International Journal of Systematic and Evolutionary Microbiology
Aspergillus asper and Aspergillus collinsii, two new species from Aspergillus section Usti --Manuscript Draft--
Manuscript Number: IJSEM-D-15-00773R2 Full Title: Aspergillus asper and Aspergillus collinsii, two new species from Aspergillus section Usti Short Title: Two new species from Aspergillus sect. Usti Article Type: Note Section/Category: New taxa - Eukaryotic micro-organisms Keywords: Systematics; fungi; section Usti; built environment Corresponding Author: Stephen W. Peterson National Center for Agricultural Utilization Research Peoria, IL UNITED STATES First Author: Zeljko Jurjevic Order of Authors: Zeljko Jurjevic Stephen W. Peterson Manuscript Region of Origin: UNITED STATES Abstract: In sampling fungi from the built environment, two isolates that could not confidently be placed in described species were encountered. Phenotypic analysis suggested that they belonged in Aspergillus sect. Usti. In order to verify the sectional placement and to assure that they were undescribed rather than phenotypically aberrant isolates, DNA was isolated and sequenced at the beta-tubulin, calmodulin, internal transcribed spacer, and RNA polymerase II loci and compared to those sequences from other species in Aspergillus. At each locus each new isolate was distant from existing species. Phylogenetic trees calculated from these data and GenBank data for section Usti species excluded the placement of these isolates in existing species, with statistical support. Because they were excluded from existing taxa Aspergillus asper (type strain NRRL 35910T=CBS140842T) and Aspergillus collinsii (type strain NRRL 66196T=CBS140843T) they were described as distinct species in sect. Usti.
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1 Aspergillus asper and Aspergillus collinsii, two new species from Aspergillus section Usti
2 Running title: Two new species from Aspergillus sect. Usti
3 Željko Jurjević
4 EMSL Analytical, Inc., 200 North Route 130, Cinnaminson, NJ 08077 USA
5 Stephen W. Peterson1
6 Mycotoxin Prevention and Applied Microbiology Research Unit, National Center for
7 Agricultural Utilization Research, Agricultural Research Service, U. S. Department of
8 Agriculture, 1815 North University Street, Peoria, IL 61604 USA
9 Key words: phylogenetic analysis, Usti, fungi, systematics, biodiversity,
10 Subject category: Eukaryotic taxonomy
11 Word count: abstract=142; main text=2968.
12 1Corresponding author: email: [email protected]; Phone: 309-681-6384
13 GenBank: novel DNA sequence accessions: Aspergillus asper BT2=KT698838,
14 CF=KT698839, ITS=KT698840, RPB2=KT698842; Aspergillus collinsii BT2=KT698843,
15 CF=KT698844, ITS=KT698845, RPB2=KT698848
16 Mycobank accessions: Aspergillus asper= Mycobank #814412, Aspergillus collinsii=
17 Mycobank #814413
18 Abbreviations: BT2, amplified fragment of the beta tubulin gene; CF amplified fragment of
19 the calmodulin gene; ITS, amplified fragment containing ITS1, 5.8S rDNA and ITS2; RPB2,
20 amplified fragment of the DNA dependent RNA polymerase II, second largest subunit.
21 Herbarium accessions: Aspergillus asper = BPI 893218, Aspergillus collinsii = BPI 893219.
22 BPI is the United States National Herbarium, USDA, Beltsville, MD.
23 Abstract
1
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 24 In sampling fungi from the built environment, two isolates that could not confidently be placed
25 in described species were encountered. Phenotypic analysis suggested that they belonged in
26 Aspergillus sect. Usti. In order to verify the sectional placement and to assure that they were
27 undescribed rather than phenotypically aberrant isolates, DNA was isolated and sequenced at
28 the beta-tubulin, calmodulin, internal transcribed spacer, and RNA polymerase II loci and
29 compared to those sequences from other species in Aspergillus. At each locus each new isolate
30 was distant from existing species. Phylogenetic trees calculated from these data and GenBank
31 data for section Usti species excluded the placement of these isolates in existing species, with
32 statistical support. Because they were excluded from existing taxa Aspergillus asper (type
33 strain NRRL 35910T=CBS140842T) and Aspergillus collinsii (type strain NRRL
34 66196T=CBS140843T) they were described as distinct species in sect. Usti.
35 Introduction
36 The Aspergillus ustus group was first recognized by Thom & Raper (1945) and
37 contained two species. Raper & Fennell (1965) recognized five species in the group. The
38 Aspergillus ustus group was provided a recognized nomenclatural designation as Aspergillus
39 section Usti (Gams et al., 1985). Houbraken and associates (Houbraken et al., 2007) added a
40 species to the section. Peterson (2008) using multilocus phylogenetic analysis of DNA
41 sequences included 15 species in the section while removing two species accepted by Raper &
42 Fennell (1965). Samson and associates (Samson et al., 2011) accepted Peterson’s analysis and
43 added more new species to the section. Visagie and others (Visagie et al., 2014) added another
44 new species A. porphyreostipitatus to the section. A notable feature of this section is that about
45 one third of the described species are known only from the type isolate or specimen.
