Updating the taxonomy of Aspergillus in South Africa.

The taxonomy and nomenclature of the genus Aspergillus and its associated sexual (teleomorphic) genera have been greatly stabilised over the last decade. This was in large thanks to the accepted species list published in 2014 and associated metadata such as DNA reference sequences released at the time. It had a great impact on the community and it has never been easier to identify, publish and describe the missing Aspergillus diversity. To further stabilise its taxonomy, it is crucial to not only discover and publish new species but also to capture infraspecies variation in the form of DNA sequences. This data will help to better characterise and distinguish existing species and make future identifications more robust. South Africa has diverse fungal communities but remains largely unexplored in terms of Aspergillus with very few sequences available for local strains. In this paper, we re-identify Aspergillus previously accessioned in the PPRI and MRC culture collections using modern taxonomic approaches. In the process, we re-identify strains to 63 species, describe seven new species and release a large number of new DNA reference sequences.

Introduction

Aspergillus is cosmopolitan fungi occurring on a wide range of substrates. Here they fulfil many different functions and have a wide-ranging influence on human and animal life. Even though most species occur as saprophytes living on dead organic material, various species have an (economic) impact on humans (Raper & Fennell 1965).

Human infections caused by Aspergillus are some of the most widely reported for all filamentous fungi (Gianni and Romano, 2004, Balajee et al., 2007). Aspergillus fumigatus, A. flavus and A. terreus attract special interest as human pathogens causing widespread aspergillosis (fungus ball) or bad allergies (Raper and Fennell, 1965, Steinbach et al., 2004, Sugui et al., 2012, de Hoog et al., 2014, Frisvad and Larsen, 2015b), while a much broader spectrum of species is known to cause less invasive and/or superficial infections (Kaur et al., 2000, Zotti and Corte, 2002, Hubka et al., 2012, de Hoog et al., 2014). Aspergillus causes widespread losses for agriculture where they spoil food or grow in agricultural produce, leading to mycotoxin contamination (Perrone et al., 2007, Pitt and Hocking, 2009, Samson et al., 2010, Frisvad and Larsen, 2015a). Aspergillus isolates produce three of the five agriculturally important mycotoxins, including aflatoxins, ochratoxins and fumonisins (Miller, 1995, Frisvad and Larsen, 2015a). The global cost of aflatoxin alone is huge and represents a major problem in developing countries where stunting in children is of major concern (Wu et al., 2008, Pitt et al., 2012, Wu, 2015). Aflatoxin is most commonly produced by A. flavus and A. parasiticus, but many other Aspergilli can produce this devastating mycotoxin. Ochratoxins are commonly produced by Aspergillus species classified in sections Circumdati and Nigri (Frisvad et al., 2004, Frisvad et al., 2011, Davolos et al., 2012, Visagie et al., 2014b), while some sect Nigri species can also produce fumonisins (Frisvad et al., 2011, Frisvad and Larsen, 2015a). On a more positive note, species have industrial applications as producers of enzymes, drugs, organic acids or are used in food fermentations. For example, A. oryzae (the domesticated form of A. flavus) is used in a koji fermentation important for the production of a wide variety of oriental foods (Raper and Fennell, 1965, Varga et al., 2000, Samson et al., 2010, Hong et al., 2013, Kim et al., 2014).

The taxonomy of Aspergillus and its nine associated sexual (or teleomorphic) genera has been greatly stabilised over the last decade. Based on a multigene phylogenetic study, Kocsube confirmed that Aspergillus is monophyletic and sister to Penicillium as originally shown by Houbraken & Samson (2011). Furthermore, they showed that the genus can be subdivided into six subgenera and several sections, which to a large degree corresponds to associated sexual states. The nomenclatural review and “accepted species list” published by Samson played a significant role in stabilizing the taxonomy of Aspergillus. It created an “open access” model in the sense that all metadata associated with species names, such as ex-type culture collection accession numbers, sectional classifications, MycoBank numbers and GenBank accession numbers to reference sequences generated from ex-type cultures, were released in the public domain. Calmodulin was proposed as a secondary identification marker to the formal, but rather conserved, ITS DNA barcode (Schoch ). All the released data resulted in more reliable species identifications, and new species discovery and its subsequent description are easier and more accurate than ever. Almost anybody with a bit of background knowledge can describe their new species. As a result, the accepted species list grew with more than 100 taxa in the space of 5 years and resulted in the so-called “broad” Aspergillus that has mostly been accepted by the community (Pitt and Taylor, 2014, Pitt and Taylor, 2016, Samson et al., 2014, Kocsube et al., 2016, Samson et al., 2017).

South Africa has great fungal diversity and makes significant contributions to international understanding of a wide range of fungi. Aspergillus is very commonly isolated across South Africa and unlike Penicillium (Schutte 1992), local mycologists were not afraid to attempt identifications down to species level (Cohen, 1950, Swart, 1959, Eicker, 1969, Eicker, 1970a, Eicker, 1970b, Eicker, 1972, Eicker, 1973, Eicker, 1974, Eicker, 1976, Eicker, 1980, van der Merwe et al., 1979, Rabie and Lübben, 1984, Allsop et al., 1987, Watson et al., 1990, Schutte, 1994, Roux and van Warmelo, 1997). These identifications were all based on morphology, meaning that diversity could easily be misidentified due to the known complexities in distinguishing between closely related species without DNA sequence data. Considering the modern methods required to identify species (Samson ), we consider Aspergillus to be grossly understudied in South Africa. To our knowledge, the only modern studies reported 23 species isolated from house dust (Visagie ) and seven species from abalone feed collected in the Western Cape (Greeff-Laubscher ). The PPRI culture collection housed at the Agricultural Research Council – Plant Health and Protection, Roodeplaat, Pretoria is the biggest repository of Aspergillus in South Africa with close to 500 accessioned strains. The PPRI also houses the old MRC (Medical Research Council) culture collection that contains several Aspergillus. Strains from these collections mostly originate from agricultural sources, but plenty was sourced from environmental collection trips across the country. The aim of this project was to recover as many strains as possible and re-identify them using modern DNA sequencing approaches in order to obtain a baseline knowledge on the diversity of Aspergillus in the country. In this paper, we report on the diversity discovered, formally introduce seven new species and release a large number of valuable DNA reference sequences in the NCBI nucleotide sequence database (GenBank).

Materials & methods

Strains

Strains were recovered from the South African National Collection of Fungi (PPRI) and the Medical Research Council (MRC) collection, both housed at the Agricultural Research Council (ARC; Plant Health and Protection, Roodeplaat). New isolates were obtained during routine identification services provided at PPRI. These originate from a wide range of sources across the country and were deposited into a working collection (CMV) and PPRI. Isolations were made using potato dextrose agar (PDA) or dichloran 18 % glycerol agar (DG18; Oxoid CM0729). Strains and its collection data are summarised in Table 1.

Table 1

Strains sequenced during the course of this project.

