Three new species of Ochrolechia (Ochrolechiaceae, Pertusariales) from Guizhou Province, China

19 Three new species of Ochrolechia (Ochrolechiaceae, Pertusariales) from Guizhou Province, China Weiwei Zheng1, Linzhi He1, Heyun Bo1, Rajesh Jeewon2, Ruvishika S. Jayawardena3,4 , Yuxian Wang5, Jie Wang1, Shaobin Fu1, Qingfeng Meng5 1 School of Pharmacy, Zunyi Medical University, Zunyi, Guizhou Province 563000, China 2 Department of Health Sciences, Faculty of Science, University of Mauritius, Reduit 80837, Mauritius 3 Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai 57100, Thailand 4 School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand 5 School of Public Health, Zunyi Medical University, Zunyi, Guizhou Province 563000, China Corresponding authors: Shaobin Fu ([email protected]); Qingfeng Meng ([email protected]) Copyright: © Weiwei Zheng 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). Research Article Abstract Based on morphological, chemical and phylogenetic analyses, three species of Ochrolechia collected from Guizhou, China, are described as new to science and named Ochrolechia guizhouensis, O. kuankuoshuiensis and O. leigongshanensis. Phylogenetic trees, based on ITS and mtSSU loci, were constructed using Maximum Likelihood analysis (ML) and Bayesian Inference (BI) methods. Ochrolechia guizhouensis and O. akagiensis are positioned close to each other in the phylogenetic tree. Ochrolechia guizhouensis is morphologically characterised by its apothecia which feature rugose to rosulate, epruinose discs resembling a floral structure. Chemically, the apothecia contain gyrophoric acid, lecanoric acid and lichesterinic acid, while the thallus contains only gyrophoric acid and lecanoric acid. Phylogenetic analysis reveals that Ochrolechia kuankuoshuiensis is closely related to O. parellula and is distinguished by its light yellow, epruinose, deeply rugose apothecial discs, the production of gyrophoric acid and lecanoric acid and the largest ascospores known in the genus. Ochrolechia leigongshanensis forms an isolated phylogenetic branch which is characterised by a soraliate thallus and apothecia with a ring of smooth, salmon-pink tissue on the inner margin. The discs are epruinose to lightly pruinose and plane. The thallus contains gyrophoric acid, lecanoric acid and a trace of atranorin, while the apothecia contain only gyrophoric acid and lecanoric acid. Additionally, O. subrosella and O. longispora were successfully sequenced for the first time and represent the first records from Guizhou Province. We found one specimen, which phylogenetically groups with O. trochophora, but is characterised by the presence of soralia, indicating that O. trochophora might be morphologically more variable than previously thought. Colour photographs are provided for all the above-mentioned species. Key words: Lichen, morphology, phylogeny, taxonomy, three new species Introduction Massalongo (1852) established the crustose lichen genus Ochrolechia by transferring six taxa from Lecanora to this genus. Ochrolechia is characterised by a crustose thallus that may be continuous or fissured and varies from thin Academic editor: Imke Schmitt Received: 15 August 2025 Accepted: 5 November 2025 Published: 2 December 2025 Citation: Zheng W, He L, Bo H, Jeewon R, Jayawardena RS, Wang Y, Wang J, Fu S, Meng Q (2025) Three new species of Ochrolechia (Ochrolechiaceae, Pertusariales) from Guizhou Province, China. MycoKeys 126: 19–40. https://doi.org/10.3897/ mycokeys.126.168652 MycoKeys 126: 19–40 (2025) DOI: 10.3897/mycokeys.126.168652 20 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species to thick, with a pale white to grey or even yellowish coloration. Soredia are often present in some species and a few taxa also produce isidia (Tonsberg 1992; Elix 2007). The apothecia are typically large, with a disc that may be epruinose or pruinose; a strongly amyloid hymenium; paraphyses are slender, branched and interwoven and asci generally contain four to eight hyaline, simple, thin-walled ascospores (Schmitt and Lumbsch 2004; Ren 2017). The taxonomy of Ochrolechia has undergone significant revisions since its establishment. Reinke (1894) initially placed Ochrolechia in the Pertusariaceae within Parmeliales. Choisy (1949) later maintained its familial placement, but transferred it to Pertusariales. Subsequently, Verseghy (1962) proposed a classification for Ochrolechia, dividing 59 species into five informal groups: Geminiparae, Parella, Upsaliensis, Tartarella and Harmandi. Advances in molecular phylogenetics later prompted a re-assessment of its systematic position, leading to the establishment of the family Ochrolechiaceae to accommodate the genus (Schmitt et al. 2006). Ochrolechiaceae is currently a monogeneric family, comprising only Ochrolechia and is placed within Pertusariales (Hyde