Crous PW, Tanaka K, Summerell BA and Groenewald JZ, Additions

ARTIC

LE

v o l u m e 2 · n o . 1 49

© 2011 International Mycological Association

You are free to share - to copy, distribute and transmit the work, under the following conditions:Attribution: Youmustattributetheworkinthemannerspecifiedbytheauthororlicensor(butnotinanywaythatsuggeststhattheyendorseyouoryouruseofthework). Non-commercial: Youmaynotusethisworkforcommercialpurposes.No derivative works: Youmaynotalter,transform,orbuilduponthiswork.For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights.

Additions to the Mycosphaerella complex

PedroW.Crous1, Kazuaki Tanaka2,BrettA.Summerell3andJohannesZ.Groenewald1

1CBS-KNAWFungalBiodiversityCentre,Uppsalalaan8,3584CTUtrecht,TheNetherlands;correspondingauthoremail:[email protected]&LifeSciences,HirosakiUniversity,Bunkyo-cho3,Hirosaki,Aomori036-8561,Japan3RoyalBotanicGardensandDomainTrust,MrsMacquariesRoad,Sydney,NSW2000,Australia

Abstract: Speciesinthepresentstudywerecomparedbasedontheirmorphology,growthcharacteristicsinculture,andDNAsequencesof thenuclear ribosomalRNAgeneoperon (including ITS1, ITS2, 5.8SnrDNAand the first900bpofthe28SnrDNA)forallspeciesandpartialactinandtranslationelongationfactor1-alphagenesequencesfor Cladosporium species.NewspeciesofMycosphaerella (Mycosphaerellaceae) introduced in this study includeM. cerastiicola (onCerastium semidecandrum,TheNetherlands),andM. etlingerae (onEtlingera elatior,Hawaii).Mycosphaerella holualoana is newly reported on Hedychium coronarium(Hawaii).EpitypesarealsodesignatedforHendersonia persooniae, the basionym of Camarosporula persooniae, and for Sphaerella agapanthi, the basionym of Teratosphaeria agapanthi comb. nov. (Teratosphaeriaceae) on Agapathus umbellatus from SouthAfrica. Thelatter pathogen is also newly recorded from A. umbellatusinEurope(Portugal).Furthermore,twosexualspeciesofCladosporium(Davidiellaceae)aredescribed,namelyC. grevilleae(onGrevilleasp.,Australia),andC. silenes(onSilene maritima,UK).Finally,thephylogeneticpositionoftwogeneraarenewlyconfirmed,namelyCamarosporula (basedonC. persooniae, teleomorph Anthracostroma persooniae),which isa leafpathogenofPersoonia spp. inAustralia, belongs to the Teratosphaeriaceae, and Sphaerulina(basedonS. myriadea),whichoccursonleavesofFagaceae(Carpinus, Castanopsis, Fagus, Quercus),andbelongstotheMycosphaerellaceae.

Article info:Submitted:5April2011;Accepted:30April2011;Published:12May2011.

Key words: AnthracostromaCamarosporulaCladosporiumMycosphaerellaphylogenySphaerulinataxonomyTeratosphaeria

doi:10.5598/imafungus.2011.02.01.08 IMA FuNgus · voluMe 2 · No 1: 49–64

INtroductIoN

The genus Mycosphaerella has in the past been recognised as one of the largest genera of ascomycetes, containing approx. 3000 species (Aptroot 2006), as well as severalthousandasexualspecies(Crouset al. 2000,2001,2004b,c,2006b,2007,Crous&Braun2003).AlthoughMycosphaerella hasbeenlinkedtomanydifferentanamorphgenera(Crous&Braun2003,Crouset al.2007),recentphylogeneticstudieshave shown that Mycosphaerella is actually polyphyletic (Crouset al.2007,2009a),withmembersbelongingtomanydifferentgenera,andevenfamilies,suchastheDavidiellaceae (Braunet al.2003,Schochet al.2006),Teratosphaeriaceae (Crous et al. 2007),Dissoconiaceae (Crous et al. 2009b),Mycosphaerellaceae and Schizothyriaceae (Aptroot 2006,Batzer et al.2008,Crous2009).

MembersoftheMycosphaerella-complexareecologicallyhighlyadaptable,andvaryfrombeingsaprobictofungicolous(Crous2009).Mycosphaerella species are also among the most common and destructive plant pathogens known,causing serious diseases on many economically important crops (Farret al.1995,Crous&Braun2003).Speciesaremainly foliicolous, although some are associated with stem cankers (Cortinaset al.2006), fruit lesions(Pretoriuset al.

2003)orblemishes,spotsandspecks(Batzeret al.2008).One of the largest barriers to understanding host

specificity and speciation in this group of organisms, hasbeentherelativeunavailabilityofauthenticcultures,asmostofthespeciesknowntodate,havebeendescribedwithoutthedepositofassociatedex-typeculturesorDNA.Theresultis that the ecological behaviour of many species remainsobscure(Crous&Groenewald2005),whilethephylogeneticposition of many other pathogens and genera remains uncertain. During the course of the present study severalpotentially novel Mycosphaerella and Mycosphaerella-like species were collected, while potential epitype specimens were also collected for older, well-established names. Theaim of this study was thus to describe these species, and also elucidate the phylogenetic relationship of genera such as Camarosporula (teleomorph Anthracostroma), andSphaerulina within the Capnodiales.

MAterIAls ANd MetHods

IsolatesLeaf and stem tissue bearing ascomata were soaked inwater for approximately 2 h, after which theywere placed

crous et al.ARTICLE

50 i m a f u n G u S

in the bottom of Petri dish lids, with the top half of the dish containing 2%malt extract agar (MEA; Oxoid,Hampshire, UK) (Crous et al. 2009c). Ascosporegermination patternswere examined after 24 h, andsingle ascospore and conidial cultures established as describedearlier(Crouset al.1991,Crous1998).Toisolate asexual fungi, host tissueswere incubated inmoist chambers for up to 2 wk, and inspected daily for microfungi, and single conidial colonies established on MEA (Crous 2002). Colonies were subcultured onto2 % potato-dextrose agar (PDA), synthetic nutrient-poor agar (SNA), MEA, and oatmeal agar (OA)(Crouset al.2009c),andincubatedundercontinuousnear-ultraviolet lightat25 °C topromotesporulation.Nomenclatural novelties and descriptions weredeposited in MycoBank (www.MycoBank.org; Crouset al. 2004a).All cultures obtained in this study aremaintainedintheopencollectionoftheCentraalbureauvoorSchimmelcultures(CBS-KNAW)andtheworkingcollection(CPC)ofP.W.CrousinCBS(Table1).

dNA phylogenyGenomic DNAwas extracted frommycelia of fungalcolonies cultivated on MEA using the UltraCleanTM Microbial DNA Isolation Kit (Mo Bio Laboratories,SolanaBeach,CA,USA).ThePrimersV9G(deHoog&GerritsvandenEnde1998)andLR5(Vilgalys&Hester1990)wereused toamplifypartof thenuclear rDNAoperon spanning the 3’ end of the 18S nrRNAgene(SSU),thefirstinternaltranscribedspacer(ITS1),the5.8SnrRNAgene,thesecondITSregion(ITS2)andthe5’endofthe28SnrRNAgene(LSU).TheprimersITS4(Whiteet al.1990)andLSU1Fd(Crouset al.2009b)were used as internal sequence primers to ensuregoodquality sequencesover theentire lengthof theamplicon.TohelpresolvespeciesofCladosporium, the ITSregionwassupplementedwithpartialsequencesof the translation elongation factor 1-a gene (EF-1a)using theprimersEF1-728F(Carbone&Kohn1999)andEF-2(O’Donnellet al.1998),andtheactingene(ACT) using the primers ACT-512F and ACT-783R(Carbone&Kohn1999).ThePCRamplificationswereperformedonaGeneAmpPCRSystem9700(AppliedBiosystems,FosterCity,CA,USA)inatotalvolumeof12.5µLsolutioncontaining10–20ngoftemplateDNA,1×PCRbuffer,2mMMgCl2,2.5pmolforeachprimer,60µMofeachdNTP(20µMforEF-1α)and0.5UTaq DNApolymerase(BiolineLuckenwalde,Germany).ForEF-1α,0.7µLdimethylsulfoxide(DMSO)wasaddedtotheamplificationreaction.PCRamplificationconditionswere set as follows: an initial denaturation temperature of94ºCfor5min,followedby40cyclesofdenaturationtemperatureof94ºCfor45s,primerannealingat48ºC(52ºCforEF-1α)for30s,primerextensionat72ºCfor90sandafinalextensionstepat72ºCfor6min.Theresulting amplicons were sequenced using the PCRprimers and a BigDye Terminator Cycle Sequencingta

ble

1. C

ollectiondetailsandGenBankaccessionnumbersofisolatesforw

hichnovelsequencesweregeneratedinthisstudy.

spec

ies

stra

in n

o.1

subs

trat

ec

ount

ryc

olle

ctor

(s)

gen

Bank

Acc

essi

on n

umbe

r (It

s, l

su, t

eF, A

ct)2

Cam

aros

poru

la p

erso

onia

e CBS112302=CPC3343

Leafspotson

Per

soon

ia sp.

