Next Article in Journal
Virulence-Associated Genes of Calonectria ilicola, Responsible for Cylindrocladium Black Rot
Next Article in Special Issue
Diversity of Ascomycota in Jilin: Introducing Novel Woody Litter Taxa in Cucurbitariaceae
Previous Article in Journal
The Characterization and the Biological Activity of Phytotoxin Produced by Paraphoma radicina
Previous Article in Special Issue
A New Cryptic Lineage in Parmeliaceae (Ascomycota) with Pharmacological Properties
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:

Two Novel Genera, Neostemphylium and Scleromyces (Pleosporaceae) from Freshwater Sediments and Their Global Biogeography

Unitat de Micologia i Microbiologia Ambiental, Facultat de Medicina i Ciències de la Salut and IISPV, Universitat Rovira i Virgili, 43201 Reus, Spain
Author to whom correspondence should be addressed.
J. Fungi 2022, 8(8), 868;
Received: 18 July 2022 / Revised: 12 August 2022 / Accepted: 15 August 2022 / Published: 17 August 2022
(This article belongs to the Special Issue Ascomycota: Diversity, Taxonomy and Phylogeny)


Although the Pleosporaceae is one of the species-richest families in the Pleosporales, research into less-explored substrates can contribute to widening the knowledge of its diversity. In our ongoing survey on culturable Ascomycota from freshwater sediments in Spain, several pleosporacean specimens of taxonomic interest were isolated. Phylogenetic analyses based on five gene markers (ITS, LSU, gapdh, rbp2, and tef1) revealed that these fungi represent so far undescribed lineages, which are proposed as two novel genera in the family, i.e., Neostemphylium typified by Neostemphylium polymorphum sp. nov., and Scleromyces to accommodate Scleromyces submersus sp. nov. Neostemphylium is characterized by the production of phaeodictyospores from apically swollen and darkened conidiogenous cells, the presence of a synanamorph that consists of cylindrical and brown phragmoconidia growing terminally or laterally on hyphae, and by the ability to produce secondary conidia by a microconidiation cycle. Scleromyces is placed phylogenetically distant to any genera in the family and only produces sclerotium-like structures in vitro. The geographic distribution and ecology of N. polymorphum and Sc. submersus were inferred from metabarcoding data using the GlobalFungi database. The results suggest that N. polymorphum is a globally distributed fungus represented by environmental sequences originating primarily from soil samples collected in Australia, Europe, and the USA, whereas Sc. submersus is a less common species that has only been found associated with one environmental sequence from an Australian soil sample. The phylogenetic analyses of the environmental ITS1 and ITS2 sequences revealed at least four dark taxa that might be related to Neostemphylium and Scleromyces. The phylogeny presented here allows us to resolve the taxonomy of the genus Asteromyces as a member of the Pleosporaceae.

1. Introduction

The Pleosporaceae is one of the largest families within the order Pleosporales (Dothideomycetes) in terms of the number of species. It was introduced by Nitschke [1] and was considered for a long time a heterogeneous group of bitunicate ascomycetes with its genera distinguished primarily by their ascospore features (i.e., shape, color, septation, pigmentation, and presence or lack of mucilaginous sheaths) [2]. According to recent taxonomic revisions of the Dothideomycetes [3,4], based on morphological investigations and phylogenetic data, the Pleosporaceae is a well-delineated family that comprises 23 genera and more than 2000 species. Alternaria, Bipolaris, Curvularia, Exserohilum, Pyrenophora, and Stemphylium are the most species-rich genera in the family [3]. They show teleomorphs characterized by black ostiolate ascomata with thick-walled peridium, cellular pseudoparaphyses and bitunicate, fissitunicate, eight-spored asci, producing melanized, phragmosporous, or muriform ascospores [3]. More commonly, they present dematiaceous hyphomycetous anamorphs producing phragmo- or dyctioconidia from tretric (poroblastic) or blastic conidiogenous cells, although coelomycetous anamorphs with phialidic or anellydic conidia have also been described [3,5]. Although Pleospora was designed as the type genus in the family, with the advent of one fungus-one name initiative in the International Code of Nomenclature for algae, fungi, and plants (ICN; Melbourne Code) [6], the name Stemphylium was retained over Pleospora by the working group on Dothideomycetes of the International Commission on the Taxonomy of Fungi [7].
Members of the Pleosporaceae are widely distributed across the environment and have a wide range of lifestyles, i.e., saprophytic, endo-/epiphytic, and parasitic on various hosts in terrestrial and aquatic environments [8]. Among them, species of Alternaria, Bipolaris, Curvularia, or Stemphylium are important pathogenic fungi to plants of various crops, resulting in yield and economic losses [8,9,10]. However, they also include human and animal pathogens that cause infections with different clinical manifestations [11]. Several metagenomic studies reveal that pleosporalean fungi are well represented in aquatic environments [12,13,14]. Although some species have been found strictly adapted to aquatic ecosystems [15], many of them are commonly found in association with terrestrial plants and, therefore, they are not considered especially adapted to freshwater habitats [16]. Results of those studies also suggest that this group of fungi has been generally overlooked and undersampled in freshwater ecosystems, particularly in rivers, despite their relevant role in ecosystem functioning as saprophytes and parasites [12,17].
In our latest efforts to expand knowledge on the diversity of culturable Ascomycota from river sediments collected in Spain, several interesting specimens of dematiaceous filamentous fungi were isolated. A preliminary sequence analysis of the nuclear ribosomal operon (i.e., the 28S large ribosomal subunit––LSU, and the internal transcribed spacer––ITS, including the 5.8S rDNA gene) revealed that those specimens would belong to the Pleosporaceae, but they could not be identified at the genus level. The aim of the present study was, therefore, to resolve the taxonomy of the above-mentioned isolates based on morphological features and multi-locus phylogenetic analysis inferred with sequences of the nuclear markers mostly represented in the different members of the Pleosporaceae. These are the LSU and ITS regions of the rDNA, and partial fragments of the RNA polymerase II largest subunit (rpb2), the translation elongation factor 1–α (tef1), and the glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes [8,18,19]. Additionally, in order to elucidate the putative global geographic distribution of those isolates and to study their diversity hidden among environmental sequences, their ITS barcodes (i.e., full-length of ITS1 and ITS2 sequences) were blasted against the GlobalFungi database [20]. This is a recently created database, which currently includes accumulated data on fungal distribution and ecology generated from more than three hundred metagenomic studies published in the last decade (GlobalFungi database, accessed on 18 May 2022).

2. Materials and Methods

2.1. Sampling and Isolates

Sediment samples were collected in 2019 from natural areas of two Spanish provinces, Lleida and Madrid. Samples from Lleida were collected from the Segre River as it passes through Camarasa, an area characterized by a continental Mediterranean climate (, accessed on 21 April 2022), with an average annual temperature of 13.5 °C, an average annual rainfall of 800 mm, an altitude of 800 m, and a vegetation dominated by holm oaks (Quercus ilex subsp. rotundifolia) (, accessed on 21 April 2022). Samples from Madrid were collected from two streams around Rascafría in the Guadarrama Natural Park. This area has a continental mountain climate with an average annual temperature of 11.8 °C, an average annual rainfall of 530 mm, an altitude of 1200 m, and a forest dominated by Cistus oromediterraneus, Juniperus communis, and Pinus sylvestris (https://www.parquenacionalsierra, accessed on 21 April 2022).
Sediments from the rivers or streams selected in the above-mentioned locations were collected randomly. Samples were obtained ca 10 cm below the surface layer from the riverbeds or edges using sterile 100 mL plastic containers, which were transported in a refrigerated container to the laboratory and processed immediately. Samples were vigorously shacked in the same containers; then, after 1 min at rest, the water was decanted and the sediment was poured into plastic trays onto several layers of sterile filter paper to remove excess water [21]. To achieve a greater fungal diversity in culture, three agar media were used: dichloran rose-bengal-chloramphenicol agar (DRBC; 2.5 g peptone, 5 g glucose, 0.5 g KH2PO4, 0.25 g MgSO4, 12.5 mg rose-bengal, 100 mg chloramphenicol, 1 mg dichloran, 10 g agar, 500 mL distilled water), DRBC supplemented with 0.01 g/L of benomyl, and potato dextrose agar (PDA; Pronadisa) supplemented with 2 g/L of chloramphenicol and 2 g/L of cycloheximide. Each sample was cultured in duplicate in each medium as follow: 0.5 g of sediment was mixed with melted medium at 45 °C in the same Petri dish and, once solidified, it was incubated at room temperature (22–25 °C) in the dark. Plates were examined weekly by stereomicroscope for 4–5 weeks. To obtain pure cultures, fragments of the colony or conidia of the fungi growing on primary cultures were transferred, using a sterile dissection needle, to plates containing PDA supplemented with chloramphenicol and incubated at 25 °C in darkness. These PDA cultures were used for a preliminary morphological identification and for extracting DNA of the fungi selected.
Living cultures of putative novel or rare fungi were preserved and deposited in the culture collection of the Faculty of Medicine in Reus (FMR, Spain) for further studies. Taxonomic information and nomenclature for the new species were deposited in MycoBank (, accessed on 23 March 2022). Cultures from ex-type strains and holotypes, which consisted of dry colonies on the most appropriate media for their sporulation, were also deposited at the Westerdijk Fungal Biodiversity Institute in Utrecht (CBS, The Netherlands) (, accessed on 22 May 2022).
In addition, the ex-type and a reference strain of Asteromyces cruciatus were also examined in the current study, because a preliminary molecular comparison revealed this species as related to some of our isolates. According to Mycobank and the Index Fungorum database, A. cruciatus represents a monotypic genus with unclarified taxonomy.

2.2. Phenotypic Study

Microscopic characterization was carried out from the isolates growing on potato carrot agar (PCA; 20 g potato, 20 g carrot, 13 g agar, 1 L distilled water) after 7–14 d at 25 °C in darkness and mounted on slides with Shear’s mounting solution (3 g potassium acetate, 60 mL glycerol, 90 mL ethanol 95%, and 150 mL distilled water) [22], using an Olympus BH-2 bright field microscope (Olympus Corporation, Tokyo, Japan). Size ranges of relevant structures in species descriptions were derived from at least 30 measurements. Micrographs were taken using a Zeiss Axio-Imager M1 light microscope (Zeiss, Oberkochen, Germany) with a DeltaPix Infinity × digital camera. Photoplates were assembled from separate photographs using PhotoShop CS6. Macroscopic characterization of the colonies was made on PDA, PCA and oatmeal agar (OA; 30 g oatmeal, 13 g agar, 1 L distilled water) after 7 days at 25 °C in darkness. Other culture media, such as OA and PCA with sterile plant debris (i.e., leaves and twigs of Dianthus caryophyllus), synthetic nutrient-poor agar (SNA; 1 g KH2PO4, 1 g KNO3, 0.5 g MgSO4 × 7H2O, 0.5 g KCl, 0.2 g glucose, 0.2 g sucrose, 14 g agar, 1 L of distilled water), and V8 medium (16 g agar, 200 mL V8 juice, 1 L distilled water), were also used specifically for the FMR 18289 in order to stimulate its sporulation. For the same purpose, this isolate was submitted to the procedure described in Nishikawa and Nakashima [23] for Alternaria sporulation. Color notations in descriptions were according to Kornerup and Wanscher [24]. Growth rates were measured in duplicate on PDA after 7 d in darkness, at 5 °C intervals from 5 to 40 °C, and also at 37 °C.

