The Enigmatic Thelebolaceae (Thelebolales, Leotiomycetes): One New Genus Solomyces and Five New Species

The family Thelebolaceae belongs to the order Thelebolales, class Leotiomycetes, and contains 22 genera. In this study, we introduce a new genus Solomyces gen. nov. in the family Thelebolaceae, which is supported by morphological observation and multilocus-based [internal transcribed spacers (ITS) + LSU and ITS + LSU+ MCM7+ EF1A+ RPB2] phylogenetic analysis. Maximum-likelihood and Bayesian phylogenetic inference analyses indicated that Solomyces is a distinct genus within this family. The new genus is compared against related Thelebolaceae genera, and its description and illustration are provided. This genus comprises one new species and one unnamed species (including two strains). We also report the addition of four new species – Pseudogymnoascus shaanxiensis, Pseudogymnoascus guizhouensis, Pseudogymnoascus sinensis, and Geomyces obovatus – in the family Thelebolaceae and present their morphological and phylogenetic characterizations.


INTRODUCTION
The class Leotiomycetes (Pezizomycotina) was erected by Eriksson and Winka (1997) to accommodate inoperculate discomycetes. Fungi in the class Leotiomycetes are ecologically diverse and include mycorrhizas, root and leaf endophytes, plant pathogens, aquatic and aeroaquatic hyphomycetes, mammalian pathogens, and saprobes (Johnston et al., 2019). Leotiomycetes currently comprises 19 orders and order-level clades, 54 families, and 13 family level clades (Ekanayaka et al., 2019;Karunarathna et al., 2020). Previously, however, this class included a wide range of taxa based on traditional morphological taxonomy (Korf, 1973;Spooner, 1987), and the current classification of Leotiomycetes is still largely based on morphologically defined taxa, especially at higher taxonomic levels (Johnston et al., 2019). Nevertheless, sexual or asexual morphs of many Leotiomycetes taxa are not recorded, and a few links between sexual and asexual morphs in Leotiomycetes have been confirmed (Sutton and Hennebert, 1995;Sati and Pathak, 2016;Ekanayaka et al., 2017;Johnston et al., 2019). Thelebolales, an order in the class Leotiomycetes, consists of Thelebolaceae and the Alatospora-Miniancora clade, in which some genera (e.g., Caccobius, Coprobolus, and Leptokalpion) are erected based on their sexual, but not asexual, morphology (Wijayawardene et al., 2017). Haeckel (1894) introduced the order Thelebolales; however, the taxonomy of this order has long been contentious. Some researchers believed that the order contained only one family (De Hoog et al., 2005), whereas others suggested that at least two families were involved (Ekanayaka et al., 2019;Johnston et al., 2019;Batista et al., 2020). The family Thelebolaceae is important in the order Thelebolales because of several species that can produce antifreeze proteins, ice-binding proteins, and some secondary metabolites with potential application values that offer valuable resources for biotechnological exploitation (Batista et al., 2020); the family was introduced by Eckblad (1968) and typified by the genus Thelebolus with Thelebolus stercoreus as the type species. Members of this family are characterized by absent, apothecial, or cleistothecial ascomata (Van Brummelen, 1985;Stchigel et al., 2001;De Menezes et al., 2017;Ekanayaka et al., 2019). During an extensive diversity survey of Geomyces and allied genera in China, we gathered a collection of fungal isolations. In this study, we introduced their morphological, cultural, and phylogenetic characterization and propose one new genus and five new species.

