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New penicillin-producing Penicillium species and an overview of section Chrysogena

Houbraken, J.; Frisvad, Jens Christian; Seifert, K. A.; Overy, David Patrick; Tuthill, D. M.; Valdez, J. G.;Samson, RamonaPublished in:Persoonia

Link to article, DOI:10.3767/003158512X660571

Publication date:2012

Document VersionPublisher's PDF, also known as Version of record

Link back to DTU Orbit

Citation (APA):Houbraken, J., Frisvad, J. C., Seifert, K. A., Overy, D. P., Tuthill, D. M., Valdez, J. G., & Samson, R. (2012). Newpenicillin-producing Penicillium species and an overview of section Chrysogena. Persoonia, 29, 78-100. DOI:10.3767/003158512X660571

http://dx.doi.org/10.3767/003158512X660571

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© 2012 Nationaal Herbarium Nederland & Centraalbureau voor Schimmelcultures

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Persoonia 29, 2012: 78–100www.ingentaconnect.com/content/nhn/pimj http://dx.doi.org/10.3767/003158512X660571RESEARCH ARTICLE

InTRoduCTIon

Penicillium sect.Chrysogena was introduced by Frisvad & Samson (2004) for specieshaving ter- or quarterverticillatebranchedconidiophores,relativelyshortphialides(<10µm)andsmoothtofinelyroughenedconidia.Fourseriesandeightspecies(P. aethiopicum, P. chrysogenum, P. confertum, P. dipodomyis, P. flavigenum, P. mononematosum, P. nalgiovense and P. persicinum) wereacceptedinthissection.Onlyspecieslackingasexualstatewereincluded,butacloseaffinitywithEupenicillium egyptiacum was suggested.Recently, singlename nomenclature was applied in Penicillium and both asexual andsexualreproducingspecieswereincludedintheredefinedgenus (Houbraken&Samson2011).Usingamultigeneap-proach, Penicilliumwas divided into 25 sections and sect.Chrysogena was expanded to include species with a sexual state(P. egyptiacum, P. kewense, P. molle and P. sinaicum),and the recently resurrected species P. rubens.With the exception of P. chrysogenum, P. nal giovense and P. rubens,thespeciesofsect.Chrysogena are primary soil-borne (Frisvad&Samson2004).Penicillium chrysogenum (andP. rubens) garnermuch research interest because ofhealth ramifications that are a consequence of their occur-

renceinvariousfoodproducts(Pitt&Hocking2009,Samsonetal.2010)andindoorenvironments,includingdampbuildingmaterials,indoorairanddust(Changetal.1995,Hunter&Lea1995,Gravesen1999,Scottetal.2004,Bekkeretal.2012).Another biotechnologically important species of this section is P. nalgiovense, which is used as a fungal starter culture for the productionoffermentedmeatproducts(Leistner1990).Penicillium chrysogenum is best known for the production of the antibiotic penicillin and for this reason its taxonomy has receivedmuchattention.Initially,Fleming’spenicillinproduc-ing strainwas identified asP. rubrum (Fleming 1929) butbecause of changing taxonomic schemes, it was often called P. notatum(Thom1945,Raper&Thom1949),P. chrysogenum (Samsonetal.1977),P. griseoroseum(Pitt1980)orP. rubens (Houbrakenetal.2011a).WhenCharlesThomwasabouttofinishhismonographin1930,hereceivedtheFlemingstrain(CBS205.57=NRRL824=IMI015378), then identifiedasP. rubrum,andre-identifieditasP. notatum(Thom1945).InthesubsequentmonographofRaper&Thom(1949),seriesChrysogena,basedonThom’s(1930)subsect.Radiata, was introduced and four species were accepted: P. chrysogenum, P. cyaneofulvum, P. meleagrinum and P. notatum.ThenameP. notatumwasmaintainedforFleming’sstrainbutthestrainstill used for the industrialproductionofpenicillin (the ‘Wis-consinstrain’=NRRL1951=CBS307.48)wasidentifiedas P. chrysogenum (Raper&Thom1949).Considerablevariationwasobservedamongstrainsofthisseries,makingitdifficultto designate distinct phenotypic differences because of inter-gradingstrains.Therefore,Samsonetal.(1977)placedP. cya neofulvum, P. meleagrinum, P. notatum and six additional species and varieties into synonymy with P. chrysogenum and asaresult,bothFleming’spenicillinproducingstrainsandtheWisconsinstrainwereclassifiedasP. chrysogenum.Althoughvarious species similar to P. chrysogenum were examined by

new penicillin-producing Penicillium species and an overview of section ChrysogenaJ.Houbraken1,J.C.Frisvad2,K.A.Seifert3,D.P.Overy2,4,D.M.Tuthill5, J.G.Valdez6,R.A.Samson1

Key words

FlemingP. chrysogenumP. rubensphylogenytaxonomy

AbstractSpeciesclassifiedinPenicilliumsect.Chrysogena are primary soil-borne and the most well-known mem-bers are P. chrysogenum and P. nalgiovense.Penicillium chrysogenum has received much attention because of its roleintheproductiononpenicillinandasacontaminantofindoorenvironmentsandvariousfoodandfeedstuffs.Another biotechnologically important species is P. nalgiovense, which is used as a fungal starter culture for the productionoffermentedmeatproducts.Previoustaxonomicstudiesoftenhadconflictingspeciescircumscriptions.Here, we present a multigene analysis, combined with phenotypic characters and extrolite data, demonstrating thatsect.Chrysogenaconsistsof18species.Sixofthesearenewlydescribedhere(P. alliisativi, P. desertorum, P. goetzii, P. halotolerans, P. tardochrysogenum, P. vanluykii)andP. lanoscoeruleum was found to be an older name for P. aethiopicum.Eachspeciesproducesauniqueextroliteprofile.Thespeciessharephenotypiccharac-ters,suchasgoodgrowthonCYAsupplementedwith5%NaCl,ter-orquarterverticillatebranchedconidiophoresandshort,ampulliformphialides(<9µm).Conidialcolours,productionofascomataandascospores,shapeandornamentationofconidiaandgrowthratesonotheragarmediaarevaluableforspeciesidentification.Eightspecies (P. alliisativi, P. chrysogenum, P. dipodomyis, P. flavigenum, P. nalgiovense, P. rubens, P. tardochrysogenum and P. vanluykii)producepenicillininculture.

Article infoReceived:20October2012;Accepted:6November2012;Published:4December2012.

1 CBS-KNAWFungalBiodiversityCentre,Uppsalalaan8,3584CTUtrecht,TheNetherlands;correspondingauthore-mail:[email protected].

2 DepartmentofSystemsBiology,Building221,SøltoftsPlads,TechnicalUniversityofDenmark,DK-2800Kgs.Lyngby,Denmark.

3 Biodiversity(MycologyandBotany),EasternCerealandOilseedResearchCentre,960CarlingAve.,Ottawa,Ontario,K1A0C6,Canada.

4 DepartmentofChemistry,UniversityofPrinceEdwardIsland,550UniversityAve.,Charlottetown,PrinceEdwardIsland,C1A4P3,Canada.

5 BerryBiodiversityConservationCenter, 1000E.UniversityAve.,Dept.4304,UniversityofWyoming,Laramie,WY82071,USA.

6 LaboratoriodeAnálisisdeSemillas‘JoséCrnko’EEALaConsulta-INTA,CC8(5567)Mendoza,Argentina.

79J.Houbrakenetal.:NewspeciesinPenicillium section Chrysogena

Samsonetal.(1977),P. griseoroseum, P. brunneorubrum and P. citreoroseumwerenotincluded.Thesespecieswerecon-sideredinthemonographofPitt(1980),butthelattertwospe-ciesweresynonymizedwithP. griseoroseum,Fleming’sstrainwasidentifiedasP. griseoroseum and the Wisconsin strain as P. chrysogenum.FollowingPitt’smonograph,variousnewap-proaches were applied to the taxonomy of P. chrysogenum.Physiological, extrolite and isozyme data suggested that P. griseoroseum andrelatedsynonymswereconspecificwith P. chrysogenum(Frisvad&Filtenborg1989,Bankeetal.1997),in which case the less commonly used name P. griseoroseum would have displaced the better known P. chrysogenum.Toavoidanamechangeforpenicillinproducingstrains,Kozakie-wiczetal.(1992)proposedformalconservationofthename P. chrysogenumand rejectionof theolderP. griseoroseum, along with its synonyms P. citreoroseum and P. brunneorubrum.TheproposalwasacceptedandthenameP. chrysogenum is currently listed as a nomen conservandum(McNeilletal.2006).Morerecently,thetaxonomyofP. chrysogenumwassubjectedtomultigenesequenceandmicrosatelliteanalysis(Scottetal.2004,Henketal.2011,Houbrakenetal.2011a).BothScottetal.(2004)andHenketal.(2011)showthepresenceoffourcladeswithinthespecies;however,thesubdivisionsaredis-cordant.Thestudiesagreeontheexistenceoftwomaincladesandbasedonapolyphasicapproach,Houbrakenetal.(2011a)named these clades P. chrysogenum and P. rubens.Interest-ingly,Fleming’sstrainandtheWisconsinstrainbothresideina clade with P. rubens(Houbrakenetal.2011a).Thefirstaimof thepresentstudywas toelucidate thephy-logenetic relationships among species belonging to sect.Chrysogena usingpartialRPB1,RPB2 (RNApolymerase IIgenes),β-tubulinandcalmodulingenesequences.A furtherobjectivewas to describe the six newspecies identifiedasbelonging to this section, usinga combination of sequencedata, pheno typic characteristics and extrolite data, including penicillinproduction.Inaddition,anoverviewofspeciesbelong-ingtosect.Chrysogenaandtheirsynonymsispresented.Thetaxonomy of P. chrysogenums.str.hasoftenbeencontroversialand the ultimate goal of this manuscript is to obtain a robust, reproducible and stable species concepts for this economically importantspecies.Anetworkanalysisbasedoneightgenes(RPB1,RPB2, calmodulin,β-tubulin, ITS, acetyl-CoA ligase(FacA), phosphoadenosine-5-phosphosulfate reductase(ParA), anthranilate synthasemultifunctional protein (TrpC))is performed in order to get insight in the haplotype diversity among P. chryso genum, P. rubensandcloselyrelatedspecies.

MATERIAL And METHodS

StrainsEx-typeandrepresentativestrainswereobtainedfromthecul-turecollectionsoftheCBS-KNAWFungalBiodiversityCentre(CBS),TechnicalUniversity ofDenmark (IBT),USDA-ARS,NationalCenter forAgriculturalUtilizationResearch(NRRL)and the working collection of the department of Applied and IndustrialMycologyhousedatCBS(DTO).AnoverviewofthestrainsisgivenTable1.Moreinformationcanbefoundintheon-linedatabaseofCBSatwww.cbs.knaw.nl/databases.

DNA extraction, PCR amplification, sequencing and data analysisTotalgenomicDNAwasextractedusingtheUltracleanTMMicro-bialDNAisolationkit(MoBio,SolanaBeach,USA)accordingto themanufacturer’s instructions.Toestimatephylogeneticrelationshipsamongspeciesofsect.Chrysogena, parts of the RPB1(RNApolymerase II largestsubunit; regionsEandF,accordingMathenyetal.2002),RPB2(polymeraseIIsecond

largestsubunit;regions5–7),calmodulin(cmd)andβ-tubulin genes (benA)wereamplifiedand sequencedaccording themethodsdescribedpreviously (Houbraken&Samson2011,Houbrakenetal.2012).TotesttheapplicabilityofITSsequenc-ingforspeciesidentification,sequencesweregeneratedofthestrainslistedinTable1usingprimersV9GandLS266(deHoog&GerritsvandenEnde1998).EachindividualdatasetwasalignedusingtheMusclesoftwareasimplementedinMEGA5(Tamuraetal.2011).Priortocom-bining datasets, each individual dataset was analysed using Neighbour Joining (NJ) analysis inMEGA5.Thenumberofbootstrap replicates was set to 1 000 and P. griseofulvumCBS185.27NTwasusedasoutgroup.ThecombinedRPB1,RPB2,benAandcmddatasetwasusedtostudythephylogenyofsect.Chrysogena.Statistical supportwasmeasuredbyBayesiantreeinference(BI)analysisusingMrBayesv.3.1.2(Ronquist&Huelsenbeck2003).Toidentifythemostsuitablesubstitu-tionmodelfortheBayesiananalyses,weusedMrModeltest v.2.3(Nylander2004),utilizingtheAkaikeinformationcriterion(AIC).TheBayesiananalysiswasperformedwithtwosetsoffourchains(onecoldandthreeheated)andtheSTOPRULEoption, stopping the analyses at an average standard deviation ofsplitfrequenciesof0.01.Thesamplefrequencywassetto100;thefirst25%oftreeswereremovedasburnin.Statisti-calsupportwasalsomeasuredbyMaximumLikelihood(ML)analysisusingtheRAxML(randomizedaxeleratedmaximumlikelihood)software(Stamatakis2008).ThephylogramobtainedwithRAxMLwasusedforpresentingthedata.

Morphological analysis and extrolite analysisFor macromorphological analysis, strains were inoculated at threepointsontoCzapekyeastagar(CYA),CYAsupplementedwith5%NaCl(CYAS),yeastextractsucroseagar(YES),maltextract agar (MEA), creatine agar (CREA), dichloran 18%glycerol agar (DG18) and oatmeal agar (OA).Plateswereincubatedinthedarkfor7dat25°C.Inaddition,CYAplateswereinoculatedandincubatedfor7dat15,30and37°Cindarkness.Afterincubation,colonydiametersweremeasuredand the degree of sporulation, obverse and reverse colony colours and the production of soluble pigments were deter-mined.Colonyphotographswere takenwithaCanon400Dcameraunderincandescentlight.Furthermore,isolateswereexaminedfortheproductionofalkaloidsreactingwithEhrlichreagent,usingthefilterpapermethoddescribedbyLund(1995).MicroscopicobservationsweremadeusingOlympusBH-2orZeissAxioskop2Plusmicroscopes.Mountsweremade in85%lacticacidandexcessconidiawerewashedawaywithadropofethanol.Manualmeasurementsweremade foratleast 20 conidia, ascospores, phialides, metulae, branches andascomata.Detailedanalysisoftheornamentationoftheascospores was performed using scanning electron microscopy (SEM)usingthemethoddescribedbyHoubrakenetal.(2011b).Forextroliteanalyses,culturesweregrownonCYAandYESfor7dat25°C.Afterincubation,fiveplugsweretakenfromeach agar medium, pooled and extracted according the method described bySmedsgaard (1997).The extractswere sub-sequentlyanalysedaccordingtheHPLC-diodearraydetectionmethod(Frisvad&Thrane1987)asmodifiedbyHoubrakenetal. (2012).Penicillinproductionwas testedaccording themethoddescribedbyAndersen&Frisvad(1994).

