Revision of the Lichen Genus Phaeophyscia and Allied Atranorin Absent Taxa (Physciaceae) in South Korea

The genus Phaeophyscia Moberg, which belongs to the family Physciaceae, includes about 50 species, with 17 species reported in South Korea. This genus is characterized by a foliose thallus, Physcia/Pachysporaria-type ascospores, a paraplectenchymatous-type lower cortex, and lacking atranorin. In this study, about 650 specimens of Phaeophyscia aligned with the atranorin-absent groups collected from South Korea were re-examined. The taxonomy of these groups in South Korea requires revision based on the analyses of the morphology, chemistry, and molecular phylogeny. We infer that (1) each genus of the main foliose groups of Physciaceae forms a monophyletic clade, which also supports the separation of Phaeophyscia species with a prosoplectenchymatous lower cortex into the genus Physciella; (2) three atranorin-lacking genera were confirmed in South Korea: Hyperphyscia, Phaeophyscia, and Physciella, including a new combination named Physciella poeltii (Frey) D. Liu and J.S. Hur, and three new records from South Korea of Phaeophyscia hunana, P. leana, and P. sonorae; and (3) four species should be excluded from the lichen flora of South Korea: Hyperphyscia adglutinata, Phaeophyscia endococcina, Phaeophyscia erythrocardia, and Phaeophyscia imbricata.

Phaeophyscia, which was first described by Moberg, originated from the genus Physcia based on the ellipsoid conidia and lacking atranorin [1]. Later, Esslinger confirmed and extended Moberg's delimitation on Phaeophyscia, for example, a paraplectenchymatous lower cortex, and Physcia/Pachysporaria type spore [2], and then placed the species with a prosoplectenchymatous lower cortex into a new genus Physciella with the following species: Physciella denigrata, Physciella melanchra, and Physciella nepalensis [10]. This definition, however, was not fully accepted by all lichenologists [4,5,8].
Microorganisms 2019, 7, 242; doi:10.3390/microorganisms7080242 www.mdpi.com/journal/microorganisms South Korea is located in the southern half of the Korean Peninsula, and is dominated by mountains in the east (Gangwon province) and south and coastal plains and river tributary coastlines in the west. Since the first species of Phaeophyscia endococcina (= P. endococcinodes) was recorded in Korea [30,31], several species of the genus Phaeophyscia have been identified and reported since the 1990s. Park [32] reported 12 species with a simple description, and an increasing number of species were later found from the mainland and islands [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47]. Hur listed 17 species of Phaeophyscia [46]; later, this number decreased to 16 species in Moon [44], and both publications recorded two species of Physciella. In 2015, a hairy species of Phaeophyscia esslingeri was discovered in Gangwon Province, a medium-latitude temperate zone mainly occupied by mountains [41]. Prior to this study, a total of three genera containing 25 species have been reported in South Korea, and these species have the following characteristics: A paraplectenchymatous upper cortex, lacking atranorin, and Physciaor Pachysporaria-type ascospores.
In this study, we combined morphology, chemistry, and phylogenetic analysis with the aim of clarifying the relationship among the foliose genera in the Physciaceae family, and to investigate the constitution, distribution, and ecology of the atranorin-absent groups in Physciaceae more comprehensively.

Morphological and Chemical Studies
We examined approximately 650 specimens that were collected in South Korea from 2003 to 2017. All were deposited in the Korean Lichen Research Institute, Sunchon National University (KoLRI). The morphological characteristics and chemical spot tests were conducted under a dissecting microscope (Nikon SMZ 745T, Tokyo, Japan) and Olympus BX 50 microscope, and photographs were taken under a Carl Zeiss MicroImaging microscope with an Axio Cam ERc 5s imaging system (Carl Zeiss MicroImaging, GmbH 37081, Göttingen, Germany). All measurements based on the sections from the thalli and apothecia were recorded in water. The ascospore dimensions ranged from 20-80 spores from a single apothecium per specimen. The secondary metabolites were studied according to the spot tests in a solution (K = 10% aqueous KOH solution; C = saturated aqueous Ca(OCl) 2 ; KC = 10% aqueous KOH solution followed by saturated aqueous Ca(OCl) 2 ; P = 5% alcoholic p-phenylenediamine solution) and thin layer chromatography (TLC) in solvent C [48,49], and zeorine and atranorin were selected as controls.

