Culturable Fungi from Urban Soils in China I: Description of 10 New Taxa

ABSTRACT An investigation of members of the soil keratinophilic fungi community in China resulted in the identification of one new monotypic genus, Zongqia, and 10 new species, 2 of which are affiliated with Solomyces, 1 with the new genus Zongqia, 4 with Pseudogymnoascus, and 3 with Scedosporium. These novel taxa form an independent lineage distinct from other species, based on morphological and multilocus phylogenetic analyses. Descriptions, illustrations, and notes are provided for each taxon. These new taxa of the soil keratinophilic fungi add to the increasing number of fungi known from China, and it is now evident that numerous novel taxa are waiting to be described. IMPORTANCE Keratinophilic fungi are a group that can degrade and utilize keratin-rich material. It is also because of this ability that many taxa can cause infections in animals or humans but remain poorly studied. In this study, we reported a novel genus and 10 novel species, 7 novel species belonging to the order Thelebolales and 3 to the genus Scedosporium, based on multilocus phylogenetic analyses combined with morphological characteristics. Our study significantly updates the taxonomy of Thelebolales and Scedosporium and enhances our understanding of this group of the keratin-degrading fungal community. The findings also encourage future studies on the artificially constructed keratin-degrading microbial consortia.

S oil microbes are the richest component of terrestrial biodiversity, and among them, soil fungi play a major role in the ecosystem processes. To date, many studies have explored fungi in ocean, caves, forests (especially pristine rainforests), extreme environments, volcanoes, mountains, deserts, freshwater aquatic systems, lakes, grasslands, indoor environments, and many other habitats (1), and they have found that fungi in different habitats have very high species diversity. At the same time, many new fungal taxa have been reported, and they have shown potential high value in the industries of agriculture and medicine. However, as global urbanization continues to expand (2,3), urban soil fungi, which are closely related to human health, have not been systematically investigated although they are a focal area for ecological and environmental issues. China has diverse urban soil types, diverse habitats, rapid urbanization, and high population mobility. Investigating the diversity of soil fungi in different cities in China will provide scientific data for understanding their ecological functions and maintaining public health safety and will enable the isolation of many new resources with potential applications.
The enrichment culture method using different substrates can often screen for the specific fungal consortium, so this method is often used for the isolation of fungal taxa with specific physiological functions. The distribution of keratinophilic fungi, as a special fungal consortium that can degrade and utilize keratin-rich materials, is greatly influenced by the activities of humans and animals, and the presence of such fungus is high in areas where humans and animals are frequently active, especially in urban parks, hospitals, and school campuses (4)(5)(6)(7). According to the habitat, keratinophilic fungi can be broadly classified into three eco-types, anthropophilic, zoophilic, and geophilic species, and are mostly pathogenic or potentially pathogenic fungi. For human health and safety, their distribution should attract the attention of governments and scientists. Keratinophilic fungi have been reported in soils of different habitats in different geographic regions of the world, so the investigation of keratinophilic fungi has epidemiological significance (8).
Since the report of the degradable keratin of Onygena equina (9), new taxa of keratinophilic fungi and their applications have been reported. Keratinophilic fungi involve a large number of taxa belonging to several orders, families, and genera, including mainly dermatophytes and some saprophytic fungi, such as some species of Arthrodermataceae and Onygenaceae in the order Onygenales (10) and some members of the genera Geomyces and Pseudogymnoascus in the order Thelebolales (11). In addition, they contain a large number of common taxa, such as some species of the genera Aspergillus, Penicillium, and Trichoderma (12,13). In the years since we investigated the members of keratin-degrading fungal communities in Chinese soils, several new taxa have been identified and reported (14)(15)(16)(17)(18)(19)(20)(21)(22). Here, we introduce one new genus, Zongqia (Thelebolales genera incertae sedis, Thelebolales), and 10 new species, 2 of which are affiliated with Solomyces, 1 with the new genus Zongqia, 4 with Pseudogymnoascus, and 3 with Scedosporium.

RESULTS
In this study, the internal transcribed spacer (ITS) regions of all isolates were sequenced, and all ITS sequences obtained were BLASTn searched in NCBI and assigned to potential genera and species. Then, strains belonging to Thelebolales and Scedosporium were screened and tested for further identification through morphological characterization and phylogenetic analyses.
In the first phylogenetic tree (Fig. 1   In the second phylogenetic tree (Fig. 2), each genus clusters into a monophyletic clade. The new genus Zongqia forms a well-supported (0.99 PP/98% BS) clade separated from other genera in Thelebolales.
