﻿Two new Rinodina lichens from South Korea, with an updated key to the species of Rinodina in the far eastern Asia

﻿Abstract Rinodinasalicis Lee & Hur and Rinodinazeorina Lee & Hur are described as new lichen-forming fungi from forested wetlands or a humid forest in South Korea. Rinodinasalicis is distinguishable from Rinodinaexcrescens Vain., the most similar species, by its olive-gray thallus with smaller areoles without having blastidia, contiguous apothecia, non-pruinose discs, paler disc color, wider ascospores in the Pachysporaria-type II, and the absence of secondary metabolites. Rinodinazeorina differs from Rinodinahypobadia Sheard by areolate and brownish thallus, non-pruinose apothecia, colorless and wider parathecium, narrower paraphyses with non-pigmented and unswollen tips, longer and narrower ascospores with angular to globose lumina, and the absence of pannarin. Molecular analyses employing internal transcribed spacer (ITS) sequences strongly support the two new species to be unique in the genus Rinodina. An updated key is provided to assist in the identification of all 63 taxa in Rinodina of the far eastern Asia.


Introduction
Rinodina, the largest genus in the family Physciaceae, comprises about three hundred species worldwide (Sheard et al. 2017;Wijayawardene et al. 2020). Several infrageneric groups have been studied since Malme (1902) introduced the ascospore-type concept for the groups in Rinodina (Poelt 1965;Grube and Arup 2001). Although the classification based on different ascospore types has been coarsely accepted, the variety of ascospores does not always correspond to the infrageneric classification. As the pattern of ascospore ontogeny is considered more important than the spore type itself, it is understood that the ascospore types should be respected in developmental stages of a spore (Giralt 1994;Grube and Arup 2001;Sheard 2010;Resl et al. 2016).
This study describes two new lichen-forming fungi in the genus Rinodina. Field surveys for the lichen biodiversity in the forested wetlands of South Korea were carried out during the summer of 2020, and a couple of specimens of Rinodina were collected from barks of Quercus and Salix, the most common genera of the substrates for corticolous Rinodina species in the far eastern Asia, in a humid forest and a forested wetland on mountains (Fig. 2). The specimens were comprehensively analyzed in ecology, morphology, chemistry and molecular phylogeny and did not correspond to any previously known species. We describe them as new species, Rinodina salicis and R. zeorina, and this discovery contributes to the taxonomy with overall 63 taxa in the genus Rinodina of the far eastern Asia. The type specimens are deposited in the herbarium of the Baekdudaegan National Arboretum (KBA, the herbarium acronym in the Index Herbariorum), South Korea.

Morphological and chemical analyses
Hand sections were prepared manually with a razor blade under a stereomicroscope (Olympus optical SZ51; Olympus, Tokyo, Japan), scrutinized under a compound microscope (Nikon Eclipse E400; Nikon, Tokyo, Japan) and pictured 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 the 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 ten to twenty 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 amplifications for the internal transcribed spacer region (ITS1-5.8S-ITS2 rDNA) RNA genes were achieved using Bioneer's AccuPower PCR Premix (Bioneer, Daejeon, Korea) in 20-μl tubes with 16 μl of distilled water, 2 μl of DNA extracts and 2 μl of the primers ITS5 and ITS4 (White et al. 1990). 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). The annealing temperature was occasionally altered by ±1 degree in order to get a better result. PCR purification and DNA sequencing were accomplished by the genomic research company Macrogen (Seoul, Korea).

Phylogenetic analyses
All ITS sequences (Table 1) were aligned and edited manually using ClustalW in Bioedit V7.2.6.1 (Hall 1999). All missing and ambiguously aligned data and parsimonyuninformative positions were removed and only parsimony-informative regions were finally analyzed in MEGA X (Stecher et al. 2020). The final alignment comprised 974 bp in which 167 variable regions were detected. The phylogenetically informative regions were 523. 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 BEAST 2.6.4 (Bouckaert et al. 2019) using the GTR 121343 model, as the appropriate model of nucleotide substitution produced by the bayesian model averaging methods with bModelTest (Bouckaert 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 2.6.4 (Bouckaert et al. 2019) 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 1.4.2 (Rambaut 2014) and edited in Microsoft Paint. The bootstrapping and posterior probability 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 28192. Overall analyses in the materials and methods were accomplished based on Lee and Hur (2020).

