683 MycoKeys MycoKeys 115: 1-18 (2025) DOI: 10.3897/mycokeys.115.138252 Research Article Non-lichenized Cytosporella, including C. fuligomixta sp. nov., and related plant-associated and fungicolous genera are close to foliicolous, lichenized fungi (Ascomycota, Graphidales) Marcin Piatek'®, Monika Stryjak-Bogacka’™®, Pawet Czachura'® 1 W. Szafer Institute of Botany, Polish Academy of Sciences, Lubicz 46, PL-31-512 Krakow, Poland Corresponding author: Marcin Piatek (m.piatek@botany.pl) OPEN Qaceess Academic editor: Pradeep Divakar Received: 30 September 2024 Accepted: 15 February 2025 Published: 6 March 2025 Citation: Pigtek M, Stryjak-Bogacka M, Czachura P (2025) Non-lichenized Cytosporella, including C. fuligomixta sp. nov., and related plant-associated and fungicolous genera are close to foliicolous, lichenized fungi (Ascomycota, Graphidales). MycoKeys 115: 1-18. https://doi.org/10.3897/ mycokeys.115.138252 Copyright: © Marcin Piatek 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 The genus Cytosporella includes non-lichenized, plant associated fungi producing eus- tromatic conidiomata, phialidic conidiophores and hyaline, ellipsoid conidia. Of the 69 names assigned to this genus in Index Fungorum, only three species are associated with sequence data. In this study, a new species: Cytosporella fuligomixta is described based ona strain isolated from the sooty mould community on Quercus robur leaves in Poland. The phylogenetic analyses including sequences of two loci (LSU, mtSSU) showed that Cytosporella species, together with members of four other non-lichenized, plant asso- ciated or fungicolous genera, namely Cladosterigma, Neoacrodontiella, Nothoramula- ria and Vanderaaea, form a sister group to lichenized and lichenicolous fungi from the family Gomphillaceae and order Graphidales. Previously, Cladosterigma was resolved as a member of Gomphillaceae using multi-locus (mtSSU, SSU, LSU, ITS, rpb2, tef7) and two-locus (LSU, mtSSU) sequence analyses, while Cytosporella, Neoacrodontiella, Notho- ramularia were shown to belong to this family using LSU sequence analyses. However, none of them resolved these genera as a sister group to lichenized members of Gomphil- laceae. The placement of the genus Vanderaaea within Gomphillaceae is shown here for the first time. Due to phylogenetic, morphological and ecological characteristics a new subfamily Cladosterigmoideae is described for these five non-lichenized genera. Key words: Ascomycota, Graphidales, Gomphillaceae, one new species, one new sub- family, sooty mould communities, taxonomy Introduction The genus Cytosporella includes non-lichenized, plant associated fungi produc- ing eustromatic conidiomata, phialidic conidiophores and hyaline, ellipsoid co- nidia (Sutton 1980; Crous et al. 2019b; Li et al. 2020). Index Fungorum (2024) includes 69 names assigned to Cytosporella but DNA sequence data are avail- able only for three species: Cytosporella calamagrostidis, C. chamaeropis and C. juncicola. The type species is Cytosporella sycina (Clements and Shear 1931; Sutton 1980; Li et al. 2020). It has been described from branches of Ficus carica in France (Saccardo 1880) but not sequenced yet, making current taxonomy of the genus tentative. The species of Cytosporella are saprobic or parasitic on Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera different hosts, mostly on branches of deciduous trees (Li et al. 2020; species data in Index Fungorum 2024). During our surveys of sooty mould communities on ornamental woody plants in southern Poland a new species of Cytosporella (described here as Cytosporella fuligomixta) was isolated from a sooty mould colony on leaves of Quercus robur (Fagaceae). The sequenced species of Cytosporella were shown to be related to three non-lichenized, plant associated or fungicolous genera: Neoacrodontiella, Nothoramularia and Vanderaaea, and altogether they were assigned to the fam- ily Acarosporaceae and order Acarosporales (Crous et al. 2019a, 2019b, 2021, 2023). This order and family contain saxicolous and terricolous lichenized fungi in the subclass Acarosporomycetidae of the Lecanoromycetes (Reeb et al. 2004; Miadlikowska et al. 2014; Westberg et al. 2015). Our initial query of sequences of Cytosporella (including new species C. fuligomixta), Neoacrodon- tiella, Nothoramularia and Vanderaaea in GenBank showed that