During the Second Tibetan Plateau Scientific Expedition and Research Program, we discovered that white terricolous lichenized fungal species of Buellia De Not. were widely distributed across the Tibetan Plateau. After examining their morphology, chemistry and phylogeny, we describe Buellia alpina Xin Y. Wang & Li S. Wang, sp. nov. as new to science. It is present in alpine meadows, and is characterized by its effigurate thallus, distinct linear marginal lobes, cover of thick white pruina and four-spored asci. This is also the first report of Buellia elegans Poelt and Buellia epigaea (Pers.) Tuck from China. The Buellia epigaea-group has previously been characterized by white and often effigurate thalli that occur mainly on soil. However, our results show that species in this group actually belong to two distinct clades. This conclusion is based on analyses of the nuITS region and the combined regions dataset (nuITS-nuLSU-mtSSU-β-tubulin). We discuss differences in morphology, anatomy, chemistry and ecology among the putative Buellia epigaea-group. Detailed descriptions and figures for the three species from China and a key for species of Buellia epigaea-group are provided.

Lichenized fungi, nuITS-nuLSU-mtSSU-β-tubulin, phylogenetic analysis, terricolous, Tibetan Plateau

The lichen genus Buellia De Not. (Caliciales, Caliciaceae) comprises approximately 400 species worldwide (Bungartz et al. 2007). Buellia s.l. is characterized by a crustose thallus, black lecideine apothecia, Bacidia-type asci, brown ascospores with one or more septate and reddish-brown or rarely hyaline hypothecia. Several other genera, which were previously included in Buellia s.l. (such as Amandinea M. Choisy, Diplotomma Flot. and Tetramelas Norman), have since been segregated based on their morphology, anatomy, chemistry and ecological environment (Scheidegger 1993; Marbach 2000; Nordin 2000). However, there remain other Buellia s.l. species with distinct morphological characters currently placed in groups instead of defined genera. Two examples are the species related to Buellia aethalea (Ach.) Th. Fr. or B. epigaea (Pers.) Tuck, which are currently treated as aethalea-group or epigaea-group, respectively (Poelt and Sulzer 1974; Scheidegger 1993). Their current classification is based solely on external morphology, without the support of molecular data, therefore strict phylogenetic relationships between Buellia s.l. species remain unclear.

More than 64 species of the genus Buellia s.l. were previously reported from China, mostly located in the Tibetan Plateau region (Wei 2020; Wang et al. 2020). During the Second Tibetan Plateau Scientific Expedition and Research Program (STEP), a large number of additional lichen specimens were collected, including Buellia epigaea-group species.

The Buellia epigaea-group contains seven species; these are characterized by white and often effigurate thalli that occur mainly on soil. Buellia epigaea, which was reported from Europe, is the core species of this group. Buellia asterella Poelt & Sulzer and B. elegans were reported from Europe, and B. zoharyi Galun was reported from Asia and Europe by Poelt and Sulzer (1974). Trinkaus and Mayrhofer (2000) published a revision of the four species above. A further three new species were described from Australia: Buellia dijiana Trinkaus, B. georgei Trinkaus, Mayrhofer & Elix, and B. lobata Trinkaus & Elix (Trinkaus et al. 2001). B. epigaea, B. dijiana and B. georgei formed a monophyletic clade, but data is still lacking for the remaining species (Grube and Arup 2001).

The aim of this study is to determine which Buellia epigaea-group species are distributed in China and whether they form a monophyletic clade. For this purpose, we carried out a phylogenetic study of the Buellia epigaea-group based on four loci.

The nuITS matrix (478bp) comprised 55 sequences including 15 newly generated sequences for new species and new records. The combined regions dataset (478bp for 43 nuITS sequences; 740bp for 27 mtSSU sequences; 899bp for 33 nuLSU sequences; 755bp for 23 β-tubulin sequences) comprised 126 terminals, including 31 newly generated sequences (Table 2). In the two phylogenetic analyses, eight representative monophyletic genera (Acolium (Ach.) Gray, Amandinea, Buellia s.str., Calicium Pers., Diplotomma, Pyxine Fr., Tetramelas, Thelomma A. Massal.) were selected from Caliciaceae Chevall. and six Physciaceae Zahlbr. species were selected as the outgroup. The results of the phylogenetic analyses showed that the species in the Buellia epigaea-group formed two clades (Figs 1, 2).