2
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 46 Section Usti species are primarily environmental fungi, some cosmopolitan and
47 common, while others are known only from one or a few isolates. Aspergillus calidoustus is
48 recognized as an opportunistic human and animal pathogen (Varga et al., 2008, Balajee et al.,
49 2009, Hubka et al., 2012, Panackal et al., 2006) and one species, reported to be A. ustus, is
50 algicolous (Liu et al., 2014). Species from sect. Usti make a variety of exometabolites (Samson
51 et al., 2011, Liu et al., 2014) but production of the aflatoxin precursor sterigmatocystin that was
52 reported from A. ustus (Rabie et al., 1977) could not be verified in subsequent studies (Samson
53 et al., 2011).
54 In the course of environmental sampling and identification, we obtained isolates of
55 Aspergillus section Usti that could not be placed in any of the described species. We
56 determined DNA sequences from multiple loci of the isolates and conducted phylogenetic
57 analysis to determine whether these isolates were morphologically abnormal isolates of known
58 species or novel taxa. Descriptions and names for two new species A. asper and A. collinsii are
59 provided based on phenotype and phylogeny of a single isolate in each species.
60 Methods
61 Fungal isolates. Fungal isolates used in this study are listed in Table 1 and are available from
62 the Agricultural Research Service Culture Collection (NRRL), Peoria, IL, USA
63 (http://nrrl.ncaur.usda.gov). The CBS Biodiversity Culture Collection, Utrecht, The
64 Netherlands and the Charles University of Prague culture collection (CCF), Prague, The Czech
65 Republic also house the type isolates of A. asper and A. collinsii.
66 Media and examination. Cultures for morphological examination were grown at 25°C for 7 d
67 in darkness on Czapek yeast autolysate agar (CYA), malt extract agar (MEA, 20 g Difco malt
68 extract, 1 g peptone, 10g glucose per liter), CYA with 20 % sucrose (CY20S), CYA
3
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 69 supplemented with 5 % NaCl (CYAS), dichloran with 18% (w/v) glycerol agar (DG18),
70 oatmeal agar (OA), potato dextrose agar (PDA), and creatine sucrose agar (CREA) (Health
71 Link®, Jacksonville, FL, USA; http://www.healthlinkinc.net) formulated as specified (Pitt
72 1980, Samson et al., 2011). Difco malt extract was used. Additional CYA and MEA cultures
73 were incubated at different temperatures (15, 20, 30, 35, 37, 40 and 43°C) for 7 d. Cultures
74 were grown in duplicate as three-point inoculations in 9 cm diam Petri dishes. Fungal isolation
75 and microscopy were performed as described by Peterson & Jurjević (2013).
76 DNA methods. For DNA extraction, cultures were grown in 25 mL malt extract broth (30g
77 Difco malt extract per liter water) with shaking at 200 rpm and collected by vacuum filtration
78 over filter paper. DNA was extracted from freeze-dried mycelium using the CTAB method
79 (Peterson et al., 2015). Beta tubulin (BT2), calmodulin (CF), internal transcribed spacer region
80 (ITS), and RNA polymerase beta second largest subunit (RPB2) were amplified from genomic
81 DNA and sequenced using published methods (Peterson & Jurjević 2013). Phylogenetic
82 analysis of data sets from each locus was carried out with Mega 6.06 (Tamura et al., 2013)
83 using maximum likelihood criterion and the model determined using the built-in model
84 selection test routine. All GenBank numbers are provided in Table S1. Trees were prepared for
85 publication as previously described (Peterson et al., 2015).
86 Results
87 Phylogenic analysis of sequence data from the RPB2 locus is presented in Fig. 1, and analyses
88 of BT2, CF and ITS loci are presented in Fig. S1. Aspergillus asper (Fig. 1, RPB2) is in a
89 statistically supported clade containing A. thesauricus, A. keveii, A. pseudodeflectus, A.
90 calidoustus, and A. insuetus. High bootstrap values (99%) support the distinction of A. asper
91 from its sibling species in the clade. Phylogenetic analysis of the BT2, CF and ITS data also
4
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 92 reveal this clade (Fig. S1). The ITS locus is the barcode region and its occasional lack of
93 resolution among closely related species is known (Peterson 2012). Even so, Aspergillus asper
94 is distinguished from other sect. Usti species using the ITS barcode. Fig. 1 shows a 73%
95 bootstrap value supporting sibling relationship between A. asper and the A. insuetus, A. keveii,
96 A. thesauricus clade. Bootstrap values greater than 70% are viewed as greater than 95%
97 statistical probability (Hillis & Bull 1993).
98 Aspergillus collinsii occurs in a 98% bootstrap supported clade containing A. deflectus, A.
99 lucknowensis, A. turkensis and A. elongatus (Fig. 1) and each of these is strongly supported
100 (85–100% bootstrap) as a distinct species by the bootstrap statistic. The single locus trees from
101 the other loci contain no contradictions with the RPB2 data that are supported by bootstrap data,
102 although A. elongatus is, by chance, excluded from the clade in the ITS and BT2 data sets (Fig.
103 S1).
104
105 Taxonomy
106 Description of Aspergillus asper Ž. Jurjević & S.W. Peterson sp. nov.
107
108 Aspergillus asper (as´.per N. L. mas. adj. asper meaning rough refers to the very rough conidial
109 surface ornamentation).
