Novel Freshwater Ascomycetes from Spain

Freshwater ascomycetes are a group of fungi of great ecological importance because they are involved in decomposition processes and the recycling of organic matter in aquatic ecosystems. The taxonomy of these fungi is complex, with representatives in several orders of the phylum Ascomycota. In the present study, we collected ninety-two samples of plant debris submerged in freshwater in different locations in Spain. The plant specimens were placed in wet chambers and developed several fungi that were later isolated in pure culture. A main phylogenetic tree using the nucleotide sequences of D1–D2 domains of the 28S nrRNA gene (LSU) was built to show the taxonomic placement of all our fungal strains, and, later, individual phylogenies for the different families were built using single or concatenated nucleotide sequences of the most suitable molecular markers. As a result, we found a new species of Amniculicola that produces a coelomycetous asexual state, a new species of Elongatopedicellata that produces an asexual state, a new species of Neovaginatispora that forms both sexual and asexual states in vitro, and the sexual states of two species of Pyrenochaetopsis, none of which have been reported before for these genera. In addition, we describe a new species of Pilidium characterized by the production of copper-colored globose conidiomata, and of Pseudosigmoidea, which produces well-developed conidiophores.


Introduction
The phylum Ascomycota is a monophyletic group of fungi that includes several taxa, commonly from freshwater habitats where they complete a part or the whole of their life cycle [1,2]. They decompose substrates such as stems, rotten wood, and decaying leaves, which then fall into bodies of water adjacent to vegetation [3]. These fungi produce a rich array of enzymes that degrade cellulose, hemicellulose, and lignin, among other natural polymers present in plant tissues. They not only provide assimilable nutrients for themselves, but also for other organisms in the same ecological niches [4]. Freshwater habitats are characterized by having a balance of organic matter controlled by the surface, location, and characteristics of the watershed [5] and can be described as lotic or lentic. Lotic environments are aquatic ecosystems with a constant flow of water (rivers, streams, springs, etc.). Lentic environments, by contrast, lack a constant flow of water (lakes, ponds, swamps, etc.) [6].
Aquatic fungi are classified into various ecological groups according to their level of adaptation and activity, and their dependence on aquatic environments [7]. Resident or native fungi are those fully adapted to an aquatic lifestyle and show morphological and physiological adaptations; most of them are also capable of sporulating below the water surface [7]. Periodic immigrant or amphibian fungi inhabit aquatic environments during parts of their life cycle and have the ability to produce spores on the land and in the water [7,8]. Versatile or facultative aquatic immigrant fungi are poorly adapted to water environments and do not sporulate below the water surface. Finally, transient fungi are those not at all adapted to aquatic environments, and are unable to sporulate or, consequently, colonize a new substrate [7].
In recent years, knowledge of the taxonomy and phylogeny of these fungi has been increasing thanks to the use of molecular techniques that mostly use PCR-based amplification and sequencing of different sorts of genes [9][10][11]. To date, ca. 3000 species of ascomycetes have been described from freshwater habitats [12]. Freshwater ascomycetes are represented in several orders and families scattered across four main classes: the Dothideomycetes (677 species), the Eurotiomycetes (276 species), the Leotiomycetes (260 species), and the Sordariomycetes (823 species) [12].
The hydrography in Spain is peculiar and inconsistent due to the richness and diversity of waterways, ecosystems, and landscapes (https://hispagua.cedex.es/en/datos/ hidrografia#1; accessed on 3 April 2022). There are four drainage areas in Spain: North, South, East, and West. Apart from the large northern rivers, the Duero and the Ebro, many have very low water flow because of low rainfall. Their profiles vary from the fast rivers in the Cantabrian slope and the Pyrenees to the slow rivers of the central plateau. Except for the Ebro, the rivers of the Atlantic slope are the longest [13]. Spain is a country where the hyphomycetes have been studied for decades, but only in recent years have the sexual states of freshwater ascomycetes and their coelomycetous asexual states begun to be investigated with any great frequency [14][15][16]. The present study describes the phenotype and taxonomy of several new species of the Ascomycota found in various freshwater habitats in Spain, all of them isolated from submerged plant debris. Their characterization and placement were achievable through the sequencing of several phylogenetically informative markers.

