Phylogeny, taxonomy and geographic distribution of novel and known fungi with holoblastic-denticulate conidiogenesis in Rhamphoriales and Pleurotheciales (Sordariomycetes)

Abstract

As part of a broader survey of lignicolous saprobic fungi, we investigated fungal taxa from the class Sordariomycetes displaying holoblastic-denticulate conidiogenesis, a distinct developmental process and phylogenetically informative trait. Although these fungi appear morphologically similar in culture, they represent distinct evolutionary lineages. This taxonomic study integrates comparative morphological analyses, phylogenetic reconstruction of five nuclear markers, and analysis of biogeographical patterns through environmental DNA data to introduce novel taxa in the Pleurotheciales and Rhamphoriales. A new genus and species Echinodenticula allantospora and three new species, Phaeoisaria parallela, Rhamphoriopsis cuprea and Rh. denticulata, are described. A rarely encountered species Rhamphoria separata is reported, along with its previously undocumented asexual morph. Furthermore, we successfully demonstrate the utility of two protein-coding genes, rpb2 and tef1, as complementary barcodes for distinguishing closely related Phaeoisaria species. Our findings highlight the significance of holoblastic-denticulate conidiogenesis as a diagnostic feature of the Rhamphoriales and a prevalent trait in the Pleurotheciales. An unknown ascomycete that produced only sterile mycelium in culture is described here as Melanocrypta curvata and placed at an incertae sedis position within the Sordariomycetes. Additionally, we present short-read whole-genome sequencing data for the ex-type strains of the newly described species, providing a valuable genomic resource for future taxonomic, phylogenetic, and functional studies. Environmental DNA data from the GlobalFungi database bring new perspective into the biogeographical patterns of Phaeoisaria, Rhamphoria, and Rhamphoriopsis. The distribution of E. allantospora and M. curvata remains poorly understood, as no records for these species were found in GlobalFungi. This study provides new insights into the molecular systematics, taxonomy, and biogeography of the Rhamphoriales and Pleurotheciales, and highlights the role of environmental DNA metabarcoding in uncovering fungal diversity and distribution patterns.

Citation: Réblová M, Nekvindová J, Hernández-Restrepo M, Hradilová M, Kolařík M (2025). Phylogeny, taxonomy and geographic distribution of novel and known fungi with holoblastic-denticulate conidiogenesis in Rhamphoriales and Pleurotheciales (Sordariomycetes). Persoonia 55: 277–311. doi: 10.3114/persoonia.2025.55.08

INTRODUCTION

The diversity and evolutionary relationships of lignicolous saprobic fungi, particularly within the class Sordariomycetes, continue to be an active area of research. Many taxa still lack living cultures and molecular DNA data, and some species are either poorly characterised, or yet to be discovered. Ascomycete fungi exhibit an extraordinary diversity of asexual reproductive strategies, where conidiogenesis, along with micro- and macromorphological characteristics, serve as important taxonomic features. As part of a broader survey of lignicolous saprobic fungi, we investigated fungal taxa from the class Sordariomycetes displaying holoblastic-denticulate conidiogenesis, a distinct developmental process and phylogenetically informative trait. In this mode of conidium ontogeny, the outer wall of the conidiogenous cell remains continuous with the wall of the developing conidium (e.g. Cole & Samson 1979: figs 3.42, 3.51). Conidial formation is typically accompanied by the sympodial extension of the fertile apical part of the conidiogenous cell, resulting in the development of several denticles or a rachis that bears conidia on denticles or minute protrusions (Fig. 1A–G).

Fig. 1.

Sympodially proliferating conidiogenous cells producing holoblastic conidia, with (A–G) or without (H) denticles, in Pleurotheciales and Pleurophragmium parvisporum (incertae sedis). A. Conidiogenous cell with conidia attached to terminal denticles (Pleurotheciella centenaria DAOM 229631). B, C. Rachis of the conidiogenous cells after conidial secession covered by minute denticles (B. Phaeoisaria fasciculata DAOM 230055, C. CBS 127885). D, E. Tips of the conidiogenous cells with conidia formed on peg-like denticles (Pleurothecium recurvatum CBS 131272). F, G. Rachis of the conidiogenous cells covered by minute protrusions (Pl. parvisporum PRA-24032). H. Penicillately arranged conidiogenous cells without denticles (Sterigmatobotrys macrocarpa PRM 915682). Scale bars: A–C = 5 µm; D–H = 10 µm.

The classification of asexually reproducing fungi has undergone a long and complex history. A milestone that revolutionised the characterisation of asexual fungi was the ontogenetic system proposed by Hughes (1953), which emphasised conidium formation patterns as a fundamental criterion. This system was further refined by Tubaki (1958, 1963), Barron (1968), Kendrick (1971), Subramanian (1971, 1972), and others, ultimately culminating in a highly detailed classification by Minter et al. (1982, 1983), which included numerous variants of conidiogenesis. The various patterns of conidial ontogeny were comprehensively described and richly illustrated by Cole & Samson (1979) and Seifert et al. (2011).

The ontogenetic classification ultimately proved to be an unsustainable approach and has gradually faded into the background of modern fungal taxonomy. Today, taxonomy and systematics are increasingly driven by an integrative framework that incorporates molecular phylogenetics, phylogenomics, morphology, physiological and biochemical characters, biogeography, and ecology. Nonetheless, conidial ontogeny remains a valuable set of diagnostic traits for identifying and distinguishing fungal taxa. In the absence of DNA sequence data, the mode of conidiogenesis, along with other asexual and sexual morphological features, can offer crucial insights for taxonomic placement. However, interpreting conidium ontogeny and its fine details can sometimes be challenging. When relying solely on natural material, specimens may be aged or immature, while in culture, morphological structures are often simplified, with conidiomata absent and conidiophores reduced to single conidiogenous cells compared to observations in vivo. Nevertheless, cultures remain indispensable for studying conidiogenesis, as demonstrated by Cole & Samson (1979). This is particularly relevant for some understudied asexually typified species that lack original cultures or recently recollected specimens. The absence of viable cultures not only impedes future taxonomic research and the study of pleomorphic taxa but also limits the discovery of asexual morphs, and potential synasexual morphs (synanamorphs) that often develop exclusively in vitro and may display a different morphotype and a mode of conidiogenesis than the asexual morph.

When working with relatively coherent taxonomic groups, conidiogenous patterns tend to be fairly consistent. Minor deviations from the typical pattern observed in a small number of species within a group can often be rationalised with some nuance. However, the main difficulties arise when comparing conidiogenesis across large phylogenetic distances. The plesiomorphic nature of certain asexual morphs, such as for example acremonium-, chalara-, chloridium-, cladosporium-, dactylaria-, idriella-, phaeoisaria-, phialocephala-, phialophora-, ramichloridium-, rhinocladiella-, sporothrix-, and verticillium-like forms, makes identification based solely on conidiogenous cell ontogeny and conidial morphology potentially misleading.

In the class Sordariomycetes, multiple types of conidium ontogeny occur, including holoblastic-denticulate conidiogenesis that can serve as a diagnostic morphological feature. This type of conidial ontogeny has been documented across multiple genera spanning several orders and families within the Sordariomycetes, including Amplistromatales (Huhndorf et al. 2009), Barbatosphaeriaceae (Samuels & Candoussau 1996, Réblová et al. 2015), Coronophorales (de Hoog 1977, Vakili 1989, Li et al. 2016, Schultes et al. 2017), Hypocreales (Barnett 1958, Samson et al. 1980, Gams et al. 1984, Yip & Rath 1989, Sun et al. 2017), Helminthosphaeriaceae (Hughes 1979, Sivanesan 1983), Magnaporthales (Klaubauf et al. 2014, Feng et al. 2024), Microascales (Gilgado et al. 2007), Myrmecridiales (Arzanlou et al. 2007), Rhamphoriales (Réblová & Štěpánek 2018, Yang et al. 2023, Chen et al. 2024), Ophiostomatales (Upadhyay & Kendrick 1975, Gebhardt et al. 2005, de Meyer et al. 2008, Harrington et al. 2010), Papulosaceae (Khemmuk et al. 2016), Pararamichloridiales (Crous et al. 2017), Pleurotheciales (de Hoog & Papendorf 1976, Réblová et al. 2016, Hernández-Restrepo et al. 2017), Pseudodactylariales (Crous et al. 2017), Sordariales (Wang et al. 2019, 2022), Xenospadicoidales (Réblová et al. 2017), Xylariales (von Nees 1817, von Höhnel 1923, Hughes 1951, 1958, Subramanian & Lodha 1968, von Arx 1982, Rogers 1984, Deighton 1985, Samuels et al. 1987, Castañeda-Ruiz & Kendrick 1990, Nonaka et al. 2013, Lin et al. 2017, Hernández-Restrepo et al. 2022), and several genera Sordariomycetes incertae sedis (uncertain placement) (Baker et al. 2001, Réblová & Seifert 2004, Crous et al. 2013). Although fungi with this type of conidiogenesis are widely distributed phylogenetically, their ecological adaptations remain insufficiently understood.

Despite this, holoblastic-denticulate conidiogenesis serves as a hallmark of the Rhamphoriales and Pleurotheciales, to which most of the fungi encountered in this study belong. Rhamphoriales (Hyde et al. 2021) consist of a single family, Rhamphoriaceae (Réblová & Štěpánek 2018), which was originally established to encompass four holomorphic genera, including Rhamphoria (von Niessl 1876), Rhodoveronaea (Arzanlou et al. 2007, Réblová 2009), as well as the newly introduced Rhamphoriopsis and Xylolentia. This classification is based on multilocus phylogenetic analyses and morphological examination and comparison on both the natural substrate and in culture. While these genera are distinguished by their sexual characteristics, they share morphologically highly similar asexual morphs phaeoisaria-, idriella-, and ramichloridium-like (Arzanlou et al. 2007, Müller & Samuels 1982, Réblová & Štěpánek 2018). Members of the Rhamphoriales are primarily saprobic lignicolous fungi, documented from terrestrial and freshwater habitats in temperate, tropical and subtropical regions.

The monotypic order Pleurotheciales (Réblová et al. 2016) was initially established to accommodate six genera and an additional three genera represented by non-type species using phylogenetic analysis of six gene markers combined with morphological comparisons. It was centred on the newly introduced genera Pleurotheciella and Melanotrigonum, along with Helicoascotaiwania (previously classified within Helicoön p. p., Dayarathne et al. 2019, Réblová et al. 2020), Phaeoisaria (von Höhnel 1909), Pleurothecium (von Höhnel 1923), and Sterigmatobotrys (Oudemans 1886, Réblová & Seifert 2011). They are characterised by holoblastic conidiogenous cells with conidia developing on denticles or minute protrusions along a sympodially elongating rachis (Fig. 1A–G). In contrast, Sterigmatobotrys lacks denticles on the polyblastic, sympodially proliferating conidiogenous cells (Fig. 1H). Since then, seven additional, asexually typified genera have been incorporated into the order introducing a new morphotype. While some conform to the previous concept and exhibit holoblastic-denticulate conidiogenous cells, such as Anapleurothecium (Hernández-Restrepo et al. 2017), others possess holoblastic conidiogenous cells that lack denticles and produce solitary, dark, muriform, macroscopic conidia, including Coleodictyospora (Charles 1929), Dematipyriforma (Sun et al. 2017), Neomonodictys (Hyde et al. 2020), Pseudosaprodesmium (Tian et al. 2024), Rhexoacrodictys (Baker et al. 2002), and Saprodesmium (Dong et al. 2021). Members of the Pleurotheciales are primarily saprobes found on decaying wood, either submerged in freshwater or thriving in terrestrial environments. Additionally, some species have been reported as endophytes (Sun et al. 2017), while others have been identified as opportunistic human pathogens (Guarro et al. 2000, Chew et al. 2010).

This research continues our long-term investigation into the taxonomy, phylogeny and fungal diversity within Pleurotheciales and Rhamphoriales (Réblová & Seifert 2011, Réblová et al. 2012, 2016, Réblová & Štěpánek 2018). This study aims to characterise novel and rare fungi, refine their phylogenetic relationships within these two groups in Sordariomycetes, and provide new insights into the biogeography and ecological roles of these fungi. It is based on an integrative approach combining morphology, molecular phylogenetics, and environmental sequencing data. For all studied fungi, we obtained axenic cultures and generated novel DNA sequences from five gene markers. To explore the biogeography, ecological aspects, and habitat preferences of the studied fungi, we utilised the GlobalFungi database (Větrovský et al. 2020), which compiles environmental ITS sequences. Additionally, we provide short-read whole-genome sequencing (WGS) data for ex-type strains of the newly introduced species, enhancing taxonomic benchmarks and improving fungal classification.

