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Unravelling species boundaries in the Aspergillus viridinutans complex (sectionFumigati) opportunistic human and animal pathogens capable of interspecifichybridization
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Citation (APA):Hubka, V., Barris, V., Dudová, Z., Sklená, F., Kubátová, A., Matsuzawa, T., Yaguchi, T., Horie, Y., Nováková, A.,Frisvad, J. C., Talbot, J. J., & Kolarik, M. (2018). Unravelling species boundaries in the Aspergillus viridinutanscomplex (section Fumigati) opportunistic human and animal pathogens capable of interspecific hybridization.Persoonia, 41, 142-174. https://doi.org/10.3767/persoonia.2018.41.08
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Aspergillus is a speciose genus with almost 400 species classi-fiedintosixsubgeneraandapproximately25sections(Samsonetal.2014,Jurjevićetal.2015,Hubkaetal.2016a,2017,Chenetal.2016a,b,2017,Kocsubéetal.2016,Sklenářetal.2017,Tanneyetal.2017).Thespeciesarewidelydistributedinnatureandhaveasignificanteconomicimpactinhumanandanimalhealth(causativeagentsofaspergillosis;allergiesandrespira-tory problems associated with presence of fungi in the indoor environment),thefoodindustry(sourceofenzymesandorganicacids for fermentation, food and feed spoilage, production of
hazardousmycotoxins), biotechnology and pharmacology(productionofbioactivesubstances,heterologousproteins)(Pitt&Hocking2009,Meyeretal.2011,Frisvad&Larsen2015b,Suguietal.2015,Gautieretal.2016).Aspergillussect.Fumigatiincludesapproximately60speciesoccurringpredominantlyinsoil(Hubkaetal.2017).Manyareof considerable medical importance as they cause human and animalinfections(Balajeeetal.2005b,2009,Katzetal.2005,Yaguchietal.2007,Hubkaetal.2012,Talbot&Barrs2018).As-pergillus fumigatus is usually reported as both the most common member of the section in soil worldwide and the most common causeofaspergillosis(Klich2002,Domschetal.2007,Mayr&Lass-Flörl2011).Aseriesofrecentstudieshighlightedthehighprevalence(11–19%)ofso-calledcrypticAspergillus species in clinicalsamples(Balajeeetal.2009,Alastruey-Izquierdoetal.2013,Negrietal.2014,Sabinoetal.2014).Theiridentificationis clinically relevant since many demonstrate drug resistance to commonly used antifungals, thus their recognition influences therapeuticmanagement.Reliableidentificationofclinicaliso-lates to the species level and susceptibility testing by reference methodsisthuswarranted(Lyskovaetal.2018).Manyoftheselesscommonpathogensbelongtosect.Fumigati and the high-est numbers of infections are attributed to A. lentulus, A. thermo- mutatus (syn.Neosartorya pseudofischeri )andspeciesfromA. viridinutansspeciescomplex(AVSC)(Balajeeetal.2005a,2006,Suguietal.2010,2014,Barrsetal.2013,Talbot&Barrs2018).
Unravelling species boundaries in the Aspergillus viridinutans complex (section Fumigati): opportunistic human and animal pathogens capable of interspecific hybridization V.Hubka1,2,3*,V.Barrs4#,Z.Dudová1,3#,F.Sklenář1,2#,A.Kubátová1,T.Matsuzawa5, T.Yaguchi6,Y.Horie6,A.Nováková2,J.C.Frisvad7,J.J.Talbot4,M.Kolařík2
Abstract Although Aspergillus fumigatus is the major agent of invasive aspergillosis, an increasing number of infections are caused by its cryptic species, especially A. lentulus and the A. viridinutansspeciescomplex(AVSC).Theiridentificationisclinicallyrelevantbecauseofantifungaldrugresistanceandrefractoryinfections.SpeciesboundariesintheAVSCareunresolvedsincemostspecieshaveuniformmorphologyandproduceinterspecifichybrids in vitro.Clinicalandenvironmentalstrainsfromsixcontinents(n=110)werecharacterizedbyDNAse-quencingoffourtosixloci.Biologicalcompatibilitiesweretestedwithinandbetweenmajorphylogeneticclades,andascosporemorphologywascharacterised.Speciesdelimitationmethodsbasedonthemultispeciescoalescentmodel(MSC)supportedrecognitionoftenspeciesincludingonenewspecies.Fourspeciesareconfirmedopportunisticpathogens;A. udagawae followed by A. felis and A. pseudoviridinutans are known from opportunistic human infec-tions, while A. felis followed by A. udagawae and A. wyomingensis areagentsoffelinesino-orbitalaspergillosis.Recentlydescribedhuman-pathogenicspeciesA. parafelis and A. pseudofelisaresynonymizedwithA. felis and an epitype is designated for A. udagawae.Intraspecificmatingassayshowedthatonlyafewoftheheterothallicspeciescanreadilygeneratesexualmorphsin vitro.Interspecificmatingassaysrevealedthatfivedifferentspeciescombinations were biologically compatible.Hybridascosporeshadatypicalsurfaceornamentationandsignificantlydifferentdimensionscomparedtoparentalspecies.ThissuggeststhatspecieslimitsintheAVSCaremaintainedbybothpre-andpost-zygoticbarriersandthesespeciesdisplayagreatpotentialforrapidadaptationandmodulationofvirulence.Thisstudyhighlightsthatasufficientnumberofstrainsrepresentinggeneticdiversitywithinaspeciesisessentialformeaningfulspeciesboundariesdelimitationincrypticspeciescomplexes.MSC-baseddelimitationmethodsarerobustandsuitabletoolsforevaluationofboundariesbetweenthesespecies.
Homothallism is a predominant reproductivemode in sect.Fumigati andmanyspeciesreadilyproduceascomata(neosar-torya-morph)inculture,whileothersareheterothallicorhaveanunknownsexualmorph(Hubkaetal.2017).Homothallicspeciesareinfrequentlypathogenic,althoughA. thermomutatus isanotableexception.Themajorityofclinicallyrelevantspeciesbelong to the A. fumigatusclade(Balajeeetal.2005b,2009,Yaguchietal.2007,Alcazar-Fuolietal.2008)ortheAVSC(Su-guietal.2010,2014,Barrsetal.2013,Novákováetal.2014)andareheterothallic.Acrypticsexualcycleofseveraloftheseopportunistic pathogens, including A. fumigatus(O’Gormanetal.2009),A. lentulus(Swilaimanetal.2013)andA. felis(Barrsetal.2013),wasdiscoveredrecentlybycrossingoppositemat-ing type isolates in vitro.Molecularmethodsareroutinelyusedforidentificationofspe-ciesfromsect.Fumigati due to overlapping morphological fea- turesoftheirasexualmorph.Incontrast,themorphologyofthesexualmorph,especiallyofascospores,isamongstthemostinformativeofphenotypiccharacteristicsinsect.Fumigati.ThetaxonomyofAVSChasdevelopedrapidlysinceeightof thecurrently11recognizedspeciesweredescribedinthelastfouryears(Barrsetal.2013,Eamvijarnetal.2013,Novákováetal.2014,Suguietal.2014,Matsuzawaetal.2015,Talbotetal.2017).Thespeciesboundariesdelimitationwasusuallybasedon comparison of single-gene phylogenies and principles of genealogicalconcordance.Inaddition,somestudiessupportedthe species concept by results of in vitromatingexperimentsbetween oppositemating type strains.With the increasingnumberofspecies,availableisolatesandnewmatingexperi-mentdata,thespeciesboundariesinAVSCbecameunclearaspointedoutbyTalbotetal.(2017)whousedthedesignation‘A. felisclade’forA. felisandrelatedspecies.Importantly,Suguietal.(2014)andTalbotetal.(2017)identifiedthatinterpretationof in vitromatingassaysinsect.Fumigati may be problematic becausedifferentphylogeneticspeciesintheAVSCwereabletoproducefertileascomatawhencrossedbetweenthemselves.Some even mated successfully with A. fumigatus s.str.Herewepresentacriticalre-evaluationofspeciesboundariesintheAVSC.Weexaminedalargesetofclinicalandenvironmen-talstrainscollectedworldwide.Wedidnotuseclassicalphylo-genetic methods or genealogical concordance phylogenetic speciesrecognitionrules(GCPSR)forspeciesdelimitationduetotheirunsatisfactoryresultsinpreviousAVSCstudies.Suchmethods, based predominantly on analysis of concatenated DNAsequencedataorcomparisonofsingle-genephylogeniesarefrequentlypronetospeciesover-delimitationorareaffectedbysubjectivejudgementsofspeciesboundaries.Instead,we usedrecentlyintroduceddelimitationtechniquesbasedoncoales- centtheoryandthemultispeciescoalescentmodel(MSC)(Flot2015).WefollowedtheapproachrecommendedbyCarstensetal.(2013)thatcombinesspeciesdelimitation,speciestreeestimationandspeciesvalidationsteps.Althoughthesemeth-ods have already been applied to other groups of organisms suchasanimalsandplantstheiruseinfungiisscarce(Stewartetal.2014,Singhetal.2015,Liuetal.2016,Sklenářetal.2017,Hubkaetal.2018).Here,theresultsofMSCmethodswere taken as a basic hypothesis for species delimitation and thenfurtherverifiedbyanalysisofintra-andinterspecificbio-logical compatibilities, as well as ascospore dimensions and ornamentation.