Samples Collection and Fungal Isolation
Ninety-two samples of plant material, consisting of small branches, leaves, and bark, were collected from different lotic environments in Spain ( Figure 1): 50 from Cascadas del Huéznar (Cazalla de la Sierra, Sevilla province, Spain); 17 from the Riaza river (near Riaza, Segovia province, Spain); 22 from de les Hortes river (on the outskirts of Capafonts, Tarragona province, Spain); three from Clot de la Mare de Déu (Burriana, Castellón province, Spain). Cascadas del Huéznar (37.993824, −5.668985) forms a part of the course of the Rivera del Huéznar river in the Sierra Norte de Sevilla natural park. The sampling location was at an altitude of around 600 m.a.s.l. It has a Csa climate (hot-summer Mediterranean climate) according to the Köppen-Geiger climate classification [17], the average annual temperature is 16 • C, and the average annual rainfall is 540 mm (https://es.climate-data.org/ europe/espana/andalucia/san-nicolas-del-puerto-828536/; accessed on 3 April 2022). Impressive travertine formations stand out in this waterfall and there is an abundance of riverside vegetation that forms a dense gallery forest of alders, ash trees, elms, and willows (https://www.juntadeandalucia.es/medioambiente/portal/web/ventanadelvisitante/ detalle-buscador-mapa/-/asset_publisher/Jlbxh2qB3NwR/content/cascadas-del-huesna-2/ 255035; accessed on 3 April 2022).
The Riaza river (41.28368, −3.47187) also has a riverside forest with an abundance of alders, ash trees, elms, and poplars. The sampling location was at around 1190 m.a.s.l. It has a Cfb climate (temperate oceanic climate without a dry season), an average annual temperature of 10.8 • C, and a total annual rainfall of around 690 mm (https://es.climatedata.org/europe/espana/castilla-y-leon/riaza-188771/; accessed on 3 April 2022). Soils are acidic, with a pH of around 5, conditioned by the presence of siliceous materials [18].
The de les Hortes river (41.28664, 1.04033) runs on a calcareous soil and is surrounded by a forest of boxwoods of considerable dimensions, holm oaks, pines, and poplars, among other trees and shrubs (http://www.valldecapafonts.com/capafonts_racons_llodriga.html; accessed on 3 April 2022). The sampling location has a Csa climate, an average annual temperature of 13 • C, and an average annual rainfall of 525 mm (https://es.climate-data. org/europe/espana/cataluna/capafonts-662465/; accessed on 3 April 2022).
Samples were placed into self-sealing sterile plastic bags, closed, and transported to the laboratory where they were stored at room temperature (20-25 • C) for four-seven days. The plant debris was rinsed twice with 500 mL sterile tap water, placed into 15 cm diameter disposable Petri dishes lined inside with two layers of filter paper moistened with sterile water (1 mL of a solution of 20 mg Dieldrin ® in 20 mL of dimethyl-ketone/L of water), incubated at room temperature (20-25 • C), and examined periodically with a stereo microscope for between four weeks and two months, until reproductive (asexual or/and sexual) structure development. A number of single fungal propagules (ascospores or conidia) were transferred using sterile disposable needles to 55 mm diameter disposable Petri dishes containing oatmeal agar (OA; 30 g of filtered oat flakes, 15 g agar-agar, 1 L tap water; [19], and then incubated at 25 ± 1 • C. This was repeated until a pure culture was obtained, and the strains of interest were deposited in the culture collection at the Faculty of Medicine (FMR, Reus, Spain) in three different ways: slant cultures on OA and potato dextrose agar (PDA; Pronadisa, Madrid, Spain) under a layer of liquid vaseline; OA blocks (where the strain had grown) immersed in sterile water in caramel-colored self-sealing vials; and lyophilized. Holotypes and cultures ex-type of the novel fungal taxa were deposited in the Westerdijk Fungal Biodiversity Institute (CBS; Utrecht, The Netherlands). The names and descriptions were deposited in MycoBank.