110 Description of colonies after 7d (Fig.2): CYA at 25°C, conidial heads radiate, splitting with
111 age, grayish brown, good sporulation, mycelium white to grayish white, floccose, at margin
112 hyphae subsurface or submerged into media, no exudate, no soluble pigment, reverse pale-
113 yellow; CYA at 30°C, abundant sporulation, abundant aggregations of Hülle cells, white to pale
114 yellow, reverse brown; MEA at 25°C, conidial heads radiate, splitting with age, grayish brown,
5
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 115 poor to good sporulation, mycelium white, floccose, plane, at margin hyphae subsurface or
116 submerged into media ca 2–3 mm, no exudate, no soluble pigments, reverse pale brown; MEA
117 at 30°C, good sporulation, abundant carpet-like aggregation of Hülle cells, white to pale yellow,
118 overgrown with white hyphae; CY20S at 25°C, conidial heads grayish-brown, poor sporulation,
119 mycelium white, subsurface or submerged into media, no exudate, no soluble pigments, reverse
120 uncolored; OA at 25°C, conidial heads grayish-brown, sporulation poor to good, mycelium
121 white to pale yellowish, floccose, clear exudate sparse, no soluble pigments, reverse pale-
122 yellow; PDA at 25°C, conidial heads grayish-brown, radiate or occasionally short columnar,
123 splitting with age, poor to good sporulation, mycelium white to pale-yellow, floccose,
124 moderately deep radial sulca, no exudate or soluble pigments, reverse yellow; DG18 at 25°C,
125 conidial heads brownish-yellow to gray-green, good sporulation, radiate, mycelium white to
126 pale-yellowish shades, at margin hyphae subsurface or submerged into media ca 3–4 mm,
127 floccose, no exudate or soluble pigments, reverse pale yellow; CYAS at 25°C, conidial heads
128 grayish-brown, sporulation poor to good at the center of colony, radiate, mycelium white,
129 floccose, no exudate or soluble pigments, reverse pale buff; CREA at 25°C, no sporulation,
130 mycelium white, subsurface or submerged in media, no acid production.
131 Stipes smooth, occasionally roughened, near crustaceous, rarely septate, hyaline becoming
132 brownish with age, 10–150 × 3–4(–5) µm if born from aerial hyphae, (150–)200–1000(–1150)
133 × (4–)5–9(–10) µm if born from substrate; vesicles globose to pyriform, (4–)5–10 µm diam if
134 born from aerial hyphae, 10–25(–29) µm diam if borne from substrate, biseriate, metulae
135 cylindrical 3–8(–10) × 2.5–4.5(–6) µm, covering ½ to 2/3 of vesicle; phialides ampulliform, (4–
136 )5–7(–8) × 2.5–3.5(–5) µm; conidia globose to subglobose, 4–5(–7) µm diam, echinulate walls,
137 abundant Hülle cells irregularly elongate, ovoid, curved to coiled 10–90 µm long.
6
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 138 Medium dependent growth characteristics assessed as colony diam after 7d growth at 25°C on:
139 CYA 38–39 mm, MEA 30–32 mm, CY20S 25–27 mm, CYAS 9–10 mm, DG18 9–10 mm, OA
140 30–31 mm, PDA 29–30 mm, and CREA 30–31 mm. Temperature dependent growth assessed
141 as colony diam on CYA and MEA respectively, after 7 d growth at: 20°C 20–21 mm/20–21
142 mm, 30°C 50–51 mm/43–45 mm, 35°C 3–4 mm/ no growth, 37°C no growth/no growth, 40°C
143 no growth/no growth, 43°C no growth/no growth.
144
145 The holotype specimen, BPI-893218, is deposited in The United States National Herbarium, US
146 Department of Agriculture, Beltsville, MD and was isolated from house air in Pennsylvania,
147 USA. The culture ex type is NRRL 35910T (=CCF 5174T =CBS 140842T). The MycoBank
148 deposit number is 814412.
149
150 Morphological comparisons. Aspergillus asper morphologically most resembles A. keveii, A.
151 porphyreostipitatus, A. pseudodeflectus, A. granulosus and A. pseudoustus in terms of rapid
152 growth (30–50 mm diam 7d, 25 °C) and colonies appearing grayish or brownish. Aspergillus
153 asper is distinguished from these species, because only A. porphyreostipitatus, A. granulosus
154 and A. pseudodeflectus grow at 37°C or higher while A. asper does not grow at 37°C.
155 Aspergillus pseudoustus, A. keveii and A. asper do not grow at 37°C and A. pseudoustus and A.
156 keveii both have CYA25 colony colors in brownish shades while A. asper makes white to
157 grayish white colonies.