Phylogenetic analyses
An independent phylogenetic tree for the genus Rinodina and related genera was produced from 67 sequences from GenBank and 11 newly generated sequences for the two new species and related species ( Table 1). The two new species were positioned in the genus Rinodina in the ITS tree. The ITS tree describes that R. salicis, a new species, is com- DNA sequences which were generated in this study, i.e., two new species such as Rinodina salicis and R. zeorina, and another compared species, R. orientalis, are presented in bold. All others were obtained from GenBank. The species names are followed by GenBank accession numbers and voucher information. ITS, internal transcribed spacer; Voucher, voucher information.
ing alone in a single clade. Several species such as R. mniaroea (Ach.) Körb., R. roscida Diagnosis. Rinodina salicis differs from R. excrescens by olive-gray thallus with smaller areoles without blastidia, contiguous apothecia, the absence of pruina on disc, paler disc color, wider ascospores in the Pachysporaria-type, and the absence of secondary metabolites. Thallus corticolous, crustose, minutely bullate, some developing to conglomerate and continuous, rarely lobulated, thin, grayish-green to olive green, margin indeter- Figure 3. Phylogenetic relationships among available species in the genus Rinodina based on a maximum likelihood analysis of the dataset of ITS sequences. The tree was rooted with the sequences of the genera Amandinea and Buellia. Maximum likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown as fatty lines. Two new species, R. salicis and R. zeorina are presented in bold as their DNA sequences were produced from this study. All species names are followed by the Genbank accession numbers. minate, vegetative propagules absent, areoles 0.1-0.2 mm diam., 85-90 μm thick; cortex hyaline, 10 μm thick, cortical cells 5-9 μm diam.; medulla 60-65 μm thick, intermixed with algal cells, without crystals (PL-); photobiont coccoid, cells globose, 5-15 μm. Prothallus absent.
Distribution and ecology. The species occurs on the bark of Salix koreensis. The species is currently known from the type collections.
Etymology. The species epithet indicates the lichen's substrate preference, namely the substrate tree Salix koreensis.
Notes. The new species is similar to R. excrescens and R. bullata Sheard & Lendemer in having bullate thallus. However, the new species differs from R. excrescens by olive-gray thallus with smaller areoles without having blastidia, contiguous apothecia, the absence of pruina on disc, paler disc color, ascospore type, larger ascospore, and the absence of secondary metabolites (Sheard 1966;Sheard et al. 2012).
The new species is closer to R. bullata in having small bullate areoles without having blastidia. However, the new species differs from the latter by olive-gray thallus, contiguous and larger apothecia, proper margin with pale brown color, crystals present in both cortex and medulla in amphithecium, larger ascospores, K-reaction on thallus, and the absence of lichen substance (Sheard et al. 2012(Sheard et al. , 2017. The new species is comparable to R. granulans Vain. as the latter represents thallus with minute areoles. However, the new species differs from the latter by thallus color, slightly smaller areoles without blastidia, abundance of apothecia without pruina, Pachysporaria-type II ascospores, K-reaction on thallus, and the absence of lichen substance Galanina et al. 2011). Reference Table 2 provides the key characteristics distinguishing R. salicis from the compared species above.  Sheard et al. 2012Sheard 1966Sheard et al. 2012Giralt et al. 1994Galanina et al. 2011 The morphological and chemical characteristics of several species close to the new species are referenced from the previous literature. All information on the new species is produced from type specimens (BDNA-L-0000558 and BDNA-L-0000560) in this study. Thallus corticolous, crustose, areolate, rimose to continuous, thin, light gray to light brownish gray, margin indeterminate or determinate with prothallus, vegetative propagules absent, 160-250 mm diam., 80-170 μm thick, areoles 0.1-0.5 mm diam.; cortex brown, 5-8 μm thick, with epinecral layer, hyaline, 3-7 μm thick; medulla 35-40 μm thick, intermixed with algal cells, without crystals (PL-); photobiont coccoid, cells globose, 5-9 μm. Prothallus absent or brownish black when present.
Distribution and ecology. The species occurs on the bark of Quercus mongolica, Tilia amurensis Rupr., and Maackia amurensis Rupr. & Maxim. The species is currently known from a humid forest and a forested wetland of two mountainous sites.
Etymology. The species epithet indicates that the lichen's substance, zeorin, is a major compound.
Notes. The new species is similar to R. hypobadia, R. sheardii, and R. sp. A in having a pigmented hypothecium. However, the new species differs from R. hypobadia by areolate, brownish thallus, apothecia without pruina, hyaline and wider parathecium, narrower paraphyses with hyaline and unswollen tips, longer and narrower ascospores with just angular to globose lumina, and the absence of pannarin (Sheard et al. 2017).
The new species differs from Rinodina sheardii by the absence of vegetative propagules, and Dirinaria-type ascospores in smaller size (Sheard et al. 2017).
The new species is distinguished from R. aff. oleae by narrower ascospores, and pigmented hypothecium (vs. hyaline hypothecium) (Sheard et al. 2017). Reference Table 3 provides the key characteristics distinguishing R. zeorina from the compared species above.
Overall, 63 taxa of Rinodina are currently recorded or expected to the far eastern Asia (Korea, Japan and Russian Far East).