et al. 2024). In a systematic revision of the Chinese species of Ochrolechia, Ren (2017) recognised 20 taxa. The genus is mainly distributed in the south-western provinces, particularly Yunnan and Sichuan, with scattered records from Fujian, Gansu, Shaanxi, Anhui and Hunan, typically occurring at elevations above 1,500 m. In that study, four new species (O. alticola, O. mexicana, O. lijiangensis, O. rugomarginata) were described, four taxa (O. arborea, O. mahluensis, O. szatalaënsi, O. trochophora var. pruinirosella) were newly recorded for China and an identification key to all 20 taxa was provided. Subsequently, O. parellula was added to the Chinese mycobiota by Fu et al. (2022). Chemical compounds of lichens, particularly secondary metabolites, such as depsides, anthraquinones and triterpenoids in lichens, have long been recognised as reliable taxonomic markers in lichen identification (Culberson 1969; Huneck and Yoshimura 1996). In Ochrolechia, the occurrence and distribution of diagnostic substances (e.g. gyrophoric acid and lecanoric acid) have been widely used for species delimitation and identification. Amongst these, gyrophoric acid is the predominant metabolite. Other depsides that produce a C + red reaction, such as olivetoric acid and lecanoric acid, are typically only in combination with gyrophoric acid (Brodo 1991). In this study, Ochrolechia specimens collected from Guizhou Province were identified using an integrative approach combining morphology, phylogeny and chemistry. Materials and methods Sample collection and morphological examination The specimens (KKS83, KKS150-2) were collected from Kuankuoshui National Nature Reserve and others (LGS40, LGS176, LGS186, LGS205, LGS213-2, LGS219) from Leigongshan National Nature Reserve, Guizhou Province, China. Macro-morphological characteristics were observed using a stereoscopic microscope (XTL-3B, Coic). The ascomatal sections were 21 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species manually prepared as temporary mounts and observed under a compound microscope (Olympus BX53), with photographic documentation obtained using an attached digital camera (Olympus DP72). Measurements were conducted using the Image Framework software (Tarosoft, version 0.9.7). The dimensions of asci and ascospores were recorded (for n ≥ 10) and presented as: (min-) [x – – SD] – [x – + SD] (max), where “min” and “max” represent extreme values, x – is the arithmetic mean and SD is the standard deviation (Meng et al. 2025). Unless otherwise specified, all measurements were taken from water-mounted sections, following the procedures outlined by Senanayake et al. (2020). Holotype specimens are deposited in the Lichen Herbarium of Kunming Institute of Botany (KUN-L), Chinese Academy of Science, Yunnan, China. Determination of secondary metabolites Secondary metabolites were examined using chemical spot tests under a stereoscopic microscope and thin-layer chromatography (TLC) for the thallus and apothecia, respectively. The spot tests included 10% aqueous potassium hydroxide (K), saturated aqueous calcium hypochlorite (C) and a sequential application of K followed by C (KC). TLC was performed using the following solvent systems: A (toluene: dioxane: acetic acid = 180: 60: 8, v/v), B’ (hexane: methyl tert-butyl ether: formic acid = 140: 72: 18, v/v), and C (toluene: acetic acid = 200: 30, v/v) (Culberson and Kristinsson 1970; Culberson 1972; Wei and Qiu 1998; Orange et al. 2001). Metabolites were identified by comparison with reference compounds in Lethariella cladonioides, which contains atranorin and norstictic acid (Dou et al. 2024). Extraction and amplification of DNA Genomic DNA was extracted from the specimens using a Fungal Genomic DNA Kit (Sangon Biotech, Shanghai) following the manufacturer’s instructions. The internal transcribed spacer region of the nuclear ribosomal DNA (ITS) was amplified using the primers pairs ITS1f/ITS4 (White et al. 1990) and the mitochondrial small subunit ribosomal RNA gene (mtSSU) was amplified using the primer pairs mrSSU1/mrSSU3r (Zoller et al. 1999). Polymerase chain reactions (PCR) were performed using a Mastercycler (Bio-RAD T-100) in a 20-µl reaction volume consisting of 10 µl of 2× Mix (Solarbio, dNTPs Mix), 6.4 µl of double-distilled water (ddH2O), 2 µl of the DNA template and 0.8 µl of each primer. The PCR amplification reaction procedures are listed in Table 1. Table 1. Primer pairs and the parameters for PCR. Locus Primers Initial Denaturation Denaturation Annealing Elongation Final Extension Hold ITS ITS1f/ITS4 95 °C/3 min 95 °C/30 s 55 °C/30 s 72 °C/90 s 72 °C/10 min 4 °C/+∞ 38 cycles mtSSU mrSSU1/mrSSU3r 95 °C/3 min 95 °C/30 s 53 °C/30 s 72 °C/1 min 72 °C/10 min 4 °C/+∞ 38 cycles 22 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species Phylogenetic analysis The quality of the