Species	Strains1	Section	Location collected / isolated, year	Host	GenBank nr
					ITS	BenA	CaM	RPB2
Aspergillus chevalieri	PPRI13427 = CMV011F5	Aspergillus	South Africa, KwaZulu-Natal, Pinetown, 2013	Soil	–	–	MK451336	–
A. chevalieri	PPRI26000 = CMV003I3	Aspergillus	South Africa, 2017	Animal feed	–	MK450979	MK451332	–
A. chevalieri	PPRI26033 = CMV012H5	Aspergillus	South Africa, Gauteng, Pretoria, 2018	Dog food	–	–	MK451338	–
A. chevalieri	PPRI26034 = CMV012H6	Aspergillus	South Africa, Gauteng, Pretoria, 2018	Dog food	–	–	MK451339	–
A. chevalieri	PPRI26348 = CMV016E5	Aspergillus	South Africa, Gauteng, Pretoria, 2019	Dog food	–	–	MN031422	–
A. chevalieri	PPRI26554 = CMV016D7	Aspergillus	South Africa, Gauteng, Pretoria, 2019	Dog food	–	–	MK951911	–
A. chevalieri	PPRI3791 = CMV011B6	Aspergillus	South Africa, 1986		–	–	MK451333	–
A. chevalieri	PPRI4908 = CMV011B7	Aspergillus	South Africa, Kwazulu Natal, 1993	Maize kernels (Zea mays)	–	–	MK451334	–
A. chevalieri	PPRI5410 = CMV012B1	Aspergillus	South Africa, Western Cape, Clanwilliam, 1994	Rooibos tea (Aspalathus linearis)	–	–	MK451337	–
A. chevalieri	PPRI6331 = CMV011B9	Aspergillus	South Africa, Gauteng, Pretoria, 1996	Dried sausage	–	–	MK451335	–
A. montevidensis	CMV012H4	Aspergillus	South Africa, Gauteng, Pretoria, 2018	Dog food	–	–	MK451446	–
A. montevidensis	MRC1250 = CMV017A6	Aspergillus	South Africa, Western Cape, Ceres, 1975	Apple juice concentrate	–	–	MK951923	–
A. montevidensis	PPRI26035 = CMV012H7	Aspergillus	South Africa, Gauteng, Pretoria, 2018	Dog food	–	–	MK451447	–
A. montevidensis	PPRI4851 = CMV011G2	Aspergillus	South Africa, Gauteng, Johannesburg, 1993	Air sample	–	–	MK451445	–
A. montevidensis	PPRI6330 = CMV011B8	Aspergillus	South Africa, Gauteng, Pretoria, 1996	Dried sausage	–	–	MK451443	–
A. montevidensis	PPRI8674 = CMV011C2	Aspergillus	South Africa, Gauteng, Johannesburg, 2007	Wheat (Triticum sp)	–	–	MK451444	–
A. porosus	PPRI3419a = CMV012A8 = CSIR980	Aspergillus	South Africa, 1988		–	–	MK451494	–
A. porosus	PPRI3419b = CMV012A9 = CSIR980	Aspergillus	South Africa, 1988		–	–	MK451495	–
A. proliferans	PPRI6735 = CMV011C1	Aspergillus	South Africa, Mpumalanga, Piet Retief, 1988	Bee larvae (Apis mellifera)	–	–	MK451496	–
A. pseudoglaucus	MRC1231 = CMV017A3	Aspergillus	South Africa, Western Cape, Elgin, 1975	Apple	–	–	MK951920	–
A. pseudoglaucus	MRC455 = CMV017E9	Aspergillus	South Africa, unknown		–	–	MN031425	–
A. pseudoglaucus	MRC462 = CMV017A1	Aspergillus	South Africa, Pretoria, unknown		–	–	MK951918	–
A. pseudoglaucus	PPRI26346 = CMV016D9	Aspergillus	South Africa, Gauteng, Pretoria, 2019	Dog food	–	–	MK951912	–
A. zutongqii	PPRI3429 = CMV011F7	Aspergillus	South Africa, Gauteng, Pretoria, 1988	Lab contaminant	–	–	MK451575	–
A. species	PPRI6060 = CMV004E8	Candidi	South Africa, Free State, Bloemfontein, 1995	Dung	MK450633	MK451000	MK451330	–
A. tritici	MRC3080 = CMV017B1	Candidi	South Africa, Mpumalanga, 1982	Maize (Zea mays)	–	–	MK951927	–
A. tritici	MRC418 = CMV016I7	Candidi	South Africa, North West Province, Brits, 1971	Sorghum malt	–	–	MK951916	–
A. ochraceus	PPRI26013 = CMV006D9	Circumdati	South Africa, Western Cape, 2018	Wheat (Triticum sp)	–	–	MK451474	–
A. ochraceus	PPRI6335 = CMV007B6	Circumdati	South Africa, Mpumalanga, Nelspruit, 1997	Cochecille insects	–	–	MK451476	–
A. ochraceus	PPRI6816 = CMV007B5	Circumdati	South Africa, North West, Potchefstroom, 1999	Cowpea (Vigna ungiculata)	–	–	MK451475	–
A. pallidofulvus	CMV012D2	Circumdati	South Africa, Limpopo, Groblersdal, 2018	Soil	MK450639	–	MK451477	–
A. sclerotiorum	PPRI8357 = CMV007B4	Circumdati	South Africa, 2006	Rat food	–	–	MK451507	–
A. westerdijkiae	PPRI5061 = CMV007B2	Circumdati	South Africa, Limpopo, Vaalwater, 1993	Chrysomelid beetle	–	–	MK451571	–
A. westerdijkiae	PPRI8700 = CMV007B7	Circumdati	South Africa, Limpopo , Kruger National Park, 2005	Mopane twigs and leaves (Colophospermum mopane)	–	–	MK451572	–
A. clavatus	PPRI13831 = CMV008F4	Clavati	South Africa, Gauteng, Bapsfontein, 2014	Barley seedling (Hordeum vulgare)	–	–	MK451347	–
A. clavatus	PPRI13832 = CMV005I8	Clavati	South Africa, Gauteng, Bapsfontein, 2014	Barley seedling (Hordeum vulgare)	–	–	MK451344	–
A. clavatus	PPRI14650 = CMV005I9	Clavati	South Africa, North Weat, Potchefstroom, 2014	Animal feed	–	–	MK451345	–
A. clavatus	PPRI17069 = CMV001F9	Clavati	South Africa, Gauteng, Near Delmas, 2014	Animal feed, maize kernels	–	–	MK451341	–
A. clavatus	PPRI21896 = CMV006A1	Clavati	South Africa, Western Cape, Malmesbury, 2016	Barley sprouted seed (Hordeum vulgare)	–	–	MK451346	–
A. clavatus	PPRI26042 = CMV013A3	Clavati	South Africa, 2018	Dragon fruit plant	–	–	MK451349	–
A. clavatus	PPRI26045 = CMV013B4	Clavati	Swaziland, 2018	Pig feed	–	–	MK451351	–
A. clavatus	PPRI26493 = CMV013A9	Clavati	Swaziland, 2018	Pig feed	–	–	MK951883	–
A. clavatus	PPRI26495 = CMV013B2	Clavati	Swaziland, 2018	Pig feed	–	–	MK451350	–
A. clavatus	PPRI4976 = CMV010D7	Clavati	South Africa, Gauteng, Magaliesburg, 1994	Soil	–	–	MK451348	–
A. clavatus	PPRI8552 = CMV005I6	Clavati	South Africa, Mpumalanga, Lydenburg, 2007	Sunflower seed (Helianthus annuus)	–	–	MK451342	–
A. clavatus	PPRI9818 = CMV005I7	Clavati	South Africa, Free State, 2008	Sunflower soil	–	–	MK451343	–
A. giganteus	MRC453 = CMV016I9	Clavati	South Africa, Pretoria, unknown		–	–	MN031424	–
A. giganteus	PPRI26019 = CMV008C9	Clavati	South Africa, Limpopo, 2018	Chicken feed	MK450637	MK451147	MK451418	–
A. seifertii	PPRI26025 = CMV011E3	Clavati	South Africa, Free State, Golden Gate, unknown	Soil	MK450648	MK451205	MK451510	MK450801
A. seifertii	PPRI3211 = CMV006F5 (ex-type)	Clavati	South Africa, Free State, Golden Gate, 1988	Grassroots	MK450647	MK451093	MK451509	MK450800
A. dimorphicus	CMV012C9	Cremei	South Africa, Limpopo, Groblersdal, 2018	Soil	MK450634	MK451246	MK451357	–
A. dimorphicus	PPRI26031 = CMV012G4	Cremei	South Africa, Limpopo, Groblersdal, 2018	Soil	MK450646	MK451263	MK451508	MK450799
A. wentii	PPRI25999 = CMV003I2	Cremei	South Africa, 2017	Animal feed	–	–	MK451569	–
A. wentii	PPRI26048 = CMV013F6	Cremei	South Africa, Mpumalanga, Barberton, 2018	Wood in mine	–	–	MK451570	–
A. wentii	PPRI26349 = CMV016E7	Cremei	South Africa, Mpumalanga, Barberton, 2018	Wood in mine	–	–	MK951914	–
A. alliaceus	PPRI6826 = CMV007B1	Flavi	South Africa, Eastern Cape, Port Elizabeth, 1999	Moth larvae (Cryptophlebia leucotreta)	–	–	MK451307	–
A. flavus	CMV015C6 = 2019-M44	Flavi	South Africa, 2019	Wood pallet	–	–	MK951894	–
A. flavus	CMV015C7 = 2019-M44	Flavi	South Africa, 2019	Wood pallet	–	–	MK951895	–
A. flavus	CMV015C9 = 2019-M44	Flavi	South Africa, 2019	Wood pallet	–	–	MK951896	–
A. flavus	MRC1317 = CMV017A2	Flavi	South Africa, Western Cape, Somerset West, 1977	Lemon (Citrus limon)	–	–	MK951919	–
A. flavus	MRC1366 = CMV017A7	Flavi	South Africa, Western Cape, Ceres, 1978	Maize (Zea mays), pathotoxicity to sheep	–	–	MK951924	–
A. flavus	MRC1745 = CMV017A8	Flavi	South Africa, North West Province, Potchefstroom, 1979	Sorghum malt	–	–	MK951925	–
A. flavus	MRC2526 = CMV017A9	Flavi	South Africa, unknown	Biltong	–	–	MK951926	–
A. flavus	MRC3732 = CMV017B3	Flavi	South Africa, Western Cape, Ceres, 1984	Apple	–	–	MK951929	–
A. flavus	MRC6979 = CMV017B5	Flavi	South Africa, Mpumalanga, Kruger National Park, unknown	Soil	–	–	MK951931	–
A. flavus	PPRI13141 = CMV002B4	Flavi	South Africa, Kwazulu Natal, Pietermaritzburg, unknown	Maize (Zea mays)	–	–	MK451376	–
A. flavus	PPRI18143 = CMV001I2	Flavi	South Africa, 2015	Rooibos (Aspalathus linearis)	–	–	MK451365	–
A. flavus	PPRI18144 = CMV001I8	Flavi	South Africa, 2015	Rooibos (Aspalathus linearis)	–	–	MK451370	–
A. flavus	PPRI18161 = CMV002B3	Flavi	South Africa, Free State, Bethlehem, 2015	Wheat (Triticum sp)	–	–	MK451375	–
A. flavus	PPRI18711 = CMV001I4	Flavi	South Africa, Northwest, Sannieshof, 2015	Frass of moth (Busseola fusca) feeding inside maize stems	–	–	MK451367	–
A. flavus	PPRI18712 = CMV001I5	Flavi	South Africa, Northwest, Sannieshof, 2015	Frass of moth (Busseola fusca) feeding inside maize stems	–	–	MK451368	–
A. flavus	PPRI18713 = CMV001I9	Flavi	South Africa, Northwest, Coligny, 2015	Frass of moth (Busseola fusca) feeding inside maize stems	–	–	MK451371	–
A. flavus	PPRI18714 = CMV001I1	Flavi	South Africa, Northwest, Coligny, 2015	Frass of moth (Busseola fusca) feeding inside maize stems	–	–	MK451364	–
A. flavus	PPRI18715 = CMV001I6	Flavi	South Africa, Northwest, Coligny, 2015	Frass of moth (Busseola fusca) feeding inside maize stems	–	–	MK451369	–
A. flavus	PPRI20581 = CMV002B1	Flavi	South Africa, Western Cape, Grabouw, 2015	Insect	–	–	MK451374	–
A. flavus	PPRI22482 = CMV001I3	Flavi	South Africa, Limpopo, Atlanta, 2016	Soya beans (Glycine max)	–	–	MK451366	–
A. flavus	PPRI23389 = CMV002A1	Flavi	South Africa, Western Cape, Stellenbosch, 2016	Animal feed	–	–	MK451372	–
A. flavus	PPRI25992 = CMV003A4	Flavi	South Africa, Western Cape, Knysna, 2017	Hominy chop animal feed	–	–	MK451379	–
A. flavus	PPRI26001 = CMV003I5	Flavi	South Africa, 2017	Animal feed	–	–	MK451380	–
A. flavus	PPRI26002 = CMV003I6	Flavi	South Africa, 2017	Animal feed	–	–	MK451381	–
A. flavus	PPRI26003 = CMV003I7	Flavi	South Africa, 2017	Animal feed	–	–	MK451382	–
A. flavus	PPRI26004 = CMV003I8	Flavi	South Africa, 2017	Animal feed	–	–	MK451383	–
A. flavus	PPRI26007 = CMV005E1	Flavi	South Africa, Gauteng, Sunninghill, 2017	Groundnut	–	–	MK451384	–
A. flavus	PPRI26022 = CMV008E3	Flavi	South Africa, Limpopo, 2018	Chicken feed	–	–	MK451385	–
A. flavus	PPRI26032 = CMV012H1	Flavi	South Africa, Gauteng, Pretoria, 2018	Dog food	–	–	MK451387	–
