Insights into Some Onygenalean Fungi from Freshwater Sediments in Spain and Description of Novel Taxa
Unitat de Micologia i Microbiologia Ambiental, Facultat de Medicina i Ciències de la Salut and IU-RESCAT, Universitat Rovira i Virgili, 43201 Reus, Spain
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Author to whom correspondence should be addressed.
J. Fungi 2023, 9(12), 1129; https://doi.org/10.3390/jof9121129
Submission received: 26 October 2023
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Revised: 18 November 2023
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Accepted: 20 November 2023
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Published: 22 November 2023
(This article belongs to the Special Issue Freshwater Fungal Diversity)
Abstract
:During the course of a project investigating culturable Ascomycota diversity from freshwater sediments in Spain, we isolated 63 strains of cycloheximide-resistant fungi belonging to the order Onygenales. These well-known ascomycetes, able to infect both humans and animals, are commonly found in terrestrial habitats, colonizing keratin-rich soils or dung. Little is known about their diversity in aquatic environments. Combining morphological features and sequence analyses of the ITS and LSU regions of the nrDNA, we identified 14 species distributed in the genera Aphanoascus, Arachniotus, Arthroderma, Arthropsis, Emmonsiellopsis, Gymnoascoideus, Leucothecium, Malbranchea, and Myriodontium. Furthermore, three novel species for the genus Malbranchea are proposed as M. echinulata sp. nov., M. irregularis sp. nov., and M. sinuata sp. nov. The new genera Albidomyces and Neoarthropsis are introduced based on Arachniotus albicans and Arthropsis hispanica, respectively. Neoarthropsis sexualis sp. nov. is characterized and differentiated morphologically from its counterpart by the production of a sexual morph. The novel family Neoarthropsidaceae is proposed for the genera Albidomyes, Apinisia, Arachnotheca, Myriodontium, and Neoarthropsis, based on their phylogenetic relationships and phenotypic and ecological traits. Pseudoamaurascopsis gen. nov. is introduced to accommodate P. spiralis sp. nov., a fungus with unclear taxonomy related to Amaurascopsis and Polytolypa. We traced the ecology and global distribution of the novel fungi through ITS environmental sequences deposited in the GlobalFungi database. Studying the fungal diversity from freshwater sediments not only contributes to filling gaps in the relationships and taxonomy of the Ascomycota but also gives us insights into the fungal community that might represent a putative risk to the health of animals and humans inhabiting or transient in aquatic environments.
1. Introduction
Interest in fungi from aquatic environments has been increasing in recent years, although their diversity and functional roles are still poorly understood [1,2]. Recent evidence indicates that fungal diversity, particularly in freshwater environments, is high [2,3,4]. In fact, fungi play a critical role in those environments, degrading organic materials (i.e., plant litter and animal tissues) and contributing to key elemental cycles by releasing CO2 into the atmosphere and inorganic nitrogen and phosphorous into the soil [1,4,5,6,7]. It is not surprising, then, to find in freshwater sediments members of the order Onygenales. They have the ability to adapt to a wide range of environments, including oligo- and eutrophic habitats, and are also specialized in the degradation of compounds like cellulose from plant material and, particularly, keratin from animal waste [8].
The order Onygenales comprises a diverse group of ascomycetous fungi that are commonly found in terrestrial habitats worldwide. They usually inhabit soil and other substrates like the dung of herbivorous and carnivorous animals, which contain different sources of keratin as the primary source of environmental carbon and nitrogen [9,10,11,12,13,14,15]. Some well-delineated groups or families in Onygenales have clear ecological preferences in their ability to cause cutaneous (Arthrodermataceae) and systemic (Ajellomycetaceae) diseases in mammals, or to live closely associated with bees (Ascosphaeraceae) [16,17,18]. However, their diversity in freshwater sediments is still poorly studied.
Several studies have contributed to investigating the diversity of onygenalean fungi in freshwater sediments from different areas of the world [19,20,21,22,23,24,25,26]. Studies by Ulfig et al. [27,28,29,30] and Vidal [31] explored the diversity of culturable keratinolytic and nonkeratinolytic species of Onygenales isolated using various culture-dependent techniques (i.e., hair-baiting and agar media supplemented with cycloheximide) from fluvial sediments collected at the mouth of various Catalonian rivers (Spain). The predominant species were Aphanoascus (Ap.) fulvescens, Ap. reticulisporus, Arthroderma (Ar.) curreyi, Chrysosporium (C.) keratinophilum, C. pannicola, C. lobatum, C. tropicum, Gymnoascus (G.) littoralis, Myriodontium (My.) keratinophilum, Narasimhella (N.) marginospora, and N. hyalinospora, among others. However, in addition to those fungi, a set of unidentified isolates, recovered also from fluvial sediments at that time, were proposed in subsequent studies as novel taxa. These were Arthropsis (A.) hispanica [32], C. fluviale [33], C. minutisporum [34], C. pilosum [35], C. submersum [34], and C. undulatum [36], and the two members of the genus Emmonsiellopsis (E.), E. coralliformis and E. terrestris [37]. All these findings and others recently published [38,39,40,41,42,43,44] indicate that freshwater sediments serve as a reservoir for a great diversity of filamentous ascomycetes, which warrant further investigation in order to address gaps in fungal phylogenetic relationships, such as has been done in the present study for some species of the order Onygenales.
Of note is that the identification of many fungi from the abovementioned reports was based only on macro- and microscopic morphology. However, with the incorporation of molecular data in taxonomic studies, the classification of the aforementioned species and, in general, of the Onygenales and related taxa has changed considerably in recent decades. For instance, based primarily on sequence analyses of some regions of the nuclear ribosomal operon (i.e., internal transcribed spacer regions–ITS, the large subunit–LSU, and/or the small subunit–SSU of the rDNA), C. pilosum was found to be related to the genus Arachnomyces and renamed Arachnomyces pilosus, the genus that belongs to the order Arachnomycetales [45,46]; C. minutisporum and C. submersum were recently transferred to the genus Keratinophyton [47], as well as C. pannicola and G. litoralis to the genus Arthroderma and Gymnascella, respectively [8]. Meanwhile, the cosmopolitan, soilborne fungus C. tropicum is currently considered conspecific with Ap. verrucosus, and N. marginospora with N. poonensis [8]. Nevertheless, the taxonomy of other species like A. hispanica or numerous Amauroascus and Chrysosporium species remains unresolved because there are few specimens for studying and due to the polyphyletic nature of those genera [8]. Since morphological features used to classify the Onygenales [9] are insufficient for delimiting their taxa, polyphasic taxonomic studies integrating morphology, physiological traits, ecology, and phylogenetic relationships based on ITS and LSU sequence data have been crucial for a fairly stable classification in this order [15,48,49,50,51] and other fungi in general. In this scenario, the most recent reevaluation of the Onygenales has been conducted by Kandemir et al. [8]. Based on an integrative taxonomy, including the genomic data of various gene markers (i.e., ITS/LSU rDNA and partial fragments of the protein-encoding genes β-tubulin–tub2, elongation factor 1-α–tef1, partial fragments of the RNA polymerase I and II largest subunit–rpb1, rpb2, and ribosomal protein 60S L10–L1-rp60s), the latter authors elucidated the taxonomy and gave nomenclatural stability to numerous species and genera in the Onygenales. Furthermore, the new families Malbrancheaeceae and Neogymnomycetaceae were added to the seven existing families in the order (Ajellomycetaceae, Arthrodermataceae, Ascosphaeraceae, Eremascaceae, Gymnoascaceae, Onygenaceae, and Spiromastigaceae).
In the course of a project focused on the study of the diversity of culturable filamentous ascomycetes from freshwater sediments collected in Spanish rivers and streams, we recovered several specimens belonging to Onygenales with the use of different culture media, including potato dextrose agar (PDA) supplemented with cycloheximide. The aim of the present work was to resolve the taxonomy of the mentioned isolates, taking into account the integrative taxonomy described in the recent revision of this group of Ascomycota [8]. We also aim to assess the global distribution and habitat affinity of the newly proposed species here through the GlobalFungi database (https://globalfungi.com, accessed on 19 November 2023), which provides information on fungal distribution and ecology from metagenomic studies published over the last decade [52].
2. Materials and Methods
2.1. Sampling and Isolates
Sediments were collected between September 2018 and March 2021. Fungi included in this study were recovered from 21 sampled points in rivers and streams that flow through natural and rural areas of various Spanish provinces (Baleares, Barcelona, Castellón, Girona, Huesca, Lleida, Navarra, and Madrid; Figure 1). While samples from the streams were collected randomly, river sediments were mainly collected at the upper and middle sections of the rivers (Table 1).
Samples were collected and processed in the laboratory according to the procedures previously published [27,44]. The culture media used for fungal isolation were 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; Condalab, Madrid, Spain) supplemented with 2 g/L of cycloheximide. The cultures were incubated at room temperature (22–25 °C) and examined weekly with a stereomicroscope for up to 4–5 weeks. To achieve pure cultures, colonies were transferred with a sterile dissection needle from isolation plate cultures to Petri dishes containing PDA with chloramphenicol and incubated at 25 °C in darkness.
All onygenalean isolates obtained in the present study (Table 1) were deposited in the fungal culture collection at the Medicine Faculty in Reus (FMR, University Rovira i Virgili, Reus, Catalonia, Spain). Holotypes, which consisted of dry cultures, and ex-type cultures of the novel species proposed here were also deposited at the fungal collection of the Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands). Nomenclatural novelties were registered, and their respective descriptions deposited in Mycobank (https://www.mycobank.org/ (accessed on 31 July 2023)).
2.2. Phenotypic Study
For the in vitro macroscopic characterization, the isolates were inoculated on PDA, potato carrot agar (PCA; potato 20 g, carrot 20 g, agar 13 g, distilled water 1 L), oatmeal agar (OA; oatmeal 30 g, agar 13 g, distilled water 1 L), and phytone yeast extract agar (PYE; Pronadisa, Madrid, Spain). Colony features were obtained at 14 days, incubated at 25 °C in the dark. Color notations in descriptions were from Kornerup and Wanscher [53].
The microscopic characterization of the isolates studied was made from cultures growing on OA at 14 days at 25 °C in darkness and mounted onto wet slides, using water and 60% lactic acid. Photomicrographs were obtained 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 (Adobe Systems, San Jose, CA, USA).
In addition, to assess the ability of the novel fungi to grow at different temperatures, they were cultured in duplicate on PDA and incubated in the dark at 5 to 40 °C, in 5 °C intervals, including at 37 °C. Colony diameter was recorded at 7 and 14 incubation days.
2.3. DNA Extraction, Sequencing, and Phylogenetic Analysis
Isolates were cultured on PDA for 7–14 days at 25 °C in darkness. The DNA was extracted through the modified protocol of Müller et al. [54]. The extracted DNA was quantified by using Nanodrop 2000 (Thermo Scientific, Madrid, Spain). The loci amplified were the ITS regions and the LSU D1/D2 domain of the nrDNA using the primer pairs ITS5/ITS4 [55] and LR0R/LR5 [56,57], respectively. In addition, to achieve an accurate identification of the Malbranchea isolates, regions of the tub2 and rpb2 genes were also amplified with the primer pairs Bt2a/Bt2b [58] and RPB2-5F2 [59]/fRPB2-7cR [60], respectively. Briefly, PCR conditions for ITS, LSU, and tub2 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, while the conditions for the rpb2 were an initial denaturation step 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 5 min at 72 °C, followed by 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. Amplified products were purified and sequenced at Macrogen (Madrid, Spain). Consensus sequences were obtained using SeqMan v. 7.0.0 (DNAStar Lasergene, Madison, WI, USA).
In addition to a preliminary morphological identification, ITS sequences of the isolates under study were compared with those deposited in the National Center for Biotechnology Information (NCBI) using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/blast.cgi (accessed on 28 December 2022)). A maximum similarity level of >98% (≥90 sequence coverage) was used for species-level identification, while lower similarity values were considered to be sequences belonging to putative novel fungi. To assess taxonomic positions and delineate novel taxa within Onygenales, we carried out two different phylogenetic analyses. The first was constructed using the ITS and LSU phylogenetic markers and included species representatives of the different families and monophyletic clades recognized in Kandemir et al. [8] (i.e., Ajellomycetaceae, Arthrodermataceae, Ascosphaeraceae, Eremascaceae, Gymnoascaceae, Malbrancheaceae, Neogymnomycetaceae, Onygenaceae, Spiromastigoidaceae, and related incertae sedis clades). A second phylogenetic reconstruction was carried out using the ITS, LSU, tub2, and rpb2 markers for members of Malbrancheaceae. Sequences of the ex-type strains and/or reference strains of accepted species and genera of the aforementioned families used in the analyses, including taxa selected as outgroups, were retrieved from GenBank (Table S1 in Supplementary Materials).