electropherograms was checked using BioEdit software. Forward and reverse sequences were assembled using the ContigExpress software (New York, USA) and the concatenated sequences were subjected to BLASTn searches in NCBI (www.ncbi.nlm.nih.gov/) for preliminary identification. The BLAST results of all newly-generated sequences indicated that these samples belong to Ochrolechia. The related taxa were retrieved from GenBank and combined with newly-generated sequences to conduct a phylogenetic analysis (Table 2). Two vouchers of Trapelia coarctata were selected as the outgroup (Park et al. 2019). Multiple alignments were performed using online MAFFT version 7 (http://mafft. cbrc.jp/alignment/-server), with default settings and trimmed using trimAl v1. (Capella-Gutiérrez et al. 2009; Katoh and Standley 2013). The ITS and mtSSU were merged in SequenceMatrix-Windows 1.7.8 and the aligned FASTA files were subsequently converted to NEXUS format using AliView v.1.27 for Bayesian Inference analysis (Glez-Peña et al. 2010). Twenty-two common DNA substitution models with rate heterogeneity were tested by ModelFinder (Kalyaanamoorthy et al. 2017). The best-fit model for each gene region, as determined by the Bayesian Information Criterion (BIC), is as follows: ITS: TIM2+F+I+G4, mtSSU: TVMu+F+R2. The resulting sequences were imported into the CIPRES Science Gateway (www.phylo. org/portal2/home.action) for RAxML analysis (Vaidya et al. 2011) and MrBayes analysis. The ML analysis was conducted with the RAxML-HPC2 tool on XSEDE (8.2.12) employing a GTRGAMMA approximation with a rapid bootstrap analysis of 1000 replicates (Stamatakis 2014). The Bayesian Inference phylogenies were inferred using MrBayes v.3.2.7a on XSEDE (Ronquist et al. 2012) (2 parallel runs, 10,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in phase. The phylogenetic trees were visualised in FigTree v.1.4.4 and edited in Adobe Illustrator CC 2019; the resulting tree only displays ML bootstrap support values ≥ 70 and BI posterior probabilities ≥ 0.90, respectively. New species are identified, based on recommendations outlined by Jeewon and Hyde (2016). Table 2. Taxa used in this study and their GenBank accession numbers, “NA” indicates unavailable sequence. Species Culture/voucher ITS mtSSU Ochrolechia akagiensis Hara Kojiro:0007 LC533077 NA O. alaskana TROM_L_60552 MK811874 NA O. alboflavescens PRA-Vondrak22558 OQ717978 OQ646357 O. alboflavescens O-L-201276 MK812244 NA O. androgyna PRA-Vondrak23666 OQ717979 OQ646358 O. androgyna PRA-Vondrak23816 OQ717525 OQ646359 O. antarctica Davey 27-3 NA KX499403 O. antarctica Davey 21-7 NA KX499401 O. arborea PRA-Vondrak25006 OQ717981 OQ646360 O. austroamericana Flakus 21197 NA KX499404 O. bahusiensis PRA-Vondrak22578 OQ717983 OQ646362 O. bahusiensis 17950 MN387044 NA O. balcanica Schmitt ESS-20968 NA AF329170 O. frigida ERCH: HS 51 OR687726 NA O. frigida P168 KR017062 KR017339 23 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species Species Culture/voucher ITS mtSSU O. guizhouensis LGS219 PV930363 PV930371 O. gowardii O-L-200100 MK812175 NA O. gowardii O-L-200283 MK812101 NA O. incarnata Ertz 10572 MH485185 NA O. incarnata GLM25533 MH485183 NA O. juvenalis AFTOL-ID 374 HQ650719 KJ766446 O. kerguelensis Ertz 18928 NA KX499406 O. kerguelensis Ertz 18906 NA KX499405 O. kuankuoshuiensis KKS83 PV930358 PV930366 O. kuankuoshuiensis LGS40 PV930359 PV930367 O. leigongshanensis LGS186 PV930364 PV930372 O. longispora LGS205 PV930362 PV930370 O. longispora LGS213-2 PV930361 PV930369 O. mahluensis PRA-Vondrak22577 OQ717985 OQ646363 O. microstictoides PRA-Vondrak23770 OQ717987 OQ646364 O. microstictoides PD032M MW325687 NA O. oregonensis CCDB-36282-A09 OQ843356 NA O. oregonensis L-793 OQ922942 NA O. pallescens J. Malicek 10146 MK778626 MK778561 O. pallescens Lumbsch, 9. Aug. 2004 NA DQ780277 O. parella K(M):202474a MZ159609 NA O. parella Ertz 10504 MH485200 KX499409 O. parellula KoLRI No.018698 KU883361 NA O. parellula KoLRI No.015662 KU883360 NA O. parellula KoLRI No.013758 KU933682 NA O. parellula KoLRI No.015650 KU883359 NA O. parellula CBM:Sakata 3456 LC489985 NA O. peruensis Lumbsch 19360c NA DQ780279 O. subathallina L-828 OQ922944 NA O. subpallescens Lumbsch 19900a NA GU980978 O. subrosella LGS176 PV930360 PV930368 O. subviridis O-L-200638 MK812495 NA O. subviridis Sadowska-Des 810H13-002_G07 MH485202 NA O. szatalaensis PRA-Vondrak23372 OQ717527 NA O. szatalaensis O-L-200097 MK811817 NA O. tartarea O-L-196041 MK812341 NA O. tartarea DNA7 JN943620 NA O. trochophora AFTOL-ID 880 NA DQ986901 O. trochophora J. Vondrak 15442 MK778627 MK778562 O. trochophora var. trochophora KKS150-2 PV930365 PV930373 O. turneri PRA-Vondrak23511 OQ717989 OQ646366 O. turneri PRA-JV23905 OK333002 OK465619 O. upsaliensis Leavitt 18-422 BRY-C MZ243924 NA O. upsaliensis O-L-195967 KY266960 NA O. xanthostoma Tonsberg 