﻿Culturable fungi from urban soils in China II, with the description of 18 novel species in Ascomycota (Dothideomycetes, Eurotiomycetes, Leotiomycetes and Sordariomycetes)

﻿Abstract As China’s urbanisation continues to advance, more people are choosing to live in cities. However, this trend has a significant impact on the natural ecosystem. For instance, the accumulation of keratin-rich substrates in urban habitats has led to an increase in keratinophilic microbes. Despite this, there is still a limited amount of research on the prevalence of keratinophilic fungi in urban areas. Fortunately, our group has conducted in-depth investigations into this topic since 2015. Through our research, we have discovered a significant amount of keratinophilic fungi in soil samples collected from various urban areas in China. In this study, we have identified and characterised 18 new species through the integration of morphological and phylogenetic analyses. These findings reveal the presence of numerous unexplored fungal taxa in urban habitats, emphasising the need for further taxonomic research in urban China.


Introduction
Biodiversity has always been a hot area of research in ecology and biology. Fungi represent one of the most diverse groups of microorganisms on the planet, with an essential role in ecosystem processes and functioning (Hyde et al. 2020a). At the same time, fungi have a significant influence on human society. On the one hand, they are able to produce a large number of secondary metabolites that can be used by humans, such as various antibiotics and enzymes (Uchida et al. 2005;Hoffmeister and Keller 2007;El-Gendi et al. 2022;Mapook et al. 2022). They also infect humans, animals and plants, bringing great harm to human health and the national economy (Fisher et al. 2012;Fisher et al. 2020;Fones et al. 2020). To date, the total number of fungal species is still a prolonged debate. While numerous studies have explored fungal diversity in marine, cave, forest, volcanic, mountain, desert, freshwater aquatic systems, lakes, grasslands and indoor environments (Hyde et al. 2020a), their distribution in urban environments seems to have been overlooked.
Urbanisation is an inevitable trend in humanity's development and is an important symbol of the progress made in science and technology (Wang et al. 2018). Urbanisation has swept across the globe over the last several decades (Berry 2008). The rate of urban expansion is currently at an unprecedented level and the migration of people from rural to urban regions leads to increased anthropogenic changes to the urban environment. These changes may include alterations in land use, the establishment of transportation networks and the management of urban soil and vegetation (Hoyt 1939;McDonnell and Pickett 1990;Antrop 2004). As urbanisation continues to expand worldwide (as noted by Rydin et al. 2012), urban soil fungi have become increasingly important in relation to human health and environmental concerns (Grimm et al. 2008). Due to the variety of urban soils, diverse habitats, rapid urbanisation and high population density, the study and investigation of the diversity of soil fungi in different cities in China will provide valuable scientific data for understanding their ecological functions and maintaining public health safety.
As the foundation of all fungal research, accurate identification and taxonomy for the fungal species are the primary and important task. Morphology is the traditional method for species classification. However, with the dramatic increase in species, it is very difficult to identify the fungal species from morphology alone. Recently, there has been an increase in the use of DNA barcoding or DNA classification methods to address the identification of specific taxa (Hebert et al. 2003;Tautz et al. 2003). ITS rDNA has been the common barcoding marker of fungal species (Schoch et al. 2012;Stielow et al. 2015;Vu et al. 2016). Polygenic phylogeny also has been used widely for species identification; for example, Wang et al. (2019) studied phylogenetic re-evaluation of Thielavia and proposed a new family and many new species. Hou et al. (2023) redisposition of acremonium-like fungi in Hypocreales combined morphological characterisation and multilocus phylogenetic analysis. Peng et al. (2023) reported eight new species of Acrophialophora, based on multilocus phylogenetic analysis. Therefore, the fungal taxonomy combining DNA-based approaches and morphological characterisation has been a widely accepted and used method (Orr et al. 2020).

