Reducing the number of accepted species in Aspergillus series Nigri

C Bian; Y Kusuya; F Sklenář; E D’hooge; T Yaguchi; S Ban; CM Visagie; J Houbraken; H Takahashi; V Hubka

doi:10.3114/sim.2022.102.03

. 2022 Dec 19;102:95–132. doi: 10.3114/sim.2022.102.03

Reducing the number of accepted species in Aspergillus series Nigri

C Bian ¹, Y Kusuya ^2,³, F Sklenář ^4,⁵, E D’hooge ⁶, T Yaguchi ², S Ban ², CM Visagie ⁷, J Houbraken ⁸, H Takahashi ^2,^9,^10,^*, V Hubka ^2,^4,^5,^*

PMCID: PMC9903907 PMID: 36760462

Abstract

The Aspergillus series Nigri contains biotechnologically and medically important species. They can produce hazardous mycotoxins, which is relevant due to the frequent occurrence of these species on foodstuffs and in the indoor environment. The taxonomy of the series has undergone numerous rearrangements, and currently, there are 14 species accepted in the series, most of which are considered cryptic. Species-level identifications are, however, problematic or impossible for many isolates even when using DNA sequencing or MALDI-TOF mass spectrometry, indicating a possible problem in the definition of species limits or the presence of undescribed species diversity. To re-examine the species boundaries, we collected DNA sequences from three phylogenetic markers (benA, CaM and RPB2) for 276 strains from series Nigri and generated 18 new whole-genome sequences. With the three-gene dataset, we employed phylogenetic methods based on the multispecies coalescence model, including four single-locus methods (GMYC, bGMYC, PTP and bPTP) and one multilocus method (STACEY). From a total of 15 methods and their various settings, 11 supported the recognition of only three species corresponding to the three main phylogenetic lineages: A. niger, A. tubingensis and A. brasiliensis. Similarly, recognition of these three species was supported by the GCPSR approach (Genealogical Concordance Phylogenetic Species Recognition) and analysis in DELINEATE software. We also showed that the phylogeny based on benA, CaM and RPB2 is suboptimal and displays significant differences from a phylogeny constructed using 5 752 single-copy orthologous proteins; therefore, the results of the delimitation methods may be subject to a higher than usual level of uncertainty. To overcome this, we randomly selected 200 genes from these genomes and performed ten independent STACEY analyses, each with 20 genes. All analyses supported the recognition of only one species in the A. niger and A. brasiliensis lineages, while one to four species were inconsistently delimited in the A. tubingensis lineage. After considering all of these results and their practical implications, we propose that the revised series Nigri includes six species: A. brasiliensis, A. eucalypticola, A. luchuensis (syn. A. piperis), A. niger (syn. A. vinaceus and A. welwitschiae), A. tubingensis (syn. A. chiangmaiensis, A. costaricensis, A. neoniger and A. pseudopiperis) and A. vadensis. We also showed that the intraspecific genetic variability in the redefined A. niger and A. tubingensis does not deviate from that commonly found in other aspergilli. We supplemented the study with a list of accepted species, synonyms and unresolved names, some of which may threaten the stability of the current taxonomy.

Citation: Bian C, Kusuya Y, Sklenář F, D’hooge E, Yaguchi T, Ban S, Visagie CM, Houbraken J, Takahashi H, Hubka V (2022). Reducing the number of accepted species in Aspergillus series Nigri. Studies in Mycology 102: 95–132. doi: 10.3114/sim.2022.102.03

Keywords: Aspergillus luchuensis; Aspergillus niger; Aspergillus tubingensis; clinical fungi; indoor fungi; infraspecific variability; multigene phylogeny, multispecies coalescence model; ochratoxin A; species delimitation

INTRODUCTION

Aspergillus niger and its relatives play important roles in food mycology (Taniwaki et al. 2018), biotechnology (Schuster et al. 2002, Yang et al. 2017), the fermentation industry (Hong et al. 2014) and medical mycology (Howard et al. 2011). Consequently, A. niger is the most frequently cited species name in the genus Aspergillus (Samson et al. 2017). Since, on most occasions, species in section Nigri have been classified as GRAS (generally regarded as safe) by the Food and Drug Administration of the US government, they are suitable organisms for genetic manipulation and thus are used by biotechnology industries to produce hydrolytic enzymes (such as amylases and lipases) or organic acids (such as citric acid and gluconic acid) (Ward 1989, Bennett & Klich 1992, Andersen et al. 2011). On the other hand, Aspergillus section Nigri species (including some A. niger strains) cause food spoilage or contaminate a wide variety of food products (Samson et al. 2019) and may produce mycotoxins such as ochratoxin A, fumonisins and oxalic acid (Frisvad et al. 2011, 2018). Aspergillus niger, A. tubingensis and their lesser known cryptic species (phylogenetically supported but phenotypically nearly identical or undistinguishable) can also act as opportunistic pathogens that cause invasive mycoses in immunocompromised patients and non-invasive mycoses (aspergilloma, allergic aspergillosis, fungal otitis externa and keratomycosis) in otherwise healthy patients (Hubka et al. 2012, D’hooge et al. 2019, Hashimoto et al. 2017, Salah et al. 2019, Vidal-Acuña et al. 2019, Gits-Muselli et al. 2021, Nargesi et al. 2022). Some previous studies also demonstrated that different antifungal susceptibility patterns to azole derivates are present between A. niger and A. tubingensis complexes (Alcazar-Fuoli et al. 2009, Hendrickx et al. 2012, Szigeti et al. 2012).

The infrageneric classification of Aspergillus has a long history with Thom & Church (1926), Thom & Raper (1945) and Raper & Fennell (1965) recognizing that its species could be classified into “groups” based on their morphological similarities. This infrageneric classification was formalised when Gams et al. (1986) introduced names as subgenera and sections in Aspergillus, e.g., all black aspergilli were classified in section Nigri in the subgenus Circumdati. Recently, Houbraken et al. (2020) reviewed this infrageneric classification with the help of multigene phylogenies, and showed that it mostly agreed with the infrageneric classification proposed by Gams et al. (1986). They introduced series rank and subdivided section Nigri into five series. The most economically important section Nigri species, including A. niger, A. tubingensis and A. luchuensis belong to the series Nigri.

The taxonomy of section Nigri at the species level has been turbulent over time. Originally, Mosseray (1934) proposed 35 species. This number was reduced by Raper & Fennell (1965), who recognized 12 species and two varieties, and provided detailed morphological characteristics to distinguish among their accepted species. Al-Musallam (1980) revised the species limits using cluster analysis based on morphological and cultural parameters, suggesting at least seven species, including A. carbonarius, A. ellipticus, A. helicothrix, A. heteromorphus, A. japonicus, A. foetidus and A. niger. In this classification, A. niger was subdivided into six varieties and two forms. Kozakiewicz (1989), who drew her taxonomic conclusions from conidial ornamentation under scanning electron microscopy, distinguished 16 taxa, among which three belong to the current series Nigri, namely, A. acidus, A. niger with six varieties and A. citricus with two varieties. Early molecular genetic studies indicated that A. niger consisted of at least two cryptic species, A. niger and A. tubingensis (Kusters-van Someren et al. 1991, Varga et al. 1994, Peterson 2000), which are morphologically undistinguishable (Pitt & Hocking 2009, Crous et al. 2009). Using a polyphasic approach combining morphological features, extrolite profiling and β-tubulin DNA sequences, Samson et al. (2004) accepted 15 species in the section, comprising eight species in the clade currently known as series Nigri, i.e., A. brasiliensis, A. costaricensis, A. foetidus, A. lacticoffeatus, A. niger, A. piperis, A. tubingensis and A. vadensis. Subsequent phylogenetic studies supported the recognition of several cryptic phylogenetic species in the series Nigri, such as A. awamori (Perrone et al. 2011), later synonymized with another cryptic species, A. welwitschiae (Hong et al. 2013). Similarly, Varga et al. (2011) recognized A. acidus, which was later synonymized with the resurrected species A. luchuensis (Hong et al. 2013). Another two cryptic species related to A. tubingensis, i.e., A. eucalypticola and A. neoniger, were described by Varga et al. (2011), who also synonymized A. foetidus and A. lacticoffeatus with A. niger. As a result, the last overview of accepted Aspergillus species (Houbraken et al. 2020) assigned ten species to the series Nigri: A. brasiliensis, A. costaricensis, A. eucalypticola, A. luchuensis, A. neoniger, A. niger, A. piperis, A. tubingensis, A. vadensis and A. welwitschiae. Four novel cryptic species have been proposed since. Silva et al. (2020) introduced A. vinaceus as a close relative of A. niger and Khuna et al. (2021) introduced A. chiangmaiensis, A. pseudopiperis and A. pseudotubingensis as close relatives of A. tubingensis.

Vesth et al. (2018) and de Vries et al. (2017) de novo sequenced the genomes of the majority of accepted section Nigri species and performed phenotypic and genomic comparative analyses. The whole genomes of the ex-neotype strain of A. niger (CBS 554.65) and 24 A. niger sensu stricto strains were subsequently sequenced, and the mating-type distribution was analysed (Ellena et al. 2021, Seekles et al. 2022). Isolates with both MAT1-2-1 and MAT1-1-1 mating-type gene idiomorphs were found among genome-sequenced strains of A. niger, in agreement with previous PCR-based detection studies (Varga et al. 2014, Mageswari et al. 2016). This fact indicates that the species might have a cryptic sexual cycle, as previously observed in A. tubingenis (Horn et al. 2013). Additionally, recent phylogenomic studies indicated that section Nigri might belong to the subgenus Nidulantes (de Vries et al. 2017, Steenwyk et al. 2019) and not subgenus Circumdati (Jurjević et al. 2015, Kocsubé et al. 2016).

The reliable identification of section Nigri species is of great importance, as evidenced by their diverse positive and negative significances for humans. However, there is an increasing number of isolates that cannot be satisfactorily classified into the currently recognized species despite using multilocus sequence data (Howard et al. 2011, Negri et al. 2014, D’hooge et al. 2019). This fact also resulted in the recent description of several cryptic species related to A. niger and A. tubingensis (Silva et al. 2020, Khuna et al. 2021). The narrow species definition in the series Nigri is also associated with unsatisfactory identification results of MALDI-TOF MS (matrix-assisted laser desorption ionization time-of-flight mass spectrometry), a widely used identification tool in diagnostic laboratories and applied spheres (Gautier et al. 2016, D’hooge et al. 2019, Ban et al. 2021). All of the abovementioned problems may indicate that the species limits are not defined correctly and that the species definitions applied in the series are too narrow. This situation prompted the initiation of this study, where we are concerned with the verification of species boundaries at the molecular level based on a large number of strains.

A common requirement of species delimitation methods is proper sampling (a high number of strains of all studied species ideally isolated from a wide range of substrates and localities) and the presence of intraspecific variability (Carstens et al. 2013). This condition is, however, frequently difficult to fulfil in fungal taxonomy (Ahrens et al. 2016). From this point of view, series Nigri represents a perfect model group for studying species limits on a large scale due to the frequent occurrence of its species in many habitats and a high representation of molecular data in public databases. To do so, we have gathered extensive sequence data and applied a wide range of phylogenetic methods. The synthesis of the resulting data and the consideration of its practical taxonomic implications have led to a significant reduction in the number of species, as detailed below.

METHODS

Molecular studies

The DNA sequences of three loci, β-tubulin (benA), calmodulin (CaM) and the RNA polymerase II second largest subunit (RPB2), were gathered for a total of 276 strains from series Nigri. For this process, we removed short sequences or those that contained obvious errors. Additional species from other series of section Nigri were selected as outgroups or reference species depending on the analysis. The sequences were either downloaded from the GenBank database and mostly originated from previous studies, or they were taken from genes amplified and sequenced in this study. Sequences downloaded from the GenBank database were mostly from studies by D’hooge et al. (2019), Peterson (2008), Jurjević et al. (2012), Fungaro et al. (2017) and Hashimoto et al. (2017). Sequence alignments from the Silva et al. (2020) study were kindly provided by the authors. Information about the provenance of all strains is listed in Table 1.

Table 1.

List of Aspergillus series Nigri strains included in the phylogenetic analyses.

Species	Strain No.¹	Country	Substrate	GenBank/ENA/DDBJ accession Nos.²
Species	Strain No.¹	Country	Substrate	*benA*	*CaM*	*RPB2*	ITS
A. brasiliensis	CBS 101740 = IMI 381727 = ATCC MYA 4553 = IBT 21946^T	Brazil	soil	genome²	genome	EF661063	MH862749