al.2005,Vinhetal.2009,Coelhoetal.2011,Shigeyasuetal.2012,Barrsetal.2013,2014,Eamvijarnetal.2013,Novákováetal.2014,Suguietal.2014,Matsuzawaetal.2015,Talbotetal.2017)andculturecollections.Thesetcomprised38clinicalstrainsand72environmentalisolates,including67fromsoil,fourfromcaveenvironmentsandonefromplantmaterial.Theprovenanceof isolatesisdetailedinTable1.Newlyisolatedstrains were deposited into the Culture Collection of Fungi at theDepartmentofBotany,CharlesUniversity,Prague,CzechRepublic(CCF).Driedherbariumspecimensweredepositedinto theherbariaof theMedicalMycologyResearchCenter,ChibaUniversity,Japan(IFM)andMycologicalDepartmentoftheNationalMuseum,Prague,CzechRepublic(PRM).
Phenotypic studiesThestrainsweregrownonmaltextractagar(MEA),CzapekYeastAutolysateAgar(CYA),Czapek-Doxagar(CZA),yeastextract sucrose agar (YES),CYA supplementedwith 20%sucrose (CY20S), and creatine sucrose agar (CREA), andincubatedat 25°C.Agarmedia compositionwasbasedonthatdescribedbySamsonetal.(2014).MaltextractandyeastextractwereobtainedfromOxoid(Basingstoke,UK)andFlukaChemieGmbH(Switzerland),respectively.Growthat42,45and47°CwastestedonMEAplatessealedwithParafilm.ColourdeterminationwasperformedaccordingtotheISCC-NBS(Inter-SocietyColorCouncil–NationalBureauofStandards)CentroidColourCharts(Kelly1964).MicromorphologywasobservedonMEA.Lacticacidwithcottonbluewasusedasamountingmedium.PhotographsweretakenonanOlympusBX-51microscope(OlympusDP72camera)usingNomarskicontrast.MacromorphologyofthecolonieswasdocumentedusingastereomicroscopeOlympusSZ61(withOlympusCamediaC-5050Zoomcamera)orCanonEOS500D.Scanningelectronmicroscopy(SEM)wasperformedusingaJEOL-6380LVscanningelectronmicroscope(JEOLLtd.Tokyo,Japan)asdescribedbyHubkaetal.(2013b).Briefly,piecesofcolonyormatureascomatawerefixedinosmiumtetroxidevapoursforonewkat5–10°CandgoldcoatedusingaBal-TecSCD050sputtercoater.Thespecimenswereobservedusing40μmspotsizeand15–25kVacceleratingvoltage.
Molecular studies ArchivePureDNAyeastandGram2+kit(5PRIMEInc.,Gaithers- burg,MD)wasused forDNA isolation from7-d-oldculturesaccording to themanufacturer’s instructions as updated byHubkaetal.(2015b).ThepurityandconcentrationofextractedDNAwasevaluatedbyNanoDrop1000Spectrophotometer.ITSrDNAregionwasamplifiedusingforwardprimersITS1orITS5(Whiteetal.1990)andreverseprimersITS4S(Kretzeretal.1996)orNL4 (O’Donnell1993);partialβ-tubulingene(benA)usingforwardprimersBt2a(Glass&Donaldson1995)orBen2f(Hubka&Kolařík2012)andreverseprimerBt2b(Glass&Donaldson1995);partialcalmodulingene(CaM)usingfor-wardprimersCF1MorCF1LandreverseprimerCF4(Peterson2008);partialactingene (act )usingprimersACT-512FandACT-783R(Carbone&Kohn1999);partialRNApolymeraseIIsecondlargestsubunit(RPB2)usingforwardprimersfRPB2-5F(Liuetal.1999)orRPB2-F50-CanAre(Jurjevićetal.2015)andreverseprimerfRPB2-7cR(Liuetal.1999);partialmcm7 gene encoding minichromosome maintenance factor 7 with primers Mcm7-709forandMcm7-1348rev (Schmittetal.2009);andpartial tsr1 gene encoding ribosome biogenesis protein with primersTsr1-1453forandTsr1-2308rev(Schmittetal.2009).Terminalprimerswereusedforsequencing.ThePCRreactionvolumeof20µLcontained1µL(50ng)ofDNA,0.3µLofbothprimers(25pM/mL),0.2µLofMyTaqTMDNAA
Polymerase(Bioline,GmbH,Germany)and4μLof5×MyTaqPCRbuffer.The ITSrDNA,benA and CaM fragments were amplifiedusingthefollowingthermalcycleprofile:93°C/2min; 30cyclesof93°C/30s;55°C/30s;72°C/60s;72°C/10min.Theannealingtemperatureforamplificationofact gene was 60°C (30cycles);and that for tsr1gene50°C (37cycles).Partial RPB2genefragmentswereamplifiedusingtheabove-mentionedcycleortouchdownthermal-cycling:93°C/2min; 5cyclesof93°C/30s,65–60°C/30s,72°C/60s;38cyclesof93°C/30s,55°C/30s,72°C/60s;72°C/10min.Thepartialmcm7genewasamplifiedusingmodifiedtouchdownthermal-cycling:93°C/2min;5cyclesof93°C/30s,65–60°C/30s,72°C/60s;38cyclesof93°C/30s,60°C/30s,72°C/60s;72°C/10min.PCRproductpurificationfollowedtheprotocolofRéblováetal.(2016).AutomatedsequencingwasperformedatMacrogenSequencingService(Amsterdam,TheNetherlands)usingbothterminalprimers.SequencesweredepositedintotheENA(EuropeanNucleotideArchive)databaseunder theaccessionnumberslistedinTable2.
Phylogenetic analysis SequenceswereinspectedandassembledusingBioeditv.7.2.5 (www.mbio.ncsu.edu/BioEdit /bioedit.html).AlignmentsofthebenA, CaM, act and RPB2 regions were performed using the G-INS-ioptionimplementedinMAFFTv.7(Katoh&Standley2013).Alignmentsweretrimmed,concatenatedandthenana-lysedusingMaximumlikelihood(ML)andBayesianinference(BI) analyses.Suitable partitioning schemeand substitutionmodels (Bayesian information criterion) for analyseswereselected using the greedy algorithm implemented in Parti-tionFinderv.1.1.1(Lanfearetal.2017)withsettingsallowingintrons,exonsandcodonpositionstobeindependentpartitions.Proposed partitioning schemes and substitution models for each datasetarelistedinTable3.ThealignmentcharacteristicsarelistedinTable4.
TheMLtreewasconstructedwithIQ-TREEv.1.4.4(Nguyenetal.2015)withnodalsupportdeterminedbynon-parametricbootstrapping (BS)with1000 replicates.Bayesianposteriorprobabilities(PP)werecalculatedusingMrBayesv.3.2.6(Ron-quistetal.2012).Theanalysesranfor107 generations, two parallel runs with four chains each were used, every 1 000th treewasretained,andthefirst25%oftreeswerediscardedasburn-in.ThetreeswererootedwithAspergillus clavatusNRRL1 and A. lentulus NRRL 35552, respectively.All alignmentsareavailable from theDryadDigitalRepository (https://doi.org/10.5061/dryad.38889).
