Aspergillus and Penicillium Species

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CHAPTER FOUR

Modern Taxonomy of Biotechnologically Important Aspergillus and Penicillium Species

Jos Houbraken1, Ronald P. de Vries, Robert A. Samson CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands 1Corresponding author: e-mail address: j [email protected]

Contents 1. Introduction 200 2. One Fungus, One Name 202 2.1 Dual nomenclature 202 2.2 Single-name nomenclature 203 2.3 Implications for Aspergillus and Penicillium taxonomy 203 3. Classification and Phylogenetic Relationships in Trichocomaceae, Aspergillaceae, and Thermoascaceae 205 4. Taxonomy of Penicillium Species and Phenotypically Similar Genera 209 4.1 Penicillium and Talaromyces 209 4.2 Rasamsonia 215 4.3 Thermomyces 216 5. Taxonomy of Aspergillus Species 219 5.1 Phylogenetic relationships among Aspergillus species 219 5.2 Aspergillus section Nigri 219 5.3 Aspergillus section Flavi 224 6. Character Analysis 225 7. Modern Taxonomy and Genome Sequencing 227 7.1 Identity of genome-sequenced strains 230 7.2 Selection of strains 231 7.3 Recommendations for strain selection 231 8. Identification of Penicillium and Aspergillus Strains 233 9. Mating-Type Genes 234 9.1 Aspergillus 236 9.2 Penicillium 238 9.3 Other genera 239 10. Conclusions 240 Acknowledgments 240 References 241

Abstract Taxonomy is a dynamic discipline and name changes of fungi with biotechnological, industrial, or medical importance are often difficult to understand for researchers in the applied field. Species belonging to the genera Aspergillus and Penicillium are commonly used or isolated, and inadequate taxonomy or uncertain nomenclature of these genera can therefore lead to tremendous confusion. Misidentification of strains used in biotechnology can be traced back to (1) recent changes in nomenclature, (2) new taxonomic insights, including description of new species, and/or (3) incorrect identifications. Changes in the recent published International Code of Nomenclature for Algae, Fungi and Plants will lead to numerous name changes of existing Aspergillus and Pen- icillium species and an overview of the current names of biotechnological important species is given. Furthermore, in (biotechnological) literature old and invalid names are still used, such as Aspergillus awamori, A. foetidus, A. kawachii, Talaromyces emersonii, Acremonium cellulolyticus, and Penicillium funiculosum. An overview of these and other species with their correct names is presented. Furthermore, the biotechnologically important species Talaromyces thermophilus is here combined in Thermomyces as Th. dupontii. The importance of Aspergillus, Penicillium, and related genera is also illustrated by the high number of undertaken genome sequencing projects. A number of these strains are incorrectly identified or atypical strains are selected for these projects. Rec- ommendations for correct strain selection are given here. Phylogenetic analysis shows a close relationship between the genome-sequenced strains of Aspergillus, Penicillium, and Monascus. Talaromyces stipitatus and T. marneffei (syn. Penicillium marneffei) are closely related to Thermomyces lanuginosus and Th. dupontii (syn. Talaromyces thermophilus), and these species appear to be distantly related to Aspergillus and Pen- icillium. In the last part of this review, an overview of heterothallic reproduction in Asper- gillus and Penicillium is given. The new insights in the taxonomy of Aspergillus, Penicillium, and related genera will help to interpret the results generated with comparative genomics studies or other studies dealing with evolution of, for example, enzymes, mating-type loci, virulence genes, and secondary metabolite biosynthetic gene clusters.

1. INTRODUCTION

Aspergillus and Penicillium are two of the most economically important genera of fungi. These genera belong to the Aspergillaceae, a family belonging to the order Eurotiales (class Eurotiomycetes,phylumAscomycota)(Houbraken & Samson, 2011). Species belonging to this family have diverse physiological properties. Some species grow at extremely low water activities due to high sugar or salt concentrations, while others can grow at low (psychrotolerant) or high temperatures (thermotolerant), low-acidity levels, and/or low oxygen levels. Aspergillaceae are predominantly saprobic and are commonly occurring in soil; however, some are known to have a positive or negative impact on Taxonomy of Aspergillus and Penicillium 201 human activities. Positive impacts include the use of these fungi in food fermentations. For example, Penicillium camemberti and Penicillium roqueforti are used in cheese production and Penicillium nalgiovense in the production of surface ripened sausages. In Asia, a larger variation of fungal fermented foods occurs and, for instance, Aspergillus oryzae and Aspergillus sojae are used in the production of miso, sake, and soy sauce, and black Aspergilli for the production of awamori liquors and Puerh tea (Mogensen, Varga, Thrane, & Frisvad, 2009; Perrone et al., 2011). Members of Aspergillaceae also produce various bioactive extrolites (¼secondary metabolites). Some of these extrolites are known as pharmaceuticals and examples are penicillin (antibiotic, Penicillium rubens), griseofulvin (antifungal, Penicillium griseofulvum), mycophenolic acid (immunosuppressant, Penicillium brevicompactum), and lovastatin (cholesterol- lowering agent, Aspergillus terreus). Other industrial applications include the production of organic acids and enzymes. Aspergilli, in particular, are known for their production of these compounds. Citric and gluconic acid are produced by Aspergillus niger, itaconic acid by A. terreus and especially members of Aspergillus section Nigri and A. oryzae arewidelyusedinextracellular enzyme production (either as donor or production organism). Less frequently exploited species for enzyme production include Aspergillus melleus, A. sojae, Talaromyces funiculosus (syn. Penicillium funiculosum), Penicillium multicolor, Rasamsonia emersonii (syn. Talaromyces emersonii), Thermoascus aurantiacus,and Thermomyces lanuginosus (van Dijck, 2008). Besides the positive interactions mentioned above, also negative aspects are linked to this family. Some species produce extrolites that can be regarded as mycotoxins and examples of regu- lated mycotoxins produced by Aspergillus and Penicillium species in food and/or feed are aflatoxins, patulin, ochratoxin, citrinin, and fumonisin (Samson, Houbraken, Thrane, Frisvad, & Andersen, 2010). In addition, Aspergillus species especially can cause a wide spectrum of diseases including mycotoxicosis, and noninvasive and invasive infections in immune- compromised patients. Aspergillus fumigatus is the principal etiological agent, but several other Aspergillus species are reported as causal agent of aspergillosis. Other adverse responses are hypersensitivity reactions (e.g., asthma, extrinsic allergic alveolitis) due to exposure to fungal fragments. Aspergillus and Penicillium are typical indoor fungi and are among the most frequently encountered genera in indoor environments (Flannigan, Samson, & Miller, 2011; Gravesen, Nielsen, Iversen, & Nielsen, 1999). These fungi produce high quantities of dry spores which can become airborne easily, resulting in exposure of humans to high spore concentrations in the air of indoor environments. 202 Jos Houbraken et al.

Naming and classifying our surroundings, especially living organisms, has likely been taking place as long as mankind has been able to communicate. The primary aim of taxonomy is to provide a classification that can be used for a wide range of purposes. It is traditionally divided into three fields: (1) classification, that is, the orderly arrangement of groups; (2) nomenclature, that is, the naming of the groups defined under 1, and (3) identification of unknown organisms, that is, the process of determining whether an organism belongs to one of the groups defined in 1, and labeled in 2 (Moore, Mihaylova, Vandamme, Krichevsky, & Dijkshoorn, 2010; Schleifer & Tru¨per, 2006). Taxonomy is a dynamic discipline but inadequate taxonomy or uncertain nomenclature can lead to tremendous confusion. The recently published International Code of Nomenclature for Algae, Fungi and Plants deleted dual nomenclature, giving anamorph names the same priority as teleomorph names, leading to several name changes (Norvell, 2011). The impact of these new nomenclatural rules on the taxonomy of biotechnologically important Aspergillus and Penicillium is addressed in this chapter and the latest develop- ments regarding the phylogeny and classification of species belonging to these genera are given. The importance of Aspergillus, Penicillium, and related genera is also illustrated by the high number of genome sequencing projects undertaken. Correct species identification and the use of valid names are the first crucial steps in these projects and in this paper we identify the currently genome-sequenced strains according to the latest taxonomic schemes.

2. ONE FUNGUS, ONE NAME 2.1. Dual nomenclature Pleomorphism in fungi was first demonstrated by Tulasne (1851) and shortly after de Bary (1854) demonstrated that Eurotium herbariorum had an Aspergil- lus anamorph, while Brefeld (1874) showed with illustrations the connection between Eupenicillium and Penicillium. In spite of the trend to apply the ana- morphic name to all of the Aspergillus and Penicillium species (Raper & Fennell, 1965; Raper & Thom, 1949; Thom & Church, 1926), the nomenclatural rules of that time forced mycologists to use the teleomorph name if a sexual state was present. Raper and Thom (1949) and Raper (1957) already noted that it is unnecessary and unjustified to recognize teleomorphs related to Aspergillus and Penicillium, since the anamorphs of these species produce similar structures as those of the strictly conidial species. Furthermore, they stated that taxonomy should be as simple as possible and taxonomy is not served with dual nomenclature. Despite their opposition, the dual Taxonomy of Aspergillus and Penicillium 203 nomenclature system gained popularity and became widely applied in Asper- gillus and Penicillium taxonomy.

2.2. Single-name nomenclature During the CBS-KNAW Fungal Diversity Centre-organized symposium “One fungus¼One name” held in Amsterdam in April 2011, ways to over- come dual nomenclature in pleomorphic fungi were discussed culminating in the “Amsterdam declaration” with recommendations on how to deal with such fungi in future (Braun, 2012; Hawksworth et al., 2011). Several proposals to emend the International Code of Botanical Nomenclature were adopted by the Melbourne Congress resulting in a new code named Inter- national Code of Nomenclature for Algae, Fungi and Plants. One of the major changes compared with the previous code is that the concept of dual nomenclature is deleted; giving anamorph names the same priority as teleomorph names (Norvell, 2011). At the generic level, anamorph-typified genus names have now equal priority as teleomorph-typified names and can be used as holomorph names, that is, for all morphs belonging to one fungus. If the anamorph genus represents the oldest valid and legitimate name, and it is the most widely used and preferred, then this name can be applied for all morphs (Braun, 2012; Norvell, 2011). In this respect, Aspergillus and Penicillium would have priority over their competing teleomorph-typified names like, for example, Eupenicillium, Neosartorya, Emericella, and Eurotium.

2.3. Implications for Aspergillus and Penicillium taxonomy On April 14, 2012, the International Commission on Penicillium and Aspergillus (ICPA) met in Utrecht, the Netherlands, and discussed the implications of the single-name nomenclature on Aspergillus and Penicillium taxonomy. Consensus for the use of the name Penicillium was established without any discussion. The use of the name Aspergillus was more contro- versial as various well-known teleomorph names are linked to this genus. One of the main concerns was that established genus names in food mycology, for example, Neosartorya and Eurotium, would disappear, causing confusion among the users in that field. These names are linked to certain physiological features and therefore have a meaning: Neosartorya ascospores are heat-resistant and Eurotium species are able to grow on low water activity products, making them important food spoilage organisms of, for example, bakery products and foodstuff with high sugar content. In contrast to the 204 Jos Houbraken et al. opinion of the users in the field of food mycology, users in Aspergillus genomics, biology, and genetics preferred the name Aspergillus. Prior to the ICPA meeting, a questionnaire was spread during Asperfest 9 held in Marburg, Germany on March 29–30, 2012, in order to get guidance from this field. Five options were given: (1) Continue with dual nomenclature system (although not allowed in the current code), (2) using the Aspergillus names for all Aspergilli (the names, e.g., Eurotium, Emericella, and Neosartorya will be lost), (3) split Aspergillus and rename all Aspergilli according to their mostly teleomorph name (the type of Aspergillus is A. glaucus and belongs to Eurotium, therefore the name Eurotium will be lost and many well-known Aspergilli (e.g., A. niger, A. flavus, A. oryzae) will be renamed), (4) similar as option 3, but select A. niger as type of genus; a part could maintain by their Aspergillus name, but other important species will be renamed, for example, Neosartorya fumigata, Emericella versicolor, (5) keep all Aspergillus names and additionally give optional names when necessary (Emericella-state of A. nidulans when dealing with expression of genes during sexual development). In total, 48 attendees filled in the questionnaire and no votes were given to option 1 and 2, 14.6% voted for option 3, 8.3% for option 4, and 77.1% for option 5. As mentioned above, no consensus among the ICPA members could be obtained during the meeting in Utrecht. The ques- tion which system to apply was brought to vote and three members voted for option 2, two for option 3 and five for option 5. In summary, the ICPA decided by vote that the genus name Aspergillus will be used for all Aspergillus species, including the teleomorph names, and therefore Aspergillus and Penicillium returns to the single named, but pleomorphic, nomenclatural, and taxonomical system as actively promoted by Thom (1930) and Raper and Thom (1949). The consequence of the single-name system is that teleomorph-based genera, such as Neosartorya, Emericella, Eurotium, and Petromyces, will be synonymized with Aspergillus. This system is being applied in the recent published Aspergillus taxonomies. Examples are Aspergillus waksmanii, A. felis, A. siamensis, A. caatingaensis, A. pernambucoensis, A. cibarius, and A. osmophilus. The first five species produce Neosartorya-type ascoma and ascospores and the latter two species produces an Eurotium-type sexual state, nevertheless, all species were classified in Aspergillus (Asgari, Zare, Zamanizadeh, & Rezaee, 2013; Barrs et al., 2013; Eamvijarn et al., 2013; Hong et al., 2012; Hubka et al., 2012; Matsuzawa et al., 2013). Furthermore, Hubka, Kolarik, Kubatova, and Peterson (2013) applied the single-name system for the revision of the genus Eurotium and all Eurotium species were transferred to their Aspergillus name. Taxonomy of Aspergillus and Penicillium 205

3. CLASSIFICATION AND PHYLOGENETIC RELATIONSHIPS IN TRICHOCOMACEAE, ASPERGILLACEAE, AND THERMOASCACEAE

Before the recently accepted Melbourne code, the name Tri- chocomaceae could only be applied to teleomorph genera, because this family was typified with the sexually reproducing genus Trichocoma (Malloch, 1985). However, from a practical point of view, also anamorph genera with phialidic structures, such as Aspergillus and Penicillium, were linked and treated in this family (Malloch & Cain, 1972). This relationship is also confirmed by phylogenetic studies showing that strict anamorphs are inter- mingled with teleomorph species (Berbee & Taylor, 1993; Berbee, Yoshimura, Sugiyama, & Taylor, 1995; Houbraken & Samson, 2011; LoBuglio, Pitt, & Taylor, 1993; Peterson, 2000). The relationships among species and genera of this family were recently studied using a four-gene phylogeny. This study showed the presence of three lineages among these investigated strains, and based on a polyphasic approach, these lineages were treated as distinct families named Aspergillaceae, Thermoascaceae, and Tri- chocomaceae (Fig. 4.1)(Houbraken & Samson, 2011). Besides their phylogenetic relationship, also differences in morphology and physiology could be observed among these three families. The Aspergillaceae includes species that produce asci inside cleistothecia, stromata, or are surrounded by Hu¨lle cells and mainly have oblate to ellipsoidal ascospores with a furrow or slit. The conidia are mostly formed on flask-shaped or cylindrical phialides. Most species belonging to the Aspergillaceae grow well on low-water-activity media such as Czapek yeast extract agar supplemented with 5% NaCl (CYAS) and many extrolites produced by species of this family are not produced in Trichocomaceae and Thermoascaceae. The Trichocomaceae are defined by having asci borne within a tuft or layer of loose hyphae and ascospores are lacking slits or furrows. The phialides of species belonging to this family are mostly lanceolate or cylindrical. In contrast to Aspergillaceae, species belonging to Trichocomaceae do not grow well on CYAS or other low water activity media. Two genera, Byssochlamys and Thermoascus, are currently classified in Thermoascaceae and both genera differ in their cleistothecia production. Thermoascus produces of firm, somewhat sclerotioid, pseudoparenchymatous cleistothecia, while Byssochlamys produces almost naked ascomata. Based on the relative branch length, Houbraken and Samson (2011) noted that both genera could represent separate families. However, there are characters 206 Jos Houbraken et al.