	PPRI 26017 = CMV 007D1	South Africa	onion	MK451119	MK451325	MK450774	MK450631
	PPRI 3388 = CMV 010B8	South Africa	garlic (Allium sativum)	MK451152	MK451326	MK450775	MK450632
	PPRI 3328 = CMV 004I8	South Africa	mangrove leaves	MK451015	MK451324	MK450773	MK450630
	NRRL 26651	USA	unknown	EF661094	EF661160	EF661064	KC796389
	NRRL 26650	USA	unknown	EF661079	EF661159	EF661062	EF661196
	NRRL 35542	USA	peanut seed	EF661096	EF661162	EU021641	EF661199
	NRRL 26652	USA	unknown	EF661095	EF661161	EF661063	EF661198
	CBS 121619 = DTO 24-D5 = ITEM 6139	Portugal	grapes	AM295185	AM295176	OP081976	AM295181
	CBS 121618 = DTO 24-D2 = ITEM 4539	Portugal	grapes (Tinta barroca)	AM295182	AM295179	OP081975	—
	NBRC 9455 = ATCC 16404 = CBS 733.88 = DSM 1387 = DSM 1988 = KCTC 6317 = MUCL 29039 = MUCL 30113 = CECT 2574 = IFO 9455 = NCPF 2275 = IHEM 3766	USA	blueberry (Vaccinium sp.)	genome	genome	genome	—
	NBRC 105650 = ATCC 9642 = CBS 246.65 = JCM 16265 = NRRL 3536 = ATHUM 2856 = CECT 2700 = DSM 63263 = IFO 6342 = IMI 091855 = MUCL 19001 = IHEM 3797	Australia	wireless set	genome	genome	genome	—
	IFM 66950 = CCF 3991	Spain	cave air	genome	genome	genome	—
	IFM 66951 = CCF 4962	Romania	microbial mat in cave	genome	genome	genome	—
	IHEM 23046	Belgium	unknown	MH614413	MH644886	OP081977	MH613107
	IHEM 23047	Belgium	unknown	MH614444	MH644887	OP081978	MH613108
	IHEM 5185 = NRRL 2276	Unknown	unknown	MH614565	MH644892	OP081982	MH613109
	IHEM 23049	Belgium	unknown	MH614414	MH644889	OP081979	MH613081
	IHEM 3766	USA	fruit, Vaccinium subg. Cyanococcus	MH614415	MH644891	OP081980	MH613079
	IHEM 3797	Australia	radio set	MH614416	MH644890	OP081981	MH613110
A. niger	CBS 554.65 = ATCC 16888 = IFO 33023 = IHEM 3415 = JCM 10254 = NRRL 326 = CCFC 222006 = IMI 050566 = NBRC 33023^T	USA	tannin-gallic acid fermentation	EF661089	EF661154	EF661058	FJ629337
	CBS 101883 = CECT 20581 = IBT 22031^T1	Indonesia	coffee bean	genome	genome	genome	—
	PPRI 8682 = CMV 005B2	South Africa	mopane debris	MK451027	MK451462	MK450792	—
	PPRI 8734 = CMV 005B7	South Africa	mopane debris	MK451032	MK451464	MK450793	—
	PPRI 26011 = CMV 005G9	South Africa	soil	MK451060	MK451468	MK450794	—
	PPRI 18719 = CMV 004I3	South Africa	frass of Busseola fusca feeding inside maize stems	MK451010	MK451458	MK450789	—
	PPRI 3640 = CMV 004I6	South Africa	old photo paper	MK451013	MK451459	MK450790	—
	IFM 63326	Japan	human sputum	MK854742	OP081973	genome	—
	IFM 63604	Japan	human sputum	MK854748	OP081974	genome	—
	IFM 59636	Japan	human abdominal drain	OP081896	OP081969	genome	—
	104	Brazil	coffee bean	OP081838	OP081913	OP082051	—
	1400	Brazil	brazil nuts	OP081842	OP081917	OP082055	—
	1551	Brazil	brazil nuts	OP081848	OP081923	OP082061	—
	2334	Brazil	brazil nuts	OP081860	OP081935	OP082073	—
	2504	Brazil	brazil nuts	OP081861	OP081936	OP082074	—
	4.17	Brazil	grapes	OP081863	OP081938	OP082076	—
	4.18	Brazil	grapes	OP081864	OP081939	OP082077	—
	643	Brazil	coffee bean	OP081878	OP081953	OP082091	—
	6504	Brazil	brazil nuts	OP081879	OP081954	OP082092	—
	7061	Brazil	brazil nuts	OP081884	OP081959	OP082097	—
	8219	Brazil	coffee bean	OP081888	OP081963	OP082101	—
	902	Brazil	brazil nuts	OP081890	OP081965	OP082103	—
	1.2	Brazil	grapes	OP081836	OP081911	OP082049	—
	1357	Brazil	brazil nuts	OP081841	OP081916	OP082054	—
	23.115	Brazil	onions	OP081859	OP081934	OP082072	—
	CBS 117.80	Unknown	unknown	OP081891	GU195632	OP082104	—
	IHEM 18069	French Guiana, France	soil under palm tree	MH614489	MH645000	OP082110	MH613155
	IHEM 2312	Belgium	cosmetics	MH614521	MH645010	OP082127	MH613218
	IHEM 17892	Belgium	chronic sinusitus	MH614520	MH644877	OP082108	KP131598
	IHEM 23979	Belgium	otitis	MH614500	MH645005	OP082139	MH613182
	IHEM 25482	France	human bronchoalveolar lavage	MH614474	MH644860	OP082150	MH613113
	IHEM 23907	Belgium	human ear	MH614492	MH645003	OP082136	MH613168
	IHEM 24459	India	otitis	MH614502	MH645006	OP082144	MH613185
	IHEM 5788	Unknown	Chinese gall	MH614437	MH645007	OP082169	MH613213
	IHEM 21604	Cuba	outdoor air	MH614478	MH644996	OP082117	MH613124
	IHEM 5844	Unknown	unknown	MH614434	MH645002	OP082170	MH613157
	IHEM 5296	Unknown	leather	MH614519	MH645009	OP082167	MH613217
	IHEM 6126	Belgium	human sputum	MH614453	MH644998	OP082171	MH613135
	IHEM 5622	Unknown	unknown	MH614451	MH645001	OP082168	MH613156
	IHEM 4023	Belgium	mycotic otitis externa	MH614496	MH645004	OP082162	KP131606
	IHEM 9673	France	dwelling environment dust	MH614523	MH645012	OP082176	MH613219
	IHEM 25481	France	human bronchoalveolar lavage	MH614473	MH644859	OP082149	MH613112
	IHEM 21605	Cuba	nickel deposit	MH614479	MH644997	OP082118	MH613125
	IHEM 6147	Belgium	human nose	MH614439	MH645008	OP082172	MH613215
	IHEM 9709	France	cement	MH614522	MH645011	OP082177	MH613084
	IFM 56816	Japan	human ear	OP081895	OP081968	genome	—
	2.6	Brazil	grapes	OP081856	OP081931	OP082069	—
	2.8	Brazil	grapes	OP081858	OP081933	OP082071	—
	102.2706	Brazil	grapes	OP081837	OP081912	OP082050	—
	13.09	Brazil	coffee bean	OP081840	OP081915	OP082053	—
	148.727	Brazil	onions	OP081846	OP081921	OP082059	—
	ITAL 47.456 = IBT 35556^T2	Brazil	surface of grape berries	MN583579	MN583580	MN583581	MN575692
	ITAL 49.577	Brazil	grapes	OP081869	OP081944	OP082082	—
	ITAL 57.1243	Brazil	grapes	OP081875	OP081950	OP082088	—
	ITAL 62.1583	Brazil	grapes	OP081877	OP081952	OP082090	—
	ITAL 66.1929	Brazil	grapes	OP081881	OP081956	OP082094	—
	ITAL 48.545	Brazil	grapes	OP081867	OP081942	OP082080	—
	47.514	Brazil	grapes	OP081865	OP081940	OP082078	—
	48.544	Brazil	grapes	OP081866	OP081941	OP082079	—
	49.580	Brazil	grapes	OP081870	OP081945	OP082083	—
	55.1175	Brazil	grapes	OP081874	OP081949	OP082087	—
	58.1292	Brazil	grapes	OP081876	OP081951	OP082089	—
	CBS 139.54^T3	Namibia	Welwitschia mirabilis	MN969369	genome	MN969100	MH857271
	PPRI 23385 = CMV 004I5	South Africa	animal feed	MK451012	MK451548	MK450816	MK450663
	PPRI 4966 = CMV 004I1	South Africa	Chrysomelidae beetle	MK451008	MK451546	MK450814	MK450661
	PPRI 13255 = CMV 004I2	South Africa	chicken house bedding, mostly sunflower husks	MK451009	MK451547	MK450815	MK450662
	PPRI 26014 = CMV 006G2	South Africa	soil	MK451097	MK451562	MK450819	MK450669
	PPRI 9362 = CMV 005D1	South Africa	twigs and leaves from Colophospermum mopane	MK451039	MK451556	MK450818	MK450666
	IFM 50267	Japan	human sputum	OP081894	OP081967	genome	—
	IFM 63309	Japan	human, bronchoalveolar lavage fluid	MK854741	OP081972	genome	—
	IFM 49718	Japan	human sputum	OP081893	OP081966	genome	—
	IFM 62618	Japan	human, bronchoalveolar lavage fluid	MK854725	OP081971	genome	—
	IFM 60653	Japan	human sputum	OP081897	OP081970	genome	—
	15477	Brazil	onions	OP081847	OP081922	OP082060	—
	15580	Brazil	onions	OP081849	OP081924	OP082062	—
	15646	Brazil	onions	OP081850	OP081925	OP082063	—
	16158	Brazil	onions	OP081851	OP081926	OP082064	—
	16285	Brazil	onions	OP081852	OP081927	OP082065	—
	171804	Brazil	onions	OP081854	OP081929	OP082067	—
	181832	Brazil	onions	OP081855	OP081930	OP082068	—
	2.7	Brazil	grapes	OP081857	OP081932	OP082070	—
	48282	Brazil	onions	OP081868	OP081943	OP082081	—
	53295	Brazil	onions	OP081871	OP081946	OP082084	—
	53a	Brazil	yerba mate	OP081872	OP081947	OP082085	—
	54298	Brazil	onions	OP081873	OP081948	OP082086	—
	6573	Brazil	brazil nuts	OP081880	OP081955	OP082093	—
	69363	Brazil	onions	OP081883	OP081958	OP082096	—
	714	Brazil	brazil nuts	OP081885	OP081960	OP082098	—
	77390	Brazil	onions	OP081887	OP081962	OP082100	—
	88430	Brazil	onions	OP081889	OP081964	OP082102	—
	127a	Brazil	yerba mate	OP081839	OP081914	OP082052	—
	145723	Brazil	onions	OP081843	OP081918	OP082056	—
	146.724	Brazil	onions	OP081844	OP081919	OP082057	—
	146725	Brazil	onions	OP081845	OP081920	OP082058	—
	361	Brazil	brazil nuts	OP081862	OP081937	OP082075	—
	670	Brazil	brazil nuts	OP081882	OP081957	OP082095	—
	716	Brazil	brazil nuts	OP081886	OP081961	OP082099	—
	17.179	Brazil	grapes	OP081853	OP081928	OP082066	—
	DTO 267-I3	Thailand	house dust	OP081892	KP330149	OP082105	—
	IHEM 22373	India	otomycosis	MH614468	MH644935	OP082121	KP131600
	IHEM 17902	Belgium	chronic sinusitus	MH614508	MH644965	OP082109	KP131599
	IHEM 25483	France	human sputum	MH614447	MH644929	OP082151	MH613114
	IHEM 3095	Mauritius	soil	MH614488	MH644942	OP082160	MH613151
	IHEM 3090	Mauritius	soil	MH614487	MH644884	OP082159	MH613150
	IHEM 20620	Cuba	soil	MH614516	MH644973	OP082115	MH613208
	IHEM 23644	India	otitis	MH614435	MH644948	OP082129	MH613171
	IHEM 6727	France	bronchopulmonary cancer	MH614450	MH644972	OP082174	KP131609
	IHEM 21970	Mauritius	soil	MH614484	MH644938	OP082120	MH613111
	IHEM 24434	India	otitis	MH614498	MH644952	OP082141	MH613179
	IHEM 18351	French Guiana, France	bark kapok tree	MH614481	MH644934	OP082112	MH613128
	IHEM 23822	India	otitis	MH614493	MH644947	OP082132	MH613169
	IHEM 23651	India	otitis	MH614491	MH644946	OP082131	MH613167
	IHEM 24707	Belgium	otitis	MH614503	MH644957	OP082145	MH613189
	IHEM 25101	Belgium	Unknown	MH614441	MH644961	OP082147	MH613194
	IHEM 21697	Belgium	onion	MH614448	MH644939	OP082119	MH613137
	IHEM 23892	Belgium	human	MH614570	MH644944	OP082134	MH613161
	IHEM 23965	France	otitis	MH614497	MH644951	OP082138	MH613178
	IHEM 15980	Belgium	ball from Pakistan	MH614518	MH644976	OP082107	MH613210
	IHEM 20621	Cuba	soil	MH614480	MH644933	OP082116	MH613126
	IHEM 18080	French Guiana, France	soil of riverside	MH614513	MH644969	OP082111	MH613077
	IHEM 25485	France	human bronchoaspiration	MH614477	MH644932	OP082153	MH613116
	IHEM 5077	Belgium	human ear	MH614515	MH644971	OP082166	MH613078
	IHEM 25340	Belgium	human toenail	MH614506	MH644962	OP082148	MH613195
	IHEM 18676	Peru	guano	MH614446	MH644928	OP082113	MH613082
	IHEM 24328	Unknown	horse (crust) - clinical sample	MH614499	MH644953	OP082140	MH613180
	IHEM 2951	Belgium	mycotic otitis externa	MH614569	MH644963	OP082156	KP131605
	IHEM 2969	India	soil	MH614449	MH644941	OP082157	MH613149
	IHEM 22432	Somalia	otitis	MH614509	MH644966	OP082126	KP131604
	IHEM 26623	Belgium	human ear	MH614486	MH644940	OP082155	MH613146
	IHEM 26214	Belgium	otitis	MH614505	MH644960	OP082154	MH613192
	IHEM 23648	India	otitis	MH614495	MH644950	OP082130	MH613174
	IHEM 24450	India	otitis	MH614469	MH644955	OP082142	MH613184
	IHEM 25484	France	human bronchoaspiration	MH614476	MH644931	OP082152	MH613115
	IHEM 22377	Belgium	conjunctivitis - human eye	MH614483	MH644937	OP082123	KP131602
	IHEM 22378	Somalia	human auditory canal	MH614510	MH644967	OP082124	MH613076
	IHEM 23913	Belgium	human ear	MH614501	MH644954	OP082137	MH613183
	IHEM 4781	Belgium	mycotic otitis externa	MH614454	MH644974	OP082165	KP131608
	IHEM 22374	India	otomycosis	MH614482	MH644936	OP082122	KP131601
	IHEM 23642	India	otitis	MH614452	MH644861	OP082128	MH613170
	IHEM 4063	Bahamas	sea water	MH614440	MH644995	OP082163	MH613152
	IHEM 23895	Belgium	human ear secretions	MH614584	MH644945	OP082135	MH613163
	IHEM 14389	Belgium	chronic obstructive bronchopneumopathy (bronchoalveolar lavage)	MH614517	MH644975	OP082106	KP131597
	IHEM 18678	Peru	guano	MH614475	MH644930	OP082114	MH613074
	IHEM 3710	Unknown	unknown	MH614490	MH644943	OP082161	MH613158
	IHEM 24767	India	otitis	MH614507	MH644964	OP082146	MH613198
	IHEM 9388	France	air	MH614438	MH644979	OP082175	MH613220
	IHEM 3019	Zaire	soil from palm grove (Elaeis guineensis)	MH614514	MH644970	OP082158	MH613206
	IHEM 24454	India	otitis	MH614470	MH644956	OP082143	MH613186
	IHEM 22379	India	otomycosis	MH614511	MH644968	OP082125	KP131603
	IHEM 4461	Belgium	mycotic otitis externa	MH614585	MH644978	OP082164	KP131607
	IHEM 23888	Belgium	otitis	MH614494	MH644949	OP082133	MH613173
	IHEM 6350	Belgium	dried food for fish	MH614471	MH644977	OP082173	MH613214
A. eucalypticola	CBS 122712 = DTO 53-A2 = IBT 29274^T	Australia	leaves of Eucalyptus sp.	genome	genome	genome	OL711732
A. luchuensis	CBS 205.80 = IFM 47726 = NBRC 4281 = IFO 428 = KACC 46772 = RIB 2642^T	Unknown	awamori-koji	JX500062	JX500071	MN969081	JX500081
	NRRL 4750 = CBS 128.52 = KACC 47005^T4	USA	unknown	EF661087	EF661152	EF661052	MH856956
	RIB 2601 = IFO 4033 = NBRC 111188 = ATCC 38854 = IFM 46994	Unknown	unknown	genome	genome	genome	LC573600
	IFO 4308 = NBRC 4308	Unknown	unknown	genome	genome	genome	AB573884
	IHEM 26285	France	human broncho-aspiration	MH614561	MH644869	OP082038	MH613141
	IHEM 25486	France	human trimming liquid	MH614563	MH644862	OP082030	MH613118
	CBS 112811 = CECT 20582 = IBT 26239^T5	Denmark	black pepper	genome	genome	genome	OL711715
	IHEM 23904	India	otitis	MH614551	MH644990	OP082022	MH613164
	PPRI 13091 = CMV 010I3	South Africa	soil	MK451175	MK451492	MK450797	—
	IHEM 5316	Belgium	human ear	MH614562	MH644893	OP082046	MH613216
	PPRI 9197 = CMV 005C9	South Africa	soil	MK451038	MK451491	MK450796	MK450641
	PPRI 13230 = CMV 011A9	South Africa	maize roots	MK451187	MK451493	MK450798	—
	PPRI 8983 = CMV 005C2	South Africa	twigs and leaves from Colophospermum mopane	MK451035	MK451490	MK450795	MK450640
A. pseudotubingensis	SDBR CMUO2^T	Thailand	rhizosphere soil	MK457206	MK457205	MK457208	—
	SDBR CMUO8	Thailand	rhizosphere soil	MW602908	MW602907	MW602909	—
	SDBR CMU20	Thailand	rhizosphere soil	MW602913	MW602912	MW602914	—
A. tubingensis	CBS 133056 = DTO 213-F6 = NRRL 4875^T	Unknown	unknown	EF661086	EF661151	EF661055	EF661193
	PPRI 8683 = CMV 005B3	South Africa	soil	MK451028	MK451452	MK450786	—
	PPRI 8733 = CMV 005B6	South Africa	plant debris from Colophospermum mopane	MK451031	MK451453	MK450787	—
	PPRI 21344 = CMV 004I4	South Africa	seed of soybean (Glycine max)	MK451011	MK451448	MK450782	—
	IFM 57258	Nagasaki, Japan	soil	OP081902	OP081909	genome	—
	IHEM 18329	French Guiana, France	rotten mango	MH614467	MH644992	OP082002	MH613202
	IHEM 18205	French Guiana, France	soil	MH614419	MH644865	OP082001	MH613127
	IHEM 21599	Cuba	rail feather	MH614458	MH644985	OP082008	MH613073
	IHEM 18097	French Guiana, France	rotten mango	MH614417	MH644981	OP081998	MH613119
	IHEM 21607	Cuba	indoor environment	MH614464	MH644988	OP082012	MH613133
	IHEM 18042	French Guiana, France	soil under mango tree	MH614462	MH644980	OP081997	MH613117
	IHEM 21597	Cuba	indoor air	MH614457	MH644984	OP082007	MH613122
	IHEM 2463	Belgium	nutmeg (Myristica fragrans)	MH614465	MH644989	OP082028	MH613147
	IHEM 21593	Cuba	cotton cloth	MH614460	MH644987	OP082006	MH613131
	IHEM 18136	French Guiana, France	soil under palm tree	MH614456	MH644983	OP082000	MH613121
	IHEM 21592	Cuba	human ear	MH614420	MH644866	OP082005	MH613130
	IHEM 21606	Cuba	indoor environment	MH614466	MH644991	OP082011	MH613199
	IHEM 21590	Cuba	indoor air	MH614418	MH644986	OP082004	MH613129
	IHEM 22375	India	otomycosis	MH614461	MH644867	OP082015	KP131633
	IHEM 22376	India	otomycosis	MH614463	MH644868	OP082016	KP131634
	IHEM 18106	French Guiana, France	leaf of mango tree	MH614455	MH644982	OP081999	MH613083
	IHEM 21602	Argentina	clinical material	MH614459	MH644864	OP082010	MH613123
	PPRI 21345 = CMV 005A8	South Africa	seed of soybean (Glycine max)	MK451024	MK451450	MK450784	—
	PPRI 21347 = CMV 005A9	South Africa	seed of soybean (Glycine max)	MK451025	MK451451	MK450785	—
	IHEM 21971	Mauritius	soil	MH614546	MH644993	OP082013	MH613134
	IHEM 25327	Belgium	laboratory contaminant	MH614421	MH644994	OP082029	MH613139
	IHEM 21601	Argentina	clinical material	MH614564	MH644917	OP082009	—
	PPRI 7393 = CMV 005A5	South Africa	unknown	MK451021	MK451542	MK450813	MK450660
	NRRL 4851 = ATCC 16879 = CBS 115.48 = CBS 558.65	Wisconsin, USA	unknown	EF661085	EF661150	EF661054	—
	CBS 134.48 = ITEM 7040	Unknown	unknown	AY820007	AJ964876	EF661055	FJ629354
	IHEM 5802	Unknown	unknown	MH614558	MH644919	OP082048	MH613204
	IHEM 22772	Belgium	human ear	MH614545	MH644871	OP082018	MH613132
	PPRI 25991 = CMV 001B7	South Africa	walnut kernels (Juglans regia)	MK450891	MK451540	MK450811	MK450658
	PW3161	Hong Kong	human nail	LC000547	LC000560	LC000573	AB987902
	PPRI 6720 = CMV 005A3	South Africa	beetle (Aspidimorpha areata)	MK451019	MK451541	MK450812	MK450659
	IFM 54640	Japan	human sputum	OP081898	OP081905	genome	—
	IFM 55763	Japan	vineyard	OP081899	OP081906	genome	—
	IFM 57143	Japan	human	OP081901	OP081908	genome	—
	IFM 56815	Japan	human ear	OP081900	OP081907	genome	—
	IFM 61612	Japan	human, bronchoalveolar lavage fluid	OP081903	OP081910	genome	—
	IHEM 22370	Belgium	otitis	MH614525	MH644894	OP082014	KP131632
	IHEM 1941	Belgium	dust from mattress	MH614550	MH644900	OP082003	MH613159
	IHEM 17170	Belgium	chronic sinusitus	MH614537	MH644923	OP081989	KP131625
	IHEM 25487	France	environment	MH614524	MH644879	OP082031	MH613120
	IHEM 23971	Belgium	mycotic otitis externa	MH614555	MH645013	OP082025	MH613181
	IHEM 17440	Belgium	chronic sinusitus	MH614540	MH644924	OP081991	KP131627
	IHEM 23890	Belgium	human ear	MH614530	MH644905	OP082020	MH613172
	IHEM 16879	Morocco	otitis human ear	MH614559	MH644921	OP081986	MH613211
	IHEM 26291	Belgium	unknown	MH614552	MH644903	OP082039	MH613165
	IHEM 17439	Belgium	chronic sinusitus	MH614539	MH644876	OP081990	KP131626
	IHEM 13662	Belgium	hospital environment	MH614553	MH644898	OP081985	MH613153
	IHEM 17033	Belgium	mycotic otomycosis externa	MH614536	MH644922	OP081987	KP131623
	IHEM 23911	Belgium	otitis	MH614566	MH644908	OP082024	MH613177
	IHEM 17904	Belgium	chronic sinusitus	MH614538	MH644875	OP081996	MH613212
	IHEM 17893	Belgium	chronic sinusitus	MH614542	MH644926	OP081994	KP131630
	IHEM 25949	France	human sputum	MH614528	MH644873	OP082034	MH613144
	IHEM 26200	Belgium	otitis	MH614568	MH644901	OP082036	MH613160
	IHEM 26645	Belgium	human ear secretions	MH614549	MH644897	OP082043	MH613148
	IHEM 26163	Belgium	otitis externa	MH614527	MH644872	OP082035	MH613140
	IHEM 23900	Belgium	otitis	MH614529	MH644904	OP082021	MH613166
	IHEM 26661	Belgium	onychomycosis human toe nail	MH614556	MH644911	OP082044	MH613193
	IHEM 17903	Belgium	chronic sinusitus	MH614543	MH644927	OP081995	KP131631
	IHEM 17441	Belgium	chronic sinusitus	MH614541	MH644925	OP081992	KP131628
	IHEM 26350	France	air	MH614548	MH644883	OP082040	MH613143
	IHEM 17168	Belgium	chronic sinusitus	MH614534	MH644920	OP081988	KP131624
	IHEM 26622	Belgium	human ear	MH614567	MH644896	OP082042	MH613145
	IHEM 25777	Belgium	human wound	MH614423	MH644913	OP082033	MH613197
	IHEM 25773	Belgium	otitis externa	MH614472	MH644912	OP082032	MH613196
	IHEM 4379	unknown	unknown	MH614554	MH644899	OP082045	MH613154
	IHEM 23985	Belgium	otitis	MH614531	MH644907	OP082026	MH613176
	IHEM 26215	Belgium	otomycosis	MH614433	MH644914	OP082037	MH613200
	IHEM 17442	Belgium	chronic sinusitus	MH614533	MH644915	OP081993	KP131629
	IHEM 10349	China	grains	MH614535	MH644918	OP081984	MH613207
	IHEM 23886	Belgium	otitis	MH614424	MH644910	OP082019	MH613188
	IHEM 5615	Unknown	unknown	MH614425	MH644916	OP082047	MH613201
	IHEM 24447	India	otitis	MH614532	MH644909	OP082027	MH613187
	CBS 115574 = IBT 23401 = ITEM 7555 = CECT 20579^T6	Costa Rica	soil	genome	genome	HE984361	MH862988
	CBS 553.65 = NRRL 5121 = ATCC 16880 = IMI 23599	Costa Rica	soil	FJ629278	OP081904	OP081983	FJ629327
	CBS 115656 = IBT 20973 = NRRL 62634^T7	Venezuela	mangrove water	KC796361	KC796377	genome	OL711719
	IHEM 23909	India	otitis	MH614422	MH644906	OP082023	MH613175
	IHEM 22394	India	conjunctivitis - human eye	MH614544	MH644870	OP082017	KP131635
	SDBR CMUI4^T8	Thailand	rhizosphere soil	MK457200	MK457199	MK457202	—
	SDBR CMU15	Thailand	rhizosphere soil	MW602898	MW602897	MW602899	—
	SDBR CMUI1^T9	Thailand	rhizosphere soil	MK457194	MK457193	MK457196	—
	SDBR CMUI7	Thailand	rhizosphere soil	MW602903	MW602902	MW602904	—
A. vadensis	CBS 113365 = IMI 142717 = CECT 20584 = IBT 24658^T	Egypt	air	AY585531	genome	HE984371	—
	IHEM 26351	France	human sputum	MH614547	MH644878	OP082041	MH613142
A. carbonarius	CBS 111.26 = ITEM 4503 = NRRL 369 = ATCC 1025 = ATHUM 2854 = CBS 556.65 = IMI 016136 = MUCL 13583 = NCTC 1325 = NRRL 1987 = NRRL 369 = ITEM 5010^T	Unknown	paper	EF661099	EF661167	EF661068	EF661204
	NRRL 67 = NBRC 5864 = ATCC 8740 = CBS 420.64 = DSM 872 = IFO 5864 = IMI 41875 = MUCL 30479 = NRRL 1737 = NRRL 605	Unknown	unknown	EF661097	EF661165	EF661066	EF661202
	NRRL 4849 = CBS 114.29	USA	unknown	EF661100	EF661168	EF661069	EF661205
	NRRL 346 = ATCC 6277 = CBS 146284	Honduras	unknown	EF661098	EF661166	EF661067	EF661203
Additional strains used only in DELINEATE and STACEY analyses
A. carbonarius	IHEM 661	France	indoor air in bakery	MH614442	MH645014	—	—
	IHEM 1931	Belgium	dust from mattress	MH61458	MH644880	—	—
	IHEM 25902	France	human sputum	MH614575	MH645015	—	—
	DTO 179-F4	South Africa	house dust	KP329846	KJ775280	—	—
	DTO 179-C6	South Africa	house dust	KP329845	KJ775278	—	—
	CCF 3388	Czech Republic	toenails, human clinical material	HE577803	HE649500	—	—
	144-3-K2	Unknown	unknown	KJ599604	KJ599576	—	—
	A-1759	Israel	grape	KC520549	KC520552	—	—
	G187	Slovakia	dried vine fruits	MT166308	MK046876	—	—
	A-2160	Spain	grape	KC520550	KC520553	—	—
A. ibericus	NRRL 35644 = CBS 121593 = IMI 391429 = DTO 24-F1 = ITEM 4776 = IHEM 23498^T	Portugal	wine grapes	EF661102	EF661163	XM_025715438	—
	NRRL 35645 = ITEM 6601 = IMI 391430	Portugal	wine grapes	EF661101	EF661164	EF661065	—
A. ellipticus	CBS 707.79 = IMI 172283^T	Unknown	unknown	AY585530	EF661170	EF661051	—
	CBS 677.79 = IMI 278383 = ITEM 4499^T10	Costa Rica	unknown	AY819993	AM117810	HE984363	—
A. heteromorphus	NRRL 4747 = CBS 117.55 = ATCC 12064 = IMI 172288 = IHEM 5801 = ITEM 7045^T	Brazil	culture contaminant	EF661103	EF661169	EF661050	—
	IHEM 18645	France, Martinique	indoor wall	MH614573	MH645030	—	—
A. sclerotiicarbonarius	CBS 121057 = IBT 121057^T	Unknown	culture contaminant	EU159229	EU159235	MN969091	—
A. sclerotioniger	CBS 115572 = IBT 22905 = ITEM 7560^T	Unknown	unknown	FJ629304	FN594557	HE984369	—
A. japonicus	CBS 114.51 = ITEM 7034^T	Unknown	unknown	HE577804	AJ964875	MN969079	—
	PPRI 4286 = CMV 005H7	South Africa	soil	MK451062	MK451430	MK450779	—
	ITEM 14787	USA	indoor air	HE984413	HE984430	HE984377	—
	ITEM 14805	USA	indoor air	HE984416	—	HE984378	—
	CBS 123.27 = NRRL 360 = ATCC 1042 = IFO 4106 = IMI 358697	Puerto Rico	soil	HE577805	EF661141	EF661047	—
	NRRL 1782 = ATCC 16873 = CBS 568.65 = IMI 211387	Panama	soil	—	EF661144	EF661048	—
	NRRL 35494 = ITEM 15926	Unknown	unknown	—	EU021690	EU021639	—
	NRRL 35541	USA	peanut field soil	EF661104	EF661143	EU021640	—
	NRRL 4839 = IHEM 5627 = NCTC 3792 = MUCL 13578 = IMI 312983 = CBS 113.48	Unknown	unknown	MH614586	EF661142	EF661049	—
	IHEM 26043	France	human sputum	MH614587	MH645026	—	—
	CBS 115571	Bahamas	marine environment	EU482434	EU482432	—	—
	AJP01	Unknown	unknown	JX103558	JX103559	—	—
	CCF 4079	Unknown	unknown	HE577811	FR751423	—	—
	ITEM 14789	USA	indoor air	HE9844174	HE984432	—	—
	NBRC 4408	Unknown	soil	LC573650	LC573708	—	—
	NBRC 32856	Japan	living leaf	LC573651	LC573709	—	—
A. aculeatus	CBS 172.66 = IHEM 5796 = NRRL 5094 = IMI 211388 = CCRC 32190 = ATCC 16872 = ITEM 7046^T	Unknown	tropical soil	HE577806	EF661148	XM020198603	—
	PPRI 7513 = CMV 005A6	Unknown	unknown	MK451022	MK451291	MK450752	—
	PPRI 26016 = CMV 007C9	South Africa	soil	MK451118	MK451296	MK450756	—
	F-719	Unknown	unknown	HE577810	HE578093	—	—
	PPRI 4070 = CMV 005F1	South Africa	soil	MK451044	MK451292	MK450753	—
	ITEM 14807	USA	indoor air	HE984409	HE984424	HE984372	—