MATERIALS AND METHODS

Isolates and morphology

Fresh specimens were collected in a temperate region of the Northern Hemisphere, in broadleaf and mixed forests in the Czech Republic. Dried collections were deposited in the Fungarium of the Institute of Botany (PRA) of the Czech Academy of Sciences in Průhonice, Czech Republic. Cultures of newly obtained strains are maintained at the culture collection housed at the Westerdijk Fungal Biodiversity Institute (CBS) in Utrecht, the Netherlands. An additional living strain was obtained from the Facultat de Medicina de Reus (FMR), Tarragona, Spain. Additional information on habitat, host associations and geographic distribution was based on environmental ITS data from the GlobalFungi database (Větrovský et al. 2020) and MyCoPortal database (Miller & Bates 2017). Taxonomic novelties were registered in MycoBank (Crous et al. 2004). Table 1 lists the studied strains, their origins, and the GenBank accession numbers for the sequences generated in this research. The abbreviations of the genera treated in the present work are: Dematipyriforma (D.), Echinodenticula (E.), Melanocrypta (M.), Phaeoisaria (P.), Pleurophragmium (Pl.), Pleurotheciella (Pt.), Rhamphoria (R.), Rhamphoriopsis (Rh.), Rhodoveronaea (Rv.), and Sterigmatobotrys (S.).

Table 1.

Species, isolate information and new sequences determined for this study (in bold) and additional sequences retrieved from GenBank.

Taxon	Source	Status1	Host	Country	GenBank accession numbers					References2
					ITS	nucLSU	nucSSU	rpb2	tef1-a
Echinodenticula allantospora	CBS 147513	T	decaying wood of Quercus cerris	Czech Republic	PV455933	PV455947	PV455960	PV483447	PV483428	This study
Melanocrypta curvata	CBS 147592	T	decaying wood of Carpinus betulus	Czech Republic	PV455934	PV455948	PV455961	PV483448	PV483429	This study
	CBS 147593		decaying wood of Ulmus sp.	Czech Republic	PV455935	PV455949	PV455962	PV483449	PV483430	This study
Phaeoisaria parallela	CBS 153403	T	decaying wood of Quercus sp.	Czech Republic	PV455936	PV455950	PV455963	PV483450	PV483431	This study
Phaeoisaria sparsa	FMR 11939		decaying wood	Spain	PV455937	PV455951	PV455964	PV483451	PV483432	This study
Pleurotheciella uniseptata	CBS 147511		decaying wood of Quercus sp.	Czech Republic	PV455938	PV455952	PV455965	PV483452	PV483433	This study
Rhamphoria delicatula	CBS 132724		decaying deciduous wood	Czech Republic	MG600391	FJ617561	JX066711	JX066702	PV483435	a, b, c
	PRA-13612		decaying wood of Fagus sylvatica	Czech Republic	MG600390	AF261068	AF242267	KT991655	PV483434	d, e, f
Rhamphoria pyriformis	CBS 153406		decaying wood of Quercus sp.	Czech Republic	PV455939	PV455953	PV455966	PV483453	PV483436	This study
	CBS 139024		decaying wood of Tilia sp.	Czech Republic	MG600392	MG600397	MG600405	MG600401	PV483437	This study, c
	CBS 139033		decaying wood of Fagus sylvatica	Germany	KT991677	KT991665	MG600406	KT991656	—	This study, c, f
Rhamphoria separata	CBS 153404		decaying wood of Quercus sp.	Czech Republic	PV455940	PV455954	PV455967	PV483454	PV483438	This study
	CBS 153405		decaying wood of Quercus sp.	Czech Republic	PV455941	PV455955	PV455968	PV483455	PV483439	This study
Rhamphoriopsis cuprea	CBS 147991	T	decaying wood of Fagus sylvatica	Czech Republic	PV455942	PV455956	PV455969	PV483456	PV483440	This study
Rhamphoriopsis denticulata	CBS 147996	T	decaying wood of Sambucus nigra	Czech Republic	PV455943	PV455957	PV455970	PV483457	PV483441	This study
	CBS 147997		decaying wood of Sambucus nigra	Czech Republic	PV455944	PV455958	PV455971	PV483458	PV483442	This study
Rhamphoriopsis muriformis	CBS 127683		decaying wood of Buxus sempervivens	France	MG600389	MG600395	MG600403	MG600399	PV483443	This study, c
	CBS 131269	T	decaying wood of Buxus sempervivens	France	PV455945	MG600396	MG600404	MG600400	PV483444	This study, c
Rhodoveronaea varioseptata	CBS 153411		decaying wood of Quercus rubra	Czech Republic	PV455946	PV455959	PV455972	PV483459	PV483445	This study
Xylolentia brunneola	PRA-13611	T	decaying wood of Fagus sylvatica	Czech Republic	MG600394	MG600398	MG600407	MG600402	PV483446	This study, c

¹T denotes ex-type culture.

²References: a) Réblová (2009); b) Réblová (2013); c) Réblová & Štěpánek (2018); d) Réblová & Winka (2000); e) Winka (2000); f) Réblová et al. (2016).

Macromorphological characters of ascomata and colonies were examined with an Olympus SZX12 dissecting microscope (Olympus America, Inc., Melville, NY, USA). Ascomata were rehydrated with tap water, and the gelatinous centrum was extracted using the tip of a needle. Microscopic slides were prepared using 90 % lactic acid, water, or Melzer’s reagent. Measurements were taken from specimens mounted in Melzer’s reagent, with means ± standard deviation (SD) calculated for asci, ascospores and conidia sizes based on 20–25 measurements. Micromorphological features were examined using an Olympus BX51 light microscope. Microphotographs were taken with an Olympus DP75 camera operated with Olympus cellSens Dimension software v. 4.3. Colony characteristics were captured with a Canon EOS 77D digital camera with Canon EF 100mm f/2.8L Macro IS USM objective (Canon Europe Ltd., Middlesex, UK) with daylight spectrum 5500K 16W LED lights. Images were processed using Adobe Photoshop CS6 software (Adobe Systems, San Jose, CA, USA).

Axenic cultures were prepared following the method described by Réblová & Nekvindová (2023). To evaluate colony features, diffusible pigments, and growth patterns, strains were grown on cornmeal dextrose agar (CMD) (cornmeal agar, Oxoid Limited, Basingstoke, UK, supplemented with 2 % w/v dextrose), malt-extract agar (MEA) (Oxoid), Modified Leonian´s agar (MLA) (Malloch 1981), oatmeal agar (OA), and potato-carrot agar (PCA) (Crous et al. 2019). Colony characteristics were assessed from 4-wk-old cultures incubated in darkness at 23 °C.

Molecular methods

Phylogenetic relationships were assessed using five gene markers: internal transcribed spacer ITS1–5.8S–ITS2 (ITS) of the nuclear rRNA cistron, nuclear large subunit (LSU) rDNA gene and the nuclear small subunit (SSU) rDNA gene, and two genes encoding the second largest subunit of RNA polymerase II (DNA-directed RNA polymerase) (rpb2) and the intermediate section of the translation elongation factor 1-α (tef1). Apart from the ITS barcode (Schoch et al. 2012), rpb2 and tef1 genes were employed as secondary barcodes. These gene markers are recognised for their effectiveness in resolving interspecific relationships (Robert et al. 2011, Stielow et al. 2015, Meyer et al. 2019) and they were successfully used in combined analyses in our previous studies (Réblová et al. 2022, Réblová & Nekvindová 2023). Protocols for DNA extraction from living tissues and PCR amplification of ITS, LSU, SSU, rpb2, and tef1 were conducted following the methods described by Réblová & Nekvindová (2023). Automated sequencing was carried out by Eurofins Genomics Europe Sequencing Service (Cologne, Germany). Analyses of raw sequence data and assembly of sequence contigs were performed using Sequencher v. 5.4.6 (Gene Codes Corp., Ann Arbor, MI, USA).

The ex-type strains of the new species: Echinodenticula allantospora CBS 147513, Melanocrypta curvata CBS 147592, Phaeoisaria parallela CBS 153403, Rhamphoriopsis cuprea CBS 147991, Rh. denticulata CBS 147996, and a non-type strain of Pleurotheciella uniseptata CBS 147511 were selected for whole-genome DNA sequencing. Genomic DNA was extracted from 5-d-old cultures grown on MEA plates using the NucleoSpin® Soil isolation kit (Macherey–Nagel, Düren, Germany). Library preparation (2 × 300 bp Illumina paired-end) was carried out, and sequencing was performed on a NextSeq 2000 instrument (Illumina) following the manufacturer’s protocol. The quality of the raw sequencing data was assessed using FASTQC v. 0.11.9 (Andrews 2010) (Accessed on 23 Aug. 2024), and low-quality reads were filtered out using Trimmomatic v. 0.39 (Bolger et al. 2014) based on the quality control results (FastQC v. 0.11.9). The high-quality reads were then assembled de novo using SPAdes v. 4.0.0 (Bankevich et al. 2012). Genome assembly quality was assessed using QUAST v. 5.2.0 (Gurevich et al. 2013), and completeness was evaluated with BUSCO v. 5.7.1.1 (Seppey et al. 2019) against the fungi_odb10.2019-11-20 dataset. The genome identities were confirmed by comparing extracted ITS barcode sequences. The annotated genomes have been deposited in the NCBI database under the bioproject code PRJNA1245676, and the quality and completeness of the assembled whole genomes are presented in Table 2.

Table 2.

Quality and completeness of the obtained whole genome sequences, BioProject PRJNA1245676.

Taxon	Strain	GenBank biosample numbers	GenBank accession number	Dataset Complete(BUSCO) (%)	Scaffold N50	Contigs N50	Number of scaffolds	Total lenght(genome size, bp)
Echinophora allantospora	CBS 147513	SAMN47761544	JBNOXNOOOOOOOOO	96.4	131890	127150	3972	39069120
Melanocrypta curvata	CBS 147592	SAMN47761542	JBOZOGOOOOOOOOO	99.1	125000	125000	1632	31705371
Phaeoisaria parallela	CBS 153403	SAMN49474292	JBPGNN000000000	98.2	359000	361472	615	37259024
Pleurotheciella uniseptata	CBS 147511	SAMN47761543	JBNOXOOOOOOOOOO	98.2	312463	309343	416	41063598
Rhamphoriopsis denticulata	CBS 147996	SAMN47761546	JBNOXLOOOOOOOOO	98.7	754807	754807	206	36497049
Rhamphoriopsis cuprea	CBS 147991	SAMN47761545	JBNOXMOOOOOOOOO	98.8	889571	847404	129	37065021

Phylogenetic analyses

Initial BLAST searches with BLASTn and megaBLAST algorithms were conducted using both ITS and LSU sequences for all studied strains to identify the most closely related fungi in the Sordariomycetes. Available ITS, LSU, SSU, rpb2, and tef1 sequences of these fungi were retrieved from the GenBank sequence database at NCBI (Sayers et al. 2022) and incorporated into the analyses. These fungi along with their GenBank accession numbers and respective references are listed in Supplementary Table S1.

Sequences were aligned in MAFFT v. 7.487 (Katoh & Standley 2013) implemented in the CIPRES Science Gateway v. 3.3 (Miller et al. 2010), checked and manually corrected in BioEdit v. 7.1.8 (Hall 1999). The program MrModeltest v. 2.4 (Nylander 2004) was used to determine the best models of nucleotide evolution for each partition (ITS, LSU, SSU, rpb2, and tef1) under Akaike information criterion. Phylogenetic analyses were reconstructed using Maximum Likelihood (ML) and Bayesian Inference (BI) methods available through the CIPRES Science Gateway v. 3.3. The identity/similarity for ITS, LSU, rpb2 and tef1 sequences in studied taxa was calculated using BioEdit.

The ML analysis was performed using RAxML-HPC v. 8.2.12 with a GTRCAT approximation (Stamatakis 2014). Rapid bootstrapping (BS) with 1000 replicates determined the node’s statistical support. The BI analysis was performed with MrBayes v. 3.2.7 (Ronquist et al. 2012). Two Bayesian searches were conducted using default parameters. The B-MCMCMC (Bayesian-Metropolis-coupled Markov chain Monte Carlo) analysis lasted until the average standard deviation of split frequencies was below 0.01, with trees saved every 1000 generations with a burn-in of 25 %. The BI and ML phylogenetic trees were compared visually regarding topological conflicts between the supported clades. Nodes supported by values of ≥ 75 % ML BS and ≥ 0.95 BI Posterior Probability (PP) were deemed well-supported. Phylogenetic trees were visualised in FigTree v. 1.4.3 (Rambaut 2010) and SeaView v. 5.0.5 (Gouy et al. 2010) and edited in Microsoft PowerPoint.

We conducted separate single-marker ML analyses. Since no conflicting clades were detected among these analyses, the individual gene alignments were manually concatenated into three final matrices. Phylogenetic reconstruction was performed using three-gene (LSU, SSU, rpb2) and two five-gene (ITS, LSU, SSU, rpb2, tef1) matrices across three separate phylogenies. Alignments are available as Supplementary File S1 and in figshare (doi: 10.6084/m9.figshare.28889915).

The first phylogenetic analysis, based on the LSU‒SSU‒ rpb2 dataset, included 126 ingroup strains representing 123 species of the Sordariomycetes (Hypocreomycetidae and Sordariomycetidae). Of 4846 characters, including gap regions, 2675 are unique character sites, identified by RAxML as 1040 of LSU, 761 of SSU, and 874 of rpb2. Three members of the order Xylariales (Xylariomycetidae), i.e. Arthrinium hysterinum ICMP 6889, Brachiampulla verticillata ICMP 15065, and Oxydothis metroxylonicola MFLUCC 15-0281, were selected as outgroup based on available sequences. The GTR+I+G models of nucleotide evolution were selected for all partitions.

