Aspergillus section Versicolores: nine new species and ... · PDF fileremoved seven species...

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© 2012 International Mycological Association

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INTRODUCTION

Aspergillus section Versicolores was originally erected as the Aspergillus versicolorgroupbyThom&Church(1926)andwassubsequentlyrevisedbyThom&Raper(1945)tocontainfour species. Raper & Fennell (1965) revised the genusAspergillus and accepted 18 species in the A. versicolor group. Gams et al.(1985)formalizedthesectionaltaxonomyofRaper&Fennell’s(1965)groups. Using scanning electron microscopy (SEM), Kozakiewicz (1989) examined conidialsurface ornamentation of most species of the section and removed seven species from section Versicolores. Klich(1993)revisedthesectionbasedonmorphologicalandothercharacteristics and accepted the seven species previously removedbyKozakiewicz (1989) fromsectionVersicolores.Peterson (2008) accepted four phylogenetically distinctspecies in the section based on multilocus DNA sequence analysis,placing theother14species indifferentcladesofAspergillus.

Aspergillus versicolor is the most widely reported and studied species in section Versicolores.Ithasbeenisolatedfromsoil(Domschet al.1980),indoorenvironments(Samsonet al.2001,Sheltonet al.2002,Engelhart et al.2002,Amendet al.2010,Andersonet al.2011),variousfoodsandfeeds(Pitt & Hocking 2009) and hypersaline water (Kis-Papo et al.2003,Mbata2008),and isassociatedwithmanyhealthissues of humans and animals (Jussila 2003, Perri et al.2005, Baddleyet al. 2009, Edmondsonet al. 2009, Pitt &Hocking2009,Moreno&Arenas2010). It isaproducerofthemycotoxinsterigmatocystinthatisaprecursorofaflatoxinB1(Mills&Abramson1986,Tuomiet al.2000,Nielsen2003,Veršilovskis&Saeger2010).

Environmental isolates of section Versicolores species exhibit great variation in macro-phenotypic ones but fewdifferences in micro-phenotypic characters (Domsch et al. 1980, Klich 2002, Raper & Fennell 1965, Thom&Church1926, Vesonder & Horn 1985), leading us to conduct aDNA-based phylogenetic study. to determine the limits of

doi:10.5598/imafungus.2012.03.01.07IMA FUNgUs · vOlUMe 3 · NO 1: 59–79

Aspergillus section Versicolores: nine new species and multilocus DNA sequence based phylogeny

ZeljkoJurjevic1,StephenW.Peterson2,andBruceW.Horn3

1EMSLAnalytical,Inc.,200Route130North,Cinnaminson,NJ080772Bacterial FoodbornePathogens andMycologyResearchUnit, NationalCenter forAgricultural UtilizationResearch,Agricultural ResearchService, U.S. Department ofAgriculture, 1815 North University Street, Peoria, Illinois 61604 USA; corresponding author email: [email protected],AgriculturalResearchService,U.S.DepartmentofAgriculture,P.O.Box509,Dawson,GA39842-0509

Abstract: β-tubulin,calmodulin,internaltranscribedspacerandpartiallsu-rDNA,RNApolymerase2,DNAreplication licensing factor Mcm7, and pre-rRNA processing protein Tsr1wereamplifiedandsequencedfromnumerous isolates belonging to Aspergillussect. versicolor.Theisolateswereanalyzedphylogeneticallyusingtheconcordancemodeltoestablishspeciesboundaries.Aspergillus austroafricanus, A. creber, A. cvjetkovicii, A. fructus, A. jensenii, A. puulaauensis, A. subversicolor, A. tennesseensis and A. venenatus are described as new species and A. amoenus, A. protuberus, A. sydowii, A. tabacinus and A. versicolor are accepted as distinct species on the basis of molecular and phenotypic differences. PCR primer pairs used to detect A. versicolor in sick building syndrome studies have a positive reaction for all of the newly describedspeciesexceptA. subversicolor.

Article info: Submitted:17January2012;Accepted:7June2012;Published:21June2012.

Key words: Aspergillus amoenusAspergillus austroafricanusAspergillus creberAspergillus cvjetkoviciiAspergillus fructusAspergillus jenseniiAspergillus protuberusAspergillus puulaauensisAspergillus subversicolorAspergillus sydowiiAspergillus tabacinusAspergillus tennesseensisAspergillus venenatusAspergillus versicolorconcordance analysisphylogenysystematics

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variationwithin species,weamplifiedandsequencedDNAfrom6lociandusedconcordanceanalysistoidentifyspeciesboundaries(Dettmanet al.2003)withinsectionVersicolores.The species described and accepted are monophyletic.

MATeRIAls AND MeTHODs

Fungal isolatesTheprovenanceoffungalisolatesexaminedinthisstudyisdetailedinTable1andtheseculturesareavailablefromtheAgricultural Research Service Culture Collection (NRRL),Peoria,Illinois(http://nrrl.ncaur.usda.gov).

Culture methodsCulturesweregrownonCzapekyeastextractagar(CYA)at5°C,25°C,and37°Candonmaltextractagar(MEA),CY20S,M40YandM60Y,allat25°Cfor10dindarkness(Pitt1980,Klich2002).M40Ycontained2%maltextract,0.5%yeastextractand40%sucrose;M60Ycontained2%maltextract,0.5% yeast extract and 60% sucrose. Colony diametersand appearance were recorded and photographs were made from10-dcultureplatesincubatedat25°C.ColornamesarefromRidgway(1912)andarereferredtowithplatenumber,e.g.R45.

MicroscopyMicroscopicexaminationwasperformedby teasingapartasmallamountofmycelium inadropof0.1%TritonX-100and examining the preparation under bright field or DIFillumination.Additionalmicroscopicsamplesweremadebygently pressing a ca 20×5mmpieceof transparent tapeontoacolony,rinsingthetapewithoneortwodropsof70%ethanolandmountingthetapeinlacticacidwithfuchsindye.ALeicaDM2500microscopewithbrightfield,phasecontrastand DIF contrast optics was used to view the slides. TheSpotcamerawithspotimagingsoftwarewasmountedonthemicroscopeandused forphotomicrography.ANikondigitalSLRcamerawithD70lenswasusedforcolonyphotography.PhotographswereresizedandfittedintoplateswithMicrosoftPowerPoint2003orAdobePhotoshop.

DNA methodsConidiafromagarslantcultureswereusedtoinoculate125-mLErlenmeyerflaskscontaining25mLofmaltextractbroth.Culturesweregrownonarotaryplatform(200rpm)for2–3dat25°C.Biomasswascollectedbyvacuumfiltration,andthenfrozen and freeze-dried in microfuge tubes. Dry myceliumwasground toapowder, rehydratedwithCTABbufferandextracted with chloroform; the phases were separated bycentrifugation and DNA was precipitated from the aqueous phase with an equal volume of isopropanol. Total nucleicacids were collected by centrifugation, the pellet was rinsed with70%ethanol,and thenucleicacidsweredissolved in100μLsteriledeionizedwater.

DNA was diluted ca 1:100 with sterile deionized waterfor use in amplifications. β-tubulin (BT2), calmodulin (CF),ITS and partial lsu-rDNA (ID), RNA polymerase 2 (RPB2),DNA replication licensing factor (Mcm7), and pre-rRNAprocessingprotein (Tsr1)wereamplifiedwithprimersused

by Peterson et al. (2010). Standard buffer and conditionswereusedwithathermalprofileof95°Cfor2minfollowedby35cyclesof96 °C for30sec;51 °C for60sec;72 °Cfor60sec;andafinalextensionphaseof72°Cfor5min.Occasionally,multipleamplificationbandswereobtainedanda higher annealing temperature was used to obtain single amplification bands. DNA sequencing was performed onbothtemplatestrandsusingdyeterminatortechnology(v3.1)andanABI3730sequencer,bothfromAppliedBiosystems(http://www.appliedbiosystems.com/). Raw sequences (bi-directional) were corrected using Sequencher (http://www.genecodes.com/). Corrected sequences were aligned forphylogenetic analysis using CLUSTALW (Thompson et al. 1994).SequencesweredepositedinGenBankasaccessionsJN853798–JN854131, EF652176, EF652178, EF652185–EF652187, EF652196, EF652203, EF652209–EF652211,EF652214–EF652216, EF652226, EF652264, EF652266,EF652273–EF652275, EF652284, EF652291, EF652297–EF652299, EF652302–EF652304, EF652314, EF652352,EF652354, EF652361–EF652363, EF652372, EF652379,EF652385–EF652387, EF652390–EF652392, EF652402,EF652440, EF652442, EF652449–EF652451, EF652460,EF652467, EF652473–EF652475, EF652478–EF652480,EF652490andJQ301889–JQ301896.

ParsimonyanalysiswasconductedusingPAUP*4.0b10(Swofford2003).Forsingle-locusdatasets,thecriterionwasparsimony, addition order was random (5000 replications),branch swapping was NNI (nearest neighbor interchange)andmaxtreeswassetat5000.Thesetoftreesgeneratedwas used as the starting point for parsimony analysis with additionorder“as is”andTBRbranchswapping.Bootstrapanalysiswasconductedwith“asis”additionorderandTBRbranchswappingfor1000replications.

Bayesian posterior probabilities were calculated usingMrBayes 3.12 (Huelsenbeck & Ronquist 2001, Ronquist &Huelsenbeck 2003). The Mcm7, Tsr1 and RPB2 data sets included only protein-coding sequences and each data set was partitionedintocodonpositions1,2,and3.TheBT2 and CF loci included protein-coding and intron regions and the data were partitioned into intron and exon data. A GTR (generaltime-reversible)modelwasusedwithaproportionofinvariantsitesandagamma-shapeddistributionofratesacrossthesites.MarkovchainMonteCarlo(MCMC)analysiswasconductedforupto5×106generationsuntilthechainsconverged.

Concordance analysis was based on the exclusionaryprinciple of Baum & Shaw (1995) and the genealogicalconcordance phylogenetic species recognition concepts of Taylor et al.(2000).Cladeswererecognizedasindependentevolutionary lineages if 1) the clade was present in themajorityofsingle-locusgenealogies(majorityruleconsensus)or 2) if a cladewas strongly supported by both parsimonyand Bayesian analysis in at least one locus, and was notcontradictedbyanotherstronglysupportedlocus(Dettmanet al.2003).Strongsupportwasassessedas>70%bootstrapand>0.95posteriorprobability(Dettmanet al.2003).TheprimersusedforidentificationofA. versicolor in a PCR amplification(Deanet al.2005)weretestedusingtheprimersequencesandamplification thermalprofile recommended,but in a uniplex rather thanmultiplex amplification system(Deanet al.2005).

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Table 1.Provenanceoffungalisolatesused.

NRRl number Provenance

Aspergillus amoenusMycoBankMB250654

226 USA:isol.exmammarygland,1913.

236 Germany:Munster,isol.exaBerberissp.fruit,1930,M. Roberg.

658 UK:isol.exbrinedmeat,1929,G. A. Ledingham.

4838 EquivalenttoNRRL236,receivedfromCentraalbureauvoorSchimmelcultures,1962,ex-type.

35600 USA:Hawaii,KapukaPauula,isol.exthebasidiomataofGandoderma australe,2005, D.T. Wicklow.

A-23228 India:Karnataka,isol.excoffeeberry,1978,B. Muthappa.