2.3. DNA Extraction, Sequencing and Phylogenetic Analysis

Total genomic DNA was extracted through the modified protocol of Müller et al. [25] and quantified using Nanodrop 2000 (Thermo Scientific, Madrid, Spain). In order to reconstruct the phylogeny of the Pleosporaceae family, the loci amplified and sequenced were the ITS barcode and the D1/D2 domains of the LSU of the rDNA, as well as gene fragments of the rpb2, tef1, and gapdh. Primers pairs for their amplification were ITS5/ITS4 [26], LR0R/LR5 [27], RPB2-5F2/fRPB2-7cR [28,29], EF1-728F/EF1-986R [30], and gpd1/gpd2 [31], respectively. Briefly, PCR conditions for ITS, LSU, gapdh, and tef1 were set as follows: an initial denaturation at 95 °C for 5 min, followed by 35 cycles of 30 s at 95 °C, 45 s at 56 °C, and 1 min at 72 °C, and a final extension step at 72 °C for 10 min. PCR conditions for the rpb2 were an initial denaturation at 94 °C for 5 min, followed by 5 cycles of 45 s at 94 °C, 45 s at 60 °C, and 2 min at 72 °C, then 5 cycles of 45 s at 94 °C, 45 s with 58 °C, and 2 min at 72 °C, later 30 cycles of 45 s at 95 °C, 45 s with 54 °C, and 2 min at 72 °C, and a final extension step at 72 °C for 7 min. PCR products were purified and sequenced at Macrogen Corp. Europe (Madrid, Spain) with the same primers used for amplification. Consensus sequences were assembled using SeqMan v. 7.0.0 (DNAStar Lasergene, Madison, WI, USA).
A preliminary species identification of the sediment isolates was carried out by comparing their ITS region with those at the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm., accessed on 5 March 2021) and with the UNITE database (, accessed on 5 March 2021). A maximum similarity level of ≥98% was used for species-level identification. Lower similarity values were considered as putative unknown fungi, and their taxonomic position was assessed from analyses of the loci mentioned above.
Sequences of related species and representatives of other genera belonging to the Pleosporaceae family were obtained from GenBank and are listed in Table 1.
Individual and combined analyses using the LSU, ITS, gapdh, rpb2, and tef1 sequences were carried out to assess the phylogenetic relationship of the unidentified isolates to the other taxa in the family. Datasets for each locus were aligned individually in MEGA (Molecular Evolutionary Genetics Analysis) software v.6.0 [46], using the ClustalW algorithm [47] and refined with MUSCLE [48] or manually adjusted, if necessary, on the same platform. Phylogenetic concordance of the five-locus datasets was tested individually in each single-locus phylogeny through visual comparison and using the Incongruence Length Difference (ILD) implemented in the Winclada program [49] in order to assess any incongruent results among nodes with high statistical support. Once their concordance was confirmed, individual alignments were concatenated into a single data matrix with SequenceMatrix [50]. The best substitution model for all gene matrices was estimated using MEGA software for Maximum Likelihood (ML) analysis, whereas for the Bayesian Inference (BI) analysis it was estimated using jModelTest v.2.1.3 following the Akaike criterion [51,52]. The phylogenetic reconstructions were performed with the combined genes using ML under RAxML-HPC2 on XSEDE v-8.2.12 [53] in CIPRES Science gateway portal [54] and BI with MrBayes v.3.2.6 [55].
For the ML analysis, phylogenetic support for internal branches was assessed by 1000 ML bootstrapped pseudoreplicates and bootstrap support (bs) ≥ 70 was considered significant [56]. The phylogenetic reconstruction by BI was carried out using 5 million Markov Chain Monte Carlo (MCMC) generations, with four runs (one cold chain and three heated chains), and samples were stored every 1000 generations. The 50% majority-rule consensus tree and posterior probability (pp) values were calculated after discarding the first 25% of samples. A pp value of ≥0.95 was considered significant [57]. The resulting trees were plotted using FigTree v.1.3.1 (, accessed on 1 June 2022). The DNA sequences and alignments generated in this study were, respectively, deposited in GenBank (Table 1) and in Zenodo (, accessed on 9 August 2022).

2.4. Phylogeny and Geographic Distribution of Allied Environmental Sequences

In order to assess putative global geographic distribution and ecology of the novel fungi detected and their hidden diversity among environmental sequences, the full length of their ITS1 and ITS2 sequences were blasted against the GlobalFungi database [20]. At the time of accession (May 2022), this dataset contained 36,684 samples from 367 studies, 213, 747, 241 unique sequences for ITS1, and 582, 264, 149 for ITS2. Since GlobalFungi has separated ITS1 and ITS2 sequences, they were analyzed separately. In order to verify generic and species boundaries among downloaded ITS environmental sequences related to our fungi, we also included in the analyses ITS sequences of known species previously obtained from the GenBank and UNITE databases. Those known species were representatives of the well-delineated monophyletic genera (i.e., Alternaria, Asteromyces, Gibbago, Paradendryphyella, Pyrenophora, and Stemphylium), which were the closest taxa to our fungi in a full length ITS analysis carried out previously (Figure S1 in Supplementary Material). ITS1 and ITS2 sequences of some of those fungi were also blasted against the GlobalFungi dataset and included in the respective analyses. In each case, we selected and downloaded environmental sequences that had a similarity of between 98 and 100% and a full-length coverage with the sediment isolates and with those related to the known species, apart from the ITS1 of the isolate FMR 18289, because its highest sequence similarity found in the database was lower, at 95%. Pleosporacean genus/species boundaries were inferred from ML trees of ITS1 and ITS2 sequences computed in RAxML. Virtual taxa, consisting of environmental sequences only, were defined as arbitrary phylotypes in the phylogenetic trees, following Réblová et al. [58,59]. Data on occurrence across environmental samples and metadata related to the particular samples (location, substrate, biome, or climatic data) were obtained for each taxon and are listed in Table S1 (Supplementary Material).

3. Results

Among pleosporacean fungi found in the freshwater sediments, we recovered five isolates (FMR 17886, FMR 17889, FMR 17893, FMR 17894, FMR 17895) exclusively from DRBC agar supplemented with benomyl. These isolates were identified initially as Stemphylium sp. because they showed similar morphological features but did not exactly fit into any of the known species described in that genus. Another interesting isolate (FMR 18289) was recovered from DRBC but could not be identified morphologically because it only produced sclerotium-like structures and failed to form fertile reproductive morphs (i.e., anamorph and/or teleomorph) despite the attempts to stimulate sporulation in various culture conditions.

3.1. Phylogeny

Molecular identification based on the BLAST query revealed that LSU sequences of the six unidentified isolates showed a high percentage of similarity with other members of the Pleosporaceae. Specifically, the stemphylium-like isolates showed a sequence identity of 99% with Stemphylium (S.) vesicarium (CBS 191.86) and Bipolaris (B.) microlaenae (CBS 280.91), while the sequence of the sclerotium-forming isolate was 99% similar to Pyrenophora (P.) seminiperda (CBS 127927) and 98% to Alternaria (A.) avenicola (CBS 121459). Similar values were obtained when sequences of species of other well-delineated genera in the Pleosporaceae were compared, which confirmed the low discriminatory power of this gene marker in the family. On the other hand, the genetic similarity was considerably lower when ITS sequences were compared with other members of the Pleosporaceae. The closest matches for the stemphylium-like isolates were Paradendryphiella (Pa.) salina (CBS 142.60 and CBS 141.60) with a similarity of 96%, followed by Pa. areniae (CBS 181.58) and S. vesicarium (CBS 191.86) with a 95%. BLAST results and the particular morphology of those isolates precluded them from being classified in the genus Stemphylium or in Paradendryphiella. The highest similarity for ITS sequence of the remaining isolate (FMR 18289) was 96% with A. avenicola (CBS 121459), followed by P. seminiperda (CBS 127927) with a similarity of 90%. BLAST searches using the remaining phylogenetic markers revealed even lower values of similarity (≤89.6%) with other members of the Pleosporaceae.
Since individual analyses with LSU, ITS, gapdh, rpb2, and tef1 were visually similar and the ILD test did not show incongruences (p = 0.33), a multi-gene analysis was carried out with the five markers. The concatenated phylogeny encompassed 59 sequences that represented 17 genera in the Pleosporaceae with 3160 bp long (531 for ITS, 892 for LSU, 865 for rpb2, 624 for gapdh, 248 for tef1), of which 1164 were variable sites (198 for ITS, 168 for LSU, 375 for rpb2, 281 for gapdh, 142 for tef1) and 905 were phylogenetically informative sites (159 for ITS, 94 for LSU, 325 for rpb2, 231 for gapdh, 96 for tef1). For the ML analyses, K2 + G + I was selected as the best fit model for ITS, LSU, and rpb2, the K2 + G for tef1 and TN93 + G for gapdh. For the BI analyses, SYM + G + I was selected as the best fit model for ITS and rpb2, the K2 + G + I for LSU, the HKY + G + I for gapdh, and the K2 + G for tef1. The RAxML tree (Figure 1) showed that FMR 17886, FMR 17889, FMR 17893, FMR 17894, and FMR 17895 clustered together in a monophyletic undescribed lineage, strongly supported (100 bs/1 pp), which was sister to a well-supported clade (83 bs/0.98 pp) that includes members of the genera Asteromyces, Paradendryphiella, and Stemphylium. Among these, Asteromyces currently has an uncertain taxonomic position among the Ascomycota. However, our analysis places its type species, A. cruciatus, into the Pleosporaceae. The undescribed lineage of the five sediment isolates represents a novel genus, which is proposed here as Neostemphylium (N.) and represented by the new species N. polymorphum (Figure 1). These isolates showed similar morphological features and had an intra-specific genetic variability ranging from 0.1 to 0.4% in the concatenated phylogenetic analysis. In the same phylogeny (Figure 1), the isolate FMR 18289 was located in a faraway single branch within a well-supported clade (99 bs/1 pp) together with other genera of the Pleosporaceae, representing a novel genus for the family. This is proposed below as Scleromyces (Sc.) and typified by the new species Sc. submersus. A detailed morphological characterization of the novel fungi is provided in the taxonomy section.

