Novel insights into the phylogenetic and metabolic diversity of preussia-like fungi

Abstract

The genus Preussia (Sporormiaceae, Pleosporales) represents one of the most diverse and metabolically active groups of ascomycetes. The genus is characterized by a complex taxonomic history involving the genera Sporormiella and Spororminula, which some authors consider synonyms of Preussia. However, the circumscription of these genera remains controversial, and the status of numerous species is unresolved. The present study aims to characterize strains of Preussia s.l. recovered during diversity surveys on marine and xerophytic substrates, explore their secondary metabolite profiles, and improve our understanding of the diversity within this group. The obtained strains, as well as strains from the CBS culture collection and exsiccata from different fungaria, were characterized morphologically, and molecular phylogenetic analyses were conducted on strains available in culture. Metabolite profiles were developed for representative strains using mass spectrometry data (MS/MS). Molecular alignments comprised the nuclear ribosomal subunits, consisting of partial sequences for the 18S small subunit (SSU) nrDNA, the internal transcribed spacer regions and intervening 5.8S nrDNA (ITS), as well as partial sequences for the 28S large subunit (LSU) nrDNA. Additional regions within protein-encoding genes were used, including the DNA-directed RNA polymerase II second-largest subunit (rpb2) and translation elongation factor 1-α (tef1-α) regions. These data were used to reconstruct a robust phylogenetic framework for Preussia s.l. and to delineate lineage structure and generic boundaries. Ten well-supported lineages were resolved and integrated with metabolite profiles to inform taxonomic decisions. Our results support the recognition of Preussia and Sporormiella as distinct genera, the re-establishment of Spororminula, and the introduction of four new genera, 11 new species, 18 new combinations, and one new name. Moreover, epi- and lectotypes have been designated to stabilize the taxonomy of some recognized species. This study provides a comprehensive framework for future taxonomic, phylogenetic, and chemical investigations within Sporormiaceae.

Taxonomic novelties: New genera: Chaetosporormiella Guerra-Mateo, Gené & Cano, Pleosporormiella Guerra-Mateo, Gené & Cano, Sporormyces Guerra-Mateo, Gené & Cano, Spororvela Guerra-Mateo, Cano & Gené. New species: Chaetosporormiella monodictyoides Guerra-Mateo, Gené & Cano, Pleosporormiella aculeata Guerra-Mateo, Gené & Cano, Pleosporormiella compacta Guerra-Mateo, González-Menéndez, Gené & Cano, Pleosporormiella polyspora Guerra-Mateo, González-Menéndez, Gené & Cano, Pleosporormiella suttonii Guerra-Mateo, Gené & Cano, Preussia gypsicola Guerra-Mateo, González-Menéndez, Cano & Gené, Sporormiella jejuensis H. Noh, H.U. Cho & S.H. Kim ex Guerra-Mateo, Gené & Cano, Sporormiella koreensis H. Noh, H.U. Cho & S.H. Kim ex Guerra-Mateo, Gené & Cano, Sporormiella solicola Guerra-Mateo, Cano & Gené, Spororminula irregularis Guerra-Mateo, Cano & Gené, Sporormyces anthraquinoniformans Guerra-Mateo, González-Menéndez & Cano. New combinations: Chaetosporormiella pilosella (Cain) Guerra-Mateo, Gené & Cano, Pleosporormiella citrullina (R.M.F. Silva et al.) Guerra-Mateo, Gené & Cano, Pleosporormiella polymorpha (Asgari & Zare) Guerra-Mateo, Gené & Cano, Pleosporormiella terricola (Cain) Guerra-Mateo, Gené & Cano, Sporormiella aegilopis (M. Mehrabi et al.) Guerra-Mateo, Gené & Cano, Sporormiella africana (Arenal et al.) Guerra-Mateo, Gené & Cano, Sporormiella bezerrensis (Ferreira-Sá et al.) Guerra-Mateo, Gené & Cano, Sporormiella elegans (D.C. Sandberg & A.E. Arnold) Guerra-Mateo, Gené & Cano, Sporormiella isabellae (Arenal et al.) Guerra-Mateo, Gené & Cano, Sporormiella japonica (Udagawa, K. Tominaga & Hamaoka) Guerra-Mateo, Gené & Cano, Sporormiella lignicola (W. Phillips & Plowr.) Guerra-Mateo, Gené & Cano, Sporormiella mediterranea (Arenal et al.) Guerra-Mateo, Gené & Cano, Sporormiella persica (Asgari & Zare) Guerra-Mateo, Gené & Cano, Sporormiella procaviae (Crous) Guerra-Mateo, Gené & Cano, Sporormiella procaviicola (Crous) Guerra-Mateo, Gené & Cano, Spororminula subticinensis (Mouton) Guerra-Mateo, Cano & Gené, Sporormyces septenarius (S.I. Ahmed & Cain) Guerra-Mateo, Gené & Cano, Spororvela tela (S. Jakob & M.J. Richardson) Guerra-Mateo, Cano & Gené. New names: Sporormiella isopartis Guerra-Mateo, Gené & Cano. New synonyms: Preussia apgariae Y.P. Tan et al., Preussia arizonica D.C. Sandberg & A.E. Arnold, Preussia crataegi Eisvand & M. Mehrabi, Preussia cylindricalis F. Liu et al., Preussia flanaganii Boylan, Preussia isomera Cain, Preussia mariae D.C. Sandberg & A.E. Arnold, Preussia mayoae Y.P. Tan et al., Preussia pianica Eisvand & M. Mehrabi, Preussia pistaciae Eisvand & M. Mehrabi, Preussia tenerifae (Arx & Aa) Kruys. Typification (basionyms): Epitypification: Perisporium funiculatum Preuss; Lectotypification: Sporormia intermedia Auersw.

Citation: Guerra-Mateo D, González-Menéndez V, Gené J, Crous PW, Bensch K, Genilloud O, Cano-Lira JF (2026). Novel insights into the phylogenetic and metabolic diversity of preussia-like fungi. Studies in Mycology 114: 437–486. doi: 10.3114/sim.2026.114.03

INTRODUCTION

The family Sporormiaceae (Pleosporales) represents a diverse group of saprobic ascomycetes that primarily inhabit dung, but also colonize soil, plant debris, wood, and even behave as endophytes (Zhang et al. 2012b, Phukhamsakda et al. 2022). Morphologically, the family is characterized by sexual morphs with bitunicate, fissitunicate asci, and thick-walled, predominantly multi-septate, dark brown ascospores, as well as pycnidial, chrysosporium- or monodictys-like asexual morphs (González-Menéndez et al. 2017, Wanasinghe et al. 2018, Crous et al. 2020a, b). Currently, Sporormiaceae comprises the genera Chaetopreussia, Forliomyces, Pleophragmia, Preussia, Sparticola, Sporormia, Sporormiella, Sporormurispora, Trichophoma, Westerdykella, and Xenomonodictys (Hyde et al. 2024). However, the taxonomic circumscription of Preussia and Sporormiella remains controversial, as phylogenetic analyses have shown that species from both genera are intermingled (Phukhamsakda et al. 2016, González-Menéndez et al. 2017).

The genus Preussia (Pr.) was established in 1866 to accommodate the basionym Perisporium funiculatum, currently Pr. funiculata, based on its saprobic behaviour and the production of ascomata with clavate asci, long stipes, and 4-celled ascospores (Fuckel 1867). The examination of additional Perisporium collections led to novel Preussia combinations, confining Perisporium to plant parasites producing pycnidial asexual morphs (Cain 1961). Before the establishment of Preussia, the genus Sporormia (Sp.) was described in 1849 based on Sp. fimetaria, a coprophilous fungus displaying ≥ 16-celled ascospores (De Notaris 1849). Later, the genus Sporormiella (Sm.) was described based on Sm. nigropurpurea, another coprophilous fungus, but displaying ascomata with short stipitate asci and 4-celled ascospores (Ellis & Everhart 1892). Over the years, fungi displaying 4–13-celled ascospores were additionally included in Sporormia. However, lignicolous species with 4-celled ascospores were later transferred to Preussia, while coprophilous species displaying between 4–13-celled ascospores were transferred to Sporormiella, restricting Sporormia to ≥ 16-celled ascospores (Cain 1961, Ahmed & Cain 1972). Using this approach, the number of Preussia and Sporormiella species increased considerably, including the description of novel species (Khan & Cain 1979). Other genera like Ohleriella and Sporormiopsis were eventually considered synonyms of Sporormiella (Ahmed & Cain 1972), and the genus Spororminula (Sn.) was ultimately considered a synonym of Preussia (Kruys & Wedin 2009).

The circumscription of Preussia and Sporormiella has been a long-standing topic of debate (Mungai et al. 2012). Preussia was reserved for saprobic fungi growing on plant debris, wood, and soil, producing cleistothecial ascomata with clavate asci and 4-celled ascospores, while Sporormiella comprised coprophilous fungi displaying perithecial ascomata with cylindrical-clavate asci and 4–13-celled ascospores (Guarro et al. 1997b, Kruys & Wedin 2009). These features lacked consistency across species, with Pr. funiculata frequently occurring on dung, several species producing both cleistothecia and perithecia depending on growth conditions in culture, and the absence of phylogenetic monophyly (Guarro et al. 1997b, Kruys & Wedin 2009). Although public databases such as Index Fungorum and MycoBank currently treat these genera as different entities, many authors have considered Sporormiella a synonym of Preussia (Kruys & Wedin 2009, Guarro et al. 2012, Zhang et al. 2012b, Kruys 2015). In fact, a large number of Sporormiella species have already been transferred to Preussia (Valldoserra & Guarro 1990, Guarro et al. 1997a, Abdullah et al. 1999, Arenal et al. 2004, Kruys & Wedin 2009).

The genus Preussia s.l. represents a heterogeneous group of fungi. This variability has also been acknowledged through secondary metabolites. These molecules represent low-molecular-weight compounds that are not essential for growth, but indicate the fungus’s ability to communicate, compete, and cooperate with other microbes in the environment (Macheleidt et al. 2016). More than 50 different metabolites have been recovered from Preussia s.l. species (González-Menéndez et al. 2017). Although some of these compounds were also reported in different microbes, up to ten secondary metabolites have been exclusively recovered from Preussia s.l., including australifungin, sporminarin A and B, and zaragozic acid B (González-Menéndez et al. 2017). These natural compounds predominantly display antifungal activity, with australifungin used against fungal causal agents of infection in humans (Mandala & Harris 2000), sporminarin A applied in the control of phytopathogens (Mudur et al. 2006, Rodríguez Sabina et al. 2025), and zaragozic acid B as a cholesterol-lowering medication inhibiting the squalene synthase (Bergstrom et al. 1993, Hornby et al. 2003). In addition, secondary compounds have been used to resolve species boundaries, an approach known as chemotaxonomy that has been successfully used in other fungal groups like Cortinarius, Lepiota, and Penicillium (Huymann et al. 2024, Overy et al. 2025, Sarawi et al. 2025).

The chemotaxonomic study of Preussia s.l. revealed 16 chemotypes characterizing different species; i.e., the exclusive production of microsphaerone B by strains of Pr. lignicola or sporminarins by different strains of Pr. africana (González-Menéndez et al. 2017). The combination of secondary metabolites with phylogenetic analyses enabled the characterization of lineages producing similar compounds and the precise identification of species, highlighting the production of unique molecules. For example, australifungin was obtained from a strain originally identified as “Sporormiella australis” (Mandala et al. 1995). However, phylogenetic analyses determined this strain as Pr. intermedia and, in addition, the metabolic profile revealed that strains of Pr. intermedia and Pr. australis produce different sets of metabolites (González-Menéndez et al. 2017). Therefore, the study of metabolites represents a powerful tool to explore the diversity of Preussia s.l. in depth, enhancing polyphasic taxonomic decisions.

Despite Preussia s.l. chemotypes, the relationships among major lineages remain unresolved with current ITS-LSU phylogenies (Kruys & Wedin 2009, González-Menéndez et al. 2017, Jakob et al. 2024). Protein-coding regions like tub2 and tef1-α have been used to discern phylogenetically close species (Sandberg et al. 2022, Eisvand & Mehrabi-Koushki 2025). However, these analyses were limited in scope due to the small number of sequences available for these regions and a focus on assessing the Pr. lignicola and Pr. minima lineages, which have been considered species complexes (González-Menéndez et al. 2017, Sandberg et al. 2022). Moreover, a large number of Preussia s.l. species lack molecular data, and the identification of many sequenced taxa relies exclusively on morphology, with type specimens not specified or only available as fungarium material. Aside from this, there has been a recent increase in the description of Preussia s.l. novel species overlooking these taxonomic limitations (Eisvand & Mehrabi-Koushki 2025, Noh et al. 2025, Phurbu et al. 2025). Thus, the taxonomy of this genus requires the re-evaluation of available strains in culture, including fungarium material when possible.

We recovered several Preussia s.l. strains as part of diversity surveys on marine and xerophytic substrates. These underexplored substrates represent sources of rare taxa that produce specialized metabolites (Imhoff 2016, Sayed et al. 2020). In this work, we aimed to revise the taxonomy and metabolic profile of these Preussia s.l. strains. To achieve this, we examined type and reference specimens and strains of the genus and allied fungi preserved at the Westerdijk Fungal Biodiversity Institute, covering the widest range possible of species with molecular data. Herein, we provide a concatenated phylogeny with protein-coding regions assessing the diversity of Preussia s.l., expand the metabolite profile of this group focused on underexplored lineages, and revise strain identification under a polyphasic approach combining phylogeny, morphology, and metabolites. In addition, we clarify the relationship between Preussia s.str. and Sporormiella, re-erect Spororminula, and propose four novel genera to stabilize the taxonomy of Sporormiaceae. Among these genera, we also propose 11 novel species, 18 novel combinations, and one new name.

MATERIALS AND METHODS

Strains and material examined

The strains included in this work comprised type and reference Preussia s.l. strains preserved in the CBS culture collection of the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands), strains recovered from gypsum soil, xerophytic plants and lichens available in the CF collection of the MEDINA Foundation (Granada, Spain), and strains recovered from marine sediments available in the FMR culture collection of the Universitat Rovira i Virgili (Faculty of Medicine and Health Sciences, Reus, Spain). The CF and FMR strains were initially identified as members of Preussia s.l. based on morphological features. However, additional strains displaying humicola-like, monodictys-like, or pycnidia asexual morphs were also included based on preliminary DNA sequence data (Supplementary material Table S1).

In addition, we examined type and reference fungarium specimens to assess strain identification when type material lacked molecular data or was not properly designated. Identification was confirmed when cultural features matched those displayed in the species protologue. The specimens examined are specified in the taxonomy section, and digital images of exsiccatae are available as Supplementary material Figs S1–S6.

DNA extraction, PCR amplification, and sequencing

Total genomic DNA was extracted from 7-d-old fungal colonies growing on malt extract agar (MEA) at room temperature. Genomic DNA was extracted using a chloroform/isopropanol protocol (Tripathy & Maharana 2017) and quantified with a Nanodrop 2000 (Thermo Scientific, Madrid, Spain). Five loci were amplified following previous works on Pleosporales: SSU, ITS, LSU, a fragment of the translation elongation factor 1-α (tef1-α) gene, and a fragment of the second-largest subunit of the RNA polymerase 2 (rpb2) gene (Thambugala et al. 2015, Hongsanan et al. 2021). The ITS and LSU regions were amplified using the primer pair ITS5/LR5 (Vilgalys & Hester 1990, Schoch et al. 2012); the SSU region with NS1/NS4 (Schoch et al. 2012); tef1-α with EF-983F/EF-2218R (Rehner & Buckley 2005), rpb2 with RPB2-5F2/RPB2-7CR (Liu et al. 1999). The PCR amplifications were performed in a total volume of 25 μL. The amplification conditions were set as follows: an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 53 °C for 1 min in ITS, LSU, and rpb2; and 57 °C for 90 s in tef1-α; followed by an extension at 72 °C for 2 min, and a final extension step at 72 °C for 7 min. In the case of SSU, the annealing step was structured in a decreasing gradient of temperature, with two sets of 5 cycles at 60 °C and 58 °C for 45 s, and 30 cycles at 54 °C for 45 s. The PCR products were subsequently purified and sequenced using either an Applied Biosystems, Hitachi 3730xl DNA analyser (Applied Biosystems Inc., Foster City, California, USA) or at Macrogen Corp. Europe (Madrid, Spain). The consensus sequences were assembled from forward and reverse sequences using Geneious Prime v. 2022.0.2 (http://www.geneious.com, Kearse et al. 2012). Novel sequences generated in this study were deposited in GenBank (http://www.ncbi.nlm.nih.gov, Table S1).

Phylogenetic analyses

Sequence alignments were initially performed using the ClustalW algorithm in MEGA v. 6.0 (Thompson et al. 1994, Tamura et al. 2013); then, refined using MUSCLE or manually adjusted as needed (Edgar 2004). Each genomic DNA region was aligned separately before being concatenated into a combined dataset. Phylogenetic concordance among individual gene trees was visually assessed to identify any topological conflicts within strongly supported clades (Supplementary material Figs S7–S10). Upon confirmation of overall concordance, the alignments were merged into a single data matrix using MEGA.

Maximum likelihood (ML) analyses were conducted with IQ-TREE v. 2.3.6, employing ultrafast bootstrap approximation to estimate branch support (Minh et al. 2013, 2020, Nguyen et al. 2015, Hoang et al. 2018). The optimal evolutionary model for each data partition was selected using ModelFinder based on the Bayesian information criterion (Chernomor et al. 2016, Kalyaanamoorthy et al. 2017). Bootstrap support values ≥ 95 % were regarded as statistically significant (Minh et al. 2013, Hoang et al. 2018). Bayesian inference (BI) analyses were performed using MrBayes v. 3.2.6 (Ronquist et al. 2012). The best-fit substitution model for each locus was determined using jModelTest v. 2.1.3 under the Akaike information criterion (Guindon & Gascuel 2003, Darriba et al. 2012). Markov chain Monte Carlo (MCMC) simulations were run for 10 million generations with four chains (one cold and three heated), starting from a random tree topology. Trees were sampled every 1000 generations, and the run was automatically terminated once the average standard deviation of split frequencies dropped below 0.01. The first 25 % of sampled trees were discarded as burn-in, and the remaining trees were used to construct a majority-rule consensus tree. Posterior probabilities (pp) ≥ 0.95 were considered statistically significant (Hespanhol et al. 2019). Resulting phylogenetic trees were visualized using FigTree v. 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/, accessed during November 2025). The alignments generated in this study were submitted to Zenodo (https://doi.org/10.5281/zenodo.19625618).

Species delimitation analyses

Species delimitation analyses were used to resolve the taxonomy of phylogenetically close species represented by several strains. We performed a coalescent-based method, Bayesian Poisson Tree Processes, bPTP (Zhang et al. 2013), and a distance-based method, Assemble Species by Automatic Partitioning, ASAP (Puillandre et al. 2021). These analyses were performed exclusively with the ITS and LSU regions to include as many strains as possible. The input trees for the PTP analyses were inferred in IQ-TREE, as explained above. The analyses were conducted on the web server for bPTP (https://species.h-its.org/ptp/) with 500000 MCMC generations, thinning set to 100, burn-in of 10 % and seed value of 123456. Convergence of the MCMC iterations was assessed by visualising the log-likelihood trace plot, where the chain’s predominant exploration of high-likelihood locations was used as indicative of convergence. The ASAP analyses were also performed for the concatenated ITS-LSU alignment using the matrix of pairwise nucleotide distances. This matrix was computed in MEGA v. 6.0 using the K2+G nucleotide substitution model. These analyses were conducted locally (https://github.com/iTaxoTools/ASAPy, accessed during January 2026). The resulting partition was determined by the lowest asap-score. Hypothetical species delimitation was established by evaluating the phylogenetic analyses and the delimitation obtained through PTP and the ASAP partition.

Morphological characterization

Macroscopic characteristics of fungal colonies were recorded after cultivation on oatmeal agar (OA), potato dextrose agar (PDA), and cornmeal agar (CMA). Colony diameters and morphological features were documented after 14 d of incubation in darkness at 25 °C. Colony colours in descriptions were based on Kornerup & Wanscher (1978). The microscopic analysis of the specimens was conducted from colonies grown on OA under the above-mentioned conditions, unless stated otherwise in species descriptions. Microscopic preparations of the reproductive structures were mounted in Shear’s solution (300 mL 2 % KCH₃CO₂ in water, 120 mL of glycerine, and 180 mL of 95 % ethanol; Chupp 1940) for long-term storage, or in water when structural features required hydration (i.e., spore release, gelatinous sheath, and germ slit). Microscopic traits were examined using an Olympus BH-2 bright-field microscope (Olympus Corporation, Tokyo, Japan), and sizes in descriptions were based on a minimum of 30 measurements per structure to establish size ranges. Photomicrographs were captured with a Zeiss Axio Imager M1 light microscope (Carl Zeiss, Oberkochen, Germany) using Nomarski differential interference contrast and phase contrast, coupled with a DeltaPix Infinity digital camera. Photoplates were compiled using Adobe Photoshop CS6 v. 13.0.

The holotypes (dried colonies) and ex-type cultures were deposited in the Westerdijk Fungal Biodiversity Institute, and nomenclatural novelties were registered in MycoBank (https://www.mycobank.org/; accessed on 7th November 2025).

Fermentation, preparation of extracts and metabolomic analyses

A subset of 28 preussia-like fungal strains was selected based on phylogenetic relationships for metabolomic characterization. Strains were grown in duplicate using EPA vials filled with 10 mL of culture media. We used five media formulations, including different carbon and nitrogen sources, and combined solid- and liquid-state fermentation conditions to promote the production of secondary metabolite diversity. For solid-state fermentations, we used rice-based medium (BRFT) and corn-based medium (F1); while for liquid-state fermentations, we used Potato Dextrose Broth (PDB), Mannitol Murashige and Skoog salts medium (MMK2), and Yeast extract Sucrose medium (YES) (González-Menéndez et al. 2014). Strains were incubated at 22 °C for 14 d in liquid-state fermentations, and 21 d in solid-state fermentations. Fermentations were harvested and extracted with acetone to generate crude extracts at 2× whole broth equivalent (WBE) final concentration following previous described protocol (González-Menéndez et al. 2014).

Fungal extracts (2 µL) were analysed by Liquid Chromatography coupled with Ultraviolet and High-Resolution Mass Spectrometry (LC-UV-HRMS). Liquid chromatography (LC) was performed on an Agilent 1200 (Santa Clara, CA), using a Zorbax SB-C8 column (2.1 × 30 mm) and 3.5 µm particle size, maintained at 40 °C with a flow rate of 300 µL/min and 210 nm UV detection. Solvent A consisted of 10 % acetonitrile and 90 % water with 0.01 % trifluoroacetic acid and 1.3 mM ammonium formate, while solvent B was 90 % acetonitrile and 10 % water with 0.01 % trifluoroacetic acid and 1.3 mM ammonium formate. Elution consisted of a gradient that shifted from 10 % solvent B to 100 % in six minutes, remained at 100 % solvent B for two minutes and returned to 10 % solvent B for two additional minutes to initialize the system. The resulting compounds were characterized through mass spectrometry on a Bruker maXis High-Resolution Quadrupole Time-of-Flight (HR-QTOF) spectrometer (Bruker Daltonics GmbH, Bremen, Germany). Ionization of the eluting solvent was obtained using the standard electrospray ionization (ESI) sources adjusted to a drying gas flow of 11 L/min at 200 °C and a nebulizer pressure of 40 psig. The capillary voltage was set to 4000 V, and the mass spectrometer was operated in data dependent positive ion mode, automatically switching between full scan MS and MS/MS acquisitions. Fragmentation spectra (m/z 50–2000) were collected in the TOF selecting the six most intensive ions above 1000 counts for fragmentation using collision-induced dissociation energy.

