Five Novel Taxa from Freshwater Habitats and New Taxonomic Insights of Pleurotheciales and Savoryellomycetidae

Pleurotheciales is the largest order in Savoryellomycetidae with a large proportion of species known from freshwater habitats. In order to investigate the phylogenetic relationships of taxa within Pleurotheciales and contribute to their diversity, submerged wood was collected from freshwater habitats in China (Yunnan Province) and Thailand. Two dematiaceous, sporodochial hyphomycetes and one annulatascales-like ascomycete with unusual morphology as compared to extant ones were discovered. They were subjected to DNA-based phylogenetic analyses and the results revealed three distinct lineages in Savoryellomycetidae. This morpho-phylo taxonomic study supports the establishment of five novel taxa including two novel genera, Obliquifusoideum and Saprodesmium, and three novel species, Coleodictyospora muriformis, Obliquifusoideum guttulatum and Saprodesmium dematiosporum. Coleodictyospora muriformis and S. dematiosporum are placed in Pleurotheciales, while O. guttulatum is referred to Savoryellomycetidae genera incertae sedis. The phylogenetic relationships are also presented for Coleodictyospora and Pseudocoleodictyospora, which raises an intriguing taxonomic issue. These two genera are positioned in two different classes, viz Sordariomycetes and Dothideomycetes, although they are quite similar except for the presence of a conidial sheath. This study expands our knowledge of the fungal diversity of freshwater fungi, and also indicates that Pleurotheciales species are mostly found in freshwater habitats.


Introduction
Savoryellomycetidae currently accommodates four orders, Conioscyphales, Fuscosporellales, Pleurotheciales and Savoryellales. This is based on evidence from phylogenetic analyses and divergence time studies with the order having a stem age estimated as 268 MYA [1]. The four orders clustered as a robust clade in all studies [1][2][3]. Pleurotheciales, with a single-family Pleurotheciaceae [4], is the largest order in Savoryellomycetidae.
Taxa of Pleurotheciaceae have perithecial ascomata with asexual dematiaceous hyphomycetous stages. Coelomyceteous asexual morphs have not been reported in the family. Pleurotheciaceae is an assemblage of genera representing a highly diverse morphology, especially in the asexual morphs. Pleurotheciella and Pleurothecium (type) generally have macronematous, unbranched conidiophores, holoblastic, sympodially proliferating conidiogenous cells with a conspicuous rachis of denticles, and hyaline, septate conidia [4,9,10,12,13]. Anapleurothecium has macronematous, unbranched conidiophores, sympodial, denticulate conidiogenous cells which are the cases of Pleurotheciella and Pleurothecium, but it has botuliform to cylindrical and brown conidia with a paler basal cell [7]. Phaeoisaria has synnematous conidiophores with tiny aseptate conidia [13]. Phragmocephala also has synnematous conidiophores, but the conidia are relatively large, ellipsoidal to subglobose, with dark brown to black central cells and paler polar cells [14]. Sterigmatobotrys are distinct in the family by their well-defined stipe and a complex penicillate conidiophore head consisting of series of penicillate branches [6]. While some other genera lack conspicuous macronematous conidiophores, and the conidia directly arise from the hyphae on the host substrate or from micronematous, subhyaline conidiophores, such as Neomonodictys with subglobose to globose, muriform conidia [8] and Helicoascotaiwania with helicoid conidia [2,15].
The diversity of morphology is also reflected in some sexual morphs of Pleurotheciaceae. The genera Adelosphaeria, Melanotrigonum, Pleurotheciella and Pleurothecium generally have superficial ascomata with a short papilla, narrowly or broadly clavate asci with a distinct, refractive apical ring and ellipsoidal-fusiform, septate ascospores [3,4,9], while Phaeoisaria has immersed ascomata with a quite long neck, cylindrical asci and filiform, multiseptate ascospores. Helicoascotaiwania is easily distinguished in the family in having generally immersed ascomata lying horizontally or obliquely to the host substrate and fusiform, versicolorous ascospores with darker central cells and paler polar cells [2,3].
