Phenotypic analyses of Circinaria contorta specimens During morphological and anatomical studies several differences were found within the studied material, that allowed us to distinguish three morphological groups. The first group was marked as “group A” (Fig. 1a–d) and included samples with convex, rounded to angular, olive-grey to olive-brown, matt, and slightly white pruinose areoles [(0.5–)0.8–1.2(–1.8) mm diam.] that were crowded in the centre and usually dispersed at the thallus margin. The apothecia are very frequent, immersed within areoles (0.2–1.2 mm diam.), crater-like, with thin and radially cracked on the inside edge, thalline margin. The discs are rounded to irregular and white pruinose. The hymenium is colourless (120–180 μm in height), with submoniliform paraphyses and an olive-brown epihymenium. Asci are 4–6-spored, with subglobose ascospores [(15–)18–25(–28) µm diam., n = 125]. Pycnidia occur rarely and contain filiform conidia (7–11 × 1 μm, n = 56). All the analysed samples of group A contained aspicilin as a secondary metabolite. This group included the largest number of examined specimens, and the group is widely distributed throughout Poland, occurring both on natural calcareous rocks and on artificial, base-rich substrata.
Circinaria contorta specimens studied; morphotypes from group A: a Poland (KRA-428, isolate C. contorta 54), b Poland (UGDA L-11404, isolate C. contorta 78), c Poland (KRAM L-64316, isolate C. contorta 17), d Poland (UGDA L-11514, isolate C. contorta 80); morphotypes from group C: e Poland (UGDA L-13404, isolate C. contorta 81), f Poland (WRSL, isolate C. contorta 217).
The second group was designated as “group B” (Fig. 2a–f). This group represents samples with flat, rounded to angular, chalky white and densely pruinose areoles (0.5–2.5 mm diam.), partially aggregated in groups or singly dispersed. Apothecia are not always developed and are immersed in the juvenile stage but sessile and distinctly lecanorine when older (0.5–2.0 mm diam.). The thalline margins of apothecia are thin initially, then distinct, thick, raised and densely white pruinose. Discs are rounded to irregular and white pruinose. The hymenium is colourless (120–200 μm in height), with submoniliform paraphyses and an olive-brown epihymenium. Asci are 4–6-spored with subglobose ascospores [(20–)24–28(–30) µm diam., n = 70]. Pycnidia are uncommon and produce short conidia (4–8 × 1 μm, n = 16). Aspicilin was detected within all of the analysed samples marked as group B. Specimens of this group were collected from warm and sunny habitats on the Wyżyna Częstochowska Upland and Pieniny Mountains, areas of natural limestone rocks occurring in Poland. None of the specimens were found on artificial substrata.
Circinaria contorta subsp. albida subsp. nov. specimens studied; morphotypes from group B: a, b Poland (WRSL, isolate C. contorta 196), c Poland (WRSL, isolate C. contorta 211), d Poland (WRSL, isolate C. contorta 213); e Apothecium section. Poland (WRSL, isolate C. contorta 189); f Apothecium section after HNO3 treatment. Poland (WRSL, isolate C. contorta 220).
The third group is described herein as “group C” (Fig. 1e, f). These consist of specimens with a moderately dark thallus divided into olive-brown to grey-brown, matt, and not pruinose areoles (0.4–1.2 mm diam.). The areoles are angular, flat to slightly convex, with slightly raised margins. The areoles are crowded in the centre and rarely dispersed at the thallus margin. The apothecia are common, sessile or rarely immersed (0.4–0.8 mm diam.). The thalline margin is thin to quite thick, radially cracked and white pruinose. The discs are rounded to irregular and also white pruinose. The hymenium is colourless (130–160 μm in height) with submoniliform paraphyses and an olive-brown epihymenium. Asci contain 4 to 6 subglobose ascospores [(18–)22–24(–26) µm diam., n = 48]. Pycnidia were not found. No chemical substances were detected in the majority of the analysed material, with aspicilin rarely observed. Specimens of this group were collected from natural limestone rocks and on artificial, base-rich substrata in different regions of Poland.
In addition to the above three groups of morphotypes whose features were relatively uniform, there were a few specimens that did not form a homogeneous group, with diverse phenotypic characteristics (marked as group X). These included, among others, specimens with a continuous, areolate, olive-grey or white-grey thallus (Fig. 3a, b), as well as specimens with a thallus composed of thick, grey-brown squamules. Therefore, it was impossible to assign them to any of the three aforementioned morphotypes. The results of morphological and chemical analyses of all specimens used in the study are summarized in the Supplementary Table S1 online.
Circinaria contorta specimens studied; morphotypes from group X: a Poland (WRSL, isolate C. contorta 230), b Ukraine (KRAM L-49450, isolate C. contorta 37).
