Four New Species of Aspergillus Subgenus Nidulantes from China

Aspergillus subgenus Nidulantes includes species with emericella-like ascomata and asexual species. Subgenus Nidulantes is the second largest subgenus of Aspergillus and consists of nine sections. In this study, agricultural soils were sampled from 12 provinces and autonomous regions in China. Based on primary BLAST analyses, seven of 445 Aspergillus isolates showed low similarity with existing species. A polyphasic investigation, including phylogenetic analysis of partial ITS, β-tubulin, calmodulin, and RNA polymerase II second largest subunit genes, provided evidence that these isolates were distributed among four new species (Aspergillus guangdongensis, A. guangxiensis, A. sichuanensis and A. tibetensis) in sections Aenei, Ochraceorosei, and Sparsi of subgenus Nidulantes. Illustrated morphological descriptions are provided for each new taxon.

Temperature growth profiles are useful characters for distinguishing species in subgenus Nidulantes [4]. In section Nidulantes, most of the sexual species with globose ascospores grow optimally at 37 • C, while most of asexual and sexual species with stellate ascospores grow optimally at 27 to 37 • C [4]. In section Aenei, all species fail to grow at or above 40 • C [28]. Ascomata and ascospores are generally produced by members in sections Nidulantes and Aenei. Hülle cells that surround ascomata are specialized thick-walled cells associated with subgenus Nidulantes. In some species, emericella-like ascomata may be absent, yet the mycelium may give rise to masses or embedded Hülle cells, e.g., species in sections Aenei, Usti, Sparsi, and occasionally in others [4,17,28,[36][37][38].
Aspergillus nidulans is the model fungus for understanding eukaryotic cell biology and molecular processes [50]. Its whole genome was sequenced in 2005 [51] to study a wide range of cellular attributes, including recombination, genome editing, DNA repair, mutation, cell cycle control, nucleokinesis, pathogenesis, secondary metabolism, and experimental evolution [52][53][54][55][56][57][58]. In addition, species of subgenus Nidulantes are well known for their complex secondary metabolism. Aspergillus nidulans FGSC A4 is arguably the most intensively studied fungal strain with regard to its secondary metabolome [59]. Aspergillus pachycristatus is the commercial source of echinocandin B, the natural product precursor for the antifungal drug anidulafungin [60] and the clinical candidate rezafungin [61].
In this study, soils were sampled from 11 provinces (Anhui, Guangdong, Guangxi, Guizhou, Hainan, Henan, Jiangsu, Shandong, Shanxi, Sichuan, Yunnan) and the Tibet autonomous region in China during 2017. After selecting strains from soil isolation plates, all isolates were sequenced, and sequences were interrogated by public database searches. Based on sequence analysis, seven isolates (CGMCC 3.19704, 3.19705, 3.19706, 3.19707, 3.19708, 3.19709, 3.19710) showed low similarity to previously sequenced species, and further phylogenetic analysis placed these strains in subgenus Nidulantes. A multi-gene phylogeny was constructed from the ITS, BenA, CaM, and RPB2 gene sequences. Morphological features have been used to corroborate their phylogenetic distinctions and to define and describe four new species.

Soil Collection
Soils were sampledfrom farmlands and forests from 11 provinces in China, including Anhui, Guangdong, Guangxi, Guizhou, Hainan, Henan, Jiangsu, Shandong, Shanxi, Sichuan, Yunnan, and the Tibet autonomous region during 2017. At each sampling site, the superficial soil layer (approximately top 5 cm) was removed, and deeper soil was carefully excavated and sealed in a sterile plastic bag. Longitude and latitude of sampling locations and vegetation information were recorded. Soil samples were transported to the lab within three days.

Fungal Isolation and Preliminary Identification
From each soil collection, approximately 10 g were sieved to remove large particles, rocks, and debris, and then suspended in 90 mL sterile water. Aliquots of 10 −3 dilutions were spread onto potato dextrose agar (PDA, Guangdong Huankai Microbiological Technology Co., Ltd. (Guangzhou, China)) and rose bengal medium (RBM, Beijing Luqiao Technology Co., Ltd. (Beijing, China)) with tetracycline hydrochloride and chloramphenicol at 100 µg/mL added after autoclaving. After three to five days, emerging colonies were removed and transferred to new PDA plates. After 1 to 2 weeks of growth, strains were sorted into approximate morphological types and identified further by microscopy. Strains belonging to the genus Aspergillus were submitted for DNA extraction using the Ultraclean TM Microbial DNA isolation Kit (MoBio Laboratories Inc., Solana Beach, CA, USA) and CaM gene sequencing for accurate identification. The living ex-type strains of described new species were deposited in China General Microbiological Culture Collection Centre, Institute of Microbiology, Chinese Academy of Sciences (CGMCC, https://cgmcc.net/english/ accessed on 1 July 2022) and dried cultures were deposited in herbarium (HMAS) located in same institute (accessed on 1st July 2022).

