Two new calcicolous caloplacoid lichens from South Korea, with a taxonomic key to the species of Huriella and Squamulea

Abstract Pyrenodesmiarugosa Lee & Hur and Huriellaaeruginosa Lee & Hur are described as new lichen-forming fungi from a calcareous mountain of South Korea. Pyrenodesmiarugosa is distinguishable from Pyrenodesmiamicromontana (Frolov, Wilk & Vondrák) Hafellner & Türk, the most similar species, by thicker thallus, rugose areoles, larger apothecia, shorter hymenium, shorter hypothecium and narrower tip cells of paraphyses. Huriellaaeruginosa, the second new species, differs from ‘Squamulea’ chelonia Bungartz & Søchting by dark greenish-grey to grey thallus without pruina, gold to yellow-brown epihymenium, larger ascospores and thallus K– and KC– reaction. Molecular analyses employing internal transcribed spacer (ITS), mitochondrial small subunit (mtSSU) and nuclear large subunit ribosomal RNA (LSU) sequences strongly support the two caloplacoid species to be distinct in their genera. A surrogate key is provided to assist in the identification of all 20 taxa in Huriella and Squamulea.


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 programme (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 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), 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 and primers ITS5 and ITS4 (White et al. 1990), mrSSU1 and mrSSU3R (Zoller et al. 1999) and LR0R and LR5 (Rehner and Samuels 1994), 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 Macrogen (Seoul, Korea).

Phylogenetic analyses
All ITS, mtSSU and LSU sequences were aligned and edited manually using ClustalW in Bioedit V.7.2.6.1 (Hall 1999). All missing and ambiguously aligned data and parsimony-uninformative positions were removed and only parsimony-informative regions were finally analysed in MEGA X (Stecher et al. 2020). The final alignment comprised 878 (ITS), 900 (mtSSU) and 1701 (LSU) columns for Pyrenodesmia. In them, variable regions were 178 (ITS), 42 (mtSSU) and 618 (LSU). The phylogenetically-informative regions were 356 (ITS), 55 (mtSSU) and 98 (LSU). The final alignment for Huriella and Squamulea comprised 693 (ITS) columns. In them, variable regions were 78 (ITS). Finally, the phylogenetically-informative region was 246 (ITS). 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 HKY (Hasegawa, Kishino and Yano) model, as the appropriate model for nucleotide substitution, based on the Bayesian Information Criterion (BIC) (Schwarz 1978) as evaluated by 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 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 28190.