46121 MN483173 MN508284 O. yasudae Hara Kojiro:0005 LC533076 NA O. yasudae KoLRI No.010572 KU883364 NA T. coarctata O-L-179924 MK812177 NA T. coarctata O-L-182063 MK812526 NA 24 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species Results and discussion Phylogenetic analysis In this study, the final aligned dataset comprised 1,256 characters, including gaps (ITS:1–541, mtSSU:542–1,256). Estimated base frequencies were as follows: A = 0.2534, C = 0.2370, G = 0.2539, T = 0.2557. Substitution rates amongst nucleotides were as follows: AC = 1.3275, AG = 4.1600, AT = 2.2572, CG = 0.8425, CT = 8.1438, GT = 1.0000. The average standard deviation of split frequencies for the BI analysis reached 0.010000, indicating good convergence. The tree topologies inferred by both ML and BI analyses were manually verified and found to be highly congruent (BP/PP = 100/1 for major supported clades). The best-scoring RAxML tree, based on combined ITS and mtSSU-sequence datasets, is presented in Fig. 1. The phylogenetic tree comprises 36 taxa of Ochrolechia. Analysis reveals that the species examined in this study cluster into a large clade with O. trochophora, O. akagiensis and O. parellula (ML Bootstrap = 90%, BI PP = 1.00). Within this clade, specimen KKS150-2 forms a distinct subclade with O. trochophora (ML Bootstrap = 100%, BI PP = 1.00), indicating a close phylogenetic relationship between them. O. kuankuoshuiensis and O. parellula form a sister clade (ML Bootstrap = 74%, BI PP = 0.93) positioned near O. longispora, suggesting a possible shared ancestry or similar evolutionary trajectory amongst these lineages. O. guizhouensis and O. akagiensis are closely related, implying a certain degree of phylogenetic affinity. However, the low support value for this relationship may reflect relatively high genetic divergence between the two species. Furthermore, O. leigongshanensis and O. subrosella each form phylogenetic lineages with low support values, indicating distant relationships to other Ochrolechia taxa, which may be attributable to their distinct genetic characteristics. Taxonomy Ochrolechia guizhouensis Zheng & Meng, sp. nov. Index Fungorum: IF902063 Facesoffungi Number: FoF18024 Fig. 2 Remark. Ochrolechia guizhouensis is characterised by apothecia with rugose to rosulate discs presenting a distinct floral morphology. Chemically, lichesterinic acid is present in the apothecia, but absent in the thallus. Type. China • Guizhou, Qiandongnan Prefecture, Leigongshan Nat. Res, on bark, 1698 m elev., 2023, B. Liu and Z. Yang, LGS219 (KUN-L 96618, holotype). Etymology. The species epithet refers to Guizhou Province, the type locality where the species was collected. Holotype. KUN-L 96618. Description. Thallus greyish-green, thick, rugose to verruculose, dull; prothallus indistinct, isidia and soredia absent. Sexual morph. Apothecia frequent, scattered, sometimes crowded, sessile, round or irregular, 0.5–0.8 mm diam.; disc light yellowish-green or light brown, rugose to rosulate, epruinose; margins thick, smooth and taller than disc when young, becoming thin, verruculose and as tall as disc when older, exciple well developed even extending to the disc surface, concolorous with 25 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species the thallus, shiny. Pycnidia are absent. Hymenium hyaline and colourless, 360–370 μm high, paraphyses are branched and anastomosing; epihymenium brown, 9–11 μm high; hypothecium pale brown (the colour of the epihymenium and hypothecium disappears in a 10% KOH solution), 30–40 μm high; coccoid green alga, algae forming a continuous layer in the margin and in scattered clumps below the hypothecium. Asci clavate, (269)272–284(288) × (48)49– 60(62) μm (n = 10). Ascospores hyaline, aseptate, broadly ellipsoid, (57)60– 78(82) × (26)28–32(34) μm (n = 31). Chemistry. Thallus contains gyrophoric acid and lecanoric acid; ascomata contain gyrophoric acid, lecanoric acid and lichesterinic acid (TLC). Thallus Figure 1. Phylogenetic relationships within the genus Ochrolechia. This is a RAxML analysis, based on combined ITS and mtSSU sequence data. Bootstrap support values of Maximum Likelihood (ML) ≥ 70% are labelled at corresponding positions; branches that simultaneously satisfy the criteria of ML bootstrap support ≥ 70% and Bayesian posterior probability (PP) ≥ 0.90 are displayed in bold. T. coarctata (O-L-179924) and T. coarctata (O-L-182063) were used as outgroup taxa. The newly-generated sequences are shown in bold font. 0.07 Ochrolechia longispora LGS205 Ochrolechia peruensis Lumbsch 19360c Ochrolechia akagiensis Hara Kojiro:0007 Ochrolechia bahusiensis PRA-Vondrak22578 Ochrolechia parella KM:202474a Ochrolechia pallescens Lumbsch 2004 Ochrolechia frigida P168 Ochrolechia alaskana TROM_L_60552 Ochrolechia incarnata GLM25533 Ochrolechia guizhouensis LGS219 