The asexual-sexual morph connections were investigated based on cultural studies with a combination of molecular data. Some hyphomycetes were linked as the life cycle of known sexual morphs. The asexual morph of Pleurothecium recurvatum was first reported from the artificial medium (WA) inoculated by an ascomycetous species Carpoligna pleurothecii [16]. Réblová et al. [9] also found the asexual morphs from another medium (PCA) inoculated by ascomycetous species Pleurotheciella rivularia and Pleurothecium semifecundum. With DNA sequence data, Luo et al. [10] linked the asexual-sexual morph of Pleurotheciella fusiformis based on two specimens collected from Erhai Lake, Yunnan, China.
Luo et al. [6] used multi-locus analysis to first report a sexual morph in Phaeoisaria, namely Ph. filiformis, which was characterized by immersed ascomata with a long, cylindrical neck, and cylindrical asci containing hyaline and filiform ascospores. Although the morphological traits associate Ph. filiformis as allied with Ceratosphaeria and Ophioceras in Magnaporthales, phylogenetic analysis placed Ph. filiformis in Pleurotheciales. Neither conidia nor conidiomatal structures were observed from the culture of Ph. filiformis [6].
The main objectives of this study were to revise the taxon diversity within Pleurotheciales, report on novel taxa and provide new insights into the systematics of Savoryellomycetidae. Two specimens of dematiaceous hyphomycetes were preliminarily identified as Pleurotheciales species with micronematous conidiophores, holoblastic conidiogenous cells and dark muriform conidia, but the morphologies were rather unusual as compared to other members of the family. One specimen resembled annulatascaceae-like taxa in Diaporthomycetidae but is similar to the taxa of Pleurotheciaceae in Savoryellomycetidae. In order to clarify the placement of these specimens, a multi-locus analysis of a concatenated nuc 28S rDNA (LSU), nuc18S rDNA (SSU), internal transcribed spacer (ITS) and second-largest subunit of RNA polymerase II (rpb2) dataset were performed, and phylogenetic relationships inferred.

Herbarium Material, Isolation and Morphology
Decayed woody twigs and branches submerged in freshwater streams in forests were randomly collected in Yunnan Province, China, as well as Satun and Songkhla provinces in Thailand where all places are in the Greater Mekong Subregion. Specimens were placed in zip-lock plastic bags containing moistened cotton and taken to the laboratory. Sediment on separated specimens was washed off with tap water and incubated in plastic boxes lined with moistened tissue paper at room temperature (20-25 • C) for 1-2 weeks. The ascomata and sporodochia developed on the specimens were examined with a Nikon SMZ-171 dissecting microscope. Fungal structures were captured with a Nikon ECLIPSE Ni compound microscope fitted with a Canon EOS 750D digital camera. Single spore isolations were made from ascospore or conidium on potato dextrose agar (PDA, Shanghai Bio-way technology Co., Ltd., Shanghai, China) at room temperature (20-25 • C). All morphological approaches used herein were modified from Chomnunti et al. [17] and Senanayake et al. [18]. Tarosoft (R) Image Frame Work program was used to measure the fungal structures. Images were processed with Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA). Herbarium specimens (dry wood with fungal materials) were deposited in the herbarium of Mae Fah Luang University (MFLU), Chiang Rai, Thailand and herbarium of Cryptogams, Kunming Institute of Botany Academia Sinica (HKAS), Kunming, China. Living cultures were deposited in the Mae Fah Luang University Culture Collection (MFLUCC) and Kunming Institute of Botany Culture Collection (KUMCC). The novel taxa were registered in the databases Facesoffungi (http://www.facesoffungi.org, accessed on 10 June 2021) [19] and Index Fungorum (http://www.indexfungorum.org/ names/names.asp, accessed on 21 June 2021).