DNA Isolation, PCR, and DNA Sequencing and Alignment
The total genomic DNA was extracted from the specimens using the NucleoSpin Plant II Kit (Clontech Laboratories, Mountain View, CA, USA) according to the manufacturer's instructions. The internal transcribed spacer (ITS) region was generated using the primers pairs ITS1F [50] and ITS4 [51]. We followed the protocols for amplification and sequencing by PCR [52]. Sequencing was conducted by the genomic research companies GenoTech (Daejeon, Korea) and Macrogen (Daejeon, Korea). Newly generated ITS sequences were complemented with the published sequences of Physciaceae from GenBank (Table S1). All raw sequences were assembled and edited using SeqMan (DNAstar packages, Madison, WI, USA) and BioEdit 7.09 (Carlsbad, CA, USA) [53], then aligned automatically with Mafft version 7.273 (Osaka, Japan) [54].

Phylogenetic Analysis
The maximum likelihood (ML) optimality criterion and Bayesian inferences (BI) were used to construct the phylogenetic trees. ML inferences were performed using RAxML v7.2.6 (Heidelberg, Germany) [55], using the GTR model. The bootstrap frequencies were estimated from the consensus tree built with 2000 trees obtained from nonparametric bootstrapping pseudoreplicates. Clades with an ML bootstrap value ≥70% were strongly supported. BI analyses were performed with MrBayes v3.1.2 (San Francisco, CA, USA) [56] using four chains and were run for 1 million generations. SYM + I + G were selected as the best-fitted substitution models based on the Akaike information criterion (AIC) using jModelTest 3.7 (San Francisco, CA, USA) [57]. The trees were sampled every 1000 generations. The phylogenetic trees were summarized using the sump and sumt commands with 20% burn-ins discarded. The Bayesian posterior probabilities (PP) were estimated from the frequencies of branches among all trees; clades with PP ≥ 0.95 were considered as being significantly supported. A phylogenetic tree was rooted with the Heterodermia species.

Genera Relationship
The ML consensus tree combined bootstrap and PP values on the nodes is presented in Figure 1. Each genus was represented by several species, which are not shown in the figure. The phylogenetic tree was rooted by the genus Heterodermia, and each clade inferring to one genus formed a single clade with high support. The genus Phaeophyscia is sister to Physciella and other atranorin-absent groups (e.g., Hyperphyscia, Anaptychia, and Physconia). All of the atranorin-absent groups with K− formed a big clade and are separate from the atranorin groups (Heterodermia and Physcia) with K+. The genus Physconia and Anaptychia are closely related to the genus Hyperphyscia. All these genera form a monophyletic group.  [56] using four chains and were run for 1 million generations. SYM + I + G were selected as the best-fitted substitution models based on the Akaike information criterion (AIC) using jModelTest 3.7 (San Francisco, CA, USA) [57]. The trees were sampled every 1000 generations. The phylogenetic trees were summarized using the sump and sumt commands with 20% burn-ins discarded. The Bayesian posterior probabilities (PP) were estimated from the frequencies of branches among all trees; clades with PP ≥ 0.95 were considered as being significantly supported. A phylogenetic tree was rooted with the Heterodermia species.