Phenotypic Study
Fungal strains were characterized macroscopically following incubation on OA, PDA, and MEA (malt extract agar; 40 g of malt extract, 15 g of agar-agar, 1 L distilled water) at 25 + 1 • C for 14 days, annotating the colony diameter and reporting the texture, topography, margins, presence of diffusible pigments and exudates. Colony color (surface and reverse) was described according to Kornerup and Wanscher [20]. The cardinal temperatures of growth were determined on PDA after 7 days of incubation in darkness, ranging from 5 to 35 • C, at 5 • C intervals. For each fungal structure of taxonomic interest, the measurements of ten specimens were carried out on Shear's mounting medium (3 g potassium acetate, 60 mL glycerol, 90 mL ethanol 95%, and 150 mL distilled water [21]). Material was taken from the natural substrate and/or from the fungal strains grown on OA in the same conditions as for colony characterization. Histological sections of the sexual or asexual reproductive bodies were made freehand with the help of a sterile 0.3 × 13 mm needle and a 15 mm wide Laseredge Scalpel. Photomicrographs were taken using a Zeiss Axio-Imager M1 microscope (Oberkochen, Germany) with a DeltaPix Infinity X digital camera using Nomarski differential interference contrast.

DNA Extraction, Amplification, and Sequencing
To extract genomic DNA, the mycelium of axenic cultures that had been grown in PDA for 7 days at 25 ± 1 • C in the dark was scraped with a sterile scalpel. The DNA was then extracted using the FastaDNA kit protocol (Bio101, Vista, CA, USA) with a Fast Prep FP120 instrument (Thermo Savant, Holbrook, NY, USA) according to the manufacturer's protocol. DNA was quantified using Nanodrop 2000 (Thermo Scientific, Madrid, Spain). The following loci were amplified and sequenced: internal transcribed spacer region (ITS), with the primer pair ITS5 and ITS4 [22]; a fragment of the 28S nrRNA gene (LSU) with the primer pair LR0R [23] and LR5 [24]; a fragment of the RNA polymerase II subunit 2 gene (rpb2) with RPB2-5F2 [25] and fRPB2-7cR [26]; a fragment of the beta-tubulin gene (tub2) with the primers TUB2Fw and TUB4Rd [27]; and translation elongation factor 1-alpha (tef1) with the primers EF1-983F and EF1-2218R [28]. Amplicons were sequenced in both directions with the same primer pair used for amplification at Macrogen Spain (Macrogen Inc., Madrid, Spain). The sequences obtained were edited and contigs were assembled using SeqMan software v. 7.0.0 (DNAStar Lasergene, Madison, WI, USA). Sequences generated in this study were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank/; accessed on 7 Juny 2022) (Table S1).

Phylogenetic Analysis
Each sequence generated in this study was subjected to an individual BLAST search to verify its identity in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast. cgi; accessed on 11 October 2021). Fungal strains were identified at species level when the nucleotide sequences of selected loci displayed a level of identity ≥ 98% with those of the ex-type or reference strains in the database. The loci used for this purpose were: ITS for Pilidium spp. and Pseudosigmoidea spp.; LSU for Elongatopendicellata spp.; rpb2 for Neovaginatispora spp. and Pyrenochaetopsis spp.; and tef-1 for Amniculicola spp. Each locus was aligned with the MEGA (Molecular Evolutionary Genetics Analysis) software v. 7.0 [29], using the ClustalW algorithm [30] and refined with MUSCLE [31] or manually, if necessary, using the same software. Phylogeny was analyzed by maximum-likelihood (ML) and Bayesian inference (BI) with RAxML v. 8.2.12 [32] software on the online Cipres Science gateway portal [33] and MrBayes v.3.2.6 [34], respectively.
A main LSU phylogenetic tree was built to display the taxonomic placement of all our fungal strains at family and genus level, using nucleotide sequences of representatives of the families Amniculicolaceae, Cucurbitariaceae, Leptosphaeriaceae, Lophiostomataceae, Neopyrenochaetaceae, Pleosporaceae, Pseudopyrenochaetaceae, Pyrenochaetopsidaceae, Roussoellaceae, Sympoventuriaceae, Torulaceae and Venturiaceae (class Dothideomycetes), and Chaetomellaceae (class Leotiomycetes), including those sequences of our strains.