Figure 1. 

Phylogenetic relationships of Caliciaceae based on a Maximum Likelihood analysis of the nuITS matrix. Species positioned in clade 1 and clade 2 belong to the Buellia epigaea-group. Maximum Likelihood bootstrap values and posterior probabilities are shown near the nodes. New species and records are shown in bold.

Figure 2. 

Phylogenetic relationships within Caliciaceae, based on a Maximum Likelihood analysis of a combined regions dataset (nuITS-nuLSU-mtSSU-β-tubulin). Species positioned in clade 1 and clade 2 belong to the Buellia epigaea-group. Maximum Likelihood bootstrap values and posterior probabilities are shown near the nodes. New species and records are shown in bold.

In clade 1: B. dijiana, B. georgei and B. epigaea formed an independent clade with strong support (100% BS and 1.00 PP in Figs 1, 2). The specimens designated as B. epigaea (including one sequence from GenBank) clustered as a single lineage with high support (96% BS and 1.00 PP in Fig. 1). These specimens had identical morphological and chemical characters to those described for B. epigaea, and thus have been confirmed as a new record for China.

In clade 2: specimens here described as B. alpina formed a well-supported sister clade to B. zoharyi (100% BS and 1.00 PP in Figs 1, 2). These two species are distinctively different in their anatomical and chemical characteristics. We therefore recognize B. alpina as a new species within Buellia epigaea-group. The collections designated as B. elegans also formed a highly supported monophyletic lineage, clustering with sequences downloaded from GenBank (96% BS and 1.00 PP in Fig. 1). This constitutes the first record of B. elegans from China. These three species (B. zoharyi, B. alpina and B. elegans) formed a monophyletic clade with strong support (100% BS and 1.00 PP in Figs 1, 2). Clade 2 was sister to the genus Tetramelas (previously included in Buellia s.l.), which also contains alpine terricolous species.

Although species in Buellia epigaea-group share common characters, there are still additional diagnostic traits which could be used to distinguish between species within this group. The monophyletic clade 1 is formed by B. dijiana and B. georgei, together with B. epigaea. These three species share the characters of having no distinct marginal lobes and lacking atranorin. Within clade 1, only B. georgei has effigurate thalli; it also has short marginal lobes which often form rosettes. Both B. georgei and B. dijiana contain arthothelin acid and were described from Australia. However, their habitat differs: B. georgei occurs primarily on soft limestone or calcareous outcrops but never directly on calcareous soil, whereas B. dijiana is present on soil in open mallee vegetation (Trinkaus et al. 2001). In contrast, B. epigaea lacks secondary metabolites and could be reliably recognized by its crusty thallus, which is often uneven to wrinkled.

We propose a new species: Buellia alpina. It was clustered with B. zoharyi and B. elegans within clade 2. The common features of clade 2 are: having slim effigurate thalli covered with granulose pruina, obvious marginal lobes and always containing atranorin. The most distinctive features of the new species B. alpina are: heavily white pruinose apothecia and four-spored asci. B. elegans is similar to B. zoharyi in its external morphology. However, B. elegans can still be reliably distinguished from B. zoharyi, based on the ornamentation of ascospores. B. elegans has a loosely regulate surface (Fig. 3B and Fig. 4B), whereas the surface of B. zoharyi is microfoveate (Fig. 3G). In addition, the two species differ in their secondary metabolites: B. zoharyi contains atranorin, stictic acid and norstictic acid, while B. elegans has four chemotypes (Trinkaus and Mayrhofer 2000). One of these chemotypes (atranorin and 2’-O-methylperlatolic acid) is widely distributed in Asia, and was detected in most of the specimens from Yunnan, Qinghai and Xizang Provinces, China.

Figure 3. 