ITS barcodingToassessthesequencediversityoftheITSlocusofstrainsbelongingtosect.Chrysogena,anUPGMA(unweightedpairgroupmethodwith arithmeticmean) dendrogrambasedonKimura2-parameterdistances(K2P,recommendedbyCBOL,www.barcoding.si.edu)wasconstructedinMEGA5.

80 Persoonia–Volume29,2012

Species

CBSno.

1 Othercollectionnumbers

2 S

ubst

rate

, loc

ality

and

rem

arks

H

aplo

type

P. a

lliis

ativ

i 131541

DTO

148-I4=IB

T15987

Mixedpigfeed;S

tora,Zagora,Bulgaria

20

131544

DTO

148-I8=IB

T18101=FR

R2818

Sorghum

malttoxictoday-oldducklings;P

otchefstroom

,SouthAfrica

21

132071

DTO

149-A5=IBT26504=LJC384

Alli

um s

ativ

um (g

arlic);Anchoris,Lujan,M

endoza,A

rgentina

24

132072

DTO

149-A6=IBT26505=LJC215

Alli

um s

ativ

um (g

arlic);LaHolanda,Lavalle,M

endoza,A

rgentina

20

132073

DTO

149-A7=IBT26506=LJC044

Alli

um s

ativ

um (g

arlic);Pocito,S

anJuan,Argentina

25

132074

T DTO

149-A8=IBT26507=LJC206

Ex-type;A

llium

sat

ivum

(garlic);Lavalle,M

endoza,A

rgentina

20

132075

DTO

149-A9=IBT26514=LJC481

Alli

um s

ativ

um (g

arlic);LaBlanca,Maipu,M

endoza,A

rgentina

26

132076

DTO

149-B1=IBT26515=LJC394

Alli

um s

ativ

um (g

arlic);Vistalba,Lujan,M

endoza,A

rgentina

27

132077

DTO

149-B2=IBT26516=LJC317

Alli

um s

ativ

um (g

arlic);Andrade,R

ivadavia,M

endoza,A

rgentina

25

132198

DTO

149-B4=IBT26518=LJC128

Alli

um s

ativ

um (g

arlic);Lavalle,M

endoza,A

rgentina

28

132207

DTO

149-F3=IBT24377=EXF633

Saltern;S

ecovljeSaltern,S

lovenia

31

P. c

hrys

ogen

um

259.29

DTO

071-G7=MUCL28649

RepresentativeofP

. cya

neof

ulvu

m;unrecordedsource

4

282.97

DTO

095-E6=IBT15162

Barley;SouthAfrica

40

289.53

DTO

148-I9=IB

T19373=IMI089373

Gelatin,U

K

22

302.67

DTO

071-H6=IBT27042=AT

CC18476=IM

I129964

Ex-typeofP

. aro

mat

icum

f.m

icro

spor

umnom

.inval.;cheese(?);Leningradregion,R

ussia

12

306.48

T DTO

012-I1=IB

T5233=NRRL807=IMI24314

Ex-lectotype;cheese,Storrs,Connecticut,U

SA

6

314.48

DTO

071-G8=AT

CC10431=IM

I039764=

Ex-typeofP

. cya

neof

ulvu

m;unrecordedsource

4

MUCL28658=MUCL29077=MUCL29143=NRRL837

355.48

DTO

098-D4=AT

CC10108=IM

I039759=IMI039759ii=NRRL821

Ex-typeofP

. not

atum

;decayingbranchesofH

ysso

pus,Norway

42

412.69

DTO

071-H9=IBT30174=IBT23022=IMI140340

Ex-typeofP

. har

mon

ense;soil;Syria

37

776.95

DTO

095-F4=IBT14462

Lechuguillacave;Carlsbad,New

Mexico;USA

111215

DTO

071-I8=IB

T21928

MouldyleavesofS

alvi

a of

ficin

alis

(sage)plant;Farum

,Denmark

11

116046

DTO

001-C2=IBT30183

Waterusedinproductionprocessofcardboard;theNetherlands

13

131516

DTO

064-E8=IBT29739=IBT30133

Airincleanroom

ofvaccineproductionplant;theNetherlands

33

131517

DTO

068-C3=IBT30182

Indoorenvironm

ent;Denmark

34

131518

DTO

068-C4=IBT30176

Indoorenvironm

ent;Finland

35

131519

DTO

068-C5=IBT30175

Indoorenvironm

ent;Finland

36

131520

DTO

078-E5=IBT29738

Indoorenvironm

entcleanroom

;theNetherlands

2

131521

DTO

087-I2

Swabsam

plefromceilinginarchive;U

trecht,theNetherlands

11

131522

DTO

091-D4

Indoorenvironm

entofpharmaceuticalcom

pany;theNetherlands

2

131524

DTO

098-E6=IBT30140=NRRL841

Ex-typeofP

. bru

nneo

rub

rum;unrecordedsource

12

131525

DTO

098-E7=IBT30146=NRRL834

Ex-typeofP

. citr

eoro

seum

;unrecordedsource

4

131526

DTO

098-E9=IBT30136=NRRL889

Ex-typeofP

. ros

eoci

treum

;unrecordedsource

4

131527

DTO

098-F1=IBT30147=NRRL817

Ex-typeofP

. chl

orop

haeu

m;unrecordedsource

12

131529

DTO

100-G4=IBT30148=NRRL819

DistributedasP

. flu

ores

censnom

.inval.;unrecordedsubstrate;C

zechRepublic

4

131530

DTO

100-G6=IBT30150=NRRL822

Sputumofawom

anwithalungdisease;unknownlocality

6

131531

DTO

100-G8=IBT30144=NRRL827

Unrecordedsource.C

apableofvolatilizingpotassium

telluride

6

131532

DTO

100-H3=IBT30138=NRRL2136

RepresentativeofP

. mel

eagr

inum

(Thom,1930;Raper&Thom,1949:366);unrecordedsource

8

131533

DTO

102-B4=IBT26889=C238

Housedust;Wallaceburg,O

N,C

anada.Representativeofgroup2inthestudyofScott

et a

l.(2004)

11

131534

DTO

102-B5=IBT26890=C71.1


N,C


et a

l.(2004)

12

131535

DTO

102-B7=IBT26892=C200


N,C


et a

l.(2004)

14

131536

DTO

103-E7=IBT30084

Unknownsubstrate;D

ryValley,Antarctica

15

131538

DTO

148-I1=IB

T6041

Dust;China

17

131545

DTO

149-A1=IBT22435

Bread;Italy

4

132068

DTO

149-A2=IBT22435

Bread;Italy

4

132199

DTO

149-B5=IBT29402

Dam

agedoilpainting;Kharkov,U

kraine

4

132201

DTO

149-C1=IBT30085

Soil;DryValley,Antarctica

15

132202

DTO

149-C2=IBT30086


15

132203

DTO

149-C3=IBT30087


15

132205

DTO

149-C5=IBT30737

Bee;U

SA

11

132208

DTO

100-H2=IBT30139=NRRL842

Representativeof

P. b

runn

eoru

brum

;unrecordedsource

7

132209

DTO

100-G5=IBT30143=NRRL820

Ex-lectotypeofP

. gris

eoro

seum

;unrecordedsource

5

132211

DTO

100-F7=DTO

086-I4=IB

T30177

Surfaceofoperatingroom

;theNetherlands

132212

DTO

102-B9=IBT27840

Indoorenvironm

ent;Wallaceburg,O

ntario,C

anada

13

Tabl

e 1

Pen

icill

iumstrainsusedinthisstudy.


132213

DTO

102-B2=IBT26887=C317.2

Indoorenvironm

ent;Wallaceburg,O

ntario,C

anada;representativeofgroup3inthestudyofScottetal.(2004)

9

132214

DTO

102-B6=IBT26891=C77.2

Indoorenvironm

ent;Wallaceburg,O

ntario,C

anada;representativeofgroup3inthestudyofScottetal.(2004)13

132215

DTO

013-E6=IBT30181

Flourforproductionoftortillas;USA

16

132216

DTO

068-B8=IBT30179

Industrialenvironm

ent;Germany

8

132217

DTO

102-B3=IBT26888=C8.18

Indoorenvironm

ent;Wallaceburg,O

ntario,C

anada;Scottetal.(2004)

10

DTO

100-F7=DTO

086-I4=IB

T30177

Unrecordedsource

2

DTO

100-H1=IBT30149=NRRL839

RepresentativeofP

. cya

neof

ulvu

m (R

aper&Thom1949:372);unrecordedsource

4

DTO

100-G9=IBT30141=NRRL837

Ex-typeofP

. cya

neof

ulvu

m;unrecordedsource

4

Con

tam

inan

t in

Pos

tia p

lace

ntaMAD698Rculture.N

ostrainavailable,fullgenomesequenced

13

P. c

onfe

rtum

171.87

T DTO

072-A9=IBT21515=IBT3098=IB

T5672=IM

I296930=

Ex-type;cheekpouch;Arizona,USA

NRRL13488=NRRLA-26904

P. d

eser

toru

m

129469

IBT20395

A1horizonsoil;Utah,USA

130050

IBT14084=IMI297544

Shrublandsoil;Wyoming,USA

131229

IBT14452

A1horizongrasslandsoil;Wyoming,USA

131514

DTO

015-H9

Soil;Chubut,Argentina

131515

DTO

016-B5

Soil;Chubut,Argentina

131542

DTO

148-I5=IB

T16313

SoilunderA

rtem

isia

trid

enta

ta,cooldesert;16kmnorthofR

awlins,W

yoming,USA

131543

T DTO

148-I6=IB

T16321

Ex-type;soilunderO

ryzo

psis

hym

enoi

des,cooldesert;20kmeastofLittleAmerica,W

yoming,USA

P. d

ipod

omyi

s 170.87

DTO

217-B4=IBT21522

Cheekpouch;A

rizona

110412

T DTO

072-B6=IBT5333=IM

I296926=NRRL13485=NRRLA-26136

Ex-type;cheekpouchofkangaroorat;Arizona,USA

110413

DTO

217-B5=IBT17759

Barley;StarrValley,W

yoming,USA

110414

DTO

217-B6=IBT12700

Kangaroorat;SocorroCounty,SevilettaNatl.WildlifeRefuge,New

Mexico,USA

110415

DTO

217-B7=IBT11425

Saddle,mouldyleather,leatherprobablyfromSaudiArabia

112570

DTO

217-B8=IBT3353

Soil;WalnutC

rater,Arizona,USA

P. e

gypt

iacu

m

137.70

DTO

092-B7=IBT14685

Unknownsource;Izm

ir,Bornova,Turkey

244.32

NT

DTO

088-F6=IBT14684=AT

CC10441=IM

I040580=NRRL2090

NeotypeofP

. egy

ptia

cum;holotypeofP

. nile

nse;soil;Cairo,E

gypt

457.72

DTO

088-G5=NRRL22307=IBT30195

Desertsoil;Egypt

458.72

IBT14687

Desertsoil;Egypt

867.70

DTO

088-G2=IBT14686

Root;Israel

456.72

DTO

088-G4=AT

CC24075=IM

I084589=IBT14682

Ex-typeofE

. mol

le a

nd P

. mol

le;soil;Pakistan

P. fl

avig

enum

419.89

T DTO

072-B4=IBT21526=IBT3091=IM

I293207

Ex-type,w

heatflour;Denmark

110406

IBT16616

SoilunderC

hrys

othm

nus

naus

eosu

s;Tablerockroad

/highw

ay80,W

yoming,USA

110407

DTO

217-C5=IBT14060

Whitebeans;U

SA

110409

DTO

217-C6=IBT3230

Sand;Tunisia

110411

DTO

217-C7=IBT11693

Barley;Canada

132247

DTO

149-C7=IBT30948

Paintingoncanvas(lining);Provostchurch,Ljubljana,S

lovenia

P. g

oetz

ii 285.73

T DTO

088-G6=IBT30199

Ex-type;soil;Calgary,Alberta

581.67

DTO

088-F8=NRRL3556=IB

T4980=IB

T4993

Soil;Lahore,P

akistan

635.70

DTO

088-F9=IBT30200

Soil;USA

812.70

DTO

088-G1=IBT30196

Culturecontaminant,inS

piro

mas

tix w

arcu

piiC

BS576.63

DTO

055-H1=IBT30198

Endophytefrom

rootsofP

inus

pon

dero

sa(ponderosapine)andP

seud

otsu

ga m

enzi

esii(Douglas-fir);M

ission

Creekwatershed,O

kanogan-WenatcheeNationalForest,north-centralW

ashingtonstate,USA

DTO

055-H2

Endophytefrom

rootsofP

inus

pon

dero


seud

otsu

ga m

enzi

esii(Douglas-fir);M

ission

Creekwatershed,O


ashingtonstate,USA

DTO

055-H3

Endophytefrom

rootsofP

inus

pon

dero


seud

otsu

ga m

enzi

esii(Douglas-fir);M

ission

Creekwatershed,O


ashingtonstate,USA

P. g

riseo

fulv

um

185.27

NT

DTO

072-A5=IBT6740=ATC

C11885=IM

I075832=IMI075832ii=

Ex-neotype;unrecordedsubstrate;B

elgium

NRRL2152=NRRL2300

P. h

alot

oler

ans

131537

T DTO

148-H9=IBT4315

Ex-type;saltm

arsh;E

gypt

P. k

ewen

se

183.72

DTO

092-B8=IBT14680

Soil;theNetherlands

344.61

IsoTDTO

088-F7=AT

CC18240=IM

I086561=NRRL3332=IB

T24547

Isotype;culturecontaminantofm

ineraloilCMI1959;Surrey,Kew

,England

P. la

noso

coer

uleu

m

215.30

T DTO

035-H4=IBT3545=ATC

C10459=CBS334.48=

Ex-type;culturecontaminantofP

. cyc

lopi

umculture;U

SA

IMI039818=NRRL888

484.84

DTO

072-A8=IBT21501=IBT5903=IM

I285524

Ex-typeofP

. aet

hiop

icum

; Hor

deum

vul

gare(barley);AddisAbeba,E

thiopia


Species

CBSno.

