In Press at Mycologia, preliminary version published on October 24, 2014 as doi:10.3852/14-059

Short title: Revision of Aspergillus flavipes Revision of Aspergillus section Flavipedes: seven new species and proposal of section Jani sect. nov. Vit Hubka1 Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, 128 01 Prague 2, Czech Republic Institute of Microbiology AS CR, v.v.i, Vídeňská 1083, 142 20 Prague 4, Czech Republic Alena Nováková Institute of Microbiology AS CR, v.v.i, Vídeňská 1083, 142 20 Prague 4, Czech Republic, and Institute of Soil Biology, Biology Centre AS CR, v.v.i., Na Sádkách 7, 370 05 České Budějovice, Czech Republic Miroslav Kolařík Department of Botany, Faculty of Science, Charles University in Prague, Benátská 2, 128 01 Prague 2, Czech Republic, and Institute of Microbiology AS CR, v.v.i, Vídeňská 1083, 142 20 Prague 4, Czech Republic Željko Jurjević EMSL Analytical Inc., 200 Route 130 North, Cinnaminson, New Jersey 08077 Stephen W. Peterson Bacterial Foodborne Pathogens and Mycology Research Unit, National Center for Agricultural Utilization Research, 1815 N. University Street, Peoria, IL 61604 Abstract: Aspergillus section Flavipedes contains species found worldwide in soils and rhizospheres, indoor and cave environments, as endophytes, food contaminants and occasionally as human pathogens. They produce many extensively studied bioactive secondary metabolites and biotechnologically relevant enzymes. The taxa were revised based on phylogenetic analysis of sequences from four loci (β-tubulin, calmodulin, RPB2, ITS

Copyright 2014 by The Mycological Society of America.

rDNA), two PCR fingerprinting methods, micro- and macromorphology and physiology. Section Flavipedes includes three known and seven new species: A. ardalensis, A. frequens, A. luppii, A. mangaliensis, A. movilensis, A. polyporicola and A. spelaeus. The name A. neoflavipes was proposed for Fennellia flavipes a distinct species from its supposed asexual state A. flavipes. Aspergillus iizukae, A. frequens and A. mangaliensis are the most common and widely distributed species, whereas A. flavipes s. str. is rare. A dichotomous key based on the combination of morphology and physiology is provided for all recognized species. Aspergillus section Jani is established to contain A. janus and A. brevijanus, species previously classified as members of sect. Versicolores, Terrei or Flavipedes. This new section is strongly supported by phylogenetic data and morphology. Section Jani species produce three types of conidiophores and conidia, and colonies have green and white sectors making them distinctive. Accessory conidia found in pathogenic A. terreus were found in all members of sects. Flavipedes and Jani. Our data indicated that A. frequens is a clinically relevant and produces accessory conidia during infection. Key words: Aspergillus flavipes, cave mycobiota, Fennellia, multilocus phylogeny, PCR fingerprinting, soil fungi INTRODUCTION The Aspergillus flavipes group (Thom and Church 1926) originally contained only A. flavipes while Raper and Fennell (1965) placed A. flavipes, A. carneus and A. niveus in the group. Gams et al. (1985) erected Aspergillus section Flavipedes to contain species of the informal A. flavipes group. Peterson (2000, 2008) using phylogenetic analysis of DNA sequence data showed that A. carneus and A. niveus are in a distinct clade and that the monophyletic sect. Flavipedes contained A. flavipes, A. aureofulgens, A. iizukae and a number of undescribed species.

The morphological species concept has been dominantt in Fungi over the centuries. The advent of relatively easy DNA sequencing has put those data at our service also. Schoch et al. (2012) proposed the use of nuclear ITS sequences for BarCode identification of fungal species. Others have included additional loci such at β-tubulin or calmodulin to confidently identify isolates (Peterson 2012). While DNA sequence analysis is perhaps the most accurate method of species identification, it is still worthwhile to provide descriptions that allow for recognition of the species with morphological criteria. Sect. Flavipedes members are distributed in soil worldwide, particularly in subtropical and tropical soils (Klich 2002, Domsch et al. 2007). Species also are isolated from foods, indoor environments and as endophytes. Several cases of human and animal infections attributed to A. flavipes have been reported, including osteomyelitis, cutaneous aspergillosis, otomycosis, onychomycosis and diskospondylitis (Stuart and Blank 1955, Roselle and Baird 1979, Barson and Ruymann 1986, Schultz et al. 2008, Gehlot et al. 2011). The ability of A. flavipes to cause invasive infections was proved experimentally (Pore and Larsh 1968). Aspergillus flavipes and related species produce a rich spectrum of secondary metabolites including the mycotoxins sterigmatocystin (Tuomi et al. 2000) and citrinin (Gorst-Allman and Steyn 1979, Greenhill et al. 2008) and the valuable cholesterol lowering drug lovastatin (Valera et al. 2005). Isolates from sect. Flavipedes, A. janus, A. brevijanus and closely related species were studied by phylogenetic analysis of DNA sequence data from four loci, PCR fingerprinting, morphology and physiology. We revised the taxonomy of these strains resulting in the recognition of 10 species in sect. Flavipedes and we described Aspergillus section Jani to contain A. janus and A. brevijanus. MATERIAL AND METHODS Phenotypic studies.—Isolates were grown at 25 C on malt extract agar (MEA; malt extract from Fluka Chemie GmbH, Switzerland, as primary medium; species also were inoculated on MEA prepared with malt extract from

Oxoid, Basingstoke, UK, for comparison), Czapek Yeast Autolysate agar (CYA; yeast extract from Fluka Chemie GmbH, Switzerland) and Czapek-Dox agar (CZA) formulated with the methods of Frisvad and Samson (2004). Lactophenol cotton blue mounting medium was used for observation of micromorphology. Microphotography was conducted with an Olympus BX-51 microscope with Olympus DP72 camera and Nomarski contrast. Macromorphology of the colonies was documented with a Canon 500D camera or an Olympus SZ61stereo microscope with camera. Staining of heat-killed fungal conidia with propidium iodide (Fluka) was used to observe nuclear numbers. Macro- and micromorphology of all species was described after 14 d incubation when the colony color was fully expressed and all typical features were present. Colonies after 7 d are mostly whitish and similar appearing across different species. Color determination was made with the ISCC-NBS Centroid Color Charts (Kelly 1964) and color numbers are mentioned only at the first occasion in each description. Ascospores and Hülle cells were measured and documented after 3–4 wk cultivation. When possible all presented dimensions were based on at least 30 measurements for each isolate (TABLE I). Ability to grow at 37, 40 and 43 C was tested on CYA plates; experiments were conducted independently in two incubators and were repeated twice. Growth at 40 C also was tested on M40Y medium (Raper and Fennell 1965). All plates incubated at temperatures greater than 37 C were sealed with parafilm and placed in a plastic bag to prevent dessication. Acid production was assessed with creatine sucrose agar (Samson et al. 2007). Molecular studies and phylogenetic analysis.—DNA was extracted from 7 d old colonies with ArchivePure DNA yeast and Gram2+ kit (5PRIME Inc., Gaithersburg, Maryland) with modified time of incubation; lytic enzyme solution (2 h, 37 C) and cell lysis solution (4 h, 64 C). PCR fingerprinting, using the phage M13-core oligonucleotide primer and primer 834t, was performed and evaluated as described by Hubka and Kolařík (2012), Nováková et al. (2012a, 2014)). The ITS region of rDNA was amplified with primers ITS5 and ITS4S; partial β-tubulin (benA) with primers Bt2a and Bt2b; partial calmodulin gene (caM) with primers CF1M or CF1L and CF4, and partial RPB2 gene (encoding RNA polymerase II) with primers fRPB2-5F and fRPB2-7cR as described by Hubka et al. (2012) except the that RPB2 gene was amplified with Touchdown cycling conditions as described by Hubka and Kolařík (2012). Sequences were inspected and assembled using BioEdit 7.0.0 (www.mbio.ncsu.edu/BioEdit/bioedit.html). DNA sequences were aligned with Clustal W implemented in MEGA 5.2 (Tamura et al. 2011). The alignments were deposited in TreeBASE (submission ID 16067). The best model for analysis was determined in

MEGA, and phylogenetic trees were calculated with maximum likelihood. Statistical support for tree nodes was calculated with 500 bootstrap iterations. Resulting trees were saved as extended metafiles and formatted for publication with CorelDraw X6.