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¹Acronyms of culture collections in alphabetic order: ATCC, American Type Culture Collection, Manassas, Virginia; ATHUM, Athens Collection of Fungi, University of Athens, Athens, Greece; CBS, Westerdijk Fungal Biodiversity Institute (formerly Centraalbureau voor Schimmelcultures), Utrecht, the Netherlands; CCF, Culture Collection of Fungi, Department of Botany, Charles University, Prague, Czech Republic; CCFC,Canadian Collection of Fungal Cultures, living fungal collection associated with the DAOM herbarium (Agriculture and Agri-Food Canada), Ottawa, Canada; CCM (F-), Czech Collection of Microorganisms, Brno, Czech Republic; CCRC (=BCRC), Bioresources Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan; CECT, Colección Española de Cultivos Tipo, Universidad de València, Edificio de Investigación, Burjassot, Spain; CMV, working and formal culture collections housed at FABI (Forestry and Agricultural Biotechnology), Innovation Africa, University of Pretoria, South Africa; DSM, Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany; DTO, internal culture collection of the Department Applied and Industrial Mycology of the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; IBT, Culture Collection at the Department of Biotechnology and Biomedicine, Lyngby, Denmark; IFM, Culture Collections for Pathogenic Fungi and Actinomycetes, Medical Mycology Research Center, Chiba University, Chiba, Japan; IFO, Institute for Fermentation, Osaka, Japan (IFO strains were transferred to the NBRC NITE collection); IHEM (BCCM/IHEM), Belgian Coordinated Collections of Micro-organisms, Fungi Collection: Human and Animal Health, Sciensano, Brussels, Belgium; IMI, CABI’s collection of fungi and bacteria, Wallingford, UK; JCM, Japan Collection of Microorganisms, Tsukuba, Japan; KACC, Korean Agricultural Culture Collection, Wanju, South Korea; KCTC, Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, South Korea; MUCL (BCCM/MUCL), Agro-food & Environmental Fungal Collection, Louvain-la-Neuve, Belgium; NBRC (NITE), National Institute of Technology and Evaluation, Biological Resource Center, Department of Biotechnology, Kisarazu, Chiba, Japan; NCTC, National Collection of Type Cultures, Central Public Laboratory Service, London, UK; ITEM, Agri-Food Toxigenic Fungi Culture Collection, Institute of Sciences of Food Production, Bari, Italy; NRRL, Agricultural Research Service Culture Collection, Peoria, Illinois, USA; PPRI, culture collection of the National Collections of Fungi, housed at the Agricultural Research Council - Plant Health and Protection, Roodeplaat, South Africa; RIB, National Research Institute of Brewing, Tax Administration Agency, Higashihiroshima, Hiroshima, Japan; SDBR, Sustainable Development of Biological Resources Laboratory, Faculty of Science, Chiang Mai University, Chiang Mai Province, Thailand. Other acronyms represent personal strain numbers (without permanent preservation).

²Accession numbers to genomic sequences analysed in this study are listed in Supplementary Table S3.

^Tex-type strain. ^T1 = A. lacticoffeatus; ^T2 = A. vinaceus; ^T3 = A. welwitschiae; ^T4 = A. nakazawae; ^T5 = A. piperis; ^T6 = A. costaricensis; ^T7 = A. neoniger; ^T8 = A. chiangmaiensis; ^T9 = A. pseudopiperis; ^T10 = A. helicothrix.

Total genomic DNA was isolated from 5-d-old cultures using the Zymo Research Fungal/Bacterial DNA Kit™ (Zymo Research, Irvin, CA, USA) or the Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany). PCR amplification of CaM was performed using the primer pairs cmd5 and cmd6 (Hong et al. 2006) or CF1L and CF4 (Peterson 2008); benA with Bt2a and Bt2b (Donaldson et al. 1995) or Ben2f (Hubka & Kolarik 2012) and Bt2b; and RPB2 with fRPB2-5F and fRPB2-7CR (Liu et al. 1999). Samples were amplified using the following cycling parameters: one initial step of 10 min at 95 °C followed by 35 cycles of 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C and a single extension step of 10 min at 72 °C. The PCRs were performed in 25 μL volume containing 2 μL of DNA (2 pM), 2 μL of forward primer, 2 μL of reverse primer, 19 μL of H₂O, and PuReTaq Ready-To-Go PCR beads (GE Healthcare UK, Little Chalfont, UK). The PCR products were sequenced using the BigDye Terminator cycle sequencing ready reaction kit (Applied Biosystems, Foster City, CA, USA) using the same primers used for PCR and analysed on an ABI Prism 3130 genetic analyser (Applied Biosystems) according to the manufacturer’s instructions.

Newly obtained DNA sequences were inspected and assembled in Geneious Prime v. 2020.2.4 (Biomatters Ltd.). Sequences were deposited into GenBank with the accession numbers listed in Table 1.

Phylogenetic analyses and species delimitation

Alignments of benA, CaM and RPB2 were performed using the FFT-NS-i option implemented in the MAFFT online service (Katoh et al. 2019). The alignments were trimmed, concatenated, and then analysed using maximum likelihood (ML), maximum parsimony (MP) and Bayesian inference (BI) methods. The final alignments are available from the DRYAD digital repository (https://doi.org/10.5061/dryad.866t1g1td).

Suitable partitioning schemes and substitution models for the analyses were selected based on the Bayesian information criterion using a greedy strategy implemented in PartitionFinder 2 (Lanfear et al. 2017) with settings allowing introns, exons and codon positions to be independent datasets (Supplementary Table S1). ML trees were constructed with IQ-TREE v. 1.4.4 (Nguyen et al. 2015) with nodal support determined by ultrafast bootstrapping (BS) with 10⁵ replicates. Bayesian posterior probabilities were calculated using MrBayes v. 3.2.6 (Ronquist et al. 2012). The analysis ran for 10⁷ generations, two parallel runs with four chains each were used, every 1 000^th tree was retained, and the first 25 % of trees were discarded as burn-in. The convergence of the runs and effective sample sizes were checked in Tracer v. 1.6 (http://tree.bio.ed.ac.uk/software/tracer). Maximum parsimony (MP) trees were calculated using PAUP* v. 4.0b10 (Swofford 2003). Analyses were performed using the heuristic search option with 100 random taxon additions; tree bisection-reconnection (TBR); maxtrees were set to 1 000. Branch support was assessed by bootstrapping with 500 replications.

The rules for the application of the GCPSR approach were adopted from Dettman et al. (2003a, b) and slightly modified for the different design of this study (different numbers of loci and methods used). To recognize a clade as an “evolutionary lineage”, it had to satisfy one of two criteria: (a) genealogical concordance - the clade was present in the majority (2/3) of the single-locus genealogies; (b) genealogical nondiscordance - the clade was well supported in at least one single-locus genealogy, as judged by ML ultrafast bootstrap proportions ≥95 %, MP bootstrap proportions ≥70 % and BI posterior probabilities ≥95 %, and was not contradicted in any other single-locus genealogy at the same level of support. When deciding which evolutionary lineages represent “phylogenetic species”, two additional criteria were applied and evaluated according to the combined phylogeny of three genes: (a) genetic differentiation - species had to be relatively distinct and well differentiated from other species to prevent minor tip clades from being recognized as a separate species; (b) all individuals had to be placed within a phylogenetic species, and no individuals were to be left unclassified.

Species delimitation analyses based on the multispecies coalescent model were performed as described previously (Sklenář et al. 2021). In brief, the haplotype function from R v. 4.0.2 (R Core Team 2016) package PEGAS (Paradis 2010) was used to retain only unique sequences in the alignments. The best fitting models were obtained in jModelTest v. 2.1.7. Parameters and the other settings of the STACEY, GMYC, bGMYC, PTP, and bPTP methods were consistent with previous studies (Sklenář et al. 2021, Glässnerová et al. 2022). Two different settings were used for the GMYC method. The ultrametric input trees for this method were calculated in BEAST v. 2.6.6 (Bouckaert et al. 2014) with a chain length of 10⁷ generations. As a model for creating input trees, we set up the coalescent constant population prior and performed two tree reconstruction methods: one with the common ancestor height (CAh) setting and a second with the median height (Mh) setting. We only show the delimitation results of both settings when they were different. The results of all analyses are graphically summarised in iTOL v. 6 (Interactive Tree of Life) (Letunic & Bork 2021).

The multilocus species delimitation method STACEY was performed in BEAST (Bouckaert et al. 2014) using the STACEY v. 1.2.5 add-on (Jones 2017). For this and the DELINEATE analysis (see below), we added several additional species from other series of section Nigri (Table 1). We set up the length of MCMC chain to 10⁹ generations, the species tree prior was set to the Yule model, the molecular clock model was set to a strict clock, the growth rate prior was set to a lognormal distribution (M = 5, S = 2), the clock rate priors for all loci were set to a lognormal distribution (M = 0, S = 1), the PopPriorScale prior was set to a lognormal distribution (M = -7, S = 2) and the relative DeathRate prior was set to a beta distribution (α = 1, β = 1 000). The substitution models were as follows: benA - K80+I; CaM - TrNef+G; RPB2 - TrNef+G. The output was processed with SpeciesDelimitationAnalyzer (Jones et al. 2015). For the presentation of the STACEY results, we first created a plot showing how the number of delimited species and the probability of the most likely scenarios changed in relation to the value of the collapseheight parameter, and then we created similarity matrices using code from Jones et al. (2015) with different values of collapseheight chosen from the plot (see the Results section).