1 Othercollectionnumbers

2 S

ubst

rate

, loc

ality

and

rem

arks

H

aplo

type

P. m

onon

emat

osum

172.87

T DTO

072-B2=IBT21535=IMI296925=NRRL13482

Ex-type;burrowsystemofD

ipod

omys

spe

ctab

ilis(banner-tailedkangaroorat);Arizona,USA

109616

DTO

217-B9=IBT4309=IB

T4310=IB

T5509

Saltm

arshsoil;Egypt

112104

DTO

217-C1=IBT3073=IB

T5521=IB

T5522=IB

T6071=

Kangaroorat;8km

eastofP

ortal,Arizona,USA

NRRLA-26910=NRRL13483

112105

DTO

217-C2=IBT11891

Squash;France

112106

DTO

217-C3=IBT11682

Jerusalemartichoke;Denmark

112575

DTO

217-C4=IBT4308=IB

T4391=IB

T5507

Marshsoil;Egypt

P. n

algi

oven

se

318.92

IBT12383

Sausage,importedfromItaly;Denmark

352.48

NT

DTO

072-A6=IBT21536=AT

CC10472=IM

I039804=NRRL911

Neotype;E

llischauercheese;CzechRepublic

109610

DTO

217-C9=IBT11965=FR

R3284

Salam

i;Germany

112438

DTO

217-D1=IBT23346

Ice;Svalbard,Norway

P. p

ersi

cinu

m

111235

T DTO

072-B8=IBT24565

Ex-type;soil;QinghaiProvince,China

P. ru

bens

197.46

DTO

065-B3

Mustcontaminant,Belgium

.Thestrainfirstusedforproducingpenicillininsubmergedculture

1

(Raper&Thom1949:368

–370)

205.57

DTO

065-B1=IBT30143=IMI015378

Culturecontaminantinbacterialculture,U

K.Fleming’soriginalpenicillinproducingstrain

1

307.48

DTO

065-B2=IBT5857=NRRL1951=IM

I40233

MouldycantaloupePeoria,Illinois,U

SA.‘Wisconsinstrain’,parentofm

osthighyieldingpenicillinproducing

1

strains;fullgenomesequenced

319.59

DTO

098-D2=AT

CC18226=IM

I068231

Ex-typeofP

. chr

ysog

enum

mut.fulvescens;soil,Japan;cinnamon-colouredconidia

39

339.52

DTO

071-H2=IBT30130=AT

CC22349=IM

I041606=IMI041606ii

Ex-typeofP

. cam

erun

ensenom

.inval.;rootofE

laei

s gu

inee

nsis

, tog

ethe

r with

Cha

lara

par

adox

a41

349.48

DTO

098-G1=IBT4350=ATC

C10468=IM

I039762=NRRL836

Unrecordedsubstrate;S

cotland.R

epresentativeofP

. mel

eagr

inum

(Thom1930,Raper&Thom1949:366)

1

401.92

DTO

001-C6

Gypsum,buildingmaterials;theNetherlands(usedasmodelorganism;e.g.B

ekkeretal.2012)

19

478.84

DTO

071-I2=IB

T21511

Airinfruitstore;D

enmark

19

111216

DTO

071-I9=IB

T22809

Saltern;S

lovenia

19

129667

T DTO

098-E8=IBT30129=NRRL792=AT

CC9783

Ex-lectotype;unrecordedsource

1

131513

DTO

015-F3=IBT30659

Tattoopaint;theNetherlands

32

131523

DTO

095-E9=IBT30661

CapofP

ETbearbottle;K

aulile,Belgium

38

131528

DTO

100-G3=IBT30145=NRRL812

Solutioncontaining4percentiron-alum;U

SA

3

131540

DTO

148-I3=IB

T14508

LechuguillaCave;Carlsbad,New

Mexico,USA

19

132069

DTO

149-A3=IBT22703

SoilunderL

arix;3kmwestofU

thoss,Russia

1

132204

DTO

149-C4=IBT30427

Unrecordedsubstrate;G

ermany

19

132206

DTO

149-C6=IBT30738

Bee;U

SA

30

132210

DTO

100-F6=NRRL843=IBT5303

Unrecordedsource;approximatedP

. bac

ulat

um(R

aper&Thom1949:363)

1

DTO

100-G7=NRRL824=IBT30142

Culturecontaminantinbacterialculture,U

K;Fleming’soriginalpenicillinproducingstrain

1

DAOM234047

Indoorair;Saskatchewan,C

anada

DAOM234052

Pipewrapinahouse;O

ntario,C

anada

DAOM234054

Housedust;Alberta,C

anada

P. s

inai

cum

279.82

T DTO

097-D3

Ex-type;m

arinesludge;S

uezCanal,30km

NofP

ortS

aid,SinaiPeninsula,E

gypt

P. ta

rdoc

hrys

ogen

um

132200

T DTO

149-B9=IBT30075

Ex-type;soil;McM

urdoDryValley,Antarctica

29

P. v

anlu

ykii

131539

T DTO

148-I2=IB

T14505

Ex-type;LechuguillaCave;Carlsbad,New

Mexico,USA

18

132070

DTO

149-A4=IBT23469

Soil;BoseJubony,Isla25daMugo,ShetlanddelSur,Antarctica

23

132197

DTO

149-B3=IBT26517=LJC005

Garlic;V

illaAberastain,Pocito,S

anJuan,Argentina

18

Pen

icill

iumsp.(nearP

. kew

ense)

103.71

Soilofw

heatfield;Kiel,Germany

227.81

DTO

103-D7=CBS653.82=NRRL2094

Unknownsource.Intermediatebetween

P. b

refe

ldia

num

and

mem

bers

of t

he C

arpe

ntel

es s

erie

s su

ch a

s

P. e

gypt

iacu

m(R

aper&Thom1949:146)

653.82

DTO

088-G7=CBS227.81=NRRL2094

Unknownsource.Intermediatebetween

P. b

refe

ldia

num

and

mem

bers

of t

he C

arpe

ntel

es s

erie

s su

ch a

s

P. e

gypt

iacu

m(R

aper&Thom1949:146)

1 CBS:culturecollectionoftheCBS-FungalBiodiversityCentre,U

trecht,TheNetherlands.

2 AT

CC:A

mericanTypeCultureCollection,Manassas,VA,U

SA;D

AOM:C

anadianCollectionofFungalC

ultures,AgricultureandAgri-FoodCanada,Ottawa,Ontario,C

anada;DTO

:internalculturecollectionofCBS-FungalB

iodiversityCentre;IBT:culturecollectionofCenterfor

MicrobialBiotechnology(C

MB)a

tDepartmentofS

ystemsBiology,TechnicalUniversityofD

enmark;IH

EM:culturecollectionoftheScientificInstituteofP

ublicHealth–Mycologysection,Brussels,Belgium

;IMI:CABIG

eneticResourcesCollection,Surrey,UK;LJC

:Coleccionde

fitopatogenosdecultivoshorticolas,Mendoza,A

rgentina;NRRL:AgriculturalR

esearchServiceCultureCollection,NationalC

enterforAgriculturalU

tilizationResearch,Peoria,Illinois,U

SA.

Tabl

e 1(cont.)


Haplotype diversityInordertostudythehaplotypediversityamongP. chrysogenum, P. rubensandcloselyrelatedspecies,theRPB1,RPB2,calmo-dulin and β-tubulinsequencedatasetswereexpandedwithITS,FacA(acetyl-CoAligase;(facA-F_Pc(TGGAAGTGGTACTTC-GAG), facA-R_Pc (ACACGACCGCGGATCCAGTA))), ParA(3-phosphoadenosine-5-phosphosulfatereductase;(parA-F_Pc (CCCGAGATTGTTTTCACCAA), parA-R_Pc (ACCTTG-GCCACCCAGTCGTA))) andTrpC (anthranilate synthasemultifunctional protein; (trpC-F_Pc (GCAGTGGAGGGT-GTTCAGTT), trpC-R_Pc (TTAACCTCGACCAGAGGCTC-CAT)))genesequences.Thesedatasetsweresupplementedwithsequencesobtainedfromthetwofullgenomeinitiatives(vandenBerg et al. 2008, http://genome.jgi.doe.gov/).The

softwareprogrammeDnaSPv.5.10(Librado&Rozas2009)wasusedtofindthedifferenthaplotypesinthealignment.Gapsandmissingdatawerenotconsideredduringthiscalculation.Networkv.4.6.1.0(www.fluxus-engineering.com)wasusedtogenerateahaplotypenetworkusingthemedian-joiningnetworkalgorithm.SequencesweredepositedintheGenBanknucleo-tidedatabaseunderaccessionnumbersJX996198–JX997117.

RESuLTS

PhylogenyThephylogeneticrelationshipamongmembersofsect.ChrysogenawasstudiedbycombiningtheRPB1,RPB2,cmdand

Fig. 1Best-scoringMaximumLikelihood (ML) treeusingRAxMLbasedonacombinationofpartialcalmodulin,β-tubulin,RPB1andRPB2sequences,showing the relationship among members of Penicillium section Chrysogena.Thebootstrap(bs)valuesoftheMLanalysisandtheBIposteriorprobabilities(pp)valuesarepresentedatthenodes(bs/pp).Valueslessthan70%supportedintheMLanalysisorlessthan0.95intheBIanalysisareomitted,whereasasterisksindicatefullsupport(100%bs,1.00pp).Thebrancheswithmorethan95%bootstrapsupportand1.00ppvaluesarethickened.Thephylogramisrooted with Penicillium griseofulvumCBS185.27NT.

CBS 484.84

P. chrysogenum

P. rubens

P. vanluykii sp. nov. P. tardochrysogenum sp. nov.

P. allii-‐sa7vi sp. nov.

P. flavigenum

P. mononematosum

P. confertum

P. desertorum sp. nov.

P. halotolerans sp. nov.

P. dipodomyis

P. nalgiovense

CBS 132213 CBS 131535

CBS 132217

92/*

CBS 131536 81/*

CBS 776.95 CBS 131526 CBS 131525 CBS 306.48T

CBS 314.48 CBS 259.29 CBS 131524 CBS 355.48 CBS 131527 CBS 302.67

85/*

CBS 412.69

89/0.99

CBS 319.59 CBS 205.57 CBS 349.48 CBS 197.46 CBS 307.48 CBS 131523 CBS 129667T

CBS 339.52

*/*

94/*

CBS 132197 CBS 131539T

*/*

CBS 132070 */*

74/0.98

CBS 132200T

95/0.96

CBS 132074T

CBS 132072 CBS 132207

70/0.99

CBS 132071 CBS 131544

*/*

*/*

CBS 132247 CBS 419.89T

*/*

CBS 110409 77/*

CBS 110406 CBS 110407

98/*

CBS 110411

*/*

CBS 112575 CBS 109616

*/*

CBS 112106 CBS 112105 */*

*/*

CBS 112104 CBS 172.87T

*/*

*/*

CBS 171.87T

*/*

95/*

CBS 131515 CBS 131514 CBS 131543T

99/0.99

CBS 131542

*/*

CBS 131537T

93/*

86/*

CBS 110414 CBS 110413

81/*

CBS 170.87 CBS 112570

85/*

CBS 110412T

CBS 110415

*/*

CBS 109610 CBS 352.48NT

CBS 318.92 CBS 112438

*/*

*/*

83/0.96

*/*

95/*

0.1

CBS 111235T

CBS 581.67 DTO 055-H3 DTO 055-H1 CBS 812.70 DTO 055-H2

93/*

CBS 285.73T

97/*

CBS 635.70

*/*

94/0.99

CBS 137.70 CBS 244.32NT

CBS 867.70 */*

CBS 456.72 89/-

CBS 457.72 CBS 458.72

*/* */*

CBS 279.82T

CBS 653.82 CBS 227.81

*/*

CBS 344.61IsoT

CBS 183.72 93/* 96/*

*/*

CBS 185.27NT P. griseofulvum

84/0.99

P. persicinum P. lanosocoeruleum CBS 215.30T

P. goetzii sp. nov.

P. egyp9acum

P. sinaicum Penicillium sp. P. kewense

-/0.98

-/*

-/0.99

-/-

Combined analysis cmd,RPB1,RPB2,benA


Species Extrolites

P. alliisativi 1)atlantinoneA;2)chrysogenamide;3)2-(4-hydroxyphenyl)-2-oxoacetaldehydeoxim;4)anaptho-γ-pyrone;5)penicillins;6)2-pyruvoylami-nobenzamide;7)roquefortineC,D&meleagrin;8)verrucosidin,normethylverrucosidin,deoxyverrucosidin&verrucosidinol;9)‘ALKONA’;

10)‘AURCH’;11)‘CRYPT’;12)‘DERH’,‘GULLA’&‘KUTZ’(atromentins?);13)‘OTOF’;14)‘SENGAX’;15)‘SNORL’;16)‘SPOFI’;17)‘VERNX’

P. chrysogenum 1)andrastrinA&B;2)chrysogine,2-pyruvoylaminobenzamide,2-acetyl-quinazolin-4(3H)-one&2-(2-hydroxypropionylamino)-benzamide;3)citreoisocoumarin; 4)penicillins; 5)roquefortineC,D&meleagrin; 6)secalonicacidD&F; 7)sorbicillins; 8)xanthocillins; 9)‘metØ’;10)‘DOLDO’

P. confertum 1)asteltoxin;2)roquefortineC,D&meleagrin;3)secalonicacidD

P. desertorum 1)austalides?;2)2-(4-hydroxyphenyl)-2-oxoacetaldehydeoxim;3)Raistrickphenols;4)‘FOL’

P. dipodomyis 1)diaporthins(citreoisocoumarin,diaportinicacid,diaportinol,dichlorodiaporthin&6-methyl-citreoisocoumarin);2)dipodazin;3)penicillins;4)‘CD’1-5&‘CRYPT’;5)‘CDU’;6)‘DI’(anindol-alkaloid);7)‘DIOR’;8)‘DIPA’;9)‘FCD’;10)‘GNALDI’;11)‘metØ’;12)‘TOLO’;13)‘VIK’

P. egyptiacum 1)10,23-dihydro-24,25-dehydroaflavinine(Wangetal.1995,alsoseeninthisstudy);2)macrophorinH(Wangetal.1995);3)molleninesA(=incl.E. molle) andB(Wangetal.1998);4)penicillicacid;5)Raistrickphenols;6)secalonicacidD&F;7)tetronicacids;8)xanthocillinX(Vesonder1979,

NRRL1022,notseeninthisstudy)

P. flavigenum 1)penicillins;2)penitremA;3)roquefortineC&meleagrin;4)sorbicillins

P. goetzii 1)andrastinA; 2)citreoisocoumarin; 3)fumitremorginA, verruculogen; 4)isoepoxydon; 5)10,23-dihydro-24,25-dehydroaflavinine&10,23,24,25-tetrahydro-24-hydroxyaflavinine;6)‘GLAD’