DNA Extraction, PCR Amplification and Sequencing
Fungal mycelia were scraped from the colonies on PDA. The Biospin Fungus Genomic DNA Extraction Kit (Bioer Technology Co., Hangzhou, China) was used to extract total genomic DNA. The polymerase chain reaction (PCR) technique was utilized for the amplification of target DNA fragments. The primer pairs LR0R/LR5, NS1/NS4, ITS5/ITS4 and fRPB2-5F/fRPB2-7cR were used to amplify LSU, SSU, ITS and rpb2 [20][21][22]. The amplifications were carried out as detailed in Dong et al. [23]. The PCR thermal cycle program for the amplification of LSU, SSU and ITS was provided as initially 94 • C for 3 min, followed by 35 cycles of denaturation at 94 • C for 30 s, annealing at 55 • C for 50 s, elongation at 72 • C for 90 s and a final extension at 72 • C for 10 min. The annealing was adjusted to 52 • C for rpb2. PCR products were checked on 1% agarose electrophoresis gels stained with Gel Red. The sequencing reactions were carried out by Shanghai Sangon Biological Engineering Technology and Services Co., Shanghai, China.

Sequence Selection and Phylogenetic Analyses Construction
The qualities of generated sequences were initially checked with Finch TV v. 1.4.0 and each gene was subjected to a BlastN search in NCBI's GenBank to assess the confidence level. Phylogenetic placements of the unidentified fungi were resolved by analyzing four different datasets: (1) two multi-locus datasets of a concatenated LSU, SSU, ITS and rpb2 sequences; (2) two separate individual LSU and ITS datasets. The first multi-locus dataset was analysed to infer the phylogenetic positions of unidentified fungi within the Savoryellomycetidae along with sequences deposited from recent relevant publications [3,6,24]. In the second multi-locus dataset, we included the other subclasses in Sordariomycetes to infer the subclass status of an unidentified genus which could not be confirmed in any orders in the subclass Savoryellomycetidae. Besides, Pseudocoleodictyospora and its related taxa were also included to show the phylogenetic relationships between Coleodictyospora and Pseudocoleodictyospora. The individual LSU and ITS phylogenetic analyses were utilized to auxiliarily assess the phylogenetic relationships of fungi in Savoryellomycetidae. All sequences used in this study were listed in Table 1.
The ex-type cultures are indicated using " T " after strain numbers and newly generated sequences are indicated in bold.

Maximum Likelihood Analyses
Each dataset was aligned with MAFFT v. 7.409 online version [25] and manually verified with BioEdit v. 7.2.5 Biological Sequence Alignment Editor (Ibis BioSciences, Carlsbad, CA, USA), and then concatenated with Mesquite v. 3.11. The maximum likelihood (ML) analyses were performed with RAxML-HPC v. 8 on XSEDE in CIPRES Science Gateway [26,27], with the following changes from the default settings: maximum hours to run: 5; model for bootstrapping phase: GTRGAMMA; analysis type: rapid bootstrap analysis/search for best-scoring ML tree (-f a); bootstrapping type: rapid bootstrapping (-x); bootstrap iterations: 1000 (the maximum value allowed).

Bayesian Inference Analyses
The Bayesian inference (BI) analyses were performed with MrBayes on XSEDE also in CIPRES Science Gateway [26,27]. In the first analysis of Savoryellomycetidae, the best-fit model was GTR+I+G for LSU, ITS and rpb2, and SYM+I+G for SSU. Six simultaneous Markov chains were run for 965,100 generations and trees were sampled every 100th generation. In total, 9651 trees were sampled and the first 25% of sampled trees representing the burn-in phase of the analyses were discarded and the remaining 7239 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (critical value for the topological convergence diagnostic is 0.01) [28].