Datasets for each locus were aligned individually in MEGA (Molecular Evolutionary Genetics Analysis) software v.6.0 [61], using the ClustalW algorithm [62] and refined with MUSCLE [63] or manually adjusted, if necessary, on the same platform. Before combining the regions, the phylogenetic concordance of the loci datasets was assessed individually for each single-locus phylogeny through visual comparison and using the Incongruence Length Difference (ILD) run in Winclada v.1.00.08 software [64] in order to determine any incongruent results among nodes with high statistical support. Once a lack of incongruence was confirmed, individual alignments were concatenated into a single data matrix with SequenceMatrix v.1.7.6 software [65]. 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 according to the Akaike criterion [66]. The phylogenetic reconstructions were made with the combined genes using ML under RaxML-HPC2 on XSEDE v-8.2.12 [67] in the CIPRES Science gateway portal [68] and BI with MrBayes v.3.2.6 [69]. The phylogenetic support of internal branches for ML analyses was assessed by 1000 ML bootstrapped pseudoreplicates and a bootstrap support (bs) of ≥70 was considered significant [70]. 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 [71]. The resulting trees were plotted using FigTree v.1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/ (accessed on 15 February 2023)).
DNA sequences and alignments generated in this study were deposited in GenBank (Table 1) and in Zenodo under the submission number 10036241 (https://doi.org/10.5281/zenodo.10036241), respectively.
2.4. Phylogeny and Geographical Distribution of the Related Environmental Sequences
To assess the global geographical distribution and habitat affiliations of the novel taxa among environmental sequences, we followed the workflow of Réblová et al. [72,73] and Torres-Garcia et al. [42]. The full length of the ITS1 and ITS2 sequences of our isolates were blasted against those deposited in the GlobalFungi database (https://globalfungi.com (accessed on 2 April 2023)) [52]. At the time of the approach, this database contained 57,184 samples from 515 studies, 791,513,743 unique sequences for ITS1, and 2,892,377,338 for ITS2. As this database has separated ITS1 and ITS2 sequences, they were processed separately. In order to verify the generic and species boundaries among the downloaded ITS environmental sequences related to our fungi, we also included in the analyses ITS sequences of known species previously obtained from GenBank. Those known species were representatives of the well-delineated closest taxa to our fungi in a full ITS analysis carried out previously (Figure S1 in Supplementary Materials). The ITS1 and ITS2 sequences of some of those fungi were also blasted against the GlobalFungi database and included in the respective analyses. For both markers, ITS1 and ITS2, we selected and downloaded environmental sequences that had a similarity between 98 and 100% and a full-length coverage with those of the sediment isolates and related known species. Due to the large numbers of environmental sequences obtained, we selected those representative of various geographical origins, biomes, and substrates. For each taxon, we obtained data on their occurrence across environmental samples. Metadata related to the particular samples (location, climatic data, biome, or substrate) is listed in Table S2 (in Supplementary Materials).
3. Results
After a preliminary morphological examination of the primary cultures, we recovered 63 isolates of onygenalean fungi from freshwater sediments (57 on PDA supplemented with 0.2% cycloheximide, four on DRBC, and two on DRBC supplemented with benomyl), of which 55 were identified at the species level through ITS sequence comparison. These isolates were representatives of 14 species of Onygenales, i.e., A. hispanica (n = 3), Ap. crassitunicatus (n = 3), Ap. fulvescens (n = 11), Ap. reticulisporus (n = 4), Ar. curreyi (n = 1), Arachniotus (Ara.) albicans (n = 12), E. tuberculata (n = 1), Gymnoascoideus (Gym.) petalosporus (n = 1), Leucothecium (L.) emdenii (n = 1), M. chinensis (n = 1), M. ostraviensis (n = 1), M. reticulata (n = 1), M. umbrina (n = 3), and My. keratinophilum (n = 12). However, eight of the isolates could not be assigned to any known species (ITS similarity values ≤ 98%) despite exhibiting a malbranchea- (FMR 17906, FMR 18266, FMR 19015, FMR 19016, FMR 19017, FMR 19030), arthropsis- (FMR 19025), and amaurascopsis-like (FMR 19014) morphology.
Table 1 includes the final identification of the isolates that resulted from the phenotypic and phylogenetic characterization.
3.1. Phylogeny
ITS and LSU sequences of the sediment isolates, apart from those considered isogenic, were analyzed phylogenetically in order to confirm identification and assess the taxonomic position of the unidentified isolates. In addition, since tub2 and rpb2 sequences were available for the currently accepted Malbranchea species (Table S1 in Supplementary Materials), we also carried out a multi-gene analysis combining the ITS, LSU, tub2, and rpb2 loci for a more precise delineation of the unidentified malbranchea-like isolates. The tree topology was in all cases similar for the ML and BI methods, so the ML trees were selected to represent our results. Bootstrap and BI posterior probability values are shown on the relevant branches.
The ITS/LSU phylogenetic tree of the order Onygenales is shown in Figure 2. It encompasses 162 isolates, representatives of most genera accepted in the order, with 318 sequences that comprised 1118 bp (520 for ITS and 598 for LSU), of which 699 (440 for ITS and 259 for LSU) corresponded to variable sites, and 589 (385 for ITS and 204 for LSU) were phylogenetically informative sites. For ML analysis, TN93+G+I was selected as the best substitution model, while for the BI analysis, GTR+G+I was selected as the best-fit model for ITS and GTR+G for LSU. This phylogeny agrees with the multi-locus analysis of the Onygenales published by Kandemir et al. [8]. There are similar topologies regarding the monophyletic clades representative of currently accepted families in the order, as well as other well-supported clades that represent putative undescribed families (Clades I–XIII). The only exceptions were the unresolved taxonomic Clades VI and VII, with the genera Leucothecium and Myotisia in the first and Shanorella in the latter, which seem to be members of the family Arthrodermataceae (Clade V).
Sediment isolates were distributed in the following clades: Clade I (Malbrancheaceae), Clade III (Onygenaceae), Clade V (Arthrodermataceae), Clade VIII (Gymnoascaceae), Clade IX (Ajellomycetaceae), and Clades IV, VI, X (incertae sedis) according to Kandemir et al. [8].
Figure 2.
Phylogenetic tree based on maximum likelihood (ML) analysis obtained by RAxML inferred from combined ITS and LSU sequences of the Onygenales and outgroups. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70%/0.95 are indicated on the nodes. Bold branches indicate bs/pp values 100/1. The tree was rooted to several members of the orders Arachnomycetales and Eurotiales. Quote marks indicate species with unresolved taxonomy. The novel taxa proposed are in red, and the known species identified in this study are in green. T= ex−type strain.
Figure 2.
Phylogenetic tree based on maximum likelihood (ML) analysis obtained by RAxML inferred from combined ITS and LSU sequences of the Onygenales and outgroups. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70%/0.95 are indicated on the nodes. Bold branches indicate bs/pp values 100/1. The tree was rooted to several members of the orders Arachnomycetales and Eurotiales. Quote marks indicate species with unresolved taxonomy. The novel taxa proposed are in red, and the known species identified in this study are in green. T= ex−type strain.
Among malbranchea-like isolates included in the Malbrancheaceae, four species were identified: M. chinensis (FMR 18267), M. ostraviensis (FMR 18693), M. reticulata (FMR 18696), and M. umbrina (FMR 17899, FMR 18695, and FMR 18766). Although the isolates FMR 17906, FMR 18266, FMR 19015, FMR 19016, FMR 19017, and FMR 19030 were confirmed as members of the genus Malbranchea, they were distributed in three distinct branches representative of putative undescribed Malbranchea species (I–III). These novelties were confirmed with phylogeny based on the ITS, LSU, rpb2, and tub2 genes, in which the accepted species of Malbranchea were compared (Figure 3). This phylotree encompasses 50 strains, including the selected outgroups. It includes 139 sequences that comprised 2749 bp (547 for ITS, 511 for LSU, 1079 for rpb2, 612 for tub2), of which 1110 (287 for ITS, 130 for LSU, 366 for rpb2, 327 for tub2) corresponded to variable sites, and 793 (211 for ITS, 113 for LSU, 295 for rpb2, 174 for tub2) were phylogenetically informative sites. For ML analysis, K2+G+I was selected as the best substitution model, while for the BI analysis, GTR+G+I was selected as the best-fit model for ITS, SYM+G for LSU, and K2+G for tub2 and rpb2. Unfortunately, rpb2 and tub2 sequences for the species M. albolutea, M. californiensis, and M. compacta were not available for comparison. Malbranchea sp. I (FMR 18266) formed a singleton distant branch into a statistically supported terminal clade (89 bs/0.99 pp) together with M. albolutea (CBS 125.77 ex-type, FMR 17679). Both specimens showed enough distance (98.8–98.9% for LSU and 95.7–95.8% for ITS) to be considered distinct species. The phylogenetic distance and morphological differences observed with its counterpart (see taxonomy section) support the proposal of the new species Malbranchea sinuata. Malbranchea sp. II (FMR 17906) was part of a supported terminal clade (81 bs/0.99 pp), but forming a singleton lineage with significant genetic distance (99.4% for LSU and 96.2% for ITS) regarding the ex-type strain of M. compacta (CBS 200.64). It is therefore proposed here as Malbranchea echinulata sp. nov. Malbranchea sp. III (FMR 19015, FMR 19016, FMR 1907, and FMR 19030) formed a fully supported terminal clade (100 bs/1 pp) closely related to the ex-type strain of M. californiensis (ATCC 15600) but representing an undescribed lineage for the genus. The phylogenetic distance from its counterpart (98.9–99.0% for LSU and 90.5–90.6% for ITS) and different morphological features (see taxonomy section) allow us to propose Malbranchea irregularis sp. nov.
The well-supported clade (96% bs/1 pp) of the family Onygenaceae (Figure 2) included the sediment isolates that were identified morphologically as belonging to the genus Aphanoascus. The species confirmed molecularly through representative sediment isolates were Ap. crassitunicatus (FMR 18700 and FMR 18701), Ap. fulvescens (FMR 18236, FMR 18704, FMR 19018, and FMR 19020), and Ap. reticulisporus (FMR 18004, FMR 19012, and FMR 19033).
Clade IV (Figure 2; 80% bs/0.97 pp) comprised a rich set of specimens belonging to various taxa that were repeatedly isolated from freshwater sediments. We recovered numerous isolates of A. hispanica, Ara. albicans, and My. keratinophilum (Table 1). These species were confirmed molecularly through representative isolates included in the concatenated ITS/LSU analysis. It is noteworthy that the protologue of A. hispanica was described based on freshwater and marine sediments collected in Spain by Ulfig et al. [32]. Another fungus included in this clade and also described from aquatic sediments was Chrysosporium (C.) undulatum [36]. These data suggest that a good number of the members of this clade seem to be well adapted to aquatic environments. Nevertheless, the species of Apinisia or Arachnotheca, two genera also included in this incertae sedis clade, have been recovered from soil, plant debris, and animal skin [8,74].
Of note is that ex-type or reference strains of several species that require taxonomic adjustment are included in Clade IV, as already reported by Kandemir et al. [8]. Those include A. hispanica, Ara. albicans, and the previously mentioned C. undulatum, as well as C. carmichaelii, C. pallidum, and C. sulfureum; several of these species are placed in phylogenetically distant branches that might represent undescribed genera per se (Figure 2). We have studied, morphologically and molecularly, the former two species. The isolates identified as A. hispanica (18694, FMR 19034, and FMR 19035) clustered in a fully supported terminal clade with the ex-type strain of the species (CBS 351.92). This terminal clade together with the separate singleton branch of the unidentified arthropsis-like isolate (FMR 19025) formed a monophyletic lineage at a significant genetic distance (88.12–88.33% of similarity for ITS and 96.29–96.92% for LSU) from the lineage representative of the monotypic genus Arachnotheca [74]. The phylogenetic distance and morphological differences observed between the members of the two lineages (see taxonomy section) provide evidence for proposing the novel genus Neoarthropsis for the Arthropsis specimens since the generic type A. truncata is allocated in the Sordariomycetes. The genus is typified with A. hispanica, and the new combination Neoarthropsis (Ne.) hispanica is proposed. The phylogenetic distance between the ex-type of Ne. hispanica and the unidentified isolate FMR 19025, with similarity values of 91.2% for ITS and 98.5% for LSU, supports the proposal of the novel species Neoarthropsis sexualis.