100 CBS 209.28 Penicillium adametzii 91 ATCC 20851 Penicillium bilaiae JGI 100 CBS 336.48 Penicillium herquei 97 CBS 125543 Penicillium glabrum 100 DAOM 239074 Penicillium glabrum JGI 84 CBS 347.59 Penicillium thomii

100 CBS 304.48 Penicillium charlesii Aspergillaceae 100 ATCC 48694 Penicillium fellutanum JGI 99 CBS 229.81 Penicillium fellutanum CBS 123361 Monascus eremophilus 100 CBS 251.56 Penicillium ramusculum 55 CBS 190.68 Penicillium ornatum 100 54 CBS 490.66 Penicillium cinnamopurpureum Aspergillaceae - CBS 341.68 Penicillium idahoense 70 100 CBS 489.66 Penicillium ochrosalmoneum CBS 247.56 Penicillium isariiforme 100 CBS 206.57 Penicillium taxi 75 CBS 334.68 Penicillium hennebertii 100 CBS 219.30 Penicillium oxalicum 114-2 “Pen. decumbens” Gb (= P. oxalicum) 74 CBS 340. 48 Penicillium janthinellum 100 CBS 341.48 Penicillium javanicum 100 68 CBS 372.48 Penicillium simplicissimum 74 CBS 315.67 Penicillium stolkiae CBS 599.73 Penicillium gracilentum CBS 203.84 Penicillium nepalense CBS 367.48 Penicillium restrictum 90 ATCC 26601 Penicillium paxilli Gb 100 CBS 290.48 Penicillium shaerii CBS 139.45 Penicillium citrinum CBS 185.65 Penicillium lagena 100 ex Postia Penicillium chrysogenum JGI 100 Wis 54-1255 P. chrysogenum JGI (= P. rubens)

100 - Aspergillaceae - Aspergillaceae - Aspergillaceae - CBS 306.48 Penicillium chrysogenum 65 CBS 352.48 Penicillium nalgiovense 100 100 CBS 221.30 Penicillium roqueforti 100 CBS 325.48 Penicillium expansum ATCC 24692 Penicillium expansum Gb 100 CBS 339.48 Penicillium italicum 80 CBS 112082 Penicillium digitatum PHI26 Penicillium digitatum Gb 100 100 CBS 527.65 Hemicarpenteles paradoxus 72 100 CBS 257.29 Penicillium brevicompactum 100 CBS 232.60 Penicillium olsonii 100 CBS 106.11 Penicillium lanosum 100 CBS 300.48 Penicillium canescens 100 ATCC 10419 JGI 56 Penicillium canescens CBS 241.56 Penicillium atrovenetum 99 CBS 231.61 Penicillium sacculum CBS 506.65 Aspergillus zonatus JGI 100 CBS 124.53 Sclerocleista ornata 99 CBS 105.25 Sclerocleista thaxteri CBS 430.64 Phialomyces macrosporus Aspergillaceae - Aspergillaceae - Aspergillaceae

Figure 4.1—Cont'd Taxonomy of Aspergillus and Penicillium 207

99 CBS 134.48 Aspergillus tubingensis JGI 82 CBS 106.47 Aspergillus luchuensis JGI (syn. A. acidus) 100 ATCC 1015 Aspergillus niger JGI 100 CBS 101740 JGI 100 Aspergillus brasiliensis ITEM 5010 Aspergillus carbonarius JGI ATCC 16872 Aspergillus aculeatus JGI 100 CBS 260.73 Aspergillus flavipedes

CBS 653.74 Aspergillus aureofulgens Aspergillaceae 98 CBS 118.45 Aspergillus janus NIH 2624 Aspergillus terreus 100 CBS 566.65 Aspergillus candidus CBS 463.65 Aspergillus arenarius 100 63 NBRC 4239 Aspergillus sojae Gb 100 CBS 100926 Aspergillus parasiticus Aspergillaceae - Aspergillaceae 100 NRRL 3557 Aspergillus flavus JGI (syn. Petromyces flavus) 100 RIB40 JGI 99 Aspergillus oryzae 100 CBS 553.77 Aspergillus coremiiformis CBS 151.66 Aspergillus leporis

54 CBS 109.46 Aspergillus avenaceus 100 CBS 108.08 Aspergillus ochraceus CBS 112812 Aspergillus steynii CBS 649.93 Aspergillus robustus 95 NRRL 1 Aspergillus clavatus 100 CBS 157.66 Dichotomomyces cejpii 100 100 Af293 Aspergillus fumigatus JGI NRRL 181 Aspergillus fischeri JGI (syn. Neosartorya fischeri) CBS 196.64 Aspergillus cervinus 100 CBS 593.65 Aspergillus sydowii JGI 55 CBS 795.97 Aspergillus versicolor JGI 92 FGSC A4 Asp. nidulans JGI (syn. Emericella nidulans) 75 CBS 121611 Aspergillus calidoustus 100 73 CBS 139.61 Aspergillus sparsus

100 CBS 468.65 Aspergillus biplanus 57 DTO 134-E9 Aspergillus wentii JGI 100 CBS 516.65 Aspergillus glaucus JGI (syn. Eurotium herbariorum) 100 NRRL 117 Aspergillus glaucus 100 ex Death Sea Aspergillus ruber JGI (syn. Eurotium rubrum) 68 CBS 518.65 Aspergillus montevidensis 100 CBS 117.33 Aspergillus restrictus DTO 011-C3 Aspergillus penicillioides 91 CBS 127.61 Aspergillus brunneo-uniseriatus CBS 578.65 Aspergillus pulvinus 100 CBS 128032 Phialosimplex canicus 100 CBS 109945 Phialosimplex chlamydosporus 100 CBS 380.74 Basipetospora halophilica 100 CBS 366.77 Phialosimplex sclerotialis 88 CBS 384.61 Polypaecilum insolitum 100 NRRL 1597 Monascus ruber JGI 100 CBS 109.07 Monascus purpureus 68 CBS 109402 Monascus arg 100 100 CBS 132.31 Chrysosporium inops CBS 236.71 Xeromyces bisporus CBS 607.74 Leiothecium ellipsoideum

81 Aspergillaceae - Aspergillaceae - Aspergillaceae - Aspergillaceae - Aspergillaceae - Aspergillaceae - 94 CBS 295.48 Hamigera avellanea CBS 377.48 Hamigera striata

Figure 4.1—Cont'd 208 Jos Houbraken et al.

100 CBS 181.67 Thermoascus crustaceus 97 CBS 528.71 Thermoascus thermophilus 100 CBS 605.74 Byssochlamys verrucosa 100 Thermoascus aurantiacus JGI CBS 891.70 Thermoascus aurantiacus 75 100 CBS 100.11 Byssochlamys nivea

CBS 101075 Byssochlamys spectabilis Thermoascaceae 99 CBS 322.48 Talaromyces duclauxii 93 ATCC 18224 Talaromyces marneffei JGI 100 CBS 310.38 Talaromyces flavus 100 CBS 272.86 Talaromyces funiculosus ATCC 10500 Talaromyces stipitatus JGI 100 100 CBS 373.48 Talaromyces trachyspermus CBS 642.68 Talaromyces mineoluteus

100 CBS 660.80 Talaromyces dendriticus 100 CBS 338.48 Talaromyces islandicus 100 CBS 643.80 Talaromyces loliensis 91 CBS 391.48 Talaromyces wortmanii 100 CBS 100536 Talaromyces emodensis 78 CBS 296.48 Talaromyces bacillisporus 100 CBS 236.58 Thermomyces dupontii 100 NRRL 2155 Tal. thermophilus GZ (= Thermomyces dupontii ) 100 65 100 Strain SSBP Thermomyces lanuginosus Gb CBS 218.34 Thermomyces lanuginosus CBS 348.51 Talaromyces luteus 100 CBS 399.69 Sagenomella diversispora 100 100 CBS 429.67 Sagenomella striatispora CBS 427.67 Sagenomella humicola 99 CBS 413.71 Rasamsonia byssochlamydoides 100 CBS 393.64 Rasamsonia emersonii 97 CBS 101.69 Rasamsonia argillacea 100 CBS 247.57 Trichocoma paradoxa Trichocomaceae - Trichocomaceae - Trichocomaceae CBS 103.73 Trichocoma paradoxa Strain RS Coccidioides immitis Gb 0.1 Figure 4.1 Best-scoring Maximum Likelihood tree using RAxML based on a combined data set of partial Cct8, Tsr1, RPB1, and RPB2 gene sequences showing the relationship among members of the Aspergillaceae, Trichocomaceae, and Thermoascaceae. Genome-sequenced strains are shown in bold font. The bootstrap percentages are presented at the nodes; values less than 70% are not shown. The tree is rooted with Coccidioides immitis. Partial Cct8, Tsr1, RPB1, and RPB2 gene sequences from genome projects were downloaded from the various databases and the origin is given after the species name (JGI¼Doe Joint Genome Institute; Gb¼NCBI Genome database; Gz¼Genomymes). shared by Thermoascus and Byssochlamys including the production of asci in croziers and the formation of smooth or finely roughened ascospores lacking a furrow or slit. Furthermore, most members of both genera are thermotolerant or thermophilic. Taxonomy of Aspergillus and Penicillium 209

4. TAXONOMY OF PENICILLIUM SPECIES AND PHENOTYPICALLY SIMILAR GENERA

The name Penicillium is derived from penicillus, which means “little brush” and was introduced by Link in 1809 (Link, 1809). Various genera have Penicillium-like conidiophores including Hamigera, Paecilomyces, Rasamsonia, Sagenomella, Talaromyces, and Trichocoma. Many of these genera are redefined due to new insights (mainly based on molecular data) and the introduction of the single-name nomenclature. For example, 18S rDNA sequences demonstrated that Paecilomyces sensu Samson (1974) is polyphyletic across two subclasses, Sordariomycetidae and Eurotiomycetidae (Luangsa- ard, Hywel-Jones, & Samson, 2004). As a consequence, Paecilomyces was redefined and the thermophilic and thermotolerant species phylogenetically related to Paec. variotii are considered as “true” Paecilomyces (Samson, Houbraken, Varga, & Frisvad, 2009). An overview of characters used for the differentiation between the biotechnologically important genera Talaromyces, Penicillium, and Rasamsonia is given in Table 4.1. This table is expanded with the phenotypically similar genera Hamigera, Trichocoma, and Sagenomella. A selection of these genera is also illustrated in Fig. 4.2. Thermomyces is also included in Table 4.1 because molecular data show that Talaromyces thermophilus actually belongs to Thermomyces. This new combination will be proposed below (Section 4.3).

4.1. Penicillium and Talaromyces Due to its economical significance, Penicillium attracted much attention and this is marked by the pioneering taxonomic studies of Dierckx (1901), Thom (1910), Westling (1911), Biourge (1923), Thom and Church (1926), and Zaleski (1927). The classification of Penicillium was the subject in various monographs (Pitt, 1980; Ramı´rez, 1982; Raper & Thom, 1949; Thom, 1930) and although the most recent monograph is published more than 30 years ago, these monographs still form the basis of many studies on Penicillium nowadays. After Pitt (1980), various new approaches, such as iso- enzyme patterns, ubiquinone systems, extrolite profiles and DNA-based techniques (sequencing, RAPD, AFLP) were used to clarify the taxonomy of Penicillium. In the last decade, combinations of these techniques were used for species delimitation (polyphasic taxonomy). Table 4.1 Overview of characters used in the differentiation between Penicillium and other genera with Penicillium-like anamorphs Genus Thermophilicity Branching pattern Phialide Color conidia Character ascomata Paecilomyces Mesophilic and Irregular Broad base with long (Olive-)brown Without distinct wall, thermotolerant gradually tapering neck born from crosiers, maturing quickly Rasamsonia Thermotolerant Regular, mostly bi- Cylindrical and (Olive-)brown Wall scanty, consisting and and terverticillate gradually tapering of a inconspicuous thermophilic toward the apices network of hyphae, maturing quickly Sagenomella Mesophilic Undifferentiated, Lanceolate, tapering White, gray, greenish, Wall of scanty layer of phialides solitary, apically, often swollen or brown interwoven hyphae occasionally in whorls at base or in center Talaromyces Mesophilic Regular, symmetrically Lanceolate Green, darker green as Walls with multiple biverticillate in Penicillium, often layers of interwoven with yellow pigmented hyphae, soft, maturation hyphae quickly Trichocoma Mesophilic Regular, mostly bi- Cylindrical and (Olive-)brown Wall consisting of and terverticillate gradually tapering hyphal masses or tufts, toward the apices up to 10–20 mm in length Penicillium Mesophilic Regular Flask-shaped or Green Sclerotium-like, rigid cylindrical wall of thick-walled, isodiametric cells, maturation generally slowly Hamigera Mesophilic Regular Flask-shaped or (Olive-)brown Covering consisting of a cylindrical, irregularly loosely interwoven formed, both terminally network of hyphae, and subterminally maturation quickly Thermomyces Thermophilic If present, mono- to Lanceolate Green, chlamydospore- Wall biverticillate, like conidia dark brown pseudoparenchymatous chlamydospore-like conidia on solitary phialides in Th. lanuginosus 212 Jos Houbraken et al.

Figure 4.2 Conidiophores of Penicillium and phenotypically similar genera. (A) Penicillium glabrum (monoverticillate). (B) Penicillium brevicompactum (terverticillate). (C) Talaromyces purpurogenus.(D)Paecilomyces variotii.(E)Hamigera avellanea.(F)Rasamsonia aegroticola. Scale bars¼10 mm. Taxonomy of Aspergillus and Penicillium 213

4.1.1 Generic classification Various studies on the phylogeny of Penicillium showed that this genus is polyphyletic and most of the species could be divided into two major clades (Berbee et al., 1995; Houbraken & Samson, 2011; LoBuglio & Taylor, 1993; LoBuglio et al., 1993; Ogawa & Sugiyama, 2000). Based on these data, Houbraken and Samson (2011) defined one clade as Penicillium sensu stricto and this clade includes Eupenicillium species and the majority of species previously assigned to Penicillium subgenus Aspergilloides, Furcatum and Penicil- lium in the classification system of Pitt (1980). The other main clade is centered on Talaromyces and includes Talaromyces and Penicillium species previously belonging to subgenus Biverticillium (Fig. 4.1). The split of Penicillium in two genera is also reflected in morphology, physiology, and extrolite production. Penicillium s.s. is most closely related to Aspergillus and these two genera share many more features with each other than they do with Talaromyces (see Chapter 3).

4.1.2 Infrageneric classification Dierckx (1901) proposed the first infrageneric classification of Penicillium and introduced the subgenera Aspergilloides, Biverticillium, and Eupenicillium. After the introduction of these subgenera, various other schemes with subgenera, sections, and series were proposed and an overview is given in Houbraken and Samson (2011). The system that gained much popularity was introduced by Pitt (1980) and included four subgenera, 10 sections, and 21 series. This division was based on a combination of phenotypic characters and physiology. This classification was challenged with molecular data and based on a four-gene phylogeny, Houbraken and Samson (2011) showed that Penicil- lium could be divided into two subgenera (Penicillium and Aspergilloides) and 25 sections. Unfortunately, characters frequently used in subgeneric and sectional classification systems, such as the branching of the Penicillium conidiophore and growth rates on agar media (Pitt, 1980; Ramı´rez, 1982; Raper & Thom, 1949; Stolk & Samson, 1985), did not correspond well with the phylogeny. Currently, it is not possible to recognize all sections without employing DNA sequence data. Ideally, a system should be formulated including phenotypic characters. The proposed classification system will serve as a starting point to investigate useful phenotypic characters for classification. Based on the structure of the conidial state, Stolk and Samson (1972) divided Talaromyces in four sections: Talaromyces, Emersonii, Thermophila, and Purpurea. Pitt (1980) followed this classification and subdivided 214 Jos Houbraken et al. section Talaromyces and introduced the series Flavi, Lutei, and Trachyspermi. This phenotype-based classification does not correlate with the phyloge- netics relationships within Talaromyces (Houbraken & Samson, 2011; Samson et al., 2011). In addition, the types of section Emersonii (T. emersonii¼Rasamsonia emersonii; Houbraken, Frisvad, & Samson, 2011a, 2011b) and section Thermophila (T. thermophilus¼Thermomyces dupontii; this study) do not belong to Talaromyces. Talaromyces is currently the subject of a taxonomic study and a new subgeneric and sectional classification scheme will be proposed in future.