Ornamentation of ascospores A Buellia alpina B Buellia elegans C Buellia epigaea D Buellia lobata E Buellia dijiana F Buellia asterella G Buellia zoharyi H Buellia georgei (A–C were drawn by Qiu Yi Zhong D–H are from Trinkaus and Mayrhofer 2000; Trinkaus et al. 2001).

Figure 4. 

Ornamentation of ascospores (6000× magnification photograph under scanning electron microscope) A Buellia alpina B Buellia elegans C Buellia epigaea Scale bars: 2 µm (A–C).

In addition to the species discussed above, Buellia epigaea-group also contains the species B. asterella and B. lobata. These have not been included in this phylogenetic study due to the lack of available sequences. Morphologically, B. asterella and B. lobata are similar to B. alpina in their possession of four mature ascospores within each ascus (Trinkaus and Mayrhofer 2000; Trinkaus et al. 2001). B. alpina differs from these two species by its heavily white pruinose apothecia, granular pruina on the thallus surface and atranorin content. Furthermore, B. alpina has Callispora-type ascospores (with lateral (subapical) thickening, always with tapering ends). However, B. asterella and B. lobata both have fine pruina on their thallus surface and Buellia-type ascospores (lacking distinct wall thickening). B. asterella contains atranorin, norstictic acid and trace quantities of stictic acid, whereas B. lobata contains atranorin and thuringione.

The Buellia species in this study have all been classified as belonging to the Buellia epigaea-group, based on their terricolous habitat and distinct morphological characters of white and effigurate thalli. However, the phylogenetic trees in this study suggest that this group is not monophyletic. Thus, the previous definition of Buellia epigaea-group may be artificial, without support from molecular data. Phylogenetic study of both a single region (nuITS) and combined regions (nuITS-nuLSU-mtSSU-β-tubulin) showed that both clades do not group together. Therefore, the fundamental concept of the Buellia epigaea-group requires further research, including additional samples from across its global distribution.

In conclusion, species of Buellia epigaea-group share common characters which can be reliably recognized. There are distinct morphological and chemical differences which could be used to distinguish between different species in this group. Ornamentation of ascospores is a useful character by which to distinguish species in Buellia epigaea-group (Figs 3, 4; Table 3).

Taxonomy

 Buellia alpina Xin Y. Wang & Li S. Wang, sp. nov.

MycoBank No: 843376

Fig. 5

Diagnosis

The species is distinguished from its closest relatives B. elegans and B. zoharyi by its linear lobate thallus, heavily pruinose apothecia and lobes, Callispora-type ascospores and four-spored asci.

Type

China. Xizang Prov.: Lasa Ci., Namucuo Nature Reserve, on soil beside a lake, 30°46'46"N, 90°52'24"E, alt. 4730 m, 28 Sep. 2016, L.S. Wang et al. 16-53720 (KUN-Holotype; SDNU-Isotype).

Description

Thallus effigurate, lobate and linear, lobes tightly aggregated, 0.5–1.5 mm wide, prothallus absent; upper surface white to grayish white, dull, covered with granulose pruina; medulla white, non-amyloid (I–). Apothecia sparse to dense, sometimes aggregate, adnate to the thallus, lecideine, margin covered with white pruina which resemble lecanorine apothecia; disc black, roundish, (0.3–)0.5–1.4(–1.6) mm in diam., heavily pruinose, roundish when immature, marginal part becoming wavy and irregular when mature; margin persistent; exciple dispersa-type (Bungartz et al. 2007), dark brown, without aeruginose pigments (HNO₃–); epihymenium brown to dark brown; hymenium hyaline, 80–100 µm tall, without oil droplets, paraphyses simple to moderately branched, apically swollen, with a brown pigment cap; hypothecium dark brown; asci oval-clavate, Bacidia-type, four-spored; spores 1-septate, hyaline when young, turning brown when mature, Callispora-type (Bungartz et al. 2007), ellipsoid, with tapering ends, proper septum narrow, not thickening during spore ontogeny, (13–)15–20(–22) × (6–)7–9(–10) µm. Pycnidia not seen.