The phylogenetic tree for the family Amniculicolaceae was built using the concatenated nucleotide sequences of the ITS region and a fragment of the tef1 gene. For the family Roussoellaceae, only the LSU was employed. For the family Lophiostomataceae, three concatenated markers-ITS, rpb2, and tef1-were used. For members of the families Chaetomellaceae and Sympoventuriaceae, the concatenated markers employed were LSU and ITS; and for the phylogenetic analysis of the family Pyrenochaetopsidaceae, the concatenated sequences of ITS, rpb2, and tub2 were employed.
The best nucleotide substitution model for BI analysis was estimated using the jMod-elTest program [35]. The best model used for the main LSU phylogenetic tree was the symmetrical model with proportion of invariable sites and gamma distribution (SYM + I+G). The best model used for the Amniculicolaceae was SYM + I+G for ITS and the general time reversible with gamma distribution (GTR + G) for LSU and tef1. The best model used for the Roussoellaceae was the general time reversible with proportion of invariable sites and gamma distribution (GTR + I+G) for LSU. For the Lophiostomataceae, the best model was Kimura 2-parameter with proportion of invariable sites and gamma distribution (K80 + I+G) for ITS and rpb2; and GTR + G for tef1. The best model substitution for the Chaetomellaceae was SYM + I+G for ITS and K80 + I+G for LSU. For the Sympoventuriaceae it was K80 + I for ITS and LSU. For the Pyrenochaetopsidaceae, the Kimura 2-parameter with proportion of invariable sites (K80 + I) was used for ITS, the SYM + I+G for rpb2, and the Hasegawa-Kishino-Yano with Gamma distribution (HKY + G) for tub2.
The parameters used in Bayesian analysis were two simultaneous runs of 5,000,000 generations. The 50% majority rule consensus tree and posterior probability values (PP) were calculated after discarding the first 25% of the resulting trees. A PP value ≥ 0.95 was considered significant [36]. For ML analysis, support for internal branches was assessed by 1000 ML bootstrapped pseudoreplicates. Bootstrap support value (BS) ≥ 70% was considered significant. Alignments and trees were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2: S29604; accessed on 7 Juny 2022). The novel nomenclature and novel taxonomic descriptions were deposited in MycoBank (http://www.mycobank.org; accessed on 7 Juny 2022).

Phylogeny
The LSU phylogenetic tree for the members of the Dothideomycetes and the Leotiomycetes classes comprised 91 ingroups of species with a total of 776 characters including gaps, from which 266 bp were parsimony-informative ( Figure 2). The ML analysis was congruent with that obtained by BI analysis, displaying trees with similar topologies. For the BI analysis, a total of 1574 trees were sampled after the burn-in with a stop value of 0.01. The phylogenetic tree ( Figure 2) shows two main clades, the first corresponding to the Dothideomycetes class (100% BS/1 PP), in which six of our strains were located, and the second to the Leotiomycetes class (90% BS/1 PP). Two of our strains (FMR 18801 and FMR 18913) were located in the clade corresponding to the genus Pyrenochaetopsis (100% BS/1 PP), close to the species P. confluens and P. decipiens. FMR 17834 was located within the Roussoellaceae family (90% BS/1 PP), together with Elongatopedicellata lignicola, the type species of the genus. Within the clade of the genus Neovaginatispora (100% BS/1 PP), the FMR 18914 strain closely related phylogenetically to N. clematidis and N. fuckelii was located. FMR 17416 was located within the Sympoventuriaceae family (100% BS/1 PP), in the terminal branch (84% BS/0.97 PP), together with Pseudosigmoidea spp. In the respective clade of the Amniculicolaceae family (100% BS/1 PP), the FMR 17946 strain was located, together with species of the Amniculicola, Fouskomenomyces, and Vargamyces genera. FMR 17839 was located in Chaetomellaceae (90% BS/1 PP), in the terminal branch (unsupported), together with species of the genus Pilidium.   To clarify the phylogenetic relationship of our isolate FMR 17946 with the species of Amniculicola, a separate phylogenetic analysis was carried out. The final concatenated nucleotide sequences comprised 20 ingroups of species with a total of 1117 characters including gaps, from which 187 bp were parsimony-informative (113 for ITS and 74 for tef1) (Figure 3). The BI analysis showed a similar tree topology and was congruent with that obtained in the ML analysis. For the BI multi-locus analysis, a total of 1141 trees were sampled after the burn-in with a stop value of 0.01. In the phylogenetic tree, the Amniculicolaceae formed a full-supported clade (100% BS/1 PP) including all genera accepted in the family: Amniculicola, Fouskomenomyces, Murispora, and Vargamyces. Our strain FMR 17946 was placed in the same terminal clade as A. guttulata. The phylogenetic analysis of the members of the Roussoellaceae included partial LSU nucleotide sequences from 18 species with a total of 845 characters including gaps, from which 80 bp were parsimony-informative. The topology of the trees inferred by the two phylogenetic methods (ML and BI) were basically the same, with minor differences in statistically supported groupings. For the BI analysis, a total of 385 trees were sampled after the burn-stop value of 0.01. In the phylogenetic tree ( Figure 4), our strain FMR 17834 was placed in the same fully-supported terminal clade as the type species of the genus Elongatopedicellata (E. lignicola).