A. flavus	PPRI26036 = CMV012H8	Flavi	South Africa, Gauteng, Pretoria, 2018	Dog food	–	–	MK451388	–
A. flavus	PPRI26044 = CMV013B3	Flavi	Swaziland, 2018	Pig feed	–	–	MK451389	–
A. flavus	PPRI26345 = CMV016D6	Flavi	South Africa, Gauteng, Pretoria, 2019	Dog food	–	–	MK951910	–
A. flavus	PPRI26347 = CMV016E4	Flavi	South Africa, Gauteng, Pretoria, 2019	Dog food	–	–	MK951913	–
A. flavus	PPRI26486 = CMV010D4	Flavi	South Africa, Limpopo, Groblersdal, 2015	Soil	–	–	MK451386	–
A. flavus	PPRI3274 = CMV002A5	Flavi	South Africa, Gauteng, Pretoria, 1988		–	–	MK451373	–
A. flavus	PPRI7977 = CMV002B5	Flavi	South Africa, 2005		–	–	MK451377	–
A. flavus	PPRI8551 = CMV002B7	Flavi	South Africa, Mpumalanga, Lydenburg, 2007	Maize (Zea mays)	–	–	MK451378	–
A. krugeri	PPRI8986 = CMV006G4 (ex-type)	Flavi	South Africa, Limpopo, Kruger National Park, 2005	Mopane debris (Colophospermum mopane)	MK450655	MK451098	MK451517	MK450808
A. krugeri	PPRI9280 = CMV002C8	Flavi	South Africa, Limpopo, Kruger National Park, 2005	Mopane debris (Colophospermum mopane)	MK450654	MK450928	MK451516	MK450807
A. magaliesburgensis	PPRI6165 = CMV007A3 (ex-type)	Flavi	South Africa, Gauteng, Magaliesburg, 1996	Antlion (Myrmeleontidae)	MK450649	MK451116	MK451511	MK450802
A. nomius	PPRI3753 = CMV002B2	Flavi	South Africa, Gauteng, Rietondale, 1989	Termites dead colony	–	MK450926	MK451473	–
A. parasiticus	PPRI14636 = CMV001H8	Flavi	South Africa, Gauteng, Bapsfontein, 2014	Spawnrun on grass	–	–	MK451478	–
A. parasiticus	PPRI14642 = CMV001H9	Flavi	South Africa, Gauteng, Bapsfontein, 2014	Spawnrun on grass	–	–	MK451479	–
A. parasiticus	PPRI23021 = CMV002C7	Flavi	South Africa, Dinaka game reserve, 2016	Animal feed	–	–	MK451483	–
A. parasiticus	PPRI26046 = CMV013B6	Flavi	Zambia, Mpangwe, Mpangwe, 2018	Wheat (Triticum sp)	–	–	MK451489	–
A. parasiticus	PPRI2885 = CMV007A7	Flavi	South Africa, 1990	Seed (Watsonin marginata)	–	–	MK451487	–
A. parasiticus	PPRI3754 = CMV007A5	Flavi	South Africa, Gauteng, Pretoria, 1989	Termites	–	–	MK451485	–
A. parasiticus	PPRI5183 = CMV007A6	Flavi	South Africa, Western Cape, Clanwilliam, 1993	Rooibos tea (Aspalathus linearis)	–	–	MK451486	–
A. parasiticus	PPRI7978 = CMV010B6	Flavi	South Africa, 2005		–	–	MK451488	–
A. parasiticus	PPRI9511 = CMV002B8	Flavi	South Africa, North West , 2008	Soil	–	–	MK451480	–
A. parasiticus	PPRI9513 = CMV002G1	Flavi	South Africa, North West , 2008	Soil	–	–	MK451484	–
A. parasiticus	PPRI9532 = CMV002C1	Flavi	South Africa, North West , 2008	Soil	–	–	MK451481	–
A. parasiticus	PPRI9534 = CMV002C2	Flavi	South Africa, North West , 2008	Soil	–	–	MK451482	–
A. pseudonomius	PPRI5063 = CMV002B6	Flavi	South Africa, Limpopo, Vaalwater, 1992	Chrysomelid beetle	–	–	MK451505	–
A. tamarii	PPRI26008 = CMV005E2	Flavi	South Africa, Gauteng, Sunninghill, 2017	Groundnut	–	–	MK451528	–
A. tamarii	PPRI26010 = CMV005E4	Flavi	South Africa, 2017	Soil	–	–	MK451529	–
A. tamarii	PPRI26023 = CMV008E4	Flavi	South Africa, Limpopo, 2018	Chicken feed	–	–	MK451531	–
A. tamarii	PPRI2812 = CMV003E1	Flavi	South Africa, 1991	Soya beans (Glycine max)	–	–	MK451527	–
A. tamarii	PPRI7392 = CMV007B3	Flavi	South Africa, 2004		–	–	MK451530	–
A. transmontanensis	PPRI14275 = CMV011A5	Flavi	Zambia, 2013	Soil	MK450657	MK451183	MK451519	MK450810
A. iizukae	PPRI4965 = CMV007B8	Flavipedes	South Africa, Gauteng, Pretoria, 1993	Chrysomelid beetle	–	–	MK451428	–
A. arcoverdensis	PPRI7491 = CMV003C4	Fumigati	South Africa, 2004		–	–	MK451311	–
A. arcoverdensis	PPRI7514 = CMV003C3	Fumigati	South Africa, 2004		–	–	MK451310	–
A. aureolus	PPRI11297 = CMV008A9	Fumigati	South Africa, Kwazulu Natal, Pinetown, 2011	Air sample	–	–	MK451321	–
A. aureolus	PPRI3451 = CMV011F8	Fumigati	South Africa, 1988		–	–	MK451322	–
A. elsenburgensis	DTO015G7	Fumigati	Argentina, La Pampa Province, Chacharramendi	Soil	MT110301	MT108410	MT108412	–
A. elsenburgensis	DTO380H5	Fumigati	Argentina, Catamarca Province	Soil	–	MT108411	MT108413	–
A. elsenburgensis	DTO381D3	Fumigati	Argentina, Catamarca Province	Soil	–	–	MT108414	–
A. elsenburgensis	DTO381D8	Fumigati	Argentina	Soil	MT110302	–	MT108415	–
A. elsenburgensis	PPRI2994 = CMV011G4 = CSIR1013 (ex-type)	Fumigati	South Africa, Western Cape, Elsenburg, 1986	Soil	MK450651	MK451215	MK451513	MK450804
A. fischeri	PPRI26026 = CMV011H6	Fumigati	South Africa, Gauteng, Pretoria, 2018	Lab contaminant	–	–	MK451359	–
A. fischeri	PPRI3418 = CMV012A7 = CSIR978	Fumigati	South Africa, 1988		–	–	MK451363	–
A. fischeri	PPRI3428 = CMV011I5 = CSIR990	Fumigati	South Africa, 1988		–	–	MK451361	–
A. fischeri	PPRI3488 = CMV012A6 = CSIR1039	Fumigati	South Africa, 1988		–	–	MK451362	–
A. fischeri	PPRI4507 = CMV011I4 = CSIR1094	Fumigati	South Africa, Eastern Cape, Butterworth, 1986	Soil	–	–	MK451360	–
A. fumigatiaffinis	PPRI13089 = CMV001G1	Fumigati	South Africa, Succulent karoo area , unknown	Soil	MK450636	MK450913	MK451390	–
A. fumigatiaffinis	PPRI13090 = CMV010I7	Fumigati	South Africa, Succulent karoo area , unknown	Soil	–	–	MK451392	–
A. fumigatiaffinis	PPRI3210 = CMV004C3	Fumigati	South Africa, Western Cape, Beaufort West, 1988	Grass	–	–	MK451391	–
A. fumigatus	CMV015C1 = 2019-M44	Fumigati	South Africa, 2019	Wood pallet	–	–	MK951890	–
A. fumigatus	CMV015C2 = 2019-M44	Fumigati	South Africa, 2019	Wood pallet	–	–	MK951891	–
A. fumigatus	CMV015C3 = 2019-M44	Fumigati	South Africa, 2019	Wood pallet	–	–	MK951892	–
A. fumigatus	CMV015C5 = 2019-M44	Fumigati	South Africa, 2019	Wood pallet	–	–	MK951893	–
A. fumigatus	CMV015D8 = 2019-M44	Fumigati	South Africa, 2019	Wood pallet	–	–	MK951904	–
A. fumigatus	CMV015D9 = 2019-M44	Fumigati	South Africa, 2019	Wood pallet	–	–	MK951905	–
A. fumigatus	MRC435 = CMV016I8	Fumigati	South Africa, Port Health, 1971	Rice	–	–	MK951917	–
A. fumigatus	PPRI10161 = CMV002G6	Fumigati	South Africa, Eastern Cape, 2009	Silage	–	–	MK451396	–
A. fumigatus	PPRI10162 = CMV002G2	Fumigati	South Africa, Eastern Cape, 2009	Silage	–	–	MK451393	–
A. fumigatus	PPRI10498 = CMV003D5	Fumigati	South Africa, Eastern Cape, Port Elizabeth, 2010	Maize silage (Zea mays)	–	–	MK451406	–
A. fumigatus	PPRI10499 = CMV002G5	Fumigati	South Africa, Eastern Cape, Port Elizabeth, 2010	Maize silage (Zea mays)	–	–	MK451395	–
A. fumigatus	PPRI12665 = CMV002G3	Fumigati	South Africa, Free State, Luchhof, 2012	Rye seed (Secale cereale)	–	–	MK451394	–
A. fumigatus	PPRI13084 = CMV002G7	Fumigati	South Africa, Gauteng, Pretoria, 2013	Pear	–	–	MK451397	–
A. fumigatus	PPRI13252 = CMV003D6	Fumigati	South Africa, 2013		–	–	MK451407	–
A. fumigatus	PPRI20934 = CMV008B7	Fumigati	South Africa, 2016		–	MK451141	MK451412	–
A. fumigatus	PPRI25993 = CMV003A5	Fumigati	South Africa, Western Cape, Knysna, 2017	Hominy chop animal feed	–	–	MK451398	–
A. fumigatus	PPRI25998 = CMV003H8	Fumigati	South Africa, 2017	Animal feed	–	–	MK451409	–
A. fumigatus	PPRI26006 = CMV005D8	Fumigati	South Africa, Gauteng, Bedfordview, 2018	Potting soil	–	–	MK451411	–
A. fumigatus	PPRI3283 = CMV003C6	Fumigati	South Africa, North West, Pella, 1993	Soil	–	–	MK451399	–
A. fumigatus	PPRI3478 = CMV004C2	Fumigati	South Africa, Gauteng, Bapsfontein, 1988	Straw	–	–	MK451410	–
A. fumigatus	PPRI3479 = CMV003C7	Fumigati	South Africa, Gauteng, Johannesburg, 1988	Compost	–	–	MK451400	–
A. fumigatus	PPRI3505 = CMV010B9	Fumigati	South Africa, Gauteng, Denneboom, 1987	Pine	–	MK451153	MK451416	–
A. fumigatus	PPRI4975 = CMV003C8	Fumigati	South Africa, Mpumalanga, Malelane, 1993	Bagasse	–	–	MK451401	–
A. fumigatus	PPRI5090 = CMV003H1	Fumigati	South Africa, Mpumalanga, Malelane, 1993	Decayed mineola	–	MK450976	MK451408	–
A. fumigatus	PPRI7394 = CMV003C9	Fumigati	South Africa, 2004		–	–	MK451402	–
A. fumigatus	PPRI8522 = CMV003D1	Fumigati	South Africa, Mpumalanga, Vlakfontein, 2006	Chickens (Gallus domesticus)	–	–	MK451403	–
A. fumigatus	PPRI8523 = CMV003D2	Fumigati	South Africa, Mpumalanga, Vlakfontein, 2006	Chickens (Gallus domesticus)	–	–	MK451404	–
A. fumigatus	PPRI8525 = CMV008F9	Fumigati	South Africa, Mpumalanga, Vlakfontein, 2006	Chickens (Gallus domesticus)	–	–	MK451414	–
A. fumigatus	PPRI8527 = CMV008G1	Fumigati	South Africa, Mpumalanga, Vlakfontein, 2006	Chickens (Gallus domesticus)	–	–	MK451415	–
A. fumigatus	PPRI8558 = CMV003D3	Fumigati	South Africa, 2007	Chickens (Gallus domesticus)	–	–	MK451405	–
A. fumigatus	PPRI8560 = CMV008F8	Fumigati	South Africa, 2007	Chickens (Gallus domesticus)	–	–	MK451413	–
A. hiratsukae	PPRI3260 = CMV012G1 = CSIR1064	Fumigati	South Africa, 1988		–	–	MK451422	–
A. hiratsukae	PPRI9172 = CMV008F5	Fumigati	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	–	MK451421	–
A. hiratsukae	PPRI9185 = CMV004E7	Fumigati	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	–	MK451420	–
A. hiratsukae	PPRI9190 = CMV004E4	Fumigati	South Africa, Limpopo, Kruger National Park, 2005	Mopane twigs and leaves (Colophospermum mopane)	–	–	MK451419	–
A. laciniosus	PPRI3197 = CMV011I6 = CSIR1050	Fumigati	South Africa, 1988		–	–	MK451440	–
A. laciniosus	PPRI3247 = CMV011G5	Fumigati	South Africa, North West, Pella, 1988		–	MK451216	MK451439	–
A. laciniosus	PPRI3417 = CMV010F8 = CSIR638	Fumigati	South Africa, 1988		–	MK451163	MK451437	–
A. laciniosus	PPRI3847 = CMV011F6	Fumigati	South Africa, Kwazulu Natal, Greytown, 1985	Maize kernels (Zea mays)	–	–	MK451438	–
A. lentulus	PPRI6170 = CMV007I3	Fumigati	South Africa, Northern Cape, Loffiesdraai, 1996	Sand	–	MK451134	MK451442	–
A. lentulus	PPRI7532 = CMV003C5	Fumigati	South Africa, 2004		–	MK450952	MK451441	–