Phylogenetic analyses
Three independent phylogenetic trees for Pyrenodesmia and one independent phylogenetic tree for Squamulea were produced from 165 sequences (96 for ITS, 37 for mtSSU and 32 for LSU) from GenBank and four new sequences (two for ITS, one for mtSSU and one for LSU) for the new species (Table 1). Pyrenodesmia rugosa, a new species, was positioned in the genus Pyrenodesmia in all ITS, mtSSU and LSU trees. The ITS tree described that the new species was solely located without any clade. Several species closely positioned with the new species were Pyrenodesmia aractina (Fr.) S.Y. Kondr., P. bicolor (H. Magn.) S.Y. Kondr. and P. haematites (Chaub. ex St.-Amans) S.Y. Kondr., represented by a bootstrap value of 84 and a posterior probability of 0.73 (not shown) for the branch (Fig. 2). The mtSSU tree showed that the new species was located in a clade with Pyrenodesmia albopruinosa (Arnold) S.Y. Kondr. and P. micromontana, represented by a bootstrap value of 72 and a posterior probability of 1.0 for the branch (Fig. 3). The LSU tree depicted that the new species was positioned solely without any clade. Several species, such as Kuettlingeria cretensis (Zahlbr.) I.V. Frolov & Vondrák, K. neotaurica (Vondrák, Khodos., Arup & Søchting) I.V. Frolov, Vondrák & Arup, Pyrenodesmia albopustulata, P. chalybaea (Fr.) A. Massal., P. helygeoides (Vain.) Arnold, P. microstepposa (Frolov, Nadyeina, Khodos. & Vondrák) Hafellner & Türk, P. molariformis (Frolov, Vondrák, Nadyeina & Khodos.) S.Y. Kondr., P. pratensis (Wetmore) Frolov & Vondrák and P. variabilis (Pers.) A. Massal. are situated close to the new species (Fig. 4). Huriella aeruginosa, the second new species, was located in Huriella in the ITS tree. The ITS tree described that the new species was positioned in a clade with 'Squamulea' subsoluta and 'Squamulea' sp., represented by a bootstrap value of 35 (not shown) without a posterior probability as the Maximum Likelihood analysis did not match with the Bayesian Inference for the clade (Fig. 5). Although the two closely located sequences were named for Squamulea in the beginning, they are close to Huriella, not Squamulea. The two sequences are arranged in the genus Huriella with the new species. The phylogenetic analyses did not designate any species identical to the two new species in each genus Pyrenodesmia and Huriella. Thallus saxicolous (calcicolous), crustose, mainly areolate or slightly rimose, rugose, greyish-brown to pale brown, often with orange spots, margin indeterminate or determinate when placodioid areoles are arranged around edge, vegetative propagules absent, areoles 0.4-1.0 mm diam., 125-200 μm thick; cortex hyaline with pale brown pigment layer, pale brown pigment K+ purple, 10-40 μm thick, cortical cells granular, 5-10 μm diam., with epinecral layer, 5-7 μm thick; medulla 60-110 μm thick below algal layer or inconspicuous and algal layer shown just above substrate; photobiont coccoid, cells globose to oval, 5-15 μm diam., algal layer 50-70 μm thick. Small crystals present between algal cells, not dissolving in K. Prothallus absent.
Distribution and ecology. The species occurs on the calcareous rock. The species is currently known from the type collections.
Etymology. The species epithet indicates the lichen's thallus texture, rugose or wrinkled, which is the key characteristic distinguished from closely-related calcicolous species in the genus Pyrenodesmia.
Notes. The new speices is similar to P. micromontana, P. microstepposa and Caloplaca micromarina Frolov, Khodos. & Vondrák in having epilithic thallus without vegetative propagules, small apothecia generally less than 0.5 mm diameter and the substrate preference to calcareous rocks. The new species differs from P.   Table 1 provides the species related to the specific GenBank accession numbers and voucher information.
Distribution and ecology. The species occurs on the calcareous rock. The species is currently known from the type collection.
Etymology. The species epithet indicates the lichen's thallus colour, dark green, which is the key characteristic distinguished from all the species in the genus Huriella.
Notes. The morphological classification of the new species is not clear between Huriella and Squamulea because the new species has some characteristics for the former genus and others for the latter, i.e. the new species represents mainly areolate thallus without lobed margin and smaller apothecia for the former, whilst showing some squamulose thallus and wider ascospores for the latter ( Table 2). The molecular results concluded the new species classification into the former genus, Huriella.
The most distinctive characteristic of the new species is the thallus colour, i.e. dark greenish-grey to grey, which is different from all comparable calcicolous species in the genus Squamulea.

Key to the species of Huriella and Squamulea (20 taxa)
Although some species of Huriella have distinct characteristics, different from Squamulea, such as mainly areolate and non-squamulose thallus without lobes at margin, smaller apothecia and narrower ascospores (Kondratyuk et al. 2017b), those morphological taxonomic keys do not clearly separate the two genera concerning all known species in the genera. The morphological characteristics are assumingly based on the comparison between type species of the comparable genera, but several species do not correspond to the characteristics (e.g. Huriella aeruginosa, H. flakusii Wilk and H. salyangiana S.Y. Kondr. & Hur with squamulose thalli), although those species are classified in the genus Huriella in molecular phylogeny. Such a discrepancy between morphology and molecular phylogeny occur in Squamulea squamosa (B. de Lesd.) Arup, Søchting & Frödén and S. subsoluta as well. Both species are considered conspecific in morphology. Both species are very similar in morphology and ecology occurring together on the same rock. Whereas the only difference between them is that the former has a thalline margin and it is lacking in the latter (Nash III et al. 2007), the latter representing a permanent thalline margin from the Galapagos Islands as well (Bungartz et al. 2020). However, the two species are separated and located distant from each other in molecular results of this study (Fig. 5). Nevertheless, those are still considered conspecific in the key below as a taxonomic key is based mainly on ecology, morphology and chemistry. The genera Huriella and Squamulea should be more studied in the future and here a preliminary key is updated from previous taxonomic keys of Wetmore (2003) and Bungartz et al. (2020).