Two new lecanoroid lichen species from the forested wetlands of South Korea, with a key for Korean Protoparmeliopsis species

Abstract Lecanoraparasymmicta Lee & Hur and Protoparmeliopsiscrystalliniformis Lee & Hur are described as new lichen species to science from the forested wetlands in southern South Korea. Molecular analyses employing internal transcribed spacer (ITS) and mitochondrial small subunit (mtSSU) sequences strongly support the two lecanoroid species to be distinct in their genera. Lecanoraparasymmicta is included in the Lecanorasymmicta group. It is morphologically distinguished from Lecanorasymmicta (Ach.) Ach., its most similar species, by areolate-rimose thallus, blackish hypothallus, larger apothecia, absence of thalline excipulum from the beginning, narrower paraphyses, larger ascospores, smaller pycnoconidia, and the presence of placodiolic acid. The second new species Protoparmeliopsiscrystalliniformis is included in a clade with Protoparmeliopsisbipruinosa (Fink) S.Y. Kondr. and P.nashii (B.D. Ryan) S.Y. Kondr., differs from Protoparmeliopsisertzii Bungartz & Elix, its most morphologically similar species, by whitish thallus, flat to concave and paler disc, longer ascospores, thallus K+ yellow reaction, presence of atranorin and rhizocarpic acid, and the substrate preference to sandstone or basalt. A key is provided to assist in the identification of Protoparmeliopsis species in Korea.

This study describes two new lichen-forming fungi species to science in the genera Lecanora, i.e., the L. symmicta group, and Protoparmeliopsis. Field surveys for the lichen biodiversity in the forested wetlands of southern South Korea were accomplished during the summer of 2020, and a few dozen specimens were collected in the wetland forests nearby seashore or in islands (Fig. 1). The collected specimens were comprehensively analyzed in ecology, morphology, chemistry and molecular phylogeny and did not correspond to any previously known species. We describe them as two new species, Lecanora parasymmicta and Protoparmeliopsis crystalliniformis, and these discoveries contributes to the taxonomy of the lecanoroid lichens of Korea by listing overall fifty three taxa of Lecanora and six taxa of Protoparmeliopsis. The 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 number of measured spores. Thinlayer 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 amplification for the internal transcribed spacer region (ITS1-5.8S-ITS2 rDNA), the mitochondrial small subunit, and the nuclear large subunit ribosomal RNA genes was 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 primers ITS5 and ITS4 (White et al. 1990), mrSSU1 and mrSSU3R (Zoller et al. 1999) or LR0R and LR5 (Rehner and Samuels 1994). 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 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 1462 (ITS) and 1058 (mtSSU) columns for Lecanora. In them, variable regions were 171 (ITS) and 117 (mtSSU). The phylogenetically informative regions were 440 (ITS) and 152 (mtSSU). The final alignment for Protoparmeliopsis comprised 945 (ITS) and 985 (mtSSU) columns. In them, variable regions were 214 (ITS) and 53 (mtSSU). Finally, the phylogenetically informative regions were 268 (ITS) and 134 (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 BEAST 2.6.4 (Bouckaert et al. 2019) using the GTR 123141 (ITS for Lecanora), the GTR 121321 (mtSSU for Lecanora), the HKY (Hasegawa-Kishino-Yano) (ITS for Protoparmeliopsis), and the GTR 123123 (mtSSU for Protoparmeliopsis) models, as the appropriate models 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 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 28189. Overall analyses in the materials and methods were accomplished based on Lee and Hur (2020).