Ochrolechia microstictoides PD032M Ochrolechia turneri PRA-Vondrak23511 Ochrolechia parellula KoLRI No.015650 Ochrolechia austroamericana Flakus 21197 Ochrolechia trochophora AFTOL-ID 880 Ochrolechia pallescens J. Malicek 10146 Ochrolechia gowardii O-L-200100 Ochrolechia androgyna PRA-Vondrak23816 Ochrolechia oregonensis L-793 Ochrolechia balcanica ESS-20968 Ochrolechia trochophora J. Vondrak 15442 Ochrolechia szatalaensis O-L-200097 Ochrolechia kerguelensis Ertz 18906 Ochrolechia oregonensis CCDB-36282-A09 Ochrolechia guizhouensis LGS219 T Ochrolechia arborea PRA-Vondrak25006 Ochrolechia kuankuoshuiensis LGS40 Ochrolechia mahluensis PRA-Vondrak22577 Ochrolechia parellula KoLRI No.015662 Ochrolechia upsaliensis O-L-195967 Ochrolechia tartarea O-L-196041 Ochrolechia upsaliensis Leavitt 18-422 Ochrolechia leigongshanensis LGS186 Ochrolechia parellula KoLRI No.018698 Ochrolechia androgyna PRA-Vondrak23666 Trapelia coarctata O-L-182063 Ochrolechia alboflavescens O-L-201276 Trapelia coarctata O-L-179924 Ochrolechia subviridis O-L-200638 Ochrolechia aff. xanthostoma Toensberg 46121 Ochrolechia subviridis FR 810H13-002-G07 Ochrolechia turneri PRA-JV23905 Ochrolechia kerguelensis Ertz 18928 Ochrolechia kuankuoshuiensis KKS83 Ochrolechia longispora LGS213-2 Ochrolechia frigida ERCH:HS 51 Ochrolechia trochophora var. trochophora KKS150-2 Ochrolechia yasudae Hara Kojiro:0005 Ochrolechia antarctica Davey 27-3 Ochrolechia subrosella LGS176 Ochrolechia subrosella LGS176 T Ochrolechia leigongshanensis LGS186 T Ochrolechia juvenalis AFTOL-ID 374 Ochrolechia parellula KoLRI No.013758 Ochrolechia incarnata Ertz 10572 Ochrolechia tartarea DNA7 Ochrolechia subpallescens Lumbsch 19900a Ochrolechia bahusiensis 17950 Ochrolechia szatalaensis PRA-Vondrak23372 Ochrolechia subathallina L-828 Ochrolechia parella Ertz 10504 Ochrolechia gowardii O-L-200283 Ochrolechia yasudae KoLRI No.010572 Ochrolechia microstictoides PRA-Vondrak23770 Ochrolechia alboflavescens PRA-Vondrak22558 Ochrolechia xanthostoma Tonsberg 46121 Ochrolechia parellula CBM:Sakata 3456 Ochrolechia antarctica Davey 21-7 100 95 100 97 100 100 99 74 100 96 100 100 100 93 90 100 93 92 100 100 100 98 86 86 72 100 100 94 81 94 100 100 97 87 100 100 100 100 100 100 82 Ochrolechia Outgroup 100 81 97 26 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species cortex: K + pale yellow, C + red, KC + red; medulla: C –; apothecia cortex: K + pale yellow, C + red, KC + yellow; medulla: C –; disc: C + red; thallus UV −. Material examined. China • Guizhou Province, Qiandongnan Prefecture City, Leigongshan National Nature Reserve, 26°22'43.24"N, 108°11'42.54"E, 1698 m elev., on bark, 27 October, 2023, Bo Liu and Ze Yang, LGS219 (KUN-L 96618, holotype). Notes. In the phylogenetic tree, O. guizhouensis (LGS219) and O. akagiensis (Hara Kojiro:0007) are closely related although with weak bootstrap support (< 70%). The ITS sequence divergence between them is 6.35% (33/520 bp). Morphologically, the two species differ distinctly: O. guizhouensis lacks isidia on the thallus and produces pale yellow, smaller apothecia (0.5–0.8 mm in diameter), whereas O. akagiensis bears isidia and develops pinkish, larger apothecia (0.8– 1.5 mm in diameter). Chemically, the apothecia of O. guizhouensis contain lichesterinic acid, while the presence of this compound has not been reported in O. akagiensis (Park et al. 2019). Although Ochrolechia guizhouensis resembles O. trochophora in some morphological and chemical characteristics, the two species differ notably in several features. The apothecia of O. guizhouensis are smaller (0.5–0.8 mm diam.), whereas Figure 2. Ochrolechia guizhouensis (LGS219). A, B. Morphology of thallus and apothecia; C. Cross-section of the ascomata; D. Algae (the white arrow points to the algae); E. Ascospores. Scale bars: 1 mm (A); 0.5 mm (B); 200 μm (C); 100 μm (D); 20 μm (E). 27 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species those of O. trochophora are larger (1–3(4) mm diam.) (Brodo 1991). In addition, the apothecia of the latter lack lichesterinic acid. Phylogenetic analyses further indicate that this new species is distantly related the two O. trochophora vouchers (AFTOL-ID 880 and J. Vondrak 15442), supported by sequence divergences of 13.76% (67/487 bp) in the ITS region and 4.97% (35/704 bp) in the mtSSU region between O. guizhouensis (LGS219) and O. trochophora (J. Vondrak 15442). The difference with another similar species O. margarita, based on the identification key of Ren (2017), is that this species has a very thin thallus and its apothecia become subglobose when mature. Moreover, the algal layer forms a continuous layer below the hymenium, containing only gyrophoric acid and a trace of lecanoric acid. Ochrolechia kuankuoshuiensis Zheng & Meng, sp. nov. Index Fungorum: IF904111 Facesoffungi Number: FoF17959 Fig. 3 Remark. Distinctive