Aspergillus asperescensStolkMycoBankMB292835

4770 Ex-type,out-groupspecies.

Aspergillus austroafricanus sp. nov.,MycoBankMB800597

233 SouthAfrica:Capetown,unknown,1922,sentby V. A. Putterill,ex-type.

Aspergillus creber sp. nov.,MycoBankMB800598

231 SouthAfrica:Capetown,unknown,1922,sentbyV. A. Putterill.

6544 AtlanticOcean:isol.exafloatingtarball,1979,A. Wellman.

25627 Japan:Ibaraki,isol.exteafieldsoil,1996,T. Goto.

58583 USA:Pennsylvania,isol.exindoorairsampler,2008,Z. Jurjevic.

58584 USA:California,isol.exindoorairsample,2008,Z. Jurjevic.


58592 USA:California,isol.exindoorairsample,2008,Z. Jurjevic,ex-type.

58597 USA:NewJersey,isol.exindoorairsample,2008,Z. Jurjevic.

58601 USA:NewJersey:isolatedfromindoorairsample,2009,Z. Jurjevic.

58606 USA:Pennsylvania,isol.exindoorairsample,2009,Z. Jurjevic.




58672 USA:Georgia,isol.exindoorairsample,2009,Z. Jurjevic.

58673 USA:Georgia,isol.exindoorairsample,2009,Z. Jurjevic.

58675 USA:Ohio,isol.exindoorairsample,2009,Z. Jurjevic.

Aspergillus cvjetkovicii sp. nov., MycoBankMB800599

227 USA:NewJersey,isol.exsoil,1915,G.W. Wilson,ex-type.

230 China:isol.exsoysauce,1917,Round.

4642 Unknown:senttoNRRL,1969,D. I. FennellasWB4642.


Aspergillus fructus sp. nov., MycoBankMB800600

239 USA:California,isol.exdatefruit,1939,Bliss,ex-type.

241 Unknown:isol.expomegranatefruit,1916,L. McCulloch.

Aspergillus jensenii sp. nov., MycoBankMB800601

225 UK:unknown,1913,senttoC.ThombyDade.

235 UK:London,isol.exparaffin,1930,H. Raistrick.

240 USA:NewYork,Ithaca,isol.extherhizosphereofpepperplants,1911,C. N. Jensen,senttoC.ThombyWhetzelastype strain of A. globosus.

58582 USA:Montana,isol.exindoorairsample,2008,Z. Jurjevic.

58600 USA:Montana,isol.exindoorairsample,2008,Z. Jurjevic,ex-type.


58674 USA:Ohio,isol.exindoorairsample,2009,Z. Jurjevic.

Aspergillus multicolorSappaMycoBankMB292849

4775 Ex-type,out-groupspecies.



Table 1.(Continued).NRRl number Provenance

Aspergillus protuberusMycoBankMB326650


3505 Yugoslavia,isol.exrubbercoatedelectricalcables,ca1968,ex-type.




58990 USA:Connecticut,isol.exindoorairsample,2009,Z. Jurjevic.

58991 USA:Connecticut,isol.exindoorairsample,2009,Z. Jurjevic.

Aspergillus puulaauensis sp. nov., MycoBankMB800602

35641 USA:Hawaii,Pu’ula’auHighway200,isol.exdeadhardwoodbranch,2003,D. T. Wicklow,ex-type.

58602 USA:WestVirginia,isol.exindoorairsample,2009,Z. Jurjevic.

62124 USA:Hawaii,mesicmountainforest,isol.exbasidiomataofInonotussp.,2003,D. T. Wicklow.

62516 Canada:Alberta,isol.exairsampleinbeehouse,ca1990,S. P. Abbot,equivalenttoUAMH7651.

Aspergillus subversicolor sp. nov., MycoBankMB800603

58999 India:Karnataka,isol.excoffeeberry,1970,B. Muthappa,ex-type.

Aspergillus sydowiiMycoBankMB279636

250 Unknown:priorto1930,senttoC.ThombyM. Swift.

254 USA:Georgia,Waycross,clinicalisolate,1940,M. M. Harris.

4768 USA:California,isol.exsoil,1969.

62450 Thailand:isol.exdeadplantstem,1977,E. G. Simmons.

Aspergillus tabacinusMycoBankMB539544


4791 Unknown:isol.extobacco,1934,Y. Nakazawa,ex-type.

5031 Unknown: type isolate of A. versicolorvar.magnusSasaki,receivedfromIFO,1962.

62481 Nepal:Kathmandu,isol.exmaize,1977.

Aspergillus tennesseensis sp. nov., MycoBankMB800604

229 Unknown:senttoC.Thom,1917,byR. Thaxter.

234 USA:Maryland,Beltsville,isol.exchestnutseed,1927,C. Thom.

13150 USA:Tennessee,isol.extoxicdairyfeed,1984,B. W. Horn,ex-type.

13152 USA:Tennessee,isol.extoxicdairyfeed,1984,B. W. Horn.

Aspergillus venenatus sp. nov., MycoBankMB800605

13147 USA:Tennessee:isolatedfromtoxicdairyfeed,1984,B. W. Horn,ex-type.



62457 USA:Missouri,isol.excorn,1989,D. T. Wicklow.

Aspergillus versicolorMycoBankMB172159

238 USA:isol.exunrecordedsubstrate,1935,V. K. Charles,ex-type.

5219 SouthAfrica:Pretoria,received1970,fromJ. P. van der Walt.




Aspergillus species, undescribed

530 EastIndies:isol.exnaturalrubber,1938,Shumard.

13151 USA:Tennessee:isol.extoxicdairyfeed,1984,B. W. Horn.

ResUlTs

Phylogenic analysis of sequence dataSixteen independent evolutionary lineages were detected

using both criteria for concordance (Dettman et al. 2003).The accepted species (Peterson 2008) A. versicolor, A. tabacinus, A. amoenus, A. protuberus and A. sydowii each were identified as independent lineages (Fig. 1). Four


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Fig. 1.PhylogenetictreecalculatedfromDNAsequencedatafromfourconcatenatedloci.ThesectionVersicolores contains three subclades, the A. versicolor subclade, the A. sydowii subclade and the A. subversicolorsubclade.Thickbranches indicate>90%bootstrapand>0.90Bayesianposteriorprobabilityforthenode.IsolateNRRL13151issimilarincolonyappearancetoA. tennesseensis but may represent a distinct species.IsolateNRRL530issimilarincolonyappearancetoA. amoenusbutalsomayrepresentadistinctspecies.

lineages contained a single isolate. Two of these single-isolate lineages, A. subversicolor and A. austroafricanus, were sufficiently distinct phenotypically from other speciesin the section and are described as new. The other twosingle-isolate lineages (NRRL13151andNRRL530)were

phenotypicallydifficulttodistinguishfromtheirsiblings,andspeciesdescriptionswerenotaccordedthem.

The section Versicolores clade contained three subclades (Fig. 1): the A. sydowii subclade containing A. sydowii, A. creber, A. venenatus, A. tennesseensis, A.



cvjetkovicii, A. jensenii and A. puulaauensis;theA. versicolor subclade containing A. versicolor, A. tabacinus, A. fructus, A. protuberus, A. amoenus and A. austroafricanus;andtheA. subversicolor subclade containing the single species A. subversicolor. Single-locus trees placed A. sydowii in the A. sydowii subclade, in the A. versicolor subclade or in a distinct clade containing only A. sydowii (FigsS1–S5,SupplementaryInformation,onlineonly)withlowconfidencelevels.TheMcm7 locus from A. sydowiiwasnot amplifieddespite numerous attempts and thus A. sydowii does not appearinFig.S3(SupplementaryInformation,onlineonly).The combined data tree (Fig. 1) depicts A. sydowii as a member of the A. sydowii subclade with strong statistical support. In thecombineddata tree,eachspecies’groupofisolatesresidesonabranchwith>90%bootstrapproportionand>0.90Bayesianposteriorprobability.

TAXONOMY

Previously described species

Aspergillus amoenus M. Roberg,Hedwigia 70:138(1931).MycoBankMB250654(Fig.2a–f)

Type: Germany:Munster,isol.exBerberissp.fruit,1930. M. Roberg(NRRL4838—exholotypeculture).

Description: Coloniesgrown10donCYAat25°C (Fig.2a–b) attained 25–40mmdiam, radially sulcate, centrally raisedor sunken 3–4 mm, one older isolate (NRRL 226) plane,sporulating moderately to well, conidial heads in grayish green

shades near tea green (R47), clear to pale orange exudatepresent in some isolates, faint reddish soluble pigment present in some isolates, reverse mostly reddish brown hues, with some isolatesuncolored.Coloniesgrown10donMEAat25°C(Fig.2c–d)attained23–33mmdiam,low,velutinous,someisolateswith shallow sulcations, colony center often with funicular hyphal aggregates, sporulation in blue-green to gray-green shades, no soluble pigment exceptNRRL 226with pale brown pigment,noexudate,reversecoloredlightorangeyellowtopaleyellowred. Incubation for 7 d onCYA at 5 °C produced no growthorgerminationofconidia.Incubationfor7donCYAat37°Ccommonlyproducedgrowthupto6mmdiam.

Stipes (Fig. 2e) smooth walled, hyaline to yellowwithbrownish shades, (35–)100–600(–1100) × (2.5–)4–7(–8)μm,vesiclespyriformtospatulate,(4–)7–17(–21)μmdiam,conidial heads biseriate, metulae covering 1/3 to entirevesicle, 3–6(–8) × 2.5–4.0(–5.5) μm, phialides (5–)6–8(–11) × 2–3 μm, fragmentary heads resembling penicillatefructifications abundant, conidia (Fig. 2f) spherical tosubspherical, occasionally ellipsoidal, 2.5–3.5(–5) μm,smoothwalled,NRRL35600producedglobosehüllecells12–22 µm diam when grown on M40Y medium, otherisolatesdidnot.

Aspergillus protuberus Muntañola-Cvetković,Mikrobiologija 5:119(1968).MycoBankMB326650(Fig.3a–h)Synonym: Aspergillus versicolorvar. protuberus(Muntañola-

Cvetković)Kozak.,Mycol.Pap.161:139(1989).

Type: Yugoslavia:isol.exrubbercoatedelectricalcables,ca 1968(NRRL3505—exholotypeculture).

Fig. 2. Aspergillus amoenus(NRRL4838),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,subglobosevesicle,andconidia,bar=10µm.f.Globose,smooth-walledconidia,bar=10µm.