3.2. Biogeography and Ecology

A BLAST search in the GlobalFungi database revealed the presence of Neostemphylium and Scleromyces among environmental sequences from samples collected worldwide. When we compared the ITS1 sequences of Neostemphylium, this resulted in 469 unique environmental ITS1 sequences (similarity 98–100%), covering 739 samples. The ITS1 sequence of the Scleromyces isolate, as mentioned before, yield the highest similarity value, that of 95%, found in the database .At that value, we obtained 500 environmental sequences, which covered 291 samples. With so many environmental sequences related to our fungi, we were able to select for the analyses representatives from a variety of locations, substrates, and biomes (Table S1 in Supplementary Material), as well as other environmental sequences from different species of the genera Alternaria, Pyrenophora, and Stemphylium. The ITS1 phylogenetic analysis included 102 sequences, 225 characters, of which 156 were variable sites and 127 were phylogenetically informative sites. The ML tree was rooted in a branch leading to Comoclathris (Co.) typhicola (CBS 132.69) and Co. sedis (CBS 366.52) (Figure 2). The environmental ITS1 sequences selected clustered into eight phylotypes, two of which, with a total of 32 sequences, were related to the genus Neostemphylium, and another two phylotypes with three sequences were related to Scleromyces.
Most environmental sequences linked to Neostemphylium formed the phylotype representative of N. polymorphum, apart from four sequences that were designated as the phylotype ITS1-ENV1 and could represent a hypothetically distinct species from N. polymorphum. On the other hand, no environmental ITS1 sequences were matched to the novel species Sc. submersus. However, this species formed a divergent branch close to the phylotypes designated as ITS1-ENV2 and ITS1-ENV3, with one and two sequences, respectively, which might also represent two hidden taxa for the genus Scleromyces.
In contrast, more environmental ITS2 sequences were found related to the genus Scleromyces than to Neostemphylium. Namely, 26 sequences 98–100% similar were linked to Scleromyces and eight unique sequences to the latter genus, covering six and nine samples, respectively. The ITS2 dereplicated dataset had 92 sequences that were representative of members of the above-mentioned genera, with 166 characters, of which 90 were variable sites and 73 phylogenetically informative sites. The ML tree was rooted to Co. typhicola (CBS 132.69) and Co. sedis (CBS 366.52) (Figure 2). The environmental sequences were distributed into seven phylotypes. Specifically, eight sequences were linked to the phylotype of N. polymorphum and one to Sc. submersus, while the remaining sequences related to Scleromyces were designated as phylotype ITS2-ENV1 because they represented a distinct species from Sc. submersus. However, this Scleromyces phylotype does not correlate with any delineated in the ITS1 analysis, since ITS2-ENV1 includes environmental sequences from the USA, while ITS1-ENV2 and ITS1-ENV3 phylotypes both have sequences from Australian samples.
Biogeography and ecological parameters of the environmental sequences related to our novel fungi and inferred in ITS1 and ITS2 phylogenetic analyses are summarized in Table 2. Briefly, Oceania (mainly Australia) has the majority of samples containing ITS1 and ITS2 sequences linked to N. polymorphum, Sc. submersus, and the hidden phylotypes identified here. Nevertheless, many sequences linked to N. polymorphum were also found in samples from areas of Europe (France and Spain) and the USA, the most-sampled areas in GlobalFungi (33.55% and 23.74%, respectively). Interestingly, the Scleromyces phylotype ITS2-ENV1 is the only sequence sampled from aquatic environments collected in the USA (Table 2). Conversely, sequences linked to N. polymorphum, Sc. submersus, and to the other phylotypes related to the novel genera were sampled from soils or roots as the most frequently inhabited substrates in different biomes (grasslands, wetlands, croplands, woodlands, shrublands, or, rarely, forests) (Table 2).

