MycoKeys 92: 45-62 (2022) er-reviewed open-access journal doi: 10.3897/mycokeys.92.83939 < MycoKkeys https://mycokeys.pensoft. net Launched to accelerate biodiversity research Taxonomy of Buellia epigaea-group (Caliciales, Caliciaceae), revealing a new species and two new records from China Min Ai'?, Li Juan Li?, Fiona Ruth Worthy'’, An Cheng Yin'?, Qiu Yi Zhong'”, Shi Qiong Wang’, Li Song Wang!?, Xin Yu Wang!” | Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, CAS, Kunming, Yunnan 650201, China 2 Yunnan Key Laboratory for Fungal Diversity and Green Development, Kunming Institute of Botany, CAS, Kunming, Yunnan 650201, China 3 Department of Botany and Molecu- lar Evolution, Senckenberg Research Institute, 60325 Frankfurt am Main, Germany 4 Kunming Botanical Garden, Kunming Institute of Botany, CAS, Kunming, Yunnan 650201, China Corresponding author: Xin Yu Wang (wangxinyu@mail.kib.ac.cn) Academic editor: Thorsten Lumbsch | Received 17 March 2022 | Accepted 16 July 2022 | Published 5 August 2022 Citation: Ai M, Li LJ, Worthy FR, Yin AC, Zhong QY, Wang SQ, Wang LS, Wang XY (2022) Taxonomy of Buellia epigaea-group (Caliciales, Caliciaceae), revealing a new species and two new records from China. MycoKeys 92: 45-62. https://doi.org/10.3897/mycokeys.92.83939 Abstract 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-6-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. Keywords Lichenized fungi, nuITS-nuLSU-mtSSU-§-tubulin, phylogenetic analysis, terricolous, Tibetan Plateau Copyright Min Ai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 46 Min Ai etal. / MycoKeys 92: 45-62 (2022) Introduction 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 morpho- logical 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 mor- phology, without the support of molecular data, therefore strict phylogenetic relation- ships between Buellia s.l. species remain unclear. More than 64 species of the genus Buellia s.|. 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 num- ber 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 re- ported 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) pub- lished 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 dis- tributed 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. Materials and methods Morphological and chemical analyses During STEP, 92 specimens of the Buellia epigaea-group were collected from the Qing- hai-Tibetan Plateau and deposited in the Lichen Herbarium, Kunming Institute of Botany, China (KUN). Morphological characteristics of thalli and apothecia were ex- amined under a dissecting microscope (Nikon SMZ 745T). Anatomical characteristics New species and records from China 47 of apothecia were examined under an optical microscope (Nikon Eclipse Ci-S). Photo- graphs were taken using a digital camera (Nikon DS-Fi2). Descriptions of the range of anatomical characteristics for each species were determined by the smallest and largest single values measured for all specimens. Thin-layer chromatography (TLC) was per- formed in order to identify secondary metabolites using solvent systems C (toluene: acetic acid = 85:15), according to Orange et al. (2001). DNA extraction, amplification and sequencing DNA was extracted from fresh apothecia or thallus pieces with a DNA secure Plant Kit (TIANGEN) according to the manufacturer’s instructions. Amplified gene mark- ers and their corresponding primers are shown in Table 1. PCR amplifications were achieved using 1.1 x T3 Super PCR Mix (TSINGKE) in a 25 uL total volume, con- taining 1 wL of genomic DNA, 1 pL of a 10 mM solution for each primer and 22 pL of 1.1 x T3 Super PCR Mix. The PCR program was: initial denaturation at 98 °C for 3 min, followed by 35 cycles of 98 °C for 10 s, 54-56 °C for 10 s, 72 °C for 15 s, fol- lowed by a final extension at 72 °C for 2 min. The PCR products were sequenced with the same amplification primers using Sanger technology by Tsingke Biotechnology Co., Ltd. (Kunming). Table |. Gene markers and primer