Independent testing of various species boundary hypotheses was performed in the DELINEATE software (Sukumaran et al. 2021). After splitting the dataset into hypothetical populations (Supplementary Table S2) using the “A10” analysis in BPP v. 4.4 (Yang 2015), the species tree for these populations was estimated in starBEAST (Heled & Drummond 2010) implemented in BEAST v. 2.6.7 (Bouckaert et al. 2014). The analysis was run in the Python (van Rossum & Drake Jr FL 2014) package DELINEATE (Sukumaran et al. 2021). In every simulated model, some populations delimited by BPP were assigned to tentative (fixedly defined) species based on the species delimitation results from previous analyses or were based on the common species definitions from previous studies. Other populations were left free to be delimited (unassigned to any species). As a result, the algorithm implemented in DELINEATE can either recognize the unassigned population as a separate species, define a wider monophyletic species composed of several unassigned populations, or merge unassigned populations with some of the predefined species.

Whole genome sequencing and assembly

Whole-genome 100 bp paired-end (PE) sequencing was performed in 18 strains with the aid of HiSeq 1500 (Illumina, San Diego, CA) using the HiSeq reagent kit v. 1, according to the manufacturer’s instructions. A 300 bp PE sequencing was performed on a MiSeq (Illumina) using the MiSeq Reagent Kit v. 3, according to the manufacturer’s instructions. Long read sequencing of IFM 61612 was performed on the MinION platform (ONT; Oxford Nanopore Technologies, Cambridge, UK). A DNA library for ONT sequencing was prepared using a short-read eliminator kit (Nippon Genetics, Tokyo, Japan) and a ligation sequencing kit (SQK-LSK109), and sequencing was performed with a FLO-MIN106D flow cell (R9.4.1) for 48 h, according to the manufacturer’s instructions. A total of 83 443 ONT reads were generated with a mean length of 12 011 bp using ONT Guppy v. 2.3.5.

Adapters and low-quality bases of Illumina reads were trimmed by Trim Galore v. 0.6.4 (Krueger 2015) with the default settings selected (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore). The mitochondrial genomes were assembled using GetOrganelle v. 1.6.4 (Jin et al. 2020) with trimmed reads. To filter the mitochondrial reads, the trimmed reads were aligned against the mitochondrial genomes by BWA v. 0.7.17-r1188 (Li & Durbin 2009), and the mapped reads were filtered by SAMtools v. 1.9 (Li et al. 2009) and SeqKit (Shen et al. 2016). For eight isolates sequenced by MiSeq, the filtered reads were used to assemble the nuclear genomes using SPAdes v. 3.14.0 (Bankevich et al. 2012) with the options ‘--cov-cutoff auto’ and ‘--careful’. For six isolates sequenced by HiSeq 1500, the assembly of nuclear genomes was carried out as follows: (1) the filtered reads were used to assemble the contigs using VelvetOptimiser v. 2.2.6 (Zerbino & Birney 2008); (2) the contigs were used to generate in silico long mate-pair reads with inserts of 3 000 ± 300 bp using Wgsim v. 0.3.1-r13 (https://github.com/lh3/wgsim) with the options ‘-e 0 -1 100 -2 100 -r 0 -R 0 -X 0 -d 3000 -s 300 -N 4000000’; (3) Illumina PE and simulated mate-pair (MP) reads were assembled by ALLPATHS-LG v. 52488 (Gnerre et al. 2011). The genome of IFM 61612 was assembled using NECAT v. 0.0.1_update20200803 (Chen et al. 2021), and polished with the ONT reads by using Racon v. 1.4.20 (Vaser et al. 2017) and Medaka v. 1.4.4 (https://github.com/nanoporetech/medaka) and with Illumina PE reads by using HyPo v. 1 (Kundu et al. 2019). Most tools were obtained through Bioconda (Grüning et al. 2018).

Genome annotation

The annotation of assembled genomes was performed using the Funannotate pipeline v. 1.7.4 (https://funannotate.readthedocs.io/en/latest/). Following the identification of repeat sequences by RepeatModeler v. 1.0.11 (http://www.repeatmasker.org/RepeatModeler.html) and RepeatMasker v. 4.0.7 (https://www.repeatmasker.org), Funannotate ab initio prediction was performed with the option ‘--busco_seed_species=aspergillus_oryzae’ by Augustus v. 3.3.3 (Stanke et al. 2006), GeneMark-ES v. 4.38 (Ter-Hovhannisyan et al. 2008), GlimmerHMM v. 3.0.4 (Majoros et al. 2004), and SNAP v. 2006-07-28 (Korf 2004) using exon hints from the proteins of A. niger CBS 513.88 and A. oryzae RIB40 downloaded from the Aspergillus Genome Database (Bairoch & Apweiler 2000, Cerqueira et al. 2014). The functional annotations of predicted genes were performed using the Swiss-Prot, InterPro v. 5.42-78.0 (Jones et al. 2014), eggNOG v. 4.5.1 (Buchfink et al. 2015, Huerta-Cepas et al. 2016), MEROPS v. 12.0 (Rawlings et al. 2018, Mitchell et al. 2019), and the dbCAN v. 8.0 (Yin et al. 2012) databases. Genome annotations of the reference strains were obtained from the Joint Genome Institute (http://genome.jgi.doe.gov/) (Supplementary Table S3). The completeness of the draft genomes and predicted proteins were evaluated using BUSCO v. 4.0.6 (Seppey et al. 2019) with the database “eurotiales_odb10”.

Phylogenetic analyses based on the genomic sequences

Orthologous genes among the 31 strains (18 newly sequenced and 13 available from previous studies) were identified by OrthoFinder v. 2.3.12 (Emms & Kelly 2019). Then, the amino acid sequences of 5 752 single-copy orthologous proteins were aligned using the G-INS-i option in MAFFT v. 7.471 and concatenated into one long protein sequence. A phylogenetic tree was constructed using multithreaded RAxML (Stamatakis 2014), selecting the PROTGAMMAWAG model and using 100 bootstrap replicates to determine node support.

The predicted protein sets of 31 strains were evaluated with BUSCO protein mode. A total of 2 754 genes present at a single copy were identified among all strains. From these, 200 genes were randomly selected for analyses using the STACEY method and randomly distributed into ten independent analyses, each containing 20 genes (Supplementary Table S4). Coding regions (exons) of the corresponding 200 nucleotide sequences were extracted for each strain from the de novo assemblies. Each sequence matrix was aligned using MAFFT, and well-aligned regions were extracted using Gblocks v. 0.91b (Talavera & Castresana 2007) with settings allowing no gap positions.

RESULTS

Combined phylogeny based on three loci

A maximum likelihood (ML) phylogenetic tree based on three loci in 280 strains (276 belonging to the series Nigri and four A. carbonarius strains used as outgroups) is presented in Fig. 1, with the geographic origin and substrate plotted on the tree in the form of colour strips. The topology of the trees inferred by the maximum parsimony (MP) and Bayesian inference (BI) methods was similar to ML and the bootstrap support values and posterior probabilities, respectively, were appended to the nodes.

Fig. 1. — Multilocus phylogeny of *Aspergillus* series *Nigri* based on three loci (*benA*, *CaM*, *RPB2*) and 276 isolates (and four *A. carbonarius* isolates as an outgroup). The best-scoring maximum likelihood (ML) tree inferred in the IQ-TREE is shown; ultrafast bootstrap support values (ML bs) are appended to nodes along with maximum parsimony bootstrap support values (MP bs) and Bayesian inference posterior probabilities (BI pp); only support values ≥95 %, ≥70 % and ≥0.95, respectively, are shown; a dash indicates lower statistical support for a specific node or the absence of a node in the phylogeny while an asterisk indicates full support; the ex-type strains are designated with a bold print; the information on geographic origin and isolation source is plotted on the tree (see legend). Alignment characteristics, partitioning schemes and substitution models are listed in Supplementary Table S1.

There are three main lineages in the tree and for practical reasons, we named them based on the priority rules. The A. brasiliensis lineage (ABL) only contains the ex-type isolate of A. brasiliensis, while the A. niger lineage (ANL) contains the ex-type isolates of A. lacticoffeatus, A. niger, A. vinaceus and A. welwitschiae (the old species names, which are usually considered synonyms, are omitted). The Aspegillus tubingensis lineage (ATL) contains ex-types of ten species: A. chiangmaiensis, A. costaricensis, A. eucalypticola, A. luchuensis, A. neoniger, A. piperis, A. pseudopiperis, A. pseudotubingensis, A. tubingensis and A. vadensis. The sequences of the three recently described species, A. chiangmaiensis, A. pseudopiperis and A. pseudotubingensis (Khuna et al. 2021), were of poor quality and contained a large number of errors (indels) in the coding regions and probably also in the noncoding regions of all three genes. Due to this fact and the absence of genomic sequences, we excluded these species from most analyses. However, we manually corrected some apparent errors in the exonic regions (partially revised sequences can be found in the alignments deposited in the Dryad digital repository: https://doi.org/10.5061/dryad.866t1g1td) and added these species to the classical phylogenetic trees based on the ML, MP and BI methods. In these phylogenies, the positions of A. chiangmaiensis and A. pseudotubingensis differ significantly from the tree presented by Khuna et al. (2021). The combined phylogeny without these species is shown in Supplementary Fig. S1.

The bootstrap support values in the combined trees (Fig. 1 and Supplementary Fig. S1) are high on many terminal branches in ATL, and the majority of the currently accepted species form their own well-supported terminal clades. On the other hand, some deeper nodes are poorly supported and the relationships among the species in ATL are mostly unresolved. If only the monophyly and statistical support of branches in these combined phylogenetic trees were considered, all currently recognized species in ATL could be accepted. Bootstrap values in the trees based on concatenated datasets are, however, often falsely high and are not a suitable criterion for deciding about species limits (Kubatko & Degnan 2007, Seo 2008). The boundaries between currently accepted species in ANL are much less clear, as there are several poorly supported clades in the proximity of A. vinaceus, A. niger and A. welwitschiae whose species identification is unclear.

The geography and substrate of isolation showed no clear patterns that could be associated with particular species, although clinical isolates from European countries (mostly strains from the study of D’hooge et al. (2019)) were overrepresented in the clade containing the A. tubingensis ex-type strain CBS 133056, while isolates from food and South America were more common throughout ANL (strains predominantly from Silva et al. (2020)).

Incongruences between single gene trees: consequences for the GCPSR approach and BLAST similarity searches

Single-gene trees based on benA, CaM and RPB2 loci are shown in Fig. 2 and Supplementary Fig. S2 (only one isolate per unique multilocus haplotype was included). The coloured connecting lines show changes in the positions of isolates between single-gene trees (Fig. 2); the branches were rotated so that the trees maximally correspond to each other; the trees without connecting lines are shown in Supplementary Fig. S2. There was no conflict in topology among the three main lineages, i.e., there was no isolate changing position between ABL, ANL and ATL in different single-gene trees. When applying the rules of the GCPSR approach in ABL and ANL, only one phylogenetic species (PS) can be recognized in each lineage because the composition of their subclades is variable in terms of the isolates resolved in them, while the statistical support of the subclades is low or unstable between phylogenies. A more complicated situation is present in the ATL, where some clades can be recognized as evolutionary lineages, as they are well supported in at least two phylogenetic trees. For example, the clade containing the two strains of A. vadensis (CBS 113365 and IHEM 26351) was present and supported in all phylogenies, while the clade containing seven strains attributed to A. luchuensis/A. piperis was present and supported in the benA and CaM trees. The singleton lineage containing the ex-type of A. eucalypticola (CBS 122712) was also present in all trees, but it cannot be evaluated by the GCPSR criteria as there are no isolates clustering with this strain. Although the abovementioned evolutionary lineages were recognized by the grouping criteria, they cannot be recognized as PS because doing so would have resulted in a situation where other strains belonging to the ATL and forming several nonmonophyletic clades in the combined tree (Fig. 1) could not be assigned to any phylogenetic species and were left unclassified. As a result, applying GCPSR criteria in the ATL would also lead to the recognition of only one phylogenetic species.

To show the practical consequences of the incongruences between single-gene datasets, we created a local BLAST database containing sequences of benA, CaM and RPB2 of the ex-type strains belonging to the series Nigri. Then, we performed BLAST searches for all sequences of these genes against this database (only one isolate per unique multilocus haplotype was used) in Geneious PRIME v. 2020.2.4 (https://www.geneious.com). The results of the BLAST searches and the closest similarities to the ex-type strains are presented in Fig. 3. It is apparent that sequences of different genes from the same strain frequently resulted in the closest sequence similarities with ex-type strains of different species and thus resulted in variable species identifications. This phenomenon does not involve A. brasiliensis.

Fig. 3. — The results of BLAST similarity searches of three loci (*benA*, *CaM* and *RPB2*) derived from strains with unique multilocus haplotypes across the genetic diversity of series *Nigri*. Coloured rectangles represent the closest hits to one of the 14 ex-type strains (every species has its unique colour; the ex-type of *A. lacticoffeatus* was omitted because it has an identical genotype to the ex-type of *A. niger*). If there was an identical similarity to two ex-type strains, the rectangles were diagonally divided. Ex-type isolates are marked with bold font and a coloured background. The phylogenetic tree was calculated in IQ-TREE using partitioned analysis and 10⁵ ultrafast bootstrap replicates.

In the ANL, different species identifications commonly resulted for different loci, which was observed in 21 out of 53 haplotypes (39.6 %). In seven haplotypes (13 %; CBS 117.80, IFM 63326, IFM 63604, IHEM 6350, 148.727, 2.8 and 102.2706), the three BLAST searches resulted in three different identifications, i.e., A. niger, A. vinaceus and A. welwitschiae. This demonstrates that there are phylogenetic conflicts throughout the entire clade and that there is no barcode gap for these species at any of these loci. These incongruences are also visible in the single-gene phylogenetic trees shown in (Fig. 2, Supplementary Fig. S2).

In ATL, unstable identification based on different genes was less prevalent than in ANL, but was still present in 14 out of 54 haplotypes (24 %). It occurred mostly between A. tubingensis, A. costaricensis, A. neoniger, A. pseudopiperis, A. luchuensis and A. piperis. Four haplotypes (7 %; IHEM 23904, IHEM 25327, IHEM 21971 and IFM 57258) were identified as a different species for each gene region. This phenomenon also has its basis in the single-gene tree incongruences (Fig. 2, Supplementary Fig. S2).

Phylogenomic tree

In Fig. 4, the topology of the phylogenomic tree was compared to the topology of the three-locus ML tree (alignment reduced to unique multilocus haplotypes; A. chiangmaiensis, A. pseudopiperis and A. pseudotubingensis were excluded; list of haplotypes in Supplementary Table S5) and to the topology of the species tree constructed in starBEAST. The phylogenomic tree was based on 5 752 protein orthologues extracted from 30 whole-genome sequences of series Nigri and one sequence of A. carbonarius as an outgroup. Nine out of 18 newly sequenced genomes (Supplementary Table S3; designated by bold print in Fig. 4) belonged to ANL, six to ATL and three to ABL. Almost all clades in the phylogenomic tree gained full statistical support, in contrast to the trees based on the benA, CaM and RPB2 loci. The topologies among the different analyses were mostly congruent, but several incongruences were observed (most of them were present at unresolved nodes with low statistical support). For example, A. vadensis was resolved as a separate branch sister to all other species in the ATL in the phylogenomic and species tree but clustered with A. costaricensis and A. neoniger with a high support of 95 % in the ML tree. In the phylogenomic and species tree, A. neoniger and A. costaricensis clustered with A. tubingensis isolates, in contrast to the ML tree, where they clustered distantly from the latter. Last, A. eucalypticola clusters with A. luchuensis and A. piperis in the phylogenomic tree, while in ML and species trees, it formed a long separate branch.

Both MAT1-2-1 and MAT1-1-1 mating-type gene idiomorphs were found in the genomes of strains belonging to the ANL and ATL, while only the MAT1-2-1 idiomorph was found in the A. brasiliensis strains (Fig. 4).

Multispecies coalescent model-based (MSC) methods

The results of the species delimitation using four single-locus species delimitation methods (GMYC, bGMYC, PTP and bPTP) and one multilocus method, STACEY, are summarised in Fig. 5. For the GMYC and STACEY methods, we showed the results of two different settings, as they differed in the number of delimited species.

Fig. 5. — Schematic representation of the results of species delimitation methods in the series *Nigri*. One multilocus method (STACEY) and four single-locus methods (GMYC, bGMYC, PTP, bPTP) were applied to a dataset of three loci (*benA*, *CaM*, *RPB2*). The results are depicted by coloured bars with different colours or shades indicating species delimited by specific methods and settings. Ex-type isolates are highlighted with a bold print. The STACEY results with two *collapseheight* parameter values, 0.005 and 0.0095, are shown. For the GMYC method, the coalescent constant population tree model was used as an input with both common ancestor heights (CAh) and median height (Mh) settings (both settings are only shown when they produced different delimitation results). The phylogenetic tree was calculated by STACEY analysis and is used solely for the comprehensive presentation of the results from different methods.

All methods and their settings delimited the entire ANL as a single species without any exception. The method STACEY and all single-locus methods except for three delimited only one species in the A. brasiliensis lineage. Three methods (GMYC with a common ancestor height (CAh) setting; bPTP based on benA, and bPTP based on CaM) recognized isolate PPRI 26017 as a singleton species separated from A. brasiliensis. All single-locus methods except for two unequivocally proposed that ATL should be considered a single broad species. The first exception was the GMYC method based on benA with a CAh setting that divided the ATL into three species: A. vadensis and another two species that are not monophyletic in any multigene phylogeny that are shown in Fig. 1 and 3. The second was the bPTP method based on CaM, which divided ATL into eight species corresponding to A. vadensis, A. eucalypticola, A. tubingensis, a species comprising both A. piperis and A. luchuensis, and four additional species whose delimitation has no support in the multigene phylogeny as they are not monophyletic (Figs 1 and 3).