Chemical profiles were carried out by comparing the data generated with the Bruker maXis; i.e., retention time, UV spectrum, and exact mass of extracted components. These data were compared to the metabolite data stored in the MEDINA Foundation internal database, which were acquired under the same exact LC-HRMS conditions explained above (Pérez-Victoria et al. 2016). The compounds that could not be identified in the database of standards were isolated by semi-preparative high-performance liquid chromatography (HPLC), following the same methodology for characterization through HR-QTOF and ESI. For these compounds, the predicted molecular formulas were compared to the Chapman & Hall Dictionary of Natural Products (v. 25.1) to identify compounds already described in the literature (González-Menéndez et al. 2017).

The metabolites detected were clustered through molecular networks based on structural similarity. The MS/MS spectra were converted to mzXML file format through Bruker Data Analysis software. The molecular network was created using the online workflow on the GNPS website (http://gnps.ucsd.edu). The dataset was filtered to exclude MS/MS noise fragment ions within +/- 17 Da of the precursor m/z, retaining the top 6 fragment ions in the +/- 50 Da window, and using a precursor ion mass tolerance of 2.0 Da, and a MS/MS fragment ion tolerance of 0.5 Da. The resulting network was refined to contain reliable spectral relationships between compounds. Edges were refined to a cosine score above 0.7 and more than 6 matched peaks. Edges connecting nodes were retained only if both appeared in each other ten most similar matches. Molecular family size was set to 100, removing low-score edges based on this threshold. This way, nodes represented compounds and edges represented similarity between fragmentation patterns, suggesting structural relatedness. Spectra included in the network were searched against GNPS libraries, with matches requiring a score above 0.7 and at least 6 matched peaks. The mzXML files were processed with MZmine v. 2.53 to develop a feature-based molecular network, including mass detection, chromatogram building, chromatogram deconvolution, isotopic peaks grouping, feature alignment, gap filling, filtering, and direct submission/export for GNPS. The resulting networks were analysed online on the GNPS platform and exported to Cytoscape v. 3.6.1 for graphical representation. GNPS data are accessible through the following links: Molecular Network (https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp) and Feature-based Molecular Networking (FBMN; https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=daff1c06c13e4dbda064d25a2c39478f).

RESULTS

Barcode distances in Sporormiaceae

Individual ITS, LSU, tef1-α, and rpb2 alignments were used to determine intra- and inter-specific distances for each locus (Fig. 1). For all loci, the mean inter-specific distance was bigger than the intra-specific distance, without overlapping. Therefore, all loci were suitable barcodes for identification at different taxonomic levels. Among them, tef1-α, ITS, and rpb2 represented appropriate barcodes for species identification with 8 %, 9 %, and 40 % inter-specific distances, respectively. The LSU region was the most conserved region with a 6 % inter-specific distance, which was indicative of different major lineages within Sporormiaceae.

Fig. 1.

Sporormiaceae inter-specific distances for the loci studied. Mean inter-specific distance is represented with*.

Sporormiaceae phylogeny

An initial ITS-LSU phylogenetic analysis was developed to infer the relationships among members of Sporormiaceae with available molecular data; i.e., Forliomyces, Preussia s.l., Sparticola, Sporormia, Sporormurispora, Trichophoma, Westerdykella, and Xenomonodictys (Fig. 2). The alignment comprised 1050 characters (583 bp for ITS, 467 bp for LSU). Among these, 606 character-sites were conserved (ITS: 275, LSU: 331), 418 were variable (ITS: 285, LSU: 133), and 301 were parsimony informative characters (ITS: 207, LSU: 94). The remaining positions included ambiguous or gap-only sites that were excluded from these categories. The models selected for the ML analysis were TIM2e+I+G4 for ITS and TNe+R3 for LSU, while for the BI analysis, the best models were GTR+I+G for ITS and TrNef+I+G for LSU. The outgroups selected were Lophiostoma rosae-ecae MFLUCC 17-0807 and Teichospora thailandica MFLUCC 13-0284. We represented the maximum-likelihood tree with the bootstrap support values of the ML analysis and the BI posterior probabilities at the nodes. The species of Preussia s.l. were labelled based on the most recent name combinations.

Fig. 2.

Phylogenetic tree inferred from a concatenated ITS–LSU alignment of 165 strains representing the family Sporormiaceae in Pleosporales. The tree is rooted to Lophiostoma rosae-ecae MFLUCC 17-0807 and Teichospora thailandica MFLUCC 13-0284. Numbers at the branches indicate support values (IQ-TREE-UFBS/BI-PP) above 90 % / 0.95. Fully supported branches (100/1) are highlighted in bold. Quotation marks indicate strains with unresolved taxonomy. Ex-type strains are indicated with ^T, ex-isotypes ^IT, reference strain ^R, and those corresponding to the accepted species are highlighted in grey. The scale bar represents the expected number of changes per site.

The strains examined in this work clustered within Preussia s.l., revealing ten independent clades with unresolved relationships (Fig. 2). These clades were defined based on the phylogenetic distances revealed by the LSU region (Fig. 1, Table 1). A threshold of 97 % LSU similarity was used to define clades, with around 92–96 % similarity between them. The relationships among clades were further explored through a concatenated analysis including protein-coding regions.

Table 1.

Percentage of similarity between Preussia s.l. lineages for the LSU region.

Lineages	Clade 1	Clade 2	Clade 3	Clade 4	Clade 5	Clade 6	Clade 7	Clade 8	Clade 9
Clade 1 Sporormiella
Clade 2 Sporormyces	95–97
Clade 3 Pleosporormiella	93–96	95–97
Clade 4 Preussia s.str.	93–95	93–95	92–96
Clade 5	95–97	94–95	95–97	92–94
Clade 6 Spororvela	93–96	92–94	93–95	91–93	95–96
Clade 7	95–97	94–95	92–95	92–94	94–95	92–94
Clade 8 Chaetosporormiella	94–97	95–96	95–97	92–95	95–97	93–96	93–95
Clade 9 Spororminula	92–95	92–94	93–95	91–93	94–95	93–94	92–95	95
Clade 10	90–93	91–92	90–93	91–92	93–95	93–95	92–94	94–95	93–95

Only ITS and LSU sequences were available for many type and reference strains (Supplementary material Table S1). Based on ITS inter-specific mean distance (Fig. 1), we used the analysis of these two gene markers to confirm strain identification at the species level (Fig. 2). The strains examined in this work represented 19 known species and nine independent lineages that could not be assigned to any known species with molecular data available. Relationships at the species level were explored in detail in the following sections.

Concatenated Preussia s.l. phylogeny

The concatenated alignment comprising SSU, ITS, LSU, tef1-α, and rpb2 sequences was constructed to provide a more robust evaluation of the phylogenetic relationships among Preussia s.l. clades. Since Pr. pulchella E: 216605 and Pr. dakotensis UPS: 2570-g (clade 10 in Fig. 2; Table 1) showed a great phylogenetic distance from other lineages in the previous ITS and LSU analysis, they were excluded from subsequent phylogenetic analyses.

Individual alignments were concatenated after confirming the congruence between the five regions. The final alignment contained a total of 3451 characters (868 bp for SSU, 567 bp for ITS, 467 bp for LSU, 837 bp for tef1-α, and 712 bp for rpb2). Among these, 2426 character-sites were conserved (SSU: 822, ITS: 308, LSU: 361, tef1-α: 610, rpb2: 326), 1004 were variable (SSU: 45, ITS: 243, LSU: 103, tef1-α: 226, rpb2: 386), and 851 were parsimony informative characters (SSU: 28, ITS: 187, LSU: 85, tef1-α: 185, rpb2: 366). The remaining positions included ambiguous or gap-only sites that were excluded from these categories. The models selected for the ML analysis were K2P+I for SSU, TIM2e+I+G4 for ITS, K2P+I+G4 for LSU, TIM+F+I+G4 for tef1-α, and TN+F+I+G4 for rpb2, while for the BI analysis, the best models were K80+I for SSU, SYM+I+G for ITS, TrNef+I+G for LSU, TIM1+I+G for tef1-α, and TrN+I+G for rpb2. The outgroups selected were Forliomyces uniseptatus MFLUCC 15-0765 and Sparticola junci MFLUCC 15-0030. We represented the maximum-likelihood tree with the bootstrap support values of the ML analysis and the BI posterior probabilities at the nodes (Fig. 3). Clades and species names were updated through a polyphasic taxonomic analysis, combining this phylogeny with the morphological traits and metabolite profiles explained in the following sections. In addition, chemotypes and the most representative metabolites were also printed in the tree as explained in the section concerning metabolites.

Fig. 3.

Phylogenetic tree inferred from a concatenated SSU-ITS-LSU-tef1-rpb2 alignment of 111 strains representing Preussia s.l. and associated genera. The tree is rooted to Forliomyces uniseptatus MFLUCC 15-0765 and Sparticola junci MFLUCC 15-0030. Numbers at the branches indicate support values (IQ-TREE-UFBS/BI-PP) above 90 % / 0.95. Fully supported branches (100/1) are highlighted in bold. Novel combinations are highlighted in brown, and novel species in green. Quotation marks indicate strains with unresolved taxonomy. Ex-type strains are indicated with ^T, ex-epitypes with ^ET, ex-isotypes ^IT, reference strains selected in the present study ^R, and those corresponding to the accepted species are highlighted in grey. The scale bar represents the expected number of changes per site. Most representative metabolites per clades are also included in the tree, and chemotypes are indicated within circles (See Table 3).

The resulting phylogenetic tree resolved the relationships among Preussia s.l. clades, defining nine monophyletic lineages (Fig. 3). Clades 3–9 revealed similar relationships to the ITS-LSU tree; however, clades 1 and 2 were displayed as sister lineages in the concatenated analysis (Figs 2, 3). The great phylogenetic distance between clades suggested independent evolutionary histories. Thus, each clade could be representing a different genus. This hypothesis was further tested through metabolite and morphology analyses in the following sections. In addition, we followed a similar procedure to resolve the taxonomy of the nine independent lineages. It is noteworthy that seven of the independent lineages were represented by strains of marine and xerophytic origin. In fact, additional marine strains represented the species Sm. irregularis, Sm. koreensis, Sm. persica, and Sn. subticinensis.

Dereplication of known compounds and identification of chemotypes

The study of metabolite production was primarily focused on clades 2–4, 8, and 9 (Figs 2, 3). Clade 1 was the main object of study in a previous work (González-Menéndez et al. 2017), while clades 5–7 remained to be assessed due to limited access to specimens. The dereplication of the fungal extracts obtained through liquid- and solid-state fermentations revealed 33 compounds. These compounds were classified into networks representing groups of similar molecular structure, with molecules produced across Preussia s.l. and molecules exclusively produced by certain clades. The complete list of metabolites identified in this work is available in Table 2, where molecules like C₁₉H₂₈O₅, C₃₂H₄₂N₄O₁₀, and C₁₁H₁₀ClO₅ could not be assigned to any known compound and may represent novel metabolites. Putative hits are included in Supplementary material Table S2.

Table 2.

Chemical characterization of the metabolites recovered.

No.	RT1 (min)	[M + H]	Proposed ion	Main secondary ions	Media	Proposed formula	Proposed compound2
1	2.42	3.070.813	C15H15O7+	209.0443; 289.0706; 325.0918	PDB	C15H14O7	Preussochromone F
2	3.91	3.010.702	C16H13O6+	283.0596; 284.06; 302.0735	YES, BRFT, F1	C16H12O6	2,8-Dihydroxy-3-methylxanthone-1-carboxylic acid
3	4.40	1.971.179	C11H17O3+	1.981.205	PDB, MMK2, YES, BRFT, F1	C11H16O3	4-Methoxy-6-pentyl-2H-pyran-2-one
4	4.27	3.212.065	C19H29O3+	303.1954; 316.1924; 322.2092	YES, MMK2, BRFT, F1	C19H28O4	Nineteen possible hits in DBs
5	7.77	7.545.823	C43H80NO9+	391.2842; 755.5958; 776.5647	YES, MMK2, BRFT, F1	C43H79NO9	Glucopyranoside
6	2.30	337.201	C19H29O5+	319.1903; 338.2042; 354.2273	YES, BRFT, MMK2, F1	C19H28O5	Seventeen possible hits in DBs
7	3.73	2.090.811	C11H13O4+	191.0701; 210.0839	YES, PDB, MMK2, BRFT, F1	C11H12O4	4-Hydroxy-5-methylmellein
8	4.29	3.091.353	C16H21O6+	223.06; 291.1226; 310.1365	PDB, BRFT, MMK2, F1	C16H20O6	Monocerin
9	3.19	2.811.746	C16H25O4+	245.1534; 263.1639; 561.3421	YES, PDB, BRFT, F1	C16H24O4	Brefeldin A
10	2.62	3.271.435	C16H23O7+	291.1225; 309.1332;	BRFT, F1	C16H22O7	Fifteen possible hits in DBs
11	3.79	3.490.916	C17H17O8+	287.0914; 299.055; 331.0818	BRFT, MMK2, F1	C17H16O8	Asterric acid
12	3.44	2.090.452	C17H17O7+	231.1376; 333.0966	BRFT, MMK2, F1	C17H16O7	Bisdechlorodihydrogeodin
13	2.22	2.401.246	C12H18NO4+	241.1262; 479.2391	YES, BRFT, MMK2, F1	C12H17NO4	Dihydroisoflavipucine
14	4.91	318.282	C21H36NO+	300.2682; 319.2821	YES, PDB, BRFT, MMK2, F1	C21H35NO	Preussin
15	4.95	2.850.755	C16H13O5+	253.0493; 286.0786	BRFT, F1	C16H12O5	Monodictyquinone
16	4.22	2.902.476	C19H32NO+	291.2507; 272.2347	YES, BRFT, MMK2, F1	C19H31NO	Preussin B
17	0.98	2.781.478	C16H24NO3+	260.164; 279.1772	PDB, MMK2	C16H23NO3	Six possible hits in DBs
18	4.16	2.761.956	C17H26NO2+	277.1986; 551.3836	YES, PDB, BRFT, MMK2	C17H25NO2	No match in DBs
19	1.80	3.091.331	C16H21O6+	291.1223; 310.1362; 331.1148	BRFT	C16H20O6	11-hydroxycurvularin
20	3.23	2.911.242	C16H19O5+	273.112; 292.1259; 581.2379	BRFT, F1	C16H18O5	Resorcylide
21	2.35	4.131.265	C19H25O8S+	245.084; 430.153	BRFT, F1	C19H24O8S	Sumalarin C
22	3.88	2.611.127	C15H17O4+	2.621.156	YES, BRFT, F1	C15H16O4	Cyperin
23	4.70	4.131.595	C23H25O7+	275.0913; 414.163; 842.3377	BRFT, F1	C23H24O7	Six possible hits in DBs
24	4.08	2.430.416	C11H12ClO4+	245.0389; 244.0447	YES, PDB, BRFT, MMK2, F1	C11H11ClO4	7-Chloro-6methoxymellein
25	1.07	280.118	C14H18NO5+	262.1073; 281.121	BRFT, MMK2, F1	C14H17NO5	Spirostaphylotrichin A
26	4.26	7.130.967	C30H29N6O7S4+	277.2159; 714.0993	YES, BRFT, MMK2, F1	C30H28N6O7S4	Leptosin analogue
27	4.95	6.432.973	C32H43N4O10+	429.202; 644.3001; 874.4223	BRFT, F1	C32H42N4O10	No match in DBs
28	4.21	2.421.172	C15H16NO2+	240.1016; 275.0911	YES, MMK2	C15H15NO2	TMC120A
29	4.80	3.711.851	C22H27O5+	297.2417; 372.1885	BRFT, F1	C22H26O5	Four possible hits in DBs
30	4.34	7.431.435	C32H35N6O7S4+	7.441.461	YES, PDB, BRFT, F1	C32H34N6O7S4	Leptosin analogue
31	3.12	2.770.028	C11H11Cl2O4+	258.9922; 279.001	YES, PDB, MMK2, BRFT, F1	C11H10Cl2O4	Antibiotic LL-N 313ε
32	2.61	3.690.972	C20H17O7+	351.0865; 370.1004	BRFT, F1	C20H16O7	Palmarumycin
33	4.64	3.031.011	C20H15O3+	285.0904; 304.1044	YES, BRFT, F1	C20H14O3	No match in DBs

¹Retention time.

²DBs: databases.

Among the networks representing structural groups, the flavipucine network revealed up to nine structural variants, with some of them detected across all media tested (Fig. 4). Flavipucines were only detected in members of clades 3 and 8. It is noteworthy that dihydroisoflavipucine was recovered from strains displaying humicola-like (CBS 217.38, FMR 20838; clade 3), monodictys-like (CBS 100.07, CBS 101.60; clade 8) or pycnidia asexual morphs (FMR 20458; clade 3). In a similar fashion, the monocerin group comprised three variants, one of them recovered in all culture media (Fig. 5A). This group of molecules were produced by members of clade 1 (CBS 468.71), clade 3 (FMR 19842, FMR 20458 and FMR 20838), and clade 9 (FMR 19850), representing unique features for each clade (Fig. 3). The compound 7-chloro-6-methoxymellein (Fig. 5B) was detected across clade 2 (CF-277801), clade 4 (CBS 318.81) and clade 9 (CBS 125.66, CBS 354.86, FMR 19850), while C₁₁H₁₀Cl₂O₄ was exclusively detected in strains from clade 9 (Fig. 5B). Other molecules were exclusively recovered from single clades. A group of unknown molecules with eight structural variants, including C₁₉H₂₈O₄, was exclusively recovered from clade 3 (Fig. 6A). The molecule 4-methoxy-6-pentyl-2H-pyran-2-one was only produced by members of clade 2 (Fig. 6B), while the compound 4-hydroxy-5-methylmellein was only detected in members of clade 9 across all five media (Fig. 7A). Two leptosin analogues (C₃₀H₂₈N₆O₇S₄ and C₃₂H₃₄N₆O₇S₄) were exclusively detected in the strains CBS 100.07 and CBS 101.60, in clade 8, across all five media tested, cyperin was only recovered from the strains CF-277822 and CBS 318.81 in clade 4, and preussochromone F from FMR 19091 in clade 1 (Fig. 7B–D).

Fig. 4.

GNPS network representing flavipucine compounds together with the strains that produced them. Each node includes the fermenting media where the compound was detected. The most abundant compound is highlighted with *.

Fig. 5.

GNPS networks representing different mellein clusters together with the strains that produced them. The most abundant compounds are highlighted with *. A. Monocerin. B. 7-chloro-6-methoxy-mellein and Antibiotic LL-N 313ε.

Fig. 6.

GNPS networks representing different clusters together with the strains that produced them. The most abundant compounds are highlighted with *. A. C₁₉H₂₈O₄. B. 4-methoxy-6-penthyl-2H-pyran-2-one.

Fig. 7.

GNPS networks representing different clusters together with the strains that produced them. The most abundant compounds are highlighted with *. A. 4-hydroxy-5-methylmellein. B. Leptosin analogues C. C. Cyperin. D. Preussochromone F.

In addition, we used these metabolites to define chemotypes based on the production/absence patterns displayed by the strains examined. We defined 19 chemotypes (A–S) that represented different species (Table 3). These structural network patterns and chemotypes were compared with the concatenated phylogenetic tree to support a polyphasic taxonomic decisions on Preussia s.l. clades, as detailed in the following section (Fig. 3).

Table 3.

Secondary metabolites detected for each strain. Chemotypes were defined based on patterns of metabolite production. Asterisks assigned to chemotypes denote minor variations in metabolite production relative to the metabolite profiles of closely related strains.

Strain	Compounds in Table 2																																	Chemotype
	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	25	26	27	28	29	30	31	32	33
FMR 19091	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	A
CF-155367	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	B
CBS 468.71	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	B
CF-295464	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	C
CF-295351	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	C
CF-282341	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	C
FMR 19618	−	−	−	+	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	D
FMR 20161	−	−	−	+	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	D
CF-277801	−	−	−	+	+	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	D*
FMR 19842	−	−	−	+	−	+	−	+	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	E
CF-287459	−	−	−	−	−	−	−	−	−	−	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	F
FMR 20838	−	−	−	−	−	−	−	+	−	−	−	−	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	G
FMR 20458	−	−	−	+	+	−	−	+	−	−	−	−	+	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	H
CBS 217.38	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	I
CBS 317.65	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	J
CBS 527.84	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	J
CBS 117679	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	K
FMR 20475	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	+	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	L
CF-295384	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	+	+	+	−	−	−	−	−	−	−	−	−	−	−	−	M
CF-277822	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	+	−	−	−	−	−	−	−	−	−	−	N
CBS 318.81	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	+	+	−	−	−	−	−	−	−	−	N*
CBS 101.60	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	+	+	−	−	−	−	−	−	O
CBS 100.07	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	+	+	−	−	−	−	−	−	O
CF-285370	−	−	−	−	−	−	−	−	−	−	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	P
CBS 396.81	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	Q
CBS 354.86	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	+	+	+	+	R
CBS 125.66	−	−	−	−	−	−	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	+	+	+	+	R
FMR 19850	−	−	−	−	−	−	+	+	−	−	−	−	−	−	−	−	−	−	−	−	−	−	−	+	−	−	−	−	−	+	+	+	+	S

Diversity of Preussia s.l. using a polyphasic approach

Based on the relationships revealed by the multi-locus phylogenetic analyses, morphological features, metabolite production, and substrate preference, we divided Preussia s.l. into nine genera, including Preussia s.str., Sporormiella, and re-erecting Spororminula (Fig. 3). Table 4 summarizes the major phenotypic differences among them.

Table 4.

Major morphological differences between genera in Sporormiaceae.