158
159 Description of Aspergillus collinsii Ž. Jurjević & S. W. Peterson, sp. nov.
160 Aspergillus collinsii (coll.ins´i.i N.L. noun in the genitive, honors Dr. Ralph P. Collins).
7
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 161
162 Description of cultures after 7d (Fig. 3): CYA at 25°C, conidial heads radiate, gray green, very
163 good sporulation at the center of colony ca 5–7 mm diam, mycelium yellow to orange or
164 orange-brown with age, floccose, lightly sulcate, rising ca 3–4 mm, at margin hyphae
165 subsurface or submerged into media ca 2–3 mm, no exudate, soluble pigment when present
166 faint reddish shades, reverse brownish-orange; MEA at 25°C, conidial heads radiate, buff, with
167 age becoming grayish-olive green at margins, poor to good sporulation, mycelium yellow to
168 buff with orange shades, white at margins, lightly floccose to funiculose, occasionally lightly
169 sulcate, at margin hyphae subsurface or submerged into media ca 2–3mm, no exudate, no
170 soluble pigments, reverse orange-brown; MEA with chloramphenicol at 25°C, conidial heads
171 orange-yellow to gray green, sporulation abundant, mycelium yellow-orange, reverse brown;
172 CY20S at 25°C, conidial heads greenish-gray, poor to good sporulation, mycelium white on
173 surface or submerged in media, no exudate, no soluble pigments, reverse uncolored; OM at
174 25°C, conidial heads gray-green, buff at margins at early stage, sporulation abundant, mycelium
175 orange, floccose, at margin hyphae subsurface or submerged into media ca 3 mm, orange
176 exudate abundant, yellow soluble pigment, reverse brownish red; PDA at 25°C, conidial heads
177 buff, very good sporulation, mycelium white to yellowish at margins, orange in the central 7–8
178 mm, floccose, no exudate or soluble pigment, reverse orange to buff at margins; DG18 at 25°C,
179 not sporulating, mycelium white, floccose, no exudate or soluble pigments; reverse uncolored;
180 CYAS at 25°C, not sporulating, mycelium white, subsurface or submerged into media, reverse
181 uncolored; CREA at 25°C, conidial heads bright green, very good sporulation, mycelium white,
182 subsurface or submerged in media, no acid production.
8
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 183 Stipes smooth walled, occasionally finely roughened, rarely septate, hyaline becoming
184 yellowish-orange with age, (45–)75–200(–260) × 3.5–7(–8) µm diam, occasionally terminating
185 with two vesicles, occasional rope-like structures seen, vesicle pyriform to globose (7–)9–18(–
186 22) µm diam, biseriate, metulae cylindrical (3–)4–6(–7) × 2–4 µm diam, covering ½ to 2/3 of
187 vesicle, phialides ampulliform, (4–)5–7(–8) × 2–2.5(–2.8) µm diam, conidia globose to
188 subglobose occasionally ellipsoidal, 2–2.5(–3.5) × 2–3 µm diam, smooth to occasionally finely
189 roughened walls.
190 Medium dependent growth characteristics assessed as colony diam after 7d growth at 25°C on:
191 CYA 14–15 mm; MEA 14–15 mm; CY20S 9–10 mm; CYAS 3–4 mm; DG18 8–9 mm; OA
192 14–15 mm; PDA 15–16 mm and CREA 13–14 mm. Temperature dependent growth assessed
193 as colony diam on CYA and MEA respectively, after 7 d growth at: 20°C 8–9 mm/9–10 mm;
194 30°C 22–23 mm/23–24 mm; 35°C 27–28 mm/35–36 mm; 40°C 14–16 mm/14–15 mm and
195 43°C no growth/no growth.
196
197 The holotype specimen, BPI 893219, is deposited in The United States National Herbarium, US
198 Department of Agriculture, Beltsville, MD and was isolated by Ž. Jurjević from an air settle
199 plate exposed in a domestic bathroom, Fair Oaks, California, August 2014. The ex type culture
200 is NRRL 66196T (=CCF 5175T =CBS 140843T). The Mycobank deposit number is MB814413.
201
202 Morphological comparisons. Among the species assigned to sect. Usti are four species whose
203 colonies on CYA attain 20 mm diam or less in 7 d at room temperature (22–24°C) or at 25°C.
204 Among those four species, pinkish or orange shaded CYA25 colonies are found in A. collinsii,
205 A. deflectus and A. turkensis while A. lucknowensis is sulfur yellow, becoming reddish in age
9
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 206 and is thus distinguished from A. collinsii. The vesicles of A. collinsii are aligned on the same
207 axis with their conidiophores while the A. deflectus vesicles are attached to the conidiophores at
208 a very distinctive and noticeable angle to the axis of the conidiophore. Aspergillus collinsii
209 colonies on CREA agar are very thin and composed of yellow hyphae while A. turkensis
210 colonies on CREA agar are plane, loose and composed of white hyphae. Neither species makes
211 acid on CREA.
212 Discussion
213 In the modern synthesis of species concepts, taxonomists describe species with the fundamental
214 idea that members of species are members of a gene pool distinct from all others ((Simpson
215 1951, de Quiroz, 2005). Other species concepts, e.g., biological species concept, phenetic
216 species concept, or species concepts based on exometabolite profiles, micromorphology, rDNA
217 sequence, or genealogical concordance, etc. are not species concepts in the theoretical sense but
218 represent species recognition systems (de Queiroz 2005). Taylor and associates (Taylor et al.,
219 2000) argued for the use of genealogical concordance as the character to determine whether
220 isolates are in the same or different species. We have shown that our new species are
221 genetically distinct, with statistical support in the bootstrap analysis of RPB2 DNA sequence
222 data.
223 Regardless of how one views species concepts there are several reasons to describe new
224 species, even when character variation in the species cannot be assessed because the species is
225 currently known only from a single isolate. In determining the phylogeny of a particular group,
226 missing taxa add uncertainty to the analysis, influencing the topology of trees (Rokas & Carroll,
227 2005). Some taxa are extinct and it may not be possible to include them in analysis, but all
228 extant species should be described and included in analysis to improve our phylogenetic
10
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 229 accuracy. Species names are also the reference point for different researchers to accumulate all
230 types of information about the species and organize the specific data sets.