Chemistry

Thallus K+ yellow, C–, PD–, UV–, medulla I–; containing atranorin.

Distribution and ecology

This species is mainly distributed in alpine meadows of the Tibetan Plateau, growing on soil within meadows, between elevations of 4700–5000 m.

Etymology

The epithet “alpina” refers to the alpine distribution of this species.

Note

This new species could be distinguished from all other Buellia species by its linear lobate thallus, covered with granulose pruina, black lecideine apothecia with heavy whitish pruina, four-spored asci and its alpine distribution. It might be misidentified as subsquamulose or subfoliose species of Squamarina Poelt, but could be distinguished by the white thickened edges and hyaline simple ascospores.

Figure 5. 

Morphology of Buellia alpina (16-53720 KUN) A thallus on soil within meadow B black lecideine apothecia covered with white pruina C the section of apothecium, exciple dispersa-type D ascospores with 1-septate, Callispora-type, with tapered ends E mature ascus containing four spores, Bacidia-type F young ascus containing four spores. Scale bars: 2 mm (A); 0.5 mm (B); 50 µm (C); 10 µm (D–F).

Specimens examined

China. Xizang Prov.: Lasa Ci., Namucuo Nature Reserve, on soil beside a lake, 30°46'46"N, 90°52'24"E, alt. 4730 m, 28 Sep. 2016, L.S. Wang et al.16-53737.

 Buellia elegans Poelt, Nova Hedwigia 25(1–2): 184–186 (1974)

Fig. 6

Type

Italy. Ad terram calcaream supra Clavennam (Madèsimo), Anzi M. (M! -Holotype).

Description

Thallus effigurate with distinct marginal lobes slim, 0.5–1 mm wide, the edge usually separated from the substrate and clearly foliaceous, thallus radiate, 1–2 cm in diam., prothallus absent; upper surface white, dull, usually covered with granular pruina; the upper cortex about 20 µm thick, with granular crystals, and the lower surface light brown to white, without cortex; medulla white, without calcium oxalate crystals. Apothecia sparse, lecideine; disc and margin black, sometimes lightly pruinose, roundish, 0.3–1.0 mm in diam., immersed and smooth when young but adnate and convex when mature; margin persistent; exciple thick, dispersa-type, without aeruginose pigments (HNO₃–); epihymenium brown to dark brown; hymenium hyaline, 70–90 µm tall, without oil droplets, paraphyses simple to moderately branched, apically swollen, with a brown pigment cap; hypothecium dark brown; asci oval-clavate, Bacidia-type, eight-spored; spores 1-septate, hyaline when young, turning brown when mature, Buellia-type, ellipsoid, not thickening during spore ontogeny, 15–22 × 7–10 µm. Pycnidia not seen.

Figure 6. 

Morphology of Buellia elegans (16-51770 KUN) A thallus on soil within meadow B lecideine apothecia C the section of apothecium, exciple dispersa-type D ascospores with 1-septate, Buellia-type E mature ascus containing eight spores, Bacidia-type. Scale bars: 2 mm (A); 1 mm (B); 50 µm (C); 10 µm (D, E).

Chemistry

Thallus K+ yellow, C–, KC–, PD–, UV+ yellow, medulla I–; containing atranorin and norstictic acid (trace) or atranorin and 2’-O-methylperlatolic acid.

Distribution and ecology

This species is mainly distributed in open and dry soil or soil over rock or within meadows between elevations of 1400–4730 m. This species has been recorded in Asia, Afghanistan, Europe and North America (Thomson, 1997). In China, it is mainly distributed in Gansu, Ningxia, Qinghai, Xizang and Yunnan Provinces.

Note

This is a new record for China, and is unique among species of Buellia due to its effigurate thallus, marginal lobes linear and slim, branched near the tips. It resembles folicolous species of Physconia Poelt, but could be differentiated by its slim lobes and lack of lower surface. It has a wide distribution across the Tibetan Plateau, especially in arid deserts and meadows. Four chemotypes of the species were previously reported (Trinkaus and Mayrhofer 2000). Only two chemotypes have been detected in Chinese materials: atranorin and 2’-O-methylperlatolic acid account for the majority, atranorin and norstictic acid (trace) constitute only a small proportion.