Phylogenetic analyses Phylogenetic analysis based on nucITS rDNA markers showed that most specimens labelled as C. contorta and collected in European countries (Austria, Estonia, Finland, Germany, Poland, Sweden, and Ukraine) and one from the USA (C. contorta 23) formed a strongly supported monophyletic clade I, closely related to C. hoffmanniana (S. Ekman & Fröberg ex R. Sant.) A. Nordin, C. serenensis (Cl. Roux & M. Bertrand) A. Nordin, and C. podoliana Szczepańska, Rodr. Flakus, Urbaniak & Śliwa (Fig. 4, Supplementary Fig. S1 online). However, some specimens labelled as C. contorta were found outside this clade. Clade II consisted of two specimens from Greece (C. contorta 4 and 5) from which only nucITS rDNA data were available, but this lineage was highly supported in both IQ-tree and Bayesian analyses; these specimens presumably represent a different taxon or even two taxa. As we did not have access to these specimens, and the sequences did not cluster with other species, we could not resolve their taxonomic affinity. Another specimen labelled as C. contorta 22 (lineage III) originated from Turkey and it also represented another unknown lineage which seems to be related to C. mansourii (Sohrabi) Sohrabi, C. pakistanica Fayyaz, M. S. Iqbal, Afshan & Khalid, and C. shimlaensis A. Noor, Saba & W. Akram. Moreover, specimens collected in the USA (clade IV) did not represent Circinaria contorta. This clade is related to Aspicilia cyanescens Owe-Larss. & A. Nordin and other species belonging to this genus (Supplementary Fig. S1 online). Therefore, these specimens probably belong to the genus Aspicilia. However, more detailed analyses of these specimens are required; this was beyond the scope of the present study.
The maximum likelihood tree based on the concatenated dataset (including aligned nucITS rDNA, mtSSU rDNA and MCM7 markers) from different Circinaria spp. Two representatives of Aspicilia cinerea (L.) Körb. were used as outgroup taxa. Bootstrap support values from IQ-tree analysis (≥ 70) and PP values from Bayesian analysis (≥ 0.95) are given near the branches on the phylogenetic tree. The names of species are followed by their sample numbers. Type and epitype specimens are marked in bold. Clades/lineages I, IA, II, III and IV are indicated on the branches. Representatives of unknown species labelled as Circinaria contorta are marked in yellow, green, or blue. Subclade IA with representatives of Circinaria contorta subsp. albida is marked in red.
Representatives of Circinaria contorta resolved in clade I, in our opinion, represent Circinaria contorta subsp. contorta, and consequently, we propose as an epitype (see Taxonomy) a specimen that was nested within this clade. However, this clade included intermingled specimens with different morphologies. We decided to recognize one highly supported lineage (subclade IA) at the subspecies level (see Taxonomy for C. contorta subsp. albida), as it comprised specimens with similar morphology (see Phenotypic analyses of Circinaria contorta specimens).
Species delimitation ABGD analysis of nucITS rDNA marker using default parameters predicted three species within the specimens of C. contorta. The largest group was formed by specimens from different European countries (Austria, Estonia, Finland, Germany, Greece, Poland, Sweden, and Ukraine) and one from the USA, while the second consisted of specimens collected in the USA (Clade IV), and the third was represented by a single specimen from Turkey (C. contorta 22 – lineage III). This delimitation was also supported by analysis of mtSSU rDNA, showing two putative species (one represented by C. contorta 16 collected in the USA and the second represented by specimens from clade I). ABGD analysis based on the MCM7 marker showed a single group, and in this case, specimen C. contorta 16 was grouped with sequences from clade I; however, this molecular marker was less variable than the nucITS rDNA and mtSSU rDNA markers.
Additional ABGD analysis of nucITS rDNA data using a gap width of 1.0 and based exclusively on sequences of C. contorta from clade I yielded initial and recursive partitions ranging from 1 to 50 OTUs. However, none of these partitions grouped specimens with similar morphologies analysed in this study, except specimens from clade IA that were usually treated as a single OTU.
Taxonomy Circinaria contorta (Hoffm.) A. Nordin, Savic & Tibell subsp. contorta (Fig. 5a, b) Mycologia 102(6), 1341 (2010). ≡ Verrucaria contorta Hoffm., Descr. Adumb. Plant. Lich. 1(4), 97 (1790). ≡ Lecanora contorta (Hoffm.) J. Steiner. Verh. Kaiserl.-Königl. zool.-bot. Ges. (Wien) 65, 199 (1915). ≡ Aspicilia contorta (Hoffm.) Körb., Syst. lich. germ. (Breslau) 166, (1855). Type: (lectotype (as iconolectotype) designated by Ekman & Fröberg 1988: 215). Hoffm. Descr. Adumb. Plant. Lich. 2, T22: 4 (1790). Epitype: (designated here) Germany, Bavaria, the southern slopes of the Altmühl river valley, above Obereichstätt town, 48°53′33″N, 11°8′6″E, on limestone rocks in xerothermic grassland, 25 Aug. 2024, K. Szczepańska 1446 (WRSL; nucITS rDNA, mtSSU rDNA and MCM7 sequences GenBank PV021014, PV021058 and PV130764, MycoBank MBT 10026460).
Epitype for Circinaria contorta subsp. contorta: a, b Thallus and apothecia (WRSL, isolate C. contorta 522); c, d Habitat – xerothermic grassland on south slopes of Altmühl river valley (Germany).