The second phylogenetic analysis, conducted on the ITS‒LSU‒SSU‒rpb2‒tef1 dataset, included 58 ingroup strains representing 56 species and 16 genera of the Pleurotheciales. Of 6424 characters, including gap regions, 2310 are unique character sites, identified by RAxML as 493 in ITS, 531 in LSU, 331 in SSU, 646 in rpb2, and 309 in tef1. Three members of Bactrodesmium, i.e. B. pallidum CBS 145349, B. diversum CBS 142448, and B. abruptum CBS 145967, were selected as outgroup fide Réblová et al. (2020). The GTR+I+G model of nucleotide evolution was selected for each partition.

The third phylogenetic analysis, conducted also with the ITS‒LSU‒SSU‒rpb2‒tef1 dataset, included an alignment of 34 ingroup strains representing 23 species across four genera of the Rhamphoriales. Of 6249 characters, including gap regions, 1640 are unique character sites, identified by RAxML, as 254 of ITS, 329 of LSU, 188 of SSU, 553 of rpb2, and 316 of tef1. Two members of the Chaetosphaeriales, i.e. Menispora uncinata ICMP 18253 and Curvichaeta curvispora ICMP 18255, were selected as outgroup based on available sequences and previous phylogeny. The following models of nucleotide evolution were selected as GTR+I+G for ITS, LSU, rpb2, and tef1, and GTR+I for SSU.

Evaluating biogeographical patterns through environmental sequence data

The geographic distribution of the studied species was assessed using published environmental ITS sequences from the GlobalFungi database (Větrovský et al. 2020, accessed on 28 Mar. 2025), release 5 (16 Nov. 2023), which includes 84972 samples from 846 studies, comprising a total of 593399355 ITS sequence variants. The assessment followed the approach described in Réblová et al. (2022). The ITS1 and ITS2 sequences were analysed separately, adhering to the GlobalFungi standard, where ITS spacers are archived independently. The relevant spacers from the newly generated ITS sequences were retrieved using the ITSx extractor within the SEED2 platform (Větrovský et al. 2018). The analysis was conducted through an exact match similarity search in GlobalFungi, comparing all unique ITS1 and ITS2 haplotypes from our study against published environmental sequences with identical length and nucleotide sequence.

For more details to each sample recorded in GlobalFungi, see Supplementary Table S2. The distribution maps were produced using the R leaflet package v. 2.0.4.1. (Cheng et al. 2021) for interactive visualisation and ggplot2 (Wickham 2016) for static outputs. The interactive maps are deposited as Supplementary Figs 1, 2.

Fig. 2.

Maximum Likelihood phylogenetic tree of the Sordariomycetes based on analysis of LSU, SSU, and rpb2 DNA sequences. Species names in bold indicate taxonomic novelties; the newly acquired strains are marked with a dark violet asterisk; T denotes ex-holotype strains. Thickened branches indicate branch support with ML BS = 100 % and PP values = 1.0. Branch support of nodes ≥ 75 % ML BS and ≥ 0.95 PP is indicated above or below branches. A hyphen (–) indicates values lower than 75 % ML BS or 0.95 PP.

RESULTS

Isolation and preliminary identification of novel and rare fungi

During our survey, we collected and successfully established axenic cultures of five previously undescribed fungi. In the Rhamphoriales, two unknown, morphologically similar species preliminarily identified as Rhamphoriopsis sp., were collected from decaying wood of Sambucus nigra and Fagus sylvatica. Additionally, we obtained cultures of several uncommon species: Rhamphoria pyriformis (von Höhnel 1913), R. separata (Munk 1948), and Rhodoveronaea varioseptata (Arzanlou et al. 2007).

An unknown fungus, collected from decaying wood of Quercus cerris, was tentatively identified as a member of the Pleurotheciales. Although the sexual morph superficially resembles Melanotrigonum ovale (Réblová et al. 2016), the culture derived from ascospores yielded a different asexual morph with conidiophores reduced to hyaline, holoblastic conidiogenous cells bearing a crown of several denticles with hyaline, allantoid conidia. A previously unknown species of Phaeoisaria was found on the inner surface of decaying bark of Quercus rubra. While it produced effuse colonies composed of loosened synnemata with conidiogenous cells restricted to the apical part on the natural substrate, conidiophores were absent in culture and reduced to single conidiogenous cells. A fifth unknown species, a perithecial ascomycete exhibiting some morphological similarity to the genera Barbatosphaeria (Réblová 2008) and Natantiella (Réblová & Štěpánek 2009), was collected from decaying wood of Carpinus betulus and Ulmus sp. Cultures derived from ascospores remained sterile, and no conidiophores or conidiogenous cells were observed on the natural substrate. Due to its simple sexual morphological characteristics, we were unable to confidently assign this fungus to a known ascomycete genus or taxonomic group.

Genome characteristics of analysed species

As part of this study, we sequenced and assembled high-quality draft genomes for six reference strains representing newly described or known fungal species from three distinct phylogenetic lineages: Pleurotheciales, Rhamphoriales, and one taxon of Sordariomycetes incertae sedis. According to the BUSCO analysis (Benchmarking Universal Single-Copy Orthologs), genome completeness ranged from 96.4 % to 99.1 %, indicating high assembly quality. The number of scaffolds ≥ 200 bp varied considerably, ranging from 129 (Rh. cuprea) to 3972 (E. allantospora), with a median of 515. The scaffold N50 values ranged from 125000 bp to 889571 bp. Genome sizes spanned from approximately 31.7 Mbp in M. curvata to 41.1 Mbp in Pt. uniseptata.

Phylogenetic analyses

The phylogenetic analysis of LSU, SSU, and rpb2 DNA sequences revealed the placement of our strains in relation to members of the Sordariomycetes. The phylogenetic trees generated using both BI and ML methods showed a high degree of concordance. Nodes with ≥ 75 % ML bootstrap (BS) values and ≥ 0.95 Bayesian posterior probability (PP) were considered well supported. The ML phylogenetic tree is shown in Fig. 2. The phylogenetic tree contained 41 well-supported lineages representing 37 families or orders and four genera incertae sedis (Ceratostomella, Melanocrypta, Natantiella, and Pseudostanjehughesia) distributed across four strongly supported clades: Diaporthomycetidae (100 % ML BS/1 PP), Hypocreomycetidae (100/1), Sordariomycetidae (99/1) and Sordariomycetidae incertae sedis (96/1). Majority of the analysed families and orders received high statistical support (100/1), with strong values also observed for Coniochaetales (87/1), Ophiostomatales (99/1), Papulosaceae (99/1), and Pleurotheciales (90/1), Rhamphoriales (91/1), and Xenospadicoidales (86/1). Support for Distoseptisporales (‒/1) and Woswasiaceae (81/‒) was limited to a single analysis.

Our strains were resolved in three distinct lineages: Rhamphoriales in the Sordariomycetidae incertae sedis and Pleurotheciales in the Hypocreomycetidae. Two strains of an undescribed fungus were positioned within the Sordariomycetidae incertae sedis as a separate lineage and represent a new genus and species, Melanocrypta curvata gen. et sp. nov. (ex-type CBS 147592, and CBS 147593). Phylogenetic relationships of the studied fungi were further assessed through subsequent analyses based on five gene markers.

Phylogenetic analyses of the Pleurotheciales, based on the ITS‒LSU‒SSU‒rpb2‒tef1 dataset, revealed 16 well-supported genera. The ML tree is presented in Fig. 3. The newly described Phaeoisaria parallela sp. nov. (ex-type strain CBS 153403) was closely related to P. sparsa (FMR 11939) with strong support (100/1), and together they formed a sister group to the clade comprising P. fasciculata (ex-type CBS 127885) and P. loranthacearum (ex-type CBS 140009). Although ribosomal DNA markers show high sequence similarity between P. parallela and P. sparsa (ITS = 99.2 %, LSU = 100 %), they are clearly separated based on protein-coding genes (rpb2 = 98.6 %, tef1 = 98.8 %).

Fig. 3.

Maximum Likelihood phylogenetic tree of members of the Pleurotheciales based on analysis of ITS, LSU, SSU, rpb2, and tef1 DNA sequences. Species names in bold and highlighted in pink indicate taxonomic novelties; the newly acquired strains and those with novel sequences are marked with a blue asterisk; T denotes ex-holotype strain. To improve visual clarity, species names in white and yellow, repeated throughout the tree, indicate members of the respective genera. Thickened branches indicate branch support with ML BS = 100 % and PP values = 1.0. Branch support of nodes ≥ 75 % ML BS and ≥ 0.95 PP is indicated above or below branches. A hyphen (–) indicates values lower than 75 % ML BS or 0.95 PP.

An undescribed fungus resembling Melanotrigonum was positioned on a distinct branch as a sister to Pleurothecium, although this relationship was not statistically supported in the ML analysis (58/0.96). It is introduced here as a new genus and species, Echinodenticula allantospora gen. et sp. nov. (ex-type strain CBS 147513). Pleurotheciella uniseptata CBS 147511 was grouped as a sister to P. uniseptata (S-936).

In the phylogenetic analyses based on ITS, LSU, SSU, rpb2, and tef1 sequences, we evaluated the relationships among members of the Rhamphoriales. The ML tree is presented in Fig. 4. The order comprised the genera Rhamphoria (100/1), Rhamphoriopsis (99/0.99), Rhodoveronaea (100/1), and Xylolentia (100/1), all forming strongly supported monophyletic clades. Although the Rhamphoriopsis clade was not statistically supported in the three-gene phylogeny (65/0.87), the genus was strongly supported based on five genes. Our strains of R. pyriformis (CBS 153406), R. separata (CBS 153404, CBS 153405), and Rhodoveronaea varioseptata (CBS 153411) were placed within the order, together with three strains of previously unknown fungi, intially classified as Rhamphoriopsis sp. They correspond to two new species: Rh. cuprea sp. nov. (ex-type CBS 147991) and Rh. denticulata sp. nov. (ex-type CBS 147996 and CBS 147997). The closest relative of Rh. denticulata is the holomorphic species Rh. muriformis (100/1), while Rh. cuprea is positioned on a separate branch as a sister to the remaining Rhamphoriopsis species. Sequence identity between Rh. denticulata and Rh. muriformis across five genetic markers was as follows: ITS = 98.2–98.6 %, LSU = 99.9 %, rpb2 = 95.2 %, and tef1 = 96.7 %.

Fig. 4.

Maximum Likelihood phylogenetic tree of members of the Rhamphoriales based on analysis of ITS, LSU, SSU, rpb2, and tef1 DNA sequences. Species names in bold and highlighted in pink indicate taxonomic novelties; the newly acquired strains and those with novel sequences are marked with a blue asterisk; T denotes ex-holotype strain. To improve visual clarity, species names in white and yellow, repeated throughout the tree, indicate members of the respective genera. Thickened branches indicate branch support with ML BS = 100 % and PP values = 1.0. Branch support of nodes ≥ 75 % ML BS and ≥ 0.95 PP is indicated above or below branches. A hyphen (–) indicates values lower than 75 % ML BS or 0.95 PP.

The AI-generated figures in the background of the phylogenetic trees (Fig. 3, 4) were created with ChatGPT (OpenAI 2025 v. Apr. 2025, retrieved on 6 Apr. 2025).

Biogeography

Biogeographic patterns were assessed for all species within the genera Phaeoisaria, Rhamphoria, and Rhamphoriopsis, to which the newly described and rare species belong, based on the availability of ITS sequence data. The genera Echinodenticula and Melanocrypta are currently monotypic. The geographic distribution of individual species, based on exact georeferenced samples in which the respective taxa were identified, is presented in Figs 5, 6 and Supplementary Figs 1, 2.

Fig. 5.

Geographic distribution of Phaeoisaria species based on environmental DNA data from the GlobalFungi database. Each dot represents an exact georeferenced sample in which the respective species was identified. The number of samples (n) for each species is indicated in parentheses.

Fig. 6.

Geographic distribution of Rhamphoria and Rhamphoriopsis species based on environmental DNA data from the GlobalFungi database. Each dot represents an exact georeferenced sample in which the respective species was identified. The number of samples (n) for each species is indicated in parentheses.

