683 MycoKeys MycoKeys 121: 329-340 (2025) DOI: 10.3897/mycokeys.121.155353 Research Article The phylogeny and taxonomy of Violella (Tephromelataceae, lichenized Ascomycota), including a new species from China Chun-jiao Zhong™, Zun-tian Zhao2, Ling Hu'® 1 College of Geography and Environment, Shandong Normal University, Jinan, Shandong, 250014, China 2 Key Laboratory of Plant Stress Research, College of Life Sciences, Shandong Normal University, Jinan, Shandong, 250014, China Corresponding author: Ling Hu (hu_ling_123@163.com) OPEN Qaceess Academic editor: Thorsten Lumbsch Received: 9 April 2025 Accepted: 7 August 2025 Published: 2 September 2025 Citation: Zhong C-jiao, Zhao Z-tian, Hu L (2025) The phylogeny and taxonomy of Violella (Tephromelataceae, lichenized Ascomycota), including anew species from China. Mycokeys 121: 329-340. https://doi. org/10.3897/mycokeys.121.155353 Copyright: © Chun-jiao Zhong et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International - CC BY 4.0). Abstract Violella yunnanensis C. J. Zhong & L. Hu is described as new to science. It is character- ized by its esorediate, areolate to weakly warted thallus, hymenium heavily pigmented with Fucatus-violet pigment, brownish inner ascospore walls, and its chemistry (atra- norin and fumarprotocetraric acid). In addition, we collected specimens of Mycoblas- tus sinensis Kantvilas and Violella wangii T. Sprib. & Goffinet from the holotype locali- ties. Mycoblastus sinensis is transferred to the genus Violella based on its morphology, chemistry and phylogeny, and it is proposed as Violella sinensis (Kantvilas) C. J. Zhong & L. Hu. The morphological descriptions, pictures and molecular phylogenetic analyses of the species are provided, along with a key to the Violella species known from world. Key words: EF1-a gene, lichenized fungi, new combination, new taxa, Yunnan Introduction Violella T. Sprib. is a genus of lichenized fungi belonging to Tephromelata- ceae, Lecanorales, Lecanoromycetes, Ascomycota (Cannon et al. 2022; Wi- jayawardene et al. 2022). The family Tephromelataceae currently includes 4 genera (Calvitimela, Mycoblastus, Tephromela and Violella) and 53 species (Wijayawardene et al. 2022). The genus Violella was a group of mainly epi- phytic species which was found in Western Europe, North America and the mountains of high Asia (Tonsberg 1992, 1993; James and Watson 2009; Spri- bille et al. 2010, 2011a, b). So far, Violella includes 2 species worldwide (Wi- jayawardene et al. 2022). The genus Violella is separated from Mycoblastus (Spribille et al. 2011b) by an abundant Fucatus-violet pigment in the hymenium and inner ascospore walls that become brownish. In contrast, Mycoblastus typically has Cinere- orufa-green pigments in the hymenium and hyaline ascospores. In addition, Mycoblastus has anastomosing paraphyses, making the hymenium coherent and fragmenting irregularly under pressure, even in K, while Violella has more columnar paraphyses with small bridges and tends to fragment along the col- umns (McCune 2007; Spribille et al. 2011b; Singh and Singh 2015; Cannon et al. 2022). In phylogenetic analysis, Violella was more closely related to Calvi- 329 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella timela and Tephromela than to Mycoblastus (Spribille et al. 2011b; Bendiksby et al. 2015; Fjelde et al. 2024). Prior to this study, only one species of Violella had been reported from China. V. wangii T. Sprib. & Goffinet was first described from Yunnan Province. During a survey of the lichen diversity in the northwest of Yunnan Province in China, we collected numerous lichen specimens including Violella wangii and Mycoblastus sinensis, which were found in the type locality at Laojun Mountain and Yulong Mountain, respectively. During our research, a new species, Violella yunnanensis was discovered from Laojun Mountain and Meili Snow Mountain. Furthermore, we proposed a new