Species delimitation and species tree inferenceSeveral species delimitation methods were applied to elucidate the species boundarieswithin theAVSC.We followed therecommendationofCarstensetal.(2013)andcomparedtheresultsofseveraldifferentmethods.Theanalysiswasdividedinto twoparts. Four genetic lociwere examined in the firstanalysiswhichcomprisedallspeciesfromtheAVSCwhilesixgeneticlociwereexaminedinthesecondanalysisfocusedonthe clade comprising Aspergillus felis, A. pseudofelis, A. para-felis and A. pseudoviridinutans(A. aureolus was used as an outgroup).ThealignmentcharacteristicsarelistedinTable4.Only unique nucleotide sequences, selectedwithDAMBEv.6.4.11 (Xia 2017)were used in the analyses.Nucleotidesubstitution models for particular loci were determined using jModeltestv.2.1.7(Posada2008)basedonBayesianinforma-tioncriterion(BIC)andwereasfollows:1stanalysis-K80+G(benA), K80+I (CaM), K80+G (act), K80+G (RPB2); 2ndanalysis-K80+I(benA),K80+G(CaM),K80(act),K80(RPB2),HKY+I+G(tsr1),K80(mcm7).Inthefirstanalysis,onlyuniquesequencesoffourlociwereused,i.e.,benA, CaM, act and RPB2.ThenumberofisolatesofA. felis and A. pseudoviridinutans was reduced to two, because thiscladewasexaminedindetailinthesecondanalysisbased
onsixloci.Threesingle-locusspeciesdelimitationmethods,i.e.,bGMYC(Reid&Carstens2012),GMYC(Fujisawa&Barra-clough2013)andPTP(Zhangetal.2013),andonemultilocusspeciesdelimitationmethodSTACEY(Jones2017)wereusedtofindputativespeciesboundaries.ThebGMYCandGMYCmethodsrequireultrametrictreesasaninput,whilePTPdoesnot.Therefore,singlelocusultrametrictreeswereconstructedusingaBayesianapproachinBEASTv.2.4.5(Bouckaertetal.2014)withbothYuleandcoalescenttreemodels.Wealsolookedatpossibledifferencesbetweenstrictandrelaxedclockmodels, but since these parameters had no effect on the number of delimited species, only the results with strict clock model are presentedhere.Chainlengthforeachtreewas1× 107 genera-tionswith25%burn-in.ThehighestcredibilitytreewasusedfortheGMYCmethodand100treesrandomlysampledthroughouttheanalysiswereusedforthebGMYCmethod.BothmethodswereperformedinRv.3.3.4(RCoreTeam2015)usingbgmyc (Reid&Carstens2012)andsplits(SPecies’LImitsbyThresholdStatistics)(Fujisawa&Barraclough2013)packages.Thenon-ultrametric trees for the PTP method were constructed using theMLapproachinRAxMLv.7.7.1(Stamatakisetal.2008)andIQ-TREEv.1.5.3(Nguyenetal.2015)with1000bootstrapreplicates.ThePTPmethodwasperformedonthewebserverhttp://mptp.h-its.org/(Kaplietal.2017)withp-valuesetto0.001.ThemultilocusspeciesdelimitationwasperformedinBEASTv.2.4.5withadd-onSTACEYv.1.2.2(Jones2017).Thechainlengthwassetto5× 108 generations, priors were set as fol-lows: the species tree prior was set to the Yule model, growth ratepriorwassettolognormaldistribution(M=5,S=2),clockratepriorsforalllociweresettolognormaldistribution(M=0,S=1),PopPriorScalepriorwassettolognormaldistribution(M=-7,S=2) and relativeDeathRate priorwas set to betadistribution(α=1,β=1000).TheoutputwasprocessedwithSpeciesDelimitationAnalyzer(Jones2017).Thespeciestreewasinferredusing*BEAST(Heled&Drummond 2010)implementedinBEASTv.2.4.5.Theisolateswereas-signed to a putative species according to the results of the above-mentionedspeciesdelimitationmethods.TheMCMCanalysis ran for 1 × 108generations,25%oftreeswerediscard-edasaburn-in.Thestrictmolecularclockwaschosenforalllociandpopulationfunctionwassetasconstant.ConvergencewasassessedbyexaminingthelikelihoodplotsinTracerv.1.6(http://tree.bio.ed.ac.uk/software/tracer).Wealsoconstructedthe phylogenetic tree based on concatenated alignment of all fourlociinIQ-TREEv.1.5.3with1000bootstrapreplicatesandthe optimal partitioning scheme determined by PartitionFinder v.2.1.1(Lanfearetal.2017).The validation of the species hypotheses was performed in BP&Pv.3.3 (Bayesianphylogenetics andphylogeography)(Yang&Rannala2010).The isolateswereassigned to thespecies based on the results of species delimitation methods andthespeciestreeinferredwith*BEASTwasusedasaguidetree.Threedifferentcombinationsofthepriordistributionsoftheparametersθ(ancestralpopulationsize)andτ0(rootage)weretestedasproposedbyLeaché&Fujita(2010),i.e.,largeancestralpopulationsizesanddeepdivergence:θ~G(1,10)andτ0~G(1,10);smallancestralpopulationsizesandshal-lowdivergencesamongspecies:θ~G(2,2000)andτ0~G(2, 2000); largeancestral populations sizesandshallowdi-vergencesamongspecies:θ~G(1,10)andτ0~G(2,2000).Thesecondanalysiswithsixprotein-coding loci, i.e.,benA, CaM, act, RPB2, mcm7 and tsr1, consisted of the same steps asdescribedabove.InsteadofPTP,weusedtheprogrammemPTP(Kaplietal.2017)withIQ-TREEandRAxMLtreesasaninput.WithinthemPTPprogrammeweusedthefollowingsettings:Maximum likelihood species delimitation inference(optionML)andadifferentcoalescentrateforeachdelimited
species(optionmulti).Rpackageggtree(Yuetal.2017)andthe programmedensitree (Bouckaert 2010)were used forvisualizationofthephylogenetictrees.
Mating experimentsTheMAT idiomorphwasdeterminedusing theprimer pairsalpha1 and alpha2 located inMAT1-1-1 locus (alpha boxdomain),andHMG1andHMG2primerslocatedinMAT1-2-1locus(high-mobility-groupdomain)asdescribedbySuguietal.(2010).TheMATidiomorphsweredifferentiatedbasedonthedifferentlengthsofPCRproductsvisualizedbygelelectro-phoresis;absenceofoppositeMATidiomorphwasalsoverifiedinall isolates.The identityofPCRproductswasprovedbyDNAsequencing in several isolates (accessionnumbers inTable1);productpurificationandsequencingwereperformedatMacrogenEurope (Amsterdam,TheNetherlands) usingterminalprimers.SelectedoppositematingtypestrainswerepairedwithinandbetweenmajorphylogeneticcladesonMEAandoatmealagar(OA;Difco,LaPontedeClaix,France)platesand incubatedat 25, 30 and37°C in the dark.TheplatesweresealedwithParafilmandexaminedweeklyfromthethirdwk of cultivation for two months under a stereomicroscope fortheproductionofascomata.Thepresenceofascosporeswasdeterminedusing lightmicroscopy.Widthandheightofascosporeswererecordedatleast35timesforeachsuccess-fulmatingpair.
Statistical analysisStatistical differences in the width and height of the asco- spores of particular species and interspecific hybridsweretestedwithone-wayANOVAfollowedbyTukey’sHSD(honestsignificantdifference)posthoctestinRv.3.3.4(RCoreTeam2015).Rpackagemultcomp (Hothornetal.2008)wasusedfor the calculation and package ggplot2(Wickham2009)forvisualizationoftheresults.
Exometabolite analysisTheextractswere prepared according toHoubraken et al.(2012).High-performanceliquidchromatographywithdiode-array detection was performed according to Frisvad & Thrane (1987,1993)asupdatedbyNielsenetal.(Nielsenetal.2011).Fungiwereincubatedfor1wkat25°CindarknessonCYAandyeastextractsucrose(YES)agarsforexometaboliteanalysis.