3.3. Taxonomy

Neostemphylium Torres-Garcia, Gené and Cano, gen. nov.
MycoBank MB 843270
Etymology: Name refers to the morphological resemblance with Stemphylium.
Subclass classification: Dothideomycetes, Pleosporomycetidae, Pleosporales, Pleosporaceae.
Type species: Neostemphylium polymorphum Torres-Garcia, Gené and Cano.
Description: Teleomorph not observed. Anamorph hyphomycetous. Conidiophores semi-macronematous or macronematous, mononematous, consisting in conidiogenous cells growing terminally or laterally often on short supporting cells from vegetative hyphae, or septate and branched towards the upper part, branches sometimes with percurrent proliferations, subhyaline to pale brown, smooth-walled or verruculose mainly towards the upper part. Conidiogenous cells mono- or polyblastic, integrated or discrete, terminal or lateral, sometimes intercalary, subglobose, subcylindrical, barrel-shaped or obclavate, pale brown, subhyaline around the conidiogenous locus, smooth-walled to verruculose. Conidia dry, acropleurogenous, solitary or in short acropetal chains, subglobose, ellipsoidal, or oblong, muriform, often constricted at the transversal septa, brown to dark brown, finely roughened to verrucose, thick-walled. Microconidiation cycle from muriform primary conidia can be present. Synanamorph state can be present, consisting in blastic fragmoconidia, growing lateral or terminal on vegetative hyphae, sessile or short stalked, cylindrical, sometimes branched, rounded apically, truncate at base, pale brown to brown, thick-walled, often remaining attached on hyphae.
Habitat and geographical distribution: In addition to our freshwater sediment isolates from Spain, the environmental data suggest that members of Neostemphylium would primarily inhabit soil, but also air, rhizosphere soils, roots, and shoots from areas of Australia, Europe (France), and the USA (Figure 2).
Neostemphylium polymorphum Torres-Garcia, Gené and Cano, sp. nov. Figure 3.
Mycobank MB 843271
Etymology: Name refers to the different structures produced by the fungus.
Type: Spain, Comunidad de Madrid, Rascafría, Arroyo del Brezal, 40°51′30.7″ N 3°54′37.6″ W, fluvial sediments, May 2019, J. Cano (holotype CBS H-24943; culture ex-type FMR 17886, CBS 149061).
Description: Mycelium immersed and superficial. Hyphae septate, branched, 2.5–4.5 μm wide, hyaline to pale brown, smooth-walled to verruculose. Conidiophores semi-macronematous or macronematous; semi-macronematous conidiophores consisting in conidiogenous cells arising terminally or often laterally from supporting cells or directly on hyphae, pale brown, verruculose; macronematous conidiophores straight to flexuous, septate, branched, up to 280 μm long, hyaline and smooth-walled towards the base, pale brown and mostly verruculose towards the branched part, with branches bearing terminally 1–2 conidiogenous cells, sometimes proliferating percurrently forming nodulose branches. Conidiogenous cells integrate and terminal or discrete growing laterally from the hyphae or conidiophore branches, sometimes intercalary due to the proliferation of the branches, subcylindrical or barrel-shaped, more commonly obclavate, 6–14.5 (–17.5) × 4.5–7 μm, pale brown to brown, usually verruculose. Conidia solitary, occasionally in short acropetal chains with up to 3 conidia, brown to dark brown, verruculose to verrucose, subglobose, ellipsoidal or oblong, (14–)18–26 (–29) × 11–16 μm, with 1–2 longitudinal or oblique septa per transversal segment and (1–)3–4 transverse septa often constricted, with a narrow cylindrical basal hylum. Microconidiation cycle was observed in some primary conidia, which give rise to secondary conidia, similar to the initial ones, from conidiogenous loci in the apical, lateral, or subterminal cells of the primary conidial body. Synanamorph present, consisting in blastic phragmoconidia, sessile or short stalked, with (1–)3–5(–7) septa, often constricted at septa, cylindrical to subcylindrical, sometimes branched, rounded apically, truncate at base, (20–)26–32(–36) × 7–10 μm, smooth- and thick-walled, brown, often remaining attached on hyphae.
Culture characteristics (7d at 25 °C): Colonies on PDA reaching 79–80 mm diam., flattened, dense, granulose, aerial mycelium scarce, sporulation abundant, dark green (30F8), yellowish green (30B7) at periphery, margins fimbriate and slightly irregular; reverse dark green (30F8) at center to golden brown (5D7) towards periphery, with a light yellow (4A4) soluble pigment. On PCA, attaining 61–62 mm diam., flattened, granulose, aerial mycelium scarce, sporulation abundant, dark green (29F5), greenish grey (28B2) at periphery, margins fimbriate and regular; reverse dull green (29E4), grey (28A2) at periphery, soluble pigment absent. On OA, reaching 52–54 mm diam., flattened, granulose, aerial mycelium scarce, sporulation abundant, dark green (28F6) at center, greenish grey (28B2) at periphery, margins fimbriate and regular; reverse dark green (28F4) to dull green (28E4) towards periphery, soluble pigment absent.
Cardinal temperatures for growth: minimum 5 °C, optimum 25 °C, maximum 35 °C.
Additional isolates examined: Spain, Comunidad de Madrid, Rascafría, Arroyo de la Umbría, 40°51′54.7″ N 3°53′40.3″ W, fluvial sediments, May 2019, J. Cano (FMR 17893, CBS 149062); Arroyo del Brezal, 40°51′31.5″ N 3°54′38.6″ W, fluvial sediments, May 2019, J. Cano (FMR 17889); Arroyo de la Umbría, 40°51′54.7″ N 3°53′40.3″ W, fluvial sediments, May 2019, J. Cano (FMR 17894); Arroyo de la Umbría, 40°51′38.6″ N 3°54′13.7″ W, fluvial sediments, May 2019, J. Cano (FMR 17895).
Distribution: Australia, France, Spain, and the USA (Figure 2, Table 2).
Notes: The multi-gene phylogeny of the Pleosporaceae presented here shows that N. polymorphum is related to the genera Asteromyces, Paradendryphiella, and Stemphylium (Figure 1). However, it is not only placed distant from the clade representative of these three genera, but Neostemphylium also differs in several diagnostic morphological features. Although Neostemphylium and Stemphylium resemble each other in their anamorphs characterized by the formation of phaeodictyospores from apically swollen conidiogenous cells, the conidiophore branching pattern is more complex in Neostemphylium than in Stemphylium species. Conidiophores in the latter genus are commonly unbranched or rarely branched [9]. In addition, Neostemphylium produces a synanamorphic state characterized by blastic, brown phragmoconidia, sometimes branched, that are not reported in any species of Stemphylium. Paradendryphiella differs in the production of exclusively cylindrical to obclavate phragmoconidia with dark septa on narrow denticles, often aggregated at the apex of lateral or terminal conidiogenous cells [18], while the conidiogenous apparatus of Asteromyces is characterized by polyblastic swollen conidiogenous cells with long denticles in a radial arrangement, giving rise to one-celled dark brown conidia [60].
Gibbago is another pleosporacean genus, represented by G. trianthemae, which also resembles Neostemphylium in its conidiogenous cells and conidial morphology [38,61]. However, like in Stemphylium species, Gibbago produces mostly unbranched or rarely branched conidiophores, and no synanamorph or microconidiation cycle have been described in G. trianthemae. In addition, our phylogeny agrees with the Pleosporaceae phylogeny presented by Pem et al. [38], placing the genus Gibbago in a fully supported clade related to Exserohilum, which are both placed far from the new genus proposed here.
Scleromyces Torres-Garcia, Dania García and Gené, gen nov.
MycoBank: MB 843291
Etymology: Name refers to the production of only sclerotium-like structures in in vitro conditions.
Subclass classification: Dothideomycetes, Pleosporomycetidae, Pleosporales, Pleosporaceae.
Type species: Scleromyces submersus Torres-Garcia, Dania García and Gené.
Description: Teleomorph and Anamorph not observed. Hyphae septate, branched, at first cylindrical, hyaline to subhyaline, smooth- and thin-walled, becoming nodose, pale olivaceous to brown and thick-walled at irregular intervals and giving rise to multicellular dark pigmented sclerotium-like structures. Sclerotium-like structures immersed to erumpent, discrete to confluent, multi-celled, globose, subglobose, or irregularly shaped, olivaceous brown to dark brown, smooth- and thick-walled.
Habitat and geographical distribution: Aside from our freshwater sediment isolate from Spain, the environmental metadata suggest that members of Scleromyces would inhabit temperate climate areas (Australia and USA), colonizing primarily soils but also it can be found associated with plant material (roots and shoots) (Figure 2, Table 2).
Scleromycessubmersus Torres-Garcia, Dania García and Gené, sp. nov. Figure 4.
MycoBank: MB 843292
Etymology: The name refers to the substrate, fluvial sediments, from which the type species was collected.
Type: Spain, Catalonia, La Noguera, Camarasa, Riu Segre, 41°53′07.4″ N 0°52′45.2″ E, fluvial sediments, December 2019, D. Torres-Garcia and J. Gené (holotype CBS H-24944, cultures ex-type FMR 18289, CBS 149025).
Description: Mycelium superficial and immersed. Hyphae septate, branched, at first cylindrical, 2.5–5.5 µm wide, hyaline to subhyaline, smooth- and thin-walled, becoming nodose at irregular intervals, with inflated cells up to 8.5–12.5 µm wide, pale olivaceous to brown and thick-walled, giving rise to multicellular dark pigmented sclerotium-like structures in all culture media tested. Sclerotium-like structures abundant, immersed to erumpent, discrete to confluent, composed of clusters of septate, pale olivaceous to brown, swollen and thick-walled cells, forming globose, subglobose, or irregularly shaped masses, up to 190 µm diam., olivaceous brown to dark brown and smooth-walled.
Based on alignments of the separate loci, Sc. submersus differs from its closest phylogenetic neighbor, A. avenicola, by having unique fixed alleles in each of the five loci: LSU positions 447 (G), 466 (T), 474 (T), 475 (C), 499 (G), 508 (C), 665 (T); ITS positions 51 (T), 59 (T), 60 (T), 62 (A), 63 (T), 64 (C), 67 (C), 69 (T), 77 (G), 97 (A), 99 (C), 100 (A), 300 (T), 312 (C), 333 (A), 348 (G), 374 (A), 376 (T), 379 (A), 382 (T), 383 (C), 384 (T), 386 (C), 388 (A); gapdh positions 48 (G), 49 (A), 56 (C), 59 (G), 60 (T), 61 (A), 62 (G), 63 (C), 65 (A), 67 (C), 68 (A), 69 (T), 70 (G), 76 (A), 79 (T), 81 (T), 85 (T), 86 (T), 91 (C), 92 (G), 115 (T), 139 (T), 145 (T), 161 (A), 163 (C), 167 (G), 169 (A), 172 (T), 173 (G), 175 (G), 178 (A), 179 (C), 182 (T), 186 (G), 187 (T), 190 (T), 194 (C), 195 (C), 196 (A), 197 (G), 198 (T), 199 (C), 201 (T), 205 (G), 207 (C), 210 (T), 211 (T), 212 (C), 214 (A), 216 (C), 217 (A), 219 (A), 221 (C), 222 (T), 223 (A), 224 (A), 226 (C), 228 (A), 229 (C), 231 (G), 233 (G), 235 (C), 236 (A), 237 (T), 238 (C), 239 (A), 242 (T), 243 (T), 244 (A), 250 (C), 253 (T), 254 (A), 255 (A), 257 (A), 258 (G), 261 (A), 271 (A), 286 (C), 289 (T), 295 (C), 313 (T), 316 (C), 325 (G), 238 (T), 331 (A), 346 (C), 358 (T), 409 (T), 433 (T), 436 (T), 487 (T), 496 (A), 511 (T), 514 (C), 541 (T), 556 (C), 565 (G), 571 (T), 577 (G), 583 (A), 586 (T), 592 (G); rpb2 positions 3 (G), 20 (T), 26 (A), 29 (C), 32 (T), 36 (T), 44 (T), 47 (G), 50 (T), 53 (T), 59 (G), 62 (C), 71 (C), 74 (G), 77 (G), 83 (A), 107 (C), 116 (C), 119 (T), 134 (A), 137 (C), 156 (T), 158 (G), 168 (C), 174 (C), 185 (T), 194 (T), 200 (C), 206 (G), 209 (G), 215 (T), 218 (C), 230 (A), 232 (T), 245 (G), 248 (G), 251 (A), 254 (G), 263 (T), 269 (C), 272 (A), 284 (A), 305 (T), 308 (T), 314 (C), 317 (C), 320 (G), 326 (A), 335 (G), 338 (A), 344 (A), 353 (A), 356 (A), 362 (A), 365 (G), 368 (C), 374 (A), 389 (A), 395 (C), 407 (T), 410 (C), 425 (T), 431 (T), 434 (C), 437 (G), 443 (T), 446 (T), 449 (C), 461 (G), 464 (C), 470 (C), 485 (C), 488 (C), 494 (A), 500 (T), 506 (T), 512 (T), 533 (T), 536 (A), 543 (G), 544 (C), 545 (G), 554 (G), 562 (A), 563 (C), 564 (T), 569 (T), 575 (C), 587 (G), 593 (T), 599 (G), 605 (G), 620 (C), 623 (T), 626 (C), 638 (G), 641 (T), 644 (G), 650 (T), 653 (T), 656 (T), 662 (A), 665 (A), 674 (T), 683 (A), 692 (C), 698 (C), 704 (A), 707 (A), 723 (A), 729 (C), 731 (A), 734 (C), 735 (A), 740 (C), 743 (C), 749 (C), 752 (A), 759 (A), 762 (G), 764 (T), 770 (C), 776 (T), 787 (C), 788 (C), 797 (T), 800 (C), 803 (T), 809 (A), 812 (T), 815 (T), 818 (C), 821 (T), 836 (T), 842 (A), 845 (G), 851 (T), 854 (C), 857 (G), 860 (C), 863 (T); tef1 positions 33 (C), 35 (C), 36 (C), 38 (A), 39 (C), 41 (A), 42 (T), 43 (C), 51 (A), 52 (C), 53 (T), 56 (C), 57 (T), 63 (T), 64 (G), 65 (G), 66 (T), 74 (A), 80 (C), 82 (T), 97 (A), 109 (T), 114 (G), 143 (G), 146 (C), 147 (T), 151 (A), 152 (A), 153 (G), 156 (C), 162 (G), 163 (A), 164 (A), 165 (A), 167 (G), 176 (C), 186 (A), 188 (C), 190 (G), 193 (C), 200 (T), 201 (C), 210 (C), 212 (T), 221 (A), 224 (C), 244 (T).
Culture characteristics (7d at 25 °C): Colonies remaining sterile in all media tested. On PDA, reaching 24–25 mm diam., slightly elevated at center, cottony and dull green (30E4) at center, velvety and yellowish grey (3B2) at periphery, margin somewhat entire, slightly fimbriate; reverse dark green (30F4) to greyish green (28E7) at center and greenish grey (28B2) at periphery. On PCA, reaching 17–18 mm diam., cottony, greyish green (29E4), margin irregular, filamentous; reverse dark green (30F5). On OA, reaching 31–33 mm diam., flattened, cottony, and dull green (28E4) at center, velvety and yellowish grey (3B2) towards periphery, margin irregular, filamentous; reverse greyish green (30E5) at center and yellowish grey (3B2) at periphery.
Cardinal temperatures for growth: minimum 5 °C, optimum 25 °C, maximum 35 °C.
Distribution: Australia and Spain (Figure 2, Table 2).
Notes: Scleromyces submersus only produced abundant sclerotium-like structures in all the culture media tested, which might resemble protoascomata. However, these never ripened nor was the type isolate able to produce conidia in any in vitro conditions to which it was submitted (see material and method section). Similar structures have been described in species of different pleosporacean genera, such as Alternaria, Curvularia, or Pyrenophora [18,33,42,62]. Some of their species have even been described as only producing these types of structures, as in the case of A. slovaca [42,63] or P. pseudoerythrospila [42]. However, these two fungi were classified and clearly distinguished from the other species of their respective genera using mostly the same phylogenic markers used to delineate Sc. submersus. The description of our fungus is clearly susceptible to emendation when new isolates of the species become available.