Genera Relationship
The ML consensus tree combined bootstrap and PP values on the nodes is presented in Figure  1. Each genus was represented by several species, which are not shown in the figure. The phylogenetic tree was rooted by the genus Heterodermia, and each clade inferring to one genus formed a single clade with high support. The genus Phaeophyscia is sister to Physciella and other atranorinabsent groups (e.g., Hyperphyscia, Anaptychia, and Physconia). All of the atranorin-absent groups with K− formed a big clade and are separate from the atranorin groups (Heterodermia and Physcia) with K+. The genus Physconia and Anaptychia are closely related to the genus Hyperphyscia. All these genera form a monophyletic group.   Consensus tree showing the phylogenetic relationships of the foliose major genera of the family Physciaceae based on the ITS (Internal Transcribed Spacer) sequence by applying maximum likelihood (ML) and Bayesian inference (BI). ML bootstrap (before the slash) and posterior probabilities from the Bayesian analysis are provided adjacent to the nodes. The species of each genus are concentrated as a single clade. Heterodermia was set as a root group. Figure 2 shows the phylogenetic tree with the species information. Seven genera with 42 species were included in the topology, which contained Phaeophyscia (17 sp.), Physciella (3 sp.), Hyperphyscia (3 sp.), Anaptychia (4 sp.), Physconia (5 sp.), Physcia (6 sp.), and Heterodermia (4 sp.). The Korean specimens were placed into three genera. Hyperphyscia crocata was strongly supported as belonging to Hyperphyscia, whereas Physciella melanchra and Physciella poeltii (= Phaeophyscia poeltii) were placed into Physciella. The specimens with a paraplectenchymatous lower cortex belonged to Phaeophyscia, but only a few internodes were highly supported.

Species Relationship of the Atranorin Absent Groups
Microorganisms 2019, 7, x FOR PEER REVIEW 4 of 24 specimens were placed into three genera. Hyperphyscia crocata was strongly supported as belonging to Hyperphyscia, whereas Physciella melanchra and Physciella poeltii (= Phaeophyscia poeltii) were placed into Physciella. The specimens with a paraplectenchymatous lower cortex belonged to Phaeophyscia, but only a few internodes were highly supported.

Taxonomy
Three genera (including 18 species), Hyperphyscia, Phaeophyscia, Physciella, were confirmed; these contained 17 species from South Korea and one species from Europe. Among them, three species, Phaeophyscia hunanna, Phaeophyscia leana, and Phaeophyscia sonorae, are new to South Korea; four species, Hyperphyscia adglutinata, Phaeophyscia endococcina, P. erythrocardia, and Phaeophyscia imbricata, were excluded from the lichen flora of South Korea. In addition, one combination, Physciella poeltii, was proposed based on the molecular and morphological characteristics. All the specimens examined are listed in List S1.
Key to the foliose genera in Physciaceae. 1a. Spore Physconia   Figure 3B). Chemistry: Thallus K−; medulla K+ violet; skyrin and unidentified substance (Rf = 6, in C system) present. Ecology: The species is found growing on bark. Distribution: Phaeophyscia hunana was recorded in China [14,15], and here reported for the first time in South Korea. Comments: This species is characterized by a small thallus with narrow lobes, lobules, orange medulla, and black lower surface, lacking atranorin, and containing skyrin and an unidentified substance.
This species is similar to P. fumosa Moberg, but differs in having lobules and in containing an unidentified substance; P. hunana resembles P. laciniata, a species that also has lobules, but the latter species has a much larger size and saxicolous. P. laciniata lacks cortical hairs on the thallus or lobe tips.  Phaeophyscia leana is most similar to P. hirtella and P. hirtuosa, but can be distinguished from the latter species by a white lower surface. This species resembles P. trichophora (Hue) Essl., but can be distinguished by the Physcia type spores, stipitate apothecia with sparse rhizines near the base, whereas P. trichophora frequently has sessile and strongly crenate apothecia with numerous rhizines near the base as well as Pachysporaria-type spores [2]. Phaeophyscia leana differs from P. sonorae by having rhizines near the apothecia base, and from P. constipate by having a foliose thallus and usually occurs on bark, whereas the latter species are usually fruticose and mixes with moss over soil.    (-23.5) × (7.5-)7.7-8.9-12(-12.2) µm, Q = 1.5-2.5, Q(m) = 2.1, n = 71; pycnidia common, brown to black spotted; conidia 2-3 × 1-1.5 µm ( Figure 3A,C-F). Chemistry: Thallus K−; medulla K−, C−, KC−, P−; no lichen substance detected. Ecology: This species was only found growing on bark. Distribution: Phaeophyscia leana was reported in North America [2] and newly reported in South Korea. Comments: Phaeophyscia leana is characterized by a slight maculate gray thallus, lacking asexual propagules, apothecia with sparse rhizines near the base, white medulla, pale to white lower surface with a paraplectenchymatous cortex, and an absence of lichen substances.