231 The phylogenetic trees (Fig. 1, Fig. S1) show that both A. asper and A. collinsii are
232 distinct from the other recognized species of sect. Usti at the four loci examined with statistical
233 support from the bootstrap. Fig. 1 also reveals representative levels of variation among known
234 sect. Usti species having multiple isolates available. Both A. asper and A. collinsii are on
235 relatively long branches showing that they have no close relatives in the tree. Within the
236 context of Fig. 1, these two species are sufficiently different genetically from other species to
237 conclude that they are distinct. The bootstrap values on the tree provide statistical evidence that
238 the differences did not occur by random chance.
239
240 Acknowledgements
241 Amy McGovern provided skilled technical assistance, Nathane Orwig provided DNA
242 sequencing services. Mention of trade names or commercial products in this publication is
243 solely for the purpose of providing specific information and does not imply recommendation or
244 endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider
245 and employer.
246 References
247 Balajee, S. A., Kano, R., Baddley, J. W., Moser, S. A., Marr, K. A., Alexander, B. D.,
248 Andes, D., Kontoyiannis, D. P., Perrone, G., Peterson, S., Brandt, M. E., Pappas, P. G., &
249 Chiller, T. (2009). Molecular identification of Aspergillus species collected for the transplant-
250 associated infection surveillance network. J Clin Microbiol 47, 3138–3141.
251
11
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 252 Gams, W., Christensen, M., Onions, A. H., Pitt, J. I. & Samson, R. A. (1985). Infrageneric
253 taxa of Aspergillus. In Advances in Penicillium and Aspergillus systematics, pp. 55–62. Edited
254 by R. A. Samson & J. I. Pitt. New York, Plenum Press.
255
256 Hillis, D. M. & Bull, J. J. (1993). An empirical test of bootstrapping as a method for
257 assessing confidence in phylogenetic analysis. Syst Biol 42, 182–192.
258
259 Houbraken, J., Due, M., Varga, J., Meijer, M., Frisvad, J. C. & Samson, R. A. (2007).
260 Polyphasic taxonomy of Aspergillus section Usti. Stud Mycol 59, 107–128.
261
262 Hubka, V., Kubatova, A., Mallatova, N., Sedlacek, P., Melichar, J., Skorepova, M., Mencl,
263 K., Lyskova, P., Sramkova, B., Chudickova, M., Hamal, P. & Kolarik, M. (2012). Rare
264 and new etiological agents revealed among 178 clinical Aspergillus strains obtained from
265 Czech patients and characterized by molecular sequencing. Med Mycol 50, 601–610.
266
267 Liu, X. H., Miao, F. P., Liang, X. R. & Ji, N. Y. (2014). Ergosteroid derivatives from an
268 algicolous strain of Aspergillus ustus. Nat Prod Res 28, 1182–1186.
269
270 Panackal, A. A., Imhof, A., Hanley, E. W., & Marr, K. A. (2006). Aspergillus ustus
271 infections among transplant recipients. Emerg Infect Dis (www.cdc.gov/eid) 12, 403–408.
272
273 Peterson, S. W. (2008). Phylogenetic analysis of Aspergillus species using DNA sequences
274 from four loci. Mycologia 100, 205–226.
12
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 275
276 Peterson, S. W. (2012). Aspergillus and Penicillium identification using DNA sequences:
277 barcode or MLST? Appl Microbiol Biotechnol 95, 339–344.
278
279 Peterson, S. W. & Jurjević, Ž. (2013). Talaromyces columbinus sp. nov., and genealogical
280 concordance analysis in Talaromyces clade 2a. PLoS One 8, e78084.
281
282 Peterson, S. W., Jurjević, Ž. & Frisvad, J. C. (2015). Expanding the species and chemical
283 diversity of Penicillium section Cinnamopurpurea. PLoS One 10, e0121987.
284
285 Pitt JI (1980). The genus Penicillium and its teleomorphic states Eupenicillium and
286 Talaromyces. New York: Academic Press.
287
288 de Queiroz, K. (2005). Ernst Mayr and the Modern Concept of Species. Proc Natl Acad Sci
289 (USA) 102, 6600–6607.
290
291 Rabie, C. J., Steyn, M. & Schalkwyk, G. C. van. (1977). New species of Aspergillus
292 producing sterigmatocystin. Appl Environ Microbiol 33, 1023–1025.
293
294 Raper, K. B. & Fennell, D. I. (1965). The genus Aspergillus. Baltimore, Williams and
295 Wilkins.
296
13
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 297 Rokas, A. & Carroll, S. B. (2005). More genes or more taxa? The relative contribution of
298 gene number and taxon number to phylogenetic accuracy. Mol Biol Evol 22, 1337–1344.
299
300 Samson, R. A., Varga, J., Meijer, M. & Frisvad, J. C. (2011). New taxa in Aspergillus
301 section Usti. Stud Mycol 69, 81–97.
302
303 Simpson, G. G. (1951). The species concept. Evolution 5, 285–298.
304
305 Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6:
306 Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 30, 2725–2729.