Phylogenetic Analysis
For strains (Table 1) unassignable to a known species, BenA, CaM, and RPB2 gene fragments were amplified using previously described primers and programs [62] and sequenced via ABI 3730XL DNA sequencer (Applied Biosystems). Reference sequences from strains of subgenus Nidulantes were used for phylogenetic reconstruction and placement of unknown isolates [4,24,29,[31][32][33]. Aligned sequences were analyzed with FindModel (http://hiv.lanl.gov/content/sequence/findmodel/findmodel.html, accessed on 1 January 2022) to select the most appropriate model of nucleotide substitution [63]. Maximum likelihood analyses with 1000 bootstrap replicates were run using RAxML [64]. Bayesian analyses were run with MrBayes v. 3.2 [65]. The sample frequency was set to 100, and the first 25% of trees were removed as a burn-in. BenA, CaM and RPB2 sequences from A. flavipes NRRL 302 were used as outgroup. The resulting trees were analyzed with FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 1 January 2022) and visualized using Adobe Illustrator CS5. BI posterior probabilities (pp) values and bootstrap (bs) percentages of analysis were labeled at the branch nodes. Values less than 0.95 pp and less than 70% bs were not shown. Branches with values more than 1.00 pp and 95% bs are thickened.

Morphology
Macroscopic and microscopic characters were observed and measured as previously described. The isolates were inoculated and incubated for 7 d on the agar media Czapek yeast autolysate agar (CYA), yeast extract sucrose agar (YES), creatine sucrose agar (CREA), dichloran 18% glycerol agar (DG18), oatmeal agar (OA), and malt extract agar (MEA; Oxoid CM0059) [4]. Colony colors were referenced to numbered color codes in parentheses [66]. Light microscope preparations were made from one wk old colonies grown on MEA. To induce ascomata formation, colonies were incubated for more than two weeks. A Zeiss Stereo Discovery V20 dissecting microscope and a Zeiss AX10 Imager A2 light microscope, both equipped with an Axiocam 506 color camera and ZEN v.2.0 software (made in Germany), were used to capture digital images.

Phylogeny
We used concatenated sequence data combining the ITS, BenA, CaM and RPB2 regions for defining relationships within subgenus Nidulantes. The total length of the aligned data set was 2772 characters, containing 626, 576, 651, and 919 bp for ITS, BenA, CaM, and RPB2 respectively. K2P + G model was used for ITS, GTR+G model was used for BenA, CaM and RPB2. The alignment sequences and the tree file were deposited in TreeBASE (S29781, https://www.treebase.org accessed on 14 October 2022).
Section Aenei (1.00 pp, 100 bs) includes A. aeneus, A. bicolor, A. crustosus, A. discophorus, A. eburneocremeus, A. foeniculicola, A. heyangensis, A. karnatakaensis, A. spectabilis, and two new species, A. sichuanensis and A. tibetensis. Aspergillus sichuanensis is represented by three strains isolated from soils under pea cultivation, Sichuan province, China. They formed a strongly supported clade closely related with A. heyangensis and A. crustosus in the multigene phylogeny and the BenA, CaM and RPB2 single-gene trees (Figures 1 and S2-S4). In the ITS phylogeny, A. sichuanensis clusters outside the clade containing A. crustosus, A. heyangensis and A. tibetensis ( Figure S1). Aspergillus tibetensis clustered outside the groups with A. crustotus, A. heyanensis and A. sichuanensis in the multi-gene phylogeny and in the BenA, CaM and RPB2 single-gene trees (Figures 1 and S2-S4). In the ITS phylogeny, A. sichuanensis formed a statistically unsupported clade with A. crustosus and A. heyanensis ( Figure S1).
Notes: In the phylogenetic analyses, the two A. guangxiensis strains were positioned at a unique branch basal to branches with A. conjunctus, A. panamensis and A. anthodesmis (Figure 1).
Notes: Aspergillus tibetensis grows restrictedly, and its hyphae develop orange pigment after one week, features that differentiate it from other species of section Aenei. According to BLAST analyses, ITS of A. tibetensis is close to A. crustosus (97% similarity), BenA, CaM and RPB2 sequences of A. tibetensis are close to A. spectabilis (94%, 86%, 97% respectively).