Phaeophyscia leana is most similar to P. hirtella and P. hirtuosa, but can be distinguished from the latter species by a white lower surface. This species resembles P. trichophora (Hue) Essl., but can be distinguished by the Physcia type spores, stipitate apothecia with sparse rhizines near the base, whereas P. trichophora frequently has sessile and strongly crenate apothecia with numerous rhizines near the base as well as Pachysporaria-type spores [2]. Phaeophyscia leana differs from P. sonorae by having rhizines near the apothecia base, and from P. constipate by having a foliose thallus and usually occurs on bark, whereas the latter species are usually fruticose and mixes with moss over soil. Phaeophyscia sonorae Essl.
Phaeophyscia sonorae is similar to P. leage, but lacks patchiness over the upper surface. This species resembles Physciella nepalensis and P. denigrata but differs in having a paraplectenchymatous lower cortex. Phaeophyscia sonorae can be distinguished from P. nashii by lacking soredia.
Thallus foliose, 2-4 cm diameter, lacking a hypothallus, closely adnate to the substratum; lobes 0.8-1.5 mm wide, often crowded or imbricated, broadened at the tip and blackened at the lobe tip margin; upper cortex grey-green to olive or brown, sorediate, soralia laminal, maculiform, occasionally orange, marginal or submarginal, frequently dense in the central part thallus; medulla golden to orange or white with an orange band in the lower medulla; lower cortex indistinct or not apparent; lower surface black, pale to brown at the lobe tips; apothecia and pycnidia not seen. Chemistry: Upper cortex K−, KC−; atranorin absent, skyrin present. Ecology: This species was found growing on trees, and together with Mikhtomia geumohdoensis and Dirinaria applanata. Distribution: The species has only been reported in Japan and South Korea. Comments: Hyperphyscia crocata is characterized by an appressed thallus without black hypothallus, contiguous lobes with orange medulla, laminal soralia, pale to brown lower surface lobe tip, and black lower surface in the center parts of the thallus. Hyperphyscia crocata is most similar to H. pandani, which has a wide distribution in North America, East Africa, and Australia [58,59]; they share similar orange medulla and a thallus with soralia, whereas the type specimen of H. pandani has a black hypothallus and narrower lobes (0.5-1 mm). Hyperphyscia crocata has been reported primarily from Jeju Island [33], and is also distributed on the mainland [45]. Phaeophyscia adiastola (Essl.) Essl.
Phaeophyscia endococcinodes is related to P. endococcina, which is common in North America, the Himalayas, Europe, and East Africa [2,3,18], but P. endococcina has Physcia-type spores, P. endococcinodes has spores of the Pachysporaria type, and the zeorine is consistently present in P. endococcina, but trace or absent in P. endococcinodes. This species may be also confused with P. erythrocardia, from which it can be distinguished by the cortex of amphithecium composed of bigger cells (more than 7 µm in diameter) [2,18], and the latter species has the spore of the Physcia type. Phaeophyscia endococcinodes can be distinguished from P. ciliata by the orange-red medulla containing skyrin.
Phaeophyscia exornatula resembles P. kairamoi (Vain.) Moberg, a species reported in North America and Europe, both have similar lobules or lobulated isidia, but the first differs from the latter by the absence of cortical hairs, which are found on the upper surface of lobes or on lobules in P. kairamoi. The species can be distinguished from P. sciastra (Ach.) Moberg by dorsiventral lobules and broader lobes, and differs from P. squarrosa by lacking zeorin. Ecology: This species is found frequently growing on the bark of Quercus spp. or sometimes on moss over bark. Distribution: Phaeophyscia hirtella is distributed in North America and South Korea [2,60]. Comments: Phaeophyscia hirtella is characterized by a whitish grey thallus without asexual propagules, slight maculate lobes with cortical hair near the margin and ends, black lower surface with black rhizines, white medulla, apothecia with numerous cortical hairs on the margin and rhizines near the base, Physcia-type spores, and an absence of lichen substance.