P. halotolerans 1)andrastinA;2)Raistrickphenols,roquefortineC,Dandmeleagrin;3)‘CUCU’andotherpolarpolyketides;4)‘PLIL’

P. kewense 1)andrastinA;2)fumitremorginA&verruculogen;3)10,23-dihydro-24,25-dehydroaflavinine&10,23,24,25-tetrahydro-24-hydroxyaflavinine;4)isoepoxydon;5)4’-oxomacrophorinA&D;6)roquefortineC;7)‘KEWS’1-3

P. cf. kewense 1)andrastinA;2)isoepoxydon;3)10,23-dihydro-24,25-dehydroaflavinine

P. lanosocoeruleum 1)griseofulvins(dechlorogriseofulvin,dehydrogriseofulvingriseofulvin,griseophenoneCetc.),de;2)isoepoxydon;3)tryptoquialanins&(=P. aethiopicum) tryptoquialanons;4)viridicatumtoxin;5)‘BR’;6)‘metU’;7)‘PRU’;8)‘RAIS’;9)‘SNOK’;10)‘VERNX’

P. mononematosum 1)andrastinA&B;2)citreoisocoumarin;3)cyclopaldicacid&derivedchromanols;4)fumitremorginA,B,C,TR-2&verruculogen;5)isochro-mantoxins;6)viriditoxin;7)‘ASTYL’;8)‘GULLA’;9)‘MER’;10)‘MONTI’;11)‘PJIM’;12)‘PLOT’;13)‘OKA’1&2(okaramins?);14)‘PAEL’;15)’PYTO’;16)‘SNAT’;17)‘TRYP’(=dehydrocurvularin?);18)‘VERNX’

P. nalgiovense 1)chrysogine,2-pyruvoylaminobenzamide,2-acetyl-quinazolin-4(3H)-one&2-(2-hydroxypropionylamino)-benzamide;2)citreoisocoumarin; 3)diaporthins(citreoisocoumarin,diaportinicacid,diaportinol,dichlorodiaporthin&6-methyl-citreoisocoumarin);4)dipodazin;5)nalgiovensin,

nalgiolaxinandbisanthron-derivativesofthose;6)penicillins

P. persicinum 1)andrastinA&B;2)chrysogine,2-pyrovoylaminobenzamide,2-acetyl-quinazolin-4(3H)-one&2-(2-hydroxypropionylamino)-benzamide;3)griseofulvins;4)roquefortineC&D;5)‘AURIN’;6)‘DOLDO’;7)‘MURA’;8)‘XYLA’

P. rubens 1)andrastinA&B;2)chrysogine,2-pyruvoylaminobenzamide,2-acetyl-quinazolin-4(3H)-one&2-(2-hydroxypropionylamino)-benzamide;3)citreoisocoumarin;4)7-deacetoxyyanuthone;5)penicillins;6)roquefortineC,D&meleagrin;7)sorbicillins(includingbisorbibutenolide,bisorbicillinol,bisvertinoquinol,bisvertinolone,2’,3’-dihydrosorbicillin,oxosorbicillinoltautomer,sorhinonesA,B,&C,rezishanonesA,B,C&D,sorbicillin);8)xanthocillins;9)PR-toxin;10)quinazoloneX(basedonUVspectrum,notyetstructureelucidated);11)‘DOLDO’

P. sinaicum 1)10,23-dihydro-24,25-dehydroaflavinine; 2)isoepoxydon or similar compound; 3)ML-236A; 4)pseurotinA; 5)indolalkaloids; 6)HO6;7)‘FOPT’;8)‘FORN’1,2&3

P. tardochrysogenum 1)asperentins;2)penicillins;3)secalonicacidD&F;4)‘metØ’

P. vanluykii 1)andrastinA;2)chrysogine;3)penicillins;4)roquefortineC,Dandmeleagrin,andtheuncharacterizedextrolites‘CRYPT’(4compounds),‘POO’,‘KNOLF’,‘TBRE’,‘FJOR’(2compounds).

Table 2OverviewofextrolitesproducedbyspeciesbelongingtoPenicillium section Chrysogena.

benAdatasets.Priortothisanalysis,NJanalysiswasperformedoneachindividualdatasettodetermineincongruences.TheindividualRPB1,RPB2, cmdandbenAdatasetswere 722,958,505and446bppositionslong,respectively.TheoptimalmodelwasdeterminedusingMrModeltest and theSYM+GmodelwasoptimalforthecalmodulinandRPB2dataset,themodelSYM+I+G for theRPB1andHKY+I+Gmodel for theBenApartition.Eighteenlineageswereobservedamongisolatesassignedtosect.Chrysogena (Fig.1)(Houbraken&Samson2011)andsix represent new species, named here as P. alliisativi, P. desertorum, P. goetzii, P. halotole rans, P. tardochrysogenum and P. vanluykii.Penicillium alliisativi, P. chrysogenum, P. rubens, P. tardochrysogenum and P. vanluykii together formed a well- supported clade in each analysis of the individual genes (>87%bootstrapsupport),exceptforcalmodulin(Fig.2).Theclustering within this clade was generally poorly supported and variedamongtheexamineddatasets;however,analysisofthecombineddatasetgeneratedhighlysupportedclades(Fig.1).Analysis of the combined dataset shows that P. alliisativi is basal to the other species of this clade, and that P. chrysogenum and P. rubens are sister species with P. vanluykiibasaltothem.ThepositionofisolatesCBS412.69(ex-typeofP. harmonense),DTO102-B2andDTO102-B7were inconflictbetween thecmd dataset and the phylogram based on combined nucleotide data.ThissetofisolatesresidesintheP. rubens clade in the cmddatasetwith statistical support (bootstrap value84%),

while they were positioned in the P. chrysogenum clade in the combinedphylogram.All P. dipodomyis, P. flavigenum and P. nalgiovense isolates formed distinct well-supported clades in all datasets, and all P. desertorum strains clustered in a well-supported clade except in the β-tubulingenetree.Penicillium dipodomyis and P. nalgiovense are sister species in all individual datasets and some variation was observed within the P. dipodomyisclade;however,thispatternwasincongruentamongdatasets.Fig.1 (combinedanalysis)showsthatP. confertum is basal to P. mono nematosum, but it renders the P. mononematosum clade para-phyletic in the β-tubulindataset.Threesubcladesareconsist-ently formed in P. mononematosumphylograms.OnecladeincludestwostrainsfromasaltmarshinEgypt(CBS109616,CBS112575)whileCBS112105andCBS112106alsoformaseparateclade,asdoCBS172.87TandCBS112104.Theex-type of P. lanoscoeruleum CBS215.30T and P. aethio picum CBS484.84T resolve in a single clade and priority is given to theformer(oldest)name.Four species are known to form ascospores and three of these, P. egyptiacum, P. kewense and P. sinaicum, group in a clade withfullsupport(100%bootstrapsupport,1.00pp).Threeline- ages were observed within the P. egyptiacumclade.Onelineage was centred on the neotype of P. egyptiacum(CBS244.32NT),anotheronCBS457.72andCBS458.72andthethirdconsistedofCBS456.72T, the ex-type strain of both Penicillium molle and Eupenicillium molle.These lineageswerepresent inall


analysed individualdatasets.Thecombinedanalysisplaced P. goetzii basal to the asexual Penicilliumspecies;however,thisisnotthecaseforindividualdatasets.IntheRPB1dataset,thisspecies was grouped together with other ascospore producing species(89%bootstrapsupport).

Morphology, physiology and extrolitesPenicillium chrysogenum, P. rubens, P. tardochrysogenum, P. vanluykii and P. alliisativi are phenotypically similar and sharecharacterssuchasafastgrowthrateonYESwithdensesporulation(exceptP. tardochrysogenum),aCYAS:CYAratiogreaterthan1,ter-orquarterverticillatedivergentlybranchedconidiophores,andrelativelyshortphialides(<9µm).PenicillinisproducedbyallspeciesandroquefortineC,Dandmeleagrinby all except P. tardochrysogenum.Therearealsodifferencesamongthespeciesof thissection.Penicillium vanluykii pro-ducesdarkgreenconidiaonMEAandCYA,yellowsolublepigments onCYA incubatedat 30°Canda series of char-acteristicunidentifiedextrolites.IncommonwithP. vanluykii, P. alliisativi also produces conidia in shades of dark green on CYA;however,thereisnoyellowsolublepigmentproductiononCYA incubatedat30°Cor in insignificantamounts.Thisspeciesalsoproducesadiagnosticarrayofextrolites(Table2) including thepotentmycotoxin verrucosidin.Penicillium tardochrysogenumisrepresentedbyonestrain(CBS132200T).It isunique in thisclade for itsmorerestrictedand floccosecoloniesonMEA,alackofsporulationonYESandtheproduc-tionoffinelyroughenedconidia.ThisspeciesdoesnotproduceyellowsolublepigmentsonCYAwhenincubatedat30°Candproduces the asperentins, a series of compounds not produced by other members of series Chrysogena.Phylogenetic analyses show that P. halotolerans and P. desertorum aresister species (Fig.1)andphenotypiccharacterssupporttheirclassificationinsect.Chrysogena(CYAS:CYAratio>1;velvetycoloniesandproductionofshort,ampulliformphialides).Penicillium halotolerans can be differentiated from P. desertorum by the production of yellow soluble pigments onCYA incubatedat30°C.Furthermore, theconidiophoresof P. desertorum have various short, divaricate branches at various levels along the stipe, while P. halotolerans has ter- or quarterverticillatebranchedconidiophoreslikeotherspeciesofsect.Chrysogena.StrainsofP. desertorum consistently produce species-specificprofilesofextrolites(Table2).Someoftheseextrolites are partially characterised and details on retention time,retentionindexandUVmaxima(nm)aregiveninTable3.Penicillium halotolerans is only known from its ex-type strain (CBS131537T)andthisisolateproducesauniquecombinationofextrolites,namelyandrastinA,roquefortineC&D,meleagrinandRaistrickphenols.Fourofthe18species(P. egyptiacum, P. goetzii, P. kewense and P. sinaicum)arecapableofformingasexualstate.Thesespecies are characterised by the production of creamish, avel-laneous or ochraceous ascomata, ter- to quarterverticillatebranchedconidiophoresandglobosetosubgloboseconidia.IsolatesgrownonCYAfor7dat25°Ctypicallyproducebrownorred-brownsolublepigments.However,theydifferfromeachotherbyvariouscharacters.Penicillium egyptiacum is a good acid producer onCREA,while the other species do not orproducelimitedamountsofacidiccompounds.Thesespeciesalsodifferinascosporesizeandornamentation(Fig.3).Theascospores of P. egyptiacummeasure2–3 × 2.5–3.5µm,butvary in theirornamentation.CBS244.32NTandCBS137.70have inconspicuous ridges and smooth-walled valves, while ascosporesofCBS457.72havecloselyseparatedequatorialridges,withprominentsecondaryridgesandroughenedvalves.Incontrast, theascosporesofP. goetziiare larger,3–4.5 × 2.5–4µm,with twodistinct equatorial ridgesandoften two

secondary ridges that are connected by transverse ribs and valvesornamentedwithareticulatepattern.Theascosporesof P. kewense take an intermediate position between those of P. egyptiacum and P. goetzii, and P. sinaicumisuniqueinhav-ingascosporeswithoutadistinctequatorialridgeandreticulatevalves(Fig.3).Penicillium egyptiacum, P. goetzii, P. kewense and P. sinaicum alsoproducespecies-specificpatternsofex-trolites.Penicillicacid,RaistrickphenolsandsecalonicacidsD & F are produced by P. egyptiacum but not by the other ascosporeproducingspecies.Ontheotherhand,andrastinA,fumitremorgin A and verruculogen are produced by P. goetzii and P. kewense,andtheuncharacterisedcompound‘GLAD’is only produced by P. goetzii.

ITS barcodingITS sequenceswere generated to assess the suitability ofthis locus forspecies identification insect.Chrysogena and 44%ofthespeciescanunequivocallybeidentifiedwiththislocus.Penicillium confertum, P. goetzii, P. halotolerans, P. lano scoeruleum, P. mononematosum, P. nalgiovense and P. percisinum can be reliably identified by ITS sequencing. FiveITSsequencevariantsarepresentinourrevisedconceptof P. chrysogenum.Atotalof61%oftheP. chrysogenum strains haveidenticalITSsequencesandthissequenceisalsopresentin P. tardochrysogenum(100%)andP. alliisativi(100%).Adifferent P. chrysogenumsequencewasobservedin15%oftheexaminedstrains (e.g.CBS776.95,CBS131522,CBS132211).ThissequenceissharedwithP. rubens, all strains of whichhaveidenticalITSsequences.ThethreeotheruniqueP. chrysogenumsequenceswererepresentedbyCBS131538(2%),CBS131516(10%)andCBS111215(12%).Thethreeinvestigated P. vanluykiiisolateshaveaITSsequencethatissharedwithNRRL3710,astrainidentifiedasP. chrysogenum byHenket al. (2011). ITS sequences could not distinguish P. dipodomyis and P. flavigenum, while P. kewense and P. sinaicumsharesequenceswithP. egyptiacumCBS456.72,CBS457.72andCBS458.72.

