New species of Aspergillus in sections Cavernicolarum and Nigri from terrestrial ecosystems of China (Eurotiales, Aspergillaceae)

275 New species of Aspergillus in sections Cavernicolarum and Nigri from terrestrial ecosystems of China (Eurotiales, Aspergillaceae) Lu-Yao Peng1,2 , Wen-Ying Zhuang1, Xin-Cun Wang1 1 State Key Laboratory of Microbial Diversity and Innovative Utilization, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China 2 University of Chinese Academy of Sciences, Beijing 100049, China Corresponding author: Xin-Cun Wang ([email protected]) Copyright: © Lu-Yao Peng et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Aspergillus species are of great industrial, agricultural, and medicinal importance. During investigations on the biodiversity of Aspergillaceae, two species of Aspergillus isolated from soil samples in China were identified as new to science based on sequence analyses and morphological comparisons. Aspergillus hebeiensis from a traditional cultural and Buddhist heritage site is the second member of the series Hainanici in section Cavernicolarum of subgenus Nidulantes, while A. xishuangbannaensis from a tropical nature reserve is classified in subgenus Circumdati, section Nigri, series Japonici. Detailed descriptions and illustrations of both species are provided, and the ecological functions of their habitats are also discussed. Key words: Aspergillaceae, biodiversity, Eurotiales, phylogeny, taxonomy Introduction Species of Aspergillus P. Micheli ex Haller are ubiquitous in various environments and have a long history of exploitation by humans. Some species have been used for food fermentations for centuries, especially in East Asia. Aspergillus oryzae (Ahlb.) Cohn and A. sojae Sakag. & K. Yamada ex Murak. play crucial roles in the production of rice wine, soybean pastes, and soy sauce (Bennett 2009), and A. niger Tiegh. is used for fermentations of baijiu (Xu et al. 2022) as well as pu-erh tea (Frisvad et al. 2007). More than 100 years ago, the ability of A. niger to produce citric acid was industrially exploited in 1919 (Schuster et al. 2002). Aspergillus terreus Thom was the first of the major statins to produce lovastatin, a medicine used to lower cholesterol (Bennett 2009). Aspergillus niger and A. terreus are also efficient agents for bioleaching of rare earth elements with low environmental impact (Mowafy 2020). Aspergillus cvjetkovicii Jurjević et al. can protect against phytopathogens through interspecies chemical signaling in the phyllosphere of rice as a biocontrol agent (Fan et al. 2024). On the other hand, some species pose severe threats to human health. Mycotoxins are produced by certain Aspergillus species, causing food contamination, e.g., aflatoxins by A. flavus Link and A. parasiticus Speare, and ochratoxin A by A. ochraceus G. Wilh. and A. niger (Xue et al. 2025). Aspergillus fumigatus Academic editor: Xinlei Fan Received: 22 September 2025 Accepted: 22 October 2025 Published: 3 November 2025 Citation: Peng L-Y, Zhuang W-Y, Wang X-C (2025) New species of Aspergillus in sections Cavernicolarum and Nigri from terrestrial ecosystems of China (Eurotiales, Aspergillaceae). MycoKeys 124: 275–290. https://doi. org/10.3897/mycokeys.124.172775 MycoKeys 124: 275–290 (2025) DOI: 10.3897/mycokeys.124.172775 276 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China Fresen. is known as the primary causative agent of aspergillosis, followed by A. flavus, A. niger, and A. terreus (Khan et