In the third phylogenetic tree (Fig. 3 Description. Sexual morph: not observed. Asexual morph: colonies on peptone-dextrose agar (PDA) slowly growing, attaining 6 to 10 mm diameter after 14 days at 25°C, velvety, short and fluffy, margins irregular, light gray to white, absent pigment and Notes. Morphologically, Pseudogymnoascus catenatus is similar to P. verrucosus in having arthroconidia but is clearly distinguished by the obovoid conidia and intercalary conidia (23). Phylogenetically, four isolates of P. catenatus formed a single clade separate from other species in Pseudogymnoascus (Fig. 1), which indicates that they are distinct species.
Pseudogymnoascus yunnanensis Zhang, Han, and Liang, sp. nov. (Fig. 6). MycoBank number: MB 840438. Etymology: refers to the region from which the fungus was isolated. Diagnosis: similar to Pseudogymnoas lindneri, Pseudogymnoas turneri, and Pseudogymnoas guizhouensis but differs in the clavate, fusiform with basal scars terminal conidia, and reniform, fusiform, truncated at both ends of intercalary conidia.  subhyaline to hyaline, smooth-walled or echinulate; obovoid, subglobose to globose, sometimes pyriform, 2.5 to 4.5 by 2.5 to 3.5 mm (n = 50); sometimes terminal conidia clavate, fusiform with basal scars, 6.5 to 9.0 by 2.5 to 4.5 mm (n = 50). Intercalary conidia are borne on the outer branches of the hyphae or verticillate hyphae, solitary or two in chains, smooth-walled or rough, reniform and fusiform truncate at both ends, 2.5 to 5.5 by 2. Notes. Morphologically, Pseudogymnoascus yunnanensis is similar to P. lindneri, P. turneri, and P. guizhouensis in having obovoid, globose conidia (27). However, P. yunnanensis can be distinguished from P. lindneri and P. turneri by the presence of its clavate, fusiform with basal scars terminal conidia and no observed sexual morph. P. yunnanensis differs from P. guizhouensis because it is reniform, fusiform, and truncated at both ends of intercalary conidia (22). Phylogenetically, three isolates of P. yunnanensis constitute a strongly supported subclade, sister to P. guizhouensis with high support values ( Fig. 1), but they can be easily distinguished.
Substrate: Soil. Distribution: Ningbo City, Zhejiang Province, China. Notes. Morphologically, Pseudogymnoascus zhejiangensis resembles P. lindneri, P. turneri, and P. yunnanensis because of the obovoid, globose conidia. However, P. zhejiangensis differs from P. lindneri, P. turneri, and P. yunnanensis in that it has obovoid, subglobose intercalary conidia (the intercalary conidia of P. linderi and P. turneri are globose to truncate, and those of P. yunnanensis are reniform, fusiform, and truncated at both ends) (27). Phylogenetically, three isolates of P. zhejiangensis formed one clade and share a sister relationship to three undescribed isolates (12NJ13, 17WV06, and 22984-1-I1) with high BS (Fig. 1). However, we did not compare morphological characteristics between P. zhejiangensis and another three isolates within Pseudogymnoascus because of the lack of morphological description of these three isolates (24).
Solomyces guizhouensis Zhang, Han, and Liang, sp. nov. (Fig. 8). MycoBank number: MB 840440. Etymology: refers to Guizhou, the province where the isolate was collected. Diagnosis: Solomyces guizhouensis differs from other species by the presence of 2 to 3 conidia in chains and 2 to 3 intercalary conidia in chains. Type: China, Guizhou  Notes. Morphologically, Solomyces guizhouensis is distinguished from other species of Solomyces by the presence of 2 to 3 conidia in chains and 2 to 3 intercalary conidia in chains. Solomyces guizhouensis is phylogenetically allied to Solomyces ramosus ( Fig. 1), but they can be easily distinguished (see notes on S. ramosus [22]).
Substrate: soil. Distribution: Shanghai City, China. Notes. Morphologically, Solomyces ramosus is distinguished from other species of Solomyces by the presence of ramose conidiophores (22). Phylogenetically, our two new isolates of S. ramosus formed one clade and share a sister relationship to S. guizhouensis with high BS (Fig. 1), which indicates that they are distinct species.