Sample collection and fungal isolation
Soil samples were collected from the green belts of hospitals, parks and university campuses in some cities in southern China. Samples were collected from 3-10 cm below the soil surface, placed in Ziploc plastic bags, brought back to the laboratory and processed immediately. The soil samples were mixed with clean, sterile, chicken feathers, approximately 2 cm long and moistened with sterile water (Li et al. 2022b). The samples were then incubated in the dark at 23-27 °C for 1 month. For fungi isolation, 2 g of each of the collected samples was suspended in 20 ml of sterile water in a 50 ml sterile conical flask. The conical flasks were thoroughly shaken using a Vortex vibration meter. The suspension was then diluted to a concentration of 10 -4 . Then, 1 ml of the diluted sample was transferred to a sterile Petri dish and modified Sabouraud's dextrose agar (SDA; peptone 10 g/l, dextrose 40 g/l, agar 20 g/l, 3.3 ml of 1% Bengal red aqueous solution) medium containing 50 mg/l penicillin and 50 mg/l streptomycin was added and mixed. The plates were incubated at 25 °C for 1-2 weeks and single colonies were selected from the plates and inoculated to new potato dextrose agar (PDA, potato 200 g/l, dextrose 20 g/l, agar 20 g/l) plates. The ITS regions of all isolates were sequenced and BLASTn searched in NCBI and assigned to a potential genus and species. According to Zhang et al. (2021a), the strains whose ITS sequences are less than 97% in the similarities to the closest strain were recognised as potential new species, which were further identified by combining morphological characterisation and phylogenetic analyses.

Morphological study
Strains of potentially new species were transferred to new plates of potato dextrose agar (PDA, Bio-way, China), malt extract agar (MEA, Bio-way, China) and oatmeal agar (OA, Bio-way, China) and were incubated at 25 °C for examining their colony morphology and microscopic morphology. The colony diameters and morphologies were determined after 14 days and the colony colours on the surface and reverse of inoculated Petri dishes were assessed according to the Methuen Handbook of Colour (Kornerup and Wanscher 1978). The characterisation 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 in MycoBank (https:// www.mycobank.org; accessed on 25 May 2022). Type collections of the novel species are deposited in the Mycological Herbarium of the Institute of Microbiology, Chinese Academy of Sciences, Beijing, China (HMAS; (https://nmdc.cn/ fungarium/). The ex-type living cultures and other isolates are deposited in the China General Microbiological Culture Collection Center (CGMCC; https://www. cgmcc.net/english/; accessed on 7 April 2022) or at the Institute of Fungus Resources, Guizhou University (GZAC), Guiyang City, Guizhou, China.