RESULTS Molecular studies.—Fourteen fingerprinting patterns, with two subtypes in pattern IV and three in pattern XI, were observed among isolates from sects. Flavipedes and Jani (TABLE I) using the M13-core primer. Fourteen patterns with two subtypes in patterns III, VI and XI and three subtypes in pattern IV were observed with primer 834t (TABLE I). The results were generally in agreement with species boundaries established by sequence data, morphology and physiology. Dissimilar fingerprinting patterns were obtained for two isolates of A. movilensis and A. janus when using either M13-core or 834t primers. The single-locus trees (benA, calmodulin, RPB2, ITS) constructed using Maximum likelihood method (ML; SUPPLEMENTARY FIGS. 1–4) had highly congruent topology and varied in the degree of statistical support at each node. The coding data from benA, calmodulin and RPB2 loci were combined into one dataset and an ML phylogenetic tree was constructed for Aspergillus subg. Circumdati (FIG. 1). Phylogenetic analysis revealed that A. janus and A. brevijanus form a strongly supported clade separate from sect. Flavipedes and basal to sect. Terrei (FIG. 1). This demonstrated the need to create a new section for these two species to maintain monophyletic sections. Ten independent lineages found among sect. Flavipedes isolates formed two main clades (designated A. flavipes and A. spelaeus clades) in the combined tree (FIG. 1) as they do in single-locus trees based on benA, calmodulin, RPB2 (SUPPLEMENTARY FIG. 1–3). Four of these lineages were represented by the described species A. flavipes, Fennellia flavipes, A. iizukae and A. aureofulgens, and the remaining lineages are described here as new species. The sequence differences between sister species measured at the benA, calmodulin and RPB2 loci often exceed 2–3% (SUPPLEMENTARY TABLE I), values commonly seen between

well defined Aspergillus sister species. The species with the lowest genetic distances were A. spelaeus and A. polyporicola at 2.1–3.7%. Both species also had different fingerprinting patterns and ecology supporting their status as separate species. BenA sequence of A. movilensis isolates CCF 4410T and NRRL 4610 exhibited 4.8% difference (24/497 bp), 3.0% calmodulin sequence difference (23/771 bp) and different fingerprinting patterns. The RPB2 locus had 0.6% difference (6/1014 bp) and the growth parameters were comparable. The morphology of NRRL 4610 that was treated as A. carneus by Raper and Fennell (1965) seems to be affected by long-term preservation and it was not possible to definitely resolve the species boundaries with our limited number of isolates. NRRL 4610 compared to CCF 4410 showed cottony colonies, thinner and septate stipes 2– 3(–5) μm, smaller vesicles, 3–7(–16) μm diam, did not create Hülle cells and its colonies have a pink tint, while CCF 4410 has white colonies that become brownish with age. Colonies diameter and color on MEA differed between two isolates of A. janus NRRL 1797T and NRRL 1936, both isolates had different fingerprinting patterns and the sequences of calmodulin and RPB2 genes revealed 2.1% (16/767 bp) and 1.6% (16/1014 bp) difference respectively. With two isolates we could not verify if they represent different species or whether these phenotypic and molecular differences were within the limits of intraspecific variability. Only four of the 10 species in sect. Flavipedes and both species in sect. Jani can be unambiguously distinguished by the ITS barcoding region. Both A. polyporicola isolates had a single unique position in the ITS region when compared with isolates of A. spelaeus; two unique positions differentiated the ex-type isolates of A. ardalensis and A. flavipes; the extype isolate of A. neoflavipes has a single unique position compared to A. frequens isolates. ITS sequencing of additional isolates will reveal the universality of these few unique positions between the species. Low discriminatory power of the ITS locus is common in Aspergillus

(Samson et al. 2011a, Nováková et al. 2012a, Jurjevic et al. 2012, Peterson 2012, Hubka et al. 2013a, Hubka and Kolařík 2012, Hubka et al. 2013b, Hubka et al. 2014). Phenotypic species differentiation.—Section Jani has strong morphological and phylogenetic support. Aspergillus janus and A. brevijanus produce three different types of conidiophores, two of them are biseriate and produce phialidic conidia and the third type is represented by micro- to semimacronematous conidiophores producing accessory conidia. Conidia produced by these different conidiophores are globose, echinulate, green, globose to elliptical, hyaline, smooth, and larger, globose to elliptical, truncate and hyaline respectively. The occurence of green conidial heads that influence the color of the colony or create green sectors together with three types of conidiophores has no equivalent in subg. Circumdati. Various characters were shown to be valuable for species recognition in sect. Flavipedes. Aspegillus spelaeus and A. polyporicola can be differentiated from all other species in sect. Flavipedes by their inability to grow at 37 C (TABLE II). The other two species in the A. spelaeus clade (FIG. 1), A. luppii and A. movilensis, grow markedly slower at 37 C than at 25 C and fail to grow on CYA at 40 C. Species from the A. flavipes clade (FIG. 1) all grow well at 37 C, three of them are able to grow on CYA at 40 C (TABLE II) and all grow at 40 C on M40Y. No species in the section grew on CYA at 43 C. Some species that fail to grow on CYA at 40 C are able to grow on M40Y at 40 C or else grow better on this medium. Colony morphology is useful in distinguishing some species. Brightly yellow colonies are present in A. neoflavipes and A. luppii on all media, colonies of A. movilensis are whitish and those of the remaining species are mostly in shades of brown. Red or brownish soluble pigment is present in some isolate of A. iizukae and A. frequens. Great variability in colony morphology (colony color, production of soluble pigment, exudate) was observed in A. frequens (FIG. 2) and A. iizukae (FIG. 3). However both species had relatively stable physiology and micromorphology.

The shape and diameter of vesicle is another important feature in species differentiation. Those of A. flavipes s. str. are predominantly spathulate, their width not exceeding 11 μm and mostly with a constriction at the base. Other species in the A. flavipes clade have predominanly globose or pyriform vesicles and those of A. iizukae commonly exceed 20 μm diam in contrast to other species. The absence of Hülle cells on all media was observed in three species, A. flavipes, A. spelaeus and A. polyporicola. Hülle cells also are absent in the majority of A. frequens isolates (FIG. 2). Their shape is variable but elongated Hülle cells are predominat across the section except A. luppi that has predominantly globose, elliptical or polygonal Hülle cells. The presence of a second type of conidiophores (a third type for sect. Jani) producing accessory conidia (synonyms in literature: aleurioconidia, secondary conidia, macroconidia) was documented in all species from sects. Flavipedes and Jani. This remarkable feature probably is restricted only to subgenus Circumdati and is typical for sects. Terrei, Flavipedes and Jani. Accesory conidia developed via blastic conidiogenesis often with sympodial proliferation. They often are multinucleated (1–3) and the number of nuclei increased with conidium size. Great variability in shape and dimensions make these structures unsuitable for species differentiation. The length of phialidic conidiophores is another uninformative character, not useful for species identification. Their length is quite variable and at least some conidiophores in almost all species exceed 1 mm. The exception is A. luppii whose stipes do not exceed 300 μm. Other features of secondary importance due to uniformity or overlapping values are length of metulae and phialides and the dimensions and ornamentation of phialidic conidia (smooth and usually 2–3 μm diam). Exposing cultures to light during cultivation notably affects colony texture, color, sporulation rate, production of exudate, accessory conidia and Hülle cells, as observed in this study and by Muntanjola-Cvetković and Vukić (1972). Both Hülle cells and accessory conidia

are absent or rare when isolates are incubated in the light and almost always present when incubated in the dark. Conidiophores producing accessory conidia are more abundant on MEA-Oxoid in contrast to MEA-Fluka, most notably in A. polyporicola; Hülle cells are not produced in A. ardalensis on MEA-Oxoid in contrast to MEA-Fluka; all isolates of A. iizukae produced Hülle cells on MEA-Oxoid in contrast to MEA-Fluka, and white conidial heads are more abundant in sect. Jani isolates grown on MEA-Oxoid versus predominantly green conidial heads on MEA-Fluka. We have not tested other sources of malt extract. The position and differentiation of species with white conidial heads has always been problematic. Misidentification with some species from sect. Candidi is also possible, but the white-spored members of sect. Candidi mostly have radiate or loosely radiate conidial heads and often produce purple, brown or black sclerotia in older cultures. In addition A. candidus and A. pragensis do not grow at 37 C, A. taichungensis grows at 37 C but is unable to grow at 40 C and A. tritici grows at 40 C and does not grow at 43 C. Conidial heads of species from sect. Flavipedes and Terrei with white colonies have mostly loose columnar to columnar conidial heads and often produce macroscopic yellow mycelial masses with Hülle cells. Aspergillus movilensis can grow at 37 C but does not grow at 40 C, and old colonies become light brown. Aspergillus carneus and A. niveus grow very well at 40 C and are able to grow even at 45 C. Older colonies of A. carneus become pink while older colonies of A. niveus are persistently white or turn brown. Some strains included in this study showed signs of degeneration including cottony colonies with less intensive sporulation, conidiophores with smaller vesicles and thinner stipes, and Hülle cells were not produced. These changes are mentioned in particular isolates following description of species in TAXONOMY.