features of O. kuankuoshuiensis are the large ascospores ((90)97–116(123) × (30)33–35(36) μm) and chemistry, which is limited to gyrophoric acid and lecanoric acid. Type. China • Guizhou, Zunyi City, Kuankuoshui Nat. Res, on bark, 1529 m elev., 2023, WW. Zheng and B. Liu, LGS219 (KUN-L 96619, holotype). Etymology. The species epithet refers to Kuankuoshui, the locality where the type species was collected. Holotype. KUN-L 96619. Description. Thallus greyish-white, thick, scaly, dull, verruculose; prothallus indistinct; isidia and soredia absent. Sexual morph. Apothecia frequent, mostly scattered, sometimes crowded, sessile, irregularly rounded or irregularly florid, 0.5–2.5 mm diam.; disc light yellow, epruinose, rough to rugose when young, with deep rugose when older, margins thick, concolorous with the thallus, shiny, verrucose and higher than disc when young, warts developing until they intersect with the folds of the disc at maturity. Pycnidia are absent. Hymenium hyaline, 410–483 μm high; paraphyses are branched, densely aggregated; epihymenium black brown (the colour disappears in a 10% KOH solution), 103–125 μm high; hypothecium 50–75 μm high; coccoid green alga, algae forming a continuous layer in the margin and below the hypothecium; asci clavate, (280)285–309(312) × (66)67–74(75) μm (n = 10), 8-spored. Ascospores (90)97–116(123) × (30)33– 35(36) μm (n = 33), aseptate, hyaline, broadly ellipsoid. Chemistry. Thallus and ascomata contain grophoric acid and lecanoric acid (TLC). Thallus cortex: K –, C + red, KC + red; medulla: C –; apothecia cortex: K + yellow, C + red, KC + red; medulla: C –; disc: C + red; thallus UV −. Material examined. China • Guizhou Province, Zunyi City, Kuankuoshui National Nature Reserve, 28°14'14.51"N, 107°9'16.55"E, 1529 m elev., on bark, 17 November, 2023, Weiwei Zheng and Bo Liu, KKS83 (KUN-L 96619, holotype); • Qiandongnan Prefecture City, Leigongshan National Nature Reserve, 26°23'6.73"N, 108°12'11.11"E, 2054 m elev., on bark, 17 October, 2023, Shaobin Fu and Ze Yang, LGS40 (KUN-L 96620). 34 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species and O. leigongshanensis, but forms a distinct, weakly-supported clade. Sequence comparison reveals that O. subrosella (LGS176) differs from O. akagiensis (Hara Kojiro: 0007) by 6.93% (37/534 bp) in ITS and from O. leigongshanensis (LGS186) by 8.49% (41/483 bp) in ITS and 1.45% (10/688 bp) in mtSSU. Morphologically, this species is characterised by mature apothecia with expanded discs bearing striated protuberances and conspicuous radial ridges, consistent with the original description of O. subrosella (Jia and Zhao 2005). Chemically, TLC confirmed the presence of gyrophoric acid and lecanoric acid in LGS176, matching the secondary metabolite profile reported for O. subrosella. Ochrolechia trochophora (Vain.) Oshio var. trochophora Fig. 7 Remark. J. Sci. Hiroshima Univ., Ser. B, Div. 2(12): 145 (1968). – Pertusaria trochophora Vain., Bot. Mag. (Tokyo) 32: 155 (1918). Type. Japan • Prov. Kozuke, Mt. Akagi. Ad corticem arboris, Yasuda 53 (TUR-V–7255 – holotype, not seen; see Brodo (1991: 762); TI – isotype, not seen; see (Oshio 1968: 145)). Description. Thallus greyish-white to greyish-green, thin, verruculose, dull; isidia absent, prothallus indistinct, soredia greyish-white. Sexual morph. Apothecia frequent, scattered, sessile, ellipsoid or round, 1.1– 1.8 mm diam.; disc pale pink, plane to lightly rugose, epruinose, with a hazy white film; margin smooth or verruculose, higher than disc, dull, concolorous with the thallus. Pycnidia are absent. Hymenium hyaline and colourless, 380– 392 μm high; epihymenium brown, 20–28 μm high; hypothecium greyish-brown (the colour of the epihymenium and hypothecium partially or completely disappears in a 10% KOH solution), 10–17 μm high; coccoid green alga, algae absent or spotty in the margin and continuous below the hypothecium. Asci clavate, 8-spored, (359)363–372(375) × (42)43–49(52) μm. Ascospores hyaline, aseptate, broadly ellipsoid, (50)55–70(75) × (22)23–26(28) μm (n = 30). Chemistry. Thallus contains gyrophoric acid, lecanoric acid and atranorin; ascomata contain gyrophoric acid and lecanoric acid. (TLC). soredia: K+yellow-green, C –, KC –; thallus cortex: K + yellow, C + red, KC + red to yellow-green; medulla: C –; apothecia cortex: K + yellow, C + red, KC + red to yellow-green; medulla: C –; disc: C + red; thallus UV –. Material examined. China • Guizhou Province, Zunyi City, Kuankoshui National Nature Reserve, 28°12'29.34"N, 107°10'24.27"E, 1418 m elev., on bark, 18 November, 2023, Weiwei Zheng and Bo Liu, KKS150-2 (KUN-L96625). Notes. Ochrolechia trochophora is a widely distributed species reported from several locations worldwide. It is primarily characterised by verruculose apothecial margins lacking or containing scattered algal cells. The