4. Discussion

The study of underexplored substrates can contribute to widening the knowledge of the Pleosporaceae diversity and, subsequently, to filling gaps in phylogenetic relationships among its taxa. In the present study, we describe two novel genera for the family, Neostemphylium and Scleromyces, sampled from Spanish freshwater sediments and cultured in vitro using the semi-selective medium DRBC. The efficacy of this medium to isolate pleosporacean fungi, such as Alternaria, Bipolaris, or Curvularia, was previously reported by Funnel-Harris et al. [64]. However, our study is the first to report that DRBC supplemented with benomyl can also be effective for culturing pleosporacean fungi of taxonomic interest, since all isolates of Neostemphylium were recovered from the latter medium. Anyway, its efficacy is well known to isolate ascomycetes from other groups, such as Microascales of clinical interest like Lomentospora or Scedosporium species [65,66].
The multi-locus phylogenetic analysis has been crucial for delimiting the novel fungi because of the resemblance of Neostemphylium to other genera, such as Stemphylium or Gibbago, and in the case of Scleromyces due to the absence of strictly sporulating structures. Neostemphylium shares with Stemphylium and Gibbago the production of phaeodictyospores from apically somewhat swollen and darkened conidiogenous cells [8,9]. However, it differs in the development of a synanamorph, which consists of blastic, cylindrical phragmoconidia, occasionally branched, that arise laterally or terminally on vegetative hyphae, and in the production of a microconidiation cycle not described in Gibbago or in Stemphylium. Similar structures to the N. polymorphum synanamorph have been described in the two species of the genus Berkeleiomyces (Be.), Be. bassicola and Be. rouxiae, although they were defined as septate chlamydospores [67]. Those fungi, however, belong to the microascaceous family Ceratocystidaceae and are phytopathogens to a wide range of plant hosts [67,68]. Conversely, the ability to produce a microconidiation cycle is known in the Pleosporaceae, since it has been reported in different species of Bipolaris and Curvularia [33,69], but the biological role of this state remains obscure. The microconidiation cycle observed in N. polymorphum is different from other genera because its mature secondary conidia resemble the primary ones (Figure 3N–Q), that is, they become dark brown dictyoconidia. Secondary conidia described in Bipolaris and Curvularia are small, globose, and usually one-celled [33,70].
Despite being limited to form sclerotium-like structures, Scleromyces is phylogenetically distinct from other genera in the Pleosporaceae, at least from those with available DNA sequence data (Figure 1). According to Hongsanan et al. [3], cultures for some accepted genera in the family (i.e., Allonecte, Diademosa, Extrawettsteinina, Platysporoides, Pleoseptum, Prathoda, and Pseudoyuconia) are not available for comparison and lack DNA sequence data for confirming their classification. However, most of them were described as associated with plant material, producing only the teleomorph and placed in the family according to their morphological features [3,4]. As mentioned before, there are other pleosporalean fungi, such as A. slovaca [18,63] or P. pseudoerythrospila [42], which only produce sclerotia or chlamydospores and have been distinguished from other members of the genus exclusively by molecular data. Another example in the Pleosporales is the recently described monotypic genus Gambiomyces, which has been delimited according to the phylogeny of LSU, ITS, rpb2, and tef1 and erected to accommodate the sterile fungus G. profunda, isolated from clinical specimens of a Gambian patient [71]. Examples from other fungal groups include the chaetothyrialean soil-inhabiting species Cyphellophora chlamydospora, which was described as producing only chlamydospores [72], and the species of the xylarialean endophytic genus Muscodor, which have been described as producing only sterile mycelia [73,74]. However, all of them have been clearly distinguished from their counterparts by their phylogeny, giving rise the possibility of naming relevant fungi like the species of Muscodor, which are important producers of volatile organic compounds with a wide range of potential applications in agriculture, medicine, and other sectors [74].
A huge number of unidentified environmental fungal sequences have been generated in the last decade by numerous metagenomic studies, with relevant information on ecology and distribution. One way to resolve their identification is currently to attempt to link them to sequences of known and well-established species [58,75,76,77,78]. In this context, following the recent studies on the phylogeny and global distribution of Zanclospora and Codinaea [58,59], we traced the novel species N. polymorphum and Sc. submersus in the GlobalFungi database [20] to explore their putative geographical distribution as well as to detect hypothetical hidden taxa related. Our results revealed that N. polymorphum is a more common worldwide fungus than Sc. submersus, since its sequences can be linked to a large set of environmental ITS1 sequences from samples collected in Australia, Europe (France and Spain), and the USA (Figure 2). This distribution is not surprising since those are the most-sampled areas given in the GlobalFungi database (7.46%, 33.55%, and 23.74%, respectively, at the time of accession). On the other hand, Sc. submersus could be defined as a rarer or more geographically restricted fungus because only one ITS2 environmental sequence, originating from Australia, matched this species. Although N. polymorphum and Sc. submersus were discovered in freshwater sediment samples, we might assume that they are more likely terrestrial fungi given that the majority of environmental sequences they were linked to originated from soils of different terrestrial biomes in regions with humid and temperate climates (Table 2). In fact, fungal communities that colonize terrestrial substrates like plants, soils, rocks, etc., will end up in the river sediments by lixiviation, where they can accumulate and survive under water conditions. That adds further support to river sediments as a suitable substrate for isolating a great diversity of fungi, including putative novel taxa.
Interestingly, our metabarcode analysis allowed us to detect four hidden phylotypes or “dark taxa”, defined by Lücking et al. [78] as “new lineages known from sequence data only but for which no individual voucher specimens or cultures exist”. Namely, one was related to the genus Neostemphylium (ITS1-ENV1) and three to Scleromyces (ITS1-ENV2, ITS1-ENV3, ITS2-ENV1) (Figure 2). However, none of them were represented by full-length ITS sequences as we did not find any correlation among the metadata from such phylotypes. That was in contrast to Réblová et al. [58], who obtained three whole ITS sequences among Zanclospora phylotypes, which were attributable to their geography and ecology data overlapping among various phylotypes found. We hope that all those “dark taxa” can be formally proposed soon following some of the options proposed by Lücking et al. [78] for naming fungi known only from environmental sequences.
Finally, our phylogenetic analysis has not only contributed to delineating two new genera but has also allowed us to confirm the taxonomic position of the monotypic genus Asteromyces [60] in the family Pleosporaceae, increasing to 26 the number of the accepted genera since its last review [3,4]. According to Mycobank, it was a genus classified in the family Dematiaceae (Helotiales) and in the Index Fungorum database as incertae sedis. We examined the morphology of the type species of the genus A. cruciatus and completed sequence data for its ex-type strain CBS 171.63 and for the reference strain CBS 536.92 (Table 1), confirming its particular features [60] and its relationship with other members of the family, such as Paradendryphyella and Stemphylium (Figure 1).

Supplementary Materials

The following supporting information can be downloaded at: Figure S1: RAxML tree of the Pleosporaceae family from ITS, including the strains recovered from freshwater sediments studied in this work. The strains studied in this work are in bold and in red in the obtained tree. Determined by MEGA software v.6, the best nucleotide substitution model for ML analysis was K2 + G + I. The aligned data set was 531 bp long, with 217 variable sites and 198 phylogenetically informative. Branch lengths are proportional to phylogenetic distance. Bootstrap support values above 50% are indicated on the nodes. The tree is rooted to Neocamarosporium chichastianum CBS 137502 and Neocamarosporium goegapense CPC 23676. T = Ex-type strain. Table S1: Environmental and biogeographical information contained in all ITS1/ITS2 sequences downloaded from the GlobalFungi database included in our analysis (see Figure 2).

Author Contributions

Conceptualization, D.T.-G. and J.G.; methodology, D.T.-G., J.G. and J.F.C.-L.; software, D.T.-G. and J.F.C.-L.; validation, D.G., J.F.C.-L. and J.G.; formal analysis, D.T.-G., D.G. and J.G.; investigation, D.T.-G. and J.G.; resources, J.G.; data curation, D.G. and J.G.; writing—original draft preparation, D.T.-G. and J.G.; writing—review and editing, D.T.-G., D.G., J.F.C.-L. and J.G.; visualization, D.G. and J.G.; supervision, D.G. and J.G.; project administration, J.G.; funding acquisition, J.G. All authors have read and agreed to the published version of the manuscript.


This study was supported by the Spanish Ministerio de Ciencia e Innovación, grant PID2021-128068NB-100.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.