Taxonomy and nomenclature.—Current nomenclature requires the use of only one name for a species (McNeill et al. 2012), whereas previously two or more names could be applied to the same fungus. We continue the trend of using Aspergillus names for pleomorphic species (Samson et al. 2011a, 2011b; Hubka et al. 2013b; Hubka et al. 2013a; Nováková et al. 2014). Fennellia flavipes is transferred to Aspergillus and the other two species of Fennellia were transferred to Aspergillus by Samson et al. (2011a, 2011b). Fennellia flavipes is not conspecific with its supposed anamorph A. flavipes (Peterson 2008) and a new name was required for this species. The new replacement name A. neoflavipes is proposed for F. flavipes. Section Flavipedes Gams et al. 1985, Adv Penicillium Aspergillus System: 59. = Aspergillus flavipes group Thom & Church 1926, The Aspergilli: 155. Aspergillus ardalensis A. Nováková, Hubka, M. Kolařík & S.W. Peterson, sp. nov. FIG. 4 MB808140 Diagnosis: This is the only species in sect. Flavipedes that grows well on CYA at 40 C, other species do not grow or grow restrictedly at this temperature (≤ 2 mm diam after 7 d). Its vesicles are mostly globose with relatively large diameter and it is able to produce Hülle cells. Typification: SPAIN. Andalucia: Ardales, near Cueva de Doña Trinidad, isol. ex soil, 3 Nov 2008, A. Nováková (holotype, dried specimen PRM 923450. Isotype PRM 923451. Exholotype culture: CCF 4031 = CCF 4426 = CMF ISB 1688 = CBS 134372 = NRRL 62824). Etymology: Named after the village of Ardales in the province of Málaga (Andalucia, southern Spain) that is located close to the place of isolation. Description: Colonies at 25 C on CYA attain 42–45 mm diam after 14 d, floccose, plane to sparsely wrinkled, light yellowish brown (ISCC-NBS No. 76) with pale orangeyellow (No. 73) margin, exudate deep orange-yellow (No. 69), reverse strong yellowish brown (No. 74) with paler margin. Colonies on MEA attain 37–42 mm diam after 14 d, plane

to delicately wrinkled, floccose, light yellowish brown, exudate deep orange-yellow, reverse strong yellowish brown. Colonies on CZA attain 38–40 mm diam after 14 d, plane, floccose with granulose colony surface, pale yellowish orange, reverse dark yellowish brown (No. 75). Colonies on CREA attain 42–45 mm diam in 14 d, plane, granulose with higher central part, light yellowish brown with pale yellow (No. 86) mycelial overgrowth in marginal part (containing Hülle cells), exudate deep brown (No. 56), no acid production. Colonies on CYA at 37 C after 7 d attain 23–25 mm diam, 4–8 mm at 40 C, able to grow on M40Y at 40 C. Conidial heads brown, radiate to short columnar, conidiophores biseriate, rarely uniseriate, diminutive conidiophores not observed, stipes mostly hyaline close to the vesicle and light brown to brown closer to the base, smooth-walled, septate, commonly exceeding 1 mm long × 3–8(–9) μm wide, vesicles predominantly globose or pyriform, rarely spathulate or subclavate, (5–)7–18.5 μm diam, metulae covering one-half to entire surface of the vesicle, (3.5–)4–8.5 μm long, phialides 4–7 μm long, conidia globose to subglobose, smooth, 2.3–2.9 μm. Accessory conidia abundantly present, sessile or on the short, hyaline micro- to semimacronematous conidiophores bearing most commonly 1–3 conidia, globose, subglobose, elliptical, commonly truncate, 3.5–7 × 3.3–5.2 μm. Hülle cells abundant on MEA and CREA, restricted to macroscopic, yellow mycelial clumps, composed of anastomosing network of hyphae 2.5–3.5 μm wide; other vegetative hyphae are 1.5–2(–3) μm wide), and Hülle cells variably shaped, globose, mostly 6–10 μm diam, elliptical, ossiform, lobate, irregular, horseshoe-shaped but most commonly long and narrow, usually nonbranched, 10– 40(–60) × 3–6 μm. No ascomata or ascospores present. Distribution: The species is known only from the ex-type isolate (ex soil, Spain); no identical sequences were found in GenBank with BLAST similarity query. Aspergillus flavipes (Bainier and Sartory) Thom and Church 1926, The Aspergilli: 155; FIG. 5 Basionym: Sterigmatocystis flavipes Bainier and Sartory 1911, Bull Soc Mycol Fr 27:90.

Typification: Plate III in Bainier et Sartory 1911, Bull Soc Mycol Fr 27: not paginated (p 96–97) (lectotype, designated here, MBT198278). Herb. IMI 171885, a dried specimen derived from the living culture NRRL 302 (epitype, designated here, MBT198279). Ex-epitype culture NRRL 302 = IMI 171885 = ATCC 24487 = FRR 0302 = CCF 3067). = Aspergillus archiflavipes Blochwitz 1934, Ann Mycol 32: 84.

Emended description:. Colonies on CYA at 25 C attain 40–52 mm diam in 14 d, white (ISCC-NBS No. 263) to yellowish white (No. 92), floccose to cottony, moderately raised, reverse pale yellowish orange (No. 73) to strong yellowish brown (No. 74). Colonies on MEA attain 36–58 mm diam, floccose, delicately wrinkled, yellowish white to light yellowish brown (No. 76), reverse deep orange-yellow (No. 72) to deep yellowish brown (No. 75), medium orange-yellow (No. 71) soluble pigment. Colonies on CZA attain 35–50 mm diam in 14 d, plane, cottony, yellowish white to light yellowish brown, reverse yellowish white to pale yellow (No. 89). Colonies on CREA attain 42–50 mm diam in 14 d, plane, floccose, yellowish white, no acid production. Colonies on CYA at 37 C attain 17–19 mm diam in 7 d, no growth on CYA at 40 C but able to grow on M40Y at 40 C (4–6 mm diam). Conidial heads white to pale brown, loosely radiate to radiate, conidiophores biseriate, rarely uniseriate, diminutive conidiophores rare, stipes hyaline to light brown, smooth-walled, nonseptate or with occasional septum, sometimes exceeding 1 mm long × 3–6 μm diam, vesicles predominantly spathulate with constriction at the base, sometimes pyriform, most commonly 12–16 μm × 7–11 μm, metulae covering one-half to two-thirds of the vesicle, 3–7 μm long, phialides 4.5–6.5 μm long, conidia globose to subglobose, smooth, 2.1–3 μm diam. Accessory conidia abundantly present, sessile or on the short, hyaline micro- to semimacronematous conidiophores bearing most commonly 1–4 conidia, mostly globose or subglobose, less frequently elliptical or truncate 3.5–5(–6.3) × 3–4.2 μm. No ascomata, ascospores or Hülle cells present.

Distribution: The species is known from only two isolates, the origin of the ex-type strain is unclear, the second strain was isolated from a dead beetle in Uruguay (Blochwitz 1934), no identical sequences were found in GenBank with BLAST similarity query. Notes: Vesicles are mostly spathulate, commonly with basal constriction. The species does not produce Hülle cells, is unable to grow on CYA at 40 C, but grows on M40Y at 40 C. The colony diameter on CYA at 37 C is approximately half in comparison to growth on 25 C. Grows rapidly on CREA. Nomenclatural notes: Original material represented by illustration is extant for A. flavipes, however the species name was lecto- and later neotypified by a specimen dried from the living culture NRRL 302 (Samson and Gams 1985, Pitt and Samson 1993, Pitt and Samson 2000). We designated above a lectotype (iconotype) to supersede neotype designated by Pitt and Samson (2000) (Art. 9.19; McNeill et al. 2012) and this former neotype is here designated as epitype. The isolate NRRL 4852 is considered to be an authentic strain of A. archiflavipes (Raper and Fennell 1965) but it was not cited in the protologue (Blochwitz 1934). The isolate shares almost identical morphology and sequence data with the ex-type of A. flavipes and it is clear that both names apply to the same organism. We designate here a neotype to fix the name A. archiflavipes and its synonymization with A. flavipes: URUGUAY. ex dead beetle, W. Herter (neotype, designated here, PRM 924051 — MBT198280, a dried specimen derived from the living culture NRRL 4852. Isotype PRM 924052. Ex-neotype culture NRRL 4852 = CCF 4836 = IMI 345934).

Aspergillus frequens Hubka, A. Nováková, M. Kolařík & S.W. Peterson, sp. nov. — MB808141; FIGS. 2, 6

Diagnosis. Vesicles of this species are mostly pyriform and usually do not exceed 15 μm diam. The species mostly does not produce Hülle cells, grows comparably at 25 and 37 C except NRRL 4263, and is unable to grow on CYA at 40 C but grows on M40Y at 40 C. Typification. HAITI. ex soil, 1960, J. Rabel (holotype, PRM 923458, a dried culture derived from the living culture NRRL 4578. Isotype PRM 923459. Ex-holotype culture NRRL 4578 = ATCC 16805 = CBS 586.65 = IMI 135423 = CCF 4555). Etymology. Named after its frequent occurence in our data set and world-wide distribution; Latin adj. frequens — „frequent“ in English. Description. Macromorphology highly variable and depending on the isolate. Colonies on CYA at 25 C attain 24–57 mm diam (NRRL 4263: 48–57 mm; other isolates: 24–46 mm) in 14 d, plane to wrinkled, velutinous, floccose to lanose with delicately granular surface, yellowish white (ISCC-NBS No. 92), light grayish yellowish brown (No. 79) to grayish brown (No. 61), dark orange yellow (No. 72), grayish yellowish brown (No. 80) to dark olive brown (No. 96), without exudate or with pale yellow (No. 86), strong yellowish brown (No. 74) to brownish black (No. 65) exudate in the colony centre, reverse light brown (No. 57) to dark yellowish brown (No. 78) with light yellowish brown (No. 76) margins and dark yellowish brown soluble pigment, in other strains dark olive brown to brownish black with strong brown (No. 55) soluble pigment. Colonies on MEA attain 26–52 mm diam in 14 d, velutinous, finely granular to floccose, plane, delicately furrowed to wrinkled, grayish yellowish brown, medium yellowish brown (No. 77), pale orange yellow (No. 73) to dark orange yellow, some strains with irregular sporulation in sectors, yellowish gray (No. 93) to grayish yellow (No. 90) in the sporulating parts, deep yellowish brown (No. 75) colored in the parts without sporulation, no exudate in some strains, strong brown in others, no soluble pigment or orange yellow to strong brown, reverse deep yellowish brown, strong yellowish brown to deep yellowish brown, strong brown or brownish orange (No. 54). Colonies on CZA