The multilocus method STACEY was performed using two different datasets. The first dataset contained unique multilocus haplotypes from the Nigri series and the second dataset was supplemented with eight additional species outside the Nigri series. The results of both analyses were identical and supported the delimitation of one or four species in the ATL depending on the value of the collapseheight parameter. Detailed results of STACEY based on the second dataset are presented in Fig. 6, where subfigure A illustrates the effect of the collapseheight parameter value on the number of delimited species. On the y-axis on the left side, there is the number of delimited species with the given collapseheight value (black line). The support for the most likely scenario (yellow line), and the support for the second most likely scenario (blue line) are shown; other less supported scenarios were omitted. The vertical dashed lines in subfigure A represent the scenarios illustrated in detail in Fig. 6B and Fig. 6C in the form of similarity matrices showing posterior probabilities of each pair of isolates being included in the same species. There are three main scenarios that gained reasonable support and which delimited 15 (collapseheight = ~0.004–0.006), 14 (collapseheight = ~0.006–0.008) and 11 species (collapseheight = ~0.009 and higher) (Fig. 6), corresponding to 3 or 7 species in the series Nigri. In all three scenarios, the ANL and ABL were always resolved as single species. At collapseheight of ~0.005, four species were delimited in the ATL: (1) A. costaricensis + A. neoniger + A. tubingensis; (2) A. luchuensis + A. piperis; (3) A. eucalypticola; and (4) A. vadensis. When the collapseheight increases and reaches values in the range 0.006–0.008, the support for the delimitation of multiple species in the ATL decreases, but four species are still supported and not lumped together. The only difference in the scenario with 14 species compared to that with 15 species is outside series Nigri, namely, A. aculeatus is delimited as one or two species, respectively. In the scenario with 11 species and a collapseheight of approximately 0.009 and higher, the whole A. tubingensis lineage is delimited as a single species.

Fig. 6. — The results of species delimitation by using the STACEY method based on the three loci: *benA*, *CaM* and *RPB2*. (A) Dependence of the delimitation results on the *collapseheight* parameter. The black solid line represents the number of delimited species (left y-axis) depending on the changing value of the *collapseheight* parameter (x-axis). The yellow line represents the probability (range from 0 to 1, right y-axis) of the most likely scenario at specific *collapseheight* value. The blue line represents the probability of the second most probable scenario at a specific *collapseheight* value; curves representing other less probable scenarios are omitted. Dashed vertical lines mark two values of the *collapseheight* parameter (0.005 and 0.0095) whose results are shown in detail by similarity matrices in subfigures B and C. The similarity matrices give the posterior probability of every two isolates belonging to the same multispecies coalescent cluster (tentative species). The darkest black shade corresponds to a posterior probability of 1, while the white colour is equal to 0. Thicker horizontal and vertical lines in the similarity matrices delimit species or their populations that gained delimitation support in some scenarios.

Species delimitation using DELINEATE

The species hypotheses were independently tested in DELINEATE software, where we set up 10 different models. These models were divided into three categories based on the broadness of the fixed species predefined within the ANL and ATL: narrow (Models 1–4), medium (Models 5–7) and broad (Models 8–10). The results are summarised in Fig. 7, where every column represents one model with individual populations either assigned into species (grey bars = predefined species), or left unassigned and free to be delimited (brown bars). The red frames show the resulting solution proposed by DELINEATE for every model, i.e., an unassigned population either recognized as a separate species, merged with other unassigned population(s), or merged with some predefined species.

Fig. 7. — Species delimitation using DELINEATE software. In total, ten models of species boundaries were set up and tested. The brown bars represent unassigned populations left free to be delimited, while the grey bars represent the predefined species. The resulting solutions suggested by DELINEATE are depicted by red frames around the bars. The populations were delimited by using BPP software; isolates belonging to each population are listed in Supplementary Table S2. The displayed tree was calculated in starBEAST based on the *benA*, *CaM* and *RPB2* loci and is only used for the comprehensive presentation of the results from different models.

Models 1, 5 and 8 focused on delimitation within the ABL, where four populations were recognized by BPP software. In all three models, these populations were left unassigned, and they were always lumped to form a single broad species regardless of whether the species in the ANL and ATL were predefined as narrow or broad.

Models 2, 6 and 9 focused on the ANL, in which all populations were left free to be delimited, while the populations of the ATL were segregated into eight narrow species (Model 2), four broader species (Model 6) and one broad species (Model 10). In all of these settings, the ANL was always recognized as a single broad species regardless of the species number and species limits in the ATL.

More variable delimitation results were obtained in the ATL. Models 3 and 4 simulated situations where A. niger populations (pop1 to pop8) were segregated into four and six species, respectively. In these cases, the ATL populations (pop1 to pop13) were arranged into four species as follows: (1) A. piperis + A. luchuensis; (2) A. tubingensis + A. costaricensis + A. neoniger; (3) A. eucalypticola; and (4) A. vadensis. In Models 7 and 10, the populations of the ANL were predefined into two and single species, respectively. In both cases, the ATL was delimited as a single species.

STACEY analysis using genomic data

To analyse species limits using MSC methods on the genome scale, we randomly selected 200 single-copy orthologous genes from the genomes. The alignments of these genes were randomly distributed into ten datasets each containing 20 genes that were analysed separately (Supplementary Table S4). The results are summarized in Fig. 8. The upper part of the figure shows the course of each analysis using different colours and the dependence of the delimitation results on the changing value of the collapseheight parameter. For every analysis, there are two curves, the first representing the number of delimited species (left y-axis) and the second representing the probability of the most likely species delimitation scenario at a specific collapseheight value (ranging from 0 to 1, right y-axis).

Fig. 8. — Species delimitation results from ten independent STACEY analyses, each based on 20 genes randomly selected from genomes of series *Nigri* species (n = 30; plus one genome of *A. carbonarius* as the outgroup). The upper part of the figure shows the dependence of the delimitation results on the changing value of the *collapseheight* parameter; each analysis is designated with a different colour. There are two curves for each analysis, the first representing the number of delimited species (left y-axis) and the second representing the probability of the most likely species delimitation scenario at a specific *collapseheight* value (ranging from 0 to 1, right y-axis). Every analysis proposed two to three taxonomic solutions having similar probabilities. In total, there were four possible scenarios (A, B, C, D) differing in the number of delimited species within the *A. tubingensis* lineage (one to four species). These delimitation results are schematically shown in the lower part of the figure. The list of genes used in every analysis can be found in Supplementary Table S4.

Surprisingly, every analysis proposed two to three taxonomic solutions having high probabilities (two to three major peaks). There were four possible scenarios (A, B, C, D) resulting in 3–6 delimited species in the Nigri series. However, none of these analyses supported the division of the ANL or ABL into more species. Thus, all of these scenarios only differed in the number of delimited species in the ATL, ranging from one to four delimited species. These delimitation results are schematically shown in the lower part of Fig. 8. All ten analyses supported the scenario with four species: (1) A. costaricensis + A. neoniger + A. tubingensis; (2) A. luchuensis + A. piperis; (3) A. eucalypticola; and (4) A. vadensis. In addition, six analyses supported scenarios with three or two species in the ATL, and only two analyses lumped all species into one at high collapseheight values (Fig. 8).

Intraspecific genetic diversity in Aspergillus and series Nigri

To determine whether the intraspecific genetic variability in the redefined species in the series Nigri falls within the range commonly found in Aspergillus, we made an intraspecific diversity comparison across Aspergillus (basic data in Supplementary Table S6). We downloaded sequences of the benA, CaM and RPB2 genes from 34 species outside section Nigri (fewer common markers, such as Mcm7, Tsr1 and Act, were available for only some species). The criteria for inclusion were as follows: (1) species boundaries were re-examined using MSC methods, and (2) the strains of the species were obtained from at least three countries (sufficient sampling).

The results of the comparison (Fig. 9) showed that dissimilarites in the common taxonomic markers were usually lower than 4 % except in benA, where a higher degree of dissimilarity was found occasionally. This was mainly caused by long intronic regions in benA. However, the two highest dissimilarity values in benA belong to A. vitricola (sect. Restricti) and A. subalbidus (sect. Candidi), which could each be a complex of two species because the species delimitation results were ambiguous (Sklenář et al. 2017, Glässnerová et al. 2022). Thus, the real maximum sequence dissimilarity in benA may be lower than that displayed in Fig. 9.

The intraspecific variability in the broadly defined A. niger (corresponding to the whole ANL) and in A. brasiliensis falls comfortably within the range frequently found in Aspergillus. The intraspecific genetic diversity of the broadly defined A. tubingensis in benA and CaM is higher than that commonly found in Aspergillus, while the variability within the narrowly defined A. tubingensis (excluding A. luchuensis/A. piperis, A. eucalypticola and A. vadensis) is within the range common for Aspergillus (Fig. 9).

TAXONOMY

The following list contains the names of accepted species in the series Nigri and their synonyms. Illegitimate and invalid names are also included as some of them are still used in the applied sphere. This list builds on previously published lists of accepted species and their synonyms in section Nigri (Thom & Church 1926, Thom & Raper 1945, Raper & Fennell 1965, Al-Musallam 1980, Kozakiewicz 1989). Compared to previous lists, this list is primarily based on available molecular data. If this data is missing, the names are included in the sub-list of unresolved or doubtful names.

One of the main goals of this list is to point out that the taxonomy of series Nigri is still unresolved and to stimulate further research to resolve names for which original material is available in the form of cultures or herbarium specimens. Some of these names may still threaten the stability of the taxonomy of series Nigri. Most importantly, some of the synonyms or unresolved names compete for priority with economically significant species such as A. tubingensis, A. luchuensis or even A. niger. Althoug A. niger Tiegh. 1867 was conserved against Ustilago ficuum Reichard 1867 and Ustilago phoenicis Corda 1840 (Kozakiewicz et al. 1992), it can still be predated by A. phaeocephalus Durieu & Mont. 1848 (no specimens available), A. nigrescens C.P. Robin 1853 (no specimens available) and A. nanus Mont. 1856 whose type is preserved in the Muséum National d’Histoire Naturelle in France but no DNA data are available. The situation is even more complicated with A. tubingensis Mosseray 1934. This species is conspecific with several older species names, such as A. pulverulentus (basionym Sterigmatocystis pulverulenta McAlpine 1896), A. schiemanniae Thom 1916, A. elatior Mosseray 1934, A. pseudoniger Mosseray 1934 and possibly some other species listed among unresolved species. Aspergillus tubingensis is the most frequently used name compared to other competing names and members of the International Commission of Penicillium and Aspergillus (ICPA) are thus currently in discussions about its possible conservation.

Accepted species and their synonyms in Aspergillus section Nigri series Nigri

1. Aspergillus brasiliensis Varga, Frisvad & Samson, Int. J. Syst. Evol. Microbiol. 57: 1929. 2007. MycoBank MB 510581. — Type: CBS 101740. Ex-type: CBS 101740 = IMI 381727 = IBT 101740. DNA barcodes: ITS = FJ629321; benA = FJ629272; CaM = FN594543; RPB2 = EF661063.

2. Aspergillus eucalypticola Varga, Frisvad & Samson, Stud. Mycol. 69: 9. 2011. MycoBank MB 560387. — Type: CBS H-20627. Ex-type: CBS 122712 = IBT 29274. DNA barcodes: ITS = EU482439; benA = EU482435; CaM = EU482433; RPB2 = MN969070.

3. Aspergillus luchuensis Inui, J. Coll. Sci. Imp. Univ. Tokyo 15: 469. 1901. MycoBank MB 151291. — Type: CBS H-24280. Ex-type: CBS 205.80 = NBRC 4281 = KACC 46772 = IFM 47726 = RIB 2642. DNA barcodes: ITS = JX500081; benA = JX500062; CaM = JX500071; RPB2 = MN969081.

Synonym: Aspergillus perniciosus Inui, J. Coll. Sci. Imp. Univ. Tokyo 15: 473. 1901. [no MycoBank record]. Synonymized by Hong et al. (2013). No living culture available.

Synonym: Aspergillus awamori Nakaz., Rep. Govt. Res. Inst. Formosa: 1. 1907. [MycoBank MB 119955]. Synonymized by Hong et al. (2014). Culture ex-type: JCM 2261 = IAM 2112 = KACC 47234.

Synonym: Aspergillus velutinus Mosseray, La Cellule 43: 252. 1934. [MycoBank MB 255342]. The benA sequence (FJ629283) derived from the ex-type strain CBS 139.48 (= NRRL 4877 = CCM F-286 = VKM F-2084 = WB 4877) is identical to the ex-type of A. luchuensis.

Synonym: Aspergillus aureus var. acidus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 960. 1936. [MycoBank MB 493809]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2013). Culture ex-type: NBRC 4121 = NRRL 4796 = CBS 564.65 = KACC 45131.

Synonym: Aspergillus aureus var. pallidus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 961. 1936. [MycoBank MB 372388]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2014). Culture ex-type: NBRC 4123 = NRRL 4797 = CBS 565.65 = KACC 47004.

Synonym: Aspergillus awamori var. piceus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 956. 1936. [MycoBank MB 250923]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2013). Representative strain: CBS 119.52 = NRRL 4846.

Synonym: Aspergillus kawachii Kitahara & Yoshida. J. Ferment. Technol. 27. 1949. [No MycoBank record]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2013). Culture ex-type: NBRC 4308 = CBS 117.80 = NRRL 4886 = KACC 46771. Hong et al. (2013) and Ban et al. (2021) examined phylogenetic position of A. kawachii based on strains KACC 46771 and NBRC 4308, respectively. Their sequences are identical with the ex-type of A. kawachii in contrast to the sequences from strain CBS 117.90 that belong to the ANL (Table 1). Our conclusion is that the strain CBS 117.90 is contaminated with A. niger.

Synonym: Aspergillus citricus var. pallidus (Nakaz., Simo & A. Watan.) Kozak., Mycol. Pap. 161: 114. 1989. [MycoBank MB 127748]. Not validly published – basionym A. aureus var. pallidus Nakaz., Simo & A. Watan. is not a validly published [Art. 41.5; Turland et al. (2018)].

Synonym: Aspergillus inuii Sakag., Iizuka & M. Yamaz., J. Agric. Chem. Soc. Japan 3: 97. 1950. [Mycobank MB 309227]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2014). Culture ex-type: JCM 22302 = IAM 2255 = CBS 125.52 = KACC 47235.

Synonym: Aspergillus nakazawae [as nakazawai] Sakag., Iizuka & M. Yamaz., J. Agric. Chem. Soc. Japan 4: 3. 1950. [Mycobank MB 309232]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2014). Culture ex-type: NRRL 4750 = CBS 128.52 = KACC 47005.

Synonym: Aspergillus foetidus var. acidus (Nakaz., Simo & A. Watan.) Raper & Fennell, The Genus Aspergillus: 326. 1965. [MycoBank MB 353273]. Synonymized by Hong et al. (2013). Not validly published – basionym A. aureus var. acidus Nakaz., Simo & A. Watan. is not a validly published name [Art. 41.5; Turland et al. (2018)].

Synonym: Aspergillus foetidus var. pallidus (Nakaz., Simo & A. Watan.) Raper & Fennell, The Genus Aspergillus: 325. 1965. [MycoBank MB 353274]. Synonymized by Hong et al. (2014). Not validly published – basionym A. aureus var. pallidus Nakaz., Simo & A. Watan. is not a validly published name [Art. 41.5; Turland et al. (2018)].

Synonym: Aspergillus niger var. awamori (Nakaz.) Al-Musallam, Revision of the black Aspergillus species: 60. 1980. Doctoral diss., Rijksuniversiteit Utrecht, Netherlands. [MycoBank MB 118405]. Hong et al. (2014) showed that the ex-type strain NRRL 4948 (= CBS 557.65 = KACC 46996 = ATCC 16877) is closely related to the ex-type of A. welwitschiae (ANL) and is synonymized here with A. niger.

Synonym: Aspergillus acidus Kozak., Mycol. Pap. 161: 110. 1989. [MycoBank MB 127765]. Synonymized by Hong et al. (2013). Culture ex-type: KACC 45131 = CBS 564.65 = ATCC 16874 = NBRC 4121 = IMI 104688 = NRRL 4796.

Synonym: Aspergillus coreanus Yu T.S. et al., J. Microbiol. Biotechnol. 14:182. 2004. [No Mycobank record]. Illegitimate name [Art. 54.1; Turland et al. (2018)] and nomen nudum. Synonymized by Hong et al. (2013). Authentic strain: KACC 41731 = CBS 119384.

Synonym: Aspergillus piperis Samson & Frisvad, Stud. Mycol. 50: 57. 2004. [MycoBank MB 500009]. Synonymized in this study.

Synonym: Aspergillus luchuensis mut. kawachii (Kitahara & Yoshida) S.B. Hong, O. Yamada & R.A. Samson, Appl. Microbiol. Biotechnol. 98: 560. 2014. [No Mycobank record]. Not validly published: basionym was not validly published [Art. 41.5; Turland et al. (2018)] and protologue does not include citation of the identifier issued for the name by a recognized electronic repository, e.g. MycoBank [Art. F.5.1; Turland et al. (2018)].

4. Aspergillus niger Tiegh., Ann. Sci. Nat., Bot. ser. 5, 8: 240. 1867. MycoBank MB 284309. Nomen conservandum taking precedence over Ustilago phoenicis Corda and Ustilago ficuum Reichard which were published earlier (Kozakiewicz et al. (1992). — Type: CBS 554.65. Ex-type: CBS 554.65 = NRRL 326 = ATCC 16888 = IFO 33023 = IHEM 3415 = IMI 050566ii = IMI 50566 = JCM 10254 = QM 9270 = QM 9946 = Thom 2766 = WB 326. DNA barcodes: ITS = EF661186; BenA = EF661089; CaM = EF661154; RPB2 = EF661058.

Synonym: Ustilago ficuum Reichardt, Verh. Zool.-Bot. Ges. Wien 17: 335. 1867. [MycoBank MB 187740]. Nomen rejiciendum, rejected name in favour of A. niger Tiegh. – see Kozakiewicz et al. (1992). The ex-type strain IHEM 3710 (= WB 364 = NRRL 364 = IMI 91881 = CBS 555.65= ATCC 16882) derived from the type IMI 91881 (Kozakiewicz et al. 1992) is located in the ANL (Fig. 1).

Synonym: Eurotium nigrum (Tiegh.) de Bary, Beiträge zur Morphologie und Physiologie der Pilze: 21. 1870. [No MycoBank record]. Basionym: A. niger Tiegh.

Synonym: Sterigmatocystis nigra (Tiegh.) Tiegh., Bull. Soc. Bot. France 24: 102. 1877. [MycoBank MB 231646]. Basionym: A. niger Tiegh.

Synonym: Ustilago welwitschiae Bres. in Sacc, Bol. Soc. Brot. 11: 68. 1893. [MycoBank MB 176748]. Synonymized in this study.

Sterigmatocystis ficuum (Reichardt) Henn., Hedwigia 34: 86. 1895. [MycoBank MB 100573]. See Ustilago ficuum Reichardt (basionym).

Synonym: Sterigmatocystis welwitschiae (Bres.) Henn. in H. Baum, Kunene-Zambesi Expedit., Berlin: 168. 1903. [MycoBank MB 233714]. See Ustilago welwitschiae Bres. in Sacc. (basionym).