The authors thank to the CBS culture collection (The Netherlands) and its curators for providing some fungal strain included in the study.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Nitschke, T.R.J. Grundlage eines systems der Pyrenomyceten. Verhandlungen des Naturhistorischen Vereins der Preussischen Rheinlande. Westfalens und des Regierungsbezirks Osnabrück 1869, 26, 70–77. [Google Scholar]
  2. Kodsueb, R.; Dhanasekaran, V.; Aptroot, A.; Lumyong, S.; McKenzie, E.H.C.; Hyde, K.D.; Jeewon, R. The family Pleosporaceae: Intergeneric relationships and phylogenetic perspectives based on sequence analyses of partial 28S rDNA. Mycologia 2006, 98, 571–583. [Google Scholar] [CrossRef] [PubMed]
  3. Hongsanan, S.; Hyde, K.D.; Phookamsak, R.; Wanasinghe, D.N.; McKenzie, E.H.C.; Sarma, V.V.; Xie, N. Refined families of Dothideomycetes: Dothideomycetidae and Pleosporomycetidae. Fungal Divers. 2020, 105, 17–318. [Google Scholar] [CrossRef]
  4. Wijayawardene, N.N.; Hyde, K.D.; Al-Ani, L.K.T.; Tedersoo, L.; Haelewaters, D.; Rajeshkumar, K.C.; Deng, C. Outline of Fungi and fungus-like taxa. Mycosphere 2020, 11, 1060–1456. [Google Scholar] [CrossRef]
  5. Zhang, Y.; Crous, P.W.; Schoch, C.L.; Hyde, K.D.  Pleosporales. Fungal Divers. 2012, 52, 1–221. [Google Scholar] [CrossRef]
  6. McNeill, J.; Barrie, F.R.; Buck, W.R.; Demoulin, V.; Greuter, W.; Hawksworth, D.L.; Herendeen, P.S.; Knapp, S.; Marhold, K.; Prado, J.; et al. International Code of Nomenclature for Algae, Fungi, and Plants (Melbourne Code): Appendices II-VIII; Turland, N., Ed.; Regnum Vegetabile: Melbourne, Australia, 2012; Volume 154, p. 492. [Google Scholar]
  7. Rossman, A.Y.; Crous, P.W.; Hyde, K.D.; Hawksworth, D.L.; Aptroot, A.; Bezerra, J.L.; Bhat, J.D.; Boehm, E.; Braun, U.; Boonmee, S.; et al. Recommended names for pleomorphic genera in Dothideomycetes. IMA Fungus 2015, 6, 507–523. [Google Scholar] [CrossRef] [PubMed]
  8. Ariyawansa, H.A.; Thambugala, K.M.; Manamgoda, D.S.; Jayawardena, R.; Camporesi, E.; Boonmee, S.; Wanasinghe, D.N.; Phookamsak, R.; Hongsanan, S.; Singtripop, C.; et al. Towards a natural classification and backbone tree for Pleosporaceae. Fungal Divers. 2015, 71, 85–139. [Google Scholar] [CrossRef]
  9. Woudenberg, J.H.C.; Hanse, B.; Van Leeuwen, G.C.M.; Groenewald, J.Z.; Crous, P.W. Stemphylium revisited. Stud. Mycol. 2017, 87, 77–103. [Google Scholar] [CrossRef]
  10. Elkhateeb, W.A.; Kolaibe, A.G.A.; Elkhateeb, A.; Daba, G.M. Allergen, pathogen, or biotechnological tool? The dematiaceous fungi Alternaria what’s for it and what’s on it? J. Pharm. Pharmacol. Res. 2021, 4, 1–6. [Google Scholar] [CrossRef]
  11. De Hoog, G.S.; Guarro, J.; Gené, J.; Ahmed, S.; Al-Hatmi, A.M.S.; Figueras, M.; Vitale, R.G. Atlas of Clinical Fungi: The Ultimate Benchtool for Diagnostics. Introductions, Lower Fungi, Basidiomicetes, Yeasts, Filamentous Ascomycetes, 4th ed.; Foundation Atlas: Hilversum, The Netherlands, 2020; p. 776. [Google Scholar]
  12. Lepère, C.; Domaizon, I.; Humbert, J.F.; Hugoni, M.; Debroas, D. Diversity, spatial distribution and activity of fungi in freshwater ecosystems. PeerJ. 2019, 7, e6247. [Google Scholar] [CrossRef]
  13. Egidi, E.; Delgado-Baquerizo, M.; Plett, J.M.; Wang, J.; Eldridge, D.J.; Bardgett, R.D.; Maestre, F.T.; Singh, B.K. A few Ascomycota taxa dominate soil fungal communities worldwide. Nat. Commun. 2019, 10, 2369. [Google Scholar] [CrossRef]
  14. Mohapatra, M.; Yadav, R.; Rajput, V.; Dharne, M.S.; Rastogi, G. Metagenomic analysis reveals genetic insights on biogeochemical cycling, xenobiotic degradation, and stress resistance in mudflat microbiome. J. Environ. Manag. 2021, 292, 112738. [Google Scholar] [CrossRef] [PubMed]
  15. Chauvet, E.; Cornut, J.; Sridhar, K.R.; Selosse, M.-A.; Bärlocher, F. Beyond the water column: Aquatic hyphomycetes outside their preferred habitat. Fungal Ecol. 2016, 19, 112–127. [Google Scholar] [CrossRef]
  16. Su, H.-Y.; Udayanga, D.; Luo, Z.; Manamgoda, D.; Zhao, Y.-C.; Yang, J.; Hyde, K. Hyphomycetes from aquatic habitats in Southern China: Species of Curvularia (Pleosporaceae) and Phragmocephala (Melannomataceae). Phytotaxa 2015, 226, 201. [Google Scholar] [CrossRef]
  17. Kambura, A.K.; Mwirichia, R.K.; Kasili, R.W.; Karanja, E.N.; Makonde, H.M.; Boga, H.I. Diversity of fungi in sediments and water sampled from the hot springs of Lake Magadi and Little Magadi in Kenya. Afr. J. Microbiol. Res. 2016, 10, 330–338. [Google Scholar] [CrossRef]
  18. Woudenberg, J.H.C.; Groenewald, J.Z.; Binder, M.; Crous, P.W. Alternaria redefined. Stud. Mycol. 2013, 75, 171–212. [Google Scholar] [CrossRef] [PubMed]
  19. Vu, D.; Groenewald, M.; De Vries, M.; Gehrmann, T.; Stielow, B.; Eberhardt, U.; Al-Hatmi, A.; Groenewald, J.Z.; Cardinali, G.; Houbraken, J.; et al. Large-scale generation and analysis of filamentous fungal DNA barcodes boosts coverage for kingdom fungi and reveals thresholds for fungal species and higher taxon delimitation. Stud. Mycol. 2019, 92, 135–154. [Google Scholar] [CrossRef]
  20. Větrovský, T.; Morais, D.; Kohout, P.; Lepinay, C.; Algora, C.; Hollá, S.A.; Bahnmann, B.D.; Bílohnědá, K.; Brabcová, V.; D’Alò, F.; et al. GlobalFungi, a global database of fungal occurrences from high-throughput-sequencing metabarcoding studies. Sci. Data 2020, 7, 228. [Google Scholar] [CrossRef]
  21. Ulfig, K.; Guarro, J.; Cano, J.; Gené, J.; Vidal, P.; Figueras, M.J. General assessment of the occurrence of keratinolytic fungi in river and marine beach sediments of Catalonian waters (Spain). Water Air Soil Pollut. 1997, 94, 275–287. [Google Scholar] [CrossRef]
  22. Chupp, C. Further notes on double cover-glass mounts. Mycologia 1940, 32, 269–270. Available online: https://10.2307/3754504 (accessed on 12 January 2022).
  23. Nishikawa, J.; Nakashima, C. Japanese species of Alternaria and their species boundaries based on host range. Fungal Syst. Evol. 2020, 5, 197–282. [Google Scholar] [CrossRef] [PubMed]
  24. Kornerup, A.; Wanscher, J.H. Methuen Handbook of Colour, 3rd ed.; Methuen: London, UK, 1978. [Google Scholar]
  25. Müller, F.M.; Werner, K.E.; Kasai, M.; Francesconi, A.; Chanock, S.J.; Walsh, T.J. Rapid extraction of genomic DNA from medically important yeasts and filamentous fungi by high-speed cell disruption. J. Clin. Microbiol. 1998, 36, 1625–1629. [Google Scholar] [CrossRef] [PubMed]
  26. White, T.J.; Bruns, T.; Lee, S.J.W.T.; Taylor, J. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications, 1st ed.; Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J., Eds.; Academic Press: San Diego, CA, USA, 1990; pp. 315–322. [Google Scholar]
  27. Vilgalys, R.; Hester, M. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 1990, 172, 4238–4246. [Google Scholar] [CrossRef] [PubMed]
  28. Sung, G.H.; Sung, J.M.; Hywel-Jones, N.L.; Spatafora, J.W. A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi): Identification of localized incongruence using a combinational bootstrap approach. Mol. Phylogenetics Evol. 2007, 44, 1204–1223. [Google Scholar] [CrossRef]
  29. Liu, Y.J.; Whelen, S.; Hall, B.D. Phylogenetic relationships among ascomycetes: Evidence from an RNA polymerse II subunit. Mol. Biol. Evol. 1999, 16, 1799–1808. [Google Scholar] [CrossRef]
  30. Carbone, I.; Kohn, L.M. A method for designing primer sets for speciation studies in filamentous ascomycetes. Mycologia 1999, 91, 553–556. [Google Scholar] [CrossRef]
  31. Berbee, M.L.; Pirseyedi, M.; Hubbard, S. Cochliobolus phylogenetics and the origin of known, highly virulent pathogens, inferred from ITS and glyceraldehyde-3-phosphate dehydrogenase gene sequences. Mycologia 1999, 91, 964–977. [Google Scholar] [CrossRef]
  32. Woudenberg, J.H.C.; Seidl, M.F.; Groenewald, J.Z.; De Vries, M.; Stielow, J.B.; Thomma, B.P.H.J.; Crous, P.W. Alternaria section Alternaria: Species, formae speciales or pathotypes? Stud. Mycol. 2015, 81, 1–21. [Google Scholar] [CrossRef]
  33. Madrid, H.; Da Cunha, K.C.; Gené, J.; Dijksterhuis, J.; Cano, J.; Sutton, D.A.; Crous, P. Novel Curvularia species from clinical specimens. Persoonia 2014, 33, 48–60. [Google Scholar] [CrossRef]
  34. Wanasinghe, D.N.; Gareth, J.E.B.; Camporesi, E.; Hyde, K.D. A new species of the genus Comoclathris (Pleosporaceae). J. Fungal Res. 2015, 13, 260–268. [Google Scholar]
  35. Ismail, S.I.; Batzer, J.C.; Harrington, T.C.; Crous, P.W.; Lavrov, D.V.; Li, H.; Gleason, M.L. Ancestral state reconstruction infers phytopathogenic origins of sooty blotch and flyspeck fungi on apple. Mycologia 2016, 108, 292–302. [Google Scholar] [CrossRef] [PubMed]
  36. Hernández-Restrepo, M.; Madrid, H.; Tan, Y.P.; Da Cunha, K.C.; Gené, J.; Guarro, J.; Crous, P.W. Multi-locus phylogeny and taxonomy of Exserohilum. Persoonia 2018, 41, 71–108. [Google Scholar] [CrossRef] [PubMed]
  37. Chilvers, M.I.; Rogers, J.D.; Dugan, F.M.; Stewart, J.E.; Chen, W.; Peever, T.L. Didymella pisi sp. nov., the teleomorph of Ascochyta pisi. Mycol. Res. 2009, 113, 391–400. [Google Scholar] [CrossRef] [PubMed]
  38. Pem, D.; Jeewon, R.; Bhat, D.J.; Doilom, M.; Boonmee, S.; Hongsanan, S.; Promputtha, I.; Xu, J.C.; Hyde, K.D. Mycosphere notes 275-324, A morpho-taxonomic revision and typification of obscure Dothideomycetes genera (incertae sedis). Mycosphere 2019, 10, 1115–1246. [Google Scholar] [CrossRef]
  39. Kumar, V.; Kumar, N.; Aneja, K.R.; Kaur, M. Gibbago trianthemae, phaeodictyoconidial genus, cause leaf spot disease of Trianthema portulacastrum. Arch. Phytopathol. Plant Prot. 2016, 49, 48–58. [Google Scholar] [CrossRef]
  40. Crous, P.W.; Shivas, R.G.; Quaedvlieg, W.; van der Bank, M.; Zhang, Y.; Summerell, B.A.; Guarro, J.; Wingfield, M.J.; Wood, A.R.; Alfenas, A.C.; et al. Fungal Planet description sheets: 214–280. Persoonia 2014, 32, 184–306. [Google Scholar] [CrossRef]
  41. Amaradasa, B.S.; Madrid, H.; Groenewald, J.Z.; Crous, P.W.; Amundsen, K. Porocercospora seminalis gen. et comb. nov., the causal organism of buffalograss false smut. Mycologia 2014, 106, 77–85. [Google Scholar] [CrossRef]
  42. Marin-Felix, Y.; Hernández-Restrepo, M.; Iturrieta-González, I.; García, D.; Gené, J.; Groenewald, J.Z.; Cai, L.; Chen, Q.; Quaedvlieg, W.; Schumacher, R.K.; et al. Genera of phytopathogenic fungi: GOPHY 3. Stud. Mycol. 2019, 94, 1–124. [Google Scholar] [CrossRef]