attain 16–48 mm diam in 14 d, plane, velutinous to floccose with granular surface, yellowish white, light grayish yellowish brown, light yellowish brown, grayish yellow to grayish brown in the colony centre, without exudate or with light orange yellow (No. 70) to strong yellowish brown exudate, medium yellowish brown to dark grayish brown (No. 62) soluble pigment, reverse grayish brown, dark grayish brown to brownish black. Colonies on CREA attain 20– 42 mm diam (NRRL 4263 and NRRL 286: 37–48 mm; other isolates: 16–33 mm) in 14 d, plane with sulcate centre in some strains, in others floccose to granular, light grayish yellowish brown to dark orange yellow or deep yellowish brown, strong yellowish brown exudate in some strains, no acid production. Colonies on CYA at 37 C attain 17–25 mm diam in 14 d, no growth on CYA at 40 C, but able to grow on M40Y at 40 C. Conidial heads white to pale brown, loosely radiate to radiate, conidiophores biseriate, diminutive conidiophores not observed, stipes hyaline to pale brown, smooth-walled, delicately roughened to roughened, sometimes with lenticular brown concretion on the surface of the wall, septate, often exceeding 1 mm in length × 2–6(–7) μm, vesicles predominantly pyriform or subglobose, most commonly (3.5–)5.5–14(–15.5) μm diam, metulae covering one half to two-thirds of the vesicle, (3.5–)4–6.5(–7.5) μm long, phialides (3.5–)4–6.5(–7.5) μm long, globose to subglobose conidia predominant in some strains, subglobose to elliptical in others, smooth, (2–)2.5–3.2(–4) μm in longer axis, conidia in chains are separated by connectives that in some strains commonly remain on free conidia. Accessory conidia regularly present in small amounts, sessile or on the short, hyaline microto semimacronematous conidiophores bearing most commonly 1–4 conidia, globose, subglobose, 3.5–6 μm diam, or elliptical, clavate, commonly truncate, (3–)4–7(–8.5) × 3.5– 5.5 μm. Hülle cells present only in strains NRRL 295, NRRL 26246 and CCF 4839 grown on MEA-Fluka (CCF 4839 also on MEA-Oxoid), CYA (the most abundant) and CREA. They are most commonly elongated, lobate to branched (mostly with three long branches) and

variously twisted (horseshoe-shaped, S-shaped), 10–35(–58) × 4–6(–8) μm, less commonly subglobose, elliptical or polygonal, 5–10 μm in long axis. No ascomata or ascospores present. Distribution. The species is distributed world-wide and is known from soil (Haiti, China, India), indoor air (Czech Republic, Trinidad and Tobago, U.S.A.) and diary products (U.S.A.). Other isolates whose sequences were found in GenBank using BLAST similarity search: INDIA, ex Halophila ovalis (endophyte), SGE15 (JN709039). U.S.A., HAWAII, ex Mycale armata, GP1 (DQ875009). PUERTO RICO, ex soil, JO20 (EU645664) — (ZuluagaMontero et al. 2010). INDIA, ex peanut, SEFN3 (JX518284). IRAN, ex straw of Triticum aestivum, IRAN2062C (KC473934, KC473913) and IRAN, ex Musa sapientum, IRAN 939C (KC473914) — (Asgari et al. 2012). SPAIN, ex roots of Dittrichia viscosa (endophyte), r347 (HQ649957) — (Maciá-Vicente et al. 2012). INDIA, ex nail infection of a woman, AS-8 (EU515154) — (Gehlot et al. 2011). Aspergillus iizukae Sugiy. 1967, J Fac Sci Univ Tokyo, Sect 3, Bot: 390; FIGS. 3, 7 Typification. JAPAN. GYMNA PREFECTURE: Fujioka, ex soil from stratigraphic drilling core, 26 Jun 1969, J. Sugiyama (holotype TI 0007. Ex-holotype culture NRRL 3750 = CBS 541.69 =IMI 141552= CCF 4548). Emended description. Macromorphology highly variable and depending on the isolate. Colonies on CYA at 25 C attain 22–46 mm diam in 14 d, velutinous to floccose with granular surface, delicately furrowed, irregularly or radially wrinkled, light grayish yellowish brown (ISCC-NBS No. 79) to grayish yellowish brown (No. 80), exudate vivid orange yellow (No. 66), deep yellowish brown (No. 75) to medium yellowish brown (No. 77), no soluble pigment or deep orange yellow (No. 69), dark orange yellow (No. 72) to deep orange (No. 51), reverse strong yellowish brown (No. 74), deep yellowish brown to strong brown (No. 55) with paler colony margin. Colonies on MEA attain 22–52 mm diam in 14 d, plane to furrowed, floccose,

granular, light grayish yellowish brown to medium yellowish brown, no exudate or brilliant yellow (No. 83), brilliant orange yellow (No. 67) to dark yellowish brown (No. 78) or deep orange to strong reddish brown (No. 40), no soluble pigment in some strains, in others strong yellowish brown to deep brown (No. 56), reverse strong yellowish brown, dark yellowish brown, dark brown (No. 59) to deep brown. Colonies on CZA attain 25–52 mm diam in 14 d, plane, velutinous, floccose to granular, yellowish white to light grayish yellowish brown, no exudate or only in colony centre, dark yellowish brown to dark brown, no soluble pigment or deep orange, reverse yellowish white (No. 92), deep yellowish brown to olive black (No. 114). Colonies on CREA attain 23–41 mm diam in 14 d, plane to irregularly furrowed, velutinous, floccose to granular, light grayish yellowish brown, deep orange yellow to dark yellowish brown, no exudate, no to weak acid production. Colonies on CYA at 37 C attain18– 21 mm diam, no growth on CYA at 40 C, but able to grow on M40Y at 40 C. Conidial heads white, pale brown to brown, radiate to columnar, conidiophores biseriate, diminutive conidiophores not observed, stipes mostly hyaline close to the vesicle and light brown to brown closer to the base, smooth-walled to delicately roughened, septate, in some strains commonly exceeding 1 mm in length, but 2 mm ................................................................................... A. ardalensis 6a) vesicles predominantly spathulate, no production of Hülle cells on MEA ........................................ A. flavipes 6b) both characteristics not combined ..................................................................................................................... 7 7a) at least some vesicles attain a diameter of 20 μm or more (MEA, 25 C, 14 d) ................................... A. iizukae 7b) vesicles do not exceed 18 μm diam .................................................................................................................. 8 8a) colonies diameter on CYA at 37 C after 7 d comparable or larger than those at 25 C ..................................... 9 8b) colonies diameter on CYA at 37 C markedly smaller than those at 25 C (the difference > 5 mm) .................... ............................................................................................................................................................... A. movilensis 9a) no growth on CYA at 40 C after 7 d ................................................................................................. A. frequens 9b) restricted growth on CYA at 40 C after 7 d ............................................................................... A. mangaliensis

DISCUSSION

Species in Section Flavipedes.—Phylogenetic analysis based on morphological comparisons did not provide a stable taxonomy in Aspergillus section Flavipedes (Thom and Church 1926; Raper and Fennell 1965; Samson 1979). Morphological analysis placed some species from section Terrei in sect. Flavipedes either as species or synonyms. DNA sequencing and phylogenetic analysis has provided a statistically testable basis for taxonomic analysis (Peterson 2000, 2008; Varga et al. 2005;) for isolates with colonies from white to brown, that often form yellow mycelial masses with Hülle cells, have conidial heads mostly columnar, biseriate conidiophores with yellow to buff stipes that up to 2–3 mm long, subglobose to elliptical vesicles with diam up to 20 × 30 μm, conidia smooth, colorless, 2–3 μm. The name section Flavipedes was introduced by Gams et al. (1985) as a nomenclaturally correct replacement for the A. flavipes group. The analysis of LSU rDNA sequences from Aspergillus species (Peterson 2000) indicated that A. carneus and A. niveus belong to sect. Terrei, and A. iizukae and A. aureofulgens are distinct species in sect. Flavipedes. Similar tree topology was obtained when ITS rDNA was analysed by Varga et al. (2005) but it was proposed that sect. Flavipedes and Terrei could be merged. Peterson (2008) based on multi-gene phylogeny showed that sect. Flavipedes is phylogenetically well supported and separated from sect. Terrei. It was also discovered that A. flavipes is distinct from its supposed sexual state F. flavipes and a new name A. neoflavipes is proposed in this study for homothallic F. flavipes. Two other Fennellia species, F. nivea (Samson 1979) and F. monodii (Locquin-Linard 1990), were placed in sects. Terrei and Usti as Aspergillus species (Samson et al. 2011a, Samson et al. 2011b). Several undescribed lineages were also discovered by Peterson (2008) that are all described in this study as new species (A. frequens, A. movilensis and A. polyporicola). The taxonomic positions of three varieties of A. terreus or A. niveus that were treated by Raper and Fennel (1965) or Samson (1979) as synonyms of A. flavipes, were resolved based on polyphasic