al. 2024). The genus Aspergillus was originally introduced in 1729 and validated in 1768. It was divided into six subgenera, 27 sections, and 75 series, with 446 species recognized (Houbraken et al. 2020). A new series, Aspergillus ser. Hainanici, was recently proposed (Wang and Zhuang 2022), and the accepted species of the genus increased to 453 (Visagie et al. 2024). Subsequently, 16 newly described species were added: one each from Europe and North America, two from South America, and the remaining 12 from Asia. Aspergillus albicolor D.S. Paiva was reported from Portugal, A. pseudoalabamensis Cañete-Gibas et al. from the USA, and A. alvaroi J.M.S. Lima et al. and A. guanovespertilionum J.M.S. Lima et al. from Brazil. Among the 12 Asian taxa, five were from China (A. cylindricus Zhi Y. Zhang et al., A. doliiformis Zhi Y. Zhang et al., A. liaoningensis C. Liu et al., A. plumeriae C. Liu et al., A. subinflatus C. Liu et al.), A. dhakephalkarii Rajeshk. et al. and A. patriciawiltshireae Rajeshk. et al. from India, A. hubkae Y.B. Zhou et al. and A. mahabadiensis Abdollahz. & O. Ghaderi from Iran, A. verrucosus R. Hagiuda & D. Hirose from Japan, A. ullungdoensis Hyang B. Lee from South Korea, and A. halopiscium V.N. Thanh et al. from Vietnam. During investigations on the biodiversity of Aspergillaceae in China, two species of Aspergillus isolated from soil were identified as new to science based on sequence analyses and morphological comparisons. Detailed descriptions and illustrations are provided. Materials and methods Fungal materials Cultures were isolated from soil samples collected from Hebei and Yunnan provinces, China. Dried cultures were preserved in the Herbarium Mycologicum Academiae Sinicae (HMAS, Beijing, China), and the living ex-type strains were deposited in the China General Microbiological Culture Collection Center (CGMCC, Beijing, China). Morphological observations Morphological characteristics were observed and recorded according to standardized methods (Samson et al. 2014). Four standard growth media were adopted: Czapek yeast autolysate agar (CYA; yeast extract, Oxoid, Hampshire, UK), malt extract agar (MEA; Amresco, Solon, OH, USA), yeast extract agar (YES), and potato dextrose agar (PDA). The methods for colonial inoculation, incubation, macroscopic and microscopic examinations, and digital capture followed our previous studies (Wang and Zhuang 2022; Wang et al. 2023; Peng et al. 2025). DNA extraction, PCR amplification, and sequencing DNA was extracted from living cultures grown on PDA for 7 days using the Plant Genomic DNA Kit (DP305; TIANGEN Biotech, Beijing, China). Polymerase chain reaction (PCR) amplifications of internal transcribed spacer (ITS), beta-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second-largest 277 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China subunit (RPB2) were conducted using routine methods (Samson et al. 2014). The forward and reverse primers used for each locus were as follows: ITS5 (5’-GGA AGT AAA AGT CGT AAC AAG G-3’) and ITS4 (5’-TCC TCC GCT TAT TGA TAT GC-3’) for ITS (White et al. 1990); Bt2a (5’-GGT AAC CAA ATC GGT GCT GCT TTC-3’) and Bt2b (5’-ACC CTC AGT GTA GTG ACC CTT GGC-3’) for BenA (Glass and Donaldson 1995); CMD5 (5’-CCG AGT ACA AGG ARG CCT TC-3’) and CMD6 (5’-CCG ATR GAG GTC ATR ACG TGG-3’) for CaM (Hong et al. 2005); and 5F (5’- GAY GAY MGW GAT CAY TTY GG-3’) and 7CR (5’-CCC ATR GCT TGY TTR CCC AT-3’) for RPB2 (Liu et al. 1999). The products were