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Description: Colonies grown 10 d on CYA at 25 °C (Fig.3a–b)attained28–34mmdiam,radiallyandconcentricallysulcate,wrinkled,centrallyraised2–4mm,clumpedaerialhyphae give a mealy appearance in some areas of some isolates, sporulation moderate with conidial heads often creamy white but sometimes patches of yellow-green conidia (celandinegreenR47)arepresent,scarletred(R1)exudatemoderatelyabundant,vinaceous-fawn(R40)topaleyellowsoluble pigment present, reverse brownish red or orange cinnamon(R20),oneisolatebrazilred(R1).Coloniesgrown10donMEAat25°C(Fig.3c–d)attained27–32mmdiam,floccose, mounded 4–5mm centrally, radially sulcate, noexudate,nosolublepigment,reverselightpinkishyellowtopinkishyellow.Incubationfor7donCYAat5°Cor37°Cproducednogrowthorgerminationofconidia.

Stipes (Fig. 3e–f) smooth to tuberose, hyaline toyellow or occasionally with brownish shades, (120–)300–800(–1250) × 4–10μm, occasionally terminatingwith twovesicles, vesicles pyriform to spatulate, rarely subspherical, (6–)10–24(–27)μmdiam,conidial heads biseriate, metulae coveringhalftoentirevesicle,(3–)4–7(–8)×2.5–4.5(–5.5)μm, phialides (4–)5–8(–11) × 2–3(–3.5) μm, fragmentaryheads resembling penicillate fructifications occasionallypresent, conidia (Fig. 3h) spherical to subspherical oroccasionally ellipsoidal to pyriform, (2.0–)2.5–3.5(–5)μm, finely roughened wall, hülle cells (Fig. 3g) globosesometimespresent.

Aspergillus sydowii (Bain.&Sart.)Thom&ChurchAspergilli:147(1926).MycoBankMB279636(Fig.4a–g)Basionym: Sterigmatocystis sydowi Bainer & Sartory,Ann.

Mycol.11:25(1913).

Type: Sine loc.: sent to C. Thom, prior to 1930,M. Swift (NRRL250—cultureexneotype).

Description: Colonies grown 10 d on CYA at 25 °C (Fig.4a–b)attained27–37mmdiam,velutinous,radiallysulcate,sporulating well, conidial heads deep bluish gray-green (R42), exudate moderate to abundant, clear to yellowishto reddish brown, reddish-brown soluble pigment, reverse tawny olive (R39) to orange cinnamon (R29) on theperiphery.Coloniesgrown10donMEAat25°C(Fig.4c–d)attained37–48mmdiam,velutinous,some isolateswithshallow sulcations, sporulating in dark grayish blue-green color, funicular hyphal aggregates often seen centrally, no exudate, no soluble pigment, reverse unpigmented tobrownishpink inNRRL4768. Incubation for7donCYAat 5°Cproducednogrowthorgerminationofconidia.Incubationat37°Cproducedcolonies10–17mmdiamin10d.

Stipes (Fig.4e)smooth,colorless,100–500µm×4–7µm, vesicles subglobose, 5–10 (–15) µm diam, conidial heads biseriate, metulaecoveringmostofthevesicle,6–7×2–3µm,phialides7–10×2.0–2.5µm,fragmentaryheads(Fig. 4f) resembling penicillate fructifications abundant, conidia (Fig.4g)globose tosubglobose,2.5–3.0 (–5)µm,spinulose.

Aspergillus tabacinus Nakaz et al., J. Agr. Chem. Soc. Japan 10:177(1934).MycoBankMB539544(Fig.5a–f)Synonym: Aspergillus versicolorvar.magnusSasaki,J. Fac.

Agric. Hokkaido Univ. 49:144(1950).

Type: Sine loc.:isol.extobacco,1934,Y. Nakazawa(NRRL4791—cultureexneotype).

Fig. 3.Aspergillus protuberus(NRRL3505),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Stipe,subglobosetoclavatevesicle,andconidia,bar=10µm.f.Roughenedsurfaceofstipe,bar=10µm.g.Globosehüllecell,bar=10µm.h.Globose,finelyroughenedconidia,bar=10µm.



Description: Colonies grown 10 d on CYA at 25 °C (Fig.5a–b) attained 30–32 mm diam, sulcate, centrally raised2–3mm,oftensporulatingheavilythroughoutbutsometimessporulationisdelayed,conidialheadsartemisiagreen(R47),sporulationfromaerialbranchespronounced,exudateclearwhen present, no soluble pigment, reverse uncolored in NRRL5031,orbrown inother isolates.Coloniesgrown10

d onMEA at 25 °C (Fig. 5c–d) attained 17–30mm diam,NRRL4791isvelutinousandcoveredwithfunicularhyphalaggregates, NRRL 5031 and NRRL 62481 are floccose,sporulation inbluish-greenshades,noexudate,nosolublepigment, reverse uncolored to cream or very pale yellow.Incubation for 7 d on CYA at 5 °C or 37 °C produced nogrowthorgerminationofconidia.

Fig. 5. Aspergillus tabacinus(NRRL4791),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,withclavatevesicle,andconidia,bar=10µm.f.Globose,smooth-walledconidia,bar=10µm.

Fig. 4. Aspergillus sydowi(NRRL250),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,subglobosevesicle,andconidia,bar=10µm.f.Penicillateconidiophorefromaerialhyphae,bar=10µm.g.Subglobose,spinuloseconidia,bar=10µm.


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Stipes smooth walled (Fig. 5e), septate, hyaline toyellow with brownish tint, (70–)300–700(–900) × 4–8(–9)μm,vesiclespyriformtospatulate,(5–)8–15(–22)μmdiam,conidial heads biseriate, metulae covering half to entire vesicle, 3–8(–9)um×2.5–4.5(–5.5) μm,phialides 5–8(–11)× 2–3(–3.5) μm, fragmentary heads resembling penicillatefructifications abundant, conidia (Fig. 5f) spherical tosubspherical, occasionally ellipsoidal, (2.5–)3–4(–7) μm,smoothwalled.

Aspergillus versicolor (Vuill.) Tirab.,Annali Bot.7:9(1908).MycoBankMB172159(Fig.6a–g)Basionym: Sterigmatocystis versicolorVuill.,inMirsky,Thèse

de médicine(Nancy)27:15(1903).

Type: Sine loc.:1935,V. K. Charles(NRRL238—cultureexneotype).

Description: Coloniesgrown10donCYAat25°C(Fig.6a–b)attained28–36mmdiam,sulcate,centrallyraised4–5mm,sporulating well, conidial heads pale grayish green near teagreen(R47),centralareamealyfromaggregatedaerialhyphae,exudatepresentinmostlycleartopalepinkshades(brownish red in one isolate), faint to very obvious pinkishsoluble pigment, reverse vinaceous or brown or scarlet (NRRL 238). Colonies grown 10 d onMEA at 25 °C (Fig.6c–d) attained 21–31mm diam, low, with funicular hyphalaggregates, sometimes dominating colony appearance, sporulating in pale to dark bluish green to gray green color, noexudateseen, solublepigment yellow in some isolates,not present in others, reverse pale yellow, yellow orange

ororange. Incubation for7donCYAat5 °Cproducednogrowthorgerminationofconidia.Incubationfor7donCYAat37°Cproducedgrowthupto8mmdiam.

Stipes (Fig.6e–f) smooth,occasionally lightly tuberose,hyaline to yellow with brownish shades, (45–)200–750(–1050) × (4–)5–8(–12) μm, vesicles pyriform to spatulate, (6–)9–17(–20)μmindiam,conidial heads biseriate, metulae coveringhalftoentirevesicle,3–6(–9)×2.5–4.5μm, phialides (4–)5–7(–11) × 2–3 μm, fragmentary heads resemblingpenicillate fructifications occasionally present, conidia (Fig.6g) spherical to subspherical, occasionally ellipsoidal, (2–)2.5–3.5(–6.5)μm,finelyroughenedwall,hülle cells globose, producedbyNRRL5219whengrownonM40Ymedium,butnototherisolates.

Observations: Theex-neotypecultureNRRL238(isolatedin1935)isquitedifferentinappearance,particularlyinproductionof dark red soluble pigment and scarlet colony reverse on CYA, from the more recent isolates that were placed in the ARSCultureCollectionbetween1970and1984.Themorerecentisolates(NRRL5219,NRRL13144,NRRL13145andNRRL 13146) are quite similar in appearance and are theprimarybasisofthephenotypicdescription.Althoughthereisphenotypicdistinction,allfiveisolatesareA. versicolor based onDNAsequenceanalysis.

New species

Aspergillus austroafricanus Jurjevic,S.W.Peterson&B.W.Horn,sp. nov. MycoBankMB800597(Fig.7a–f)Etymology: IsolatedfromsoilinSouthAfrica.

Fig. 6. Aspergillus versicolor(NRRL238),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Bifurcatingstipeproducingtwoconidiophores,bar=50µm.f.Smoothstipe,subglobosevesicle,andconidia,bar=10µm.g.Globoseconidiawithroughenedwalls,bar=10µm.



Type: south Africa:Capetown, sent toC.Thom,1922,V. A. Putterill (BPI880914–holotype [fromdriedcoloniesofNRRL233grown7dat25°ConCYAandMEA]).

Diagnosis: Conidia smooth-walled, no growth at 37 °C,producesreddishbrownsolublepigmentwhengrownonCYA.

Description: Coloniesgrown10donCYAat25°C(Fig.7a–b) attained 23–24mmdiam,mounded, shallowly sulcate,

overgrowth by clumped hyphae making surface appear mealy, sporulating well, conidial heads near sage green (R47),sparseclearexudate,solublepigmentreddishbrown,reversedullbrown.Coloniesgrown10donMEAat25°C(Fig. 7c–d) attained 27 mm diam, velutinous, sporulationpalebluegreen,centralhyphaltufts,noexudate,nosolublepigment, reverse yellowish orange. Incubation for 7 d onCYAat5 °Cor37 °Cproducednogrowthorgerminationofconidia.

Fig. 8. Aspergillus creber(NRRL58583),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,subglobosevesicle,andconidia,bar=10µm.f.Globose,finelyroughenedconidia,bar=10µm.

Fig. 7. Aspergillus austroafricanus(NRRL233),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolony reverse.e.Smoothstipe,subglobosevesicleandconidia,bar=10µm. f.Globose,smooth-walledconidia,bar=10µm.


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Stipes (Fig. 7e) smooth walled, hyaline to yellowish,(40–)100–350(–500)µm×3–5(–6)μm,vesicles pyriform to spatulate,(4–)6–12(–15)μmdiam,conidial heads biseriate, metulaecovering1/3toentirevesicle,3–7(–9)um×2.5–4.5μm,phialides (4–)5–7(–9)×(2–)2.5–3(–4)μm,fragmentaryheads resembling penicillate fructifications occasionallypresent, conidia (Fig.7f)spherical tosubspherical,2.5–3.5(–4.5)μm,smoothwalled.

Aspergillus creber Jurjevic,S.W.Peterson&B.W.Horn,sp. nov. MycoBankMB800598(Fig.8a–f)Etymology:FromtheLatinwordcreber meaning numerous orfrequent.

Type: UsA: California: isol. ex air sample, Nov. 2008, Z. Jurjevic (BPI 800912 – holotype; [from dried colonies ofNRRL58592grown7dat25°ConCYAandMEA]).