analysis (Samson et al. 2011a). Aspergillus terreus var. subfloccosus was synonymized with A. terreus, and the remaining two varieties were raised to species level as A. floccosus and A. neoindicus (Samson et al. 2011a). The placement of A. janus and its variety A. janus var. brevis (now A. brevijanus) has always been problematic. Raper and Thom (1944) and also Raper and Fennell (1965) placed these taxa in sect. Versicolores because the green conidial heads resembled those of A. sydowii. Molecular phylogenetic studies showed affinity of these taxa to sects. Terrei and Flavipedes (Peterson 2000, Varga et al. 2005) and A. janus was placed into this section by Peterson (2000). Peterson (2008) raised A. janus var. brevis to the species level as A. brevijanus and placed both species to sect. Flavipedes. However, sect. Flavipedes is paraphyletic when A. janus and A. brevijanus are included. In this study, we proposed a new section for these two species that is not only based on strong molecular support (FIG. 1) but is also well supported by morphological data. The position of A. rehmii (Zukal 1893), listed as a possible synonym of A. flavipes by Raper and Fennell (1965), is not fully resolved. The asexual state of A. rehmii resembles the species from sect. Flavipedes in the original description (but placement in several other sections can not be excluded), however the sexual state is dissimilar to the fennellia-morph and other Aspergillus species teleomorphs, so the position of this species remains enigmatic. The taxonomic revision presented in this study based on molecular, morphological and physiological data showed that many undescribed lineages representing new species are hidden behind the broad common name A. flavipes. Based on the species concept presented here, A. flavipes s. str. is an uncommon species. Section Flavipedes encompasses ten species, seven of which are described here. Ecology.—The species from sect. Flavipedes are distributed world-wide in many types of soils and rhizospheres (Zhou et al. 2004, Rochfort et al. 2005, Ahmet 2004, Miller et al. 1957,

Panwar 1970, Mehrotra and Kakkar 1972). They are especially common in subtropical and tropical soils (Domsch et al. 2007, Klich 2002) with lower frequencies in temperate climate, where they are isolated particularly from soils with steppe-type of vegetation (this study, Orput and Curtis 1957, Panwar 1970, Fassatiová 1969). They occasionally cause spoilage of food (fruit, cassava, groundnuts) or are isolated as food contaminants (fruit, manioc, black beans, coriander, diary products, peanuts, sugar products, wheat, barley, mayze, seeds, rice, rice fodder, smoked fish) (Pitt and Hocking 2009, Asgari et al. 2012, McDonald 1970, Castillo et al. 2004, Horn 2006, Lilabati and Viswanath 2000, Saini et al. 2012, this study) and have been reported from the indoor environment (Samson et al. 2001, Tuomi et al. 2000, Bogomolova and Kirtsideli 2009), including hospital air (this study, Karalti and Çolakoğlu 2012, Ahmet 2004, Singh and Singh 1999). These reports are mostly associated with the name A. flavipes and it is in general not possible to classify these isolates according to the new species concepts presented here due to missing molecular and detailed morphological data. Reports from cave environments are also restricted to the name „A. flavipes“ and these records originated from cave air, cave sediment or cave walls in Bahamas, Czech Republic, Russia, South Africa and U.S.A. (Vanderwolf et al. 2013). However, at least A. spelaeus described in this study was common in Nerja cave (Spain) and another record of this species came from Kartchner Caverns (U.S.A.) (Vaughan et al. 2011). Other species isolated directly in caves were A. iizukae (TABLE I), and A. movilensis (this isolate was identified only based on morphology and is no longer viable). Aspergillus flavipes isolates tolerate relatively high concentrations of osmotically active solutes in media, being able to grow on media with 40% (w/v) sucrose and 25% (w/v) NaCl (Tresner and Hayes 1971, Moustafa and Al-Musallam 1975) and were isolated from natural habitats with high NaCl concentration such as salterns (Moustafa 1975, Butinar et al. 2011, Cantrell et al. 2006), brakish water (Pawar and Thirumalachar 1966), mangrove swamp

(Lee and Baker 1972) or coastal sand of the Dead Sea (Grishkan et al. 2003). Other record of A. flavipes involved e.g. wastewater (Ahmet 2004), bedding material (Cooke and Foter 1958), macerated carton material (Narciso and Parish 1997), but the the complete list of substrates and references exceeds the possibilities of this publication. Aspergillus iizukae, A. frequens and A. mangaliensis are species distributed worldwide based on our data and sequence data deposited in GenBank. These species were, except for soil and rhizosphere, isolated as herb and tree endophytes (Anitha et al. 2013, El-Elimat et al. 2014, Yuan et al. 2011, Shan et al. 2012, Maciá-Vicente et al. 2012). Aspergillus frequens and A. mangaliensis were also isolated from marine sponges (Wiese et al. 2011). Aspergillus brevijanus is probably a rare species and only the authentic strain examined by Raper and Fennel (1965) was available for this study. An additional record of this species originated from forest soil in Turkey (Azaz and Pekel 2002). Numerous records of A. janus world-wide exist that are based on morphology, namely from soil in Egypt, Czech and Slovak Republics and U.S.A. (Hodges 1962, Miller et al. 1957, Nováková et al. 2012b, Migahed 2003), agricultural soil and foodstuff in Turkey (Ahmet 2004), blue pine seeds and soil in Pakistan (Farooq 2000, Mirza and Bajwa 2005), Arubotaim Cave in Israel (Grishkan et al. 2004), indoor air and hospital air in India (Singh and Singh 1999), aeciospores of Cronartium comandrae in Canada (Powell 1971), sand from a beach and digestive tract of Panstrongylus megistus in Brazil (Moraes et al. 2001, Gomes et al. 2008), keyboard in Italy (Piccoli et al. 2001) and macerated carton material in the U.S.A. (Narciso and Parish 1997). Pathogenicity and accessory conidia.—The members of sect. Flavipedes are uncommon human pathogens that were repeatedly isolated in hospital environments (CCF 4005, this study; BCCM/IHEM collection catalogue; Karalti and Çolakoğlu 2012, Ahmet 2004, Singh and Singh 1999). Human and animal infections attributed to A. flavipes include lumbar vertebral osteomyelitis in 54-year-old man treated for 17 days with dexamethasone (Roselle

and Baird 1979), cutaneous aspergillosis in 15-year-old boy with acute lymphocytic leukemia (Barson and Ruymann 1986), otomycosis in 71-year-old male (Stuart and Blank 1955), onychomycosis in 25-year-old female (Gehlot et al. 2011), diskospondylitis in dog (Schultz et al. 2008), additional isolates from sputum and lung of patients are deposited in BCCM/IHEM collection, the species was also reported as contamination of clinical material (MartínMazuelos et al. 2003, Ainley and Smith 1965). Cerebral and cerebellar aspergilloma and disseminated infection resulted from intravenous inoculation of mice by conidia of A. flavipes (Pore and Larsh 1968). The infection foci in mice contained accessory conidia, the feature that is only present is several related species (A. terreus, A. carneus, A. niveus) (Pore and Larsh 1968). One of the isolates used in these experimental infections (NRRL 286) was examined by us and was reidentified as A. frequens. The isolate of A. flavipes AS-8 associated with human onychomycosis can be identified based on deposited sequence (EU515154) as A. frequens, similar to CCF 4005 (TABLE I) from hospital air. These data suggest that A. frequens is a clinically relevant species that could in fact cause the majority of the abovementioned cases of infection. Only a single report of pathogenity of A. janus was associated with a case of otomycosis (Vennewald et al. 2003). Accessory conidia were first reported in A. terreus (Raper and Fennell 1965) and subsequently found in several other species in sect. Terrei (A. carneus, A. niveus, A. alabamensis) and in A. flavipes (Pore and Larsh 1967, Burrough et al. 2012). All these species are rare human and animal pathogens (Hubka et al. 2014, Barson and Ruymann 1986, Burrough et al. 2012, Morquer and Enjalbert 1957, Roselle and Baird 1979, Schultz et al. 2008, Gehlot et al. 2011, Auberger et al. 2008, Wadhwani and Srivastava 1984) that are able to produce diagnostic accessory conidia in tissue during infection (Pore and Larsh 1968, Burrough et al. 2012, Balajee 2009, Tracy et al. 1983, Walsh et al. 2003). These spores develop mostly under oxygen-limiting conditions, such as submerged mycelium and it was