Figure 3.
Phylogenetic tree based on a maximum likelihood (ML) analysis obtained by RAxML inferred from combined ITS, LSU, tub2, and rpb2 sequences of members of the genus Malbranchea and outgroups. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70%/0.95 are indicated on the nodes. Bold branches indicate bs/pp values 100/1. The tree was rooted to Paracoccidioides brasiliensis UAMH 8037 and Blastomyces percursus CBS 139878. The novel taxa proposed are in red, and the known species identified in this study are in green. T = ex−type strain.
Figure 3.
Phylogenetic tree based on a maximum likelihood (ML) analysis obtained by RAxML inferred from combined ITS, LSU, tub2, and rpb2 sequences of members of the genus Malbranchea and outgroups. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70%/0.95 are indicated on the nodes. Bold branches indicate bs/pp values 100/1. The tree was rooted to Paracoccidioides brasiliensis UAMH 8037 and Blastomyces percursus CBS 139878. The novel taxa proposed are in red, and the known species identified in this study are in green. T = ex−type strain.
Representative sediment isolates of Ara. albicans (FMR 17576, FMR 18029, FMR 18698, and FMR 19026) grouped with the ex-type strain of this species (CBS 151.65) to form, along with a reference strain of C. sulfureum, an undescribed lineage that can be distinguished easily from the others in Clade IV. Considering that the type species of the genus Arachniotus is placed in the family Gymnoascaceae [75] (Clade VIII), we proposed the new genus Albidomyces, typified by Albidomyces (Al.) albicans comb. nov. Since Clade IV comprises a highly supported group of taxa distantly related to other families in Onygenales, whose members share morphological (see taxonomy section) and ecological traits as mentioned above, it deserves to be recognized as a novel family, named Neoarthropsidaceae. Taxa delineation in this new family is also supported by an additional ITS analysis (Figure S2 in Supplementary Materials), in which more isolates from different origins were added for several species.
The ITS/LSU analysis also confirmed the identity and taxonomic position of Ar. curreyi (FMR 19038) in the Arthrodermataceae (Clade V), L. emdenii (FMR 18703) in the incertae sedis Clade VI, Gym. petalosporus (FMR 19036) in the Gymnoascaceae (Clade VIII), and E. tuberculata (FMR 17582) in the Ajellomycetaceae (Clade IX). The asexual morph of L. emdenii, under the name Arthrographis alba, was repeatedly isolated from freshwater and marine sediments also collected in Spain [76].
The remaining unidentified isolate amaurascopsis-like strain (FMR 19014) clustered into the well-supported incertae sedis Clade X (Figure 2; 99% bs/1 pp). This clade included the ex-type strain of Amaurascopsis (Am.) perforata (FMR 3882), but also those of Polytolypa (Po.) hystricis (UAMH 7299) and C. chiropterorum (MUCL 45495), as well as a reference strain of C. lobatum (CBS 275.77). The closest species to the unidentified isolate were Am. perforata and Po. hystricis, but both fungi showed a sequence similarity of 89.2% for ITS and of 97.4% for LSU in the case of Po. hystricis. The LSU sequence of Am. perforata was not available for comparison. The phylogenetic distance as well as morphological differences, mainly regarding growth temperatures and conidial morphology (see taxonomy section), support the proposal of the new genus Pseudoamaurascopsis to accommodate Pseudoamaurascopsis (Ps.) spiralis sp. nov. Chrysosporium species included in the clade deserve further studies to clarify their taxonomy. Although the phylogeny of Clade X suggests that it could represent an undescribed family, the small number of taxa and the long phylogenetic distance between them preclude a formal taxonomic proposal for the time being.
3.2. Distribution of Species in the Rivers and Streams Sampled
The isolates under study were mainly recovered from samples collected in the rivers (n = 50; 79.4%) as they pass through various Spanish provinces, i.e., Llobregat (Barcelona) (n = 20; 31.8%), Segre (Lleida) (n = 17; 27.0%), and Ter (Barcelona and Girona) (n = 13; 20.6%). Meanwhile, the isolates obtained from streams (n = 13; 20.6%) were distributed as follows: Torrent de la Coma (Lleida; n = 4, 6.3%), Mare de Déu Stream (Castellón; n = 3, 4.7%), Remáscaro Stream (Huesca; n = 2, 3.2%), Brezal Stream (Madrid; n = 1, 1.6%), Esca Stream (Navarra, n = 1, 1.6%), Pont d’en Blai Stream (Mallorca; n = 1, 1.6%), and Umbría Stream (Madrid; n = 1, 1.6%). The difference in the number of isolates between rivers and streams is probably influenced by the greater number of sampling points in the rivers.
Although the number of isolates was rather similar among the three rivers, samples from the rivers Llobregat and Ter exhibited the greatest number of species identified, i.e., 10 species in the Llobregat (i.e., Al. albicans, Ap. fulvescens, Ap. reticulisporus, Ar. curreyi, Gym. petalosporus, M. irregularis, My. keratinophilum, Ne. hispanica, Ne. sexualis, and Ps. spiralis) and eight in the Ter (i.e., Al. albicans, Ap. crassitunicatus, Ap. fulvescens, L. emdenii, M. ostraviensis, M. reticulata, M. umbrina, and Ne. hispanica). Meanwhile, only four species were recovered in the river Segre (i.e., Ap. fulvescens, M. chinensis, M. sinuata, and My. keratinophilum). Curiously, five out of the seven novel taxa proposed here (i.e., Al. albicans, M. irregularis, Ne. hispanica, Ne. sexualis, and Ps. spiralis) were isolated from samples collected in the river Llobregat at the Balsareny locality (Catalonia).
As mentioned previously, Al. albicans, Ap. fulvescens, and My. keratinophilum were the most common species isolated, with 11 or 12 isolates for each species. However, their distribution across the sampling points showed some differences. While Al. albicans was found in 7 out of the 21 sampled points from different rivers and streams, and mainly recovered along the rivers Llobregat and Ter, Ap. fulvescens and My. keratinophilum were found in 6 and 5 sampled points, respectively, but both species were primarily recovered from samples collected along the river Segre, in particular the latter species (Table 1).
3.3. Global Biogeography and Ecology of the Novel Taxa
According to BLAST results with ITS sequences in the GlobalFungi database, we were able to plot the global distribution of the following novel taxa through environmental sequences of samples collected around the world: Al. albicans, M. sinuata, Ne. hispanica, Ne. sexualis, and Ps. spiralis (Figure 4). However, hindered environmental sequences of undescribed hypothetical species related to the aforementioned taxa were not detected in our ITS 1 and ITS 2 analyses (Figures S3–S5 in Supplementary Materials).
ITS1 sequences of Al. albicans, Ne. sexualis, and Ps. spiralis were recovered from 173, 84, and 270 environmental samples, respectively (similarity 89–100%). The high amounts of environmental sequences obtained after BLAST regarding our studied fungi allowed us to select the sequences that originated from various environmental samples. Additionally, other environmental sequences of the closest species were added to the reconstructed phylogeny (Table S2 and Figures S3 and S4 in Supplementary Materials). ITS1 sequences of these three species were retrieved from soil samples collected from biomes in various European countries: in particular, those linked to Al. albicans were from grassland, forest, and urban environments in Estonia, the Czech Republic, and Latvia; those linked to Ne. sexualis were retrieved from grassland soils in Estonia; and those of Ps. Spiralis were found in samples collected from soil, roots, and rhizosphere soil of croplands, grasslands, and urban environments in Denmark, Estonia, Spain, and the UK. In addition, environmental sequences of the hypothetical species Ne. sexualis were also detected in soil samples from Australia, and of Ps. spiralis, in soil from Australia and China.
On the other hand, the ITS2 sequences give us additional biogeography and ecological information for those three species, which were recovered from 478, 20, and 632 environmental samples, respectively (similarity 98–100%). Environmental ITS2 sequences of Al. albicans were obtained from samples of soil and rhizosphere soil of different environments (grassland, forest, and woodland) exclusively in Europe (the Czech Republic, Estonia, Germany, and the UK). Meanwhile, environmental ITS2 sequences linked to Ne. sexualis and Ps. spiralis revealed that both are soil-inhabiting fungi, but those of the former species were found mainly in samples of forest soil in the USA, while those of Ps. spiralis were associated with cropland soil samples from Australia, China, and the USA (Figure 4).
The ITS sequences of M. echinulata and M. irregularis did not match any ITS1/ITS2 environmental sequences deposited in the GlobalFungi database. As mentioned previously, only environmental ITS2 sequences deposited in the database corresponded with the ITS2 sequence of M. sinuata (Table S2 and Figure S5 in Supplementary Materials), which were recovered from 66 environmental samples (98–100% similarity). Its geographical distribution was limited to samples from soil and rhizosphere soil of shrublands in Spain (Figure 4).
3.4. Taxonomy
This section is organized according to the phylogenetic position of the family shown in our concatenated ITS and LSU analysis, and in alphabetical order of the respective novel genera and species proposed.
- Malbrancheaceae (Figure 2, Clade I)
- Malbranchea echinulata Torres-Garcia, Cano & Gené, sp. nov. Figure 5.
MycoBank MB848037
Etymology: Referring to the wall ornamentation of the peridial hyphae and ascospores.
Type: Spain, Comunidad de Madrid, Rascafría, Guadarrama Natural Park, Arroyo de la Umbría, N 40°51′54.7′′ W 3°53′40.3′′, from fluvial sediments, May 2019, J. Cano (holotype CBS H-25255; cultures ex-type FMR 17906, CBS 149936).
Description: Mycelium immersed and superficial, composed of hyaline, septate, branched, smooth-walled, 1.5–2.5 μm wide hyphae. Asexual morph with well-differentiated fertile hyphae arising laterally from vegetative hyphae, branched, sinuous and forming randomly intercalary and terminally arthroconidia, 1.5–2.5 μm wide. Arthroconidia enteroarthric, 0–1-septate, hyaline, smooth- and thin-walled, cylindrical, subcylindrical or T-shaped, 2.5–6 × 1–3 μm; secession rhexolytic. Sexual morph with gymnothecial ascomata, globose, single to confluent, brown, 182–432 μm diam. (excluding appendages); peridial hyphae branched, septate, pale orange-brown, finely echinulate, thick-walled, 2–3.5 μm wide, with short and long lateral appendages; short appendages blunt, spine-like, with both subacute or truncate ends; long appendages 2–5, arising from peridium at acute angles, unbranched, straight and with subacute ends, orange-brown, paler toward apex, smooth-walled, mostly with one basal knuckle-joint, 127–778 μm long. Asci 8-spored, evanescent, hyaline, irregularly disposed, globose to subglobose, 6–8.5 × 6–8 μm. Ascospores hyaline, coarsely echinulate, thick-walled, globose, 2.5–3 μm diam.
Culture characteristics (14 d at 25 °C): Colonies on PDA reaching 43–44 mm diam., slightly elevated, dense, cottony, concentrically zonate, pastel yellow (1A4) at the center, yellowish white (1A2) to white toward the periphery, margins regular, sporulation abundant, reverse olive brown (4D4) at the center, yellowish white (5F6) toward the periphery, diffusible pigment absent. On PCA, reaching 47–48 mm diam., slightly elevated, floccose, brownish gray (8C2), white (1A1) at the periphery, margins regular, sporulation abundant, reverse brownish gray (8D2) at the center, yellowish white (1A2) to white (1A1) toward the periphery, diffusible pigment absent. On OA, reaching 29–30 mm diam., flattened, aerial mycelium sparse, granulose due to presence of ascomata, brown (7E5), whitish at the periphery, margins irregular, sporulation abundant, reverse grayish orange (5B4) at the center, diffusible pigment absent. On PYE, reaching 20–21 mm diam., elevated, cottony, slightly concentrically sulcate, yellowish white (1A2) at the center, colorless toward the periphery, margins fimbriate, sporulation sparse to moderate; reverse yellow (5A6) at the center, light yellow (4A6) toward the periphery, diffusible pigment absent.