A. udagawae	PPRI11324 = CMV010I9	Fumigati	South Africa, Eastern Cape, Port Elizabeth, 2011	Mealy bug on Citrus	–	MK451179	MK451543	–
A. udagawae	PPRI26030 = CMV012F7	Fumigati	South Africa, Limpopo, Groblersdal, 2018	Soil	–	MK451259	MK451544	–
A. wyomingensis	PPRI5178 = CMV007I4	Fumigati	South Africa, Western Cape, Clanwilliam, 1993	Rooibos tea (Aspalathus linearis)	–	–	MK451574	–
A. wyomingensis	PPRI5573 = CMV007I2	Fumigati	South Africa, Western Cape, Clanwilliam, 1994	Rooibos tea (Aspalathus linearis)	–	–	MK451573	–
A. amoenus	PPRI26021 = CMV008E2	Nidulantes	South Africa, Limpopo, 2018	Chicken feed	–	–	MK451308	–
A. amoenus	PPRI26047 = CMV013F4	Nidulantes	South Africa, Mpumalanga, Barberton, 2018	Wood in mine	–	–	MK451309	–
A. creber	PPRI13168 = CMV002A9	Nidulantes	South Africa, North West, Mafikeng, 2013	Chicken house bedding	–	–	MK451352	–
A. creber	PPRI3737 = CMV002G9	Nidulantes	South Africa, Gauteng, Pretoria, 1989	Orange (Citrus sinensis)	–	–	MK451353	–
A. creber	PPRI3869 = CMV011F9	Nidulantes	South Africa, Free State, Bloemfontein, 1990	Honey flower seed (Melianthus comosus)	–	–	MK451356	–
A. creber	PPRI5081 = CMV002H1	Nidulantes	South Africa, Mpumalanga, Hazyview, 1993	Lemon (Citrus limon)	–	–	MK451354	–
A. creber	PPRI9900 = CMV008C2	Nidulantes	South Africa, Kwazulu Natal, Pinetown, 2008		–	–	MK451355	–
A. jensenii	PPRI13238 = CMV001F7	Nidulantes	South Africa, KwaZulu-Natal, Pinetown, 2013	Environmental sample	–	–	MK451433	–
A. jensenii	PPRI2806 = CMV011G1	Nidulantes	South Africa, 1991		–	–	MK451436	–
A. jensenii	PPRI5384 = CMV007A9	Nidulantes	South Africa, 1993	Flower (Gladiolus corms)	–	–	MK451435	–
A. jensenii	PPRI6329 = CMV003H2	Nidulantes	South Africa, Kwazulu Natal, 1996	Contaminant bioproduct	–	MK450977	MK451434	–
A. nidulans	PPRI20935 = CMV010I1	Nidulantes	South Africa, 2016		–	–	MK451456	–
A. protuberus	PPRI26350 = CMV016F8	Nidulantes	South Africa, Mpumalanga, Barberton, 2018	Wood in mine	–	–	MK951915	–
A. protuberus	PPRI5575 = CMV008B2	Nidulantes	South Africa, 1994	Diesel fuel filters	–	–	MK451497	–
A. purpureocrustaceus	PPRI3840 = CMV008B3 (ex-type)	Nidulantes	South Africa, Limpopo, 1990	Plant debris	MK450653	MK451138	MK451515	MK450806
A. purpureocrustaceus	PPRI5548 = CMV008B1	Nidulantes	South Africa, Western Cape, Cape Town, 1994	Spider (Palystes castaneus)	MK450652	MK451137	MK451514	MK450805
A. quadrilineatus	PPRI26342 = CMV015B3	Nidulantes	South Africa, Mpumalanga, Marble Hall, 2019		–	–	MK951887	–
A. recurvatus	PPRI3165 = CMV010C4	Nidulantes	South Africa, 1988		MK450645	MK451157	MK451506	–
A. rugulosus	MRC3329 = CMV017B2	Nidulantes	South Africa, Free State, Clocolan, 1983	Oats	–	–	MK951928	–
A. sydowii	CMV008E1 = 2018-M76/352	Nidulantes	South Africa, Limpopo, 2018	Chicken feed	–	–	MK451524	–
A. sydowii	CMV015B9 = 2019-M44	Nidulantes	South Africa, 2019	Wood pallet	–	–	MK951889	–
A. sydowii	PPRI12668 = CMV008C6	Nidulantes	South Africa, KwaZulu-Natal, Pinetown, 2012	Environmental sample	–	–	MK451523	–
A. sydowii	PPRI13067 = CMV008B4	Nidulantes	South Africa, KwaZulu-Natal, Pinetown, 2012	Environmental sample	–	–	MK451521	–
A. sydowii	PPRI13241 = CMV001D6	Nidulantes	South Africa, KwaZulu-Natal, Pinetown, 2013	Environmental sample	–	–	MK451520	–
A. sydowii	PPRI3810 = CMV008F1	Nidulantes	South Africa, Free State, Bloemfontein, 1990	Honey flower seed (Melianthus comosus)	–	–	MK451525	–
A. sydowii	PPRI3839 = CMV008F2	Nidulantes	South Africa, 1990	Watsonia marginata	–	–	MK451526	–
A. sydowii	PPRI6542 = CMV008C5	Nidulantes	South Africa, Kwazulu Natal, Pinetown, 1997	Lab shelf	–	–	MK451522	–
A. alabamensis	PPRI25994 = CMV003A6	Terrei	South Africa, Western Cape, Knysna, 2017	Hominy chop animal feed	–	MK450947	MK451300	MK450758
A. alabamensis	PPRI25996 = CMV003A9	Terrei	South Africa, Western Cape, Knysna, 2017	Hominy chop animal feed	–	MK450948	MK451301	MK450759
A. alabamensis	PPRI26028 = CMV012E2	Terrei	South Africa, Limpopo, Groblersdal, 2018	Soil	–	–	MK451299	–
A. aureoterreus	PPRI13096 = CMV010F6	Terrei	South Africa, Succulent karoo area , unknown	Soil	–	MK451161	MK451323	MK450772
A. carneus	PPRI13094 = CMV010F7	Terrei	South Africa, Succulent karoo area , unknown	Soil	–	MK451162	MK451331	MK450778
A. cf alabamensis	PPRI7492 = CMV004A7	Terrei	South Africa, 2004		–	MK450983	MK451312	MK450765
A. cf alabamensis	PPRI8696 = CMV004D7	Terrei	South Africa, Limpopo , Kruger National Park, 2005	Soil	–	MK450993	MK451318	MK450770
A. cf alabamensis	PPRI8741 = CMV004C9	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	MK450990	MK451315	MK450768
A. cf alabamensis	PPRI8747 = CMV004D2	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	MK450991	MK451316	MK450769
A. cf alabamensis	PPRI8979 = CMV004D5	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	MK450992	MK451317	–
A. cf alabamensis	PPRI9150 = CMV004C8	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Mopane debris (Colophospermum mopane)	–	MK450989	MK451314	MK450767
A. cf alabamensis	PPRI9184 = CMV004C7	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	MK450988	MK451313	MK450766
A. cf alabamensis	PPRI9189 = CMV004E1	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Mopane twigs and leaves (Colophospermum mopane)	–	MK450995	MK451320	–
A. cf alabamensis	PPRI9206 = CMV004D9	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Mopane twigs and leaves (Colophospermum mopane)	–	MK450994	MK451319	MK450771
A. cf allahabadii	PPRI5574 = CMV004C1	Terrei	South Africa, Western Cape, Clanwilliam, 1994	Rooibos tea (Aspalathus linearis)	–	MK450987	MK451302	MK450760
A. cf allahabadii	PPRI7534 = CMV004E6	Terrei	South Africa, Limpopo, Kruger National Park, 2003	Soil	–	MK450999	MK451306	MK450764
A. cf allahabadii	PPRI8751 = CMV004E3	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Soil	MK450629	MK450997	MK451304	MK450762
A. cf allahabadii	PPRI8987 = CMV004E2	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Mopane debris (Colophospermum mopane)	MK450628	MK450996	MK451303	MK450761
A. cf allahabadii	PPRI9194 = CMV004E5	Terrei	South Africa, Limpopo, Kruger National Park, 2005	Mopane debris (Colophospermum mopane)	–	MK450998	MK451305	MK450763
A. citrinoterreus	PPRI7464 = CMV004A6	Terrei	South Africa, North West, Welwitschia, 2004		–	–	MK451340	–
A. heldtiae	PPRI4229 = CMV004A2 (ex-type)	Terrei	South Africa, 1991	Millet seed	MK450656	MK450981	MK451518	MK450809
A. hortai	PPRI25995 = CMV003A8	Terrei	South Africa, Western Cape, Knysna, 2017	Hominy chop animal feed	–	–	MK451423	–
A. hortai	PPRI5864 = CMV004A5	Terrei	South Africa, Gauteng, Onderstepoort, 1995	Animal tissue	–	–	MK451424	–
A. hortai	PPRI7533 = CMV004A9	Terrei	South Africa, Limpopo, Kruger National Park, 2003	Soil	–	MK450985	MK451425	–
A. hortai	PPRI8707 = CMV004C5	Terrei	South Africa, Limpopo , Kruger National Park, 2005	Mopane (Colophospermum mopane)	–	–	MK451427	–
A. hortai	PPRI9902 = CMV004B1	Terrei	South Africa, Gauteng, Pretoria, 2008	Industrial food colourant	–	–	MK451426	–
A. species CBS142751	PPRI9903 = CMV004A8	Terrei	South Africa, Gauteng, Pretoria, 2008	Industrial food colourant	MK450635	MK450984	MK451358	–
A. terreus	PPRI10373 = CMV007A8	Terrei	South Africa, Eastern Cape, Port Elizabeth, 2010	Maize silage (Zea mays)	–	–	MK451535	–
A. terreus	PPRI13086 = CMV011A4	Terrei	South Africa, Succulent karoo area , unknown	Soil	–	–	MK451537	–
A. terreus	PPRI20932 = CMV004B3	Terrei	South Africa, 2016		–	–	MK451533	–
A. terreus	PPRI25997 = CMV003H4	Terrei	South Africa, 2017	Animal feed	–	–	MK451532	–
A. terreus	PPRI26027 = CMV012E1	Terrei	South Africa, Limpopo, Groblersdal, 2018	Soil	–	–	MK451538	–
A. terreus	PPRI26029 = CMV012E3	Terrei	South Africa, Limpopo, Groblersdal, 2018	Soil	–	–	MK451539	–
A. terreus	PPRI8282 = CMV010F9	Terrei	South Africa, 2006		–	–	MK451536	–
A. terreus	PPRI8672 = CMV004B9	Terrei	South Africa, 2007	Kenaf (Hibiscus cannabinus)	–	–	MK451534	–
A. calidoustus	PPRI15353 = CMV006B3	Usti	South Africa, KwaZulu-Natal, Pinetown, 2014		–	–	MK451329	–
A. insuetus	MRC5597 = CMV017B4	Usti	South Africa, Western Cape, Cape Town, unknown	Direct scraping off Castle wall	–	–	MK951930	–
A. insuetus	PPRI3456 = CMV006F8	Usti	South Africa, 1988	Grass	–	–	MK451429	–
A. pseudodeflectus	PPRI5177a = CMV006F9	Usti	South Africa, Western Cape, Clanwilliam, 1993	Rooibos tea (Aspalathus linearis)	MK450644	MK451096	MK451503	–
A. pseudodeflectus	PPRI5177b = CMV010G3	Usti	South Africa, Western Cape, Clanwilliam, 1993	Rooibos tea (Aspalathus linearis)	–	–	MK451504	–
A. pseudodeflectus	PPRI8971 = CMV005H9	Usti	South Africa, Limpopo, Kruger National Park, 2005	Mopane debris (Colophospermum mopane)	MK450642	MK451064	MK451498	–
A. pseudodeflectus	PPRI8976 = CMV005I1	Usti	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	–	MK451499	–
A. pseudodeflectus	PPRI9168 = CMV006A6	Usti	South Africa, Limpopo, Kruger National Park, 2008	Mopane twigs and leaves (Colophospermum mopane)	–	–	MK451502	–
A. pseudodeflectus	PPRI9203 = CMV005I2	Usti	South Africa, Limpopo, Kruger National Park, 2005	Mopane twigs and leaves (Colophospermum mopane)	MK450643	MK451065	MK451500	–
A. pseudodeflectus	PPRI9404 = CMV005I3	Usti	South Africa, Limpopo, Kruger National Park, 2005	Soil	–	–	MK451501	–
A. pseudoustus	MRC1233 = CMV017A5	Usti	South Africa, Western Cape, Drakenstein, 1975	Apple juice concentrate	–	–	MK951922	–
A. pseudoustus	MRC1234 = CMV017A4	Usti	South Africa, Western Cape, Drakenstein, 1975	Apple juice concentrate	–	–	MK951921	–
A. sigurros	PPRI15889 = CMV005I4 (ex-type)	Usti	South Africa, KwaZulu-Natal, Pinetown, 2014	Environmental sample	MK450650	MK451066	MK451512	MK450803