suggested that this type of conidia may play a role during dissemination of infection (Raper and Fennell 1965, Deak et al. 2009). This hypothesis was supported by faster dissemination of infection in mice inoculated solely by accessory conidia in comparison to phialidic conidia (Pore and Larsh 1968). Accessory conidia have higher viability, faster germination, lower ergosterol content in the cell wall rendering them less susceptible to amphotericin B (Deak et al. 2009). These conidia were found in this study in all species in sect. Flavipedes and Jani with some differences in abundance between species and isolates. Accessory conidia have more nuclei than the other conidia, similar to reports on A. terreus (Deak et al. 2011). The discovery of such conidia can be expected in other species in sect. Terrei in addition to A. terreus, A. niveus and A. carneus. The dimensions, shape, metabolism, cell wall utrastructure and antigenicity of accessory conidia are different from phialidic conidia (Pore et al. 1969, Deak et al. 2009, Deak et al. 2011). Bioprospecting.—Species from sect. Flavipedes are a rich source of secondary metabolites, many of them show various biological activities and have biotechnological and pharmacological potential. Almost all of these metabolites were attributed to A. flavipes but it was possible in several cases to classify the examined isolate according to the present taxonomy based on deposited sequence data (see below). It was shown that the culture extract from A. flavipes had antimycotic activity against A. fumigatus, Trichophyton and Scopulariopsis (Blunt and Baker 1968), and grain contamined with A. flavipes was toxic to Pekin ducklings (De Scott 1965). Only two mycotoxins in a strict sense, sterigmatocystin (Tuomi et al. 2000) and citrinin (Gorst-Allman and Steyn 1979, Greenhill et al. 2008), were detected in A. flavipes. Other reported metabolites were the cholesterol lowering drug lovastatin (Valera et al. 2005); antitumor and antimalarial butyrolactones (Nagia et al. 2012); antibacterial flavimycins (Kwon et al. 2012); antifungal flavipin (Raistrick and Rudman 1956) and terreic acid (Li et al. 2001); antibiotic flavipucine (Casinovi et al. 1968), glutamycin

(Casinovi et al. 1969), dihydroisobenzofurans (Kwon et al. 2009) and TMC-69 (Kohno et al. 1999); cytotoxic and antiviral cytochalasins (Zhou et al. 2004, Rochfort et al. 2005); cytotoxic flavicerebrosides (Jiang et al. 2004); antifungal, analgesic and herbicidal methylsalicylic acid (Nagia et al. 2012); antitumor terrein (Nagia et al. 2012) and asperphenamate (Clark et al. 1976); neuromodulatory spiroquinazoline (Barrow and Sun 1994), and others (Clark et al. 1976, Barrow and Sun 1994, Delmotte et al. 1956, Laskin and Lechevalier 1973, Frisvad and Samson 1991). Three flavonolignans, the components of silymarin, were produced by A. iizukae (El-Elimat et al. 2014), aflaquinolones were produces by A. polyporicola (Neff et al. 2012). In case of standardisation of metabolite extraction and analysis, the metabolite profiles can be used to support species concept (Frisvad et al. 2007). Not surprisingly, many of metabolites mentioned above were also detected in species belonging to sibling sect. Terrei (Samson et al. 2011a, Bräse et al. 2009). Aspergillus flavipes is also a significant producer of many biotechnologically important enzymes such as amylase, α-galactosidase, keratinase, L-methioninase, pectinase, pectin lyase and others (El-Sayed and Shindia 2011, Solís et al. 2009, Martínez-Trujillo et al. 2011, El-Ayouty and Salama 2012, Frolova et al. 2002, Ozsoy and Berkkan 2003). The ability of A. flavipes to degrade some herbicids in soil was described in the past (Kaufman et al. 1963, Kaufman and Blake 1970). Only a few reports exist on metabolites found in species from sect. Jani. Aspergillus janus produced antimalaric janoxepin and brevicompanine B (Sprogøe et al. 2005), antiinflammatory desacetoxy-wortmannin (D. Hauser, patent, 1972) and antitumor asperphenamate (= asjanin) (Nakashima et al. 1983). Antitumor survivin was produced by hitherto undescribed species close to A. janus (Felix et al. 2013). ACKNOWLEDGMENTS

This study was supported by the GAČR projects (“Complex study of the endemic earthworm Allolobophora hrabei and its effects on soil and soil organisms in steppe ecosystems” and “Bat adaptations to the fungal disease geomycosis”) and the project RNM-5137 of the Junta de Andalucía, Spain. This study was also partially supported by the Ministry of Education, Youth and Sports (CZ.1.07/2.3.00/20.0055, CZ.1.07/2.3.00/30.0003 and SVV project). Molecular genetics analyses were supported by the projects GAUK 607812. The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned. Prof Karol Marhold’s consultation on nomenclatural issues is gratefully acknowledged. We thank Dr MJ Vaughan for providing several isolates from American caves, D. Kozáková for the lyophilisation of the cultures, Dr A. Kubátová for deposition of cultures into the CCF collection, Dr M. Chudíčková and A. Gabrielová for their invaluable assistance in laboratory. LITERATURE CITED Ahmet A. 2004. Aspergillus, Penicillium, and related species reported from Turkey. Mycotaxon 89:155–157.

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LEGENDS FIG. 1 Maximum likelihood tree based on combined benA, calmodulin and RPB2 sequence data, coding region only, 1671 characters. Tree calculated using MEGA 5.2 and the GTR+G+I model. Thick branches have 90–100% bootstrap proportions, numbers above internodes report 70–89% boostrap proportion. Section markers are placed on the tree. The ex-type isolates are designated by a superscript T, the type species of each section is underlined. FIG. 2 Variability of colonies of A. frequens on CYA at 37 C after 7 d (row CYA37) and on different media after 14 d at 25 C (CYA, MEA, CZA, CREA). Column a. NRRL 295 isolate (producing Hülle cells on CYA, MEA and CREA), detail of colony on MEA in the last row; column b. CCF 4005 isolate, detail of colony on CYA in the last row; column c. NRRL 4263 isolate, detail of colony on CZA in the last row; column d. NRRL 4578T isolate, detail of colony on MEA in the last row; column e. CCF 2026 isolate, detail of colony on CZA in the last row. FIG. 3 Variability of colonies of A. iizukae on CYA at 37 C after 7 d (row CYA37) and on different media after 14 d at 25 C (CYA, MEA-Fluka, CZA, CREA). Column a. NRRL 3750T isolate, detail of colony on CZA in the last row; column b. CMF ISM 2417 isolate, detail of colony on MEA in the last row; column c. CCF 4032 isolate, detail of colony on MEA in the last row; column d. CMF ISB 2620 isolate, detail of colony on CZA in the last row; column e. CMF ISB 2619 isolate, detail of colony on CYA in the last row; column f. CMF ISB 2616 isolate, detail of colony on MEA in the last row.

FIG. 4 Aspergillus ardalensis CCF 4031T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on CYA; f. detail of colony on MEA; g-h. Hülle cells; i-l. micro- to semimacronematous conidiophores producing accessory conidia; m. phialidic conidia; n-r. biseriate conidiophores. — Scale bars 10 µm. FIG.5 Aspergillus flavipes NRRL 302T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on CYA; f-i. micro- to semimacronematous conidiophores producing accessory conidia; j. phialidic conidia; k-o. biseriate conidiophores. — Scale bars 10 µm. FIG. 6 Aspergillus frequens NRRL 4578T (parts f-i. NRRL 259). a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on CYA; f. detail of colony on MEA; g-i. Hülle cells; j-l. micro- to semimacronematous conidiophores producing accessory conidia; m. phialidic conidia; n-r. biseriate conidiophores. — Scale bars 10 µm. FIG. 7 Aspergillus iizukae NRRL 3750T (parts e-f. CMF ISB 2417). a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on CYA; f. detail yellow masses containing Hülle cells on MEA-Oxoid; g-h. Hülle cells; i-k. micro- to semimacronematous conidiophores producing accessory conidia; l. phialidic conidia; m-o. biseriate conidiophores. — Scale bars 10 µm. FIG. 8 Aspergillus luppii NRRL 6326T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on MEA after 4 weeks; f. detail conidial heads and yellow mycelial masses with Hülle cells on MEA; g-h. Hülle cells. i-k. micro- to semimacronematous conidiophores producing accessory conidia; l. phialidic conidia; m-r. biseriate conidiophores. — Scale bars 10 µm. FIG. 9 Aspergillus mangaliensis CCF 4698T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of conidial heads on MEA; f. detail of