Synonym: Aspergillus batatas [as batatae] Saito, Zentralbl. Bakteriol. Parasitenkn. Abt. II, 18: 34. 1907. [MycoBank MB 214829]. Synonymized by Hong et al. (2014). Culture ex-type: NRRL 4785 = CBS 115.34 = NBRC 4034 = KACC 46995.

Synonym: Aspergillus welwitschiae (Bres.) Henn. in Wehmer, Zentrbl. Bakteriol. ParasitK. Abt. II, 18: 294. 1907. [MycoBank MB 490584]. See Ustilago welwitschiae Bres. in Sacc. (basionym).

Synonym: Aspergillopsis nigra (Tiegh.) Speg., Anales Mus. Nac. Hist. Nat. Buenos Aires, ser. 3, 13: 435. 1911. [MycoBank MB 210007]. Basionym: A. niger Tiegh.

Synonym: Rhopalocystis nigra (Tiegh.) Grove, J. Econ. Biol. 6: 41. 1911. [MycoBank MB 432029]. Basionym: A. niger Tiegh.

Synonym: Aspergillus niger var. altipes E. Schiemann, Z. Indukt. Abstamm. Vererbungsl. 8: 1–35. 1912. [Mycobank MB 493817]. This variety belongs to the ANL based on the ITS sequence (MH854603) derived from the ex-type strain CBS 102.12 (= ATCC 10549 = IBT 19348 = IBT 26343 = IFO 4067 = MUCL 13608 = NRRL 4863 = WB 4863).

Synonym: Sterigmatocystis batatas [as batatae] (Saito) Sacc. Syll. Fung. 22: 1261. 1913. [MycoBank MB 209678]. See A. batatas Saito (basionym).

Synonym: Aspergillus aureus Nakazawa, Rep. Govt. Res. Inst. Formosa 4: 215–222. 1915. [No MycoBank record]. Synonymized by Hong et al. (2014). Culture ex-type: CBS 121.28 = NRRL 4784 = NBRC 4031 = RIB 2014 = KACC 47003 = IFO 4031 = IMI 104687 = WB 4784. Illegitimate name [Art. 54.1; Turland et al. (2018)]. Not A. aureus Berk. 1836 [MycoBank MB 214770].

Synonym: Aspergillus ficuum (Reichardt) Thom & Currie, J. Agric. Res. 7: 12. 1916. [MycoBank MB 101909]. See Ustilago ficuum Reichardt (basionym).

Synonym: Aspergillus citricus Mosseray, Ann. Univ. Sci. Budap. Rolando Eotvos Nominatae Biol. 43: 262. 1934. [MycoBank MB 251457]. Synonymized by Frisvad et al. (2011) and Houbraken et al. (2014). The genotype of the ex-type strain IHEM 5622 (= CBS 126.48 = WB 337 = NRRL 337 = NCTC 1692 = MUCL 28130 = IMI 15954 = IFO 6428 = DSM 734 = CCRC 30206 = ATCC 10254) is identical to the ex-type strains of A. niger (Fig. 1).

Synonym: Aspergillus bainieri Mosseray, Ann. Soc. Sci. Brux. 54: 79. 1934. [No MycoBank record]. The description is based on the same strain like A. longobasidia Bainier ex Mosseray (CBS 121.48).

Synonym: Aspergillus longobasidia Bainier ex Mosseray, La Cellule 43: 227. 1934. [MycoBank MB 253086]. The ex-type strain CBS 121.48 (= NRRL 4857) belongs to the ANL (Hong et al. 2013).

Synonym: Aspergillus pseudocitricus Mosseray, La Cellule 43: 228. 1934. [Mycobank MB 254148]. The ex-type strain CBS 127.48 (= NRRL 4869) can be identified as A. niger based on benA (Hong et al. 2013).

Synonym: Aspergillus niger mut. fusca Blochwitz, Ann. Mycol. 32: 87. [No MycoBank record]. The ex-type strain CBS 113.33 (= NRRL 4864 = ATCC 10548 = WB 4864) can be identified as A. niger based on benA (Hong et al. 2013).

Synonym: Aspergillus hennebergii Blochwitz, Ann. Mycol. 33: 238. 1935. [MycoBank MB 266969]. Not validly published [Art. 39.1; Turland et al. (2018)]. The ex-type strain CBS 118.35 (= NRRL 4883 = CECT 2801 = QM 9704 = WB 4883 = VKM F-4392) can be identified as A. niger based on benA (Hong et al. 2013).

Synonym: Aspergillus aureus var. brevior [as brevius] Nakaz., Simo & A. Watan. Bull. Agric. Chem. Soc. Japan 12: 962. 1936. [MycoBank MB 493810]. Not validly published [Art. 39.1; Turland et al. (2018)]. The representative strain CBS 113.52(= WB 4841) can be identified as A. niger based on benA (Hong et al. 2013).

Synonym: Aspergillus awamori var. fuscus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 959. 1936. [MycoBank MB 250921]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2013, 2014). Representative strain: CBS 117.52 = NRRL 4844 = WB 4844.

Synonym: Aspergillus awamori var. minimus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 955. 1936. [MycoBank MB 250922]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2013). Representative strain: CBS 118.52 [the whole-genome sequence was generated by Seekles et al. (2022)].

Synonym: Aspergillus miyakoensis Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 963. 1936. [MycoBank MB 253392]. Not validly published [Art. 39.1; Turland et al. (2018)]. Synonymized by Hong et al. (2014). Culture ex-type: NRRL 4859 = CBS 117.51 = KACC 46998.

Synonym: Aspergillus pyri W.H. English, Res. Stud. State Coll. Wash. 8: 127. 1940. [MycoBank MB 453796]. Nomen nudum. Subsequent work by English led to recognition of the name as a synonym of A. niger and withdrawal of the name (Thom & Raper 1945, Raper & Fennell 1945).

Synonym: Aspergillus foetidus Thom & Raper, A manual of the Aspergilli: 219. 1945. [Mycobank MB 284300]. Synonymized by Varga et al. (2011). Culture ex-type: CBS 121.28 = NRRL 4784 = NBRC 4031 = RIB 2014 = KACC 47003 = IFO 4031 = IMI 104687 = WB 4784.

Synonym: Aspergillus usamii Sakag., Iizuka & M. Yamaz., J. Appl. Mycol. Japan 4: 1. 1950. [No MycoBank record]. Not validly published [Art. 39.1; Turland et al. (2018)]. Validly published later – see A. usamii Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy.

Synonym: Aspergillus usamii mut. shirousamii Iizuka & Yamaguchi. J. Gen. Appl. Microbiol. 1: 194–200. 1954. [No MycoBank record]. Not validly described. Synonymized by Hong et al. (2014). Culture ex-type: NRRL 4889 = KACC 47001.

Synonym: Aspergillus usamii Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy., J. Jap. Bot.: 232. 1965. [MycoBank MB 326664]. Synonymized by Yamada et al. (2011) and Hong et al. (2014). Culture ex-type: CBS 139.52 = NRRL 4760 = ATCC 11364 = ATCC 14331 = NBRC 4388 = RIB 2602 = IAM 2185 = KACC 47000 = IFO 4388 = QM 8164 = WB 4760.

Synonym: Aspergillus niger f. hennebergii Blochwitz ex Al-Musallam, Revision of the black Aspergillus species: 68. 1980. Doctoral diss., Rijksuniversiteit Utrecht, Netherlands. [MycoBank MB 118404]. See A. hennebergii Blochwitz (basionym). Synonym: Aspergillus niger var. usamii (Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy.) Al-Musallam, Revision of the black Aspergillus species: 64. 1980. Doctoral diss., Rijksuniversiteit Utrecht, Netherlands. [MycoBank MB 118400]. See A. usamii Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy. (basionym).

Synonym: Aspergillus niger var. ficuum (Reichardt) Kozak., Mycol. Pap. 161: 112. 1989. [MycoBank MB 127745]. See Ustilago ficuum Reichardt (basionym).

Synonym: Aspergillus lacticoffeatus Frisvad & Samson, Stud. Mycol. 50: 52. 2004. [Mycobank MB 500008]. Synonymized by Varga et al. (2011) and confirmed in this study.

Synonym: Aspergillus vinaceus Ferranti et al., J. Fungi 6: 371 - p14. 2020. [MycoBank MB 833399]. Synonymized in this study.

5. Aspergillus tubingensis Mosseray, La Cellule 43: 245. 1934. [MycoBank MB 255209]. — Type: CBS H-24288. Ex-type: NRRL 4875 = CBS 133056 = QM 8904 = WB 4875. DNA barcodes: ITS = EF661193; benA = EF661086; CaM = EF661151; RPB2 = EF661055.

Synonym: Sterigmatocystis pulverulenta McAlpine, Agric. Gaz. N.S.W., Sydney 7: 302. 1896 (1897). [MycoBank MB 194998]. Synonymized by Houbraken et al. (2014). The ex-type strain IHEM 5615 (= CBS 558.65 = CBS 115.48 = MUCL 13592 = MUCL 15630 = NRRL 4851 = WB 4851 = IMI 211396 = ATCC 16879) is close to the ex-type of A. tubingensis (Fig. 1).

Synonym: Aspergillus pulverulentus (McAlpine) Wehmer, Zentralbl. Bakteriol. Parasitenkd., Abt. II, 18: 394. 1907. [MycoBank MB 121243]. See Sterigmatocystis pulverulenta McAlpine (basionym).

Synonym: Aspergillus cinnamomeus E. Schiemann, Z. Indukt. Abstamm. Vererbungsl. 8: 1–35. 1912. [MycoBank MB 122195]. The benA sequence (FJ629307) derived from the ex-type strain CBS 103.12 = ATCC 1027 = IBT 16906 = IBT 28139 = IBT 29892 = IFO 4043 = IMI 016148 = LSBH Ac42 = MZKI A-76 = NCTC 3774 = NRRL 348 = QM 326 = Thom 3534B = WB 348) is identical with the ex-type of A. tubingensis.

? Synonym: Aspergillus fuscus E. Schiem., Z. Indukt. Abstamm. Vererbungsl. 8: 1–35. 1912. [MycoBank MB 450741]. Illegitimate name [Art. 54.1; Turland et al. (2018)]. Not A. fuscus Bonord. 1861 [MycoBank MB 206602]. Illegitimate name A. fuscus was replaced by nomen novum A. schiemanniae Thom 1916 (see A. schiemanniae).

? Synonym: Aspergillus proteus E. Schiem., Z. Indukt. Abstamm. Vererbungsl. 8: 1–35. 1912. [No MycoBank record]. Illegitimate name – not accepted by the author itself in the original publication [Art. 36.1; Turland et al. (2018)].

Synonym: Aspergillus pulverulentus (McAlpine) Thom, J. Agric. Res. 7: 11. 1916. [No MycoBank record]. Illegitimate name [Art. 54.1; Turland et al. (2018)] - a later homonym of A. pulverulentus (McAlpine) Wehmer 1907.

? Synonym: Aspergillus schiemanniae Thom [as schiemanni], J. Agric. Res. 7: 11. 1916. [MycoBank MB 102113]. The ITS sequence (MH854950) derived from the ex-type strain CBS 122.28 (= ATCC 1040 = NRRL 361 = IFO 4091 = IMI 091895 = IMI 16269 = LSHB AC.37 = NCTC 3781 = QM 327 = Thom 3534C = WB 361) belongs to the ATL.

Synonym: Aspergillus elatior Mosseray, Ann. Univ. Sci. Budap. Rolando Eotvos Nominatae Biol. 43: 253. 1934. [MycoBank MB 251950]. The ex-type strain IHEM 5615 (= CBS 558.65 = CBS 115.48 = MUCL 13592 = MUCL 15630 = NRRL 4851 = WB 4851 = IMI 211396 = ATCC 16879) is similar to the ex-type of A. tubingensis (Fig. 1).

Synonym: Aspergillus pseudoniger Mosseray, La Cellule 43: 256. 1934. [MycoBank MB 472168]. The ex-type strain IHEM 4379 (= CBS 128.48 = IMI 313491 = WB 4870 = MUCL 31312) is similar to A. tubingensis (Fig. 1).

Synonym: Aspergillus niger mut. cinnamomeus (E. Schiemann) Thom & Raper, A manual of the Aspergilli: 223. 1945. [MycoBank MB 440825]. See A. cinnamomeus E. Schiemann (basionym).

? Synonym: Aspergillus niger mut. schiemanniae (Thom) Thom & Raper [as schiemanni], A manual of the Aspergilli: 224. 1945. [MycoBank MB 123940]. See A. schiemanniae Thom (basionym).

Synonym: Aspergillus saitoi Sakag., Iizuka & M. Yamaz., J. Appl. Mycol. Japan 3: 68. 1950. [No MycoBank record]. Not validly published [Art. 39.1; Turland et al. (2018)]. Validly published later – see A. saitoi Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy.

Synonym: Aspergillus saitoi var. kagoshimaensis Sakag., Iizuka & M. Yamaz., J. Appl. Mycol. Japan 4: 3. 1950. [No MycoBank record]. Not validly published [Art. 39.1; Turland et al. (2018)]. Validly published later – see A. saitoi var. kagoshimaensis Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy.

Synonym: Aspergillus awamori var. hominis Bat. & Maia, An. Soc. Biol. Pernambuco 15: 186. 1957. [MycoBank MB 351895]. The benA sequence (FJ629318) derived from the ex-type strain CBS 107.55 (= NRRL 4740 = ATCC 12074 = WB 4740) is almost identical (419/420 bp) to the ex-type of A. tubingensis.

Synonym: Aspergillus saitoi Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy., J. Jap. Bot. 40: 230. 1965. [Mycobank MB 326655]. Synonymized by Hong et al. (2014). Culture ex-type: CBS 136.52 = CBS 552.65 = NRRL 4757 = IAM 2209 = ATCC 11362 = IMI 211395 = KACC 46993 = WB 4757.

Synonym: Aspergillus saitoi var. kagoshimaensis Sakag., Iizuka & M. Yamaz. ex Iizuka & K. Sugiy., J. Jap. Bot. 40: 231. 1965. [MycoBank MB 349043]. Synonymized by Hong et al. (2014). Culture ex-type: CBS 137.52 = NRRL 4758 = IMI 214827 = ATCC 11363 = KACC 46994 = QM 8162 = IAM 2190 = WB 4758.

Synonym: Aspergillus niger f. pulverulentus (McAlpine) Al-Musallam, Revision of the black Aspergillus species: 58. 1980. Doctoral diss., Rijksuniversiteit Utrecht, Netherlands. [MycoBank MB 118403]. See Sterigmatocystis pulverulenta McAlpine (basionym).

Synonym: Aspergillus niger var. pulverulentus (McAlpine) Kozak., Mycol. Pap. 161: 113. 1989. [MycoBank MB 127747]. See Sterigmatocystis pulverulenta McAlpine (basionym).

Synonym: Aspergillus niger var. tubingensis (Mosseray) Kozak., Mycol. Pap. 161: 112. 1989. [MycoBank MB 127746]. Basionym: A. tubingensis Mosseray.

Synonym: Aspergillus costaricensis [as costaricaensis] Samson & Frisvad, Stud. Mycol. 50: 52. 2004. [MycoBank MB 369151]. Synonymized in this study.

Synonym: Aspergillus neoniger Varga, Frisvad & Samson, Stud. Mycol. 69: 16. 2011. [MycoBank MB 560390]. Synonymized in this study.

Synonym: Aspergillus chiangmaiensis S. Khuna, N. Suwannarach & S. Lumyong, Front. Microbiol. 12: 705896 - p6. 2021. [Mycobank MB 830887]. Synonymized in this study.

Synonym: Aspergillus pseudopiperis S. Khuna, N. Suwannarach & S. Lumyong, Front. Microbiol. 12: 705896 - p6. 2021. [MycoBank MB 830888]. Synonymized in this study.

6. Aspergillus vadensis Samson, de Vries, Frisvad & Visser, Antonie van Leeuwenhoek 87: 201. 2005. [MycoBank MB 340234]. — Type: CBS 113365. Ex-type: CBS 113365 = CECT 20584 = IMI 313493 = IBT 24658. DNA barcodes: ITS = AY585549; benA = AY585531; CaM = FN594560; RPB2 = HE984371.

Synonym: Aspergillus vadensis de Vries et al., Appl. Environ. Microbiol. 70: 3954. 2004. [MycoBank MB 560390]. Not validly described (nomen nudum).

Unresolved or doubtful names most probably belonging to the series Nigri

Aspergillus phaeocephalus Durieu & Mont., Exploration scientifique de l’Algérie 1: 342. 1848. [MycoBank MB 179669]. No specimens are available.

Synonym: Sterigmatocystis phaeocephala (Durieu & Mont.) Sacc., Syll. Fung. 4: 76. 1886. [MycoBank MB 233071].

Aspergillus nigrescens C.P. Robin, Histoire naturelle des végétaux parasites: 518. 1853. [MycoBank MB 182212]. Considered either A. fumigatus or A. niger by various authors (Wilhelm 1877, Wehmer 1901, Raper & Fennell 1965), no material is available (species described from pathological specimens only).

Aspergillus nanus Mont., Syll. Gen. Sp. Crypt. (Paris): 300. 1856. [MycoBank MB 193617]. Typus in Muséum National d’Histoire Naturelle, France, Paris (PC); no DNA sequence available. The strains of A. niger var. nanus examined by Al-Musallam (1980) belong either to the ANL or ATL, e.g., CBS 105.47 (MH856174; ATL), CBS 106.47 (FJ629281; ATL), CBS 110.30 (MH855092; ANL), CBS 115.50 (MH856565; ANL), CBS 131.52 (the whole-genome sequence was generated by Seekles et al. (2022); ANL).

Synonym: Aspergillus niger var. nanus (Mont.) Al-Musallam, Revision of the black Aspergillus species: 62. 1980. Doctoral diss., Rijksuniversiteit Utrecht, Netherlands. [MycoBank MB 118406].

Sterigmatocystis antacustica C.E. Cramer, Vierteljahrsschr. Naturforsch. Ges. Zürich 4: 325. 1859. [MycoBank MB 209963].

Synonym: Rhopalocystis antacustica (C.E. Cramer) Grove, J. Econ. Biol.: 41. 1911. [MycoBank MB 503610].

Aspergillus nigricans Wreden, C. R. Hebd. Seances Acad. Sci. 65: 368. 1867. [No MycoBank record]. No specimen is available.