Acronyms of culture collections: PPRI, culture collection of the National Collections of Fungi, housed at the Agricultural Research Council - Plant Health and Protection (ARC), Roodeplaat, South Africa; MRC, culture collection of the Medical Research Council housed at PPRI; CSIR, culture collection of the Council for Scientific and Industrial Research; CMV, working collection housed at the PPRI; DTO, working collection of the Applied and Industrial Mycology group housed the Westerdijk Institute, Utrecht, the Netherlands.

DNA extraction, sequencing, and phylogenetic analysis

DNA was extracted from 7 d old colonies grown on Blakeslee’s (1915) malt extract agar (MEAbl) using the Quick-DNATM Fungal/Bacterial Miniprep Kit (Zymo Research, CA, USA). The 5.8S rDNA internal transcribed spacer regions (ITS), beta-tubulin (BenA), calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) genes were amplified in a 25 μl PCR master mix containing 12.5 μl OneTaq® 2X Master Mix with GC Buffer (New England Biolabsinc, MA, USA), 0.5 μl for each primer (10 μM), 10.5 μl milliQ H2O, and 1 μl template DNA. PCR conditions and primers were used as suggested by Samson . Automated sequencing was done at Inqaba Biotechnical Industries (Pty) Ltd (Pretoria, South Africa) using the same primers used for PCR amplification. For RPB2, additional sequencing reactions were performed with internal sequencing primers RPB2-527R (Peterson 2008), RPB2-388F (Peterson 2008), RPB2-F311 (Houbraken & Samson 2011) and RPB2-R310 (Houbraken & Samson 2011).

Contigs were assembled and edited in Geneious Prime v. 2019.2.1 (BioMatters Ltd., Auckland, New Zealand), and new sequences deposited to GenBank (www.ncbi.nlm.nih.gov/genbank/). Accession numbers are listed in Table 1. Sequences were compared to a locally curated reference sequence dataset based on the ex-type sequences published in Samson . Preliminary identifications were made using this dataset in a local BLAST search tool in Geneious. Subsequent reference sequences were selected (Supplementary Table 1) based on these results, with GenBank accession numbers also shown on phylogenetic trees.

All datasets were aligned in MAFFT v. 7.427 (Katoh & Standley 2013) selecting the G-INS-I option, with alignments manually trimmed, adjusted and concatenated in Geneious where needed or appropriate. Aligned datasets were analysed using Maximum Likelihood (ML) and Bayesian tree Inference (BI). For concatenated phylogenies, each gene was treated as separate partitions. ML was performed using IQtree v. 1.6.11 (Nguyen ). For each dataset or partition, the most suitable model was calculated using Modelfinder (Kalyaanamoorthy ) and ultrafast bootstrapping approximation done using UFBoot2 (Hoang ), both integrated into IQtree. BI was performed using MrBayes v. 3.2.7 (Ronquist ). The most suitable model for each dataset or partition was selected based on the Akaike information criterion (Akaike 1974) using MrModeltest v. 2.4 (Nylander 2004). Analyses were performed with three sets of four chains (1 cold and three heated) and were stopped at an average standard deviation for split frequencies of 0.01 using the stoprule. Trees were visualised in Figtree v. 1.4.4 (https://github.com/rambaut/figtree/releases) and visually prepared for publication in Affinity Designer v. 1.7.1 (Serif (Europe) Ltd, Nottingham, UK). ML and BI tree topologies did not differ, and thus the former was chosen to present results with both boostrap values and posterior probabilities shown for supported branches.

Several phylogenetic analyses were prepared. Firstly, a total phylogeny based on ITS, BenA, CaM and RPB2 sequence data was calculated which covered all sections detected in this study. Secondly, smaller datasets were prepared based on observed relationships, which allowed for more reliable alignments and more presentable trees. Thirdly, single gene trees were calculated in the case of putative new species to apply the genealogical concordance phylogenetic species recognition concept (GCPSR) (Taylor ).

Morphology

Morphological characterisation and species descriptions were made using standardised protocols published in Samson . Colony characters were captured on Czapek yeast autolysate agar (CYA), CYA with 5 % NaCl (CYAS), DG18, MEAbl (Oxoid LP0039 malt extract, Oxoid LP0034 peptone), MEA (Samson ), oatmeal agar (OA), yeast extract sucrose agar (YES) and creatine sucrose agar (CREA). Strains were three-point inoculated on these media in 90 mm Petri dishes. Plates were incubated in darkness for 7 d at 25 °C, with additional CYA plates incubated at 30 and 37 °C. Colour names and codes used in descriptions follow Kornerup & Wanscher (1967). Microscopic observations were made using a Zeiss AXIO Imager.M2 compound and Zeiss AXIO Zoom.V16 microscopes equipped with AxioCaM MRc5 and 512 cameras driven by Zen Blue v. 2.3 software (Carl Zeiss CMP GmbH, Göttingen, Germany). Colonies were captured with a Sony NEX-5N camera. Extended Depth of Field analysis and stacking of colony texture micrographs were performed in Helicon Focus v. 7.5.4 (HeliconSoft, Kharkiv, Ukraine). Plates were prepared in Affinity Photo v. 1.7.1 (Serif (Europe) Ltd, Nottingham, UK). For aesthetic purposes, micrographs were adjusted using the "inpainting brush tool" without altering areas of scientific significance.

Results

Of the ±320 PPRI strains selected for this study, ±250 were viable with 218 selected for sequencing. Eighteen MRC strains were sequenced. New isolations resulted in 65 strains, of which 51 were deposited in PPRI. DNA reference sequences (350 total: 24 ITS, 52 BenA, 250 CaM, 28 RPB2) were generated and submitted to GenBank during this study. Identified strains belonged to 63 species, representing 11 sections of Aspergillus. Seven of the species were found to be novel species and are described below in the Taxonomy section.

Phylogeny

For a general overview of results, a total phylogeny was calculated including all sequences generated during this study and reference sequences summarised in Supplementary Table 1. Results were summarised as a circular tree (Supplementary Fig. 1) and subsequently used as a baseline to calculate more focused phylogenies used to confirm final identifications and show relationships of the novel species.

Sections Aspergillus and Cremei (Fig. 1) — We identified six section Aspergillus species including A. chevalieri, A. montevidensis, A. porosus, A. proliferans, A. pseudoglaucus and A. zutongqii. This section was reviewed recently and two recently described species A. porosus and A. zutongqii are detected here (Chen ). From section Cremei, we identified A. wentii and A. dimorphicus. Aspergillus dimorphicus and A. sepultus are phylogenetically identical. Since A. dimorphicus (Mehrotra & Prasad 1969) is the older name, A. sepultus (Tuthill & Christensen 1986) is synonymised with the former.

Fig. 1

Multigene phylogeny of Aspergillus sect Aspergillus and Cremei based on a combined ITS, BenA, CaM and RPB2 dataset. Strains identified during this study are shown in black text and reference strains in grey text. GenBank accession numbers for ITS (green), BenA (blue), CaM (maroon) and RPB2 (purple) are given behind strain numbers. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Sections Candidi and Circumdati (Fig. 2) — In section Candidi, only two species were identified. One strain represented A. tritici, while PPRI6060 resolved in a unique clade closely related to A. subalbidus, which represents a new species that will be described in a different paper. Section Circumdati typically contains ochratoxin A producing species (Visagie et al., 2014b, Frisvad and Larsen, 2015a). Our study respectively identified strains as A. ochraceus, A. pallidofulvus, A. sclerotiorum and A. westerdijkiae.

Fig. 2

Multigene phylogeny of Aspergillus sect Circumdati and Candidi based on a combined ITS, BenA, CaM and RPB2 dataset. Strains identified during this study are shown in black text and reference strains in grey text. GenBank accession numbers for ITS (green), BenA (blue), CaM (maroon) and RPB2 (purple) are given behind strain numbers. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Section Flavi (Fig. 3) — Among strains identified during this study, section Flavi was well represented. Seven known (A. alliaceus, A. flavus, A. nomius, A. parasiticus, A. pseudonomius, A. tamarii and A. transmontanensis) and two new species were detected. PPRI14275 consistently grouped basal to the A. transmontanensis clade. This single strain was morphologically identical to latter and we, therefore, identified it as A. transmontanensis. PPRI8986 and PPRI9280 formed a well-supported clade basal to the A. parasiticus clade and is described below as A. krugeri. PPRI6165 represented a unique lineage in the A. vandermerweii, A. lanosus, A. alliaceus and A. neoalliaceus clade, and is described as A. magaliesburgensis below.

Fig. 3

Multigene phylogeny of Aspergillus sect Flavi based on a combined ITS, BenA, CaM and RPB2 dataset. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. GenBank accession numbers for ITS (green), BenA (blue), CaM (maroon) and RPB2 (purple) are given behind strain numbers. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Sections Fumigati and Clavati (Fig. 4) — Section Fumigati was well represented amongst strains identified during this study. Strains were identified into 11 known (A. arcoverdensis, A. aureolus, A. fischeri, A. fumigatiaffinis, A. fumigatus, A. hiratsukae, A. laciniosus, A. lentulus, A. udagawae and A. wyomingensis) and one new species described below as A. elsenburgensis. The multigene phylogeny resolved this strain as sister species to A. australensis. Strains previously identified as A. laciniosus resolved in two distinct clades. One clade containing the ex-type (CBS 117721T) for A. laciniosus and the other the ex-type (CBM-FA884T) for A. takakii. Three species were identified in section Clavati, including A. clavatus, A. giganteus and a new species described below as A. seifertii.

Fig. 4

Multigene phylogeny of Aspergillus sect Fumigati and Clavati based on a combined ITS, BenA, CaM and RPB2 dataset. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. GenBank accession numbers for ITS (green), BenA (blue), CaM (maroon) and RPB2 (purple) are given behind strain numbers. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Section Nidulantes (Fig. 5) — Ten section Nidulantes species were identified during this study: A. amoenus, A. creber, A. jensenii, A. nidulans, A. protuberus, A. quadrilineatus, A. recurvatus, A. rugulosus, A. sydowii, and one new species described below as A. purpureocrustaceus. The new species resolved as a close relative of A. tumidus.