Phaeophyscia hirtuosa is most similar to P. hirtella, but can be distinguished from the latter species by the following characteristics: The absence of cortical hairs on the lobe or lobe margin or tips, whereas they are usually present in the latter species; in addition, the lobe of P. hirtuosa is much broader. This species resembles P. spinellosa, but differs in having larger spores and corticolous. Phaeophyscia hispidula (Ach.) Essl.
Thallus grey, orbicular to irregular, up to 7 cm in diameter; lobes irregularly branched, contiguous or imbricate, flat to concave, 1-3 mm wide; upper surface sorediate, soralia laminal, starting as pustules and sometimes reaching the margins, then becoming capitate; medulla white; upper surface black, covered with long black rhizines; upper and lower cortex paraplectenchymatous, cells isodiametric with dark brown walls in the lower cortex; apothecia not seen. Chemistry: Thallus K−; medulla K−, C−, KC−, P−; no lichen substance detected. Ecology: This species is found frequently growing on bark, and occasionally on rocks. Distribution: Phaeophyscia hispidula is widely distributed in Japan, China, North America, and South Korea. Comments: Phaeophyscia hispidula is characterized by broad lobes with capitate marginal or laminal soralia, white medulla, and a black lower surface. Representative specimens examined: Gangwon Province. Jeongseong County, Gangneung City, tourist pass toward peak Seokbyeongsan, 37 • (25.8) × (7.5)8.5-11.1-13.6(15.5) µm, Q = 1.6-2.4, Q(m) = 2.0, n = 27; pycnidia common, immersed into the thallus, brown to black; conidia ellipsoid, 2.5-3 × 1-1.5 µm long. Chemistry: Thallus K−; medulla K−; lichen substance absent. Ecology: The species is found growing on bark, moss, or rock. Distribution: Phaeophyscia primaria is distributed widely in China, Nepal, Japan, and South Korea [4,6,32]. Comments: This species is characterized by the distinctly concave, branched, and broad lobes (2-4 mm wide) without cortical hairs, marginal or sub-marginal granular to isidioid soredia, black lower surface with simple black rhizines, white medulla, apothecial margin without cortical hairs, Physcia-type spores, and an absence of lichen substance. The soredia of this species are variable, ranging from granular, elongate to isidiod, but usually numerous growing on the lobe margin, and if spread to the upper surface, it usually presents frequently on the young lobes and is much smaller than elongate or isidiod ones.
This species resembles Phaeophyscia ciliata, and was also treated as P. ciliata in Japan for a long time until the revision on this species, and three species were isolated [6]. These are similar in having a greyish thallus without asexual propagules, but the lobes of the P. primaria are concave and much broader (3-7 mm wide) than those of P. ciliata (<1.5 mm wide). The present species was recorded in the Sorak Mountain and Chiri Mountain in South Korea [32,34] and extends to Nepal, China, and Japan [4,6]. The species usually grows on the moss over bark or rocks.
Ecology: The species is usually found growing on the bark of Abies, Betula, and Quercus; one specimen occurs on rocks in South Korea. Distribution: Phaeophyscia pyrrhophora is distributed widely in China, Japan, South Korea, Nepal, and the eastern parts of Russia [4,14,18,33,46]. Comments: This species is characterized by broad, contiguous, or imbricate lobes (up to 5 mm in length), lacking asexual propagules, a black lower surface with a paraplectenchymatous lower cortex, apothecial margin without cortical hairs, spores of the Pachysporaria type, and containing skyrin, but lacking zeorine.