most closely related sequences belong to members of the lichenized family Gomphillaceae. Also, non-lichenized, fungicolous Cladosterigma clavariellum, which was re- cently included in Gomphillaceae (Guterres et al. 2020), was amongst the resul- tant related sequences. This family includes mostly foliicolous lichens and is included either in Graphidales or Ostropales within subclass Ostropomycetidae of the Lecanoromycetes (Baloch et al. 2010; Miadlikowska et al. 2014; Kraichak et al. 2018). Indeed, a recent phylogenetic tree, based on LSU sequences, pub- lished in Crous et al. (2024), placed Cladosterigma, Cytosporella, Neoacrodon- tiella and Nothoramularia inside the family Gomphillaceae. The genus Vander- aaea was not included in these analyses. Similarly, multi-locus (using mtSSU, SSU, LSU, ITS, rpb2 and tef1 sequences) and two-locus (using LSU and mtSSU sequences) trees, published by Guterres et al. (2020), placed Cladosterigma inside the family Gomphillaceae with Vezdamyces vulgaris (syn. Gyalideopsis vulgaris) being the most basal species. However, in these cases (based on GenBank accession numbers of sequences included by Guterres et al. 2020 in their tables), sequences of “Gyalideopsis vulgaris” used in the multi-locus tree belonged to Gyalidea praetermissa that is a member of Gyalectaceae (Ertz et al. 2021), while LSU sequence used in two-locus tree belonged to other species (closest hits in GenBank are species of Eurotiomycetes) that could have been responsible for such a result. In this study, we describe and illustrate a new species of Cytosporella isolat- ed from a sooty mould colony on leaves of Quercus robur. Additionally, based on publicly available sequences, we reassess phylogenetic placement of spe- cies of Cladosterigma, Cytosporella, Neoacrodontiella, Nothoramularia and Van- deraaea using two-locus (LSU, mtSSU) sequence analyses. Materials and methods Strain and morphological analyses The strain was obtained from sooty mould communities during the study of sooty moulds on ornamental woody plants cultivated in municipal greenery in southern Poland (Pigtek et al. 2023). Macroscopic features of cultures were documented using 2-week-old colonies grown on malt extract agar (MEA — Blakeslee’s formula), potato dextrose agar (PDA) and oatmeal agar (OA) at 6 °C, MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 2 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera 15 °C and 25 °C. Growth at different temperatures was assessed by measur- ing the colony diameter after 2 weeks and 4 weeks. Microscopic features were studied using colonies grown on MEA and OA at 15 °C after 7 weeks and 6 weeks, respectively. The characteristics of hyphae were observed on MEA and characteristics of conidiomata, conidiophores and conidia were studied on OA. Hyphae taken from the edge of the colony and mature conidiomata were mount- ed in lactic acid (80%) on microscope slides and analysed under Nikon Eclipse 80i light microscope. Microscopic structures were measured and photographed using NIS-Elements BR 3.0 imaging software. Holotype is a dried specimen ob- tained from culture and is stored in the fungal collection of the W. Szafer Insti- tute of Botany, Polish Academy of Sciences, Krak6w (KRAM F). Culture is depos- ited in the culture collection of the Westerdijk Fungal Biodiversity Institute (CBS) and in the W. Szafer Institute of Botany, Polish Academy of Sciences, Krakéw. DNA isolation, amplification and sequencing Genomic DNA was extracted from four-week-old MEA culture using DNeasy® Plant Mini Kit (Qiagen, Germany), according to the manufacturer's protocol. A total of five loci were amplified: ITS1-5.8S-ITS2 rRNA (= ITS), partial large sub- unit rRNA (28S D1—D2 = LSU), small subunit mtRNA (=mtSSU), partial DNA-di- rected RNA polymerase II second largest subunit (rpb2) and translation elon- gation factor 1-alpha (tef7). To amplify the regions of ITS, LSU, mtSSU, rpb2 and tef1, five different primer pairs were used, namely ITS1-ITS4 (White et al. 1990), LSU1Fd-LR5 (Vilgalys and Hester 1990; Crous et al. 2009), mrSSU1- mrSSU3R (Zoller et al. 1999), fRPB2-5F-fRPB2-7cR (Liu et al. 1999) as well as EF1-983F and EF1-2218R (Rehner and Buckley 2005), respectively. Polymerase chain reaction mixtures were performed in a total volume of 25 uL as explained in Pigtek et al. (2023). Amplification conditions for