Diagnosis: Producesrough-walledconidia,nogrowthat37°C, no soluble pigments formed on CYA or MEA, conidialcolorpeagreenorsagegreenonCYAandMEA.

Description: Colonies grown 10 d on CYA at 25 °C (Fig.8a–b)attained18–26mmdiam,radiallysulcate,raised3–5mm centrally, peripheral areas white or yellow, central area sporulating well, conidial heads pea green to artemisia green (R47),exudatewhenpresentyellowishtoreddish,nosolublepigment, reverse clay colored to cinnamon or reddish brown (R29). Colonies grown 10 d onMEA at 25 °C (Fig. 8c–d)attained 18–22mm diam, low to 1–2mmmounded, oftenovergrown centrally with hyphae aggregated into funicles, sporulationinyellow-greenshades(peagreentosagegreen

R47),withca 1mmwhite border, one isolate (NRRL231)with vivid brown soluble pigment, other isolates no soluble pigment, no exudate, reverse pale yellow orange or olivedrabororangebrown.Incubationfor7donCYAat5°Cor 37°Cproducednogrowthorgerminationofconidia.

Stipes(Fig.8e)smoothwalled,(10–)70–450(–650)x(3–)4–7(–8)μm,vesicles pyriform to spatulate and occasionally subglobose,(4–)7–17(–25)μmdiam,conidial heads biseriate, metulae (3–)4–6(–8)x2.5–4.5(–5)μm, phialides (4–)5–8(–10)x2–3(–4)μm,conidia(Fig.8f) spherical to subspherical, occasionallyellipsoidaltopyriform,(2.5–)3–4(–9)μm,finelyroughenedwall.

Aspergillus cvjetkovicii Jurjevic,S.W.Peterson&B.W.Horn,sp. nov.MycoBankMB800599(Fig.9a–h)Etymology: NamedinhonorofBogdanCvjetković(UniversityofZagreb);pronunciation\`chet-kO-``vi-chi\.

Type: UsA: New Jersey:isol.exsoil,1915,W. Wilson(BPI880909–holotype[fromdriedcoloniesofNRRL227grown7dat25°ConCYAandMEA]).

Diagnosis: Producesspinuloseconidia,nogrowthat37°C,coloniesproducingredexudateandredsolublepigmentonCYA.

Description: Coloniesgrown10donCYAat25°C(Fig.9a–b)attained24–29mmdiam,radiallysulcate,eithercentrallysunkenorraised(2–3mm),sporulatingwell,conidialheadswhite tocream inmost isolates,peagreen (R47) inNRRL58593,exudategenerallyabundant,reddishbrowntoorangecinnamon, reddish brown soluble pigment, reverse yellowish

Fig. 9. Aspergillus cvjetkovicii(NRRL4642),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Numerousconidiophoresarisingfromthebasalcolony,bar=50µm.f.Stipe,subglobosevesicle,andconidia,bar=10µm.g.Globose,spinuloseconidia,bar=10µm.h.Globosehüllecell,bar=10µm.



red shades near orange cinnamon (R29) or tawny olive(R39). Colonies grown 10 d onMEA at 25 °C (Fig. 9c–d)attained 17–36mm diam, low, slightly sulcate, sporulatingthroughout in creamy yellow shades,NRRL 58593 conidiaare yellowish green, NRRL 227 and NRRL 230 producebrownsolublepigmentwhileNRRL4642and58593donotproduce soluble pigment, reverse brownish orange or pale creamyyellow. Incubation for7donCYAat5°Cor37°Cproducednogrowthorgerminationofconidia.

Stipes (Fig. 9e–f) smooth walled, hyaline to yellow,(40–)200–700(–850) × (3–)4–7(–8) μm, vesicles pyriform to spatulate, rarely subspherical, (5–)9–18(–23) μm diam,conidial heads biseriate, metulae covering half to entire vesicle, 3–6(–8)×2.5–4.5μm,phialides 5–8(–10)×2–3(–4) μm, occasionally solitary phialides present up to 32 μmlong,fragmentaryheadsresemblingpenicillatefructificationsoccasionally present, conidia (Fig. 9g) spherical tosubspherical, occasionally ellipsoidal, (2–)2.5–3.5(–5) μm,spinulose, hülle cells(Fig.9h)globose,sometimespresent.

Aspergillus fructus Jurjevic,S.W.Peterson&B.W.Horn,sp. nov.MycoBankMB800600(Fig.10a–g)Etymology:Fromfruit.

Type: UsA: California: isol. ex date fruit, 1939,Bliss (BPI880915–holotype[fromdriedcoloniesofNRRL239grown7dat25°ConCYAandMEA]).

Diagnosis: Resembling A. versicolor growth at 37 °C, butformingshorterconidiophores150–400µmversus200–750µmconidiophoresinA. versicolor.

Description: Colonies grown10 d onCYAat 25 °C (Fig.10a–b)attained29–39mmdiam,sulcate,centrallyraised4–5 mm, funicular clumps of aerial hyphae abundant,sporulating well, conidial heads celandine green (R47),exudate clear to yellow, moderately abundant, solublepigmentclear,orangeredinNRRL239,reverseuncoloredormahogany red to orange-rufous (R2).Colonies grown10 d onMEA at 25 °C (Fig. 10c–d) attained 22–32mmdiam, slightly sulcate, centrally covered by hyphal tufts, sporulation in yellow-green hues near artemisia green (R47),noexudate,nosolublepigment,reverseuncoloredordraborange.NRRL241wasfloccoseonMEA.Incubationfor7donCYAat5°Cproducednogrowthorgerminationof conidia. Incubation for7donCYAat37 °Cproducedgrowthupto4mmdiam.

Stipes(Fig.10e)smoothwalled,hyalinetoyellow,(50–)150–400(–500) × 4–7 µm, vesicles pyriform to spatulate, (6–)9–17(–21) μm diam, conidial heads biseriate, metulae coveringhalftoentirevesicle,(2–)3–7(–9)×2.5–4.5(–7)μm,phialides (5–)6–8(–11) × 2–3(–4) μm, fragmentary headsresemblingpenicillate fructificationsabundant,conidia (Fig.10f) spherical tosubspherical,occasionallyellipsoidal, (2–)2.5–3.5(–4.5)μm,finelyroughenedwall,hülle cells(Fig.10g)globose,sometimespresent.

Aspergillus jensenii Jurjevic,S.W.Peterson&B.W.Horn,sp. nov.MycoBankMB800601(Fig.11a–g)Etymology: Named in honor of C. N. Jensen who firstreported this species as Aspergillus globosusJensen,alaterhomonym of A. globosusLink.

Fig. 10. Aspergillus fructus(NRRL239),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,spathulatevesicle,andconidia,bar=10µm.f.Globose,finelyroughenedconidia,bar=10µm.g.Globosehüllecell,bar=10µm.


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Type: UsA: Montana: isol. ex air sample, Oct. 2008, Z. Jurjevic(BPI880910–holotype[fromdriedcoloniesofNRRL58600grown7dat25°ConCYAandMEA]).

Synonym: Aspergillus globosus Jensen, Cornell UniversityAgriculturalExperimentStationBulletin315:482(1912);nonLink1809.

Diagnosis: Conidial walls roughened, no growth at 37 °C,conidial color near celandine, tawny olive to dark umber colonyreverseonCYA.

Description: Colonies grown 10 d on CYA at 25 °C (Fig.11a–b) attained20–27mmdiam, radially sulcate, centrallyraised or sunken, with clumped hyphal aggregates common in some isolates, sporulating moderately well, conidial heads celandine (R47) centrally and often white peripherally,exudate when present reddish brown or yellow brown,soluble pigment faint or intense yellow brown, in one case reddishbrown,reversetawnyolive(R39)todarkbrownneardarkumber(R3).Coloniesgrown10donMEAat25°C(Fig.11c–d) attained17–30mmdiam, low, plane,most isolateshave funicular tufts of aerial hyphae centrally, sporulating well in yellowish blue-green shades, no exudate seen, solublepigment either light brown or reddish brown, brownish orange in one isolate, reverse pale yellow or orange or brownish red.Incubationfor7donCYAat5°Cor37°Cproducednogrowthorgerminationofconidia.

Stipes (Fig. 11e) smooth walled, hyaline to yellow,occasionally with brownish shades, (45–)200–700(–1000)× (–3)4–7(–8) μm, vesicles pyriform to spatulate, rarely subspherical, (5–)7–16(–22) μm diam, conidial heads biseriate, metulaecovering1/3toentirevesicle,3–8×2.5–4(–5) μm,phialides (4–)5–8(–11) × 2–3 μm, rarely solitary

phialidespresentupto32μmlongandupto4.5μmdiam,fragmentary heads resembling penicillate fructifications(Fig.11f)commonlypresent,conidia (Fig.11g) spherical to subspherical, occasionally ellipsoidal to pyriform, (2.5–)3–4.5(–7)μm,finelyroughenedwall,globosehüllecells15–20µmdiamproducedbyNRRL58582butnototherisolates.

Aspergillus puulaauensis Jurjevic,S.W.Peterson&B.W.Horn,sp. nov.MycoBankMB800602(Fig.12a–h)Etymology.IsolatednearthePu’ula’auHighwayonHawaii;pronunciation\pU-U-la-U-en-sis\

Type: UsA: Hawaii: isol. ex dead hardwood branch, 2003,D.T. Wicklow(BPI880911–holotype[fromdriedcoloniesofNRRL35641grown7dat25°ConCYAandMEA]).

Diagnosis: IsolatesproduceabundanthüllecellswhengrownonM40Yagar,nogrowthat37°C,.

Description: Coloniesgrown10donCYAat25°C(Fig.12a–b)attained22–25mmdiam,sulcate,centrallyraised5–6mmwith funicular hyphal clumps, sporulation light, conidial heads artemisiagreen(R47),exudatewhenpresentclearorreddish,soluble pigment when present brown, reverse yellowish to clay color(R39)orcinnamon(R29).Coloniesgrown10donMEAat25°C(Fig.12c–d)attained21–25mmdiam,sulcateorplane,low,velutinous,deepgreen(artemisia to lilygreenR47),noexudateseen,nosolublepigment, reversepaleyellownearchamoisorpaleorange.Incubationfor7donCYAat5°Cand37°Cproducednogrowthorgerminationofconidia.

Stipes(Fig.12e)smoothwalled,hyalinetoyellow,(35–)100–500(–700)×(3–)4–7μm,vesicles pyriform to spatulate,

Fig. 11. Aspergillus jensenii(NRRL58671),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,subglobosevesicle,metulaeandphialides,bar=10µm.f.Penicillateconidiogenouscellsfromaerialhyphae,bar=10µm.g.Globose,finelyroughenedconidia,bar=10µm.



occasionallysubspherical,(5–)8–18(–21)μmdiam,conidial heads biseriate, metulaecoveringhalftoentirevesicle,(3–)4–7(–9)×2.5–4μm,phialides5–7(–10)×2–3μm,fragmentaryheads resembling penicillate fructifications occasionallypresent, conidia (Fig.12h)spherical toellipsoidal, (2.5–)3–4(–5.5) μm, finely roughened wall, hülle cells (Fig. 12f–g)spherical11–19µmdiamseeninallisolateswhengrownonM40Ymedium.

