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Research Article
A survey of xerophilic Aspergillus from indoor environment, including descriptions of two new section Aspergillus species producing eurotium-like sexual states
expand article infoCobus M. Visagie§, Neriman Yilmaz§, Justin B. Renaud|, Mark W. Sumarah|, Vit Hubka, Jens C. Frisvad#, Amanda J. Chen¤«, Martin Meijer«, Keith A. Seifert§
‡ University of Ottawa, Ottawa, Canada
§ Biodiversity (Mycology), Agriculture and Agri-Food Canada, Ottawa, Canada
| London Research & Development Centre, Agriculture & Agri-Food Canada, London, Canada
¶ Charles University, Prague, Czech Republic
# Technical University of Denmark, Kgs. Lyngby, Denmark
¤ Institute of Medicinal Plant Development, Chinese Academy of medical Sciences and Peking Union Medical College, Beijing, China
« Applied and Industrial Mycology, CBS-KNAW Fungal Biodiversity Centre, Utrecht, Netherlands
Open Access

Abstract

Xerophilic fungi grow at low water activity or low equilibrium relative humidity and are an important part of the indoor fungal community, of which Aspergillus is one of the dominant genera. A survey of xerophilic fungi isolated from Canadian and Hawaiian house dust resulted in the isolation of 1039 strains; 296 strains belong to Aspergillus and represented 37 species. Reference sequences were generated for all species and deposited in GenBank. Aspergillus sect. Aspergillus (formerly called Eurotium) was one of the most predominant groups from house dust with nine species identified. Additional cultures deposited as Eurotium were received from the Canadian Collection of Fungal Cultures and were also re-identified during this study. Among all strains, two species were found to be new and are introduced here as A. mallochii and A. megasporus. Phylogenetic comparisons with other species of section Aspergillus were made using sequences of ITS, β-tubulin, calmodulin and RNA polymerase II second largest subunit. Morphological observations were made from cultures grown under standardized conditions. Aspergillus mallochii does not grow at 37 °C and produces roughened ascospores with incomplete equatorial furrows. Aspergillus megasporus produces large conidia (up to 12 µm diam) and roughened ascospores with equatorial furrows. Echinulin, quinolactacin A1 & A2, preechinulin and neoechinulin A & B were detected as major extrolites of A. megasporus, while neoechinulin A & B and isoechinulin A, B & C were the major extrolites from A. mallochii.

Key words

BenA, CaM, indoor environments, mycotoxin, RPB2

Introduction

Species of Aspergillus section Aspergillus, the “A. glaucus” group of Thom and Raper (1941) and Raper and Fennell (1965), typically produce yellow cleistothecia (white in A. leucocarpus) with lenticular ascospores and the section includes species that were traditionally classified in the genus Eurotium. Species of section Aspergillus have a broad distribution in nature, but their xerophilic physiology makes them significant for the built environment and the food industry. In the built environment, species of section Aspergillus are among the primary colonizers of building materials (Flannigan and Miller 2011). Modern heating systems are designed to remove humidity from buildings, creating opportunities for xerophiles to dominate indoor fungal communities. Also of concern is the growth of these fungi in museums or libraries on historic artefacts such as books, carpets or paintings. They also commonly grow on/in leather, dust, softwood, a variety of textiles and even dried specimens in herbaria (Cavka et al. 2010; Micheluz et al. 2015; Pinar et al. 2013; Pinar et al. 2015; Pitt and Hocking 2009; Raper and Fennell 1965; Samson et al. 2010). For the food industry, these species have an economic impact because they can grow on stored grain, cereals or preserved foods with high sugar (i.e. jams, maple syrup) or salt content (i.e. biltong, dried fish) (Pitt and Hocking 2009; Samson et al. 2010).

Xerophily is a common physiological property of many Aspergillus species from several subgenera and sections, enabling those species to grow at low water activity (aw) or equilibrium relative humidity (ERH) (Flannigan and Miller 2011; Pitt 1975). Water activity is a measure of available water in liquid or solid substrates that has a significant effect on which organisms can grow on foods or other matrices, including building materials (Scott 1957). Reducing aw is widely used in the food industry to reduce spoilage (Pitt and Hocking 2009). For the built environment, however, it is very difficult and often impractical to measure aw and as a result relative humidity (RH) is often used as a proxy. Because RH measures moisture in air rather than available water in a substrate, it is not considered a reliable indication of whether growth will actually occur on surfaces in the built environment (Flannigan and Miller 2011). A better measure is ERH because it is more representative of available water and is numerically proportional to aw (Flannigan and Miller 2011; Pitt and Hocking 2009).

Species of section Aspergillus produce many extrolites exhibiting a wide range of biological activities (Frisvad and Larsen 2015a; Gomes et al. 2012; Kanokmedhakul et al. 2011; Li et al. 2008a; Li et al. 2008b; Slack et al. 2009; Smetanina et al. 2007). Most notably, compounds from A. chevalieri were shown to be active against Plasmodium falciparum (malaria), Mycobacterium tuberculosis and cancer cell lines (Kanokmedhakul et al. 2011), an antitumor compound was reported from A. cristatus (Almeida et al. 2010), while many compounds are known to be antioxidants. They also produce mycotoxins, especially echinulin, flavoglaucin and physcion, which are toxic to animals (Ali et al. 1989; Bachmann et al. 1979; Cole and Cox 1981; Greco et al. 2015; Nazar et al. 1984; Rabie et al. 1964; Semeniuk et al. 1971; Slack et al. 2009; Vesonder et al. 1988), but toxicity has not been reported in humans. These species are not considered significant human pathogens, because most infections are superficial, with few cases of invasive infections known (de Hoog et al. 2014). Species commonly grow as saprobes on clinical specimens, such as skin and nails (Hubka et al. 2012). The biggest concern to humans, or nuisance, is the growth of these species inside homes, where exposure to spores and fragments, which contains β-(1, 3)-D-glucan, and other metabolites, cause allergies (Green et al. 2006; Slack et al. 2009).