key distinction between O. trochophora var. trochophora and O. trochophora var. pruinirosella lies in the fact that the latter has a pruinose apothecial disc and the vast majority of its specimens contain variolaric acid. In contrast, O. trochophora var. trochophora occasionally exhibits a hazy white film on the disc, but this is not pruina and very few specimens contain variolaric acid or atranorin (Brodo 1991). As reported by Kukwa (2009), European and Turkish specimens of O. trochophora var. trochophora possess a relatively thin thallus. Furthermore, the detection of atranorin in one 35 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species Figure 7. Ochrolechia trochophora (Vain.) Oshio var. trochophora, J. Sci. Hiroshima Univ (LGS150-2). A. Morphology of thallus and apothecia; B, C. Morphology of ascomata; D. Cross-section of the ascomata in 10% KOH (the white arrow points to the algae); E–G. Asci; H–J. Ascospores. Scale bars: 3 mm (A); 0.7 mm (B); 1 mm (C); 500 μm (D); 50 μm (E–G); 20 μm (H–J). 36 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species Chinese specimen aligns with these findings. Collectively, this evidence supports the identification of specimen KKS150-2 as O. trochophora var. trochophora. The specimen also bears soredia, which is a relatively distinctive variation, as according to previous reports, no records of soredia have been documented in O. trochophora (whether the typical variety or other known varieties) (Brodo 1991; Kukwa 2009). This indicates that O. trochophora might be morphologically more variable than previously thought. In Ochrolechia, many species (e.g. O. africana, O. antillarum, O. gowardii, O. trochophora) contain small crystalline structures in the apothecial medulla (Brodo 1991). In the species we describe, the brownish appearance of both the epihymenium and hypothecium in apothecial sections is more likely a structural colour rather than a pigment-based one. This conclusion is primarily based on the partial or complete fading of the brown colour when treated with 10% KOH, likely due to the dissolution of abundant granular or crystalline substances in the tissues. Traditionally, the classification of species within the genus Ochrolechia has primarily relied on a combined analysis of morphological characteristics and chemical compounds. Morphologically, key diagnostic features include thallus thickness, the presence or absence of isidia and soredia, apothecial morphology, hymenium height, spore size and the position of the algal layer. Chemically, species in this genus exhibit a remarkable diversity of secondary metabolites, which serve as critical taxonomic markers. These compounds mainly belong to the following classes: Orcinol depsides (e.g. gyrophoric acid, lecanoric acid and olivetoric acid), orcinol depsidones (e.g. variolaric acid and alectoronic acid), higher aliphatic acids (e.g. lichesterinic acids, protolichesterinic acids and murolic acids) and xanthones, which can induce yellow fluorescence in the thallus under long-wave ultraviolet light. Additionally, trace amounts of atranorin have been detected in some species (Brodo 1991; Kukwa 2009). In our study, thin-layer chromatography revealed significant levels of the lichen secondary metabolite atranorin in both the newly-described species O. leigongshanensis and the known species O. trochophora var. trochophora. This finding contrasts with the previous understanding that atranorin exists only in trace amounts within the genus Ochrolechia. Notably, these two species share a key morphological characteristic, the presence of soredia. The combination of this chemical and morphological feature provides valuable new insights into the evolutionary relationships within Ochrolechia. Soredia are rarely observed in Ochrolechia and atranorin is not usually a major metabolite in this genus. The discovery that both O. leigongshanensis and O. trochophora var. trochophora possess these two characteristics strongly suggests that they may belong to a distinct phylogenetic lineage previously unrecognised. The production of soredia, as an asexual reproductive structure, is linked to specific genotypes, while the substantial synthesis of atranorin indicates the activation of particular biochemical pathways. The stable co-existence of these two independent traits, both morphological and chemical, within a limited taxonomic group is unlikely to be coincidental and likely represents synapomorphies inherited from a common ancestor. Thus, we hypothesise that these two species may share a most recent common ancestor within Ochrolechia, potentially forming a monophyletic group. With the rapid advancement of molecular techniques, phylogenetic analysis has become an indispensable tool in species identification and evolutionary 37 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species