DNA extraction, PCR amplification and sequencing
Total genomic DNA was extracted from fungal mycelia using a BioTeke fungus genomic DNA extraction kit (DP2032, BioTeke, Beijing, China) following the manufacturer's instructions. The internal transcribed spacers (ITS) are widely used in fungal biodiversity and phylogenetic studies (Schoch et al. 2012;Stielow et al. 2015;Vu et al. 2016) and combining ITS and LSU improves species discrimination (Heeger et al. 2018;Vu et al. 2019); thus, ITS and the 28S nrRNA locus (LSU) sequences of all isolates were sequenced. In addition, more loci are often needed for specific taxa to obtain higher accuracy, so this study also amplified different loci for different taxa, such as β-tubulin (TUB), RNA polymerase II subunit (RPB2) and Twenty S rRNA accumulation (TSR1), translation elongation factor 1-alpha gene region (EF1A), calmodulin gene (CaM), partial γ-actin (ACT), DNA replication licensing factor (MCM7), translation elongation factor 3 (TEF3) and 60S ribosomal protein L10 (RP 60S L1) etc. (Table 1). The amplification reactions were performed in 25 μl final volumes consisted of 2 µl DNA template (10 ng/μl), 1 µl forward primer (10 µM), 1 µl reverse primer (10 µM), 12.5 µl 2× SanTaq PCR Master Mix (containing Taq polymerase, dNTP and Mg 2+ ; Sangon Biotech Co., Ltd, Shanghai, China) and 8.5 µl sterile water. The PCR was run using a T100 (Bio-Rad, California, USA) Thermal Cycler and the resulting amplified PCR products were sequenced in both directions using PCR primers. After amplification, the PCR products were visualised on a 1% agarose gel stained with ethidium bromide and the positive PCR products were then sent for sequencing to Sangon Biotech (Shanghai, China). The primer pairs and amplification conditions for each of the above-mentioned gene regions are provided in Table 1. All of the new sequences generated were deposited to GenBank (Suppl. material 1).
Additional specimens examined.  (Zhang et al. 2021a). In addition, the distinction between A. doliiformis and A. cylindricus is shown in the notes of A. cylindricus.
Notes. Pseudogymnoascus papyriferae is nested in clade B (Fig. 14). Clade B is composed of three species (P. shaanxiensis, P. australis and P. griseus) and six other strains that remain unidentified species (Minnis and Lindner 2013;Zhang et al. 2020bZhang et al. , 2021bVillanueva et al. 2021). Phylogenetic analysis clearly shows that P. papyriferae forms a distinct lineage (Fig. 14). Pseudogymnoascus papyriferae can be distinguished from P. shaanxiensis by the presence of arthroconidia (Zhang et al. 2020b). Pseudogymnoascus papyriferae can be differentiated from P. australis by the shape of intercalary conidia (drum-shaped, barrel-shaped, pyriform to elongated vs. subglobose to elongated and barrel-shaped, respectively) and rarely arthroconidia (Villanueva et al. 2021). In addition, P. papyriferae differs from P. griseus in the size and shape of its intercalary conidia (3.5-5.5 × 2.5-3.5 µm, drum-shaped, barrel-shaped, pyriform to elongated vs. 3.5-9.6 × 1.7-3.9 µm, subglobose to elongated and barrel-shaped, respectively) (Villanueva et al. 2021).   Figure 19. Concatenated phylogeny of the ITS and TUB gene regions of species in Clonostachys. Sixty strains are used. The tree is rooted in Fusarium acutatum (CBS 402.97) and Calonectria ilicicola (CBS 190.50). The tree topology of the BI was similar to the ML analysis. Bayesian posterior probability (≥ 0.7) and ML bootstrap values (≥ 70%) are indicated along branches (PP/ML). Novel species are in blue and bold font and "T" indicates type derived sequences. Description. Culture characteristics (14 d at 25 °C): Colony on PDA 13-15 mm diam., pale orange (6A3) to white (6A1) from centre to margin, fluffy, flocculent, nearly round, margin slightly sunken, exudates absent, diffusible pigments transparent and inconspicuous; reverse brownish-grey (6C2) to light orange (6A5) from centre to margin. Colony on MEA 12-13 mm diam., light yellow (4A4) to white (4A1) from centre to margin, hyphae kink into bundles, raised at the centre, nearly round, margin regular, exudates and diffusible pigments absent; reverse chrome yellow (5B8) to light yellow (4A4) from centre to margin. Colony on OA 12 mm diam., grey (5C1) to white (5A1) from centre to margin, flocculent, dense at the centre, sparse at margins, nearly round, margin regular, exudates absent, diffusible pigments transparent and inconspicuous; reverse raw umber (5F8) to grey (5D1) from centre to margin.
Notes. Pseudogymnoascus zongqii was placed as a member of clade J (Fig. 14). Clade J is composed of P. sinensis and many other strains that remain unidentified species (Minnis and Lindner 2013;Zhang et al. 2020b). Phylogenetically, P. zongqii forms a distinct lineage with strong support (Fig. 14). Morphologically, P. zongqii can be distinguished from P. sinensis by its subglobose conidia and absence of drum-or irregularly shaped intercalary conidia (Zhang et al. 2020b).
The genus Niesslia was established in 1869, with the type species N. chaetomium (Auerswald 1869). Niesslia is characterised by tuberculate perithecia, surrounded by brown, septate setae, clavate asci and filiform ascospores (Auerswald 1869). This genus is one of the more species-rich genera of ascomycetes, but has received relatively little taxonomic attention. Members of the genus are mostly saprophytic and globally distributed (Gams et al. 2019). Etymology. In reference to Guizhou, the Province where the type specimen was obtained.