RESULTS
Phylogenetic definition of AVSCInthephylogeneticanalysis,76combinedbenA, CaM, act and RPB2sequenceswereassessedformembersofsect.Fumigati.TheanalysiswasbasedonthemodifiedalignmentpreviouslyusedbyHubkaetal.(2017)andenrichedbytaxafromAVSC.IntheBayesiantreeshowninFig.1,membersofsect.Fumi-gati areresolvedinseveralmonophyleticclades.TheanalysisshowedthatAVSCisaphylogeneticallywell-definedgroupandthecladegainedfullsupport.Similarly,someothercladesarewell-supportedbybothBIandMLanalysesincludingA. spino-sus clade, A. brevipes clade, A. tatenoi clade, A. thermomutatus clade and A. fennelliaeclade;A. spathulatus forms a single-specieslineagedistantlyrelatedtootherclades.Othercladeshave moderate or low support and the species represented therein may differ based on genetic loci used for phylogenetic reconstructionandtaxaincludedintheanalysis.Heterothallicspeciesaredispersedacrosssect.Fumigati (Fig.1) but the majorityofthemclusterinAVSCandA. fumigatusclades.Thesetwo clades also encompass the highest number of human and animalpathogensinsect.Fumigati not only in terms of their numberbutalsotheirclinicalrelevance.
■ A. pseudoviridinutans CBS 458.75■ A. arcoverdensis IFM 61334T
■ A. arcoverdensis IFM 59922
■ A. frankstonensis CBS 142233T
■ A. frankstonensis CBS 142234
● A. fischeri NRRL 181T
■ A. fumisynnematus IFM 42277T
● A. lentulus NRRL 35552T
● A. laciniosus KACC 41657T
■ A. marvanovae CCM 8003T
● A. turcosus IBT 27921T
■ A. brevistipitatus CCF 4149T
■ A. conversis CCF 4190T
● A. wyomingensis IMI 133982● A. siamensis IFM 59793T
● A. siamensis IFM 61157● A. felis CBS 130245T
● „A. pseudofelis“ NRRL 62903T
● „A. parafelis“ NRRL 62900T
● A. felis FRR 5679■ A. pseudoviridinutans NRRL 62904T
■ A. pseudoviridinutans IMI 182127
● A. fumigatus NRRL 163T
■ A. oerlinghausensis CBS 139183T
● A. paulistensis CBM-FA-0690T
● A. takakii CBM-FA-884T
● A. aureolus NRRL 2244T
● „A. indohii“ IFM 53615T
■ A. acrensis IFM 57291T
■ A. acrensis CCF 4959● A. udagawae IFM 46972T
● A. udagawae IFM 54131● A. udagawae CCF 4475 ● A. udagawae CCF 4478
● A. wyomingensis CCF 4417T
● A. wyomingensis CCF 5640
0.02
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A. neoglaber clade
A. fumigatus clade
A. spinosus clade
A. brevipes clade
A. tatenoi clade
A. thermomutatus / A. spathulatus clades
A. unilateralis clade
A. auratus clade
A. fennelliae clade
A. viridinutans clade
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Fig. 1Phylogenetic relationshipsof thesect.Fumigati members inferred from Bayesian analysis of the combined, 4-gene dataset of β-tubulin (benA),calmodulin(CaM ),actin(act )andRNApolymeraseIIsecondlargestsubunit(RPB2)genes.Bayesianposteriorprobabilities(PP)andMaximumlikelihoodbootstrapsupports(BS)areappendedtonodes;onlyPP≥95%andBS≥70%areshown;lowersupportsareindicatedwithahyphen,whereasasterisksindicatefullsupport(1.00PPor100%BS);ex-typestrainsaredesignatedbyasuperscriptT;speciesnamesinquotesareconsideredsynonyms;thebarindicatesthenumberofsubstitutionspersite.ThetreeisrootedwithAspergillus clavatusNRRL1T.Thereproductivemodeofeachspeciesisdesignatedbyiconsbeforethespeciesname(seelegend).
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A. udagawae clade 1 A. udagawae clade 2 A. udagawae clade 3 A. acrensis A. aureolus A. wyomingensis A. siamensis A. pseudoviridinutans A. felis
A. frankstonensis
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Fig. 2 Schematic representation of results of species delimitation methods in Aspergillus viridinutansspeciescomplexbasedonfourgeneticloci.Theresultsofmultilocusmethod(STACEY)arecomparedtoresultsofsingle-locusmethods(PTP,bGMYC,GMYC).TheresultsofSTACEYareshownastreebrancheswithdifferentcolours,whiletheresultsofsingle-locusmethodsaredepictedwithcolouredbarshighlightingcongruenceacrossmethods.ThedisplayedtreeisderivedfromIQ-TREEanalysisbasedonaconcatenateddatasetandisusedsolelyforthecomprehensivepresentationoftheresultsfromdifferentmethods.Thespeciesvalidationanalysisresults(BP&P)areappendedtonodesandshowningreyborderedboxes;thevaluesrepresentposteriorprobabilitiescalcu-latedinthreescenarioshavingdifferentpriordistributionsofparametersθ(ancestralpopulationsize)andτ0(rootage).Thetopvaluerepresentstheresultsofanalysiswithlargeancestralpopulationsizesanddeepdivergence:θ~G(1,10)andτ0~G(1,10);themiddlevaluerepresentstheresultsofanalysiswithlargeancestralpopulationssizesandshallowdivergencesamongspecies:θ~G(1,10)andτ0~G(2,2000);andthebottomvaluesmallancestralpopulationsizesandshallowdivergencesamongspecies:θ~G(2,2000)andτ0~G(2,2000).
ex-type of A. pseudoviridinutans ex-type of A. fumigatus var. sclerotiorum
Fig. 3 Schematic representation of results of species delimitation methods in Aspergillus felis cladebasedonsixgenetic loci.Theresultsofmultilocusmethod(STACEY)arecomparedtoresultsofsingle-locusmethods(mPTP,bGMYC,GMYC).TheresultsofSTACEYareshownastreebrancheswithdifferent colours,whiletheresultsofsingle-locusmethodsaredepictedwithcolouredbarshighlightingcongruenceacrossmethods.ThedisplayedtreeisderivedfromIQ-TREEanalysisbasedonaconcatenateddatasetandisusedsolelyforthecomprehensivepresentationoftheresultsfromdifferentmethods.Thespeciesvalidationanalysisresults(BP&P)areappendedtonodesandshowningreyborderedboxes;thevaluesrepresentposteriorprobabilitiescalculatedinthreescenarioshavingdifferentpriordistributionsofparametersθ(ancestralpopulationsize)andτ0(rootage).Thetopvaluerepresentstheresultsofanalysiswithlargeancestralpopulationsizesanddeepdivergence:θ~G(1,10)andτ0~G(1,10);themiddlevaluerepresentstheresultsofanalysiswithlargeancestralpopulationssizesandshallowdivergencesamongspecies:θ~G(1,10)andτ0~G(2,2000);andthebottomvaluesmallancestralpopulationsizesandshallowdivergencesamongspecies:θ~G(2,2000)andτ0~G(2,2000).
154 Persoonia–Volume41,2018
Species delimitation and validation in AVSCIn thefirstanalysis, fourgenetic lociwereexaminedacrossspecies ofAVSC, isolates ofA. felis and its close relatives were reduced to two individuals, because a separate analysis basedonsixlociwasperformedforthisclade.Elevententativespeciesweredelimited inAVSCusingSTACEY.TheresultsaresummarisedinFig.2,thedifferencesinthecolourofthetreebranchesreflectspeciesdelimitedby theanalysis.Theanalysis supported recognition of three putative species in A. udagawae lineage, delimitation of A. acrensis (describedbelow)fromA. aureoluswasnotsupported,otherAVSCspe-ciesweresupportedbySTACEYwithoutdifferencesfromtheircurrentconcept.The results derived fromSTACEYwere compared to thosefromthreesingle-locusspeciesdelimitationmethods.Thecon-sensual results from single-locus species delimitation methods aregenerallyinagreementwiththeresultsofSTACEYforthemajority of species but vary greatly for A. udagawae, A. aureolus and A. acrensislineages(Fig.2).Recognitionofthreeputativespecies in A. udagawae lineage was supported only based on the CaM locus, while based on benA locus, none of these three sublineagesgainedsupport.Variousdelimitationschemeswereproposed by different single-locus species delimitation methods in the A. udagawae lineage based on the RPB2gene(resultseven varied between the analyses based on different input trees forthePTPandGMYCmethods),whilefiveputativespecieswere identically delimited based on the actlocus.Themethodsrelatively consistently supported delimitation of the A. acrensis lineage based on the RPB2locusandsimilarly,bGMYCandGMYCmethodssupportedthisspeciesbasedontheactlocus.In contrast, lineages of A. acrensis and A. aureolus were not splitbyanymethodwhenanalyzingbenA and CaM loci.The species validation analysis results are appended to nodes ofthetreeinFig.2.Areasonablesupportisdefinedbyposteriorprobabilities≥0.95underallthreescenariossimulatedbydif-ferentpriordistributionsofparametersθ(ancestralpopulationsize)andτ0(rootage).Delimitationofallputativespecies(thosedelimitedbySTACEY,A. acrensis and A. aureolus)weresup-portedbytheposteriorprobability0.98orhigherbasedontheanalysisinBP&Pv.3.1(Yang&Rannala2010)underallthreescenarios.TheonlyexceptionwaslowersupportforsplittingofA. acrensis and A. aureolus;thisscenariowassupportedbytheposteriorprobabilities0.84,0.88,1.00,respectively.
