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Research Article
Two new calcicolous caloplacoid lichens from South Korea, with a taxonomic key to the species of Huriella and Squamulea
expand article infoBeeyoung Gun Lee, Jae-Seoun Hur§
‡ Baekdudaegan National Arboretum, Bonghwa, Republic of Korea
§ Sunchon National University, Suncheon, Republic of Korea
Open Access

Abstract

Pyrenodesmia rugosa Lee & Hur and Huriella aeruginosa Lee & Hur are described as new lichen-forming fungi from a calcareous mountain of South Korea. Pyrenodesmia rugosa is distinguishable from Pyrenodesmia micromontana (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. Huriella aeruginosa, the second new species, differs from ‘Squamuleachelonia 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.

Keywords

Biodiversity, phylogeny, saxicolous, taxonomy, Teloschistaceae

Introduction

Many lichens are only detected in calcareous areas, particularly for crustaceous lichens, as many plants are never found, except on calcareous rocks and soils (Watson 1918; Kossowska 2008; Pykälä et al. 2017). Caloplacoid lichens have been discovered in calcareous areas, such as Pyrenodesmia albopustulata (Khodos. & S.Y. Kondr.) I.V. Frolov & Vondrák, P. badioreagens (Tretiach & Muggia) Søchting, Arup & Frödén, P. concreticola (Vondrák & Khodos.) Søchting, Arup & Frödén, P. erodens (Tretiach, Pinna & Grube) Søchting, Arup & Frödén, ‘Squamuleachelonia, Squamulea galactophylla (Tuck.) Arup, Søchting & Frödén,‘Squamuleahumboldtiana Bungartz & Søchting, Squamulea parviloba (Wetmore) Arup, Søchting & Frödén and S. subsoluta (Nyl.) Arup, Søchting & Frödén (Khodosovtsev et al. 2002; Tretiach et al. 2003; Wetmore 2003; Tretiach and Muggia 2006; Vondrák 2008; Arup 2013; Bungartz et al. 2020). Many lichens have been introduced from the calcareous areas in Korea, such as Anema decipiens (A. Massal.) Forssell, Astroplaca loekoesiana S.Y. Kondr., Farkas, J.J. Woo & Hur, Caeruleum heppii (Nägeli ex Körb.) K. Knudsen & Arcadia, Clauzadea metzleri (Körb.) Clauzade & Cl. Roux, Clauzadea monticola (Ach.) Hafellner & Bellem., Collema auriforme (With.) Coppins & J.R. Laundon, C. cristatum (L.) Weber ex F.H. Wigg., Endocarpon pallidum Ach., Halecania pakistanica van den Boom & Elix, Heppia adglutinata A. Massal., Ionaspis epulotica (Ach.) Blomb. & Forssell, Lecania turicensis (Hepp) Müll. Arg., Lecanora albescens (Hoffm.) Branth & Rostr., L. semipallida H. Magn., Lemmopsis arnoldiana (Hepp) Zahlbr., Lichinella cribellifera (Nyl.) P.P. Moreno & Egea, L. stipatula Nyl., Placynthium tantaleum (Hepp) Hue, Porina fluminea P.M. McCarthy & P.N. Johnson, Psorotichia frustulosa Anzi, P. schaereri (A. Massal.) Arnold, Pterygiopsis affinis (A. Massal.) Henssen, Pyrenocarpon aff. thelostomum (Ach. ex J. Harriman) Coppins & Aptroot, Rufoplaca aesanensis S.Y. Kondr. & Hur, Staurothele frustulenta Vain., Synalissa ramulosa (Hoffm.) Körb., Thyrea confusa Henssen, Toninia poeltiana S.Y. Kondr., Lőkös & Hur, T. tristis (Th. Fr.) Th. Fr. and Verrucaria muralis Ach. (van den Boom and Elix 2005; Joshi et al. 2009; Schultz and Moon 2011; Aptroot and Moon 2014, 2015, Kondratyuk et al. 2016a, 2016b, 2017a, 2020). Although calcicolous caloplacoid lichens were little reported from Korea in the past, for example, Rufoplaca aesanensis, it is assumed that diverse caloplacoid lichens inhabit calcareous rocks and soils which were previously reported from just rock or soil without specifying specific rock or soil types.