For the Lophiostomataceae, the combined alignment of the three loci datasets (ITS-rpb2 and tub2) encompassed 15 ingroups of species with a total of 2265 characters including gaps, of which 609 bp were parsimony-informative sites (178 for ITS, 298 for rpb2, 129 for tub2). Phylogenies obtained by ML and BI showed a topological congruence. For the BI multi-locus analysis, a total of 402 trees were sampled after the burn-stop value of 0.01. In the Lophiostomataceae (Figure 5), the genus Neovaginatispora (100% BS/1 PP) included the two previously accepted species of the genus plus our strain FMR 18914.
We carried out a combined phylogenetic analysis with ITS and LSU sequences to resolve the taxonomic position of our strain FMR 17839 in Chaetomellaceae. A concatenated dataset from 18 sequences contained a total of 1246 characters, from which 277 bp were parsimony-informative sites (172 for ITS and 105 for LSU). Bayesian inference and ML analyses of the concatenated dataset yielded similar topologies. For the BI multilocus analysis, a total of 134 trees were sampled after the burn-stop value of 0.01. In that phylogenetic tree (Figure 6), the genus Pilidium (100%BS/1PP) included all species with sequences available as well as our strain FMR 17839, which was placed alone in a basal terminal branch.
The phylogenetic analysis of the Sympoventuriaceae included sequences from eight species, with 1325 characters including gaps, from which 161 bp were parsimony-informative sites (93 for ITS and 68 for LSU). The topology of the tree identified by ML analysis was almost identical to that found by the Bayesian analyses. For the BI multi-locus analysis, a total of 29 trees were sampled after the burn-stop value of 0.01. In our phylogenetic tree (Figure 7), the genus Pseudosigmoidea formed a well-supported clade (99% BS/1 PP) and included all accepted species (except the type species P. cranei) plus our strain FMR 17416.
Diagnosis: Up to the present work, the genus Elongatopedicellata was considered to be monospecific. Elongatopedicellata lignicola, its type species was described as producing an asexual state only on wood [37]. In our study, we report a new species, E. aquatica, characterized by the production of an asexual coelomycetous state on the natural substrate as well as in vitro.
Notes: The difference in nucleotides between LSU sequences of E. lignicola and E. aquatica is 27 bp.  Because the asexual state of Neovaginatispora has not been reported and described up to this paper, we have emended the description of this genus as follows: Neovaginatispora A. Hashim., emended by V. Magaña-Dueñas, Cano, and Stchigel.
Diagnosis: Neovaginatispora aquadulcis is the only species of the genus that produces both asexual and sexual morphs in vitro. Furthermore, our species is characterized by the production of ascospores 1-3-septate, unlike the two previously reported species (1-septate).