307
308 Taylor, J. W., Jacobson, D. J., Kroken, S., Kasuga, T., Geiser, D. M., Hibbett, D. S. &
309 Fisher, M. C. (2000). Phylogenetic species recognition and species concepts in fungi. Fungal
310 Genet Biol 31, 21–32.
311
312 Thom, C. & Raper, K. B. (1945). Manual of the Aspergilli. Baltimore, Williams and
313 Wilkins.
314
315 Varga, J., Houbraken, J., Van Der Lee, H. A. L., Verweij, P. E. & Samson, R. A. (2008).
316 Aspergillus calidoustus sp. nov., causative agent of human infections previously assigned to
317 Aspergillus ustus. Eukaryot Cell 7, 630–638.
318
14
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 319 Visagie, C. M., Hirooka, Y., Tanney, J. B., Whitfield, E., Mwange, K., Meijer, M., Amend,
320 A. S., Seifert, K. A. & Samson, R. A. (2014). Aspergillus, Penicillium and Talaromyces
321 isolated from house dust samples collected around the world. Stud Mycol 78, 63–139.
322
323 Figure legends.
324 Fig. 1. Phylogenetic analysis of Aspergillus section Usti based on maximum likelihood
325 analysis of the RPB2 locus; A. sparsus is the outgroup species. Numbers near nodes are
326 bootstrap percentages based on 1000 bootstrap samplings. Aspergillus collinsii and Aspergillus
327 asper are presented in bold text and each is distinguished from other section Usti species with
328 high statistical probability. Intraspecific variability can be assessed from some species that are
329 represented by two isolates. Two distinct species, A. pseudodeflectus and A. calidoustus are not
330 different in this tree, although differences exist at other loci.
331
332 Fig. 2. Aspergillus asper NRRL 35910. (a) Conidiophore with globose to pyriform vesicle,
333 metulae, phialides and conspicuously roughened conidia, (b) elongated hülle cells; 7d growth at
334 25°C on, (c) CYA, (d) MEA, (e) CYS20, (f) OA, (g) PDA and (h) CREA. Bar=10 µm for
335 microphotographs.
336
337 Fig. 3. Aspergillus collinsii NRRL 66196. (a) Conidiophores with globose to pyriform
338 vesicles, metulae, phialides and smooth to finely roughened spherical conidia; growth for 7 d at
339 25°C on (b) CYA, (c) MEA, (d) CYS20, (e) OA, (f) PDA and (g) CREA agar. Bar=10 µm for
340 microphotograph.
341 Table legends.
15
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 342 Table 1. Provenance of isolates examined and sequenced in this study.
NRRL Provenance number Aspergillus asper 35910 T USA, Pennsylvania, isol ex air sampler, January 2008, Ž. Jurjević; =Z-345, culture ex type. Aspergillus baeticus 62501 T Spain, Gruta de las Maravillas cave, isol ex cave sediment, April 2012, Vit Hubka. culture ex type=CCF 4226 62487 Spain, Gruta de las Maravillas cave, isol ex cave sediment, April 2012, Vit Hubka. culture ex type=CCF 4226 Aspergillus calidoustus 26162 USA, Peoria, isol ex NRRL 3361 as a contaminant, September 1968. Aspergillus californicus IBT 16748 T USA, California, ex chamise chaparral, 1978, Jeff S. La Favre. Aspergillus carlsbadensis IBT 14493 T USA, New Mexico, Lechuguilla Cave, soil, 1992, D. E. Northrup. Culture ex type. Aspergillus cavernicola 6327T Romania, isol ex cave soil, Caprei grotto, Defileul Nature Reserve, culture ex type 5813 USSR, isol ex wheat starch, culture ex type of A. amylovorous Aspergillus collinsii 66196 T USA, isol ex air sample, November 2014, Ž. Jurjević, culture ex type Aspergillus compatibilis 5097 T Costa Rica, Esparta, isol ex forest soil, J. C. Cavender 5096 Costa Rica, Esparta, isol ex forest soil, J. C. Cavender Aspergillus deflectus 2206 T Brazil, Rio de Janeiro, isol ex soil, 1949, A. Cury, isolate ex type 4235 USA, Nebraska, isol ex potting soil, 1955, R. W. Embree Aspergillus egyptiacus 5920 T Egypt, isol ex sandy soil in olive grove, 1974, culture ex type Aspergillus elongatus 5176 T India, isol ex alkaline soil, 1970 Aspergillus funiculosus 4744 T Nigeria, isol ex soil, 1965 Aspergillus germanicus DTO 179B7 South Africa, isol ex house dust Aspergillus granulosus 1932 T USA, Arkansas, Fayetteville, isol ex soil, isolate ex type 1931 USA, Texas, Greenville, isol ex soil, 1942 Aspergillus insuetus 279 T South Africa, Capetown, isol ex soil, Putterill, isolate ex type Aspergillus kassunensis 3752 T Syria, isol ex soil, A. Onions Aspergillus keveii 1974 Panama, isol ex soil, 1942 Aspergillus lucknowensis 3491 India, Lucknow, isol ex soil, 1969, J. N. Rai Aspergillus porphyreostipitatus