This study describes two new calcicolous caloplacoid lichens in the genera Pyrenodesmia and Huriella. Qualified field surveys for the lichen diversity on the Baekdudaegan Mountains, the main mountain range stretching across the entire Korean Peninsula, were accomplished during the summer of 2020 and a few dozen specimens of caloplacoid lichens were collected in Mt. Seokbyung, a calcareous mountain (Fig. 1). We describe them as two new species, Pyrenodesmia rugosa and Huriella aeruginosa. The specimens are deposited in the herbarium of the Baekdudaegan National Arboretum (KBA), South Korea.

Figure 1. 

Specific collection site for two new species, representing the habitat/landscape and the location (black star mark).

Materials and methods

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.

Results and discussion

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 Squamuleasubsoluta 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.

Table 1.

Species list and DNA sequence information employed for phylogenetic analysis.

No Species ID (ITS) ID (mtSSU) ID (LSU) Voucher
1 Amundsenia approximata KJ789965 L08179 (LD)
2 Amundsenia austrocontinentalis KJ789962 21966 (HO)
3 Athallia holocarpa MG954144 Vondrak 18072
4 Athallia vitellinula FJ346556 Arup L03052
5 Caloplaca monacensis MG773668 MG773679 Malicek 8255
6 Caloplaca sp. KC611244 CBFS:JV6943
7 Erichansenia sauronii KC179120 Sochting 7654
8 Huriella aeruginosa MW832829 BDNA-L-0001072
9 Huriella flakusii MT967442 Bungartz 4131 (CDS 28162)
10 Huriella flakusii MT967443 Bungartz 4157 (CDS 28188)
11 Huriella flakusii MT967444 Aptroot 65261 (CDS 31847)
12 Huriella loekoesiana KY614406 KoLRI 15423
13 Huriella loekoesiana KY614407 KoLRI 19017
14 Huriella loekoesiana KY614408 KoLRI 40141
15 Huriella loekoesiana KY614409 KoLRI 40236
16 Huriella loekoesiana KY614410 KoLRI 40238
17 Huriella loekoesiana MK499351 HKAS 102112
18 Huriella sp. MN108089 KRAM-L-70242
19 Kuettlingeria albolutescens KC179423 KC179502 MT952898 Arup L09030 (LD)
20 Kuettlingeria areolata MN305805 MN305825 MN305847 Vondrak 10843
21 Kuettlingeria atroflava MH104921 MH100775 Vondrak 8723 (PRA)
22 Kuettlingeria cretensis MH104925 MH100783 MH100751 Frolov s.n.
23 Kuettlingeria diphyodes MH104926 MH100785 MH100753 Frolov 1430
24 Kuettlingeria emilii KC416102 MH100787 MH100754 JV9358
25 Kuettlingeria erythrocarpa KC179427 KC179506 KC179173 Arup L07109 (LD)
26 Kuettlingeria neotaurica MN305807 MN305829 MN305849 Vondrak 7213
27 Kuettlingeria percrocata MH104931 MH100794 Vondrak 4634 (PRA)
28 Kuettlingeria soralifera MN305808 MN305830 MN305850 Vondrak 10813
29 Kuettlingeria aff. soralifera JN641781 CBFS:JV8325
30 Kuettlingeria teicholyta MH104935 MH100797 MH100767 Vondrak 6943 (PRA)
31 Kuettlingeria xerica MN305809 MN305831 MN305851 Vondrak 14544
32 Kuettlingeria aff. xerica HQ611275 CBFS:JV7618
33 Lendemeriella borealis MG954129 Vondrak 11073
34 Lendemeriella exsecuta MG954227 Spribille 24441
35 Lendemeriella nivalis MG954222 Spribille 29306
36 Lendemeriella reptans MH104934 MH100796 MH100766 Lendemer 48186 (NY)
37 Lendemeriella sorocarpa MG954132 Vondrak12695
38 Lendemeriella tornoensis MG954221 Spribille 29473
39 Olegblumia demissa KT220203 KT220221 KT220212 SK C65
40 Pyrenodesmia aetnensis EU639590 KT291476 X. Llimona (BCN)
41 Pyrenodesmia albopruinosa EF093577 MH100770 TSB 37658