sequenced on an ABI 3730 DNA Sequencer (Applied Biosystems, Foster City, CA, USA). Phylogenetic analyses The newly generated forward and reverse sequences in this study were assembled using SeqMan v. 7.1.0 (DNASTAR Inc., Madison, WI, USA). The assembled sequences were deposited in GenBank, with accession numbers shown in bold (Tables 1, 2). The additional sequences used for phylogenetic analyses are also listed. Sequences were aligned using MAFFT v. 7.221 (Katoh and Standley 2013), either as individual single-gene datasets (ITS, BenA, CaM, and RPB2) or concatenated datasets. They were then manually edited and concatenated in BioEdit v. 7.1.10 (Hall 1999) and MEGA v. 11.0.13 (Tamura et al. 2021). Maximum likelihood (ML) analyses were performed using the IQ-TREE web server (Trifinopoulos et al. 2016) with the default automatic substitution model and bootstrap (BP) iteration (1,000 replicates) settings. Bayesian inference (BI) analyses were conducted with MrBayes v. 3.2.7 (Ronquist et al. 2012). Modeltest v. 3.7 (Posada and Crandall 1998) was adopted to determine appropriate nucleotide substitution models and parameters. Four MCMC chains (three heated and one cold) were run for at least 1 million generations, and posterior probability (PP) values were calculated based on the remaining 75% of trees after the burn-in phase. The consensus trees were viewed using FigTree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree, accessed on 28 December 2023). Table 1. Species and sequences used in the phylogenetic analyses for Aspergillus subgenus Nidulantes section Cavernicolarum. Series Species Strain Locality Substrate ITS BenA CaM RPB2 Cavernicolarum A. californicus Frisvad et al., 2011 CBS 123895 T USA chaparral of Adenostoma fasciculatum FJ531153 FJ531180 FJ531128 MN969065 A. cavernicola Lörinczi, 1969 CBS 117.76 T Romania on walls of cave EF652508 EF652332 EF652420 EF652244 A. kassunensis Baghd., 1968 CBS 419.69 T Syria soil EF652461 EF652285 EF652373 EF652197 A. subsessilis Raper & Fennell, 1965 CBS 502.65 T USA desert soil EF652485 EF652309 EF652397 EF652221 Egyptiaci A. egyptiacus Moub. & Mustafa, 1972 CBS 656.73 T Egypt sandy soil EF652504 EF652328 EF652416 EF652240 Hainanici A. hainanicus X.C. Wang & W.Y. Zhuang, 2022 CGMCC 3.20888 T China: Hainan sandy soil OM414846 OM475626 OM475630 OM475634 A. hebeiensis X.C. Wang, L.Y. Peng & W.Y. Zhuang, sp. nov. JJJ40-31 T China: Hebei soil under Platycladus orientalis PV883250 PV877068 PV877070 PV877073 JJJ40-12 China: Hebei soil under Platycladus orientalis PV883251 n.a. PV877071 PV877074 GenBank accession numbers in bold indicate the newly generated sequences. “n.a.” is the abbreviation for “not available.” 278 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China Results To determine the species identities of the investigated strains, the single-gene datasets (ITS, BenA, CaM, and RPB2) and the concatenated three-locus (BenA + CaM + RPB2) dataset were compiled and analyzed. The detailed characteristics of the datasets are listed in Table 3. In the phylogeny of section Cavernicolarum (Fig. 1), the strains JJJ40-12 and JJJ40-31, representing the same species, were located in series Hainanici as sister to Aspergillus hainanicus. The close relationship between these two species was strongly supported by statistical values, inferred either from the concatenated dataset (MLBP = 100, BIPP = 1.00; Fig. 1) or from the single-gene datasets (MLBP = 100; Suppl. material 1: figs S1–S4). In the