Cardinal temperatures for growth on PDA after 14 d (mm): Minimum 15 °C (18–20), optimum 25 °C (42–43), maximum 30 °C (12–14).
Habitat and geographical distribution: Freshwater sediments, Spain.
Notes: Malbranchea echinulata is related to M. compacta (=Auxarthron compactum) but with a phylogenetic distance far enough to be considered a distinct species (Figure 3). Both species are similar in forming ascomata with pale orange-brown peridial hyphae that show a dense, net-like reticulum with short and long appendages. However, M. echinulata has smaller ascomata (182–432 μm diam vs. 310–675 μm diam. in M. compacta) and considerably longer (127–778 μm) and straight appendages, while those of M. compacta are up to 600 μm long and bent to coiled apically [74,77]. The ascospores of M. compacta are slightly larger (up to 3.6 μm diam), hyaline to pale brown, and delicately asperulate to echinulate [77], while those of M. echinulata are 2.5–3 μm, hyaline, and coarsely echinulate. It is difficult to establish differences between their asexual morphs because of the variable size and shape of the arthorconidia, although they are hyaline to pale yellow in M. compacta [77], while strictly hyaline in M. echinulata.
- Malbranchea irregularis Torres-Garcia, Gené & Dania García, sp. nov. Figure 6.
MycoBank MB848039
Etymology: Referring to the irregular shape of the colonies on PYE and PDA.
Type: Spain, Catalonia, Berguedà, Castellar de n’Hug, Llobregat River, N 42.27298° E 1.99177°, from fluvial sediments, March 2021, D. Torres-Garcia & J. Gené, (holotype CBS H-25256; cultures ex-type FMR 19016, CBS 149937).
Description: Mycelium immersed and superficial, composed of hyaline, septate, branched, smooth-walled, 1–2.5 μm wide hyphae. Sexual morph with gymnothecial ascomata, globose, 134–317 μm diam. (excluding appendages), rosaceous initially, becoming brown to orange-brown, peridial hyphae asperulate to verrucose, 3–5 μm wide, with short and long lateral appendages, short appendages orange-brown, verruculose, spine-like, with subacute to rounded ends, 23–29 μm long, long appendages arising from peridium at acute and subacute angles, unbranched, straight, cylindrical, progressively tapering terminally, orange-brown, thick-walled, and asperulate toward the base, paler and smooth to asperulate terminally, with a rounded or subacute apex and a basal knuckle-joint, 42.5–230.5 μm long. Asci 8-spored, evanescent, irregularly disposed, hyaline, globose to subglobose, 6–9 × 5.5–7.5 μm. Ascospores hyaline, minutely echinulate to echinulate, thick-walled, globose, 2.5–3 μm diam. Asexual morph not observed.
Figure 5.
Malbranchea echinulata (ex-type FMR 17906). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C,D) Ascomata. (E) Peridial hyphae, asci, and ascospores. (F) Ascospores. (G) Fertile hyphae. (H) Arthroconidia. Scale Bars: (C) = 75 μm; (D) = 25 μm; (E−H) = 10 μm.
Figure 5.
Malbranchea echinulata (ex-type FMR 17906). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C,D) Ascomata. (E) Peridial hyphae, asci, and ascospores. (F) Ascospores. (G) Fertile hyphae. (H) Arthroconidia. Scale Bars: (C) = 75 μm; (D) = 25 μm; (E−H) = 10 μm.
Culture characteristics (14 d at 25 °C): Colonies on PDA reaching 21–22 mm diam., slightly raised at the center, velvety, yellowish white (3A2) to light yellow (3A4), margins irregularly lobulated, sporulation absent, reverse light brown (5D6) to golden yellow (5B7), diffusible pigment absent. On PCA, reaching 11–12 mm diam., slightly raised at the center, velvety, grayish orange (5B5) at the center, pale yellow (4A3) toward the periphery, margin lobulated, sporulation absent to sparse, reverse yellowish brown (5E6) at the center, orange (5A2) at the periphery, diffusible pigment absent. On OA, reaching 19–20 mm diam., flattened, granular, light brown (6D4) to brownish orange (6C4) at the center, yellowish white (1A2) at the periphery, margins fimbriate, sporulation moderate to abundant, reverse grayish yellow (4B4), diffusible pigment absent. On PYE, reaching 22–23 mm diam., irregularly elevated, radially sulcate at the periphery, velvety, grayish yellow (4B4) at the center, yellow (4A6) to pale yellow (4A3) toward the periphery, margins irregularly lobulated, sporulation absent, reverse dark brown (6F8) at the center, pale brown (5D7) toward the periphery, diffusible pigment absent.
Cardinal temperatures for growth on PDA after 14 d (mm): Minimum 5 °C (2–3), optimum 25 °C (25–26), maximum 30 °C (6–7).
Additional specimens examined: Spain, Catalonia, Berguedà, Balsareny, Llobregat River, N 41.87818° E 1.88900°, from river sediments, March 2021, D. Torres-Garcia & J. Gené (FMR 19015); ibid. Castellar de n’Hug, Llobregat River, N 42.27967° E 2.00535°, from river sediments, March 2021, D. Torres-Garcia & J. Gené (FMR 19017); ibid. (FMR 19030).
Habitat and geographical distribution: Freshwater sediments, Spain.
Notes: Malbranchea irregularis is phylogenetically close to M. californiensis (Figure 3). Both species are morphologically well differentiated. Malbranchea californiensis (=Auxarthron californiense) produces larger ascomata (135–435 μm diam) with longer (72–375 μm), terminally uncinated and occasionally branched appendages, which are entirely smooth and arise at irregular angles around the periphery of the ascomata [74,77]. This latter feature is also observed in our new species, but the elongate appendages produced by M. irregularis are shorter (42.5–230.5 μm), unbranched, terminally straight, and asperulate toward the base. In addition, M. californiensis shows slightly bigger asci (10.6 × 7.2–8 μm vs. 6–9 × 5.5–7.5 μm in M. irregularis) and ascospores that are globose to ovoid (3.2–4 × 2.4–4.8 μm) and hyaline to pale yellow-brown [74,77]. The ascospores in M. irregularis are globose (2.5–3 μm) and hyaline. Another difference is that M. californiensis produces both sexual and asexual morphs, while M. irregularis only shows the sexual morph.
- Malbranchea sinuata Torres-Garcia, Gené & Cano, sp. nov. Figure 7.
Etymology: Referring to the sinuous fertile hyphae produced by the species.
Type: Spain, Catalonia, El Segrià, La Granja d’Escarp, Segre River, N 41.42754° E 0.35020°, from fluvial sediments, December 2019, D. Torres-Garcia & J. Gené, (holotype CBS H-25257; cultures ex-type FMR 18266, CBS 149938).
Description: Mycelium immersed and superficial composed of straight to sinuous, septate, branched, hyaline, thin- and smooth-walled, 1–2 μm wide hyphae. Racquet hyphae present. Asexual morph with fertile hyphae well-developed, arising as lateral branches from vegetative hyphae, right or sinuous, contorted or arcuate at apical part, 1–2 μm wide, or developing at extremes of the vegetative hyphae, in both cases forming arthroconidia randomly intercalary and terminally. Arthroconidia enteroarthric, 0-septate, hyaline, smooth- and thin-walled, mostly cylindrical, sometimes barrel-shaped, 1.5–3 × 1.5–2.5 μm, when mature become subglobose or irregularly shaped, secession rhexolytic. Sexual morph not observed.
Figure 6.
Malbranchea irregularis (ex-type FMR 19016). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C,D) Ascomata. (E) Peridial hyphae. (F) Peridial hyphae and asci. (G) Asci. (H) Ascospores. Scale Bars: (C,D) = 25 μm; (E–H) = 10 μm.
Figure 6.
Malbranchea irregularis (ex-type FMR 19016). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C,D) Ascomata. (E) Peridial hyphae. (F) Peridial hyphae and asci. (G) Asci. (H) Ascospores. Scale Bars: (C,D) = 25 μm; (E–H) = 10 μm.
Culture characteristics (14 d at 25 °C): Colonies on PDA reaching 54–55 mm diam., cottony, umbonate and pale yellow (4A3) to orange (5A6) at the center, flat and light orange (5A4) toward the periphery, margins regular, sporulation sparse, reverse reddish orange (7B8) at the center, orange (6A7) toward the periphery, diffusible pigment absent. On PCA, reaching 41–42 mm diam., floccose, raised, grayish orange (5B4) at the center, orange (5A6), pale orange (5A3) toward the periphery, margins regular, sporulation abundant, reverse orange (5A7) at the center, reddish yellow (4A6) at the periphery, diffusible pigment absent. On OA, reaching 40–41 mm diam., slightly raised at the center, granulose due to the production of irregular mycelial tufts, which are pale orange (5A7) to light yellow (4A4), margins irregular, sporulation abundant in the mycelial tufts, reverse brownish orange (7C8) at the center, pale orange (5A3–7) toward the periphery, diffusible pigment absent. On PYE, reaching 46–47 mm diam., cottony, slightly raised, pale yellow (1A3) to deep orange (5A8) at the center, light orange (5A4) toward the periphery, margins fimbriate, sporulation moderate, reverse reddish brown (8E6) at the center, reddish orange (7A7) toward the periphery, diffusible pigment absent.
Cardinal temperature for growth on PDA after 14 d (mm): Minimum 15 °C (13–14), optimum 25 °C (49–52), maximum 37 °C (14–15).
Habitat and geographical distribution: Freshwater sediments, soil, and rhizosphere soil in scrublands, Spain (Figure 4, Table S2 in Supplementary Materials).
Notes: Malbranchea sinuata is phylogenetically close to M. albolutea. The main difference observed between the species is that M. sinuata only produces the asexual morph in the media and conditions tested, while M. albolutea presents both sexual/asexual morphs [78]. The asexual morph of the two species produces hyaline arthroconidia by the segmentation of broader primary hyphae and of arcuate or curved lateral fertile branches, which give rise to cylindrical, barrel-shaped, subglobose or irregularly shaped conidia at maturity. However, both species differ in arthroconidial size, which is smaller in M. sinuata (1.5–3 × 1.5–2.5 μm) than in M. albolutea (2.5–3(–4) × (1.5–)2–5(–6.5) μm) [78], and in colony color; our new species shows colonies of yellow to orange and reddish brown shades in PYE and OA, while in M. albolutea, they are only of yellow shades in the same culture media previously mentioned [78]. In addition, M. sinuata grew at 37 °C, while M. albolutea was not able to grow at that temperature, except for the strain UAMH 1846 according to Sigler and Carmichael [78].
- Neoarthropsidaceae Torres-Garcia & Gené, fam. Nov. (Figure 2, Clade IV).
MycoBank MB850654
Type genus: Neoarthropsis Torres-Garcia, Cano & Gené.
Included genera: Albidomyces, Apinisia, Arachnotheca, Myriodontium, and Neoarthropsis.
Description: Colonies white, yellowish, or cream. Asexual morph arthropsis-like, chrysosporium-like, and myriodontium-like. Sexual morph with gymnothecial ascomata, superficial, single or aggregated, white, globose, with or without appendages, peridium composed of a network of loosely interwoven undifferentiated hyphae, septate, branched, hyaline, thin- or thick-walled, peridial appendages helical when present. Asci unitunicate, 8-spored, evanescent, globose, subglobose or oval, in clusters or in chains. Ascospores one-celled, globose, subglobose or oblate, hyaline, subhyaline, yellowish, rarely pale brown, smooth-, punctate- or punctate-reticulate wall, with or without a sheath.
Notes: The family Neoarthropsidaceae is based on a well-supported clade (80% bs/0.97 pp) in our concatenated LSU/ITS analysis (Figure 2), which correlated with the fully supported lineage, composed of practically the same taxa, shown in the multi-gene analysis previously published by Kandemir et al. [8]. However, in addition to being a highly supported lineage clearly separated from other families of the order, Neoarthropsidaceae is also supported because its members share morphological traits, as described above, and similar physiological features, such as having maximum temperatures for growth at 25–30 °C and the lack of, or weak, keratinolytic activity [32,36,74,75,79,80,81]. The only member reported to have strong keratinolytic activity is My. keratinophylum [80], which is the only species in the family described as a human opportunist by a case of sinusitis [17]. Therefore, according to the reviewed data [32,36,74,75,79,80,82,83], the species in Neoarthropsidaceae can be defined ecologically as saprobic, commonly inhabiting soil, rarely animal dung or plant debris, and able to survive in aquatic environments like freshwater and marine sediments. Currently, the family includes 12 species, from which those found in aquatic sediments are Al. albicans, C. undulatum, My. keratinophylum, Ne. hispanica, and Ne. sexualis.