In the second analysis, the best-fit model was GTR+I+G for all datasets. Six simultaneous Markov chains were run for 685,100 generations and trees were sampled every 100th generation. In total, 6851 trees were sampled and the first 25% of sampled trees representing the burn-in phase of the analyses were discarded and the remaining 5139 trees were used for calculating posterior probabilities (PP) in the majority rule consensus tree (critical value for the topological convergence diagnostic is 0.01) [28]. Phylogenetic

Phylogenetic Analyses
In the first phylogenetic analysis (Figure 1), the representative homologous sequences of Conioscyphales, Fuscosporellales and Savoryellales and sequences from all genera of Pleurotheciales representing 90 isolates and two outgroup taxa (Doratomyces stemonitis AFTOL-ID 1380 and Microascus trigonosporus AFTOL-ID 914) were included. The matrix had 2205 distinct alignment patterns, with 41.06% of completely undetermined characters and gaps. In the RAxML tree, three distinct independent lineages were identified: (1)    In the second multi-locus phylogenetic analysis (Figure 2), a total of seven subclasses (Diaporthomycetidae, Hypocreomycetidae, Lulworthiomycetidae, Pisorisporiomycetidae, Savoryellomycetidae, Sordariomycetidae and Xylariomycetidae) in Sordariomycetes, as well as Pseudocoleodictyospora and its relatives in Dothideomycetes were included in the dataset, representing 55 isolates and one outgroup taxon (Cheilymenia stercorea AFTOL 148). The matrix had 2068 distinct alignment patterns, with 45.12% of completely undetermined characters and gaps. In the RAxML tree, Coleodictyospora was phylogenetically distant from Pseudocoleodictyospora, although their morphology was quite similar [29]. The relationships of Obliquifusoideum were weak with four orders in Savoryellomycetidae (Figure 1), but it was shown to be a genus in Savoryellomycetidae with strong bootstrap support.  In the second multi-locus phylogenetic analysis (Figure 2), a total of seven subclasses (Diaporthomycetidae, Hypocreomycetidae, Lulworthiomycetidae, Pisorisporiomycetidae, Savoryellomycetidae, Sordariomycetidae and Xylariomycetidae) in Sordariomycetes, as well as Pseudocoleodictyospora and its relatives in Dothideomycetes were included in the dataset, representing 55 isolates and one outgroup taxon (Cheilymenia stercorea AFTOL 148). The matrix had 2068 distinct alignment patterns, with 45.12% of completely undetermined characters and gaps. In the RAxML tree, Coleodictyospora was phylogenetically distant from Pseudocoleodictyospora, although their morphology was quite similar [29]. The relationships of Obliquifusoideum were weak with four orders in Savoryellomycetidae (Figure 1), but it was shown to be a genus in Savoryellomycetidae with strong bootstrap support.

Figure 2.
RAxML tree with taxa from two classes, Dothideomycetes and Sordariomycetes, to show the phylogenetic relationships between Coleodictyospora and Pseudocoleodictyospora. The illustrations of species in Coleodictyospora and Pseudocoleodictyospora are displayed near the generic names. The multi-locus tree is generated from combined LSU, SSU, ITS and rpb2 sequence data. Bootstrap support values for maximum likelihood (the first value) equal to or greater than 60% and Bayesian posterior probabilities (the second value) equal to or greater than 0.95 are placed near the branches as ML BS/BI PP. The asterisk "*" represents bootstrap support values with 100% ML BS and 1.00 BI PP. The tree is rooted to Cheilymenia stercorea AFTOL 148. The ex-type cultures are indicated using " T " after strain numbers and the new species introduced in this study are indicated in bold.
In order to assess the phylogenetic position of Obliquifusoideum in Savoryellomycetidae, we constructed individual LSU and ITS phylogenetic trees (shown as Supplementary   Figure 2. RAxML tree with taxa from two classes, Dothideomycetes and Sordariomycetes, to show the phylogenetic relationships between Coleodictyospora and Pseudocoleodictyospora. The illustrations of species in Coleodictyospora and Pseudocoleodictyospora are displayed near the generic names. The multi-locus tree is generated from combined LSU, SSU, ITS and rpb2 sequence data. Bootstrap support values for maximum likelihood (the first value) equal to or greater than 60% and Bayesian posterior probabilities (the second value) equal to or greater than 0.95 are placed near the branches as ML BS/BI PP. The asterisk "*" represents bootstrap support values with 100% ML BS and 1.00 BI PP. The tree is rooted to Cheilymenia stercorea AFTOL 148. The ex-type cultures are indicated using " T " after strain numbers and the new species introduced in this study are indicated in bold.