Aus

tralia

P.W.C

rous&B.A.S

ummerellJF770447,JF770459,—

,—

CBS112494=CPC3350

Leafspotson

Per

soon

ia sp.

Aus

tralia

P.W.C

rous&B.A.S


,—

CBS116258=CPC3344

Leafspotson

Per

soon

ia sp.

Aus

tralia

P.W.C

rous&B.A.S


,—

Cla

dosp

oriu

m g

revi

lleae

CBS114271=CPC2913

LeavesofG

revi

lleasp.

Aus

tralia

P.W.C

rous&B.A.S

ummerellJF770450,JF770462,JF770472,JF770473

Cla

dosp

oriu

m s

ilene

sCBS109082

StemsofexposedS

ilene

mar

itim

aUK

A.Aptroot

EF679354,JF770463,EF679429,EF679506

Myc

osph

aere

lla c

eras

tiico

laCBS115913=CPC11290

DeadleavesandstemsofC

eras

tium

se

mid

ecan

drum

Netherlands

A.Aptroot

JF770451,JF770464,—

,—

Myc

osph

aere

lla e

tling

erae

CBS129062=CPC12274

DeadleavesofE

tling

era

elat

ior

Haw

aii

W.G

ams

JF770452,JF770465,—

,—

CPC12277

DeadleavesofE

tling

era

elat

ior

Haw

aii

W.G

ams

JF770453,JF770466,—

,—

Myc

osph

aere

lla h

olua

loan

a CBS129063=CPC12286

DeadleavesofH

edyc

hium

cor

onar

ium

Haw

aii

W.G

ams

JF770454,JF770467,—

,—

Sph

aeru

lina

myr

iade

a CBS124646=JC

M15565

LeavesofQ

uerc

us d

enta

taJa

pan

K.Tanaka

JF770455,JF770468,—

,—

Tera

tosp

haer

ia a

gapa

nthi

CBS129064=CPC18332

Leafspotson

Aga

pant

hus

umbe

llatu

sP

ortu

gal

P.W.C

rous

JF770456,JF770469,—

,—

CBS129192=CPC18304

Leafspotson

Aga

pant

hus

umbe

llatu

sSouthAfrica

P.W.C

rous

JF770457,JF770470,—

,—

CPC18266

Leafspotson

Aga

pant

hus

umbe

llatu

sSouthAfrica

P.W.C

rous

JF770458,JF770471,—

,—1 CBS:C

BS-KNAW

FungalBiodiversityCentre,U

trecht,TheNetherlands;CPC:C

ulturecollectionofPedroCrous,housedatCBS.

2 ITS:Internaltranscribedspacers1and2togetherwith5.8SnrDNA;LSU:partial28S

nrDNA;TEF:partialtranslationelongationfactor1-alphagene;ACT:partialactingene.

Additions to the Mycosphaerella complex ARTIC

LE

51v o l u m e 2 · n o . 1

Kitv.3.1(AppliedBiosystems)andanalysedonanABIPrism3730xlDNASequencer(Perkin-Elmer,Norwalk,CN,USA).

The generated sequences were compared with otherfungal DNA sequences from NCBI’s GenBank sequencedatabase using a megablast search; sequences with highsimilaritywereaddedtothealignment(LSU)ordiscussedinthespeciesnoteswhereapplicable (ITS,EF-1αandACT).TheadditionalGenBanksequencesweremanuallyalignedusingSequenceAlignmentEditorv.2.0a11(Rambaut2002).The phylogenetic analyses of the aligned sequence datawere performed using PAUP (PhylogeneticAnalysis UsingParsimony) v. 4.0b10 (Swofford 2003) and consisted ofneighbour-joining analyses with the uncorrected (‘p’), theKimura2-parameterandtheHKY85substitutionmodelsandparsimonyanalyses.Alignmentgapsweretreatedasmissingdataandallcharacterswereunorderedandofequalweight.Any ties were broken randomly when encountered. Forparsimonyanalyses,alignmentgapsweretreatedasafifth(“new”) character state and all characters were unorderedand of equal weight. Maximum parsimony analysis wasperformedusingtheheuristicsearchoptionwith100randomsimple taxaadditionsand treebisectionandreconstruction(TBR)as thebranch-swappingalgorithm.Branchesofzerolengthwerecollapsedandallmultiple,equallyparsimonioustreesweresaved.Therobustnessofthetreesobtainedwasevaluatedby1000bootstrapreplications(Hillis&Bull1993).Treelength(TL),consistencyindex(CI),retentionindex(RI)and rescaled consistency index (RC) were calculated andtheresultingtreeswereprintedwithTreeViewv.1.6.6(Page1996).Newsequenceswere lodged inGenBank (Table 1)and the alignments and phylogenetic trees in TreeBASE(www.treebase.org).

MorphologyCulturecharacteristicswererecordedfromcoloniesgrownonMEA,OAandPDAplatesafter2–4wkincubationindarknessat25ºC.Colonycolours(surfaceandreverse)wereassessed

using the colour charts ofRayner(1970).Preparationsweremountedinlacticacidandstudiedunderalightmicroscope(×1000magnification).The95%confidenceintervalsofsporeswerederivedfrom30observations(unlessstatedotherwise),withextremesgiveninparentheses.

results

dNA phylogenyApproximately 1700 bases, spanning the ITS and LSUregions,wereobtained for isolates listed inTable1.Thesetwo regions were analysed separately; ITS to determinespecies level relationships (presented in the speciesnotesbelow,whereapplicable)andLSUforthegenericplacement.Approximately300and220basesweredeterminedforEF-1αandACT, respectively,and their respectiveblast resultsare discussed under the Cladosporiumspeciesnotesbelow.

ThemanuallyadjustedLSUalignmentcontained49taxa(includingtheoutgroupsequence)and,ofthe825characters

used in the phylogenetic analysis, 147 were parsimony-informative,103werevariableandparsimony-uninformative,and575wereconstant.Neighbour-joininganalysisusingthethreesubstitutionmodelsonthesequencedatayieldedtreeswithsimilartopologyandbootstrapsupportvalues.Twenty-fiveequallymostparsimonioustreeswereobtainedfromtheheuristicsearch, thefirstofwhich isshown inFig.1 (TL=551,CI=0.657,RI=0.872,RC=0.573).ThephylogenetictreeoftheLSUregion(Fig.1)showedthreedistinctgroupsof fungal isolates; the first was the Davidiellaceae clade (70%bootstrapsupport),thesecondtheTeratosphaeriaceae clade (98 % bootstrap support), and the third was theMycosphaerellaceaeclade(93%bootstrapsupport).

tAxoNoMy

Duringthepresentstudyseveralnoveltaxaweredelineated,or newly collected cultures linked to established names.These species are treated per family below:

Davidiellaceae

cladosporium grevilleae Crous & Summerell, sp. nov.MycoBankMB560082(Figs2,3)

Etymology:Namedafterthehostonwhichitwascollected,Grevillea.

Ascifasciculati,bitunicati,subsessiles,obovoideivellateellipsoidei,octospori, 35–45 × 9–12 µm. Ascosporae tri- ad pluriseriatas,hyalinae, guttulatae cum inclusionibus angularibus, crassitunicatae, rectaevel lenitercurvatae,fusoides-ellipsoideae,utrinqueobtusae,mediane1-septatae,(9–)11–12(–13)×3.5–4(–4.5)µm.