42 Pyrenodesmia albopustulata MH104918 MH100771 MH100741 Vondrak 10463 (PRA)
43 Pyrenodesmia alociza EF090931 MH100772 MH100742 TSB 37735
44 Pyrenodesmia aractina GU723415 Bornholm 5907
45 Pyrenodesmia aractina GU723418 Bornholm 6911
46 Pyrenodesmia aractina MH104919 MH100773 Vondrak 6702 (PRA)
47 Pyrenodesmia atroalba MH104920 MH100774 Spribille s.n.
48 Pyrenodesmia badioreagens EF081035 MH100776 MH100745 TSB 36422
49 Pyrenodesmia bicolor MH104922 MH100777 MH100746 Vondrak 10373 (PRA)
50 Pyrenodesmia ceracea HQ234603 BM-6656
51 Pyrenodesmia chalybaea KC884498 MH100779 MH100747 CBFS:JV4059
52 Pyrenodesmia circumalbata MH104923 MH100780 MH100748 Halici s.n.
53 Pyrenodesmia concreticola KC884506 MH100781 MH100749 CBFS:JV9443
54 Pyrenodesmia duplicata HQ611272 TUR-V-7513
55 Pyrenodesmia erodens MH104927 MH100788 MH100755 Vondrak 12733 (PRA)
56 Pyrenodesmia haematites GU723420 MH100789 MH100756 Vondrak 7278 (PRA)
57 Pyrenodesmia haematites GU723421 JS280
58 Pyrenodesmia haematites MH104928 Vondrak 7278 (PRA)
59 Pyrenodesmia helygeoides MH104929 MH100790 MH100757 Frolov 1414
60 Pyrenodesmia micromarina NR_156257 CBFS:JV8199
61 Pyrenodesmia micromarina MH100791 MH100758 Vondrak 7236 (PRA)
62 Pyrenodesmia micromontana NR_158297 MH100792 MH100759 CBFS:JV9467
63 Pyrenodesmia microstepposa NR_156260 MH100760 CBFS:JV9141
64 Pyrenodesmia molariformis KC416145 MH100793 MH100761 Nadyeina 132 (KW)
65 Pyrenodesmia obscurella MH104938 MH100762 Vondrak 7641 (PRA)
66 Pyrenodesmia peliophylla MG733135 Jason Hollinger:16476
67 Pyrenodesmia pratensis MH104933 MH100795 MH100765 MIN 891605
68 Pyrenodesmia rugosa MW832828 MW832825 MW832904 BDNA-L-0001099
69 Pyrenodesmia transcaspica MH104936 MH100799 MH100768 Vondrak 9430 (PRA)
70 Pyrenodesmia variabilis KT291466 KT291514 KT291561 Ulf Arup L07196 (LD)
71 Shackletonia buelliae KC179117 Sochting 7583
72 Shackletonia siphonospora KC179121 Sochting 7883
73 Squamulea galactophylla KC179122 Morse 10997 (LD)
74 Squamulea kiamae KC179123 Kondratyuk 20480 (LD)
75 Squamulea parviloba KC179124 Wetmore 87830 (LD)
76 Squamulea squamosa MT967462 Moberg 8782 (UPS)
77 Squamulea squamosa KC179125 Karnefelt AM960105 (LD)
78 Squamuleasquamosa MT967465 Bungartz 7428 (CDS 37915)
79 Squamulea subsoluta AF353954 Arup L97072
80 Squamulea subsoluta DQ173238 Arup L97829
81 Squamulea subsoluta KJ133480 KoLRI 011067
82 Squamuleachelonia MT967448 Bungartz 4521 (CDS 28607)
83 Squamuleachelonia MT967451 Bungartz 9251 (CDS 46069)
84 Squamuleachelonia MT967452 Bungartz 6146 (CDS 34358)
85 Squamuleahumboldtiana MT967439 Buck 29560 (MIN)
86 Squamuleahumboldtiana MT967440 Bungartz 4711B (CDS 56235)
87 Squamuleahumboldtiana MT967441 Bungartz 9985 (CDS 47354)
88 Squamuleaoceanica MT967445 Yánez-Ayabaca 2023 (CDS 48373)
89 Squamuleaoceanica MT967446 Bungartz 10152 (CDS 47571)
90 Squamuleaoceanica MT967447 Bungartz 9857 (CDS 47195)
91 Squamuleaosseophila MT967455 Aptroot 65489 (CDS 32078)
92 Squamuleaphyllidizans MT967456 Aptroot 65468 (CDS 32057)
93 Squamuleaphyllidizans MT967457 Bungartz 4710 (CDS 28808)
94 Squamuleaphyllidizans MT967458 Bungartz 4158 (CDS 28189)
95 Squamuleasubsoluta KJ133481 KoLRI 012491
96 Squamulea’ sp. MG954160 Vondrak 18682
97 Usnochroma carphineum KC179468 KC179598 KC179259 Roux s.n.
98 Usnochroma scoriophilum JQ301664 JQ301496 JQ301560 P. & B. v.d. Boom 38386
Overall 98 38 33
Figure 2. 