4.1.3 Recent name changes of biotechnologically important Penicillium species Penicillium species are commonly occurring and have given us penicillin, mycophenolic acid, compactin, fungal steroid transformations, white and blue cheeses, fermented salamis, pigments, and extracellular enzymes. Most of the well-known Penicillium species belong to the newly defined genus and are known under their Penicillium name. These changes in taxonomy due to the new nomenclatural rules therefore do not have a large impact in the field of biotechnology and applied research. However, this is not the case for Pen- icillium species that belong to the redefined Talaromyces. For example, Pen- icillium purpurogenum and P. funiculosum belong to the redefined genus Talaromyces and are named T. purpurogenus and T. funiculosus, respectively (Samson et al., 2011). Both species are important species in biotechnology for their ability to produce extracellular enzymes (e.g., xylanases and cellu- lases) and pigments, which are used as natural colorants (Belancic, Scarpa, Peirano, & Diaz, 1995; Jeya et al., 2010; Mapari, Meyer, Thrane, & Frisvad, 2009; Steiner, Socha, & Eyzaguirre, 1994; Zou et al., 2012). Recent revision of the taxonomy of P. purpurogenum showed that this species is a complex consisting of four taxa: T. purpurogenus, T. ruber (syn. P. rubrum), T. amestolkiae, and T. stollii. From a biotechnological point of view, it is rec- ommended to use T. ruber for enzyme production, because T. purpurogenus produces four types of mycotoxins and T. amestolkiae and T. stollii are poten- tially pathogenic to immuno-compromised persons (Yilmaz et al., 2012). Another potential pigment producer is the recently described species Talaromyces atroroseus, a species forming azaphilone biosynthetic families mitorubrins and Monascus pigments without any production of mycotoxins (Frisvad et al., submitted for publication). The most well-known example of incorrect identification of a Penicillium strain is that of Fleming’s penicillin producing strain. In 1929, Fleming Taxonomy of Aspergillus and Penicillium 215 reported his penicillin producing strain as P. rubrum (Fleming, 1929). Later, Thom (1945) reidentified this strain as P. notatum, Samson, Hadlok, and Stolk (1977) as P. chrysogenum and Pitt (1980) as P. griseoroseum. Recent studies on P. chrysogenum and related species using extrolites, micro- satellites and multigene sequence data show that this species is actually a complex consisting of five species, P. chrysogenum, P. rubens, P. vanluykii, P. tardochrysogenum, and P. allii-sativi. Application of this new taxonomic scheme shows that Fleming’s strain and the classic strain used for the production of penicillin (the Wisconsin strain) are actually P. rubens (Henk et al., 2011; Houbraken et al., 2011b; Houbraken, Frisvad, et al., 2012). Another example of a wrong connection between species and drug production is compactin, an anticholesterolemic agent. Originally, the production strain was identified as Penicillium brevicompactum (Brown, Smale, King, Hasenkamp, & Thompson, 1976), and later compactin producers were reported as, for example, P. citrinum, P. cyclopium, and P. aurantiogriseum (Doss et al., 1986; Endo, Kuroda, & Tsujita, 1976; Wagschal, Yoshizawa, Witter, Liu, & Vederas, 1996). These reports were based on incorrect identifications and although the correct name for these producers was shown to be P. solitum (Frisvad & Filtenborg, 1989), the incorrect name P. citrinum persists in literature (Barrios-Gonza´lez & Miranda, 2010; Xing, Deng, & Hu, 2010). Penicillium album, P. candidum, and P. glaucum are three names commonly encountered in (popular) articles on cheese manufacturing. However, these names are not valid, and the former two are synonyms of P. camemberti, while P. glaucum is incorrectly used for P. roqueforti.

4.2. Rasamsonia Based on phenotypic, physiological, and molecular data, the genus Rasamsonia was introduced to accommodate the species Geosmithia argillacea, Talaromyces emersonii, Talaromyces byssochlamydoides. Members of this genus form Penicillium-like conidiophores but differ from Penicillium by the formation of cylindrical phialides usually gradually tapering toward the apices, distinctly rough walled stipes and metulae, olive-brown conidia and ascomata, if present, with a scanty covering (Fig. 4.2). Furthermore, Rasamsonia species are thermotolerant or thermophilic, while Penicillia are generally mesophiles (Table 4.1). Currently, Rasamsonia consists of nine species: R. aegroticola, R. argillacea (syn. Geosmithia argillacea), R. brevistipitata, R. byssochlamydoides (syn. Talaromyces byssochlamydoides), R. cylindrospora, R. eburnea (syn. Talaromyces eburneus, Geosmithia eburnea), R. emersonii (syn. 216 Jos Houbraken et al.

Talaromyces emersonii), R. composticola, and R. piperina. Among those species, Rasamsonia emersonii is commercially used for the production of thermostable enzymes. Rasamsonia byssochlamydoides NRRL 3658 (as Paecilomyces byssochlamydoides) and R. emersonii NRRL 3221 (as Talaromyces emersonii) are being genome-sequenced in the Genozymes project (www. fungalgenomics.ca). Besides the biotechnological applications, Rasamsonia species can also cause invasive mycosis in patients with chronic granulomatous disease and cause spoilage in heat-treated foods (De Ravin et al., 2011; Machouart et al., 2011). Until now, no R. emersonii isolates are associated with human infections. Cimon et al. (1999) isolated R. emersonii (as Penicil- lium emersonii) from respiratory secretions of a CF patient, however, this identification was wrong and this strain is reidentified as R. aegroticola (Houbraken et al., 2013).

4.3. Thermomyces First studies on Thermomyces date back to 1899 when P. Tsiklinsky reported on a thermophilic hyphomycete incidentally encountered on a potato inoc- ulated with garden soil. This fungus was grown on bread kept at 52–53 C and its thermophilic nature was assessed (Mouchacca, 1997; Tsiklinsky, 1899). Currently, four species are accommodated in Thermomyces: Th. lanuginosus, Th. ibadanensis, Th. stellatus, and Th. verrucosus. Our phylogenetical studies showed that these species belong to different families. Thermomyces lanuginosus and Th. ibadanensis belong to the Trichocomaceae (Eurotiomycetes, Eurotiomycetidae, Eurotiales), Th. stellatus to the Microascaceae (Sordariomycetes; Hypocreomycetidae; Microascales), and Th. verrucosus to the Chaetomiaceae (Sordariomycetes; Sordariomycetidae; Sordariales)(Fig. 4.3). The paraphyletic nature of this genus is also reflected by the thermophilicity of the species: Th. lanuginosus and Th. ibadanensis are true thermophiles, Th. stellatus is thermotolerant and Th. verrucosus is mesophilic (Morgenstern et al., 2012; Mouchacca, 1997). Recently, Houbraken and Samson (2011) showed that Th. lanuginosus is phylogenetically closely related to Tal. thermophilus. Both species share the ability to grow at high temperatures, but seem phenotypically unrelated. Talaromyces thermophilus has a Penicillium-type anamorph and produces a sexual state while Th. lanuginosus reproduces only asexually by thick-walled, brown conidia. Interestingly, T. thermophilus also produces thick-walled chlamydospores (single and in short chains) on phialide-shaped structures, similar to those observed in Th. lanuginosus (Pitt, 1980; Stolk, 1965). Because these two species are phylogenetically closely related, it is CBS 153.75 Thermomyces lanuginosus

CBS 152.75 Thermomyces lanuginosus

CBS 224.63 Thermomyces lanuginosus

CBS 281.67 Thermomyces lanuginosus (type of Th. ibandensis)

CBS 218.34 Thermomyces lanuginosus

CBS 395.62 Thermomyces lanuginosus

CBS 632.91NT Thermomyces lanuginosus

ATCC 200065 Thermomyces lanuginosus JF412006 (FGS) 94 CBS 288.54 Thermomyces lanuginosus Trichocomaceae

CBS 630.91 Thermomyces lanuginosus

NT CBS 236.58 = NRRL 2155 Thermomyces thermophilus (FGS)

CBS 110455 Thermomyces thermophilus 84 CBS 161.71 Thermomyces thermophilus

CBS 393.64 Rasamsonia emersonii JF417478

NRRL 2098 Talaromyces flavus EU021596

CBS 103.73 Trichocoma paradoxa JN899399

UAMH 929 Sagenomella diversispora GQ169318

CBS 130296 Yunnania penicillata JN831359

CBS 398.54 Scopulariopsis brevicaulis

CBS 241.64 Thermomyces stellatus (FGS) Microascaceae

CBS 272.61T Thermomyces stellatus

CBS 218.31T Microascus trigonosporus

CBS 113533 Thermomyces verrucosus

T CBS 116.64 Thermomyces verrucosus Chaetomiaceae C96 Chaetomium murorum HM365268

CBS 137.58 Chaetomium angustispirale JN209862

CBS 377 Saccharomyces uvarum EU145770 0.1 Figure 4.3 Best-scoring Maximum Likelihood tree using MEGA5 based on ITS sequences, showing the relationships among species belonging to Thermomyces. Strains that are (in the process of being) genome-sequenced are presented in bold font. Numbers at the nodes are bootstrap values; values less than 70% are not shown and branches with >95% support are thickened. The phylogram is rooted with Saccharomy- ces uvarum CBS 377. 218 Jos Houbraken et al. likely that these structures are derived from a common ancestor. This would imply that Th. lanuginosus lost its Penicillium-like and sexual state during evolution or that other cultivation conditions are needed in order to produce these states. In addition, both species also share the ability to grow at high temperatures. Figure 4.3 shows a phylogram based on ITS sequences of the four Ther- momyces species, Tal. thermophilus and other related species. These data con- firm that Tal. thermophilus is phylogenetically closely related to the type species of Thermomyces, Th. lanuginosus, and not to the type of Talaromyces, T. flavus. Furthermore, the ex-type strain of Th. ibadanensis CBS 281.67T (¼ATCC 22716¼IHEM 3336¼IMI 096473) shares ITS sequences with Th. lanuginosus and this species is placed in synonymy with Th. lanuginosus. The taxonomical position of Th. stellatus and Th. verrucosus will be the subject of a future study. Based on these data and Houbraken and Samson (2011), we here com- bine Tal. thermophilus as Th. dupontii in Thermomyces. Thermomyces dupontii (Griffon and Maublanc) Houbraken and Samson, comb. nov., Mycobank MB 805186. Basionym:Penicilliun dupontii Griffon and Maublanc, Bull. Trimmest. Soc. Mycol. Fr. 27: 73. 1911. Synonyms: Talaromyces dupontii (Griffon and Maublanc) Apinis, Nova Hedwigia 5: 72. 1963. (nom inval., art. 36). Talaromyces dupontii (Griffon and Maublanc) Cooney and Emerson, Thermophilic Fungi: 38. 1964. (nom inval., art. 36). Talaromyces dupontii (Griffon and Maublanc) Emerson apud Fergus, Mycologia 56: 277. 1964. (nom inval., art. 33 and 36). Talaromyces thermophilus Stolk, Antonie van Leeuwenhoek 31: 268. 1965. With the exclusion of Talaromyces emersonii, T. byssochlamydoides, and T. thermophilus from Talaromyces, no true thermophiles are currently accommodated in Talaromyces. Talaromyces leycettanus is the only thermotolerant to thermophilic species in Talaromyces, but this species is phylogenetically unrelated to Talaromyces and belongs to the Aspergillaceae. Consequently, T. leycettanus will be accommodated in another genus in future. These taxonomical changes have impact on various applied fields. Thermomyces lanuginosus and Th. dupontii gain much attention because of their thermophilic nature and these species are commercially used for the production of various enzymes. Furthermore, two strains of Th. lanuginosus (strain SSBP and ATCC 20065) and one strain of Th. dupontii (as Tal. thermophilus; Taxonomy of Aspergillus and Penicillium 219 strain NRRL 2155) are (in the progress of being) genome-sequenced (McHunu et al., 2013; Morgenstern et al., 2012). Also Thermomyces stellatus CBS 241.64 is in the process of being genome-sequenced, but as mentioned above, this species belongs to the Microascaceae. Candidates for future genome comparisons with this strain are, for example, Microascus trigonosporus CBS 218.31 and Scopulariopsis brevicaulis LF580.

5. TAXONOMY OF ASPERGILLUS SPECIES 5.1. Phylogenetic relationships among Aspergillus species The classification of Aspergillus is traditionally based on morphological characters. The defining characteristic of Aspergillus is the aspergillum-like spore- bearing structure. The size and arrangement of the conidial heads, the color of the conidia, growth rate on agar media, and physiological characteristics (temperature, water activity) are important features for identification of Aspergilli. For example, species belonging to Aspergillus section Nigri generally grow fast on agar media and produce black-colored conidia, while those of section Candidi grow more restricted and form white-colored colonies. Based on these characteristics, Raper and Fennell (1965) divided Aspergillus in 18 groups. More recently, Peterson (2008), Peterson, Varga, Frisvad, and Samson (2008), and Houbraken and Samson (2011) studied the relationship among Aspergilli using a multigene phylogeny. These studies show that the phenotype-based groups of Raper and Fennell (1965) largely correspond with the classifications nowadays. Currently, 4 subgenera and 19 sections are accepted in Aspergillus (Table 4.2). These sections form a single mono- phyletic clade, however, statistical support was low (1.00 posterior probabil- ity; 55% bootstrap support) in the study of Houbraken and Samson (2011). Furthermore, the subgenera Ornati and Warcupi do not belong to Aspergillus and should be transferred to other genera.

5.2. Aspergillus section Nigri Among the Aspergilli, species belonging to section Nigri (black Aspergilli) and section Flavi (A. oryzae, A. sojae) are frequently used in biotechnology for the production of (extracellular) enzymes, organic acids (citric acid, kojic acid) and applied in food fermentations such as miso, soy sauce, awamori liquors, and Puerth tea. The taxonomy of section Nigri and the classification of strains belonging to this section have been studied various times since the introduction of molecular techniques and currently 26 species are accepted 220 Jos Houbraken et al.