Typus: Australia: New South Wales:MountAnnanBotanicalGarden,on leavesofGrevillea sp.,Aug.1999,P.W. Crous & B.A. Summerell JT 974(DAR74881–holotypus;culturesex-typeCPC2913–2916=CBS114271).

In vivo: Leaf spotsabsent.Ascomata occurring in leaf litter, amphigenous,black,subepidermal,erumpenttosuperficial,globose, to100µmdiam,withcentral,periphysateostiole,10–15 µm diam; wall of 3–4 layers of brown texturaangularis. Asci aparaphysate, fasciculate, bitunicate with fissitunicate discharge, subsessile, obovoid to broadlyellipsoid, slightly curved, 8-spored, 35–45 × 9–12µm,withvisible apical apiculus. Ascospores tri- to multi-seriate, hyaline, guttulate with angular inclusions, thick-walled, straighttoslightlycurved,fusoid-ellipsoidwithobtuseends,medianly 1-septate, widest in middle of apical cell, slightly constricted at the septum, tapering towards both ends, but slightlymoretolowerend,(9–)11–12(–13)×3.5–4(–4.5)µm;ascospores surrounded with a thin sheath when mounted in water,becomingbrownandverruculosewithage;ascospore

crous et al.ARTICLE


10 changes

2x

70

90

98

63

93

9850

58

93

57

55

54

90

90

77

77

95

89

69

100

100

100

100

100

Saccharomyces cerevisiae Z73326Cladosporium sp. EU167586

Cladosporium bruhnei GU214408Cladosporium grevilleae CPC 2913Cladosporium silenes CBS 109082

“Pseudocercospora” sp. GU214681Teratosphaeria cryptica GU214505

CPC 18332CPC 18304 Teratosphaeria agapanthiCPC 18266

Dothideomycetes sp. GU323983Teratosphaeria encephalarti FJ372414Teratosphaeriaceae sp. GU323219

Phaeothecoidea melaleuca HQ599595CBS 116258CBS 112494 Camarosporula persooniaeCBS 112302

Mycosphaerella colombiensis DQ204745CPC 12277 CPC 12274

Mycosphaerella irregulariramosa GQ852609Mycosphaerella holualoana GU214440CPC 12286 Mycosphaerella holualoanaMycosphaerella crystallina EU167579Mycosphaerella waimeana AY260083Mycosphaerella acaciigena GU214432Mycosphaerella heimioides GU214439Mycosphaerella heimii GU214438Pseudocercospora eucommiae GU253742

Septoria obesa GU214493CBS 124646 Sphaerulina myriadeaMycosphaerella latebrosa GU214444Septoria aceris GQ852673Mycosphaerella berberidis EU167603Mycosphaerella harthensis EU167602Mycosphaerella flageoletiana EU167597

Ramularia endophylla AY490776Ramularia proteae EU707899Ramularia aplospora EU040238Ramularia acroptili GU214689Ramularia sp. GU214693CBS 115913 Mycosphaerella cerastiicolaCercosporella centaureicola EU019257Mycosphaerella fragariae GU214691Ramularia miae DQ885902Ramularia pratensis var. pratensis EU019284Ramularia sp. GU214690Ramularia coleosporii GU214692Ramularia uredinicola GU214694

Mycosphaerella etlingerae

79

63

Mycosphae-rellaceae

Teratosphae-riaceae

Davidiellaceae

Fig. 1. Thefirstof25equallymostparsimonioustreesobtainedfromaheuristicsearchwith100randomtaxonadditionsoftheLSUsequencealignmentusingPAUPv.4.0b10.Thescalebarshows10changes,andbootstrapsupportvaluesfrom1000replicatesareshownatthenodes.Thickenedlinesindicatethestrictconsensusbranchesandnovelsequencesareprintedinboldface.Thedifferentfamiliesareindicatedwithcolouredblocks.ThetreewasrootedtoSaccharomyces cerevisiae(GenBankno.Z73326).


LE

53v o l u m e 2 · n o . 1

germinationwithgermtubesparallel tothe longaxisof thespore,butdistortingprominently(originalsporecellsupto8µmwide), germinatingwith numerousgerm tubes, formingdense clusters of hyphae, but mostly remaining hyaline after 24honMEA.

Culture characteristics: Colonies after 2 wk at 24 °Cspreading, reaching 15–20 mm diam. On MEA erumpent,witheven,lobedmargins;surfacefolded,withsparseaerialmycelium, olivaceous-grey; reverse iron-grey. On OA flat,with sparse aerial mycelium, and lobed, somewhat feathery margins;surfacepaleolivaceous-greyinmiddle,iron-greyinouterregion.OnPDAerumpent,withlobed,featherymargins;surfacefolded,withsparseaerialmycelium,grey-olivaceous;reverseiron-grey.

Notes: The genus Cladosporium1816islinkedtoteleomorphsthat are placed in Davidiella2003(Braunet al.2003,Schubertet al.2007).Inmovingtoasinglenomenclatureforpleomorphicfungi,wegivepreferencetotheoldestgenusname,namelyCladosporium. Cladosporium grevilleae only forms the sexual state of the life-cycle.The ascospores are typical ofa Cladosporium teleomorph,having thickwalls,andangularinclusions (Aptroot 2006), becoming brown and verruculosewithage.Presentlywearenotawareofanysexualorasexualspecies on Grevillea that represent this fungus, and thus wedescribe ithereasnew.Althoughall four loci supportedthe association of the species with Cladosporium, it did not match any of the Cladosporiumsequencescurrentlyavailableon theGenBanknucleotidedatabase(closestmatchon ITSwas Davidiella macrospora GenBank EU167591 with 95%

Fig. 2. Cladosporium grevilleae(DAR74881).A, B.Asci.c.Ostiolarregion(arrowed).d.Ascuswithascospores.Bars=10µm.

Fig. 3. Cladosporium grevilleae(DAR74881).Asciwithascospores.Ascosporesshowingsheath,andangularinclusions.Bar=10µm.

crous et al.ARTICLE


identity,onEF-1αwasCladosporium myrtacearumGenBankHM148360with97%identityandonACTitwasCladosporium iranicumGenBankHM148599with89%identity).

cladosporium silenes Crous, sp. nov.MycoBankMB560083(Fig.4)

Etymology:Namedafterthehostonwhichitwascollected,Silene maritima.

Cladosporii cladosporioidis similis, sed conidiophoris brevioribus,non ramosis, cellulis conidiogenois longioribus, ramo-conidiis et conidiisintercalaribusbrevioribusdiscernitur.

Typus: uK: Pembrokeshire:SkomerIsland,stemsofexposedSilene maritima, 22 Aug. 2000, A. Aptroot 49319) (CBSH-19874–holotypus;cultureex-typeCBS109082).

In vivo: Ascomataoccurringinexposedtwigs,amphigenous,black to dark brown, subepidermal, to 70µmdiam, visibleby an erumpent, central, periphysate ostiole, 5–10 µm;wall of 2–3 layers of red-brown textura angularis. Asci aparaphysate, fasciculate, bitunicate with fissitunicate

discharge, subsessile,obovoid tobroadlyellipsoid, straighttoslightlycurved,8-spored,25–35×10–12µm,withvisibleapicalapiculus.Ascospores tri- to multiseriate, hyaline, non-guttulate with angular inclusions, thick-walled, straight to slightly curved, fusoid-ellipsoidwith obtuse ends,medianly1-septate, widest in middle of apical cell, not to slightly constricted at the septum, tapering towards both ends, but slightly more to lower end, (10–)11–13(–14) × (3–)3.5(–4)µm; turning brown once discharged, and some containingremnants of a mucoid layer; germinating from both ends,distorting,becomingbrownandfinelyverruculose.

Mycelium consisting of branched, septate, pale to medium brown 2(–4) µm wide hyphae, without any swellings andconstrictions,smoothtominutelyverruculose,wallsunthickened.Conidiophores solitary, macronematous or micronematous, arising terminally from ascending hyphae or laterally, straight to somewhat flexuous, narrowly cylindrical to cylindrical-oblong,non-nodulose,notgeniculate-sinuous,15−100(−200)× 3−4(−4.5)µm,unbranched,pluriseptate,usuallynotconstrictedatsepta,paletomediumolivaceous-brown,smoothtominutelyverruculose, especially towards the base, walls unthickened,base sometimes swollen, up to 8 µm wide; micronematousconidiophores shorter, unbranched, 10−30 × 3−4 µm.Conidiogenous cells integrated, usually terminal, sometimes

Fig. 4. Cladosporium silenes(CBSH-19874).A.Ascomataonhosttissue(arrows).B.Asci.c.Ascospores(arrowdenotesmucoidappendage;A–CfromBenschet al.2010).d–F.Conidiophoreswithconidialchains.Bars=10µm.