Phylogenetic relationships amongst available species in the genus Pyrenodesmia, based on a Maximum Likelihood analysis of the dataset of ITS sequences. The tree was rooted with the sequences of the genera Caloplaca, Lendemeriella, Olegblumia and Usnochroma. Maximum Likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Pyrenodesmia rugosa is 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.

Figure 3. 

Phylogenetic relationships amongst available species in the genus Pyrenodesmia, based on a Maximum Likelihood analysis of the dataset of the mitochondrial small subunit (mtSSU) sequences. The tree was rooted with five sequences of the genera Caloplaca, Lendemeriella, Olegblumia and Usnochroma. Maximum Likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Pyrenodesmia rugosa is 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.

Figure 4. 

Phylogenetic relationships amongst available species in the genus Pyrenodesmia , based on a Maximum Likelihood analysis of the dataset of the nuclear large subunit ribosomal RNA (LSU) sequences. The tree was rooted with three sequences of the genera Lendemeriella and Usnochroma. Maximum Likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Pyrenodesmia rugosa is 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.

Figure 5. 

Phylogenetic relationships amongst available species in the genera Huriella and Squamulea, based on a Maximum Likelihood analysis of the dataset of ITS sequences. The tree was rooted with the sequences of the genera Amundsenia, Erichansenia and Shackletonia. Maximum Likelihood bootstrap values ≥ 70% and posterior probabilities ≥ 95% are shown above internal branches. Branches with bootstrap values ≥ 90% are shown in bold. The new species Huriella aeruginosa is 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.

Taxonomy

Pyrenodesmia rugosa B.G. Lee & J.-S. Hur, sp. nov.

MycoBank No: 839184
Fig. 6

Diagnosis

Pyrenodesmia rugosa differs from P. micromontana by thicker thallus (125–200 μm vs. 95–125 μm), rugose areoles (vs. flat areoles), larger apothecia (0.2–0.7 mm diam. vs. 0.2–0.4 mm diam.), shorter hymenium (60–70 μm vs. 80–100 μm), shorter hypothecium (50–55 μm vs. 80–100 μm) and narrower tip cells of paraphyses (3–4.5 μm vs. 5–6 μm).

Figure 6. 

Pyrenodesmia rugosa (BDNA-L-0001102, holotype) in morphology A–C habitus and apothecia. Rugose thallus brown with orange spots and without pruina, but black apothecia often white pruinose D–E zeorine apothecia with well-developed parathecium. Algal layers continue to the base and underlying the hypothecium F epihymenium K+ purple and tiny granules not dissolving in K G–K asci oblong to narrowly clavate with eight spores K in the lactophenol cotton blue L ascospores simple in the beginning and developed polarilocular at maturity M paraphyses anastomosing in lactophenol cotton blue. Paraphysis tips slightly swollen. Scale bars: 1 mm (A–C); 100 μm (D); 50 μm (E, F); 10 μm (G–M).

Type

South Korea, Gangwon Province, Gangneung, Okgye-myeon, Mt. Seokbyung (summit), 37°35.21'N, 128°53.87'E, 1,072 m alt., on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000902, with Athallia cf. vitellinula (Nyl.) Arup, Frödén & Søchting, Bagliettoa baldensis (A. Massal.) Vězda, Catillaria lenticularis (Ach.) Th. Fr. and Staurothele aff. succedens (Rehm) Arnold (holotype: BDNA-L-0001102!); same locality, on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000899, with Athallia cf. holocarpa (Hoffm.) Arup, Frödén & Søchting and Staurothele cf. rupifraga (A. Massal.) Arnold (paratype: BDNA-L-0001099; GenBank MW832828 for ITS, MW832825 for mtSSU and MW832804 for LSU).

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.