Genera	Ascomata		Asci		Ascospores		Asexual morph						Main habitat(s)
	Shape	Hairs	Shape	Stipe	Cells	Sheath	Pycnidia	C-like1	H-like2	M-like3	Holob.4	Holot.5
Sporormiella (Clade 1)	Perithecia or Cleistothecia	Absent	Cylindrical-clavate (subglobose)	Short	4–13-celled	Subglobose	Present	NA	NA	NA	NA	Present	Dung, soil, and as endophytes
Sporormyces (Clade 2)	Perithecia	Absent	Cylindrical-clavate	Short	7-celled	Subglobose	NA	NA	NA	NA	NA	NA	Dung, soil, and as endophytes
Pleosporormiella (Clade 3)	Perithecia or Cleistothecia	Absent	Subcylindrical	Short	4-celled	Subglobose	Present	Present	Present	NA	NA	NA	Soil and sediments
Preussia s.str. (Clade 4)	Cleistothecia or Perithecia	Absent	Subglobose or broadly clavate	Long (short)	4-celled	Absent or subglobose	Present	Present	NA	NA	NA	NA	Soil and sediments
Clade 5	Perithecia	Absent	Cylindrical-clavate	Short	4–8-celled	Subglobose	Present	NA	NA	NA	NA	NA	Dung, soil, and sediments
Spororvela (Clade 6)	Perithecia	Absent	Cylindrical-clavate	Long	8-celled	Subglobose	NA	NA	NA	NA	NA	NA	Dung
Clade 7	Perithecia	Absent	Cylindrical-clavate	Long (short)	4–8-celled	Subglobose	NA	NA	NA	NA	NA	NA	Dung
Chaetosporormiella (Clade 8)	Perithecia	Present or absent	Subcylindrical	Short	4-celled	Subglobose	Present	NA	NA	Present	NA	NA	As endophyte, dung, and soil
Spororminula (Clade 9)	Perithecia	Absent	Subcylindrical	Short	8-celled	Absent	Present	NA	NA	NA	NA	NA	Dung, soil and sediments
Sporormia	Perithecia	Absent	Subcylindrical	Short	16-or more celled	Lobated	NA	NA	NA	NA	NA	NA	Dung
Westerdykella	Perithecia or cleistothecia	Absent	Globose to subcylindrical	Short	1–4-celled	Absent	Present	NA	NA	NA	NA	NA	Soil, mud, and plant material
Sporormurispora	Cleistothecia	Absent	Subcylindrical	Short	1-celled	Absent	NA	NA	NA	NA	NA	NA	Plant material
Sparticola	Perithecia	Absent	Cylindrical-clavate	short	4–8-celled	Subglobose	NA	NA	NA	NA	Present	NA	Plant material
Forliomyces	NA	NA	NA	NA	NA	NA	Present	NA	NA	NA	NA	NA	Plant material
Trichophoma	NA	NA	NA	NA	NA	NA	Setose	NA	NA	NA	NA	NA	Plant material
Xenomonodictys	NA	NA	NA	NA	NA	NA	NA	NA	NA	Present	NA	NA	Wood

¹Chrysosporium-like asexual morph.

²Humicola-like asexual morph.

³Monodictys-like asexual morph.

⁴Holoblastic conidiogenous cells.

⁵Holothallic conidiogenesis.

NA: not available.

Clade 1 comprised members of Sporormiella, displaying short-stipitate asci with 4–13-celled ascospores, and primarily pycnidial asexual morphs. This lineage comprised 22 known species, including 11 proposed new combinations into this genus, as well as one new name and one novel species, Sm. solicola. It is noteworthy that this clade included the species complexes of “Preussia lignicola” and “Preussia minima”. The first complex was determined as a single taxon, while the latter represented a set of five species (Figs 2, 3), including the recent addition to the genus Preussia, Pr. japonica (Li et al. 2025; Supplementary material Fig. S11).

Clade 2 represented an independent lineage that we proposed as Sporormyces (Sc.), typified by the novel combination Sc. septenarius. This genus was characterized by short-stipitate asci with 7-celled ascospores, and producing 4-methoxy-6-pentyl-2H-pyran-2-one among other compounds. In addition, we proposed the novel species, Sc. anthraquinoniformans, represented by strains of xerophytic origin.

We proposed clade 3 as the novel genus Pleosporormiella (Ps.), characterized by short-stipitate asci with 4-celled ascospores, pycnidial and humicola-like asexual morphs, and producing preussins and C₁₉H₂₈O₅. Three new combinations were proposed for this genus, along with four novel species, Ps. aculeata, Ps. compacta, Ps. polyspora, and Ps. suttonii, the last three species described from strains of marine and xerophytic substrates.

Clade 4 included the type species of the genus Preussia, Pr. funiculata, another known species, and a novel species of xerophytic origin, Pr. gypsicola. We defined this lineage as Preussia s.str., which is characterized by displaying predominantly long-stipitate asci with 4-celled ascospores, pycnidial and chrysosporium-like asexual morphs. In addition, Pr. funiculata was characterized by producing cyperin.

Clade 6 represented an independent lineage that we proposed as Spororvela (Sv.), characterized by long-stipitate asci with 7–12-celled (primarily 8-celled) ascospores. Only one species is known, the novel combination Sv. tela.

We also proposed clade 8 as the novel genus Chaetosporormiella (Cp.), characterized by hairy ascomata, short-stipitate asci with 4-celled ascospores, pycnidial and monodictys-like asexual morphs, and producing two leptosin analogues. A known species was recombined into this genus, and we proposed an additional novel species, Cp. monodictyoides.

Clade 9 accommodated the type species of the genus Spororminula, Sn. tenerifae (proposed here as Sn. subticiensis), and one novel species of marine origin, Sn. irregularis. This lineage is characterized by short-stipitate asci with 8-celled ascospores, pycnidial asexual morph, and producing 7-chloro-6-methoxymellein.

The complete morphological descriptions and discussions on synonyms and novel species are provided in the taxonomy section. Unfortunately, we could not examine the strains included in clades 5, 7, and 10, so they remained as unresolved lineages in Sporormiaceae.

Taxonomy

We resolved the classification of Preussia s.l. using an integrative taxonomic approach that combined phylogenetic, morphological, ecological, and secondary metabolite data. In this section, we provide the information necessary to stabilize the nomenclature of the recognized taxa, including comments on the unresolved clades. Our taxonomic decisions are further supported by the examination of type-specimen exsiccatae. When the original authors did not clearly designate type specimens, or when these were lost or poorly preserved, we addressed the issue by examining original collections and designating lectotypes and/or epitypes when needed. In cases where typification was not possible (i.e., when available specimens differed in substrate and/or geographic origin), and in accordance with best-practice recommendations, we designated a reference strain whose characters matched the protologue of the species undergoing nomenclature stabilization (Lendemer 2020). In addition, two non-sporulating species were proposed based only on DNA sequence data, following the procedures established by previous authors (Gomes et al. 2013, Lombard et al. 2016, Hou et al. 2023). Genera are presented here according to their position in the phylogenetic tree (Fig. 3), and species are arranged alphabetically.

Clade 1. Sporormiella Ellis & Everh., N. Amer. Pyrenomyc.: 136. 1892.

Mycelium immersed and superficial, composed of hyaline, septate, branched, smooth-walled, sometimes anastomosed hyphae. Sexual morph: Ascomata perithecial, sometimes cleistothecial, immersed to semi-immersed, solitary or aggregated in small groups, globose to subglobose or pyriform, glabrous, carbonaceous-membranaceous to coriaceous; peridium smooth, dark brown to black, textura angularis; neck short, papilliform to rostrate, long, cylindrical, sometimes 2–3 necks. Paraphyses filiform, septate, intermixed with asci and longer. Asci 8-spored, bitunicate, cylindrical-clavate, sometimes subglobose, tapering into a short stipe. Ascospores predominantly biseriate, subcylindrical, 4–13-celled, septa transverse to oblique, deeply constricted, frequently cells easily separable at maturity, and variable in size and shape, thick-walled, hyaline when young, dark brown at maturity; germinal slit elongated extending the full length of each cell, parallel to diagonal, or transverse to the longitudinal axis of the ascospore; gelatinous sheath hyaline, subglobose, surrounding each ascospore. Coelomycetous asexual morph: Conidiomata pycnidial, solitary or aggregated, immersed to erumpent, globose to subglobose or irregularly shaped, textura angularis, brown, ostiolate, with up to 4 ostioles. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, hyaline, smooth. Conidia aseptate, solitary, ellipsoid with obtuse ends, hyaline, smooth, guttulate, aggregated in mucous masses. Chlamydospores aseptate, solitary or sometimes in short chains, intercalary or terminal, globose to ellipsoidal or variable in shape, grey to brown, smooth-walled.

Type species: Sporormiella nigropurpurea Ellis & Everh., N. Amer. Pyrenomyc.: 136. 1892.

Habitat: Predominantly on dung, but also on soil, wood, and as endophytes in leaves.

Notes: The genus Sporormiella was described based on a coprophilous fungus growing on cow dung in Newfield (New Jersey, USA), Sm. nigropurpurea (Ellis & Everhart 1892). The type specimen is available in the New York Botanical Garden (NY 01047198) without available living cultures or DNA sequences. Despite this, the fungus remains recognizable and displays the main morphological features of the genus; i.e., short-stipitate asci with 4-celled ascospores covered by a gelatinous sheath. In fact, an additional specimen of this species was recently identified based on morphology (Wergen 2025), but cultures or DNA sequence data were not generated.

In our phylogenetic analyses (Fig. 3), clade 1 formed a well-supported monophyletic lineage comprising predominantly coprophilous species, including species with 4-celled (e.g., Sm. minimoides and Sm. similis), 8-celled (e.g., Sm. bipartis and Sm. minipascua), and 13-celled ascospores (i.e., Sm. antarctica). This circumscription matched the definition of the genus in the sense of Ahmed & Cain (1972). Although we could not include Sm. nigropurpurea in the analyses, we recognize this clade as the genus Sporormiella based on morphology. The species Sm. lignicola, formerly in Preussia, also clustered here as anticipated by Kruys & Wedin (2009). This way, Sporormiella is defined as a different lineage from Preussia s.str. and Sporormia, comprising 22 phylogenetic species. Among them, three species are only known for their pycnidial asexual morph.

It is worth mentioning that the genus Ohleriella was considered a synonym of Sporormiella based on the morphological resemblance between its type species, Ohleriella neomexicana, and Sm. octomera (Ahmed & Cain 1972). Our phylogenetic analyses resolved the specimen of Sm. octomera with available molecular data (strain F: 2579) as a member of clade 7 (Fig. 3). Although we could not examine this specimen, our results suggest that Ohleriella should not be considered a synonym of Sporormiella. In addition, Ohleriella is characterized by producing long trabeculate pseudoparaphyses (Earle 1902). Therefore, we follow previous works that considered this genus as a member of Delitschiaceae (Zhang et al. 2012b).

Sporormiella aegilopis (M. Mehrabi et al.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862060.

Basionym: Preussia aegilopis M. Mehrabi et al., Nova Hedwigia 114: 152. 2022.

Typus: Iran, Kermanshah province, Sonqor, 34°50’20.65” N, 47°09’52.08” E, seed endophyte of Aegilops umbellulata, Aug. 2018, M. Mehrabi (holotype IRAN 17713F, ex-type strain IRAN 3454C).

Description and illustration: Mehrabi et al. (2022).

Notes: Our phylogenetic analyses revealed the basionym Pr. aegilopis as a member of Sporormiella, sister to Sm. australis and Sm. bipartis (Figs 2, 3). Sporormiella aegilopis is only known by its coelomycetous asexual morph, while Sm. australis and Sm. bipartis display sexual morphs with ascospores that differ in being 4-celled and 8-celled, respectively (Ahmed & Cain 1972, Mehrabi et al. 2022).

Sporormiella africana (Arenal et al.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862061.

Basionym: Preussia africana Arenal et al., Fungal Diversity 20: 6. 2005.

Typus: Spain, Canary Islands, Tenerife, Llano de los Viejos, on leaves of Viburnum tinus, 2 Apr. 1995, A. Santos (holotype AH32767, ex-type strain S17).

Description and illustration: Arenal et al. (2005).

Additional materials examined: Kenya, on elephant dung, Jan. 1973, R.F. Cain (CBS 166.73). Mozambique, Inhaca Island, from mangrove mud, Nov. 1953, H.J. Swart (CBS 405.54). South Africa, Limpopo province, Mookgophong (Naboomspruit), from Prunus salicina, Aug. 2008, U. Damm (CBS 120881).

Notes: The ex-type strain of the basionym Pr. africana clustered as a member of Sporormiella, forming a lineage with Sm. procaviae (Figs 2, 3). Only the sexual morph is known for Sm. africana, while Sm. procaviae only produces a pycnidial asexual morph (Crous et al. 2020a).

Sporormiella antarctica (Speg.) S.I. Ahmed & Cain, Canad. J. Bot. 50(3): 434. 1972.

Basionym: Sporormia antarctica Speg., Bol. Acad. Nac. Ci. 11(2): 224. 1888.

Synonym: Preussia antarctica (Speg.) J.H. Chang & Y.Z. Wang, Nova Hedwigia 88: 252. 2009.

Typus: Argentina, Tierra del Fuego, Ushuaia, on dung of Bernicla antarctica, Jun. 1882, C. Spegazzini (holotype LPS 3500).

Description and illustration: Ahmed & Cain (1972).

Additional material examined: Norway, Spitsbergen, from soil, May 1989, F. Seigle-Murandi (CBS 222.89).

Notes: Sporormiella antarctica was transferred to Preussia based on morphology, with both genera considered as synonyms (Chang & Wang 2009). Our phylogenetic analyses support this species as a member of Sporormiella, characterized by producing 13-celled ascospores (Fig. 3, Supplementary material Fig. S1).

Sporormiella australis (Speg.) S.I. Ahmed & Cain, Canad. J. Bot. 50: 434. 1972.

Basionym: Sporormia australis Speg., Bol. Acad. Nac. Ci. 11: 50. 1887.

Synonym: Preussia australis (Speg.) Arx, Proc. Kon. Ned. Akad. Wetensch., C 76: 294. 1973.

Typus: Argentina, Santa Cruz, Misioneros area, on dung of Cavia australis, Aug. 1882, C. Spegazzini (holotype LPS 3521).

Description and illustration: Ahmed & Cain (1972).

Additional materials examined: Kuwait, from soil, unknown collection date and collector (CBS 123149); ibid., from saline desert soil, Sep. 1973, A.F. Moustafa (CBS 806.73; proposed here as reference strain). Morocco, from soil, Jul. 1987, F. Seigle-Murandi (CBS 387.87). South Africa, Western Cape Province, Bonnievale, from wood symptoms of Prunus armeniaca, Aug. 2005, U. Damm (CBS 120880).

Notes: Our phylogenetic analyses clustered the strains examined in this work with strains previously used to characterize Sm. australis (Fig. 2; Arenal et al. 2005, Kruys & Wedin 2009, González-Menéndez et al. 2017). We compared size ranges with the protologue and confirmed the identification (Supplementary material Fig. S2). Since DNA data of the type specimen is not available and metadata of the examined specimens do not match with that of the protologue, we propose CBS 806.73 as reference strain for the species in phylogenetic analyses.

Sporormiella bezerrensis (Ferreira-Sá et al.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862062.

Basionym: Preussia bezerrensis Ferreira-Sá et al., Persoonia 49: 315. 2022.

Typus: Brazil, São Domingos, Goiás, Lapa do Bezerra cave, from air, Dec. 2020, A.S. Ferreira-Sá, et al. (holotype HUEG 15380/SXS 693, ex-type strain URM 8508).

Description and illustration: Tan et al. (2022).

Notes: Sporormiella bezerrensis, formerly known as Pr. bezerrensis, formed an independent lineage sister to the Sm. minima species complex (Figs 2, 3). For a detailed morphological comparison with related species, see Tan et al. (2022).

Sporormiella bipartis (Cain) S.I. Ahmed & Cain, Canad. J. Bot. 50: 435. 1972.

Basionym: Sporormia bipartis Cain, Stud. Sphaer. Ontario: 106. 1934.

Synonym: Preussia bipartis (Cain) Kruys, Syst. Biodiv. 7 (4): 476. 2009.

Typus: Canada, Ontario, Nipissing District, Lake Timagami, on rabbit dung (holotype TRTC 5320).

Description and illustration: Ahmed & Cain (1972).

Notes: Based on the phylogeny of the strain S: 17250-a, which had been morphologically identified as Sm. bipartis, this species was transferred to Preussia following the synonymization of the two genera (Kruys & Wedin 2009). Our concatenated phylogenetic analysis confirmed this strain as a member of Sporormiella (Fig. 3). This species is characterized by producing 8-celled ascospores (Ahmed & Cain 1972).

Sporormiella elegans (D.C. Sandberg & A.E. Arnold) Guerra-Mateo, Gené & Cano comb. nov. MB 862063.

Basionym: Preussia elegans D.C. Sandberg & A.E. Arnold, Pl. Fungal Syst. 67: 69. 2022.

Typus: USA, Arizona, Pima County, Tucson Mountains, as endophyte in healthy photosynthetic stems of Ephedra trifurca, 2009, D.C. Sandberg & A.E. Arnold (holotype ARIZ-AEA-DS0014, ex-type strain DS0014).

Description and illustration: Sandberg et al. (2022).

Notes: Our phylogenetic analyses determined the basionym Pr. elegans as a member of Sporormiella (Figs 2, 3), forming a well-supported clade with Sm. minipascua (UPS: 306). These species differ in ascospore cell number, with Sm. elegans producing 4-celled ascospores, while Sm. minipascua displays 8-celled ascospores (Kruys & Wedin 2009, Sandberg et al. 2022).

Sporormiella intermedia (Auersw.) S.I. Ahmed & Cain ex Kobayasi, Bull. Natl. Sci. Mus., Tokyo n.s. 12: 339. 1969.

Basionym: Sporormia intermedia Auersw., Hedwigia 7: 67. 1868.

Synonym: Preussia intermedia (Auersw.) S. Ahmad, Monogr. Biol. Soc. Pakistan 8: 49. 1979.

Typus: Germany, isolated from cow, deer, hare, and rabbit dung (holotype was not clearly indicated); lectotype designated here MBT 10031132: UK, near Batheaston, on rabbit dung, Oct. 1863, C.E. Broome, No. 644 (under Sphaeria stercoris), Rabenhorst, Fungi Eur. Exs., examined by Auerswald, included in the protologue of Sp. intermedia, and preserved at the Martin-Luther-Universität Halle-Wittenberg (HAL), Germany.

Description and illustration: Ahmed & Cain (1972).

Additional materials examined: Greece, Cephalonia Island, on goat dung, unknown collection date, F. Arenal, et al. (S4; proposed here as reference strain); ibid., (S3). USA, Minnesota, Cook County, on moose dung, unknown collection date and collector (MF5672).

Notes: The taxonomic history of the basionym Sm. intermedia is based on non-type specimens recovered worldwide (Ahmed & Cain 1972, Arenal et al. 2005). In addition, it is important to highlight that the holotype specimen was not properly indicated in the protologue of the species. Auerswald (1868) described Sp. intermedia based on several collections that were distributed in exsiccatal series and deposited in different herbaria under other names. Consequently, these materials can be regarded as original specimens suitable for lectotypification. At the Martin-Luther-Universität Halle-Wittenberg (HAL, Germany), we located one of Auerswald’s specimens (No. 644), preserved in good condition, but incorrectly labelled as Sp. stercoris, which is here designated as the lectotype of the species (Supplementary material Fig. S3). Although the cultures examined in this study are morphologically consistent with Sm. intermedia, none of them is suitable for designation as an epitype. Therefore, we propose strain S4 as a reference strain to stabilize the phylogenetic framework of this species. We selected this strain based on its morphological resemblance to the protologue and its long history of use as a representative strain of Sm. intermedia in phylogenetic analyses (Arenal et al. 2005, González-Menéndez et al. 2017).

The species has been transferred to both Sporormiella and Preussia (Kobayasi et al. 1969, Ahmad 1979). However, morphologically it resembles Sporormiella, and our phylogenetic analyses confirmed Sm. intermedia as a monophyletic species (Figs 2, 3). It is noteworthy that the original producer of australifungin and australifunginol, the strain MF5672, was formerly identified as Sm. australis, but phylogenetic analyses using both strains (MF5672 and S4) determined the former strain as Sm. intermedia (Fig. 2; González-Menéndez et al. 2017).

Furthermore, our phylogenetic analyses revealed the strain S: 16745-c, labelled as Pr. borealis (Kruys & Wedin 2009) as conspecific with Sm. intermedia (Figs 2, 3). These species differ in ascospore length, 48–59 µm in Sm. intermedia vs 92–114 µm in Pr. borealis (Ahmed & Cain 1972).

Sporormiella isabellae (Arenal et al.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862064.

Basionym: Preussia isabellae Arenal et al., Fungal Diversity 20: 7. 2005.

Typus: Puerto Rico, from woody plant debris, Jan. 1996, J. Guarro (holotype AH32771, ex-type strain S25).

Description and illustration: Arenal et al. (2005).

Notes: The ex-type strain of Pr. isabellae was determined as a member of Sporormiella, forming an independent lineage within the Sm. minima species complex (Figs 2, 3). Morphological differences among members of this group are subtle and Sm. isabellae is primarily distinguished through phylogenetic analyses (Fig. 3).

Sporormiella isopartis Guerra-Mateo, Gené & Cano, nom. nov. MB 862065.

Replaced synonym: Preussia isomera Cain, Canad. J. Bot. 39: 1643. 1961.

Etymology: Greek “iso-“, equal, and latin “part”, portion, referring to the ascospores’ septation in equal portions.

Typus: USA, Florida, near Tallahassee, on unidentified dung, Mar. 1952, S.L. Meyer (holotype TRTC 32089, ex-type strain CBS 318.65).

Description and illustration: Cain (1961).

Additional materials examined: Italy, from Avena sativa grain, Jul. 1962, G. Vegni (CBS 251.62). Japan, Miyazaki Prefecture, Toimisak, on horse dung, Jul. 1974, K. Furuya (CBS 671.77). Venezuela, Edo Monagas, 5 km North of El Zamuro, along route 10, on cow dung, Jul. 1972, J.C. Krug (CBS 415.82); ibid., Jusepín, on cow dung, Jun. 1978, J.C. Krug (CBS 388.78).

Notes: The name Sm. isopartis is a replacement name for Pr. isomera (Cain 1961), as the epithet “isomera” is unavailable in Sporormiella due to prior valid publication of Sm. isomera (Ahmed & Cain 1972). Therefore, a new name is proposed for Pr. isomera.

Although Sm. isomera and Sm. isopartis differ in ascospore size, 32–38(–40) × 5.5–7(–8) µm vs 30–33 × 4–4.5 µm, respectively, both fungi were described from dung collected in North America (Cain 1961, Ahmed & Cain 1972). Given that ascospore size may vary within a species, they may represent a single taxon. However, no molecular data are available for Sm. isomera for comparison. Therefore, we regard this species as taxonomically uncertain (Supplementary material Fig. S2) until further studies are conducted.

Sporormiella isopartis formed an independent lineage sister to Sm. jejuensis and Sm. minimoides (Figs 2, 3), differing from Sm. minimoides by producing smaller asci, 43–55 µm vs 90–110 µm in length, respectively (Cain 1961, Ahmed & Cain 1972). For Sm. jejuensis only a coelomycetous asexual morph is known (Noh et al. 2025).

Sporormiella japonica (Udagawa et al.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862375.

Basionym: Scytalidium japonicum Udagawa et al., Mycotaxon 25(1): 281. 1986.

Synonym: Preussia japonica (Udagawa et al.) D.W. Li & N.P. Schult., Fungal Biol. 130 (1, no. 101691): 15. 2025.

Typus: Japan, Yamaguchi, Kagawa-ninowari, Yamaguchi-ken Chubu livestock Hygiene service Center, isolated from bronchiole lesions on domestic cow (Bos taurus var. domesticus), Jun. 1984, K. Tominaga (holotype NHL 2954, ex-type strain CBS 494.88).

Description and illustration: Udagawa et al. (1986).

Additional descriptions and illustrations: De Hoog et al. (2020).