Haplotype diversityA detailed analysis was performed on 88 P. alliisativi, P. chrysogenum, P. rubens, P. tardochrysogenum and P. vanluykii isolates, including ex-type and authentic strains, supplemented withisolatesusedinothertaxonomicstudies(Samsonetal.1977,Scottetal.2004,Houbrakenetal.2011a)andotherrep-resentativestrainsfromculturecollections.Haplotypicgroupsweredefinedbasedonthecombinedsequencealignmentofeightloci(cmd,RPB1,RPB2,benA,TrpC,parA,FacA,ITS).Forty-three haplotype groups were detected, most containing onlyonestrain.ThehaplotypenetworkisshowninFig.4andthehaplotypeassignmentofeachstrainisincludedinTable1. ThisdatademonstratesthathaplotypediversityamongP. chry sogenum strains is higher than among P. rubensstrains.ThefullgenomesequencedP. rubensstrainWisconsin54-1255belongstohaplotype1.Thishaplotypeincludesmostof theother P. rubens strains, including the ex-type strainCBS129667T (9/20P. rubens isolates).Serendipitously, a strainof P. chrysogenum for which no culture is available, had its fullgenomesequencedunexpectedlyasacontaminantofaPostia placentaMAD698Rculture(http://genome.jgi.doe.gov/Pench1/Pench1.info.html).Ourhaplotypeanalysisshowsthatthisstrainbelongstohaplotypegroup13,togetherwithstrainsCBS132214,CBS132212andCBS116046;perhapsoneofthesestrainscouldbeselectedas‘epitype’kindofvouchertorepresentthisgenomestrain.However,CBS116046isagoodpenicillin producer, but no penicillin production was observed in CBS132214andCBS132212.Incontrast,bothCBS132214andCBS132212produce roquefortineC, butCBS116046


0.01

CBS 131526 P. chrysogenum CBS 355.48 P. chrysogenum CBS 314.48 P. chrysogenum CBS 131524 P. chrysogenum CBS 131525 P. chrysogenum CBS 302.67 P. chrysogenum CBS 132217 P. chrysogenum CBS 259.29 P. chrysogenum CBS 131527 P. chrysogenum CBS 306.48T P. chrysogenum CBS 776.95 P. chrysogenum CBS 131536 P. chrysogenum

CBS 132213 P. chrysogenum CBS 131535 P. chrysogenum

CBS 412.69 P. chrysogenum CBS 339.52 P. rubens

CBS 205.57 P. rubens CBS 349.48 P. rubens CBS 197.46 P. rubens CBS 307.48 P. rubens CBS 319.59 P. rubens CBS 129667T P. rubens CBS 131523 P. rubens

CBS 131539T P. vanluykii CBS 132070 P. vanluykii CBS 132197 P. vanluykii

CBS 132200T P. tardochrysogenum CBS 132072 P. allii-‐sa7vi CBS 132207 P. allii-‐sa7vi CBS 132074T P. allii-‐sa7vi

CBS 131544 P. allii-‐sa7vi CBS 132071 P. allii-‐sa7vi CBS 132247 P. flavigenum

CBS 419.89T P. flavigenum CBS 110409 P. flavigenum

CBS 110411 P. flavigenum CBS 110406 P. flavigenum CBS 110407 P. flavigenum CBS 109616 P. mononematosum CBS 112575 P. mononematosum CBS 112105 P. mononematosum CBS 112106 P. mononematosum

CBS 112104 P. mononematosum CBS 172.87T P. mononematosum

CBS 131542 P. desertorum CBS 131543T P. desertorum CBS 131515 P. desertorum CBS 131514 P. desertorum

CBS 131537T P. halotolerans CBS 171.87T P. confertum

CBS 110412T P. dipodomyis CBS 110414 P. dipodomyis CBS 170.87 P. dipodomyis CBS 110413 P. dipodomyis CBS 112570 P. dipodomyis CBS 110415 P. dipodomyis

CBS 109610 P. nalgiovense CBS 352.48NT P. nalgiovense CBS 112438 P. nalgiovense CBS 318.92 P. nalgiovense

CBS 484.84 P. lanosocoeruleum CBS 215.30T P. lanosocoeruleum

CBS 111235T P. persicinum DTO 055H3 P. goetzii CBS 812.70 P. goetzii DTO 055H1 P. goetzii DTO 055H2 P. goetzii CBS 581.67 P. goetzii

CBS 285.73T P. goetzii CBS 635.70 P. goetzii

CBS 244.32NT P. egyp9acum CBS 867.70 P. egyp9acum

CBS 137.70 P. egyp9acum CBS 457.72 P. egyp9acum CBS 458.72 P. egyp9acum

CBS 456.72 P. egyp9acum CBS 227.81 Penicillium sp. CBS 653.82 Penicillium sp.

CBS 183.72 P. kewense CBS 344.61IsoT P. kewense

CBS 279.82T P. sinaicum CBS 185.27NT P. griseofulvum

82

93

84

77 82

99

96

77 94

71 98

85

78

72

92

84

95

81

75

88

99

92 92

99

74

99 77

88

83

99

0.01

CBS 131524 P. chrysogenum CBS 131525 P. chrysogenum CBS 776.95 P. chrysogenum CBS 314.48 P. chrysogenum CBS 302.67 P. chrysogenum CBS 355.48 P. chrysogenum CBS 131536 P. chrysogenum CBS 306.48T P. chrysogenum CBS 131527 P. chrysogenum CBS 132213 P. chrysogenum CBS 259.29 P. chrysogenum CBS 131526 P. chrysogenum CBS 132217 P. chrysogenum CBS 131535 P. chrysogenum

CBS 132200T P. tardochrysogenum CBS 412.69 P. chrysogenum

CBS 131544 P. allii-‐sa7vi CBS 132207 P. allii-‐sa7vi CBS 132071 P. allii-‐sa7vi CBS 132072 P. allii-‐sa7vi CBS 132074T P. allii-‐sa7vi

CBS 131539T P. vanluykii CBS 132197 P. vanluykii CBS 132070 P. vanluykii

CBS 131523 P. rubens CBS 205.57 P. rubens CBS 307.48 P. rubens CBS 319.59 P. rubens CBS 197.46 P. rubens CBS 339.52 P. rubens CBS 129667T P. rubens CBS 349.48 P. rubens

CBS 110407 P. flavigenum CBS 419.89T P. flavigenum CBS 132247 P. flavigenum CBS 110411 P. flavigenum

CBS 110409 P. flavigenum CBS 110406 P. flavigenum

CBS 109616 P. mononematosum CBS 11257 P. mononematosum CBS 112105 P. mononematosum CBS 112106 P. mononematosum

CBS 112104 P. mononematosum CBS 172.87T P. mononematosum

CBS 171.87T P. confertum CBS 110413 P. dipodomyis CBS 110414 P. dipodomyis CBS 110412T P. dipodomyis CBS 170.87 P. dipodomyis CBS 112570 P. dipodomyis

CBS 110415 P. dipodomyis CBS 112438 P. nalgiovense CBS 318.92 P. nalgiovense

CBS 352.48NT P. nalgiovense CBS 109610 P. nalgiovense

CBS 131514 P. desertorum CBS 131515 P. desertorum CBS 131543T P. desertorum CBS 131542 P. desertorum

CBS 131537T P. halotolerans CBS 484.84 P. lanosocoeruleum CBS 215.30T P. lanosocoeruleum

CBS 111235T P. persicinum CBS 244.32NT P. egyp9acum

CBS 867.70 P. egyp9acum CBS 137.70 P. egyp9acum

CBS 456.72 P. egyp9acum CBS 457.72 P. egyp9acum CBS 458.72 P. egyp9acum

CBS 279.82T P. siniacum CBS 227.81 Penicillium sp. CBS 653.82 Penicillium sp.

CBS 344.61IsoT P. kewense CBS 183.72 P. kewense

DTO 055H1 P. goetzii DTO 055H3 P. goetzii DTO 055H2 P. goetzii CBS 635.70 P. goetzii CBS 581.67 P. goetzii CBS 812.70 P. goetzii CBS 285.73T P. goetzii


90

86

99

95

72 82

83

94 85

99

96 81

70

99

82 98

99

99

99

89

99

94

92 96

99

Cmd RPB1

Fig. 2Best-scoringNeighbourJoining(NJ)phylogramsbasedoncalmodulin,RPB1,RPB2andβ-tubulindatasetsusingMEGA5.Well-supportedbranches(>95%bootstrapsupported)areinbold,valueslessthan70%bootstrapsupportarenotshown.Penicillium griseofulvumCBS185.27NTwasusedasoutgroup.

cmd RPB1


0.01

CBS 131523 P. rubens CBS 339.52 P. rubens CBS 319.59 P. rubens CBS 349.48 P. rubens CBS 307.48 P. rubens CBS 197.46 P. rubens CBS 129667T P. rubens CBS 205.57 P. rubens

99

CBS 131539T P. vanluykii CBS 132197 P. vanluykii

90

CBS 132213 P. chrysogenum CBS 131535 P. chrysogenum CBS 132217 P. chrysogenum

83

CBS 131536 P. chrysogenum CBS 776.95 P. chrysogenum 70

CBS 412.69 P. chrysogenum CBS 131524 P. chrysogenum CBS 355.48 P. chrysogenum CBS 131526 P. chrysogenum CBS 131527 P. chrysogenum CBS 302.67 P. chrysogenum CBS 314.48 P. chrysogenum CBS 259.29 P. chrysogenum CBS 306.48T P. chrysogenum CBS 131525 P. chrysogenum

CBS 132070 P. vanluykii CBS 132200T P. tardochrysogenum

90

CBS 132074T P. allii-‐sa7vi CBS 132207 P. allii-‐sa7vi CBS 132072 P. allii-‐sa7vi CBS 132071 P. allii-‐sa7vi

73

CBS 131544 P. allii-‐sa7vi

98

100

CBS 109616 P. mononematosum CBS 112575 P. mononematosum

CBS 112106 P. mononematosum CBS 112105 P. mononematosum CBS 112104 P. mononematosum CBS 172.87T P. mononematosum

88

CBS 171.87T P. confertum

80

CBS 132247 P. flavigenum CBS 419.89T P. flavigenum

98


94


73

98

CBS 131537T P. halotolerans CBS 131514 P. desertorum CBS 131515 P. desertorum CBS 131543T P. desertorum

71

CBS 131542 P. desertorum

100

CBS 110413 P. dipodomyis CBS 110414 P. dipodomyis CBS 112570 P. dipodomyis CBS 170.87 P. dipodomyis

85

CBS 110412T P. dipodomyis CBS 110415 P. dipodomyis 96

93

CBS 109610 P. nalgiovense CBS 352.48NT P. nalgiovense

CBS 112438 P. nalgiovense CBS 318.92 P. nalgiovense

100

100

70

CBS 484.84 P. lanosocoeruleum CBS 215.30T P. lanosocoeruleum

100

DTO 055H2 P. goetzii DTO 055H3 P. goetzii CBS 812.70 P. goetzii DTO 055H1 P. goetzii CBS 285.73T P. goetzii CBS 581.67 P. goetzii CBS 635.70 P. goetzii

100

CBS 111235T P. persicinum

87

80

CBS 244.32NT P. egyp9acum CBS 867.70 P. egyp9acum CBS 137.70 P. egyp9acum CBS 457.72 P. egyp9acum CBS 458.72 P. egyp9acum

74

78

CBS 456.72 P. egyp9acum

99


CBS 227.81 Penicillium sp. CBS 653.82 Penicillium sp.

97 73

CBS 279.82T P. sinaicum

100

CBS 185.27NT P. griseofulvum 0.01

CBS 132213 P. chrysogenum CBS 132217 P. chrysogenum CBS 131535 P. chrysogenum CBS 131536 P. chrysogenum

96

CBS 259.29 P. chrysogenum CBS 412.69 P. chrysogenum CBS 131526 P. chrysogenum CBS 355.48 P. chrysogenum

CBS 132074T P. allii-‐sa7vi CBS 132071 P. allii-‐sa7vi CBS 132207 P. allii-‐sa7vi CBS 131544 P. allii-‐sa7vi CBS 132072 P. allii-‐sa7vi

CBS 314.48 P. chrysogenum CBS 306.48T P. chrysogenum CBS 776.95 P. chrysogenum CBS 302.67 P. chrysogenum CBS 131524 P. chrysogenum CBS 131525 P. chrysogenum CBS 131527 P. chrysogenum

70

CBS 129667T P. rubens CBS 339.52 P. rubens CBS 349.48 P. rubens CBS 205.57 P. rubens CBS 307.48 P. rubens CBS 319.59 P. rubens CBS 131523 P. rubens CBS 197.46 P. rubens

88

CBS 132070 P. vanluykii

CBS 132200T P. tardochrysogenum CBS 132197 P. vanluykii CBS 131539T P. vanluykii

71

88



CBS 132247 P. flavigenum CBS 419.89T P. flavigenum

96

CBS 131543T P. desertorum CBS 131515 P. desertorum CBS 131514 P. desertorum

CBS 131542 P. desertorum CBS 109616 P. mononematosum CBS 112575 P. mononematosum CBS 112105 P. mononematosum CBS 112106 P. mononematosum

85

CBS 171.87T P. confertum CBS 112104 P. mononematosum CBS 172.87T P. mononematosum

100

CBS 131537T P. halotolerans CBS 110413 P. dipodomyis

CBS 170.87 P. dipodomyis CBS 110414 P. dipodomyis CBS 110415 P. dipodomyis CBS 112570 P. dipodomyis CBS 110412T P. dipodomyis CBS 109610 P. nalgiovense CBS 318.92 P. nalgiovense CBS 112438 P. nalgiovense

CBS 352.48NT P. nalgiovense

89

CBS 111235T P. persicinum CBS 484.84 P. lanosocoeruleum CBS 215.30T P. lanosocoeruleum

100

CBS 137.70 P. egyp9acum CBS 244.32NT P. egyp9acum



83


CBS 279.82T P. sinaicum CBS 227.81 Penicillium sp. CBS 653.82 Penicillium sp.

99 78

DTO 055H1 P. goetzii DTO 055H2 P. goetzii DTO 055H3 P. goetzii CBS 581.67 P. goetzii CBS 635.70 P. goetzii CBS 285.73T P. goetzii CBS 812.70 P. goetzii

99


RPB2 BenA

Fig. 2(cont.)

BenARPB2


Extrolite Retentiontime Retentionindex UVmaxima(nm)(sh:shoulder) Ref.