Apothecia abundant, scattered or concentrated in centre, rounded, often contiguous or even coalescent when mature, emerging on the surface of thallus, immersed or adnate, slightly constricted at the base, 0.2–0.7 mm diam. Disc flat when young and flat or concave when mature, often white pruinose, black, 200–300 μm thick; zeorine, margin persistent, slightly prominent, generally entire or rarely slightly crenulate, thalline margin paler to disc and showing brown colour, often inconspicuous due to locating below proper margin, proper margin concolorous to disc. Amphithecium present, with small crystals between algal cells, not dissolving in K, 80–130 μm wide laterally, algal layers continuous to the base and underlying the hypothecium, algal cells 5–15 μm diam., cortical layer hyaline with pale brownish pigment at periphery, 10–40 μm thick. Parathecium well-developed, hyaline, but grey with slightly brown pigment concolorous to epihymenium at periphery, 20–40 μm wide laterally and 50–90 μm wide at periphery. Epihymenium grey with slightly brown pigment, K+ purple, tiny granules abundant on surface, not dissolving in K, 5–10 μm high. Hymenium hyaline, 60–70 μm high. Hypothecium hyaline, base open and extending downwards, 50–55 μm high. Oil droplets present in upper hypothecium, but absent in hymenium. Paraphyses septate, often anastomosing, 2–2.5 μm wide, generally simple, but occasionally branched at tips, tips slightly swollen, not pigmented, 3.0–4.5 μm wide. Asci oblong to narrowly clavate, 8-spored, 52–60 × 14–18 μm (n = 5). Ascospores ellipsoid, 1-septate, polarilocular when mature or narrow septum remaining, hyaline permanently, 11–18 × 5.5–11 μm (mean = 14.1 × 7.6 μm; SD = 1.6(L), 1.0(W); L/W ratio 1.5–2.5, ratio mean = 1.9, ratio SD = 0.3; n = 105), septum 1.5–3.0 μm. Pycnidia not detected.

Chemistry

Thallus K–, KC–, C–, Pd–. Epihymenium K+ purple. Hymenium I+ blue. UV–. No lichen substance was detected by TLC.

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. micromontana by thicker thallus (125–200 μm vs. 95–125 μm), rugose areoles (vs. flat areoles), larger apothecia (0.2–0.7 mm diam. vs. 0.2–0.4 mm diam.), shorter hymenium (60–70 μm vs. 80–100 μm), shorter hypothecium (50–55 μm vs. 80–100 μm) and narrower tip cells of paraphyses (3–4.5 μm vs. 5–6 μm) (Frolov et al. 2016).

The new species is different from P. microstepposa by darker thallus (greyish-brown to pale brown vs. ochre, grey or grey-white), rugose thallus (vs. flat thallus), thinner thallus (125–200 μm vs. 85–370 μm), smaller algal cells (5–15 μm diam. vs. 13.5–20.5 μm diam.), presence of pruina on disc (vs. absence of it), absence of oil droplets in hymenium (vs. presence of it), greyish epihymenium (vs. brownish epihymenium), wider ascospores (11–18 × 5.5–11 μm with the L/W ratio of 1.5–2.5 vs. 13.6–18.4 × 6–7.9 μm with the ratio of 1.9–2.9) (Frolov et al. 2016).

The new species is distinguished from C. micromarina by darker thallus (greyish-brown to pale brown vs. ochre to grey), rugose thallus (vs. flat thallus), absence of pruina on thallus (vs. presence of it), shorter hymenium (60–70 μm vs. 90–100 μm), shorter septum (1.5–3 μm vs. 2.6–3.4 μm) and the habitat preference to mountain rocks (vs. coastal rocks) (Frolov et al. 2016).

Additional specimens examined: South Korea, Gangwon Province, Okgye-myeon, Mt. Seokbyung (summit), 37°35.21'N, 128°53.87'E, 1,072 m alt., on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000889, with Bagliettoa baldensis, Catillaria lenticularis, Fulgogasparrea decipioides (Arup) S.Y. Kondr., M.H. Jeong, Kärnefelt, Elix, A. Thell & Hur and Laundonia flavovirescens (Wulfen) S.Y. Kondr., Lőkös & Hur (BDNA-L-0001089); same locality, on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000909, with Bagliettoa baldensis, Rusavskia elegans (Link) S.Y. Kondr. & Kärnefelt and Verrucaria nigrescens Pers. (BDNA-L-0001109); same locality, on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000910, with Bagliettoa baldensis, Catillaria lenticularis and Laundonia flavovirescens (BDNA-L-0001110); same locality, on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000911, with Athallia cf. vitellinula, Bagliettoa baldensis, Lichenella sp. and Rusavskia elegans (BDNA-L-0001111); same locality, on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000913, with Athallia cf. vitellinula, Bagliettoa baldensis, Endocarpon sp., Laundonia flavovirescens, Lichenella sp. and Rusavskia elegans (BDNA-L-0001113); same locality, on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000916, with Caloplaca sp., Endocarpon sp., Lichenella sp. and Rusavskia elegans (BDNA-L-0001116).