Xerophilic fungi are well studied from a morphological point of view, but much work remains to develop reference sequence data for them. In this paper, we report on the diversity of Aspergillus isolated from house dust using media with low aw that select for the growth of xerophiles. Reference sequences are released for all species, including those received as Eurotium from the Canadian Collection of Fungal Cultures and re-identified here. Furthermore, we describe two new species and report on their extrolite production.

Materials and methods

Strains/sampling and isolations

House dust samples were received from various areas in North America. A modified dilution-to-extinction method (Collado et al. 2007) was used to isolate cultures, as described in Visagie et al. (2014a). Modifications included the use of 48-well titre plates rather than 96-well microtube plates and the use of Dichloran 18% Glycerol agar (DG18; (Hocking and Pitt 1980)), Malt extract yeast extract 10% glucose 12% NaCl agar (MY10-12) and Malt extract yeast extract 50% glucose agar (MY50G) (Samson et al. 2010) isolation media to select for xerophilic fungi.

In addition to newly obtained house dust isolates, several strains, including unidentified isolates and some reference or ex-type cultures, of Aspergillus sect. Aspergillus were obtained from the Canadian Collection of Fungal Cultures, Canada (DAOMC) and the CBS-KNAW Fungal Biodiversity Centre, the Netherlands (CBS).

Morphology

Colony characters were recorded from cultures grown for 7 d on various media, including CYA (Czapek yeast autolysate agar), MEA (Blakeslee’s malt extract agar), CREA (Creatine sucrose agar), CY20S (CYA with 20% sucrose agar), MEA20S (MEA with 20% sucrose agar), DG18, YES (yeast extract sucrose agar), M40Y (Harrold’s agar; 2% malt, 0.5% yeast extract, 40% sucrose), MY50G and MY10-12 (Harrold 1950; Pitt and Hocking 2009; Samson et al. 2014). Plates were incubated upside down in the dark at 25 °C and left unwrapped. Additional CY20S, DG18 and MEA20S plates were wrapped and incubated at 37 °C. Colour names and codes in descriptions are from Kornerup and Wanscher (1967). Microscopic preparations were made from colonies growing on DG18 and observations made using an Olympus SZX12 dissecting and Olympus BX50 compound microscopes equipped with Infinity3 and InfinityX cameras using Infinity Analyze v. 6.5.1 software (Lumenera Corp., Ottawa, Canada). Variation of conidia and ascospores was evaluated by measuring at least 50 structures and presented as mean +/- standard deviation. Photographic plates were prepared in Pixelmator iOS v. 2.3 (http://www.pixelmator.com/ios), with photomicrographs modified for aesthetic purposes using the repair tool, without altering scientifically significant areas.

DNA extraction, sequencing and phylogenetic analysis

DNA was extracted from 8–10 d old colonies grown on DG18 using the UltracleanTM Microbial DNA isolation Kit (MoBio Laboratories Inc., Solana Beach, USA). Loci chosen for amplification included ITS barcodes (internal transcribed spacer rDNA region, including ITS1-5.8S-ITS2) (Schoch et al. 2012), BenA (partial β-tubulin), CaM (partial calmodulin) and RPB2 (RNA polymerase II second largest subunit). Thermocycler programs used for amplification followed Samson et al. (2014) and employed primer pairs V9G & LS266 (ITS; (de Hoog and Gerrits van den Ende 1998; Masclaux et al. 1995), Bt2a & Bt2b (BenA; (Glass and Donaldson 1995)), CF1 & CF4 or sometimes CMD5 & CMD6 (CaM; (Hong et al. 2006; Peterson et al. 2005)) and 5F & 7CR (RPB2; (Liu et al. 1999)). Sequencing was done as described in Visagie et al. (2016). Contigs were assembled in Geneious v. 8.1.8 (Biomatters Ltd, New Zealand) and newly generated sequences submitted to GenBank.

As a preliminary step in identification, CaM sequences derived from the newly isolated cultures were compared to an ex-type reference sequence database published by Samson et al. (2014). Then, gene sequences of the two presumed to be new sect. Aspergillus species were compared to reference datasets obtained from Peterson (2008), Hubka et al. (2013) and Visagie et al. (2014a). All datasets were aligned in MAFFT v. 7.221 (Katoh and Standley 2013) using the L-INS-i option for ITS and G-INS-i option for the other genes. All alignments were trimmed in Geneious and then analysed as single and concatenated datasets using Maximum Parsimony (MP) and Bayesian Inference of phylogenetic trees (BI). For concatenated phylogenies, a partitioned dataset of ITS, BenA, CaM and RPB2 regions was used.

MP analyses were run in PAUP* v. 4.0b10 (Swofford 2002) using heuristic searches with 100 random taxon additions and gaps treated as missing data. Support in nodes was calculated using a bootstrap analysis with the heuristic search option and 1000 replicates.

BI analyses were run in MrBayes v. 3.2.5 (Ronquist et al. 2012). Model selections for BI were made for each gene based on the lowest Akaike Information Criterion (AIC) value, calculated in MrModeltest v. 2.3 (Nylander 2004). Analyses were run with two sets of four chains and stopped at a split frequency of 0.01. The sample frequency was set at 100 and 25 percent of trees removed as burnin. Trees were visualized in FigTree v. 1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) and prepared for publication in Adobe® Illustrator® CS6. Aligned datasets, command blocks and trees were uploaded to TreeBase (www.treebase.org) under submission number 19771.