‘CRYPT1’ 10.62 769 200,271 A1

‘CRYPT2’ 10.87 774 200,271 A‘CRYPT3’ 12.66 812 200,271 A‘CRYPT4’ 14.66 855 200,271 A‘FJOR1’ 6.43 799 200,335 A‘FJOR2’ 6.67 810 200,228,270,330 A‘KNOLF’ 16.76 900 202,235sh,270,337 A‘POO’ 15.07 864 202,266,319 A‘TBRE’ 7.60 705 221,267,331 A‘KEWS1’ 2.67 692 220,275,297,380,400sh A‘KEWS2’ 6.96 802 200,225,250,275,311sh,378,400sh A‘KEWS3’ 13.11 967 226,250sh,260,276sh,355sh,376,385 A‘AURIN’ 6.48 814 200,235,311 A‘DOLDOX’ 2.86 710 265 A‘MURA’ 25.39 1479 201,212sh,265,310 A‘XYLA’ 5.03 773 201,231,281,320 A‘FOPT’ 12.54 976 200,240sh,319 A‘FORN1’ 20.84 1280 204,239,292 A‘FORN2’ 25.57 1494 204,239,292 A‘FORN3’ 16.58 1111 204,239,292 A‘HO6’ 8.22 847 200,225,242,274sh,323 A

‘DOLDO’ 4.63 710 280 B2

‘metØ’ 7.95 852 210,255,275sh B‘ALKONA’ 11.60 1974 200,215sh,265,287sh B‘AURCH’ 6.89 796 200,228,310 Ba naphtho-γ-pyrone 6.77 814 202,232,280,328,338,405 Bchrysogenamide 9.87 963 221,273,280sh B‘DERH’ 8.02 869 223,280,359,440sh B‘GULLA’ 8.15 875 220,272,359,481sh B‘KUTZ’ 12.70 1057 220,269,320,412 B‘OTOF’ 12.48 1038 217,271,315 B‘SENGAX’ 15.48 1360 220,277,330 B‘SNORL’ 15.84 1380 210,225,264,323 B‘SPOFI’ 12.21 1106 200,227sh B

‘CD1’ 11.460 892 200,273 C3

‘CD2’ 12.782 919 200,273 C‘CD3’ 13.612 935 200,273 C‘CD4’ 13.972 942 200,273 C‘CD5’ 15.960 981 200,273 C‘CDU’ 9.854 858 200,220,275 C‘CRYPT’ 10.769 877 200,269 C‘DI’ 11.891 886 200,240,270,325 C‘DIOR’ 14.143 947 200,261,425 C‘DIPA’ 17.554 995 200,213,236,259,295,331 C‘FCD’ 7.359 808 200,215,280,341 C‘GNALDI’ 10.174 865 200,224,335 C‘TOLO’ 16.828 990 207,250,281,376 C‘VIK’ 25.092 1163 200,210sh,280sh,330-375 CTetronicacid,P. egyptiacum1 1.390 6974 227,261,322sh CTetronicacid,P. egyptiacum1 1.623 7014 200,223,270sh,303 CTetronicacid,P. egyptiacum1 1.918 7084 200,225sh,275 C‘BR’ 3.846 733 200,225,271,320sh,421 C‘metU’ 2.679 711 200,230+,263,364 C‘PRU’ 1.892 697 200,235,280 C‘RAIS’ 3.51 716 214,222sh,270,310 C‘SNOK’ 14.15 911 (200),275 C‘VERNX’ 2.285 704 202,285 C‘ASTYL’ 16.819 994 263,359 C‘GULLA’ 15.283 964 220,272,359,431sh C‘MER’ 7.351 798 222,225sh,263,318 C‘MONTI’ 17.416 992 200,210sh,266,280sh,372,440sh C‘PJIM’ 13.656 993 200,218,270 C‘PLOT’ 17.571 987 202,265,281.360 C‘PAEL’ 29.533 1291 230 C‘PYTO’ 4.384 741 200,276,370 C‘SNAT’ 19.394 1043 200,224sh,275 C‘TRYP’ 12.304 893 202,225,279,300sh C‘VERNX2’ 1.877 694 202,285 C‘CUCU’ 1.519 683 202,222sh,277,300sh C‘PLIL’ 23.405 1218 200,223sh,299 C

Table 3 A partial characterisation of extrolites from Penicillium section ChrysogenawhichhavenotyetbeenfullystructureelucidatedbasedonHPLC-DAD.

1A:Nielsen&Smedsgaard2003;2B:Nielsenetal.2011;3C:Frisvad&Thrane1987;4InaccurateRIvalues,aschromatographicpeakswerebroad.


doesnot.CBS132214wastheonlystrainproducingtheun-characterisedcompound‘metØ’.Theseresultssuggestthateven with 8 loci, the resulting haplotype assignments may not bepreciseenoughtocorrelatewithaprecisegenome.

TAxonoMy

Penicillium allii-sativi Frisvad, Houbraken & Samson, sp. nov.—MycoBankMB801873;Fig.5

Typus.ArgentinA,Mendoza,Lavalle,Col3deMayo,onbulbsofAllium sativum (garlic),M. Makuch & J. Valdez(CBSH-21058holotype,culturesex-typeCBS132074=IBT26507=DTO149-A8=LJC206).

Etymology. ReferringtoAllium sativum (garlic),thesubstratewherethetypestrainwasisolatedfrom.

SporulationonCYAdense;coloniesslightlypolygonalinoutline,velvety;myceliumwhite,sporulationinshadesofdarkgreen,

exudatedropletslarge,clear,paleyelloworlightbrown;solublepigmentsabsentoroccasionallypresent,lightbrown;colonyreversepalebrown.SolublepigmentsonYESabsent;myce-liumwhite;sporulationdense;sporulationdarkgreen;exudateabsent,reversebeige.SporulationonDG18dense;sporulationgrey-greenordullgreen;reversepale.ColoniesonMEAvelvetyorslightlyfloccose;sporulationvariable,grey-green,darkgreenordullgreen;exudatedropletslarge,clear,paleyelloworlightbrown,reverseyellow-brown.NovioletreactionwithEhrlichreagents.Sclerotiaabsent.Conidiophores borne from the agar surface,ter-orquarterverticillate,divaricate.Stipes200–400 × 3–4µm,smoothwalled.Branches15–25(–35) × 3–4µm.Metulaeunequalinlength,inverticilsof3–8,10–12(–16) × 2.5–3.5µm.Phialidesampulliform,inverticilsof4–10,closelypacked,7.5–8.5 × 2–5.5µm.Conidia globose to subglobose, smooth,2.5–3.5µm. Diagnosis — Penicillium alliisativi is phenotypically similar to P. chrysogenum and P. vanluykii. Isolatesof thisspeciesproduce conidia in shades of dark green on CYA and yellow solublepigmentusuallyabsentonCYAincubatedat30°C. Colony morphology — Colony diam, 7 d, in mm: CYA 26–38;CYA15°C 18–25;CYA30°C 22–32;CYA37°C:nogrowth–4;MEA31–42;YES45–58;DG1826–40;CYAS37–45(–60);creatineagar18–30,weakormoderategrowth,weakacidproduction. Extrolites—Penicillins,AtlantinoneA, chrysogenamide,2-(4-hydroxyphenyl)-2-oxo acetaldehydeoxim, a naptho-γ-pyrone,2-pyruvoylaminobenzamide,roquefortineC,D,melea-grin, verrucosidin, normethylverrucosidin, deoxyverrucosidin, verrucosidinolandtheuncharacterisedcompounds‘ALKONA’,‘AURCH’,‘CRYPT’,‘DERH’,‘GULLA’,‘KUTZ’(atromentins?),‘OTOF’,‘SENGAX’,‘SNORL’,‘SPOFI’,‘VERNX’. Distribution&Ecology —Thisspecieshasabroaddistribu-tion(Argentina,Bulgaria,France,Portugal,SouthAfrica,UK)and has been isolated from garlic, soil, salterns, sorghum malt andmixedpigfeed(Henketal.2011,thisstudy).Thisspeciesis not a pathogen on garlic like P. allii(Valdezetal.2009). Barcode&MolecularbasedID — ITSsequencingisimpre-ciseforspeciesidentificationbecauseallinvestigatedstrainsof P. alliisativi and P. chrysogenumCBS306.48T share the sameITSsequence(GenBankJX997021).

Fig. 4 Haplotype network of P. chrysogenum (yellow),P. rubens (blue),P. vanluykii (purple),P. alliisativi(green)andP. tardochrysogenum(black)strains based on cmd, benA,RPB1,RPB2,TrpC,ParA, FacAand ITSsequences.Intotal,43differenthaplotypesweredetectedandadetailedlistisgiveninTable1.Redcolouredcirclesrepresentmedianvectors.Thelines between the groups connecting the haplotypes show the number of nucleotidesdiffering.

Fig. 3ScanningElectronMicrographsofascosporesofspeciesbelongingtoPenicillium section Chrysogena.a.P. egyptiacumCBS244.32;b.P. egyptiacum CBS457.72;c.P. sinaicumCBS279.82;d.P. goetziiCBS285.73;e.P. kewenseCBS344.61;f.Penicilliumsp.CBS103.71—Scalebars=2µm.

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Penicillium desertorum Frisvad, Houbraken & Samson, sp. nov.—MycoBankMB801874;Fig.6

Typus. USA,Wyoming,20kmeastofLittleAmerica,excooldesertsoilunder Oryzopsis hymenoides, J.C. Frisvad(CBSH-21056holotype,culturesex-typeCBS131543=IBT16321=DTO148-I6).

Etymology. Referringtodesert;becausethisspeciesiscommonindesertsoil.

SporulationonCYAdense;coloniesentireorslightlypolygonalinoutline,velvety,radiallysulcate;myceliumwhite,conidiadullgreen or greyish dull green, exudate absent or sparsely pro-

Fig. 5 Penicillium alliisativi,CBS132198.a.7doldculturesat25°Cunlessstatedotherwise,lefttoright,firstrow,allobverse,CYA,YES,DG18,MEA;secondrow,CYAreverse,YESreverse,CYAincubatedat30°Cobverse,CREAobverse;b–f.conidiophores;g.conidia.—Scalebars=10µm.

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duced;solublepigmentsabsent;colonyreversebrown.SolublepigmentsonYESabsent;myceliumwhite;sporulationdense;sporulationdarkgreen;exudateabsent,reversebeigewithabrowncentreorbrown,withcerebriformsulcations.SporulationonDG18dense;sporulationgreyen masse;reversepale,trans-parent.ColoniesonMEAvelvetyorslightlyfloccose;sporulation

dense,conidiagrey-greenwithablueshade;exudatedropletsabsent,reverseunaffectedorbecomingbrown.Novioletreac-tionwithEhrlichreagent.Sclerotiaabsent.Conidio phores borne fromsurface,with(short)divaricatebranchesatvariouslevelsalongthestipe.Stipeslong,200–400 × 2.5–3.5µm,smoothwalledandoccasionallyveryfinelyroughened.Branches8–15

Fig. 6 Penicillium desertorum,CBS131543T.a.7doldculturesat25°Cunlessstatedotherwise,lefttoright,firstrow,allobverse,CYA,YES,DG18,MEA;secondrow,CYAreverse,YESreverse,CYAincubatedat30°Cobverse,CREAobverse;b–f.conidiophores;g.conidia.—Scalebars=10µm.

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(–25) × 2.5–3.5 µm.Metulae equal in length, occasionallyinflated,denselypacked,3–8,8–10(–15) × 2.5–3.5µm.Phialidesampulliform,inverticilsof4–10,closelypacked,6–7.5 × 2–3µm.Conidiaglobose,smooth,2.5–3(–3.5)µm. Diagnosis—IsolatesofP. desertorum do not produce yellow pigmentsonCYAincubatedat25°Cand30°CandcoloniesonYEShaveabeige-brownorbrown,cerebriform,sulcatereverse.Thisspeciesisuniqueinsect.Chrysogena by the production of conidiophores that have several short, divaricate branches atvariouslevelsalongthestipe. Colony morphology — Colonydiam,7d,inmm:CYA(20–) 24–37;CYA15°C17–25;CYA30°C(15–)20–32;CYA37°C:nogrowth–4;MEA20–37;YES37–55;DG1820–30;CYAS24–38;creatineagar10–23,weakgrowth,weaktomoderateacidproduction. Extrolites — 2-(4-hydroxyphenyl)-2-oxoacetaldehydeoxim,Raistrickphenols,austalides?,‘FOL’. Distribution&Ecology — This species has aworld-widedistributionandhasbeenfoundinArgentina,Iran,USA(Wyo-ming,NewMexico),Canada(BritishColumbia),PuertoRicaandCostaRica.Aridordesertsoilseemstobetheprimarysubstrateofthisspecies.OnlyaselectednumberofstrainsareincludedinTable1. Barcode&MolecularbasedID — TwoITSsequencetypesare detected in P. desertorum.DTO016-B5,DTO148-I5andDTO148-I6 share thesame ITSsequenceand this type isspeciesspecific(GenBankJX997010).DTO015-H9sharesitsITSsequencewiththetypeofP. chrysogenumCBS306.48T (GenBank JX997038) and therefore ITS sequencing is im-precise for identification ofP. desertorum.Partialβ-tubulin, calmodulin,RPB1orRPB2sequencesarerecommendedforspeciesidentification.

Penicillium goetzii J.Rogers, Frisvad, Houbraken & Samson, sp. nov. —MycoBankMB801876;Fig.7

Typus.CAnAdA, Calgary, ex soil, J. Bissett(CBSH-21061holotype,cul-turesex-typeCBS285.73=DTO088-G6).

Etymology. Named after JohnRichardGoetz III, a student of Jack Rogerswhoisolatedthisspecies(isolatesDTO055-H1,DTO055-H2andDTO055-H3)andperformedexperimentswithit.

Sporulation onCYA variable, absent to dense; velvety orslightlyfloccose,colonieswithafeatheryedge,radiallysulcate;mycelium white and occasionally pinkish, conidia grey-green, exudatesparselyproduced,clear,lightbrownorreddishbrown;solublepigmentsbrownorreddishbrown;colonyreversebeige,sometimeswithareddishbrowncentre.SolublepigmentsnotproducedonYES;myceliumwhite;sporulationoftenabsent,occasionally present and dense, grey green en masse;exudateabsent, reverse yellow, sometimes with a yellow-orange cen-tre.SporulationonDG18absentorpoor;sporulationgreyen masse;myceliumwhite,reversepaleorbrightyellow.ColoniesonMEAfloccose;sporulationvariable,absenttodense,conidiagrey-green en masse;myceliumwhite,exudatedropletsabsentor produced as clear or light brown droplets, reverse unaffected orbecomingyellow.Noviolet reactionwithEhrlich reagent.Ascomata white when young, becoming creamish brown in time,maturingwithin 3–6wk, 150–350µm.Asci 6.5–11 × 5.5–8µm.Ascosporesellipsoidal,withtwodistinctequatorialridges and often two secondary ridges which are connected by transverse ribs, valves ornamented with a reticulate pat-tern,3–5 × 3–4.5µm.Conidiophores borne from surface and aerialmycelium,ter-toquarterverticillate,200–400 × 2.5–3.5µm,smoothwalled.Branches 12–20 × 2.5–3.5µm.Metulae equal in length, slightly inflated, 2–6, 8–12(–15) × 2.5–3.5µm.Phialidesampulliform,inverticilsof4–10,closelypacked,

7–9(–10)×2–3µm.Conidiabroadlyellipsoidal,smooth,2–2.5 × 2–3µm. Diagnosis — Penicillium goetzii is characterised by fast growth rate on CYA, production of brown soluble pigments onCYA and ascosporesmeasuring 3–4.5 × 2.5–4 µm. ItformslargercoloniesonDG18after7dofincubationat25°C(22–30mm) thanP. kewense (12–19mm)anddiffers from P. egyptiacumbyascosporesizeandornamentation. Colony morphology — Colonydiam,7d,inmm:CYA(30–) 33–42;CYA15°C18–28;CYA30°C(10–)15–27;CYA37°C:nogrowth;MEA33–42;YES40–55;DG1822–30;CYAS30– 40;creatineagar15–30,weakgrowth,acidproductionabsentorweak. Extrolites — Andrastin A, citreoisocoumarin, fumitremorgin A,verruculogen,isoepoxydon,10,23-dihydro-24,25-dehydroaf-lavinine&10,23,24,25-tetrahydro-24-hydroxyaflavinineandtheuncharacterisedcompound‘GLAD’. Distribution&Ecology — Theprimarysubstrateseemstobe soil, but this species was also isolated as an endophyte inconiferousroots(Goetz2006)andculturecontaminantofa Spiromastix warcupiiculture.Thespecieshasabroaddis-tributionandhasbeenisolatedfromCanada(Alberta,BritishColombia),PakistanandtheUSA. Barcode&MolecularbasedID— Thisspeciescanbeiden- tified reliably by ITS sequencing.Two ITS sequence typesweredetected.CBS581.67,812.70,285.73TandDTO055-H1,DTO055-H2andDTO055-H3sharethesameITSsequence(e.g.GenBankJX997042)andCBS635.70hasauniqueITSsequencetype(GenBankJX997112).