Selected specimens examined

China. Gansu Prov.: Jiayuguan Ci., Xigou, mineral, on soil, 39°39'34"N, 97°56'15"E, alt. 2198 m, 28 May 2018, L.S. Wang et al. 18-59611; Yumen Ci., meadow along the route from Yumen to Yuerhong, on soil, 39°57'45"N, 96°39'23"E, alt. 2395 m, 27 May 2018, L.S. Wang et al. 18-59513. Ningxia Prov.: Zhongwei Ci., Shanpotou, Mengjiawan, on soil, 37°36'12"N, 104°55'06"E, alt. 1403 m, 18 Sep. 2010, D.L. Niu et al. 10-0089. Qinghai Prov.: Wulan Co., desert along the route from Wulan to Delingha, on soil, 37°02'08"N, 98°12'29"E, alt. 3072 m, 20 May 2018, L.S. Wang et al. 18-58303; Dulan Co., Xiangjia Vil., on sandy rock, 36°00'53"N, 97°44'36"E, alt. 3056 m, 15 Sep. 2020, L.S. Wang et al. 20-68266. Xizang Prov.: Dazi Dis., Bangdui Vil., on soil, 29°44'06"N, 91°24'55"E, alt. 3709 m, 16 Jul. 2019, L.S. Wang et al. 19-64615; Basu Co., beside Ranwu Lake, on soil over rock, 29°23'34"N, 96°50'20"E, alt. 3901 m, 15 Jul. 2019, X.Y. Wang et al. (XY19-278; XY19-272); Geji Co., beside S301 road, on soil, 32°14'47"N, 82°10'27"E, alt. 4514 m, 21 Jul. 2019, L.S. Wang et al. 19-63808; Bomi Co., along the route to Basu Co., on soil, 29°40'31"N,96°12'38"E, alt. 2920 m, 10 Nov. 2018, L.S. Wang et al. 18-62336; Langkazi Co., Simila Mt., on soil over rock, 28°50'37"N, 89°51'54"E, alt. 4343 m, 24 Jul. 2019, X.Y. Wang et al. XY19-1372; Sangri Co., Sangri Town, on soil over rock, 29°17'29"N, 92°05'30"E, alt. 3595 m, 30 Jul. 2019, X.Y. Wang et al. (XY19-1899; XY19-1907); Jangzi Co., Simila Mt., on soil over rock, 28°50'30"N, 89°51'48"E, alt. 4223 m, 24 Jul. 2019, X.Y. Wang et al. XY19-2308; Jiangda Co., Kakong Vil., on soil over rock, 31°20'22"N, 98°08'01"E, alt. 3785 m, 23 Sep. 2020, L.S. Wang et al. 20-68931. Yunnan Prov.: Deqin Co., Benzilan Vil., on soil over rock, 28°10'27"N, 99°22'53"E, alt. 2007 m, 26 Sep. 2020, L.S. Wang et al. 20-69241; Deqin Co., Benzilan Vil., beside JinSha river, on soil, 28°11'36"N, 99°21'08"E, alt. 2108 m, 19 Aug. 2018, L.S. Wang et al. 18-60340; Deqin Co., Benzilan Vil., on soil, 28°13'38"N, 99°19'20"E, alt. 2110 m, 3 Jul. 2012, L.S. Wang et al. 12-34754.

 Buellia epigaea (Pers.) Tuck. Gen. lich.: 185 (1872)

Fig. 7

Type

Germany. Hesse, ad terram inter muscos non procul a Monte Meissner, 1794, Persoon (H-Ach-Isotype, not seen).

Description

Thallus terricolous, tightly attached to the substrate, upper surface white or greyish white, usually with white fine pruina, thallus crusty, uneven to wrinkled, 0.2–1 mm thick, prothallus absent; the upper cortex 60–150 µm thick, with granular crystals, pith completely interspersed with Ca oxalate crystals; medulla white. Apothecia sparse to dense, lecideine, but usually surrounded by a thalline collar (pseudolecanorine); disc black, always with finely white pruina, roundish, 0.5–1.0 mm in diam., mostly flat, rarely slightly convex; young apothecia immersed and margin with finely white pruina breaking out broadly, mature apothecia adnate and margin absent or not obvious; exciple aethalea-type, up to 50 µm thick, without aeruginose pigments (HNO₃–); epihymenium brown to dark brown; hymenium hyaline, 60–80 µm tall, without oil droplets, paraphyses simple to moderately branched, apically swollen, with a brown pigment cap; hypothecium hyaline to light brown, up to 100 µm high; asci oval-clavate, Bacidia-type, eight-spored; spores 1-septate, hyaline when young, turning brown when mature, with tapering ends, Callispora-type, often curved, not thickening during spore ontogeny, 12–20 × 6–10 µm. Pycnidia not seen.

Figure 7. 

Morphology of Buellia epigaea (XY19-2294 KUN) A thallus growing on the soil B lecideine apothecia with white pruina, surrounded by a thalline collar (pseudolecanorine) C the section of apothecium, exciple aethalea-type D ascospores with 1-septate, Callispora-type E ascus Bacidia-type. Scale bars: 2 mm (A); 1 mm (B); 50 µm (C); 10 µm (D, E).

Chemistry

Thallus K–, C–, KC–, PD–, UV–; without secondary metabolites.

Distribution and ecology

This species mainly occurs on open and dry soil, soil within meadows or on soil over rock, between elevations of 2300–4700 m. This species has been recorded in Asia, Europe and North America (Trinkaus and Mayrhofer 2000). In China, it is mainly distributed in Gansu, Qinghai and Xizang Provinces.

Note

The species is a new record for China; it could be distinguished from all the other terricolous Buellia species reported in China by the combination of the following characteristics: thallus white crustose, uneven to wrinkled, always covered by finely white pruina, apothecia pseudolecanorine, the ornamentation of the ascospore surface densely areolate and rough, pycnidia rare, Callispora-type ascospores and absence of secondary metabolites. This species is close to Tetramelas species in phylogeny and similar in morphology, but could be distinguished by absence of the secondary metabolites 6-O-methylarthothelin or related xanthones, and pseudolecanorine apothecia covered with white pruina.

Selected specimens examined

China. Gansu Prov.: Sunan Co., along the route from Linze to Sunan, on soil over rock, 38°52'26"N, 99°44'21"E, alt. 2294 m, 29 May 2018, L.S. Wang et al. 18-58766; Sunan Co., along the route from Linze to Sunan, on soil over rock, 38°52'47"N, 99°43'57"E, alt. 2296 m, 29 May 2018, L.S. Wang et al. 18-59699. Qinghai Prov.: Gonghe Co., meadow beside Qinghai Lake, on soil within meadow, 36°33'26"N, 100°28'45"E, alt. 3431 m, 18 May 2018, L.S. Wang et al. 18-59162. Xizang Prov.: Qushui Co., Niedang Vil., on soil, 29°30'24"N, 90°56'17"E, alt. 3527 m, 22 Jul. 2019, X.Y. Wang et al. XY19-1234; Qushui Co., Niedang Vil., on soil over rock, 29°30'22"N, 90°56'15"E, alt. 3624 m, 22 Jul. 2019, X.Y. Wang et al. XY19-1218; Langkazi Co., entrance to Karuola Glacier, on soil over rock, 28°53'54"N, 90°13'32"E, alt. 4774 m, 24 Jul. 2019, X.Y. Wang et al. XY19-2294.

We express our sincere thanks to the M herbarium for providing type specimens and digital images. This study was supported by grants from the Flora Lichenum Sinicorum (31750001), the Second Tibetan Plateau Scientific Expedition and Research Program (STEP) (2019QZKK0503), Youth Innovation Promotion Association CAS (2020388), Yunnan Young & Elite Talents Project, National Natural Science Foundation of China (31970022) and State Key Laboratory of Phytochemistry and Plant Resources in West China (P2020-KF08).