Notes: Differences between the ITS-LSU nucleotide sequences of P. robusta and the other species of the genus are: from P. alnicola, 28 bp; from P. excentricum, 25 bp; and from P. ibarakiensis, 27 bp.  Because the sexual state of the family has not been reported and described until now, we have emended the description of this family as follows: Sexual state: Ascomata immersed to semi-immersed, brown to dark brown, ostiolate, outer wall of textura angularis. Hamathecium comprising hyaline, septate, filamentous paraphyses. Asci 8-spored, bitunicate, stipitate, cylindrical to clavate. Ascospores 3-6 septate, hyaline, fusiform. Asexual state: Conidiomata pycnidial, pale brown to brown, solitary or confluent. Conidiomata wall of textura angularis, glabrous or setose, subglobose to ovoid, with a non-papillate or papillate ostiolar neck. Conidiogenous cells phialidic, hyaline, discrete or integrated into the septate, acropleurogenous conidiophores. Conidia aseptate, hyaline, smooth, thin-walled, ovoid, cylindrical to allantoid, guttulate. Since the sexual state of the genus Pyrenochaetopsis has not been reported before, below we emended the generic description.
Diagnosis: Similarly to the other species of the genus, P. perfecta produces pycnidia ornamented with brown setae (especially abundant around and near the ostiole) and an abundant amount of hyaline, one-celled small phialoconidia. Pyrenochaetopsis perfecta is easily distinguishable from the other species of the genus because is the only one that produces a sexual state. Unlike its closest species, P. globosa, P. perfecta produces bigger pycnidia (240-380 × 260-300 µm vs. 50-220 × 140-190 µm) covered by abundant long setae (absent in P. globosa).
The genus Amniculicola was introduced by Zhang et al. [43] to accommodate A. lignicola. Until now, six species were accepted in the genus (http://www.indexfungorum.org/ names/Names.asp; accessed on 3 April 2022), and all of them were discovered in freshwater. Most of these species stain the substrate in purple tinges [43,44], with the exceptions of A. aquatica, A. guttulata [10,44], and the new species, A. asexualis. Amniculicola longissima was the only species previously reported as producing an asexual state, characterized by the formation of long curved or sigmoid conidia on short coniodiophores of sympodial development [45]. Amniculicola asexualis is the only species producing a coelomycetous asexual state and lacking a sexual state.
Elongatopedicellata was introduced by Zhang et al. [37] to accommodate E. lignicola. This genus only comprises the type species, isolated from a dead tree branch in Mae Chang Hot Spring, Thailand. Elongatopedicellata lignicola produces papillate ascomata, filiform pseudoparaphyses, bitunicate, fissitunicate, and fusiform-clavate asci, and hyaline, 1-septate, fusiform ascospores constricted at the septum. The authors did not report the production of any fertile structure in pure culture [37]. The new species, E. aquatica, is the only one from aquatic habitats that is characterized by the production of pyrenochaeta-like or pyrenochaetopsis-like conidiomata, an undescribed feature for the genus.
Hashimoto et al. [46] performed a phylogenetic study of the Lophiostomataceae and introduced the genus Neovaginatispora to accommodate N. fuckelii. This genus differs from Vaginatispora in having thinner, sub-carbonaceous peridium of uniform thickness [45,46]. Currently, Neovaginatispora comprises two species, N. clematidis and N. fuckelii, both isolated from decaying plants (Clematis viticella and oak tree, and Mangifera indica, respectively) [46][47][48]. Neovaginatispora fuckelii has also been reported from wood submerged in freshwater [49]. The asexual state in both species is unknown. Neovaginatispora aquadulcis, recovered from plant debris in freshwater produce both sexual and asexual states in vitro. The coelomycetous asexual state in N. aquadulcis is a novel feature for that genus.
Pilidium is a genus introduced by Kunze and Schmidt [50]. Species of Pilidium are commonly found as plant-associated fungi or isolated from soil, and they are known to produce two kinds of conidiomata: sporodochia and pycnidia [51]. Until recent years, the genus included P. acerinum, P. eucalyptorum, P. lythri, P. pseudoconcavum, and P. septatum [38,39,42]. Subsequently, Crous et al. [40,41] introduced P. anglicum and P. novae-zelandie. From all known species, only P. septatum has been reported in freshwater environments [42]. The new species, Pilidium cuprescens, also found in freshwater, is easily distinguished from other species of the genus by the production of closed, copper-colored globose conidiomata.