Notes: Sporormiella japonica is an opportunistic pathogen originally described as Scytalidium japonicum from bronchiole lesions on a domestic cow (Udagawa et al. 1986, De Hoog et al. 2020). The basionym was recently transferred to Preussia based on phylogenetic analyses; however, the authors acknowledged the complex taxonomy of Preussia s.l. and the need for further studies (Li et al. 2025). Although this species is not represented in Figs 2 and 3, an additional LSU-ITS analysis (Supplementary material Fig. S11) allowed us to resolve the taxonomic position of the type strain (CBS 494.88) as a member of Sporormiella, forming an independent lineage within the Sm. minima species complex. Sporormiella japonica is characterized by producing holothallic, cylindrical, 0–1-septate, hyaline arthroconidia with truncated ends, which may sometimes be clavate to pyriform, as well as dark brown chlamydospores arranged in chains (Udagawa et al. 1986). This species represents the first report of holoarthric conidiogenesis in Sporormiella.

Sporormiella jejuensis H. Noh, H.U. Cho & S.H. Kim ex Guerra-Mateo, Gené & Cano, sp. nov. MB 862066.

[originally described as Preussia jejuensis H. Noh et al., Mycobiology 53(2): 209. 2025. nom. inval., Art. F.5.2 (Madrid)].

Typus: Republic of Korea, Jeju Island, Seopjikoji (33°25′48.5″N 126°56′03.5″E), on horse dung, Oct. 2023, unknown collector (holotype NIBRFGC000512615, ex-type strain DUCC 15669).

Description and illustration: Noh et al. (2025).

Notes: The species Pr. jejuensis was invalidly published without citing an identifier for the new name (Noh et al. 2025). Herein, we validated this lineage as a new species of Sporormiella, sister to Sm. isopartis and Sm. minimoides (Figs 2, 3). Sporormiella jejuensis is characterized by the production of pycnidia, while the asexual morph remains unknown in the other two species (Cain 1961, Kruys & Wedin 2009).

Sporormiella koreensis H. Noh, H.U. Cho & S.H. Kim ex Guerra-Mateo, Gené & Cano, sp. nov. MB 862067.

[originally described as: Preussia koreensis H. Noh et al., Mycobiology 53(2): 210. 2025. nom. inval., Art. F.5.2 (Madrid)].

Typus: Republic of Korea, Jeju Island, Seopjikoji (33°25′48.5″N, 126°56′03.5″E), on horse dung, Oct. 2023, unknown collector (holotype NIBRFGC000512614, ex-type strain DUCC 15668).

Description and illustration: Noh et al. (2025).

Additional materials examined: Panama, Santa Rosa, on goat dung, Apr. 1948, S.L. Mayer (CBS 524.50). South Africa, on Podocarpus falcatus fruit, unknown collection date, U. Damm (CBS 120888). Spain, Catalonia, Mediterranean coast, Estartit, discharging area of the Ter River, from sediments at 27 m depth, Oct. 2023, P. Rojas & D. Guerra-Mateo (FMR 21125).

Notes: The species Pr. koreensis was invalidly published without citing an identifier for the new name (Noh et al. 2025). Our phylogenetic analyses confirmed this taxon as a member of Sporormiella, forming an independent lineage within the Sm. minima species complex and we thus validate the name here (Figs. 2, 3). Morphological differences among members of this group are subtle and Sm. koreensis is primarily distinguished through phylogenetic analyses.

Sporormiella lignicola (W. Phillips & Plowr.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862068.

Basionym: Sporormia lignicola W. Phillips & Plowr., Grevillea 6 (37): 29. 1877.

Synonyms: Preussia lignicola (W. Phillips & Plowr.) Kruys, Syst. Biodiv. 7 (4): 476. 2009.

Preussia mariae D.C. Sandberg & A.E. Arnold, Pl. Fungal Syst. 67 (2): 70. 2022, syn. nov.

Typus: England, Norfolk, King’s Lynn, Tilney St Lawrence, from wood of Fraxinus excelsior, Mar. 1877, unknown collector [holotype K(M) 131910].

Description and illustration: Phillips & Plowright (1872).

Additional materials examined: Ethiopia, from seeds of wheat, unknown collection date and collector (CBS 110384). Netherlands, Texel, De Muy area, on rabbit dung, May 1969, unknown collection date, G.S. de Hoog (CBS 363.69; proposed here as reference strain); ibid., Texel, De Geul area, unknown collection date and collector (CBS 264.69). Spain, Granada, from leaves of Retama sphaerocarpa, Mar. 2012, G.F. Bills (CF-279765).

Notes: Sporormia lignicola was transferred to Preussia based on phylogenetic analyses using the strain CBS 363.69 (Kruys & Wedin 2009). Although those authors considered the genera Preussia and Sporormiella as synonyms, they stated that this species displayed the morphological characters of Sporormiella. Our phylogenetic analyses confirmed this strain as a member of the latter genus (Figs 2, 3).

We examined the holotype of Sp. lignicola and could see that the morphological features of the species match those of CBS 363.69, confirming the identity of the strain (Supplementary material Fig. S4). We tried to obtain DNA from the herborized material but, unfortunately, poor quality hampered further analyses. Therefore, we propose CBS 363.69 as a reference strain to stabilize nomenclature until suitable material is found for epityfication.

In previous works, the Sm. lignicola clade was referred to as a species complex (González-Menéndez et al. 2017, Sandberg et al. 2022). However, our phylogenetic analyses displayed a single taxon with minor intraspecific variability (Fig. 3). Moreover, the ex-type strain of the recently described species Pr. mariae (Sandberg et al. 2022) clustered in this lineage with a 99 % ITS identity with CBS 363.69. Therefore, we consider this species a synonym of Sm. lignicola.

Sporormiella mediterranea (Arenal et al.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862069.

Basionym: Preussia mediterranea Arenal et al., Fungal Diversity 25: 10. 2007.

Synonym: Preussia mayoae Y.P. Tan et al., Index Austral. Fungi 42: 14. 2024, syn. nov.

Typus: Spain, Cáceres, Monfragüe National Park, from leaves of Cistus albidus, Nov. 1996, F. Arenal (holotype AH 32774, ex-type strain S23).

Description and illustration: Arenal et al. (2007).

Additional materials examined: Netherlands, Texel, on sheep dung, Oct. 1969, unknown collector (CBS 263.69). UK, no additional data available (CBS 338.35).

Notes: The ex-type strain of Pr. mediterranea clustered in the Sporormiella clade, forming an independent lineage within the Sm. minima species complex (Figs 2, 3). Although morphological features are conserved among members of this group, Sm. mediterranea differs by producing the largest ascospores in the group, 32–50 × 6–10 µm (Arenal et al. 2007). In addition, our phylogenetic analysis resolved Pr. mayoae as conspecific with Sm. mediterranea (Fig. 2). Of note is that Tan et al. (2024) avoided the morphological characterization of this species and reported a 98 % ITS similarity to other members of the Sm. minima complex.

Sporormiella minimoides S.I. Ahmed & Cain, Canad. J. Bot. 50 (3): 450. 1972.

Synonym: Preussia minimoides (S.I. Ahmed & Cain) Valldos. & Guarro, Bol. Soc. Micol. Madrid 14: 88. 1990.

Typus: Canada, Ontario, Algoma District, near Aubinadong River, on wolf dung, Aug. 1960, R.F. Cain (holotype TRTC 36242).

Description and illustration: Ahmed & Cain (1972).

Notes: Sporormiella minimoides was considered a member of Preussia, accepting both genera as synonyms (Valldoserra & Guarro 1990). This species is currently represented in phylogenetic analyses through two strains, S10 and S18 (Arenal et al. 2005). Here, we determined these strains as members of Sporormiella as originally stated in the protologue (Figs 2, 3).

Sporormiella minipascua S.I. Ahmed & Cain, Canad. J. Bot. 50 (3): 451. 1972.

Synonym: Preussia minipascua (S.I. Ahmed & Cain) J.H. Chang & Y.Z. Wang, Nova Hedwigia 88 (1–2): 250. 2009.

Typus: Canada, Ontario, Nipissing District, Pickerel Lake Portage from Lake Timagami, on deer dung, Aug. 1933, R.F. Cain (holotype TRTC 5390).

Description and illustration: Ahmed & Cain (1972).

Notes: Sporormiella minipascua was transferred to Preussia based on the phylogenetic analysis of the strain UPS: 306, accepting both genera as synonyms (Kruys & Wedin 2009). However, our analyses confirmed this strain as a Sporormiella species (Figs 2, 3).

Sporormiella pascua (Niessl) S.I. Ahmed & Cain, Canad. J. Bot. 50(3): 454. 1972.

Basionym: Sporormia pascua Niessl, Oesterr. bot. Z. 28: 45. 1878.

Synonym: Preussia pascua (Niessl) Valldos. & Guarro, Bol. Soc. Micol. Madrid 14: 88. 1990.

Typus: Czech Republic, Brno, Lellekowitz, on sheep dung, Jun. 1874, Niessl (holotype M-0378428).

Description and illustration: Ahmed & Cain (1972).

Additional material examined: Spain, Baleares Islands, Palma de Mallorca, Secar de la Real, on horse dung, Mar. 2021, J.F. Cano-Lira (FMR 19091).

Notes: Sporormiella pascua is a poorly represented species in living cultures. The best representative is the strain NBRC 30183, which was identified based on morphological features (Ahmed & Cain 1972; Udagawa & Furuya 1976). Unfortunately, only the LSU sequence is available for phylogenetic analyses (Supplementary material Table S1). Here, we also included the strain FMR 19091, which remained sterile in all culture media. Although the type specimen of this species is well preserved in the Bavarian State Collection for Botany in Munich (M-0378428), no living culture is available for sequencing. We chose to retain this lineage as Sm. pascua based on the phylogenetic placement of NBRC 30183 within Sporormiella (Fig. 2), which is consistent with the morphological features of the holotype (Supplementary material Fig. S5). Future studies should aim to stabilize the taxonomy of this species by incorporating additional metabolically active strains and barcode sequences.

Sporormiella persica (Asgari & Zare) Guerra-Mateo, Gené & Cano, comb. nov. MB 862070.

Basionym: Preussia persica Asgari & Zare, Nova Hedwigia 90 (3–4): 541. 2010.

Synonym: Preussia arizonica D.C. Sandberg & A.E. Arnold, Pl. Fungal Syst. 67 (2): 66. 2022, syn. nov.

Typus: Iran, East Azerbaijan, Sarab, from dead barley leaves, Jan. 2003, B. Asgari (holotype IRAN 12397 F, ex-type strain CBS 117680).

Description and illustration: Asgari & Zare (2010).

Additional materials examined: Germany, Giessen, from colon content of freshly shot deer, Jun. 1975, R. Hadlok (CBS 471.75). Papua New Guinea, Northern Prov., Owen Stanley Range, Myola, near guesthouse along Iora creek, on sheep dung, Oct. 1995, A. Aptroot (CBS 767.96). Spain, Catalonia, Mediterranean coast, Estartit, discharging area of the Ter River, from sediments at 27 m depth, Oct. 2023, P. Rojas & D. Guerra-Mateo (FMR 21126). Venezuela, Monagas state, Caripe, on donkey dung, Jul. 1978, J.C. Krug (CBS 442.78).

Notes: Our phylogenetic analyses revealed Pr. persica as a member of Sporormiella, forming an independent lineage within the Sm. minima species complex (Figs 2, 3). Although morphological features are conserved among members of this group, Sm. persica differs by producing ascomata with up to three necks (Asgari & Zare 2010). Moreover, the ex-type strain of the recently described species Pr. arizonica (Sandberg et al. 2022) clustered in this lineage with a 99 % ITS identity with CBS 117680. Therefore, we consider this species a synonym of Sm. persica.

Sporormiella procaviae (Crous) Guerra-Mateo, Gené & Cano, comb. nov. MB 862071.

Basionym: Preussia procaviae Crous, Persoonia 45: 321. 2020.

Synonyms: Preussia crataegi Eisvand & M. Mehrabi, Antonie van Leeuwenhoek 118 (52): 6. 2025, syn. nov.

Preussia pianica Eisvand & M. Mehrabi, Antonie van Leeuwenhoek 118 (52): 10. 2025, syn. nov.

Preussia pistaciae Eisvand & M. Mehrabi, Antonie van Leeuwenhoek 118 (52): 12. 2025, syn. nov.

Typus: Namibia, Gobabeb-Namib Research Institute, Mirabib Rock, on dung of Procavia capensis, Nov. 2019, P.W. Crous (holotype CBS H-24446, ex-type strain CBS 146827).

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 1.5–2 µm wide hyphae. Sexual morph not observed. Coelomycetous asexual morph: Conidiomata pycnidial, solitary or aggregated in clusters of 2- to several pycnidia, immersed to erumpent, rarely superficial or on aerial mycelium, pale brown to brown, globose, sub-globose or ellipsoidal, sometimes irregular in shape, glabrous or with some hyphal outgrowths, 22–200 μm diam., papillate or non-papillate, ostiolate, with 1–4 ostioles, 10 μm diam.; conidiomatal wall of 3–6 layers, formed by brown cells of textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, 4–5 × 2.5–3 μm, hyaline, smooth-walled. Conidia aseptate, solitary, ellipsoid with obtuse ends, 3–4 × 2 μm, hyaline, smooth, with one to two guttules. Chlamydospores unicellular, solitary or sometimes in short chains, intercalary or terminal, globose to ellipsoidal or variable in shape, 4–14 × 4–11 μm, grey to brown, thick- and smooth-walled. (Adapted from Crous et al. 2020, Eisvand & Mehrabi-Koushki 2025).

Culture characteristics (after 14 d at 25 °C). Colonies on PDA reaching 50 mm diam., flat, surface folded, with sparse aerial mycelium or floccose in the centre, olivaceous grey to cream towards the margin, margin lobate, smooth to filiform; reverse iron-grey to cream. On OA, 50 mm diam., flat, with sparse aerial mycelium or floccose to cottony, pale brown with cream margin, often yellow-pigmented, with paler concentric zones of sporulation; reverse brown to cream.

Notes: The ex-type strain of Pr. procaviae clustered with the recently described species Pr. crataegi, Pr. pianica, and Pr. pistaciae (Eisvand & Mehrabi-Koushki 2025), forming a well-supported clade in Sporormiella (Figs 2, 3). The ITS barcode sequences of the four species showed a 100 % of identity. In addition, the original authors of those species also provided partial sequences of the tef1-α region, revealing a 99 % of identity among them; therefore, they are considered conspecific. Since Pr. procaviae is the earliest described species, it is proposed as a new combination into Sporormiella. Here, we provide an updated description of Sm. procaviae, incorporating subtle morphological variations reported in the protologues of the synonymised species (Eisvand & Mehrabi-Koushki 2025).

Sporormiella procaviicola (Crous) Guerra-Mateo, Gené & Cano, comb. nov. MB 862072.

Basionym: Preussia procaviicola Crous, Persoonia 46: 387. 2021.

Synonym: Preussia apgariae Y.P. Tan et al. [as ‘apgarae’], Index Austral. Fungi 42: 13. 2024, syn. nov.

Typus: Namibia, Gobabeb Namib Research Institute, Mirabib, on dung of Procavia capensis, Nov. 2019, P.W. Crous (holotype CBS H-24493, ex-type strain CBS 146981).

Description and illustration: Crous et al. (2021).

Additional material examined: Netherlands, Texel, De Muy area, from rabbit dung, Oct. 1968, G.S. de Hoog (CBS 364.69).

Notes: Preussia procaviicola and Pr. apgariae clustered together forming a well-supported clade in Sporormiella (Fig 2). We consider this clade to represent a single species that is proposed as Sm. procaviicola. It is of note that the authors of Pr. apgariae did not provide a morphological characterization and reported a 99 % ITS identity with Pr. procaviicola (Tan et al. 2024).

Sporormiella similis R.S. Khan & Cain, Canad. J. Bot. 57(10): 1178. 1979.

Synonym: Preussia similis (R.S. Khan & Cain) Arenal, Mycotaxon 89(1): 140. 2004.

Typus: Kenya, Lake Amboseli, 2º33´S, 37º10´E, on rabbit dung at 1200 m above sea level, Aug. 1966, R.F. Cain, et al. (holotype TRTC 66.1777a).

Description and illustration: Khan & Cain (1979).

Additional materials examined: Kuwait, from saline desert soil, Sep. 1973, A.F. Moustafa (CBS 804.73); ibid., (CBS 805.73). Spain, Catalonia, Mediterranean coast, Tarragona, Miracle Beach, from sediments at 27 m depth, Jun. 2021, G. Quiroga-Jofre & D. Guerra-Mateo (FMR 19464). USA, Wyoming, Albany Co., Bamforth Lake, from saline soil, May 1975, D.E. Davidson (CBS 272.75).

Notes: Sporormiella similis was transferred to Preussia based on the morphological features of the strain S19, which was later used for phylogenetic analyses (Arenal et al. 2004, Kruys & Wedin 2009). However, our phylogenetic analyses confirmed this strain as a member of Sporormiella (Fig 2). In addition, the ex-type strain of the recently described species Pr. iranica (IRAN 4962C; Eisvand & Mehrabi-Koushki 2025) clustered into this lineage with a 98.5 % identity to S19. These two species seem to be conspecific, but due to the lack of DNA sequence data for the type of Sm. similis, further studies are necessary to confirm this synonymy. Therefore, Pr. iranica is included in this work among the list of Sporomiella species with uncertain status (Supplementary material Table S3).

Sporormiella solicola Guerra-Mateo, Cano & Gené, sp. nov. MB 862040.

Etymology: Referring to the substrate where the species has been reported, soil.

Typus: USA, Alaska, from soil, Jun. 1971, P.W. Flanagan (holotype CBS H-18421, ex-type strain CBS 468.71 = FMR 21346).

Culture sterile. Sporormiella solicola differs from Sm. bipartis, represented by the culture: S: 17250-a, by unique fixed alleles in two of the loci studied. ITS, 16 positions: 94 (gap), 95 (gap), 98 (A), 103 (A), 122 (A), 130 (A), 134 (A), 139 (gap), 345 (C), 349 (T), 357 (T), 390 (A), 429 (A), 430 (C), 435 (C), 437 (T). LSU, six positions: 99 (T), 411 (T), 431 (C), 432 (C), 449 (T), 451 (T). It differs from Sm. australis (culture: CBS 806.73) by unique fixed alleles in four of the loci studied. ITS, 33 positions: 58 (gap), 97 (A), 108 (C), 120 (T), 121 (A), 133 (A), 141 (gap), 226 (A), 328 (T), 344 (C), 350–353 (gap), 358–363 (gap), 366 (T), 381 (A), 399 (A), 426 (gap), 435 (C), 436 (C), 438 (G), 439 (A), 443 (A), 444 (T), 447 (T), 454 (G). LSU, 5 positions: 432 (C), 440 (T), 450 (T), 455 (T), 456 (A). tef1-α, 32 positions: 22 (C), 43 (T), 46 (C), 121 (C), 124 (C), 128 (C), 130 (G), 154 (C), 220 (C), 235 (C), 238 (C), 243 (G), 265 (C), 284 (T), 298 (C), 304 (C), 346 (C), 382 (G), 418 (A), 430 (T), 487 (A), 499 (C), 521 (G), 522 (T), 664 (T), 697 (C), 715 (C), 730 (G), 760 (T), 763 (C), 775 (T), 811 (T). rpb2, 132 positions: 9 (A), 12 (G), 42 (A), 48 (C), 57 (C), 66 (G), 69 (G), 72 (T), 78 (A), 84 (C), 90 (T), 99 (T), 102 (A), 129 (T), 141 (C), 144 (G), 153 (C), 156 (T), 177 (C), 183 (T), 184 (A), 186 (C), 195 (T), 201 (C), 207 (C), 222 (T), 231 (T), 235 (C), 237 (T), 246 (C), 249 (C), 252 (G), 255 (A), 258 (G), 264 (T), 279 (C), 282 (C), 288 (C), 294 (G), 303 (T), 315 (T), 318 (C), 319 (T), 320 (C), 321 (A), 331 (T), 333 (T), 336 (G), 339 (G), 348 (C), 351 (G), 354 (G), 363 (G), 375 (G), 384 (T), 385 (A), 387 (C), 390 (C), 405 (T), 423 (C), 426 (T), 429 (C), 432 (T), 444 (C), 448 (T), 450 (G), 453 (T), 456 (G), 459 (G), 462 (A), 464 (C), 465 (G), 467 (A), 471 (A), 474 (T), 475 (A), 477 (A), 479 (C), 481 (gap), 483 (A), 484 (C), 490 (A), 491 (G), 493 (T), 498 (T), 499 (A), 508 (G), 511 (A), 517 (T), 518 (G), 519 (G), 521 (T), 522 (A), 523 (T), 529 (C), 531 (C), 532 (T), 535 (C), 537 (A), 541 (C), 543 (T), 544 (T), 550 (C), 558 (A), 562 (T), 565 (C), 574 (G), 579 (T), 580 (C), 581 (A), 583 (G), 592 (A), 601 (A), 604 (C), 611 (A), 612 (G), 613 (T), 622 (T), 625 (G), 637 (C), 649 (G), 661 (T), 670 (T), 673 (G), 674 (G), 684 (C), 685 (G), 688 (A), 693 (A), 697 (A), 700 (C), 706 (A).

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 28–38 mm diam., slightly elevated, crateriform, glabrous, radially sulcate, yellowish white (2A2), margin slightly lobated and entire; reverse concolourous. On OA, 35–37 mm diam., flat, glabrous, colourless, pale red (11A3) to colourless in CF-155367, margin diffusing in medium; reverse colourless. On CMA, 45 mm diam., flat, glabrous, colourless, margin diffusing in medium; reverse colourless.

Additional material examined: Spain, from soil, 2002, unknown collector (CF-155367 = FMR 21494).

Cardinal temperatures for growth: Minimum 5 °C (10 mm), optimum 25 °C (36–38 mm), maximum 30 °C (1 mm).

Secondary metabolite: C₁₆H₁₂O₆, (possible 2,8-dihydroxy-3-methylxanthone-1-carboxylic acid).

Notes: We crossed the strains CBS 468.71 and CF-155367 in culture, but unfortunately, no sporulation was obtained in any of the media tested. Strain CBS 468.71 was in the past morphologically identified as Sm. australis. This suggests that at that time the culture produced 4-celled ascospores resembling Sm. australis. However, our phylogenetic analyses revealed these strains as different taxa (Figs 2, 3). In addition, CBS 468.71 and CF-155367 produced C₁₆H₁₂O₆, a possible 2,8-dihydroxy-3-methylxanthone-1-carboxylic acid that has not been detected in other Sporormiella species. Based on this data, we propose both strains as representatives of a novel Sporormiella species.

Clade 2. Sporormyces Guerra-Mateo, Gené & Cano, gen. nov. MB 862043.

Etymology: Referring to the morphological similarity to Sporormiella and Greek múkēs (μύκης), fungus.

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled hyphae. Sexual morph perithecial, immersed to semi-immersed, solitary or aggregated in small groups, subglobose to nearly pyriform, glabrous; peridium thin, membranaceous, dark brown to nearly black, with cells of textura angularis; neck small, papilliform. Paraphyses filiform, hyaline, longer than asci. Asci 8-spored, bitunicate, clavate, tapering into a short stipe. Ascospores bi- or tri-seriate above, uni- or bi-seriate below, subcylindrical, 7-celled, thick-walled, septa transverse, constrictions at septa moderate, segments separable, rounded at the ends, hyaline at first, ranging through yellowish brown to dark brown when mature; third cell from the upper end larger than the remaining cells; lower end cells longer than the other cells; germ slit oblique; gelatinous sheath hyaline, narrow. Asexual morph not observed.