Aspergillus subversicolor Jurjevic,S.W.Peterson&B.W.Horn,sp. nov.MycoBankMB800603(Fig.13a–f)Etymology:BeneathoratthefootofAspergillus versicolor.

Type: India: Karnataka: isol.exgreencoffeeberries,1970,B. Muthappa(BPI880918–holotype[fromdriedcoloniesofNRRL58999grown7dat25°ConCYAandMEA]).

Fig. 13. Aspergillus subversicolor(NRRL58999),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,subglobosevesicle,andconidia,bar=10µm.f.Subglobose,finelyroughenedconidia,bar=10µm.

Fig. 12. Aspergillus puulaauensis(NRRL35641),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,vesicle,andconidia,bar=10µm.f.Massofhüllecells,bar=50µm.g.Hüllecell,bar=10µm.h.Globoseconidiawithfinelyroughenedwalls,bar=10µm.


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Diagnosis:Conidiarough-walled,nogrowthat37°C,growingslowly on all media, producing yellow soluble pigment on CYAbutnoexudate.

Description: Colonies grown 10 d on CYA at 25 °C (Fig.13a–b) attained 18–20mm diam, sulcate, raised 5–6mmcentrally, wrinkled, sporulating sparsely, conidial heads artemisia green (R47), no exudate, soluble pigment faintyellow,reversetawny(R15)toochraceousorange.Coloniesgrown10donMEAat25°C(Fig.13c–d)attained12–14mmdiam, low, plane, velutinous, sporulating in bluish green color (artemisia R47), no exudate, no soluble pigment, reversebrownishorange.Incubationfor7donCYAat5°Cor37°Cproducednogrowthorgerminationofconidia.

Stipes (Fig. 13e) smooth walled, hyaline to slightlybrownish, (60–) 250–450 (–550) × 4–7(–10) μm, vesicles pyriform to subglobose (6–)10–17(–22) μm diam, conidial heads biseriate, metulaecoveringhalftoentireorrarely1/3ofvesicle,(3–)4–7(–9)×(2–)2.5–4μm,bearing2–3ampuliform phialides,5–8(–10)×2–3μm,fragmentaryheadsresemblingpenicillate fructifications occasionally present, conidia (Fig.13f) spherical to subspherical, occasionally ellipsoidal topyriform,(2.5–)3–4(–7)μm,finelyroughenedwall.

Aspergillus tennesseensis Jurjevic,S.W.Peterson&B.W.Horn,sp. nov. MycoBankMB800604(Fig.14a–f)Etymology:IsolatedinTennessee.

Type: UsA: Tennessee:isol.extoxicdairyfeed,1984,B.W. Horn(BPI880917–holotype[fromdriedcoloniesofNRRL13150grown7dat25°ConCYAandMEA]).

Diagnosis:Producingrough-walledconidia,nogrowthat37°C,conidialcolorslategreenwhengrownonMEA.

Description: Coloniesgrown10donCYAat25°C(Fig.14a–b) attained 22–30mm diam, composed of a loose hyphalmat, radially sulcate, centrally raised or sunken, overgrown by clumps of aerial hyphae in some isolates, sporulating well centrally, pea green to artemisia green (R47), scant clearexudate usually present, soluble pigment absent, reversein brownish orange shades near honey yellow or chamois (R30).Coloniesgrown10donMEAat25 °C (Fig.14c–d)attained20–46mmdiam,low,plane,velutinous,sporulatingin dark green color near slate green (R47), no exudate,no soluble pigment, reverse uncolored, pale lemon yellow, orpalebrown. Incubation for7donCYAat5°Cor37°Cproducednogrowthorgerminationofconidia.

Stipes(Fig.14e)smoothwalled,hyalinetoyellowishwithbrownish shades, (35–)100–300(–400) × 4–7 μm, vesicles pyriform,(7–)10–16(–18)μmdiam,conidial heads biseriate, metulaecoveringhalftoentirevesicle,4–6(–8)×2.5–4μm,phialides5–8(–11)×2–3μm,fragmentaryheadsresemblingpenicillate fructifications occasionally present, conidia (Fig.14f) spherical to subspherical, occasionally ellipsoidal topyriform,(2.5–)3–4(–8)μm,finelyroughenedwall.

Aspergillus venenatus Jurjevic,S.W.Peterson&B.W.Horn,sp. nov.MycoBankMB800605(Fig.15a–h)Etymology:Producingtoxins.

Type: UsA: Tennessee:isol.extoxicdairyfeed,1984, B.W. Horn(BPI880916–holotype[fromdriedcoloniesofNRRL13147grown7dat25°ConCYAandMEA]).

Fig. 14. Aspergillus tennesseensis(NRRL13150),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,pyriformvesicle,andconidia,bar=10µm.f.Globose,finelyroughenedconidia,bar=10µm.



Diagnosis:Producingspinuloseconidia,nogrowthat37°C,producingnoexudateorsolublepigmentsonCYAorMEA.

Description: Colonies grown 10 d on CYA at 25 °C (Fig.15a–b)attained22–31mmdiam,radiallysulcate,sporulatingcentrallyinartemisiagreen(R47)todeepbluishgray-green(R42)inoneisolate,noexudate,nosolublepigment,reversedeepolivebufftotawnyorbrown(R15).Coloniesgrown10donMEAat25 °C (Fig.15c–d)attained17–24mmdiam,lightly sulcate, low, central tufted funicular aggregates of aerial hyphae, sporulating well in deep green color near slate green (R47),noexudate,nosolublepigment, reversepalelemonyellow,chamois,orlightolivedrab.Incubationfor7donCYAat5°Cor37°Cproducednogrowthorgerminationofconidia.

Stipes (Fig. 15e) smoothwalled, hyaline to yellowwithbrownish shades, (20–)100–400(–500) × 4–7 µm, vesicles pyriform to spatulate, (6–)9–17(–21) μm diam, conidial heads biseriate, metulaecoveringhalftoentirevesicle,(3–)4–7(–9)×2.5–4(–5)μm,phialides (5–)6–8(–11)×2–3(3.5)μm,fragmentaryheadsresemblingpenicillatefructifications(Fig.15f)commonlypresent,hülle cells(Fig.15g)spherical,present in some isolates, conidia (Fig. 15h) spherical tosubspherical,occasionallyellipsoidaltopyriform,3–4(–6)μmdiam,spinulose.

Phenotypic species recognition.Growth rates of species on different media are presented in Table2.

Phenotypic recognition of species in section Versicolores is based on smooth, roughened or spinulose conidia, conidial color,exudateandsolublepigmentcolorsonCYAandMEA,growthratesandabilitytogrowat37°C,andontheuniformpresenceofhüllecellsinonespecies.

Aspergillus cvjetkovicii, A. sydowii and A. venenatus isolatesproducespinuloseconidia.A. sydowii isolates grow at37°C,whileA. cvjetkovicii and A. venenatus isolates do not. A. cvjetkovicii isolates produce reddish exudate andsoluble pigment on CYA, while A. venenatus isolates produce noexudateorsolublepigment.

Aspergillus amoenus, A. austroafricanus and A. tabacinus producesmooth-walled conidia.Of theseonlyA. amoenus isolates grow at 37 °C. A. tabacinus isolates produce no soluble pigment and A. austroafricanus produces reddish brownsolublepigmentwhengrownonCYA.

The remaining eight species produce conidia with noticeably roughened walls, but the ornamentation is not pronouncedenoughtobeconsideredspinulose.Twooftheeight species, A. versicolor and A. fructus, have roughened conidialwallsandgrowat37°C.Thesetwospeciesareverysimilarbuthavesomewhatdistinctstipelengthsof150–400µm inA. fructus versus 200–750 µm inA. versicolor. WeexaminedonlytwoA. fructus isolatesandfiveA. versicolor isolates and while separation of these species using phenotype on standard media appears possible, until more isolatesareseen,itisrecommendedthatstrainsbeidentifiedfrom gene sequences such as beta tubulin or calmodulin.Genealogical concordance species recognition clearly distinguishesthesesiblingspecies(Fig.1).

Specieswithroughenedconidiathatdonotgrowat37°Care A. protuberus, A. creber, A. jensenii, A. puulaauensis, A. subversicolor and A. tennesseensis.Aspergillus protuberus isolates on CYA produce a red exudate (near scarletR1) and a vinaceous or yellow soluble pigment, andMEAcultures are floccose. A. jensenii isolates produce brown CYA colony reverse colors from tawny olive to dark umber, and conidial color is near celandine green (R47). All A. puulaauensis isolates produce spherical hülle cells when

Fig. 15. Aspergillus venenatus(NRRL13147),cultureplatesare9cmdiam,coloniesgrownat25°Cfor10d.a.CYAcolonies.b.CYAcolonyreverse.c.MEAcolonies.d.MEAcolonyreverse.e.Smoothstipe,spathulatevesicle,andconidia,bar=10µm.f.Penicillateconidiogenouscellsonaerialhyphae,bar=10µm.g.Globosehüllecells,bar=10µm.h.Globose,spinuloseconidia,bar=10µm.


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grownonM40Ymediumandthespeciesisdistinguishedbythis consistent character.One isolateeachofA. versicolor and A. amoenus(bothgrowat37°C)andoneisolateofA. jenseniialsoproducedhüllecellsonM40Y.A. subversicolor isolates are relatively slow growing on MEA and M40Y(Table2)andproducefaintyellowsolublepigmentonCYA.A. tennesseensis,whengrownonMEAproduce very darkgreenconidialareas(nearslategreenR47)notproducedbyotherrough-sporedspeciesinthesection.Aspergillus creber isolatesproducenosolublepigmentoneitherCYAorMEA,and conidial color on either medium is pea green to sage green(R47).

There is considerable variation in colony appearance within species and considerable overlap in colony appearance between species, making species separation within section Versicolores challenging. In addition, some of the isolatesincluded in this study were propogated in vitro for several

decadespriortopreservationbylyophilization.Amongthoseisolates, several appear to have mutated and consequently produce colonies that have a wet appearance when grown on CYA or produce only moist aerial aggregates of hyphae with little sporulation. Identification of these degenerate strainsrelies onDNA sequence analysis.DNA sequence analysisis the most reliable means for identifying species within this section.

ITSregiongenotypesfromspeciesinsectionVersicolores arepresentedinTable3.Somegenotypesaresharedbytwoormore species.GenotypeA is present in isolates of fivedifferent species and genotype D is present in four different speciesofthesection.Isolatesofsomespecies,suchasA. creber(genotypesI,K,LN),displaytwotofourITSgenotypeswithinspecies.