Aspergillus fuliginosus Peck, Bull. Buffalo Soc. Nat. Sci. 1: 69. 1873. [MycoBank MB 208679]. Typus in New York State Museum, USA (NYSf 1265); no DNA sequence available.

Synonym: Sterigmatocystis fuliginosa Bainer, Bull. Soc. Bot. France 28: 78. 1881. [No MycoBank record].

Aspergillus nigricans Cooke, Grevillea 6: 127. 1878. [MycoBank MB 182128]. Illegitimate name. Not A. nigricans Wreden 1867.

Alliospora sapucaya [as sapucayae] Pim, J. Bot. London 21: 234. 1883. [Mycobank MB 215122].

Aspergillus cookei Sacc., Syll. Fung. 4: 71. 1886. [MycoBank MB 206435]. A replaced synonym for illegitimate name A. mucoroides Cooke.

Basionym: Aspergillus mucoroideus Cooke, Grevillea 12: 9. 1883. [MycoBank MB 187861]. Illegitimate name [Art. 54.1; Turland et al. (2018)]. Not A. mucoroides Corda 1838 [MycoBank MB 187771].

Aspergillus subfuscus Johan-Olsen, Nordiskt Med. Ark. 18: 14. 1886. [MycoBank MB 184212].

Basionym: Sterigmatocystis subfusca (Johan-Olsen) Sacc., Syll. Fung. 10: 526. 1892. [MycoBank MB 226258].

Aspergillus nigriceps Berk. & Curt., Grevillea 17: 21. 1888. [No MycoBank record]. A slide from material in the Harvard Collection (Curtis collection, Wright no. 927) showed species inseparable from A. niger (Raper & Fennell 1965).

Aspergillus ustilago Beck in H. Wawra, Itinera Principum S. Coburgi 2: 148. 1888. [MycoBank MB 161832].

Synonym: Sterigmatocystis ustilago (Beck) Sacc., Syll. Fung. 10: 526. 1892. [MycoBank MB 198044].

Sterigmatocystis castanea F. Patt., Bull. Torrey Bot. Club 27: 284. 1900. [MycoBank MB 195664]. Typus in BPI Herbarium (BPI 410863).

Aspergillus gallomyces Calmette, Germ. Pat. Abstr. 129: 164. 1902. [No MycoBank record].

Aspergillus strychni Lindau, Hedwigia 43: 306. 1904. [MycoBank MB 183848].

Synonym: Sterigmatocystis strychni (Lindau) Sacc. & D. Sacc., Syll. Fung. 18: 516. 1906. [MycoBank MB 225824].

Sterigmatocystis pseudonigra Costantin & Lucet, Bull. Soc. Mycol. France 19: 33. 1903. [MycoBank MB 195028]. No specimen available.

Synonym: Aspergillus pseudoniger (Costantin & Lucet) Saincl., Centralbl. Gesammte Forstwesen: 103. 1949. [MycoBank MB 292856]. Illegitimate name. Not A. pseudoniger Mosseray 1934. [MycoBank MB 472168].

Sterigmatocystis luteonigra M.L. Lutz, Bull. Soc. Bot. France 53: 50. 1907. [MycoBank MB 228634].

Synonym: Aspergillus luteoniger (M.L. Lutz) Thom & Church, The Aspergilli: 166. 1926. [MycoBank MB 269972].

Aspergillopsis intermedia Speg., Anales Mus. Nac. Hist. Nat. Buenos Aires, ser. 3, 13: 435. 1911. [MycoBank MB 209760]. Typus deposited in the Herbarium of Museo de La Plata, Argentiva (LPS 12691); no DNA sequence is available. The strains considered A. niger var. intermedius by Al-Musallam (1980) belong either to the ANL or ATL, e.g., CBS 117.32 (MH855233; ATL), CBS 115.29 (MH855018; ATL), CBS 117.52 (MH856951; ANL), CBS 130.52 (FJ629359, FJ629310; ATL), CBS 107.55 (FJ629367, FJ629318; ATL).

Synonym: Aspergillus niger var. intermedius (Speg.) Al-Musallam, Revision of the black Aspergillus species: 66. 1980. Doctoral diss., Rijksuniversiteit Utrecht, Netherlands. [MycoBank MB 118402].

Aspergillus phoenicis (Corda) Thom, J. Agric. Res. 7: 14. 1916. [MycoBank MB 101952].

Basionym: Ustilago phoenicis Corda, Icones fungorum hucusque cognitorum 4: 9. 1840. [MycoBank MB 169060]. Nomen rejiciendum, rejected name in favour of A. niger Tiegh. (Kozakoewict et. al. 1992). Type located in Corda′s herbarium (PRM, Czech Republic) but no sequence derived from the original material is available. Representative strains NRRL 363, NRRL 365 and NRRL 1956 do either belong to ANL or ATL (Houbraken et al. 2014).

Synonym: Sterigmatocystis phoenicis (Corda) Pat. & Delacr., Bull. Soc. Mycol. France 7: 119. 1891. [MycoBank MB 232882].

Synonym: Aspergillus niger var. phoenicis (Corda) Al-Musallam, Revision of the black Aspergillus species: 56. 1980. Doctoral diss., Rijksuniversiteit Utrecht, Netherlands. [MycoBank MB 118401].

Aspergillopsis tropicalis Speg., Bol. Acad. Nac. Cienc. Córdoba 23: 588. 1918. [MycoBank MB 209817].

Aspergillus fumaricus Wehmer ex Thom & Church, The Aspergilli: 181. 1926. [MycoBank MB 122538].

Sterigmatocystis gigantea Mattlet, Ann. Soc. Belge Méd. Trop. 6: 31. 1926. [MycoBank MB 252349].

Synonym: Aspergillus giganteus (Mattlet) C.W. Dodge, Medical Mycology: 629. 1935. [No MycoBank record]. Illegitimate name. Not A. giganteus Wehmer 1901 [MycoBank MB 206765].

Synonym: Aspergillus mattletii Hendr., Publ. Inst. Nat. Étude Agron. Congo Belge 35: 7. 1948. [MycoBank MB 284307].

Aspergillus atropurpureus Blochwitz, Ann. Mycol. 32: 86. 1934. [No MycoBank record]. Illegitimate name. Not A. atropurpureus Zimm. 1902 [MycoBank MB 214593].

Aspergillus biourgei Mosseray, La Cellule 43: 241. 1934. [MycoBank MB 251009].

Aspergillus buntingii Mosseray, La Cellule 43: 236. 1934. [MycoBank MB 251145].

Aspergillus churchii Mosseray, La Cellule 43: 242. 1934. [MycoBank MB 251413].

Aspergillus densus Mosseray, La Cellule 43: 232. 1934. [MycoBank MB 251792].

Aspergillus granulatus Mosseray, La Cellule 43: 249. 1934. [MycoBank MB 252446]. Culture ex-type: CBS 120.48 = NRRL 4855 = WB 4855 (no DNA sequence available).

Aspergillus guttifer Mosseray, La Cellule 43: 235. 1934. [MycoBank MB 252493].

Aspergillus microcephalus Mosseray, La Cellule 43: 225. 1934. [MycoBank MB 253336]. The strain CBS 122.48 is considered the ex-type strain (Al-Musallam 1980) but no DNA sequence is available.

Aspergillus niger var. fermentarius Nakaz., Simo & A. Watan., J. Agric. Chem. Soc. Japan 10: 171. 1934. [No MycoBank record].

Aspergillus olivaceofuscus Mosseray, La Cellule 43: 258. 1934. [MycoBank MB 253675].

Aspergillus praecox Mosseray, Ann. Soc. Sci. Brux. 54: 79. 1934. [No MycoBank record].

Aspergillus pseudoelatior Mosseray, La Cellule 43: 255. 1934. [MycoBank MB 254152].

Aspergillus rutilans Mosseray La Cellule 43: 234. 1934. [MycoBank MB 254522].

Aspergillus sclerotifer Mosseray, Ann. Soc. Sci. Brux. 54: 79. 1934. [No MycoBank record].

Aspergillus variegatus Mosseray La Cellule 43: 238. 1934. [MycoBank MB 255332].

Aspergillus macfiei C.W. Dodge, Medical Mycology: 629. 1935. [MycoBank MB 253139]. Not validly described [Art. 39.1, 40.1; Turland et al. (2018)].

Aspergillus aureus var. minor Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 958. 1936. [MycoBank MB 534300]. Not validly published [Art. 39.1; Turland et al. (2018)].

Aspergillus aureus var. murinus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 959. 1936. [MycoBank MB 534301]. Not validly published [Art. 39.1; Turland et al. (2018)].

Aspergillus awamori var. ferrugineus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 957. 1936. [Mycobank MB 250919]. Not validly published [Art. 39.1; Turland et al. (2018)].

Aspergillus awamori var. fumeus Nakaz., Simo & A. Watan., Bull. Agric. Chem. Soc. Japan 12: 961. 1936. [MycoBank MB 250920]. Not validly published [Art. 39.1; Turland et al. (2018)]. Representative strain: CBS 116.52 = NRRL 4843 = WB 4843 = IBT 16908 (no DNA sequence available).

Aspergillus luchuensis var. rubeolus Y.K. Shih, Lingnan Sci. J. 15: 374. 1936. [Mycobank MB 253092]. The ITS sequence (DQ196202) derived from the representative strain CBS 114.37 (= IAM 2185 = IBT 4944 = IBT 29898 = IMI 313493 = NRRL 4856 = WB 4856) belongs to the ATL.

Cladosarum olivaceum E. Yuill & J.L. Yuill, Trans. Brit. Mycol. Soc. 22: 199. 1938. [MycoBank MB 272878]. Culture ex-type: CBS 147.38 (no DNA sequence available).

Aspergillus niger var. arecae Lal & Ram Chandra, J. Scient. Res. Banaras Hindu Univ. 3: 123. 1953. [MycoBank MB 349040]. Not validly published [Art. 39.1; Turland et al. (2018)].

Aspergillus niger var. taxi D.P. Zhou, K. Zhao & Ping, J. Appl. Microbiol. 107: 1206. 2009. [MycoBank MB 542212]. Not validly described [Art. 39.1, 40.1; Turland et al. (2018)]. The ITS sequence (EU853157) derived from the original strain belongs to the ATL.

Aspergillus pseudotubingensis S. Khuna, N. Suwannarach & S. Lumyong, Front. Microbiol. 12: 705896 - p9. 2021. [MycoBank MB 830889]. See comments in sections Results and Discussion.

DISCUSSION

Taxonomic studies on A. niger, A. tubingensis and their related species have been the subject of many taxonomic controversies, rearrangements, name resurrections and repeated synonymizations. Currently, the species number in series Nigri is at one of its historical peaks and involves 14 accepted species (Houbraken et al. 2020, Silva et al. 2020, Khuna et al. 2021). Distinguishing among these species using morphology is impossible, and DNA sequencing is the gold standard for reliable classification and species identification (Howard et al. 2011, Varga et al. 2011). Therefore, these species can be considered cryptic in the truest sense. However, even when using molecular methods, some isolates cannot be identified satisfactorily at the species level (Howard et al. 2011, Negri et al. 2014, D’hooge et al. 2019). Contradictory species identifications in significant part of isolates using BLAST similarity searches with different genes (Fig. 3) can serve as evidence of taxonomic issues and may indicate the need for a taxonomic reclassification. To verify this assumption, we gathered a large dataset of a series Nigri sequences from three common phylogenetic markers and de novo sequenced 18 genomes and employed various species delimitation methods. All of these methods unequivocally suggested species number reduction.

Phylogenetic support of species reduction

We demonstrated that MSC delimitation methods using three genes broadly agreed on the distinction of only three species in the series Nigri (A. brasiliensis, A. niger and A. tubingensis). Only four analyses from among the 15 depicted in Fig. 5 proposed the delimitation of more species. These conclusive results, especially in the A. niger lineage (ANL) and A. brasiliensis lineage (ABL), are supported by the collection of a large number of strains well representing the intraspecific genetic variability.

In the A. tubingensis lineage (ATL), the multilocus method STACEY gave similar support to the delimitation of one or four species. This dilemma has not been satisfactorily resolved even at the genome level because ten STACEY analyses based on 20 genes each proposed several solutions ranging from one to four species in the ATL (Fig. 8), with four species being most preferred. The datasets containing 20 genes (and not higher) were selected due to the relatively high computational requirements of the method and because 20 genes are usually enough to resolve the phylogeny with a similar level of accuracy to the genome-wide data (Rokas et al. 2003, Yang & Rannala 2010, 2014). The uncertainty regarding the number of delimited species can probably be attributed to the underrepresentation of isolates related to A. vadensis and A. eucalypticola and, to a lesser extent, other species in the ATL, with the exception of A. tubingensis itself. Generally, it is thought that when a dataset contains a few isolates with large genetic distances, the delimitation methods may be prone to over delimitation, but with a large set of isolates representing genetic variability of every species, the probability of methods finding real species boundaries increases (Pante et al. 2015, Chambers & Hillis 2020). Another reason for the conflicting results can be the widely present incongruences between single-gene genealogies as typically present in recently speciating species or in populations that have not completed their speciation process. In recently diverged species, it is typically caused by retaining ancestral polymorphisms (incomplete lineage sorting), or it can be caused by past hybridization events (Hubka et al. 2018, Steenkamp et al. 2018, Matute & Sepúlveda 2019).

At this point, it is appropriate to mention that the single-gene phylogenies based on benA, CaM and RPB2 loci were highly incongruent (Fig. 2, Supplementary Fig. S2) and that the topology of the resulting multigene phylogeny shows several conflicts or poorly resolved clades compared to the whole-genome phylogeny, as we demonstrated in Fig. 4. Suboptimal phylogenetic signals contained in these three loci can further contribute to the discrepancy between the results of the MSC methods (support of a single species in the ATL) and those of the STACEY analyses based on 20 loci randomly selected from genomes (predominant support of four species in the ATL) (Fig. 8). When species limits are evaluated using the GCPSR approach (Taylor et al. 2000, Dettman et al. 2003a) using the benA, CaM and RPB2 loci only, the obvious conclusion is the recognition of only three species in the series Nigri: A. brasiliensis, A. niger and A. tubingensis. The recently proposed A. vinaceus (Silva et al. 2020) from the ANL was also described based on the GCPSR approach, but the phylogeny lacked some isolates with intermediate genotypes included here. The presence of these strains in the phylogenies contradicts the delimitation of A. vinaceus as a separate phylogenetic species.

Another independent testing of species hypotheses was performed in the recently released software DELINEATE (Sukumaran et al. 2021). The software needs some a priori defined species that are correctly delimited in the dataset. This requirement was fulfilled by the inclusion of some species belonging to the other series of section Nigri. In addition, A. brasiliensis is well defined and can serve as a “reference species” when dealing with delimitation in A. niger and A. tubingensis lineages. The results of the various models were relatively stable, always supporting the broad concept of A. niger (comprising A. niger, A. welwitschiae and A. vinaceus) and A. brasiliensis (Fig. 7). In the ATL a broad species definition (comprising all species in the lineage) gained support in Models 7 and 10 when A. niger was defined as a broad species as well. In models where the ANL was divided into four and six species, the unassigned ATL populations were segregated into four tentative species. This scenario is, however, improbable because the ANL was never divided into several species in Models 2, 6 and 9. Model 6 shows the situation where populations of the ATL are predefined into four species identical to those delimited by Models 3 and 4, while populations of the ANL are unassigned. In this situation, A. niger was also delimited as a single broad species. In summary, DELINEATE analysis convincingly supported that the series Nigri contains only three species based on benA, CaM and RPB2.

Phenotype and sexual reproduction in series Nigri

Species in the series Nigri are all biseriate and present colonies with brown, dark brown to black sporulating areas and white to yellowish white mycelial areas. Generally, the species exhibit largely overlapping characteristics in terms of both macromorphology (colony diameter and colour) and micromorphology (size, shape and ornamentation of conidia, diameter and shape of vesicle, and length and width of stipes) (Table 2). Not only particular species but also the main lineages of A. niger and A. tubingensis are indistinguishable from each other using morphology. Additionally, species identification is sometimes complicated by the occurrence of atypical strains exhibiting unusual morphological characteristics. For example, A. lacticoffeatus was described based on its different colony colour and a specific extrolite pattern (Samson et al. 2004). However, it was later synonymized with A. niger, because it was phylogenetically inseparable (Varga et al. 2011). Within the ATL, A. vadensis stands out among the others for its slow growth and short stipes (Table 2), but the original description is based on only one isolate (Vries et al. 2005).

Table 2.

Overview of macro- and micromorphological features of species belonging to the series Nigri.