Table 4.2 Subgeneric and sectional classification of Aspergillus based on the studies of Peterson (2008), Peterson et al. (2008), Varga, Frisvad, and Samson (2010), Houbraken and Samson (2011), and current review Subgenus Section Aspergillus Aspergillus (Eurotium) Restricti (Eurotium) Circumdati Candidi Circumdati (Neopetromyces) Flavi (Petromyces) Flavipedes (Fennellia) Nigri Terrei Fumigati Cervini Clavati (Neocarpenteles, Dichotomomyces) Fumigati (Neosartorya) Nidulantes Aeni (Emericella) Bispori Cremeia (Chaetosartorya) Nidulantes (Emericella) Ochraceorosei Silvati Sparsi Usti (Emericella) aSection Cremei is placed in subgenus Aspergillus by Houbraken and Samson (2011) and was resolved in subgenus Circumdati in the study of Peterson (2008). Our phylogenetic analysis (Figs. 4.1 and 4.7) shows with moderate bootstrap support that A. wentii, a member of this section, belongs to subgenus Nidulantes. The teleomorph forms that are associated with each section are mentioned between brackets. in this section (Fig. 4.4; Hong et al., 2013; Jurjevic´ et al., 2012; Varga, Frisvad, Kocsube´, et al., 2011) Aspergilli known as black- and white-koji molds that are used for food and beverage fermentations (e.g., awamori, shochu, makgeolli) are reported in the literature as A. luchuensis, A. awamori, A. kawachii, and A. acidus. The taxonomic position of these species was investigated and A. acidus and A. kawachii were placed in synonymy Taxonomy of Aspergillus and Penicillium 221

Aspergillus luchuensis RIB 2604 (FGS as A. awamori) Aspergillus luchuensis CBS 106.47 (FGS as A. acidus) Aspergillus luchuensis NBRC 4308 (FGS as “A. kawachii”) 83 Aspergillus luchuensis CBS 564.65 (type of A. acidus) Aspergillus luchuensis RIB 2642T Aspergillus piperis CBS 112811T 88 Aspergillus eucalypticola CBS 122712T Aspergillus costaricaensis CBS 115574T 99 Aspergillus tubingensis CBS 134.48T (FGS) Aspergillus tubingensis CBS 558.65 (type of A. pulverulentus) Aspergillus tubingensis CBS 136.52 (type of A. saitoi) Aspergillus neoniger CBS 115657T Aspergillus vadensis CBS 113365T Aspergillus niger CBS 513.88 (FGS) 100 Aspergillus niger ATCC 1015 (FGS) Aspergillus niger CBS 121.28 (type of A. foetidus) Aspergillus niger 554.65T Aspergillus niger RIB 2602 (type of A. usamii) 100 Aspergillus niger IHEM 5622 (type of A. citricus) epiT 78 Aspergillus welwitschiae CBS 139.54 Aspergillus welwitschiae CBS 557.65 (neotype of A. awamori sensu Perrone) 80 Aspergillus welwitschiae IHEM 3710 (representative of A. ficuum, IMI 91881) Aspergillus brasiliensis CBS 101740T (FGS) 100 Aspergillus carbonarius CBS 111.26T 71 70 Aspergillus carbonarius ITEM 5010 (FGS) 99 Aspergillus sclerotioniger CBS 115572T 74 Aspergillus ibericus CBS 121593 Aspergillus sclerocarbonarius CBS 121057T Aspergillus ellipticus CBS 707.79 Aspergillus heteromorphus CBS 117.55 Aspergillus aculeatinus CBS 121060T Aspergillus trinidadensis ITEM 14821 90 Aspergillus brunneoviolaceus CBS 621.78 Aspergillus brunneoviolaceus CBS 313.89 (type of A. fijensis) 72 Aspergillus floridensis ITEM 14783 Aspergillus sp.CBS 620.78 ATCC 16872T (FGS) 95 Aspergillus aculeatus 96 Aspergillus japonicus CBS 114.51 88 Aspergillus japonicus CBS 123.27 (as “A. violaceofuscus”) 100 Aspergillus indologenus CBS 114.80 100 Aspergillus uvarum CBS 121591 Aspergillus saccharolyticus CBS 127449 Aspergillus homomorphus CBS 101889 Aspergillus clavatus NRRL 1 0.1 Figure 4.4 Best-scoringMaximum Likelihood tree usingMEGA5 based onpartial b-tubulin sequences,showingthe relationships among speciesbelonging to Aspergillussection Nigri. (Continued) 222 Jos Houbraken et al. with A. luchuensis based on priority (Hong et al., 2013). There is confusion around the identity of A. awamori. No living-type material of A. awamori exists and this species was neotypified with CBS 557.65 (¼NRRL 4948) (Al-Musallam, 1980). The name A. awamori implies that this species is associated with black koji fermentations and awamori production, however, the neotype strain does not originate from awamori koji. Based on this neotype, Perrone et al. (2011) reestablished A. awamori as a phylospecies in Aspergillus section Nigri and in addition demonstrated that the A. awamori strains used in the Japanese koji fermentation do not belong to this phylospecies. Hong et al. (2013) showed that the neotypification of A. awamori is incorrect and this species is probably a synonym of A. niger or A. luchuensis, two species commonly found in awamori liquors (Yamada et al., 2011). Figure 4.4 shows that the epitype of A. welwitschiae resides in the clade with CBS 557.65, the neotype of A. awamori sensu Perrone, and IMI 91881 (¼CBS 555.65¼NRRL 364), a culture received by C. Thom from J. Westerdijk as A. ficuum. Although some reported IMI 91881 as the type of A. ficuum, we could not verify this and consider this species doubtful (Frisvad et al., 1990; Kozakiewicz et al., 1992). Other species names of black Aspergilli still used in today’s literature include A. citricus, A. fijensis, A. foetidus, A. phoenicis, A. pulverulentus, A. saitoi, A. usamii, and A. violaceofuscus (e.g., Jurjevic´ et al., 2012; Kozlakidis et al., 2013; Kumar, Kumar, & Reddy, 2012; Riul, Gonc¸alves, Jorge, & Guimara˜es, 2013; Zhang, Wu, Li, Gao, & Yang, 2012). These names are not in use and an overview of the correct names is given in Table 4.3. However, it needs to be noted that the correct names listed in Table 4.3 are based on the identity of the ex-type or representative strains. Isolates with old incorrect names should not be automatically transferred to the correct corresponding name listed in Table 4.3 and to ensure correct identification a detailed molecular analysis is needed. Aspergillus niger is one of the most important industrial filamentous fungal species used in biotechnology. This species is considered to be nontoxic under industrial conditions and is therefore regarded as a safe production

Figure 4.4—Cont'd The genome-sequenced strains are presented in bold font and in between brackets are incorrect or invalid names that are reported in recent literature (A. acidus, A. awamori, A. citricus, A. ficuum, A. fijensis, A. foetidus, A. kawachii, A. pulverulentus, A. saitoi, A. usamii, A. violaceofuscus). Numbers at the nodes are bootstrap values; values less than 70% are not shown. The phylogram is rooted with Asper- gillus clavatus NRRL 1. Taxonomy of Aspergillus and Penicillium 223

Table 4.3 Overview of invalid black Aspergilli in recent biotechnological literature and their current taxonomic status Species name Correct classification and remarks A. acidus A. luchuensis (Hong et al., 2013) A. awamori sensu No type material is saved of this species and strains Nakazawa originating from awamori liquor are either A. niger or A. luchuensis (Yamada et al., 2011) A. awamori sensu (Perrone A. welwitschiae (Hong et al., 2013) et al. 2011) A. citricus A. niger (Frisvad et al., 2011; Fig. 4.4) A. ficuum A. welwitschiae (Fig. 4.4) A. fijensis A. brunneoviolaceus A. foetidus A. niger (Varga, Frisvad, Kocsube´, et al., 2011) A. foetidus var. acidus A. luchuensis (Hong et al., 2013) A. kawachii nom. inval. A. luchuensis (Hong et al., 2013) A. phoenicis The type of A. phoenicis (from dates from Istanbul) is located in the Corda herbarium (PRM) and representative strains of this species (NRRL 363, NRRL 365, NRRL 1956) do either belong to A. niger and A. tubingensis. This name is rejected over the conserved species A. niger (Frisvad et al., 1990; Kozakiewicz et al., 1992) A. pulverulentus A. tubingensis (Fig. 4.4) A. saitoi nom. inval. A. tubingensis (Fig. 4.4) A. usamii A. niger (Yamada et al., 2011) A. violaceofuscus Gasperini (1887) described A. violaceofuscus as a biseriate Aspergillus with very short phialides. Several authors considered it as being uniseriate. No type material is available and the exact identity of this species remains unsolved. Based on the neotype of A. violaceofuscus (CBS 123.27NT), Varga, Frisvad, Kocsube´, et al. (2011) reestablished this species and Hubka and Kolarik (2012) subsequently treated A. japonicus as a synonym of A. violaceofuscus. Because no type material is present and confusion exist around the seriation of the species, A. violaceofuscus is considered here as a doubtful species 224 Jos Houbraken et al. organism (Schuster, Dunn-Coleman, Frisvad, & van Dijck, 2002). How- ever, Frisvad et al. (2011) showed that some of the industrially used A. niger strains can produce ochratoxin A and fumonisin at conditions mim- icking industrial citric acid production conditions. Careful analyses of production processes involving A. niger are needed to ensure absence of these mycotoxins. Other black Aspergilli used in food fermentation, citric acid and enzyme production such as A. aculeatus, A. brasiliensis, A. japonicus, A. luchuensis, and A. tubingensis do not produce ochratoxin A and fumonisins (Frisvad et al., 2011) and might be better candidates for biotechnological use than A. niger.

5.3. Aspergillus section Flavi Aspergillus section Flavi currently includes 27 species and taxa belonging to this section are characterized by the production of uni- or biseriate conidial heads, conidia in shades of yellow-green to brown and dark-colored sclerotia (Gonc¸alves et al., 2012; Soares, Rodrigues, Peterson, Lima, & Venaˆncio, 2012; Taniwaki et al., 2012; Varga, Frisvad, & Samson, 2011). Members of this section, such as A. flavus and A. parasiticus are important producers of aflatoxins in (sub)tropical food commodities, while their domesticated counterparts A. oryzae and A. sojae are used in oriental food fermentations and as hosts for heterologous gene expression. Although evidence suggests that A. sojae and A. oryzae are morphological variants of A. parasiticus and A. flavus, respectively, these species are separated because of the regulatory confusion that conspecificity might generate (Geiser, Pitt, & Taylor, 1998). The main difference between A. oryzae and A. flavus is the presence of muta- tions in the aflatoxin biosynthesis gene cluster of A. oryzae, leading to the absence of this mycotoxin in this species (Lee, Liou, & Yuan, 2006; Tominaga et al., 2006). However, the lack of aflatoxin production is not a unique feature for A. oryzae as approximately 60% of the A. flavus are also nonproducers (Cotty, Bayman, Egel, & Elias, 1994). Gibbons et al. (2012) studied the genome-wide sequence and functional variation between the A. oryzae and A. flavus, and discovered dramatic changes in the sequence variation and abundance profiles of genes, and wholesale primary and secondary metabolic pathways during growth on rice. For example, all A. oryzae isolates possess two or three copies of a-amylase, compared to a single copy in A. flavus. This makes A. oryzae a more efficient starch degrader and gives an advantage in, for example, rice fermentations. In addition, differences in sequence and genome architecture of the glutaminase and Taxonomy of Aspergillus and Penicillium 225 sesquiterpene loci were detected, and a glycosyl transferase (a member of a broad sugar modifier family involved in the making of many sweeteners) and an asparaginase gene (an enzyme used commercially to reduce acrylamide levels in starch-rich foods, such as rice) were upregulated.

6. CHARACTER ANALYSIS

The redefined family Trichocomaceae is split into three separate families, namely, Aspergillaceae, Trichocomaceae, and Thermoascaceae (Fig. 4.1). Most members of these families produce complex, branched conidiophores with monophialides and conidia borne in dry chains. An exception is the Basipetospora-type anamorph in Monascus, a genus producing aleurioconidia from simple conidiogenous cells. In this case, the family concept does not conform to phylogenetic relationships. The conidia of Mon- ascus are formed, similarly to other members of Aspergillaceae, in a basipetal manner, but differ by the progressive shortening of the conidiogenous cell (retrogression, Cole & Samson, 1979). They have a truncated base and resemble chlamydospores. It can be speculated that the Monascus “conidia” are actually chlamydospores and in that scenario, the conidial state of Mon- ascus is considered absent. Phialosimplex and Polypaecilum s. str., two genera that resolve in Aspergillus, are other exceptions in Aspergillaceae, due to their ability to form polyphialides. Unpublished data (J. Varga) show that mutagenesis of Aspergillus species can result in various deviating conidiophores types; however, in these experiments a Polypaecilum form was never detected. Interestingly, the polyphialides of Polypaecilum differ from those produced in other genera in having simultaneously functioning phialidic apertures (Cole & Samson, 1979). Whether this feature is unique for Aspergillaceae and also present in Phialosimplex needs to be studied further. Phylogenetically, Aspergillus and Penicillium species are closely related. Species belonging to those genera are characterized by flask-shaped or cylindrical phialides and the conidia borne in a basipetal manner in dry chains. They differ from each other on various characters. The stipes of Aspergilli are heavy walled, usually nonseptated, have a footcell and end in a vesicle. In contrast, Penicillium species lack a footcell, the stipes are often septated and do not have a distinct vesicle (Fig. 4.2A and B vs. Fig. 4.5D–F). Furthermore, the phialides in Aspergillus are produced simultaneously, while those of Pen- icillium are formed successively. Houbraken and Samson (2011) redefined Penicillium following the new nomenclatural rules, and the genera Eupenicillium, Chromocleista, Eladia, Hemicarpenteles, Thysanophora, and 226 Jos Houbraken et al.

Figure 4.5 (A–C) Aspergillus species with atypical Penicillium-like conidiophores. (A) Penicillium inflatum; this species phylogenetically belongs to Aspergillus sect. Cremei and will be combined in Aspergillus in future (R. A. Samson, unpublished results). (B) Pen- icillate conidiophores in Aspergillus candidus. (C) Aspergillus restrictus with diminutive vesicles resembling a monoverticillate Penicillium species. (D–F) Typical conidiophores of biotechnological important Aspergilli. (D) Aspergillus flavus. (E) Aspergillus niger. (F) Aspergillus nidulans. Scale bars¼10 mm. Taxonomy of Aspergillus and Penicillium 227

Torulomyces were placed in synonymy with Penicillium s. str. Species previously assigned to Torulomyces lack conidiophores (Fig. 4.6D). The type species of Torulomyces (T. lagena) has a Eupenicillium-type of sexual state, confirming the close relationship with Penicillium s. str. and indicating loss of conidiophore branching. The transfer of Hemicarpenteles and Thysanophora to Penicillium might be more confusing when solely using phenotypic characters. Hemicarpenteles paradoxa produces typical Aspergillus conidiophores (Fig. 4.6C) although the sexual state resembles a Penicillium. It has been shown that morphologically similar (anamorph) genera have evolved similar traits as a result of adaptation to similar environments or ecological niches (convergence) or due to sharing a common ancestor (divergence) (Crous, Braun, & Groenewald, 2007). Penicillium and Aspergillus are sister genera (Fig. 4.7), and therefore it is likely that an Aspergillus-type of conidiophore in Penicillium s. str. is present due to divergent evolution. Members of Thysanophora form a separate clade in Penicillium s. str. This genus is unique in having dark-colored colonies, melanized stipes and secondary growth of the stipe by means of the proliferation of an apical penicillus. This combination of characters is not present in other Penicillium species and it could be speculated that this genus should be treated separately. However, that would create a paraphyletic clade in Penicillium or the need of at least eight genera to restore monophyly. In order to avoid many name changes and to keep all species with a Penicillium-type conidiophore in one genus, it was decided to transfer Thysoanophora species to the monophy- letic Penicillium (Houbraken & Samson, 2011). There are also Aspergilli that look similar to Penicillium. An example is Penicillium inflatum, which phylogenetically belongs to Aspergillus section Cremei and will therefore be transferred from Penicillium to Aspergillus in future (R.A. Samson, unpublished data; Fig. 4.5A). Other examples are Aspergillus sydowii, A. candidus, and A. restrictus. These species can produce diminutive vesiculate monoverticillate stipes and resemble in appearance conidiophores of some Pen- icillium species (Fig. 4.5B and C).

7. MODERN TAXONOMY AND GENOME SEQUENCING

The recent insights in the relationships between members of the Trichocomaceae, Aspergillaceae, and Thermoascaceae have implications in the interpretation of comparative genome data. The genome-sequenced strains of Aspergillus, Penicillium s. str., Monascus, and Xeromyces are closely related to each other and treated in the revived family Aspergillaceae. 228 Jos Houbraken et al.