combination Violella sinensis (Kantvilas) C. J. Zhong & L. Hu. In the present paper, we characterize these species using mor- phological, chemical, and molecular evidence and provide a key to the Violella species known from world. Materials and methods Morphological and chemical study All the specimens were deposited in the Lichen Section of the Botanical Herbarium, Shandong Normal University, Jinan, China (SDNU). A dissecting microscope, Nikon SMZ 745T, was used to observe the morphological fea- tures. Apothecia and thalli were sectioned by hand with razor blade and their microscopic traits were observed and measured using an Olympus CX21 op- tical stereomicroscope. Photographs were taken by the Olympus SZX16 and BX16 microscope with DP72 camera system. Lugol's iodine (I) was used to examine the apical structure of asci. Spots tests were conducted by K (a 10% aqueous solution of potassium hydroxide), C (a saturated aqueous solution of sodium hypochlorite), P (a saturated solution of p-phenylenediamine in 95% ethylalcohol). Secondary metabolites were detected by thin-layer chro- matography (TLC) as described by Orange et al. (2001) in solvent C (tolu- ene: acetic acid =170: 30). The dimensions of ascospores and the ascospore length/width ratio are presented as (minimum) mean+SD (maximum); n= the number of measurements. DNA extraction, amplification and sequencing DNA was extracted from dry or fresh specimens. The voucher numbers refer to Table 1. The Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit (St Louis, Mis- souri, USA) was used to isolate DNA, following the manufacturer’s instructions, except only 30 ul of extraction buffer and 30 ul dilution buffer were used. We se- lected two loci for this study: the nuclear ribosomal internal transcribed spac- er region (nriTS) and one protein-coding gene, namely translation elongation factor 1-a (EF1-a). PCR amplification for nriTS and EF1-a was achieved using primers ITS1F and ITS4 (White et al. 1990), EF983 and EF2218R (Rehner and Buckley 2005), respectively. The 50 ul PCR mixture consisted of 2 ul DNA, 2ul of each primer, 25 ul 2x Taq PCR MasterMix (Taq DNA Polymerase [0.1 unit/ul]; 3 mM MgCL,; 100 mM KCI; 0.5 mM dNTPs; and 20mM Tris-HCl [pH 8.3]) (Tiangen, Beijing, China) and 19 yl ddH,O0. Sequencing was performed by BioSune Biolog- ical Technology (Shanghai). Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 330 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella Table 1. Specimens and sequences used for phylogenetic analyses. Newly generated sequences are in bold. Species Calvitimela armeniaca 1 C. armeniaca 2 Tephromela atra 1 T. atra 2 Violella fucata 1 V. fucata 2 V. fucata 3 V. sinensis 1 V. sinensis 2 V. sinensis 3 V. wangii 1 V. wangii 2 V. wangii 3 V. wangii 4 V. yunnanensis 1 V. yunnanensis 2 V. yunnanensis 3 GenBank numbers Locality Voucher specimens are ei Norway O L-225775 OR763878 OR738421 Norway O L-225781 OR763865 OR738420 Italy, Campania, Napoli, Capri Island Muggia (TSB37119) EU558648 JNO009698 Greece, Crete, Herakleion, Kameraki Muggia (TSB37924) EU558688 JN009697 Germany, Bavaria, Bayerischer Wald Spribille 32112 (GZU) JN009732 = USA, Mt. Greylock Spribille 32161 (GZU) JF744968 JNO009703 Slovenia, Sneznik area Spribille 30276 & Mayrhofer (GZU) | JN009733 = China, Mt. Yulong SDNU20232737 PQ586228 = China, Mt. Yulong SDNU20232538 PQ586227 PQ602071 China, Mt. Meili Snow SDNU20232001 PQ586226 PQ602070 China, Mt. Laojun Goffinet 10029 (KUN) JNO009734 JNO009704 China, Mt. Laojun Goffinet 10033 (UPS) JNO009735 = China, Mt. Laojun SDNU20232930 PQ586229 PQ602072 China, Mt. Laojun SDNU20232975 PQ586230 PQ602073 China, Mt. Meili Snow SDNU20235026 PQ586223 PQ602076 China, Mt. Laojun SDNU20232875 PQ586231 PQ602074 China, Mt. Laojun SDNU20232882 PQ586232 PQ602075 Phylogenetic analysis The raw sequences were initially checked with the BLAST tool on the NCBI online service (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to