Of the 38 species of Phaeoisaria listed in MycoBank, molecular DNA data are available for 20 species, and 19 of these had an exact match in the GlobalFungi database. The only species without an exact match was P. siamensis, which showed a best match of 97 % similarity to deposited ITS1 or ITS2 environmental sequences. The sampled taxa showed similar ecological preferences, being predominantly associated with forest soil. Of the 756 environmental samples, the majority originated from forests and woodlands biomes (85.4 %), while croplands (6.6 %), grasslands (1.9 %), and shrublands (0.5 %) represented less common habitats. Soil was the principal substrate (89.9 %), with roots (2.4 %) and deadwood (1.9 %) occurring only occasionally. The distribution patterns of Phaeoisaria species reveal a clear biogeographical differentiation and can be grouped into three categories: (1) species that are globally widespread across all climatic zones except the boreal, such as P. annesophieae, P. goiasensis, and P. loranthacearum; (2) species predominantly found in tropical and humid subtropical regions, either with a broad global range, e.g. P. clematidis, P. dalbergiae, P. ellipsoidea, P. guttulata, P. obovata, P. pseudoclematidis, P. sedimenticola, and P. synnematica, or restricted to Southeast Asia such as P. aquatica, P. filiformis, P. laianensis, P. microspora, and P. motuoensis; and (3) species typically associated with temperate regions, with occasional records in boreal zones, including P. fasciculata, P. parallela, and P. sparsa. The most dominant species was P. synnematica, detected in 377 samples, followed by P. sparsa (74 samples) and P. loranthacearum (71 samples). Moderately abundant species included P. annesophieae (59 samples), P. goiasensis (44 samples), and P. ellipsoidea (30 samples). The remaining species were recorded at low frequencies, with sample counts ranging from 1 to 17. For detailed information on occurrences by country, refer to Fig. 5 and Supplementary Fig. 1.

Out of the 14 recognised species and varieties of Rhamphoria listed in MycoBank, molecular DNA data are available for only R. delicatula, R. pyriformis, and R. separata, all of which occur on decayed wood (von Niessl 1876, Munk 1948, Réblová & Štěpánek 2018, this study). The analysed Rhamphoria species exhibited a predominantly temperate distribution, with occurrences in broadleaf and mixed forest biomes, as well as extending into the boreal zone within subpolar coniferous forests, across multiple European countries (Fig. 6). The most frequently recorded species, R. delicatula, was present in 60 samples, mostly from deadwood, rarely identified in soil and roots, within broadleaf, mixed, and subpolar coniferous forests biomes in the Czech Republic, Finland, Germany, Hungary, and the Netherlands. Rhamphoria pyriformis was detected in 16 samples, primarily from deadwood, less frequently from litter and soil, in broadleaf, mixed, and subpolar coniferous forests across the Czech Republic, Finland, Germany Hungary, and the Netherlands. Rhamphoria separata was found in eight samples from soil, deadwood, and shoots within broadleaf forests, with records from the Czech Republic, Estonia, Finland, France, Germany, and Italy.

DNA data were available for all six known Rhamphoriopsis species. They displayed primarily temperate distribution, with most records originating from Europe, East Asia (particularly China), and less often from Africa (Fig. 6). Rhamphoriopsis aquimicrospora, initially described from submerged wood in China (Yang et al. 2023), was the most frequently detected species. It was recorded in 38 samples from soil, air, and water across aquatic, anthropogenic, cropland, forest, grassland, and shrubland ecosystems. While most samples originated from China, it was also identified in Italy, Ivory Coast, Japan, and Zambia. Based on its occurrence in 25 samples, Rh. muriformis is the next most common species. Originally described from decaying wood of Buxus sempervivens in France (Réblová & Štěpánek 2018), it has been primarily identified in soil (rhizosphere soil, topsoil), deadwood, shoots, with occasional detections in air, spanning anthropogenic, forest, grassland, and shrubland ecosystems in Austria, Belgium, China, Czech Republic, Italy, Poland, Spain, Tunisia, and the UK. Rhamphoriopsis cuprea (18 samples) and Rh. denticulata (five samples) were primarily detected in soil, air, and roots across Europe. Their distribution suggests a strong association with terrestrial habitats, particularly soil and plant-associated environments. For further details, see the biogeography notes for each species. Rhamphoriopsis hyalospora, originally described from decaying wood in China (Lin et al. 2023), was identified in two samples from dust and soil in anthropogenic and forest environments in China and South Korea. Rhamphoriopsis synnematosa, collected on decaying wood in South Africa (Crous et al. 2023), was present in a single sample from forest soil in China. Environmental data for Rh. sympodialis (Hyde et al. 2020), which was described from decaying wood in China, are absent from the Global Fungi database. The ITS sequence data of Rh. glauca are not available.

No records of the newly described species, E. allantospora and M. curvata, were found in the GlobalFungi database.

Taxonomy

New and previously known species accepted in this study are listed alphabetically.

Echinodenticula Réblová, gen. nov. MB 858886.

Etymology: From Latin echīnus derived from the Ancient Greek word ekhînos (hedgehog or sea urchin), and denticula diminutive of dēns (small tooth). Referring to conidiogenous cells with numerous denticles.

Type species: Echinodenticula allantospora Réblová

Description: Sexual morph. Ascomata perithecial, non-stromatic, immersed to semi-immersed, becoming superficial, papillate, subglobose to conical, glabrous, dark brown to black. Ostiole periphysate. Ascomatal wall leathery to carbonaceous, two-layered. Paraphyses abundant, septate, hyaline, longer than the asci. Asci unitunicate, persistent, cylindrical-clavate, stipitate, broadly rounded to truncate at the apex, with a non-amyloid apical annulus, 8-spored. Ascospores fusiform, straight or slightly curved, hyaline, septate, mono- to biseriate within the ascus. Asexual morph observed only in culture. Colonies effuse, mycelium hyaline. Conidiophores reduced to conidiogenous cells. Conidiogenous cells integrated, intercalary, mono- and polyblastic with 1 to several denticles, oval, subglobose to ampulliform, hyaline; conidiogenesis holoblastic-denticulate. Conidia allantoid, hyaline, with and without septa, secession schizolytic.

Echinodenticula allantospora Réblová, sp. nov. MB 858890. Fig. 7.

Fig. 7.

Echinodenticula allantospora (ex-type strain CBS 147513). A, B. Ascomata on natural substrate. C. Longitudinal section of the ascomatal wall. D–F. Asci with ascospores, and paraphyses. G–N. Conidiogenous cells with conidia. O, P. Conidia. Q. Colony morphology after 4 wk on CMD, MLA, OA, and PCA (from left to right). A–F. On natural substrate. G–P. On MLA. Scale bars: A, B = 500 µm; C = 20 µm; D–P = 10 µm; Q = 1 cm.

Etymology: From Greek allantos (sausage-shaped) and -spora (spore or seed). Referring to the shape of the conidia.

Typus: Czech Republic, South Moravian Region, Břeclav district, Valtice, Rendezvous National Nature Monument, 200 m.a.s.l., on decaying wood of Quercus cerris, 28 Oct. 2018, M. Réblová, M.R. 4023 (holotype PRA-22500, culture ex-type CBS 147513).

Description on the natural substrate: Sexual morph. Ascomata perithecial, non-stromatic, immersed to semi-immersed, becoming superficial, gregarious, 180–250 μm diam., subglobose to conical, papillate, brown to black, glabrous, opening by a rounded pore. Ostiole periphysate. Ascomatal wall leathery to fragile, 32–38 μm thick, 2-layered. Outer layer consisting of dark brown, polyhedral cells of textura prismatica with opaque walls; towards the interior grading into paler, more elongated cells. Inner layer consisting of several rows of thin-walled, hyaline, polyhedral cells. Paraphyses abundant, persistent, septate, hyaline, 2.5–3.5 μm wide, longer than the asci. Asci 59.5–77.5 μm long in the sporiferous part, 8–9(–10) μm wide (mean ± SD = 68.4 ± 5.9 × 8.7 ± 0.5 μm), with a stipe 20–46.5 μm long, cylindrical, broadly rounded to truncate at the apex, apex with a non-amyloid apical annulus ca 2.5 μm wide, 1–1.5 μm high, 8-spored. Ascospores (11.5–)12.5–15.5(–16.5) × 3.5–4.5 μm (mean ± SD = 13.9 ± 0.9 × 4.1 ± 0.3 μm), fusiform, straight to slightly curved, hyaline, 3-septate, not constricted at the septa, smooth, arranged obliquely uniseriate, sometimes partially 2-seriate. Asexual morph not observed.

Culture characteristics: On CMD colonies 6–7 mm diam., circular, margin entire, flat, cobwebby to mucoid, isabelline, reverse creamy. On MLA 7–10 mm diam., circular, margin slightly lobate, raised, deeply furrowed with crack in the folds, mucoid, sparsely funiculose at the inoculation block, cobwebby at the margin, pink-creamy, reverse of the same colour. On OA 8–9 mm diam., circular, flat, margin entire, mucoid to cobwebby, whitish to isabelline, reverse isabelline. On PCA 7–8 mm diam., circular, flat, margin filiform to rhizoidal, velvety, pale salmon, reverse of the same colour. Sporulation was abundant on MLA, OA and PCA, absent on CMD.

Description in culture: Colonies on MLA effuse. Mycelium composed of hyaline, septate hyphae, 1–2 μm wide. Conidiophores reduced to single conidiogenous cells. Conidiogenous cells 3.7–9.6(–13.5) × 2–6(–7.5) μm, integrated, terminal and intercalary, mono- and polyblastic, sympodially extending, oval, subglobose to ampulliform, hyaline, smooth, with one to several hyaline denticles, 1.5–2 × 0.5 μm; conidiogenesis holoblastic-denticulate. Conidia 6–10(–10.5) × 2–2.5 μm (mean ± SD = 8.2 ± 0.9 × 2.1 ± 0.2 μm), allantoid, hyaline, broadly rounded at the ends, mostly aseptate, occasionally with 1–3 septa, not constricted at the septa, smooth, secession schizolytic; sporulation associated with the aerial part of the colony. Sexual morph not observed.

Habitat and geographical distribution: Saprobic on decaying wood of Quercus cerris in the Czech Republic. No identical ITS sequences were found in the GlobalFungi database.

Notes: Within the Pleurotheciales, E. allantospora resembles M. ovale (Réblová et al. 2016) in its dark, glabrous non-stromatic ascomata, stipitate non-amyloid asci, fusiform 3-septate ascospores, and conidiophores that are reduced to single conidiogenous cells bearing denticles in culture. However, in M. ovale, the paraphyses disintegrate at maturity, the conidiogenous cells are pigmented, mostly monoblastic with a single apical denticle, and produce oval to beanshaped, 1-septate pigmented conidia that leave a pore upon detachment.

Melanocrypta Réblová, gen. nov. MB 858893.

Etymology: From Greek melano- derived from mélās (black), and -crypta derived from kruptós (hidden or concealed). Referring to the dark immersed ascomata.

Type species: Melanocrypta curvata Réblová

Description: Sexual morph. Ascomata perithecial, non-stromatic, immersed with protruding necks, venter globose to subglobose, glabrous, dark brown, neck rostrate, central, cylindrical, straight to slightly flexuous, sulcate at the apex. Ostiole periphysate. Ascomatal wall leathery to carbonaceous, two-layered. Paraphyses abundant, septate, hyaline, tapering, longer than the asci. Asci unitunicate, persistent, cylindrical-clavate, stipitate, broadly rounded at the apex, contracted below the sporiferous part into a long tapering stipe, floating freely within the centrum at maturity, with a non-amyloid apical annulus, 8-spored. Ascospores ellipsoidal to oblong, sometimes slightly curved, hyaline, aseptate, biseriate within the ascus. Asexual morph not observed.

Melanocrypta curvata Réblová, sp. nov. MB 858895. Fig. 8.

Fig. 8.

Melanocrypta curvata (ex-type strain CBS 147592). A, B. Ascomata on natural substrate. C. Longitudinal section of the ascomatal wall. D–H. Asci with ascospores, and paraphyses. I. Colony morphology after 4 wk on CMD, MLA, OA, and PCA (from left to right). A–H. On natural substrate. Scale bars: A, B = 500 µm; C = 20 µm; D–H = 10 µm; I = 1 cm.

Etymology: From Latin curvatus (curved). Referring to the slightly curved ascospores.

Typus: Czech Republic, South Moravian region, Břeclav district, Lanžhot, Ranšpurk National Nature Reserve, ca 150 m.a.s.l., on decaying wood of Carpinus betulus, 26 Oct. 2018, M. Réblová, M.R. 3998 (holotype PRA-22506, culture ex-type CBS 147592).

Description on the natural substrate: Sexual morph. Ascomata perithecial, non-stromatic, scattered, immersed with protruding necks, venter 430−500 µm diam., globose to subglobose, glabrous, dark brown, neck 97−130 µm wide, up to 850 µm long, rostrate, central, cylindrical, straight to slightly flexuous, sulcate at the apex. Ostiole periphysate. Ascomatal wall leathery to carbonaceous, 20−25 µm thick, two-layered. Outer layer consisting of thick-walled, brown, cells of textura epidermoidea to prismatica, cells tend to be more flattened and paler towards the interior. Inner layer consists of several rows of thin-walled, hyaline, flattened cells. Paraphyses abundant, septate, hyaline, 2.5–4 µm wide, tapering to ca 1.5 µm, longer than the asci. Asci 92–116.5 × 9.5–10.5 µm (mean ± SD = 103.9 ± 7.3 × 9.9 ± 0.4 μm), 64–77 µm (mean ± SD = 70.7 ± 5.2 μm) long in the sporiferous part, broadly rounded at the apex, cylindrical-clavate, contracted below the sporiferous part into a slender, tapering, thread-like stipe that partially disintegrate, floating freely within the centrum at maturity, base of the stipe oval to bulbous, apical annulus non-amyloid, 2.5–3 µm wide, 1–1.5 µm high, 8-spored.