mycelial masses with Hülle cells on MEA. g-h; Hülle cells; i-k. micro- to semimacronematous conidiophores producing accessory conidia; l. phialidic conidia; m-o. biseriate conidiophores. — Scale bars 10 µm. FIG. 10 Aspergillus movilensis CCF 4410T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on CYA; f. detail of colony on MEA; g-h. Hülle cells; i-k. micro- to semimacronematous conidiophores producing accessory conidia; l. phialidic conidia; m-p. biseriate conidiophores. — Scale bars 10 µm. FIG. 11 Aspergillus neoflavipes NRRL 5504T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of 28-d-old colony on MEA-Oxoid; f. detail of 28-d-old colony on MEA-Fluka with conidial heads; g-h. Hülle cells; i-k. micro- to semimacronematous conidiophores producing accessory conidia; l. phialidic conidia; m-o. biseriate conidiophores. p. 4-spored asci; r. 8-spored asci; s. ascospores. — Scale bars 10 µm. FIG. 12 Aspergillus polyporicola CCF 32683T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on MEA-Fluka with dominant biseriate conidiophores producing phialidic conidia; f. detail of colony on MEA-Oxoid with dominant micro- or semimacronematous conidiophores producing accessory conidia; g-j. micro- to semimacronematous conidiophores producing accessory conidia; k. phialidic conidia; l-p. biseriate conidiophores. — Scale bars 10 µm. FIG. 13 Aspergillus spelaeus CCF 4425T (parts f-h. CCF 4699). a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on MEAFluka; f. detail of yellow mycelial masses with Hülle cells on MEA-Fluka; g-h. Hülle cells; i. micro- to semimacronematous conidiophores producing accessory conidia; j. phialidic conidia; k-n. biseriate conidiophores. — Scale bars 10 µm. FIG. 14 Aspergillus brevijanus NRRL 1787T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of green and white conidial heads

sporulating in sectors; f-g. micro- to semimacronematous conidiophores producing accessory conidia; h. phialidic conidia produced by white conidial heads; i. phialidic conidia produced by green conidial heads; j-k. biseriate white sporulating conidiophores; l-m. biseriate green sporulating conidiophores. — Scale bars 10 µm. FIG. 15 Aspergillus janus NRRL 1787T. a-d. Colonies incubated 14 d at 25 C on CYA, MEA, CZA and CREA, from left to right; e. detail of colony on MEA; f. detail of green and white sporulating conidial heads with mycelial masses containing Hülle cells; g. Hülle cells; h-j. micro- to semimacronematous conidiophores producing accessory conidia; k. phialidic conidia produced by green conidial heads; l. phialidic conidia produced by white conidial heads; m-o. biseriate white sporulating conidiophores; p-r. biseriate green sporulating conidiophores. — Scale bars 10 µm.

FOOTNOTES 1

Corresponding author. E-mail: [email protected]

TABLE II. Colony diam (mm) or growth abilitied of sects. Flavipedes and Jani species on various media Colony diam after 7 d (14 d) at 25 C

Growth on CYA after 7 d

M40Y after 7 d

Species CYA

MEA

CZA

CREA

37 C

40 C

43 C

at 40 C

A. ardalensis

25–28 (42–45)

25–28 (37–42)

20–28 (38–40)

28–30 (42–45)

23–25

+

—

+

A. flavipes

25–35 (40–52)

25–35 (36–58)

25–28 (35–50)

27–30 (42–50)

17–19

—

—

+

A. frequens

14–30 (24–57)

18–28 (26–52)

9–25 (16–48)

11–20 (20–42)

17–25

—

—

+

A. iizukae

16–35 (22–46)

13–30 (22–52)

13–25 (25–52)

12–24 (23–41)

18–21

—

—

+

A. luppii

18–20 (35–38)

20–22 (33–35)

17–21 (35–40)

15–16 (26–28)

9–10

—

—

—

A. mangaliensis

21–30 (30–52)

24–30 (40–52)

21–28 (32–42)

14–22 (23–36)

23–27

±a

—

+

A. movilensis

22–25 (44–52)

25–30 (48–55)

19–20 (36–46)

15–16 (25–42)

10–17

—

—

±

A. neoflavipes

17–21 (30–33)

18–22 (34–35)

18–20 (41–45)

16–17 (24–28)

20–22

±

—

+

A. polyporicola

17–25 (30–37)

22–32 (28–35)

14–20 (18–35)

12–14 (13–15)

—

—

—

—

A. spelaeus

12–30 (16–45)

15–32 (38–45)

6–26 (11–38)

7–22 (9–28)

—

—

—

—

A. brevijanus

20–27 (28–44)

15–18 (28–35)

18–19 (30–31)

12–16 (21–22)

10–13

—

—

—

A. janus

22–32 (32–52)

22–25 (33–42)

18–22 (28–35)

5–18 (20–36)

—

—

—

—

sect. Flavipedes

sect. Jani

a

± Very restricted growth (≤ 2 mm).

TABLE I. Aspergillus strains from sects. Flavipedes and Jani examined in this study Strain No.a

Species

Provenance (substrate, locality, year of isolation, collector)

Flavipedes A. ardalensis

CCF 4031T = CCF 4426T = NRRL T

T

62824 = CMF ISB 1688 = CBS 134372 A. flavipes

T

T

171885 = ATCC 24487 = FRR

A. frequens

M13

834t

I

I

Nováková

GenBank/EMBL accession Nos. ITS

benA

caM

RPB2

FR733808

HG916683

HG916725

HG916704

EF669591

EU014085

EF669549

EF669633

LM999909

LM644261

LM644241

LM644260

EF669602

EU014082

EF669560

EF669644

HG915893

HG916684

HG916726

HG916705

EF669588

EU014081

EF669546

EF669630

T

CCF 3067T = NRRL 302T = IMI

0302

Spain, Andalucia, Ardales, near Cueva de Doña Trinidad, isol ex soil, 2008, A.

Fingerprinting

Received by Charles Thom in 1922 from Da Fonseca as Bainier’s culture of

II

II

Sterigmatocystus flavipes

T

NRRL 4852 = IMI 345934 = CCF

Uruguay, isol ex dead beetle, received in NNRL from CBS as Blochwitz’s strain

4836

of A. archiflavipes, W. Herter

NRRL 4578T = ATCC 16805T = CBS

Haiti, isol ex soil, 1960, J. Rabel

II

II

III

IIIa

586.65T = IMI 135423T = CCF 4555T CCF 2026

CRb, Prague, isol ex archive material, 1986, O. Fassatiová

III

IIIa

NRRL 295 = ATCC 16814 = CBS

USA, Minnesota, isol ex dairy products, 1933, H. Macy

III

IIIa

585.65 = IMI 135422 = CCF 4554 = FRR 0295 CCF 4005

CR, Hradec Králové, isol ex hospital indoor air, 2005, V. Buchta

III

IIIb

FR727135

HG916685

HG916727

HG916706

NRRL 4263 = CCF 4556

India, Dehradun New Forest, isol ex soil, 1955, K.B. Bakshi

III

IIIb

EF669600

EU014083

EF669558

EF669642

NRRL 286 = ATCC 1030 = FRR

Received in NNRL from Dr. J. Westerdijk (CBS)

AY373849

LM644262

LM644246

LM644258 LM644257

—

—

0286 NRRL 26246 = CCF 4838

China, isol ex soil, 1944

—

—

LM999905

LM644263

LM644247

NRRL 58660 = CCF 4839

Trinidad & Tobago, isol ex indoor air of a home, 2009, Ž. Jurjević

—

—

LM644239

LM644264

LM644248

NRRL 58682 = CCF 4840

Puerto Rico, isol ex indoor air of a home, 2009, Ž. Jurjević

—

—

LM644240

LM644265

LM644251

NRRL 58899 = CCF 4841

USA, New York, isol ex indoor air of a home, 2009, Ž. Jurjević

—

—

LM999903

LM644266

LM644249

A. iizukae

NRRL 58598 = CCF 4842

USA, New Jersey, isol ex indoor air of a home, 2008, Ž. Jurjević

—

NRRL 3750T = CBS 541.69T = IMI

Japan, Gymna Prefecture, Fujioka, isol ex soil from stratigraphic drilling core,

LM999904

LM644267

LM644250

141552T = CCF 4548T

1969, J. Sugiyama

EF669597

EU014086

EF669555

EF669639

CCF 1895

CR, Most, isol ex soil of spoil bank, 1984, M. Černý

IVb

IVb

FR727134

FR775336

HG916728

HG916707

CCF 4033 = CMF ISB 1551

CR, Most brown lignite district, isol ex soil of spoil tip, 2004, A. Nováková

IVa

—

FR733809

HG916686

HG916729

CCF 4032 = CMF ISB 1245

Germany, Weissagker Berg near Cottbus, Lusatian brown lignite district, isol ex

IVa

— HG915894

HG916687

HG916730

HG916708

HG915895

HG916694

HG916731

HG916709

HG915896

HG916688

HG916732

HG916710

HG915899

HG916689

HG916733

HG916711

HG915897

HG916692

HG916734

HG916712

HG915898

HG916693

HG916735

HG916713

HG915900

HG916690

HG916736

HG916714

HG915901

HG916691

HG916737

HG916715

IVa

— IVa

soil of spoil bank, 1999, A. Nováková CMF ISB 2544

Romania, Dobrogea, Mangalia, isol ex soil near Movile Cave, 2011, A.

IVb

—

Nováková CMF ISB 2417

Romania, National Park Apuseni Mountains, Meziad Cave, isol ex earthworm

IVb

IVb

casts, 2009, A. Nováková CMF ISB 2616

CR, Ječmeniště, National Nature Monument, isol ex soil, 2012, A. Nováková

IVb

—

CMF ISB 2617

CR, Kolby, National Reservation Pouzdřanská step, isol ex soil, 2012, A.

IVb

—

Nováková CMF ISB 2618

CR, Kolby, National Reservation Pouzdřanská step, isol ex soil, 2012, A.