Penicillium halotolerans Frisvad, Houbraken & Samson, sp. nov. —MycoBankMB801875;Fig.8

Typus. egypt, ex salt marsh, A.H. Moubasher(CBSH-21060holotype,culturesex-typeCBS131537=IBT4315=DTO148-H9=MOUSS42).

Etymology. Namedafteritsabilitytogrowwellinthepresenceof5%NaCl.

SporulationonCYAdense;coloniesentire,velvety;myceliumwhite,sporulationdullgreenwithabluetinge;exudatedrop-letsclear,small;solublepigmentsabsent;colonyreverselightbrown.Soluble pigments onYESabsent;myceliumwhite;sporulationmoderatetodense;sporulationdullgreentoblue-green;exudateabsent,reversecream.SporulationonDG18moderatedense;sporulationblue-green;reversepale.ColoniesonMEAvelvety,slightlyfloccoseincentre;sporulationgreentogrey-green, reverseyellow-brown.Noviolet reactionwithEhrlichreagent.Sclerotiaabsent.Conidiophores borne from surface; stipes (100–)200–300(–500) × 2–3.5µm, smoothwalled, ter- toquarterverticillate,bearing terminalverticilsof2–4metulae.Branchesdivaricate,10–20(–40) × 2–3.5µm.Metulae unequal in length, (8–)10–15 × 2–3µm.Phialides ampulliformtocylindrical, inverticilsof2–6,7.5–9 × 2–2.5µm.Conidiaglobose,smooth,2–3µm. Diagnosis — Penicillium halotolerans can be distinguished from P. desertorum by the production of yellow soluble pigments onCYAwhenincubatedat30°C,slightlysmallerconidiaandtheproductionoftheextrolitesandrastinA,roquefortineC&D,meleagrinandRaistrickphenols. Colony morphology — Colonydiam,7d,inmm:CYA27–35;CYA15°C19–23;CYA30°C20–25;CYA37°C:germination(0–2);MEA31–39;YES41–51;DG1826–32;CYAS32–38;creatineagar16–22,weakgrowth,noacidproduction. Extrolites — AndrastinA, roquefortineC&D,meleagrin,Raistrickphenolsand theuncharacterisedcompoundssuchas‘CUCU’and‘PLIL’. Distribution&Ecology — Thisspeciesisknownonlyfromitstype,isolatedfromasaltmarchinEgypt.AnITSsequence


depositedinGenBank(HQ607840)andobtainedfromastrain(ATT111)isolatedfromanestoftheantAtta texanainTexas,USA,wasidenticaltothatgeneratedfromCBS131537T. Barcode&MolecularbasedID — Thisspeciescanbereli-ablyidentifiedusingITSbarcoding(GenBankJX997005).

Penicillium tardochrysogenum Frisvad, Houbraken & Sam-son, sp. nov. —MycoBankMB801877;Fig.9

Typus. AntArCtiCA,McMurdoDryValley,S. Onofri(CBSH-21057holotype,culturesex-typeCBS132200=IBT30075=DTO149-B9.

Fig. 7 Penicillium goetzii,CBS581.67.a.7doldculturesat25°Cunlessstatedotherwise,lefttoright,firstrow,allobverse,CYA,YES,DG18,MEA;secondrow,CYAreverse,YESreverse,CYAincubatedat30°Cobverse,CREAobverse;b.ascomata;c–f.conidiophores;g.conidia;h.ascospores.—Scalebars=10µm.

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Etymology. Named after its resemblance to P. chrysogenum and its rela-tiveslowgrowthrate.

SporulationonCYAdense;coloniesentire,velvetytoslightlyfloccose,distinctly radiallysulcate;myceliumwhite,sporula-tiongreygreen;exudatedropletsclearorpalebrown,large;solublepigmentsabsent;colonyreversebrown.Solublepig-

ments onYESabsent;myceliumwhite; sporulation absent;exudateabsent,reverseyellow-brown.SporulationonDG18dense;conidiagreygreenen masse;reversepale.ColoniesonMEA floccosewith awide, non-sporulating edge (4–8mm); exudate droplets large in centre, smaller towards therimofcolony,hyaline;sporulationbluishgreygreen,reverse

Fig. 8 Penicillium halotolerans,CBS131537T.a.7doldculturesat25°Cunlessstatedotherwise,lefttoright,firstrow,allobverse,CYA,YES,DG18,MEA;secondrow,CYAreverse,YESreverse,CYAincubatedat30°Cobverse,CREAobverse;b–f.conidiophores;g.conidia.—Scalebars=10µm.

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brownandinyellow-browninvalvesofsulcations.NovioletreactionwithEhrlichreagent.Sclerotiaabsent.Conidiophores mainly borne from aerial mycelium, sometimes direct from agarsurface,ter-toquarterverticillate;stipes150–400 × 2–3µm,smoothwalled.Branchesdivaricate,10–20(–25) × 2–3µm.Metulaeequalinlength,occasionallyunequal,inverticils

of2–4,10–13(–18) × 2.5–3.5µm.Phialides ampulliform, in verticilsof3–8,closelypacked,short,7–9 × 2–3µm.Conidia globose,finelyroughened,2.7–3.5µm. Diagnosis — Penicillium tardochrysogenum differs from other members of series Chrysogena by more restricted and floccose colonies onMEA, lack of sporulation onYESand

Fig. 9 Penicillium tardochrysogenum,CBS132200T.a.7doldculturesat25°Cunlessstatedotherwise,lefttoright,firstrow,allobverse,CYA,YES,DG18,MEA;secondrow,CYAreverse,YESreverse,CYAincubatedat30°Cobverse,CREAobverse;b–f.conidiophores;g.conidia.—Scalebars=10µm.

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finelyroughenedconidia.ItdoesnotproduceyellowsolublepigmentsonCYAincubatedat30°C.Thespeciesproducesthe asperentins, a series of compounds not produced by other members of series Chrysogena.

Colony morphology — Colony diam, 7 d, in mm: CYA 29–37;CYA15°C 16–20;CYA30°C 20–25;CYA37°C:germination(0–2);MEA18–24;YES35–45;DG1834–40;CYAS36–44;creatineagar8–12,weakgrowth,noorpooracidproduction.

Fig. 10 Penicillium vanluykii,CBS132070.a.7doldculturesat25°Cunlessstatedotherwise,lefttoright,firstrow,allobverse,CYA,YES,DG18,MEA;secondrow,CYAreverse,YESreverse,CYAincubatedat30°Cobverse,CREAobverse;b–f.conidiophores;g.conidia.—Scalebars=10µm.

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Extrolites — Penicillins, secalonic acids D & F, asperentins andtheuncharacterisedextrolitemetØ. Distribution&Ecology — Thisspeciesisonlyknownfromitstype,whichwasisolatedfromtheMcMurdoDryValley,Ant-arctica. Barcode&MolecularbasedID — ThisspeciessharesITSsequenceswiththetypeofP. chrysogenumCBS306.48T(Gen-BankJX997093).Partialβ-tubulin,calmodulin,RPB1orRPB2canbeusedforspeciesidentification.

Penicillium vanluykii Frisvad, Houbraken & Samson, sp. nov. —MycoBankMB801878;Fig.10

Typus. USA,NewMexico,Carlsbad,exLechuguillaCave,D. Northup (CBSH-21059holotype,culturesex-typeCBS131539=IBT14505=DTO148-I2).

Etymology. NamedafterAbrahamvan Luyk, aCBSmycologistwhoworked on the antibiotic activity of Penicilliuminthe1940s.

Sporulation onCYAdense; colonies entire, velvety, sulcateradially;myceliumwhite, sporulation dark green to green;exudatedropletshyaline,lightbrownorabsent,small;solublepigmentsabsent,in149A4yellowsolublepigmentsproduced;colonyreversepalebrownoryellow-brown.SolublepigmentsonYESabsent;myceliumwhite;sporulationdense;conidiadarkgreen or green en masse;exudateabsent,reversegreenishbrownincentrewithpalebrownedge.SporulationonDG18moderatedense;conidiagreenordullgreenen masse;reverseunaffectedorpalebrown.ColoniesonMEAvelvety;exudatedropletsabsent;sporulationgreentodarkgreen,reverseunaf-fected,sometimeswithdarkbrowncentre.NovioletreactionwithEhrlich reagent.Sclerotiaabsent.Conidiophores borne fromsurface; quarterverticillate.Stipes 100–300× 2.5–3.5µm,smoothwalled.Branchesdivaricate,15–25 × 2.5–3.5µm.Metulaeequalinlength,3–8,8–12 × 3–3.5(–4)µm.Phialides ampulliform,inverticilsof4–10,closelypacked,short,6.5–8.5 × 2–3µm.Conidia globose to subglobose, smooth, with distinct connectives,2.5–3.5µm. Diagnosis — Penicillium vanluykii is phenotypically similar to P. alliisativi and P. chrysogenum.ThisspeciesischaracterisedbytheproductionofdarkgreenconidiaonMEAandCYA,yellowsolublepigmentproductiononCYAincubatedat30°Candaseriesofincompletelycharacterisedextrolites. Colony morphology — Colonydiam,7d,inmm:CYA30–45;CYA15°C18–25;CYA30°C18–27;CYA37°C: germina-tion–4;MEA30–40;YES50–65;DG1835–47;CYAS40–55;creatineagar 15–20,weak tomoderategrowth,weakacidproduction. Extrolites — Penicillins, chrysogine, roquefortineC andmeleagrin, and andrastin A and the uncharacterised extrolites ‘CRYPT’(4compounds),‘POO’,‘KNOLF’,‘TBRE’,‘FJOR’(2compounds). Distribution&Ecology — Thisspecieshasaworld-widedis-tributionandisfoundintheUSA(Florida,NewMexico,Ohio),SouthShetlandIslands,Antarctica,Argentina(SanJuan),theUK(Henketal.2011,thisstudy). Barcode&MolecularbasedID— DTO148-I2,DTO149-A4andDTO149-B3sharethesameITSsequence,whichcanbeusedforprecisespeciesidentification(GenBankJX997025).

LIST oF SpECIES CuRREnTLy ACCEpTEd In pEnICILLIuM SECTIon CHRySogEnA

Thefollowinglistincludesacceptedspeciesinsect.Chrysogena andtheirpresentlyacceptedsynonyms.Ourdataindicatethatmorespeciesmightexistinthissection.Forexample,threephy-logenetic species are present in P. mononematosum(accordingthePSC)andalsoP. egyptiacummightrepresentthreetaxa.

Penicillium allii-sativi Frisvad, Houbraken & Samson, this study.

Typus. ArgentinA,Mendoza,Lavalle,Col3deMayo,garlic,M. Makuch & J. Valdez(CBSH-21058).

Penicillium chrysogenumThom,Bull.Bur.Anim.Ind.USDA118:58,1910;nom.cons.

Typus.USA,Connecticut,Storrs,excheese,1904,C. Thom(IMI24314typ.cons.).

= Penicillium citreoroseumDierckx,Ann.Soc.Sci.Bruxelles25:86.1901;nom.rej. = Penicillium griseoroseumDierckx,Ann.Soc.Sci.Bruxelles 25: 86.1901;nom.rej. = Penicillium brunneorubrumDierckx,Ann.Soc.Sci.Bruxelles25:88.1901;nom.rej. = Penicillium notatumWestling,Ark.Bot.11,1:95.1911. =Penicillium cyaneofulvumBiourge,Cellule33:171.1923. =Penicillium roseocitreumBiourge,Cellule33:184.1923. =Penicillium chlorophaeumBiourge,Cellule33:249.1923. ?=Penicillium chrysogenumvar.brevisterigmaForster,Brit.Pat.691:242.1953;(nom.inval.Art.36.1;withoutLatindiagnosis). =Penicillium aromaticumf.microsporumRomankova,Uchen.Zap.Lenin.Univ.Zhdanov191:102.1955;(nom.inval.Art.36.1;withoutLatindiagnosis). =Penicillium harmonenseBaghdadi,NovostiSist.Nizsh.Rast.5:102.1968.

Notes — Penicillium brunneorubrum, P. citreoroseum and P. griseoroseum predate P. chrysogenum but these names are formally nomina rejicienda(McNeilletal.2006).No(ex-type)material was available of P. chrysogenumvar.brevisterigma and this invalidly described species is tentatively placed in synonymy with P. chrysogenum.

Penicillium confertum(Frisvadetal.)Frisvad,Mycologia81:852.1989.

Typus: USA,Arizona,6kmeastofPortal,cheekpouchofDipodomys spectabilis(IMI296930).

=Penicillium glandicolavar.confertumFrisvad,Filt.&Wicklow,Canad.J.Bot.65:769.1987.

Penicillium desertorum Frisvad, Houbraken & Samson, this study.

Typus: USA,Wyoming,20kmeastofLittleAmerica, cooldesert soilunder Oryzopsis hymenoides, J.C. Frisvad(CBSH-21056).

Penicillium dipodomyis (Frisvad, Filt. &Wicklow)Banke,Frisvad&S.Rosend., Int.Mod.Meth.Pen.Asp.Clas.:270.2000.

Typus.USA,Arizona,6kmeastofPortal,cheekpouchofDipodomys spectabilis(IMI296926).

=Penicillium chrysogenum var.dipodomyis Frisvad, Filt. &Wicklow,Canad.J.Bot.65:766.1987. =Penicillium dipodomyis (Frisvad, Filt. &Wicklow)Banke,Frisvad& S.Rosend.,Mycol.Res.101:622.1997(nom.inval.Art.33.3,basionymnotcited).