Species	CYA, 7 d (mm)		MEA, 7 d, 25 °C (mm)	Prevailing colony colour(s) on CYA	Vesicle diam (μm), shape	Stipe (μm)		Conidia: diam (μm), shape, surface	Sclerotia (mm), colour	References ¹
Species	25 °C	37 °C	MEA, 7 d, 25 °C (mm)	Prevailing colony colour(s) on CYA	Vesicle diam (μm), shape	Length	Width	Conidia: diam (μm), shape, surface	Sclerotia (mm), colour	References ¹
A. brasiliensis	71–76	71–76	52–70	cream-coloured to light brown	30–45, nearly globose	700–1 700	8–13	3.5–4.8, subglobose, echinulate	1–1.5, white	Varga et al. (2007)
	70–85	70–85	n/a	black	45–80, globose	1 500–3 000	15–20	3.5–5.5, globose, finely to distinctly roughened	0.5–0.7, cream	Crous et al. (2009), Samson et al. (2010), Frisvad et al. (2014)
A. lacticoffeatus	71–76	59–75	52–70	cream to light brown	40–65, nearly globose	(200–)300–1 200	(7–)10–15(–18)	3.5–4.1 × 3.4–3.9, subglobose, usually smooth to very finely roughened	not observed	Samson et al. (2004)
A. vinaceus	64–66	60–63.5	35–39	black	63–75, globose to subglobose	1 300–1 800	16–21	3–5.5, globose to subglobose, finely roughened to echinulate	0.9–1.5, white to cream	Silva et al. (2020)
A. welwitschiae	similar to A. niger	similar to A. niger	similar to A. niger	black	45–85, globose	similar to A. niger	n/a	3.5–5.5, globose, finely to distinctly roughened	not observed	Hong et al. (2013), Silva et al. (2020)

A. chiangmaiensis	62–63	57–66	68–70	dark brown to black	35–68, globose to ellipsoidal	370–1 430	10–15	3–4.5, globose to subglobose, echinulate	0.4–1.4, white	Khuna et al. (2021)
A. costaricensis	63–78	58–62	26–62	white with sparse black-sporulating areas	40–90, globose	(800–)1 000–1 700(–1 900)	(12–)13–20(–22)	3.1–4.5, globose to subglobose, smooth to distinctly roughened	1.2–1.8, pink to yellow	Samson et al. (2004)
A. eucalypticola	68–72	30–50	46–51	beige to cream yellow	30–55, globose	n/a	8–14	2.5–3.5, globose, smooth to coarsely roughened	not observed	Varga et al. (2011)
A. luchuensis ²	37–80	30–67	43–68	white, gray, brown, black	15–90, subglobose to globose	1500	10–22	3–4.5, globose, smooth	0.8–1.3, cream	Samson et al. (2010), Varga et al. (2011), Hong et al. (2013), Frisvad et al. (2014)
A. neoniger	72–80	37–67	54–61	gray to black	30–50, globose	n/a	8–12	3.5–5, globose, coarsely roughened to echinulate	observed (without description)	Varga et al. (2011), Frisvad et al. (2014)
A. piperis	60–75	64–82	59–78	cream-coloured with sparse black-sporulating areas	40–55, subglobose	(300–)400–3 000	(7–)12–15(–20)	2.8–3.6, subglobose to broadly ellipsoidal, distinctly roughened	1–1.7, yellow to pink brown	Samson et al. (2004)
A. pseudopiperis	63–65	61–64	55–60	greenish brown to dark brown	15–39, globose to ellipsoidal	200–2 125	10–18	3–5, globose to subglobose, roughened to spinulose	0.2–1 × 0.2–0.8, light yellow to pinkish orange	Khuna et al. (2021)
A. pseudotubingensis	51–58	55–68	62–63	grayish brown to dark brown	20–70, globose to ellipsoidal	740–3 060	10–18	3–6, globose to subglobose, fine spiny surface	not observed	Khuna et al. (2021)
A. tubingensis	n/a	n/a	40–65	beige, grayish brown, brown to black	27–80, globose	550–2 300(–6 000)	10–35	3–5, globose, coarsely roughened	0.4–1.8, cream to pinkish tan	Samson et al. (2007), Varga et al. (2007), Hong et al. (2013), Horn et al. (2013), Ismail (2017)
A. vadensis	25	n/a	30	light brown to olive green brown	25–35, globose	up to 150	6–15	3–4, globose, rough-walled to finely echinulate	not observed	de Vries et al. (2005)

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n/a - exact data not available.

¹if there are multiple studies in the column “references”, the description and dimensions are shown as a summary from all these sources.

²the dimensions shown here represent summary from description of A. luchuensis and its synononym A. acidus as they appeared in the publications of Varga et al. (2011) and Hong et al. (2013).

Series Nigri species are known for producing a wide range of extrolites, and their spectra were summarised by Samson et al. (2007), Nielsen et al. (2009) and Frisvad et al. (2018). It is often asserted that species in series Nigri could be identified by different secondary metabolite patterns. However, the extrolite profiles can vary among strains, which makes its use to distinguish between species complicated. Additionally, the usability of secondary metabolite production for taxonomy and evolutionary biology in general might be limited due to the potential for horizontal gene transfer of secondary metabolism gene clusters between species of the same genus or even between fungal genera (Richards 2011, Slot & Rokas 2011, Szöllősi et al. 2015). The high frequency of this phenomenon is also suggested from the genomes of series Nigri species (Vesth et al. 2018). Considering our proposed reclassification of series Nigri, the extrolite profiles of the six species we accepted need to be reassessed to determine if they support the delineation proposed here based on a phylogenetic species concept.

Sexual reproduction in heterothallic fungi is governed by mating-type (MAT) genes (Dyer & O’Gorman 2011). In section Nigri, the development of asci and ascospores occurs in the stroma of sclerotia (Horn et al. 2013). Therefore, sclerotia formation is considered a prerequisite for sexual reproduction. In the ATL, sclerotia were reported in most species except for A. eucalypticola, A. pseudotubingensis and A. vadensis. In the ANL, sclerotia were formed in some strains of A. vinaceus and A. niger (Frisvad et al. 2014, Silva et al. 2020). Sclerotia in the ATL were mostly pink to yellow, while those in the ANL were white to cream (Table 2). Despite the heterogeneity in sclerotia production among series Nigri species, most of them were proven to be heterothallic or protoheterothallic, which means that mating-type gene idiomorph(s) associated with heterothallism have been detected among the examined strains of these species (Houbraken et al. 2020). Currently, A. tubingensis is the only species in series Nigri with an experimentally proven sexual cycle (Horn et al. 2013). In this study, we detected both MAT1-1-1 and MAT1-2-1 idiomorphs among isolates of ANL and ATL (Fig. 4), in agreement with previous studies (Horn et al. 2013, Varga et al. 2014, Mageswari et al. 2016, Seekles et al. 2022). An interesting observation was made by Horn et al. (2013), who sequenced five genes in the parental isolates of A. tubingensis and their progeny resulting from sexual reproduction. The authors found that A. neoniger cannot be separated from A. tubingensis in the benA or tsr1 phylogenies, while in the other phylogenies (CaM, Mcm7 and RPB2), it is resolved outside the least inclusive clade containing all A. tubingensis strains and its ex-type. In our opinion, this fact demonstrates gene flow between A. tubingensis and A. neoniger and supports the synonymization made in this study. Aspergillus costaricensis was not included by Horn et al. (2013), but it is a close relative of A. neoniger and must be synonymized together to retain the monophyly of the redefined A. tubingensis (Fig. 5).

Updated taxonomy of series Nigri and species identification

We showed based on various independent species delimitation analyses that the number of accepted species in the Nigri series is too high and needs to be reduced. The methods broadly agreed that the ABL and ANL contain only one species each. The only uncertainty was about the species number in the ATL, where single-gene MSC methods and the GCPSR approach based on the commonly used phylogenetic markers benA, CaM and RPB2 supported only one species, while STACEY analyses supported up to four species: A. tubingensis, A. luchuensis, A. eucalypticola and A. vadensis. After considering all results and practical consequences, we decided on a conservative solution by retaining four species in the ATL. This newly proposed taxonomic treatment of series Nigri with six accepted species overall is shown in Fig. 10.

Fig. 10. — Taxonomic rearrangement of series *Nigri* into six species with marked synonymizations. The proposed taxonomy is schematically shown in the form of a radial tree (species tree based on *benA*, *CaM* and *RPB2* calculated in starBEAST). The positions of *A. chiangmaiensis* and *A. pseudopiperis* are approximated based on the tree shown in Figs 1, 3 as they were not included in this phylogenetic analysis.

The following findings played a major role in our decision-making process. Aspergillus luchuensis is industrially extremely important (Hong et al. 2013, 2014), and its imprudent synonymization without further confirmation could cause unnecessary taxonomic instability. The phylogenetic signal from the dataset containing only benA, CaM and RPB2 loci may be suboptimal when compared to the predominant signal obtained from whole genome data (Fig. 4); thus, the results should be considered with caution before data from more genes/genomes are evaluated. We also showed that the intraspecific genetic diversity of the broadly defined A. tubingensis would be higher than that commonly found for Aspergillus species, while the variability within A. tubingensis, excluding A. luchuensis, A. eucalypticola and A. vadensis comfortably falls within this range (Fig. 9).

We believe that the user community will benefit from the simplified taxonomy of series Nigri with a lower number of species. This new classification will facilitate species identification that is currently complicated by inconsistent identification results when using sequence data of different genes (Fig. 3) and the impossibility of finding species-specific mass spectra for some narrow species when using the MALDI-TOF method (Gautier et al. 2016, D’hooge et al. 2019, Ban et al. 2021). DNA sequence identification of these newly defined species is possible by using benA, CaM and RPB2 markers, although in a minority of cases, there can be conflicting identification in the ATL (Fig. 3). Because of this, we recommend that if identification to a species level is required in the ATL, it should be done through DNA sequencing of all three markers and performing phylogenetic analysis. The ITS barcoding sequence can only distinguish among ABL, ANL and ATL (Supplementary Fig. S3).

As mentioned above, we excluded three newly described species, A. chiangmaiensis, A. pseudopiperis and A. pseudotubingensis (Khuna et al. 2021), from the analyses based on the MSC model because of the poor quality of their available sequences. However, we propose that A. chiangmaiensis and A. pseudopiperis are synonyms of A. tubingensis, as their phylogenetic position in the combined ML, MP and BI trees is within the redefined A. tubingensis: the A. pseudopiperis ex-type strain (SDBR-CMUI1) is located in a clade sister to the clade containing the A. tubingensis ex-type (CBS 133056), and A. chiangmaiensis is closely related to A. costaricensis and A. neoniger (Figs 1, 3), which are also synonymized with A. tubingensis. The status of A. pseudotubingensis remains uncertain and needs to be revised in the future as it forms a relatively separate lineage in the combined phylogeny based on three loci similar to A. eucalypticola (Figs 1, 3). The phylogeny presented by Khuna et al. (2021) is affected by numerous errors in the DNA sequences of the newly described species and it shows clear signs of the long branch attraction phenomenon (Kück et al. 2012). We believe that a more reliable position is shown in our phylogenies constructed based on partially corrected sequences (Figs 1, 3).

The species reduction proposed in this study is in line with some other studies using MSC model-based methods, genomic data or more complex approaches for species delimitation in fungi. For example, species number was significantly reduced in the plant pathogens from the Diaporthe eres complex (Hilário et al. 2021) and Alternaria section Alternaria (Woudenberg et al. 2015), the edible mushroom Flammulina (Wang et al. 2018), the lichen-forming fungus Bryoria (Boluda et al. 2019), the indoor fungi from Aspergillus series Versicolores (Sklenář et al. 2022) and the opportunistic human and animal pathogens from Aspergillus series Viridinutantes (Hubka et al. 2018). Numerous examples can also be found outside the Fungi kingdom (Li et al. 2019, Feng et al. 2021, Parker et al. 2021). These studies demonstrate that species over splitting similar to that observed in series Nigri is relatively common in extensively studied groups and species number reduction can become an increasingly common trend in taxonomy.

Future prospects

Further reduction of species number in the series Nigri cannot be ruled out in the future after collecting more strains and more whole genome sequences from underrepresented taxa such as A. eucalypticola and A. vadensis. Inclusion of more variability from these rare taxa should resolve persistent ambiguities in the definition of species limits and increase the probability of methods to delimit species more conclusively. As mentioned above, the position of A. pseudotubingensis should be re-examined, and higher quality DNA sequences should be obtained, ideally whole genome sequences. For now, we recommend avoiding using this name until the abovementioned issues are resolved.

Advanced phylogenetic methods spanning the whole genome will allow for a systematic and comparable metric of species differentiation. For example, reciprocal monophyly and a high level of concordance between thousands of markers on the genome scale should allow better differentiation between processes such as recombination, incomplete lineage sorting and speciation in most cases (Kobmoo et al. 2019, Matute & Sepúlveda 2019, Mavengere et al. 2020). A dense population-level representation in genome-scale phylogenetic studies is important for accurate identification of species boundaries and for resolving evolutionary relationships between species and higher-level taxonomic ranks in Aspergillus (Steenwyck et al. 2022). Our efforts in the following years will be directed towards achieving this goal.

Acknowledgments

Vit Hubka was supported by the Czech Ministry of Health (grant NU21-05-00681), the Charles University Research Centre program no. 204069 and the Czech Academy of Sciences Long-term Research Development Project (RVO: 61388971). František Sklenář was supported by the project of Charles University Grant Agency (GAUK 380821). This work has been partly supported by JST SPRING awarded to Cai Bian (JPMJSP2109). CMV acknowledges the Plant Health and Protection - Agricultural Research Council (ARC-PHP), where research on the PPRI strains included in this study was initiated and financially supported by a grant (nr 110441; reference FBIS170406226088) received from the Foundational Biodiversity Information Programme (FBIP) of the National Research Foundation of South Africa. VH is grateful for the support from the Japan Society for the Promotion of Science - Postdoctoral Fellowships for Research in Japan (Standard) and Grant-in-aid for a JSPS research fellow (No. 20F20772). We are grateful to Jan Karhan for the help with graphical adjustments of analysis outputs. We thank Kateřina Glässnerová for her assistance in the laboratory and Osamu Yamada for providing copies of some old taxonomic studies.

DECLARATION ON CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

Supplementary Material: https://studiesinmycology.org/

Fig. S1.

Multilocus phylogeny of Aspergillus series Nigri based on three loci (benA, CaM and RPB2) without species A. chiangmaiensis, A. pseudopiperis and A. pseudotubingensis. Best-scoring Maximum Likelihood (ML) tree inferred in the IQ-TREE is shown; ultrafast bootstrap support values (ML bs) are appended to nodes; only support values ≥95 % are shown; the ex-type strains are designated with a bold print; the information on geographic origin and isolation source is plotted on the tree (see the legend).

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sim-2022-102-3-SF1-2.jpg^{(637KB, jpg)}

Fig. S2.

Comparison of single-gene genealogies based on the benA, CaM and RPB2 loci and created by three different phylogenetic methods (only one isolate per unique multilocus haplotype is included in each phylogeny). Best-scoring single-locus maximum likelihood (ML) trees are shown; ML ultrafast bootstrap support values (ML bs), maximum parsimony bootstrap support values (MP bs) and Bayesian inference posterior probabilities (BI pp) are appended to nodes. Only support values ≥95 %, ≥70 % and ≥0.95, respectively, are shown. A dash indicates lower statistical support for a specific node, or the absence of a node in the phylogeny, while an asterisk indicates full support. The ex-type strains are designated with a bold print. Alignment characteristics, partitioning schemes and substitution models are listed in Supplementary Table S1.

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sim-2022-102-3-SF2-2.jpg^{(952.1KB, jpg)}

Fig. S3.

Best scoring maximum likelihood tree inferred in IQ-TREE based on ITS rDNA region sequences of series Nigri members (Table 1). It is apparent from the tree that a limited variability present in this locus can only differentiate the three main lineages, and more detailed differentiation is not possible. The ITS sequence of the A. vadensis ex-type was excluded from the analysis because it contains numerous errors and unresolved positions (accession number AY585549). Alignment characteristics, partitioning schemes and substitution models are listed in Supplementary Table S1.

sim-2022-102-3-SF3.jpg^{(563.3KB, jpg)}

Table S1.

Characteristics of alignments, partition-merging results and best substitution model for each partition according to the Bayesian information criterion.

sim-2022-102-3-SD1.jpg^{(280.1KB, jpg)}

Table S2.

Assignment of strains into populations for analysis in DELINEATE based on BPP v. 4.3.

sim-2022-102-3-SD2.jpg^{(190KB, jpg)}

Table S3.

Aspergillus series Nigri genomes analyzed in this study and their basic characteristics.

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Table S4.

Distribution of 200 orthologous genes into ten separate STACEY analyses.

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Table S5.

List of unique multilocus haplotypes.

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Table S6.

Maximum sequence dissimilarity between isolates of the same Aspergillus species whose species limits have been delimited using methods based on a multispecies coalescent model; only species represented by isolates from at least three countries were included.

sim-2022-102-3-SD6-1.jpg^{(57.3KB, jpg)}

sim-2022-102-3-SD6-2.jpg^{(753.5KB, jpg)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1.

sim-2022-102-3-SF1-1.jpg^{(610.5KB, jpg)}

sim-2022-102-3-SF1-2.jpg^{(637KB, jpg)}

Fig. S2.

sim-2022-102-3-SF2-1.jpg^{(978.6KB, jpg)}

sim-2022-102-3-SF2-2.jpg^{(952.1KB, jpg)}

Fig. S3.

sim-2022-102-3-SF3.jpg^{(563.3KB, jpg)}

Table S1.

Characteristics of alignments, partition-merging results and best substitution model for each partition according to the Bayesian information criterion.

sim-2022-102-3-SD1.jpg^{(280.1KB, jpg)}

Table S2.

Assignment of strains into populations for analysis in DELINEATE based on BPP v. 4.3.

sim-2022-102-3-SD2.jpg^{(190KB, jpg)}

Table S3.

Aspergillus series Nigri genomes analyzed in this study and their basic characteristics.

sim-2022-102-3-SD3.jpg^{(240.7KB, jpg)}

Table S4.

Distribution of 200 orthologous genes into ten separate STACEY analyses.

sim-2022-102-3-SD4.jpg^{(162.1KB, jpg)}

Table S5.

List of unique multilocus haplotypes.

sim-2022-102-3-SD5.jpg^{(100.4KB, jpg)}

Table S6.

sim-2022-102-3-SD6-1.jpg^{(57.3KB, jpg)}

sim-2022-102-3-SD6-2.jpg^{(753.5KB, jpg)}

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PERMALINK

Reducing the number of accepted species in Aspergillus series Nigri

C Bian

Y Kusuya

F Sklenář

E D’hooge

T Yaguchi

S Ban

CM Visagie

J Houbraken

H Takahashi

V Hubka

Abstract

INTRODUCTION

METHODS

Molecular studies

Table 1.

Phylogenetic analyses and species delimitation

Whole genome sequencing and assembly

Genome annotation

Phylogenetic analyses based on the genomic sequences

RESULTS

Combined phylogeny based on three loci

Fig. 1.

Incongruences between single gene trees: consequences for the GCPSR approach and BLAST similarity searches

Fig. 2.

Fig. 3.

Phylogenomic tree

Fig. 4.

Multispecies coalescent model-based (MSC) methods

Fig. 5.

Fig. 6.

Species delimitation using DELINEATE

Fig. 7.

STACEY analysis using genomic data

Fig. 8.

Intraspecific genetic diversity in Aspergillus and series Nigri

Fig. 9.

TAXONOMY

Accepted species and their synonyms in Aspergillus section Nigri series Nigri

Unresolved or doubtful names most probably belonging to the series Nigri

DISCUSSION

Phylogenetic support of species reduction

Phenotype and sexual reproduction in series Nigri

Table 2.

Updated taxonomy of series Nigri and species identification

Fig. 10.

Future prospects

Acknowledgments

DECLARATION ON CONFLICT OF INTEREST

Supplementary Material: https://studiesinmycology.org/

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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