Phaeophyscia pyrrophora is similar to Physcia erythrocardia in having a similar thallus containing skyrin, but it can be distinguished from the latter species by a Pachysporaria-type spore rather than a Physcia type. In addition, this species lacks zeorin, which is constantly produced in Physcia erythrocardia [2,18]. This species differs from P. endococcinodes in having broader lobes, and is usually corticolous, whereas the latter is saxicolous and zeorine is occasionally present. Based on these points, the specimens recorded as P. endococcinodes [60] were found to be P. pyrrophora according to the morphological and chemical analyses. Phaeophyscia pyrrophora is apparently closely related to P. fumosa Moberg, a species reported in Kenya; however, the lobes of P. pyrrophora are concave and broader, whereas they are rather flat and narrower in P. fumosa. Thallus foliose, 1-3 cm in diameter; lobes dichotomously or irregularly branched, 0.5-1.5 mm wide; upper surface greenish gray or greenish brown, plane or more or less concave, epruinose, sorediate, soralia usually terminal or marginal, lip-shaped, occasionally laminal or capitate; thallus 110-200 µm thick; upper cortex paraplectenchymatous, 20-30 µm thick; algal layer continuous, about 25-40 µm thick, green alga, cells 7-14 µm in diameter; medulla orange or white with an orange band in the lower medulla, loose, 70-90 µm thick; lower cortex paraplectenchymatous, 25-30 µm thick; lower surface black or becoming pale to white toward the lobe tip, rhizinate; rhizines rather dense, black, occasionally pale to even white at tip, up to 2 mm long; apothecia not seen; pycnidia immersed into thallus, brown to black; conidia ellipsoid, 2-3.5 × 1.5-2.4 µm. Chemistry: Thallus K−; medulla K+ violet; skyrin present. Ecology: The species is found growing on the rocks and bark of Acer spp., Cornus controversus, Fraxinus sp., Lindera obtusata, Meliosma myriantha, Cedrus deodara, Cerasus, and Pinus densiflora. Distribution: Phaeophyscia rubropulchra is distributed widely in Asia, North America, Russia, and East Asia [2,4,7,41]. Comments: Phaeophyscia rubropulchra is characterized by well-developed lobes with terminal or marginal soralia, and orange-red medulla. Phaeophyscia rubropulchra is apparently closely related to P. endococcina and P. erythrocardia from which it can be distinguished by the presence of soredia. The species can be saxicolous and corticolous in Korea, when it occurs in a saxicolous and shading environment; the thallus is much darker and thicker; the orange color of the medulla is also much more obvious; the lower surface is almost black even toward to the lobe tips, whereas the thallus is usually gray to pale greenish, the medulla light orange, or even the center and lower part of medulla orange; that of the tip and upper parts are white, the lower surface is usually brown to black, or white at the lobe tip occasionally if found in the corticolous and exposed surroundings. Representative specimens examined: Gangwon Province. Jeongseong County, Gangneung City, tourist pass toward peak Seokbyeong Mt., 37 •  Thallus foliose, whitish grey, loosely attached substrate, 5-10 cm in diameter; lobes irregularly or dichotomously branched, discrete or imbricate, concave, 1-2 mm wide; thallus 100-200 µm thick; upper surface soredia, isidia and lobules absent, slightly pruinose sometimes; upper cortex paraplectenchymatous, 30-45 µm thick; algal layer 30-55 µm thick, algae cells green, 5-14 µm in diameter; medulla white, loose, 60-120 µm thick; lower surface black, usually becoming pale to brown toward the lobe tips, rhizinate; rhizines dense, black, simple, 1-4 mm long; lower cortex paraplectenchymatous, blackish brown, 20-30 µm thick; apothecia numerous if present, up to 4 mm in diameter; margin entire, cortical hair sparsely present on apothecia margin; disc reddish brown, flat to concave; amphithecium occasionally with retrosely hair near the base; hymenium 50-65 µm high; hypothecium hyaline or very pale straw-yellow, 40-50 µm thick; asci clavate, 8-spored, 56-73 × 10-13 µm; spores brown, Physcia type, 16.4-19.4-23 × 7.3-8.9-11.4 µm, Q = 2.0-2.6, Q(m) = 2.2, n = 65; pycnidia common, immersed into thallus, black; conidia ellipsoid, 2.5-3 × 1-1.5 µm. Chemistry: Thallus K−; medulla K−; lichen substance absent. Ecology: The species is found growing on saxicolous mosses. Distribution: Phaeophyscia spinellosa is distributed widely in Japan and South Korea [6,30]. Comments: Phaeophysica spinellosa is characterized by a whitish grey thallus without asexual propagules, a black lower surface with black rhizines, white medulla, apothecia with sparse cortical hairs on the margin, spores Physcia type, and an absence of lichen substances.