phylogenetic tree of section Nigri series Japonici (Fig. 2), strain ZYN0501 was grouped into a small clade associated with the following species: A. japonicus, A. indologenus, and A. uvarum. A similar tree topology was shown in the BenA analysis (Suppl. material 1: fig. S5), but it differed somewhat from the other two single-gene inferences. Strain ZYN05-01 clustered with A. uvarum in the CaM tree (MLBP = 82; Suppl. material 1: fig. S6), but it was closely related to A. japonicus in the RPB2 phylogeny (MLBP = 94; Suppl. material 1: fig. S7). Table 2. Species and sequences used in the phylogenetic analyses for Aspergillus subgenus Circumdati section Nigri. Series Species Strain Locality Substrate ITS BenA CaM RPB2 Japonici A. aculeatinus Noonim et al., 2008 CBS 121060 T Thailand dried parchment and green beans of Coffea arabica EU159211 EU159220 EU159241 HF559233 A. aculeatus Iizuka, 1953 CBS 172.66 T Japan unknown EF661221 HE577806 EF661148 EF661046 A. brunneoviolaceus Bat. & H. Maia, 1955 CBS 621.78 T Brazil gills of Osteichthyes AJ280003 EF661105 EF661147 EF661045 A. dhakephalkarii Rajeshk. et al., 2025 NFCCI 5750 T India rhizosphere soil associated with Anthurium andraeanum PP741453 PP739067 PP739063 PP739059 A. floridensis Jurjevic et al., 2012 NRRL 62478 T USA air MN431366 HE984412 HE984429 HE984376 A. hydei Doilom, 2020 KUMCC 18-0196 T China: Yunnan air under Quercus variabilis MT152332 MT161679 MT178247 MT384370 A. indologenus Frisvad et al., 2011 CBS 114.80 T India soil AJ280005 AY585539 AM419750 HE984366 A. japonicus Saito, 1906 CBS 114.51 T unknown unknown AJ279985 HE577804 FN594551 MN969079 A. labruscus Fungaro et al., 2017 IBT 33586 T Brazil fruit of Vitis labrusca KU708544 KT986014 KT986008 MN969196 A. oxumiae C.N. Figueiredo et al., 2020 CCDCA 11546 T Brazil soil cultivated with Agave sisalana MN431160 n.a. MN531842 MN521389 A. patriciawiltshireae Rajeshk. et al., 2025 NFCCI 5959 T India soil PQ826401 PQ855384 PQ855382 PQ855386 A. saccharolyticus A. Sørensen et al., 2011 CBS 127449 T Denmark toilet seat of treated Quercus wood HM853552 HM853553 HM853554 HF559235 A. serratalhadensis L.F. Oliveira et al., 2018 URM 7866 T Brazil soil MH169127 LT993222 LT993223 LT995971 A. trinidadensis Jurjevic et al., 2012 NRRL 62479 T TrinidadTobago air MN431380 HE984420 HE984434 HE984379 A. uvarum G. Perrone et al., 2008 CBS 121591 T Italy grape berries AM745757 AM745751 AM745755 HE984370 A. xishuangbannaensis X.C. Wang, L.Y. Peng & W.Y. Zhuang, sp. nov. ZYN05-01 T China: Yunnan soil in limestone seasonal rainforest PV883252 PV877069 PV877072 PV877075 Nigri A. niger Tiegh., 1867 CBS 554.65 T France unknown EF661186 EF661089 EF661154 EF661058 GenBank accession numbers in bold indicate the newly generated sequences. “n.a.” is the abbreviation for “not available.” 279 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China Taxonomy Aspergillus hebeiensis X.C. Wang, L.Y. Peng & W.Y. Zhuang, sp. nov. Fig. 3 Fungal Names: FN 572829 Etymology. The specific epithet refers to the type locality. In Aspergillus subgenus Nidulantes section Cavernicolarum series Hainanici. Typification. China • Hebei Province, Handan City, Fengfeng Mining District, Xiangtangshan Caves, one of the First Batch of Key Cultural Relics Units under National Protection of China, Northern Xiangtangshan, 36°32'2"N, 114°9'40"E, in soil under Platycladus orientalis (L.) Franco, 17 July 2023, Xin-Cun Wang, culture, Lu-Yao Peng, JJJ40-31 (holotype HMAS 354080, ex-type strain CGMCC 