Figure 4.6 Conidiophores (A–E and G) and sclerotia (F) of Penicillium species previously classified in other genera. (A) Penicillium sacculum (syn. Eladia saccula). (B) Penicillium glaucoalbidum (syn. Thysanophora penicillioides). (C) Aspergillus paradoxus (¼“Penicillium paradoxum”); this species phylogenetically belongs to Penicillium but produces typical Aspergillus conidiophores. (D) Penicillium lagena (syn. Torulomyces lagena). (E and F). Pen- icillium kewense (syn. Eupenicillium crustaceum). (G) Penicillium malachiteum (syn. Chro- mocleistha malachitea). Scale bars¼10 mm. Taxonomy of Aspergillus and Penicillium 229

IFO 4308 Aspergillus luchuensis (as “A. kawachii” )JGI

CBS 134.48 Aspergillus tubingensisJGI

ATCC 1015 Aspergillus nigerJGI

CBS 101740 Aspergillus brasiliensisJGI

ITEM 5010 Aspergillus carbonariusJGI

95 ATCC 16872 Aspergillus aculeatusJGI

NRRL 3557 Aspergillus flavusJGI

RIB40 Aspergillus oryzaeJGI 91 NIH 2624 Aspergillus terreus JGI Aspergillus Af293 Aspergillus fumigatusJGI

NRRL 181 Aspergillus fischeri (as Neosartorya fischeri)JGI 68 NRRL 1 Aspergillus clavatusJGI

CBS 593.65 Aspergillus sydowii JGI

CBS 795.97 Aspergillus versicolorJGI 75 FGSC A4 Aspergillus nidulans JGI

DTO 134-E9 Aspergillus wentii JGI

CBS 516.65 Aspergillus glaucusJGI

“ex dead sea” Aspergillus ruber JGI (as Eurotium herbariorum)

ATCC 20851 Penicillium bilaiaeJGI

DAOM 239074 Penicillium glabrumJGI

87 ATCC 48694 Penicillium charlesii (as P. fellutanum)JGI

Strain 114-2 Penicillium oxalicum (as P. decumbens)JGI

ATCC 26601 Penicillium paxilli GB

“contaminant of Postia culture” Penicillium chrysogenumJGI Penicillium

Wisconsin 54-1255 Penicillium rubens (as P. chrysogenum)JGI

PHI26 Penicillium digitatumGB

ATCC 24692 Penicillium expansum GB

ATCC 10419 Penicillium canescens JGI

CBS 506.65 Aspergillus zonatus JGI

NRRL 1597 Monascus ruber JGI

Strain SSBP Thermomyces lanuginosusGB

NRRL 2155 Thermomyces dupontii (as Talaromyces thermophilus)GZ

ATCC 18224 Talaromyces marneffei (as Penicillium marneffei )GB

ATCC 10500 Talaromyces stipitatus GB

Thermoascus aurantiacusJGI

Strain RS Coccidioides immitisGB 0.1 Figure 4.7 Best-scoring Maximum Likelihood tree using MEGA5 based on a selection of 25 loci. The total length of the data set was 56.7 kb; 27,511 characters were parsimony informative. The data set was analyzed without partitions. The bootstrap percentages (100 bootstraps) are presented at the nodes; values less than 50% are not shown; well-supported branches are double-thickened. The tree is rooted with Coccidioides immitis. Data were downloaded from the various databases and the origin is given in superscript after the species name (JGI¼Doe Joint Genome Institute; Gb¼NCBI Genome database; Gz¼Genomymes). 230 Jos Houbraken et al.

Talaromyces stipitatus, T. marneffei (syn. P. marneffei), T. funiculosus (syn. P. funiculosum), Rasamsonia emersonii (¼T. emersonii), Thermomyces dupontii (syn. T. thermophilus), and Thermomyces lanuginosus are (in the process of being) genome-sequenced and belong to the narrowed Trichocomaceae, which is a sister family of the Aspergillaceae (Fig. 4.7). These new insights in the relationship among Aspergillus, Penicillium, and related genera will help to interpret the results generated with comparative genomic studies or other studies dealing with evolution of, for example, enzymes, mating- type loci, virulence genes and secondary metabolite biosynthetic gene clusters.

7.1. Identity of genome-sequenced strains The importance of Aspergillus, Penicillium, and related genera is also illustrated by the high number of genome sequencing projects undertaken. Cor- rect species identification should be the first crucial step in these projects. Currently, a selection of genome-sequenced strains is incorrectly identified and the underlying reasons can be divided into three: (1) introduction of new taxonomic schemes, (2) recent changes in nomenclatural rules (single- name nomenclature), and (3) use of invalid names. An example of the use of invalid names is the white-koji mold so-called “A. kawachii” (IFO 4308¼NBRC 4308). This strain has been genome sequenced by Futagami et al. (2011); however, A. kawachii is not a valid name (Art. 36) and based on partial b-tubulin sequence data, this isolate actually belongs to Aspergillus luchuensis (Fig. 4.4). Interestingly, the genomes of “A. awamori” (NRBC 4314¼RIB 2604; Machida et al., 2010) and “A. acidus” (CBS 106.47) are also sequenced and these strains are also reidentified as A. luchuensis (Fig. 4.4). Besides the incorrect identifications, also the recent advances in taxonomy influence the naming of genome-sequenced strains. Aspergillus glaucus CBS 516.65 and Eurotium rubrum (e.g., Dead Sea), the two xerophilic species, are genome-sequenced. Although the generic names do not suggest a close relationship, both species are actually sister species and both belong to Aspergillus section Aspergillus (Fig. 4.7). In the current single-name system, these species are listed under their Aspergillus name. In addition, the correct name of Penicillium marneffei is Talaromyces marneffei, Talaromyces emersonii is combined in Rasamsonia and Penicillium chrysogenum strain Wisconsin 54-1255 is reclassified as P. rubens (Houbraken et al., 2011b; Houbraken, Frisvad, et al., 2012; Samson et al., 2011). Serendipi- tously, a strain of P. chrysogenum for which no culture is available, had its Taxonomy of Aspergillus and Penicillium 231 genome sequenced unexpectedly as a contaminant of a Postia placenta MAD 698R culture (http://genome.jgi.doe.gov/Pench1/Pench1.info. html). Haplotype analysis on eight loci showed that this uncultured organism shares the same haplotype as strains CBS 132214, CBS 132212, and CBS 116046. One of these strains could be selected as an “epitype” kind of voucher to represent this genome-sequenced strain. However, CBS 116046 is a good penicillin producer, but no penicillin production was observed in CBS 132214 and CBS 132212. In contrast, both CBS 132214 and CBS 132212 produce roquefortine C, but CBS 116046 does not and CBS 132214 was the only strain producing the uncharacterized compound “met Ø.” These results suggest that even with eight loci, the resulting haplotype assignments may not be precise enough to correlate with a precise genome (Houbraken, Frisvad, et al., 2012). In Table 4.4 an overview of incorrectly named genome-sequenced Aspergillus and Penicillium strains is given, together with their correct identity.

7.2. Selection of strains The selection of a good representative of a species for genome sequencing is important. One can choose the ex-type strain of a species; however, some- times these strains are degenerated and do not exhibit their typical phenotype. For P. rubens, strain Wisconsin 54-1255, an ancestor of the strain used for penicillin production, was genome-sequenced. This is a logical choice from a biotechnological point of view, but this strain underwent several steps of mutagenesis and has a different phenotype than wild-type isolates (Barreiro, Martı´n, & Garcı´a-Estrada, 2012). Another example is the genome-sequenced Aspergillus oryzae isolate RIB 40 (Machida et al., 2005). Aspergillus oryzae is used as a koji (starter) mold for Asian fermented foods; however, the genome-sequenced strain was isolated from cereals and probably not from an industrial environment. This strain produces abundant sclerotia and is phenotypically similar to A. flavus. It would therefore be rec- ommended to genome sequence an Aspergillus oryzae strain used for koji fermentation, for example, the ex-type culture (CBS 100925) (Varga, Frisvad, & Samson, 2011b).

7.3. Recommendations for strain selection The number of genome sequencing projects has increased tremendously in the last years. In order to avoid incorrect identification and ensure a selection 232 Jos Houbraken et al.

Table 4.4 Overview of incorrectly identified genome-sequenced strains and important species used in biotechnology Genome- sequenced Correct Reason name strain Reference identity change Remarks/reference Aspergillus JGI Aspergillus New Hong et al. (2013) acidus CBS luchuensis taxonomic 106.47 scheme Aspergillus Machida Aspergillus Uncertain Hong et al. (2013) awamori RIB et al. luchuensis name, new 2604 (2010) taxonomic scheme Aspergillus Futagami Aspergillus Invalid name This study, Fig. 4.4 kawachii IFO et al. luchuensis 4308 (2011) Eurotium JGI Aspergillus 1F¼1N This study, Hubka rubrum ex. ruber et al. (2013) Dead Sea Neosartorya Fedorova Aspergillus 1F¼1N This study fischeri NRRL et al. fischeri 181 (2008) Penicillium Chooi, Penicillium New Houbraken, Frisvad, aethiopicum Cacho, and lanosocoeruleum taxonomic et al. (2012) IBT 5753 Tang scheme (2010) Penicillium van den Penicillium New Houbraken et al., chrysogenum Berg et al. rubens taxonomic 2011b, Houbraken, Wisconsin (2008) scheme Frisvad, et al., 2012 54-1255 Penicillium Liu et al. Penicillium Incorrect This study, Fig. 4.1 decumbens (2013) oxalicum identification strain 114-2 Penicillium JGI Penicillium Incorrect This study, Fig. 4.1 fellutanum charlesii identification ATCC 48694 Penicillium Llanos et al. Talaromyces 1F¼1N This study funiculosum (2012) funiculosus Taxonomy of Aspergillus and Penicillium 233

Table 4.4 Overview of incorrectly identified genome-sequenced strains and important species used in biotechnology—cont'd Genome- sequenced Correct Reason name strain Reference identity change Remarks/reference Penicillium Woo et al. Talaromyces New Samson et al. (2011) marneffei (2011) marneffei taxonomic ATCC 18224 scheme, 1F¼1N

1F¼1N: single-name nomenclature. JGI: name listed in the Doe Joint Genome Institute (JGI) database (Grigoriev et al., 2011). of a good representative of a species, we recommend the following rules before the start of a genome sequencing project: 1. The strains should be deposited in two or more recognized, public culture collections (from two countries). This would guarantee that the strain is easily accessible for other researchers and for future research purposes. Ideally, this procedure should be mandatory for all microbial, biochemical, and chemical journals. 2. Perform an identification of the strain prior genome sequencing. If needed, contact a taxonomist who can advise on the current identity of the strain. 3. If the project involves sequencing a representative of a species, make sure that the selected strain is typical for the species. Type strains (and other strains in culture collections) are not always the best choice because these strains might be preserved over a long time and could be deteriorated.

8. IDENTIFICATION OF PENICILLIUM AND ASPERGILLUS STRAINS

Identification of a species is an important step in biological research. A correct name is vital for optimal communication, and is often the link between studies in various fields. It is therefore important that taxonomy is clear and stable. Ideally, identification should be unequivocal, accurate, simple, and immutable. In the last decade, new insights have resulted that certain well-known species belonging to Aspergillus or Penicillium appear to be species complexes. This might lead (initially) to confusion; however, a correct identification has a function: certain species of these complexes have unique properties such as higher resistance to certain antifungals, 234 Jos Houbraken et al. production of different mycotoxins and/or have unique enzyme profiles (Balajee, Gribskov, Hanley, Nickle, & Marr, 2005; de Vries et al., 2004; Meijer, Houbraken, Dalhuijsen, Samson, & de Vries, 2011; Samson et al., 2009). Various erroneous identifications of Aspergilli and Penicillia used in biotechnology are present in recent literature and an overview of strains and/or species names is given in Table 4.5. Also the number of hits in Scopus is given in Table 4.5 and interestingly, the number of publication using the incorrect names Penicillium occitanis, Penicillium griseoroseum, and Acremonium cellulolyticus has increased in the last 5 years. Hopefully, these isolates are maintained in a culture collection and will be reidentified, otherwise the data presented in these publications will become more difficult to interpret for future researchers. In the past, Aspergillus and Penicillium identification has primary been based on phenotypic and physiological characters. Identification solely based on the phenotype is often difficult and requires well-trained staff. Correct identification by routine laboratories solely based on phenotypic characters is therefore becoming difficult and nowadays, molecular-based techniques, especially DNA sequencing, are frequently used for identification. Recently, the ITS region was accepted as the prime fungal barcode (Schoch et al., 2012); however, various studies showed that this locus cannot be used for identification of Aspergillus and Penicillium species. Protein- coding genes are widely used in mycology for identification and have generally a higher interspecies variability than the ITS region. There is no standard choice of protein-coding gene in the fungal kingdom, but b-tubulin and calmodulin sequences are frequently used for identification of Aspergillus and Penicillium species and are better species markers than ITS (e.g., Geiser et al., 2007; Houbraken et al., 2011a; Samson, Seifert, Kuijpers, Houbraken, & Frisvad, 2004; Skouboe et al., 1999).

9. MATING-TYPE GENES

There are two main types of sexual breeding systems in fungi, hetero- thallism and homothallism. In Ascomycetes, the master regulators of sexual reproduction are the “mating-type” (MAT) genes that reside in MAT loci (Turgeon & Yoder, 2000). Heterothallic ascomycetes have a bipolar mating-type system, with isolates possessing one of two nonallelic versions (idiomorphs) of a single MAT locus, termed MAT1-1 and MAT1-2. MAT1-1 isolates contain a characteristic MAT1-1 gene encoding a protein with a MATa_HMG domain, whereas MAT1-2 isolates contain a MAT1-2 Taxonomy of Aspergillus and Penicillium 235

Table 4.5 Selection of incorrect species names used literature (with exception of section Nigri; see Table 4.3) Correct Reason name Number hits in Remarks/ Incorrect name identity change Scopusa reference Acremonium Talaromyces Invalid name 20 (2008–current) The ITS cellulolyticum pinophilus 4 (2002–2007) sequence (¼A. cellulolyticus) deposited in GenBank (AB474749) shows that this strain is actually Talaromyces pinophilus Emericella nidulans Aspergillus 1F¼1N N/A This study nidulans Geosmithia Rasamsonia 1F¼1N, new 7 (2012–current) Houbraken, argillacea argillacea taxonomic Spierenburg, scheme and Frisvad (2012) Penicillium Talaromyces 1F¼1N, new 28 (2012–current) Samson et al. funiculosum funiculosus taxonomic (2011) scheme Penicillium Penicillium Incorrect 15 (2008–current) Houbraken griseoroseum chrysogenum identification; 11 (2002–2007) et al. (2011b), sensu latob new Houbraken, taxonomic Frisvad, et al. scheme (2012) Penicillium occitanis Unknown Invalid name 13 (2008–current) Exact 4 (2002–2007) identity remains unknown, no material was available for examination Penicillium Talaromyces 1F¼1N, new 1 (2012–current) Samson et al. purpurogenum purpurogenusc taxonomic (2011), scheme Yilmaz et al. (2012) Continued 236 Jos Houbraken et al.

Table 4.5 Selection of incorrect species names used literature (with exception of section Nigri; see Table 4.3)—cont'd Correct Reason name Number hits in Remarks/ Incorrect name identity change Scopus reference Talaromyces Rasamsonia 1F¼1N, new 8 (2012–current) Houbraken, emersonii emersonii taxonomic Spierenburg, scheme and Frisvad (2012) Talaromyces Thermomyces 1F¼1N, new N/A This study thermophilus dupontii taxonomic scheme aNumber of hits in Scopus on July 26, 2013. Hits referring to recently updated names are excluded from this number. bPenicillium chrysogenum sensu lato is a complex of five species (Houbraken, Frisvad, et al., 2012). cT. purpurogenus is a complex consisting of four species (Yilmaz et al., 2012). gene encoding a protein with a MATA_HMG domain. By contrast, homothallic (self-fertile) species typically contain MAT loci with genes encoding both MATa_HMG and MATA_HMG-domain proteins present on the same chromosome (Martin et al., 2010; Paoletti et al., 2007). However, even if both mating types are present in culture, then real mating can still be blocked by other genetic barriers (Debuchy & Turgeon, 2006). Dyer and O’Gorman (2012) compiled a list of over 75 genes associated with sexual reproduction in Aspergillus species and some of these genes are essential for sexual development to occur.