Species delimitation and validation in A. felis clade and its relativesInthesecondanalysis,sixgeneticlociwereexaminedacrossisolates of A. felis, A. parafelis, A. pseudofelis and A. pseudo-viridinutans.Onlytwotentativespecies,A. felis and A. pseudo-viridinutans,weredelimitedinthiscladeusingSTACEY.Theresults are shown as branches designated by different colours in Fig.3.TheanalysisdidnotsupportseparationofA. pseudofelis and A. parafelis from A. felis;A. fumigatusvar.sclerotiorum is included in the lineage of A. pseudoviridinutans.The results of three single-locus species delimitation methods werecomparedto thosefromSTACEY,andtheconsensualresultsshowedageneralagreement(Fig.3).DelimitationofA. pseudofelis from A. felis was not supported by any of the usedmethods.Onlyanegligiblenumberofanalysessupporteddelimitation of basal clades in A. felisastentativespecies(des-ignatedasclade2and3inFig.3).Butevenintheseminorityscenarios, there were no clear consensual delimitation patterns that would support delimitation of A. parafelis. Interestingly,mPTP analysis based on act, benA, CaM (withRAxMLtreesasaninputonly),mcm7 and tsr1locitogetherwithGMYCanalysisbased on benA(onlyinputtreebasedoncoalescenttreemodel)
and act(onlyinputtreebasedonYuletreemodel)locididnotsupport delimitation of A. pseudoviridinutans from a robust clade of A. felis.AnincompletelineagesortingwasobservedbetweenA. felis and A. pseudoviridinutans(Fig.3)evidencingthattherewasprobablyanancestralgeneflowbetweentheselineages.Two isolates from A. felislineage(IFM59564andCCF5610)have benA sequencesthatclusterwithA. pseudoviridinutans whilesequencesof the remaining5 lociplaced them in theA. felislineage(single-genetreesnotshown).The species validation analysis results are appended to nodes of the tree inFig.3.DelimitationofA. felis and A. pseudo-viridinutansgainedabsolutesupportinBP&Panalysis(Yang&Rannala2010)underall threescenariossimulatedbydif-ferentpriordistributionsofparametersθ(ancestralpopulationsize)andτ0(rootage).Delimitationofthreeputativespecieswithin A. felislineagegainednosupport(posteriorprobability0.51)underthescenariowithsmallancestralpopulationsizesandshallowdivergencesamongspecies:θ~G(2,2000)andτ0~G(2,2000).
Species treeThespeciestreetopologywasinferredwith*BEAST(Heled&Drummond2010)andisshowninFig.4.Itwasusedasaguidetree during species validation using BP&P but it also represents the most probable evolutionary relationships between species intheAVSC.Theanalysisconfirmedrecombinationbetweenthree subclades of A. felis(Fig.4)whichincludealsorecentlyproposed species A. parafelis and A. pseudofelis thus repre-senting the synonyms of A. felis.Similarly,therecombinationbetween three subclades of A. udagawae rejected the hypoth-esisthattheycouldbeconsideredseparatespecies(Fig.4).
A. lentulus (outgroup)
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Fig. 4Speciestreeinferredwith*BEASTvisualizedbyusingDensiTree(Bouckaert 2010).All trees created in theanalysis (except 25%burn-inphase)aredisplayedontheleftside.Treeswiththemostcommontopologyare highlighted by blue, trees with the second most common topology by red, trees with the third most common topology by pale green and all other treesbydarkgreen.Ontherightside,theconsensustreesofthethreemostcommontopologiesaredisplayed.
Fig. 5 Phylogenetic relationships of the Aspergillus viridinutans speciescomplex members inferred from Bayesian analysis of the combined, 4-gene dataset of β-tubulin(benA),calmodulin(CaM),actin(act)andRNApolymeraseIIsecondlargestsubunit(RPB2)genes.Bayesianposteriorprobabilities(PP)andMaximumlikelihoodbootstrapsupports(BS)areappendedtonodes;onlyPP≥90%andBS≥70%areshown;lowersupportsareindicatedwithahyphen,whereasasterisksindicatefullsupport(1.00PPor100%BS);ex-typestrainsaredesignatedbyasuperscriptT;speciesnamesinquotesareconsideredsynonyms;thebarindicatesthenumberofsubstitutionspersite.ThetreeisrootedwithAspergillus lentulusNRRL35552T.ThegeographicoriginandreproductivemodewithMATidiomorph(ifknown)isdesignatedbyiconsbeforetheisolatenumberwhilesubstrateoforiginisdesignatedbyiconsafterisolatenumber(seelegend).
❺ M2 IFM 57289 ❷ M2 CBS 458.75 „A. fumigatus var. sclerotiorum“
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Continent of origin❶ Europe❷ Asia❸ Australia❹ North America❺ South America❻ Africa
Source of isolationanimalhumansoil/cave sedimentother
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Fig. 6 Schematic depiction ofresultsofintraspecificmatingexperimentsbetweenoppositematingtypeisolatesofheterothallicmembersoftheAspergil-lus viridinutansspeciescomplex.Onlysuccessfulmatingexperimentsaredisplayedbyconnectinglinesbetweenoppositematingtypeisolates;remainingmatingexperimentswerenegative.Isolatesmarkedbyasteriskwereonlycrossedwithex-typestrainsofA. felis(CBS130245T),A. parafelis(NRRL62900T)and A. pseudofelis(NRRL62903T).BoxplotandviolingraphswerecreatedinR3.3.4(RCoreTeam2015)withpackageggplot2(Wickham2009)andshowthe differences between the width and height of ascospores of A. udagawae, A. wyomingensis and A. felis.Differentlettersabovetheplotindicatesignificantdifference(P<0.05)inthesizeoftheascosporesbetweendifferentspeciesbasedonTukey’sHSDtest.Boxplotsshowmedian,interquartilerange,valueswithin±1.5ofinterquartilerange(whiskers)andoutliers.
The remaining species delimited in previous steps (Fig. 4),including A. pseudoviridinutans and A. acrensis(introducedinthisstudy),weresupportedby*BEASTanalysis.ThespeciestreehadidenticaltopologywiththetreesinferredbyMLandBIanalysesoftheconcatenatedandpartitioneddataset(Fig.5),andallspeciessupportedby*BEASThad100%MLboot-strapsupport(MLBS)and1.00BIposteriorprobabilities(BIPP).SeveraldeepnodesinthespeciestreehadonlylimitedsupportsimilarlytoMLandBIanalyses.Thusclearpositionsof A. wyomingensis and A. siamensis within the clade also containing A. udagawae, A. acrensis and A. aureolus remains unresolved, while A. acrensis with A. aureolus form a sister clade to A. udagawae(thistopologygainedabsolutesupportinallfurtheranalyses–seebelow).Anotherrobustcladecon-tained sister species A. felis and A. pseudoviridinutans.Theremainingspecies,i.e.,A. viridinutans, A. frankstonensis and A. arcoverdensis,formedabasalcladeintheAVSCandtheirpositionswithinthecladearefullyresolved(Fig.4).