Extrolite analysis

For extrolite analysis, all strains were grown on 9 cm polystyrene Petri dishes on MEA supplemented with 7.5% NaCl at 25 °C for 14 d. Six agar plugs from each fungal isolate were removed with a sterilized 7 mm cork borer and placed into a 13 mL polypropylene tube. Ethyl acetate (2 mL) was added to the tubes and vortexed for 30 s, followed by 1 h of sonication at 30 °C and vortexed again for 30 s. The supernatants were transferred into clean polypropylene tubes and dried on a centrifugal vacuum concentrator at 35 °C. Extracts were reconstituted in 1 mL of 8:2 methanol:water and filtered into 2 mL amber glass HPLC vials using a 0.45 µm PVDF syringe filter. Extracts were immediately stored at -20 °C until LC-MS analysis. Extracts were analyzed on a Q-Exactive orbitrap coupled to a 1290 Agilent HPLC in both positive and negative polarities. Chemical formula of observed extrolites were determined with Xcalibur® software using accurate mass measurements and manually verified by isotopic pattern. Chemical formulae were searched against AntiBase2013 and Scifinder and putatively confirmed by comparing product ions observed with those published in the literature or though manual interpretation. The fungi were also analysed using the HPLC-DAD method described by Frisvad and Thrane (1987) as modified by Nielsen et al. (2011), by taking two agar plugs from each of the following media: DG18, CYA20S and YES agar, and extracting the combined 6 agar plugs of the colonies of Aspergillus with ethylacetate / isopropanol (3:1, vol./vol.) with 1% (vol.) formic acid added to that mixture. The retention indices and UV spectra were compared to those given in the supplementary material of the Nielsen et al. (2011) paper.

Results

Sampling, isolations and identification

Isolations from house dust collected in Canada and Hawaii resulted in 1039 isolates of xerophilic/xerotolerant fungi. 296 isolates were identified as Aspergillus, of which members from sections Aspergillus, Nidulantes (A. versicolor clade) and Restricti were most abundant. Strains were identified to species using CaM sequences and identities confirmed by morphological examination. They include A. chevalieri, A. cibarius, A. montevidensis, A. proliferans, A. pseudoglaucus, A. ruber and A. tonophilus from sect. Aspergillus. In section Nidulantes (A. versicolor clade), A. jensenii and A. sydowii were isolated most frequently, while A. creber, A. fructus, A. protuberus, A. tenneseensis and A. versicolor were also recovered. A large degree of sequence diversity was observed in sect. Restricti and will be presented in a separate study. Other Aspergillus species identified include A. aureolatus, A. candidus, A. calidoustus, A. flavus, A. japonicus, A. lentulus, A. luchuensis, A. micronesiensis, A. niger, A. pragensis, A. tamarii, A. terreus, A. tubingensis, A. welwitschiae and A. westerdijkiae. Reference sequences, mostly CaM, obtained for these species were uploaded to GenBank under accession numbers KX894565KX894666 and KY351765KY351785, and are included in Suppl. material 1: Table 1 to assist with future identifications. This table also include additional information with regard to strains’ location and growth medium used for their isolations. During this survey, two sect. Aspergillus species with eurotium-like sexual states could not be identified as known species and are described below as new species, based on growth characters on a wide range of culture media. The new species are compared with their close relatives and notes are provided on their diagnostic phenotypic characters, including extrolite production.

Phylogeny

To demonstrate genealogical concordance for the two new species, phylogenies for all known species of sect. Aspergillus were prepared (Table 1) using alignments of ITS, BenA, CaM, and RPB2 (Fig. 1) and overall phylogenetic relationships considered as a concatenated dataset (Fig. 2).

Figure 1.

One of the most parsimonious trees of Aspergillus sect. Aspergillus based on ITS, CaM, BenA and RPB2. Trees were rooted to A. xerophilus, A. leucocarpus and A. osmophilus. Support in nodes higher than 80% bootstrap values and 0.95 posterior probabilities are shown above thickened branches. New species are shown in bold and colour, while ex-type strains are followed by T.

Figure 1.

Continued.

Figure 1.

Continued.

Figure 1.

Continued.

Figure 2.

One of the most parsimonious trees of Aspergillus sect. Aspergillus based on a combined dataset of ITS, BenA, CaM and RPB2. The tree was rooted to A. xerophilus, A. leucocarpus and A. osmophilus. Support in nodes higher than 80% bootstrap values and 0.95 posterior probabilities are shown above thickened branches. New species are shown in bold and colour, while ex-type strains are followed by T.

Strains used for phylogenetic analyses.

Species Strains Origin GenBank accession numbers
ITS CaM BenA RPB2
Aspergillus appendiculatus CBS 374.75T; DAOMC 231665; IMI 278374; ETH 8286 Smoked sausage, Switserland HE615132 HE801318 HE801333 HE801307
Aspergillus appendiculatus CBS101746; AS 3.4673 Sheep dung, China HE615133 HE801319 HE801334 HE801308
Aspergillus brunneus CBS 112.26T; NRRL131; ATCC 1021; IMI 211378; MUCL 15646 Fig, USA EF652060 EF651998 EF651907 EF651939
Aspergillus brunneus CBS 113.27; NRRL124; ATCC 1036; IMI 029188 Unknown EF652056 EF651997 EF651904 EF651938
Aspergillus brunneus NRRL 133 Unknown EF652061 EF651999 EF651908 EF651940
Aspergillus chevalieri CBS 522.65T; NRRL 78; ATCC 16443; IMI 211382 Coffee beans, USA EF652068 EF652002 EF651911 EF651954
Aspergillus chevalieri NRRL 4755 Contaminated culture, USA EF652071 EF652004 EF651913 EF651956
Aspergillus chevalieri NRRL 79 Unknown, USA EF652069 EF652003 EF651912 EF651955
Aspergillus cibarius KACC 46346T Meju, Korea JQ918177 JQ918183 JQ918180 JQ918186
Aspergillus cibarius KACC 46764 Meju, Korea JQ918178 JQ918184 JQ918181 JQ918187
Aspergillus cibarius KACC 46765 Meju, Korea JQ918179 JQ918185 JQ918182 JQ918188
Aspergillus costiformis CBS 101749T; AS 3.4664 Rotten paper, China HE615136 HE801320 HE801338 HE801309
Aspergillus cristatus CBS 123.53T; NRRL 4222; ATCC 16468; IMI 172280; MUCL 15644 Unknown, South Africa EF652078 EF652001 EF651914 EF651957
Aspergillus cumulatus KACC 47316T Rice straw, Korea KF928303 KF928300 KF928297 KF928294
Aspergillus cumulatus KACC 47513 Indoor air from meju fermentation room, Korea KF928304 KF928301 KF928298 KF928295
Aspergillus cumulatus KACC 47514 Indoor air from meju fermentation room, Korea KF928305 KF928302 KF928299 KF928296
Aspergillus glaucus CBS 516.65T; NRRL 116; ATCC 16469; IMI 211383 Unpainted basement board, USA EF652052 EF651989 EF651887 EF651934
Aspergillus glaucus NRRL 117; ATCC 66470 Unpainted basement board, USA EF652053 EF651990 EF651888 EF651935
Aspergillus glaucus NRRL 120; ATCC 16925; FRR 120 Unknown EF652054 EF651991 EF651889 EF651936
Aspergillus glaucus NRRL 121; IMI 313756 Unknown EF652055 EF651992 EF651890 EF651937
Aspergillus intermedius CBS 377.75; IMI 278376; ETH 8277 Soil, Spain HE974459 HE974437 HE974432 HE974425
Aspergillus intermedius CBS 523.65T; NRRL 82; ATCC 16444; IMI 089278; IMI 089278ii; DSM 2830 Unknown, United Kingdom EF652074 EF652012 EF651892 EF651958
Aspergillus intermedius NRRL 4817; IMI 313754 Unknown EF652072 EF652014 EF651894 EF651960
Aspergillus intermedius NRRL 84 Unknown EF652070 EF652013 EF651893 EF651959
Aspergillus leucocarpus CBS 353.68T; NRRL3497; IMI 278375 Raw sausage, Germany EF652087 EF652023 EF651925 EF651972
Aspergillus mallochii DAOMC 146054T = CBS 141928 = DTO 357A5 = KAS 7618 Pack rat dung, USA KX450907 KX450902 KX450889 KX450894
Aspergillus mallochii CBS 141776 = DTO 343G3 'Chocolat miroir' icing for cake, the Netherlands KX450908 KX450903 KX450890 KX450895
Aspergillus megasporus DAOMC 250799T = CBS 141929 = DTO 356H7 = KAS 6176 House dust, Canada KX450910 KX450905 KX450892 KX450897
Aspergillus megasporus DAOMC 250800 = DTO 356H1 = KAS 5973 House dust, Canada KX450909 KX450904 KX450891 KX450896
Aspergillus megasporus CBS 141772 = DTO 048I3 Dutch chocolate butter, the Netherlands KX450911 KX450906 KX450893 KX450898
Aspergillus montevidensis CBS 491.65T; NRRL 108; ATCC 10077; IMI 172290; IHEM 3337 Human tympanic membrane, unknown EF652077 EF652020 EF651898 EF651964
Aspergillus montevidensis CBS 518.65; NRRL90; ATCC 16464; IMI 229971; IFO 33018 Unknown, USA EF652076 EF652017 EF651897 EF651963
Aspergillus montevidensis NRRL 4716; IMI 350348 Candied grapefruit rind, USA EF652079 EF652018 EF651899 EF651965
Aspergillus montevidensis NRRL 89; ATCC 10065; IMI 211806 Unknown EF652075 EF652016 EF651896 EF651962
Aspergillus neocarnoyi CBS 471.65T; NRRL126; ATCC 16924; IMI 172279 Unknown EF652057 EF651985 EF651903 EF651942
Aspergillus niveoglaucus CBS 101750; AS 3.4665 Soil, China HE615135 HE801323 HE801331 HE801312
Aspergillus niveoglaucus CBS 114.27T; NRRL127; NRRL 129; NRRL 130; ATCC 10075; CBS 517.65; IMI 032050; IMI 032050ii Unknown EF652058 EF651993 EF651905 EF651943
Aspergillus niveoglaucus NRRL 128; FRR 128; IMI 091871 Unknown EF652059 EF651994 EF651906 EF651944
Aspergillus niveoglaucus NRRL 136 Unknown EF652062 EF651995 EF651909 EF651945
Aspergillus niveoglaucus NRRL 137; IMI 091872 Unknown EF652063 EF651996 EF651910 EF651946
Aspergillus osmophilus CBS 134258T; IRAN 2090C Leaf of Triticum aestivu, Iran KC473921 KC473918 KC473924 KX512310
Aspergillus proliferans CBS 121.45T; NRRL 1908; CBS 528.65; ATCC 16922; IMI 016105; IMI 016105ii; IMI 016105iii; MUCL 15625 Cotton yarn, United Kingdom EF652064 EF651988 EF651891 EF651941
Aspergillus proliferans NRRL 114; ATCC 10076; IMI 211808 Unknown, USA EF652051 EF651987 EF651886 EF651933
Aspergillus proliferans NRRL 62482; CCF 4096 Palm skin, Czech Republic FR848827 HE650908 FR775375 HE801303
Aspergillus proliferans NRRL 62494; CCF 4146 Toenail, Czech Republic HE578067 HE650909 HE578076 HE801304
Aspergillus proliferans NRRL 62497; CCF 4115 Toenail, Czech Republic FR851850 HE578090 FR851855 HE578107
Aspergillus proliferans NRRL 71 Leafhoppers, USA EF652047 EF651986 EF651885 EF651932
Aspergillus pseudoglaucus CBS 123.28T; NRRL 40; ATCC 10066; IMI 016122; IMI 016122ii; MUCL 15624 Unknown EF652050 EF652007 EF651917 EF651952
Aspergillus pseudoglaucus CBS 379.75; IMI 278373; ETH 8218; DSM 1370 Leaf from Vaccinium myrtillus, Switserland HE615131 HE801322 HE801336 HE801311
Aspergillus pseudoglaucus CBS 529.65; NRRL13; NRRL 24; ATCC 9294; IMI 016114; IMI 016114ii; MUCL 15649 Prunus domestica, France EF652048 EF652005 EF651915 EF651950
Aspergillus pseudoglaucus CBS101747; AS 3.4674 Animal dung, China HE615130 HE801321 HE801335 HE801310
Aspergillus pseudoglaucus NRRL 17; ATCC 10079; UAMH 6580 Skin from wrist, USA EF652049 EF652006 EF651916 EF651951
Aspergillus ruber CBS 101748; AS 3.4632 Soil, China HE615134 HE801325 HE801337 HE801315
Aspergillus ruber CBS 464.65; NRRL5000; ATCC 16923; IMI 32048 Coffee beans, United Kingdom EF652080 EF652010 EF651922 EF651949
Aspergillus ruber CBS 530.65T; NRRL 52; ATCC 16441; IMI 211380 Unknown EF652066 EF652009 EF651920 EF651947
Aspergillus ruber NRRL 76; IMI 91868 Unknown EF652067 EF652011 EF651921 EF651948
Aspergillus sloanii CBS 138176; DTO 244-I8 House dust, United Kingdom KJ775539 KJ775308 KJ775073 KX463364
Aspergillus sloanii CBS 138177T; DTO 245-A1 House dust, United Kingdom KJ775540 KJ775309 KJ775074 KX463365
Aspergillus sloanii CBS 138178; DTO 245-A8 House dust, United Kingdom KJ775542 KJ775313 KJ775076 KX450900
Aspergillus sloanii CBS 138179; DTO 245-A9 House dust, United Kingdom KJ775543 KJ775314 KJ775077 KX450901
Aspergillus sloanii CBS 138231; DTO 245-A6 House dust, United Kingdom KJ775541 KJ775311 KJ775075 KX450899
Aspergillus tonophilus CBS 405.65T; NRRL 5124; ATCC 14567; ATCC 16440; ATCC 36504; DSM 3462; IFO 6529; IMI 108299; IMI 108299ii Binocular lens, Japan EF652081 EF652000 EF651919 EF651969
Aspergillus xerophilus CBS 938.73T; NRRL6131; FRR 2804; IMI 278377 Desert soil, Egypt EF652085 EF651983 EF651923 EF651970
Aspergillus xerophilus NRRL 6132 Desert soil, Egypt EF652086 EF651984 EF651924 EF651971

The ITS alignment was 535 bp long and contained 68 variable characters, of which 27 were parsimony informative. MP analysis resulted in two equally parsimonious trees (length 79 steps, CI = 0.987, RI = 0.992). HKY+I was found to be the most suitable model for BI analysis. ITS is highly conserved in sect. Aspergillus, as demonstrated in the phylogenetic analysis, making it uninformative as an identification barcode in section Aspergillus. Of the 22 species, including the two new species described here, only A. cumulatus, A. leucocarpus, A. osmophilus and A. xerophilus have unique ITS barcodes. The alignments for the BenA, CaM and RPB2 datasets were respectively 389 (151 variable, 136 parsimony informative), 556 (221 variable, 177 parsimony informative) and 871 bp (202 variable, 162 parsimony informative) long. MP analyses resulted in 84 (length 287 steps, CI = 0.728, RI = 0.923), 12 (length 275 steps, CI = 0.7, RI = 0.904), 28 (length 364 steps, CI = 0.648, RI = 0.911) and 24 (length 798 steps, CI = 0.692, RI = 0.907) equally parsimonious trees for BenA, CaM, RPB2 and concatenated dataset. K80+G (BenA), SYM+G (CaM) and SYM+I+G (RPB2) were the most suitable models for BI.

Tree topologies did not differ for respective genes between MP and BI; therefore, MP trees were used to present results. Some species are consistently resolved as sister species such as A. proliferans and A. glaucus, A. brunneus and A. niveoglaucus, A. montevidensis and A. intermedius, and A. osmophilus and A. xerophilus. On a deeper level, however, the backbones in all gene trees were generally poorly supported, resulting in inconsistent clades among different gene trees. The addition of more newly discovered species of section Aspergillus in future may result in better backbone support. With regards to the new species, A. mallochii was sister to A. appendiculatus, although RPB2 placed it on a unique branch. Aspergillus megasporus resolves in different positions depending on gene analyzed, but based on the concatenated phylogeny belongs in a clade with A. brunneus, A. niveoglaucus, A. neocarnoyi, A. glaucus and A. proliferans. For species identifications, it is clear that all three of these genes are superior to ITS and distinguish between all 22 accepted species in sect. Aspergillus.

Extrolites

Aspergillus mallochii and A. megasporus produced several related tryptophan derived alkaloids including, echinulins, neoechinulins and isoechinulins, but in varying amounts (Table 2). Aspergillus mallochii (DAOMC 146054) was a major producer of neoechinulin A & B, while also producing isoechinulin A, B & C (Fig. 3a). Quinolactacin A1, A2 & B were among the major extrolites produced by A. megasporus (Fig. 3b). The other was echinulin produced by DAOMC 250799, although it was not detected in DAOMC 250800. The latter strain was generally a poor extrolite producer. The chemical structures of major extrolites produced by A. megasporus and A. mallochii are shown in Fig. 4.

Figure 3.

Base peak chromatograms observed in positive ionization mode. a Aspergillus mallochii (DAOMC 146054 = KAS 7618) b Aspergillus megasporus (DAOMC 250799 = KAS 6176). Both species show some production of echinulin class of alkaloids to varying amounts. Quinolactacin A1, A2 and B were not detected in A. mallochii.

Figure 4.

Chemical structure of major compounds produced by A. mallochii and A. megasporus.

Overview of the major extrolites detected and product ions.

Extrolite m/z Formula RT (min) Product ions m/z
echinulin 462,311 C29H39N3O2 4,16 338,186 266,190 198,128 210,128 270,124
isoechinulin A 392,233 C24H29N3O2 3,63 268,108 336,170 256,108 69,071
isoechinulin B 390,217 C24H27O2N3 3,75 266,092 322,155 334,155 254,092 306,123
isoechinulin C 406,212 C24H27O3N3 3,37 334,155 266,092 237,138 338,150
neoechinulin A 324,171 C19H21O2N3 3,14 256,108 268,108 185,071 69,071
neoechinulin B 322,155 C19H19O2N3 3,26 254,092 266,095 69,071 226,097
preechinulin 326,186 C19H23O2N3 2,99 130,065 198,128 270,123 258,124
quinolactacin A1 271,144 C16H18N2O2 2,66 214,073
quinolactacin A2 271,144 C16H18N2O2 2,72 214,073
quinolactacin B 257,129 C15H16N2O2 2,54 214,073
questin* 283,061 C16H12O5 3,39 268,038 240,042

Taxonomy

Aspergillus mallochii Visagie, Yilmaz & Seifert, sp. nov.

MycoBank No: 819025
Fig. 5

Etymology

Latin, mallochii, named after Prof. David Malloch, a Canadian specialist in ‘Plectomycetes’ who first collected this species in the 1960’s.

Typus

USA, California, San Mateo, pack rat dung, added to DAOMC in 1969, collected by David Malloch, Holotype DAOM 740296, culture ex-type DAOMC 146054 = CBS 141928 = DTO 357-A5 = KAS 7618.

Additional material examined

The Netherlands, ‘chocolat miroir’ icing for a cake, unknown date and collector, culture CBS 141776 = DTO 343-G3.

ITS barcode

KX450907. Alternative identification markers: BenA = KX540889, CaM = KX450902, RPB2 = KX450894.

Colony diam

7 d (in mm), 25 °C. CYA 6–8; CY20S 14–17; MEA 3–4; MEA20S 29–31; DG18 48–50; YES 9–10; M40Y 48–50; MY50G 35–40; MY10-12 29–30; CY20S, DG18, MEA20S at 37 °C no growth; CREA no growth.

Colony characters

CYA: Colonies with restricted growth; conidiophores sparse; cleistothecia absent. CY20S: Colonies grow faster than on CYA; sporulation sparse to moderately dense, greyish to dark green (30E5–F5); cleistothecia dark yellow, abundant at colony centre. MEA: Colonies with restricted growth; conidiophores and cleistothecia absent. MEA20S: Colonies grow faster than on MEA; sporulation sparse, greyish to dark green (30E5–F5); cleistothecia yellow to orange, abundant. DG18: Colonies very fluffy with aerial mycelia giving rise to conidiophores; sporulation sparse to moderately dense, greyish to dark green (30E5–F5); cleistothecia abundant at colony centre, yellow to orange. Homothallic.

Figure 5.

Aspergillus mallochii (DAOMC 146054). a Colonies on MEA, MEA20S, MY10-12 (top row, from left to right), DG18, CY20S, MY50G (bottom row, from left to right) b Texture on DG18 c Asci d Ascospores e Cleistothecium f, g Conidiophores h Conidia. Scale bars: e = 50 µm, c, d, f–h = 10 µm.

Micromorphology on DG18

Cleistothecia eurotium-like, wall consisting of one layer of flattened cells, yellow to orange, turning deep brown with age, globose, 95–250 μm diam. Asci eight-spored, globose, ellipsoidal to pyriform, 10–15 μm diam, maturing after 7–14 d. Ascospores lenticular, equatorial crest present but incomplete, convex surface roughened, 4.5–6 × 3.5–4.5 μm (5.1±0.3 × 3.9±0.3), n = 52. Conidiophores radiate and columnar, uniseriate; stipes smooth, 200–1000 × 7.5–17(–19) μm; vesicle globose, (25–)40–65 μm diam; phialides ampulliform, covering 80–100% of vesicle, 7–11 × 3–5 μm; conidia roughened to spiny, ellipsoidal, connectives easily visible, 4.5–6.5 × 4–5.5 μm (5.4±0.4 × 4.5±0.3), average width/length = 0.83, n = 68.

Extrolites

Isoechinulin A, B & C; neoechinulin A & B; unknowns C20H18O9, C19H32O3N2, C19H21O3N3, C24H30O3N3, C39H43O6N5. Additionally, echinulin, erythroglaucin, auroglaucin, flavoglaucin, dihydroauroglaucin, tetrahydroauroglaucin were found in CBS 141776. Some extrolites tentatively identified as tetracyclic compounds were detected in CBS 141776.

Notes

Aspergillus mallochii is phylogenetically and morphologically most similar to A. appendiculatus. Both are unable to grow at 37 °C and both have ascospores with incomplete equatorial furrows. Ascospores of the new species, however, are generally smaller and at least finely roughened compared to the smoother ascospores of A. appendiculatus.

Aspergillus megasporus Visagie, Yilmaz & Seifert, sp. nov.

MycoBank No: 819028
Fig. 6

Etymology

Latin, megasporus, in reference to the large conidia produced by this species.

Typus

Canada, Nova Scotia, Wolfville, house dust, 29 January 2015, collected by Allison Walker, isolated by Cobus M. Visagie, holotype DAOM 741781, culture ex-type DAOMC 250799 = CBS 141929 = DTO 356-H7 = KAS 6176.

Additional material examined

Canada, New Brunswick, Little Lepreau, house dust, 29 January 2015, collected by Allison Walker, isolated by Cobus M. Visagie, culture DAOMC 250800 = DTO 356-H1 = KAS 5973. The Netherlands, Dutch chocolate butter, August 2007, collected and isolated by Martin Meijer, culture CBS 141772 = DTO 048-I3.

ITS barcode

KX540910. Alternative identification markers: BenA = KX450892, CaM = KX450905, RPB2 = KX450897.

Colony diam

7 d (in mm), 25 °C. CYA 3–8; CY20S 30–35; MEA 3–5; MEA20S 24–35; DG18 47–50; YES 15–16; M40Y 45–47; MY50G 35–40; MY10-12 40–44; CY20S, DG18, MEA20S at 37 °C no growth, CREA no growth.

Colony characters

CYA: Colonies with restricted growth; conidiophores and cleistothecia absent. CY20S: Colonies grow faster than on CYA; sporulation moderately dense, greyish to dark green (30E5–F5); cleistothecia yellow, sparse. MEA: Colonies with restricted growth; conidiophores and cleistothecia absent. MEA20S: Colonies grow faster than on MEA; sporulation moderately dense, greyish to dark green (30E5–F5); cleistothecia yellow, moderately abundant. DG18: Colonies very fluffy with abundant aerial mycelia giving rise to conidiophores; sporulation moderately dense, dull to dark green (28E3–F3); cleistothecia abundant, dark yellow to orange. Homothallic.

Micromorphology on DG18

Cleistothecia eurotium-like, wall consisting of one layer of flattened cells, yellow to orange, globose, 115–205 μm diam. Asci eight-spored, globose, ellipsoidal to pyriform, 14–19.5 μm diam. Ascospores lenticular, equatorial crest roughened, convex surface smooth, 5–8 × 3.5–6 μm (6.4±0.6 × 4.9±0.5), n = 51. Conidiophores radiate and columnar, uniseriate; stipes smooth, (30–)60–1000 × (9–)13–20 μm; vesicle globose, (8.5–)20–60 μm diam; phialides ampulliform, covering 70–100% of vesicle, (9–)11–15 × 5–7 μm; conidia roughened to spiny, ellipsoidal, connectives often visible, 7–12 × 6–8.5 μm (9.5±1.0 × 6.9±0.5), average width/length = 0.72, n = 85.

Figure 6.

Aspergillus megasporus (DAOMC 250799). a Colonies on MEA, MEA20S, MY10-12 (top row, from left to right), DG18, CY20S, MY50G (bottom row, from left to right) b Texture on DG18 c Asci d Ascospores e Cleistothecium f, g Conidiophores h Conidia. Scale bars: e = 50 µm, c, d, f–h = 10 µm.

Extrolites

Echinulin; neoechinulin A & B; preechinulin; quinolactacin A1 & A2; unknowns C15H20O2, C21H37N, C24H30O6, C29H37O2N3, C21H44O2. In addition, asperflavin, emodin, erythroglaucin, physcion and bisanthron were found in CBS 141772. Some additional extrolites, tentatively identified as tetracyclic compounds, were detected in CBS 141772

Notes

The concatenated phylogeny of BenA, CaM and RPB2 resolves A. megasporus in a clade with A. brunneus, A. niveoglaucus, A. neocarnoyi, A. glaucus and A. proliferans. None of these species are able to grow on CY20S at 37 °C. Aspergillus niveoglaucus and A. megasporus can be distinguished from other species by their large conidia, which are up to 11 and 12 μm in the longest axis respectively. Aspergillus megasporus colonies grow faster than A. niveoglaucus on DG18.

Discussion

Species of Aspergillus section Aspergillus are xerophilic and widespread in nature. Indoor environments, including homes and public buildings, are designed to be as dry as possible, especially in temperate countries, and these conditions select for these xerophiles to thrive. This partially explains the dominance of Aspergillus, Penicillium, Cladosporium and Wallemia in indoor fungal communities (Amend et al. 2010; Flannigan and Miller 2011; Samson et al. 2010; Visagie et al. 2014a). In our isolations of xerophiles occurring in Canadian and Hawaiian house dust, these genera were also found to be dominant. Xerophily is spread broadly across Aspergillus. Thirty Aspergillus species were isolated in our survey, excluding the many section Restricti species that will be addressed in another study. All Aspergillus are capable of growth on DG18, but MY10-12 and MY50G have much lower water activities. Species isolated from these selective media included species of sections Aspergillus, Candidi, Flavipedes, Nidulantes, Nigri, Restricti and Versicolores (summarized in Suppl. material 1: Table 1). One new species was discovered from our house dust samples, described as A. megasporus. We also re-identified all cultures from DAOMC deposited as Eurotium. Among these, we discovered an additional species that we described as A. mallochii using morphology, extrolite and phylogenetic analyses.

Aspergillus megasporus was isolated from Canadian house dust collected in Wolfville, Nova Scotia and Little Lepreau, New Brunswick, and was also isolated from chocolate butter in the Netherlands. Phylogenetically, the position of this species varies depending on which gene is analysed; CaM resolves it in its own distinct clade, BenA in a clade with a poorly supported branch with A. glaucus and A. proliferans, and RPB2 closest to A. niveoglaucus. The multigene phylogeny places it in a large clade, including A. brunneus, A. niveoglaucus, A. neocarnoyi, A. glaucus and A. proliferans. Both A. niveoglaucus and A. megasporus produces conidia respectively reaching 11 and 12 µm, easily distinguishing them from other species of section Aspergillus. Aspergillus megasporus can be distinguished from A. niveoglaucus based on its faster growth on DG18. Aspergillus megasporus produces extrolites commonly detected in species of section Aspergillus, including echinulin, neoechinulin and preechinulin. However, we also detected quinolactacin, a first report for the group. In an independent study using different methods and media, compounds detected from CBS 141772 include asperflavin, auroglaucin, bisanthrons, dihydroauroglaucin, echinulin, emodin, erythroglaucin, flavoglaucin, isoechinulins, neoechinulins, preechinulin, physcion, quinolactacin, tetracyclic compounds, and tetrahydroauroglaucin (Frisvad, personal communication).

Aspergillus mallochii was isolated from pack rat dung collected from San Mateo, California, USA. An additional strain was recently isolated from ‘Chocolat miroir’ icing for a cake in the Netherlands. Phylogenetically, it has A. appendiculatus as sister species, originally described by Blaser (1975) from German smoked sausages. These two species share identical ITS sequences that are distinct from all others in the section (Fig. 2). All other genes, especially RPB2, easily distinguish the two. Morphologically they are also similar, but the roughened ascospores of A. mallochii are distinct from the smoother ascospores of A. appendiculatus. In an independent study using different methods and media, compounds detected from CBS 141776 included auroglaucin, dihydroauroglaucin, echinulins, erythroglaucin, flavoglaucin, isoechinulins, neoechinulins, tetracyclic compounds and tetrahydroauroglaucin. Comparisons revealed that A. appendiculatus produced several compounds not observed in A. mallochii, such as asperflavin, asperentins, bisanthrons, 5-farnesyl-5,7-dihydroxy-4-methylphthalide, mycophenolic acid, physcion and questin (Nielsen et al. 2011). None of the extrolites identified in A. mallochii are unique to the species.