9.1. Aspergillus The ability to determine the presence of MAT1-1 and/or MAT1-2 loci in formerly asexual species belonging to Penicillium, Aspergillus and related genera (formerly known as Trichocomaceae) speeded up the discovery of sexual states in species. Directed crosses of opposite mating partners showed that assumed asexual species form cleistothecia and fertile progeny. This was first demonstrated in Paecilomyces variotii, a species described more than a century ago by Bainier (1907). Soon afterwards, sexual states were discovered in the “asexual” Aspergillus species A. fumigatus (O’Gorman, Fuller, & Dyer, 2009), A. flavus (Horn, Moore, & Carbone, 2009), A. parasiticus (Horn, Ramirez-Prado, & Carbone, 2009b, 2009c), A. nomius (Horn, Moore, & Carbone, 2011), and A. tubingensis (Horn et al., in press). Around 70% of the accepted Aspergillus species and Penicillium species have no known sexual state (Dyer & O’Gorman, 2012). The majority of the Aspergillus, Penicillium, and Paecilomyces species that reproduce sexually are homothallic, and Taxonomy of Aspergillus and Penicillium 237

Table 4.6 Overview of heterothallic species in Aspergillaceae, Trichocomaceae, and Thermoascaceae Structure Species ascomata References Aspergillus felis Neosartorya Barrs et al. (2013) Aspergillus fennelliae Neosartorya Kwon-Chung and Kim (1974) Aspergillus flavus Petromyces Horn, Moore, and Carbone (2009a) Aspergillus fumigatus Neosartorya O’Gorman et al. (2009) Aspergillus heterothallicus Emericella Raper and Fennell (1965) Aspergillus lentulus Neosartorya Swilaiman, O’Gorman, Balajee, and Dyer (2013) Aspergillus nishimurae Neosartorya Takada, Horie, and Abliz (2001) Aspergillus nomius Petromyces Horn et al. (2011) Aspergillus parasiticus Petromyces Horn, Ramirez-Prado, and Carbone (2009b, 2009c) Aspergillus sclerotiicarbonarius Petromyces Darbyshir, van de Vondervoort, and Dyer (2013) Aspergillus spathulatus Neosartorya Takada and Udagawa (1985) Aspergillus terreus Fennellia Arabatzis and Velegraki (2013) Aspergillus tubingensis Petromyces Horn, Olarte, Peterson, and Carbone (2013) Aspergillus udagawae Neosartorya Horie, Miyaji, Nishimura, Franco, and Coelho (1995) Aspergillus wyomingensis Neosartorya Nova´kova´ et al. (2013) Paecilomyces variotii Byssochlamys Houbraken, Varga, Rico-Munoz, Johnson, and Samson (2008) Penicillium rubens (reidentified Eupenicillium Bo¨hm et al. (2013) here; reported as P. chrysogenum) Talaromyces derxii Talaromyces Takada and Udagawa (1988) heterothallic mating has been demonstrated in 16 Aspergillus, one Penicillium and one Talaromyces species (Table 4.6). Strikingly, the majority of heterothallic Aspergillus species that are able to recombine belong to section Fumigati. The reason for this bias is unknown. The main reason might be that many species in this section are intensively studied as they have clinical 238 Jos Houbraken et al. importance. Another reason is that species belonging to the section Fumigati produce their cleistothecia relatively fast on agar media such as oatmeal agar, as is observed in their homothallic counterparts. If that is the case, then it is likely that mating experiments with members of section Nidulantes will also be successful as these species also form cleistothecia easily on oatmeal agar. In contrast, species with a Petromyces or Neopetromyces-morph produce their cleistothecia slowly on regular agar media and therefore the discovery of fertile mating partners will be more difficult.

9.2. Penicillium In comparison to Aspergillus, the presence of heterothallic species is low in other related genera. One heterothallic species is described in Penicillium (P. rubens, described as P. chrysogenum; Bo¨hm et al., 2013)(Table 4.6). In 2008, Hoff et al. discovered that mating-type genes in P. chrysogenum are transcriptionally expressed. The 12 examined strains showed a 1:1 distribution of the MAT1 and MAT2 regions, indicating that (occasionally) sexual reproduction occurs in P. chrysogenum. A population study of P. chrysogenum (>200 isolates) confirmed that the 1:1 mating-type ratio, and in addition, recombination among loci supported a sexual or sexual-like reproductive mode in P. chrysogenum (Henk et al., 2011). More recently, the attempt to induce a teleomorph in P. chrysogenum was successful, leading to the production of cleistothecia and ascospores, similar to those described recently for P. kewense (Bo¨hm et al., 2013). In Penicillium, evidence suggests that cryptic/covert sexuality occurs in also P. dipodomyis, P. verrucosum, P. commune, P. roqueforti, P. miczynskii, and P. camemberti (Eagle, 2009; Frisvad, Lund, & Elmholt, 2005; Henk & Fisher, 2011; Henk et al., 2011; Lund, Nielsen, & Skouboe, 2003; Tuthill, 2004). Taken together, these data suggest that more Penicillium species have the potential to reproduce heterothallically and future studies might reveal a sexual state in these species as well. The limited number of successful mating experiments in Pen- icillium (Eagle, 2009; Henk & Fisher, 2011; Henk et al., 2011; Hoff, Po¨ggeler, & Ku¨ck, 2008; J. Houbraken unpublished data) might be explained by the strains used in these experiments. In some cases, mating experiments were conducted with strains that were maintained for a long period in culture collections. These strains could have lost their fertility. For example, the heterothallic Histoplasma capsulatum lost fertility rapidly during laboratory passage and it was suggested that selective pressures may serve to maintain fertility in the environment (Fraser et al., 2007; Kwon- Chung, Weeks, & Larsh, 1974). For the heterothallic and heat-resistant Taxonomy of Aspergillus and Penicillium 239

P. variotii (¼B. spectabilis), it was shown that only strains derived from pas- teurized products were fertile (Houbraken et al., 2008). It will therefore be promising to repeat the mating experiments with Penicillium strains directly isolated from nature. Another possibility for the unsuccessful mating experiments is the stringent conditions required for successful mating. Various growth factors induce formation of cleistothecia, such as temperature, light, nutrients, and oxygen levels (Han et al., 2003). Recently, Houbraken, Frisvad, and Samson (2010) showed that P. psychrosexualis, a species related to P. roqueforti, produces abundant cleistothecia at low temperatures (9–15 C). The production of a sexual stage at low temperatures might be more widespread in Penicillium, and mating experiments at this temperature might result in the discovery of a sexual stage in other species.

9.3. Other genera In the light of the single-name nomenclature, Samson et al. (2011) expanded the concept of Talaromyces and included teleomorph and anamorph characters. The majority of sexually reproducing Talaromyces species is homothallic and Talaromyces derxii is the only heterothallic species described in Talaromyces (Takada & Udagawa, 1988), however, there is evidence that also P. pinophilum is able to form ascospores in a heterothallic manner. Lo´pez- Villavicencio et al. (2010) crossed P. pinophilum strains of opposite mating types resulting in immature cleistothecia. There was no compelling evidence of efficient sexual reproduction, although sexuality in this species cannot be completely ruled out. It was suggested that the asexual Talaromyces species may have lost sex only very recently and/or that the MAT genes are involved in other functions. An ancestral state reconstruction analysis indi- cated several events of putative loss of sexuality in the genus. Alternatively, it is possible that the supposedly asexual Talaromyces species may have retained a cryptic sexual stage (Lo´pez-Villavicencio et al., 2010). Currently, nine species are described in Rasamsonia and the presence of a sexual cycle is described in three species (R. emersonii, R. byssochlamydoides, R. eburnea). The former two species produce numerous ascospores in a homothallic manner; however, no ascospores were detected in the latter species by Houbraken, Spierenburg, and Frisvad (2012b). Although not reported in the original description of R. eburnea (as T. eburneus; Yaguchi, Someya, & Udagawa, 1994), De Ravin et al. (2011) mentioned that R. eburnea is heterothallic and fails to form a teleomorph without mating appropriate strains. Unfortunately, no additional details were given for this observation in their publication. Similar to R. eburnea, also R. argillacea is 240 Jos Houbraken et al. isolated from heat-treated products. The presence of this species in heat- treated food products suggests the potential presence of a teleomorph. This suggests that this species might have a heterothallic mating system; or it could be that specific conditions are required for homothallic reproduction to occur.

10. CONCLUSIONS

Aspergillus and Penicillium species are common saprobes and have impact, positive and negative, in various fields, including fungal taxonomy, food and indoor mycology, biotechnology, ecology, medical mycology, and genomics. The new insights in the phylogeny and classification of Aspergilli and Penicillia will influence these fields and correct strain identification is a crucial step in each research. Like in many other fields, incorrect species identification also occurs in the field of biotechnology and genomics. Due to these misidentifications it is difficult to compare studies with each other leading to confusion and misinterpretation of results. It is rec- ommended to consult a taxonomist to ensure correct species identification. The availability of fully sequenced genomes resulted in large amounts of sequence data, and will inevitably also have an impact on taxonomy. Geno- mics can aid taxonomy by serving as a source of novel and unprecedented quantitative comparative data and to provide molecular tools for a more accurate delineation of species boundaries (Gibbons et al., 2012; Rokas et al., 2007). However, it remains to be seen if it will provide a conclusive answer on the definition of a species. Initial studies show variations among the genomes of the same species (Andersen et al., 2011; Fedorova et al., 2009, 2008), and it is questionable to assign every genetic variant to a novel taxonomic rank. The issue remains whether a strain represents a species or is an individual within the species. Genes and genomes do not function on their own and they can only unfold their potential within a cell. It is the phenotype that in combination with the natural selection that “drives” evolution in an environment.

ACKNOWLEDGMENTS The authors thank Henk Spierenburg and Martin Meijer for their work on Thermomyces, Neriman Yilmaz for the identification of the Acremonium cellulolyticum strain, and Jan Dijksterhuis for his valuable suggestions and being of great importance for this contribution. Genome sequence data downloaded from the JGI Website (http://genome. jgi.doe.gov/) were produced by the US Department of Energy Joint Genome Institute (http://www.jgi.doe.gov/) in collaboration with the user community. Taxonomy of Aspergillus and Penicillium 241

REFERENCES Al-Musallam, A. (1980). Revision of the black Aspergillus species. PhD Thesis, Utrecht: Rijksuniversiteit Utrecht. Andersen, M. R., Salazar, M. P., Schaap, P. J., van de Vondervoort, P. J., Culley, D., Thykaer, J., et al. (2011). Comparative genomics of citric-acid-producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88. Genome Research, 21, 885–897. Arabatzis, M., & Velegraki, A. (2013). Sexual reproduction in the opportunistic human pathogen Aspergillus terreus. Mycologia, 105, 71–79. Asgari, B., Zare, R., Zamanizadeh, H. R., & Rezaee, S. (2013). Aspergillus osmophilus sp. nov., and a new teleomorph for A. proliferans. Mycoscience, http://dx.doi.org/10.1016/ j.myc.2013.05.005. Bainier, G. (1907). Mycothe`que de l’ećole de Pharmacie XL: Paecilomyces, genre nouveau de Muce´dineés. Bulletin trimestriel de la Socie´te´ mycologique de France, 23, 26–27. Balajee, S. A., Gribskov, J. L., Hanley, E., Nickle, D., & Marr, K. A. (2005). Aspergillus lentulus sp. nov., a new sibling species of A. fumigatus. Eukaryotic Cell, 4, 625–632. Barreiro, C., Martıń, J. F., & Garcıá-Estrada, C. (2012). Proteomics shows new faces for the old penicillin producer Penicillium chrysogenum. Journal of Biomedicine and Biotechnology, 2012, 105–109. Barrios-Gonza´lez, J., & Miranda, R. U. (2010). Biotechnological production and applications of statins. Applied Microbiology and Biotechnology, 85, 869–883. Barrs, V. R., van Doorn, T. M., Houbraken, J., Kidd, S. E., Martin, P., Pinheiro, D. M., et al. (2013). Aspergillus felis sp. nov., an emerging agent of invasive aspergillosis in humans, cats and dogs. PLoS One, 8(6), e64871. http://dx.doi.org/10.1371/journal. pone.0064871. Belancic, A., Scarpa, J., Peirano, A., & Diaz, R. (1995). Penicillium purpurogenum produces several xylanases: Purification and properties of two of the enzymes. Journal of Biotechnol- ogy, 41, 71–79. Berbee, M. L., & Taylor, J. W. (1993). Ascomycete relationships: Dating the origin of asexual lineages with 18S ribosomal RNA gene sequence data. In D. R. Reynolds & J. W. Taylor (Eds.), The fungal holomorph: Mitotic, meiotic, and pleomorphic speciation in fungal systematics (pp. 67–78). Wallingford: CAB International. Berbee, M. L., Yoshimura, A., Sugiyama, J., & Taylor, J. W. (1995). Is Penicillium monophy- letic? An evaluation of phylogeny in the family Trichocomaceae from 18S, 5.8S and ITS ribosomal DNA sequence data. Mycologia, 87, 210–222. Biourge, P. (1923). Les moisissures du groupe Penicillium Link. Cellule, 33, 7–331. Bo¨hm, J., Hoff, B., O’Gorman, C. M., Wolfers, S., Klix, V., Binger, D., et al. (2013). Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum. PNAS, 110, 1476–1481. Braun, U. (2012). The impacts of the discontinuation of dual nomenclature of pleomorphic fungi: The trivial facts, problems, and strategies. IMA Fungus, 3, 81–86. Brefeld, O. (1874). Botanische Untersuchungen uber Schimmelpilze. Heft 2 “Die Entwicklungsgeschichte von Penicillium” Leipzig: A. Felix. Brown, A. G., Smale, T. C., King, T. J., Hasenkamp, R., & Thompson, R. H. (1976). Crys- tal and molecular structure of compactin, a new antifungal metabolite from Penicillium brevicompactum. Journal of the Chemical Society Perkin Transactions I, 1976, 1165–1170. Chooi, Y. H., Cacho, R., & Tang, T. (2010). Identification of the viridicatumtoxin and griseofulvin gene clusters from Penicillium aethiopicum. Chemistry & Biology, 17, 483–494. Cimon, B., Carrere, J., Chazalette, J. P., Vinatier, J. F., Chabasse, D., & Bouchara, J. P. (1999). Chronic airway colonization by Penicillium emersonii in a patient with cystic fibro- sis. Medical Mycology, 37, 291–293. Cole, G. T., & Samson, R. A. (1979). Patterns of development in conidial fungi. London: Pittman. 242 Jos Houbraken et al.