ascertain that all the new sequences were reliable. All raw sequences were assembled and edited using Geneious 9.0.2. Sequences extracted from new materials were aligned with the additional sequence data from GenBank, using an online version of MAFFT v. 7.0.26. The algorithm of MAFFT chose Auto (FFT-NS-1, FFT-NS-2, FFT-NS-i or L-INS-i; depending on data size). We used Tephromela atra and Cal- vitimela armeniaca as the outgroup. Phylogenetic relationships were inferred using maximum likelihood (ML) and Bayesian inference (BI). The best substitution models were estimated using ModerFinder v2.2.0 for the subsequent ML and BI analyses (Kalyaanamoorthy et al. 2017). In ML anal- ysis, the best-fit model for nrlTS and EF1-a were SYM+G and TRNEF+6G, respec- tively. In BI analysis, GTR+I+G was the best-fit model for both sequences. The ML analyses were performed in IQ-TREE v2.2.0 with 1000 standard bootstrap replicates (Nguyen et al. 2014). The BI analyses were performed in MrBayes v.3.2.6 (Ronquist et al. 2012), using three Markov chains running for 2 million generations for the concatenated dataset. The trees were sampled every 100 generations, and the first 25% of the trees were discarded as burn-in. Boot- strap support (BS) =70 and posterior probability (PP) =0.95 were considered significant support values. The datasets/alignments were deposited in Tree- Base (http://purl.org/phylo/treebase/phylows/study/TB2:S32226). All of the above analysis software were implemented in PhyloSuite v. 1.4.2 (Zhang et al. 2020; Xiang et al. 2023). The phylogenetic trees generated were visualized with FigTree v. 1.4.2 (Rambaut 2012). Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 33] Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella 100/1 0.04 100/1 Results and discussion In the present study we generated eight new nrITS and seven new EF1-a se- quences (Table 1). We constructed ML and BI topologies based on these new sequences and fifteen additional sequences downloaded from GenBank, most- ly from Spribille et al. (2011b) and Fjelde et al. (2024). The phylogenetic trees obtained from ML and BI exhibited consistent topologies, so only the ML tree was provided here (Fig. 1). Within the phylogenetic tree, specimens of the putative new species formed a single clade with bootstrap support of 100 and a posterior probability of 1. This clade was strongly supported as sister to V. wangii. Based on the combina- tion of morphological characters, chemistry and phylogenetic analysis, which are described in detail below (Table 2), we propose a new species named Vi- olella yunnanensis. The new combination, Violella sinensis, is phylogenetically close to V. fucata, which is the type of the genus; they all contain atranorin and fumarprotocetraric acid as secondary metabolites, but V. sinensis is unequiv- ocally non-sorediate and has well developed areolate, verruculose to papillose thallus, which distinguish it from V. fucata. 1001) Calvitimela armeniaca 1 Calvitimela armeniaca 2 io [- Fephromela atra 1 Tephromela atra 2 Violella yunnanensis 2 Violella yunnanensis 1 woo | §¥°°L_ Violella yunnanensis 3 Violella wangjii 3 Violella wangii 1 97'_| Violella wangii 2 Violella wangii 4 _ Violella sinensis 2 Violella sinensis 3 96/1 Il Violella sinensis 1 san Violella fucata 2 i001 | Violella fucata 3 Violella fucata 1 Figure 1. Phylogenetic tree generated from maximum likelihood (ML) analysis based on nrITS and EF1-a sequences. ML bootstrap values (left) and Bayesian posterior probabilities (right) are indicated at the nodes. The scale bar indicates the number of substitutions per site. Newly generated sequences from China are shown in bold. Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 332 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella Table 2. Comparison of the characters of Violella species. (Spribille et al. 2011b; Kantvilas 2011; Cannon et al. 2022). Species V. fucata V. wangii V. sinensis V. yunnanensis Thallus Apothecia (mm) Ascospores (um) Chemistry sorediate, soredia bluish grey often absent = atranorin, fumarprotocetraric acid thin, effuse rare, 0.5-1.5 (25-)30-48(-52) x atranorin, fumarprotocetraric acid 15-21 sorediate, soredia white often absent a atranorin, roccellic/angardianic acid granular and corticate (0.7—)1.3-2.6(-4.1) | (35-)41.7-54.2(-65) x | atranorin, roccellic/angardianic acid areolate (15-)20.8-30.8(-35) esorediate, areolate, 0.5-1.25(-2.25) | (38—-)45.95+6.18(-58) x | atranorin, fumarprotocetraric acid verruculose to papillose (18-)25.65+4.63(-33) esorediate, areolate to (0.4-)0.8-1.75(-—2) | (43-)50.4+5.37(-63) x | atranorin, fumarprotocetraric acid weakly warted (23-)28.15+3.5(-38) Taxonomy Violella sinensis (Kantvilas) C. J. Zhong & L. Hu, comb. nov. Fungal Names: FN 572208 Fig. 2 = Mycoblastus sinensis Kantvilas, J. Jap. Bot. 86(2): 59 (2011). Diagnosis. The species is characterized by having esorediate, areolate, ver- ruculose to papillose thallus, hymenium heavily pigmented with Fucatus-violet pigment, brownish inner ascospore walls, and its chemistry (atranorin and fu- marprotocetraric acid). Specimens. CHINA. Yunnan Prov.: * Yulong Co., Baisha Vil., entrance of Al- pine Botanical Garden, 27°0'10.80"N, 100°10'49.50"E, 3204 m, on bark, 28 April 2023, L. Hu et al. SDNU 20232538. Description. Thallus crustose, chalk white, ochre to grey, areolate, consist- ing of discrete convex to bullate areoles (0.25—)0.37—0.5 mm diam., forming a verruculose to papillose, widespread crust. Medulla white, with crystals. Sore- dia and isidia absent. Prothallus visible, dark. Photobiont chlorococcoid, cells rounded to irregularly angular, 8-16 um diam. Apothecia rounded, single or clustered in groups of 2-3 and becoming con- fluent, 0.5-1.25(-2.25) mm diam., base broadly adnate; disc + flat to strongly convex, black and shiny, becoming cracked when old; margin indistinct. Proper exciple reduced, similar in structure to the hymenium, hyphae radiate, similar to paraphyses. Epihymenium not differentiated as a distinct layer. Hymenium 80- 150 um tall, densely inspersed with minute oil droplets, strongly infused with Fu- catus-violet pigment, especially in the upper part, sometimes also infused with Cinereorufa-green, K+ peacock green. Subhymenium consisting of a thin layer of ascogenous hyphae, 17.5-27.5 um tall, filled like the hymenium with Fuca- tus-violet pigment but sometimes also infused with Cinereorufa-green. “thalline cushion” in section prosoplectenchymatous, variable in thickness, 12.5-137.5 um thick. Hypothecium absent. The amyloid reaction is restricted to the asci and surrounding gel, and does not occur through the whole hymenium. Paraphy- ses branched and anastomosing, 2-4 um wide, arranged vertically and linked to each other in their lower halves by thin bridges; paraphyses tips not or scarcely expanded. Asci clavate, Biatora-type. Ascospores ovate to broadly ellipsoid, 2 MycoKeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 333 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella # OS. s Figure 2. Violella sinensis (SDNU 20232538). A. Thallus; B. Apothecia; C. Apothecium section; D. Crystals; E. Ascus; F. Amyloid reaction of ascus; G. Ascospores. Scale bars: 1 mm (A); 500 um (B); 100 um (C, D); 20 um (E); 10 um (F, G). per ascus, beginning colourless and apparently with a single wall, eventually developing a secondary inner wall, which quickly turns brown while still in ascus; outer wall thick, to 4 um in some cases, the inner brown wall thin, healthy as- cospores (38-)45.95+6.18(—58) x (18-)25.65+4.63(-33) um in water, length/ width ratio (1.37—)1.83+0.29(—2.28); n=20. Pycnidia not seen in our materials. Chemistry. Spot test: thallus K+ yellow, C-, P+ orange red, UV-. TLC: atra- norin and fumarprotocetraric acid. Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 334 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella Ecology and distribution. Found on twigs of Rhododendron and bark in sub- alpine and alpine regions. Collections came from elevations of 3200-3800 m in the northwest of Yunnan Province in China. Some collections were associated with Mycoblastus affinis and Ochrolechia. Notes. The species is characterized by having esorediate, areolate, verruculose to papillose thallus, hymenium heavily pigmented with Fucatus-violet pigment, brownish inner ascospore walls, and its chemistry (atranorin and fumarprotoce- traric acid). It is distinct from V. wangii in its chemistry (fumarprotocetraric acid in- stead of roccellic/angardianic acid) and thallus morphology (esorediate and ver- ruculose to papillose, instead of sorediate and composed of granular, corticate areoles) (Spribille et al. 2011b). V. sinensis was also different with the chemically concordant V. fucata. Apothecia were always present in the former, while rarely found in the later; thallus was thick, esorediate, areolate and verruculose to papil- lose in the former, while thin, sorediate, effuse in the later (Spribille et al. 2011b). Mycoblastus sinensis was originally collected in Yunnan Province at Yulong Mountain. It was placed in the genus Mycoblastus based on its relatively large apothecia and Biatora-type asci with two, thick-walled ascospores (Kantvilas 2011). After the collection of fresh specimens from the same locality and comparison with the photograph and description of the type specimen, we confirmed that our collections were morphologically identical to the holotype, except that M. sinensis had larger (0.7—2.5(-3.5) mm) white-rime apothecia (Kantvilas 2011). However, phylogenetic analyses demonstrated that this spe- cies should be placed within the genus Violella rather Mycoblastus. In addi- tion, this species is more closely matches the description of Violella (Kantvilas 2011). We consider that the type specimens were previously misassigned as species of Mycoblastus. It is likely that Kantvilas didn’t recognize the establish- ment of this new genus Violella because it was published in the same year as the other one (Spribille et al. 2011b; Kantvilas 2011). Based on the morphology, chemistry, and phylogeny of specimens from the type locality, a new combina- tion is proposed here, Violella sinensis. Specimens examined. CHINA. Yunnan Prov.: + Diging Tibetan Autono- mous Prefecture, Deqin Co., on the way from Yubeng Upper Village to Glacier Lake beside the observation platform in Meili Snow Mountain National Park, 28°23'56.37'N, 98°46'8.97"E, 3506 m, on bark, 22 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232001, 20235022, 20235025. + Lijiang, Yulong Co., on the mountain next to Yulong Lake in Gaoshan Botanical Garden, 27°0'12.89'N, 100°11'8.85"E, 3228 m, on bark, 27 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232503, 20235064, 20235065. + Baisha Vi., entrance of Alpine Botani- cal Garden, 27°0'10.80"N, 100°10'49.50"E, 3204 m, on bark, 28 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232538, 20232542. * Alpine Botanical Gar- den, 27°0'25.17"N, 100°10'34.51"E, 3399 m, on bark, 28 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232559. * hidden slope of Alpine Botanical Gar- den, 27°0'23.91"N, 100°10'41.05"E, 3421 m, on bark, 29 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20235063, 20232737, * same location, 27°0'30.65'N, 100°10'29.75"E, 3475 m, on bark, 29 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232753. * same location, 27°0'39.77'N, 100°10'39.12°"E, 3511 m, on bark, 29 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232767. * Yulong Co., next to Laojun Mountain Homestay, 26°37'54.05'"N, 99°43'37.23"E, 3821 m, on bark, 30 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232873. Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 335 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella Violella yunnanensis C. J. Zhong & L. Hu, sp. nov. Fungal Names: FN 572207 Fig. 3 Diagnosis. The species is characterized by having esorediate, areolate to weak- ly warted thallus, hymenium heavily pigmented with Fucatus-violet pigment, brownish inner ascospore walls, and its chemistry (atranorin and fumarproto- cetraric acid). Type. CHINA. Yunnan Prov.: * Deqin Co., on the way from Yubeng Upper Vil- lage to Glacier Lake beside the observation platform in Meili Snow Mountain National Park, 28°23'56.37"N, 98°46'8.97"E, 3506 m, on bark, 22 April 2023, L. Hu et al. SDNU 20235026 (holotype). Description. Thallus crustose, areolate, weakly warted, consisting of dis- crete flattened to convex areoles (0.18—)0.23-0.28 mm diam., color grey to pale ocher, with crystals in the medulla. Soredia and isidia absent. Hypothallus not observed. Photobiont chlorococcoid, cells rounded to irregularly angular, (7-)8-10(-12) um diam. Apothecia rounded to irregular, single or clustered in groups of 2-3 and becom- ing confluent, (0.4—)0.8—1.75(—2) mm diam., base broadly adnate; disc + flat to weakly convex, jet black and shiny; margin indistinct; “thalline cushion” present, visible from above and forming a thin white line at least when young, in section prosoplectenchymatous, variable in thickness, 25-100(-—137.5) um thick, clearly differentiated from subhymenium above and medulla below. Proper exciple simi- lar in structure to the hymenium, hyphae radiate, similar to paraphyses, filled with Fucatus-violet pigment and often suffused with Cinereorufa-green. Epihymenium not differentiated. Hymenium 125-137.5 um tall, densely inspersed with minute oil droplets; the Fucatus-violet pigment is concentrated at the base and the top of the hymenium and largely diffuse. Subhymenium consisting of a thin layer of ascogenous hyphae, 30-32.5 pm tall, filled like the hymenium with Fucatus-vio- let pigment but sometimes also infused with Cinereorufa-green pigment; differ- entiated hypothecium absent. Paraphyses branched, anastomosing, 2-—2.5 um wide; paraphyses’ tips not or scarcely expanded, 2.5-3 um wide. Asci clavate, inner and outer walls staining blue, tholus strongly I+ blue, pierced by a broad, conical non-amyloid structure, thus similar to the Biatora-type. Ascospores el- lipsoidal, 2 per ascus (occasionally 1), beginning colourless and apparently with a single wall, eventually developing a secondary inner wall, which quickly turns brown while still in the ascus; outer wall thick, to 3 um in some cases, the inner brown wall thin, often collapsing, live, healthy ascospores also with brown endo- spore, (43—)50.4+5.37(-63) x (23—)28.15+3.5(-38) um in water, length/ width ratio (1.53—)1.8+0.14(—2); n=20. Pycnidia not seen in Chinese materials. Chemistry. Spot test: thallus K+ yellow, C-, P+ orange-red, UV-; TLC: atra- norin and fumarprotocetraric acid. Ecology and distribution. Found on twigs of Rhododendron and bark in sub- alpine and alpine regions. Collections came from elevations of 3500-3800 m in the northwest of Yunnan Province in China. Etymology. The specific epithet ‘yunnanensis’ refers to the Yunnan Province, where this species was found. Notes. Violella yunnanensis is characterized by having esorediate, areolate to weakly warted thallus, hymenium heavily pigmented with Fucatus-violet pigment, Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 336 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella wie ma Figure 3. Violella wangii ( 20235026). C. Thallus; D. Apothecia; E. Apothecium section; F. Crystals; G. Ascus; H. Amyloid reaction of ascus; I. Asco- spores. Scale bars: 1 mm (A, C); 500 um (B, D); 100 um (E, F); 10 um (G-l). Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 337 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella brownish inner ascospore walls, and producing atranorin and fumarprotocetraric acid. Violella wangii has a similar habitat to this new species. Specimens of V. wangii collected at Laojun Mountain are found growing on bark of Rhododendron sp. or on wood of Pinus at elevations ranging from 3400-3900 m. However, Violella wangii differs in its chemistry (atranorin and roccellic/angardianic acid), thallus morphol- ogy (white, granular corticate areoles, sorediate) and larger apothecia [(0.7—)1.3- 2.6(-4.1) mm] (Fig. 3A, B). V. fucata differs from V. yunnanensis in possessing a generally thin, effuse, sorediate thallus, apothecia rare and smaller ascospores [((38.5+6.7x18.5+3.3um, n=24) vs (43-)50.4+5.37(-63) x (23-)28.15+3.5(-38) um, n=20)] (Spribille et al. 2011b). Furthermore, Violella yunnanensis differs from Violella sinensis in its weakly warted thallus and larger ascospores. Specimens examined. CHINA. Yunnan Prov.: * Diging Tibetan Autono- mous Prefecture, Deqin Co., on the way from Yubeng Upper Village to Glacier Lake beside the observation platform in Meili Snow Mountain National Park, 28°23'56.37'N, 98°46'8.97"E, 3506 m, on bark, 22 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20231997, 20232019, 20235026. * Lijiang, Yulong Co., Baisha Vi., Alpine Botanical Garden, 27°1'2.33"N, 100°10'27.11"E, 3708 m, on bark, 28 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232618. + Yulong Co., next to Laojun Mountain Homestay Vi., 26°37'54.05"N, 99°43'37.23"E, 3821 m, on bark, 30 April 2023, L. Hu, C.J. Zhong & J.X. Wang. SDNU 20232874, 20232841, 20232885, 20232828, 20232875, 20232890, 202328872. Key to the species of Violella in the world 1 Soralia present; apothecia often ADSeNt......... eee eee cceesseeceeseeeeeseeeeesees 2 Ta” SOfaliasabsSent-apotirecia. PLOSOM ey .ccsxacscvcaccstte-verespnrpemverassessipesnnpesee reso 3 2 Soredia often bluish grey; thallus Pd+orange-red ...............ccceeee V. fucata 2. ~Sorediarwhite: thallussP a= Ashasecsnsrhevcedensecn ne varareagipiennn oavinheade V. wangii ou ertihalusKAVelOW POE... cic. costs caren alacertindlceteasac Met acarehunate V. wangii 3 = Thallus K+yellow, Pd+orange-red .............ccccccccsccceessccessssceeeesseecessseeeesseaeees 4 4 Thallus thin, effuse; ascospores (25—)30-48(—52) x 15-21 um. V. fucata 4 Thallus well developed; ascospores largel...............ccccccceesseeeeesteceeeseeesesaes 5 5 Areolate, verruculose to papillose thallus; ascospores (38-)45.95+6.18(- DO) SH) 2 SO OEA, GAO Sy PM es, 5 cy hee ela dresses vsdese tons deiadeacss ores V. sinensis 5 Areolate to weakly warted thallus; ascospores (43-)50.4+5.37(-63) x (23) 28 cS#S -S(3 8s Nites Serle ws. rors As ests, Soot esas V. yunnanensis Acknowledgments We are very grateful to (SDNU) J.X. Wang for helping us to collect specimens in this study. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Mycokeys 121: 329-340 (2025), DOI: 10.3897/mycokeys.121.155353 338 Chun-jiao Zhong et al.: The phylogeny and taxonomy of Violella Use of Al No use of Al was reported. Funding This research was funded by the National Natural Science Foundation of China (No.31900010). Author contributions Conceptualization, C.Z. and L.H.; methodology, C.Z. and L.H.; software, C.Z. and L.H.; valida- tion, C.Z. and L.H.; formal analysis, C.Z. and L.H.; investigation, C.Z. and L.H.; data curation, C.Z. and L.H.; writing — original draft preparation, C.Z.; writing — review and editing, C.Z. and L.H.; visualization, C.Z.; supervision, L.H.; project administration, L.H.; funding acquisition, L.H. and Z.Z. All authors have read and agreed to the published version of the manuscript. Author ORCIDs Chun-jiao Zhong © https://orcid.org/0009-0001-51 59-238X Ling Hu © https://orcid.org/0009-0008-4568-6639 Data availability All of the data that support the findings of this study are available in the main text. References Bendiksby M, Haugan R, Spribille T, Timdal E (2015) Molecular phylogenetics and taxon- omy of the Calvitimela aglaea complex (Tephromelataceae, Lecanorales). 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