Description—thallus lichenized, crustose, areolate. Areoles convex, rounded to angular, (0.2–)0.8–1.2(–1.7) mm in diam., olive-grey to olive-brown, matt, white pruinose, crowded in the centre and dispersed at the thallus margin. Apothecia immersed, 1–2 per areole, 0.2–0.8 mm in diam., crater-like, thalline margin thin, white pruinose and radially cracked on the inside edge, disc rounded to irregular, white pruinose. Hymenium colourless, 140–180 μm in height, paraphyses submoniliform (2–3 globose apical cells), epihymenium olive-brown, N + clearly green, K + orange-brown (Caesiocinerea-green), hypothecium colourless, 25–35 μm tall. Asci 4–6-spored, ascospores hyaline, subglobose, (15–)18–25(28) µm diam. Conidia filiform, 7–11 × 1 μm.
Chemistry—aspicilin.
Ecology—on natural calcareous rocks and on artificial, base-rich substrata, in the lowlands and lower mountain localities, in warm and sunny places.
Distribution—widely distributed and very common species, occurring in the Northern Hemisphere including Asia, Europe and North America; reported from Austria15, Belarus16, the Czech Republic17, Denmark18, Finland19, France20, Germany13, Italy21, Netherlands22, Norway19, Poland23, Portugal24, Russia9, Spain24, Sweden19, Switzerland15, Syria25, and Ukraine26, as well as the United States and Canada27. Molecularly confirmed from Austria, Estonia, Finland, Germany, Poland, Sweden, Ukraine, and the USA.
Circinaria contorta subsp. albida Szczepańska & Guzow-Krzem. subsp. nov. (Fig. 2c). Holotype: Poland, Wyżyna Krakowsko-Częstochowska Upland, Zegarowe Skały Rocks, 50°26′12″N, 19°41′27″E, alt. 450 m, on limestone, 4 June 2014, K. Szczepańska 1221 (WRSL; nucITS rDNA and MCM7 sequences GenBank PV021004 and PV130754, MycoBank MB859222).
Diagnosis—differing from Circinaria contorta subsp. contorta in the larger chalky white, flat areoles (0.5–2.5 mm), larger apothecia (0.5–2.0 mm) and spores (24–28 μm), as well as higher hymenium (120–200 μm).
Etymology—the subspecific epithet refers to the densely pruinose, white areoles.
Description—thallus lichenized, crustose, areolate. Areoles flat, rounded to angular, chalky white and densely pruinose, 0.5–2.5 mm diam., partially aggregated in groups or singly dispersed. Apothecia immersed, older sessile and distinctly lecanorin, 1–2 per areole, 0.5–2.0 mm diam., thalline margin thin initially, then distinct and thick, raised, dense white pruinose, disc rounded to irregular, white pruinose. Hymenium colourless, 120–200 μm tall, paraphyses submoniliform (2–3 globose apical cells), epihymenium olive-brown, N + clearly green, K + orange-brown (Caesiocinerea-green), hypothecium colourless. Asci 4-6-spored, ascospores subglobose, (20–)24–28(–30) µm diam. Conidia 4–8 × 1 μm.
Chemistry—aspicilin.
Ecology—on natural calcareous rocks, in the lower mountain localities, in warm and sunny places.
Distribution—the area of natural limestone rocks in Poland (Wyżyna Częstochowska Upland and Pieniny Mountains). Probably frequent in Europe.
Comments—among the European taxa of Megasporaceae growing on limestone rocks with a white pruinose thallus, the new subspecies could be confused with Aspicilia candida (Anzi) Hue, A. subfarinosa (J. Steiner) Şenkard. & Sohrabi, Circinaria calcarea (L.) A. Nordin, Savić & Tibell, C. coronata (A. Massal.) Wirth, Hauck & M. Schultz ex Paukov & Alverdiyeva, C. serenensis (Cl. Roux & M. Bertrand) A. Nordin, Lobothallia farinosa (Flörke) A. Nordin, Savić & Tibell and Oxneriaria permutata (Zahlbr.) S.Y. Kondr. & Lőkös. Nevertheless, Aspicilia candida has smaller ascospores (14–16 × 10–16 μm) and a thalline margin that shows a K + yellow reaction13. A. subfarinosa has similarly very large spores, however, they are only 4 per ascus, in addition its thallus is continuous to finely rimose, and its conidia distinctly longer (6–18 × 0.6–1 μm)21. C. calcarea also has a continuous, finely rimose, not areolate thallus, with a distinctly visible prothallus12, while C. serenensis possesses a continuous thallus without dispersed areoles at the margin, black, epruinose or rarely faintly pruinose apothecial discs, as well as longer conidia (8.5–13.5 × 1 μm)21. C. coronata differs by an endolithic to hemiendolithic, poorly visible thallus and apothecia immersed in the rock21. Lobothallia farinosa is distinguished from the new subspecies by 8-spored asci and the distinctly smaller ascospores (10–16 × 7.5–10.5 μm)39. Oxneriaria permutata also has smaller ascospores (17–20 × 9–12 μm), as well as longer conidia (16–18 × 0.5 μm), and substictic acid is present in the thallus21.