ITS and LSU were described in Czachura et al. (2021), while conditions for subsequent loci were set as fol- lows: an initial denaturation at 94 °C for 3 min, followed by 35 cycles (mtSSU, rpb2) or 40 cycles (tef7) of denaturation at 94 °C for 60 sec (mtSSU, rpb2) or 30 sec (tef7); annealing at 52 °C (mtSSU) or 54 °C (rpb2) or 55 °C (tef7) for 60 sec (mtSSU) or 90 sec (rpb2) or 50 sec (tef7); and extension at 72 °C for 1 min (mtS- SU, tef7) or 2 min (rpb2). The process was finished with the final extension at 72 °C for 7 min (mtSSU) or 10 min (rpb2, tef7). PCR amplifications were confirmed on 1% agarose electrophoresis gels stained with SimplySafe (EURx, Poland). Amplicons were enzymatically cleaned using Exo-BAP Mix (EURx, Poland). The same primers as given above were used in sequencing reactions which were carried out by Macrogen Europe B.V. (Amsterdam, The Netherlands). Obtained sequences were assembled and trimmed in Geneious Prime 2020.0.4. Con- sensus sequences were deposited in the NCBI's GenBank nucleotide database (https://www.ncbi.nim.nih.gov/genbank/). Phylogenetic analyses The affinities of obtained Cytosporella sequences and sequences of related genera and species were determined in the NCBIs GenBank nucleotide da- tabase using the megablast search tool (Zhang et al. 2000). For the phylo- genetic analyses only LSU and mtSSU sequences were used due to limited MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 3 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera sampling of reference sequences available for members of Gomphillaceae. The assembled concatenated LSU-mtSSU alignment contained available sequences of all sequenced species of Cladosterigma, Cytosporella, Neo- acrodontiella, Nothoramularia and Vanderaaea, and sequences of selected members of Gomphillaceae and members of its most closely related family Graphidaceae used as an outgroup (Table 1). In the case of problematic se- quences of Vezdamyces vulgaris (syn. Gyalideopsis vulgaris) used by Guterres et al. (2020), the correct sequences obtained from two different specimens were taken following Xavier-Leite et al. (2022). Sequence reads which we obtained were checked for quality and assem- bled. The complete LSU and mtSSU sequences were separately aligned for each single-gene dataset using MAFFT algorithm (Katoh et al. 2005) in Ge- neious 11.1.5. Phylogenetic trees were constructed by using the Maximum likelihood (ML) and Bayesian inference (BI) analysis. For both ML and BI analyses, the model of DNA evolution that best fitted the dataset was deter- mined using the ModelTest-NG v. 0.2.0 under the Bayesian Information Crite- rion (BIC) (Darriba et al. 2020). Maximum likelihood (ML) analyses were per- formed using the RAXxML-NG v. 1.1.1 (Kozlov et al. 2019). Branch support was inferred with 1000 bootstrap replicates. Bayesian analyses were conducted using a MrBayes v. 3.2.6 (Ronquist et al. 2012). One million generations were run, sampling every 100 generations. Four parallel chains, one cold and three heated, were used. A consensus tree was generated after discarding the first 25% of trees as burn-in. Average standard deviations of split frequencies dropped below 0.01 at the end of the runs. The final phylogenetic trees were graphically visualised using FigTree v.1.4.3. Results Phylogenetic analyses The concatenated LSU-mtSSU alignment contained sequences belonging to 41 species. The alignment comprised a total of 1779 characters (LSU: 969, mtSSU: 810), including alignment gaps. The best matching substitution models select- ed for single locus alignments in the ML analysis were as follows: TIM2+I+G4 for LSU and TPM3uf+I+G4 for mtSSU. The BI analysis was performed with the following substitution model: GTR+I+G4 for LSU and mtSSU. ML and BI anal- yses resulted in similar tree topologies. The best scoring maximum likelihood phylogenetic tree is shown on Fig. 1. Maximum likelihood bootstrap (MLB) sup- port values above 70% and Bayesian posterior probabilities (BPP) above 0.95 are shown at the nodes. Representatives of the family Gomphillaceae formed strongly supported monophyletic lineage (MLB/BPP = 100/1). The strain of the new Cytosporel- la species clustered with members of the genus Cytosporella as sister to C. chamaeropis and C. juncicola (MLB/BPP = 98/1). Representatives of the gen- era Cladosterigma, Cytosporella, Neoacrodontiella, Nothoramularia