Isolates and Morphology
Soil samples were obtained from Dali City, Yunnan Province; Guiyang City, Guizhou Province; Xi'an and Hanzhong City, Shaanxi Province; and Yichang City, Hubei Province, China. Samples were treated according to the method described by Zhang et al. (2019). Fungi were isolated using a modified baiting technique with chicken feathers as the substrate (Vanbreuseghem, 1952). The feathers were washed, thoroughly rinsed with distilled water, dried, cut into 2-cm fragments, and autoclaved. Plates containing soil material and sterile feathers were incubated at room temperature for 1 month. Fungi were isolated and purified using a conventional dilution technique described by Zhang et al. (2019), as follows: 2 g of soil sample was suspended in 9 ml of distilled water, and the prepared suspension was vortexed, diluted to 1:10,000, and cultured on Sabouraud's dextrose agar (SDA; 10 g of peptone, 40 g of dextrose, 20 g of agar, 1 L of ddH 2 O) supplemented with chloramphenicol and cycloheximide. The plates were incubated at 25 • C until fungal colony growth was observed. The axenic strains were then transferred to potato dextrose agar (PDA; Shanghai Bioway Technology Co., Ltd., China) plates for purification. Dried holotypes were deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS), or the Institute of Fungus Resources, Guizhou University (GZUIFR, formally the Herbarium of Guizhou Agricultural College; code, GZAC). Ex-type strains and other strains were deposited in the China General Microbiological Culture Collection Center (CGMCC) or the GZUIFR.
The pure strains were incubated on PDA at 25 • C for 14 days in the dark to determine the macroscopic characteristics, diameters, and colony colors (surface and reverse). The characterization and measurement of fungal microscopic characteristics were performed in 25% lactic acid. Images were obtained using an optical microscope (OM, DM4 B, Leica, Germany) with differential interference contrast (DIC). The taxonomic descriptions and names of the new taxa were introduced into MycoBank 1 and Faces of Fungi 2 .
Sequence alignment and maximum-likelihood (ML) and Bayesian inference (BI) phylogenetic analyses were performed according to the methodology described by Zhang et al. (2019). Maximum-likelihood (ML) analyses were constructed with IQ-TREE v. 1.6.11 (Nguyen et al., 2015). The best-fit model of substitution for each locus was estimated using IQ-TREE's ModelFinder function (Kalyaanamoorthy et al., 2017) under the Bayesian information criterion (BIC). Bootstrap analysis was performed using the ultrafast bootstrap approximation (Minh et al., 2013) with 1,000 replicates, and bootstrap support (BS) ≥90% was considered as statistically significant. For   (Posada and Crandall, 1998). After the BI analyses, both runs were examined with Tracer v.1.5 (Drummond and Rambaut, 2007) to determine burn-in and check for convergence. Two multilocus phylogenies were analyzed to evaluate the various generic placements and establish the phylogenetic relationships in Thelebolaceae. The genus placement was evaluated based on a concatenated ITS and LSU dataset. This phylogeny was constructed to assess if Solomyces is a welldelimited genus. The second multilocus phylogeny was based on a concatenated alignment of ITS, LSU, MCM7, RPB2, and EF1A sequences and included 49 isolates of Geomyces and its allied genera. This analysis was performed to evaluate the generic boundaries and species groupings within the genera Solomyces, Geomyces, and Pseudogymnoascus. All the trees were displayed in FigTree 1.4.2 3 and edited in Microsoft Paint. DNA alignments have been deposited at TreeBASE.

Phylogenetic Analyses
The first concatenated matrix contains 1,220 nucleotides, i.e., 445 from the ITS and 775 from the LSU. The second concatenated alignment contained 2,808 nucleotides (ITS: 434, LSU: 806, MCM7: 503, RPB2: 437, and EF1A: 628). The best-fit evolutionary model for each locus in the two datasets is listed in Table 2.
The tree topology of the Bayesian inference agrees with that of the ML tree (Figures 1, 2). The phylogenies indicated that each genus clusters into a monophyletic clade (Figure 1). In the phylogenetic tree, the new genus Solomyces forms a wellsupported (1 BYPP/100 MLBS) clade separated from other genera in Thelebolaceae (Figure 1) (Figure 3).
Etymology: Refers to Shaanxi, the province where the isolate was collected.
Holotype: permanently preserved in a metabolically inactive state, HMAS 255395.
Etymology: In reference to China, the country where the type specimen was obtained.
Holotype: permanently preserved in a metabolically inactive state, HMAS 255394.
Notes: Morphologically, P. sinensis is similar to P. linderi and P. turner, based on its obovoid conidia. However, P. sinensis differs from P. linderi and P. turneri as it has drum, obovoid, pyriform, or irregularly shaped intercalary conidia (the intercalary conidia of P. linderi and P. turneri are globose to truncate) (Crous et al., 2019). Phylogenetically, our isolates CGMCC 3.18493 and CGMCC 3.18494 cluster together very well and form a single clade separated from other Pseudogymnoascus species (Figure 1) Notes: Solomyces is introduced to accommodate Solomyces sinensis and another unnamed species (containing two strains, 15PA02 and 17WV02, from Hibernacular soil in Pennsylvania and West Virginia, respectively; Minnis and Lindner, 2013). The morphology of Solomyces species is similar to that of Geomyces and the asexual morphs of Pseudogymnoascus. However, Geomyces differ in having terminal and lateral conidia borne on hyphae, short protrusions or side branches; intercalary conidia barrel shaped, and conidiophores abundant, always forming verticillate and opposite branches with an acute angle to the axis near the apex (Van Oorschot, 1980;Chen et al., 2017). As can be seen in Figures 1, 2, strains of these genera appear in distinct clades in a phylogeny based on multiple strains, thereby justifying the erection of the new genus Solomyces.  (Figure 7).

Solomyces sinensis
Etymology: In reference to China, the country where the type specimen was obtained.
Holotype: permanently preserved in a metabolically inactive state, HMAS 255397.
Sexual morph: not observed. Notes: Solomyces sinensis was isolated from soil in Guizhou Province, China. We did not compare morphological characteristics between S. sinensis and another two strains within Solomyces owing to the lack of morphological description of these two strains (Minnis and Lindner, 2013). However, S. sinensis is phylogenetically distinct from these strains with high statistical support (1.00 BYPP, 100% MLBS) (Figures 1, 2).