Figure 7.
Malbranchea sinuata (ex-type FMR 18266). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C) Colony on OA (front) at 25 °C after 14 days. (D) Hyphae on tufts. (E) Racquet hyphae. (F) Main fertile hyphae. (G,H) Sinuous fertile hyphae. (I) Arthroconidia. Scale Bars: (D) = 25 μm; (E–I) = 10 μm.
Figure 7.
Malbranchea sinuata (ex-type FMR 18266). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C) Colony on OA (front) at 25 °C after 14 days. (D) Hyphae on tufts. (E) Racquet hyphae. (F) Main fertile hyphae. (G,H) Sinuous fertile hyphae. (I) Arthroconidia. Scale Bars: (D) = 25 μm; (E–I) = 10 μm.
- Albidomyces Torres-Garcia, Dania García, Cano & Gené, gen. nov.
MycoBank MB850655
Etymology: Referring to the white colony color of the type species on all agar media tested.
Type species: Albidomyces albicans (Apinis) Torres-Garcia, Dania García, Cano & Gené.
Description: Colonies white or cream, floccose or felty, composed of hyaline, septate, and branched hyphae. Racquet hyphae present. Asexual morph chrysosporium-like, with undifferentiated conidiophores. Fertile hyphae straight to flexuose, septate, branched, lateral branches arising at right angles, throughout conidia borne sessile or in short cylindrical or conical protrusions. Conidia terminal or lateral, one-celled, subglobose or pyriform, hyaline, smooth-walled, secession rhexolytic. Sexual morph gymnothecial ascomata, superficial, single or aggregated, white, globose, without peridial appendages, peridium composed of loosely interwoven undifferentiated hyphae, septate, branched, hyaline, smooth- and thin-walled. Ascomata initials developing in a side branch spirally twisted around ascogonium. Asci 8-spored, evanescent, oval, in clusters on short stalks. Ascospores unicellular, hyaline, and slightly punctate, globose to subglobose.
- Albidomyces albicans (Apinis) Torres-Garcia, Dania García, Cano & Gené, comb. nov.
MycoBank MB850658
≡ Arachniotus albicans Apinis, Mycological papers 96: 45, 1964; ≡ Amauroascus albicans (Apinis) Arx, Persoonia 6: 376, 1971; ≡ Arachnotheca albicans (Apinis) Arx, The genera of fungi sporulating in pure culture, 2nd ed., J Cramer, Veduz.: 98, 1974; ≡ Nannizziopsis albicans (Apinis), Guarro, Cano & de Vroey, Mycotaxon 42: 195, 1991.
Type: UK, Lakenham, Norfolk, waterlogged alluvial pasture soils, April 1950, Apinis, (holotype IMI 100875, cultures ex-type CBS 151.65, ATCC 22478, BDUN 264, IHEM 4423, NRRL 5141, RV 2280).
Description: Mycelium immersed and superficial, composed of hyaline, septate, branched, smooth-walled hyphae, 1.5–3 μm wide. Racquet hyphae present. Asexual morph with fertile hyphae branched, hyaline, smooth-walled, and conidia borne sessile or in short cylindrical or conical protrusions. Conidia solitary, terminal, and lateral, unicellular, hyaline, smooth-walled, pyriform, 3.5–4.5(–6.5) × 4.5–7.5 μm. Sexual morph with gymnothecial ascomata, superficial, single or confluent, white or cream, globose, 188–435 μm diam, peridial hyphae composed of a conspicuous network of hyphae loosely interwoven, undifferentiated, septate, branched, hyaline, smooth, 2.5–3 μm wide. Asci 8-spored, evanescent, in clusters, oval, 8–11 × 11–17 μm. Ascospores unicellular, hyaline, punctate to reticulate, globose to subglobose, 3–4.5(–5) × 3–4.5 μm (adapted from Apinis [84]).
Culture characteristics (14 d at 25 °C): Colonies on PDA reaching 30–31 mm diam., velvety, umbonate, radially sulcate and white (1A1) to yellowish white (1A2) at the center, flat and gray (1B1) toward the periphery, margins lobulate, sporulation absent, reverse light yellow (4A4) at the center, yellowish white (4A2) toward the periphery, diffusible pigment absent. On PCA, reaching 26–29 mm diam., velvety, concentrically sulcate and yellowish white (1A2) at the center, flat and grayish white (1B1) toward the periphery, margins lobulate, sporulation absent, reverse yellowish white (1A2) at the center, yellowish gray (2B2) toward the periphery, diffusible pigment absent. On OA, reaching 42–27 mm diam., velvety, concentrically sulcate and grayish beige (4C2) across the entire colony, immersed colony, margins entire, sporulation sparse, reverse grayish beige (4C2), diffusible pigment absent. On PYE, reaching 35–36 mm diam., velvety, umbonate, radially sulcate and white (1A1) at the center, flat and yellowish white (1A2) to white (1A1) toward the periphery, margins lobulate, sporulation absent, reverse brownish orange (5C4) at the center, grayish orange (5B5) toward the periphery, diffusible pigment absent.
Cardinal temperature for growth on PDA after 14 d (mm): Minimum 15 °C (21–22), optimum 25 °C (30–31), maximum 30 °C (8–10).
Additional specimens examined: Spain, Aragon, Huesca, Cerler, Remáscaro Stream, N 42.58698° E 0.54993°, from river sediments, September 2018, J.F. Cano-Lira (FMR 17576); ibid. Catalonia, Barcelona, Bages, Balsareny, Llobregat River, N 41.87818° E 1.88900°, from river sediments, March 2021, D. Torres-Garcia & J. Gené (FMR 19029); ibid. Berguedà, Castellar de n’Hug, Llobregat River, N 42.24012° E 1.93742°, from river sediments, March 2021, D. Torres-Garcia & J. Gené (FMR 19026); ibid. (FMR 19027); ibid. (FMR 19028); ibid. Osona, Vilanova de Sau, Ter River, N 41.97084° E 2.38108°, from river sediments, September 2020, D. Torres-Garcia & J. Gené (FMR 18697); ibid. Girona, Llanars, Ter River, N 42.32383° E 2.32989°, from river sediments, September 2020, D. Torres-Garcia & J. Gené (FMR 18698); ibid. Sant Joan de les Abadesses, Ter River, N 42.24138° E 2.28830°, from river sediments, September 2020, D. Torres-Garcia & J. Gené (FMR 18699); ibid. Lleida, Berguedà, Gósol, Torrent de la Coma Stream, N 42.21889° E 1.65159°, from river sediments, November 2019, J. Gené (FMR 18029); ibid. (FMR 18030); ibid. (FMR 18031); ibid. (FMR 18033).
Habitat and geographical distribution: Freshwater sediments, soil, and rhizosphere soil from different environments (forest, grassland, urban, and woodland). Czech Republic, Estonia, Germany, Latvia, Spain, and the UK (Figure 4, Table S2 in Supplementary Materials).
Notes: Arachniotus albicans was proposed by Apinis [84] based on a group of isolates from which the selected type strain was found in alluvial soils. However, because of its inconspicuous morphological traits (i.e., white colonies, gymnothecia with undifferentiated peridial hyphae, small and globose ascospores, and an asexual morph producing terminal and lateral conidia on hyphae), its taxonomy has been confused. Arx [75] transferred the species to the genus Amauroascus on the basis of its ascomatal initials, spore shape and ornamentation, and its keratinolytic activity, but later transferred the species to Arachnotheca [85]. The last taxonomic proposal was rejected by Guarro et al. [82] because the morphological features of Ara. albicans, such as hyaline and sheathed ascospores or its chrysosporium-like asexual morph, did not fit with the Arachnotheca concept. In fact, they considered it to be a more likely species of Nannizziopsis [82]. The recent phylogenetic analysis of the Onygenales [8], which correlates with that presented here (Figure 2), shows that the generic types of the aforementioned genera are placed in other families (Arachniotus ruber in the Gymnoascaceae; Amauroascus niger and Nannizziopsis vriesii in the Onygenaceae), and the type of Arachnotheca, Arach. glomerata, although belonging to the same clade as Ara. albicans, is placed phylogenetically distant from the lineage representative of Ara. albicans. Since this later lineage, which includes the ex-type strain of Ara. albicans (CBS 151.65), was separated from the other lineages that comprised the new family Neoarthropsidaceae, we describe the monotypic genus Albidomyces. The other accepted genera included in the family can be distinguished morphologically from Albidomyces by the following main features: Apinisia by its helical appendages in the ascomata, Arachnotheca and Myriodontium by their sheathed ascospores and type of conidiogenous apparatus [8,82], and Neoarthropsis also by its asexual morph with enteroarthric conidia (see below). Of note is that close to the Al. albicans clade is the ex-type strain of Chrysosporium sulfureum. This species, originally described as Isaria sulfurea, did not form a sexual morph, and it was transferred to Chrysosporium by Oorschot based on the morphology of its asexual morph [79]. It has conidia growing directly on hyphae, sessile or on short cylindrical or conical protrusions. These conidial features and the ontogenesis are similar to the chrysosporium-like asexual morph present in Al. albicans, suggesting that it is probably another species of Albidomyces. However, we prefer to delay making any taxonomic changes until further investigation with more isolates of the genus.
- Neoarthropsis Torres-Garcia, Cano & Gené, gen. nov.
MycoBank MB847965
Etymology: Referring to the morphological resemblance with the genus Arthropsis.
Type species: Neoarthropsis hispanica (Ulfig et al.) Torres-Garcia, Cano & Gené.
Description: Mycelium septate, hyaline to subhyaline, smooth- and thin-walled, variable in width. Racquet hyphae usually present. Asexual morph arthropsis-like, consisting of fertile hyphae, single or aggregated in tufts, straight to flexuous, with lateral branches arising at right angles, straight or recurved, septating basipetally to form enteroartric conidia, often joined by distinct, narrow connectives with rhexolytic secession. Arthroconidia hyaline to subhyaline, straight or curved, smooth- and thin-walled, cylindrical, cubic, barrel-shaped, often broader than long, usually truncate at both ends, showing occasionally remnants of outer wall. Sexual morph with gymnothecial ascomata, superficial, single or confluent, growing as ascal clusters surrounded by distinct peridial hyphae, white to yellowish white, globose to subglobose, peridium composed of a conspicuous network of septate, branched, anastomosed hyphae when mature, hyaline to pale yellow, thin- and smooth-walled. Ascomatal initials consisting of crozier formations. Asci 8-spored, evanescent, borne singly or in chains, hyaline, smooth-walled, globose to subglobose or pyriform. Ascospores unicellular, hyaline, thick-walled, punctate (minutely echinulate), and globose.
- Neoarthropsis hispanica (Ulfig, Gené & Guarro) Torres-Garcia, Cano & Gené, comb. nov.
MycoBank MB850653
Arthropsis hispanica Ulfig, Gené & Guarro, Mycotaxon 54: 282, 1995.
Type: Spain, Catalonia, Girona, Estartit Beach, from marine sediments, September 1991, J. Gené, K. Ulfig & J. Guarro (holotype IMI 353700; cultures ex-type FMR 4059, CBS 352.92).
Additional specimens examined: Spain, Catalonia, Barcelona, Bages, Balsareny, Llobregat River, N 41.87818° E 1.88900°, March 2021, D. Torres-Garcia & J. Gené (FMR 19034); ibid. (FMR 19035); ibid. Osona, Les Masies de Voltregà, Ter River, N 42.02951° E 2.25359°, from river sediments, September 2020, D. Torres-Garcia & J. Gené (FMR 18694).
Habitat and geographical distribution: Air, fluvial and marine sediments, human clinical specimens, rhizosphere soil, roots, soil from different environments (aquatic, cropland, forest, shrubland, urban, and woodland). Australia, China, Estonia, Japan, South Korea and Spain (Figure 4, Table S2 in Supplementary Materials).