  43. Deng, J.X.; Paul, N.C.; Park, M.S.; Yu, S.H. Molecular characterization, morphology, and pathogenicity of Alternaria panax from araliaceous plants in Korea. Mycol. Prog. 2013, 12, 383–396. [Google Scholar] [CrossRef]
  44. Thambugala, K.M.; Daranagama, D.A.; Phillips, A.J.; Bulgakov, T.S.; Bhat, D.J.; Camporesi, E.; Bahkali, A.H.; Eungwanichayapant, P.D.; Liu, Z.L.; Hyde, K.D. Microfungi on Tamarix. Fungal Divers. 2017, 82, 239–306. [Google Scholar] [CrossRef]
  45. Crous, P.W.; Schumacher, R.K.; Akulov, A.; Thangavel, R.; Hernández-Restrepo, M.; Carnegie, A.J.; Cheewangkoon, R.; Wingfield, M.J.; Summerell, B.A.; Quaedvlieg, W.; et al. New and interesting fungi. 2. Fungal Syst. Evol. 2019, 3, 57–134. [Google Scholar] [CrossRef]
  46. Tamura, K.; Stecher, G.; Peterson, D.; Filipski, A.; Kumar, S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol. Biol. Evol. 2013, 30, 2725–2729. [Google Scholar] [CrossRef] [PubMed]
  47. Thompson, J.D.; Higgins, D.G.; Gibson, T.J. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. [Google Scholar] [CrossRef] [PubMed]
  48. Edgar, R.C. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004, 32, 1792–1797. [Google Scholar] [CrossRef]
  49. Farris, J.S.; Kallersjo, M.; Kluge, A.G.; Bult, C. Testing significance of incongruence. Cladistics 1994, 10, 315–319. [Google Scholar] [CrossRef]
  50. Vaidya, G.; Lohman, D.J.; Meier, R. SequenceMatrix: Concatenation software for the fast assembly of multi-gene datasets with character set and codon information. Cladistics 2011, 27, 171–180. [Google Scholar] [CrossRef]
  51. Darriba, D.; Taboada, G.L.; Doallo, R.; Posada, D. jModelTest 2: More models, new heuristics and parallel computing. Nat. Methods 2012, 9, 772. [Google Scholar] [CrossRef]
  52. Guindon, S.; Gascuel, O. A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood. Syst. Biol. 2003, 52, 696–704. [Google Scholar] [CrossRef]
  53. Stamatakis, A. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 2014, 30, 1312–1313. [Google Scholar] [CrossRef]
  54. Miller, M.A.; Pfeifferm, W.; Schwartz, T. The CIPRES science gateway: Enabling high-impact science for phylogenetics researchers with limited resources. In Proceedings of the 1st Conference of the Extreme Science and Engineering Discovery Environment: Bridging from the Extreme to the Campus and Beyond, Chicago, IL, USA, 16–20 July 2012; Association for Computing Machinery: New York, NY, USA, 2012; pp. 1–8. [Google Scholar] [CrossRef]
  55. Ronquist, F.; Teslenko, M.; van der Mark, P.; Ayres, D.L.; Darling, A.; Höhna, S.; Larget, B.; Liu, L.; Suchard, M.A.; Huelsenbeck, J.P. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 2012, 61, 539–542. [Google Scholar] [CrossRef]
  56. Hillis, D.M.; Bull, J.J. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 1993, 42, 182–192. [Google Scholar] [CrossRef]
  57. Hespanhol, L.; Vallio, C.S.; Costa, L.M.; Saragiotto, B.T. Understanding and interpreting confidence and credible intervals around effect estimates. Braz. J. Phys. Ther. 2019, 23, 290–301. [Google Scholar] [CrossRef] [PubMed]
  58. Réblová, M.; Kolařík, M.; Nekvindová, J.; Miller, A.N.; Hernández-Restrepo, M. Phylogeny, global biogeography and pleomorphism of Zanclospora. Microorganisms 2021, 9, 706. [Google Scholar] [CrossRef] [PubMed]
  59. Réblová, M.; Kolařík, M.; Nekvindová, J.; Réblová, K.; Sklenář, F.; Miller, A.N.; Hernández-Restrepo, M. Phylogenetic reassessment, taxonomy, and biogeography of Codinaea and similar Fungi. J. Fungi 2022, 7, 1097. [Google Scholar] [CrossRef] [PubMed]
  60. Hennebert, G.L. Wardomyces and Asteromyces. Can. J. Bot. 1962, 40, 1203–1216. [Google Scholar] [CrossRef]
  61. Simmons, E.G. Gibbago, a new phaeodictyoconidial genus of hyphomycetes. Mycotaxon 1986, 27, 107–111. [Google Scholar]
  62. Campbell, M.A.; Medd, R.W.; Brown, J.F. Growth and sporulation of Pyrenophora semeniperda in vitro: Effects of culture media, temperature and pH. Mycol. Res. 1996, 100, 311–317. [Google Scholar] [CrossRef]
  63. Svobodová, Y. Chmelia slovaca gen. nov., a dematiaceous fungus, pathogenic for man and animals. Biológia 1966, 21, 81–88. [Google Scholar]
  64. Funnell-Harris, D.L.; Prom, L.K.; Pedersen, J.F. Isolation and characterization of the grain mold fungi Cochliobolus and Alternaria spp. from sorghum using semi-selective media and DNA sequence analyses. Can. J. Microbiol. 2013, 59, 87–96. [Google Scholar] [CrossRef]
  65. Gilgado, F.; Cano, J.; Gené, J.; Guarro, J. Molecular phylogeny of the Pseudallescheria boydii species complex: Proposal of two new species. J. Clin. Microbiol. 2005, 43, 4930–4942. [Google Scholar] [CrossRef]
  66. Pham, T.; Giraud, S.; Schuliar, G.; Rougeron, A.; Bouchara, J.P. Scedo-Select III: A new semi-selective culture medium for detection of the Scedosporium apiospermum species complex. Med. Mycol. 2015, 53, 512–519. [Google Scholar] [CrossRef]
  67. Nel, W.J.; Duong, T.A.; Wingfield, B.D.; Wingfield, M.J.; De Beer, Z.W. A new genus and species for the globally important, multihost root pathogen Thielaviopsis Basicola. Plant Pathol. 2018, 67, 871–882. [Google Scholar] [CrossRef]
  68. Wang, X.W.; Bai, F.Y.; Bensch, K.; Meijer, M.; Sun, B.D.; Han, Y.F.; Crous, P.W.; Samson, R.; Yang, F.Y.; Houbraken, J. Phylogenetic re-evaluation of Thielavia with the introduction of a new family Podosporaceae. Stud. Mycol. 2019, 93, 155–252. [Google Scholar] [CrossRef] [PubMed]
  69. Bhunjun, C.S.; Dong, Y.; Jayawardena, R.S.; Jeewon, R.; Phukhamsakda, C.; Bundhun, D.; Hyde, K.D.; Sheng, J. A polyphasic approach to delineate species in Bipolaris. Fungal Divers. 2020, 102, 225–256. [Google Scholar] [CrossRef]
  70. Raza, M.; Zhang, Z.F.; Hyde, K.D.; Diao, Y.Z.; Cai, L. Culturable plant pathogenic fungi associated with sugarcane in southern China. Fungal Divers. 2019, 99, 1–104. [Google Scholar] [CrossRef]
  71. Valenzuela-López, N.; Martin-Gómez, M.T.; Los-Arcos, I.; Stchigel, A.M.; Guarro, J.; Cano-Lira, J.F. A new pleosporalean fungus isolated from superficial to deep human clinical specimens. Med. Mycol. 2021, 59, 278–288. [Google Scholar] [CrossRef]
  72. Madrid, H.; Hernández-Restrepo, M.; Gené, J.; Cano, J.; Guarro, J.; Silva, V. New and interesting chaetothyrialean fungi from Spain. Mycol. Prog. 2016, 15, 1179–1201. [Google Scholar] [CrossRef]
  73. Chen, J.J.; Feng, X.X.; Xia, C.Y.; Kong, D.D.; Qi, Z.Y.; Liu, F.; Chen, D.; Lin, F.C.; Zhang, C.L. Confirming the phylogenetic position of the genus Muscodor and the description of a new Muscodor species. Mycosphere 2019, 10, 187–201. [Google Scholar] [CrossRef]
  74. Saxena, S.; Strobel, G.A. Marvellous Muscodor spp.: Update on their biology and applications. Microb. Ecol. 2021, 82, 5–20. [Google Scholar] [CrossRef]
  75. Hawksworth, D.L.; Hibbett, D.S.; Kirk, P.M.; Lücking, R. Proposals to permit DNA sequence data to serve as types of names of fungi. Taxon 2016, 65, 899–900. [Google Scholar] [CrossRef]
  76. Tedersoo, L.; Bahram, M.; Puusepp, R.; Nilsson, R.H.; James, T.Y. Novel soil-inhabiting clades fill gaps in the fungal tree of life. Microbiome 2017, 5, 42. [Google Scholar] [CrossRef]
  77. Ryberg, M.; Nilsson, R.H. New light on names and naming of dark taxa. MycoKeys 2018, 30, 31–39. [Google Scholar] [CrossRef] [PubMed]
  78. Lücking, R.; Aime, M.C.; Robbertse, B.; Miller, A.N.; Aoki, T.; Ariyawansa, H.A.; Cardinali, G.; Crous, P.W.; Druzshinina, I.S.; Geiser, D.M.; et al. Fungal taxonomy and sequence-based nomenclature. Nat. Microbiol. 2021, 6, 540–548. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Phylogenetic analysis of the Pleosporaceae based on maximum likelihood analysis obtained by RAxML inferred from the combined ITS, LSU, rpb2, tef1, and gapdh loci. Branch lengths are proportional to phylogenetic distance. Bold branches indicate bs/pp values 100/1. Bootstrap support values/Bayesian posterior probability scores above 70%/0.95 pp are indicated on the nodes. Isolates corresponding to the new genera are shown in red. Sequences of isolates generated in this study are in bold. The tree is rooted to Neocamarosporium chichastianum CBS 137502 and Neocamarosporium goegapense CPC 23676 (Neocamarosporiaceae). T = ex-type strain, ET = ex-epitype strain, NT = ex-neotype strain.
Figure 1. Phylogenetic analysis of the Pleosporaceae based on maximum likelihood analysis obtained by RAxML inferred from the combined ITS, LSU, rpb2, tef1, and gapdh loci. Branch lengths are proportional to phylogenetic distance. Bold branches indicate bs/pp values 100/1. Bootstrap support values/Bayesian posterior probability scores above 70%/0.95 pp are indicated on the nodes. Isolates corresponding to the new genera are shown in red. Sequences of isolates generated in this study are in bold. The tree is rooted to Neocamarosporium chichastianum CBS 137502 and Neocamarosporium goegapense CPC 23676 (Neocamarosporiaceae). T = ex-type strain, ET = ex-epitype strain, NT = ex-neotype strain.
Jof 08 00868 g001
Figure 2. Phylogenetic relationships among Neostemphylium (N.) polymorphum, Scleromyces (Sc.) submersus, and related ITS1/ITS2 environmental sequences deposited in the GlobalFungi database. Titles of sequences contain sequence and sample codes taken from GlobalFungi. ITS1/ITS2 sequences of sediment isolates of N. polymorphum and Sc. submersus are written in red. ITS1/ITS2 sequences of known pleosporacean species are written in green. Bootstrap support values above 50% are indicated on the nodes. The trees are rooted to Comoclathris typhicola CBS 132.69 and Comoclathris sedis CBS 366.52. The trees also include data on the origin of the samples.
Figure 2. Phylogenetic relationships among Neostemphylium (N.) polymorphum, Scleromyces (Sc.) submersus, and related ITS1/ITS2 environmental sequences deposited in the GlobalFungi database. Titles of sequences contain sequence and sample codes taken from GlobalFungi. ITS1/ITS2 sequences of sediment isolates of N. polymorphum and Sc. submersus are written in red. ITS1/ITS2 sequences of known pleosporacean species are written in green. Bootstrap support values above 50% are indicated on the nodes. The trees are rooted to Comoclathris typhicola CBS 132.69 and Comoclathris sedis CBS 366.52. The trees also include data on the origin of the samples.
Jof 08 00868 g002