The genus Pseudosigmoidea was created by Ando and Nakamura [52] to place the fungal strain 85B-65 (= ATCC 16660) isolated from freshwater in Maryland (USA). Originally, this fungus was identified as Sigmoidea (≡ Flagellospora) prolifera. Later, Ando and Nakamura [52] distinguished P. cranei (the type species of the new genus) from S. prolifera because the former produces enteroblastic conidia from polyphialides, and the second one holoblastic conidia on a sympodialy proliferative conidiogenous cell. Jones et al. [53], in a phylogenetic study based on the analysis of the nucleotide sequences of the small subunit (SSU) of the ribosomal DNA, confirmed P. cranei as a distinct taxon from S. prolifera, despite both fungi being placed in the family Phaeosphaeriaceae. Later, Diene et al. [54] described the second species, P. ibarakiensis, from forest soil in Honshu (Japan). Despite the SSU sequences displaying a high similarity (99 %) with those of Troposporella fumosa, Helicoma monilipes, and H. olivaceum, the fungus was placed as a new species of Pseudosigmoidea because it showed a high alignment score (100%) and 97 % similarity with the SSU sequence of P. cranei, but also based on morphological similarities (P. cranei produce long subcylindrical to obclavate conidia and P. ibarakiensis scolecoid conidia, whereas these spores are helical in T. fumosa, H. monilipes, and H. olivaceum). However, the authors [54] did not build a phylogenetic tree to show the relationships between these taxa. Pseudosigmoidea alnicola, isolated from leaf litter near Berlin (Germany), was the third species of the genus. Despite a phylogenetic tree being built, only the ITS and LSU nucleotide sequences of the new species were used, and consequently, P. alnicola falls into the same terminal clade as T. fumosa, Scolecobasidium excentricum, Sympoventuria capensis, and Sympoventuria melaleucae. However, the authors mentioned that the ITS sequence of P. alnicola displays 95 % similarity with that of P. ibarakiensis [55]. The morphological similarity between the reproductive structures of P. alnicola, P. cranei, and P. ibarakiensis was probably responsible for the final placement at genus level. The fourth species, P. excentrica [56], was originally erected as Scolecobasidium excentricum, being isolated from leaf litter in Santiago de las Vegas (Cuba) [57]. The morphology of P. excentrica differs from the other species of the genus because it produces larger conidiophores bearing sympodially proliferating conidiogenous cells (described as polyphialidic in the other species) and because it produces shorter, cylindrical to allantoid conidia with rounded ends (scolecoid in the remaining species). The morphology of our new species, P. robusta, is similar to P. excentrica, because it produces short cylindrical to obclavate conidia and conidiophores bearing integrated sympodially proliferating conidiogenous cells. However, P. robusta produces longer and narrower conidia than P. excentrica (8-65 × 2.5-3 µm vs. 14-22 × 3-5 µm), and in the apical part of the conidia, it can develop a conidiogenous locus (feature not reported for P. excentrica).
Pyrenochaetopsis was introduced by De Gruyter et al. [58] for several phoma-like species placed together in the same clade in a phylogenetic study. These authors introduced P. decipiens, P. indica, P. leptospora, P. microspora, and P. pratorum to that genus. Valenzuela-Lopez et al. [59], thanks to a multilocus phylogenetic analysis, transferred Pyrenochaetopsis from the family Cucurbitariaceae to the new family Pyrenochaetopsidae. The genus contains 19 species (http://www.indexfungorum.org/names/Names.asp; accessed on 3 April 2022). The members of this genus have been isolated from terrestrial, marine, and freshwater environments, and also from human clinical specimens (superficial tissue, bronchial washing, and blood) [16,[58][59][60][61]. Up to now, only the asexual (coelomycetous) reproductive state has been described for the members of the genus and the family. In the present study, we describe two new species of Pyrenochaetopsis. Pyrenochaetopsis perfecta is the first one to produce a sexual reproductive state, characterized by the production of perithecial ascomata, cylindrical to clavate bitunicate asci and hyaline, 3-6-septate fusiform ascospores. In comparison with the second new species, P. globosa, P. perfecta produces bigger pycnidia covered by abundant long setae (structures absent in P. globosa).