Discussion
In this study, soil samples were collected from 11 provinces and the Tibet autonomous region and ranged across temperate and tropical regions, high and low altitude areas of China. After sorting the isolates into morphological types, preliminary identification revealed 445 Aspergillus strains of 6840 fungal strains. These Aspergillus species belonged to six subgenera, and among them 93 isolates belonged to subgenus Nidulantes, and seven isolates were identified as four new specie belonging to sections Aenei, Ochraceorosei, and Sparsi of subgenus Nidulantes, respectively (Table S1).
Some of the species in subgenus Nidulantes produce a sexual state. These species with emericella-like ascomata mostly belong to section Nidulantes (A. stellatus, A. aurantiobrunneus, A. nidulans and A. multicolor clades) [4] and section Aenei (A. bicolor, A. discophorus, A. foeniculicola and A. spectabilis) [21,28,68]. Two new species (A. sichuanensis and A. tibetensis) assigned to section Aenei in this study only produced Hülle cells in culture, but not ascospores. In section Aenei, all species produce subglobose to subclavate, biseriate conidial heads, however, the length of conidiophores, size of vesicles and conidial ornamentation can be used to distinguish species in this section [17,28].
Species in sections Sparsi and Ochraceorosei generally originate from tropical or subtropical soils [17,[36][37][38][69][70][71]. Two new species (A. guangdongensis and A. guangxiensis) reported in this study were isolated from adjacent subtropical provinces (Guangdong and Guangxi provinces). Section Sparsi species are characterized by thin, sparse and submerged mycelia [17], such as observed in A. guangxiensis. Other features common in this section include globose to subglobose, biseriate conidial heads that are fertile over the entire surface. Most species in section Sparsi produce smooth to finely roughened conidia. The only exception is A. biplanus, which produces conspicuously echinulate conidia [17].
Species assigned to section Ochraceorosei clustered in two main branches, A. guangdongensis clustered together with A. funiculosus, while the other two species, A. ochraceoroseus and A. rambellii, clustered together. The classification of A. funiculosus in a species group was originally doubtful, even though Raper and Fennell (1965) accepted A. funiculosus as the only uniseriate species in section Sparsi (A. sparsus group) [17]. Phylogenetic classifications showed that A. funiculosus belonged to section Ochraceorosei even though bootstrap and Bayesian support values were low [4,34]. Introducing a second uniseriate species, A. guangdongensis, from this study, has clarified and stabilized the phylogenetic position of A. funiculosus. The two species form a distinct pair of sister species both with uniseriate vesicles. Aspergillus guangdongensis produces microcolonies on CYA at 37 • C, however, the growth characteristics of A. funiculosus at 37 • C were not mentioned in its original description [72]. Shumi et al. (2004) reported that profuse aerial mycelium was present at 37 • C during an enzyme screening experiment [73]. Not all species in section Ochraceorosei species produce Hülle cells. Aspergillus ochraceoroseus and A. rambellii, which appeared as sister species on a separate branch (Figure 1), are biseriate and do not grow at 37 • C [36,37].
To date, thirty-six species of subgenus Nidulantes have been recorded in China. The China General Microbiological Culture Collection Centre (CGMCC) maintains 11 species [4,74,75] from 15 provinces, and most of them originated from soil, air, and moldy materials. The first extensive study of subgenus Nidulantes and related species in China [74] was based on morphological identifications and recorded 17 species in subgenus Nidulantes and eight species in Emericella (assigned in sections Aenei, Nidulantes, Covernicolarum according to current classification). Sun and Qi described Aspergillus heyangensis [67], Li et al. (1998) collected and examined 1402 soil samples from northeastern (Jiamusi and Harbin), northern (Beijing), and northwestern (Yunchuan and Xian) China, and recorded eight species and one variety: Emericella acristata, E. corrugate, E. foeniculicola, E. miyajii, E. nidulans var. lata, E. quadrilineata, E. rugulosa and E. undulata [76]. Later, Aspergillus keveioides and A. sigarelli in section Usti and Emericella miraensis in section Nidulantes were newly described [33,77,78]. With four new species described in this study, subgenus Nidulantes is now extended to 40 species in China and includes species in sections Sparsi and Ochraceorosei. The findings of this study will improve our understanding of the distribution of Aspergillus species in China and provide a basis for the further development and application of species in the subgenus Nidulantes.