Ascospores 11.5–15.5 × 3.5–4.5 µm (mean ± SD = 13.3 ± 1.2 × 3.9 ± 0.3 μm), ellipsoidal to oblong, sometimes slightly curved, hyaline, aseptate, biseriate within the ascus. Asexual morph not observed.

Culture characteristics: On CMD colonies 7–8 mm diam., circular, flat, margin entire, lanose, beige-brown, brown at the margin, reverse dark brown. On MLA colonies 8–10 mm diam., circular, convex, margin entire, lanose, beige-brown, reverse almost black. On OA colonies 8–9 mm diam., circular, raised, margin entire, lanose with numerous colourless exudates, grey-brown, reverse dark brown. On PCA colonies 8–9 mm diam., circular, raised, margin entire, lanose, beige-brown, dark brown at the margin, reverse dark brown. Sporulation absent on all media.

Description in culture: Colonies on MLA effuse. Mycelium composed of subhyaline to pale brown, septate hyphae, 1.5–3 μm wide. Asexual morph. Conidiophores, conidiogenous cells and conidia absent. Sexual morph not observed.

Additional material examined: Czech Republic, South Moravian region, Břeclav district, Lanžhot, Ranšpurk National Nature Reserve, ca 150 m.a.s.l., on decaying wood of Ulmus sp., 26 Oct. 2018, M. Réblová, M.R. 4000 (PRA-22507, culture CBS 147593).

Habitat and geographical distribution: Saprobic on decaying wood of Carpinus betulus and Ulmus sp. in the Czech Republic. No identical ITS sequences were found in the GlobalFungi database.

Notes: Melanocrypta curvata closely resembles other lignicolous fungi, N. ligneola (Réblová & Štěpánek 2009) and members of Barbatosphaeria (Réblová 2008, Réblová et al. 2015). However, N. ligneola can be distinguished by its glabrous, narrower neck, ascospores with rounded ends and the distinctive shape of its ascus. The sporiferous region is typically swollen in the middle and tapers below into a long, slender stipe, while the upper part, just below the apex, becomes abruptly narrower. Barbatosphaeria differs in having ascomata usually adorned with short flexuous hairs, arranged in circular or oval groups or nests, as well as in the morphology of elongating ascogenous hyphae and septate ascospores.

Phaeoisaria parallela Réblová, sp. nov. MB 858896. Fig. 9.

Fig. 9.

Phaeoisaria parallela (ex-type strain CBS 153403). A, B. Synnemata. C–E. Conidiogenous cells with a sympodially extending rachis bearing denticles. F, M, N. Conidia. G–L. Conidiogenous cells with conidia. O. Colony morphology after 4 wk on CMD, MLA, OA, and PCA (from left to right). A–F. On natural substrate. G–N. On PCA. Scale bars: A–N = 10 µm; O = 1 cm.

Etymology: From Latin parallelus (parallel or side by side). Referring to the arrangement of the synnematous conidiophores, which grow closely aligned, parallel to one another.

Typus: Czech Republic, South Bohemian Region, Novohradské hory Mts, Horní Stropnice, Meziluží, ca 600 m.a.s.l., on inner side of decaying bark of Quercus rubra, 11 Oct. 2024, M. Réblová, M.R. 4108 (holotype PRA-22501, culture ex-type CBS 153403).

Description on the natural substrate: Sexual morph not observed. Asexual morph. Colonies effuse, tufted, dark brown, grey to olivaceous-grey when sporulating. Synnemata up to 70 µm high, 10–14 µm wide, indeterminate, cylindrical, in the upper part loosely arranged, brown. Conidiophores 45.5–70 × 2.5–3.5 µm, macronematous, synnematous, sparsely branched towards the apices, cylindrical, septate, brown, parallel along most of the length, flexuous and loosened up at the top. Conidiogenous cells (9.5–)11–15.5(–19) × 2.5–3.5 µm, slightly tapering below the apex 1.5–2.5 µm, slightly swollen at the apex 2–2.5 µm wide, integrated, terminal, polyblastic, sympodially extending, cylindrical, subhyaline to pale brown, paler at the apex, smooth, with several denticles, ca 1 × 0.5 µm; conidiogenesis holoblastic-denticulate. Conidia (6.5–)7–9(–10) × 1.5–2 µm (mean ± SD = 7.9 ± 0.7 × 1.8 ± 0.2 μm), solitary, dry, fusiform, oblong to elongate obovoid, with a truncate basal scar, rounded at the apex, hyaline, aseptate, smooth, secession schizolytic.

Culture characteristics: On CMD colonies 21–22 mm diam., circular, convex with flat margin, margin entire to rhizoidal, farinose, floccose, beige-brown, creamy and mucoidcobwebby at the margin, reverse beige. On MLA colonies 17–19 mm diam., circular, raised, margin slightly lobate, floccose, lanose towards the periphery, zonate, olivaceous grey, whitish grey at the centre, dark olivaceous grey at the margin, reverse dark olivaceous grey. On OA colonies 28–29 mm diam., circular, flat, margin entire, floccose, farinose, partially mucoid at the centre, olivaceous grey, darker at the margin, reverse olivaceous grey. On PCA colonies 29–30 mm diam., circular, flat, margin entire, lanose, cobwebby at the margin, camel brown, creamy to pale ochre towards the periphery, reverse dark ochre. Sporulation was abundant on CMD, MLA, and OA, absent on PCA.

Description in culture: Colonies on MLA effuse. Mycelium composed of hyaline, septate hyphae, 1.5–2.5 μm wide. Synnemata absent. Conidiophores reduced to single conidiogenous cells. Conidiogenous cells 3.5–6 × 2–2.5 µm, integrated, terminal and intercalary, polyblastic, sympodially extending, oval to subglobose, hyaline, smooth, with several denticles 1 × 0.5 µm; conidiogenesis holoblastic-denticulate. Conidia solitary, dry, hyaline, aseptate, smooth, with one or two large guttules, with a truncate basal scar, secession schizolytic, of two types (formed on CMD, MLA and OA): 4.5–8.5(–9.5) × 2–3 µm (mean ± SD = 6.6 ± 1.1 × 2.5 ± 0.1 μm), oblong to dacryoid, straight or slightly curved; (3.5–)4–5.5 × 2–2.5 µm (mean ± SD = 4.7 ± 0.4 × 2.5 ± 0.2 μm), obovoid to somewhat guttuliform; sporulation associated with the aerial part of the colony. Sexual morph not observed.

Habitat and geographical distribution: Saprobe on decaying bark of Quercus rubra in the Czech Republic. According to GlobalFungi, P. parallela is an uncommon species, detected in five environmental samples primarily from soil, and occasionally from air and deadwood within broadleaf forest and, less frequently, cropland biomes in the Czech Republic, Italy, and Turkey.

Notes: Among the known species, P. parallela resembles P. aguilerae (Castañeda-Ruiz et al. 2002) and P. sparsa (Sutton 1973) in conidial shape but differs from both by its shorter, aseptate conidia and by the arrangement of conidiophores. Although no molecular data are available for P. aguilerae, phylogenetic analyses support a close relationship between P. parallela and P. sparsa, for which DNA sequences are available.

Morphologically, P. parallela and P. sparsa share similarly shaped conidia on the natural substrate, ranging from fusiform to oblong-clavate, but they clearly differ in conidial size, and other morphological features of synnemata, denticles, and conidiogenous cells. On the natural substrate, P. parallela produces smaller, aseptate conidia (6.5–)7–9(–10) × 1.5–2 µm on denticles 1 µm high, whereas P. sparsa has larger, 0–3-septate conidia 10–15.5 × 2.5–3 µm arising from longer denticles 2 µm high (Sutton 1973). On the natural substrate, the synnemata of P. parallela are paler, shorter (up to 70 × 10−14 µm), composed of loosely arranged conidiophores, apically sparsely branched bearing cylindrical conidiogenous cells with slightly swollen apices, whereas synnemata of P. sparsa are distinctly opaque, taller (up to 200 × 30 µm), composed of tightly arranged conidiophores bearing subulate conidiogenous cells (Sutton 1973).

In culture, P. parallela produced conidia that differed in shape and size from those observed on the natural substrate. The conidia were broader, larger conidia were oblong to dacryoid, while smaller conidia were obovoid to somewhat guttuliform and typically contained one or two guttules. Synnemata were absent in culture, and conidiophores were reduced to single conidiogenous cells.

Phaeoisaria sparsa B. Sutton, Mycol. Pap. 132: 87. 1973. Fig. 10.

Fig. 10.

Phaeoisaria sparsa (FMR 11939). A. Synnemata. B, M–O. Conidia. C. Apex of the synnema showing conidiogenous cells with attached conidium. D, E. Details of colony showing funiculose hyphae with sporulating conidiophores. F. Conidiogenous cells with conidia. G–L. Conidiophores with conidiogenous cells and conidia. P. Colony morphology after 4 wk on CMD, MLA, OA, and PCA (from left to right). A–C. On natural substrate. D–O. On CMD. Scale bars: A = 20 µm; B, C, M–O = 5 µm; D, E = 500 µm; F–L = 10 µm; P = 1 cm.

Description on the natural substrate: See Sutton (1973) and Hughes (1978).

Culture characteristics: On CMD colonies 27–29 mm diam., circular, raised with a flat margin, margin entire, funiculose, cobwebby to mucoid towards the periphery, white-beige, margin isabelline, reverse isabelline. On MLA colonies 20–21 mm diam., circular, slightly convex, margin finely undulate, velvety, furrowed, grey, margin olivaceous grey, reverse dark olivaceous grey. On OA colonies 31–33 mm diam., circular, flat, margin entire, cobwebby to mucoid, farinose with sparse funiculose ropes of hyphae at the margin, olivaceous brown, beige towards the periphery, reverse olivaceous brown. On PCA colonies 28–30 mm diam., circular, raised with a flat margin, margin entire, velvety, funiculose at the inoculation block, white-beige, creamy at the margin, reverse pale brown. Sporulation was abundant on CMD, MLA and PCA, moderate on OA.

Description in culture: Colonies on CMD effuse. Mycelium composed of hyaline, subhyaline to brown, septate hyphae, 1.5–2.5 μm wide. Synnemata absent. Conidiophores macronematous, semi-macronematous or reduced to a single conidiogenous cell. Conidiogenous cells 5–18 × 2–3.5 µm, integrated, terminal and intercalary, polyblastic, sympodially extending, oval, cylindrical to subulate, hyaline, smooth, with several denticles, 1.5 × 0.5 µm; conidiogenesis holoblastic-denticulate. Conidia solitary, dry, hyaline, aseptate, smooth, with one or two large guttules, with a truncate basal scar, secession schizolytic, of two types (formed on all media): 5.5–8 × 2–2.5 µm (mean ± SD = 7.2 ± 0.7 × 2.2 ± 0.2 μm), on MLA 4.5–7.5 × 2–3 µm (mean ± SD = 6.1 ± 0.8 × 2.6 ± 0.3 μm), oblong to elongate clavate, straight or slightly curved, rounded at the apex, hyaline, aseptate; 3–3.5 × 1.5–2 µm (mean ± SD = 3.4 ± 0.3 × 1.7 ± 0.2 μm), on MLA 3.5–4.5 × 1.5–3 µm (mean ± SD = 3.8 ± 0.4 × 2.2 ± 0.4 μm), guttuliform to dacryoid; sporulation associated with the aerial part of the colony. Sexual morph not observed.

Material examined: Spain, Aragón, Ordesa y Monte Perdido National Park, Torla, on dead twig, 23 Mar. 2011, M. Hernández-Restrepo & J. Gené, culture FMR 11939.

Habitat and geographical distribution: Saprobe, originally described from decaying bark of Acer spicatum, with additional reports from decaying wood of various deciduous trees, e.g. Alnus sp., Betula papyrifera, Corylus sp., Populus tremuloides, Populus balsamifera, Salix sp. in Canada, New Zealand, and Spain (Sutton 1973, MyCoPortal, this study). According to GlobalFungi database, this species was the second most abundant within the genus, detected in 66 samples. Most records originated from forests and woodlands (83 %), followed by anthropogenic habitats (10 %), croplands (5.7 %), and to a lesser extent, grasslands and desert biomes (1.3 %). The majority of samples were soil-related (66 %), with additional occurrences from dead organic matter (17 %), air (9 %), and roots or shoots (7 %). The samples predominantly originated from temperate zones across three continents: North America (USA), Europe (Czech Republic, Belgium, Germany, Latvia, Estonia, Italy, Spain, Sweden), and East Asia (China, South Korea). Two exceptions included records from Mediterranean climates (Seville, Spain) and a single occurrence in the Antarctic polar zone.