and Vander- aaea formed a well-supported clade (MLB/BPP = 73/1) as a sister group to the well-supported clade (MLB/BPP = 78/1) of the remaining Gomphillaceae that contained lichenized species and two non-lichenized lichenicolous genera (Corticifraga, Taitaia). MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 4 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera Table 1. List of species, with country of origin, host/substrate, strain/voucher, GenBank accession numbers and refer- ences, used in phylogenetic analyses. Species Aderkomyces heterellus Asterothyrium longisporum Aulaxina quadrangula Aulaxinella minuta Chroodiscus defectus Cladosterigma clavariellum Cladosterigma clavariellum Clandestinotrema stylothecium Corticifraga peltigerae Corticifraga peltigerae Cruentotrema thailandicum Cytosporella calamagrostidis Cytosporella fuligomixta Cytosporella juncicola Cytosporella chamaeropis Dyplolabia afzelii Echinoplaca sp. Fissurina comparimuralis Fissurina marginata Fissurina rufula Gomphillus calycioides Gomphillus ophiosporus Graphis scripta Gyalectidium catenulatum Gyalectidium imperfectum Gyalidea fritzei Gyalidea hyalinescens Monocalenia monospora Monocalenia monospora Country Host/substrate Strain/voucher Brazil = Caceres & Aptroot 11953 Costa Rica = Licking s.n., F sample no. 4 Costa Rica = Licking s.n., F sample no. 66 Costa Rica = E. Baloch HK2 (GZU) Thailand o Papong 5118 Brazil Phyllachora sp. on UB 23227 leaves of Eugenia florida Brazil Phyllachora sp. on UB 23228 leaves of Eugenia florida Nicaragua i Licking 28636 Luxembourg Peltigera cf. Marson 2015-05- rufescens 02-1 India Peltigera elisabethae | Zhurbenko 1353 (LE 260537) Thailand = Lumbsch 19955d Netherlands Calamagrostis CPC 46236 arenaria Poland sooty mould G107 = CBS 152343 community on Quercus robur leaves USA Juncus effusus CPC 38040 Italy Chamaerops humilis CBS 355.71 USA = Luicking 26509a Nicaragua = Lticking 28550 El Salvador = Luicking 28103 Thailand od Lticking 24122 Fiji E Lumbsch 205211 UK = Lumbsch 20100d (F) (Scotland) Costa Rica = Will-Wolf 10006a (F) Germany a Staiger 982 Costa Rica = Licking 032b Costa Rica = Licking s.n., F sample no. 2 Sweden Nordin 6022 (UPS) Costa Rica = AFTOL-ID 332 Costa Rica = Lticking 032h Costa Rica = Liicking 032e MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 GenBank acc. no. LSU KF833330 AY341349 AY341350 FJ708490 MK933757 MK933758 JX421470 KY462801 KY661661 JF828975 PP791461 PQ001665 MN9567660 MH871929 JX421483 KF833328 JX421492 JX421493 JX421497 KF833329 AY341357 DQ431937 KF833323 AY341358 HM244767 DQ973046 KF833327 KF833325 mtSSU KF833342 AY341363 AY341364 AY648887 FJ708497 MK910849 MK910850 HQ639597 KY661684 JF828960 PP999744 JX421027 KF833340 JX421042 HQ639613 JX421053 KF833341 AY341371 AY648904 KF833335 AY341372 HM244744 DQ972996 KF833339 KF833337 References I. Schmitt, T. Lumbsch, E. Kraichak (unpubl.) Licking et al. 2004 Licking et al. 2004 Grube et al. 2004 Guterres et al. 2020 Guterres et al. 2020 Rivas Plata et al. 2011, 2013 H.-O. Baral, G. Marson (unpubl.) Pino-Bodas et al. 2017 Rivas Plata and Lumbsch 2011 Crous et al. 2024 this study Crous et al. 2019b Vu et al. 2019 Rivas Plata et al. 2013 |. Schmitt, T. Lumbsch, E. Kraichak (unpubl.) Rivas Plata et al. 2013 Rivas Plata et al. 2011, 2013 Rivas Plata et al. 2013 |. Schmitt, T. Lumbsch, E. Kraichak (unpubl.) Licking et al. 2004 |. Schmitt, T. Lumbsch, E. Kraichak (unpubl.) Licking et al. 2004 Baloch et al. 2010 Miadlikowska et al. 2006 |. Schmitt, T. Lumbsch, E. Kraichak (unpubl.) |. Schmitt, T. Lumbsch, E. Kraichak (unpubl.) Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera Species Myriotrema olivaceum Neoacrodontiella eucalypti Nothoramularia ragnhildianicola Ocellularia oculata Phaeographis lobata Psorotheciopsis cf. premneella Pycnotrema pycnoporellum Rolueckia aggregata Rolueckia conspersa Taitaia aurea Taitaia aurea Thelotrema subtile Thelotrema suecicum Tricharia longispora Tricharia longispora Vanderaaea ammophilae Vezdamyces vulgaris GenBank acc. no. Country Host/substrate Strain/voucher References LSU mtSSU Australia = Lumbsch 19113f | EU075627 | EU075579 = Malaysia | Eucalyptus urophylla CBS 145561 MK876437 = Crous et al. 2019a Germany Ragnhildiana CBS 149076 0Q990069 = Crous et al. 2023 ferruginea on leaves of Artemisia vulgaris Australia = Mangold 33a EU075613 | EU075564 = Bermuda = Berger 19598 DQ431944 | DQ431984 =z Cuba a Licking et al. MZ851727 = Xavier-Leite et al. 2022 41885b USA = Licking 26545 HQ639658 | HQ639584 | Rivas Plata et al. 2011 Brazil Caceres & Aptroot | MZ851690 o Xavier-Leite et al. 2022 28665a Brazil = Xavier-Leite etal. | MZ851644 Se Xavier-Leite et al. 2022 2803 Kenya Crocodia cf. Kirika 5103 (EA) | MF372797 | MF372799 Suija et al. 2018 clathrata Kenya Crocodia cf. aurata Rikkinen 16259 MF509277 + Suija et al. 2018 Australia = Mangold 3j (F) EU075651 | EU075607 | Mangold et al. 2008 Turkey = Palice (ESS 21521) | AY300867 | AY300917 | Lumbsch etal. 2004 Costa Rica 3 Liicking 033a KF833326 | KF833338 | |. Schmitt, T. Lumbsch, E. Kraichak (unpubl.) Costa Rica bs Lticking s.n., F AY341360 | AY341374 | Licking et al. 2004 sample no. 37 Netherlands dead leaves of CBS 886.68 MH878416 car Crous et al. 2021 Ammophila arenaria Brazil/Costa = Xavier-Leite 1476/ | MZ851481 | AY584618 Lutzoni et al. 2004; Rica AFTOL ID 105 Xavier-Leite et al. 2022 Taxonomy Gomphillaceae Walt. Watson ex Hafellner, Beih. Nova Hedwigia 79: 280. 1984 Gomphilloideae Rivas Plata, Liicking & Lumbsch, Fungal Diversity 52(1): 108. 2012 Notes. Nominative subfamily includes current members of the family Gomphil- laceae, excluding Cladosterigma, Cytosporella, Neoacrodontiella, Nothoramula- ria and Vanderaaea. The development of hyphophores with their diahyphae is a unique feature of this subfamily. Hyphophores with diahyphae are present in many, though not all, members of this lineage (Ferraro 2004; Licking et al. 2004; Xavier-Leite et al. 2022, 2023). Cladosterigmoideae Pigtek, Stryjak-Bogacka & Czachura, subfam. nov. MycoBank No: 857024 Etymology. Named after the genus Cladosterigma. Description. Non-lichenized, plant associated or fungicolous fungi. Conidiomata sporodochial, synnematal, eustromatic or conidiophores arising directly from hyphae. Conidiophores hyaline, smooth, subcylindrical, MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 6 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera 100/1 Gomphillus ophiosporus AY341357/AY 341371 87/1 Gomphillus calycioides KF833329/KF 833341 72/1 Aderkomyces heterellus KF833330/KF 833342 82/1 100/1 Tricharia longispora KF833326/KF 833338 93/1 Echinoplaca sp. KF833328/KF 833340 g9/- — Rolueckia aggregata MZ851690/— Rolueckia conspersa MZ851644/— 100/1 Gyalectidium imperfectum AY341358/AY 341372 Gyalectidium catenulatum KF 833323/KF833335 -/0.95 99/1 Monocalenia monospora KF833327/KF 833339 Monocalenia monospora KF833325/KF 833337 -/1 Aulaxinella minuta —/AY648887 Aulaxina quadrangula AY 341350/AY 341 364 -10.95 Asterothyrium longisporum AY341349/AY341363 100/11, Corticifraga peltigerae KY462801 /— Corticifraga peltigerae KY661661/KY661684 78/1! joo — Taitaia aurea MF372797/MF372799 Taitaia aurea MF509277/— 97/1 Gyalidea fritzei HM244767/HM244744 100/1 Gyalidea hyalinescens DQ973046/DQ972996 Psorotheciopsis cf. premneella MZ851727/— 100/1 97/1, Cytosporella chamaeropis MH871929/— 99/1 [| Cytosporella juncicola MN567660/— 98/1|L Cytosporella fuligomixta PQ001665/PP999744 93/1 Cytosporella calamagrostidis PP791461/— 100/1 , Cladosterigma clavariellum MK933758/MK910850 3/1 Cladosterigma clavariellum MK933757/MK910849 88/1 Vanderaaea ammophilae MH878416/— Neoacrodontiella eucalypti MK876437/— -/0.99 Nothoramularia ragnhildianicola O0Q990069/— 100/1 Thelotrema subtile EU075651/EU075607 99/1 Thelotrema suecicum AY300867/AY 300917 Soe Chroodiscus defectus FJ708490/F J708497 Phaeographis lobata DQ431944/DQ431984 100/1 Graphis scripta DQ431937/AY648904 Ocellularia oculata EU075613/EU075564 91/1 Myriotrema olivaceum EU075627/EU075579 Koen 100/1 Fissurina rufula JX421497/JX421053 87/1 Fissurina marginata JX421493/HQ639613 Pycnotrema pycnoporellum HQ639658/HQ639584 Clandestinotrema stylothecium JX421470/HQ639597 99/1 Cruentotrema thailandicum JF828975/JF828960 76/0.97 Dyplolabia afzelli JX421483/JX421027 Fissurina comparimuralis JX421492/JX421042 0.07 Figure 1. Phylogenetic tree of selected representatives of Gomphillaceae, including all sequenced species of Cladoste- rigma, Cytosporella, Neoacrodontiella, Nothoramularia and Vanderaaea, obtained from a maximum likelihood analysis of the combined two-locus alignment (LSU, mtSSU). Representatives of Graphidaceae are used as an outgroup. The posi- tions of Cytosporella fuligomixta