In order to assess the phylogenetic position of Obliquifusoideum in Savoryellomycetidae, we constructed individual LSU and ITS phylogenetic trees (shown as Supplementary  Figures S1 and S2) to enable topological comparison with those derived from the multilocus datasets. The matrix of the LSU sequence comprised 92 isolates and had 537 distinct alignment patterns, with 14% of undetermined characters or gaps. The matrix of ITS sequence comprised 70 isolates and had 615 distinct alignment patterns, with 30.9% of undetermined characters or gaps. The phylogenetic position of Obliquifusoideum was different in all RAxML trees: it clustered with Savoryellales in individual LSU tree (Supplementary Figure S1), clustered with Savoryellales and Pleurothecium species of Pleurotheciales in individual ITS tree (Supplementary Figure S2), and clustered with Pleurotheciales in the multi-locus phylogenetic tree ( Figure 1); but without bootstrap support in all trees.  [31] validated this genus and characterized it as having cylindrical, simple, septate and hyaline conidiophores, monoblastic conidiogenous cells, and transversely oblong or inverse reniform, muriform conidia surrounded by a semi-gelatinous, hyaline sheath. Berkleasmium micronesicum was then transferred to Coleodictyospora as C. micronesiaca based on its very similar morphological traits with C. cubensis, but it differs in having smaller conidia (30-42 × 15-18 µm vs. 42-50 × 20-22 µm) and reduced conidiophores [31].

Taxonomy of Fungi
Coleodictyospora cubensis was initially collected from North America [30] and subsequently recorded in Brunei [32] and Japan [33]. Nakagiri and Ito [33] named their specimen IFO 32,660 as C. cubensis based on the dimensions of conidiophores, conidia and conidial sheaths, as well as the number of conidial septa and the conidiophore attaching point, although it had overlapping conidial size with C. micronesiaca. Nakagiri and Ito [33] emphasized that IFO 32,660 might be a novel species considering the thinner conidia (28-48 × 13-19 µm vs. 42-50 × 20-22 µm) and less number of septa (7-11 vs. 8-14) than the type specimen of C. cubensis. However, this hypothesis could not be tested without the re-examination of the specimen IFO 32,660 or the molecular data from similar specimens in the same locality (Ishigaki Island, Japan).
In this study, we isolated a Coleodictyospora species from decaying wood submerged in freshwater and provide sequence data for it. Since C. cubensis and C. micronesiaca lack sequence data in GenBank, we identified our new collection as a novel species in Coleodictyospora based on the comparison of their morphology.
Culture characteristics: on PDA, colony irregular, reaching 15 mm diam. in 25 days at room temperature (25-30 • C), surface rough, with dense mycelia, velvety, dry, umbonate in the middle from the side view, edge undulate; from above, dark gray at the margin, pale gray at the middle; from below, dark brown to black at the margin, pale gray at the middle; not producing pigmentation in culture. Habitat and distribution: stream is located in tropical rainforest in Southern Thailand with hot and humid climate conditions, shallow and clear, flowing slowly from the Panan Waterfall, surrounded by angiosperms.
Dematipyriforma is an endophytic genus comprising a single species D. aquilaria [43]. Dematipyriforma shares similar morphological characteristics with Saprodesmium in having micronematous conidiophores, holoblastic conidiogenous cells and septate conidia. However, they are entirely different genera in the following aspects. The conidiophores of Dematipyriforma are hypha-like [43], while they are vesiculate in Saprodesmium which are also unique in the family Pleurotheciaceae. The conidia of Dematipyriforma are elongate pyriform, 4-5 transverse septate, sometimes 1-2 longitudinal septate, pale grey olivaceous to pale brown, and has rhexolytic conidial secession [43]. In contrast, the conidia of Saprodesmium are obovoid to ellipsoidal, irregularly muriform and olivaceous when young, becoming quite blackish with age and obscuring the septa, with several subhyaline basal cells, and has schizolytic conidial secession. In addition, Saprodesmium species is saprobe, while Dematipyriforma species is endophyte [43].