Fig. 5

Multigene phylogeny of Aspergillus sect Nidulantes based on a combined ITS, BenA, CaM and RPB2 dataset. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. GenBank accession numbers for ITS (green), BenA (blue), CaM (maroon) and RPB2 (purple) are given behind strain numbers. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Sections Terrei and Flavipedes (Fig. 6) — Ten section Terrei species were identified during this study: A. alabamensis, A. aureoterreus, A. carneus, A. citrinoterreus, A. hortai, A. terreus and four new species. One of these new species is described below as A. heldtiae, which consistently resolved as a sister species to A. pseudoterreus. The remaining three species or clades were temporarily named A. cf. alabamensis, A. cf. allahabadii and Aspergillus sp. CBS 142751 as they will be described in a separate paper. Aspergillus iizukae was the only species identified from section Flavipedes.

Fig. 6

Multigene phylogeny of Aspergillus sect Terrei and Flavipedes based on a combined ITS, BenA, CaM and RPB2 dataset. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. GenBank accession numbers for ITS (green), BenA (blue), CaM (maroon) and RPB2 (purple) are given behind strain numbers. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Section Usti (Fig. 7) — Five section Usti species were identified during this study and included A. calidoustus, A. insuetus, A. pseudodeflectus and A. pseudoustus, while one new species is described below as A. sigurros. The new species resolved in a clade with A. carlsbadensis and A. contaminans. Based on phylogenetic results, the more recently described A. fuscicans (Romero ) should be considered a synonym of the older A. pseudodeflectus (Samson & Mouchacca 1975).

Fig. 7

Multigene phylogeny of Aspergillus sect Usti based on a combined ITS, BenA, CaM and RPB2 dataset. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. GenBank accession numbers for ITS (green), BenA (blue), CaM (maroon) and RPB2 (purple) are given behind strain numbers. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Section Nigri — The PPRI collection contained a large number of black Asperillus strains classified in section Nigri. Full results will be published elsewhere. Strains were identified into nine species as A. aculeatus, A. brasiliensis, A. brunneoviolaceus, A. japonicus, A. neoniger, A. niger, A. piperis, A. tubingensis and A. welwitschiae.

We introduce seven new species in the Taxonomy section below. These species belong to sections Clavati, Flavi, Fumigati, Nidulantes, Terrei and Usti based on the phylogenetic analyses. Strains conformed to the general morphological characters previously observed for species accepted in these sections. All of the new species were compared with respective close relatives, with notes provided on distinguishing characters after each species description in the Taxonomy section.

Taxonomy

Visagie, S.M. Romero & Houbraken, MycoBank MB834199. Fig. 14.

Fig. 14

Aspergillus elsenburgensis. A. Colonies, from left to right, CYA, MEAbl, DG18. B–E. Close-up of colonies on DG18 (B, C), CYA (D) and OA (E). F. Ascoma. G, H. Asci and ascospores. I–L. Conidiophores. M. Conidia. Scale bars: B, D, E = 1 mm; C = 0.2 mm; F, G = 20 μm; H–M = 10 μm.

Etymology: Latin, elsenburgensis, named after Elsenburg, the town the ex-type was collected from.

Classification: Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus section Fumigati.

Diagnosis: Colonies showing faster growth at 37 °C than at 25 °C, white and floccose, ascomata produced in aerial hyphae after prolonged incubation, white to cream colored, sporulation very sparse, conidiophores with short stipes (10–70 μm) and small globose conidia (1.5–2 μm).

Typus: South Africa, Western Cape, Elsenburg, soil, June 1986, (holotype PREM 62313, culture ex-type PPRI 2994 = CMV 011G4 = CSIR1013).

ITS Barcode: MK450651 (alternative identification markers: BenA = MK451215; CaM = MK451513; RPB2 = MK450804).

Colony diam (7 d, in mm): CYA 35–40; CYA 30 °C 50–53; CYA 37 °C 50–60; CYAS 8–12; MEAbl 55–60; MEA 40–45; DG18 20–25; YES 45–50; OA 45–50; CREA 35–36.

Colony characters (25 °C, 7 d): CYA colonies surface floccose, mycelial areas white, ascomata present after prolonged incubation, produced in aerial hyphae, sparse sporulation present after >2 wk incubation, greenish, soluble pigment absent, exudate clear, reverse pigmentation yellowish white to pale yellow (3A2–3A3). MEAbl colonies surface floccose, mycelial areas white, ascomata present after prolonged incubation, produced in aerial hyphae, sporulation absent, sparse sporulation present after >2 wk incubation, greenish, soluble pigment absent, exudate clear, reverse pigmentation yellowish white to pale yellow (3A2–3A3). YES colonies surface floccose, mycelial areas white, sporulation absent, soluble pigment absent, exudate clear, reverse pigmentation yellowish white to pale yellow (3A2–3A3). DG18 colonies surface floccose, mycelial areas white, sporulation sparse, white but becomes greenish with age, soluble pigment absent, exudate clear, reverse pigmentation yellowish white to pale yellow (3A2–3A3). CREA colonies weak growth, acid not produced.

Micromorphology: Conidial heads radiate. Conidiophores uniseriate. Stipes hyaline, smooth, 10–70 × 2.5–4(–4.5) μm. Vesicles subclavate, phialides cover 50 % of head, 5–8 μm wide. Phialides ampulliform, 4.5–6 × 2–3 μm. Conidia globose, smooth, 1.5–2.5 × 1.5–2.5 μm, (2.06 ± 0.15 × 2 ± 0.16, n = 56) μm, length/width 1.03 ± 0.05. Ascomata neosartorya-like, white to cream, abundant on OA, 70–270 μm. Asci 8-spored, 9–15 μm. Ascospores smooth, with 2 prominent equatorial furrow, globose to subglobose from the top, 4–5 × 3.5–5 μm (4.5 ± 0.2 × 4.2 ± 0.3, n = 43) μm, length/width 1.08 ± 0.07.

Notes: The multigene phylogeny resolves A. elsenburgensis as a close relative of A. australensis and A. galapagensis in section Fumigati (Fig. 4, Fig. 8). The new species grows faster on MEAbl and have somewhat longer stipes than A. australensis (55–60 vs 40–45 mm; up to 70 μm vs up to 30 μm (Samson )). Compared to A. galapagensis, A. elsenburgensis shows slightly faster growth on most media, while it also produces smaller conidia (1.5–2 vs 2.5–3 μm (Samson )).

Fig. 8

Single gene phylogenies of Aspergillus sect Fumigati based on ITS, BenA, CaM and RPB2. Strains from new species are shown in orange text and reference strains in grey text. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Visagie, MycoBank MB834200. Fig. 15.

Fig. 15

Aspergillus heldtiaeA. Colonies, from left to right, CYA, MEAbl, DG18. B–F. Close-up of colonies on CYA (B), DG18 (C, D) and MEAbl (E, F). G–L. Conidiophores. M. Conidia. Scale bars: B–C = 2 mm; D, E = 0.5 mm; F = 0.2 mm; G–I = 20 μm; J–M = 10 μm.

Etymology: Latin, heltdiae, named after Margaret Vinci Heldt, the creator of the beehive hairstyle that was popular during the 1960s and famously Marge Simpson’s choice of hairstyle. This species resembles the beehive when observed through a dissection microscope.

Classification: Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus section Terrei.

Diagnosis: Colonies showing rapid growth, bright yellow mycelial areas, cinnamon sporulation, conidiophores biseriate, vesicle 17–28 μm, stipes hyaline with a small proportion darkened, conidia smooth, globose to subglobose, 2–2.5 μm.

Typus: South Africa, unknown, Millet seed, June 1991, (holotype PREM 50864, culture ex-type PPRI 4229 = CMV 004A2).

ITS Barcode: MK450656 (alternative identification markers: BenA = MK450981; CaM = MK451518; RPB2 = MK450809).

Colony diam (7 d, in mm): CYA 54–58; CYA 30 °C 53–56; CYA 37 °C 60–65; CYAS 50–55; MEAbl 55–60; MEA 36–38; DG18 55–65; YES > 70; OA 33–35; CREA 30–32.

Colony characters (25 °C, 7 d): CYA colonies surface floccose, mycelial areas greenish yellow (1A8), sporulation sparse, cinnamon colored, soluble pigment absent, exudate absent, reverse pigmentation olive brown (4B8), light yellow (3A5). MEAbl colonies surface floccose, mycelial areas yellow (2A7), sporulation sparse, cinnamon colored, soluble pigment absent, exudate absent, reverse pigmentation olive brown (4B8), light yellow (3A5). YES colonies surface floccose, mycelial areas greenish yellow (1A8), sporulation sparse, cinnamon colored, soluble pigment absent, exudate absent, reverse pigmentation olive brown (4B8), light yellow (3A5). DG18 colonies surface floccose, mycelial areas greenish yellow (1A8), sporulation sparse, cinnamon colored, soluble pigment absent, exudate absent, reverse pigmentation olive brown (4B8), light yellow (3A5). CREA colonies strong growth, weak acid production.

Micromorphology: Conidial heads columnar. Conidiophores biseriate. Stipes hyaline, small proportion darkened, smooth, 140–330 × 5–8 μm. Vesicles globose, metulae cover 100 % of head, 17–28 μm wide. Metulae 6.5–8.5 × 3–4 μm. Phialides ampulliform, 5.5–7.5 × 2–2.5 μm. Conidia globose to subglobose, smooth, 2–2.5 × 2–2.5 μm, (2.4 ± 0.1 × 2.1 ± 0.1, n = 52) μm, length/width 1.15 ± 0.07. Ascomata not observed.

Notes: Phylogenies resolve A. heldtiae as a close relative of A. pseudoterreus in section Terrei (Fig. 6, Fig. 9). Both species produce bright yellow colonies with cinnamon colored sporulation. However, A. pseudoterreus produce conidiophores in distinctive loosely bundled synnema (Samson ), which is absent in the new species. Aspergillus heldtiae produces a minor proportion of darkened stipes, which are not reported for A. pseudoterreus.

Fig. 9

Single gene phylogenies of Aspergillus sect Terrei based on ITS, BenA, CaM and RPB2. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Visagie, MycoBank MB834203. Fig. 16.

Fig. 16

Aspergillus krugeri. A. Colonies, from left to right, CYA, MEAbl, DG18. B–D. Close-up of colonies on MEAbl (B), DG18 (C) and CYA (D). E–I. Conidiophores. J. Conidia. Scale bars: B, D = 2 mm; C = 0.5 mm; F, G = 20 μm; H–M = 10 μm.

Etymology: Latin, krugeri, named after the Kruger National Park, the National Park where the ex-type was collected from.

Classification: Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus section Flavi.

Diagnosis: Colonies on CYA showing rapid growth at 25 °C and moderate growth at 37 °C, dense sporulation, greyish to dark green, dark brown sclerotia abundant, conidial heads radiate, splitting into 3 or more columns, conidiophores uni- to biseriate, stipes rough, vesicle 40–80 μm wide, conidia broadly ellipsoid, rough, 4–7 × 3.5–6.5 μm.

Typus: South Africa, Kruger National Park, Mopane tree debris (Colophospermum mopane), October 2005, collected by E.J. vd Linde (holotype PREM 62309, culture ex-type PPRI 8986 = CMV 006G4).

ITS Barcode. MK450655 (alternative identification markers: BenA = MK451098; CaM = MK451517; RPB2 = MK450808).

Colony diam (7 d, in mm): CYA 60–70; CYA 30 °C 65–70; CYA 37 °C 40–47; CYAS 55–58; MEAbl > 70; MEA > 70; DG18 > 70; YES > 70; OA 52–56; CREA 33–36.