Notes: Arthropsis was established by Sigler et al. [86] and typified by A. truncata, to accommodate species with dark arthroconidia, joined by adjacent connectives, and developed from undifferentiated conidiogenous hyphae [87]. Later, Ulfig et al. [32] introduced A. hispanica based on its morphological characteristics regarding the conidial connectives and the absence of differentiated conidiophores. However, in contrast to other Arthropsis species, which exhibited dark pigmented mycelium and conidia, A. hispanica showed hyaline to subhyaline arthroconidia, fast-growing rates, and strongly tufted colonies in culture [32]. Later, based on the LSU analysis of the type strain A. hispanica (CBS 351.92), Giraldo et al. [87] placed the species in the order Onygenales (Eurotiomycetes), which was far away from A. truncata (Sordariomycetes). As a result, based on phylogenetic data together with the distinct morphological traits of its closely related genus Arachnotheca (Figure 2), we introduce the genus Neoarthropsis in Onygenales with two species, Ne. hispanica as the type of the genus and Ne. sexualis proposed and discussed below. The key morphological features to distinguish Neoarthropsis from Arachnotheca are the presence of narrow connectives in the arthroconidia of Neoarthropsis species (absent in the asexual morph of Arachnotheca) and the lack of sheath in their ascospores (presence of an irregular furrowed sheath in the ascospores of Arachnotheca) [74].
- Neoarthropsis sexualis Torres-Garcia, Cano & Gené, sp. Nov. Figure 8
MycoBank MB847966
Etymology: Referring to the presence of a sexual morph in the type strain.
Type: Spain, Catalonia, Barcelona, Bages, Balsareny, Llobregat River, N 41.87818° E 1.88900°, from fluvial sediments, March 2021, D. Torres-Garcia & J. Gené, (holotype CBS H-25259, cultures ex-type FMR 19025, CBS 149940).
Description: Mycelium immersed and superficial, composed of hyaline, septate, branched, smooth-walled, 1.5–3 μm wide hyphae. Racquet hyphae abundant. Asexual morph with undifferentiated conidiophores. Fertile hyphae produced mainly in tufts, hyaline, smooth- and thin-walled, main hyphae straight to flexuose, 2.5–3 μm wide, lateral branches arising at right angles, often recurved, septating basipetally to form enteroarthric conidia joined by narrow connectives. Arthroconidia unicellular, hyaline, thin- and smooth-walled, cylindrical or barrel-shaped, straight or curved, 2.5–7 × 1.5–3 μm. Sexual morph with gymnothecial ascomata, superficial, single or confluent, white to yellowish white, globose to subglobose, 365–810 μm diam, peridium composed of a conspicuous network of hyphae, septate, branched, anastomosed, hyaline to pale yellow, thick- and smooth-walled, cylindrical, 3–4 μm wide. Asci 8-spored, evanescent, borne singly or in chains, hyaline, smooth-walled, globose to subglobose or pyriform, 4–6 × 4–5.5 μm. Ascospores unicellular, hyaline, thick-walled, punctate, globose, 1.5–2.5 μm diam.
Culture characteristics (14 d at 25 °C): Colonies on PDA reaching 40–41 mm diam., raised, floccose, and white (1A1) at the center, flat, cottony, and yellowish white (1A2) toward the periphery, margins slightly irregular and fimbriate, sporulation abundant, reverse pastel yellow (3A4) at the center, yellowish white (3A2) toward the periphery, diffusible pigment absent. On PCA, reaching 32–33 mm diam., slightly raised, granulose, and white (1A1) at the center, floccose to cottony and yellowish gray (2B2) toward the periphery, margins fimbriate, sporulation abundant, reverse pale yellow (4A3) at the center, yellowish white (3A2) toward the periphery, diffusible pigment absent. On OA, reaching 33–34 mm diam., granulose and white (1A1) at the center, velvety and grayish beige (4C2) toward the periphery, margins irregular, sporulation abundant, reverse grayish yellow (4B3), diffusible pigment absent. On PYE, reaching 44–45 mm diam., slightly raised at the center, floccose, white (1A1) to yellowish white (3A2), margins regular and slightly fimbriate, sporulation moderate at the center, reverse yellow (4A6) at the center, pale yellow (4A3) at the periphery, diffusible pigment absent.
Cardinal temperature for growth on PDA after 14 d (mm): Minimum 15 °C (16–17), optimum 25 °C (40–41), maximum 37 °C (2–3).
Habitat and geographical distribution: Fluvial sediment, forest, and grassland soil. Australia, Estonia, Spain, and the USA (Figure 4, Table S2 in Supplementary Materials).
Notes: In our reconstructed phylogeny, Ne. sexualis is allocated in a well-supported clade with Ne. hispanica. The two species were recovered from aquatic environments (freshwater/marine sediments) in Spain; although Ne. hispanica has also been found in clinical samples in the USA, its role in disease has never been demonstrated [81]. Both species produce a practically indistinguishable asexual morph. The main difference between them is the presence of ascomata in Ne. sexualis, produced on OA and PCA agar, which is absent in Ne. hispanica under the same in vitro conditions [32,81]. In addition, Ne. sexualis shows more restricted growth on PDA at 25 °C after 7d (12–13 mm) and is able to grow at 37 °C, while the colonies of Ne. hispanica reach 17–23 mm on the same culture medium and do not grow at that temperature [81].
- Onygenales, Incertae sedis (Figure 2, Clade X).
- Pseudoamaurascopsis Torres-Garcia, Dania García & Gené, gen. nov.
MycoBank MB848131
Etymology: Referring to the morphological resemblance with the asexual morph of the genus Amaurascopsis.
Type species: Pseudoamaurascopsis spiralis Torres-Garcia, Dania García & Gené.
Figure 8.
Neoarthropsis sexualis (ex-type FMR 19025). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C) Colony on OA (front) at 25 °C after 14 days. (D) Racquet and fertile hyphae. (E) Fertile hyphae. (F) Arthroconidia with narrow connectives. (G) Initial forms of asci. (H) Detail of a gymnothecium showing peridial hyphae and ascospores. (I) Asci and ascospores. (J) Ascospores. Scale Bars: (D) = 25 μm; (E–J) = 10 μm.
Figure 8.
Neoarthropsis sexualis (ex-type FMR 19025). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C) Colony on OA (front) at 25 °C after 14 days. (D) Racquet and fertile hyphae. (E) Fertile hyphae. (F) Arthroconidia with narrow connectives. (G) Initial forms of asci. (H) Detail of a gymnothecium showing peridial hyphae and ascospores. (I) Asci and ascospores. (J) Ascospores. Scale Bars: (D) = 25 μm; (E–J) = 10 μm.
Description: Mycelium septate, hyaline, smooth- and thin-walled. Spiral hyphae present, growing laterally from vegetative hyphae. Asexual morph chrysosporium-like, consisting of fertile hyphae usually with intercalary and terminal thallic conidia, occasionally lateral on a short stalk, conidial secession rhexolytic. Terminal conidia arising on straight or slightly curved branches, single, hyaline to subhyaline, smooth-walled to verruculose, thick-walled, subglobose or short clavate, with a truncate end, intercalary conidia predominant, single, alternate, hyaline to subhyaline, smooth-walled to verruculose, thick-walled, cylindrical to barrel-shaped. Sexual morph not observed.
- Pseudoamaurascopsis spiralis Torres-Garcia, Dania García & Gené, sp. nov. Figure 9.
MycoBank MB848132.
Etymology: Referring to the ability of the species to produce spiral hyphae on vegetative hyphae.
Type: Spain, Catalonia, Barcelona, Bages, Balsareny, Llobregat River, N 41.87818° E 1.88900°, from fluvial sediments, March 2021, D. Torres-Garcia & J. Gené, (holotype CBS H-25260; cultures ex-type FMR 19014, CBS 149941).
Description: Mycelium partly immersed and partly superficial, composed of hyaline, septate, branched, smooth-walled, 1.5–2.5 μm wide hyphae. Spiral vegetative hyphae abundantly produced on OA after 14 d at 25 °C. Asexual morph chrysosporium-like, with terminal and intercalary thallic conidia, occasionally lateral on short stalks, terminal conidia on straight or slightly curved branches, single, hyaline to subhyaline, thick-walled, smooth-walled when young, becoming slightly echinulate, subglobose, obpyriform or short clavate, 3–5 × 2–3 μm, intercalary conidia single, alternate, hyaline to subhyaline, smooth-walled to echinulate, thick-walled, cylindrical or barrel-shaped, 7–15(–22.5) × 2–3 μm. Sexual morph not observed.
Culture characteristics (14 d at 25 °C): Colonies on PDA reaching 23–24 mm diam., slightly raised, cottony and pinkish white (7A2) at the center and yellowish white (3B2) at the periphery, margins entire, sporulation abundant, reverse brownish orange (5C4) at the center, pale yellow (4A3) toward the periphery, diffusible pigment absent. On PCA, reaching 22–23 mm diam., slightly raised, cottony, white (1A1) to pinkish white (7A2) at the center, velvety and yellowish gray (4B2) toward the periphery, margins entire, sporulation abundant, reverse grayish orange (5B3) at the center, brownish orange (5C4) and orange-gray (5B2) toward the periphery, diffusible pigment absent. On OA, reaching 22–23 mm diam., flattened, velvety, yellowish gray (4B2), margins lobulate, sporulation absent, reverse orange-gray (5B2), diffusible pigment absent. On PYE, reaching 21–22 mm diam., raised, velvety, yellowish white (1A2) to white (1A1), margins fimbriate, sporulation sparse to moderate, reverse golden blond (5C4) at the center, pale yellow (4A4) toward the periphery, diffusible pigment absent.
Cardinal temperature for growth on PDA after 14 d (mm): Minimum 15 °C (5–6), optimum 25 °C (23–24), maximum 37 °C (2–3).
Habitat and geographical distribution: Fluvial sediments, rhizosphere soil, roots, and soil of different natural environments (cropland, grassland, and urban). Widespread distribution (Australia, Belgium, China, Denmark, Estonia, Germany, Spain, the UK, and the USA) (Figure 4, Table S2 in Supplementary Materials).
Notes: Our phylogenetic analysis placed Ps. spiralis as being related to Am. perforata and Po. hystricis, but the three taxa were phylogenetically very distant and representatives of distinct genera. Pseudoamaurascopsis spiralis differs morphologically from Am. perforata and Po. hystricis mainly by the lack of a sexual morph and by the production of spiral hyphae from vegetative hyphae, which are absent at least in Am. perforata [88], since Po. hystricis shows coiled appendages but they are associated with the ascomata [89]. Despite attempts to induce the sexual morph in Ps. spiralis, it was never observed. The three species produce a chrysosporium-like morph with terminal and intercalary conidia, from which we only observed subtle morphological differences. The conidia of Po. spiralis are longer (3–11 μm) and finely echinulate, while those of Am. perforata are (3–)4–6.5(–7.5) μm long and roughened to tuberculate [88]. Polytolypa hystricis shows smooth conidia, and it is unable to grow at 37 °C [89]. Pseudoamaurascopsis spiralis and Am. perforata grow at this temperature but the latter grows faster (28–30 mm diam on PDA after 14 days) [88].
Figure 9.
Pseudoamaurascopsis spiralis (ex-type FMR 19014). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C) Colony on OA (front) at 25 °C after 14 days. (D) Spiral hyphae. (E,F) Fertile hyphae with intercalary and terminal arthroconidia. (G−J) Arthroconidia. Scale Bars: (D–J) = 10 μm.
Figure 9.
Pseudoamaurascopsis spiralis (ex-type FMR 19014). (A) Colonies on PDA (front and reverse) at 25 °C after 14 days. (B) Colonies on PYE (front and reverse) at 25 °C after 14 days. (C) Colony on OA (front) at 25 °C after 14 days. (D) Spiral hyphae. (E,F) Fertile hyphae with intercalary and terminal arthroconidia. (G−J) Arthroconidia. Scale Bars: (D–J) = 10 μm.
4. Discussion
Although the species of Onygenales are not considered freshwater fungi, they are able to inhabit substrates in aquatic environments such as freshwater sediments. Among onygenalean fungi found in this substrate are, for instance, the species of the thermally dimorphic nonpathogenic genus Emmonsiellopsis, E. coralliformis, and E. terrestris. This genus belongs to the Ajellomycetaceae and represents a sister lineage to the thermally dimorphic human pathogens of the family (i.e., Blastomyces, Emergomyces, Histoplasma, and Paracoccidioides (Figure 2)) [8]. In fact, the study of isolates from Spanish freshwater sediments —curiously of the same origin as the third species of the genus, E. tuberculata—led to the proposal of Emmonsiellopsis [37]. Emmonsiellopsis tuberculata was isolated from a sediment sample taken from an oligotrophic stream during our survey in the Aragonese Pyrenees [38]. In the present work, the study of isolates from fluvial sediments has not only allowed us to propose three novel genera (i.e., Albidomyces, Neoarthorpisis, and Pseudoamaurascopsis) and five new species (M. echinulata, M. irregularis, M. sinuata, Ne. sexualis, and Ps. spiralis), but also to delineate the new family Neoarthropsidaceae. According to our records, this family includes species like Al. albicans, My. keratinophilum, and Ne. hispanica that appear to be common in freshwater sediments. These findings and others previously reported [27,28,29,30,32,33,34,35,36,38,76] suggest that fluvial sediments are a good source of onygenalean fungi, and further investigation on their diversity is required. On that point, special attention should be paid to fluvial sediments in humid regions in the Northern Hemisphere, particularly sites in Eastern Europe, which have recently been defined as important hotspots of Onygenales [90].