Type species: Sporormyces septenarius (S.I. Ahmed & Cain) Guerra-Mateo, Gené & Cano

Habitat: Dung, soil, and plant endophytes.

Notes: Sporormyces represents a lineage sister to Sporormiella (Fig. 3). The morphological features of Sporormyces overlap with those displayed by different species of Sporormiella. Despite this, these genera can be distinguished through phylogeny (Figs 2, 3) and metabolite production, with Sporormyces producing anthraquinones and 4-methoxy-6-pentyl-2H-pyran-2-one, compounds that have not been detected across species of Sporormiella (González-Menéndez et al. 2017). Unfortunately, the second species included in Sporormyces remained sterile.

Sporormyces anthraquinoniformans Guerra-Mateo, González-Menéndez & Cano, sp. nov. MB 862045.

Etymology: Referring to the production of 1,7-dihydroxy-3-(hydroxymethyl) anthraquinone.

Typus: Spain, Andalusia, Granada, Escúzar quarry, from gypsum soil associated with Squamarina lentigera, Nov. 2016, V. González-Menéndez (holotype FMR H-20558, ex-type (CF-295351 = FMR 20558).

Culture sterile. Sporormyces anthraquinoniformans differs from its closest phylogenetic species Sporormyces septenarius (specimen: S: 00036) by unique fixed alleles in two of the loci studied. ITS, 31 positions: 45 (gap), 54 (G), 56 (C), 83 (A), 89 (C), 90 (A), 95 (A), 100–103 (gap), 105 (T), 106 (T), 108 (T), 112 (C), 116 (gap), 131 (A), 139 (C), 141 (A), 306 (T), 335 (C), 345 (T), 351 (C), 352 (G), 356 (G), 358 (T), 426 (G), 428 (G), 433 (T), 434 (T), 439 (T). LSU, 12 positions: 96 (C), 101 (G), 166 (C), 171 (G), 382 (A), 387 (T), 390 (C), 394 (A), 401 (T), 430 (C), 431 (C), 433 (A).

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 42–60 mm diam., crateriform, radially sulcate, velvety, white (1A1), margin fimbriate, slightly lobated; reverse concolourous. On OA, 50–65 mm diam., flat, velvety, white (1A1), margin submerged, entire, slightly lobated; reverse colourless. On CMA, 38–50 mm diam., flat, velvety, white (1A1), margin submerged, entire, slightly lobated; reverse colourless.

Additional materials examined: Spain, Andalusia, Granada, Alhendin business park, from Dittrichia viscosa, Jul. 2012, V. González-Menéndez (CF-282341); ibid., Escúzar quarry, from gypsum soil associated with Squamarina lentigera, Nov. 2016, V. González-Menéndez (CF-295464 = FMR 20557).

Cardinal temperatures for growth: Minimum 5 °C (6–8 mm), optimum 25 °C (42–60 mm), maximum 35 °C (1 mm).

Secondary metabolites: 4-Methoxy-6-pentyl-2H-pyran-2-one, 1,7-dihydroxy-3-(hydroxymethyl) anthraquinone, SPF 32629B.

Notes: The three strains representing Sc. anthraquinoniformans were crossed in vitro on different media, but cultures remainded sterile. This species can be distinguished through phylogenetic analyses and is characterized by the production of 4-methoxy-6-pentyl-2H-pyran-2-one (Figs. 2, 3). The ex-type strain of the species, CF-282341, was also reported as a producer of SPF 32629B and 7-dihydroxy-3-(hydroxymethyl) anthraquinone (González-Menéndez et al. 2017). Future studies should address the morphological description of the new fungus.

Sporormyces septenarius (S.I. Ahmed & Cain) Guerra-Mateo, Gené & Cano, comb. nov. MB 862073. Fig. 8.

Fig. 8.

Sporormyces septenarius (S: 00036). A. Fungarium label and collection data. B. Preserved specimen. C. Perithecia. D. Peridium. E. Asci and paraphyses. F. Ascus. G, H. Ascospores at the ascus bottom. I. Released ascospore. Scale bars: D, G–I = 10 μm; E, F = 20 μm.

Basionym: Sporormiella septenaria S.I. Ahmed & Cain, Canad. J. Bot. 50: 458. 1972.

Typus: Mexico, Villa Hidalgo, on dung of Capra hircus, Aug. 1960, R.F. Cain (holotype TRTC 36550).

Description adapted from Ahmed & Cain (1972) and the exsiccatal specimen of S: 00036. Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 1.5–2 µm wide hyphae. Sexual morph: Ascomata perithecial, immersed to semi-immersed, solitary or aggregated in small groups, subglobose to nearly pyriform, glabrous, 270–310 × 225–250 µm; peridium thin, membranaceous, dark brown to nearly black, with cells of textura angularis; neck small, papilliform. Paraphyses filiform, hyaline, longer than asci. Asci 8-spored, bitunicate, clavate, tapering into a short stipe, 130–160 × 18–20 µm. Ascospores bi- or tri-seriate above, uni- or bi-seriate below, subcylindrical, 43–54 × 8–10 µm, 7-celled, thick-walled, septa transverse, constrictions at septa moderate, segments separable, rounded at the ends, hyaline at first, ranging through yellowish brown to dark brown when mature; third cell from the upper end larger than the remaining cells, 7–8 × 8.5–10 µm; lower end cell longer than the other cells, 10–11.5 × 6.7–7 µm; germ slit oblique; gelatinous sheath hyaline. Asexual morph not observed.

Additional material examined: Argentina, Río Negro, Pilcomayo, on sheep dung, Feb. 1987, L. Espigores (S: 00036; proposed here as reference material).

Notes: Sequences of the Argentinian specimen, S: 00036, have been used to represent Sm. septenaria in previous phylogenentic analyses (Kruys & Wedin 2009, González-Menéndez et al. 2017). Notably, no living cultures of this species are available in any public collections. In our phylogeny, this specimen was resolved as phylogenetically distant from the genus Sporormiella (Fig. 3); in fact, it represents a novel genus as proposed here. We examined the exsiccate deposited in the Swedish Museum of Natural History, and confirmed that its morphology exactly matches the protologue, including shape and arrangement of the ascospore cells (Fig. 4). Although no culture exists for the specimen, DNA sequence data are available in GenBank (Supplementary material table S1). Therefore, sequence data together with the fungarium material of S: 00036 is designated as reference material for the species, and we introduce the new combination Sc. septenarius. Further collections from the type locality are needed for nomenclatural stabililization of the species.

Clade 3. Pleosporormiella Guerra-Mateo, Gené & Cano, gen. nov. MB 862049.

Etymology: Referring to the morphological resemblance of the sexual morph to the genus Sporormiella and the production of different asexual morphs.

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled hyphae. Sexual morph: Ascomata perithecial or cleistothecial, solitary or aggregated, immersed to semi-immersed, globose to subglobose; peridium with polyhedral to isodiametric cells (textura angularis and globosa) and slightly coriaceous; neck short, papilliform, to rostrate. Paraphyses filiform, sometimes branched, hyaline, and longer than asci. Asci 8-spored, bitunicate, subcylindrical to clavate, tapering into a short stipe. Ascospores bi- to tetra-seriate, subcylindrical, 4-celled, thick-walled, transversely septate, deeply constricted, cells subcylindrical to subglobose, at first hyaline, dark brown in maturity; germ slit parallel to slightly diagonal; gelatinous sheath hyaline, subglobose, surrounding each ascospore. Asexual morph: pycnidial, humicola-like or chrysosporium-like. Pycnidia solitary, immersed to erumpent, brown, globose, with a central ostiole; wall brown, with cells of textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, hyaline, smooth, guttulate, aggregated in mucous masses. Humicola-like conidiophores usually reduced to conidiogenous cells that laterally extend to form a single conidium, sessile or arising from a short lateral pedicel on the hyphae. Conidiogenous cells intercalary, lateral or terminal, integrated, monoblastic, cylindrical or slightly swollen as doliiform, hyaline. Conidia solitary, sometimes forming clusters, terminal or lateral, 1–2-celled, or multi-celled, olivaceous brown with hyaline to sub-hyaline basal cell, sometimes upper cell tightly coiled in three dimensions resembling dictyospores; conidial secession rhexolytic. Chrysosporium-like conidiophores reduced to conidiogenous cells arising laterally or on branched supporting cells. Conidiogenous cells holoblastic. Conidia solitary, clavate to obovate, smooth, hyaline, with a truncated base.

Type species: Pleosporormiella compacta Guerra-Mateo, González-Menéndez, Gené & Cano

Habitat: Primarily on soil and marine sediments, but also on dung, on dead leaves, or as endophytes.

Notes: Pleosporormiella represents a well-supported, independent lineage in Sporormiaceae (Fig. 3). This genus is characterized by short-stipitate asci with 4-celled ascospores, and the production of three different asexual morphs, pycnidia, humicola-like and chrysosporium-like. Pycnidia resemble those produced by other Preussia s.l. lineages, but differ on longer conidiogenous cells (6–10 µm vs 4–5 µm in Sporormiella). The chrysosporium-like morph has only been observed in Preussia s.str. (See below), displaying similar subglobose to obovoid conidia (Asgari & Zare 2010, Phurbu et al. 2025). The humicola-like morph is reported here for the first time in association with the family Sporormiaceae. Our phylogeny reveals that Pleosporormiella comprises seven species.

The strain UPS: 305 “Preussia megalospora” formed an additional independent lineage that probably represents another species of Pleosporormiella. However, we could not examine this strain.

Pleosporormiella aculeata Guerra-Mateo, Gené & Cano, sp. nov. MB 862051. Fig. 9.

Fig. 9.

Pleosporormiella aculeata (ex-type CBS 217.38). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B, C. Conidiophore disposition. D. Sessile conidia. E, F. Conidia with spiky mucoid sheath. Scale bars = 10 μm.

Etymology: Latin “aculeus”, spine, referring to the spiny gelatinous sheath covering the conidia.

Typus: Netherlands, Rotterdam, from soil, Jan. 1938, N.V. Unilever (holotype CBS H-25952, ex-type strain CBS 217.38 = FMR 20561).

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 2–4.5 µm wide hyphae. Sexual morph not observed. Asexual morph humicola-like. Conidiophores often reduced to conidiogenous cells that form lateral sessile conidia or arising from a short lateral pedicel on the hyphae, hyaline; sometimes slightly differentiated conidiophores present as branched supporting conidial cells. Conidiogenous cells intercalary, lateral or terminal, integrated, monoblastic, cylindrical or slightly swollen as doliiform, hyaline, smooth. Conidia solitary, 1–2-celled, 12–18 × 6–10 μm, hyaline at first, then upper cell dark brown and basal cell hyaline to subhyaline; upper cell, globose to subglobose, 8–11 × 6–10 μm, thick-walled, smooth, with gelatinous sheath forming mucilaginous spikes in mature conidia, hyaline; basal cell cylindrical or slightly swollen as doliiform, 4.5–7 × 3–6.5 μm, often remaining attached to the supporting cells.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 80–85 mm diam., slightly raised, velvety, medium grey (1E1) with violet brown (10E8) patches to white (1A1) at periphery, margin diffusing in the medium; reverse dark grey (1F1) to white (1A1). On OA, 80–85 mm diam., flat, velvety to glabrous, colourless, margin diffusing in medium; reverse colourless. On CMA, 85 mm diam., flat, velvety, greyish white (1B1) to colourless, margin diffusing in medium; reverse colourless.

Cardinal temperatures for growth: Minimum 5 °C (2 mm), optimum 30 °C (78 mm), maximum 37 °C (12–14 mm).

Secondary metabolite: Dihydroisoflavipucin.

Notes: The strain representing Ps. aculeata was previously labelled as Humicola brunnea var. brunnea based on the morphology of the conidia. Previous phylogenetic analyses determined that an additional variety of this species, Humicola brunnea var. africana, was related to Lophiotremataceae; while the genus Humicola s.str. represented a member of Chaetomiaceae (Wang et al. 2019). Pleosporormiella aculeata represents another case of misidentification based on the conserved and cryptic morphological features displayed by these fungi. Our phylogenetic analyses determined Ps. aculeata as a novel species in Pleosporormiella (Figs 2, 3). This genus is characterized by the production of pycnidia, chrysosporium-like and humicola-like asexual morphs. Another species in the genus with a similar humicola-like morph is Ps. polyspora. However, it produces tightly coiled conidia without sheath (Fig. 11), while Ps. aculeata exhibits subglobose conidia covered by a smooth or spiny gelatinous sheath (Fig. 9).

Fig. 11.

Pleosporormiella polyspora (A, C–F. Ex-type FMR 19842. G, H. FMR 20838). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B. Colony on OA after 14 d at 25 °C. C, D. Pycnidia. E. Pycnidial conidiogenous cells. F. Pycnidial conidia. G, H. Humicola-like conidia disposition. Scale bars: E, F, H = 10 μm; G = 20 μm.

Pleosporormiella citrullina (R.M.F. Silva et al.) Guerra-Mateo, Gené & Cano, comb. nov. MB 862074.

Basionym: Preussia citrullina R.M.F. Silva et al., Persoonia 40: 313. 2018.

Typus: Brazil, Petrolândia municipality, Pernambuco state, as endophyte from leaves of Citrullus lanatus (Cucurbitaceae), Jul. 2016, R.M.F. Silva (holotype URM 91190, ex-type strain URM 7865).

Description and illustration: Crous et al. (2018).

Notes: Pleosporormiella citrullina, formerly known as Pr. citrullina, is only known to produce a pycnidial asexual morph (Crous et al. 2018). Although morphological features among pycnidia are conserved, Ps. citrullina can be distinguished through phylogenetic analyses, forming an independent and distant lineage in the genus (Fig. 3).

Pleosporormiella compacta Guerra-Mateo, González-Menéndez, Gené & Cano, sp. nov. MB 862052. Fig. 10.

Fig. 10.

Pleosporormiella compacta (ex-type FMR 20161). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B. Perithecium. C. Peridium with textura angularis. D. Peridial wall layers. E. Paraphyses. F. Straight and curved asci. G. Ascospores. H. Pycnidia. I. Pycnidial peridium with textura angularis. J. Conidiogenous cells and conidia. Scale bars = 10 μm.

Etymology: Referring to the compacted disposition of the ascospores inside the asci.

Typus: Spain, Catalonia, Mediterranean coast, Tarragona, Platja de l’Arrabassada, from sediments at 20 m depth, Jun. 2022, G. Quiroga-Jofre & D. Guerra-Mateo (holotype CBS H-25953, ex-type strains FMR 20161 = CF-308079).

Mycelium immersed and superficial, composed of hyaline, septate, branched, smooth-walled, 1.5–2 µm wide hyphae. Sexual morph: Ascomata perithecial, solitary or aggregated, immersed to semi-immersed, subglobose, 270–500 × 230–360 μm, black, ostiole 55–80 μm diam.; peridium 5–8-layered, brown, with cells of textura angularis, 5–6 × 4–5 μm. Paraphyses filiform, sometimes branched, longer than asci, 1–1.5 μm wide, hyaline, smooth. Asci 8-spored, bitunicate, subcylindrical, often curved, 80–110 × 7–9 μm, with short stipe, 10 μm in length. Ascospores biseriate and compacted within asci, cylindrical, straight or slightly curved, 4-celled, transversely septate, septa constricted, broad and shallow, 36–42(–45) × 3.5–5 μm, hyaline at first then pale green, dark brown in maturity, thick- and smooth-walled; middle cells barrel-shaped with obtuse angles; terminal cells tapering apically in obtuse angles, generally longer than middle cells (10–13.5 vs 7–10 μm in length); germ slit parallel to slightly diagonal; gelatinous sheath hyaline. Coelomycetous asexual morph: Conidiomata pycnidial, solitary, immersed to erumpent, brown, globose, 90–150 μm diam., ostiolate, central ostiole 20–28 μm diam.; wall of 3–6 layers, brown, with cells of textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, 6–8 × 2.5–3.5 μm, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, 2.5–4.5 × 2 μm, hyaline, smooth, 1-guttulate, aggregated in pink mucous masses.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 16–28 mm diam., umbonate, velvety, pastel red (9A4) to yellowish white (4A2) and white (1A1) at periphery, margin entire, slightly lobated; reverse pinkish white (9A2) to white (1A1); in CF-277965 and CF-277801 radially sulcate, funiculate at centre to glabrous towards periphery, greyish orange (5B4) to brownish grey (5C2), white (1A1) at periphery and reverse orange grey (5B2). On OA, 23–30 mm diam., flat, aerial mycelium scarce, arranged in white threads of long aerial mycelium, colourless to pinkish white (9A2) over time, margin submerged, entire and slightly lobated; reverse concolourous. On CMA, 18–27 mm diam., flat, aerial mycelium scarce, arranged in long white hyphae, pinkish white (13A1) or yellowish grey (4B2), margin submerged, entire; reverse concolourous.

Additional materials examined: Spain, Catalonia, Mediterranean coast, Tarragona, Platja del Miracle, from sediments at 27 m depth, Jun. 2021, G. Quiroga-Jofre & D. Guerra-Mateo (FMR 19618); ibid., Andalusia, Granada, Fuente Vaqueros, agricultural soil, Oct. 2010, J. Siles (CF-277801, CF-277965).

Cardinal temperatures for growth: Minimum 5 °C (4 mm), optimum 25 °C (16–28 mm), maximum 30 °C (4–10 mm).

Secondary metabolites: 4-Hydroxy-5-methylmellein, C₁₉H₂₈O₄, C₄₃H₇₉NO₉, C₁₉H₂₈O₅.

Notes: Pleosporormiella compacta represents a phylogenetic independent lineage that produces both a perithecial sexual morph and a pycnidial asexual morph. The sexual morph resembles those of other species with 4-celled, cylindrical ascospores distributed among Preussia and Sporormiella, like Pr. constricta, Sm. capybarae, Sm. cylindrospora, Sm. ovina and Sm. teretispora. Although the taxonomic position of these species remains obscure due to the lack of genetic barcodes for phylogenetic comparison, they differ in ascospore cell morphology. The above-mentioned species display ascospores with round-edge cells, while Ps. compacta is characterised by ascospores with barrel-shaped middle cells and terminal cells tapering apically in obtuse angles (Ahmed & Cain 1972, Guarro et al. 1997a). The morphology of the ascospore cells also distinguishes Ps. compacta from other species of Pleosporormiella that produce ascospores with round-edge cells, Ps. polymorpha and Ps. terricola (Cain 1961, Asgari & Zare 2010). In addition, these species differ in ascospore size, with Ps. polymorpha displaying bigger ascospores [45–50(–58) × 10.5–12.5 µm], followed by Ps. compacta [36–42(–45) × 3.5–5 μm], and Ps. terricola with the smallest ascospores (26–32 × 5–5.5 μm). For the remaining species of Pleosporormiella only the asexual morph is known; i.e., Ps. citrullina, Ps. compacta, and Ps. suttonii produce pycnidial conidiomata with subtle morphological differences, while Ps. aculeata and Ps. polyspora display humicola-like morphs, and Ps. polymorpha was described as chrysosporium-like (Asgari & Zare 2010, Crous et al. 2018). In addition, Ps. compacta differs metabolically from other species of the genus by the production of C₁₉H₂₈O₄, C₄₃H₇₉NO₉, and C₁₉H₂₈O₅ (Table 3).

It is possible that Ps. compacta has also been recovered from dung under the provisional name “Sporormiella catenatispora” (Wergen 2025). This identification is based on the characteristic compacted disposition of the ascospores inside asci.

Pleosporormiella polymorpha (Asgari & Zare) Guerra-Mateo, Gené & Cano, comb. nov. MB 862075.

Basionym: Preussia polymorpha Asgari & Zare, Nova Hedwigia 90(3–4): 539. 2010.

Typus: Iran, East Azerbaijan Province, Osku, from dead barley leaves, Mar. 2003, B. Asgari (holotype IRAN 12398 F, ex-type strains CBS 117679 = IRAN 845 C).

Description and illustration: Asgari & Zare (2010).

Secondary metabolite: Brefeldin.

Notes: We introduce Ps. polymorpha, formerly known as Pr. polymorpha, based on phylogenetic analyses (Figs 2, 3). This species is characterized by asci variable in shape and producing a chrysosporium-like morph (Asgari & Zare 2010). For a detailed comparison with other Pleosporormiella species, see notes section under Ps. compacta.

Pleosporormiella polyspora Guerra-Mateo, González-Menéndez, Gené & Cano, sp. nov. MB 862053. Fig. 11.

Etymology: Referring to the production of different asexual morphs.

Typus: Spain, Catalonia, Mediterranean coast, Tarragona, Platja del Miracle, from sediments at 20 m depth, Jun. 2021, G. Quiroga-Jofre & D. Guerra-Mateo (holotype CBS H-25954, ex-type strains FMR 19842 = CF-308080).

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 2–3 µm wide hyphae. Sexual morph not observed. Asexual morph pycnidial or humicola-like. Pycnidia solitary or aggregated, immersed to erumpent, globose, 120–200 μm diam., ostiolate, central ostiole, 9–18 μm diam; wall of 3–6 layers, brown, with cells of textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, 8 × 3.5 μm, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, 2.5–4.5 × 1.5–2 μm, hyaline, smooth, 2-guttulate, aggregated in white mucous masses. Humicola-like conidiophores reduced to conidiogenous cells forming lateral sessile conidia or arising as short lateral pedicels on hyphae, hyaline, smooth. Conidiogenous cells intercalary, lateral or terminal, monoblastic, cylindrical or slightly swollen as doliiform, 5–8 × 2.5–3 μm, hyaline. Conidia solitary, sometimes two in chain or several in cluster, usually 1–2-celled, sometimes multi-celled, 10–13.5(–15.5) × 9–11 μm, hyaline at first, then olivaceous brown with hyaline or subhyaline basal cell, smooth; upper cell globose to subglobose, very tightly coiled in three dimensions resembling dictyospores, 9–11.5 × 9–11 μm, thick- and smooth-walled; basal cell when present cylindrical or slightly swollen as doliiform, 3.5–7.5 × 3.5–6 μm, thin-walled; conidial secession rhexolytic.

Culture characteristics (after 14 d at 25 °C): On PDA, reaching 22–44 mm diam., crateriform, radially sulcate, velvety, pale violet (15A3) to yellowish white (4A2), margin entire, slightly lobated; reverse olive brown (4B8) to yellowish grey (4B2); white (1A1) in FMR 19842, CF-287459, and greyish yellow (4B3) in FMR 20838. On OA, reaching 40–60 mm diam., flat, velvety, pink (12A4) to colourless towards periphery, margin submerged, entire and lobated; reverse concolourous. On CMA 34–50 mm diam., flat, velvety in patches, pink (12A4) to colourless towards periphery, margin submerged, entire and lobated; reverse concolourous. FMR 20838 with abundant pale grey (1B1) aerial sporulation in the two latter culture media.

Additional materials examined: Spain, Andalusia, Almería, Tabernas, associated with Launaea arborescens, May 2014, V. González-Menéndez (CF-287459); ibid., Catalonia, Mediterranean coast, discharging area of the Ebro River, from sediments at 13 m of depth, Oct. 2022, Q. Roca & D. Guerra-Mateo (FMR 20458); ibid., from sediments at 27 m depth, May 2023, Q. Roca & D. Guerra-Mateo (FMR 20838).