Colony characters (25 °C, 7 d): CYA colonies surface granular and velutinous, mycelial areas white, sporulation moderately dense to dense, greyish green to dark green (29E7–F7) colored, sclerotia abundant, white when young becoming brown to almost purplish, soluble pigment absent, exudate clear, reverse pigmentation pale yellow to dull yellow (3A3–B3), olive brown (4D4) below sclerotia. MEAbl colonies surface granular and velutinous, mycelial areas white, sporulation moderately dense to dense, greyish green to dark green (29E7–F7) colored, sclerotia abundant, white when young becoming brown to almost purplish, soluble pigment absent, exudate clear, reverse pigmentation pale yellow to dull yellow (3A3–B3). YES colonies surface velutinous, granular and floccose, mycelial areas white, sporulation dense, greyish green (29E7–30E7) colored, covering white to brown to almost purplish sclerotia, soluble pigment absent, exudate clear, reverse pigmentation greyish yellow (4B5), pale yellow to light yellow (4A3–5). DG18 colonies surface velutinous, mycelial areas white, sporulation dense, greyish green (29E7–30E7) colored, covering white to brown sclerotia, soluble pigment absent, exudate absent, reverse pigmentation pale yellow to dull yellow (3A3–B3). CREA colonies weak growth, weak acid production.

Micromorphology: Conidial heads radiate, splitting into 3 or more columns. Conidiophores uniseriate to biseriate with an equal ratio. Stipes hyaline, rough, 350–1000(–1300) × 10–18(–21) μm. Vesicles globose to spathulate, metulae/phialides cover 100 % of head, 40–80 μm wide. Metulae 11–22 × 5–10 μm. Phialides ampulliform, 10–15 × 4.5–7 μm. Conidia broadly ellipsoid, rough, 4–7 × 3.5–6.5 μm, (5.5 ± 0.7 × 5.1 ± 0.6, n = 72) μm, length/width 1.08 ± 0.04. Ascomata not observed. Sclerotia white when young, becoming dark brown with age, 370–850 μm.

Notes: Aspergillus krugeri belongs to the A. flavus-clade (Frisvad ) and is closely related to A. arachidicola, A. parasiticus, A. novoparasiticus, A. sergii and A. transmontanensis (Fig. 3, Fig. 10). These species are morphologically similar, but colony growth rates can distinguish between them. Aspergillus krugeri grows faster than A. parasiticus (40–60 mm), A. sergii (<55 mm) and A. transmontanensis (55–57 mm) on CYA (Soares ). On CYA at 37  °C, Aspergillus krugeri grows more restricted than A. arachidicola (60–70 mm), A. novoparasiticus (58–63 mm), A. sergii (<60 mm) and A. transmontanensis (55–57 mm) (Pildain et al., 2008, Gonçalves et al., 2012).

Fig. 10

Phylogenies of Aspergillus sect Flavi based on ITS, BenA, CaM and RPB2. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Visagie, MycoBank MB834204. Fig. 17.

Fig. 17

Aspergillus magaliesburgensis. A. Colonies, from left to right, CYA, MEAbl, DG18. B–E. Close-up of colonies after prolonged incubation on CYA (B–D) and MEAbl (E). F–J. Conidiophores. K. Conidia. Scale bars: B, E = 2 mm; C = 0.2 mm; D = 0.5 mm; F–L = 20 μm; J, K = 10 μm.

Etymology: Latin, magaliesburgensis, named after Magaliesburg, the town the ex-type was collected from.

Classification: Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus section Flavi.

Diagnosis: Colonies pale, sparse intense yellow sporulation becoming cinnamon with age. Stipes yellow, conidia smooth, globose to subglobose, 2.5–3.5 × 2.5–3.5 μm. Sclerotia present.

Typus: South Africa, Gauteng, Magaliesburg, from an Antlion (Myrmeleontidae), April 1996, collected by J. Pieterse (holotype PREM 62314, culture ex-type PPRI 6165 = CMV 007A3).

ITS Barcode: MK450649 (alternative identification markers: BenA = MK451116; CaM = MK451511; RPB2 = MK450802).

Colony diam (7 d, in mm): CYA 65–70; CYA 30 °C 60–65; CYA 37 °C 50–55; CYAS 65-70; MEAbl > 70; MEA 53–56; DG18 > 70; YES > 70; OA 55–60; CREA 55–60.

Colony characters (25 °C, 7 d): CYA colonies surface floccose, mycelial areas white, some yellow aerial mycelia present, sporulation absent after 7 d, intense yellow when present, with age cinnamon, sclerotia present, black when mature, soluble pigment absent, exudate absent, reverse pigmentation yellowish white to pale yellow (2A2–3). MEAbl colonies surface floccose, mycelial areas white, sporulation absent after 7 d, intense yellow when present, with age cinnamon, sclerotia present, black when mature, soluble pigment absent, exudate absent, reverse pigmentation pale yellow to dull yellow (3A3–B3). YES colonies surface floccose, mycelial areas white, sporulation very sparse, bright yellow, black when mature, soluble pigment absent, exudate absent, reverse pigmentation pale yellow to greyish yellow (4A3–B3). DG18 colonies surface floccose, mycelial areas white, some yellow aerial mycelia present, sporulation absent, soluble pigment absent, exudate absent, reverse pigmentation yellowish white to pale yellow (2A2–3). CREA colonies weak growth, acid not produced.

Micromorphology: Conidial heads radiate. Conidiophores biseriate. Stipes yellow, smooth, (350–)900–1150 × (6–)8–12 μm. Vesicles globose, metulae cover 100 % of head, 40–85 μm wide. Metulae 8–12(–16) × 3.5–5.5 μm. Phialides ampulliform, 7.5–10 × 2–3 μm. Conidia globose to subglobose, smooth, 2.5–3.5 × 2.5–3.5 μm, (3.1 ± 0.2 × 2.9 ± 0.3, n = 52) μm, length/width 1.08 ± 0.12. Ascomata not observed. Sclerotia black when mature, 550–1500 μm.

Notes: Phylogenies resolve A. magaliesburgensis in section Flavi in the A. alliaceus clade (Frisvad ), containing A. alliaceus, A. lanosus, A. neoalliaceus and A. vandermerwei (Fig. 3, Fig. 10). BenA, CaM and RPB2 can be used to identify the new species. Aspergillus magaliesburgensis produces sclerotia, and these structures are absent in A. vandermerwei, while A. lanosus typically produces bright yellow colonies. The new species is distinct from A. neoalliaceus based on the subglobose to ellipsoid conidia of the latter. Morphologically, A. magaliesburgensis and A. alliaceus could not be distinguished from each other. We do note that “faintly yellow conidiophores” were previously observed for A. alliaceus (Raper & Fennell 1965), while A. magaliesburgensis produce conidiophores with distinctly yellow stipes.

Visagie, MycoBank MB834205. Fig. 18.

Fig. 18

Aspergillus purpureocrustaceus. A. Colonies, from left to right, CYA, MEAbl, DG18. B–E. Close-up of colonies on MEAbl (B), DG18 (C, E) and CYA (D). F. Hülle cells. G. Potential immature asci. H–K. Conidiophores. L. Conidia. Scale bars: B, C, D = 2 mm; E = 0.5 mm; F = 20 μm; G–K = 10 μm.

Etymology: Latin, named purpureocrustaceus, meaning purple and crust, in reference to the colonies on CYA and MEAbl that turn purple and crust-like with age.

Classification: Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus section Nidulantes.

Diagnosis: Colonies crust-like and very hard due to abundant Hülle cells produced on surface, having a reddish brown to purple color, sporulation sparse to absent, conidiophores biseriate, stipes 130–310 μm, conidia globose to subglobose, rough, 3.5–4.5(–5) × 3–4.5 μm.

Typus: South Africa, Limpopo, plant debris, January 1990, (holotype PREM 62264, culture ex-type PPRI 3840 = CMV 008B3).

Additional material examined: South Africa, Western Cape, Cape Town, Huntsman spider (Palystes castaneus), January 1994, collected by N. Larsen & H. Robertson PPRI 5548 = CMV 008B1.

ITS Barcode: MK450653 (alternative identification markers: BenA = MK451138; CaM = MK451515; RPB2 = MK450806).

Colony diam (7 d, in mm): CYA 40–41 (25–26); CYA 30  C 10–15; CYA 37 °C no growth; CYAS 25–28; MEAbl 45–47 (33–35); MEA 38–41; DG18 35–40; YES 53–60; OA 25–30; CREA 27–30.

Colony characters (25 °C, 7 d): CYA colonies surface floccose, mycelial areas yellow to grey, sporulation absent, Hülle cells abundant, reddish brown, becoming purple and crust-lie with age, soluble pigment absent, exudate reddish brown and clear, reverse pigmentation dark brown (6F8), yellowish brown (5D4–5). MEAbl colonies surface floccose, mycelial areas yellow to grey, sporulation absent, Hülle cells abundant, reddish brown, becoming purple and crust-lie with age, soluble pigment absent, exudate reddish brown and clear, reverse pigmentation dark brown (6F8), yellowish brown (5D4–5). YES colonies surface floccose, mycelial areas yellow to grey, sporulation absent, Hülle cells abundant, reddish brown, soluble pigment absent, exudate reddish brown and clear, reverse pigmentation olive brown (4F8), pale yellow (4A2). DG18 colonies surface floccose, mycelial areas yellow to grey, sporulation sparse, greyish green (28D6), Hülle cells abundant, reddish brown, soluble pigment absent, exudate reddish brown and clear, reverse pigmentation dark brown (6F8), yellowish brown (5D4–5). CREA colonies weak growth, acid not produced.

Micromorphology: Conidial heads radiate. Conidiophores biseriate. Stipes hyaline, smooth, 130–310 × 5–7.5 μm. Vesicles subclavate, metulae cover 75–100 % of head, 10–20 μm wide. Metulae 6–11.5 × 3–5.5 μm. Phialides ampulliform, 7–10 × 3–4 μm. Conidia globose, rough, 3.5–4.5(–5) × 3–4.5 μm, (4.1 ± 0.4 × 3.9 ± 0.4, n = 28) μm, length/width 1.05 ± 0.04. Hülle cells globose to subglobose, occurring in hard crusts with reddish purple color, 13–25 μm. Ascomata not observed.

Notes: Phylogenies resolve A. purpureocrustaceus in a clade of section Nidulantes with A. multicolor, A. mulundensis, A. pluriseminatus and A. tumidus (Fig. 5, Fig. 11). This group of species typically produce abundant Hülle cells, often giving the colony a reddish to purple color with age (Roy et al., 1987, Stchigel and Guarro, 1997, Chen et al., 2016, Crous et al., 2018). Comparing these species, only A. multicolor and A. mulundensis are capable of growth on CYA at 37 °C. Aspergillus pluriseminatus can be distinguished from the other species in this clade by the presence of a sexual state and absence of asexual state. Compared to the new species, A. tumidus grows more restricted on MEA (38–41 vs 22–23 mm), grows more rapidly on CYA at 30 °C (10–15 vs 32–34 mm), with its colony appearance dominated by good sporulation.

Fig. 11

Single gene phylogenies of Aspergillus sect Nidulantes based on ITS, BenA, CaM and RPB2. Strains from new species are shown in orange text and reference strains in grey text. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Visagie & N. Yilmaz, MycoBank MB834206. Fig. 19.

Fig. 19

Aspergillus seifertii. A. Colonies, from left to right, CYA, MEAbl, DG18. B–E. Close-up of colonies on DG18 (B) and CYA (C–E). F–H. I. Conidiophores. I. Conidia. Scale bars: B–D = 0.5 mm; E = 2 mm; F, G = 20 μm; H–I = 10 μm.