Phylogenetic analyses
Four independent phylogenetic trees for the genera Lecanora and Protoparmeliopsis were produced from 117 sequences (71 for ITS, and 30 for mtSSU) from GenBank and, 16 new sequences (11 for ITS and 5 for mtSSU) from the new and compared species (Table 1). Lecanora parasymmicta, one of the new species, is positioned in the L. symmicta group in both ITS and mtSSU trees. The ITS tree illustrates that the new species is located in its own clade without any species close to it. Lecanora symmicta, the most similar species, is positioned in a clade with L. confusa Almb. and L. compallens Herk & Aptroot, situated far from the new species (Fig. 2). The mtSSU tree shows that the new species is located in a clade with L. symmicta and L. strobilina Ach., represented by a bootstrap value of 100 and a posterior probability of 1.0 for the branch (Fig. 3). The second new species, Protoparmeliopsis crystalliniformis, was positioned in Protoparmeliopsis in both ITS and mtSSU trees. The ITS tree explains that the new species is located in a clade with P. bipruinosa (Fink) S.Y. Kondr. and P. nashii (B.D. Ryan) S.Y. Kondr., represented by a bootstrap value of 92 and a posterior probability of 1.0 for the branch (Fig. 4). The mtSSU tree shows that P. crystalliniformis is located in its own clade (Fig. 5). The phylogenetic analyses, and according to the included taxa, did not indicate any such species to the two new proposed in Lecanora and Protoparmeliopsis. DNA sequences which were generated in this study, in bold the new species Lecanora parasymmicta and Protoparmeliopsis crystalliniformis and newly generated sequences of Lecanora symmicta and Polyozosia sp. specimens. 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. The new sequences of Lecanora parasymmicta and Lecanora symmicta produced from this study are presented in bold, and all species names are followed by the GenBank accession numbers. Reference Table 1 provides the species related to the specific GenBank accession numbers and voucher information.  Table 1 provides the species related to the specific GenBank accession numbers and voucher information.  Table 1 provides the species related to the specific GenBank accession numbers and voucher information.  Diagnosis. Lecanora parasymmicta differs from L. symmicta, the most similar species, by its areolate-rimose thallus (vs. areolate to leprose thallus), blackish hypothallus (vs. hypothallus indistinct), larger apothecia (up to 1.7 mm diam. vs. up to 1 mm diam.), absence of thalline excipulum from the beginning (vs. presence of thalline excipulum when young at least), narrower paraphyses (1-1.5 μm vs. 2-2.5 μm), larger ascospores (11-18 × 4-7 μm vs. 9-15.5 × 4-5 μm), smaller pycnoconidia ( Description. Thallus corticolous, crustose, areolate to rimose but not leprose, light olivish gray to light gray, margin determinate, not pruinose, 60-200 μm thick; cortex hyaline, 5-10 μm thick; medulla often intermixed with algae and even with bark layer, small crystals in cortex or between algae, dissolving in K; photobiont coccoid, cells globose to ellipsoid, 5-15 μm. Hypothallus blackish. Apothecia abundant, rounded, often contiguous or even coalescent, emerging on the surface of thallus and sessile when mature but margin generally attached to thallus surface, constricted at the base, 0.3-1.7 mm diam. Disc flat in the beginning and soon convex, smooth or becoming rugose by apothecia adjoining, not pruinose or slightly pruinose, pale yellow in the beginning and slightly darker when mature, sometimes with dark spots (algae), 180-400 μm thick; biatorine. Thalline excipulum absent from the beginning, proper excipulum present and sometimes slightly paler than disc, more distinctive when young, hyaline but yellowish brown to pale brown at periphery with granules which dissolving in K, periphery color same to epihymenium, ca. 90 μm wide laterally and 70-80 μm wide at periphery, disappearing to the base. Epihymenium yellowish brown to pale brown, granular, dissolving in K, 10-20 μm high. Hymenium hyaline, 70-90μm high. Subhymenium hyaline, 30-50 μm high. Hypothecium hyaline, prosoplectenchymatous (irregular), 50-60 μm high. Crystals and oil droplets absent in apothecial section. Paraphyses septate, anastomosing, 1-1.5 μm wide, simple or branched at tips, tips not swollen or slightly swollen, not pigmented, epihymenium pigmented by granules, not by paraphysial tips, ca. 1.5 μm wide. Asci clavate, 8-spored, 50-60 × 13-21 μm (n = 7). Ascospores constantly simple but rarely 1-septate, coarsely biseriate or irregularly arranged, 11-18 × 4-7 μm (mean = 13.8 × 5.8 μm; SD = 1.62(L), 0.63(W); L/W ratio 1.8-4.0, ratio mean = 2.4, ratio SD = 0.3; n = 105). Pycnidia immersed, ostiolar region slightly projected with a thalline excipulum, round to irregularly asymmetric, brown to black, 220 × 180 μm. Pycnoconidia threadlike, generally curved, 12-21 × 0.5-0.8 μm.
Chemistry. Thallus K-or K+ slightly yellowish, KC-, C-, Pd-. Hymenium, epihymenium and ascus tholus I+ blue. UV-. Usnic acid, zeorin, and placodiolic acid were detected by TLC.  (paratype) Smith et al. 2009Nath III et al. 2004Smith et al. 2009Brodo et al. 2001Smith et al. 2009Brodo et al. 2001Nash III et al. 2004;Smith et al. 2009; BDNA-L-0000547, BDNA-L-0000548, and BDNA-L-0000551 The morphological and chemical characteristics for several species close to the new species are referenced mainly from the previous literature. All information on the new species is measured from type specimens (BDNA-L-0001218, BDNA-L-0001220, and BDNA-L-0001235) in this study. Particularly the asci of the closest species, Lecanora symmicta, was not described from the previous literature and the asci and the ascospores for the species are measured from selected specimens (BDNA-L-0000547, BDNA-L-0000548, and BDNA-L-0000551) in this study, represented with asterisk marks(*).
Distribution and ecology. The species occurs on the rock (sandstone or basalt) nearby coast. The species is currently known from two localities in the southern coast of South Korea.
Etymology. The species epithet indicates the insoluble large crystals present in the thalline excipulum of the lichen.
Notes. The new species is morphologically similar to Protoparmeliopsis ertzii in having insoluble large crystals in the thalline excipulum and the absence of usnic acid, which are the key characteristics distinguishing them from all other species in the genus Protoparmeliopsis. However, the new species differs from P. ertzii by whitish thallus, flat to concave disc, paler disc color, longer ascospores, chemical reaction, presence of atranorin and rhizocarpic acid, and the substrate preference (Bungartz et al. 2020).
The new species is compared with P. bipruinosa and P. nashii as those are closest to the new species in molecular results (Figs 4 and 5). However, the new species differs from P. bipruinosa by whitish thallus, absence of pruina, presence of large crystals, and the presence of atranorin and rhizocarpic acid (Nash III et al. 2004).
The new species is different from P. nashii by whitish thallus, absence of pruina, presence of large crystals, and the presence of atranorin and rhizocarpic acid (Nash III et al. 2004). Reference Table 3 provides specific characteristics distinguishing P. parasymmicta from closely related species above in Protoparmeiliopsis.