Cotty, P. J., Bayman, D. S., Egel, D. S., & Elias, K. S. (1994). Agriculture, aflatoxins and Aspergillus. In K. A. Powell, A. Renwick, & J. F. Peberdy (Eds.), The genus Aspergillus: From taxonomy and genetics to industrial applications (pp. 1–27). New York: Plenum Press. Crous, P. W., Braun, U., & Groenewald, J. Z. (2007). Mycosphaerella is polyphyletic. Studies in Mycology, 58, 1–32. Darbyshir, H. L., van de Vondervoort, P. J. I., & Dyer, P. S. (2013). Discovery of sexual reproduction in the black Aspergilli [abstract]. In Pacific Grove, California: 10th Interna- tional Aspergillus meeting (p. 290). de Bary, A. (1854). Ueber die Entwickelung und den Zusammenhang von Aspergillus glaucus und Eurotium. Botanische Zeitung, 12, 425–471. De Ravin, S. S., Challipalli, M., Anderson, V., Shea, Y. R., Marciano, B., Hilligoss, D., et al. (2011). Geosmithia argillacea: An emerging cause of invasive mycosis in human chronic granulomatous disease. Clinical Infectious Diseases, 52, e136–e143. de Vries, R. P., Burgers, K., van de Vondervoort, P. J., Frisvad, J. C., Samson, R. A., & Visser, J. (2004). A new black Aspergillus species, A. vadensis, is a promising host for homologous and heterologous protein production. Applied and Environmental Microbiol- ogy, 70, 3954–3959. Debuchy, R., & Turgeon, B. (2006). Mating-type structure, evolution, and function in Euascomycetes.InU.Ku¨es & R. Fischer (Eds.), The mycota, growth, differentiation, and sexuality, Vol. 1. (pp. 293–323). Berlin: Springer. Dierckx, R. P. (1901). Un essai de revision du genre Penicillium Link. Annales de la Socie´te´ scientifique de Bruxelles, 25, 83–89. Doss, S. L., Chu, C. K., Mesbah, M. K., Cutler, H. G., Cole, P. D., Arrendale, R. F., et al. (1986). Isolation of compactin (a hypocholesterolemic metabolite) from a new source— Penicillium cyclopium. Journal of Natural Products, 49, 357–358. Dyer, P. S., & O’Gorman, C. M. (2012). Sexual development and cryptic sexuality in fungi: Insights from Aspergillus species. FEMS Microbiology Reviews, 36, 165–192. Eagle, C. E. (2009). Mating-type genes and sexual potential in the Ascomycete genera Aspergillus and Penicillium. PhD thesis, University of Nottingham. Eamvijarn, A., Manoch, L., Chamswarng, C., Piasai, O., Visarathanonth, N., Luangsa-ard,- J. J., et al. (2013). Aspergillus siamensis sp. nov. from soil in Thailand. Mycosciencehttp://dx. doi.org/10.1016/j.myc.2013.01.005. Endo, A., Kuroda, M., & Tsujita, Y. (1976). ML-236A, ML-236B, and ML-236C, new inhibitors of cholesterogenesis produced by Penicillium citrinum. Journal of Antibiotics, 29, 1346–1348. Fedorova, N. D., Harris, S., Chen, D., Denning, D. W., Yu, J., Cotty, P. J., et al. (2009). Using a CGH to study intraspecific genetic variability in two pathogenic molds, Asper- gillus fumigatus and Aspergillus flavus. Medical Mycology, 47(Suppl.), S34–S41. Fedorova, N. D., Khaldi, N., Joardar, V. S., Maiti, R., Amedeo, P., Anderson, M. J., et al. (2008). Genomic islands in the pathogenic filamentous fungus Aspergillus fumigatus. PLoS Genetics, 4, e1000046. Flannigan, B., Samson, R. A., & Miller, J. D. (2011). Microorganisms in home and indoor work environments (2nd Ed.). Boca Raton, Florida: CRC Press. Fleming, A. (1929). On the antibacterial action of cultures of a Penicillium, with special reference to their use in the isolation of B. influenzae. British Journal of Experimental Pathology, 10, 226–236. Fraser, J. A., Stajich, J. E., Tarcha, E. J., Cole, G. T., Inglis, D. O., Sil, A., et al. (2007). Evo- lution of the mating type locus: Insights gained from the dimorphic primary fungal path- ogens Histoplasma capsulatum, Coccidioides immitis, and Coccidioides posadasii. Eukaryotic Cell, 6, 622–629. Frisvad, J. C., & Filtenborg, O. (1989). Terverticillate Penicillia: Chemotaxonomy and mycotoxin production. Mycologia, 81, 837–861. Taxonomy of Aspergillus and Penicillium 243

Frisvad, J. C., Hawksworth, D. L., Kozakiewicz, Z., Pitt, J. I., Samson, R. A., & Stolk, A. S. (1990). Proposals to conserve important species names in Aspergillus and Penicillium.In R. A. Samson & J. I. Pitt (Eds.), Modern concepts in Penicillium and Aspergillus classification (pp. 83–88). New York: Plenum Press. Frisvad, J. C., Larsen, T. O., Thrane, U., Meijer, M., Varga, J., Samson, R. A., et al. (2011). Fumonisin and ochratoxin production in industrial Aspergillus niger strains. PLoS One, 6, e23496. Frisvad, F., Lund, F., & Elmholt, S. (2005). Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability. Journal of Applied Micro- biology, 98, 684–692. Futagami, T., Mori, K., Yamashita, A., Wada, S., Kajiwara, Y., Takashita, H., et al. (2011). Genome sequence of the white koji mold Aspergillus kawachii IFO 4308, used for brewing the Japanese distilled spirit shochu. Eukaryotic Cell, 10, 1586–1587. Gasperini, G. (1887). La biologia e piu` specialmente il polimorfismo di varie specie d’Ifomiceti. Atti della Societa` toscana di scienze naturali, residente in Pisa, 6, 326. Geiser, D. M., Klich, M. A., Frisvad, J. C., Peterson, S. W., Varga, J., & Samson, R. A. (2007). The current status of species recognition and identification in Aspergillus. Studies in Mycology, 59, 1–10. Geiser, D. M., Pitt, J. I., & Taylor, J. W. (1998). Cryptic speciation and recombination in the aflatoxin-producing fungus Aspergillus flavus. PNAS, 95, 388–393. Gibbons, J. G., Salichos, L., Slot, J. C., Rinker, D. C., McGary, K. L., King, J. G., et al. (2012). The evolutionary imprint of domestication on genome variation and function of the filamentous fungus Aspergillus oryzae. Current Biology, 22, 1403–1409. Gonc¸alves, S. S., Stchigel, A. M., Cano, J. F., Godoy-Martinez, P. C., Colombo, A. L., & Guarro, J. (2012). Aspergillus novoparasiticus: A new clinical species of the section Flavi. Medical Mycology, 50, 152–160. Gravesen, S., Nielsen, P. A., Iversen, R., & Nielsen, K. F. (1999). Microfungal contamina- tion of damp buildings—Examples of risk constructions and risk materials. Environmental Health Perspectives, 107, S505–S508. Grigoriev, I. V., Cullen, D., Goodwin, S. B., Hibbett, D., Jeffries, T. W., Kubicek, C. P., et al. (2011). Fueling the future with fungal genomics. Mycology, 2, 192–209. Han, K.-H., Lee, D.-B., Kim, J.-H., Kim, M.-S., Han, K.-Y., Kim, W.-S., et al. (2003). Environmental factors affecting development of Aspergillus nidulans. Journal of Microbiol- ogy, 41, 34–40. Hawksworth, D. L., Crous, P. W., Redhead, S. A., Reynolds, D. R., Samson, R. A., Seifert, K. A., et al. (2011). The Amsterdam declaration on fungal nomenclature. IMA Fungus, 2, 105–112. Henk, D. A., Eagle, C. E., Brown, K., van den Berg, M. A., Dyer, P. S., Peterson, S. W., et al. (2011). Speciation despite globally overlapping distributions in Penicillium chrysogenum: The population genetics of Alexander Fleming’s lucky fungus. Molecular Ecology, 20, 4288–4301. Henk, D. A., & Fisher, M. C. (2011). Genetic diversity, recombination, and divergence in animal associated Penicillium dipodomyis. PLoS One, 6, e22883. Hoff, B., Po¨ggeler, S., & Ku¨ck, U. (2008). Eighty years after its discovery, Fleming’s Peni- cillium strain discloses the secret of its sex. Eukaryotic Cell, 7, 465–470. Hong, S.-B., Lee, M., Kim, D.-H., Meijer, M., Majoor, E., Vankuyk, P. A., et al. (2012). Aspergillus cibarius sp. nov., from traditional meju in Korea. Journal of Microbiology, 50, 712–714. Hong, S.-B., Lee, M., Kim, D.-H., Varga, J., Frisvad, J. C., Perrone, G., et al. (2013). Aspergillus luchuensis, an industrially important black Aspergillus in East Asia. PLoS One, 8, e63769. Horie, Y., Miyaji, M., Nishimura, K., Franco, M. F., & Coelho, K. I. R. (1995). New and interesting species of Neosartorya from Brazilian soil. Mycoscience, 36, 199–204. 244 Jos Houbraken et al.

Horn, B. W., Moore, G. G., & Carbone, I. (2009). Sexual reproduction in Aspergillus flavus. Mycologia, 101, 423–429. Horn, B. W., Moore, G. G., & Carbone, I. (2011). Sexual reproduction in aflatoxin- producing Aspergillus nomius. Mycologia, 103, 174–183. Horn, B., Olarte, R., Peterson, S. W., & Carbone, I. (2013). Sexual reproduction in Asper- gillus tubingensis from section Nigri. Mycologia, 105, 1153–1163. Horn, B. W., Ramirez-Prado, J. H., & Carbone, I. (2009a). The sexual state of Aspergillus parasiticus. Mycologia, 101, 275–280. Horn, B. W., Ramirez-Prado, J. H., & Carbone, I. (2009b). Sexual reproduction and recombination in the aflatoxin-producing fungus Aspergillus parasiticus. Fungal Genetics and Biol- ogy, 46, 169–175. Houbraken, J., Frisvad, J. C., & Samson, R. A. (2010). Sex in Penicillium series Roqueforti. IMA Fungus, 2, 171–180. Houbraken, J., Frisvad, J. C., & Samson, R. A. (2011a). Taxonomy of Penicillium section Citrina. Studies in Mycology, 70, 53–138. Houbraken, J., Frisvad, J. C., & Samson, R. A. (2011b). Fleming’s penicillin producing strain is not Penicillium chrysogenum but P. rubens. IMA Fungus, 2, 87–92. Houbraken, J., Frisvad, J. C., Seifert, K. A., Overy, D., Tuthill, D. M., Valdez, J. G., et al. (2012). New penicillin-producing Penicillium species and an overview of section Chrysogena. Persoonia, 29, 78–100. Houbraken, J., Giraud, S., Meijer, M., Bertout, S., Frisvad, J. C., Meis, J. F., et al. (2013). Taxonomy and antifungal susceptibility of clinically important Rasamsonia species. Journal of Clinical Microbiology, 51, 22–30. Houbraken, J., & Samson, R. A. (2011). Phylogeny of Penicillium and the segregation of Tri- chocomaceae into three families. Studies in Mycology, 70, 1–51. Houbraken, J., Spierenburg, H., & Frisvad, J. C. (2012). Rasamsonia, a new genus comprising thermotolerant and thermophilic Talaromyces and Geosmithia species. Antonie van Leeu- wenhoek, 101, 403–421. Houbraken, J., Varga, J., Rico-Munoz, E., Johnson, S., & Samson, R. A. (2008). Sexual reproduction as the cause of heat resistance in the food spoilage fungus Byssochlamys spectabilis (anamorph Paecilomyces variotii). Applied and Environmental Microbiology, 74, 1613–1619. Hubka, V., & Kolarik, M. (2012). b-tubulin paralogue tubC is frequently misidentified as the benA gene in Aspergillus section Nigri taxonomy: Primer specificity testing and taxonomic consequences. Persoonia, 29, 1–10. Hubka, V., Kolarik, M., Kubatova, A., & Peterson, S. W. (2013). Taxonomic revision of the genus Eurotium and transfer of species to Aspergillus. Mycologia, 105, 912–937. Hubka, V., Peterson, S. W., Frisvad, J. C., Yaguchi, T., Kuba´tova´, A., & Koları´k, M. (2012). Aspergillus waksmanii sp. nov. and Aspergillus marvanovae sp. nov., two closely related species in section Fumigati described using polyphasic approach. International Journal of Sys- tematic and Evolutionary Microbiology, 63, 783–789. Jeya, M., Joo, A. R., Lee, K. M., Tiwari, M. K., Lee, K. M., Kim, S. H., et al. (2010). Char- acterization of b-glucosidase from a strain of Penicillium purpurogenum KJS506. Applied Microbiology and Biotechnology, 86, 1473–1484. Jurjevic´,Zˇ ., Peterson, S. W., Stea, G., Solfrizzo, M., Varga, J., Hubka, V., et al. (2012). Two novel species of Aspergillus section Nigri from indoor air. IMA Fungus, 3, 159–173. Kozakiewicz, Z., Frisvad, J. C., Hawksworth, D. L., Pitt, J. I., Samson, R. A., & Stolk, A. C. (1992). Proposals for nomina specifica conservanda and rejicienda in Aspergillus and Pen- icillium (Fungi). Taxon, 41, 109–113. Kozlakidis, Z., Herrero, N., Ozkan, S., Kanhayuwa, L., Jamal, A., Bhatti, M. F., et al. (2013). Sequence determination of a quadripartite dsRNA virus isolated from Aspergillus foetidus. Archives of Virology, 158, 267–272. Taxonomy of Aspergillus and Penicillium 245

Kumar, Y. S., Kumar, P. V., & Reddy, O. V. S. (2012). Pectinase production from mango peel using Aspergillus foetidus and its application in processing of mango juice. Food Bio- technology, 26, 107–123. Kwon-Chung, K. J., & Kim, S. J. (1974). A second heterothallic Aspergillus. Mycologia, 66, 628–638. Kwon-Chung, K. J., Weeks, R. J., & Larsh, H. W. (1974). Studies on Emmonsiella capsulata (Histoplasma capsulatum). II. Distribution of the two mating types in 13 endemic states of the United States. American Journal of Epidemiology, 99, 44–49. Lee, C. Z., Liou, G. Y., & Yuan, G. F. (2006). Comparison of the aflR gene sequences of strains in Aspergillus section Flavi. Microbiology, 152, 161–170. Link, H. F. (1809). Observationes in Ordines plantarum naturales, Dissertation 1ma (Berlin Ges. NatKde 3: 1–42), Berlin. Liu, G., Zhang, L., Wei, X., Zou, G., Qin, Y., Ma, L., et al. (2013). Genomic and secretomic analyses reveal unique features of the lignocellulolytic enzyme system of Penicillium decumbens. PLoS One, 8(2), e55185. Llanos, A., Neugnot-Roux, V., Parrou, J.-L., Alcocer, M., Franc¸oi, J. M., & Archer, D. (2012). Regulation of the expression of genes encoding glycoside hydrolases in Pen- icillium funiculosum.In11th European Conference on fungal genetics, programme & abstract book. LoBuglio, K. F., & Taylor, J. W. (1993). Molecular phylogeny of Talaromyces and Penicillium species in subgenus Biverticillium. In D. R. Reyolds & J. W. Taylor (Eds.), The fungal holomorph: mitotic, meiotic and pleomorphic speciation in fungal systematic (pp. 115–119). Surrey: C.A.B., International. LoBuglio, K. F., Pitt, J. I., & Taylor, J. W. (1993). Phylogenetic analysis of two ribosomal DNA regions indicates multiple independent losses of a sexual Talaromyces state among asexual Penicillium species in subgenus Biverticillium. Mycologia, 85, 592–604. Lo´pez-Villavicencio, M., Aguileta, G., Giraud, T., de Vienne, D. M., Lacoste, S., Couloux, A., et al. (2010). Sex in Penicillium: Combined phylogenetic and experimental approaches. Fungal Genetics and Biology, 47, 693–706. Luangsa-ard, J. J., Hywel-Jones, N. L., & Samson, R. A. (2004). The polyphyletic nature of Paecilomyces sensu lato based on 18S generated rDNA phylogeny. Mycologia, 96, 773–780. Lund, F., Nielsen, A. B., & Skouboe, P. (2003). Distribution of Penicillium commune isolates in cheese dairies mapped using secondary metabolite profiles, morphotypes, RAPD and AFLP fingerprinting. Food Microbiology, 20, 725–734. Machida, M., Asai, K., Sano, M., Tanaka, T., Kumagai, T., et al. (2005). Genome sequencing and analysis of Aspergillus oryzae. Nature, 438, 1157–1161. Machida, M., Koike, H., Kikuzato, I., Fujimori, K. E., Teruya, M., Tsukahara, M., et al. (2010). Comparative analysis of koji mold’s genomes. In The 7th International Aspergillus Meet- ing “Asperfest 7”, NH Conference Centre, Leeuwenhorst, The Netherlands, Poster abstract 19, http://www.fgsc.net/Aspergillus/7thAsperfest/Asperfest7Program.pdf. Machouart, M., Garcia-Hermoso, D., Rivier, A., Hassouni, N., Catherinot, E., Salmon, A., et al. (2011). Emergence of disseminated infections due to Geosmithia argillacea in patients with chronic granulomatous disease receiving long-term azole antifungal prophylaxis. Journal of Clinical Microbiology, 49, 1681–1683. Malloch, D. (1985). The Trichocomaceae: Relationships with other Ascomycetes.In R. A. Samson & J. I. Pitt (Eds.), Advances in Penicillium and Aspergillus systematics (pp. 365–382). New York: Plenum Press. Malloch, D., & Cain, R. F. (1972). New species and combinations in cleistothecial Ascomy- cetes. Canadian Journal of Botany, 50, 61–72. Mapari, S. A. S., Meyer, A. S., Thrane, U., & Frisvad, J. C. (2009). Identification of poten- tially safe promising fungal cell factories for the production of polyketide natural food colorants using chemotaxonomic rationale. Microbial Cell Factories, 8, 24. 246 Jos Houbraken et al.