sp. nov. and Cladosterigmoideae subfam. nov. are indicated in bold. GenBank accession numbers (LSU/mtSSU) are given after species name. Numbers above branches indicate maximum likelihood bootstrap (MLB) support values > 70% and Bayesian posterior probabilities (BPP) > 0.95, respectively (MLB/BPP). The scale bar represents the expected number of changes per site. conical, ampulliform or subglobose, branched or not, with terminal and/or intercalary conidiogenous cells, sometimes reduced to conidiogenous cells. Conidia solitary or rarely in chains, hyaline, smooth, 0-1-septate, ellipsoid, fusoid or subcylindrical. Sexual morph undetermined [based on generic descriptions in Sutton 1980; Guterres et al. 2020; Crous et al. 2019a, 2021, 2023]. Type genus. Cladosterigma Pat. Notes. This subfamily includes genera Cladosterigma, Cytosporella, Neo- acrodontiella, Nothoramularia and Vanderaaea. Members of this subfamily are different morphologically (absence of hyphophores with diahyphae), phylogenetically (distinct, sister lineage in molecular analyses) and ecologi- cally (non-lichenized and non-lichenicolous species) from representatives of the nominative subfamily. MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 Tricharia longispora AY 341360/AY341374 Vezdamyces vulgaris MZ851481/AY584618 Gomphilloideae Cladosterigmoideae Gomphillaceae Graphidaceae Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera Cytosporella fuligomixta Piatek, Stryjak-Bogacka & Czachura, sp. nov. MycoBank No: 857025 Figs 2-4 Etymology. Name refers to the isolation of this fungus from sooty mould com- munities. DNA barcodes. ITS (PQ001666), LSU (PQ001665), mtSSU (PP999744), rpb2 (PP997507) and tef7 (PP997508). Typus. POLAND + Matopolska Province, Krakow County: Krakow-Czyzyny (Park Lotnikéw), municipal greenery (city park), isolated from sooty mould community on Quercus robur leaves, 10 Oct. 2018, leg. M. Piatek, W. Bartoszek & P. Czachura (holotype KRAM F-59995; culture ex-type G107 = CBS 152343). Figure 2. Morphology of cultures of Cytosporella fuligomixta (strain G107 = CBS 152343) after 4 weeks of growth at 15 °C: a-c general view, upper side and reverse side of colony on MEA d-f general view, upper side and reverse side of colony on PDA g-i general view, upper side and reverse side of colony on OA. Scale bars: 10 mm (a, d, g); 5 mm (b, ¢, e, f, h, i). MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 9 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera Description. Mycelium composed of sparsely branched, septate, hyaline, straight to curved, thin-walled hyphae, 1.0-1.5 um wide; sometimes with swell- ings, 2.0-3.0 um wide. Hyphae sometimes anastomose, intertwine or form fas- cicles [description on MEA]. Conidiomata flat, erumpent, separate, eustromatic, brown, disintegrating at the top during maturation, up to 500 um diam, exuding a creamy conidial mass, partly enclosed by a wall of greenish-olive textura an- gularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, hyaline, smooth, ampulliform or subcylindrical, phialidic, 3.5-8 x 3-5 um. Co- nidia solitary, aseptate, hyaline, smooth, cylindrical, rarely slightly allantoid, apex obtuse, base bluntly rounded, (4—)5-8.5 x 2—2.5(—3) um [description on OA]. Culture characteristics. Colonies on MEA erumpent, spreading, convex, rosa- ceous, reaching 4 mm diam after 2 weeks at 6 °C, 9 mm diam after 2 weeks at 15 °C and 6 mm diam after 2 weeks at 25 °C, reaching 6 mm diam after 4 weeks at 6 °C, 18 mm diam after 4 weeks at 15 °C and 8 mm diam after 4 weeks at 25 °C, surface cerebriform, with sparse aerial mycelium, margin finely crenate. Reverse rosaceous. Colonies on PDA erumpent, spreading, umbonate, slimy rosaceous, reaching 4 mm diam after 2 weeks at 6 °C, 11 mm diam after 2 weeks at 15 °C and 4mm diam after 2 weeks at 25 °C, reaching 7 mm diam after 4 weeks at 6 °C, 20 mm diam after 4 weeks at 15 °C and 7 mm diam after 4 weeks at 25 °C, sur- face with radial furrows starting from centre towards margin, with sparse aerial - Figure 3. Morphology of Cytosporella fuligomixta on MEA (strain G107 = CBS 152343): a, b hyphae, white arrows show swellings and black arrow shows anastomosing hyphae c intertwined hyphae d hyphal fascicles. Scale bars: 10 um. MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 9 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera Figure 