Rhexoacrodictys, typified by R. erecta, was introduced for several hyphomycetes characterized by macronematous, long cylindrical conidiophores with percurrent proliferating, monoblastic, integrated, terminal conidiogenous cells, and obovoid, oval or subspherical, muriform, brown to dark brown conidia often with a paler basal cell bearing a small marginal frill derived from the upper portion of the conidiophores and with rhexolytic conidial secession [44]. Saprodesmium shares some morphological traits with Rhexoacrodictys especially with regards to its muriform and obovoid conidia. Saprodesmium, however, has olivaceous conidia and when mature it has quite a blackish pigmentation obscuring the conidial septa. Rhexoacrodictys is featured by rhexolytic conidial secession with conidia that have a conspicuous paler basal cell bearing a small marginal frill, while the conidia of Saprodesmium secede schizolytically and it instead has several subhyaline, depressed subglobose cells at the base. Rhexoacrodictys has macronematous, long cylindrical conidiophores with percurrent proliferating [44,45], whereas Saprodesmium has micronematous, short, vesiculate, determinate conidiophores.
Based on the multi-locus and individual phylogenetic analyses, as well as the morphological comparison with the similar taxa in the family, we introduce Saprodesmium as a novel genus in Pleurotheciaceae. Culture characteristics: on PDA, colony circular, reaching 50 mm diam. in 30 days at room temperature (25-30 °C), surface rough, with dense mycelia, dry, raised from the side view, edge entire; from above, dark gray at the margin, pale gray to white at the middle; from below, black at the margin, dark olivaceous at the middle; not producing pigmentation in culture.
Culture characteristics: on PDA, colony circular, reaching 50 mm diam. in 30 days at room temperature (25-30 • C), surface rough, with dense mycelia, dry, raised from the side view, edge entire; from above, dark gray at the margin, pale gray to white at the middle; from below, black at the margin, dark olivaceous at the middle; not producing pigmentation in culture. The placement of Obliquifusoideum guttulatum is different in multi-locus and individual LSU and ITS phylogenetic trees and lacks significant support in all trees. Obliquifusoideum guttulatum is revealed as a sister taxon of Pleurotheciales in the multi-locus analysis of concatenated LSU-SSU-ITS-rpb2 matrix (Figure 1), while it clusters with Savoryellales in the individual LSU phylogeny (Supplementary Figure S1); Savoryellales and Pleurothecium species in the individual ITS phylogeny (Supplementary Figure S2). Obliquifusoideum is similar to Helicoascotaiwania in Pleurotheciales, and Ascotaiwania, Neoascotaiwania and Savoryella in Savoryellales. They generally have dark ascomata with a lateral neck, which is oblique or horizontal to the host substrate, and septate ascospores. However, the ascospores of the four genera are mostly ellipsoidal and versicolorous with dark middle cells and hyaline polar cells [2][3][4]46,47]. In contrast, Obliquifusoideum has fusoid and evenly hyaline ascospores. The morphological differences and the independent lineage in the multi-locus and individual phylogenetic trees therefore support Obliquifusoideum as a new genus.
Although the relationships of Obliquifusoideum were weak with four orders in Savoryellomycetidae (Figure 1), it was shown to be a genus in Savoryellomycetidae with strong bootstrap support ( Figure 2). We consider it is wise to refer Obliquifusoideum to Savoryellomycetidae genera incertae sedis for now, until its phylogeny is better resolved with additional taxon sampling followed by divergence time estimates studies.
Culture characteristics: on PDA, colony circular, reaching 8 mm diam. in 48 days at room temperature (25-30 • C), surface rough, with dense mycelia, dry, rigid, umbonate from the side view, edge entire; from above, creamy at the margin, dark grey to dark brown at the middle, brown at the center; dark brown from below; not producing pigmentation in culture.
Habitat and distribution: stream is located in tropical rainforest in Southern Thailand with hot and humid climate conditions, shallow and clear, flowing slowly from the Borriphat Waterfall, surrounded by angiosperms.