Figure 3. Neostemphylium polymorphum gen. et sp. nov. (FMR 17886). (A,B) Colonies on PDA (front and reverse). (C) Colony on PCA. (D) Colony on OA, after two weeks at 25 °C. (E,IL) Conidiophores and conidia, some arranged in short acropetal chains. (FH) Conidiophores showing phragmoconidia of the synanamorph. (M) Conidia with narrow cylindrical basal hylum (black arrow). (NQ) Conidia showing different states of the microconidiation cycle. Scale bars: (EG) = 25 μm. (HQ) = 10 μm.
Figure 3. Neostemphylium polymorphum gen. et sp. nov. (FMR 17886). (A,B) Colonies on PDA (front and reverse). (C) Colony on PCA. (D) Colony on OA, after two weeks at 25 °C. (E,IL) Conidiophores and conidia, some arranged in short acropetal chains. (FH) Conidiophores showing phragmoconidia of the synanamorph. (M) Conidia with narrow cylindrical basal hylum (black arrow). (NQ) Conidia showing different states of the microconidiation cycle. Scale bars: (EG) = 25 μm. (HQ) = 10 μm.
Jof 08 00868 g003
Figure 4. Scleromyces submersus gen. et sp. nov. (FMR 18289). (A) Colony on PDA. (B) Colony on PCA. (C) Colony on OA, after two weeks at 25 °C. (DG) Sclerotium-like structures. Scale bars: (DG) = 10 μm.
Figure 4. Scleromyces submersus gen. et sp. nov. (FMR 18289). (A) Colony on PDA. (B) Colony on PCA. (C) Colony on OA, after two weeks at 25 °C. (DG) Sclerotium-like structures. Scale bars: (DG) = 10 μm.
Jof 08 00868 g004
Table 1. Details of the strains of the species included in the multi-locus phylogenetic analysis.
Table 1. Details of the strains of the species included in the multi-locus phylogenetic analysis.
SpeciesStrain NumberSubstrateCountryGenBank Accession Number 1Citation
Alternaria abundansCBS 534.83 TFragaria stolonNew ZealandMH861639MH873354KC584448KC584707KC584154[18]
Alternaria alternataCBS 104.26UnknownUknownKP124299KP124450KP124767KP125074KP124156[32]
Alternaria anigozanthiCBS 121920 TAnigozanthus cultivarAustraliaKC584180NG069123KC584376KC584635KC584097[18]
Alternaria argyranthemiCBS 116530Argyranthemum sp.New ZealandKC584181KC584254KC584378KC584637KC584098[18]
Alternaria avenicolaCBS 121459 TAvena sp.NorwayKC584183KC584256KC584380KC584639KC584100[18]
Alternaria infectoriaCBS 210.86 TTriticum aestivumUSAFM958526MH873633KC584404KC584662KY038017[18]
Alternaria macrosporaCBS 117228 TGossypium barbadenseUSANR136045NG069135KC584410KC584668KC584124[18]
Alternaria nobilisCBS 116490 TDianthus caryophyllusNew ZealandKC584208KC584291KC584415KC584673KC584127[18]
Alternaria obclavataCBS 124120 TAirUSANR165505MH874877KC584443KC584701KC584149[18]
Alternaria penicillataCBS 116607 TPapaver rhoeasAustriaKC584229KC584322KC584447KC584706-[18]
Alternaria slovacaCBS 567.66 THuman clinical specimenSlovakiaKC584226KC584319KC584444KC584702KC584150[18]
Alternaria solaniCBS 116651Solanum tuberosumUSAKC584217KC584306KC584430KC584688KC584139[18]
Asteromyces cruciatusCBS 171.63 TSand of dunesFranceMH858254MH869856ON703247ON542234ON542232[19]; this study
CBS 536.92Composting seaweedUSAON773141ON773155ON703248ON542235ON542233This study
Bipolaris cynodontisCBS 285.51Cynodon transvaalensisKenyaMH856862MH868380HF934831-HG779081[33]
Bipolaris maydisCBS 136.29 ETZea maysJapanMH855024MH866491HF934828-HG779086[33]
Bipolaris microlaenaeCBS 280.91 TMicrolaena stipoidesAustraliaNR137073HF934877HF934835-HG779092[33]
Clathrospora elynaeCBS 196.54Carex curvulaSwitzerlandMH857290MH872973KC584496--[18]
Comoclathris incomptaCBS 467.76Olaea europaea branchGreceKY940770MH871007KC584504--[18]
Comoclathris linisMFLUCC 145047 TDead stems of Linum sp.ItalyNR153904NG058917---[34]
Comoclathris sedisCBS 366.52UnknownUSAKY940748MH871007KT216533--[35]
Curvularia heteropogonisCBS 284.91 THeteropogon contorusAustraliaMH862253LT631396HF934821-HG779121[33]
Curvularia lunataCBS 730.96 NTHuman lung biopsyUSAMG722981LT631416HF934813-LT715821[36]
Decorospora gaudefroyiCBS 332.63UnknownFranceMH858305MH869915---[19]
CBS 250.60UnknownUKMH857974MH869526---[19]
Dichotomophthora luteaCBS 145.57 TUnknownUnknownMH857676NG069497LT990634-LT990663[19]
Dichotomophthora portulacaeCBS 174.35 TUnknownUnknownNR158421MH867137LT990638LT990668-[19]
Didymella exiguaCBS 183.55Rumex arifoliusFranceMH857436MH871007EU874850--[37]
Exserohilum monocerasCBS 239.77Echinochloa colonaAustraliaLT837474LT883405LT852506-LT883547[36]
Exserohilum rostratumCBS 128061Zea maysUSAKT265240MH877986LT715752-LT715900[36]
Exserohilum turcicumCBS 387.58Zea maysUSAMH857820LT883412LT852514-LT883554[36]
Gibbago trianthemaeNFCCI 1886Trianthema portulacastrumIndiaHM448998MH870931---[19,38]
GT-VMTrianthema portulacastrumPakistanKJ825852MH870931---[39]
Johnalcornia aberransCBS 510.91UnknownAustraliaMH862272KM243286LT715737-KM257056[36]
Neocamarosporium chichastianumCBS 137502UnknownIranKJ869163MH877648---[40]
Neocamarosporium goegapenseCPC 23676Mesembryanthemum sp.South AfricaKJ869163KJ869220---[40]
Paradendriphyella arinariaeCBS 181.58 TUnknownFranceMH857747KC793338DQ435065--[19]
Paradendriphyella salinaCBS 302.84 TCancer pagurus shellDenmarkMH873443KC584325KC584450KC584709-[19]
CBS 142.60Stem of Spartina sp.EnglandMH857928MH869472DQ435066--[19]
Pheosphaeria oryzaeCBS 110110Oryza sativaKoreaMH862850MH871007---[19]
Porocercospora seminalisCBS 134907Bouteloua dactyloidesUSAHF934941HF934862HF934843--[41]
CPC 21349B. dactyloidesUSAHF934945HF934861HF934845--[41]
Neostemphylium polymorphumFMR 17886 TFluvial sedimentSpainOU195609OU195892OU196009ON368192OU195960This study
FMR 17889Fluvial sedimentSpainOU195610OU195914OU196957ON368193OU195977This study
FMR 17893Fluvial sedimentSpainOU195631OU195915OU197255ON368194OU195978This study
FMR 17894Fluvial sedimentSpainOU195879OU195937OU196956ON368195OU195998This study
FMR 17895Fluvial sedimentSpainOU195878OU195936OU197545ON368196OU195999This study
Pyrenophora avenicolaCBS 307.84Avena seedSwedenMK539972MK540042--MK540180[42]
Pyrenophora chaetomioidesCBS 314.68Avena sativaGermanyMK539979MH870853MK540105-MK540187[42]
Pyrenophora cynosuriCBS 127918Seeds of CynosurusNew ZealandMK539980MK540047MK540106-MK540188[42]
Pyrenophora novozelandicaCBS 127934Seeds of Triticum sp.New ZealandMK539997MK540061MK540125-MK540209[42]
Pyrenophora phaecomesDAOM 222769UnknownUnknownJN943649JN940093DQ497614--[18]
Pyrenophora pseudoerythrospilaCBS 127931 TLolium sp.GermanyNR164465NG066344--MK540212[42]
Pyrenophora seminiperdaCBS 127927UnknownUnknownMK540001MH877966MK540128-MK540213[42]
Scleromyces submersusFMR 18289 TFluvial sedimentSpainOU195893OU195959OU197244OU196982OU196008This study
Stemphylium botryosumCBS 714.68 TMedicago sativaCanadaMH859208MH870931-KC584729MH206176[18]
Stemphylium lycopersiciCNU 070067Capsicum annumKoreaJF417683-JF417698JX213347JF417693[43]
Stemphylium vesicariumCBS 191.86Medicago sativaIndiaMH861935JX681120KC584471KC584731-[18]
Tamaricicola muriformisMFLUCC 150488Tamarix sp.ItalyKU752187KU561879KU820870--[44]
MFLUCC 150489Tamarix sp.ItalyKU752188KU729857---[44]
Typhicola typharumCBS 145043 NTLeaf of Typha sp. GermanyMK442590MK442530MK442666MK442696-[45]
CBS: Culture Collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands; JAC: Culture Collection of J.A. Cooper, New Zealand; MFLUCC: Culture Collection of the Mae Fah Luang University, Chiang Rai, Thailand; NFCCI: National Fungal Culture Collection of India, Agharkar Research Institute, New Delhi, India; GT-VM: Culture Collection of V. Kumar and K.R. Aneja, Pakistan; CPC: Culture Collection of P.W. Crous, The Netherlands; FMR: Facultat de Medicina i Ciències de la Salut, Reus, Spain; DAOM: Canadian Collection of Fungal Cultures, Ottawa Research and Development Centre, Ottawa, Canada. CNU: Culture Collection of the Chungnam National University, Chungnam, South Korea; T Indicates ex-type strains; ET Indicates ex-epitype strains; NT Indicates ex-neotype strains. 1 ITS: Internal transcribed spacer region of the rDNA and 5.8S gene; LSU: 28S large ribosomal subunit; rpb2: the DNA dependent RNA polymerase II largest subunit; tef1: translation elongation factor 1–α; gapdh: glyceraldehyde-3-phosphate dehydrogenase. Novelties and sequences generated in this study are in bold.
Table 2. The biogeography, substrate, and habitat affinity of Neostemphylium and Scleromyces environmental sequences in the GlobalFungi database.
Table 2. The biogeography, substrate, and habitat affinity of Neostemphylium and Scleromyces environmental sequences in the GlobalFungi database.
AbundanceOther Data 4Geographical Origin 5Substrate 6Biomes 7
TaxaSamples 1Reads 2FR 3MATMAPpHUSAEuropeAustraliaSoilRhiz. Soil *RootOthers 8WetlandCroplandForestWoodlandShrublandGrasslandAquatic
polymorphum ITS1
polymorphum ITS2
submersus ITS2
Scleromyces ITS1-ENV212.000.01215.0681.05.800110000001000
Scleromyces ITS1-ENV312.000.01220.1563.06.700110000000100
Scleromyces ITS2-ENV1226.870.04416.8731.4NA 910000110000001
1 Environmental samples containing sequences belonging to a particular taxon. 2 Mean values of reads of a sequence belonging to a particular taxon appears across the environmental samples. 3 FR: Frequency of Reads, mean values of a particular taxon across samples where the taxon was found. 4 Average values across all samples are represented; MAT: Mean Annual Temperature (°C), MAP: Mean Annual Precipitation (mm), pH (AVG). 5 Presence (1) or absence (0) of a sequence of a particular taxon according to the geographical origin of environmental samples. 6 Presence (1) or absence (0) of sequences of a particular taxon according to the kind of substrate of a sample. 7 Presence (1) or absence (0) of sequences of a particular taxon according to the biome conforming the environment where samples were collected. 8 Other substrates in the GlobalFungi database represent air or shoots. 9 No data available. * Rhizosphere soil.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Torres-Garcia, D.; García, D.; Cano-Lira, J.F.; Gené, J. Two Novel Genera, Neostemphylium and Scleromyces (Pleosporaceae) from Freshwater Sediments and Their Global Biogeography. J. Fungi 2022, 8, 868.

AMA Style

Torres-Garcia D, García D, Cano-Lira JF, Gené J. Two Novel Genera, Neostemphylium and Scleromyces (Pleosporaceae) from Freshwater Sediments and Their Global Biogeography. Journal of Fungi. 2022; 8(8):868.

Chicago/Turabian Style

Torres-Garcia, Daniel, Dania García, José F. Cano-Lira, and Josepa Gené. 2022. "Two Novel Genera, Neostemphylium and Scleromyces (Pleosporaceae) from Freshwater Sediments and Their Global Biogeography" Journal of Fungi 8, no. 8: 868.

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop