Aspergillus, Penicillium and Talaromyces isolated from house dust samples collected around the world

As part of a worldwide survey of the indoor mycobiota, dust was collected from nine countries. Analyses of dust samples included the culture-dependent dilution-to-extinction method and the culture-independent 454-pyrosequencing. Of the 7 904 isolates, 2 717 isolates were identified as belonging to Aspergillus, Penicillium and Talaromyces. The aim of this study was to identify isolates to species level and describe the new species found. Secondly, we wanted to create a reliable reference sequence database to be used for next-generation sequencing projects. Isolates represented 59 Aspergillus species, including eight undescribed species, 49 Penicillium species of which seven were undescribed and 18 Talaromyces species including three described here as new. In total, 568 ITS barcodes were generated, and 391 β-tubulin and 507 calmodulin sequences, which serve as alternative identification markers.


INTRODUCTION
Indoor environments provide humans with a protective habitat in which they spend up to 90 % of their time (Höppe & Martinac 1998). These environments reportedly have unique microbial communities, which have adapted to the specific carbon, temperature and humidity constraints of these environments (Flannigan et al. 2011). Indoor environments, especially in first world countries, are well regulated with regards to temperature and are generally dry. Carbon sources available to fungi include damaged building materials (Flannigan et al. 2011), textiles, various food products and dust , Flannigan et al. 2011. When actively growing on these substrates, fungi often release high concentrations of spores and fungal fragments into the air that could affect humans as pathogens (Li et al. 1998, de Hoog 2000, Garber 2001), allergens (Horner et al. 1995, Terr 2009, Aimanianda et al. 2009, Green et al. 2009, Jaakkola et al. 2010, Karvala et al. 2011, food spoilers (Pitt & Hocking 2009 or cause structural damage to building materials (Kauserud et al. 2007, Schmidt 2007, Chunduri 2014. Combined with the number of immuno-compromised individuals rising worldwide, many common fungi are being reported as causing infections (Vartivarian et al. 1993, Latg e 1999, Lin et al. 2001, Lyratzopoulos et al. 2002. This has lead to increased concern in the western world concerning indoor fungal communities, with much focus on Stachybotrys (commonly referred to as the "black toxic-mould") and sick building syndrome (Mahmoudi & Gershwin 2000, Wilson & Straus 2002, Terr 2009, Straus 2009, Adams et al. 2013a. Samson et al. (2010) and Flannigan et al. (2011), based on years of experience in isolating and identifying fungi from food and indoor environments, published a list of 100 species that are commonly isolated from indoor environments in the western world. Next-generation sequencing, however, has changed the way we perceive these communities, because it allows us to detect the "unculturable" fungi (Amend et al. 2010). Culture-independent techniques rely on curated sequences (mostly from the ITS region) for correct identifications. The number of reliable databases is currently limited (Kõljalg et al. 2005(Kõljalg et al. , 2013. This can be attributed to the large volume of alpha taxonomy necessary to create reliable identification databases (Peterson 2012). It is crucial to get these in place, because obtaining an accurate name unlocks a large amount of associated information.
Aspergillus, Penicillium and Talaromyces (Eurotiomycetes) are considered among the commonest genera found indoors (Pitt & Hocking 2009, Amend et al. 2010. These species are often associated with specific food items (Frisvad & Samson 2004) and colonies can produce millions of conidia. This profuse sporulation may account for the ease with which some species are isolated. Very common indoor Aspergillus species include A. calidoustus, A. flavus (common aflatoxin producers; Codner et al. 1963, Schroeder & Boller 1973, Pildain et al. 2008, Ezekiel et al. 2014, A. fumigatus (human pathogen and most common causative agent of aspergillosis and mycetoma; Latg e 1999, de Hoog 2000, Grosjean & Weber 2007, A. penicillioides, A. restrictus, A. sydowii (a common cause of human mycoses; Takahata et al. 2008, A. versicolor and A. westerdijkiae (common ochratoxin A producer; Frisvad et al. 2004a). Many Penicillium species are associated with biodeterioration of specific foods and are thus commonly reported from indoor surveys. Examples include P. expansum, commonly associated with apple rot and patulin production (Frisvad et al. 2004b), while P. digitatum and P. italicum cause rots of citrus (Frisvad & Samson 2004). Other species considered very common in indoors include P. brevicompactum (produces mycophenolic acid; Frisvad et al. 2004b), P. chrysogenum (penicillin producer; Houbraken et al. 2012), P. citrinum, P. commune, P. glabrum, P. olsonii, P. oxalicum and P. rubens (penicillin producer; Houbraken et al. 2012), while common Talaromyces species include T. funiculosus, T. rugulosus (produces rugulosin; Samson et al. 2010) and T. wortmanii (produces rugulosin and wortmanin; Brian et al. 1957. The two species, A. versicolor and P. chrysogenum were very common in buildings with water damage and have been suggested as indicators for sick building syndrome (Andersen et al. 2011).
As of 1 January 2013, single name nomenclature for fungi was enforced in the International Code of Nomenclature for algae, fungi, and plants (ICN) (McNeill & Turland 2011, McNeill et al. 2012. The abandonment of dual nomenclature resulted in significant changes in the taxonomy and nomenclature of Aspergillus, Penicillium and Talaromyces. Based on a four gene phylogeny,  showed that species formerly classified in Penicillium subgenus Biverticillium are resolved in a monophyletic clade with the former teleomorph genus Talaromyces, while the remaining Penicillium species are associated with the younger teleomorph genus name Eupenicillium. As such, Samson et al. (2011) transferred the accepted species of Penicillium subgenus Biverticillium into Talaromyces and  transferred Eupenicillium species into Penicillium. This was well received by the general community working on these fungi (Houbraken et al. 2011a,b, Visagie & Jacobs 2012, Yilmaz et al. 2012, Manoch et al. 2013, Visagie et al. 2013, Peterson & Jurjevic 2013, Sang et al. 2013, Fujii et al. 2013, Frisvad et al. 2013, Devi et al. 2014, Dufoss e et al. 2014, Kanse et al. 2014. The single name solution for Aspergillus and its large number of younger associated teleomorphic genera (such as Emericella, Neosartorya, Eurotium etc.) is controversial, although phylogenetic data seems to suggest that Aspergillus and its associated teleomorphic genera collectively represent a monophyletic clade based on Bayesian analysis of a four gene ) and 25 gene  dataset. The International Commission of Penicillium and Aspergillus (ICPA) voted on this nomenclatural issue on 14 April 2012 and chose to retain a broad but monophyletic concept of Aspergillus, rather than splitting the genus into smaller clades correlating with the teleomorphic names.
To achieve stability in names, the opportunity exists to have them protected in the ICN. With this in mind, lists for Aspergillus, Penicillium and Talaromyces were published in Visagie et al. (2014a), Samson et al. (2014) and Yilmaz et al. (2014), with updates to these lists available on ICPA's website (http://www. aspergilluspenicillium.org). In addition to providing accepted species lists, it also provides information such as culture collection numbers for living ex-type material and GenBank accession numbers for sequences linked to these ex-types. This is considered an important step towards enabling correct species identifications in these large genera; there are currently 331 species in Aspergillus, 319 in Penicillium and 85 in Talaromyces. The ITS barcodes from this list are incorporated in the RefSeq data set intended to enhance reliable fungal identifications using the GenBank database (Schoch et al. 2014).
This paper is focused on the identification of Aspergillus, Penicillium and Talaromyces species isolated from house dust collected from nine countries and the creation of a DNA barcode database for these species. We describe 18 new species, including eight in Aspergillus, seven in Penicillium, and three in Talaromyces. This work contributes to the Alfred P. Sloan research network on the Microbiology of the Built Environment, by providing authoritative taxonomic and molecular data to be used for metagenomic studies, thereby helping bridge the gap between culture-dependent and -independent detection techniques. This is the second of a series of reports on the taxonomy of fungi isolated using dilution to extinction in the survey, the first being the brief description of a new species of Rasamsonia (Tanney & Seifert 2013). The taxonomy of the other fungi isolated, reporting a taxonomically broad and diverse range of fungi, will be the subject of future publications.

Isolations and identifications
Settled dust was collected in April of 2009 using sterilised Duststream ® collectors (Indoor Biotechnologies) attached to vacuum cleaners. Buildings from nine countries, including Australia, Indonesia, Mexico, Micronesia, New Zealand, South Africa, Thailand, United Kingdom and Uruguay, were included in the survey. Samples from Canada and the United States were not included in this taxonomic study. Isolations were made using a dilution-to-extinction (d2e) method modified from Collado et al. (2007), using microplates composed of capped 1.5 mL micro tubes instead of 48-well microplates (Seifert et al. unpubl.). Malt extract agar (MEA, 20 g malt extract, 15 g agar, 1 000 mL dH 2 O) and 20 % sucrose MEA (20SMEA) with chloramphenicol were used as isolation media and 1.0 mL of medium was dispensed into each micro tube using a multichannel pipette. House dust was suspended in a carboxymethylcellulose solution and diluted stepwise up to 1:64; the dilution yielding the maximum number of single-species colonies in the isolation was selected for subsequent study. For preliminary screening, cultures that appeared to represent Penicillium, Aspergillus, or Talaromyces were plated onto MEA in 6 cm Petri dishes and purified as necessary. All selected cultures from each country were sorted into putative species groups based on colony characters on MEA, and then up to five strains per culture-group per country were selected for more detailed study.
For our analyses of the prevalence of particular species in specific countries (Table 1), samples collected from different sites were considered to represent one sample.
Isolates from each country were placed into morpho-groups based on their characters on Czapek Yeast Autolysate agar Table 1. Aspergillus, Penicillium and Talaromyces species distribution in house dust from around the world.
ASPERGILLUS, PENICILLIUM AND TALAROMYCES ISOLATED FROM HOUSE DUST www.studiesinmycology.org (CYA) and Malt Extract agar (MEA), with Dichloran 18 % Glycerol agar (DG18) added for Aspergillus. From here, 5-10 strains from each morpho-group were selected for sequencing. The β-tubulin gene (BenA) and internal transcribed spacer regions of the nrDNA operon (ITS) were sequenced for Penicillium and Talaromyces, with calmodulin (CaM) and ITS sequenced for Aspergillus. Phylogenetic comparisons of newly generated sequences with a reliable reference sequence database were used for making identifications. This reference database was compiled based on the accepted species list described in the introduction. This list provides GenBank accession numbers to ITS, BenA and CaM sequences of all extype strains for accepted species in Aspergillus, Penicillium and Talaromyces. In the case of new species, CaM was added for Penicillium and Talaromyces, while BenA was added for new Aspergillus species.
Isolates were deposited into the working collection of the Applied and Industrial Mycology department (DTO) housed at the CBS-KNAW Fungal Biodiversity Centre, Utrecht, the Netherlands. Strains representing new species were deposited into the public collection of the CBS-KNAW (CBS).

DNA extraction, sequencing and phylogenetic analysis
DNA was extracted from 10-day-old strains grown on MEA (some Aspergillus species on DG18) using the Ultraclean™ Microbial DNA isolation Kit (MoBio, Solano Beach, USA), with DNA preps stored at −20°C. PCR reactions were prepared as described in . Amplification of ITS was done using the primer set ITS1 and ITS4 (White et al. 1990), primer pair Bt2a and Bt2b for BenA (Glass & Donaldson 1995) and cmd5 and cmd6 for CaM (Hong et al. 2006). A standard amplification cycle was used, which ran 35 cycles with an annealing temperature of 55°C. Sequencing reactions were set up using the BigDye Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, CA) with the same primer sets used for amplification. Sequences were determined on an ABI PRISM 3730xl genetic analyser (Applied Biosystems, California, USA). Sequence contigs were assembled using Seqman Pro v. 9.0.4 (DNAstar Inc.) and newly generated sequences deposited into GenBank.
The phylogenies presented here were prepared using a subset of representative strains of each species. The ITS phylogenies for Aspergillus, Penicillium and Talaromyces were used to direct the allocation of sequences into the correct genera and into smaller clades (indicated by different colours in the large scale trees), to allow more robust alignments of the alternative genes BenA and CaM.
Data sets were aligned in MAFFT v. 7.058b (Katoh & Standley 2013) using the L-INS-i algorithm. When needed, manual adjustments to alignments were made in MEGA v. 5.2.2 (Tamura et al. 2011). Maximum-likelihood (ML) trees were calculated for aligned data sets using MEGA. For the multigene phylogenies presented in the Taxonomy section for new species below, data sets were concatenated in Seaview v. 4.4.1 (Gouy et al. 2010). The most suitable model was determined in MEGA based on the lowest Bayesian Information Criterion (BIC). ML analyses were run by calculating the initial tree with the Bio-Neighbour-Joining (BioNJ) option, followed by a Heuristic search with the Nearest-Neighbour-Interchange (NNI) option. Support in nodes was calculated using a bootstrap analysis with 1 000 replicates. In the phylogenies presented, thickened branches indicate bootstrap support above 80 %.

Morphology
Species were characterised using standard growth conditions (Okuda et al. 2000, Visagie et al. 2013, Visagie et al. 2014a. Strains were inoculated in three-point fashion onto CYA, MEA, Yeast Extract Sucrose agar (YES), DG18, CYA with 5 % NaCl (CYAS), Oatmeal agar (OA) and Creatine Sucrose agar (CREA). Plates were incubated in plastic boxes for 7 d in the dark at 25°C. Additional CYA plates were incubated at 30 and 37°C. Colour names and alphanumeric codes used in descriptions refer to Kornerup & Wanscher (1967).
Microscopic preparations were made from colonies grown on MEA, with DG18 also used for Aspergillus, after 1 to 2 wk. Lactic acid (60 %) was used as mounting fluid and excess conidia were washed away with 70 % ethanol. Characters were captured using a Zeiss SteREO Discovery.V20 dissecting microscope and Zeiss AX10 Imager.A2 compound microscope, both equipped with AxioCam MRc5 cameras using AxioVs40 v. 4.8.2.0. Microscopic measurements were done using Nikon NISelements D v. 4.0. Photo plates were prepared in Adobe ® Photoshop ® CS6 with photomicrographs cleaned up using the healing brush tool, for aesthetic reasons, without altering areas of scientific significance.

Isolations and identifications
D2E dust isolations resulted in 7 904 isolates, including 1 160 Aspergillus, 1 459 Penicillium and 98 Talaromyces isolates. Isolates represented 59 Aspergillus, 49 Penicillium and 18 Talaromyces species. Of these, 18 displayed unique characters deviating from known species of these genera and are described below as new species in the taxonomy section. Species identities and their presence/absence at a country scale are provided in Table 1.
Aspergillus diversity was high in Thailand (25 species), South Africa (23 species), Mexico (23 species) and Micronesia (19 species), while no Aspergillus species were isolated from Australian house dust. Penicillium species richness was highest in New Zealand (22 species) and South Africa (16 species), with no Penicillium species isolated from Uruguay. For Talaromyces, Thailand (10 species) and South Africa (8 species) had the highest species richness, while none were isolated from Australia, Indonesia, the United Kingdom and Uruguay.
Several species were common in the house dust (Table 1), with 13 Aspergillus species isolated from more than two countries. Aspergillus sydowii occurred in dust from six countries, A. fumigatus in five and A. subramanianii, A. niger and A. versicolor in four. Six Penicillium species were isolated from more than two countries. Penicillium brevicompactum, P. citrinum VISAGIE ET AL.

Aspergillus phylogeny
An ITS phylogeny ( Fig. 1) was used to place Aspergillus isolates into their respective sections. The aligned data set included 347 strains and was 622 bp long, and the analysis employed the General Time Reversible (GTR) model with Gamma distribution (+G), with a certain fraction of sites that are evolutionary invariable (+I) selected. The analysis distributed the 59 Aspergillus species into 10 clades. The black Aspergillus species of section Nigri were resolved in two clades (clades 5 & 8), the first containing species closely related to A. nigri and the other relatives of A. aculeatus. The section is considered monophyletic following the three gene phylogeny of Peterson (2008) and four gene phylogeny of . For accurate identification, CaM phylogenies were prepared for each of the 10 clades and are presented in Figs 2-11. Clade 1 consisted of species classified in section Circumdati (Fig. 2). The aligned data set was 554 bp long, with Kimura 2parameter (K2 + G + I) the most suitable model. This group of species is generally recognised by their ochre coloured conidiophore heads and a large number of species produce ochratoxins (Frisvad et al. 2004a). One of these ochratoxin producers is A. westerdijkiae, which was isolated in very high numbers from the South African house dust. A monographic treatment on the section is published in this issue of Studies in Mycology and includes descriptions for two new species, A. occultus and A. pulvericola isolated in this study. We note that A. elegans and A. steynii have identical CaM sequences (Fig. 2), even though ITS (Fig. 1a) and BenA distinguishes them.
Clade 2 represents section Candidi (Fig. 3). The aligned data set was 527 bp long, with K2 + G the most suitable model. Within the clade, we identified a new species with similar morphological features to A. candidus. Phylogenetically it is distinct and is described as A. subalbidus in the taxonomy section below. Two isolates identified as A. taichungensis had sequence variation compared to the ex-type strain, but morphologically they were all identical. As such, the sequence variation was considered insufficient to justify describing a new species. Sequences for A. candidus are highly variable based on Varga et al. (2007). A new species, A. pragensis, was recently described in the A. candidus complex (Hubka et al. 2014). However, a number of strains analysed in Varga et al. (2007) do not phylogenetically conform to the clades accepted by Hubka et al. (2014)   ASPERGILLUS, PENICILLIUM AND TALAROMYCES ISOLATED FROM HOUSE DUST www.studiesinmycology.org DTO 244F1 as A. candidus, even though it most probably represents a new species.
Aspergillus section Flavi is resolved in clade 3 (Fig. 4). The CaM alignment was 517 bp long and K2 + G selected for ML analysis. The species isolated from dust include A. flavus, A. nomius, A. pseudonomius and A. tamarii.
Clade 4 contains sections Terrei and Flavipedes (Fig. 5). The aligned data set was 571 bp long with the K2 + G model selected for ML analysis. In section Terrei, we isolated A. terreus and A. allahabadii. In section Flavipedes, the three isolated species are considered new and described as A. capensis, A. micronesiensis and A. templicola in the taxonomy section.
Clade 5, labelled section Nigri clade 1 (Fig. 6), contains the black Aspergilli closely related to A. niger. The aligned data set was 442 bp long, with the K2 + G model selected for ML analysis. Five species were identified in this clade as A. carbonarius, A. neoniger, A. niger, A. tubingensis and A. welwitschiae.
Sections Fumigati, Clavati and Cervini all occurred in clade 6 ( Fig. 7). The aligned data set was 578 bp long and the K2 + G model was selected for ML analysis. Strains were identified as A. clavatus, A. hiratsukae, A. lentulus and A. fumigatus. The latter species was isolated in high numbers from five countries, namely Mexico, Micronesia, New Zealand, South Africa and Thailand.
Clade 7 resolves sections Restricti and Aspergillus in one clade (Fig. 8). The aligned data set was 610 bp long and the K2 + G + I model was used for ML analysis. Within the clade, we isolated one new species closely related to A. glaucus and A. proliferans, the latter also found in the dust samples together with A. chevalieri, A. montevidensis, A. penicillioides, A. pseudoglaucus, A. restrictus and A. ruber.
Clade 8 contains the black Aspergillus species closely related to A. aculeatus, labelled as Nigri clade 2 ( Fig. 9). Even though the ITS phylogeny places A. arenarius closest to this clade, its taxonomic placement is currently uncertain and will be the focus of a future paper. The aligned data set was 470 bp long and the most suitable model was K2 + I. Two black species were identified as A. aculeatinus and A. brunneoviolaceus. A new species, closely related to A. arenarius, is described as A. arenarioides in the taxonomy section.
Clade 9 contains section Usti species (Fig. 10). The aligned data set was 510 bp long and K2 + I selected for the ML analysis. Five species were identified as A. germanicus, A. minutus, A. puniceus, A. ustus and a new species described as A. porphyreostipitatus.
Clade 10 contains sections Versicolores and Nidulantes (Fig. 11). The aligned data set was 545 bp long and K2 + G was selected as the most suitable model. Aspergillus versicolor is often isolated from indoor environments. Jurjevi c (2012) considered it to represent a complex and accepted nine species. We isolated and identified all of the new species they accepted, namely A. amoenus, A. austroafricanus, A. creber, A. fructus, A. jensenii, A. protuberus, A. puulaausensis, A. tennesseensis, and A. versicolor. Aspergillus sydowii was abundant and had a wide distribution in the house dust. In addition, we introduce a new species in the section as A. griseoaurantiacus. In section Nidulantes, we identified strains as A. unguis, A. montenegroi and A. nidulans.

Penicillium phylogeny
An ITS phylogeny was used to place Penicillium house dust isolates in their respective sections (Fig. 12). The aligned data set included 380 strains and was 585 bp long. The GTR + G + I model was the most suitable for the ML analysis. The phylogeny resolved the 49 house dust species, distributed among 12 clades. Clades corresponded well with the sections proposed by . To obtain more accurate identifications, BenA gene trees were analysed for each ITS clade and are presented in Figs 13-24.
Clade 1 contains section Citrina (Fig. 13), a group of species of wide distribution and isolated from a wide range of sources (Houbraken et al. 2011b). The aligned data set was 448 bp long, with the K2 + G model selected for ML analysis. Species were identified as P. citrinum, P. pancosmium, P. roseopurpureum, P. sanguifluum, P. sizovae, P. steckii and P. sumatraense. Penicillium pancosmium was abundant in samples collected from South Africa, Indonesia and Micronesia. It is also extremely common in isolations from house dust samples collected in Regina, Canada (Hirooka, Tanney & Seifert, unpubl.).
Clade 2 corresponds with the recently revised section Sclerotiora ( Fig. 14) (Rivera et al. 2012, Visagie et al. 2013). The aligned data set was 374 bp long, with the K2 + G model selected for ML analysis. Penicillium brocae was the only species isolated from house dust that belongs to the section.
Clade 3 includes section Ramigena (Fig. 15). The aligned data set was 402 bp long and K2 + I was the most suitable model for ML analysis. Two species, P. hispanicum and P. ramusculum, were identified from house dust. BenA also shows that P. cyaneum, P. dierckii and P. sublateritium are synonyms, with P. cyaneum (Bainier & Sartory) Biourge, Cellule 33: 102. 1923 representing the oldest name.
Clade 4 includes species classified in section Cinnamopurpurea (Fig. 16). The aligned BenA data set was 390 bp long, with the K2 + G model selected for the ML analysis. One species was identified as P. incoloratum, while a second is described as P. infrapurpureum below.
Clade 5 contains the section Aspergilloides (Fig. 17), which is reviewed in Houbraken et al. (2014b). The alignment was 459 bp long and K2 + G was selected as the most suitable model for ML analysis. Three species were identified, including P. glabrum and two new species, P. sublectatum prov. nom. and P. longicatenatum prov. nom., described in Houbraken et al. (2014b).
Clade 6 contains section Exilicaulis (Fig. 18). The aligned data set was 448 bp long and K2 + G was selected for ML analysis. Species isolated include P. atrosanguineum, P. citreonigrum, P. corylophilum, P. decumbens, P. melinii, P. restrictum and P. rubefaciens. From the phylogeny, it is clear that some species need further study. The P. restrictum complex, including five species, represents one of these. This will be the focus of a future paper. We thus tentatively identify isolates in this complex as P. restrictum, mainly based on their morphological characters.
Clade 7 includes species of section Lanata-Divaricata (Fig. 19). The aligned data set was 472 bp long, with the K2 + G model selected for ML analysis. Isolates were identified as P. oxalicum and a new species is described here as P. singorense.
Clade 8 contains species classified in section Canescentia (Fig. 20). The BenA alignment was 403 bp long, with K2 + G selected for the ML analysis. We describe one new species in this section as P. dunedinense.
Sections Brevicompacta and Ramosa are resolved in clade 9 (Fig. 21). The aligned data set was 394 bp long and K2 + G was selected for the ML analysis. One of the more common species   ASPERGILLUS, PENICILLIUM AND TALAROMYCES ISOLATED FROM HOUSE DUST www.studiesinmycology.org found in dust was P. brevicompactum. Our data suggest that the recently described P. kongii (Wang & Wang 2013) is a synonym of P. brevicompactum. The remaining isolates were identified as P. buchwaldii and P. olsonii. In section Ramosa, we isolated P. swiecickii and one new species closely related to P. soppii, described below as P. lenticrescens.
The species in clade 10 are classified in sections Paradoxa and Turbata (Fig. 22). The aligned data set was 394 bp long and K2 + G was selected for ML analysis. Within section Paradoxa, we describe two new species in the P. atramentosum species complex as P. mexicanum and P. magnielliptisporum. In section Turbata, we identified one of the species as P. madriti.
Clade 11 comprises the recently reviewed section Chrysogena   (Fig. 23). This group of species was well represented in dust samples, especially P. rubens and to a lesser degree P. chrysogenum. The aligned data set was 444 bp long and the K2 + G model selected for the ML analysis. Isolates were identified as P. allii-sativii, P. chrysogenum, P. halotolerans, P. lanosocoeruleum and P. rubens.  Clade 12 mostly includes species classified in sections Penicillium and Fasciculata (Fig. 24). The alignment was 322 bp long and the K2 + G model was selected for ML analysis. Isolates were identified as P. biforme, P. commune, P. coprophilum, P. crustosum, P. cyclopium, P. italicum, P. melanoconidium, P. palitans and P. solitum.

Talaromyces phylogeny
An ITS phylogeny was used to place Talaromyces house dust isolates into their respective sections (Fig. 25), as described by Yilmaz et al. (2014). The aligned ITS data set was 605 bp long and included 125 strains. The ML analysis was done with the GTR + G + I model selected. The phylogeny resolved house dust isolates into four sections, with BenA gene trees subsequently calculated for each section.
Clade 1 contains species classified in section Talaromyces (Fig. 26). The aligned BenA data set was 413 bp long, with the K2 + G + I model most suitable for ML analysis. Isolates were identified as the newly described T. cnidii (Sang et al. 2013) and T. amestolkiae (Yilmaz et al. 2012), the previously described T. siamensis and T. verruculosus, and two new species described here as T. sayulitensis and T. oumae-annae.
Clade 2 contains species that typically produce synnemata after more than one week of growth, which are classified in section Purpurei (Fig. 27). The aligned data set was 389 bp long and K2 + G was selected for ML analysis. Talaromyces ramulosus was isolated from the South African house dust, a species originally described from soil, apples (from the Fynbos biome in South Africa) and moth-damaged grapes (Ontario, Canada) (Visagie et al. 2009).
Clade 3 contains species of section Trachyspermi (Fig. 28). The aligned data set was 373 bp long, with the K2 + G model selected for ML analysis. Isolates were identified as T. albobiverticillius, T. atroroseus, T. diversus and T. minioluteus. Frisvad et al. (2013) recently introduced T. atroroseus, a species that produces large amounts of red pigmentation. In the same paper, T. albobiverticillius was shown to have genetic and phenotypic variation, with either white or green-pigmented conidia produced. Our house dust isolates produced the green phenotype. Phylogenetic data suggest that T. minioluteus represents a species complex. The dust isolates were thus tentatively identified as T. minioluteus, with strains that will be part of a future study on this complex.
Notes: Aspergillus subalbidus is morphologically almost identical to A. candidus. The new species also includes strains (CBS 567.65 and CBS 112449) previously identified as A. candidus and most recently, as A. taichungensis     (Fig. 30). Both A. subalbidus and A. candidus are distinguished from these species by their typical white colonies, smooth conidia and inability to grow at 37°C. Minor differences were observed when comparing the new species with A. candidus. The new species grew slightly slower on CYA, YES and DG18. The purple to black sclerotia common in A. subalbidus when grown on OA were not observed in A. candidus, as previously reported by Varga et al. (2007). These minor differences make morphological identification very difficult. However, phylogenetically this species is distinct and these minor phenotypic differences warrant describing it as new.  Colony characters: Colony surface floccose; sporulation and mycelial areas yellowish white to pale yellow (4A2-3); soluble pigment brown to absent; exudate clear; reverse brown to dark brown (5F8-6F8). CYA 30°C, 7 d: Colonies similar to CYA at 25°C, except for yellowish colour in colonies in DTO 267H4. CYA 37°C, 7 d: Colonies similar to CYA at 25°C. MEA 25°C, 7 d: Colony surface floccose; mycelial areas orange white (5A2); sporulation brownish grey (4C2); soluble pigment absent; exudate absent; reverse brown (6E8). YES 25°C, 7 d: Colony surface floccose; sporulation and mycelial areas yellowish white to yellowish grey to greyish yellow (4A2-B2-3); soluble pigment brown; exudate absent; reverse brown (6D8-7E8). DG18 25°C, 7 d: Colony surface floccose; sporulation and mycelial areas pale orange to greyish orange (5A3-B3); soluble pigment absent; exudate absent; reverse light yellow to greyish orange (4A4-5C5). OA 25°C, 7 d: Colony surface velutinous to somewhat floccose; sporulation and mycelial areas pale orange (5A3); soluble pigment olive, inconspicuous; exudate absent; reverse brownish orange (5C5). CREA 25°C, 7 d: Colony surface velutinous to somewhat floccose, orange white to pale orange (5A2-3); acid not produced.
Notes: Penicillium infrapurpureum is classified in section Cinnamopurpurea with other species that grow slowly on CYA and MEA (Fig. 46). The new species produces a striking purple to bluish reverse on CYA. A similar colouration was reported for P. cinnamopurpureum (Pitt 1979). Phylogenetically, P. infrapurpureum is resolved in a clade with P. idahoense and P. ellipsoideosporum. Penicillium idahoense also produces a purple reverse and smooth walled conidia (Paden 1971), but a cleistothecial morph is commonly observed, whereas P. ellipsoideosporum produces similar conidia, but lacks the colourful reverse (Wang & Kong 2000). Penicillium ellipsoideosporum also produces shorter phialides, 6.5-8.5 μm, than P. infrapurpureum and P. idahoense. Penicillium infrapurpureum does not grow on CYA at 30°C or above, with P. idahoense sometimes growing at 37°C. Two species described from China, P. guizhouanum and P. jiangxiense, were thought to be close relatives of P. cinnamopurpureum (Kong 2000, Kong & Liang 2003. Sequences from the ex-type strains show that this is not the case with P. guizhouanum, which has almost identical sequences to Talaromyces funiculosus. Penicillium jianxiense is tentatively placed in section Cinnamopurpurea. Based on its ITS barcode, it does not belong in the section, but BenA and CaM is most similar to other species in the section (Fig. 46).
Notes: Penicillium singorense is a close relative to P. penajorense and P. vanderhammenii (Fig. 48). The latter species do not grow on CYA at 37°C, in contrast to the fast growing colonies of P. singorense. In addition, yellow cleistothecia were reported for P. vanderhammenii ). This was not observed in P. singorense.
Notes: Penicillium mexicanum is closely related to P. atramentosum and P. magnielliptisporum (described below) in section Paradoxa (Fig. 54). Frisvad & Samson (2004) considered the smooth walled globose conidia, good growth on CREA and absence of growth at 30°C diagnostic for P. atramentosum. Phylogenetically, the strains identified as P. atramentosum in Frisvad & Samson (2004) represent a species complex. In our studies, 0-6 mm growth was observed on CYA at 30°C for strains previously assigned to P. atramentosum. Slightly faster growth, 12-14 mm, was seen in CBS 109588, the closest relative of P. mexicanum. Penicillium mexicanum and P. magnielliptisporum grew 19-21 mm and 9-10 mm respectively at 30°C. In addition, conidia of the two new species were consistently larger than those of P. atramentosum (< 3 μm). Except for the faster growth of P. mexicanum at 30°C, its growth is more restricted on most media than P. magnielliptisporum. In addition, P. magnielliptisporum produces much bigger conidia than P. mexicanum. Etymology: Latin, magnielliptisporum, meaning large ellipsoidal conidia, in reference to the conidia of this species, which are larger than those of its closest relatives.

The genus
Diagnosis: Growing restrictedly on CYA and DG18, grows well on other media, conidiophores biverticillate with some subterminal branches formed, stipes smooth walled, conidia rough walled and ellipsoidal.    not allow unequivocal morphological identification of the new species. Phylogenetically it is distinct and this justifies introducing it as a new species.
Notes: Talaromyces yelensis is closely related to T. tratensis in section Islandici (Fig. 60). The latter species typically produces a sexual state with roughened ascospores and ellipsoidal smooth walled conidia. Talaromyces yelensis produces subglobose to broadly ellipsoidal conidia that have rough walls and lacks a sexual state.

DISCUSSION
Phylogenetic species recognition ITS is the most commonly sequenced gene for fungi and was recently accepted as the official DNA barcode (Schoch et al. 2012). Curated reference data sets are currently limited, with several publications addressing the issue (Kõljalg et al. 2005, Santamaria et al. 2012, Kõljalg et al. 2013, Schoch et al. 2014. With regards to Aspergillus, Penicillium and Talaromyces, the accepted species list endorsed by ICPA provides accession numbers of ITS barcodes to all ex-type strains. Although ITS does not distinguish among all species, with some species sharing identical sequences (Skouboe et al. 1999, Peterson 2000a,b, Samson et al. 2011, it does provide valuable information on sectional classification and often provides enough information for making a species identification. In order to compensate for the lack of variability in ITS, the ICPA list also include accession numbers for BenA and CaM sequences, meant to serve as secondary identification markers. Our data shows that BenA works well for Penicillium and Talaromyces identifications, while CaM performs well in Aspergillus. However, some problems were experienced. Aspergillus steynii and A. elegans share identical CaM sequences (Fig. 2), something very uncommon in Aspergillus. In this case, these two species have unique ITS and BenA sequences.
In Penicillium, BenA has limitations in the P. chrysogenum (Fig. 23) and P. camembertii (Fig. 24) species complexes. Houbraken et al. (2012) reviewed section Chrysogena and distinguished several phylogenetically closely related species, and showed that different genes suggest different phylogenies. For example, although P. chrysogenum has unique BenA sequences, variation among strains makes distinguishing it from P. allii-sativii complicated. On the other hand, CaM does not distinguish well between P. chrysogenum and P. rubens ), but BenA easily distinguishes the two. As such, a combination of the two genes is often required for identifying isolates within the clade. Another difficult clade is the P. camemberti complex. The ex-type cultures for P. commune, P. camemberti and P. caseifulvum have identical ITS, BenA and CaM sequences, as also reported by Giraud et al. (2009) for elongation factor-1α. The importance and different roles of these species in the cheese industry makes it unsatisfactory to synonymise them, and as a result the white sporulating P. camembertii are considered a domesticated form of P. commune or P. caseifulvum (green sporulating). A similar situation exists in Aspergillus, where A. oryzae is considered a domestic form of A. flavus (Varga et al. 2011). None of these problems were experienced for Talaromyces, where BenA worked very well for identifications.
In some cases, an ex-type sequence alone is insufficient reference data for making a conclusive identification, a reflection of intraspecific variation, for example in P. italicum (Fig. 24), P. sumatraense (Fig. 13) (Houbraken et al. 2011b) and in Talaromyces section Trachyspermi (Fig. 28) (Frisvad et al. 2013). As such, a verified reference data set that includes non-ex-type strains representing the sequence diversity within phylogenetically delineated species is the next crucial step for sequencebased identifications in these genera. Samson et al. (2010) and Flannigan et al. (2011) listed 100 fungal species common in indoor environments. From this list, we also found A. fumigatus, A. sydowii, P. brevicompactum and P. citrinum to be common in the collected house dust. Of significance is the effect of taxonomic revisions on this type of information. For example, A. versicolor used to be considered very common in indoor environments. However, it was recently shown to represent a species complex, with nine new species introduced (Jurjevi c et al. 2012). From our data, A. versicolor was still isolated from four different countries and A. creber was isolated in higher numbers from three countries. From unpublished data, we are also noting that most of the "A. versicolor" strains collected from indoor environments over many years in the DTO collection housed at CBS should now be identified as A. creber. Another example is Aspergillus section Circumdati. The ochratoxin producer A. westerdijkiae is reported to have a wide distribution indoors. From dust, we could only recover this species from Mexico and South Africa, whereas A. subramanianii was found in high numbers from four countries. Penicillium chrysogenum is also considered to have a worldwide distribution from indoor environments. However, after Houbraken et al. (2011a) reintroduced P. rubens as the name for the commercial penicillin producing strain closely related to P. chrysogenum, we are finding P. rubens to be very common indoors and not P. chrysogenum.

Fungi in house dust
The origin of common indoor species is difficult to determine. Aspergillus sydowii is a good example. We found A. sydowii to be one of the most common species in collected dust samples and the species is generally considered as widespread. The species is often isolated from soil (Domsch et al. 1980), is very common on mouldy gypsum wallboard, dust, paint and various foods (Gorbushina et al. 2007, Flannigan et al. 2011) and is commonly found in marine environments where it acts as an opportunistic pathogen of sea corals (Roth et al. 1964, Smith et al. 1996, Geiser et al. 1998, Toledo-Hern andez et al. 2008, Rypien 2008, Kirkwood et al. 2009). The source or origin of this species is still unknown, even though most studies suggest it being a terrestrial soil-borne fungus. The suggestion thus is that A. sydowii, along with a number of other soil-borne fungi, gets carried into indoor environments. Its ability to grow in such a wide range of niches is intriguing and needs further studies.
Recent studies suggested that the indoor fungal communities as observed with metagenomic analyses exploiting next generation sequencing are mostly determined by the outdoor fungal communities (Adams et al. 2013a,b). In our study, the highest diversity was observed in countries that are also listed as biodiversity hotspots of the world (Myers et al. 2000). This might suggest that at least a considerable proportion of these species isolated from house dust originated from outdoors. However, the prevalence of specific species commonly isolated from indoor surveys suggests that the indoor environments do select for the growth of specific species. In addition, much of the metagenomics diversity may come from transient, dormant or dead spores.
From various indoor culture-independent surveys, it is apparent that the ITS database is not yet sufficient for identification of Aspergillus, Penicillium and Talaromyces (Amend et al. 2010, Adams et al. 2013a. These studies often cite Aspergillus sp. and Penicillium sp. as the most abundant. It would be valuable, even if species identification were not feasible, to identify to which clade or taxonomic section or series the sequences belong. The Last Common Ancestor (LCA) analysis commonly employed for identifying OTU's in metagenomic studies employ the GenBank taxonomic hierarchy to assign query sequences to taxonomic nodes. This hierarchy generally lacks ranks between genus and species, which means that the analysis suffers from a regrettable lack of precision for large genera, such as those studied here. In order to at least partly alleviate this kind of issue, our ITS barcodes of ex-type sequences and reference barcodes created from dust isolates will be uploaded into the UNITE database as part of a planned curated set on indoor moulds. As part of a future study, these reference sequences will be used for comparing d2e and 454-pyrosequencing data (Amend et al. 2010) in order to better understand the communities of Aspergillus, Penicillium and Talaromyces in indoor environments.