Penicillium egyptiacumJ.F.H.Beyma,Zentralbl.Bakteriol.,2.Abt.,88:137.1933.

Typus.egypt, Cairo, soil, Y.S. Sabet(CBS344.32).

=Eupenicillium egyptiacum(J.F.H.Beyma)Stolk&D.B.Scott,Persoonia4:401.1967. =Eupenicillium molleMalloch&Cain,Canad.J.Bot.50:62.1972. =Penicillium nilensePitt,ThegenusPenicillium:145.1980,‘1979’. = Penicillium mollePitt,ThegenusPenicillium:148.1980,‘1979’.


Penicillium flavigenumFrisvad&Samson,Mycol.Res.101:620.1997.

Typus. denmArk, wheat flour, J.C. Frisvad,1985(CBS419.89).

Penicillium goetziiJ.Rogers,Frisvad,Houbraken&Samson,thisstudy.

Typus. CAnAdA, Calgary, soil, J. Bissett(CBSH-21061).

Penicillium halotolerans Frisvad, Houbraken & Samson, thisstudy.

Typus. egypt, salt marsh, A.H. Moubasher (CBSH-21060).

Penicillium kewenseG.Sm.,Trans.Brit.Mycol.Soc.44:42. 1961.

Typus. Contaminant of a culture stored under mineral oil, G. Smith (LSHTMBB400).

=Eupenicillium crustaceumF.Ludw.,Lehrb.Nied.Krypt.:263.1892.

Notes — Penicillium crustaceum was described by Fries (1829:407).CrustsofconidiaareformedbyseveralspeciesinPenicilliumandFries’descriptionofthisspeciesisnotinforma-tiveenoughfor tocharacterise it inmodernterms.Althoughitsexactidentitycannotbeestablished,Raper&Thom(1949:515)indicatedthatthisspeciescouldbethesameasP. expansum.Brefeld(1874)describedtheformationofsclerotioidcleistotheciaindetail,inaspeciesheidentifiedas“Penicillium crustaceum Fries, Penicillum glaucumLink”.ItisunlikelythatBrefeld’sfungusrepresentedthespeciesdescribedbyLinkandFries.TheillustrationsoftheconidialstatestronglysuggestthatBrefelddealtwithmixedcultures(Stolk&Scott1967).Winter(1887)includedBrefeld’sfungusinhisworkonascomycetes(asP. crustaceum) and later, Ludwig (1892) introduced thegeneric name Eupenicillium based on the name used by Winter and named this species Eupenicillium crustaceum.Penicillium kewense most closely resembles the species described by Brefeld (Scott&Stolk 1967) and therefore, applying singlename nomenclature, we use this epithet for strains formerly identifiedas E. crustaceum.

Penicillium lanosocoeruleum Thom,thePenicillia:322.1930. Typus.USA,culturecontaminantofP. cyclopium culture, C. Thom(NRRL888).

= Penicillium aethiopicumFrisvad,Mycologia81:848.1990.

Notes — Penicillium aethiopicumCBS484.84T and P. lanosocoeruleumCBS215.30Tareconspecific.Thisissupportedbymoleculardata,phenotypiccharacteristicsandextrolitedata.Both species formellipsoidal conidia (Raper&Thom1949:436)andproducetheextrolitesgriseofulvin,tryptoquialaninsandviridicatumtoxin (Frisvadetal.2004,Chooietal.2010,Gaoetal.2011).StrainIBT5753isfullygenomesequenced(Chooietal.2010).

Penicillium mononematosum(Frisvadetal.)Frisvad,Myco-logia81:857.1990.

Typus.USA,Arizona,6kmeastofPortal,burrowsystemofDipodomys spectabilis(IMI296925).

=Penicillium glandicola var.mononematosa Frisvad,Filt.&Wicklow,Canad.J.Bot.65:767.1987. =Penicillium granulatumvar.mononematosa(Frisvad,Filt.&Wicklow)Bridge,Kozak.&R.R.M.Paterson,Mycol.Pap.165:38.1992.

Notes—Ourphylogeneticanalyses(Fig.1,2)revealthreedistinct clades within P. mononematosum.Theoccurrenceoftwotypes(IandII)wasdescribedbyFrisvad&Samson(2004:

126).Both‘typeII’ isolates(CBS112575,CBS10916)wereisolatedfromsaltmarshsoilinEgyptandclustertogetherinourphylogeneticanalysis.

Penicillium nalgiovenseLaxa,Zentralbl.Bakteriol.,2.Abt.,86:160.1932.

Typus. CzeCh repUbliC,Ellischauercheese(CBS352.48neotype).

Penicillium persicinumL.Wang,H.B.Zhou,Frisvad&Sam-son,AntonievanLeeuwenhoek86:177.2004.

Typus. ChinA,Qinghai:soil(HMAS80638-1-4).

Penicillium rubensBiourge,Cellule33:265.1923. Typus.Unrecordedsource,P. Biourge,CBSH-20595(NRRL792=IBT30129=ATCC9783=CBS129667).

?=Penicillium baculatumWestling,SvenskBot.Tidskr.14:139.1910. ?=Penicillium meleagrinumBiourge,Cellule33:184.1923. = Penicillium camerunenseR.Heim,Nouvel&Saccas,Bull.Acad.Belg.C1.Sci.,Ser.5,35:52.1949(nom.inval.Art.36,withoutLatindiagnosis). =Penicillium chrysogenummut. fulvescensTakash.,Arima&S.Abe,J.Gen.Appl.Microbiol.2,1-2:92.1956(nom.inval.Art.36,withoutLatindiagnosis). =Penicillium chrysogenummut.fulvescensTakash.,Arima&S.AbeexC.Ramírez,Man.AtlasPenicil.:364.1982.

Notes—Raper&Thom(1949:363)statedthatNRRL843(=CBS132210=DTO100-F6= IBT5303)was similar to P. baculatum, but no ex-type of P. baculatum has been saved in culture collections. Therefore,wedecidedtoplacethisspe-cies in synonymy with P. rubens.Similarly,notypematerialofP. meleagrinumisavailable.Raper&Thom(1949)basedtheirdescription of P. meleagrinumonNRRL836(=CBS349.48=DTO098-G1=IBT4350)andNRRL2136(=CBS131532=DTO100-H3=IBT30138=NRRL2136).Theformerstrainisre-identifiedhereasP. rubens and the latter as P. chrysogenum.TheexactpositionofP. meleagrinumisuncertain.

Penicillium sinaicumUdagawa&S.Ueda,Mycotaxon14:266. 1982.

Typus. egypt,SinaiPeninsula,SuezCanal,30kmnorthfromPortSaid,marine sludge, H. Komatsu(NHL2894).

=Eupenicillium sinaicumUdagawa&S.Ueda,Mycotaxon14:266.1982.

Penicillium tardochrysogenum Frisvad, Houbraken & Sam-son,thisstudy.

Typus. AntArCtiCA,DryValley,S. Onofri(CBSH-21057).

Penicillium vanluykii Frisvad, Houbraken & Samson, this study.

Typus. USA,NewMexico,Carlsbad,LechuguillaCave,D. Northup (CBSH-21059).

dISCuSSIon

With this revision, Penicillium sect.Chrysogena now consists of 18 phylogenetic and phenotypic species, most of which are alsodiagnosablemorphologically.Comparedwiththeclassi-ficationofHoubraken&Samson(2011),sixnewspeciesare added to this section and P. molle is synonymised with P. egyptiacum and P. aethiopicum with P. lanosocoeruleum.Recenttaxonomic studies on P. chrysogenum determined the presence offourlineageswithinthisspecies(Scottetal.2004,Henketal.2011,Houbrakenetal.2011a).OurresultsconfirmthoseofHoubrakenetal.(2011a),demonstratingthatonelineageis


centred on the ex-type strain of P. chrysogenum CBS306.48 (=‘clade1’inScottetal.(2004))andanotheronP. rubens CBS 129667 (=‘Flemingspecies’fideHenketal.(2011);‘clade4’inScottetal.(2004)).TheothertwolineagesfoundbyScottetal.(2004)andHenketal.(2011)donotcorrespondwitheachother.OurdatasupportthoseofHenketal.(2011)andshowthatthetwootherlineagesrecognisedbyScottetal.(2004;‘clade2’and‘clade3’)stillrepresentP. chrysogenum.Acom-parisonofsequencesdepositedinGenBankshowthatthetwogroupsofisolateslistedas‘speciesA’and‘speciesB’byHenketal.(2011)correspondwiththenewlydescribedspeciesP. vanluykii and P. alliisativi.AlargenumberofspeciesresemblingP. chrysogenumhavebeendescribedhistorically(Samsonetal.1977,Pitt1980)andallareplacedhereinsynonymywithP. chrysogenum or P. rubens.Houbrakenetal.(2011a)focusedon penicillin producing strains and also included the ex-type strains of P. griseoroseum, P. notatum and P. rubens.Varioussynonyms of P. chrysogenum were included in the study of Henk etal.(2011)andtheirspeciesdesignationslargelycorrespondwiththecurrentstudy.TheonlyexceptionistheplacementofP. camerunenseCBS339.52T in synonymy with P. rubens, whereasHenketal.(2011)treatedthisspeciesasP. chrysogenum.Ourmultigenephylogeny(Fig.1)andthehaplotypenetworkanalysis (Fig.4)demonstrate thatP. aromaticum f.microsporum(nom.inval.),P. brunneorubrum, P. chlorophaeum, P. citreorosum, P. cyaneofulvum, P. griseoroseum, P. harmonense, P. notatum and P. roseocitreum, are all synonyms of P. chrysogenum.Additionally,P. camerunense (nom. inval.)and P. chrysogenum mut.fulvescensshouldbeplacedinsyn-onymy with P. rubens.AnoverviewofacceptedspeciesandtheirsynonymsisgivenintheTaxonomypartofthispaper.Pitt(1974,1980)treatedE. egyptiacum, P. kewense(asE. crus taceum)andE. molle as distinct species based on the orna-mentation and size of the ascospores. In contrast,Stolk&Samson(1983)definedP. kewense(asE. crustaceum)asonevariablespecies.AlthoughStolk&Samson(1983)includedfiveascospore patterns in their circumscription of P. kewense, they treated E. molle and E. egyptiacum as small-spored strains of P. kewense.Theyalsoobservedthesameornamentation,butthe ribs and ridges on ascospores of E. egyptiacum were less pronounced.Our results show thatP. kewense sensu Stolk &Samson(1983)canbedividedintoatleastthreespecies: P. egyptiacum, P. goetzii and P. kewense, while P. molle is placed in synonymy with P. egyptiacum.Ourstudyalsoshowsthat the isolates with large ascospores represent a separate species, here named P. goetzii.Phylogeneticanalysesindicatethat this group of related species probably contains additional newspecies.Forexample,threelineagesoccurinP. egyptiacum,whichmightrepresentdistinctspecies.Also,CBS653.82(=CBS227.81=NRRL2094)formsasinglestrainlineageandRaper&Thom(1949:146)notedthatthisstrainisintermediatebetween P. brefeldianum and P. egyptiacum.Thedescriptionofthis species is deferred until more strains of this tentative new speciesarecollected.Polyphasic characterisation of Penicillium species allows iden- tificationusingseveraldifferenttypesofdata,includingcolonycharacters andmicromorphology (morphological speciesconcept),extroliteprofiles(phenotypicspeciesconcept)andcorrelationsamongmultigenephylogenies(phylogeneticspe-cies concept).Thenewspecies describedheremeet all ofthese criteria as distinct species, although their morphological characters are similar to other species of Penicillium sect.Chrysogena,whichare notoriously difficult to identify usingclassicaltaxonomictechniques.Incommonwithotherspecieslevel studies of Penicillium subgenus Penicillium,sequencesoftheITSregionhaveminimalresolutionfordistinguishingcloselyrelated species in sect.Chrysogena (Skouboe et al. 1999,

Houbrakenetal.2011b).TheindividualgenetreesbasedonRPB1andβ-tubulinsequencedatageneratedthebestcluster-ing of species, and these genes are therefore promising loci for barcodingwithinthisgenus.Neithergenetreecorrelateswellwiththeseriesproposedwithinsect.Chrysogena by Frisvad &Samson(2004),asalreadynotedbySamsonetal.(2004).Bothsexualandasexualspeciesareaccommodated in thecurrentlydefinedsect.Chrysogena.Thesexuallycompetentmembers(P. kewense, P. goetzii, P. egyptiacum, P. sinaicum)are all homothallic and there are indications that P. chrysogenum, P. dipodomyis and P. rubens may reproduce in a hete-rothallicmanner(Hoffetal.2008,Henketal.2011,Henk&Fischer2011).Repeatedattemptstoinduceasexualstatein P. chrysogenumandrelatedspecieswereunsuccessful(Hoffetal.2008,Eagle2009,Henk&Fischer2011,Henketal.2011,J.Houbrakenunpubl.res.,K.A.Seifertunpubl.res.).However,some unpublished crossing experiments with P. chrysogenum isolates have apparently resulted in the production of cleisto-thecia and ascospores, similar to those described recently for P. kewense (Böhmetal.2012).Thelimitednumberofsuccess-ful mating experiments in P. chrysogenum might be explained bythestrainsusedintheseexperiments.Perhapsstrainsmain-tainedforlongperiodsinculturecollectionslosetheirfertility.For example, the heterothallic Histoplasma capsulatum lost fertility rapidly during laboratory passage, leading to speculation that selective pressures might serve to maintain fertility in the environment(Kwon-Chungetal.1974,Fraseretal.2007).Forthe heterothallic and heat resistant Byssochlamys spectabilis (syn. Paecilomyces variotii), itwas shown that only strainsderivedfrompasteurisedproductswerefertile(Houbrakenetal.2008). Itwill thereforebepromisingtorepeat thematingexperiments with Penicilliumstrainsfreshlyisolatedfromnature.Another possible reason for unsuccessful crossing experiments maybe stringent conditions required for successfulmating.Variousgrowth factors inducetheformationofcleistothecia,suchas temperature, light,nutrientandoxygen levels (Hanetal.2003).Recently,Houbrakenetal. (2010)showed that P. psychrosexualis, a species related to P. roqueforti, produces abundant cleistothecia at low temperatures (9–15°C).Theproduction of a sexual stage at low temperatures might be more widespread in Penicillium, and mating experiments at this temperature might result in the discovery of a sexual stage inotherspecies.

Acknowledgements WearegratefultoUweBraunforhissuggestionsonthespeciesnames.JelleBosisthankedforgeneratingsequencesofvariousP. chrysogenumisolatesandSharonGroenenisacknowledgedforeditingthehaplotypenetworkfigure.

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