Zongqia Zhang and Han, gen. nov. MycoBank number: MB 840447. Typification: Zongqia sinensis Zhang and Han. Etymology: in honor of Zong-Qi Liang, acknowledging his contributions to our group. Diagnosis: in addition to the phylogenetic distinctions ( Fig. 1 to 2), Zongqia differs from Pseudeurotium by the presence of chains of conidia, conidiophores degenerated into conidiophore cells, clavate conidiophores cells.
Notes. The new genus Zongqia is introduced here based on phylogeny and morphological evidence. Until now, the Thelebolales consisted of 23 genera (22,28). In five-loci (ITS, LSU, MCM7, RPB2, and EF1A; Fig. 1) and two-loci (ITS and LSU; Fig. 2) phylogenetic analyses, Zongqia was related to Pseudeurotium with high support values (1 PP/100% BS). However, because no ITS, LSU, MCM7, RPB2, and EF1A sequence data were reported for Ascophanus, Ascozonus, Caccobius, Coprobolus, Leptokalpion, Neelakesa, and Pseudascozonus (22), we could not compare the phylogenetic relationships between these genera and Zongqia. Morphologically, because there is no record of the asexual stage of Ascophanus, Ascozonus, Caccobius, Coprobolus, Leptokalpion, Neelakesa, and Pseudascozonus in the literature (29), we could not compare the morphology between these genera and Zongqia. Of the remaining genera, Zongqia is similar to Pseudeurotium, but there are still noteworthy differences between them. Zongqia is distinguished from Pseudeurotium by the presence of chains of conidia, conidiophores degenerated into conidiophore cells, clavate conidiophores cells, and no observed sexual morph.
Zongqia sinensis Zhang and Han, sp. nov. (Fig. 10 Description. Sexual morph: not observed. Asexual morph: colonies grow slowly on PDA, reaching 11 to 13 mm diameter after 14 days at 25°C, suborbicular, white, floccose, margins regular; reverse white, no growth at 37°C. Hyphae hyaline, branched, septate, smooth, 1.5 to 3.5 mm wide. Conidiophores not observed but degenerated into conidiophore cells. Conidiophore cells hyaline, cylindrical, clavate, arising directly from the aerial hyphae, smooth-walled, solitary. Conidia aseptate, smooth-walled, one-celled, solitary, obovate to subobovoid, 5 to 9 by 3 to 5 mm (n = 50); or 2 to 20 in chains, obovate, subglobose, fusiform and obtuse at apex and base, sometimes cylindrical, clavate, 3.5 to 8.5 Notes. Based on multilocus phylogenetic analyses ( Fig. 1 and 2) and similar morphological characteristics, the four strains are regarded as the same species, which cluster together very well and form a single clade separated from other species of Thelebolales. Morphologically, Zongqia sinensis is the only species that produces the conidia chains in this order. Therefore, based on both morphological and phylogenetic evidence, Z. sinensis is proposed as a novel species as a type of Zongqia.
Scedosporium multisporum Zhang, Han, and Liang, sp. nov. (Fig. 13) Description. Sexual morph: not observed. Asexual morph: colonies on PDA attaining 45 to 50 mm diameter after 5 days at 25°C, cottony, powdery at the center; reverse white, light yellow at the center; absent pigment and exudates. Colonies on PDA attaining 70 to 73 mm diameter after 5 days at 37°C. Hyphae hyaline, branched, septate, smooth-walled, 1.0 to 4.0 mm wide. Conidiophores solitary, often consisting of a single conidiogenous cell, or arranged in whorls of 2 to 3 conidiogenous cells, arising terminally or laterally from hypha, undifferentiated hypha, short-stalked, or inside branches. Conidiogenous cells annellidic, hyaline, thin-and smooth-walled, lateral or terminal, cylindrical or slightly broad at the base, sometimes with several annellations at the top with the age, 0.  (30). However, in our phylogenetic study, S. sanyaense is placed in the genus Parascedosporium. Therefore, we propose a new combination for that species.

DISCUSSION
The hair baiting technique was first used to isolate keratinophilic fungi from the soil by Vanbreuseghem (34) and has become applied widely. So far, the investigation of such resources is still dominated by traditional isolated cultures and baiting with materials of human or animal origin, such as feathers (35), horsehair (4), wool (36), human hair (37), and human nails (38). Only a small number of studies have used next-generation sequencing technologies (39).