LE

55v o l u m e 2 · n o . 1

intercalary, cylindrical-oblong, not geniculate, non-nodulose, (10−)20−60µmlong,withuptothreelocicrowdedattheapex,subdenticulate to denticulate, protuberant, 2(−2.5) µm diam,centraldomemostlyflat,somewhat thickenedanddarkened-refractive.Ramoconidia straight to slightly curved, cylindrical-oblong,15−20(–25)×3−4(–4.5)µm,aseptate,paleolivaceous-brown, concolorous with tips of conidiophores, smooth, base not cladosporioid,2−2.5µmwide,thickened,somewhatrefractive.Secondary ramoconidia aseptate, smooth, pale olivaceousbrown,cylindrical-oblong,(8–)10–15(–20)×3.5–4µm.Conidia numerous, catenate, in branched chains of up to 6 in theupperunbranchedpart,branching inalldirections. Intercalary conidia limoniform, ellipsoid-ovoid, 7−8(−10) × (2−)2.5−3µm, aseptate,with up to 3 distal hila.Small terminal conidia aseptate, subglobose, obovoid, ovoid to limoniform, 4−5(−6)× (2.5−)3(−3.5) µm; hila darkened and somewhat thickened,0.5–1µmdiam.

Culture characteristics:Coloniesafter2wkat24 °Cspreadingwith moderate aerial mycelium and smooth, lobate margins, reaching 40 mm diam after 2 wk. On MEA surface paleolivaceous-greytoolivaceous-grey,reverseiron-grey.NoOAsurfacegrey-olivaceoustoolivaceous-grey.OnPDAsurfacegrey-olivaceous,reverseolivaceous-grey.

Notes:StrainCBS109082representsanascosporeisolate,obtained from material of Silene maritima and initially identifiedasMycosphaerella tassianavar.arthopyrenioides.MorphologicallytheCBSstrainissimilartoC. cladosporioides, but is a member of a distinct clade (C. cladosporioides s.lat. Lineage 3; see fig. 1, partA in Bensch et al. 2010).Cladosporium sileniae isthefirstsexualspeciesknownfromthe cladosporioidescomplex.Giventhattheseascomataarerather inconspicuous(immersed,approx.100µmdiam), itsnotsurprisingthattheyhavebeenlargelyoverlookedinthepast.Cladosporium sileniae differs from C. cladosporioides in having shorter, unbranched conidiophores, longerconidiogenous cells, shorter ramo- and intercalary conidia (Benschet al.2010).

Mycosphaerellaceae

Mycosphaerella cerastiicola Crous,sp. nov.MycoBankMB560084(Fig.5)Anamorph: Septoria-like

Etymology:Namedafterthehostonwhichitwascollected,Cerastium semidecandrum.

Fig. 5. Mycosphaerella cerastiicola (CBS 115913).A, B. Leaveswith black ascomata and conidiomata.c, d.Asci.e, F.Ascospores.g. Conidiophoresgivingrisetoconidia.H.Conidia.Bars=10µm.

crous et al.ARTICLE


Ascosporae imbricatae, hyalinae, guttulatae, tenuitunicatae, rectae vel leniter curvatae, fusoides-ellipsoideae, constrictae ad septamediana,utrinqueattenuatae,(11–)15–20(–23)×(3–)3.5(–4)µm.

Typus: Netherlands: Flevoland: Noordoostpolder, nearUrk, km block 20-26-24, coordinations 168,9, 523,5, ondykeofformersea,ondeadleavesandstemsofCerastium semidecandrum, 2May 2004,A. Aptroot (CBSH-20549 –holotypus;culturesex-typeCPC11290=CBS115913;CPC11291,11292).

In vivo: Leaf spots absent. Ascomata amphigenous on leavesandstems,black,subepidermal,becomingerumpent,solitary, globose, up to 150 µm diam; central ostiole 5–10µm diam; wall consisting of 2–3 layers of medium browntextura angularis.Asci aparaphysate, fasciculate, bitunicate withfissitunicatedischarge,subsessile,narrowlyellipsoidtosubcylindrical,straight toslightlycurved, (2–)8-spored,35–45×8–10µm;apicalchamber1–1.5µmdiam.Ascospores bi- to tri-seriate,overlapping,hyaline,guttulate, thin-walled,straight to somewhat curved, fusoid-ellipsoidalwithacutelyroundedends,widestatseptumwhenimmature,justabovemedian septum when mature, constricted at the septum, tapering towardsbothends, (11–)15–20(–23)× (3–)3.5(–4)µm;ascosporesgerminatefrombothends,withgermtubesparalleltothelongaxisofthespore.

Septoria-like state developing on SNA and OA.Conidiomata pycnidial on host, pycnidial to sporodochial on agar, black under the dissecting scope, globose, immersed in agar to superficial; colonies also sporulating profuselyon aerial mycelium via solitary loci, 1.5 µm diam, 1 µmtall, thickened along the rim. Stromata dark brown under the compound microscope, to 200 µm diam, 100 µm tall,consistingofdarkbrown, thickened,ovoidtoglobosecells,5–10 µm diam; upper layers fertile, giving rise to a densenetwork of intermingled brown, hyphal-like elements, that in turn give rise to an upper layer of conidiophores.Conidiophorescylindrical,0–1-septate,palebrowntohyalineatbase,hyalineatapex,smooth,rarelybranchedatbulbousbase,20–30×2µm.Conidiogenous cells terminal, hyaline, 10–15 × 1.5–2 µm, proliferating sympodially with flattenedloci,1.5–2µmdiam,unthickenedandnotdarkened;onhostmaterial conidiogenous cells ampulliform, 10–15 µm long,4–5µmwide at bulbous base; sympodial or phialidic,withpericlinalthickeningvisible.Conidia solitary, hyaline, smooth, guttulate, cylindrical with obtuse apex and truncate base,at times somewhat long obconically subtruncate on host material,1–3-septate,straighttoflexuous,(15–)50–60(–65)×(1.5–)2µm(av.55×2µm)in vitro.

Culture characteristics: Colonies after 2 wk at 24 °Cspreading, erumpent, with folded surface, splitting agar, with sparse aerial mycelium and smooth, lobate margins, reaching15–20mmdiam.OnMEAsurfacesalmontofleshtodirtywhite,reversechestnut(centre),toapricot(margin).OnOAsurfaceolivaceousbuff,fertile.OnPDAsurfacedirtywhitewithpatchesof olivaceousgrey; reverse iron-grey in

middle,salmoninouterregion.ColoniesonSNAdevelopingadiffuseredpigmentinagar.

Notes: Mycosphaerella cerastiicola is presently the only Mycosphaerella species known from Cerastium (Caryophyllaceae) (Aptroot 2006). Both ITS and LSU data(Fig. 1) support the placement of this genus within theRamularia clade. A blast search using the ITS sequenceobtains as closest hits (maximum identity of 97–98%) anundescribedspeciesof“Pseudocercosporella”(strainsKACC42395, KACC 42363, CPC 11297; GenBank EF600945,EF600956, GU214693), which appears to belong to thisgenus.ThegenusRamularia has thus far been assumed to be monophyletic, and thus the description of M. cerastiicola with its Septoria-like to Pseudocercosporella-like anamorph isanenigma.Presently there isnobootstrapsupport for itto represent another genus than Mycosphaerella/Ramularia, raising the question if the anamorph could represent apossible Ramulariasynanamorph?Furthercollectionswouldberequiredtoaddressthisissue.

Mycosphaerella etlingerae Crous,sp. nov. MycoBankMB560085(Fig.6)

Etymology:Namedafterthehostonwhichitwascollected,Etlingera elatior.

Ascosporae imbricatae, hyalinae, granulatae, tenuitunicatae, rectae vel leniter curvatae, fusoides-ellipsoideae, mediane 1-septatae,leniterconstrictaeadsepta,(7–)9–10(–12)×3(–3.5)µm.