DISCUSSION
In this study, Solomyces gen. nov. is introduced with an asexual morph. Five new species are also described. All the new taxa belong in the order Thelebolales, the members of which are ubiquitous in the environment. Several taxa belonging to this order have been isolated from tropical to arctic regions. They are often coprophilous and frequently isolated from freshwater and saline lakes (De Hoog et al., 2005). They have also been recorded from soils, epiphytic soils in tree holes (Chen et al., 2017), mine sediments (Crous et al., 2019), and sponges (Bovio et al., 2018). Thelebolales have been recorded as saprobic on dead plant material, rarely as plant-parasitic, and also as animal pathogens, e.g., the well-known white-nose disease of bats (Lorch et al., 2011). In addition, several members of Thelebolales are keratinophilic; i.e., they can invade and degrade keratin material (Saxena et al., 2005). All the proposed new taxa in our study were isolated using the baiting technique, a method specifically designed for the isolation of keratinophilic fungi. Consequently, additional studies are needed to assess whether our new taxa can also degrade keratin material.
Recent studies demonstrated that the Thelebolales comprise at least two families or groups (Ekanayaka et al., 2019;Johnston et al., 2019;Batista et al., 2020). Some studies have proposed that the Thelebolales consists of Pseudeurotiaceae and Thelebolaceae based on three distinct pieces of evidence (568 ITS sequences, 15 genes concatenated from 279 species, and a phylogenomic approach from 51 complete genomes) and on a phylogenomic approach, respectively (Johnston et al., 2019;Batista et al., 2020). The Thelebolaceae, represented by the Thelebolus and Antarctomyces, share a common ancestor with the family Pseudeurotiaceae, represented by the genus Pseudogymnoascus (Johnston et al., 2019;Batista et al., 2020). However, a different study indicated that Pseudeurotiaceae was nested within Thelebolaceae based on phylogenetic analysis and synonymized Pseudeurotiaceae under Thelebolaceae (Ekanayaka et al., 2019). The same authors discovered that several genera, previously classified as Leotiaceae and Leotiomycetes genera incertae sedis, clustered within Thelebolales as a sister clade to the Thelebolaceae and defined them as the Alatospora-Miniancora clade (Ekanayaka et al., 2019). The studies by Ekanayaka et al. contained more genus taxa in Thelebolales; therefore, we continued the phylogenetic analysis in Thelebolales based on this study.
Recent reports have indicated that the Thelebolales contained 22 genera. However, because no ITS and LSU sequence data were available for Ascophanus, Ascozonus, Caccobius, Coprobolus, Leptokalpion, Neelakesa, and Pseudascozonus (Ekanayaka et al., 2019), we could not compare the phylogenetic relationships between these genera and Solomyces. In our phylogenetic analysis, our three isolates (CGMCC 3.18498, CGMCC 3.18499, and CGMCC 3.18500; Solomyces sinensis) and two isolates of Minnis and Lindner (2013) (15PA02 and 17WV02, from Hibernacular soil in Pennsylvania and West Virginia, respectively) formed an independent clade with strong statistical support (BYPP 1/MLBS 100%) and were close to Geomyces. Morphologically, the asexual stage of Ascophanus, Ascozonus, Caccobius, Coprobolus, Leptokalpion, Neelakesa, and Pseudascozonus is not recorded in the literature (Wijayawardene et al., 2017). Therefore, no morphological comparison can be done between these genera and Solomyces. However, Solomyces differs from Geomyces by terminal and lateral conidia borne on hyphae, short protrusions or side branches, olivary, subglobose to globose intercalary conidia, and absence of the forming verticillate and opposite branches with an acute angle to the axis near the apex of conidiophores (Sigler and Carmichael, 1976).
Based on morphological characteristics, it was difficult to distinguish closely related species, and even genera, using traditional taxonomy, and modern phylogenetic methods were a very important adjunct. Although Crous et al. (2019) described the new species, P. linderi and P. turneri, based on the similarity of morphological characteristics between these two new species and P. bhattii, they did not compare the phylogenetic relationship. Our phylogenetic analysis indicated that P. bhattii (type strain CBS 760.71) was nested within Gymnostellatospora (Figure 1), and we, therefore, transferred P. bhattii to the Gymnostellatospora and named it G. bhattii.

AUTHOR CONTRIBUTIONS
YH and JH were responsible for conceptualization and funding acquisition. ZZ, CD, WC, QM, and XL were responsible for data acquisition. ZZ, CD, and WC did the formal analysis. ZZ wrote the first draft. YH and ZL wrote, reviewed, and edited the manuscript. All authors have read and agreed to the published version of the manuscript.