Clustering of isolates by origin and mating-type idiomorphIn the phylogenetic analysis, 111 combined benA, CaM, act and RPB2sequenceswereassessed formembersofAVSC.Allspecies delimited by methods based on the coalescent model werefullysupportedbyBIandMLanalyses(Fig.5).The A. udagawaelineageincluded25isolatesthatclusteredinthreemainclades.MatingtypegeneidiomorphMAT1-1-1wasdetectedin10isolateswhile14strainshadMAT1-2-1idio-morph(MATidiomorphwasnotdeterminedinonestrain).ThemajorityofNorthAmericanisolates(10/14)clusteredinclade1togetherwithonestrainfromAustralia;clade2comprisedonlythreestrainsoriginatingfromAsia;isolatesfromfourdifferentcontinentswerepresent inclade3.Therewasnoapparentclustering based on clinical or environmental origin of strains, ortheirMATidiomorph.AllthreeclinicalisolatesfromAsiahadan identical haplotype based on four studied protein-coding loci(Fig.5)butonestrainhadMAT1-2-1idiomorphincontrasttoMAT1-1-1idiomorphdetectedintheremainingtwostrains.The A. acrensislineageincludedfivestrainsisolatedfromsoil(Brazil)orcavesediment(Romania),twoofwhichhadMAT1-1-1 idiomorphandthreehadMAT1-2-1idiomorph.Thislineageisvery closely related to a homothallic species A. aureolus rep-resentedbyfourstrainsinouranalysis.Theonlyknownclini-cal isolate of A. aureolus(IHEM22515)wasisolatedfromthecorneaofapatientinPeru.Wewereunabletosourcefurtherinformation about this case and thus the clinical relevance of thisisolatecannotbeconfirmed.The mutual phylogenetic position of homothallic A. siamensis and heterothallic A. wyomingensisremainsunresolved.Asper-gillus siamensis was represented in our analysis by only two isolates from soil in Thailand, which were included in the original description(Eamvijarnetal.2013).TheA. wyomingensis line-ageincluded15isolates;12ofthemcamefromWyoming(USA)and were closely related to each other and to one isolate from China,whiletwoisolatesfromAustraliaandEuropedisplayedahighernumberofuniquepositions.TheratioofMAT1-1-1isolates toMAT1-2-1 isolateswas8:7, and themajorityofMAT1-1-1isolatesfromtheUSA(6/7)clusteredinaseparatesubcladethatwasonlysupportedintheBIanalysis.The A. felislineagecomprised35isolatesthatclusteredinthreemainclades.MatingtypegeneidiomorphMAT1-1-1wasde-tectedin12isolates,while20strainshadMAT1-2-1idiomorph(MATidiomorphwasnotdeterminedinthreestrains).Therewasno clustering based on geographic origin as all three clades includedisolatesfromtwotofourcontinents.Clade3containedonlyclinicalisolates(n=4).Clinicalstrainswerepredominantinclade1(18:2)whereasenvironmentalstrainsdominated
inclade2(7:4).TheratioofMAT1-1-1isolatestoMAT1-2-1isolatesinclade1wasbalanced(10:9)butwasbiasedtowardMAT1-2-1idiomorphinclades2(1:7)and3(1:3).Eightiso-lates of A. pseudoviridinutans, a sister species of A. felis, were examinedinthisstudy;MAT1-1-1idiomorphwasdeterminedinfiveofthemandMAT1-2-1idiomorphinthreeofthem.Therewas no apparent clustering based on clinical or environmental originofstrains,ortheirMATidiomorph(Fig.5).Abasal cladeofAVSCcomprises three soil-borne species.Whilst A. viridinutans and A. frankstonensis are known only from onelocalityinAustralia,13A. arcoverdensis strains included intheanalysiswereisolatedonthreecontinents, i.e.,SouthAmerica,AsiaandAustralia.Both,A. viridinutans and A. frank-stonensis were represented only by one and two isolates, re-spectively,includedintheoriginaldescriptions(McLennanetal.1954,Talbotetal.2017),andonlyisolatesofonematingtypeareknownforeachofthesespecies.Isolatesofbothmatingtypes were present in A. arcoverdensis(MAT1-1-1:MAT1-2-1 ratio,8:5).AgeographicalclusteringwasapparentinA. arco- verdensisstrains;twostrainsfromChinaandonestrainfromAustralia formed sublineages separate from theBrazilianstrains(Fig.5).
Mating experiments and morphology of sporesTheMAT1-1-1andMAT1-2-1idiomorphsweredeterminedfor100of104isolatesrepresentingheterothallicspeciesinAVSC(Table1).SystematicmatingexperimentswerefirstperformedwithinmajorphylogeneticcladesoftheAVSC.Oppositematingtype strains representing genetic and geographic diversity for eachheterothallic specieswere selected formatingexperi-ments and crossed in all possible combinations if not otherwise indicated(Fig.6).Successfulmatingwasobservedinlineagesof A. felis, A. udagawae and A. wyomingensis.Atleastsomeindividuals representing all three phylogenetic subclades of A. felis(Fig.3,5)andA. udagawae(Fig.2,5)crossedsuc-cessfullywithindividualsfromtheothersubclades.Thematingcapacityofindividualisolateswasunequal.Whilstsomeisolatesof a particular species were able to mate with a broad spectrum of opposite mating type strains of the same species, others produced fertile ascomata with only a limited set of strains ordidnotmateatall.Themorphologyofascosporesamongdifferentcrossesinthesethreespecieswasconsistent(Fig.7).Theexceptionwasgreatvariabilityintheconvexsurfaceornamentation of A. wyomingensis ascospores among and as well as within pairings of different isolates ranging from almost smooth,tuberculatetoechinulate(Fig.7).Althoughboththewidth and height of ascospores of A. felis, A. udagawae and A. wyomingensis overlapped significantly, their dimensionswere statistically significantly different (Tukey’sHSD test,pvalue<0.05)(Fig.6).Nofertilecleistotheciawereproducedby crossing opposite mating type isolates of A. pseudoviridi-nutans, A. acrensis and A. arcoverdensis.Matingexperimentswere not performed in A. viridinutans and A. frankstonensis due totheabsenceofoppositematingtypestrains.Oppositematingtypeisolatesofeachheterothallicspecieswerealsoselectedforinterspecificmatingassaysandcrossedinallpossiblecombinations.MorphologicalcharacteristicsofAVSCascosporesandinducedhybridsaresummarisedinTable5.Onlythreeof12selectedA. udagawae isolates produced fertile ascomata with some isolates of A. felis, A. wyomingensis or A. acrensis (Fig.8).Thehighestmatingcapacitywasobservedintheex-typestrainofA. udagawaeIFM46972thatproducedfertile ascomata when crossed with A. felis(CCF5609,CCF4171andCCF5611),A. wyomingensis(CCF4416andCCF4411)andA. acrensis(IFM57290).Thewidthandheightofascospores of interspecific hybrids betweenA. udagawae and A. acrensisweresignificantlydifferent(Tukey’sHSDtest,
158 Persoonia–Volume41,2018
Fig. 7ComparisonofmorphologyofsexualmorphsofA. felis, A. udagawae and A. wyomingensis.a.FertilecleistotheciaofA. felis as a result of crossing ofisolatesIFM60053×FRR5680;b.ascosporesinlightmicroscopy;c–d.ascosporesinscanningelectronmicroscopy:CBS130245T ×CCF5627(c),CBS130245T ×IFM60053(d);e.fertilecleistotheciaofA. udagawaeasaresultofcrossingofisolatesIFM46972T ×IFM46973;f.ascosporesinlightmicroscopy;g–h.ascosporesinscanningelectronmicroscopy;i.fertilecleistotheciaofA. wyomingensisasaresultofcrossingofisolatesCCF4416× CCF 4417T;j.ascosporesinlightmicroscopy(CCF4416×CCF4169);k–n.ascosporesinscanningelectronmicroscopy:CCF4416× CCF 4417T(k–l),CCF4417T × CCF 4419(m–n).—Scalebars:b,f,j=5μm;c–d,g–h,k–n=2μm.