Discussion
Doilom et al. [29] established a novel genus Pseudocoleodictyospora to accommodate three hyphomycetous species collected from the bark of living Tectona grandis (teak) and distinguished them from Coleodictyospora by the absence of a hyaline sheath. This establishment, however, lacks the support from the DNA sequence data of Coleodictyospora. The

Discussion
Doilom et al. [29] established a novel genus Pseudocoleodictyospora to accommodate three hyphomycetous species collected from the bark of living Tectona grandis (teak) and distinguished them from Coleodictyospora by the absence of a hyaline sheath. This establishment, however, lacks the support from the DNA sequence data of Coleodictyospora. The presence of conidial sheath as a criterion for delimiting two genera is interesting as this is often used for species delimitation in classification, such as species in Astrosphaeriella, Dictyosporium, Kirschsteiniothelia and Natipusilla [48]. In this study, we collected a freshwater hyphomycetous species which has very similar morphs to Pseudocoleodictyospora, but is characterized by a hyaline sheath. This peculiar phenotype further confirms it as a novel species in Coleodictyospora, namely C. muriformis (see notes of C. muriformis). On the basis of DNA-based phylogeny, Coleodictyospora muriformis is phylogenetically distant from Pseudocoleodictyospora (Pseudocoleodictyosporaceae, Pleosporales) and clusters as a member of the Pleurotheciales (Figure 2). This study further confirms the Doilom et al. [29] taxonomic assumption of establishing a novel genus based on the presence of conidial sheath with the support from the DNA sequence data. Amazingly, Coleodictyospora and Pseudocoleodictyospora are positioned in two different classes Sordariomycetes and Dothideomycetes, respectively (Figure 2), though they are quite similar except in terms of the conidial sheath. Nevertheless, it is not advisable to use conidial sheath as a criterion segregating species at a higher taxonomic level as it is often an unstable characteristic, especially among freshwater species such as Caryospora submersa and Pseudoastrosphaeriella bambusae [48].
In our multi-locus phylogenetic tree (Figure 1), Coleodictyospora is affiliated to Neomonodictys muriformis and Pleurothecium obovoideum. Coleodictyospora is similar to Neomonodictys in having muriform conidia, but they are entirely different genera. The conidia of Neomonodictys are subglobose to globose, comprising several subglobose cells, which are irregularly arranged in the conidia, pale brown when immature, producing black pigmentation and obscuring the conidial septa, with a protruding basal cell which attaches to the conidiophore [8]. In contrast, Coleodictyospora has cylindro-ellipsoidal conidia, with (7-)8-9 transverse and (2-)3 longitudinal septa, deeply constricted and with dark brown bands at the transverse septa, generally produced in the middle position and are perpendicular on the conidiophore. Pleurothecium obovoideum was proposed based on a known species, Ramichloridium obovoideum [49]. In the phylogenetic tree of Arzanlou et al. [49], they showed that the strain CBS 209.95 of R. obovoideum clustered with the sexual morph of Pleurothecium recurvatum (type species) and its morphological characteristics fit well with Pleurothecium and R. obovoideum was therefore transferred to Pleurothecium, namely P. obovoideum. However, with more species in Pleurothecium, P. obovoideum was reported to be distant from P. recurvatum and clustered with Neomonodictys in a well-supported clade [6,8]. The reassessment of P. obovoideum is pending, however, its pleurothecium-like morphological characteristics [49] warrant it cannot be congeneric with Coleodictyospora.
Interestingly, we found that the conidia of Coleodictyospora are quite similar to the ascospores of a sexual species Boerlagiomyces websteri. Boerlagiomyces were recognized in Tubeufiaceae [11,50,51] and confirmed with DNA sequence data derived from a reference specimen of Boerlagiomyces macrospora [29]. However, B. websteri represented by a putative strain BCC 3834 clustered with several apothecial taxa in Pezizomycotina [52], and Boonmee et al. [50] had some doubts on this species because of its perithecial characteristic. Therefore, the accurate phylogenetic position of B. websteri is still questionable. Although the soft, membranous, setose ascomata and large dictyosporous ascospores of B. websteri fit with the features of Boerlagiomyces [50], the two-spored asci are unusual compared with the eight-spored asci of the type species B. velutinus [50]. Whether B. websteri has close phylogenetic relationships with Coleodictyospora in Savoryellomycetidae is pending and has to be resolved.