4. Morphology of Cytosporella fuligomixta on OA (strain G107 = CBS 152343): a conidiomata b wall of textura angularis c—e conidiophores f, g conidia. Scale bars: 10 um. mycelium, margin finely crenate. Reverse rosaceous. Colonies on OA spreading, flat, rosaceous, reaching 4 mm diam after 2 weeks at 6 °C, 10 mm diam after 2 weeks at 15 °C and 5 mm diam after 2 weeks at 25 °C, reaching 8 mm diam after 4 weeks at 6 °C, 22 mm diam after 4 weeks at 15 °C and 5mm diam after 4 weeks at 25 °C, surface with indistinct radial furrows starting from the centre towards the margin, without aerial mycelium, margin entire. Reverse roSaceous. Notes. Cytosporella fuligomixta is well delimited morphologically and ecolog- ically from four other Cytosporella species described on Quercus hosts. These are Cytosporella mendax, C. pisiformis, C. quercus and C. sphaerosperma. All of them were described from branches or wood of Quercus sp. or Quercus robur and differ from C. fuligomixta in shape and sizes of conidia. The conidia are glo- bose-ellipsoid, hyaline, 4-5 x 3.5—4 um in C. mendax (Saccardo 1884; Saccardo and Roumeguére 1884), globose, yellowish, 3-4 um in C. pisiformis (Saccardo 1884), perfectly globose, hyaline, 9-12 um in C. quercus (Saccardo and Sydow 1902), and globose and hyaline in C. sphaerosperma (Saccardo 1884). Other than being phylogenetically distinct, Cytosporella fuligomixta differs also morphologically from three sequenced species of this genus: C. calama- grostidis, C. chamaeropis and C. juncicola (Crous et al. 2019b, 2024). Cytosporel- la calamagrostidis described from old leaves of Calamagrostis arenaria has slightly shorter conidia, (5-)6-7 um long (Crous et al. 2024), C. chamaeropis MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 10 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera described from rotten Chamaerops humilis has globose conidia (Passerini 1888) and C. juncicola described from culms of Juncus effusus has slightly shorter and narrower conidia, (4—)5—6(—7) x 2 um (Crous et al. 2019b). Discussion Cytosporella is an understudied genus without modern revision and only with few available DNA sequence data (Sutton 1980; van der Aa et al. 2001; Crous et al. 2019b, 2024; Li et al. 2020). The type species Cytosporella sycina has not been sequenced yet, making current taxonomy of the genus tentative. However, all sequenced species of Cytosporella form monophyletic lineage. All species assigned to this genus in Index Fungorum (2024) are described from different host plants or differ morphologically if described from the same host plant, which suggests that Cytosporella species might be host specific. Currently, sparse DNA sequence data does not exclude host specialization of members of this genus. Cytosporella fuligomixta described here from the sooty mould community on Quercus robur leaves is well delimited morphologically and eco- logically from four other species described on Quercus hosts. The phylogenetic analyses including sequences of two loci (LSU, mtSSU) showed that sequenced species of Cytosporella, together with four other non-li- chenized, plant associated or fungicolous genera, namely Cladosterigma, Neo- acrodontiella, Nothoramularia and Vanderaaea, form sister group to lichenized and lichenicolous fungi in the family Gomphillaceae. Thus, these five genera belong to the family Gomphillaceae and order Graphidales and not to the family Acarosporaceae and order Acarosporales where they were previously includ- ed in most studies (Crous et al. 2019a, 2019b, 2021, 2023). The placement of Cladosterigma inside the Gomphillaceae was previously reported by Guterres et al. (2020). Recently, Crous et al. (2024) in a phylogenetic tree based on LSU sequences showed placement of these genera (except Vanderaaea that was not included in their analyses) inside the Gomphillaceae that was assigned to Ostropales. Most lichenized clades within Ostropales s.