pvalue <0.05) fromA. udagawae (Fig. 8).Approximately50%ofhybridascosporesfrommatingCMFISB2190withIFM57290lackedvisibleequatorialcrestsandifpresent,theywere frequently interruptedorstellate (Fig.9) incontrast toA. udagawae(visiblecrestspresentin>90%ofascospores,crests continuous).The ascomata frommating IFM46972with IFM57290containedonly lownumbersof ascosporesthatweregloboseorsubgloboseandglabrous(withoutcrestsandornamentationonconvexsurface).Thisobservationsup-ported the hypothesis that A. acrensis is a separate species despite its close phylogenetic relationships to A. udagawae.The ascospore dimensions of hybrids between A. udagawae and A. wyomingensis were similar to those of A. udagawae andbothwidthandheightweresignificantlydifferent(Tukey’sHSDtest,pvalue<0.05)fromA. wyomingensis (Fig.8).Thesehybridascosporeshadwell-definedequatorialcreststhatweremostcommonly0.5–1µmbroadandsimilartothoseofA. uda-gawae (Fig.9).ThehybridsofA. udagawae and A. felis had ascosporeswithsimilarequatorialcrestlengthandbodywidthto A. udagawaebutweresignificantlydifferent fromA. felis, and theirheightwassignificantlydifferent frombothA. felis and A. udagawae (Fig.8).TheascomataofhybridsbetweenA. udagawae with A. wyomingensis and A. felis, respectively, usuallycontainedonlylownumbersofascospores.Nomatingor production of infertile ascomata only was observed between crosses of A. udagawaeandtheremainingheterothallicAVSCmembers(Fig.8).Interestingly,themajorityofinterspecifichy-bridsproducedapproximately1–10%ofgloboseorsubgloboseasco-sporeswithabnormallylargedimensions,approximately6.5–10.5 µmdiam (their dimensionswere not included forcalculationsof statisticalmeasures inFig.8and10,and in Table5).Thesecellshadthickwallssimilartonormalasco-spores,butlackedequatorialcrestsandhadaglabrousorechi-nulatesurface.Theirdimensionswereintermediatebetweennormal ascospores and asci but their walls were dissimilar to thoseofthin-walledasci.Thesecellswerenotobservedamongprogenyoftheintraspecificcrosses(intraspecificmatingassay)and their presence probably indicates a defect in meiosis and ascosporedevelopment.
TwoMAT1-1-1isolatesofA. pseudoviridinutans selected for interspecificmatingassays,namelytheex-typestrainNRRL62904andstrainIFM59502,wereabletomatewitharelativelyhighnumber ofMAT1-2-1 isolatesofA. felis (Fig. 10).The ascosporesofthesehybridswerestatisticallysignificantlydif-ferent in their width and height from A. felis.Equatorialcrestswereabsentinapproximately5–20%hybridascosporesand,if present, they were shorter than those of A. felis(Table5).These observations suggest that A. pseudoviridinutans should be treated as a separate species as proposed by species delimi-tation methods despite the close phylogenetic relationships of both species and incomplete lineage sorting detected between thesetwospecies(Fig.3).Onlyoneinterspecifichybridwasinduced in our assay between A. wyomingensis CCF4169andA. felisNRRL62900.Theascosporebodywidthandheightofthishybridwassignificantlydifferentfrombothparentalspecies(Fig.10).IncontrasttoA. wyomingensis,equatorialcrestswerepresent in the majority of hybrids and they were occasionally interruptedandstellate(Fig.11).Infertileascomatawereob-served in some crosses between A. felis and following species: A. acrensis, A. wyomingensis and A. viridinutans.Aspergillus aureolus and A. siamensis are the only two homo-thallicspeciesintheAVSCandreadilyproduceascomataonabroad spectrum of media and growth temperatures and are eas-ilydistinguishablefromtheeightheterothallicAVSCmembers.MostA. aureolus isolates produce distinctive yellow colonies in contrast to the whitish colonies of A. siamensis (Fig.12).Theascosporesofbothspecieshavesimilardimensions,convexsurfaceornamentationandequatorial crest length (Table5,Fig.12)andmostcloselyresemblethoseofA. felis from among heterothallicspecies.Themacromorphologyofcolonies,micromorphologyofasexualmorphs and physiology have only limited discriminatory power inAVSCmembers,asrecognizedinpreviousstudies(Nováková etal. 2014,Matsuzawaetal. 2015).Wecomparedsurfaceornamentationofconidia inall currently recognizedspeciesusingSEM.Theornamentationshowedamicro-tuberculatepatternandwasbroadlyidenticalacrossallspecies(Fig.13).
A. felis 4.4±0.5 3.9±0.6 0.5–1.5(–2) crestspresent3;CStuberculatetoechinulate(SEM)
A. siamensis 4.5±0.5 3.7±0.4 (0.5–)1–1.5 crestspresent3;CStuberculate,echinulatetoreticulate(SEM)
A. udagawae 4.8±0.4 4.2±0.4 (0–)0.5(–1) visiblecrestsabsentin<10%ofascospores(LM); CStuberculatetoreticulate(SEM)
A. wyomingensis 4.2±0.4 3.4±0.4 0–0.5 visiblecrestsabsentin>50%ofascospores(LM); CSalmostsmooth,tuberculate,echinulate(SEM)
A. felis × A. pseudoviridinutans 4.9±0.4 4.2±0.5 (0–)0.5–1 visiblecrestsabsentin5–20%ofascospores(LM)depending onparentalisolates;CStuberculatetoechinulate(SEM)
A. felis × A. wyomingensis 4.8±0.5 4.3±0.3 (0–)0.5–1 visiblecrestsabsentin~10%ofascospores(LM); CStuberculate(SEM)
A. felis × A. udagawae 5.1±0.5 4.5±0.5 0–0.5(–1) visiblecrestsabsentin~20%ofascospores(LM); CSechinulate,tuberculatetoreticulate(SEM)
A. udagawae × A. wyomingensis 5.0±0.4 4.6±0.3 0–1 visiblecrestsabsentin~15%ofascospores(LM); CStuberculate(SEM)
A. udagawae × A. acrensis 5.2±0.5 4.4±0.5 0–0.5 visiblecrestsabsentin~50%ofascospores(LM)inCMF ISB2190×IFM57290andin100%ofascosporesin IFM46972×IFM57290;CStuberculatetoechinulateinCMF ISB2190×IFM57290(SEM)andglabrousinIFM46972× IFM57290(LM)1Valuesinparenthesesarelesscommon(lessthan10%ofmeasurements).2LM=lightmicroscopy;SEM=scanningelectronmicroscopy;CS=convexsurface.3Crestsmayabsentin<1%ofascosporesinsomeisolates/crosses.
Table 5 Ascospores characteristics of Aspergillus viridinutanscomplexspeciesandinterspecifichybrids.
160 Persoonia–Volume41,2018
A. felis
A. udagawae
A. wyomingensis
As
co
sp
ore
bo
dy w
idth
(μm
)A
sco
sp
ore
bo
dy h
eig
ht
(μm
)
3
4
5
6
A. udagawae ×A. acrensis
A. udagawae ×A. wyomingensis
A. udagawae ×A. felis
a b c d ab e
3
4
5
2
CCF 4479
IFM 54131
IFM 54132
CCF 4476
CCF 4478
IFM 62155
CCF 5634
CMF ISB 2190
IFM 51744
IFM 55207
IFM 46972T
IFM 46973
CBS 121595
IFM 47045T
CCF 4411
IFM 57291T
IFM 59922
CBS 142233T
CBS 142234
CCF 5610CCF 5611
CCF 5632 IFM 57289 CBS 458.75
CCF 4169
IFM 57290
IFM 61345
CCF 4171
IMI 182127
CCF 4417T
CCF 4959
IFM 61338
CCF 5609IFM 60053 NRRL 62900 NRRL 62901
NRRL 62904T
IFM 59502
CCF 4416 CCF 4419
CCF 4960 CCF 4961
IFM 61334T
IFM 61362
CCF 5619CBS 130245T
CCF 5622CCF 4148 NRRL 62903
CCF 5617CCF 5626 CCF 5628
CCF 5625 CCF 5627
MAT1-1-1MAT1-2-1
A. udagawae
A. felis
A. pseudoviridinutans
A. acrensis
A. wyomingensis
A. arcoverdensis
A. frankstonensis
A. viridinutans
a b ab c b d
Fig. 8 Schematic depiction ofresultsof interspecificmatingexperimentsbetweenoppositemating type isolatesofA. udagawae and other heterothallic members of Aspergillus viridinutansspeciescomplex.Onlysuccessfulmatingexperimentsaredisplayedbycolouredconnecting linesbetweenoppositematingtypeisolates(differentcolourscorrespondtohybridsbetweendifferentspecies);greydashedlinesindicateproductionofinfertileascomata;remain-ingmatingexperimentswerenegative.BoxplotandviolingraphswerecreatedinR3.3.4(RCoreTeam2015)withpackageggplot2 (Wickham2009)andshow the differences between the width and height of ascospores of particular species andtheirhybrids.Differentlettersabovetheplotindicatesignificantdifference(P<0.05)inthesizeoftheascosporesbasedonTukey’sHSDtest.Boxplotsshowmedian,interquartilerange,valueswithin±1.5ofinterquartilerange(whiskers)andoutliers.