Cardinal temperatures for growth: Minimum 15 °C (2 mm), optimum 30 °C (35–38 mm), maximum 18–20 °C (40 mm).

Secondary metabolites: Asterric acid, bisdechlorodihydrogeodin (C₁₇H₁₆O₇), brefeldin, dihydroisoflavipucin, monocerin, C₁₉H₂₈O₄, C₄₃H₇₉NO₉, C₁₉H₂₈O₅, C₁₆H₂₂O₇, C₂₁H₃₅NO, C₁₆H₁₂O₅.

Notes: Our phylogenetic analyses resolved FMR 19842, FMR 20458, FMR 20838, and CF-287459 as a single species (Fig. 2). Nevertheless, these strains exhibited distinct asexual morphs; i.e., pycnidia in FMR 20458 and 19842, humicola-like in FMR 20838 and CF-287459 remained sterile. Their phylogenetic divergence ranged from 7–9 % for rpb2, and less than 2–3 % for ITS, LSU, and tef1-α. Moreover, the speciation analysis based on the ITS-LSU regions determined these strains as a single evolutionary lineage (Supplementary material Fig. S12). The metabolite profile revealed this group is characterized by the production of brefeldin; additionally, FMR 20838 and FMR 20458 produced dihydroisoflavipucin, a molecule predominantly detected in strains with humicola- and monodictys-like strains (Fig. 3, Table 3). Altogether, the available phylogenetic evidence supports their recognition as a single species exhibiting intraspecific phenotypic variability.

Pycnidia have been observed in other species of Pleosporormiella, Ps. citrullina, Ps. compacta, and Ps. suttonii, but the morphological differences among their conidiomata are subtle. Humicola-like asexual morph has also been observed in Ps. aculeata, but Ps. polyspora produces tightly coiled conidia without a gelatinous sheath, while the conidia in Ps. aculeata are covered with a spiky gelatinous sheath (Fig. 9E, F).

We attempted to induce the sexual morph through in vitro crossings among the aforementioned strains, but no ascomata were observed.

Pleosporormiella suttonii Guerra-Mateo, Gené & Cano, sp. nov. MB 862054. Fig. 12.

Fig. 12.

Pleosporormiella suttonii (ex-type FMR 20475). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B, C. Pycnidia. D. Conidiogenous cells and conidia. Scale bars: C = 20 μm; D = 10 μm.

Etymology: Named in honour of Brian Charles Sutton for his enormous contributions to our knowledge of coelomycetous fungal diversity.

Typus: Spain, Catalonia, Mediterranean coast, discharging area of the Ebro River, 40°43’26” N, 0°54’35” E, from sediments at 6 m depth, Oct. 2022, Q. Roca & D. Guerra-Mateo (holotype CBS H-25955, ex-type strains FMR 20475 = CF-309230).

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 1.5–2 µm wide hyphae. Sexual morph not observed. Coelomycetous asexual morph: Conidiomata pycnidial, solitary, immersed to erumpent, globose, 100–120 μm diam., ostiolate, with central ostiole 10–20(–30) μm diam.; wall of 3–6 layers, with brown cells of textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, 8–10(–11) × 2.5–3.5 μm, hyaline, smooth-walled. Conidia aseptate, ellipsoid with obtuse ends, 3.5–4.5 × 2 μm, hyaline, smooth, 2-guttulate, aggregated in white mucous masses.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 12–15 mm diam., flat, cottony, white (1A1) to pale yellow (4A3), margin submerged irregular; reverse greyish yellow (4B5) to white (1A1). On OA, reaching 10–18 mm diam., flat, glabrous and colourless with velvety, dark grey (1F1) patches, margin submerged and entire; reverse dark grey (1F1) and colourless. On CMA, 20 mm diam., flat, velvety, greyish white (1B1), margin submerged, entire and lobated; reverse colourless.

Cardinal temperatures for growth: Minimum 15 °C (10 mm), optimum 25 °C (12–15 mm), maximum 30 °C (8 mm).

Secondary metabolites: C₂₁H₃₅NO, C₁₆H₂₃NO₃, C₁₇H₂₅NO₂.

Notes: Pleosporormiella suttonii represents a novel species that, in our phylogeny, formed an independent lineage sister to Ps. polymorpha (Fig. 3). These species differ in asexual morph morphology, with Ps. suttonii producing pycnidial conidiomata, while Ps. polymorpha displays a chrysosporium-like morph (Asgari & Zare 2010). Other species in the genus that display pycnidia are Ps. citrullina, Ps. compacta and Ps. polyspora. Pleosporormiella suttonii can be identified through phylogenetic analyses and by the production of C₁₇H₂₅NO_2.

Pleosporormiella terricola (Cain) Guerra-Mateo, Gené & Cano, comb. nov. MB 862076.

Basionym: Preussia terricola Cain, Canad. J. Bot. 39: 1641. 1961.

Typus: Honduras, Olnollita farm, from banana rhizosphere soil, Jul. 1958, R.D. Goos (holotype TRTC 36955, ex-type strain CBS 317.65).

Description and illustration: Cain (1961).

Additional material examined: Tanzania, Mount Kilimanjaro, North of Lyamungu at 2000 m altitude, on elephant dung, Aug. 1966, R.F. Cain, et al. (CBS 527.84).

Secondary metabolite: C₂₁H₃₅NO.

Notes: The ex-type strain of Pr. terricola from Honduras and the isolate from Tanzania formed an independent lineage in Pleosporormiella, representative of a single species (Figs 2, 3). This species is included in a well-supported clade together with Ps. citrullina, Ps. polymorpha and Ps. suttonii. Pleosporormiella terricola is only known through its sexual morph, which differs from that of Ps. polymorpha in ascospore size (26–32 × 5–5.5 μm vs 45–50 × 13–15 μm, respectively). On the contrary, Ps. citrullina and Ps. suttonii are only known by their pycnidial asexual morphs (Cain 1961, Asgari & Zare 2010, Crous et al. 2018).

Clade 4. Preussia Fuckel, Fungi Rhen. Exs., Suppl. Fasc. 3: no. 1750. 1866.

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled hyphae. Sexual morph: Ascomata cleistothecial, rarely perithecial, immersed to semi-immersed, solitary or aggregated in groups, globose to subglobose, glabrous, carbonaceous-membranaceous to coriaceous; peridium dark brown to black, textura angularis. Paraphyses filiform, septate, usually present in early stages of development but disappearing at maturity. Asci 8-spored, bitunicate, subglobose or broadly clavate with long, rarely short stipe. Ascospores arranged in a parallel fascicle or irregularly disposed in a subglobose mass, subcylindrical, 4-celled, septa transverse, deeply constricted, segments easily separable at maturity, at first hyaline, dark brown in maturity; germinal slit elongated, extending the full length of each cell; gelatinous sheath rarely present, hyaline, surrounding each ascospore. Asexual morph pycnidial or chrysosporium-like. Pycnidia solitary or aggregated, immersed to erumpent, globose to subglobose; wall brown, with cells of textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, hyaline, smooth, guttulate, aggregated in mucous masses. Chrysosporium-like conidiophores reduced to conidiogenous cells arising laterally or on branched supporting cells. Conidiogenous cells holoblastic. Conidia solitary, clavate to obovate, smooth, hyaline, with a truncated base.

Type species: Preussia funiculata (Preuss) Fuckel, Fungi Rhen. Exs., Suppl. Fasc. 3: no. 1750. 1866.

Habitat: Predominantly on soil and marine sediments, but also on dung.

Notes: We defined clade 4 as Preussia s.str. based on morphology and phylogeny. Our phylogenetic analyses determined this clade as a distant, independent, and well-supported lineage displaying long-stipitate asci, rarely short-stipitate, that produce 4-celled ascospores. These features match the original and later morphological circumscription of Preussia before considering Sporormiella a synonym (Fuckel 1867, Cain 1961). In addition, we emended the generic description to include pycnidia and chrysosporium-like asexual morphs in the genus (Figs 2, 3; von Arx & Storm 1967, Phurbu et al. 2025).

Although Preussia s.str. comprises reference and ex-type strains of several species, we recognise only three species (Figs 2, 3). See Pr. funiculata notes section for a detailed discussion on strain identification.

Preussia funiculata (Preuss) Fuckel, Fungi Rhen. Exs., Suppl. Fasc. 3: no. 1750. 1866. Fig. 13.

Fig. 13.

Preussia funiculata (ex-epitype CBS 127.66). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B. Cleistothecia. C. Cleistothecium under light microscopy. D. Peridium with textura angularis. E. Subglobose long-stipitate ascus. F. Sub-cylindrical short-stipitate ascus. G. Ascospores with oblique septa. H. Ascospores with straight septa. I. Pycnidia with peridium of textura angularis. J. Conidia. Scale bars: C = 25 μm; D–J = 10 μm.

Basionym: Perisporium funiculatum Preuss, Linnaea 24: 143. 1851.

Synonyms: Sporormia aemulans var. ostiolata Arx, Persoonia 4(4): 411. 1967, syn. nov.

Preussia cylindricalis F. Liu, S. Song & L. Cai, Mycology 16(2): 676. 2024 [2025], syn. nov.

Preussia flanaganii Boylan, Canad. J. Bot. 48(1): 163. 1970, syn. nov.

Typus: Germany, Hoyerswerda, on rotten branches of angiosperms, 1851, Preuss (holotype B 700132488); epitype designated here MBT 10031131: Germany, Kiel-Kitzeberg, from wheat field soil, Jan. 1966, W. Gams (epitype CBS H-25950, ex-epitype strain CBS 127.66).

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 1.5–2 µm wide hyphae. Sexual morph: Ascomata cleistothecial or perithecial, solitary or aggregated, immersed to semi-immersed, subglobose, slightly depressed from above, black, glabrous, 120–700 μm diam.; peridium carbonaceous-membranaceous to slightly coriaceous when young and fresh, becoming very fragile and brittle when dry, 5–8-layered, black, with cells of textura angularis, 3–10 μm diam. Paraphyses septate, filiform, 1–1.5 μm wide, hyaline, abundant in young ascomata and disappearing at maturity, with cylindrical or swollen cells, 3–8 μm diam. Asci 8-spored, subglobose or broadly clavate, 75–200 × 20–28 μm, with long stipe, 15–60(–130) μm in length. Ascospores overlapping, lying parallel with ascus, nearly cylindrical, straight or slightly curved, 4-celled, 22–38 × 5–7 μm, thick-walled, transverse to obliquely septate, deeply constricted, segments easily separable at maturity, hyaline at first, then yellow and finally very dark brown and opaque at maturity; middle cells subglobose, 6–8 × 6.5–7 μm, equal in size and shape, only slightly broader and slightly shorter than end-cells; terminal cells conical, 7–11 × 6 μm, narrowed toward the end and broadly rounded; germ slit parallel to slightly oblique; gelatinous sheath absent; ascospores rarely with fused cells. Coelomycetous asexual morph: pycnidial, solitary or aggregated, globose, 50–170 μm diam.; wall 3–6-layered, with cells of brown textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, 2.5–4 × 1.5–3.5 μm, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, 2–3 × 1.5 μm, hyaline, smooth, 1-guttulate, aggregated in pink mucous masses.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 20–26 mm diam., slightly umbonate, radially sulcate, velvety, white (1A1), margin entire and slightly lobated; reverse orange white (5A2); diffusible pigment orange white (5A2). On OA, attaining 45–50 mm diam., flat, with sparse, white aerial mycelium, and abundant dark grey sporulation (1F1), pink (12A4) to hyaline at margin, margin diffusing in medium; reverse concolourous. On CMA, reaching 35–50 mm diam., flat, glabrous, subtly pinkish white (12A2) to hyaline at margin, margin slightly lobated; reverse concolourous.

Cardinal temperatures for growth: Minimum 5 °C (2–4 mm), optimum 25 °C (20–26 mm), maximum 30 °C (2 mm).

Secondary metabolites: Cyperin, 7-chloro-6methoxymellein, C₂₃H₂₄O₇, C₁₄H₁₇NO₅.

Additional materials examined: Germany, Kiel-Kitzeberg, from wheat field soil, unknown collection date, K.H. Domsch (CBS 565.63 = CBS 126.66, received as Preussia fleischhakii); ibid., W. Gams (CBS 120.66, type of Sporormia aemulans var. ostiolata). Japan, Hokkaido, Otaru-shi, from deer dung, Aug. 1967, S. Udagawa (CBS 107.69, received as Preussia typharum). Mexico, southwest of Pueblo, from sandy soil in a dry irrigation ditch, Apr. 1967, unknown collector (CBS 112.73, isotype of Preussia flanaganii). Netherlands, Wageningen, from greenhouse soil, unknown collection date and collector (CBS 287.67, received as Preussia aemulans); ibid., from steamed soil from potato field, Oct. 1980, A. Cholil (CBS 318.81, received as Preussia aemulans); ibid., Groningen, from human nail, unknown collection date, A.E. Beute (CBS 361.49, received as Preussia fleischhakii). Spain, Granada, Fuente Vaqueros, from agricultural soil, Oct. 2010, J. Siles (CF-277822, received as Preussia funiculata). USA, Wyoming, 11 km west of Rock Springs, A1 horizon soil, sagebrush, shrubland, 1978, unknown collector (CBS 126548, received as Preussia funiculata).

Notes: Perisporium funiculatum was described from rotten branches of angiosperms collected in Hoyerswerda, Germany (Preuss 1851). Although Preuss did not indicate a type specimen, his fungarium was rediscovered in the Berlin herbarium (Jülich 1974). A single collection is preserved in this collection under the name Perisporium funiculatum (https://herbarium.bgbm.org/object/B700132488), which represents original material and can be interpreted as the holotype (Supplementary material Fig. S6). This species was later erected as the type of the genus Preussia, and Pr. funiculata has been represented in phylogenetic analyses with several strains (Figs 2, 3; Kruys & Wedin 2009, González-Menéndez et al. 2017, Sandberg et al. 2022, Noh et al. 2025). Among these, CBS 127.66 was recovered from angiosperm field soil in Kiel-Kitzeberg, Germany, and displayed morphological features typical of Pr. funiculata, such as subglobose, long-stipitate asci with 4-celled ascospores with oblique septa (Fig. 13). Therefore, to stabilize the nomenclature of the species and, ultimately, the genus Preussia, this strain is selected here as epitype based on its morphological resemblance to the protologue.

In accordance with previous works, our phylogenetic analyses clustered CBS 127.66 with representatives of Pr. aemulans, Pr. alloiomera, Pr. cylindricalis, Pr. flanaganii, and Pr. typharum (Figs 2, 3). The phylogenetic distance within this clade was around 3–5 % in rpb2, 1–3 % in tef1-α, and smaller than 1 % for ITS, LSU, and SSU. The speciation analysis of the ITS-LSU regions determined the clade as a single species (Supplementary material Fig. S13). This analysis included the ex-type strains of Sp. aemulans var. ostiolata, Pr. cylindricalis, and Pr. flanaganii; therefore, they are considered conspecific with Pr. funiculata. Although Pr. aemulans s.str., Pr. alloiomera, Pr. fleischhakii and Pr. typharum likely also represent Pr. funiculata, the lack of sequence data from their type specimens prevents the resolution of their taxonomy. Consequently, these taxa are regarded as species of uncertain status in the genus until further studies are conducted.

In this context, Pr. funiculata encompasses strains that are phylogenetically closely related, but exhibit morphological variability. The strain CBS 107.69, received as Pr. typharum, showed asci with long stipes and ascospores bearing transverse septa. CBS 361.49 was identified as Pr. fleischhakii, a species often confused with Pr. funiculata, and characterized by short-stipitate asci and ascospores with transverse septa (Cain 1961). Similarly, Pr. flanaganii was described as a species resembling Pr. fleischhakii in the mentioned characters (Boylan 1970). In addition, the ex-type strain of Pr. flanaganii (CBS 112.73) clustered with strains displaying Pr. funiculata morphology (Figs 2, 3). The strain S: 21345-p was identified as Pr. alloiomera by producing perithecia, short-stipitate asci and ascospores that exhibit oblique septa (Ahmed & Cain 1972, Kruys & Wedin 2009). The type of Sp. aemulans var. ostiolata (CBS 120.66) was characterized by forming perithecia with subcylindrical long-stipitate asci, ascospores exhibiting transverse septa, and pycnidial conidiomata as asexual morph (von Arx & Storm 1967). Moreover, the strain NMC 1013, which exclusively produces pycnidia, was recently described as Pr. cylindricalis (Phurbu et al. 2025). Therefore, based on our phylogenetic analyses, we have updated the morphological description of Pr. funiculata.

Finally, the metabolite profile on strains CF-277822 and CBS 318.81 revealed the production of cyperin, a molecule exclusively detected in this lineage (Table 3). It is noteworthy that the strain TTI-1095, previously identified as “Pr. typharum”, clustered within Pr. funiculata (Fig. 2). This strain was characterized by the production of leptosin C (Perlatti et al. 2021), a molecule that in the present work has not been detected.

Preussia gypsicola Guerra-Mateo, González-Menéndez, Cano & Gené, sp. nov. MB 862055. Fig. 14.

Fig. 14.

Preussia gypsicola (ex-type CF-295384). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B, C. Pycnidia. D. Conidiogenous cells and conidia. Scale bars: C = 20 μm; D = 10 μm.

Etymology: Referring to the substrate where the species has been reported, gypsum soil.

Typus: Spain, Andalusia, Granada, Escúzar quarry, from gypsum soil associated with Acarospora reagens, Nov. 2016, V. González-Menéndez (holotype CF-H-295384, ex-type strains CF-295384 = FMR 20555).

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 1.5–3 µm wide hyphae. Sexual morph not observed. Coelomycetous asexual morph: Conidiomata pycnidial, solitary, immersed to erumpent, globose to subglobose, 50–100 × 50–100 μm, ostiolate, with central ostiole, 10–35 μm diam.; wall of 2–3 layers, with cells of brown textura angularis. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, 4–5 × 2.5–3 μm, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, 3 × 1.5–2 μm, hyaline, smooth, 1-guttulate, aggregated in pale yellow mucous masses.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 23–26 mm diam., crateriform, radially sulcate, velvety, white (1A1), margin entire and lobated; reverse greyish red (7B3) to yellowish white (3A2). On OA, 30 mm diam., flat, velvety, pink (12A4) to colourless, margin submerged, entire and slightly lobated; reverse concolourous. On CMA, 26–28 mm diam., flat, velvety, pink (12A4) to colourless, margin submerged, entire and slightly lobated; reverse concolourous.

Cardinal temperatures for growth: Minimum 15 °C (9 mm), optimum 25 °C (23–26 mm), maximum 30 °C (18 mm).

Secondary metabolites: C₄₃H₇₉NO₉, C₁₆H₂₀O₆, C₁₆H₁₈O₅, C₁₉H₂₄O₈S.

Notes: Preussia gypsicola represents an independent lineage in the Preussia s.str. clade (Figs 2, 3). This species produced pycnidial conidiomata that are difficult to discern from those of Pr. funiculata, but differs from Pr. sedimenticola, which produces a chrysosporium-like asexual morph (Phurbu et al. 2025). These species can be distinguished through phylogenetic analyses, with Pr. gypsicola showing a 96–97 % ITS identity to both Pr. funiculata and Pr. sedimenticola.

Preussia sedimenticola F. Liu, S. Song & L. Cai, Mycology 16 (2): 678. 2025.

Typus: China, Xizang, Dagze Co lake (31°49’25.32” N, 87°28’16.68” E), from lake sediment, Aug. 2021, J.E. Huang (holotype HMAS 352881, ex-type strain CGMCC 3.27276).

Description and illustration: Phurbu et al. (2025).

Notes: This species is characterized by the production of a chrysosporium-like morph, and represents the first report of this asexual morphology in Preussia s.str. A similar asexual morph has been observed in Ps. polymorpha; however, Pr. sedimenticola differs in producing smaller conidia (2.5–4.5 μm vs 3–7.5 μm in length, respectively) (Asgari & Zare 2010, Phurbu et al. 2025).

Clade 5.

Notes: Clade 5 comprised strains identified as “Pr. dubia”, “Pr. irregularis”, “Pr. splendens”, and “Sm. muskokensis”, but lacked ex-type strains for these species (Fig. 3). Based on these identifications, the clade would be characterized by short, sometimes long-stipitate asci producing 4–8-celled ascospores (Ahmed & Cain 1972). The combination of these morphological features with the great phylogenetic distance to other Preussia s.l. lineages, suggest that clade 5 represents another novel genus. However, we could only examine two of their strains, CBS 111.77 and FMR 20357, and preferred to maintain this lineage without a formal name until additional strains can be examined.

According to our phylogeny (Figs 2, 3), these two strains are placed in different lineages. Strain FMR 20357, recovered from marine sediments of the Mediterranean Sea, remained sterile in culture but clustered with the strain S: 16568-f identified as “Sm. irregularis”. Interestingly, CBS 111.77 was received as “Sm. australis”, but clustered with S: 19562-G, a reference strain representative of “Pr. dubia” (Kruys & Wedin 2009). Both strains were recovered from horse dung in the Netherlands and Iceland, respectively. Preussia dubia was characterized by producing short-stipitate asci with 4-celled ascospores (Ahmed & Cain 1972). Strain CBS 111.77 produced a similar sexual morph in culture, but also a coelomycetous asexual morph characterized by subglobose pycnidial conidiomata and phialidic conidiogenous cells that produce conidia aggregated in white mucous masses.

Clade 6. Spororvela Guerra-Mateo, Cano & Gené, gen. nov. MB 862056.

Etymology: The name refers to the morphological resemblance to the genus Sporormiella and Latin velum (plural vela), veil, referring to the production of ascomata under a mycelial veil or weft.

Sexual morph: Ascomata perithecial, semi-immersed, covered by a mycelial weft, globose to subglobose, glabrous, coriaceous; peridium smooth, brown-black, thick-walled, with cells of textura angularis, sometimes with spikes of hyphae and occasional setae at the base appearing anchor hairs; neck long. Pseudoparaphyses filiform to moniliform, septate, hyaline, longer than the asci and mixed with them. Asci 8-spored, bitunicate, cylindrical-clavate, tapering into a long stipe. Ascospores fusiform, bi- to triseriate, fusiform, straight or slightly curved, primarily 8-celled, sometimes 7–12-celled, septa transverse, deeply constricted, segments variable in size and shape, end cells conical and rounded at the tip; at first hyaline, dark brown in maturity, thick-walled; germinal slit diagonal, extending the full length of each cell; gelatinous sheath hyaline, subglobose, surrounding each ascospore. Asexual morph not observed.

Type species: Spororvela tela (S. Jakob & M.J. Richardson) Guerra-Mateo, Cano & Gené

Habitat: Only known from dung.

Notes: We propose the novel genus Spororvela to accommodate Sm. tela, which represents a monotypic, independent lineage in Sporormiaceae (Fig. 3). Spororvela is characterized by ascomata covered by a mycelial weft and long-stipitate asci that produce up to 12-celled ascospores (Jakob & Richardson 2024).

Spororvela tela (S. Jakob & M.J. Richardson) Guerra-Mateo, Cano & Gené, comb. nov. MB 862077.

Basionym: Sporormiella tela S. Jakob & M.J. Richardson, N. E. Naturalist 31 (3, C1): 1. 2024.

Typus: USA, Van Cortlandt Park, New York, 40°53’24” N, 73°53’24” W, on dung of Branta canadensis (Canada Goose) in grassy field, Feb. 2023, S. Jakob (holotype NY 04449436, ex-type strain iNa152236789).

