSSU) columns. In them, variable regions were 265 (nuITS), 532 (nuLSU), and 305 (mtSSU). The phylogenetically informative regions were 734 (nuITS), 638 (nuLSU), and 307 (mtSSU). A concatenation was carried out for combining all nuITS, nuLSU, and mtSSU gene loci. They were manually combined for the informative regions. Four problematic sequences were removed when conflicting results occurred in the internal branches with the bootstrap values ≥ 70% and the posterior probabilities ≥ 95% in the concatenated tree. Phylogenetic trees with bootstrap values were obtained in RAxML GUI 2.0.6 [10] using the maximum likelihood method with a rapid bootstrap with 1000 bootstrap replications and GTR GAMMA (SYM + I + G4) for the substitution matrix as the best models produced by the model test in the software. The posterior probabilities were obtained in BEAST 2.7.4 [11] using the GTR 123141 model as the appropriate model of nucleotide substitution produced by the Bayesian model averaging methods with bModelTest [12], 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 2.7.4 [11] with no discard of burnin, no posterior probability limit, a maximum clade credibility tree for the target tree type, and median node heights. All trees were displayed in FigTree 1.4.4 [13] and edited in Microsoft Paint. Overall analyses in the materials and methods were accomplished based on Lee and Hur [14].

Phylogenetic Analyses
The new species is positioned in the Lendemeriella-clade in the concatenated tree. The tree describes L. luteoaurantia, the new species, being nested with L. aureopruinosa, supported by a bootstrap value of 100 and a posterior probability of 1.00 for the branch (Figure 2). Although neighboring L. aureopruinosa, L. luteoaurantia is located in another branch distant from L. aureopruinosa. The new species is included in the subfamily Caloplacoideae and positioned closer to the genera Bryoplaca, Pyrenodesmia, Rufoplaca and Usnochroma Søchting, Arup and Frödén than other genera such as Caloplaca s. str., Leproplaca (Nyl.) Nyl., and Seirophora Poelt in the subfamily. This result on the phylogeny for Lendemeriella and closely related genera corresponds to Frolov et al. [3]. Their phylogeny also describes Lendemeriella neighboring with Pyrenodesmia, Rufoplaca, and Usnochroma, and other genera such as Caloplaca s. str., Leproplaca, and Seirophora are located in a different clade.  [4]. Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown as fatty lines. The new species, L. luteoaurantia, is presented in bold as its DNA sequences were produced from this study. All species names are referred to Table 1.    Description: thallus saxicolous, crustose, generally continuous but discontinuous or isolated locally, rarely rimose, smooth to slightly rugose, rarely with local thickenings, thin, brown-gray to olive-gray, the margin determinate, 70-150 µm thick; cortex indistinct, up to 10 µm thick; medulla mainly indistinct because of domination of algal layer and substrateoriginated large rock crystals dispersed on and below algal layer, up to 150 µm thick; photobiont coccoid, cells globose to subglobose, 5-15 µm thick, algal layer 60-100 µm thick. Small crystals present between algal cells, dissolving in K. Prothallus black, endosubstratal.
Etymology: the species epithet indicates the yellow-orange color of the apothecia. Notes: the new species is similar to the saxicolous members of the genus, i.e., L. aureopruinosa, L. exsecuta, and L. reptans. However, the new species differs from L. aureopruinosa and L. exsecuta by pale, small apothecia, colorless hypothecium, little swollen tips of paraphyses, larger ascospores, and the absence of Cinereorufa-green pigment and teloschistin ( Table 2).
The new species differs from L. reptans by the absence of soredia and thalline margin and the presence of emodin, fallacinal, parietin, and parietinic acid ( Table 2).
Without regard to the substrate preference for rock, the new species is similar to the corticolous L. borealis in having yellow-orange and small (<0.5 mm diam.) apothecia, black prothallus, epihymenium with K+ red reaction, and the presence of emodin, fallacinal, parietin, and parietinic acid. However, the new species differs from L. borealis by darker and thicker thallus, higher hypothecium, larger ascospores with wider septum width, and the absence of teloschstin [15][16][17].

Conflicts of Interest:
The authors declare no conflict of interest.