On the basis of morphology, we initially considered Obliquifusoideum as a member of Annulatascales due to its black ascomata with a lateral neck which is oblique or horizontal to the host substrate, hypha-like paraphyses with tapering apex, cylindrical asci with a distinct, refractive apical ring, and fusoid, hyaline ascospores. It is of interest, however, that Obliquifusoideum clusters in Savoryellomycetidae with relationships to Pleurotheciales and Savoryellales, which is distant from Annulatascales and annulatascales-like taxa in Diaporthomycetidae (Figure 2). It is not unexpected that Obliquifusoideum with annulatascales-like morphology can be discovered in another subclass Savoryellomycetidae as Annulatascales is commonly recognized to be polyphyletic and the species are often encountered from freshwater habitats [53,54]. It is reasonable that Obliquifusoideum is placed in Savoryellomycetidae due to its dark ascomata with an oblique or horizontal neck, and phragmoseptate conidia which are the sexual features of the other two members Pleurotheciales and Savoryellales.
DNA-based phylogeny has helped to provide better insights into the taxonomy of Pleurotheciales and a recommendation of species boundaries was established [55], leading to recent classification updates. Rhexoacrodictys, which was treated as a genus in Savoryellales by Xia et al. [56], was accepted in Pleurotheciales in a later phylogenetic study [6]. Our phylogenetic results corroborate those of Luo et al. [6], and our new genus Saprodesmium forms a well-supported lineage basal to Rhexoacrodictys in Pleurotheciales ( Figure 1). The BlastN search of Saprodesmium using SSU sequence in NCBIs GenBank reveals a high similarity (99.49%) with a hyphomycetous species Dematipyriforma aquilaria. Dematipyriforma, typified by D. aquilaria, was isolated as an endophyte from the trunk of Aquilaria crassna, producing dark muriform conidia [43]. Dematipyriforma was placed in Savoryellales in the phylogenetic tree of Sun et al. [43], however, they did not include other related orders (Conioscyphales, Fuscosporellales and Pleurotheciales) in Savoryellomycetidae. Our multi-locus phylogeny places Dematipyriforma as a sister genus to Rhexoacrodictys and Saprodesmium with good bootstrap support in Pleurotheciales (99% ML BS/1.00 BI PP, Figure 1). On the other hand, the muriform conidia of Dematipyriforma are similar to Neomonodictys in Pleurotheciales. According to this morphological trait and phylogenetic result, we accept Dematipyriforma in Pleurotheciales. Besides saprobes and few opportunistic human pathogens, Dematipyriforma is the only presently known endophytic genus in the order, which increases our understanding of different life modes of Pleurotheciales.
Freshwater fungi are a unique group of organisms with a special ability to survive and grow on submerged wood in water by producing soft rot cavities [57,58]. There is very little overlap between the fungi growing on wood submerged in freshwater and those on adjacent stream sides [59,60]. Thus, we are continually finding novel taxa from this unique habitat and since streams are often disparate elements, we are likely to discover many more which will improve our understanding of fungal classification [61]. Freshwater appears to be an ecologically important niche for species in Pleurotheciales [62]. Almost all Pleurotheciella species were collected from freshwater [3,4,6,9,10,13], as well as some species from Helicoascotaiwania, Phaeoisaria, Pleurothecium and Sterigmatobotrys [6,10,12,15,63].
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jof7090711/s1, Figure S1: Phylogenetic tree generated from maximum likelihood analysis (RAxML) based on LSU sequence data, Figure S2: Phylogenetic tree generated from maximum likelihood analysis (RAxML) based on ITS sequence data.