Quinolactacin A1, A2 & B were the major compounds produced by A. megasporus, the only species of section Aspergillus that produces these. These quinolone structures with a γ-lactam ring were first characterized from fermentations of an unknown Penicillium species (Kakinuma et al. 2000; Takahashi et al. 2000) and further characterized by Kim et al. (2001) in Penicillium citrinum, where they were demonstrated to be acetylcholinesterase inhibitors. Quinolactacins have since been reported from multiple Penicillium species from sections Citrina (Houbraken et al. 2011), Brevicompacta (Frisvad et al. 2013; Perrone et al. 2015) and Robsamsonia (Houbraken et al. 2016); Aspergillus quadricinctus, A. stramenius (section Fumigati) (Frisvad and Larsen 2015b; Samson et al. 2007), A. karnatakaensis (section Aenei) (Varga et al. 2010); and from the distantly related marine derived Xylariaceae (Nong et al. 2014). Based on current knowledge, the echinulins (including echinulin, neoechinulin and isoechinulin) detected in both A. megasporus and A. mallochii seem specific to Aspergillus sections Aspergillus and Restricti (Frisvad and Larsen 2015a). Echinulin was first discovered in A. brunneus (= E. echinulatum) by Quilico and Panizzi (1943). It was subsequently detected in many more section Aspergillus species (Ali et al. 1989; Almeida et al. 2010; Greco et al. 2015; Li et al. 2008b; Slack et al. 2009; Smetanina et al. 2007; Vesonder et al. 1988) and shown to be toxic to animal cells (Ali et al. 1989; Umeda et al. 1974), while swine and mice respectively refused feed and water containing echinulin (Vesonder et al. 1988). The presence of echinulin in the environment is not well documented however. In contrast to the negative effects of echinulin, neoechinulin has anti-oxidant properties (Yagi and Doi 1999) and protected PC12 cell lines, used in neurological research, against cell death by peroxynitrite (Kimoto et al. 2007; Maruyama et al. 2004).

Visagie et al. (2014a) emphasized that despite the existence of comprehensive ITS barcode reference databases, this marker is insufficient for identifying most Aspergillus, Penicillium and Talaromyces to species level in culture-independent surveys such as those of Amend et al. (2010) and Adams et al. (2013a; 2013b). The reference sets include sequences for multiple genes obtained from ex-type cultures for all accepted species in these genera (Samson et al. 2014; Visagie et al. 2014b; Yilmaz et al. 2014) and are invaluable as anchoring points for species. Curating databases is laborious and has many complications, but both UNITE and NCBI have ongoing curation projects involving ITS barcodes (Kõljalg et al. 2013; Nilsson et al. 2015; Schoch et al. 2014). ITS will always have limited resolution for species identification. In Aspergillus and Penicillium, ITS is highly conserved in many sections, as is observed in our phylogeny of section Aspergillus (Fig. 2). Barcode-based metagenomic studies commonly use Last Common Ancestor (LCA) analyses for assigning OTU’s to GenBank taxonomic nodes. In LCA, when the analysis cannot identify an operational taxonomic unit (OTU) at a taxonomic rank, it will move up one level until it can make a confident assignment. As now implemented, most species of sect. Aspergillus species would be identified only to the generic level. For species-rich genera such as Aspergillus and Penicillium, this is problematic. Different ecologies, functions, extrolites etc. are often associated with specific groups (i.e. true xerophily in at least three sections of Aspergillus), and much potentially important information is lost because of this imprecision. To circumvent this problem, a few recent studies have used alternative genes combined with next generation sequencing (NGS) for making “mass” identifications in Aspergillus. Lee and Yamamoto (2015) assessed the accuracy of high-throughput amplicon sequencing using ITS, BenA and CaM, and identified OTU’s using the ex-type sequences published by Samson et al. (2014). Results were promising with both BenA and CaM, which are obviously more accurate than ITS. Unfortunately, amplifications of these alternative barcodes were sometimes problematic, perhaps because they are single copy genes or undocumented sequence variation, especially considering comparisons to only ex-type sequences. Similar results were obtained in a subsequent study by Lee et al. (2016). Even though these types of studies are promising, considerable optimisation is required to amplify and sequence low copy markers from a complex matrix, and shotgun sequencing may be more effective. No matter what the experimental approach used by ecologists, taxonomists need to make identifications as easy as possible, not only in the traditional morphological sense, but also by generating reference data that will enhance the robustness of analyses of data generated using new technologies such as NGS. Surveys such as ours are thus important not only for discovering previously unnamed species, but for providing more reference sequences in public databases that capture infraspecies sequence variation for multiple barcodes.

Recently, the International Code of Nomenclature for algae fungi and plants (ICN, Melbourne Code; (McNeill et al. 2012)) adopted single name nomenclature for pleomorphic fungi, meaning decisions are needed to choose either the teleomorphic (sexual morph) or anamorphic (asexual morph) name to represent the genus. In anticipation of this change, Houbraken and Samson (2011) reviewed the taxonomy and phylogeny Trichocomaceae, of which Penicillium and Aspergillus are the largest groups, using a four gene combined analysis. The situation with the generic concept and name for Aspergillus is complicated and controversial, partly because of conflicting interpretations of phylogenies, and partly because of differing opinions on how much taxonomic weight to apply to sexual states in generic concepts (Houbraken and Samson 2011; Pitt and Taylor 2014; Taylor et al. 2016). In this paper, we have followed the traditional broad concept of Aspergillus advocated by the International Commission of Penicillium and Aspergillus (ICPA), which includes species formerly classified in the sexual genera Eurotium, Emericella, Neosartorya and Petromyces in Aspergillus. The section of Aspergillus that is the focus of our paper includes the type species of both Aspergillus (A. glaucus) and Eurotium (E. herbariorum). With the community decision to respect the priority of Aspergillus in both the nomenclatural and practical sense, the new species described here would be described in Aspergillus whether a broad or narrow generic concept is applied. The recent proposal by Taylor et al. (2016) to conserve Aspergillus with the type species changed to A. niger is still being discussed, but at this time seems unlikely to be accepted. If the proposal is implemented, along with the narrower generic concept endorsed by these authors, approximately 180 Aspergillus species would be renamed, including those described in this paper.

Acknowledgements

This research was supported by a grant from the Alfred P. Sloan Foundation Program on the Microbiology of the Built Environment. We thank our dust collectors, Allison Walker, Bryce Kendrick and Anthony Amend.

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