Table 2.Colonydiameters(mm)ofsectionVersicoloresspeciesonvariousmediaafter7d.Incubationat25°Cexceptwherenoted.

species CYA MeA CY20 M40Y M60Y CYA at 37 °CA. amoenus 20–29 11–15 11–20 14–22 15–24 6

A. austroafricanus 18–19 16–17 24–25 18–19 17–18 -

A. creber 17–23 11–15 15–22 18–23 19–24 -

A. cvjetkovicii 15–21 14–17 17–20 16–24 15–25 -

A. fructus 13–20 10–16 9–17 10–23 10–20 4

A. jensenii 16–20 9–13 15–20 21–26 22–28 -

A. protuberus 17–25 11–18 14–22 21–24 20–24 -

A. puulaauensis 18–21 11–12 17–19 19–22 18–21 -

A. subversicolor 13–14 6–7 10–11 15–16 16–18 -

A. sydowii 20–25 20–25 21–26 23–26 23–27 8

A. tabacinus 21–26 12–16 21–26 8–23 8–23 -

A. tennesseensis 20–22 12–14 17–19 19–22 19–22 -

A. venenatus 14–17 8–10 15–16 19–23 17–21 -

A. versicolor 20–26 10–18 19–22 21–26 22–25 8

Table 3.PredictedspeciesidentitybasedonITSgenotypeandcorrelationofITSgenotypesandspeciesinsectionVersicolores.ITSgeneotypeswereassignedarbitraryletterdesignationsandspeciesaredeterminedbygenealogicalconcordance.

ITs genotype Predicted speciesA A. amoenus, A. fructus, A. protuberus, A. tabacinus, A. versicolor

B A. subversicolor

C A. austroafricanus

D A. cvjetkovicii, A. jensenii, A. tennesseensis, A. venenatus

E A. sydowii

F A. sydowii

G A. amoenus

H A. tabacinus

I A. creber, A. versicolor

J A. puulaauensis

K A. creber

L A. creber

M A. jensenii

N A. creber



DIsCUssION

Initial phenotypic examination of Aspergillus section Versicolores isolates was made using CYA cultures grown for 7dat25°C(Klich&Pitt1988).Thoseculturesdidnotprovidesufficientdatatoreliably identifythespecies.Subsequentlywe triedculturing the isolates for10dat25 °ConCYA toallowforfurtherdevelopmentofexudate,solublepigmentandconidialcolor.Raper&Fennell(1965)usedincubationtimesofgenerally10–14d.We found that incubation for10d isnecessaryforcharacterizingisolatesofsectionVersicolores.

Only four of the available genetic loci were used inpreparing the combined data tree (Fig. 1). The ITS regionwas not included because it contained few informative nucleotides and because its veracity as a phylogenetic indicatorisquestionable(Galaganet al. 2005).TheITSdatathemselves however may be of interest for bar-coding studies (discussed later).Thebeta tubulinsequences fromsectionVersicolores are of the “two intron” type and probably have a different evolutionary origin than the “three intron” type of betatubulinfoundintheout-groupspecies(Peterson2008).Becauseofthesuspectedparalogyofthismolecule, itwasnot included in thecombineddata tree. Itwas includedasa possible target forDNA sequence-based identification ofisolates.

Henig (1966) in his work on systematics required thatall taxa be monophyletic. When working with phenotypiccharacters in section Versicolores, itwasdifficult to identifythe informative characters that could satisfy Henig’srequirement. Analysis of DNA sequences from unlinkedloci using concordance (Taylor et al. 2000, Dettman et al. 2003) makes it possible to define monophyletic groups.Phylogenetic recognition of species occasionally makes it necessary to accept cryptic species (Perrone et al. 2011)because the phenotypic characters of the species overlap withtheirsiblingstosuchanextentthatthespeciescannotbereliablyidentifiedwithoutmoleculartools.ForNRRL530andNRRL13151 that formsingle isolate lineages, reliablecharacters to define the species have not been found, butwiththeidentificationofadditionalisolatesitmaybepossibletophenotypicallycharacterizeandsubsequentlynamethesespecies.ForA. versicolor and A. fructus the limited number of isolates and the observed intraspecific variation reduceconfidence in the current phenotypic recognition of thespecies, but the phylogenetic data are unequivocal and so A. fructuswasdescribedasnew.

Prior to this publication A. versicolor was a species with documented genetic and phenotypic variation that did not resolve intoclearly recognizablespecies.Fourteenspeciesare now known in section Versicolores and the ITS regionvariation isca.3%ascalculated from thedataherein.Bycomparison ca. 4 % variation is found in the Petromyces clade (Aspergillus sect. Flavi) between P. flavus and P. nomius and 14 species have been named (Varga et al.2009). In thePetromyces clade, one species may possess a phenotype very similar to another species (Kurtzman et al. 1987,Petersonet al. 2001,Soareset al., 2012).Whilethe validity of some species in the Petromyces clade have beenquestioned(Vargaet al.2009),phylogeneticdistinctionhas served to validate species (Peterson 2008, Varga

et al. 2009) regardless of the phenotypic similarities oroverlappingcharacterstatesofthespecies.Peterson(2008)suggested that sect.Versicolores could easily be dropped from Aspergillus taxonomy. This much broader study ofA. versicolor sensu lato isolates suggests that section Versicolores should be retained as a monophyletic and useful subgenericdesignation.

Aspergillus versicolor is the most reported fungal species in section Versicolores from damp indoor environments (Jussila2003,Rydjordet al.2005)anditspresenceisusedasanindicatorofSickBuildingSyndrome(SBS)(Schwab&Straus 2004).We amplified each newly described speciesusing A. versicolor-specificprimers (Deanet al.2005;datanotshown)andobtainedapositivesignalinallcasesexceptfor A. subversicolor and A. sydowii;thereforetheprimersetretains its usefulness. In A. creber and A. jensenii some isolates did not amplify even though the genotypes were identical with isolates that did amplify, suggesting degradation orincorrectquantitationofthegenomicDNA.

Twenty-foursect.Versicolores isolates in this study were obtainedbyoneofus(ZJ)fromairsamplesinbuildings,butnone comprised A. versicolor sensu stricto(Table1).OfthefiveA. versicolorisolatesexamined,threewereisolatedfromasinglelotoftoxiccattlefeedintheUSAandthesubstratefortheothertwoisolates,onefromtheUSAandtheotherfromSouthAfrica,wasnot recorded.Thespecies iswidespreadgeographically, but was not commonly encountered among the isolates used in this study.Aspergillus creber was the most frequently isolated species from indoor air samples in theUSA(13strainsfromsixstates),followedbyA. protuberus (fivestrainsfromtwostates)andA. jensenii(fourstrainsfromthreestates).Aspergillus versicolor sensu stricto may not be commoninbuildings.Twootherspecies,A. cvjetkovicii and A. puulaauensis,wereeachisolatedoncefromindoorair.Theother newly described species, A. fructus, A. austroafricanus, A. subversicolor, A. tennesseensis and A. venenatus, were isolatedfromplantmaterialorhadunknownsources(Table1). Amend et al. (2010) reported that fungi isolated fromindoor air sources (e.g., dust, carpet) are highly diverse inthe temperate regions of the world and are much less diverse in tropical regions. Therefore, our strains from indoor airsamplesfromtheUSAinadditiontoculturecollectionstrainsfrom many regions of the world may represent much of the diversity present in section Versicolores.

There is considerable interest in using ITS sequencesfor bar-coding identification of fungi, particularly for large-scaleecologicalstudies(Begerowet al.2010,Schochet al.2012). In section Versicolores species, one particular ITSgenotype ispresent in isolatesoffivedifferentspeciesandanothergenotypeisfoundinfourdifferentspecies(Table3).Because ITSgenotypesdonotuniquely identifyspecies inthis section, use of multiple loci is the most reliable means ofDNAsequence-basedidentificationinsectionVersicolores (Peterson2012).

Viable propagules of A. versicolor have been recovered from the highly saline Dead Sea (Kis-Papo et al. 2003),showinganabilitytosurviveconditionsofsalinityordrying.The ARS Culture Collection contains a few putative A. versicolor isolates obtained from brined meats in the UK.Upon sequence and phenotypic analysis, these isolates were


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77v o l u m e 3 · n o . 1

identifiedasthreespecies,A. amoenus, A. tabacinus and A. protuberus, all of which occur in the A. versicolor subclade (Fig. 1). Additionally, A. creber NRRL 6544, from the A. sydowiisubclade,wasisolatedfromatarballfloatingintheAtlanticOcean.Hightolerancetosalinitymayextendtootherspecies in section Versicolores. Aspergillus versicolor has alsobeen identified fromdustcollected in the InternationalSpaceStation (Vesperet al.2008). Inaddition toCYAandMEA we used high sugar content media (CY20S, M40YandM60Y)containing20,40or60%sucrose,respectively.All isolatesgrewwellonallof themedia(Table2),withnonoticablereductioningrowthratesevenonM60Ymedium.SpeciesfromsectionVersicolores have a remarkably broad toleranceforawiderangeofwateractivityoftheirsubstrates.

Aspergillus versicolor isolates produce the aflatoxinprecursor sterigmatocystin, a compound that is mutagenic and tumorigenic (Veršilovskis&Saeger2010).Animal feedinfested with three morphotypes of A. versicolor, all of which produce sterigmatocystin, have been implicated in dairy animaltoxicosis,butitisunknownwhethersterigmatocystincaused the toxicosis (Vesonder & Horn 1985). Thosethree morphotypes are now identified as A. versicolor, A. tennesseensis and A. venenatus, and as these species occur in the two main subclades of section Versicolores (Fig. 1), sterigmatocystin production may be present inadditional species. The distribution of sectionVersicolores species in agricultural commodities and their role in stigmatocystintoxicosesrequireadditionalstudy.Inadditionto sterigmatocystin, recent studies have revealed numerous metaboliteswithbiologicalactivities(Finefieldet al.2011,Leeet al.2011) fromA. versicolor sensu latoJaioet al. (2007)discovered novel nucleotide analogs from A. puulaauensis which was reported under the name A. versicolor.

Aspergillus versicolor has been implicated as the causitiveagentofdisseminatedaspergillosisindogs(Zhanget al.2012),hasprobablycausedaspergillosisintransplantrecipients (Baddley et al. 2009), and has been isolatedfrom the infectedeyeofapatientsuffering fromHIV (Perriet al. 2005).We included two sectionVersicolores clinical isolatesinourstudy.NRRL254wasidentifiedasA. sydowii andNRRL226,originallyidentifiedasA. versicolor, is here identifiedasA. amoenus. Becauseof different sensitivitiesof fungal species to fungal antibiotics, a more detailed study of A. versicolor clinical isolates might be of value to guide appropriatetherapeuticregimens(Pfalleret al.2011).

ACKNOWleDgeMeNTs

WethankAmyE.McGovernforvaluabletechnicalsupport.PatriciaEckel kindly advised us on Latin usage. David L. Hawksworthadvised us on some nomenclatural issues. The authors thankDonaldT.Wicklow(USDA,Peoria,IL)andLynneSigler(UniversityofAlberta,Edmonton,Alberta)forthegiftofHawaiianandCanadianisolates.Mention of a trade name, proprietary product, or specificequipment does not constitute a guarantee or warranty by the United StatesDepartmentofAgricultureanddoesnotimplyitsapprovaltothe exclusion of other products thatmay be suitable.USDA is anequalopportunityproviderandemployer.