This species is similar to Phaeophyscia ciliata (Hoffm.) Moberg, but it can be separated clearly from P. ciliata by the broader and imbracate lobes and the presence of cortical hairs on the apothecial margin; Phaeophysica spinellosa resembles P. hirtuosa, an Eastern Asiatic species, both of them lack asexual propagules, have apothecia with cortical hairs on the margins, and produce spores of the Physcia type. In addition, P. spinellosa differs from P. hirtuosa in the much sparser hair around the apothecia and usually grows on rocks, whereas P. hirtuosa usually has very dense hairs on the apothecial margin and grows on bark in the mountains in Eastern Asia. Ecology: This species is found frequently growing on the bark of Quercus spp. or sometimes on moss over bark. Distribution: Phaeophyscia squarrosa is distributed in North America, Japan, and South Korea [17,60]. Comments: This species is characterized by broad lobes with numerous dorsiventral lobules near the margin, white medulla, brown to black lower surface of lobes covered with squarrosely branched rhizines together with simple ones, cup shaped apothecia with retrorsely hairy amphithecium at the base, Physcia-type spores, and contain zeorine as constant substances in the thallus.
The present species is closely related to P. ciliata and P. hispidula, from which it can be distinguished by the presence of lobules; Phaeophyscia squarrosa is most similar to P. exornatula, which has a wide distribution in Asia and lobules, but the lobules of the latter species come from submarginal pustules that usually break into soralia. In addition, P. exornatula lacks zeorin and can grow on rock or saxicolous mosses, whereas P. squarrosa has zeorine and grows frequently on the bark; this species may be confused with P. laciniata Mull. Arg., a species known from Costa Rica and North America; all of them have lobules, but it can be distinguished from the latter species by white medulla due to lacking skyrin. This species might be confused with P. primaria because they both have larger thalli without asexual propagules and share a similar habitat, but they differ in having squarrosely branched rhizines and constantly containing zeorine.
Phaeophyscia imbricata (Vain.) Essl., is a species recorded in Japan and North America [2,17,19,60], and recorded as containing zeorine with lobes, but the type specimen examined by Moberg [4] is not well-developed and does not contain zeorine. Consequently, the species was treated as P. imbricata (Vain.) Essl. as a variation of P. hispidula. Some lichenologists have used erect lobules as the identical characteristics for P. imbricata [2,60], but these erected lobules are also quite common in P. exornatula, which lacks zeorine. Eventually, all of the specimens cited by Wei & Hur. [60] and regarded as P. imbricata were placed into P. squarrosa in this study. Physciella melanchra (Hue) Essl.

Phylogeny Analysis
The foliose genera within the family Physciaceae were traditionally distinguished by their thallus, cortical structure, ascospores, and chemical substances [1,2,19,21,61,62]. Nordin et al. [63] selected parts of representative genera including the fruticose, crustose, and foliose groups, and combined the molecular and morphological data. Their results suggested that the foliose groups were Chemistry: Thallus K−; medulla K−, C−, KC−, P−; lacking secondary substances. Ecology: The species is found growing on rocks in South Korea, together with Phaeophyscia primaria, P. orbicularis, and P. exornatula.