Taxonomy and phylogenetic identification of fungi remain significant challenges (40). One of the main fundamental needs in fungal ecology is a strong taxonomic basis, which is dependent on advances in nucleic acid sequence technology. However, some researchers have relied too much on these techniques to the complete exclusion of fungal isolation and characterization using classical methods. While bacterial microbiome studies have relatively reliable taxonomic identification using 16S ribosomal DNA (rDNA) and even metagenome sequencing, mycobiome studies are still few and far between, with limited taxonomic interpretation capabilities. Indeed, phenotypic and culture-based studies remain an invaluable tool for fungal biology and ecology (41). The advantage of placing these organisms in pure culture is, of course, that almost all aspects of their biology can be studied, which may help to understand how they function in their natural ecological context. Thus, many challenges remain in studying the hundreds of niches on Earth that may be inhabited by fungi, not only to demonstrate their presence in these niches but also to culture them in pure form and store them properly for further study (42).
The ability of microorganisms to degrade recalcitrant materials has been widely explored for environmental remediation and industrial production. Significant success has been achieved with single strains, but the focus is now on the use of microbial consortia because of their functional stability and efficiency (43). The keratin degradation process requires the synergistic action of different enzymes, such as endoproteases, exoproteases, oligopeptidases, and disulfide reductases (44); thus, this process involves the synergistic cooperation of multiple species. We did not isolate purified fungal strains directly from feathers after enrichment using hair bating but did isolate members of the fungal community from the soil. Therefore, we could not determine whether the obtained strains are keratinophilic fungi and whether they are able to degrade and utilize keratin. However, numerous studies have shown that many members of Thelebolales and Scedosporium are indeed keratinophilic fungi (45)(46)(47)(48). Hence, we think that our obtained strains are the keratinophilic fungi and should at least be constituent members of the keratin-degrading fungal consortia, although it is not clear what role they play in this consortium. In this study, 10 new species were identified and introduced, not only contributing to the further understanding of the keratin-degrading fungal community but also accumulating strains for future artificially constructed keratin-degrading microbial consortia.

MATERIALS AND METHODS
Sampling, fungal isolation, and morphology. Soil samples were collected from Guizhou, Hunan, Zhejiang, Yunnan, Fujian, Hainan, Jiangxi, Guangdong, and Zhejiang provinces in southern China and transported to the laboratory in Ziploc plastic bags. The soil samples were processed using the method we described previously (22). Briefly, clean and sterile chicken feathers were placed in a sterile petri dish after the soil sample was added, wetted with distilled water, and incubated at room temperature for 1 month. Fungi were isolated using a conventional dilution technique based on Sabouraud's dextrose agar (SDA; 10 g of peptone, 40 g of dextrose, 20 g of agar, 1 liter of ddH 2 O) supplemented with chloramphenicol and cycloheximide, and the purification of the strains was performed using potato dextrose agar (PDA; Shanghai Bioway Technology Co., Ltd., China) (20,22). Colonies on PDA were incubated after 14 days at 25°C, and the cultures were placed to slowly dry at 50°C to produce the holotype. Holotypes were deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS). All strains were deposited in the Institute of Fungus Resources, Guizhou University (GZUIFR, the Herbarium of Guizhou Agricultural College, code GZAC), and the ex-type strains were also deposited in the China General Microbiological Culture Collection Center (CGMCC). The living cultures were stored in a metabolically inactive state, i.e., kept in sterile 30% glycerol in a 280°C freezer. Macroscopic and morphological characterization of the colonies was performed on PDA incubated for 14 days in the dark at 25°C. 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). Taxonomic descriptions and nomenclature were deposited at MycoBank (https://www.mycobank.org/). DNA extraction, PCR amplification, and sequencing. Total genomic DNA was extracted from fungal mycelia using the BioTeke fungus genomic DNA extraction kit (DP2032, BioTeke, Beijing, China) following the manufacturer's instructions. Multiple loci were amplified and sequenced for each new isolate, and the primer sets are listed in Table 2. Amplification conditions were carried out as in the original literature where the primers were reported. The PCR thermal cycle programs for each locus amplification were performed as in the original literature where the primers were reported. The PCR products were sequenced with the amplified primers at a commercial sequencing service provider (Shanghai Sangon Biological Engineering Technology & Services Co., Shanghai, China) in an ABI 3730xl DNA analyzer using the Sanger method. The consensus sequences were obtained using the SeqMan software v. 7 (DNASTAR Lasergene, Madison, WI, USA).
Data availability. The sequences generated in this study can be found in GenBank. The accession numbers of the sequences deposited in GenBank are listed in Table 3. a Accession numbers for these strains generated from this study.