Etymology: Latin, seifertii, named after Dr. Keith A. Seifert, a prominent Canadian mycologist specialised on mycotoxigenic genera and other hyphomycetes.

Classification – Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus section Clavati.

Diagnosis — Colonies greyish to dark green, producing large conidiophores with clavate heads, stipes up to 6 mm long, vesicles 26–60 μm wide, up to 210 μm long.

Typus: South Africa, Free State, Golden Gate National Park, Grassroots, January 1988, collected by R. Anelich (holotype PREM 49066, culture ex-type PPRI 3211 = CMV 006F5).

Additional material examined: South Africa, Free State, Golden Gate National Park, Soil, 2018, collected by R. Jacobs, PPRI 26025 = CMV 011E3; CMV 011E4.

ITS Barcode: MK450647 (alternative identification markers: BenA = MK451093; CaM = MK451509; RPB2 = MK450800).

Colony diam (7 d, in mm): CYA 33–35; CYA 30 °C 35–38; CYA 37 °C 2–3; CYAS 10–12; MEAbl 40–45; MEA 38–40; DG18 25–28; YES 45–50; OA 28–35; CREA 20–25.

Colony characters (25 °C, 7 d): CYA colonies surface floccose, mycelial areas white, sporulation moderately dense, greyish green to dark green (25D5–F5), soluble pigment absent, exudate clear, reverse pigmentation pale green to pale yellow (30A3–1A3–2A3). MEAbl colonies surface floccose, mycelial areas white, sporulation sparse, dark green (25F5), soluble pigment absent, exudate clear, reverse pigmentation pale green to pale yellow (30A3–1A3–2A3). YES colonies surface floccose, mycelial areas white, sporulation dense, greyish green to dark green (25D5–F5), soluble pigment absent, exudate clear, reverse pigmentation yellowish white to yellow (3A2–6). DG18 colonies surface floccose, mycelial areas white, sporulation moderately dense, dull green to dark green (25D4–F5), soluble pigment absent, exudate absent, reverse pigmentation pale green to pale yellow to light yellow (30A3–1A3–2A3–3A4). CREA colonies weak growth, acid not produced.

Micromorphology: Conidial heads clavate, with age splitting into 3–4 divergent columns. Conidiophores uniseriate. Stipes hyaline, smooth, up to 6 mm × 17–24 μm. Vesicles clavate, phialides cover 100 % of head, 26–60 μm wide, up to 210 μm long. Phialides ampulliform, 7–9.5 × 2.5–3.5 μm. Conidia globose, smooth, 3–4 × 3–4 μm, 3.4 ± 0.2 × 3.3 ± 0.19, n = 59) μm, length/width 1.03 ± 0.05. Ascomata not observed.

Notes: Phylogenies resolves Aspergillus seifertii as a unique lineage in section Clavati (Fig. 4, Fig. 12). Generally, species from this section produce blue-green conidia and clavate conidiophores (except for A. posadasensis for which only a sexual reproductive state was reported (Marin-Felix )). The stipe and vesicle length are generally good characters to distinguish between these species (Varga ). Aspergillus clavatus and A. seifertii produce conidiophores with stipes of up to 3 and 6 mm, respectively, while A. giganteus and A. longivesica can grow several cm in length. The remaining section Clavati species have stipes shorter than 1 mm. Vesicle length is also a useful character. Aspergillus clavati, A. seifertii, A. giganteus and A. longivesica have vesicles up to 200, 210, 600 and 3200 μm, respectively.

Fig. 12

Single gene phylogenies of Aspergillus sect Terrei based on ITS, BenA, CaM and RPB2. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Visagie, MycoBank MB834207. Fig. 20.

Fig. 20

Aspergillus sigurros. A. Colonies, from left to right, CYA, MEAbl, DG18. B–E. Close-up of colonies on CYA (B), DG18 (C, D) and MEAbl (E). F. Hülle cells. G–K. I. Conidiophores. I. Conidia. Scale bars: B, C, E = 2 mm; D = 0.5 mm; F–H = 20 μm; I–K = 10 μm.

Etymology: Latin, sigurros, named after Sigurrós, one of Keith A. Seifert’s favourite music groups.

Classification: Eurotiomycetes, Eurotiales, Aspergillaceae, Aspergillus section Usti.

Diagnosis: Colonies with grey to brownish moderately dense sporulation, growth on CYA at 30 °C 10–14 mm; conidiophores with brown stipes, vesicles 11–25 μm wide, conidia spiny, globose 3–4 × 3–4 μm.

Typus: South Africa, KwaZulu-Natal, Pinetown, unknown environmental sample, April 2014, collected by M. Truter (holotype PREM 62308, culture ex-type PPRI 15889 = CMV 005I4 = 2014-M62/147).

ITS Barcode: MK450650 (alternative identification markers: BenA = MK451066; CaM = MK451512; RPB2 = MK450803).

Colony diam (7 d, in mm): CYA 34–35; CYA 30 °C 10–15; CYA 37 °C no growth; CYAS 27–29; MEAbl 29–31; MEA 28–30; DG18 28–32; YES 39–42; OA 32–35; CREA 25–26.

Colony characters (25 °C, 7 d): CYA colonies surface floccose, mycelial areas white, sporulation moderately dense, brownish grey to brown (5E2–5–6E5–2), soluble pigment absent, exudate clear, minute droplets, reverse pigmentation olive (2D4), yellowish white (3A2). MEAbl colonies surface floccose, mycelial areas white, sporulation moderately dense, grey (5E1–6E1) to brown (6E6), soluble pigment absent, exudate absent, reverse pigmentation olive (2D4), yellowish white (3A2). YES colonies surface floccose, mycelial areas white, sporulation sparse to moderately dense, greyish brown (5D3–6D3), soluble pigment absent, exudate absent, reverse pigmentation brownish orange (5C5), yellowish white (3A2). DG18 colonies surface floccose, mycelial areas white, sporulation moderately dense, brownish grey to brown (5E2–5–6E5–2), soluble pigment absent, exudate clear, minute droplets, reverse pigmentation brown (5E5), olive (2D4), yellowish white (3A2). CREA colonies strong growth, acid not produced.

Micromorphology: Conidial heads radiate. Conidiophores biseriate. Stipes brown, smooth, (85–)120–360 × 2.5–6.5 μm. Vesicles globose, metulae cover 50–75 % of head, 11–25 μm wide. Metulae 6–10 × 3–4 μm. Phialides ampulliform, 6–8.5 × 2.5–3.5 μm. Conidia globose, spiny to somewhat wart-like, some covered in sheath, 3–4 × 3–4 μm, (3.2 ± 0.2 × 3.2 ± 0.2, n = 53) μm, length/width 1.03 ± 0.04. Hülle cells irregularly elongated, in scattered groups, 22–60 × 11–20 μm. Ascomata not observed.

Notes: Aspergillus sigurros resolves as a close relative of P. carlsbadensis and P. contaminans in section Usti (Fig. 7, Fig. 13). Compared to A. carlsbadensis, the new species produces conidiophores with broader vesicles (11–25 vs 10–14 μm), larger conidia (3–4 vs 2.5–3 μm) and grows more restricted on CYA at 30 °C (10–15 vs 28–32 mm) (Samson ). Microscopically A. contaminans and A. sigurros are very similar. However, the new species grows faster on CYA at 30 °C (4–5 vs 10–14 mm) (Crous ).

Fig. 13

Single gene phylogenies of Aspergillus sect Usti based on ITS, BenA, CaM and RPB2. Strains from new species are shown in orange text, strains identified during this study in black text and reference strains in grey text. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; ∗ = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp).

Discussion

With this project, we aimed to re-identify strains previously lodged in the PPRI and MRC culture collections as Aspergillus or its old associated sexual state genera (e.g. Eurotium, Emericella etc.). Unfortunately, a large proportion of strains in PPRI were either badly contaminated or not viable (±35 %). As a result, only 250 strains were included in this particular study, with 354 new DNA reference sequences (ITS 24; BenA 52; CaM 250; RPB2 28) generated and published on GenBank. South African Aspergillus was found to be relatively diverse with 63 species identified belonging to 11 sections (sections Aspergillus, Candidi, Circumdati, Clavati, Cremei, Flavi, Flavipedes, Fumigati, Nidulantes, Terrei and Usti). This does not include the 11 Aspergillus sect Nigri species that will be published elsewhere. Among the 63 species, seven were found to be new and are described in the Taxonomy section above. One problem experienced during this project was that for the new species, very few strains were available, e.g. four new species were represented by only one strain, while the remaining three new species had only two strains. This situation is frequent when sequencing smaller collections around the world. For A. elsenburgensis we were fortunate that CBS had several additional strains sequenced. Even though not ideal, comparisons based on morphology, multigene phylogenies and single gene trees applying genealogical concordance phylogenetic species recognition (Taylor ), leaves little doubt about the novelty of the new species introduced here.

Sequence based identifications of PPRI and MRC strains was relatively straight forward thanks to the secondary identification marker CaM and associated database (Samson ). Throughout the genus and between different sections, the primer pairs cmd5&cmd6 performed (Hong ) well. For only a minor proportion of strains, additional ITS, BenA and/or RPB2 sequences were needed to confirm the CaM based identifications. ITS and BenA posed no problems in terms of amplification using proposed methods of (Samson ), but RPB2 was difficult to amplify using either 5F&7CR (Liu ) or 5FEur&7CREur (Houbraken ). Both primer sets provided intermitted hits and misses, with the internal sequencing primers F310, R310, 388F and 527R (Houbraken & Samson 2011) at the end needed to obtain high quality sequences contigs.

Several Aspergillus strains belonging to sect Terrei were tentatively identified during this study. Both A. allahabadii and A. alabamensis appears to contain a large degree of infraspecies variation and potentially contain a large number of new species. Even though the multigene phylogeny (Fig. 6) appears to indicate that several new species may exist, we did not feel comfortable introducing new species in a difficult clade without having more data from other regions of the world. Similarly, PPRI 14275 potentially represents a new species closely related to A. transmontanensis from section Flavi. However, since no consistent morphological differences were observed in this strain, we decided to not introduce a phylogenetic species for this single strain.

One of the big challenges we face in Aspergillus is to discover the missing biodiversity. This can either be in the form of new species discovery and/or isolation of additional strains of already known species. Phylogenetic approaches and their incorporation into our species concepts resulted in rather aggressive approaches. It is not ideal to introduce new species based on one or two strains, but as is obvious from this study, often one is left with that as the only option. Monotypic species are frequent in Aspergillus with 118 of the 415 accepted species represented by a single strain (the ex-type), while 80 species are represented by two strains. Within a modern taxonomy like that employed in Aspergillus, this creates problems on several levels, but most pressing is infraspecies variation for species that are often not captured. This is true from a morphological and DNA sequence perspective, but especially concerning the latter, it creates difficulties with identifications. It is not uncommon to find strains that show a few nucleotide differences from the ex-type sequence. Trying to identify such a sequence becomes very complicated amongst monotypic species as one will often not know if the strain belongs to a new species or if they found infraspecies variation within a known species. Studies that generate a lot of additional reference sequences are thus of great importance, not only to discover new species but also to discover infraspecies variation which ultimately makes future species delineations and thus identifications easier. For taxonomic revisions it is crucial to have as much data as possible available, as recently illustrated for the A. viridinutans species complex, where it was found that A. parafelis and A. pseudofelis should be considered synonyms of the genetically diverse A. felis (Hubka ). Expanded efforts to isolate and identify fungi should thus remain a priority in important genera such as Aspergillus.