Huriella aeruginosa B.G. Lee & J.-S. Hur, sp. nov.

MycoBank No: 839185
Fig. 7

Diagnosis

Huriella aeruginosa differs from ‘Squamuleachelonia by dark greenish-grey to grey thallus without pruina (vs. yellow orange to deep orange thallus with white pruina), gold to yellow-brown epihymenium (vs. orange epihymenium), larger ascospores (7.5–12 × 4.5–7.5 μm vs. 8–10.4 × 4.7–6.0 μm) and the chemistry (thallus K–, KC– and no substance vs. thallus K+ purple, KC± purplish and the presence of parietin, teloschistin, fallacinal, parietinic acid and emodin).

Figure 7. 

Huriella aeruginosa (BDNA-L-0001072, holotype) in morphology A–C habitus and apothecia. Thallus dark greenish-grey to grey with no pruina. Thalline margin of apothecia concolorous to disc D apothecia adnate or rarely sessile. Amphithecium well-developed, but parathecium inconspicuous. E thallus with dark green pigment layer under cortex F–G clavate asci containing 8-spores H ascospores generally ellipsoid, but occasionally globose, developing polarilocular in both types. Two blue coloured spores in lactophenol cotton blue. Scale bars: 1 mm (A–C); 100 μm (D);10 μm (E–H).

Type

South Korea, Gangwon Province, Gangneung, Okgye-myeon, Mt. Seokbyung (summit), 37°35.21'N, 128°53.87'E, 1,072 m alt., on calcareous rock, 17 June 2020, B.G.Lee & H.J.Lee 2020-000872, with Bagliettoa baldensis, Catillaria lenticularis, Endocarpon subramulosum Y. Joshi & Hur, Laundonia flavovirescens, Rusavskia elegans and Verrucaria nigrescens (holotype: BDNA-L-0001072!; GenBank MW832829 for ITS).

Thallus saxicolous (calcicolous), crustose, mainly areolate or slightly rimose, placodioid around edge, but without distinct lobes, thin, dark greenish-grey to grey, occasionally pale yellowish-grey when young, margin indeterminate or determinate when placodioid areoles are arranged around edge, vegetative propagules absent, areoles 0.3–0.7 mm diam., 150–200 μm thick; cortex hyaline with dark green pigment layer, 15–25 μm thick, cortical cells granular, coarsely anticlinally arranged, 5–10 μm diam., with epinecral layer, up to 5 μm thick; medulla 80–100 μm thick, below algal layer, with large crystals (materials of substrate possibly) and brown cells (dead algal cells possibly); photobiont coccoid, cells globose to oval, 5–25 μm. Small crystals in cortex, medulla and between algal cells, dissolving in K. Prothallus absent.

Apothecia abundant, scattered and not concentrated in centre, rounded, often contiguous when mature, emerging on the surface of thallus, immersed, adnate or rarely sessile, constricted at the base, 0.2–0.4 mm diam. Disc flat when young and flat or slightly convex when mature, not pruinose, orange from the beginning, 110–230 μm thick; margin persistent, even to disc or slightly prominent, generally entire or slightly crenulate, thalline margin concolorous to disc, proper margin inconspicuous. Amphithecium well-developed, with small crystals between algal cells, dissolving in K, 50–55 μm wide laterally, algal layers continuous to the base or solitarily remaining in amphithecium, algal cells 5–25 μm diam., cortical layer hyaline with gold to yellow-brown pigment concolorous to epihymenium at periphery, 15–20 μm thick. Parathecium inconspicuous, hyaline but gold to yellow-brown at periphery, ca. 10 μm wide laterally and ca. 20 μm wide at periphery. Epihymenium gold to yellow-brown, granular, pigment K+ wine red and dissolved, 10–20 μm high. Hymenium hyaline, 45–55 μm high. Hypothecium hyaline, 35–45 μm high. Oil droplets present, small, along paraphyses and more in the base of hymenium and hypothecium. Paraphyses septate, anastomosing, 2–3 μm wide, simple or branched at tips, tips swollen or slightly swollen, not pigmented, 3.5–5.5 μm wide. Asci clavate, 8-spored, 35–48 × 14–17 μm (n = 5). Ascospores generally ellipsoid, occasionally globose, 1-septate, polarilocular or narrow septum remaining, hyaline permanently, 7.5–12 × 4.5–7.5 μm (mean = 9.9 × 5.7 μm; SD = 0.9(L), 0.6(W); L/W ratio 1.2–2.3, ratio mean = 1.8, ratio SD = 0.2; n = 104), globose spores 7.5–9 × 7.0–9.2 μm (mean = 8.0 × 7.7 μm; SD = 0.8(L), 0.9(W); L/W ratio 1.0–1.1, ratio mean = 1.0, ratio SD = 0.1; n = 11). Pycnidia not detected.