Martin, T., Lu, S. W., van Tilbeurgh, H., Ripoll, D. R., Dixelius, C., Turgeon, B. G., et al. (2010). Tracing the origin of the fungal a1 domain places its ancestor in the HMG-box superfamily: Implication for fungal mating-type evolution. PLoS One, 5(12), e15199. Matsuzawa, T., Campos Takaki, G. B., Yaguchi, T., Okada, K., Gonoi, T., & Horie, Y. (2013). Two new species of Aspergillus section Fumigati isolated from caatinga soil in the State of Pernambuco, Brazil. Mycoscience, http://dx.doi.org/10.1016/j.myc.2013.04.001. McHunu, N. P., Permaul, K., Abdul Rahman, A. Y., Saito, J. A., Singh, S., & Alam, M. (2013). Xylanase superproducer: Genome sequence of a compost-loving thermophilic Fungus. Thermomyces lanuginosus strain SSBP. Genome Announcements, http://dx.doi. org/10.1128/genomeA.00388-13. Meijer, M., Houbraken, J., Dalhuijsen, S., Samson, R. A., & de Vries, R. P. (2011). Growth and hydrolase profiles can be used as characteristics to distinguish Aspergillus niger and other black aspergilli. Studies in Mycology, 69, 19–30. Mogensen, J. M., Varga, J., Thrane, U., & Frisvad, J. C. (2009). Aspergillus acidus from Puerh tea and black tea does not produce ochratoxin A and fumonisin B2. International Journal of Food Microbiology, 132, 141–144. Moore, E. R. B., Mihaylova, S. A., Vandamme, P., Krichevsky, M. I., & Dijkshoorn, L. (2010). Microbial systematics and taxonomy: Relevance for a microbial commons. Research in Microbiology, 161, 430–438. Morgenstern, I., Powlowski, J., Ishmael, N., Darmond, C., Marqueteau, S., Moisan, M. C., et al. (2012). A molecular phylogeny of thermophilic fungi. Fungal Biology, 116, 489–502. Mouchacca, J. (1997). Thermophilic fungi: Biodiversity and taxonomic status. Cryptogamie Mycologie, 18, 19–69. Norvell, L. L. (2011). Fungal nomenclature. 1. Melbourne approves a new code. Mycotaxon, 116, 481–490. Nova´kova´, A., Hubka, V., Dudova´, Z., Matsuzawa, T., Kuba´tova´, A., Yaguchi, T., & Kolarˇı´k, M. (2013). New species in Aspergillus section Fumigati from reclamation sites in Wyoming (U.S.A.) and revision of A. viridinutans complex. Fungal Diversity, http:// dx.doi.org/10.1007/s13225-013-0262-5. O’Gorman, C. M., Fuller, H. T., & Dyer, P. S. (2009). Discovery of a sexual cycle in the opportunistic fungal pathogen Aspergillus fumigatus. Nature, 457, 471–474. Ogawa, H., & Sugiyama, J. (2000). Evolutionary relationships of the cleistothecial genera with Penicillium, Geosmithia, Merimbla and Sarophorum anamorphs as inferred from 18S rDNA sequence divergence. In R. A. Samson & J. I. Pitt (Eds.), Integration of modern taxonomic methods for Penicillium and Aspergillus classification (pp. 149–161). New York: Plenum Press. Paoletti, M., Seymour, F. A., Alcocer, M. J., Kaur, N., Calvo, A. M., Archer, D. B., et al. (2007). Mating type and the genetic basis of self-fertility in the model fungus Aspergillus nidulans. Current Biology, 17, 1384–1389. Perrone, G., Stea, G., Epifani, F., Varga, J., Frisvad, J. C., & Samson, R. A. (2011). Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biology, 115, 1138–1150. Peterson, S. W. (2000). Phylogenetic analysis of Penicillium species based on ITS and LSU- rDNA nucleotide sequences. In R. A. Samson & J. I. Pitt (Eds.), Integration of modern taxonomic methods for Penicillium and Aspergillus classification (pp. 163–178). New York: Plenum Press. Peterson, S. W. (2008). Phylogenetic analysis of Aspergillus species using DNA sequences from four loci. Mycologia, 100, 205–226. Peterson, S. W., Varga, J., Frisvad, J. C., & Samson, R. A. (2008). Phylogeny and subgeneric taxonomy of Aspergillus. In J. Varga & R. A. Samson (Eds.), Aspergillus in the genomic era (pp. 33–56). Wageningen: Wageningen Academic Publishers. Taxonomy of Aspergillus and Penicillium 247

Pitt, J. I. (1980). The genus Penicillium and its teleomorphic states Eupenicillium and Talaromyces. London: Academic Press. Ramı´rez, C. (1982). Manual and atlas of the Penicillia. Amsterdam: Elsevier Biomedical Press. Raper, K. B. (1957). Nomenclature in Aspergillus and Penicillium. Mycologia, 49, 644–662. Raper, K. B., & Fennell, D. I. (1965). The genus Aspergillus. Baltimore: Williams & Wilkins Co. Raper, K. B., & Thom, C. (1949). A manual of the Penicillia. Baltimore: Williams & Wilkins co. Riul, A. J., Gonc¸alves, H. B., Jorge, J. A., & Guimara˜es, L. H. S. (2013). Characterization of a glucose- and solvent-tolerant extracellular tannase from Aspergillus phoenicis. Journal of Molecular Catalysis B: Enzymatic, 85–86, 126–133. Rokas, A., Payne, G., Fedorova, N. D., Baker, S. E., Machida, M., Yu, J., et al. (2007). What can comparative genomics tell us about species concepts in the genus Aspergillus? Studies in Mycology, 59, 11–17. Samson, R. A. (1974). Paecilomyces and some allied hyphomycetes. Studies in Mycology, 6, 1–119. Samson, R. A., Hadlok, R., & Stolk, A. C. (1977). A taxonomic study of the Penicillium chrysogenum series. Antonie van Leeuwenhoek, 43, 169–175. Samson, R. A., Houbraken, J., Thrane, U., Frisvad, J. C., & Andersen, B. (2010). Food and indoor fungi, CBS laboratory manual series 2. Utrecht: CBS-Fungal Biodiversity Centre. Samson, R. A., Houbraken, J., Varga, J., & Frisvad, J. C. (2009). Polyphasic taxonomy of the heat resistant ascomycete genus Byssochlamys and its Paecilomyces anamorphs. Persoonia, 22, 14–27. Samson, R. A., Seifert, K. A., Kuijpers, A. F. A., Houbraken, J. A. M. P., & Frisvad, J. C. (2004). Phylogenetic analysis of Penicillium subgenus Penicillium using partial b-tubulin sequences. Studies in Mycology, 49, 175–200. Samson, R. A., Yilmaz, N., Houbraken, J., Spierenburg, H., Seifert, K. A., Peterson, S. W., et al. (2011). Phylogeny and nomenclature of the genus Talaromyces and taxa accommodated in Penicillium subgenus Biverticillium. Studies in Mycology, 70, 159–183. Schleifer, K.-H., & Tru¨per, H. G. (2006). Prokaryote characterization and identification. In M. Dworkin, S. Falkow, E. Rosenberg, K.-H. Schleifer & E. Stackebrandt (Eds.), The prokaryotes: Vol. 1. (pp. 58–79) (3rd ed.). New York: Springer. Schoch, C. L., Seifert, K. A., Huhndorf, S., Robert, V., Spouge, J. L., Levesque, C. A., et al. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the United States, 109, 6241–6246. Schuster, E., Dunn-Coleman, N., Frisvad, J. C., & van Dijck, P. W. (2002). On the safety of Aspergillus niger—A review. Applied Microbiology and Biotechnology, 59, 426–435. Skouboe, P., Frisvad, J. C., Lauritsen, D., Boysen, M., Taylor, J. W., & Rossen, L. (1999). Nucleotide sequences from the ITS region of Penicillium species. Mycological Research, 103, 873–881. Soares, C., Rodrigues, P., Peterson, S. W., Lima, N., & Venaˆncio, A. (2012). Three new species of Aspergillus section Flavi isolated from almonds and maize in Portugal. Mycologia, 104, 682–697. Steiner, J., Socha, C., & Eyzaguirre, J. (1994). Culture conditions for enhanced cellulase production by a native strain of Penicillium purpurogenum. World Journal of Microbiology and Biotechnology, 10, 280–284. Stolk, A. C. (1965). Thermophilic species of Talaromyces Benjamin and Thermoascus Miehe. Antonie van Leeuwenhoek, 31, 262–276. Stolk, A. C., & Samson, R. A. (1972). Studies on Talaromyces and related genera II: The genus Talaromyces. Studies in Mycology, 2, 1–65. Stolk, A. C., & Samson, R. A. (1985). A new taxonomic scheme for Penicillium anamorphs. In R. A. Samson & J. I. Pitt (Eds.), Advances in Penicillium and Aspergillus systematic (pp. 163–192). New York: Plenum Press. 248 Jos Houbraken et al.

Swilaiman, S. S., O’Gorman, C. M., Balajee, S. A., & Dyer, P. S. (2013). Discovery of a sexual cycle in Aspergillus lentulus, a close relative of A. fumigatus. Eukaryotic Cell, 12, 962–969. Takada, M., Horie, Y., & Abliz, P. (2001). Two new heterothallic Neosartorya from African soil. Mycoscience, 42, 361–367. Takada, M., & Udagawa, S.-I. (1985). A new species of heterothallic Neosartorya. Mycotaxon, 24, 395–402. Takada, M., & Udagawa, S. (1988). A new species of heterothallic Talaromyces. Mycotaxon, 31, 417–425. Taniwaki, M. H., Pitt, J. I., Iamanaka, B. T., Sartori, D., Copetti, M. V., Balajee, A., et al. (2012). Aspergillus bertholletius sp. nov. from brazil nuts. PLoS One, 7, e42480. Thom, C. (1910). Cultural studies of Penicillium (Vol. 118, pp. 1–109). Washington: Govern- ment Printing Office: Bureau of Animal Industry, US Department of Agriculture. Thom, C. (1930). The Penicillia. Baltimore: Williams & Wilkins, 1–644. Thom, C., & Church, M. B. (1926). The Aspergilli. Baltimore: Williams & Wilkins Co., 1–272. Thom, C. (1945). Mycology presents penicillin. Mycologia, 37, 460–475. Tominaga, M., Lee, Y. H., Hayashi, R., Suzuki, Y., Yamada, O., Sakamoto, K., et al. (2006). Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Applied and Environmental Microbiology, 72, 484–490. Tsiklinsky, P. (1899). Sur les mucedinees thermophiles. Annales de l’Institut Pasteur, Paris, 13, 500–504. Tulasne, L. R. (1851). Note sur l’appareil reproducteur dans les lichens et les champignons (1ere partie). Comptes rendus de l’Acade´mie des Sciences, Paris, 32, 427–430. Turgeon, B. G., & Yoder, O. C. (2000). Proposed nomenclature for mating type genes of filamentous ascomycetes. Fungal Genetics and Biology, 31, 1–5. Tuthill, D. (2004). Genetic variation and recombination in Penicillium miczynskii and Eupenicillium species. Mycological Progress, 3, 3–12. van den Berg, M. A., Albang, R., Albermann, K., Badger, J. H., Daran, J. M., Driessen, A. J., et al. (2008). Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nature Biotechnology, 26, 1161–1168. van Dijck, P. W. M. (2008). The importance of Aspergilli and regulartory aspects of Asper- gillus nomenclature in biotechnology. In J. Varga & R. A. Samson (Eds.), Aspergillus in the genomic era (pp. 249–257). Wageningen: Wageningen Academic Publishers. Varga, J., Frisvad, J. C., Kocsube´, S., Brankovics, B., To´th, B., Szigeti, G., et al. (2011). New and revisited species in Aspergillus section Nigri. Studies in Mycology, 69, 1–17. Varga, J., Frisvad, J. C., & Samson, R. A. (2010). Aspergillus sect. Aeni sect. nov., a new section of the genus for A. karnatakaensis sp. nov. and some allied fungi. IMA Fungus, 1, 197–205. Varga, J., Frisvad, J. C., & Samson, R. A. (2011). Two new aflatoxin producing species, and an overview of Aspergillus section Flavi. Studies in Mycology, 69, 57–80. Wagschal, K., Yoshizawa, Y., Witter, D. J., Liu, Y., & Vederas, J. C. (1996). Biosynthesis of ML-236C and the hypocholesterolemic agents compactin by Penicillium aurantiogriseum and lovastatin by Aspergillus terreus: Determination of the origin of carbon, hydrogen and oxygen atoms by 13C NMR spectrometry and observation of unusual labelling of acetate-derived oxygens by 18O2. Journal of the Chemical Society Perkin Transactions I, 1996, 2357–2363. Westling, R. (1911). U¨ ber die Gru¨nen Spezies der Gattung Penicillium. Arkiv før Botanik, 11, 1–156. Woo, P. C., Lau, S. K., Liu, B., Cai, J. J., Chong, K. T., Tse, H., et al. (2011). Draft genome sequence of Penicillium marneffei strain PM1. Eukaryotic Cell, 10, 1740–1741. Taxonomy of Aspergillus and Penicillium 249

Xing, W., Deng, C., & Hu, C. H. (2010). Molecular cloning and characterization of the global regulator LaeA in Penicillium citrinum. Biotechnological Letters, 32, 1733–1737. Yaguchi, T., Someya, A., & Udagawa, S. (1994). Two new species of Talaromyces from Tai- wan and Japan. Mycoscience, 35, 249–255. Yamada, O., Takara, R., Hamada, R., Hayashi, R., Tsukahara, M., & Mikami, S. (2011). Molecular biological researches of Kuro-Koji molds, their classification and safety. Journal of Bioscience and Bioengineering, 112, 233–237. Yilmaz, N., Houbraken, J., Hoekstra, E. S., Frisvad, J. C., Visagie, C. M., & Samson, R. A. (2012). Delimitation and characterisation of Talaromyces purpurogenus and related species. Persoonia, 29, 39–54. Zaleski, K. M. (1927). U¨ ber die in Polen gefundenen Arten der Gruppe Penicillium Link. I, II and III Teil. Bulletin de l’Acade´mie Polonaise des Sciences et des Lettres, Classe des Sciences Mathe´matiques et Naturelles—Se´rie B: Sciences Naturelles, 1927, 417–563, pls 36–44 (printed in 1928). Zhang, H., Wu, M., Li, J., Gao, S., & Yang, Y. (2012). Cloning and expression of a novel xylanase gene (Auxyn11D) from Aspergillus usamii E001 in Pichia pastoris. Applied Biochem- istry and Biotechnology, 167, 2198–2211. Zou, S., Xie, L., Liu, Y., Kaleem, I., Zhang, G., & Li, C. (2012). N-linked glycosylation influences on the catalytic and biochemical properties of Penicillium purpurogenum b-d-glucuronidase. Journal of Biotechnology, 157, 399–404. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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