16
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 DTO 266 Mexico, Sayulita, isol ex church dust, 2010, Ed Whitfield & Kalima Mwange Aspergillus pseudodeflectus 6135 T Egypt, isol ex desert soil, 1974. Aspergillus pseudoustus IBT 28161 South Africa, isol ex stored maize, ca. 1973. Aspergillus puniceus 1852 USA, Louisiana, isol ex soil near greenhouse, 1942 5077 Costa Rice, isol ex soil Aspergillus subsessilis 4905 T USA, California, isol ex Mojave desert soil, 1961, G. F. Orr. 4907 USA, California, isol ex Mojave desert soil, 1961, G. F. Orr. Aspergillus thesauricus CCF 4152 T Spain, Gruta de las Maravillas cave, isol ex cave sediment, April 2012, Vit Hubka. CCF 4150 Spain, Gruta de las Maravillas cave, isol ex cave sediment, April 2012, Vit Hubka. Aspergillus turkensis 4993T Turkey, Ankara, isol ex soil, 1950, culture ex type Aspergillus ustus 275 T USA, Connecticut, isol ex A. sydowii culture, 1914, C. Thom. 4991 Brasil, isol ex soil, 1969, D.I. Fennell 343
17
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 T Figure 1 93ClickNRRL here to 275downloadA. ustus Figure Fig1.eps
99 NRRL 4991 A. ustus 100 NRRL 62487 A. baeticus 100 NRRL 62501T A. baeticus
100 NRRL 1852 A. puniceus 86 NRRL 5077T A. puniceus NRRL 1931 100 A. granulosus NRRL 1932T A. granulosus NRRL 1974 100 A. keveii 100 NRRL 58663T A. keveii CCF4152 A. thesauricus 99 CCF4166T A. thesauricus 73 NRRL 279T A. insuetus 99 100 T NRRL 35910 A. asper
100 NRRL 6135 A. pseudodeflectus NRRL 26162 A. calidoustus
T 100 NRRL 5096 A. compatibilis 97 NRRL 5097 A. compatibilis
T 100 NRRL 2206 A. deflectus 100 NRRL 4235 A. deflectus 85 NRRL 3491T A. lucknowensis
98 T NRRL 4993 A. turkensis
100 T NRRL 66196 A. collinsii
NRRL 5176T A. elongatus NRRL 4905T 100 A. subsessilis 100 NRRL 4907 A. subsessilis
NRRL 3752T A. kassunensis
100 NRRL 5813 A. cavernicola NRRL 6327T A. cavernicola NRRL 5920T A. egyptiacus Downloaded from www.microbiologyresearch.org by T NRRL 1933 A. sparsusIP: 130.88.242.12 10 On: Thu, 12 May 2016 13:41:06 Figure 2 Click here to download Figure Fig. 2.tif
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 Figure 3 Click here to download Figure Fig. 3.tif
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 Supplementary Material Files Click here to download Supplementary Material Files supplementary data.pdf
( a ) ( b ) ( c )
T CCF 4152T 85 NRRL 2206 Aspergillus deflectus NRRL 279 81 Aspergillus thesauricus Aspergillus insuetus NRRL 3491T DTO 179-B7 Aspergillus germanicus Aspergillus lucknowensis NRRL 1974 Aspergillus keveii 94 T NRRL 1974 Aspergillus keveii NRRL 66196 Aspergillus collinsii DTO 179-B7 Aspergillus germanicus 82 T NRRL 6135T NRRL 4993 Aspergillus turkensis T 95 Aspergillus pseudodeflectus CCF 4152 Aspergillus thesauricus NRRL 5176T NRRL 26162 Aspergillus calidoustus Aspergillus elongatus NRRL 26162 Aspergillus calidoustus T NRRL 279 Aspergillus insuetus NRRL 3752 Aspergillus kassunensis NRRL 6135T Aspergillus pseudodeflectus 100 T T 100 NRRL 4905 T NRRL 35910 Aspergillus asper Aspergillus subsessilis NRRL 35910 Aspergillus asper
T IBT 14493T IBT 16748 Aspergillus californicus T Aspergillus carlsbadensis 81 NRRL 62501 Aspergillus baeticus T T NRRL 5176 Aspergillus elongatus NRRL 6327 Aspergillus cavernicola DTO 266D9 Aspergillus porphyreostipitatus T NRRL 2206T Aspergillus deflectus NRRL 5920 Aspergillus egyptiacus IBT 28161T Aspergillus pseudoustus
T NRRL 66196T Aspergillus collinsii 97 CCF 4152 Aspergillus thesauricus NRRL 5097 Aspergillus compatibilis 100 T DTO 179-B7 NRRL 3491 Aspergillus lucknowensis 73 Aspergillus germanicus NRRL 1852 Aspergillus puniceus 99 NRRL 4993T Aspergillus turkensis NRRL 279 Aspergillus insuetus NRRL 275T Aspergillus ustus 98 IBT 28161T NRRL 1974 Aspergillus keveii T Aspergillus pseudoustus 82 IBT 14493 Aspergillus carlsbadensis T T NRRL 62501 Aspergillus baeticus NRRL 35910 Aspergillus asper CBS 434.95T Aspergillus monodii