ReFeReNCes

AmendAS,SeifertKA,SamsonR,BrunsTD(2010) Indoor fungalcomposition is geographically patterned and more diverse in temperatezonesthaninthetropics.Proceedings of the National Academy of Sciences,USA107:13748–13753.

AndersonB,FrisvadJC,SondergaardI,RasmussenIB,LarsenLS(2011)Associationsbetweenfungalspeciesandwater-damagedbuildingmaterials.Applied and Environmental Microbiology 77: 4180–4188.

Baddley JW, Marr KA, Andes DR, Walsh TJ, Kauffman CA,KontoyiannisDP,ItoJI,BalajeeSA,PappasPG,MoserSA(2009)Patterns of susceptibility of Aspergillus isolates recovered from patients enrolled in the transplant-associated infection surveillance network.Journal of Clinical Microbiology 47: 3271–3275.

BaumDA,ShawKL(1995)Genealogicalperspectivesonthespeciesproblem. In:Experimental and Molecular Approaches to Plant Biosystematics(PCHoch&AGStephenson,eds):289–303.StLouis.Mo:MissouriBotanicalGarden.

BegerowD,NilssonH,UnterseherM,MaierW.(2010)CurrentstateandperspectivesoffungalDNAbarcodingandrapididentificationprocedures. Applied Microbiology and Biotechnology 87: 99–108.

Dean TR, Roop B, Betancourt D, Menetrez MY (2005) A simplemultiplexpolymerasechainreactionassayfortheidentificationoffourenvironmentallyrelevantfungalcontaminants.Journal of Microbiological Methods 61:9–16.

Dettman JR Jacobson DJ, Turner E, PringleA, Taylor JW (2003)Reproductive isolation and phylogenetic divergence in Neurospora: comparing methods of species recognition in a modeleukaryote.Evolution 57:2721–2741.

Domsch KH,GamsW,Anderson TH (1980)Compendium of Soil Fungi Vol.1. AcademicPress,UK.

EdmondsonDA,BarriosCS,BraselTL,StrausDC,KurupVP,FinkJN(2009)Immuneresponseamongpatientsexposedtomolds.International Journal of Molecular Sciences 10:5471-5484.

EngelhartS,LoockA,SkutlarekD,SagunskiH,LommelA,FarberH,ExnerM(2002)OccurrenceoftoxigenicAspergillus versicolor isolates and sterigmatocystin in carpet dust from damp indoor environments. Applied and Environmental Microbiology 68: 3886–3890.

Finefield JM, Sherman DH, Tsukamoto S, Williams RM (2011)Studiesonthebiosynthesisofthenotoamides:synthesisofanisotopomerof6-hydroxydeoxybrevianamideEandbiosyntheticincorporationintonotoamideJ.Journal of Organic Chemistry 76: 5954–5958.

GalaganJE,CalvoSE,CuomoC,MaL-J,WortmanJR,BatzoglouS,LeeS-I,BatürkmenM,SpevakCC,ClutterbuckJ,KapitonovV, Jurka J, Scazzocchio C, Farman M, Butler J, Purcell S,Harris S, Braus GH, Draht O, Busch S, D’Enfert C, BouchierC, Goldman GH, Bell-Pedersen D, Griffiths-Jones S, DoonanJH,YuJ,VienkenK,PainA,FreitagM,SelkerEU,ArcherDB,PeñalvaMA,OakleyBR,MomanyM,TanakaT,KumagaiT,AsaiK,MachidaM,NiermanWC,DenningDW,CaddickM,HynesM,PaolettiM,FischerR,MillerB,DyerP,SachsMS,OsmaniSA,BirrenBW(2005)SequencingofAspergillus nidulans and comparative analysis with A. fumigatus and A. oryzae. Nature 438:1105–1115.

GamsW,ChristensenM,OnionsAHS,Pitt JI,SamsonRA (1985)InfragenerictaxaofAspergillus.In:Advances in Penicillium and



Aspergillus Systematics(RASamson&JIPitt,eds):55–64.NewYork:PlenumPress.

Hennig W (1966) Phylogenetic Systematics. [English translation.]Urbana,IL:UniversityofIllinoisPress.

HuelsenbeckJP,RonquistF (2001)MRBAYES:Bayesianinferenceofphylogenetictrees.Bioinformatics 17:754–755.

Jiao P, Murdur SV, Gloer JB, Wicklow DT (2007) Kipukasins,nucleoside derivatives from Aspergillus versicolor. Journal of Natural Products 70:1308–1311.

JussilaJ(2003)Inflammatory Responses in Mice after Intratracheal Instillation of Microbes Isolated from Moldy Buildings.Helsinki,Finland:NationalPublicHealthInstitute.

Kis-Papo T, Kirzhner V, Wasser SP, Nevo, E (2003) Evolution ofgenomicdiversityandsexatextremeenvironments: fungal lifeunderhypersalineDeadSeastress.Proceedings of the National Academy of Sciences, USA 100:14970–14975.

Klich MA (1993) Morphological studies of Aspergillus section Versicolorandrelatedspecies.Mycologia 85:100–107.

Klich MA (2002) Identification of Common Aspergillus Species. Utrecht:CentraalbureauvoorSchimmelcultures.

KlichMA,PittJI(1988)A Laboratory Guide to Common Aspergillus Species and Their Teleomorphs.NorthRyde,NSW:CSIRO.

Kozakiewicz Z (1989) Aspergillus species on stored products.Mycological Papers 161:1–188.

KurtzmanCP,HornBW,HesseltineCW(1987)Aspergillus nomius, anewaflatoxin-producingspeciesrelatedtoAspergillus flavus and Aspergillus tamarii. Antonie van Leeuwenhoek 53: 147–158.

LeeYM,DangHT,LiJ,ZhangP,HongJ,LeeCO,JungJH(2011)Acytotoxicfellutamideanaloguefromthesponge-derivedfungusAspergillus versicolor.Bulletin of the Korean Chemical Society 32:3817–3820.

MbataTI(2008) Isolationof fungi inhypersalineDeadSeawater.Sudanese Journal of Public Health 3:170–172.

Mills JT, Abramson D (1986) Production of sterigmatocystin byisolates of Aspergillus versicolor from western Canadian storedbarleyand rapeseed/canola.Canadian Journal of Plant Pathology 82:151–153.

MorenoG,ArenasR. (2010)Other fungi causing onychomycosis.Clinics in Dermatology 28:160–163.

Nielsen KF (2003)Mycotoxin production by indoormolds.Fungal Genetics and Biology 39:103–117.

Perri P, Campa C, Incorvaia C, Parmeggiani F, Lamberti G,Costagliola C, Sebastian A (2005) Endogenous Aspergillus versicolorendophthalmitisinanimmuno-competentHIV-positivepatient.Mycopathologia 160: 259–261.

PerroneG,SteaG,EpifaniF,VargaJ,FrisvadJC,SamsonRA(2011)Aspergillus niger contains the cryptic phylogenetic species A. awamori. Fungal Biology 115:1138–1150.

PetersonSW(2008)PhylogeneticanalysisofAspergillus species usingDNAsequencesfromfourloci.Mycologia 100:205–226.

PetersonSW(2012)Aspergillus and PenicilliumidentificationusingDNAsequences:barcodeorMLST?Applied Microbiology and BiotechnologyDOI10.1007/s00253-012-4165-2.

PetersonSW,ItoY,HornBW,GotoT.(2001)Aspergillus bombycis, anewaflatoxigenic speciesandgenetic variation in its siblingspecies, A. nomius.Mycologia 93:689–703.

PetersonSW,JurjevicZ,BillsGF,StchigelAM,GuarroJ,VegaFE(2010) The genusHamigera, six new species and multilocusDNAsequencebasedphylogeny.Mycologia 102:847–864.

PfallerMA,CastanheiraM,DiekemaDJ,MesserSA,JonesRN(2011)TriazoleandechinocandinMICdistributionswithepidemiologicalcutoff values for differentiation of wild-type strains from non-wild-type strains of six uncommon species ofCandida. Journal of Clinical Microbiology 49:3800–3804.

Pitt JI (1980) [“1979”]The Genus Penicillium and Its Teleomorph States Eupenicillium and Talaromyces.London:AcademicPress.

PittJI,HockingAD(2009)Fungi and Food Spoilage.3rdedn.London:SpringerVerlag.

RaperKB,FennellDI(1965)The Genus Aspergillus.Baltimore,MD:Williams&Wilkins.

Ridgway R (1912) Color Standards and Color Nomenclature.Washington,DC:Ridgway.

RonquistF,HuelsenbeckJP(2003)MrBayes3:Bayesianphylogeneticinferenceundermixedmodels.Bioinformatics 19:1572–1574.

Rydjord B, Hetlandy G, Wikerz HG (2005) Immunoglobulin Gantibodies against environmental moulds in a Norwegian healthy population shows a bimodal distribution for Aspergillus versicolor.Scandinavian Journal of Immunology 62:281–288.

Samson RA, Houbraken J, Summerbell RC, Flannigan B, MillerJD (2001) Common and important species of fungi andactinomycetes in indoor environments. In: Microorganisms in Home and Indoor Work Environments(B.Flannigan,RASamson&JDMiller,eds):287–292.London:TaylorandFrancis.

SchochCL,SeifertKA,HuhndorfS,RobertV,SougeJL,LevesqueCA, Chen W, Fungal Barcoding Consortium (2012) Nuclearribosomalinternaltranscribedspacer(ITS)regionasauniversalDNA barcode marker for Fungi. Proceedings of the National Academy of Sciences, USA 109:6241–6246.

SchwabCJ,StrausDC(2004)TherolesofPenicillium and Aspergillus insickbuildingsyndrome.Advances in Applied Microbiology 55: 215–238.

SheltonBG,KirklandKH,DanaW(2002)ProfilesofairbornefungiinbuildingsandoutdoorenvironmentsintheUnitedStates.Applied and Environmental Microbiology 68:1743–1753.

SoaresC,RodriguesP,PetersonSW,LimaN,VenâncioA (2012)Three new species of Aspergillus section Flavi isolated from almondsandmaizeinPortugal.Mycologia:doi:10.3852/11-088.

SwoffordDL(2003)PAUP*: Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4. Sunderland, MA: SinauerAssociates.

TaylorJW,JacobsonDJ,KrokenS,KasugaT,GeiserDM,HibbettDS, Fisher MC (2000) Phylogenetic species recognition andspecies concepts in fungi. Fungal Genetics and Biology 31: 21–32.

ThomC,ChurchMB(1926)The Aspergilli.Baltimore,MD:Williams&Wilkins.

ThomC,RaperKB(1945)Manual of the Aspergilli.Baltimore,MD:Williams&Wilkins.

ThompsonJD,HigginsDG,GibsonTJ(1994)CLUSTALW:improvingthe sensitivity of progressive multiple sequence alignments throughsequenceweighting,position-specificgappenaltiesandweightmatrixchoice.Nucleic Acids Research 22: 4673–4680.