IVb

—

Nováková CMF ISB 2619

CR, Kolby, National Reservation Pouzdřanská step, isol ex earthworm casts,

IVb

IVc

2012, A. Nováková CMF ISB 2620

CR, Kolby, National Reservation Pouzdřanská step, isol ex earthworm casts,

IVb

IVb

2012, A. Nováková

A. luppii

NRRL 58963 = CCF 4843

USA, Illinois, isol ex indoor air of a home, 2009, Ž. Jurjević

—

—

LM644237

LM644268

LM644245

CCF 4844

USA, Idaho, Boise, isol ex indoor air of a home, 2012, Ž. Jurjević

—

—

LM644238

LM644269

LM644244

CCF 4845

Romania, Movile Cave, isol ex cave sediment, 2013, A. Nováková

—

—

LM999906

LM644270

LM644243

NRRL 6326T = CBS 653.74T = CCF

France, Provence, near Aups, isol ex natural truffle soil, 1972, A.M. Luppi-

EF669617

EU014079

EF669575

EF669659

HG915902

HG916695

HG916738

HG916716

4545 A. mangaliensis

T

CCF 4698T = CMF ISB 2662T =

V

V

Mosca Romania, Mangalia, isol ex soil near Moville Cave, 2012, A. Nováková

VIa

VIa

NRRL 62825T CCF 869 = NRRL 62823

China, isol ex industrial material, 1955, V. Zánová

VIb

VIb

NRRL 4893 = IMI 343701 = CCF

Japan, isol ex soil

—

—

HG915903

HG916696

HG916739

HG916717

LM999907

LM644271

LM644242

LM644256

HG915904

HG916697

HG916740

HG916718

EF669604

EU014080

EF669562

EF669646

EF669614

EU014084

EF669572

EF669656

EF669595

EU014088

EF669553

EF669637

HQ288052c

LM644274

LM644252

LM644254

HG915905

HG916698

HG916741

HG916719

HG915906

HG916699

HG916742

HG916720

HG915907

HG916700

HG916743

HG916721

4846 A. movilensis

CCF 4410T = CMF ISB 2614T =

Romania, Mangalia, Dobrogea, isol ex soil near Movile Cave, 2011, A.

NRRL 62819T = CBS 134395T

Nováková

NRRL 4610 = IMI 350352 = CCF

Haiti, Fons Parisien, isol ex soil, unrecorded date or collector

VII

VIII

VII

VIII

4551 A. neoflavipes

NNRL 5504T = ATCC 24484T = CBS T

T

260.73 = IMI 171883 = IFM 40894 = CCF 4552 A. polyporicola

T

Thailand, Pak Thong Chai, isol ex cellulosic material buried in forest soil, 1968,

IX

IX

C. Klinsukont

T

NRRL 32683T = CCF 4553T

USA, Hawaii, Hilo, Alien Wet Forest Zoo, isol ex basidioma of Earliella

X

X

scabrosa (Polyporales), 2003, D.T. Wicklow NRRL 58570 = CCF 4828

USA, Hawaii, Alien Wet Forest, isol ex basidioma of Rigidoporus microporus

X

X

(Polyporales), 2003, D.T. Wicklow A. spelaeus

CCF 4425T = CMF ISB 2615T = CBS T

134371 = NRRL 62826

Spain, Andalusia, Nerja Cave, isol ex cave sediment, 2011, A. Nováková

XIa

XIa

T

CCF 544 = NRRL 62827

CR, Doutnáč hill near Srbsko, Bohemian Karst, isol ex soil, 1961, O. Fassatiová

XIb

XIb

CCF 4699 = CMF ISB 2659

CR, Hostěradice, National Nature Monument U Kapličky, isol ex Allolobophora

XIb

XIb

hrabei intestine, 2012, A. Nováková CCF 4679 = CMF ISB 2663

CR, Ječmeniště, National Nature Monument, isol ex soil, 2012, A. Nováková

XIc

XIb

HG915908

HG916701

HG916744

HG916722

CCF 4680

Spain, Andalusia, Nerja Cave, isol ex cave sediment, 2012, A. Nováková

XIc

XIb

HG915909

HG916702

HG916745

HG916723

CCF 4697

Spain, Andalusia, Nerja Cave, isol ex cave air, 2012, A. Nováková

XIc

XIb

HG915910

HG916703

HG916746

HG916724

CCF 4830

Spain, Andalusia, Nerja Cave, isol ex cave sediment, 2012, A. Nováková

XIc

XIb

S716

Spain, Andalusia, Nerja Cave, isol ex cave sediment, 2012, A. Nováková

XIc

XIb

S718−S723

Spain, Andalusia, Nerja Cave, isol ex cave air, 2012, A. Nováková

XIc

XIb

HG916747 LM999908

LM644272

HG916748 HG916749–

LM644259

916753 BMP 3043 = CCF 4829

USA, Arizona, Benson, Kartchner Caverns, isol ex speleothem surface, 2008,

XIc

XIb

HQ832962d

LM644273

LM644253

LM644255

EF669582

EU014078

EF669540

EF669624

EF669578

EU014076

EF669536

EF669620

EF669583

EU014077

EF669541

EF669625

M.Vaughan Jani A. brevijanus

NRRL 1935T = ATCC 16828T = CBS T

T

Mexico, Alameda, isol ex soil, 1942, W.B. Roos

XII

XII

T

111.46 = IMI 16066 = CCF 4546 = FRR 1935 A. janus

T

NRRL 1787T = ATCC 16835T = CBS T

T

118.45 = IHEM 4374 = IMI 16065 T

= CCF 4549 = FRR 1787

Panama, isol ex soil, 1942 (soil was collected in 1941), J.T.Bonner

XIII

XIII

T

T

NRRL 1936 = IMI 362226 = CCF

Panama, isol ex soil, 1942, R.D. Coghill

XIV

XIV

4550 a

Acronyms of culture collections in alphabetic order: ATCC, American Type Culture Collection, Manassas, Virginia.; BMP, Barry M. Pryor laboratory culture collection; CBS, Centraalbureau voor Schimmelcultures, Utrecht, the

Netherlands; CCF, Culture Collection of Fungi at the Department of Botany of Charles University in Prague; CMF ISB, Collection of Microscopic Fungi of the Institute of Soil Biology of the Academy of Sciences of the Czech Republic in České Budějovice; FRR, Food Fungal Culture Collection, North Ride, Australia; IFM, Collection at the Medical Mycology Research Center, Chiba University; IHEM, Belgian Coordinated Collections of Micro-organisms (BCCM/IHEM); IMI, CABI’s collection of fungi and bacteria, Egham, UK; NRRL–Agricultural Research Service Culture Collection, Peoria, Illinois. b

CR, Czech Republic.

c

Neff et al. (2012).

d

Vaughan et al. (2011)

benA + calmodulin + RPB2 loci, exons only, GTR+G+I model

A. spelaeus clade

sect. Terrei 82 80

76

sect. Candidi

subg. Circumdati

NRRL 313 A. tritici T NRRL 3174 A. westerdijkiae NRRL 419 A. ochraceus NRRL 4850T A. elegans

sect. Circumdati NRRL 458 A. flavus T NRRL 502 A. parasiticus NRRL 20818T A. tamarii NRRL 13137T A. nomius NRRL 6599 A. leporis T NRRL 5025 A. cervinus (sect. Cervini) 70

sect. Flavi 0.1

NRRL 303T A. candidus

Jani

sect. Nigri

Flavipedes

A. flavipes clade

CMF ISB 2617 CMF ISB 2619 CCF 4032 NRRL 3750T CCF 1895 CMF ISB 2618 CMF ISB 2620 A. iizukae CMF ISB 2616 CMF ISB 2544 CMF ISB 2417 81 CCF 869 A. mangaliensis sp. nov. CCF 4698T T A. ardalensis sp. nov. CCF 4031 T NRRL 302 A. flavipes 89 CCF 2026 NRRL 295 NRRL 4578T A. frequens sp. nov. CCF 4005 NRRL 4263 A. neoflavipes nom. nov. NRRL 5504T CCF 4697 CCF 4679 CCF 4680 CCF 4699 A. spelaeus sp. nov. CCF 544 CCF 4425T NRRL 32683T A. polyporicola sp. nov. CCF 4410T A. movilensis sp. nov. NRRL 4610 T NRRL 6326 A. luppii sp. nov. NRRL 2399T A. neoafricanus NRRL 255T A. terreus T NRRL 4017 A. pseudoterreus NRRL 29810 A. alabamensis NRRL 1923T A. aureoterreus NRRL 4539T A. allahabadii T NRRL 527 A. carneus T NRRL 6134 A. neoindicus NRRL 4737T A. ambiguus NRRL 4749T A. microcysticus T NRRL 5299 A. neoniveus T NRRL 1787 A. janus NRRL 1936 T NRRL 1935 A. brevijanus NRRL 2053 Aspegillus sp. NRRL 4912T A. brunneoviolaceus NRRL 4747T A. heteromorphus NRRL 326T A. niger T NRRL 4875 A. tubingensis T NRRL 369 A. carbonarius NRRL 13001T A. campestris