3.29151). Table 3. Detailed characteristics of the datasets. Dataset Gene fragment No. of seq. Length of alignment (bp) No. of variable sites No. of parsimony-informative sites Model for BI Cavernicolarum ITS 8548 64 44 BenA 7471 150 83 CaM 8553 204 129 RPB2 81071 241 151 BenA+CaM+RPB2 82095 595 363 TIMef+I Japonici BenA 16 530 235 108 CaM 17 580 245 128 RPB2 17 1052 273 155 BenA+CaM+RPB2 17 2162 753 391 TrN+I+G Abbreviations of the model: TIMef+I (equal-frequency transition model with invariant sites), TrN+I+G (Tamura–Nei model with invariant sites and gamma distribution). Figure 1. Maximum likelihood phylogeny of Aspergillus subgenus Nidulantes section Cavernicolarum inferred from the combined BenA, CaM, and RPB2 dataset. Bootstrap values ≥ 70% (left) or posterior probability values ≥ 0.95 (right) are indicated at nodes. Asterisks denote 100% bootstrap or 1.00 posterior probability. 0.04 Aspergillus californicus CBS 123895 T Aspergillus hebeiensis JJJ40-12 Aspergillus cavernicola CBS 117.76 T Aspergillus hainanicus CGMCC 3.20888 T Aspergillus subsessilis CBS 502.65 T Aspergillus egyptiacus CBS 656.73 T Aspergillus kassunensis CBS 419.69 T Aspergillus hebeiensis JJJ40-31 T */* -/* */* 92/* ser. Cavernicolarum ser. Hainanici ser. Egyptiaci 280 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China DNA barcodes. ITS PV883250, BenA PV877068, CaM PV877070, RPB2 PV877073. Colony diam. 7 days, 25 °C (unless stated otherwise): CYA 15–17 mm; CYA 37 °C 12–15 mm; MEA 19–20 mm; YES 18–19 mm; PDA 16–17 mm. Colony characteristics. On CYA 25 °C, 7 days: Colonies nearly circular, slightly concave at centers; margins moderately wide, entire; mycelia white and then yellow; texture velutinous; sporulation sparse; conidia en masse greyish; soluble pigments absent; exudates absent; reverse buff to yellow brown. On CYA 37 °C, 7 days: Colonies nearly circular or irregular, slightly protuberant at centers; margins narrow to moderately wide, fimbriate; mycelia white and Figure 2. Maximum likelihood phylogeny of Aspergillus subgenus Circumdati section Nigri series Japonici inferred from the combined BenA, CaM, and RPB2 dataset. Bootstrap values ≥ 70% (left) or posterior probability values ≥ 0.95 (right) are indicated at nodes. Asterisks denote 100% bootstrap or 1.00 posterior probability. 0.04 Aspergillus oxumiae CCDCA 11546 T Aspergillus trinidadensis NRRL 62479 T Aspergillus niger CBS 554.65 T Aspergillus indologenus CBS 114.80 T Aspergillus serratalhadensis URM 7866 T Aspergillus uvarum CBS 121591 T Aspergillus labruscus IBT 33586 T Aspergillus hydei KUMCC 18-0196 T Aspergillus aculeatinus CBS 121060 T Aspergillus brunneoviolaceus CBS 621.78 T Aspergillus saccharolyticus CBS 127449 T Aspergillus aculeatus CBS 172.66 T Aspergillus xishuangbannaensis ZYN05-01 T Aspergillus patriciawiltshireae NFCCI 5959 T Aspergillus floridensis NRRL 62478 T Aspergillus japonicus CBS 114.51 T Aspergillus dhakephalkarii NFCCI 5750 T */* 87/* */* 72/- 99/* 91/* 96/* 81/0.98 78/* 91/* 87/* 82/* 92/* 281 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China Figure 3. Aspergillus hebeiensis (JJJ40-31). A. Colonies: top row left to right, obverse CYA, MEA, YES, and PDA; bottom row left to right, reverse CYA, MEA, YES, and PDA; B–F. Conidiophores; G. Conidia. Scale bars: 17.5 µm (B, C); 15 µm (D); 12.5 µm (E); 10 µm (G, F). 282 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China then buff; texture velutinous; sporulation sparse; conidia en masse brownish; soluble pigments absent; exudates absent; reverse buff to yellow brown. On MEA 25 °C, 7 days: Colonies irregular, protuberant; margins narrow, entire; mycelia white and then cream; texture velutinous; sporulation sparse; conidia en masse creamish; soluble pigments absent; exudates absent; reverse buff to yellow brown. On YES 25 °C, 7 days: Colonies nearly circular, concave at centers, radially sulcate; margins narrow, entire; mycelia pale; texture velutinous; sporulation sparse; conidia en masse greyish; soluble pigments absent; exudates absent; reverse yellow brown to orange brown. On PDA 25 °C, 7 days: Colonies nearly circular, protuberant; margins narrow, entire; mycelia white and then cream; texture velutinous; sporulation sparse; conidia en masse creamish to brownish; soluble pigments yellow; exudates absent; reverse buff to yellow brown, occasionally with dark brown sectors. Micromorphology. Conidial heads radiate; stipes short, 65–110 (–140) × 4.0–7.0 µm, not septate, walls thick, smooth, brown; vesicles 8.5–13 × 8.5– 13 µm, subglobose to globose; biseriate; metulae 5.0–8.5 × 3.5–6.5 µm, cylindrical to obovate, covering almost a half to two-thirds surface of the vesicle; phialides 5.5–8.0 × 3.5–4.0 µm, flask-shaped; conidia 6.0–7.5 µm, subglobose, vivid green, strongly echinulate. Additional strain examined. China • Hebei Province, Handan City, Fengfeng Mining District, Xiangtangshan Caves, one of the First Batch of Key Cultural Relics Units under National Protection of China, Northern Xiangtangshan, 36°32'2"N, 114°9'40"E, in soil under Platycladus orientalis (L.) Franco, 17 July 2023, Xin-Cun Wang, culture, Yi-Fan Wang, JJJ40-12. Notes. This species is the second member of series Hainanici and sister to A. hainanicus (Fig. 1; Suppl. material 1: figs S1–S4). It differs from the latter by 29 bp for BenA (93.76% sequence identity), 16 bp for CaM (97.03%), and 21 bp for RPB2 (98.04%). Morphologically, although both species have short stipes, biseriate conidiophores, and strongly echinulate conidia, the new species is easily distinguished from A. hainanicus by growth on CYA at 37 °C, vivid green, and smaller conidia (6.0–7.5 vs. 6.0–9.5 µm, Table 4). Aspergillus xishuangbannaensis X.C. Wang, L.Y. Peng & W.Y. Zhuang, sp. nov. Fig. 4 Fungal Names: FN 572923 Etymology. The specific epithet refers to the type locality. In Aspergillus subgenus Circumdati section Nigri series Japonici. Typification. China • Yunnan Province, Xishuangbanna Dai Autonomous Prefecture, Mengla County, Menglun Town, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Green Stone Forest, 21°54'39"N, 101°17'00"E, in soil of limestone seasonal rainforest, 28 May 2024, Zhao-Qing Zeng, culture, Xiao Mou, ZYN05-01 (holotype HMAS 354081, ex-type strain CGMCC 3.29152). DNA barcodes. ITS PV883252, BenA PV877069, CaM PV877072, RPB2 PV877075. 283 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China Figure 4. Aspergillus xishuangbannaensis (ZYN05-01). A. Colonies: top row left to right, obverse CYA, MEA, YES, and PDA; bottom row left to right, reverse CYA, MEA, YES, and PDA; B–F. Conidiophores; G. Conidia. Scale bars: 22.5 µm (B); 20 µm (C–F); 10 µm (G). 290 MycoKeys 124: 275–290 (2025), DOI: 10.3897/mycokeys.124.172775 Lu-Yao Peng et al.: New species of Aspergillus in China Supplementary material 1 Мaximum likelihood phylogenies Authors: Lu-Yao Peng, Wen-Ying Zhuang, Xin-Cun Wang Data type: zip Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/mycokeys.124.172775.suppl1