studies. Molecular data provide objective genetic evidence that enables the effective differentiation of morphologically similar and cryptic species, clarifying taxonomic uncertainties, such as synonymy and facilitating the discovery of new taxa (Spatafora et al. 2006; Miadlikowska et al. 2014). Relying solely on phenotypic characteristics often fails to resolve taxonomic ambiguities, as these features are influenced by environmental factors and developmental stages. In contrast, genetic data offer greater stability. Therefore, this study adopts an integrative taxonomic approach, combining phylogenetic analysis, morphological examination and chemical profiling to provide a comprehensive and accurate classification of new Ochrolechia species. The phylogenetic analysis in this study revealed that all newly-described species form a single clade (Fig. 1). Although bootstrap support for some branches within this clade was relatively low (< 70%), we interpret this not to ambiguous taxon delimitation, but rather to the substantial genetic distances amongst these new species or it might be due to the insufficient sampling in this clade. As more sequences of Ochrolechia species are published in the future, the topology may become more stable. Such considerable interspecific genetic divergence may result in a higher number of ambiguous alignment sites, leading to reduced nodal support in phylogenetic reconstructions. This phenomenon strongly suggests that the present study may have only uncovered a fraction of the diversity within the genus Ochrolechia in Guizhou, China. Limitations in sampling scope and specimen numbers currently hinder a full assessment of the true morphological variation and geographical distribution ranges of these species, which may also contribute to the temporarily unresolved phylogenetic relationships. It is important to note that, while molecular support could be further strengthened through additional gene loci or expanded sampling in the future, all new species described in this study exhibit unique combinations of morphological and chemical characteristics, enabling clear distinction from all known related species. In conclusion, the distinct clade formed by the new species described in this study not only enhances our understanding of the phylogenetic framework of Ochrolechia, but also highlights significant genetic variation within the clade, suggesting that the region may harbour an underexplored diversity of Ochrolechia. Future studies should prioritise more extensive and systematic specimen collection from this region and neighbouring areas, combined with multi-locus genomic data, to thoroughly elucidate the speciation mechanisms and the broader diversity of this genus. Acknowledgements We would like to express our deepest gratitude to Dr. Qiang Ren for the invaluable assistance in specimen confirmation. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. 38 MycoKeys 126: 19–40 (2025), DOI: 10.3897/mycokeys.126.168652 Weiwei Zheng et al.: Three new Ochrolechia species Use of AI No use of AI was reported. Funding This research is supported by the National Natural Science Foundation of China (No. 32560002), Zunyi Scientific and Technological Innovation Talent Team Training Project (ZSK-RC[2023]8), Innovation and Entrepreneurship Project for College Students (202210661208, ZYDC202302260 & S2024106612235) and Summer Practicum Project for Graduate Students (zy-ysh202410). Author contributions Weiwei Zheng designed the experiments, analysed the data, structured the manuscript framework and drafted the initial manuscript. Linzhi He participated in methodology and Heyun Bo contributed to data analysis. Yuxian Wang and Jie Wang participated in investigation. Ruvishika S. Jayawardena, Rajesh Jeewon, Qingfeng Meng and Shaobin Fu reviewed and edited the manuscript. All authors reviewed, revised and approved the final version for publication. Author ORCIDs Weiwei Zheng https://orcid.org/0009-0006-4099-7599 Linzhi He https://orcid.org/0009-0008-3621-7339 Heyun Bo https://orcid.org/0009-0003-8641-1619 Rajesh Jeewon https://orcid.org/0000-0002-8563-957X Ruvishika S. Jayawardena https://orcid.org/0000-0001-7702-4885 Yuxian Wang https://orcid.org/0009-0004-8810-3641 Jie Wang https://orcid.org/0009-0000-7788-4471 Shaobin Fu https://orcid.org/0000-0001-9932-1346 Qingfeng Meng https://orcid.org/0000-0001-9814-8238 Data availability All of the data that support the findings of this study are available in the main text. References Brodo IM (1991) Studies in the lichen genus Ochrolechia. 2. Corticolous species of North America. Revue Canadienne De Botanique 69: 733–772. https://doi.org/10.1139/b91-099 Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T (2009) trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. 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