Chemistry

Thallus K–, KC–, C–, Pd–. Apothecia K+ wine red. Epihymenium K+ wine red. Epihymenium and hymenium I+ blue. UV–. No lichen substance was detected by TLC.

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.

Table 2.

Comparison of the new species with two type species in Huriella and Squamulea.

Species Huriella aeruginosa Huriella loekoesiana Squamulea subsoluta
Thallus mainly areolate, rimose or placodioid around edge, but without lobes areolate (not squamulose) squamulose, areolate or subsquamulose, margin slightly lobed
Apothecia (mm in diam.) 0.2–0.4 0.2–0.4(–0.5) 0.1–0.6
Ascospores (μm) 7.5–12 × 4.5–7.5 (8.5–)9–11(–12) × (4.5)5–6 9.5–12.5 × 5.5–7
Molecular phylogeny Huriella Huriella Squamulea
Reference Kondratyuk et al. 2017b Nash III TH et al. 2007; Arup et al. 2013

The new species is unique with the key characteristics of green pigmented thallus (with a distinct green layer in a section) and the substrate preference to calcareous rocks amongst all Huriella species.

The new species is similar to ‘Squamuleachelonia, Squamulea galactophylla,‘Squamuleahumboldtiana, S. parviloba and S. subsoluta in the substrate preference to calcareous rocks. However, the new species is different from ‘Squamuleachelonia by dark greenish-grey to grey thallus without pruina (vs. yellow orange to deep orange thallus with white pruina), gold to yellow-brown epihymenium (vs. orange epihymenium), larger ascospores (7.5–12 × 4.5–7.5 μm vs. 8–10.4 × 4.7–6.0 μm) and the chemistry (thallus K–, KC– and no substance vs. thallus K+ purple, KC± purplish and the presence of parietin, teloschistin, fallacinal, parietinic acid and emodin) (Bungartz et al. 2020).

The new species differs from S. galactophylla by thallus colour (dark greenish-grey to grey vs. dirty white to yellowish-brown), flat to convex disc (vs. flat disc only), yellowish-orange apothecia (vs. cinnamon-brown apothecia), smaller ascospores (7.5–12 × 4.5–7.5 μm vs. 10–15 × 5–7 μm) (Fink 1935; Arup 2013).

The new species is distinguished from‘Squamuleahumboldtiana by dark greenish-grey to grey thallus without pruina (vs. yellow-orange to deep orange thallus with pruina), absence of prothallus (vs. presence of prothallus), larger ascospores (7.5–12 × 4.5–7.5 μm vs. 8.1–9.9 × 4.8–5.9 μm) and the chemistry (thallus K–, KC– and no substance vs. thallus K+ purple, KC± purplish and the presence of parietin, teloschistin, fallacinal, parietinic acid and emodin) (Bungartz et al. 2020).

The new species differs from S. parviloba by dark greenish-grey to grey thallus (vs. yellow-orange to orange thallus), absence of lobes (vs. short narrow elongated lobes around edge), convex and yellow-orange disc (vs. flat and deep orange disc), smaller ascospores (7.5–12 × 4.5–7.5 μm vs. 11–14 × 5.5–7 μm) and the chemistry (thallus K– vs. thallus K+ red) (Wetmore 2003; Nash III TH et al. 2007).

The new species is different from S. subsoluta by dark greenish-grey to grey thallus (vs. yellow-orange, orange to reddish-orange thallus), absence of prothallus (vs. black prothallus), flat to convex, yellow-orange apothecia (vs. flat to concave, deep orange apothecia) and the chemistry (thallus K– and no substance vs. thallus K+ red, the presence of parietin, fallacinal, emodin and teloschistin) (Wetmore 2003; Nash III TH et al. 2007).

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 TH 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).

