New Species of Aspergillus (Aspergillaceae) from Tropical Islands of China

Aspergillus species are cosmopolitan and ubiquitous, closely related to human daily life. They are also of food, industrial and medical importance. From the examination of cultures isolated from soil samples collected on tropical islands of China, four new species of the genus were discovered based on phylogenetic analyses and morphological comparisons. Aspergillus xishaensis sp. nov. and A. neoterreus sp. nov. belong to sections Flavipedes and Terrei of subgenus Circumdati, and A. hainanicus sp. nov. and A. qilianyuensis sp. nov. are in sections Cavernicolarum and Nidulantes of subgenus Nidulantes. To accommodate A. hainanicus, a new series Hainanici was proposed. Detailed descriptions and illustrations of the new taxa were provided.


Introduction
Species of Aspergillus P. Micheli ex Haller are cosmopolitan and ubiquitous. Some of them are closely related to human daily life. Strains of A. niger Tiegh. and A. oryzae (Ahlb.) Cohn were used for the fermentation of food for more than two millennia and the manufacturing of food enzymes for over 50 years [1]. Aspergillus niger is also a workhorse and cell factory for the production of citric acid, an organic acid with high economic importance, which is widely used in beverage, food, detergents, cosmetics and pharmaceutical industries [2]. Aflatoxins, produced by A. flavus Link and other aspergilli, are highly toxic secondary metabolites and severely contaminate food supplies of humans and animals, resulting in health hazards and even death [3]. Some black aspergilli were reported to be postharvest pathogens of economically important crops, e.g., A aculeatus Iizuka, A. japonicus Saito and A. uvarum G. Perrone et al. infecting the fruits of grapes [4]. Aspergillosis infections caused by Aspergillus species are of significant morbidity and mortality. Mostly, they are attributed to A. fumigatus Fresen., followed by A. flavus and A. terreus Thom [5].
During the examinations of the cultures isolated from sandy soil collected on tropical islands of China, four new species were discovered based on phylogenetic analyses and morphological comparisons. They belong to sections Flavipedes and Terrei of subgenus Circumdati and sections Cavernicolarum and Nidulantes of subgenus Nidulantes, respectively. The detailed descriptions and illustrations of the new taxa are provided.

Fungal Materials
Cultures were isolated from sandy soil collected on tropical islands of China in 2015. Dried cultures were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS), and living ex-type strains were preserved in the China General Microbiological Culture Collection Center (CGMCC).

Molecular Experiments
DNA was extracted from the cultures grown on PDA for 7 days using the Plant Genomic DNA Kit (DP305, TIANGEN Biotech, Beijing, China). Polymerase chain reaction (PCR) amplifications of the internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) gene regions were conducted with the routine methods [21][22][23][24]. The products were purified and subject to sequencing on an ABI 3730 DNA Sequencer (Applied Biosystems). Although the ITS region is proposed as the universal DNA barcode for fungi, it is not sufficient to distinguish species of Aspergillus. The ITS sequences provided in this study might be helpful for other researchers in case of need.

Phylogenetic Analyses
Forward and reverse sequences newly generated in this study were assembled using Seqman v. 7.1.0 (DNASTAR Inc., Madison, WI, USA). The assembled sequences were deposited at GenBank. The sequences used for phylogenetic analyses are listed in Tables 1 and 2. Sequences of the combined loci (BenA, CaM and RPB2) of each of the two subgenera were aligned using MAFFT v. 7.221 [25] and then manually edited and combined in BioEdit v. 7.1.10 [26] and MEGA v. 6.0.6 [27]. The combined datasets of individual subgenera were analyzed to infer their phylogeny. Maximum likelihood (ML) analyses were conducted using RAxML-HPC2 [28]

Phylogenetic Analysis
To determine the positions of the isolates, two combined datasets (BenA + CaM + RPB2) of Aspergillus subgenera Nidulantes and Circumdati were compiled and analyzed. The detailed characteristics of the datasets are listed in Table 3. In the phylogeny of Aspergillus subg. Nidulantes (Figure 1), the strains ZC79 and ZC101 were located in sect. Cavernicolarum and Nidulantes, respectively. The strain ZC79 was sister to the species of ser. Cavernicolarum and Egyptiaci, and a new series was proposed as ser. Hainanici to accommodate it. The strain ZC 101 formed a distinct lineage in ser. Versicolores. As shown in the phylogenetic tree of Aspergillus subg. Circumdati (Figure 2), the strain ZC108 was a member of sect. Flavipedes ser. Flavipedes, and clustered with A. micronesiensis and A. neoflavipes. The strain ZC111 was revealed to be affiliated to sect. Terrei ser. Terrei, and shared a close relationship with A. citrinoterreus.
Note: This species is phylogenetically related to A. californicus, A. cavernicola, A. kassunensis and A. subsessilis of ser. Cavernicolarum and A. egyptiacus of ser. Egyptiaci (Figure 1), but differs from the former four species in its brown stipe and larger, strongly echinulate conidia, and differs from the latter one due to no growth on CYA at 37 • C, slower growth rates on MEA and YES, brown stipe and larger and strongly echinulate conidia (Table 4).
Note: This species is phylogenetically related to A. citrinoterreus ( Figure 2) but differs in slower growth rate on CYA and smaller conidia (Table 4).
Note: This species is phylogenetically related to A. micronesiensis and A. neoflavipes ( Figure 2) but differs from them in slower growth rates on CYA, MEA and YES and larger conidia (Table 4).

Discussion
Aspergillus is a large genus with more than 400 accepted species and more than 1000 names. A comprehensive taxonomic treatment of the genus was recently established on the basis of molecular data and morphological characteristics [6]. Six subgenera, twentyseven sections and seventy-five series were currently accepted, among which five new sections and seventy-three new series were erected. Based on the above treatment, researchers are able to quickly position their materials to specific ranks of series, sections and subgenera. Three of the four new species described in this study were classified into the known series, except for A. hainanicus, for which the new series Hainanici is proposed. Along with future discovery of new taxa, the current classification system may be updated.
In the Flavi, Fumigati, Nigri and Terrei sections of Aspergillus, some species cause the infectious disease aspergillosis, such as the most frequently occurred and well-known pathogen A. fumigatus [36]. In sect. Nidulantes, A. versicolor (Vuill.) Tirab., a close relative of A. qilianyuensis, was isolated from the skin [37] and nails [38] of humans and also invasively infected multiple organs of dogs [39,40]. Aspergillus hongkongensis C.C. Tsang et al. causes onychomycosis [41]. In subgen. Circumdati, A. citrinoterreus J. Guinea et al. and A. suttoniae J.P.Z. Siqueira et al. were isolated from the sputum of humans [35,42], and A. alabamensis Balajee et al. from the wounds of humans [43]. Whether others of these sections are potentially pathogenic requires future investigation.
Tropical islands represent a unique ecosystem. Due to their extremely isolated location and special environmental conditions, some of them are considered as the world's biodiversity hotspots. Several species of Aspergillus were recorded from similar geographical origins, such as A. griseoaurantiacus Visagie et al. and A. micronesiensis Visagie et al. from Micronesia [33], and A. puulaauensis Jurjević et al. from Hawaii [32]. The four new species were all derived from the soil samples of the Xisha Islands, which seem to exhibit high species diversity. Further explorations on tropical islands are desperately needed, and we certainly expect to find more new fungi there.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The sequences newly generated in this study have been submitted to the GenBank database.