T T 73 NRRL 275 Aspergillus ustus 98 IBT 14493 Aspergillus carlsbadensis NRRL 1932T Aspergillus granulosus 96 DTO 266D9 NRRL 6135T NRRL 2206T 83 Aspergillus porphyreostipitatus Aspergillus pseudodeflectus 98 Aspergillus deflectus 96 NRRL 1852 Aspergillus puniceus NRRL 26162 Aspergillus calidoustus 70 NRRL 3491T Aspergillus lucknowensis
T NRRL 5097 Aspergillus compatibilis CBS 434.95 Aspergillus monodii 97 NRRL 4993T Aspergillus turkensis
T T NRRL 1932 Aspergillus granulosus IBT 28161 Aspergillus pseudoustus NRRL 66196T Aspergillus collinsii T DTO 266D9 T 81 CBS 434.95 Aspergillus monodii Aspergillus porphyreostipitatus NRRL 5176 Aspergillus elongatus
T 97 T NRRL 3752 Aspergillus kassunensis NRRL 275 Aspergillus ustus IBT 16748T Aspergillus californicus 84 T 100 T T 99 IBT 16748 Aspergillus californicus NRRL 62501 Aspergillus baeticus 99 NRRL 4905 Aspergillus subsessilis NRRL 4905T Aspergillus subsessilis NRRL 1852 Aspergillus puniceus NRRL 3752T Aspergillus kassunensis
81 T T NRRL 6327 Aspergillus cavernicola NRRL 1932 Aspergillus granulosus NRRL 6327T Aspergillus cavernicola NRRL 5920T Aspergillus egyptiacus NRRL 5097 Aspergillus compatibilis NRRL 5920T Aspergillus egyptiacus NRRL 4744T Aspergillus funiculosus NRRL 4744T Aspergillus funiculosus NRRL 4744T Aspergillus funiculosus
T NRRL 1933T Aspergillus sparsus NRRL 1933 Aspergillus sparsus NRRL 1933T Aspergillus sparsus 0.1 0.1 0.1
Fig. S1. Phylogenetic analysis of DNA sequences using maximum likelihood criterion for sequences of (a) beta tubulin, (b) calmodulin and (c) internal transcribed spacer region. Numbers near nodes are bootstrap values. The new species are marked in bold font. Some species are not present in all trees because the authors did not provide sequences from that particular locus. Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06 Table S1. GenBank numbers for isolates used in phylogenetic analysis.
Species Isolate BT2 CF ITS RPB2 Aspergillus asper NRRL 35910 KT698838 KT698839 KT698840 KT698842 Aspergillus baeticus CCF 4226 HE615092 HE615117 HE615086 HE615124 CCF 4150 HE615093 HE615118 HE615087 HE615125 Aspergillus calidoustus NRRL 26162 EF652276 EF652364 EF652452 EF652188 A. californicus IBT 16748 FJ531180 FJ531128 FJ531153 no data A. carlsbadensis IBT 14493 FJ531169 FJ531124 FJ531148 no data Aspergillus cavernicola NRRL 6327 EF652332 EF652420 EF652508 EF652244 NRRL 5813 EF652327 EF652415 EF652503 EF652239 Aspergillus collinsii NRRL 66196 KT698843 KT698844 KT698845 KT698848 Aspergillus compatibilis NRRL 5097 EF652324 EF652412 EF652500 EF652236 NRRL 5096 EF652323 EF652411 EF652499 EF652235 Aspergillus deflectus NRRL 2206 EF652261 EF652349 EF652437 EF652173 NRRL 4235 EF652287 EF652375 EF652463 EF652199 Aspergillus egyptiacus NRRL 5920 EF652328 EF652416 EF652504 EF652240 Aspergillus elongatus NRRL 5176 EF652326 EF652414 EF652502 EF652238 A. germanicus DTO 179B7 KP329849 KJ775277 KJ775491 no data Aspergillus granulosus NRRL 1931 EF652253 EF652341 EF652429 EF652165 NRRL 1932 EF652254 EF652342 EF661078 EF652166 Aspergillus insuetus NRRL 279 EF652281 EF652369 EF652457 EF652193 Aspergillus kassunensis NRRL 3752 EF652285 EF652373 EF652461 EF652197 Aspergillus keveii NRRL 1974 EF652256 EF652344 EF652432 EF652168 A. lucknowensis NRRL 3491 EF652283 EF652371 EF652459 EF652195 A. monodii CBS 435.95 FJ531171 FJ531142 FJ531150 no data A. porphyreostipitatus DTO266D9 KJ775080 KJ775338 KJ775564 no data A. pseudodeflectus NRRL 6135 EF652331 EF652419 EF652507 EF652243 A. pseudoustus IBT 28161 FJ531168 FJ531129 FJ531147 no data Aspergillus puniceus NRRL 1852 EF652249 EF652337 EF652425 EF652161 NRRL 5077 EF652322 EF652410 EF652498 EF652234 Aspergillus subsessilis NRRL 4905 EF652309 EF652397 EF652485 EF652221 NRRL 4907 EF652311 EF652399 EF652487 EF652223 Aspergillus thesauricus CCF 4152 HE615096 HE615121 HE615089 HE615127 CCF 4166 HE615095 HE615120 HE615088 HE615126 Aspergillus turkensis NRRL 4993 EF652318 EF652406 EF652494 EF652230 Aspergillus ustus NRRL 275 EF652279 EF652367 EF652455 EF652191 Aspergillus ustus NRRL 4991 EF652316 EF652404 EF652492 EF652228 Aspergillus sparsus NRRL 1933 EF661125 EF661173 EF661181 EF661071
Downloaded from www.microbiologyresearch.org by IP: 130.88.242.12 On: Thu, 12 May 2016 13:41:06