1 Not directly on rock, but on lichen or bone 2
On rock 4
2 On lichen (Aspicilia) living on rock Squamulea nesodes
On bone 3
3 Thallus generally areolate, without blastidia, not pruinose Squamuleaosseophila
Thallus generally (sub)squamulose, blastidia abundant, not pruinose or faintly orange pruinose on thallus Squamuleaphyllidizans
4 On calcareous rocks 5
On siliceous rocks 10
5 Thallus pruinose 6
Thallus not pruinose 7
6 Thallus angular, areolate to subsquamulose, prothallus absent Squamuleachelonia
Thallus areolate or bullate, prothallus black when present Squamuleahumboldtiana
7 Thallus whitish, greyish or greenish 8
Thallus yellow-orange to orange 9
8 Thallus dirty whitish, disc cinnamon-brown Squamulea galactophylla
Thallus dark greenish-grey to grey, disc orange Huriella aeruginosa
9 Areole margins with small lobules Squamulea parviloba
Areole margins without lobules Squamulea squamosa (S. subsoluta)
10 With blastidia or soredia 11
Without blastidia or soredia 13
11 Thallus brownish-orange, apothecia rare, disc reddish to reddish-brown, ascospores 11–16 × 6–8 μm, isthmus 1–3 μm Squamulea kiamae
Thallus yellowish-orange to deep orange, apothecia common, disc concolorous to thallus or slightly deeper, ascospores 8.4–13.3 × 5–7.1 μm, isthmus 2.5–4.6 μm 12
12 Blastidia abundant, sometimes faintly orange pruinose on thallus, but not pruinose on disc Squamuleaphyllidizans
Soredia rarely present, rarely white pruinose on disc, but not pruinose on thallus Squamulea squamosa (S. subsoluta)
13 Thallus areolate to (sub)squamulose 14
Thallus areolate or bullate, but not squamulose 21
14 Prothallus distinctly blackened Squamuleaoceanica
Prothallus absent 15
15 Disc brownish to reddish or blackish 16
Disc orangish 19
16 Thallus orange, disc reddish, ascospores 11–14.2 × 5.9–7.5 μm Huriella flakusii
Thallus brownish, disc pale brown, brownish-orange to blackish-brown 17
17 Disc dark brown-orange to black-brown, hypothecium 20–30 μm high, ascospores 7–9 × 4.5–6.5 μm Huriella salyangiana
Disc pale brown to brownish-orange, hypothecium 50–150 μm high, ascospores 9–13 × 4.5–6 μm 18
18 Disc 0.4–0.9 mm diam., hypothecium 50–100 μm high, ascospores 9–13 × 5–6 μm Squamulea coreana
Disc 0.2–0.4 mm diam., hypothecium 100–150 μm high, ascospores 10–10.5 × 4.5–6 μm Squamulea uttarkashiana
19 Areole margins with small lobules Squamulea parviloba
Areole margins without lobules 20
20 Ascospores 8–10.4 × 4.7–6 μm, isthmus 2.1–3.3, not pruinose on disc Squamuleachelonia
Ascospores 8.4–13.3 × 5.2–7 μm, isthmus 2.5–4 μm, rarely pruinose on disc Squamulea squamosa (S. subsoluta)
21 Thallus yellow-orange to deep orange, prothallus black when present, ascospores 8.1–9.9 × 4.8–5.9 μm, isthmus 2.7–3.2 μm Squamuleahumboldtiana
Thallus yellow-brownish or yellow-greenish, prothallus absent, ascospores 9–15 × 5–8 μm, isthmus 2–5 μm 22
22 Apothecia 0.2–0.3 mm diam., disc dull brown, dull yellow to bright yellow 23
Apothecia 0.3–1 mm diam., disc orange, brownish-yellow to reddish-orange 24
23 Disc dull yellow to bright yellow, hymenium 50–60 μm high, hypothecium 20–30 μm high, ascospores 9–11 × 5–6 μm, isthmus 4–5 μm Huriella loekoesiana
Disc dull brown, hymenium 80–100 μm high, hypothecium 80–110 μm high, ascospores 13–14.5 × 7–8 μm, isthmus 3–4 μm Huriella upretiana
24 On mountain, thallus yellow-brown, disc orange, isthmus 3–4 μm Squmulea micromera
On coast, thallus dull green-yellow to yellow-brown, disc orange to red-orange, isthmus 2–3 μm Huriella pohangensis

Acknowledgements

This work was supported by a grant from the Korean National Research Resource Center Program (NRF-2017M3A9B8069471).

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