TuomiT,ReijulaK,JohnssonT,HemminkiK,HintikkaEL,LlindroosO,KalsoS,Koukila-KähköläP,Mussalo-RauhamaaH,HaahtelaT (2000) Mycotoxins in crude building materials from water-damagedbuildings.Applied and Environmental Microbiology 66: 1899–1904.

Varga J, Frisvad JC, Samson RA. (2009) A reappraisal of fungiproducingaflatoxins.World Mycotoxin Journal 2:263−277


LE

79v o l u m e 3 · n o . 1

Veršilovskis A, Saeger SD (2010) Sterigmatocystin: occurrencein foodstuffs and analyticalmethods – an overview.Molecular Nutrition and Food Research 54:136–147.

Vesonder RF, Horn BW (1985) Sterigmatocystin in dairy cattlefeed contaminated with Aspergillus versicolor. Applied and Environmental Microbiology 49:234–235.

VesperSJ,WongW,KuoCM,PiersonDL(2008)MoldspeciesindustfromtheInternationalSpaceStationidentifiedandquantifiedby

mold-specificquantitativePCR.Research in Microbiology 159: 32–435.

ZhangS,CorapiW,QuistE,GriffinS,ZhangM(2012)Aspergillus versicolor, a new causative agent of canine disseminated aspergillosis.Journal of Clinical Microbiology 50:187–191.


i m a f U N G U S

Supplementary InformatIon

79-S1

1

NRRL 58600T

NRRL 58671

NRRL 58674

NRRL 240

NRRL 237

NRRL 235

NRRL 225A. jensenii

NRRL 35641T

NRRL 58602A. puulaauensis

NRRL 227T

NRRL 58593NRRL 4642

NRRL 230

A. cvjetkovicii

NRRL 13147T

NRRL 13148

NRRL 13149 A. venenatus

NRRL 229

NRRL 13152

NRRL 13151 sp.Aspergillus

NRRL 13150T

NRRL 234

A. tennesseensis

NRRL 6544

NRRL 58984

NRRL 58673

NRRL 58607

NRRL 58606NRRL 58601

NRRL 58597

NRRL 58592T

NRRL 58587


NRRL 58583

NRRL 58675


NRRL 231

A. creber

NRRL 238T

NRRL 13146

NRRL 13145

NRRL 13144

NRRL 5219

NRRL 4791T

NRRL A-23173

NRRL 5031

NRRL 239T

NRRL 241

NRRL 58747NRRL 58748NRRL 58613

NRRL 3505T


NRRL 226NRRL 530 spAspergillus

NRRL 236

NRRL 35600

NRRL 4838T

A. versicolor

A. tabacinus

A. fructus

A. protuberus

A. amoenus

NRRL 233 A. austroafricanusT

NRRL 250T

NRRL 5585

NRRL 4768

NRRL 254

A. sydowii

NRRL 58999T A. subversicolor

NRRL 4770T A. asperescens

BT2 locus, 643 characters: 534 are constant45 variable but parsimony-uninformative

are parsimony-informative; <100 mp trees,CI=0., RC=0.

are64

100

82

76

99

75

93

96

98

75

98

90

94

Supplemental Fig 1

.93

.99

.99

1.0

78

.98

1.0

.95

1.0

.98

1.0

1.0

Fig. S1. Phylogenetic tree based on beta tubulin sequences; Bayesian posterior probabilities and bootstrap values based on 1000 replicates are placed on the internodes where values are significant.


LE

v o l U m e 3 · N o . 1


79-S2

1

NRRL 6544

NRRL 58984

NRRL 58673

NRRL 58607

NRRL 58606

NRRL 58601

NRRL 58597

NRRL 58592T

NRRL 58587

NRRL 58584

NRRL 25627

NRRL 58583

NRRL 58675

NRRL 58672

NRRL 58612

NRRL 231

A. creber

NRRL 35641T

NRRL 58602 A. puulaauensis

NRRL 227T

NRRL 58593

NRRL 4642

NRRL 230

A. cvjetkovicii

NRRL 13147T


A. venenatus

NRRL 229

NRRL 13152


NRRL 13150T

NRRL 234A. tennesseensis

NRRL 58600T

NRRL 58671

NRRL 58674

NRRL 240

NRRL 237

NRRL 235

A. jensenii

NRRL 225

NRRL 238T

NRRL 13146


NRRL 5219

NRRL 4791T

NRRL A-23173

NRRL 5031

NRRL 239T

NRRL 241

NRRL 58747

NRRL 58748

NRRL 58613

NRRL 3505T

NRRL 58942

NRRL 58991

NRRL 58990

NRRL 226


NRRL 236

NRRL 35600

NRRL 4838T

A. versicolor

A. tabacinus

A. fructus

A. amoenus


NRRL 250T

NRRL 5585NRRL 4768

NRRL 254A. sydowii

A. protuberus



Calmodulin locus, 694 characters: 510 are constant86 are variable but parsimony-uninformative

are parsimony-informative; 18 mp treesCI=0.8617, RC=0.8337

98

84

87

84

100

100

86

100

97

93

100

100

80

Supplemental Fig. 2

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

-1.0

-

1.0

Fig. S2. Phylogenetic tree based on calmodulin sequences; Bayesian posterior probabilities and bootstrap values based on 1000 replicates are placed on the internodes where values are significant.


i m a f U N G U S


79-S3

1

NRRL 58600T

NRRL 58671

NRRL 58674

NRRL 240

NRRL 237

NRRL 235

A. jensenii

NRRL 225

NRRL 35641T


NRRL 227T

NRRL 58593

NRRL 4642

NRRL 230

A. cvjetkovicii

NRRL 13147T

NRRL 13148

NRRL 13149

A. venenatus

NRRL 229

NRRL 13152


NRRL 13150T


NRRL 6544

NRRL 58984

NRRL 58673

NRRL 58607

NRRL 58606

NRRL 58601

NRRL 58597

NRRL 58592T

NRRL 58587

NRRL 58584

NRRL 25627

NRRL 58583

NRRL 58675

NRRL 58672

NRRL 58612

NRRL 231

A. creber

NRRL 238T

NRRL 13146

NRRL 13145

NRRL 13144

NRRL 5219

NRRL 4791T

NRRL A-23173

NRRL 5031

NRRL 239T

NRRL 241

NRRL 58747

NRRL 58748

NRRL 58613

NRRL 3505T

NRRL 58942

NRRL 58991

NRRL 58990

NRRL 226


NRRL 236

NRRL 35600

A. versicolor

A. tabacinus

A. fructus

A. amoenus


A. protuberus



NRRL 4838T

Mcm7 locus, 616 characters: 476 are constant,72 are variable but parsimony-uninformative,

are parsimony-informative; 2 mp trees,CI=0.8324, RC=0.8074

68

86

93

95

100

99

87

85

94

100

100

Supplemental Fig. 3

.93

.99

1.0

1.0

1.0

1.0

1.0

871.0

1.0

1.0

1.0

Fig. S3. Phylogenetic tree based on Mcm7 locus sequences; Bayesian posterior probabilities and bootstrap values based on 1000 replicates are placed on the internodes where values are significant.


LE

v o l U m e 3 · N o . 1


79-S4

10

NRRL 58600T

NRRL 58671

NRRL 58674


A. jensenii

NRRL 225

NRRL 35641T


NRRL 227T

NRRL 58593

NRRL 4642NRRL 230

A. cvjetkovicii

NRRL 13147T

NRRL 13148

NRRL 13149 A. venenatus

NRRL 229

NRRL 13152


NRRL 13150T

NRRL 234

A. tennesseensis

NRRL 6544

NRRL 58984

NRRL 58673

NRRL 58607

NRRL 58606

NRRL 58601

NRRL 58597

NRRL 58592T

NRRL 58587

NRRL 58584

NRRL 25627

NRRL 58583

NRRL 58675

NRRL 58672

NRRL 58612

NRRL 231A. creber

NRRL 238T

NRRL 13146

NRRL 13145

NRRL 13144

NRRL 5219

NRRL 4791T

NRRL A-23173

NRRL 5031

NRRL 239T

NRRL 241

NRRL 58747

NRRL 58748

NRRL 58613

NRRL 3505T

NRRL 58942

NRRL 58991

NRRL 58990

NRRL 226

NRRL 530 sp.AspergillusNRRL 236

NRRL 35600

NRRL 4838T

A. versicolor

A. tabacinus

A. fructus

A. amoenus


NRRL 250T

NRRL 5585

NRRL 4768NRRL 254

A. sydowii

A. protuberus



RPB2 locus, 1011 characters: 801 are constant,88 are variable but parsimony-uninformative,

are parsimony-informative; 6 mp trees,CI=0.7935, RC=0.7585.

122

100

100

89

100

82

100

97

98

89

94

100

100

99

100

100

95

100

Supplemental Fig. 4

941.0

1.0

1.0

.96

1.0

1.0

1.0

1.0

1.0

-

1.0

1.0

1.0

1.0

1.0

1.0

1.0

1.0

Fig. S4. Phylogenetic tree based on RPB2 locus sequences; Bayesian posterior probabilities and bootstrap values based on 1000 replicates are placed on the internodes where values are significant.


i m a f U N G U S


79-S5

1

NRRL 58600T

NRRL 58671

NRRL 58674NRRL 240

NRRL 237NRRL 235

A. jensenii

NRRL 225

NRRL 35641T


NRRL 227T

NRRL 58593NRRL 4642

NRRL 230

A. cvjetkovicii

NRRL 13147T


A. venenatus

NRRL 229

NRRL 13152


NRRL 13150T


NRRL 6544

NRRL 58984

NRRL 58673

NRRL 58607

NRRL 58606

NRRL 58601

NRRL 58597

NRRL 58592T

NRRL 58587

NRRL 58584

NRRL 25627

NRRL 58583

NRRL 58675

NRRL 58672

NRRL 58612

NRRL 231A. creber

NRRL 238T

NRRL 13146

NRRL 13145

NRRL 13144

NRRL 5219

NRRL 4791T

NRRL A-23173NRRL 5031

NRRL 239T

NRRL 241

NRRL 58747

NRRL 58748

NRRL 58613

NRRL 3505T

NRRL 58942

NRRL 58991

NRRL 58990

NRRL 226


NRRL 236

NRRL 35600

NRRL 4838T

A. versicolor

A. tabacinus

A. fructus

A. amoenus


NRRL 250T

NRRL 5585

NRRL 4768

NRRL 254

A. sydowii

A. protuberus



Tsr1 locus, 841 characters: 638 are constant,90 are variable but parsimony-uninformative,

are parsimony-informative; >100 mp trees,CI=0.7882, RC=0.7523

113

98

95

98

94

9396

99

71

78

100

97

99

93

99

96

97

100

Supplemental Fig. 5

1.0

1.0

1.0

1.0

1.0 1.0

1.0

.98

.99

1.0

1.0

1.0

1.0

1.0

1.01.0

.90

Fig. S5. Phylogenetic tree based on Tsr1 locus sequences; Bayesian posterior probabilities and bootstrap values based on 1000 replicates are placed on the internodes where values are significant.

Aspergillus section Versicolores: nine new species and ... · PDF fileremoved seven species...

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