|. are also recognised as distinct orders (Graphidiales, Gyalectales, Odontotrematales, Ostropales s.str. and Thelenellales) (Kraichak et al. 2018; Licking 2019). Therefore, if that concept is accepted, Cladosterigma, Cytosporella, Neoacrodontiella, Nothora- mularia and Vanderaaea belong to the family Gomphillaceae and order Graphi- dales. Moreover, the current analyses showed for the first time that they form distinct, sister lineage to remaining, mostly lichenized genera and species. Biologically and ecologically, the above mentioned lineage of Gomphillaceae represents a coherent group of non-lichenized species occurring on plants or other fungi. The genus Cladosterigma contains only one species Cladosterig- ma clavariellum that is fungicolous hyphomycete (hyperarasite) occurring on Phyllachora species infecting Eugenia species (Myrtaceae) in Paraguay and Brazil (Seifert and Bandoni 2001; Guterres et al. 2020). Cytosporella, as stated above, includes species forming eustromatic conidiomata and is associated with diverse host plants. The genus Neoacrodontiella is typified with Neoac- rodontiella eucalypti that produces conidiophores aggregated in sporodochia and occurs on leaves of Eucalyptus urophylla (Myrtaceae) in Malaysia (Crous et al. 2019a). The only known species in the genus Nothoramularia, namely Nothoramularia ragnhildianicola, is a fungicolous hyphomycete (hyperparasite) MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 11 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera occurring on cercosporoid Ragnhildiana ferruginea, which in turn is parasitic on Artemisia vulgaris (Asteraceae) in Germany (Crous et al. 2023). Vanderaaea is typified with Vanderaaea ammophilae that forms sporodochial conidiomata with curved, 0-1-septate conidia and occurs on dead leaves of Ammophila are- naria (Poaceae) in the Netherlands (Crous et al. 2021). So far, the family Gomphillaceae included mostly foliicolous lichenized fungi, which are predominantly known from the tropics (Xavier-Leite et al. 2022, 2023, 2024). Lichenized fungi from this family are unique in that they form a special type of asexual conidiomata called hyphophores, which pro- duce diahyphae (Ferraro 2004; Liicking et al. 2004; Xavier-Leite et al. 2022, 2023). The family also contained three lichenicolous genera Corticifraga, Paragyalideopsis and Taitaia (Pino-Bodas et al. 2017; Suija et al. 2018; Liick- ing and Kalb 2002; Xavier-Leite et al. 2022, 2024; Diederich et al. 2024). Cur- rent confirmation or inclusion of non-lichenized, plant associated or fungic- olous genera Cladosterigma, Cytosporella, Neoacrodontiella, Nothoramularia and Vanderaaea enlarge the concept of this predominantly lichenized family Gomphillaceae. Due to phylogenetic, morphological (notably: absence of hyphophores with diahyphae) and ecological characteristics a new subfami- ly Cladosterigmoideae is described for these five non-lichenized genera. It is worthy to note that within Graphidales a very similar situation is found in the lichenized family Graphidaceae, where two non-lichenized genera (Furcaspo- ra, Rubikia) are now included therein as subfamily Rubikioideae (Caceres et al. 2020), Additionally, non-lichenized genus Papilionovela is a member of the core Graphidaceae (Caceres et al. 2020). Thus, Gomphillaceae and Graphidaceae, along with still other families of the subclass Ostropomyceti- dae (e.g. Stictidaceae), constitute good models to study transitions between lichenized and non-lichenized lifestyles. Acknowledgements We are grateful to Wactaw Bartoszek (Krakow, Poland) for help in the field work. Additional information Conflict of interest The authors have declared that no competing interests exist. Ethical statement No ethical statement was reported. Funding This study was funded by the National Science Centre, Poland, under the project 2017/27/B/NZ9/02902. Author contributions MP: conceptualization, investigation, formal analyses, visualisation, writing — original draft preparation; MSB: investigation, formal analyses, visualisation, writing - review and editing; PC: investigation, writing — review and editing. All authors have read and approved the final version of the manuscript. MycoKeys 115: 1-18 (2025), DOI: 10.3897/mycokeys.115.138252 12 Marcin Piatek et al.: Cytosporella (including C. fuligomixta sp. nov.) and related genera Author ORCIDs Marcin Pigtek © https://orcid.org/0000-0002-4968-2861 Monika Stryjak-Bogacka © https://orcid.org/0000-0003-2845-9975 Pawet Czachura ® https://orcid.org/0000-0002-3562-8776 Data availability The data that support the findings of this study are available in GenBank (https://www. ncbi.nim.nih.gov/genbank/) and in culture collections and fungal herbarium, as shown in Table 1 and the text. 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