Description and illustration: Jakob et al. (2024).

Notes: Spororvela represents a monotypic genus phylogenetically related to clades 5 and 7 in Preussia s.l. (Figs 2, 3). The species Sv. tela is characterized by long-stipitate asci producing predominantly 8-celled, but also 7–13-celled ascospores, while the species included in clades 5 and 7 display short-, sometimes long-stipitate asci producing 4–7(–8)-celled ascospores (Ahmed & Cain 1972, Jakob & Richardson 2024).

Clade 7.

Notes: Clade 7 comprised strains identified as “Sm. affinis”, “Sm. heptamera”, “Sm. leporina”, “Sm. octomera”, and “Sm. vexans” (Figs 2, 3). Based on these identifications, the clade would be characterized by long-, sometimes short-stipitate asci producing primarily 7–8-celled ascospores (Ahmed & Cain 1972). Our phylogeny, together with these morphological features, suggests clade 7 as a novel genus in Sporormiaceae. However, since we could not examine any specimens, this clade remains unresolved pending further studies.

Additional species without molecular data, and displaying short- to long-stipitate asci producing 8-celled ascospores like Pr. alpina, Pr. octocylindrospora, Pr. octonalis, and Sm. corynespora could also represent members of this group (Ahmed & Cain 1972, Kruys 2015). However, this hypothesis needs to be tested through molecular analyses.

Clade 8. Chaetosporormiella Guerra-Mateo, Gené & Cano, gen. nov. MB 862057.

Etymology: Greek chaítē (χαίτη), long hair, referring to the production of hairy ascomata and the morphological resemblance to Sporormiella in microscopic features.

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled hyphae. Sexual morph: Ascomata perithecial, solitary or aggregated, immersed to semi-immersed, subglobose to pyriform, bare or hairy, dark brown to black; neck short or long, papilliform, conical or cylindrical, stout, bare or hairy, black; peridium membranaceous, covered by dark brown hairs. Paraphyses septate, filiform, occasionally branched, longer than the asci and mixed with them, hyaline. Asci 8-spored, cylindrical-clavate, tapering into a short stipe. Ascospores bi- or tri-seriate, 4-celled, fusiform-cylindrical, curved, septa transverse, constrictions at septa shallow to deep, dark brown, smooth-walled; cells almost equal in size, or terminal cells longer and narrower than the middle cells; germ slit parallel to diagonal; gelatinous sheath hyaline. Asexual morph: pycnidial or monodictys-like. Pycnidia solitary, immersed to erumpent, brown, globose, with a central ostiole; wall brown, with cells of textura angularis and hairs. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, hyaline, smooth, guttulate, aggregated in mucous masses. Monodictys-like conidiophores reduced to conidiogenous cells that laterally extend to form sessile or pedicillate conidia on hyphae. Conidiogenous cells lateral or terminal, integrated, cylindrical or slightly swollen as doliiform, monoblastic, hyaline. Conidia solitary, bi-cellular to muriform, black, globose to subglobose, rarely subcylindrical or pyriform, dark brown, thick-walled, conspicuously verrucose, basal cell paler and verrucose: secession rhexolytic.

Type species: Chaetosporormiella pilosella (Cain) Guerra-Mateo, Gené & Cano

Habitat: Predominantly as plant endophyte, but also on dung and soil.

Notes: Chaetosporormiella represents an independent lineage in Sporormiaceae characterized by hairy or bare ascomata, short-stipitate asci with 4-celled ascospores, and monodictys-like asexual morph. Our phylogeny determined four lineages within the genus representing different species (Figs 2, 3). However, the singleton lineage with the strain S: 16551-g identified as “Pr. longisporopsis”, and that with the strains S37 and CF-285370 identified as “Pr. grandispora”, remain unresolved because we were not able to examine these fungi in the present study.

Chaetosporormiella monodictyoides Guerra-Mateo, Gené & Cano, sp. nov. MB 862058. Fig. 15.

Fig. 15.

Chaetosporormiella monodictyoides (ex-type CBS 101.60). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B. Conidiophore disposition. C. Conidia in chains. D, E. Single-celled conidia. Scale bars: B, C = 10 μm; D, E = 5 μm.

Etymology: Referring to conidial morphology, which resembles the genus Monodictys.

Typus: Netherlands, from a pillow, Jun. 1960, unknown collector (holotype CBS H-25951, ex-type strain CBS 101.60 = FMR 20560).

Mycelium immersed and superficial, composed of hyaline, septate, branched, anastomosed, smooth-walled, 1.5–3 µm wide hyphae. Sexual morph not observed. Asexual morph monodictys-like. Conidiophores reduced to a hyphal cell that laterally extends to form a sessile conidium or arising as short lateral pedicels on the hyphae, sometimes branched, 10–50 × 1.5–2 μm, hyaline, finely warted. Conidiogenous cells lateral or terminal, integrated, monoblastic, cylindrical or slightly swollen as doliiform, 2–6 × 1.5–2 μm, hyaline, smooth-walled. Conidia solitary, bi-cellular to muriform, globose to subglobose, pyriform, rarely subcylindrical, 12–20(–50) × 12–16 μm, hyaline at first, then dark brown to black in mass, thick-walled, conspicuously verrucose, basal cell pale, thin-walled, smooth to verrucose; secession rhexolytic. Chlamydospores in chains, composed of aseptate to muriform cells, brown, thick-walled, and verrucose.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 22–30 mm diam., slightly elevated, woolly, growth in rings, orange white (5A2) with light grey (1C1) areas due to sporulation, margin entire; reverse dark orange (5A8) to light yellow (4A4); diffusible pigment yellowish white (4A2). On OA, 45–55 mm diam., flat, woolly, dark grey (1F1) to white (1A1), margin entire; reverse concolourous; diffusible pigment pale red. On CMA, 28 mm diam., flat, slightly floccose with rings of growth, dark grey (1F1) to white (1A1), margin entire; reverse colourless.

Additional material examined: Unknown origin, 1907, C.A.J.A. Oudemans (CBS 100.07).

Cardinal temperatures for growth: Minimum 5 °C (1 mm), optimum 20–25 °C (22–38 mm), maximum 30 °C (6 mm).

Secondary metabolites: Dihydroisoflavipucin, leptosins (C₃₀H₂₈N₆O₇S₄ and C₃₂H₃₄N₆O₇S₄), C₃₂H₄₂N₄O₁₀.

Notes: Chaetosporormiella monodictyoides was originally identified as Monodictys castaneae based on the morphological similarity to the protologue of this species (Wallroth 1833). However, several phylogenetic analyses revealed Monodictys as a polyphyletic genus, and many species have been transferred to different orders in Dothideomycetes, Sordariomycetes, and Leothiomycetes (Han et al. 2014, Tanaka et al. 2015, Réblová et al. 2020). Monodictys-like asexual morphs have been reported throughout the order Pleosporales, comprising genera in different families such as Alpestrisphaeria in Lophiostomataceae, Pleomonodictys in Pleomonodictydaceae, or Xenomonodictys in Sporormiaceae (Zhang et al. 2012b, Hernández-Restrepo et al. 2017, Yeh & Kirschner 2019). Chaetosporormiella monodictyoides resembles the conidia displayed by the species Ohleria brasiliensis (Ohleriaceae), Antealophiotrema brunneosporum (incertae sedis), and the genus Paradictyoarthrinium (Paradictyoarthriniaceae). These fungi display globose to pyriform dictyoconidia that differ in size and ornamentation. Antealophiotrema brunneosporum produces smooth and large conidia of up to 85 µm diam. (Hashimoto et al. 2017). Species of Paradictyoarthrinium and Cp. monodictyoides produce smooth to verrucose, medium sized conidia, around 20 µm in length (Htet et al. 2023), while O. brasiliensis is characterized by verruculose and small conidia of 15 µm diam. (Samuels 1980). In Sporormiaceae, the only species displaying monodictys-like conidia is Xenomonodictys iranica. Chaetosporormiella monodictyoides differs in conidial cell organization, showing three rows of cells, while X. iranica only produces two rows of cells (Crous et al. 2020a). In addition, these two species can be distinguished through phylogenetic analyses (Figs 2, 3).

Chaetosporormiella pilosella (Cain) Guerra-Mateo, Gené & Cano, comb. nov. MB 862078. Fig. 16.

Fig. 16.

Chaetosporormiella pilosella (CBS 396.81). A. Pycnidium. B. Pycnidial wall and hairs. C. Conidiogenous cells and conidia. Scale bars: A = 100 μm; B = 50 μm; C = 10 μm.

Basionym: Sporormia pilosella Cain, Stud. Sphaer. Ontario: 93. 1934.

Synonyms: Sporormiella pilosella (Cain) S.I. Ahmed & Cain, Canad. J. Bot. 50(3): 455. 1972.

Preussia pilosella (Cain) Guarro & Gené, Mycol. Res. 101(3): 308. 1997.

Typus: Canada, Ontario, Muskoka District, Stoneleigh, from porcupine dung (holotype TRTC 5316).

Description adapted from Ahmed & Cain (1972, under Sporormiella pilosella). Mycelium immersed and superficial, composed of septate, branched, anastomosed, hyaline, smooth-walled, 2–3.5 µm wide hyphae. Sexual morph: Ascomata perithecial, solitary or aggregated, immersed to semi-immersed, subglobose to pyriform, 450–550 × 300–450 μm, black; ostiolar beak short, papilliform or conical; peridium 5–8-layered, dark brown, covered with septate, thick-walled, flexuous, dark brown hairs. Paraphyses septate, filiform, occasionally branched, 2–3 μm wide, hyaline. Asci 8-spored, subcylindrical-clavate, (160–)190–230 × 17–20.5 μm, with short stipe, 20–30 μm in length. Ascospores 4-celled, fusiform-cylindrical, curved, (29–)32–37 × 8–9 μm, septa transverse, constrictions at septa moderately shallow, hyaline at first, dark brown and thick-walled in maturity; terminal cells ovoid-conical, comparatively longer and narrower than the middle cells; germ slit diagonal; gelatinous sheath hyaline. Coelomycetous asexual morph: Conidiomata pycnidial, solitary, immersed to erumpent, brown, globose, 170–200 μm diam., ostiolate, central ostiole 23–25 μm diam.; wall of 3–6 layers, brown, with cells of textura angularis, covered by septate, erect, thick-walled setae, 150–180 × 3–4 μm, brown, smooth. Conidiophores reduced to conidiogenous cells lining the inner cavity, phialidic, ampulliform, 7.5–12 × 2.5–3 μm, hyaline, smooth. Conidia aseptate, ellipsoid with obtuse ends, 2.5–3 × 1.5–2.5 μm, hyaline, smooth, 1-guttulate, aggregated in white mucous masses.

Additional descriptions and illustrations: Ahmed & Cain (1972).

Additional materials examined: Spain, Murcia, Sierra Espuña, from soil, Sep. 2002, D. Garcia (CBS 113807; proposed here as reference strain). Switzerland, Montorge, from needles of Juniperus communis, 1978, O. Petrini (CBS 396.81, received as Sporormiella cymatomera).

Secondary metabolite: C₂₂H₂₆O₅.

Notes: In our phylogeny, Cp. pilosella is represented by two strains, CBS 113807 and CBS 396.81 (Fig. 3). Strain CBS 113807, identified as Pr. pilosella, exhibits the typical features described in the protologue of the species, namely hairy ascomata with short-stipitate asci, producing 4-celled ascospores (Ahmed & Cain 1972). Given the absence of ex-type cultures and the good preservation of the holotype in the Royal Ontario Museum (TRTC 5316), we select this strain (CBS 113807) as the reference strain for the species and typified Chaetosporormiella with Cp. pilosella. Nevertheless, cultures of the other strain, CBS 396.81, produced bare ascomata and hairy pycnidia, suggesting that Cp. pilosella can actually produce both hairy and bare sexual morphs, together with a hairy coelomycetous asexual morph. These features have been incorporated into the concept of the new genus.

Chaetosporormiella pilosella differs from other species with hairy ascomata by showing longer asci (190–230 × 17–20.5 μm vs 130–174 × 16–19 μm in Pr. aquilirostrata), and smaller ascospores (32–37 × 8–9 μm vs 62–76 × 10–12 μm in Pr. lasiocarpa) (Lorenzo 1994, Guarro et al. 1997a). Moreover, hairy pycnidia in Sporormiaceae have only been observed in Trichophoma cylindrospora. However, the conidia of this latter species are cylindrical and considerably larger (18–20 × 2–3 μm; Crous et al. 2020b) than those produced by Cp. pilosella.

Clade 9. Spororminula Arx & Aa, Trans. Brit. Mycol. Soc. 89(6): 117. 1987.

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled hyphae. Sexual morph: Ascomata perithecial, immersed to semi-immersed, solitary or aggregated in small groups, globose to subglobose, glabrous, carbonaceous-membranaceous to coriaceous; peridium smooth, dark brown to black, with cells of textura angularis; neck papilliform. Paraphyses filiform, septate, hyaline, longer than the asci and mixed with them. Asci 8-spored, bitunicate, subcylindrical, tapering into a short stipe. Ascospores biseriate, 8-celled, subcylindrical, curved, septa transverse, deeply constricted, cells easily separable at maturity, at first hyaline, brown in maturity, thick- and smooth-walled; germinal slit diagonal; gelatinous sheath absent. Coelomycetous asexual morph: Conidiomata pycnidial, solitary or aggregated, globose to subglobose or irregularly shaped; peridium dark brown, with cells of textura angularis. Conidiogenous cells lining the inner cavity, phialidic, hyaline, thin- and smooth-walled. Conidia aseptate, ellipsoid or cylindrical, with obtuse ends, hyaline, smooth, guttulate, aggregated in mucous masses.

Type species: Spororminula subticinensis (Mouton) Guerra-Mateo, Cano & Gené

Habitat: On dung, soil, and marine sediments.

Notes: The genus Spororminula was established to accommodate the species Sn. tenerifae based on morphological features (von Arx & van der Aa 1987). Although this species was later transferred to Preussia (Kruys & Wedin 2009), our phylogenetic analyses resolved this lineage as an independent clade (Figs 2, 3). Therefore, we consider Spororminula as a valid genus and accept two species, Sn. irregularis and Sn. subticinensis. The latter species comprises Sn. tenerifae as a synonym; for a detailed discussion, see the species notes section.

Additional Preussia s.l. species exhibiting short-stipitate asci with 8-celled ascospores could be representatives of this genus; i.e., Pr. octocylindrospora, Sm. insignis and Sm. platymera (Kruys 2015). However, these species lack molecular data, and this hypothesis needs to be evaluated in future works.

Spororminula irregularis Guerra-Mateo, Cano & Gené, sp. nov. MB 862059. Fig. 17.

Fig. 17.

Spororminula irregularis (ex-type CF-295384). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B, C. Pycnidia. D. Conidiogenous cells and conidia. Scale bars: C = 25 μm; D = 10 μm.

Etymology: Referring to the irregular shape of the pycnidial conidiomata.

Typus: Spain, Catalonia, Mediterranean coast, Tarragona, Platja del Miracle, 41°6’19” N, 1°15’37” E, from sediments at 20 m depth, Jun. 2021, G. Quiroga-Jofre & D. Guerra-Mateo (holotype CBS H-25956, ex-type strains FMR 19850 = CF-308081).

Mycelium immersed and superficial, composed of septate, branched, hyaline, smooth-walled, 1.5–2.5 µm wide hyphae. Sexual morph not observed. Coelomycetous asexual morph: Conidiomata pycnidial, solitary or aggregated, immersed to erumpent, dark brown, globose to subglobose or irregular in shape, 45–150 × 45–100 μm, ostiolate, ostiole 18–23 μm diam.; wall of 2–3 layers, with brown cells of textura angularis. Conidiogenous cells phialidic, cylindrical, 13 × 3.5 μm, hyaline, thin- and smooth-walled. Conidia aseptate, cylindrical with obtuse ends, 3.5–4.5 × 2 μm, hyaline, smooth, 2-guttulate, aggregated in white mucous masses.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 26–30 mm diam., slightly crateriform, radially sulcate, velvety, white (12A1) with pinkish white (9A2) areas, margin entire; reverse reddish grey (9B3) to yellowish white (4A2). On OA, 38–49 mm diam., flat, filamentous, white (1B1), with abundant pycnidia, margin submerged, entire; reverse pale grey (1B1) to colourless; diffusible pigment pale red in old cultures. On CMA, 42–45 mm diam., flat, filamentous, pale grey (1B1) to white (1A1), margin submerged, entire; reverse colourless.

Cardinal temperatures for growth: Minimum 5 °C (4 mm), optimum 25 °C (26–30 mm), maximum 40 °C (4 mm).

Secondary metabolites: Antibiotic LL-N 313ε, monocerin, palmarumycins, 4-hydroxy-5-methylmellein, 7-chloro-6methoxymellein, C₁₁H₁₀ClO₅, C₂₀H₁₄O₃.

Notes: Spororminula irregularis represents the second species described in the genus (Fig. 3), but unlike Sn. subticinensis, it only produces a pycnidial asexual morph in culture. This differs from the pycnida produced by Sn. subticinensis in showing slightly longer conidiogenous cells (13 µm vs 8.5 µm in length) and conidia (3.5–4.5 µm vs 2.5–4 µm in length). In addition, Sn. irregularis is particularly characterized by the production of monocerin (Table 3).

Spororminula subticinensis (Mouton) Guerra-Mateo, Cano & Gené, comb. nov. MB 862079. Fig. 18.

Fig. 18.

Spororminula subticinensis (FMR 20474). A. Colonies on PDA, OA, and CMA after 14 d at 25 °C. B. Perithecium. C. Peridium with textura angularis. D. Asci and paraphyses. E. Ascospores. F, G. Pycnidia. H. Conidiogenous cells and conidia. Scale bars = 10 μm.

Basionym: Sporormia subticinensis Mouton, Bull. Soc. Roy. Bot. Belgique 36: 14. 1897.

Synonyms: Preussia subticinensis (Mouton) Arx, Proc. Kon. Ned. Akad. Wetensch., C 76(3): 294. 1973.

Sporormiella subticinensis (Mouton) Dugan & R.G. Roberts, Mycologia 87(5): 717. 1995.

Sporormia perplexans Nicot & Rouch, Compt. Rend. Hebd. Séances Acad. Sci., Sér. D 262: 1694. 1966.

Spororminula tenerifae Arx & Aa, Trans. Brit. Mycol. Soc. 89(6): 117. 1987.

Preussia tenerifae (Arx & Aa) Kruys, Syst. Biodiv. 7(4): 476. 2009, syn. nov.

Typus: Belgium, Beaufays (Vierset), near Liège, from wood, 1897, V. Mouton 427 (holotype BR 5020105315705).

Description adapted from Mouton (1897, under Sporormia subticinensis). Mycelium immersed and superficial, composed of septate, branched, anastomosed, hyaline, smooth-walled, 2–3.5 µm wide hyphae. Sexual morph: Ascomata perithecial, solitary or aggregated, immersed to semi-immersed, pyriform, 260–400 μm, black; ostiolar beak 120–160 × 200–250 μm; peridium 5–8-layered, smooth, dark brown, with cells of textura angularis, 2.5–5 μm, sometimes covered with some pigmented hyphae. Paraphyses septate, filiform, 2 μm wide, hyaline. Asci 8-spored, subcylindrical-clavate, 130–160 × 20–30 μm (150–220 × 30–40 μm in CBS 354.86), with short stipe, 12–19 μm in length. Ascospores 8-celled, subcylindrical, curved, 60–70 × 10 μm (72–108 × 14–19 μm in CBS 354.86), septa transverse, constrictions at septa deep, easily separating into single cells, hyaline at first, brown and thick-walled in maturity; central cells rectangular in vertical section, triangular in transverse section, 10 × 8 μm; apical cells longer than broad, 12 × 9 μm (15–18 × 12–25 μm in CBS 354.86); germ slit diagonal, faint, slightly sinuous; gelatinous sheath absent; sometimes cells remain aseptate, reaching up to 20 μm in length. Coelomycetous asexual morph: Conidiomata pycnidial, solitary, immersed to erumpent, brown, subglobose, 150 μm diam., with central ostiole, 10–15 μm diam.; wall of 3–6 layers, with cells of brown textura angularis. Conidiogenous cells phialidic, cylindrical, 8.5 × 3 μm, hyaline, smooth. Conidia aseptate, subglobose, 2.5–4 × 2 μm, hyaline, smooth, guttulate, aggregated in white mucous masses.

Culture characteristics (after 14 d at 25 °C): Colonies on PDA reaching 28 mm diam., crateriform, velvety, light grey (1D1) to white (1A1) towards periphery, margin entire; reverse concolourous. On OA, attaining 33–35 mm diam., flat, floccose, dark grey (1F1) to greyish green (1D3) and colourless towards periphery, margin submerged and entire; reverse concolourous. On CMA, reaching 34–38 mm diam., flat, velvety, dark grey (1F1) to greyish green (1D3) and colourless towards periphery, margin submerged and entire; reverse concolourous.

Additional descriptions and illustration: Von Arx & van der Aa (1987).

Additional materials examined: Bolivia, from soil, Oct. 1987, J.B. Speakman (CBS 539.87, received as Sn. tenerifae). France, Toulouse region, from vineyard soil with copper accumulation, unknown collection date, J. Nicot (CBS 155.67, ex-isotype strains of Sp. perplexans). Germany, Kiel-Kitzeberg, from wheat field soil, Jan. 1966, W. Gams (CBS 125.66, received as Sp. subticinensis, proposed here as reference strain). Spain, Canary Islands, Tenerife, Las Cañadas, from dung of unknown origin, Mar. 1986, J.A. von Arx (CBS 354.86, ex-type strain of Spororminula tenerifae); ibid., Catalonia, Mediterranean coast, discharging area of the Ebro River, from marine sediments at 27 m of depth, Oct. 2022, J. Gené & D. Guerra-Mateo (FMR 20474).

Cardinal temperatures for growth: Minimum 5 °C (4 mm), optimum 25 °C (28–30 mm), maximum 40 °C (4 mm).

Secondary metabolites: Antibiotic LL-N 313ε, palmarumycins, 4-hydroxy-5-methylmellein, 7-chloro-6methoxymellein, C₁₁H₁₀ClO₅, C₂₀H₁₄O₃.

Notes: Sporormia subticinensis was erected to represent a fungus characterized by subcylindrical, curved, 8-celled ascospores (Mouton 1897). This species was later transferred to Preussia based on morphology (von Arx 1973). Although molecular data from the ex-type strain of this fungus are not available, early phylogenetic analyses of Preussia s.l. using representative strains of Pr. subticinensis suggested a close relationship with the ex-type strain of Sn. tenerifae (Kruys & Wedin 2009). Despite morphological differences between strains of Pr. subticinensis examined in this study and the ex-type strain of Sn. tenerifae (von Arx & van der Aa 1987), our concatenated phylogenetic analysis both clustered species as a single conspecific taxon (Fig. 3). In addition, the isotype of Sp. perplexans (CBS 155.67) also clustered within this lineage, confirming they are the same species (von Arx & Storm 1967, Ahmed & Cain 1972). Since Sp. subticinensis represents the oldest name for this taxon, we propose the novel combination Sn. subticinensis in the benefit of nomenclatural stability and the preservation of extant names. In addition, we propose the strain CBS 125.66 as the reference strain for the species in phylogenetic analyses.

Here, we updated the morphological description of Sn. subticinensis with FMR 20474, which resembled the protologue in asci and ascospores shape and size ranges and, in addition, produced a pycnidial conidiomata in culture. See Sn. irregularis notes section for a detailed morphological comparison of asexual morphs. The ex-type strain of Sn. tenerifae (CBS 354.86) exhibited larger asci and ascospores and, according to von Arx & van der Aa (1987), they did not show a germinal slit. However, the ITS comparison of this strain with other strains in this lineage revealed a 100 % identity. Therefore, we interpret these morphological differences as intraspecific variability.

It is worth mentioning that Sn. tenerifae was transferred to the genus Preussia as Pr. tenerifae (Kruys & Wedin 2009). In Index Fungorum, the same name appears as Pr. tenerifae (von Arx & Guarro) Kruys, representing an update on the species Asordaria tenerifae. However, this synonym is incorrect as Asordaria tenerifae is a sordariomycetous species typically characterized by dark brown, aseptate ascospores (von Arx et al. 1987). Therefore, this should be regarded as a mistake resulting from the identical epithet and similar authorship of the two species.

Species of uncertain status

We have compiled a brief morphological description of the Preussia s.l. species with uncertain taxonomic status due to lack of molecular data or additional representative strains (Supplementary material Table S3). In particular, we have referred to the Sporormiella minima species complex throughout the manuscript. Our phylogenetic analyses confirmed the species Sm. isabellae, Sm. koreensis, Sm. mediterranea, and Sm. persica; however, we could not define Sm. minima in the phylogeny. Accordingly, the taxonomic status of this species remains unresolved until further strains are examined.

DISCUSSION

This work aimed to characterize Preussia s.l. strains of marine and xerophytic origin and assess the diversity within the genus through a polyphasic approach, combining morphology, phylogeny, and metabolite profiles. The five-region concatenated phylogeny confirmed the genera Preussia, Sporormiella, and Spororminula as different lineages. In addition, we described four novel genera and 11 novel species in Sporormiaceae. The marine and xerophytic strains primarily represented novel species, provided additional asexual morphs, and expanded metabolite profiles. These results highlight that the diversity associated with unusual substrates can help resolve complex taxonomic groups.

Unravelling the diversity within Preussia s.l.

The confusing circumscription of Preussia and Sporormiella led to databases organizing species under both genera regardless of synonyms. In consequence, novel species were recently described in either genus indistinctively; i.e., Pr. jejuensis, Pr. koreensis, and Sm. tela (Jakob et al. 2024, Noh et al. 2025). Our results agree with recent phylogenies addressing Sporormiaceae, where Preussia s.l. was displayed as a polyphyletic genus (Thambugala et al. 2015, Crous et al. 2020a, Hongsanan et al. 2021). However, the limited representation of type specimens in the phylogeny hampered taxonomic decisions. We addressed this common situation in fungal taxonomy, aiming for the most natural classification possible. For this reason, we defined the genus Sporormiella based on the consistency of morphological features in clade 1, and delimited clade 4 as Preussia s.str. based on CBS 127.66, which we designated as the epitype culture of Pr. funiculata based on morphology and geographical origin (Cain 1961, Ahmed & Cain 1972). Among the remaining clades of Preussia s.l., clade 9 was determined as Spororminula based on its type species Sn. subticinensis, and we managed to describe four additional genera; i.e., Chaetosporormiella, Pleosporormiella, Sporormyces, and Spororvela. In particular, the genera Pleosporormiella and Sporormyces were described based on the strains recovered from marine and xerophytic substrates, which filled phylogenetic gaps in Preussia s.l. (Fig. 3). This way, our phylogenetic tree updated Preussia s.l. diversity and enabled the recognition of traditional and novel genera.

The taxonomic rank of genus indicates definable units with biological meaning that result from evolutionary processes (Crous et al. 2021). In this sense, genera can be recognized through morphological, metabolic, and ecological patterns. In Table 4, we provide a polyphasic characterization of the genera proposed in this work. This classification provides a robust backbone to understand Preussia s.l. diversity and stabilizes nomenclature. It is worth noting that these taxonomic decisions resulted in 18 novel combinations of species and one new name. Although best practices indicate that taxonomic decisions should lead to as few name changes as possible, we think that the complex taxonomic history of this group and its limited circumscription in previous phylogenetic analyses support these nomenclatural changes (Kruys & Wedin 2009, González-Menéndez et al. 2017, Aime et al. 2021).

Up to 13 novel species of Preussia s.l. have been proposed in the last couple of years (Sandberg et al. 2022, Jakob et al. 2024, Eisvand & Mehrabi-Koushki 2025, Li et al. 2025, Noh et al. 2025, Phurbu et al. 2025). We proposed one of them as the novel genus Spororvela, five represented novel Sporormiella and Preussia s.str. species, while the rest clustered with representative strains of known species: i.e., Pr. funiculata, Sm. lignicola, Sm. procaviae and Sm. similis (Fig. 3). Most of these species lack molecular data derived from type specimens, which promoted the description of superfluous novel taxa taking advantage of intraspecific variability. Our phylogenetic analyses revealed the ITS region as a suitable barcode for species identification in Sporormiaceae (Figs 1, 2). Interestingly, the authors of some of the recent novel species overlooked the ITS region and relied on tef1-α despite more than 98 % similarity between strains (Sandberg et al. 2022, Eisvand & Mehrabi-Koushki 2025). Based on these minor phylogenetic distances accounting for intraspecific variability (Figs 2, 3), we defined reference strains to stabilize the taxonomy of older species and prevent the description of further superfluous taxa. Following best practices and previous works on different fungal groups, we refrained from designating unnecessary epitypes (Ariyawansa et al. 2014, Lendemer 2020, Bradshaw et al. 2024). Reference strains were selected based on morphological similarity to the protologue, availability of the culture, and historical use in phylogenies. We selected a lectotype or designated an epitype culture only when the original material was not indicated in the protologue, or when only herbarium material was available from the ex-type strain (See Sm. intermedia and Pr. funiculata taxonomy section).

This conservative approach, favouring nomenclature stability, also helped understand morphological diversity and ecological roles across lineages. Aside from sexual morphological differences, each genus produced different sets of asexual structures, with coelomycetous conidiomata as the most common across lineages (Table 4). It is noteworthy that Pleosporormiella comprised species with chrysosporium- and humicola-like asexual morphs, and Sporormiella included Sm. japonica, a holothallic species that, to the extent of our knowledge, represents the first report of an opportunistic animal pathogen in Preussia s.l. (Li et al. 2025). Animal pathogens are rare in Sporormiaceae, with only the species Westerdykella reniformis reported as a causal agent of skin lesions in dogs that can disseminate to the kidney and vertebral disks (Hurst 2016).

Despite our current effort to disentangle the diversity within Preussia s.l., more than 70 known species remain to be assessed with molecular data. For now, we treat these species as taxa of uncertain classification (Supplementary material Table S3). Hopefully, projects targeting the sequencing of fungarium material will provide novel insights in the near future.

Chemotaxonomy: secondary metabolites across genera and species

Secondary metabolite profiles represent a powerful tool to aid in disentangling complex taxonomic groups, supplementing morphological and phylogenetic evidence. Metabolites have been successfully applied to confirm genus circumscriptions, like the genus Pochonia (Clavicipitaceae) by the production of pochonins, Conoideocrella (Clavicipitaceae) by zeorins, and Pseudorhypophila (Naviculisporaceae) by zopfinol (Stadler et al. 2003, Isaka et al. 2011, Harms et al. 2021). This approach has also been useful to resolve species boundaries in cryptic groups like Amesia, Ascochyta, Aspergillus, Cladobotryum, Cortinarius, Penicillium, and Talaromyces (Frisvad 2015, Kim et al. 2016, Milic et al. 2022, Charria-Girón et al. 2023, Huymann et al. 2024). In this work, we combined metabolite profiles with morphology and phylogeny to understand the diversity within Preussia s.l. Since this group has been extensively assessed in search for novel bioactive molecules, we compiled these compounds in Table 5, where we updated species identification when possible. Overall, more than 30 compounds have been detected across Preussia s.l. lineages, many of them biologically active, while others still await a characterization (Table 5). In fact, here we recovered up to 15 molecules that should be studied in depth in future works.

Table 5.

Bioactive secondary metabolites detected in Preussia s.l. with updated species identification.

Metabolite	Current ID	Original ID	Major nutrient source	Fermentation state	Biological activity	Strain1	Barcode available	Reference
Asterric acid	Sporormiella persica	Preussia sp.	Rice-based	Liquid	Endothelin binding inhibitor	CAD4	Yes	Talontsi et al. (2014)
Preussiafuran A, B	Sporormiella persica	Preussia sp.	Rice-based	Liquid	Cytotoxic	CAD4	Yes	Talontsi et al. (2014)
Cissetin	Sporormiella persica	Preussia sp.	Rice-based	Liquid	Antibacterial	CAD4	Yes	Talontsi et al. (2014)
Auranticins A, B		Preussia aurantiaca	Potato Dextrose Broth	Liquid	Antifungal and antibacterial	ATCC 14745	No	Poch & Gloer (1991)
7-Chloro-6-methoxymellein		Preussia affinis	Corn starch and molassses	Liquid	Antifungal	Lederle culture N313	No	McGahren & Mitscher (1968)
Antifungal FR 173945		Sporormiella minima	Starch flour	Lquid	Antifungal	15604	No	Hatori et al. (2004)
Antifungal FR 171456		Sporormiella minima	Starch flour	Lquid	Antifungal	15604	No	Hatori et al. (2004)
Australifungin	Sporormiella intermedia	Sporormiella australis	Corn-based	Solid	Antifungal	MF5672	Yes	Hensens et al. (1995)
C28H42N4O8		Sporormiella minimoides	Corn-based	Liquid	Antifungal	MF5867	No	Clapp-Shapiro et al. (1998)
Cryptosporiopsin		Sporormia affinis	Corn starch and molassses	Lquid	Antifungal	N313	No	McGahren et al. (1969)
Cyperin		Preussia fleischhakii	Potato Dextrose Broth	Liquid	Antifungal	NRRL 6460	No	Weber & Gloer (1988)
Hyalopyrone		Sporormiella teretispora	Potato Dextrose Broth	Liquid	Antifungal	JS 148	No	Wang et al. (1995)
Terezines A, B, C, D		Sporormiella teretispora	Soybean flour	Liquid	Antifungal and antibacterial	JS 148	No	Wang et al. (1995)
Leptosin A, C	Preussia funiculata	Preussia typharum	MMK2	Liquid	Cytotoxic and antifungal	TTI-1095	Yes	Perlatti et al. (2021)
Preussiadin A, B	Preussia funiculata	Preussia typharum	Cheerios breakfast cereal	Solid	Cytotoxic	JX143871	Yes	Du et al. (2014)
Microsphaeropsone A		Sporormiella minima	Rice-based	Solid	Antifungal and antibacterial	66342	No	Xiong et al. (2014)
Preussin		Preussia sp.	Soluble starch and glucose	Liquid	Antifungal	NA	NA	Johnson et al. (1989)
Preussochromone A, B, C, D	Sporormiella africana	Preussia africana	Rice-based	Solid	Cytotoxic	DN136	Yes	Zhang et al. (2012)
Preussomerin A, D, E, F	Sporormiella isopartis	Preussia isomera	Potato Dextrose Broth	Liquid	Antifungal and antibacterial	CBS 415.82	Yes	Weber & Gloer (1991)
Similin A, B		Preussia similis	Soybean flour	Liquid	Antifungal	DM 87	No	Weber et al. (1992)
Spiropreussione		Preussia sp.	Wheat bran and glucose	Liquid	Cytotoxic	CGMCC 2022	No	Chen et al. (2009)
Sporminarin A, B	Sporormiella africana	Sporormiella minimoides	Rice-based	Solid	Antifungal	NRRL 37629	Yes	Mudur et al. (2006)
Sporostatin		Sporormiella sp.	Glucose and peptone broth	Liquid	Cytotoxic	M5032	No	Kinoshita et al. (1997)
Sporovexins A, B, C		Sporormiella vexans	Potato Dextrose Broth	Liquid	Antifungal and antibacterial	JS 306	No	Soman et al. (1999)
Zaragozic acid B		Sporormiella intermedia	Cerelose and cotton seed	Liquid	Antifungal	Several strains	No	Bills et al. (1994)
5-hydroxy-2,7-dimethoxy-8-methylnaphthoquinone	Sporormiella africana	Sporormiella minimoides	Rice-based	Solid	hCaM inhibition	MEXU 26355	Yes	Leyte-Lugo et al. (2013)
C26H22O8		Preussia sp.	Potato Dextrose Broth	Liquid	Antioxidant and antibacterial	NA	NA	Paudel et al. (2018)
Preuisolactone A	Sporormiella africana	Preussia isomera	Malt extract broth	Liquid	Antifungal and antibacterial	XL-1326	Yes	Xu et al. (2019)
Verbalide A–F	Sporormiella africana	Preussia africana	Potato Dextrose Broth	Liquid	Antiviral	CPCC 400972	No	Zhang et al. (2023)
Preussilides A, C	Sporormiella similis	Preussia similis	Oatmeal and molasses	Liquid	Cytotoxic and antifungal	DSM 104666	Yes	Noumeur et al. (2017)

¹ NA: not available.

The metabolite profiles developed in this work relied on the GNPS platform and MS/MS data, which enabled the creation of networks representing metabolite production across strains. This way, metabolites could be traced across the phylogeny to uncover patterns of production. Melleins represented the most common group of polyketide metabolites detected across the lineages studied (Table 3). We detected monocerin, 4-hydroxy-5-methylmellein, and 7-chloro-6-methoxymellein across Chaetosporormiella (clade 8), Pleosporormiella (clade 3), Preussia (clade 4), and Spororminula (clade 9). In particular, 7-chloro-6-methoxymellein was the only compound already associated with Preussia s.l., recovered from a strain identified as “Sporormiella affinis” (McGahren & Mitscher 1968, Reveglia et al. 2020). Although we detected this molecule in the aforementioned genera, our phylogeny located Sm. affinis in clade 7, suggesting a transversal production across lineages 3–9. It is also noteworthy that monocerin was primarily obtained from strains of marine origin in Pleosporormiella and Spororminula (Fig. 5A). These compounds are typically associated with common plant pathogens displaying phytotoxic activity, but also antifungal and antibacterial properties (Reveglia et al. 2020, Gomes et al. 2023). However, the production of this compound in vivo should be tested before drawing ecological conclusions.

Dihydroisoflavipucine represented another compound detected across lineages (Fig. 4). This molecule was described from a phoma-like fungus (Loesgen et al. 2011). Interestingly, we exclusively recovered it from strains of Chaetosporormiella and Pleosporormiella displaying phoma-like, humicola-like, and monodictys-like asexual morphs (Table 3). Sporulation processes affect the production of metabolites, and the asexual stage of these strains may have triggered the production of this molecule across lineages (Calvo et al. 2002).

Networks also enabled the identification of metabolites exclusively produced by certain genera that could potentially represent chemotaxonomic markers. Regarding the genus Sporormiella, we included three strains in the metabolite profile, and one of them produced preussochromone F (Fig. 7D). The previous chemotaxonomic study of Preussia s.l. detected preussochromones as common compounds across Sm. africana, Sm. isopartis, and Sm. koreensis using the MMK2 medium (González-Menéndez et al. 2017). Other compounds with similar patterns were microsphaerones, detected in Sm. africana and Sm. lignicola with the same medium. We think these compounds could be used to characterize members of Sporormiella (Fig. 3). In fact, we also used MMK2 in the profiling of sister genera, but none produced these molecules (Table 3). In a similar manner, we only detected the production of the antibiotic LL-N 313ε, and the compounds C₁₁H₁₀ClO₅ and C₂₀H₁₄O₃ in Spororminula species. Moreover, Sporormyces could be characterized by the production of 4-methoxy-6-pentyl-2H-pyran-2-one (Fig. 6B), but also 1,7-dihydroxy-3-(hydroxymethyl)-anthraquinone, which have been exclusively obtained in this lineage (González-Menéndez et al. 2017). The remaining genera produced different sets of secondary compounds, without a clear characteristic metabolite at the genus level. However, Pleosporormiella outstands as a metabolically active group with 16 different metabolites (Table 3).

We defined chemotypes to analyse the sets of metabolites produced by different strains (Table 3). In some cases, these chemotypes could be related to phylogenetic species to support species boundaries; i.e., Pr. gypsicola produced C₁₆H₂₀O₆, C₁₆H₁₈O₅, C₁₉H₂₄O₈S and C₄₃H₇₉NO₉, molecules that were not detected in Pr. funiculata, Ps. suttonii produced C₁₆H₂₃NO₃ and C₁₇H₂₅NO₂, while these molecules were absent in Ps. polymorpha fermentations, and Sn. irregularis differed from Sn. tenerifae by the production of monocerin (Table 3). We stated these metabolite relationships of production or absence of production based on the methodology followed here, which ensured that all strains fermented under the same conditions, ensuring the possibility of comparison (Frisvad 2012). However, some species were particularly prolific in metabolite production, like Ps. polyspora, which produced up to 12 different metabolites by different strains with only three of them produced simultaneously across strains. A similar pattern was observed in other species like Pr. funiculata and Sm. africana, with up to 17 secondary metabolites obtained from different strains of Sm. africana (Mudur et al. 2006, Zhang et al. 2012a, 2023, Leyte-Lugo et al. 2013, González-Menéndez et al. 2017).

In the case of Pr. funiculata, ten different metabolites have been detected, including leptosin C (Table 3, Du et al. 2014, González-Menéndez et al. 2017, Perlatti et al. 2021). In this work, we expected to obtain this compound across representatives of Pr. funiculata based on the strain TTI-1095 (Fig. 2), which was used to define the gene cluster of the compound (Perlatti et al. 2021). In fact, leptosin C had also been recovered from an additional strain, JX143871 (Du et al. 2014). Surprisingly, we detected cyperin and 7-chloro-6-methoxymellein, among two other undetermined molecules, from the strains of Pr. funiculata included in the metabolite profile, but only recovered leptosin analogues from strains of Cp. monodictyoides (Table 3). Leptosin C was detected in strains of Pr. funiculata using MMK2 and Cheerios breakfast cereal (Du et al. 2014, Perlatti et al. 2021). Although we also used MMK2 under similar incubation conditions, the substrate of isolation and intraspecific variability may have played a key role. The strains producing leptosin C were recovered from dung, while the strains examined in this work were obtained from soil samples. In addition, the clade of Pr. funiculata is characterized by morphological variability in asci and ascospore septa (See taxonomy section). We do not consider these morphological differences as indicative of a species complex, but they evidence intraspecific diversity, which may also be associated with metabolite production.

Finally, the strains of xerophytic and marine habitats examined here confirmed previous works promoting the exploration of these unusual substrates (Imhoff 2016, Sayed et al. 2020). These strains expanded the phylogenetic diversity associated with Preussia s.l., but also yielded a wide array of secondary metabolites. Assessing the biological activity of these compounds was beyond the scope of this work. However, we think that these novel insights into the diversity of Preussia s.l. represent the first step towards a more natural classification that ultimately will enable metabolite prediction and ease the search for novel bioactive compounds.

Supplementary Material: https://studiesinmycology.org/

Fig. S1

Sporormiella antarctica (holotype LPS 3500). A. Fungarium label and collection data. B. Original drawings. C. Dung of Bernicla antarctica colonized by Sm. antarctica. D. Perithecia. E. Asci with mature ascospores.

Fig. S2

Sporormiella australis (holotype LPS 3521). A. Fungarium label and collection data. B. Dung of Cavia australis colonized by Sm. australis. C. Original drawings.

Fig. S3

Sporormiella intermedia (lectotype Rabenhorst Fungi Europaei No. 644). A. Fungarium label and collection data. B. Rabbit dung.

Fig. S4

Sporormiella lignicola (holotype K(M)131910). A. Fungarium label and collection data and wood of Fraxinus excelsior colonized by Sm. lignicola. B. Ascus. C. Ascospores. Scale bars: B = 25 μm; C = 10 μm.

Fig. S5

Sporormiella pascua (holotype M-0378428). A. Fungarium label, collection data, and dung colonized by Sm. pascua. B. Perithecia. C. Ascospores on dung. D–E. Ascospores. Scale bars: D = 25 μm; E = 10 μm.

Fig. S6

Perisporium funiculatum (holotype B700132488). A. Fungarium label. B. Ascomata on angiosperm pieces. C–D. Detail of the ascomata. Scale bars = 2 μm.

Fig. S7

Phylogenetic tree inferred from an ITS alignment of 165 strains representing the family Sporormiaceae in Pleosporales. The tree is rooted to Lophiostoma rosae-ecae MFLUCC 17-0807 and Teichospora thailandica MFLUCC 13-0284. Numbers at the branches indicate support values (IQ-TREE-UFBS) above 90 %. The scale bar represents the expected number of changes per site.

Fig. S8

Phylogenetic tree inferred from the LSU alignment of 165 strains representing the family Sporormiaceae in Pleosporales. The tree is rooted to Lophiostoma rosae-ecae MFLUCC 17-0807 and Teichospora thailandica MFLUCC 13-0284. Numbers at the branches indicate support values (IQ-TREE-UFBS) above 90 %. The scale bar represents the expected number of changes per site.

Fig. S9

Phylogenetic tree inferred from a tef1-α alignment of 111 strains representing Preussia s.l. and associated genera. The tree is rooted to Sparticola junci MFLUCC 15-0030. Numbers at the branches indicate support values (IQ-TREE-UFBS) above 90 %. The scale bar represents the expected number of changes per site.

Fig. S10

Phylogenetic tree inferred from a rpb2 alignment of 111 strains representing Preussia s.l. and associated genera. The tree is rooted to Sparticola junci MFLUCC 15-0030. Numbers at the branches indicate support values (IQ-TREE-UFBS) above 90 %. The scale bar represents the expected number of changes per site.

Fig. S11

Phylogenetic tree inferred from a concatenated ITS–LSU alignment of 28 strains, representing the Sporormiella minima species complex and related taxa. The tree is rooted to representative strains of the genus Westerdykella. Numbers at the branches indicate support values (IQ-TREE-UFBS) above 90 %. Ex-type strains are indicated with ^T, and those corresponding to the accepted species are highlighted in grey. Novel combinations are highlighted in brown. The scale bar represents the expected number of changes per site.

Fig. S12

Speciation analysis of Pleosporormiella. The tree represents the bPTP analyses of the concatenated ITS-LSU alignment. Blue lines indicate speciation events and red lines comprise species. The topology obtained through the ASAP analysis is represented on the side. Incongruent topologies are highlighted in red.

Fig. S13

Speciation analysis of Preussia s.str. The tree represents the bPTP analyses of the concatenated ITS-LSU alignment. Blue lines indicate speciation events and red lines comprise species. The topology obtained through the ASAP analysis is represented on the side. Incongruent topologies are highlighted in red.

Table S1

Collection details from the strains studied in the present work and GenBank accession numbers of the barcodes used for identification.

Table S2

Potential metabolite hits obtained in the Chapman & Hall Dictionary of Natural Products (v. 25.1) for those compounds unassigned in Table 2.

Table S3

Species of Preussia s.l. with uncertain taxonomic status.