Despite the fact that Onygenales are considered uncommon fungi in environmental surveys [91], using appropriate methods for their isolation can change this premise. In our survey, including PDA supplemented with cycloheximide among the culture media for fungal isolation has allowed us to isolate a set of onygenalean isolates that would otherwise have been difficult to detect, due mainly to their slow growth relative to other common environmental fungi like Aspergillus, Penicillium, or Mucorales in general. We identified 19 species of Onygenales, including the novel species mentioned previously (Table 1), using phenotypic traits and sequence analyses of the ITS and LSU regions. Recent molecular phylogenetic studies on the Onygenales have analyzed sequences of other nuclear genes like tef1, tub2, rpb2, or actin as the most commonly used loci to resolve the taxonomy in some groups [8,92,93,94,95,96]. However, sequences of those loci for many members of the order are not yet available for comparison. According to our results, the phylogenetic reconstruction with the barcode regions shows enough phylogenetic resolution to classify and delineate species of fresh isolates of this group of fungi. The ITS regions should be mandatory not only for identification purposes but also to assess the global distribution of the new fungi, as has been done here (see below) and in other taxonomic studies [42,73,74], including for novel species in the Arthrodermataceae [96].
In our study, the ITS sequence comparison of the novel taxa proposed here with the ITS environmental sequences deposited in the GlobalFungi database revealed that, with exception of the Malbranchea species, the other new taxa (i.e., Al. albicans, Ne. hispanica, Ne. sexualis, and Ps. spiralis) seem to be worldwide fungi that inhabit different types of soil (i.e., mainly forest, agricultural, rhizosphere soil) in various European countries and in Australia, China, South Korea, and the USA. Neoarthropsis hispanica was the only species with ITS environmental sequences derived from aquatic sediments different from those of the Spanish ones, namely from Japan (Figure S3, Table S1 in Supplementary Materials). These results suggest that, even though they have been found in fluvial sediment, they are terrestrial fungi, the main origin of the Onygenales [9,10,11,12,13,14,15]. They probably arrive in river and stream sediments by lixiviation, where they then are able to adapt to aquatic environments. Evidence of this is that most of the abovementioned fungi shows cardinal temperatures ranging from 5 to 30 °C and are unable to growth at higher temperatures. Regarding the three new Malbranchea species, we only found environmental sequences related to M. sinuata but limited to sequences from shrubland rhizosphere soil from Spain, revealing that these Malbranchea species are rare fungi with a very limited distribution. However, further studies are required to confirm this approach.
Apart from the novel species, we confidently identified a set of known species, which belong to other families (Figure 1): namely, Al. albicans (= Arachniotus or Amauroascus albicans), My. Keratinophilum, and Ne. hispanica (= Arthropsis hispanica) in the Neoarthropsidaceae; Ap. Crassitunicatus, Ap. Fulvescens, and Ap. Reticulisporus in the Onygenaceae; Ar. Curreyi in the Arthrodermataceae; E. tuberculata in the Ajellomycetaceae; Gym. Petalosporus in Gymnoascaceae; M. chinensis, M. ostraviensis, M. reticulata, and M. umbrina in the Malbrancheaceae; and finally, L. emdenii, which is regarded as a species with an unclear taxonomic position despite being close to the Arthrodermataceae [8]. From those known species identified, Ap. Fulvescens, Ap. Reticulisporus, Ar. Curreyi, Ne. hispanica, Gym. Petalosporus, L. emdenii, M. reticulata, and My. Keratinophilum have already been reported in Spanish freshwater sediments in previous studies [27,29,30,32,76,80].
As we stated in the results, the most prevalent species in our survey were Al. albicans, Ap. fulvescens, and My. keratinophilum, which represented 55.5% of the fungi identified. Of note is that the latter two species, together with Narasimhella marginospora (currently N. poonensis), not isolated in the present survey, were found to be the most abundant species in fluvial sediments collected at the mouth of various Catalonian rivers (Besós, Ebro, Francolí, Fluvià, Llobregat, Muga, Ter, and Tordera) in the studies of Ulfig et al. [27,28,29,30]. Even in those studies, Ap. fulvescens and, particularly, N. marginospora were associated with badly polluted rivers, mainly by sewage contamination, such as Besós, Francolí, Llobregat, and Tordera. Our results are not comparable with those of Ulfig’s research, primarily because we have no data on the state of contamination of the rivers sampled in our surveys and also because sediments were collected from the upper and middle sections of the rivers rather than from the river mouth as in Ulfig’s surveys. Nevertheless, in our study, the majority of Ap. fulvesces and My. keratinophilum strains were primarily isolated from sediments of the river Segre that were collected close to rural populations and farmlands, which may have contributed to the eutrophication of the sediments. Because of the lack of experimental data, it can only be hypothesized that these two species might be bioindicators of water pollution, as stated previously by Ulfig et al. [28] regarding keratinolytic species in general. However, further studies are still needed to verify this or a similar approach. It is relevant to mention that both fungi have been described as human opportunists. Although they only cause cutaneous infections [17,97], they should be regarded as a risk to human health, particularly in river areas used for bathing purposes.
This is the first report of Al. albicans in fluvial sediments, although the ex-type strain (CBS 151.65) was found to be associated with wetlands, as it was isolated from pasture alluvial soils in the UK [85]. As mentioned in the taxonomy section, the classification of this species was confused; it was classified in Amauroascus, Arachniotus, Arachnotheca, and Nannizziopsis [9,75,82,85,98], and even Kuehniella racovitzae (currently Apinisia racovitzae) was considered by several authors a synonym of Al. albicans—at that time classified in the genus Arachnotheca [75,99]. As a consequence of this nomenclatural instability, data on its habitat and distribution in the literature or even in databases like the Global Biodiversity Information Facility (GBIF.org) might be confusing. After the delineation of the species in the genus Albidomyces (Figure 2) together with metadata on the ITS environmental sequences corresponding to Al. albicans deposited in the GlobalFungi database, it was revealed to be a species inhabiting mainly forests limited to European countries (Table S2 in Supplementary Materials). Our data also show that Al. albicans is widespread in the rural and natural areas of Catalonia, where it exhibits a clear capacity to disperse and survive in both oligotrophic and eutrophic fluvial sediments. It would be interesting to verify the role of the species in aquatic environments.
The most diverse genus found in our freshwater sediment was Malbranchea, from which seven species were identified, including the newly proposed (Table 1). This is not so surprising since it is currently one of the most species-rich genera in the Onygenales, comprising nearly 30 species, many of them resistant to cycloheximide like other onygenalean fungi [8,51,78]. Ulfig et al. [30], focused on the occurrence of cycloheximide-resistant fungi from sediments collected at river mouths along the Catalonian coast, isolated several Malbranchea species (formerly under the Auxarthron genus) using Sabouraud agar supplemented with that antimicrobial agent. They were M. arcuata, M. conjugata, M. pulchella, M. reticulata, M. umbrina, and M. zuffiana, in addition to several unidentified Malbranchea isolates. Culture media with cycloheximide might help to investigate the diversity of Malbranchea in unexplored areas or substrates that would be challenging to find and isolate using other culture-dependent techniques. Taking into account that species of Malbranchea can usually inhabit animal dung or eutrophic environments like soil enriched by dung [8], this might lead us to think that Malbranchea could also be bioindicators of fecal contamination, although we lack more field surveys and laboratory tests to confirm this approach. Among the species identified here, M. chinensis, which was recently described from cave soil in China [83], was initially considered a rare species, but data obtained from the GlobalFungi database has revealed it is not so. ITS2 environmental sequences of M. chinensis were found to be associated with other types of soils, such as desert sand and rhizosphere soil, or roots from Chile and Spain; it was even detected from marine samples in China (Table S1, Figure S5-ITS2 in Supplementary Materials). Using the GlobalFungi database, we were also able to see the ubiquity in the soil of the three other species identified from Spanish fluvial samples (i.e., M. ostraviensis, M. reticulata, and M. umbrina), confirming the usefulness of this database for tracing the geographical distribution of fungal species over the world. It is also worth noting that M. ostraviensis and M. umbrina have been reported in cases of onychomycosis [13,77], and the latter has also been identified from various human specimens (i.e., bronchoalveolar lavage, nail, sinus, and wound), although without a demonstrated pathogenic role in disease [51].
5. Concluding Remarks
Although the order Onygenales is a well-known group of Ascomycota, widely investigated mainly because of its association with mammals and ability to cause superficial and systemic diseases to humans, its diversity and prevalence in aquatic environments is still poorly known. Research on its diversity in the underexplored areas of the world or poorly studied substrates, like freshwater sediments, might provide new insights and interesting specimens that allow to clarify the taxonomy of the group and also the role of these fungi in a particular habitat.
Knowing the variety and prevalence of Onygenales in freshwater sediments would help to determine whether they pose a risk not only to human health but also to wild animals that inhabit aquatic environments, like reptiles, in which certain onygenalean species are able to cause infections [17,95,98,100,101]. This insight would be particularly relevant in the current context of climate change [102]. It is estimated that, for instance, the rainfall patterns in the Mediterranean region will be greatly impacted by a more extreme climate [103]. Areas that are currently covered by water are likely to dry up, allowing potential pathogenic fungi like Emmonsiellopsis species to be exposed to the air. As a result of that and considering the ability of onygenalean fungi to adapt to extreme conditions [8], climate change may lead to an increase in human and animal infections by species previously described as nonpathogens.
Supplementary Materials
The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/jof9121129/s1, Table S1: Strain information and GenBank/EMBL accession numbers of the species included in the phylogenies of this study, Table S2: Environmental and biogeographical information contained in all ITS1/ITS2 sequences downloaded from the GlobalFungi database included in our analysis (see Figure 4), Figure S1: RAxML ITS tree of the order Onygenales, including the strains recovered from freshwater sediments studied in this work, Figure S2: RAxML ITS tree of the family Neoarthropsidaceae, including the strains recovered from freshwater sediments studied in this work, Figure S3: RAxML phylogenetic tree showing the relationships among species investigated in the new family Neoarthropsidaceae and related ITS1/ITS2 environmental sequences deposited in the GlobalFungi database, Figure S4: RAxML phylogenetic tree showing the relationships among species investigated in Clade X (incertae sedis at the family level) and related ITS1/ITS2 environmental sequences deposited in the GlobalFungi database, Figure S5: RAxML phylogenetic tree showing the relationships among some species in the Malbrancheaceae and related ITS1/ITS2 environmental sequences deposited in the GlobalFungi database. Refs. [8,14,15,36,37,38,46,48,49,50,51,81,83,87,95,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128] are cited in the supplementary materials.
Author Contributions
Conceptualization, D.T.-G. and J.G.; methodology, D.T.-G., D.G., J.G. and J.F.C.-L.; software, D.T.-G. and J.F.C.-L.; validation, J.F.C.-L. and J.G.; formal analysis, D.T.-G., J.F.C.-L. and J.G.; investigation, D.T.-G. and J.G.; resources, J.F.C.-L. and J.G.; data curation, D.T.-G., J.F.C.-L. 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.T.-G. and J.G.; supervision, D.G., J.F.C.-L. and J.G.; project administration J.G.; funding acquisition, J.G. All authors have read and agreed to the published version of the manuscript.
Funding
This study was supported by the grant PID2021-128068NB-100 funded by MCIN/AEI/10. 13039/501100011033/and by “ERDF A way of making Europe”.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The data presented in this study are available on request from the corresponding author.
Conflicts of Interest
The authors declare no conflict of interest.
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Figure 1.
Rivers and streams sampled in Spain (red points). Altitude is represented in shades of gray. Scale bar: 100 km.
Figure 1.