Notes: In our phylogenetic analyses, P. sparsa is represented by strain FMR 11939, isolated from a dead twig in Spain (Hernández-Restrepo et al. 2017). Our material corresponds well with the protologue (Sutton 1973) based on observations on natural substrate: synnemata 220–250 × 17–28 µm, conidiogenous cells 15–20.5 × 3–3.5 µm with denticles 1.5–2 × 0.5 µm, and conidia 12–17 × 2.5–3.5 µm. It is well defined by its 0–3-septate, fusiform to oblong-clavate conidia and subulate conidiogenous cells, which are mostly confined to the apices of the synnemata, giving them a somewhat penicillate appearance. In culture, the synnemata were absent and the fungus produced two types of conidia differing in shape and size (oblong to elongate clavate and guttuliform to dacryoid), a feature also observed in P. parallela. In culture, the conidia were consistently aseptate. Furthermore, in culture, the size and shape of both conidial types were comparable across all media, although on MLA, the oblong to elongate conidia were slightly shorter, while the guttuliform to dacryoid conidia were slightly longer (see description). The longer conidia, although comparable in shape to those on the natural substrate, were shorter in culture (5.5–8 × 2–2.5 µm on CMD, 4.5–7.5 × 2–3 µm on MLA).

Phaeoisaria sparsa is similar to P. parallela in conidial characteristics; however, both species are morphologically well differentiated. As only ITS and LSU sequences of the strain FMR 11939 were available in GenBank, we generated sequences from other gene regions to complete its molecular dataset. The combined morphological and molecular differences support the recognition of P. parallela and P. sparsa as distinct species. For a detailed morphological comparison of both species, see the notes under the description of P. parallela.

Rhamphoria separata Munk, Dansk Bot. Ark. 12: 13. 1948. Fig. 11.

Fig. 11.

Rhamphoria separata (CBS 153404). A, B. Ascomata on natural substrate. C. Longitudinal section of the ascomatal wall. D, E. Asci with ascospores and paraphyses. F–H. Ascospores. I–M. Conidiophores, conidiogenous cells and conidia. N. Conidia. O. Detail of the colony. P. Colony morphology after 4 wk on CMD, MLA, OA, and PCA (from left to right). A–H, L–N. On natural substrate. I–K, O. On MLA. Scale bars: A = 100 µm; B = 500 µm; C–N = 10 µm; O = 0.5 cm; P = 1 cm.

Description on the natural substrate: Sexual morph. Ascomata perithecial, non-stromatic, grouped or solitary, superficial, venter 200−320 µm diam., conical to pyriform, often laterally collapsed upon drying, dark brown, glabrous, with a distinct papilla or a short beak. Ostiole periphysate. Ascomatal wall leathery, 21−36 µm thick, two-layered. Outer layer consisting of thick-walled, brown, polyhedral cells with opaque walls of textura prismatica, cells tend to be more brick-like and flattened and paler towards the interior. Inner layer consists of several rows of thin-walled, hyaline, polyhedral to flattened cells. Paraphyses abundant, septate, hyaline, 2–3.5 µm wide, longer than the asci. Asci 148–210(–223) × 11–12.5 µm (mean ± SD = 184.9 ± 23.5 × 12.2 ± 0.5 μm), 119–152 µm (mean ± SD = 134.2 ± 13.3 μm) long in the sporiferous part, unitunicate, persistent, truncate at the apex, cylindrical, with a tapering stipe, apical annulus non-amyloid, 4.5–5 µm wide, 1.5–2.5 µm high, 8-spored. Ascospores 18–24 × 7.5–9.5 µm (mean ± SD = 20.9 ± 1.7 × 8.2 ± 0.6 μm), ellipsoidal, ellipsoidal-fusiform, slipper-shaped, occasionally clavate, hyaline, very irregularly muriform, usually with 4−9 transverse septa and 1−3 incomplete longitudinal septa, sometimes slightly constricted at the septa, with a large guttule at each cell, smooth-walled, obliquely uniseriate within the ascus. Sexual morph not observed asexual morph. Colonies effuse, scattered, tufted, brown, whitish-grey when sporulating. Conidiophores 35−93 × 2–3 µm, basal cell bulbous or slightly curved 3.5–6.5 µm wide, macronematous, mononematous, in loose fascicles or solitary, cylindrical, flexuous, unbranched, brown, paler towards the apex, smooth-walled. Conidiogenous cells 10–14.5(–16) × 2–3 µm, holoblastic, integrated, terminal, polyblastic, sympodially extending, subulate, sometimes slightly swollen, tapering to ca 1.5 μm, apex often swollen, 2.5–3 µm wide, pale brown to subhyaline, paler at the apex, smooth, with several denticles, ca 0.5 × 0.5 µm; conidiogenesis holoblastic-denticulate. Conidia 4–5.5 × 1.5 µm (mean ± SD = 4.9 ± 0.3 × 1.4 ± 0.1 μm), oblong, straight, slightly tapering towards the base, rounded at the apex, hyaline, aseptate, with a basal scar, smooth-walled, secession schizolytic.

Culture characteristics: On CMD colonies 10.5–12 mm diam., circular, flat, margin entire to slightly lobate, mucoid, isabelline, reverse of the same colour. On MLA colonies 10.5–11 mm diam., circular, convex, margin entire to slightly lobate, mucoid, furrowed becoming cerebriform with multiple cracks in the folds, creamy to isabelline, reverse creamy. On OA colonies 11–12 mm diam., circular, slightly raised with flat margin, margin entire, mucoid, creamy to pale yellow at the centre, isabelline towards the periphery, reverse creamy. On PCA colonies 8–9.5 mm diam., circular, flat, margin entire, mucoid, creamy at the centre, isabelline towards the periphery, reverse isabelline. Sporulation was abundant on MLA, absent on CMD, OA and PCA.

Description in culture: Colonies on MLA effuse. Mycelium composed of subhyaline to pale brown, septate hyphae, 1–2.5 μm wide, often monilioid with thick-walled cells, 3.5–5.5 μm wide. Conidiophores and conidiogenous cells are like those on the natural substrate. Conidiophores 53.5–90 × 2.5–3.5 µm. Conidiogenous cells 8–16.5 × 2.5–3 µm, tapering to ca 1.5 μm, subulate, apex sometimes swollen, subhyaline, paler towards the apex, with several denticles, 0.5 × 0.5 µm. Conidia hyaline, aseptate, of two kinds: oblong to suballantoid 3.5–4.5 × 1–1.5 µm (mean ± SD = 4.2 ± 0.3 × 1.2 ± 0.2 μm), guttuliform 2. 3–3.5(–5) × 1.5–2(–2.5) µm (mean ± SD = 2.8 ± 0.3 × 2.0 ± 0.2 μm), smooth-walled; sporulation associated with the aerial part of the colony.

Materials examined: Czech Republic, South Bohemian Region, Novohradské hory Mts., Horní Stropnice, Bedřichovský potok Natural Monument, on decaying wood of Quercus sp., 7 Oct. 2024, M. Réblová, M.R. 4109 (PRA22502, culture CBS 153404); ibid., associated with R. pyriformis and N. lignicola, M.R. 4116A (PRA-22503, culture CBS 153405).

Habitat and geographical distribution: Saprobe on decaying wood of Fagus sylvatica and Quercus sp. in the Czech Republic, Denmark, France, and the Netherlands (Munk 1948, 1957, MyCoPortal, this study). According to the GlobalFungi database, samples with identical ITS sequences originate from temperate climates and were isolated from deadwood, soil, and shoots in montane, mixed broadleaf, and broadleaf forest biomes across the Czech Republic, Estonia, Finland, France, Germany, and Italy.

Notes: Munk (1948, 1957) distinguished R. separata from R. pyriformis by ascospores that lack the ability to produce ascoconidia. Ascoconidia represent a type of asexual spores that are produced by budding from the surface of ascospores while still inside the ascus. Munk noted that both species were found in the same locality and their ascomata occurred on the same branch. However, this distinction was later questioned by Müller & Samuels (1982). Réblová & Štěpánek (2018) examined a specimen of R. pyriformis CBS 139033, which did not produce ascoconidia from ascospores. Phylogenetic analysis, which included another strain of R. pyriformis CBS 139024 that regularly formed budding ascospores, revealed that they are conspecific. Based on Sivanesan’s (1976) holotype’s revision of R. separata and phylogenetic analysis of R. pyriformis strains with and without ascoconidia, Réblová & Štěpánek (2018) concluded that R. separata is a synonym of R. pyriformis.

However, recent findings of R. separata, represented by strains CBS 153404 and CBS 153405, have provided further clarity on this issue. Phylogenetic analyses of five gene markers confirmed that R. pyriformis and R. separata are distinct species (Fig. 4), supporting the original hypothesis that budding ascospores are a species-specific trait.

Munk (1948) described R. separata on Quercus sp. with asci 110–135 µm long in the sporiferous part and 10–13 µm wide, containing slipper-shaped or clavate, very irregularly muriform ascospores ranging from 16–27 × 5–8 µm. In our material, the asci were slightly longer in the sporiferous part compared to those in the Danish material, and the ascospores exhibited variable shapes, even within a single ascoma, which appeared to correlate with different stages of maturity. We also confirm that R. separata and R. pyriformis were found in the same locality and even on the same branch, as originally observed by Munk (1948, 1957). Rhamphoria pyriformis, occurring on the same sample, is part of the specimen PRA-22503 and is included in this study as CBS 153406.

Rhamphoriopsis cuprea Réblová, sp. nov. MB 858897. Fig. 12.

Fig. 12.

Rhamphoriopsis cuprea (ex-type strain CBS 147991). A. Conidiophores on empty ascomata of an unidentified ascomycete. B, C. Detail of the colony with aerial hyphae and sporulating conidiophores. D–H, L–O. Conidiophores, conidiogenous cells and conidia. I–K. Conidia P. Colony morphology after 4 wk on CMD, MLA, OA, and PCA (from left to right). A, D–I. On natural substrate. B, C, J–O. On MLA. Scale bars: A, C = 500 µm; B = 1000 µm; D–O = 10 µm; P = 1 cm.

Etymology: From Latin cupreous derived from cuprum (coppery). Referring to the copper colour of aerial mycelium formed in vitro.

Typus: Czech Republic, Pardubice region, Chrudim district, Železné hory Mts Protected Landscape Area, Horní Bradlo, Malá Střítež settlement, Polom National Nature Reserve, ca 590 m.a.s.l., on decaying wood of Fagus sylvatica and old perithecial ascomata of unidentified fungus, 9 Oct. 2020, M. Réblová, M.R. 4084 (holotype PRA-22504, culture ex-type CBS 147991).

Description on the natural substrate: Sexual morph not observed. Asexual morph. Colonies effuse, hairy, reddish-brown to brown, whitish when sporulating. Conidiophores 30−90 × 2–3(–3.5) µm, basal cell slightly swollen 4–5.5 µm wide, macronematous, mononematous, solitary, cylindrical, unbranched, pale brown to red brown, subhyaline towards the apex, smooth-walled. Conidiogenous cells 13–22 × 2–2.5 µm, tapering to ca 1.5 μm, integrated, terminal, polyblastic, sympodially extending, cylindrical, pale brown near the base, hyaline to subhyaline towards the apex, smooth, with numerous denticles, 0.5–1 × 0.5 µm; conidiogenesis holoblastic-denticulate. Conidia 4–6.5 × 1.5–2 µm (mean ± SD = 4.7 ± 0.8 × 1.9 ± 0.2 μm), solitary, dry, obovoid to dacryoid, straight or slightly curved appearing somewhat lunate from the side view, rounded at the apex, hyaline, aseptate, smooth, secession schizolytic.

Culture characteristics: On CMD colonies 9–10 mm diam., circular, flat to slightly raised, margin undulate with a prominent irregular submerged growth, velvety-lanose, mucoid towards the margin, whitish, beige centrally and at the colony margin, reverse beige. On MLA colonies 18–19 mm diam., circular, raised, margin fimbriate, funiculose to cobwebby centrally, mucoid towards the periphery, furrowed, zonate, ochre-beige with a brown zone, reverse ochre. On OA colonies 15–17 mm diam., circular, flat, margin entire to partially lobate, cobwebby to mucoid, dark brown to dark reddish-brown with irregular white spots, white-beige at the margin, reverse dark brown. On PCA colonies 15–16 mm diam., circular, raised, margin entire, sparsely floccose, cobwebby towards the margin, whitish-beige centrally, dark brown at the margin, beige at the margin, reverse beige-brown. Sporulation was abundant on CMD and OA, moderate on MLA, absent on PCA.

Description in culture: Colonies on MLA effuse. Mycelium composed of hyaline to subhyaline, septate hyphae, 1.5–3.5 μm wide. Conidiophores 24.5–109 × 2–2.5 µm in 4 wk, prolonging due to the repeated extension of the conidiogenous cells, becoming 115−159 µm long in 8 wk, macronematous or reduced to single conidiogenous cells, mononematous, cylindrical, branched, primary branches 20.5–64.5 µm long, pale brown to reddish-brown, subhyaline towards the apex, smooth-walled. Conidiogenous cells 22–35 × 2–2.5 µm, tapering to ca 1.5 μm, integrated, terminal and intercalary, polyblastic, cylindrical, pale brown near the base, hyaline to subhyaline towards the apex, smooth, with sympodially extending rachis bearing numerous denticles, 0.5–1 × 0.5 µm, after extension of the conidiogenous cell, denticles remain active along the whole length of the conidiophore; conidiogenesis holoblastic-denticulate. Conidia 3.5–5 × 1.5–2(–2.5) µm (mean ± SD = 4.3 ± 0.4 × 1.9 ± 0.2 μm), on OA 3–4.5(–5) × 2–2.5 µm (mean ± SD = 3.7 ± 0.4 × 2.4 ± 0.2 μm), solitary, dry, shortly obovoid to guttuliform, straight or curved, with a truncate basal scar, rounded at the apex, hyaline, aseptate, smooth; sporulation associated with the aerial part of the colony. Sexual morph not observed.