Typus: usA: Hawaii:ondeadleavesofEtlingera elatior,14Aug.2005,W. Gams(CBSH-20550–holotypus;culturesex-typeCBS129062=CPC12274,12275–12279).

In vivo: Leaf spots absent. Ascomata amphigenous, black, subepidermal, becoming erumpent, globose, up to 90 µmdiam; central ostiole 5–10 µm diam; wall consisting of 2–3layers of medium brown textura angularis.Asci aparaphysate, fasciculate,bitunicatewithfissitunicatedehiscence,subsessile,narrowlyellipsoidtosubcylindrical,straighttoslightlycurved,8-spored, 35–45 × 6–7 µm. Ascospores bi- to tri-seriate, overlapping,hyaline,granular,thin-walled,straighttocurved,fusoid-ellipsoidal with obtuse ends, widest in middle of apical cell, medianly 1-septate, slightly constricted at the septum, tapering towards both ends, but more prominently towards the lowerend,(7–)9–10(–12)×3(–3.5)µm;ascosporesgerminatefrom both ends, with germ tubes growing parallel to the long axis, developing lateral branches; ascospores remaininghyaline,becomingconstricted,to4µmwide.

Culture characteristics: Coloniesafter2wkat24 °Cspreading,erumpent, surface irregular, folded, margin smooth, lobate, withsparseaerialmycelium,reaching15mmdiam.OnMEAsurface pale mouse grey with patches of olivaceous greyandscarlet,withwhitemargin;reverseiron-grey;redcrystals


LE

57v o l u m e 2 · n o . 1

produced inagar.OnOAsurfaceolivaceousgrey.OnPDAsurfacesmoke-greywithpatchesofpaleolivaceous-greyandscarlet.

Notes: No Mycosphaerella species have previously beenreported from Etlingera elatior (Zingiberaceae), though sixspecieshavebeenreportedonZingiberaceae.TheseincludeM. alpiniae and M. alpiniicola on Alpinia inChina,M. amomi on AmomuminChina,M. hedychii on Hedychium in Brazil, M. zingiberi on ZingiberfromChinaandKorea,andM. zingiberis on Zingiber from Japan (www.nt.ars-grin.gov/fungaldatabases).Other than for M. hedychii(ascospores8–11×2µm;Soares&Barreto2008),theotherspeciesaresomewhatobscure,andAptroot (2006) could not trace anymaterial for comparison.MorethanonespeciesofMycosphaerellawasobservedontheleavesofEtlingera elatiorcollectedinHawaii,butascosporesof M. etlingerae could be distinguished from other taxa bybeing fusoid-ellipsoidal,andwidestinmiddleoftheapicalcell.Mycosphaerella etlingerae is 100% identical to sequencesof M. thailandica and M. colombiensisforbothITSandLSU.Mycosphaerella colombiensis, described from Eucalyptus leaf spotsinColombia,hasascosporesthatareobovoid, (11–)12–14(–15)×3–3.5(-4)µm(Crous1998),thusquitedistinctfromM. etlingerae. Mycosphaerella thailandica has ascospores that are fusoid-ellipsoidal, (9–)10–11(–12) × (2–) 2.5–3 µm,thus being very similar to those ofM. etlingerae. However,M. thailandica causes a disease of Acacia in Thailand, and has a Pseudocercospora state in vivo and in vitro (Crouset al. 2004c),whichisdifferentfromthatobservedinM. etlingerae.

Mycosphaerella holualoana Crous et al., Mycotaxon 78:458(2001).(Fig.7)

In vivo: Leaf spots absent.Ascomata amphigenous, black, subepidermal, becoming erumpent, aggregated in clusters, globose, to 70 µm diam; central ostiole 5–10 µm diam;wall consisting of 2–3 layers of medium brown textura angularis. Asci aparaphysate, fasciculate, bitunicate with fissitunicate dehiscence, subsessile, narrowly ellipsoid tosubcylindrical,straighttoslightlycurved,8-spored,35–45× 6–7µm.Ascospores bi- to tri-seriate, overlapping, hyaline,prominently guttulate, thin-walled, straight, fusoid-ellipsoidal with obtuse ends, widest just above septum, medianly1-septate, constricted at the septum, tapering towards both ends,butmoreprominentlytowardsthelowerend,(10–)11–12(–13)×(2.5–)3(–3.5)µm;ascosporesgerminatefrombothends,withseveralgermtubesthatareirregularinwidthandgrowthdirection;ascosporesbecomingslightlyconstrictedatseptum,to4µmwide,remaininghyaline,smooth.

Culture characteristics:Coloniesafter2wkat24 °Cspreading,erumpent, surface folded with sparse aerial mycelium and even,lobatemargin,reaching20mmdiam.OnMEAsurfacepaleolivaceousgreywithpatchesofolivaceous-grey;reverseolivaceous-greytoiron-grey.OnOAsurfaceolivaceousgrey,reverse olivaceous-grey with patches of scarlet. On PDAsurfacegrey-olivaceoustoolivaceousgrey,reverseiron-greywithpatchesofscarletincentre.

Fig. 6. Mycosphaerella etlingerae(CPC12274).A.Colonyonmaltextractagar.B, c.Asci.d.Ascospores.e–g.Germinatingascospores.Bars=10µm.

crous et al.ARTICLE


Specimen examined: usA: Hawaii: ondead leavesofHedychium coronarium,14Aug.2005,W. Gams(CBSH-20551;culturesCBS129063=CPC12286,12287,12288).

Notes: Mycosphaerella holualoana, which is part of the M. heimii-complex(Crous1998)basedonitsLSUandITSsequences,was initially described from leaf spots of Leucospermumspp.collected in Hawaii (Taylor et al. 2001). Ascospores weresomewhat larger than in the present collection (12–15 × 2.5–3µm),but thegeneralshape,culturecharacteristicsandgermination patterns suggest that this is the same species, occurringondead leavesofHedychium coronarium.Anotherspecies of Mycosphaerella, M. hedychii, is also known from thishostinHawaiiandBrazil(Stevens1925,Soares&Barreto2008). Itdiffers fromM.holualoana by being associated with brownleafspots,3–10mmdiam,havingshorterasci(25–35× 5–8µm),andsmallerascospores(8–11×2µm).

sphaerulina myriadea(DC.)Sacc.,Michelia 1(4):399(1878).(Fig.8)

Leaf spots epiphyllous, round to irregular shaped, grey to black,1.2–8mmdiam(av.3.8mm).Ascomata pseudothecial, immersed,subepidermal,erumpentatthetop,singleto2–3grouped, globose in longitudinal section, glabrous, without prominentbeak,100–165µmtall(av.128µm),90–150µmdiam (av. 115 µm).Ostiole central, 7.5–15 µm wide, withhyaline innerperiphysesof2–2.5µmwide.Ascomatal wall textura angularis in surface view; in longitudinal section5–15 µm thick at side and base, composed of 2–4 layersofpolygonal tosubglobosebrowncells (6–18×3.5–9µm),25–35 µm thick around apical ostiole. Interascal filaments notseen.Ascibitunicateinstructure,dischargefissitunicate,clustered,arisingfromthecentrumbaseof12–15µmthick,cylindrical toobclavate, roundedatapex,withorwithoutashallowapical chamber (ca.0.5µmhigh),withaknob-likestipe(7–10µmlong)orsessile,with8tri-seriatetobi-seriateascospores,57–82×10–13(–14.5)µm(av.69.0×12.2µm).Ascosporescylindrical, roundedatapex,slightly taperedat

below, straight or slightly curved, 3-septate,withaprimaryseptumnearlymedian(0.48–0.52,av.0.50),hyaline,smooth,withoutsheathorappendages,(28–)30–38(–40.5)×(2.5–)3–3.5(–4)µm(av.34.4×3.2µm),L/W9.7–12.4(av.10.8).

Culture characteristics:Coloniesafter2wkat24 °Cspreading,erumpent, with sparse aerial mycelium and feathery margins, reaching 8 mm diam. On MEA surface saffron, reverseluteous.OnOAsurfacesaffron.OnPDAsurfacedirtywhitetopaleluteous;reversepaleluteous.

Specimens examined: uK: sine loc., on leaves ofQuercus robur, J.E. Vize[MicrofungiBrit.Ex.No.195](exIMI57186,K(M)167735).– Japan: Aomori: Tsugaru, Kidukuri, Bense-marsh (40°51’53’’ N,140°17’42’’E),onleavesofQuercus dentata,21Apr.2007,K. Tanaka 2243 (HHUF29940;singleascosporecultureCBS124646=JCM15565).–germany:Driesen,Lasch [Rabenhorst,FungiE u r . no.149](L).–usA: California:SequoiaNationalPark.alt.2590m,onleavesofCastanopsis sempervirens,18Jun.1931,H.E. Parks(BPI623686);LakeCo.,Hoberg’sResort,onleavesofQuercus kelloggii, 15May1943,V. Miller(BPI623707).Maryland:Marlboro,onleavesof Quercus alba, 26Apr. 1929,C.L. Shear ( BPI 623705).Texas: Houston,onleavesofQuercus alba, 8Apr.1869,H.W. Ravenel(BPI623704).

Notes: The genus Sphaerulina, which is based on S. myriadea with 3-septate ascospores, was distinguished from Mycosphaerellawith1-septateascospores.Thesetwogenera have traditionally been separated on the basis ofascosporeseptationalone.However,severalspecieswithaSphaerulina ascomatal anatomy and 3-septate ascospores are known that do not belong to Sphaerulina s. str., suggesting thatascosporeseptationaloneisinsufficientlyrobusttoinferphylogeneticrelatedness(Crouset al.2003).Thematter isfurther complicated as S. myriadea, which occurs on hosts in the Fagaceae,appearstobeaspeciescomplex.Becauseofthis, no epitype is designated here, pending further collections ofauthenticEuropeanmaterialonQuercusfromFrance.

Inourstudy,S. myriadea clusters in Mycosphaerellaceae, as sister to Septoria s.str. (Fig. 1). Although the name

Fig. 7. Mycosphaerella holualoana(CPC12286).A, B.Asci.c.Ascospores.d, e.Germinatingascospores.Bars=10µm.


LE

59v o l u m e 2 · n o . 1

Fig. 8. Sphaerulina myriadea (HHUF29940).A.LeafspotofQuercus dentata.B.Close-upoferumpentascomata.c, d.Longitudinalsectionofascomata.e.Fasciculateasci.F. Fissitunicateascus.g, H.Asci.I.Apicesofasci.J–N.Ascospores.o.Germinatingascospore.Bars:A=1mm;B,C=200µm;D,E,O=20µm;F–N=10µm.

crous et al.ARTICLE


Fig. 9. Camarosporula persooniae(CBS116258).A.Leafspotwithascostromata.B, c.Acervuli.d, e.Asci.F.Ascospores.g, H.Conidia.Bars=10µm.

Sphaerulina 1878 predates that of Mycosphaerella 1884,Mycosphaerellaceaehaverecentlybeenshowntorepresenta generic complex (Crous et al. 2007, 2009a, b), in whichSphaerulina appears to represent a distinct lineage.Mycosphaerella is restricted to species with Ramularia anamorphs (Verkley et al. 2004), and remains distinct fromSphaerulina.AmegablastsearchusingtheITSsequenceofS. myriadea places it with members of Mycosphaerella, but withahighestquerycoverageof90% forMycosphaerella brassicicola (GenBank EU167607; Identities = 434/487 (89%),Gaps=37/487(8%)).

Teratosphaeriaceae

camarosporula persooniae (Henn.)Petr.,Sydowia 8: 99(1954).(Fig.9)Basionym: Hendersonia persooniaeHenn.,Hedwigia 40:97(1901).Synonym:Dichomera persooniae (Henn.)Henn.,Hedwigia 42:87(1903).

Teleomorph: Anthracostroma persooniae (Henn.) Petr.,Sydowia 8:97(1954).Basionym: Mycosphaerella persooniaeHenn.,Hedwigia 42: 81(1903).Synonyms:Sphaerella persooniae(Henn.)Sacc.&D.Sacc.,

Syll. Fung. 17:639(1905).Pseudosphaerella persooniae (Henn.) Hansf., Proc. Linn. Soc. New South Wales 79:123(1954).

Leaf spots amphigenous, irregular, dark brown to black, specksof0.5mm,coalescingtolargerspotsto1cmdiam.Ascostromata amphigenous, predominently epiphyllous, subcuticular, uni-to multi-locular, erumpent, solitary to aggregated,to300µmdiam,withsingle,central,periphysateostiole, 10–15 µm diam; wall consisting of brown, thick-walled, textura angularis; basal stroma on the epidermisconsisting on a single layer of brown, thick-walled cells.Interascal filaments pseudoparaphysoids, filamentous,branched, anastomosed, hyaline, indistinct, constricted at septa, 3–5 µm. Asci sessile, stipitate, obovoid, 8-spored,30–50 × 12–15 µm, with well developed apical chamber,1–2 µm diam, and multi-layered endotunica. Ascospores fusoid-ellipsoid, hyaline, smooth, thick-walled, prominently guttulate,widestjustabovethemedianseptum,prominentlyconstrictedatseptum,(12–)13–15(–17)×(3–)4–5(–5.5)µm;becomingpalebrowninolderasci.

Conidiomata acervular, amphigenous, subcuticular,epidermal tosubepidermal,separateorconfluent, to1mmdiam, composed of dark brown, thick-walled, globose to angularcells,dehiscingirregularlyattheapex,visibleassmallblackspotsonsuperficialstromata.Conidiophores reduced to conidiogenous cells. Conidiogenous cells determinate,


LE

61v o l u m e 2 · n o . 1

integrated, cylindrical to doliiform, hyaline to pale brown, smooth tofinelyverruculose, lining theconidiomatalcavity,3–10×3–5µm;minutepericlinalthickeningvisibleatapex,whichinsomecasesappearstoalsoproliferatepercurrently.Conidia dictyoseptate, distoseptate, with 3 transverseseptaand1–2verticalorobliquesepta, thick-walled,finelyverruculose, irregular or clavate toobclavate,withabroadtruncatebase,(10–)13–15(–17)×(5–)7–8(–10)µm.

Culture characteristics:Coloniesafter2wkat24 °Cspreading,erumpent with folded surface, and smooth, lobate margins, reaching7mmdiam.OnMEAsurfaceumbertoolivaceousgrey; reverse iron-grey.OnOA surface olivaceous rey.OnPDAsurfaceandreverseolivaceousgrey.

Specimens examined: Australia: Western Australia: Perth, on Persoonia elliptica,1900,Pritzel 104a(B–holotypeofHendersonia persooniae). New South Wales: Boudhi National Park, coastalunderstory,on leavesofPersoonia levis,25Sept.1983,C. Liddell (K(M)167734,exIMI281194);teleomorphandanamorphpresent;Ku-ring-gai Chase National Park, on leaves ofPersoonia sp., 16Nov. 1999,P.W. Crous & B.A. Summerell, epitype (CBSH-20548–epitypus hic designatus of Hendersonia persooniae;culturesex-epitypeCPC3344=CBS116258,CPC3343=CBS112302).

Notes: The teleomorph genus Anthracostroma is based on a species initially described in Mycosphaerella as M. persooniae, which is in accordance to its Mycosphaerella-likemorphology.Within the Teratosphaeriaceae this genus is distinct in that the ascomata are situated in a subcuticular stroma, and the asciare intermingledamongnumerouspseudoparaphyses.The Camarosporula anamorph is reminiscent of Dichomera, which is again allied to Botryosphaeriales(Barberet al.2005,Crous et al. 2006a).Camarosporula is thus distinct within Teratosphaeriaceae. Both the phylogenetic analysis of theLSUsequences(Fig.1)andthemegablastsearchesoftheITSsequencesplacePhaeothecoidea melaleuca as closest sister species to Camarosporula persooniae.

teratosphaeria agapanthi(Kalchbr.&Cooke)Crous,comb. nov.MycoBankMB560086(Fig.10)Basionym: Sphaerella agapanthi Kalchbr.&Cooke,Grevillea 9:31(1880).Synonym: Mycosphaerella agapanthi (Kalchbr. & Cooke)Lindau, inEngler&Prantl,Natürlichen Pflanzenf. 1(1):426(1897).

In vivo: Leaf spotsamphigenous,ellipsoid,large,developingwhere plants are grown in wet, shady areas, pale to medium brown,withared-brownborder,coalescing,becomingvisibleasleaftipblightsymptoms.Ascomata amphigenous, black, substomatal, erumpent, predominantly arranged in tight, roundclusters,2–6mmdiam;ascomata80–150µmdiam,withcentral,periphysateostiole,10–15µmdiam;periphyses0–1-septate,10–15×1.5–2µm;ascomatalwallof3–4layers

of brown textura angularis. Interascal filaments absent.Asci fasciculate, bitunicate with fissitunicate dehiscence,subsessile, fusoid-ellipsoid, straight to slightly curved,8-spored,25–45×15–25µm,withasmallapicalapiculus.Ascospores multi-seriate, hyaline, thick-walled, with angular cellular inclusions, fusoid-ellipsoid, medianly 1-septate, becoming constricted at the septum, widest in middle of apical cell, with rounded ends, granular, (17–)18–20(–21)×4.5–5(–6)µm;ascosporegermination irregular, frombothendsormiddleofthecell,paralleloratangletothelongaxis;sporebecomingbrown, verruculose,6–7µmwide,but notswellinganddistortingafter24honMEA.

Culture characteristics: Colonies after 14 d at 24 °Cspreading,with sparse aerialmyceliumand even, smooth,lobate margins, reaching 30 mm diam. On MEA surfacedark mouse-grey, reverse greenish black. On OA surfaceolivaceous grey.OnPDA surface olivaceous grey, reverseiron-grey.

Specimens examined: south Africa: Western Cape Province: on uppersurfaceofdeadleavesofAgapanthus umbellatus(Alliaceae),Kalchbrenner 1342 (K (M) – holotype); Kirstenbosch BotanicalGarden,atentrancetolowergate,onleafspotsofA. umbellatus,8May2010,P.W. Crous(CBSH-20552–epitypus hic designatus;culturesex-epitypeCPC18304–18305=CBS129192).–Portugal: Braga,RiadoSouto,ondeadleafofA. umbellatus,8June2010,P.W. Crous(CBSH-20553;cultureCPC18332–18333=CBS129064).

Notes: Ascospores in the holotype specimen were similar (17–21 × 5–6 µm) to those observed in the presentcollections. Aptroot (2006) regarded this as a species ofDavidiella, but probably only observed immature asci,given that his observations referred to somewhat smallerascospores (10–12×4–5µm).Furthermore,healso refersto other African collections, which again match the ascospore dimensionsonthetype.Theseobservations,togetherwithitsoccurrence on Agapanthus in Portugal, suggest this species to probably be host-specific. Mycosphaerella agapanthi-umbellati,describedfromthesamehost inIndia,maybealatersynonym,withasci25–50×14µm,andascospores14–25×4–7µm(Corlett1991).BothLSUandITSdataplaceT. agapanthi in Teratosphaeria(Fig.1),withITSsequencesofT. considenianae(GenBankGQ852792),T. miniata(GenBankGQ852803)andT. stellenboschiana (GenBankGQ852825)havingthehighestidentity(94%)inamegablastsearchoftheGenBanknucleotidedatabase.

dIscussIoN

The present study resolves the phylogenetic position ofseveralgeneraofwhichtheclassificationhasbeenthetopicofpastspeculation.ThegenusCamarosporula(teleomorphAnthracostroma)isshowntobelongtotheTeratosphaeriaceae, whichisnottotallysurprisinggiventhefactthatitsteleomorphwas originally described as Mycosphaerella persooniae.

crous et al.ARTICLE


However,basedon itsuniqueascostromatawitherumpentascomata, and acervular conidiomata with muriformlyseptate, brown conidia, Camarosporula appears to represent a distinct genus within this family, which is also supported byitsDNAphylogeny(Fig.1).ThephylogeneticpositionofSphaerulina, and potential taxonomic implications thereofhasbeendiscussedindetailelsewhere(Crouset al.2003),as the genus predates Mycosphaerella.Resultsobtainedinthisstudy,however,haveshownthatwhileMycosphaerella-like taxa with 3-septate ascospores have evolved morethan once in the Mycosphaerellaceae (Crous et al. 2003),Sphaerulina s.str., typified by S. myriadea, represents a lineage embedded in the Pseudocercospora/Septoriaclade.Although Sphaerulina may represent a genus in its own right, thepresentdataarestillinsufficienttoresolveitsphylogenywithin the Mycosphaerellaceae.

This study also introduces two species of Cladosporium that have teleomorphs, C. silenes which is a member of the C. cladosporioides complex (Bensch et al. 2010), andC.grevilleaewhichdoesnot formananamorph in culture.

Furthermore,wemanagedtorecollectaspeciesassociatedwith leaf spots of AgapanthusinSouthAfrica,M. agapanthi, which Aptroot (2006) suspected to represent a possiblespecies of Davidiella.Ascanbeseenfromitsphylogeneticposition(Fig.1),however,it isaspeciesofTeratosphaeria, which appears to be closely associated with its host, has beenintroducedintoEurope(Portugal),andmayalsooccurinothercontinentswherethisplantiscultivated.

Two species of Mycosphaerella known from leaf litter collected in Hawaii have been treated; M. etlingerae on Etlingera elatior, and M. holualoana on Hedychium coronarium.Thelatterspecieshasuntilnowbeenacceptedas a leaf pathogen of LeucospermuminHawaii(Tayloret al. 2001),anditsoccurrenceonleaf litterofanotherhostmaysuggest that more stringent plant hygiene practices need to be followed in ProteafieldsinHawaii,asdeadleavesofHedychiumappeartoactasanalternatehostforthisfungus.

Although this study resolved several long standingquestions related to the phylogeny of genera such asCamarosporula/Anthracostroma and Sphaerulina, it also

Fig. 10. Teratosphaeria agapanthi(CPC18304).A, B.LeafspotsandblightonAgapanthus.c.Circularaggregationofascomata.d.Evenlydistributedascomatawithostiolarregionsvisible.e, g–J.Asci.F.Ostiolewithperiphyses(arrow).K.Ascospores.l.Germinatingascospores.Bars=10µm.


LE

63v o l u m e 2 · n o . 1

raisedsomenewquestions.This isspecifically true for thespecies described here as M. cerastiicola, collected on Cerastium semidecandrum in The Netherlands. While theteleomorphisratheroddinthesensethatitcanhavelessthaneightascospores,theanamorphisverypeculiarinhavingaSeptoria- or Pseudocercosporella-likemorphology(pycnidiato sporodochia, and sympodial to phialidic proliferation, with conidia also formed individually on aerial mycelium). Theoddity of this species lies in the fact that it clusters in the middle of the Ramularia clade, which has hitherto been accepted asmonophyletic(Crouset al.2009a,b).WhetherRamularia would eventually be reavealed as paraphyletic, or if thisSeptoria-like anamorph in fact represents a synanamorph of a Ramularia species, can only be resolved once moreMycosphaerella species with Ramularia-like anamorphs havebeencollectedandsubjectedtoDNAanalysis.

AcKNowledgeMeNts

We thank the technical staff, Arien van Iperen (cultures), MarjanVermaas (photographic plates), and Mieke Starink-Willemse(DNA isolation, amplification and sequencing) for their invaluableassistance. Walter Gams and André Aptroot (both formerlyCBS) are thanked for several of the collections treated here.ThecuratorsofB(BotanischeGartenundBotanischesMuseum,Berlin-Dahlem,Germany),BPI(USDA,Beltsville,USA),andKew(UK)areacknowledgedformakingseveralvaluablespecimensavailableforstudy.

reFereNces

AptrootA(2006)Mycosphaerellaanditsanamorphs:2.Conspectusof Mycosphaerella.CBS Biodiversity Series 5:1–231.

BarberPA,BurgessT,HardyGStJ,SlippersB,KeanePJ,WingfieldMJ(2005)Botryosphaeria species from Eucalyptus in Australia are pleoanamorphic, producing Dichomera synanamorphs in culture.Mycological Research 109:1347–1363.

Batzer JC, Mercedes DiazArias M, Harrington TC, Gleason ML,Groenewald JZ,CrousPW (2008) Four species ofZygophiala (Schizothyriaceae, Capnodiales)areassociatedwith thesootyblotchandflyspeckcomplexonapple.Mycologia 100:246–258.

Bensch K, Groenewald JZ, Dijksterhuis J, Starink-Willemse M,AndersenB,SummerellBA,ShinH-D,DuganFM,SchroersH-J,BraunU,CrousPW(2010)Speciesandecologicaldiversitywithinthe Cladosporium cladosporioides complex (Davidiellaceae, Capnodiales).Studies in Mycology 67:1–94.

BraunU,CrousPW,DuganF,GroenewaldJG,HoogSGde(2003)Phylogeny and taxonomyofCladosporium-like hyphomycetes, including Davidiellagen.nov., theteleomorphofCladosporium s.str.Mycological Progress 2:3–18.

CarboneI,KohnLM(1999)Amethodfordesigningprimersetsforspeciation studies in filamentous ascomycetes.Mycologia 91: 553–556.

Corlett M (1991) An annotated list of the published names inMycosphaerella and Sphaerella, Mycologia Memoir 18:1–328.

CortinasMN,CrousPW,WingfieldBD,WingfieldMJ(2006)Multi-gene phylogenies and phenotypic characters distinguish two species within the Colletogloeopsis zuluensis complexassociated with Eucalyptusstemcankers.Studies in Mycology 55:133–146.

Crous PW (1998) Mycosphaerella spp. and their anamorphsassociated with leaf spot diseases of Eucalyptus. Mycologia Memoirs 21:1–170.

Crous PW (2002) Taxonomy and Pathology of Cylindrocladium (Calonectria) and allied genera.StPaul,MN:APSPress.

Crous PW (2009) Taxonomy and phylogeny of the genusMycosphaerellaanditsanamorphs.Fungal Diversity 38:1–24.

CrousPW,AptrootA,KangJC,BraunU,WingfieldMJ(2000)Thegenus Mycosphaerellaanditsanamorphs.Studies in Mycology 45:107–121.

CrousPW,BraunU (2003)Mycosphaerellaand itsanamorphs.1.NamespublishedinCercospora and Passalora.CBS Biodiversity Series 1:1–571.

Crous PW, Braun U, Groenewald JZ (2007) Mycosphaerella is polyphyletic.Studies in Mycology 58:1–32.

CrousPW,GamsW,Stalpers JA,RobertV,StegehuisG (2004a)MycoBank:anonlineinitiativetolaunchmycologyintothe21stcentury.Studies in Mycology 50:19–22.

CrousPW,Groenewald JZ (2005)Hosts, species and genotypes:opinionsversusdata.Australasian Plant Pathology 34:463–470.

CrousPW,GroenewaldJZ,MansillaJP,HunterGC,WingfieldMJ(2004b)PhylogeneticreassessmentofMycosphaerellaspp.andtheir anamorphs occurring on Eucalyptus. Studies in Mycology 50:195–214.

CrousPW,GroenewaldJZ,PongpanichK,HimamanW,ArzanlouM,WingfieldMJ(2004c)Crypticspeciationandhostspecificityamong Mycosphaerella spp. occurring on Australian Acacia speciesgrownasexoticsinthetropics.Studies in Mycology 50: 457–469.

Crous PW, Groenewald JZ, Wingfield MJ, Aptroot A (2003) ThevalueofascosporeseptationinseparatingMycosphaerella from Sphaerulina in theDothideales: aSaccardoanmyth?Sydowia 55:136–152.

Crous PW, Kang JC, Braun U (2001) A phylogenetic redefinitionof anamorph genera in Mycosphaerella based on ITS rDNAsequenceandmorphology.Mycologia 93:1081–1101.

Crous PW, Schoch CL, Hyde KD, Wood AR, Gueidan C, HoogGS de, Groenewald JZ (2009a) Phylogenetic lineages in theCapnodiales.Studies in Mycology 64:17–47.

Crous PW, Slippers B, Wingfield MJ, Rheeder J, MarasasWFO,Philips AJL, Alves A, Burgess T, Barber P, Groenewald JZ(2006a) Phylogenetic lineages in the Botryosphaeriaceae.Studies in Mycology 55:235–253.

Crous PW, Summerell BA, Carnegie AJ, Wingfield MJ, HunterGC,BurgessTI,AndjicV,BarberPA,Groenewald JZ (2009b)Unravelling Mycosphaerella: do you believe in genera?Persoonia 23:99–118.

CrousPW,VerkleyGJM,GroenewaldJZ,SamsonRA(eds)(2009c)Fungal Biodiversity. [CBS Laboratory Manual Series no. 1.]CentraalbureauvoorSchimmelcultures,Utrecht.

CrousPW,WingfieldMJ,MansillaJP,AlfenasAC,GroenewaldJZ(2006b)PhylogeneticreassessmentofMycosphaerellaspp.and

i m a f u n G u S

ARTICLE

64

crous et al.

their anamorphs occurring on Eucalyptus.II.Studies in Mycology 55:99–131.

CrousPW,WingfieldMJ,ParkRF(1991)Mycosphaerella nubilosa a synonym of M. molleriana.Mycological Research 95:628–632.

FarrDF,BillsGF,ChamurisGP,RossmanAY(1995)Fungi on plants and plant products in the United States.StPaul,MN:APSPress.

Hillis DM, Bull JJ (1993)An empirical test of bootstrapping as amethod for assessing confidence in phylogenetic analysis.Systematic Biology 42:182–192.

HoogGSde,GerritsvandenEndeAHG(1998)Moleculardiagnosticsof clinical strains of filamentousBasidiomycetes.Mycoses 41: 183–189.

O’Donnell K, Kistler HC, Cigelnik E, Ploetz RC (1998) Multipleevolutionaryoriginsof the fungus causingPanamadiseaseofbanana: concordant evidence from nuclear and mitochondrialgene genealogies. Proceedings of the National Academy of Sciences, USA 95:2044–2049.

PageRDM(1996)TREEVIEW:Anapplicationtodisplayphylogenetictrees on personal computers. Computer Applications in the Biosciences 12:357–358.

PretoriusMC,CrousPW,GroenewaldJZ,BraunU(2003)Phylogenyof some cercosporoid fungi from Citrus.Sydowia 55:286–305.

RambautA(2002)Sequence Alignment Editor.Version2.0.Oxford:DepartmentofZoology,UniversityofOxford.

RaynerRW(1970)A Mycological Colour Chart.Kew:CommonwealthMycologicalInstitute.

SchochCL,ShoemakerRA,Seifert,KA,HambletonS,SpataforaJW,CrousPW(2006)AmultigenephylogenyoftheDothideomycetes usingfournuclearloci.Mycologia 98:1041–1052.

SchubertK,GroenewaldJZ,BraunU,DijksterhuisJ,StarinkM,HillCF,ZalarP,HoogGSde,CrousPW(2007)Biodiversity intheCladosporium herbarumcomplex(Davidiellaceae, Capnodiales),with standardisation of methods for Cladosporiumtaxonomyanddiagnostics.Studies in Mycology 58:105–156.

Soares DJ, Barreto RW (2008) Fungal pathogens of the invasiveriparian weed Hedychium coronarium from Brazil and their potentialforbiologicalcontrol.Fungal Diversity 28:85–96.

Stevens FL (1925) Hawaiian fungi. Bernice P. Bishop Museum Bulletin 19:1–189.

SwoffordDL(2003)PAUP*: phylogenetic analysis using parsimony (*and their methods). Version 4. Sunderland, MA: SinauerAssociates.

Taylor JE, Crous PW, Palm ME (2001) Foliar and stem fungalpathogens of ProteaceaeinHawaii.Mycotaxon 78:449–490.

Verkley GJM, Crous PW, Groenewald JZ, Braun U, Aptroot A(2004) Mycosphaerella punctiformis revisited: morphology,phylogeny,andepitypificationof the typespeciesof thegenusMycosphaerella (Dothideales, Ascomycota). Mycological Research 108:1271–1282.

Vilgalys R, Hester M (1990) Rapid genetic identification andmappingofenzymaticallyamplifiedribosomalDNAfromseveralCryptococcusspecies.Journal of Bacteriology 172:4238–4246.

WhiteTJ,BrunsT, Lee J,Taylor J (1990)Amplification anddirectsequencing of fungal ribosomalRNAgenes for phylogenetics.In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: a guide to methods and applications:315–322.SanDiego,CA:AcademicPress.

Documents

Crous PW, Tanaka K, Summerell BA and Groenewald JZ, Additions