Rivers and streams sampled in Spain (red points). Altitude is represented in shades of gray. Scale bar: 100 km.
Figure 4.
Global geographical distribution and substrate affinity of newly described taxa in Onygenales based on the ITS1/ITS2 environmental sequence data deposited in the GlobalFungi database (additional information in Supplementary Materials—Figures S3–S5 and Table S2). The European continent has been expanded for better observation of the species distribution. Polygonal shapes in different colors represent the novel taxa and their distribution according to the ITS environmental sequences detected.
Figure 4.
Global geographical distribution and substrate affinity of newly described taxa in Onygenales based on the ITS1/ITS2 environmental sequence data deposited in the GlobalFungi database (additional information in Supplementary Materials—Figures S3–S5 and Table S2). The European continent has been expanded for better observation of the species distribution. Polygonal shapes in different colors represent the novel taxa and their distribution according to the ITS environmental sequences detected.
Table 1.
Information and GenBank accession numbers of the isolates of the species identified in the present study.
Table 1.
Information and GenBank accession numbers of the isolates of the species identified in the present study.
| Species | Strain Numbers 1 | Rivers and Streams in Spain (Coordinates) | GenBank Accession Numbers | |||
|---|---|---|---|---|---|---|
| ITS | LSU | tub2 | rpb2 | |||
| Albidomyces albicans (≡Arachniotus albicans) | FMR 17576 | Remáscaro Stream, Cerler, Huesca (N 42.58698° E 0.54993°) | ON720226 | ON720765 | - | - |
| FMR 18029 | Torrent de la Coma Stream, Lleida (N 42.21889° E 1.65159°) | ON721307 | ON720780 | - | - | |
| FMR 18030 | Torrent de la Coma Stream, Lleida (N 42.21889° E 1.65159°) | ON720223 | ON720762 | - | - | |
| FMR 18031 | Torrent de la Coma Stream, Lleida (N 42.21889° E 1.65159°) | ON720231 | ON720770 | - | - | |
| FMR 18033 | Torrent de la Coma Stream, Lleida (N 42.21889° E 1.65159°) | ON720228 | ON720767 | - | - | |
| FMR 18697 | Ter River, Vilanova de Sau, Barcelona (N 41.97084° E 2.38108°) | OP373730 | OP373734 | - | - | |
| FMR 18698 | Ter River, Llanars, Girona (N 42.32383° E 2.32989°) | ON720224 | ON720763 | - | - | |
| FMR 18699 | Ter River, Sant Joan de les Abadesses, Girona (N 42.24138° E 2.28830°) | OP373731 | OP373735 | - | - | |
| FMR 19026 | Llobregat River, Castellar de n’Hug, Barcelona (N 42.24012° E 1.93742°) | ON720229 | ON720768 | - | - | |
| FMR 19027 | Llobregat River, Castellar de n’Hug, Barcelona (N 42.24012° E 1.93742°) | ON720225 | ON720764 | - | - | |
| FMR 19028 | Llobregat River, Castellar de n’Hug, Barcelona (N 42.24012° E 1.93742°) | ON720227 | ON720766 | - | - | |
| FMR 19029 | Llobregat River, Balsareny, Barcelona (N 41.87818° E 1.88900°) | ON720230 | ON720769 | - | - | |
| Aphanoascus crassitunicatus | FMR 18700 | Ter River, Vilanova de Sau, Barcelona (N 41.97084° E 2.38108°) | ON720203 | ON720742 | - | - |
| FMR 18701 | Ter River, Roda de Ter, Barcelona (N 41.96974° E 2.31211°) | ON720204 | ON720743 | - | - | |
| FMR 18702 | Ter River, Ripoll, Girona (N 42.17258° E 2.19372°) | ON721311 | ON720784 | - | - | |
| Aphanoascus fulvescens | FMR 18235 | Segre River, Aitona, Lleida (N 41.97084° E 2.38108°) | ON720208 | ON720747 | - | - |
| FMR 18236 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON720210 | ON720749 | - | - | |
| FMR 18241 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON721308 | ON720781 | - | - | |
| FMR 18242 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON720211 | ON720750 | - | - | |
| FMR 18243 | Segre River, Camarassa, Lleida (N 41.89834° E 0.88110°) | ON942224 | ON942226 | - | - | |
| FMR 18704 | Ter River, Les Masies de Voltregà, Barcelona (N 42.02951° E 2.25359°) | ON720207 | ON720746 | - | - | |
| FMR 19018 | Mare de Déu Stream, Borriana, Castellón (N 39.87727° W 0.05374°) | ON720212 | ON720751 | - | - | |
| FMR 19019 | Mare de Déu Stream, Borriana, Castellón (N 39.87727° W 0.05374°) | ON720209 | ON720748 | - | - | |
| FMR 19022 | Mare de Déu Stream, Borriana, Castellón (N 39.87727° W 0.05374°) | ON720206 | ON720745 | - | - | |
| FMR 19020 | Llobregat River, Cercs, Barcelona (N 42.10512° E 1.88044°) | ON721312 | ON720785 | - | - | |
| FMR 19021 | Llobregat River, Cercs, Barcelona (N 42.10512° E 1.88044°) | ON720205 | ON720744 | - | - | |
| Aphanoascus reticulisporus | FMR 18004 | Esca Stream, Navarra (N 42.81466° W 0.95221°) | ON720200 | ON720739 | - | - |
| FMR 19012 | Llobregat River, Cercs, Barcelona (N 42.10512° E 1.88044°) | ON720201 | ON720740 | - | - | |
| FMR 19013 | Llobregat River, Cercs, Barcelona (N 42.10512° E 1.88044°) | ON720202 | ON720741 | - | - | |
| FMR 19033 | Llobregat River, La Pobla de Lillet, Barcelona (N 42.24294° E 1.96998°) | OP373732 | OP373736 | - | - | |
| Arthroderma curreyi | FMR 19038 | Llobregat River, Balsareny, Barcelona (N 41.87818° E 1.88900°) | ON720238 | ON720777 | - | - |
| Emmonsiellopsis tuberculata | FMR 17582T | Remáscaro Stream, Cerler, Huesca (N 42.58698° E 0.54993°) | LR598892 | LR598891 | - | - |
| Gymnoascoideus petalosporus | FMR 19036 | Llobregat River, Cercs, Barcelona (N 42.10512° E 1.88044°) | ON720236 | ON720775 | - | - |
| Leucothecium emdenii | FMR 18703 | Ter River, Sant Joan de les Abadesses, Girona (N 42.24138° E 2.28830°) | ON720239 | ON720778 | - | - |
| Malbranchea chinensis | FMR 18267 | Segre River, Camarassa, Lleida (N 41.89834° E 0.88110°) | ON720190 | ON720729 | OP425706 | OP425715 |
| Malbranchea echinulata (=Malbranchea sp. II) | FMR 17906 T = CBS 149936 | Umbría Stream, Rascafría, Madrid (N 40.86011° W 3.90849°) | ON720198 | ON720737 | OP425705 | - |
| Malbranchea irregularis (=Malbranchea sp. III) | FMR 19016 T = CBS 149937 | Llobregat River, Castellar de n’Hug, Barcelona (N 42.24012° E 1.93742°) | ON720191 | ON720730 | OP425710 | OP425719 |
| FMR 19017 | Llobregat River, Castellar de n’Hug, Barcelona (N 42.24012° E 1.93742°) | ON720192 | ON720731 | OP425713 | OP425722 | |
| FMR 19030 | Llobregat River, Castellar de n’Hug, Barcelona (N 42.24012° E 1.93742°) | ON720193 | ON720732 | OP425712 | OP425721 | |
| FMR 19015 | Llobregat River, Balsareny, Barcelona (N 41.87818° E 1.88900°) | ON720194 | ON720733 | OP425711 | OP425720 | |
| Malbranchea ostraviensis | FMR 18693 | Ter River, Roda de Ter, Barcelona (N 41.96974° E 2.31211°) | ON720199 | ON720738 | OP425707 | OP425716 |
| Malbranchea reticulata | FMR 18696 | Ter River, Roda de Ter, Barcelona (N 41.96974° E 2.31211°) | ON721310 | ON720783 | - | - |
| Malbranchea sinuata (=Malbranchea sp. I) | FMR 18266 T = CBS 149938 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON720195 | ON720734 | OP425704 | OP425714 |
| Malbranchea umbrina | FMR 17899 | Ter River, Sant Joan de les Abadesses, Girona (N 42.24138° E 2.28830°) | ON721306 | ON720779 | - | - |
| FMR 18695 | Ter River, Sant Joan de les Abadesses, Girona (N 42.24138° E 2.28830°) | ON720196 | ON720735 | OP425709 | OP425718 | |
| FMR 18766 | Brezal Stream, Rascafría, Madrid (N 40.86011° W 3.90849°) | ON720197 | ON720736 | OP425708 | OP425717 | |
| Myriodontium keratinophilum | FMR 17624 | Pont d’en Blai Stream, Mallorca, Balears Islands (N 39.76907° E 2.88418°) | ON720220 | ON720759 | - | - |
| FMR 18244 | Segre River, Aitona, Lleida (N 41.48444° E 0.46446°) | ON720222 | ON720761 | - | - | |
| FMR 18247 | Segre River, Aitona, Lleida (N 41.48444° E 0.46446°) | ON721309 | ON720782 | - | - | |
| FMR 18248 | Segre River, Aitona, Lleida (N 41.48444° E 0.46446°) | ON720215 | ON720754 | - | - | |
| FMR 18245 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON720213 | ON720752 | - | - | |
| FMR 18246 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON720221 | ON720760 | - | - | |
| FMR 18251 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON720217 | ON720756 | - | - | |
| FMR 18258 | Segre River, La Granja d’Escarp, Lleida (N 41.42754° E 0.35020°) | ON720219 | ON720758 | - | - | |
| FMR 18249 | Segre River, Camarassa, Lleida (N 41.89834° E 0.88110°) | ON720216 | ON720755 | - | - | |
| FMR 18250 | Segre River, Camarassa, Lleida (N 41.89834° E 0.88110°) | ON720218 | ON720757 | - | - | |
| FMR 18257 | Segre River, Camarassa, Lleida (N 41.89834° E 0.88110°) | ON720214 | ON720753 | - | - | |
| FMR 19031 | Llobregat River, Guardiola del Berguedà, Barcelona (N 42.24012°E 1.93742°) | OP070725 | OP077087 | - | - | |
| Neoarthropsis hispanica (≡Arthropsis hispanica) | FMR 18694 | Ter River, Les Masies de Voltregà, Barcelona (N 42.02951° E 2.25359°) | ON720234 | ON720773 | - | - |
| FMR 19034 | Llobregat River, Balsareny, Barcelona (N 41.87818° E 1.88900°) | ON720232 | ON720771 | - | - | |
| FMR 19035 | Llobregat River, Balsareny, Barcelona (N 41.87818° E 1.88900°) | ON720233 | ON720772 | - | - | |
| Neoarthropsis sexualis | FMR 19025T = CBS 149940 | Llobregat River, Balsareny, Barcelona (N 41.87818° E 1.88900°) | ON720235 | ON720774 | - | - |
| Pseudoamaurascopsis spiralis | FMR 19014T = CBS 149941 | Llobregat River, Balsareny, Barcelona (N 41.87818° E 1.88900°) | ON720237 | ON720776 | ||
1 CBS: culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; FMR: Facultat de Medicina i Ciències de la Salut, Reus, Spain; the taxonomic novelties proposed are in bold; T indicates ex-type strains.
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MDPI and ACS Style
Torres-Garcia, D.; Gené, J.; García, D.; Cano-Lira, J.F. Insights into Some Onygenalean Fungi from Freshwater Sediments in Spain and Description of Novel Taxa. J. Fungi 2023, 9, 1129. https://doi.org/10.3390/jof9121129
AMA Style
Torres-Garcia D, Gené J, García D, Cano-Lira JF. Insights into Some Onygenalean Fungi from Freshwater Sediments in Spain and Description of Novel Taxa. Journal of Fungi. 2023; 9(12):1129. https://doi.org/10.3390/jof9121129
Chicago/Turabian StyleTorres-Garcia, Daniel, Josepa Gené, Dania García, and Jose F. Cano-Lira. 2023. "Insights into Some Onygenalean Fungi from Freshwater Sediments in Spain and Description of Novel Taxa" Journal of Fungi 9, no. 12: 1129. https://doi.org/10.3390/jof9121129
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