Habitat and geographical distribution: Saprobe on decaying wood of Fagus sylvatica and old perithecial ascomata of an unknown fungus, known from the Czech Republic. According to Global Fungi database, Rh. cuprea was identified in 18 samples primarily isolated from soil, occasionally from air, shoots and roots in forest and grassland, rarely in anthropogenic and cropland habitats in Belgium, France, Germany, Italy, Spain, Sweden, Turkey and the UK.

Notes: Conidia formed in vitro were slightly smaller than those on the natural substrate, often appearing shortly obovoid to guttuliform. Additionally, conidia produced on MLA were somewhat larger than those formed on OA. Conidiophores in culture exhibited distinct elongation due to the repeated sympodial extension of the conidiogenous cell. In culture, Rh. cuprea produced dense tufts of conidiophores with a characteristic copper colouration.

Rhamphoriopsis cuprea is readily distinguished from other species by its solitary conidiophores on the natural substrate and by conidia, which are obovoid to dacryoid, often slightly curved, and appear lunate in lateral view.

Rhamphoriopsis denticulata Réblová, sp. nov. MB 858899. Fig. 13.

Fig. 13.

Rhamphoriopsis denticulata (ex-type strain CBS 147996). A. Upper part of an immersed ascoma with protruding neck. B. Immersed ascomata with broken neck apices exposing the ostiole. C. Longitudinal section of the ascomatal wall. D. Asci with ascospores. E, F. Ascospores. G. Ascal apex with apical ring. H. Empty asci with paraphyses. I–L. Conidiophores with conidiogenous cells and conidia. M–O. Conidiogenous cells with conidia. P. Colony morphology after 4 wk on CMD, MLA, OA, and PCA (from left to right). A–H. On natural substrate. I–O. On MLA. Scale bars: A, B = 200 µm; C, D, H = 20 µm; E–G, I–O = 10 µm; P = 1 cm.

Etymology: From Latin denticulātus (finely dentate, bearing many small tooth-like structures). Referring to the denticulate conidiogenous cells.

Typus: Czech Republic, Central Bohemian region, Úvaly, Nature Park Škvorecká obora – Králičina, ca 270 m.a.s.l., on decaying wood of Sambucus nigra, 28 Aug. 2020, M. Réblová, M.R. 4102 (holotype PRA-22505, culture ex-type CBS 147996).

Description on the natural substrate: Sexual morph. Ascomata perithecial, non-stromatic, grouped or solitary, immersed with protruding neck, venter 300–350 µm diam., globose to subglobose, dark brown, glabrous, neck 80–95 µm wide, up to 600 µm long, rostrate, central, cylindrical, slightly flattened, upright, glabrous, brittle. Ostiole periphysate. Ascomatal wall leathery, 46–57 µm thick, two-layered. Outer layer consisting of thick-walled, reddish brown, polyhedral cells with opaque walls of textura epidermoidea to prismatica, cells tend to be more brick-like and flattened and paler towards the interior. Inner layer consists of several rows of thin-walled, hyaline, flattened cells. Paraphyses abundant, septate, hyaline, 2.5–3.5 µm wide, longer than the asci. Asci 221–243 × 11–14.5 µm (mean ± SD = 230.4 ± 9.4 × 13.2 ± 1.2 μm), 165–205(–240) µm (mean ± SD = 195.3 ± 31.6 μm) long in the sporiferous part, unitunicate, persistent, truncate at the apex, cylindrical, with a short tapering stipe, apical annulus non-amyloid, 4.5–5 µm wide, 2.5–3 µm high, 8-spored. Ascospores 17–25 × 7–10 µm (mean ± SD = 20.6 ± 1.4 × 8.7 ± 0.7 μm), fusiform, hyaline, with 6−8(–9) transverse septa and 6−10 incomplete longitudinal septa, slightly constricted at the septa, smooth-walled, obliquely uniseriate within the ascus. Asexual morph not observed.

Culture characteristics: On CMD colonies 12–13 mm diam., circular, flat, margin lobate, sparsely floccose becoming mucoid, deep reddish brown centrally, pale salmon towards the periphery, reverse ochre-beige. On MLA colonies 13–14 mm diam., circular, slightly raised, margin entire, submerged, floccose, similar to those on CMD but covered by whitish aerial mycelium to obscure the pigmented vegetative mycelium (dark reddish brown, salmon at the margin), reverse reddish-brown. On OA colonies 14–15 mm diam., circular, flat, margin fimbriate to somewhat undulate, floccose, reddish brown, pale salmon-beige towards the margin, whitish on the inoculation block, ochre pigment diffusing into the agar, reverse deep apricot. On PCA colonies 11–13 mm diam., circular, flat to slightly raised, margin lobate, lanose, similar to those on MLA with a prominent whitish aerial mycelium covering the pigmented vegetative mycelium (dark reddish-brown, salmon towards the periphery), pale orange pigment diffusing into the agar, reverse apricot. Sporulation was absent on CMD and OA, abundant on MLA and PCA.

Description in culture: Colonies on MLA effuse. Mycelium composed of hyaline to pale brown, septate hyphae, 1–2.5 μm wide. Conidiophores 26−45 × 2.5–3 µm in 4 wk, prolonging due to the repeated extension of the conidiogenous cells, becoming 86−120 µm long in 8 wk, macronematous or reduced to single conidiogenous cells, mononematous, cylindrical, seldom branched, pale brown to reddish-brown, subhyaline towards the apex, smooth-walled. Conidiogenous cells 22–39 × 2–2.5 µm, tapering to ca 1.5 μm, integrated, terminal and intercalary, polyblastic, cylindrical, with sympodially extending rachis bearing numerous denticles, 0.5–1 × 0.5 µm, rachis can bear several short denticulate branches up to 9.3 µm long, pale brown near the base, hyaline to subhyaline towards the apex, after extension of the conidiogenous cell meristem, denticles remain active, especially in the upper part of the conidiophore, or conidiogenous cells discrete on vegetative hyphae, ampulliform to lageniform with elongated rachis, 12–17.5 × 3–3.5 µm, tapering to 1–1.5 μm, hyaline or subhyaline at the base, hyaline towards the apex, smooth; conidiogenesis holoblastic-denticulate. Conidia hyaline, aseptate, smooth-walled, with a basal scar, of two types (formed on MLA and PCA): ellipsoidal-fusiform to obovate 3.5–4.5 × 1.5(–2) μm (mean ± SD = 4.0 ± 0.3 × 1.6 ± 0.2 µm), straight, narrowly rounded at the apex, tapering towards the base; guttuliform (2.5–)3–4.5 × (1.5–)2–2.5 μm (mean ± SD = 3.6 ± 0.4 × 2.2 ± 0.3 µm), broadly rounded at the apex, slightly tapering towards the base, secession schizolytic; sporulation associated with the aerial part of the colony. Sexual morph not observed.

Additional material examined: Czech Republic, Central Bohemian region, Úvaly, Nature Park Škvorecká obora – Králičina, ca 270 m.a.s.l., on decaying wood of Sambucus nigra, 28 Aug. 2020, M. Réblová, M.R. 4103B, culture CBS 147997.

Habitat and geographical distribution: Saprobe on decaying wood of Sambucus nigra, known from the Czech Republic. According to the GlobalFungi database, Rh. denticulata was identified in five samples isolated from air, soil and roots from forest, grassland, cropland and anthropogenic biomes in Italy, Spain, Sweden, and the UK.

Notes: Two distinct conidial types were observed in culture, i.e. ellipsoidal-fusiform to obovate and guttuliform. The ellipsoidal-fusiform conidia, produced on terminal, integrated conidiogenous cells of macronematous conidiophores, correspond to the phaeoisaria-like morphotype. In contrast, the guttuliform conidia developed exclusively on discrete conidiogenous cells bearing a short or elongated rachis, respectively. In Fig. 13N, several young conidia still attached to the rachis appear narrowly fusiform but become inflated and guttuliform as they mature. The discrete, ampulliform conidiogenous cells with an elongated rachis resemble the idriella-like conidiogenous apparatus described in R. pyriformis (Müller & Samuels 1982) and R. delicatula (Réblová & Štěpánek 2018).

The closest relative of Rh. denticulata is Rh. muriformis; they are the only known holomorphic species within the genus that produce both sexual and asexual morphs. They are clearly distinguishable based on both molecular and morphological data. Rhamphoriopsis muriformis differs from the new species by its broader asci 210–250 × 13.5–16.5(–19) μm, and larger ellipsoidal to fusiform ascospores (20–)23–29 × 8.5–10(–10.5) μm with a greater number of transverse septa (8–12) (Réblová & Štěpánek 2018).

DISCUSSION

Taxonomic placement and novelty within the Pleurotheciales and Rhamphoriales

Molecular DNA data from three- and five-gene phylogenies confirmed the systematic placement and taxonomic novelty of four species exhibiting holoblastic-denticulate conidiogenesis. In axenic culture, they displayed similar morphotypes of conidia, conidiogenous cells and conidiophores, making it difficult to assign them to a genus based solely on these characteristics. This study highlights the importance of integrating sexual and asexual characters, along with observations from both culture and natural substrates, to capture the complete range of morphological variation.

The two new species, Rh. cuprea and Rh. denticulata, together with R. pyriformis, R. separata, and Rhodoveronaea varioseptata, were placed within the Rhamphoriales. The three latter species are relatively uncommon based on known literature records, environmental data, living strains and available DNA sequences (von Höhnel 1913, Munk 1948, 1957, Arzanlou et al. 2007, Réblová 2009, Réblová & Štěpánek 2018), with R. separata being rediscovered nearly 80 years after its original description by Munk (1948). Its asexual morph has been observed for the first time (this study). Echinodenticula allantospora was more challenging to assign to previously known genera using only morphological characteristics. Its tentative placement pointed towards the Pleurotheciales, where it superficially resembles Melanotrigonum. The five-gene phylogenies confirmed the placement of Echinodenticula as a new genus within this order. Although the new species Phaeoisaria parallela is clearly distinguishable from the closely related P. sparsa based on the morphology of its conidiomata, conidiogenous cells, and conidia, the similarity in their ITS and LSU sequences was insufficient for reliable differentiation. However, the use of the protein-coding genes rpb2 and tef1 as secondary barcodes provided clear support for distinguishing the two species. The lack of distinguishing sexual characteristics and absence of asexual morphology complicated the identification of the ascomycete, which was described as M. curvata at an incertae sedis position within the Sordariomycetes. Although its placement was close to Atractosporales, Conlariales and Xenospadicoidales, it lacked statistical support.

Genomic resources for Pleurotheciales and Rhamphoriales

This study contributes significantly to the genomic resources available for fungi within the Sordariomycetes by presenting high-quality draft genomes of six reference strains. Notably, it includes the first published genome for a representative of the order Rhamphoriales, expanding our ability to study this understudied lineage at the genomic level.

Within the Pleurotheciales, only a single genome, Phaeoisaria clematidis from Vitis vinifera (Bruez et al. 2021), had previously been available. Our dataset therefore significantly broadens the phylogenetic coverage for this order by adding E. allantospora, P. parallela and Pt. uniseptata, enabling deeper comparative and evolutionary studies. In addition, the genome of M. curvata adds to the pool of high-quality genomic data for taxa currently of incertae sedis position, offering insights for future phylogenomic frameworks.

The availability of these genomes will support comparative analyses across taxa and orders, especially among morphologically similar or convergently evolved forms. They also create a foundation for integrating environmental DNA datasets with taxonomically validated genomes. Importantly, the genomic data can help inform ongoing efforts to characterize ‘dark taxa’ (Nilsson et al. 2019) and may contribute to future developments in DNA-based typification (e.g. Nilsson et al. 2023), particularly where cultures and type specimens are lacking.

Conidiogenesis and morphological complexity in the Pleurotheciales and Rhamphoriales

The order Pleurotheciales exhibits two types of holoblastic conidiogenesis. The prevalent mode involves mono- or polyblastic sympodially elongating conidiogenous cells bearing one to several denticles or a rachis with multiple conidiogenous loci, occasionally the cells lack denticles. The second morphotype is characterised by determinate, monoblastic conidiogenous cells with a flat locus which produces darkly pigmented, muriform or rarely transversely septate macroconidia. Interestingly, the combination of both modes of conidiogenesis within a life cycle has been documented in two genera, Dematipyriforma and Sterigmatobotrys. Dematipyriforma americana, representing the morphotype with dark, muriform macroconidia, was reported to produce secondary conidiophores and conidia through holoblastic-denticulate conidiogenesis in culture (Crous et al. 2024), aligning with the typical mode of conidial ontogeny in Pleurotheciales. Sterigmatobotrys offers another illustrative example. While the type species, S. macrocarpa, along with other members of the genus, produces dark, macronematous conidiophores differentiated into a stipe and a penicillate head bearing polyblastic, sympodial conidiogenous cells that produce conidia holoblastically (Fig. 1H), S. rudis (formerly Taeniolella rudis, Réblová et al. 2012, Heuchert et al. 2018) differs markedly. It forms dark brown, multiseptate macroconidia arranged in acropetal chains, with sterigmatobotrys-like penicillate conidiophores arising from the terminal macroconidium (Hughes 1980, Jones et al. 2002). The holoblastic-denticulate conidiogenesis functions as the predominant mode of conidial ontogeny in the Pleurotheciales. Therefore, it serves as a consistent character but not a diagnostic one.

Phaeoisaria is one of the genera within the Pleurotheciales that exhibit holoblastic-denticulate conidiogenesis and is currently the only representative in the order known to form synnematous conidiophores. The hyphomycete genus was originally established by von Höhnel (1909) for what was presumed to be the asexual morph of Neopeckia bambusae, based on their close physical association. It is characterised by indeterminate synnemata composed of parallel conidiophores and polyblastic, sympodially extending conidiogenous cells with denticles that produce aseptate or septate, hyaline conidia. Based on four representative species, the genus was assigned to the Pleurotheciales by Réblová et al. (2016). According to MycoBank, 38 species and varieties have been described in Phaeoisaria to date. The life cycle and sexual traits of the genus were clarified with the discovery of P. filiformis (Luo et al. 2019), the only known sexually reproducing species, whose asexual morph was subsequently documented by Xu et al. (2025). The sexual morph is defined by non-stromatic ascomata with a cylindrical neck, unitunicate asci, and filiform, hyaline ascospores.

The presence of synnemata and other forms of conidiophore arrangement on the substrate in nature varies among species of Phaeoisaria, ranging from reduced forms consisting of fasciculate conidiophores, as seen in P. fasciculata (Réblová et al. 2016), to loosely organised, parallelly arranged but not tightly appressed conidiophores in P. parallela (this study), to well-developed synnematous structures in typical species such as P. clematidis, P. clavulata, P. filiformis, and P. sparsa (Hughes 1958, Sutton 1973, de Hoog & Papendorf 1976, Xu et al. 2025), among others. The arrangement of conidiogenous cells also varies; in some species they are confined to the apices of the synnemata giving them a somewhat penicillate appearance, while in others they are distributed along the sides.

In the Rhamphoriales, the holoblastic-denticulate conidiogenesis is the only known mode of conidiogenesis and holds a diagnostic value. Moreover, Rhamphoria is characterised by three types of conidial structures: i) ascoconidia budding from ascospores within the ascus and observed only in R. pyriformis, ii) conidia of the asexual morph, and iii) conidia of the synasexual morph, which occurs exclusively in culture and develops early in colony formation (Müller & Samuels 1982, Réblová & Štěpánek 2018). The asexual morphotype of Rhamphoria in culture has been compared to solitary phaeoisaria-like conidiophores, accompanied by secondary conidiophores of the idriella-like morphotype (Müller & Samuels 1982, Réblová & Štěpánek 2018). Members of Rhamphoria and Rhamphoriopsis are particularly difficult to differentiate based on asexual characteristics. On the natural substrate, their asexual morphs appear similar and exhibit a myrmecridium-like appearance but without the wing-like mucilaginous sheath on conidia. On the natural substrate, the conidiogenous cell is terminal, with a rachis that extends sympodially to a limited extent. In culture, however, as the conidiogenous cell undergoes repeated sympodial elongation, the transverse septa are formed and the meristem bearing denticles or minute protrusions becomes intercalary and remains active along most of the length of the conidiophore (Fig. 12, 13).

In the Rhamphoriales, conidial polymorphism within a single species has been documented not only in Rhamphoria, but also in two species of Rhamphoriopsis, i.e. Rh. denticulata (this study) and Rh. glauca (de Hoog & Papendorf 1976). Secondary conidia of varying shapes and sizes, produced either on conidiophores or on discrete conidiogenous cells, are formed exclusively in vitro. The idriella-like conidiogenous apparatus morphotype was also observed in Rh. denticulata on discrete conidiogenous cells.

Although the genus Rhamphoriopsis was originally established based on both sexual and asexual characters of Rh. muriformis (Réblová & Štěpánek 2018), it has since been expanded to include only asexually typified species: Rh. aquimicrospora and Rh. glauca (Yang et al. 2023), Rh. hyalospora (Lin et al. 2023), Rh. sympodialis (Hyde et al. 2020), and Rh. synnematosa (Crous et al. 2023). Our study contributes with the addition of the holomorphic Rh. denticulata and the asexual Rh. cuprea. Rhamphoriopsis closely resembles Rhamphoria in sexual and asexual characteristics but can be primarily distinguished by its smaller, globose and immersed ascomata with a cylindrical, often flattened necks. While Rhamphoriopsis species are morphologically similar in their conidial and conidiogenous cell characteristics, they exhibit considerable variation in the arrangement of their conidiophores on the natural substrate, a feature that resembles the pattern observed in Phaeoisaria. This diversity ranges from typical synnemata in R. aquimicrospora, to fasciculate or subfasciculate conidiophores in Rh. glauca, Rh. hyalospora and Rh. muriformis, to solitary conidiophores in Rh. cuprea and Rh. sympodialis. Rhamphoriopsis synnematosa is known only from culture and exhibits solitary conidiophores. In the phylogenetic analyses, the two holomorphic species, Rh. denticulata and Rh. muriformis, clustered together in a strongly supported clade, forming a sister group to other Rhamphoriopsis species.

Yang et al. (2023) proposed a new combination for Phaeoisaria glauca (de Hoog & Papendorf 1976) in Rhamphoriopsis, based on a non-type strain CBS 480.75, with an available partial LSU sequence (GenBank: MH872702, Vu et al. 2019). However, no additional morphological or molecular studies were conducted to support this reallocation. In our preliminary analysis, the inclusion of this sequence in the five-gene phylogeny resulted in branch collapse within the genus (results not shown), leading to its exclusion from the final phylogeny. Further analysis using additional gene markers (Réblová & Hernández-Restrepo, in prep.) suggests that R. glauca represents a distinct species.

Global distribution patterns of Phaeoisaria, Rhamphoria and Rhamphoriopsis

To gain a better understanding of the global distribution and ecological preferences of the studied fungi, we utilised the GlobalFungi database. The limited number of records in GlobalFungi for certain species may reflect their rarity, specific habitat preferences, or gaps in current environmental sequencing efforts. This is especially true for several Phaeoisaria species, i.e. P. filiformis, P. microspora, P. motuoensis, P. pseudoclematidis, and P. sedimenticola, and Rh. synnematosa, which were each detected in only a single sample.

The genus Phaeoisaria displays distinct biogeographical and ecological patterns, with its species grouped into three main distributional categories. Some taxa, such as P. annesophieae and P. loranthacearum, exhibit nearly global distributions, occurring across all climatic zones except for boreal regions. In contrast, other species show more restricted climatic preferences, either to tropical and humid subtropical regions or to temperate zones, with only occasional occurrences in boreal climates. This distributional trend is consistent with patterns observed in species of Chloridium, Codinaea, and related genera (Réblová et al. 2021, 2022), suggesting similar ecological requirements among these groups. Although Phaeoisaria species have traditionally been reported from dead plant material (bark, wood, bamboo culms) in terrestrial and less frequently in freshwater habitats, with rare occurrence in soil (P. annesophieae Crous et al. 2017), marine sediments (P. sedimenticola, Cheng et al. 2014), and as opportunistic human pathogens (P. clematidis, Guarro et al. 2000, Chew et al. 2010), our study emphasises their strong association with forest soils.

While GlobalFungi reports that 53 % of all fungal samples originate from soil (and only 2.7 % from dead wood), and 57 % are from forest or woodland habitats, our findings reveal an even more pronounced trend for Phaeoisaria. Specifically, 90 % of the samples were recovered from soil and 85.4 % from forest or woodland environments, with only 2 % associated with deadwood. These results underscore a strong ecological link between Phaeoisaria and forest ecosystems, with an unexpectedly high prevalence in soil habitats. The near absence of records from other biomes suggests a narrow ecological niche, likely specialised for forest ecosystems. Furthermore, the notable scarcity of boreal representatives, which is in sharp contrast to the abundance of this genus in temperate and tropical regions, may reflect physiological limitations that hinder adaptation to colder climates.

The biogeographic data suggest that Rhamphoria species are strongly associated with forest ecosystems in the temperate zone, particularly broadleaf and mixed forests, where they primarily inhabit deadwood and, to a lesser extent, soil and plant material. Rhamphoria delicatula appears to be the most widespread and abundant species, suggesting a broader ecological range or greater detection frequency in environmental samples. In contrast, R. separata has the most limited number of records, potentially indicating a more restricted distribution or habitat preferences. The presence of R. pyriformis and R. delicatula in subpolar coniferous forests suggests that some species within the genus can adapt to cooler climates. Overall, these findings highlight the ecological role of Rhamphoria species as saprobes in temperate and boreal forests, contributing to the decomposition of wood and organic material.

Environmental sequence data from GlobalFungi indicate that Rhamphoriopsis species are predominantly found in temperate regions, with less frequent occurrences in subtropical and tropical areas. These findings also suggest that Rhamphoriopsis species predominantly occupy soilassociated habitats, with some species exhibiting broader ecological plasticity. This is particularly interesting, as all previously described species have been isolated from decaying wood. Rhamphoriopsis aquimicrospora shows adaptability to both terrestrial and aquatic environments, unlike other species in the genus, which have so far been found exclusively in terrestrial habitats. This ecological flexibility is supported by the range of substrates from which samples have been collected (Yang et al. 2023). Members of Rhamphoriopsis were primarily recorded in numerous countries in Europe and Asia, particularly in eastern and south-eastern China, with occasional records from Japan and Turkey, and a few records from Africa. Notably, this genus is absent from the boreal zone. Both Rhamphoria and Rhamphoriopsis have predominantly Euroasian distribution as they were not detected in samples from North and South America or Australasia. No records of E. allantospora and M. curvata were found in GlobalFungi.

CONCLUSIONS

This study offers new perspectives on the molecular systematics, taxonomy, and biogeography of the Rhamphoriales and Pleurotheciales, contributing to the description of novel taxa and the refinement of species boundaries within the studied genera. Our findings emphasise the significance of holoblastic-denticulate conidiogenesis as a distinguishing feature of the Rhamphoriales and a prevalent trait in the Pleurotheciales, reinforcing its importance in fungal systematics. Additionally, this study highlights the need for cultivation studies and in vitro morphological observations, as they enable the discovery of synasexual morphs and modes of conidiogenesis that might otherwise be overlooked and absent in protologues. Such observations enhance the taxonomic value of conidial ontogeny in taxa at higher ranks, as demonstrated by the examples of D. americana and S. rudis.

The introduction of E. allantospora broadens our understanding of holomorphic genera within the Pleurotheciales. The discovery of two new species, Rh. cuprea and Rh. denticulata, expands the known diversity of Rhamphoriopsis. Furthermore, the recognition of R. separata as a distinct species, supported by phylogenetic analyses of multiple gene markers, resolves a long-standing taxonomic uncertainty regarding its relationship with R. pyriformis. The distinction of P. sparsa from the new species P. parallela is supported by morphology and dual barcoding using rpb2 and tef1 as secondary barcodes. The identification of M. curvata as a new genus and species further contributes to elucidating phylogenetic relationships within Sordariomycetes, particularly among taxa with unresolved affinities.

Environmental DNA data highlight pronounced association of Phaeoisaria with forest soils despite its species have been traditionally documented on dead plant material, and occurrence in three distinct biogeographical patterns. The environmental data revealed that members of Rhamphoria are primarily associated with deadwood in temperate and boreal forests in Europe, while members of Rhamphoriopsis are predominantly soil-inhabiting fungi in temperate regions, with less frequent occurrences in subtropical and tropical areas and have much broader distribution. The absence of records for E. allantospora and M. curvata in GlobalFungi indicate that their distribution remains poorly understood, likely due to limited sampling or underrepresentation in environmental sequencing databases.

Overall, this study underscores the value of integrating morphological characteristics, both sexual and asexual, with molecular evidence in fungal taxonomy. Additionally, it highlights the role of environmental DNA data in uncovering fungal diversity and refining our understanding of distribution patterns.

Supplementary material

Fig. S1

Geographic distribution of Phaeoisaria species based on environmental DNA data from the GlobalFungi database. Each dot represents an exact georeferenced sample in which the respective species was identified.

Fig. S2

Geographic distribution of Rhamphoria and Rhamphoriopsis species based on environmental DNA data from the GlobalFungi database. Each dot represents an exact georeferenced sample in which the respective species was identified.

File S1

Alignments of LSU–SSU–rpb2 and ITS–LSU–SSU–rpb2–tef1 used to generate the phylogenies in Figs 2–4.

Table S1

Taxa, isolate information and sequences retrieved from GenBank

Table S2

Biogeographical distribution, sample type, habitat and other detailed metadata for members of Phaeoisaria, Rhamphoria and Rhamphoriopsis inferred from the GlobalFungi database