pairs used in this study. Gene markers Primers Sequences of Primers 5’-3’ References nulTS ITS1F CTTGGTCATTTAGAGGAAGTAA Gardes and Bruns 1993 ITS4 TCCTCCGCTTATTGATATGC White et al. 1990 nuLSU LROR GTACCCGCTGAACTTAAGC Rehner and Samuels 1994 LR5 ATCCTGAGGGAAACTTC Vilgalys and Hester 1990 mtSSU SSU1 AGCAGTGAGGAATATTGGTC Zoller et al. 1999 SSU3R ATGTGGCACGTCTATAGCCC 6-tubulin Bt3-LM GAACGTCTACTTCAACGAG Myll 2001 Br10-LM TCGGAAGCAGCCATCATGTTCTT eS ye Phylogenetic analyses All newly obtained original sequences were edited manually using GENEIOUS v8.0.2. Their taxon name, voucher and GenBank accession number are shown in Ta- ble 2. All sequences, including those downloaded from GenBank, were aligned using MAFFT v7 with the option of E-INS-I (Katoh et al. 2005). Ambiguous regions were excluded using GBLOCKS (Talavera and Castresana 2007) with the default settings. Congruence between different gene regions was analyzed before combining. Bayesian inference (BI) and maximum likelihood (ML) were employed to determine the phy- logenetic relationships. The best-fit partition substitution models were selected based on the lowest Bayesian information criterion (BIC) using PARTITION FINDER 2 48 Min Ai etal. / MycoKeys 92: 45-62 (2022) (Guindon et al. 2010; Lanfear et al. 2012, 2017): nuITS dataset (TIM2e+G4) and the combined regions dataset (GTR+G for ITS1, ITS2 and mtSSU; GTR+I+G for 5.88, nuLSU and §-tubulin), respectively. ML analyses were performed with RAxML v8.2.12 (Stamatakis 2006). Bootstrap support values (MLBS) were estimated from the 70% majority rule tree of all saved trees obtained from 2000 non-parametric bootstrapping pseudo-replicates. BI analyses were performed with MrBayes v3.2.7 (Ronquist et al. 2012) running for 2 million gen- erations. The trees were sampled every 100 generations and the first 25% of the trees were discarded as burn-in, since the average SD of split frequencies had converged at the step of 20% of the total. Bayesian posterior probabilities (BPP) were obtained from the 95% majority rule consensus tree of all saved trees. The final trees were visualized in Fig Iree v1.4.0 (Rambaut 2012). The final matrices were submitted to TreeBASE: TB2: 29562 for nulTS and TB2: 29563 for the combined regions dataset. Results The nulTS matrix (478bp) comprised 55 sequences including 15 newly generated sequences for new species and new records. The combined regions dataset (478bp for 43 nulTS sequences; 740bp for 27 mtSSU sequences; 899bp for 33 nuLSU sequenc- es; 755bp for 23 $-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 Calici- aceae 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). 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 morpho- logical 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 distinc- tively 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 se- quences 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.1.), which also contains alpine terricolous species. New species and records from China 49 Table 2. Specimens used in this study, with taxon name, voucher and GenBank accession number. Newly obtained sequences are in bold font. ° os) gions’ dataset analysis. “NA” indicates that there is no sequence available. ’ indicates that the sample was not included in the combined re- Taxon Voucher Accession number nulTS mtSSU nuLSU 6-tubulin Acolium inquinans Wedin 6352 (UPS) AY450583 AY143404 AY453639 KX529023 Ac. karelicum Hermansson 16472 (UPS) KX512897 NA KX512879 NA Amandinea punctata 1 18-60759 (KUN) OL467351 NA NA NA Am. punctata 2 AFTOL 1306 HQ650627.1 NA DQ986756.1 NA Buellia alpina 16-53720 (KUN) OM914626 NA NA NA B. alpina 16-53737 (KUN) OM914627 NA OP060154 OM925561 B. dijiana - AF250788 NA NA NA B. disciformis \ EDNA09-01524 FR799139 NA NA NA B. disciformis 2 EDNAO09-02095 FR799136 NA NA NA B. disciformis 3 EDNA09-02116 FR799138 NA NA NA B. elegans 18-60340 (KUN) OM914622 NA OM935566 OM925559 B. elegans 20-68266 (KUN) OM914634 NA OM935569 OM925562 B. elegans XY19-272 (KUN) OM914624 NA OM935567 OM925560 B. elegans 18-59513 (KUN) OM914623 NA NA NA *B. elegans 18-62336 (KUN) OM914630 / / / *B. elegans XY19-1907 (KUN) OM914632 / / / *B. elegans XY19-1372 (KUN) OM914631 / / / *B. elegans XY19-2308 (KUN) OM914633 / / / *B. elegans 12-34754 (KUN) OM914625 / / / *B. elegans 10-0089 (KUN) OM914636 / / / *B. elegans 16-0084 (NXAC) MN103116 / / / *B. elegans Beck 242 (GZU) AY143411 / / / *B. elegans Leavitt 19085 MZ922074 / / / B. epigaea XY19-1218 (KUN) OM914628 OM913210 OM935568 NA B. epigaea XY19-2294 (KUN) OM914629 OM913211 NA NA *B. epigaea - AF250785 / / / *B. epigaea 18-59162 (KUN) OM914635 / / / B. georgei Trinkaus 356a (GZU) AJ421416 NA NA NA B. zoharyi \ SA2 MG592314 MG592321 MG592328 MG592346 B. zoharyi 2 MT30 MG592315 MG592322 MG592329 MG592347 B. zoharyi 3 SAG MG592316 MG592323 MG592330 MG592348 B. zoharyi 4 TE13 MG592317 MG592324 MG592331 MG592349 Calicium nobile 1 Tibell 21968 (UPS) KX512913 KX512988 KX529070 NA C. nobile 2 Tibell 23396 (UPS) KX512914 KX512987 KX529071 NA Diplotomma alboatrum | 18-60034 (KUN) MNG15696 OL467286 OL444781 OM925557 Di. alboatrum 2 18-60448 (KUN) MZ224658 OL467287 OL444782 OM925558 Di. venustum 1 18-58557 (KUN) OL467349 OL467284 OL444779 OM925555 Di. venustum 2 18-58102 (KUN) OL467350 OL467285 OL444780 OM925556 * Di. venustum 3 XY19-252 (KUN) OL467353 / / / Heterodermia speciosa Wetmore (S) KX512927 KX512975 KX512868 KX529000 He. vulgaris Frisch 11/Ug1226 (UPS) KX512928 KX512989 KX512857 NA Phaeophyscia ciliata Prieto (S) KX512929 KX512958 KX512886 KX529012 Ph. orbicularis Prieto 3012 (S) KX512930 KX512967 KX512876 NA Physcia aipolia Wedin 6145 (UPS) KX512931 AY143406 AY300857 KX529021 P tenella Odelvik and Hellstrém 0827 (S) KX512932 KX512974 KX512869 NA Pyxine coccoes Prieto (S) KX512936 KX512964 NA KX529010 Py. subcinerea - HQ650705 NA DQ883802 NA Py. sorediata Wetmore 91254 (S) KX512937 KX512973 KX512870 KX529001 Tetramelas chloroleucus Westberg 10-001 (S) KX512938 NA KX512875 KX529006 50 Min Ai et al. / MycoKeys 92: 45-62 (2022) Taxon Voucher Accession number nulTS mtSSU nuLSU 6-tubulin Te. geophilus 20-67496 (KUN) OL467354 OL467291 OL444785 OM925563 Te. pulverulentus Nordin 6368 (UPS) KX512940 KX512983 KX512860 KX528990 Thelomma mammosum 1 Tibell 23775 (UPS) KX512942 KX512954 KX512888 KX529016 Th. mammosum 2 Hernandez et al. 2002 (UPS) KX512943 KX512953 KX512851 KX529017 Th. santessonii 1 Nordin 4011 (UPS) KX512944 KX512951 KX512889 NA Th. santessonii 2 Nash 38262 (UPS) KX512945 KX512950 KX512890 NA Discussion Although species in Buellia epigaea-group share common characters, there are still ad- ditional 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 efhigurate thalli; it also has short marginal lobes which often form rosettes. Both B. georgei and B. dijiana con- tain 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. \t 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 atra- norin, 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. 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 New species and records from China 51 Py. sorediata Py. subcinerea | Pyxine Py. coccoes 96/1 Di. venustum 1. | 97/1) ' Di. venustum 2 1001} 'Di. venustum 3 Diplotomma Di. alboatrum 1 99/1'Dj. alboatrum 2 Ccliciaweae 1400/1 Ac. karelicum Ageia 97/1 Ac. inguinans 98/1 Th. mammosum 1 Th.mammosum 2 | theiomma 4100/1 Th. santessonii 2 Th. santessonii 1 99/1 B. disciformis 1 100/1 |B. disciformis 2 Buellia s. str. 82/0.67 B. disciformis 3 1400/1 Am. punetata 1 Am. punctata 2 400/1 ©. nobile 1 C. nobile 2 Te. geophilus 99/1 Te. chloroleucus ‘Tetramelas’ Te. pulverulentus — | B. zoharyi 1 100/41) 5. zoharyi 2 96/1 B. zoharyi 3 98/1 B. zoharyi 4 B. alpina 16-53720 100/1!B, alpina 16-53737 elegans 18-60340 . elegans 18-59513 . elegans XY19-272 . elegans 20-68266 Clade 2 elegans 18-62336 elegans XY19-1372 elegans XY19-1907 elegans XY19-2308 B. elegans 12-34754 B. elegans 10-0089 B. elegans 16-0084 B. elegans Leavitt 19085 B. elegans Beck 242 Amandinea Calicium 4100/1 DOOD DDD 96/1 gai B. dijiana B. georgei B. epigaea XY19-1218 B. epigaea XY19-2294 Clade 1 B. epigaea AF250785 B. epigaea 18-59162 99/1 He. speciosa He. vulgaris 100/1 96/1 P. aipolia 95/1L- P. tenella 4100/1 Ph. ciliata Ph. orbicularis Physciaceae 97/1 0.05 Figure |. Phylogenetic relationships of Caliciaceae based on a Maximum Likelihood analysis of the nul TS 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. 52 Min Ai et al. / MycoKeys 92: 45-62 (2022) Di. venustum 1 100/1]= Di. venustum 2 100/1 ¢ Di. alboatrum 1 Diplotomma 87/091 Di. alboatrum 2 89/0.77 Py. coccoes 56/0.76 100/1 Py. subcinerea | Pyxine Py. sorediata 100/1)C. nobile 1 aes C. nobile 2 | Calicium . B. disciformis 1 San cene 100/L L00/1\' B. disciformis 2 Buellia s.str. B. disciformis 3 100/1p—Am. punctata 1 | a eananba Am. punctata 2 B. zoharyi | 100/1f B. zoharyi 2 B. zoharyi 3 al B. zoharyi 4 B. alpina 16-53720 100/1} \00/1!B. alpina 16-53737 Clade 2 B. elegans 18-60340 100/1| B. elegans 20-68266 100/1 B. elegans 18-59513 B. elegans XY19-272 Te. geophilus 53/0.78 100/1 Te. chloroleucus ‘Tetramelas’ Te. pulverulentus 97/1) B. dijiana 100/1 B. georgei B. epigaca XY19-2294 Clade 1 96/0.98L B. epigaea XY19-1218 49/0.90 Th. mammosum 1 100/1 Th. mammosum 2 ; Thelomma 100/1 Th. santessonii 2 100/1 Th. santessonii 1 99/1p— Ac. Enquiniaias Acolim Ac. karelicum 100/1 He. speciosa 100/1 He. vulgaris 100/1 P. aipolia : 100/1 phe at Physciaceae 100/1 p— PA. ciliata Ph. orbicularis 0.03 Figure 2. Phylogenetic relationships within Caliciaceae, based on a Maximum Likelihood analysis of a combined regions dataset (nuITS-nuLSU-mtSSU--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. (lacking distinct wall thickening). B. asterella contains atranorin, norstictic acid and trace quantities of stictic acid, whereas B. /obata 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 (nulTS) and combined regions (nuI[TS-nuLSU-mtSSU-f-tubulin) showed that both clad- es do not group together. Therefore, the fundamental concept of the Buellia epigaea-group requires further research, including additional samples from across its global distribution. New species and records from China 25 me feast ors oe oe oo) == + 2 eetes = Gece a on oe Pree ee oe a > » “- » , PE eet wed hey “S « ie 7 at? as #ee, a ae Figure 3. Ornamentation of ascospores A Buellia alpina B Buellia elegans © 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 (6000x magnification photograph under scanning electron mi- croscope) A Buellia alpina B Buellia elegans C Buellia epigaea Scale bars: 2 um (A=C). 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. Ornamen- tation of ascospores is a useful character by which to distinguish species in Buellia epigaea-group (Figs 3, 4; Table 3). 54 Min Ai et al. / MycoKeys 92: 45-62 (2022) Table 3. Key characteristics of the Buellia epigaea-group. Species Apothecia| Exciple | Spores | Ornamentation| Major chemistry | Pycnidia| Parasitic of spores fungi B. alpina effigurate, lobes linear, flat, dispersa- | Callispora- | densely rugulate, atranorin not seen | not seen closely aggregate; covered | margin type | type; four- |} resulting in with granulose pruina | wavy and spored rough surface irregular B. asterella | effigurate, lobes short and || convex | aethalea-| Buellia- microfoveate | atranorin, stictic acid, not seen connected; surface with type | type; often norstictic acid fine pruina four well developed spores B. dijiana | not effigurate, crustose to soon | aethalea- | Buellia-type warty, arthothelin filiform rare granulose-squamulose; | irregularly | type microrugulate conidia surface with fine pruina; | convex dispersive B. elegans | effigurate, lobes short to flat to Buellia-type Loosely a) atranorin; b) not seen | common slender, multi-forked; convex rugulate, atranorin and covered with granulose resulting in | 2'-O-methylperlatoric pruina rough surface (in Asia) B. epigaea not effigurate, crusty, aethalea-| Callispora- | surface densely no secondary rare uneven to wrinkled; type type areolate and metabolite surface with fine pruina rough B. georgei | effigurate, marginal lobes | flat or | aethalea- | Buellia-type| surface densely arthothelin filiform | common short and often forming |sometimes| type areolate and conidia rosettes; surface with white | slightly rough granulose pruina convex B. lobata | effigurate, marginal lobes | apothecia | aethalea-| Buellia- warty, arthothelin, filiform | common distinct but short, the tips | disc below] type | type; often | microrugulate thuringione conidia of lobes dark; surface with | margin four well lightly fine pruina developed spores B. zoharyi | effigurate, lobes obvious; | flat to Buellia-type| microfoveate | atranorin, norstictic |common] rare covered with granulose convex acid, stictic acid pruina 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. zo- haryi by its linear lobate thallus, heavily pruinose apothecia and lobes, Callispora-type ascospores and four-spored asci. Type. Cuina. 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 New species and records from China 55 lecideine apothecia covered with white pruina C the section of apothecium, exciple dispersa-type D as- cospores 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 um (C); 10 um (D-F). 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 (EIN@3—); epihymenium brown to dark brown; hymenium hyaline, 80-100 pm tall, without oil droplets, paraphyses simple to moderately branched, apically swollen, with a brown pigment cap; hypothe- cium dark brown; asci oval-clavate, Bacidia-type, four-spored; spores 1-septate, hya- line when young, turning brown when mature, Callispora-type (Bungartz et al. 2007), ellipsoid, with tapering ends, proper septum narrow, not thickening during spore on- togeny, (13—)15—20(—22) x (6—)7—9(—10) um. 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 “a/pina” 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 56 Min Ai et al. / MycoKeys 92: 45-62 (2022) 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. Specimens examined. Cuina. 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. Itary. Ad terram calcaream supra Clavennam (Madésimo), Anzi M. (M! -Holotype). Description. Thallus efiigurate 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 pm thick, with granular crystals, and the lower surface light Figure 6. Morphology of Buellia elegans (16-51770 KUN) A thallus on soil within meadow B lecideine ap- othecia 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 um (C); 10 pm (D, E). New species and records from China 57 brown to white, without cortex; medulla white, without calcium oxalate crystals. Apothe- cia 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 (HNO,-); epihymenium brown to dark brown; hymenium hyaline, 70-90 um tall, without oil droplets, paraphyses simple to moderately branched, apically swollen, with a brown pig- ment 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 x 7-10 um. Pycnidia not seen. Chemistry. Thallus K+ yellow, C-, KC-, PD-, UV+ yellow, medulla I-; contain- ing 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 (Ihomson, 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 efhgurate 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. Cuina. Gansu Prov.: Jiayuguan Ci., Xigou, min- eral, 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 etal. 18-59513. Ningx- ia 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., de- sert 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 $301 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. 58 Min Ai et al. / MycoKeys 92: 45-62 (2022) 20-68931. Yunnan Prov.: Degin 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; Degin Co., Ben- zilan 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 ym 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 slight- ly 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 um thick, without aeruginose pigments (HNO,—); epihymenium brown to dark brown; hymenium hyaline, 60-80 pm tall, without oil droplets, paraphyses simple to mod- erately branched, apically swollen, with a brown pigment cap; hypothecium hyaline to light brown, up to 100 um 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 x 6-10 pm. Pycnidia not seen. 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 spe- cies 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 charac- teristics: 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 Tétramelas 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. Cuina. 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 New species and records from China aD 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 apothe- cium, exciple aethalea-type D ascospores with 1-septate, Callispora-type E ascus Bacidia-type. Scale bars: 2 mm (A); 1 mm (B); 50 um (C); 10 pm (D, E). 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. Key to species of Buellia epigaea-group 1 Thallus efhgurate and marginal lobes long and obvious; containing atranor- DN se nny ea yg nd 9d en en neem NN eos eel eS SRE Re {Yad bs nae RS o> i RS | 2 = Thallus either not effigurate or efhgurate but with marginal lobes short and closelyvageresate sachin Gad ANON Ak. cov duer Ue lzerhder a tualea redeem ceeauedeole 4, 60 Min Ai et al. / MycoKeys 92: 45-62 (2022) 2 PASCUS HOUESGOLGA 5 5,4. .0h eM cnc vest neu scdseeazeetbe ates ses thapaaiers Buellia alpina PASCUS CLE Ibs OTC cis, 5 sleet Ae departs an Rede esaaic ss eondced nhs deol vndtei brid estamtee abi aeengad 3 3 Spores large, up to 23 um long, ornamentation of spores rugulate ................ Pe abwcchisid Marcela SEser tat schbus balks sachets gbdse tay bbchls pshsite SacetaRaie seat Buellia elegans = Spores smaller, less than 17 um long, ornamentation of spores microfoveate. Be osicB ponent eenislah saci lete de eae atu ncene' cctaenecstalior sotteede agit aveustnate ean Buellia zoharyi 4 dinallissn@ rere UPAtee.. Miaka ac stedosh nedeaprsn terriers bsllt Aenusce wes Renciamy barn ved: 5 Thallus-eftiourate, ustvall y-withs Short: LODeS-.. os: onsen sonrsisnsoneetpicones vaeanadacinenss 6 5 Thallus crustose to granulose-squamulose; containing arthothelin...........0..... slated, Goheascts that btrad ea neeistand, cai Siva coteda an vesenaanedserreaaatectesatals Buellia dijiana = Thallus crusty, uneven to wrinkled; lacking secondary metabolites................ Rie ot: VN Te A RN REIT CS uae a Bone: Oe ty PR eee Buellia epigaea 6 On rock; ascus eight-spored; marginal lobes forming rosettes...... Buellia georgei - On soil; usually four mature spores in cach aSCUS ..........eseeeeeeeeseeeneeeeeeeeeeeee 7, vi Containing atranorin and thuringione; ornamentation of spores warty, mi- SPOR SW ALE. denver tees en qosieeeecudeesucuneteeren tcdvetrereustentnuetetinedsigdete Buellia lobata — Containing atranorin, norstictic acid and stictic acid (trace); ornamentation OleS POLES MUCLOLOVEATE: uecctecc teueraeeet ee arent aes ab eaend neuer Buellia asterella Acknowledgements 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 Sini- corum (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 Founda- tion of China (31970022) and State Key Laboratory of Phytochemistry and Plant Resources in West China (P2020-KFO08). 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Lichenologist 31(5): 511-516. https://doi.org/10.1006/lich.1999.0220 Supplementary material | Ornamentation of ascospores Authors: Min Ai, Li Juan Li, Fiona Ruth Worthy, An Cheng Yin, Qiu Yi Zhong, Shi Qiong Wang, Li Song Wang, Xin Yu Wang Data type: Images Explanation note: Figure $1. Ornamentation of ascospores (6000x magnification photograph under scanning electron microscope). A-D Buellia alpina. Scale bars: 2 pm. Figure $2. Ornamentation of ascospores (6000x magnification photograph under scanning electron microscope). A—D Buellia elegans. Scale bars: 2 um. Figure $3. Ornamentation of ascospores (6000x magnification photograph under scan- ning electron microscope). A-D Buellia epigaea. Scale bars: 2 um. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.92.83939.suppl1