Distribution: This species has a wide distribution in North America, Asia, Europe, and South Korea. Comments: Physciella melanchra is characterized by a gray thallus lacking atranorin and with laminal, punctiform soralia, a prosoplectenchymatous lower cortex, and white to tan lower surface. Physciella melanchra is similar to Hyperphyscia adglutinata by having soralia, pale lower surface, and prosoplectenchymatous lower cortex, but differs in having ellipsoid conidia; Physciella melanchra can be distinguished from P. chloantha by laminal soralia, whereas P. chloantha has marginal or terminal soralia. Representative specimens examined: Gyeongnam. Namhae, Mt. Mangeun, 34 •

Phylogeny Analysis
The foliose genera within the family Physciaceae were traditionally distinguished by their thallus, cortical structure, ascospores, and chemical substances [1,2,19,21,61,62]. Nordin et al. [63] selected parts of representative genera including the fruticose, crustose, and foliose groups, and combined the molecular and morphological data. Their results suggested that the foliose groups were gathered together, but species of Physciella were not included. We supplemented the ITS sequences of several species in Physciella with Heterodermia, Hyperphyscia, Physcia, Physconia, and Phaeophyscia, and then four characteristics (atranorin, ascospores type, and upper and lower cortex type) were used to examine the relationship among species. Seven genera separated from each other and formed a single clade. All were monophyletic. The genus Physciella is strongly supported as being a sister to Phaeophyscia and can be distinguished from the latter by having a prosoplectenchymatous lower cortex, which also provides support for separating these two genera based on the difference in the lower cortex [4,20]. Morberg [20] combined the species Physcia poeltii Frey into Phaeophyscia as Phaeophyscia poeltii (Frey) Nimis based on having a white lower surface and a prosoplectenchymatous lower cortex. According to our phylogenetic analysis, Phaeophyscia poeltii is in the Physciella clade, and by combining the cortex characters from Morberg's identification [20], we propose treating the new combination as Physciella poeltii (Frey) D. Liu and J.S. Hur.
Korean specimens lacking atranorin and with Physciaor Pachysporaria-type ascospores belong to three clades: Phaeophyscia, Physciella, and Hyperphyscia; the squamulose species with light orange medulla, Hyperphyscia crocata is close to Hyperphyscia confusa. This species shares the same type of lower cortex and conidia with H. confusa and Hyperphyscia adglutinata. In contrast, the specimens with ellipsoid conidia and a prosoplectenchymatous lower cortex formed a clade with the type species Physciella chloantha, which was not well separated from P. melanchra in terms of laminal soralia. The genus was divided into two large clades without support, and the species had orange medulla mixed with white medulla; however, more information was difficult to obtain due to the lesser support. Phaeophyscia squarrosa and Phaeophyscia constipata had white medulla, but P. pyrrhophora, P. encoccinodes, P. endococcina, and P. rubropulchra had orange medulla.

Taxonomy Revision of Phaeophyscia, Hyperphyscia and Physciella in South Korea
The Phaeophyscia species was first reported in South Korea [30] with two species, Physcia endococcina (= Phaeophyscia endococcina) and P. lithotea var. sciastra (Phaeophyscia sciastra), whereas Korean specimens of Phaeophyscia endococcina were re-identified as P. endococcinodes [42,60]. Previous studies have reported that the Phaeophyscia encoccinodes specimens were P. pyrrhophora based on a re-examination from the morphology and TLC results; P. encoccinodes was saxicolous and had smaller lobes [2,18], and specimens of P. erythrocardia were assigned to two different species: P. pyrrhophora and P. endococcinodes. These two species can be distinguished from P. erythrocardia by Pachysporaria-type spores, whereas P. erythrocardia has Physcia-type spores, zeorine is constantly present, and there are smaller amphithecium cortical cells [2,18]. As a result, Phaeophyscia endococcina and P. erythrocardia do not occur in South Korea. Phaeophyscia trichphora is characterized by Pachysporaria-type spores, whereas the specimens cited in Wei & Hur [60] have Physcia-type spores; all are now referred to as P. primaria.
The genera Hyperphyscia and Physciella share a similar lower cortex, prosoplectenchymatous if present, but Hyperphyscia can be distinguished from Physciella by the filiform conidia, whereas those of the latter species are ellipsoid. Hyperphyscia adglutinata is characterized by a small thallus with an indistinctly prosoplectenchymatous lower cortex and filiform conidia, and was reported in South Korea [43]. All the specimens cited are now referred to as Physciella melanchra based on the morphology and molecular data, and Hyperphyscia adglutinata should be excluded from the lichen flora of South Korea. The morphology of Physciella melanchra varies considerably in different substrates. The thallus is often orbicular on the rock, but is frequently irregular and smaller when found on bark.