Fig. 9AscosporemorphologyofinterspecifichybridsbetweenA. udagawaeandotherspecies.a–g.HybridofA. udagawaeCMFISB2190× A. acrensis IFM57290;a–b.ascosporesinlightmicroscopy;c–g.ascosporesinscanningelectronmicroscopy;h–r.hybridofA. udagawaeCCF4479× A. felis NRRL62901;h–k.ascosporesinlightmicroscopy;l–r.ascosporesinscanningelectronmicroscopy;s–v.hybridofA. udagawaeIFM46972T × A. wyomingensis CCF4411;s–t.ascosporesinlightmicroscopy;u–v.ascosporesinscanningelectronmicroscopy.—Scalebars:a–b,h–k,s–t=5μm;c–g,l–r,u–v=2μm.
162 Persoonia–Volume41,2018
A. felisA. wyomingensis
A. felis × A. pseudoviridinutansA. wyomingensis × A. felis
Asco
sp
ore
bo
dy w
idth
(μm
)
Asco
sp
ore
bo
dy h
eig
ht
(μm
)
3
4
5
6
2
5
4
3
a b c ba b c b
IFM 47045T
CBS 121595
CCF 5632
CCF 4169
CCF 4417T
IFM 57291T
CCF 4961
IFM 61334T
IFM 59922
NRRL 62904T
IFM 59502
CCF 5610
NRRL 62901
CCF 5625
IFM 60053
CCF 4148
CCF 5617
CCF 5627
IFM 61362
CBS 142233T
CBS 142234
IMI 182127
IMI 133982
IFM 57290
IFM 61345
IFM 61338
IFM 57289
CBS 458.75
CCF 4416
CCF 4419
CCF 4959
CCF 4960
CCF 5609
NRRL 62900
CCF 4171
NRRL 62903
CCF 5622
CCF 5619
CBS 130245T
CCF 4497
CCF 4498
FRR 5680
A. felis
A. pseudoviridinutans
A. acrensis
A. wyomingensis
A. arcoverdensis
A. frankstonensis
A. viridinutans
MAT1-2-1MAT1-1-1
Fig. 10 Schematic depiction ofresultsofinterspecificmatingexperimentsbetweenoppositematingtypeisolatesofheterothallicmembersofAspergillus viridinutansspeciescomplexexceptofA. udagawae.Onlysuccessfulmatingexperimentsaredisplayedbycolouredconnectinglinesbetweenoppositemat-ingtypeisolates(differentcolourscorrespondtohybridsbetweendifferentspecies);greydashedlinesindicateproductionofinfertileascomata;remainingmatingexperimentswerenegative.BoxplotandviolingraphswerecreatedinR3.3.4(RCoreTeam2015)withpackageggplot2(Wickham2009)andshowthe differences between the width and height of ascospores of particular species andtheirhybrids.Differentlettersabovetheplotindicatesignificantdiffer-ence(P<0.05)inthesizeoftheascosporesbasedonTukey’sHSDtest.Boxplotsshowmedian,interquartilerange,valueswithin±1.5ofinterquartilerange(whiskers)andoutliers.
Fig. 11AscosporemorphologyofinterspecifichybridsbetweenA. felis, A. pseudoviridinutans and A. wyomingensis.a–e.HybridofA. felis × A. pseudovir-idinutans;a–c.ascosporesofhybridCCF4497×IFM59502inlightmicroscopy;d–e.ascosporesinscanningelectronmicroscopy:CCF4497×IFM59502(d),CCF4171×IFM59502(e);f– l.hybridofA. felisNRRL62900× A. wyomingensisCCF4169;f–g.ascosporesinlightmicroscopy;h–l.ascosporesinscanningelectronmicroscopy.—Scalebars:a–c,f–g=5μm;d–e,h–l=2μm.
Myceliumcomposedofhyaline,branched,septate,smooth-walledhyphae.Conidialheadsgreyishgreen,looselycolumnar,upto140μmlong,15–25μmdiam.Conidiophoresuniseriate,arising from aerial hyphae or the basal mycelium, hyaline to paleyellowishbrown,frequentlynodding,smooth,150–600μmlong;stipes3–5.5(–8)μmwideinthemiddle;vesicleshyalinetogreyishgreen,pyriform,subclavatetoclavate,(6–)9–16(–20)μmdiam;phialidesampulliform,hyalinetogreyishgreen,4.5–6(–7.5)×1.5–2.5(–3)μm,coveringapproximatelytheapicalhalfofthevesicle.Conidiahyalinetogreyishgreen,globose,subglobose to broadly ellipsoidal, smooth-walled to delicately roughened,microtuberculateinSEM,2.5–3×2–2.5μm(mean±standarddeviation,2.8±0.2×2.4±0.2;length/widthratio1.1–1.3,1.2±0.1).Heterothallic,sexualmorphunknown.
Culturecharacteristics(7dat25°C,unlessotherwisestated)—ColoniesonMEAattained51–62mmdiam,sparselylanose,slightly raised, flat, yellowishwhite (ISCC–NBSNo. 92) topalegreen(No.149),noexudate,solublepigmentlightgrey-ishyellow(No.101),reverselightgreenishyellow(No.101)tobrilliantgreenishyellow(No.98).ColoniesonCYAattained33–48mmdiam,floccose,slightlyraised,flattoslightlyradi-allyfurrowed,yellowishwhite(No.92)togreenishwhite(No.153),sporulationinthecolonycentrepalegreen(No.149)togreyish green (No. 150), no exudate, soluble pigment darkgreyishyellow (No.91), reversedeepyellow (No.85), lightolivebrown (No.94) tomoderateolivebrown (No.95)withlightyellow(No.86)margin.ColoniesonCYAat37°Cgrowmorerapidlycomparedto25°Candattained60–70mmdiam,lanose, slightly raised, flat to radially furrowed, white mycelium inmargins,sporulationlightolivegrey(No.112)toolivegrey(No.113),noexudate,nosolublepigment,reversecolourless,moderateyellow(No.87)togreyishyellow(No.90).ColoniesonCZAattained36–48mmdiam,lanose,slightlyraised,flat,yellowishwhite(No.92),noexudate,noorlightgreyishyel-low(No.101)solublepigment,reverselightyellow(No.86),
lightgreenishyellow(101)tobrilliantgreenishyellow(No.98).ColoniesonYESlanose,yellowishwhite(No.92),irregularlyfurrowed,noexudate,solublepigmentbrilliantyellow(No.83),reversebrilliantyellow(No.83).ColoniesonCY20Sattained58–65mmdiam,lanose,slightlyraised,flat,yellowishwhite(No.92),noexudate,nosolublepigment,reversemoderatebrown(No.58)tomoderatereddishbrown(No.43).ColoniesonCREAattained32–35mmdiam,sparsely lanose,plane,mycelium yellowish white, no visible sporulation, reverse strong brown(No.55),noacidproduction.GrowthonMEAat45°C,nogrowthonMEAat47°C. Exometabolites— IsolateIFM57291producedanaszona-pyrone,afumigatin,tryptoquivalines,tryptoquivalones;isolateIFM57290anaszonapyrone, fumagillin, fumigatins,helvolicacid,pseurotinA,tryptoquivalines,andatrytoquivalone;iso-lateCCF4959pseurotinA, viriditoxin and several potentialnaphtho-gamma-pyrones;CCF4960antafumicins,fumagillin,afumigatin,helvolicacid,pseurotinA,andatryptoquivalone;and CCF 4961 an aszonapyrone, fumagillin, fumigatins,pseurotinA,tryptoquivalinesandtryptoquivalones.Ingeneral,similar metabolites are also produced by the two most closely relatedspecies,i.e.,A. aureolus and A. udagawae.Aspergillus aureolus produces fumagillin, helvolic acid, pseurotin A, trypto-
Notes— The morphology of A. acrensis strongly resembles that of several other A. viridinutans complexmembers.ThecloselyrelatedtaxaA. aureolus and A. siamensis are readily distinguished from A. acrensis by the production of ascomata understandardcultivationconditions(botharehomothallic).Aspergillus viridinutans and A. frankstonensis grow more slowly at25°Candhavesmallervesicles.Themacromorphologyofcoloniesandmicromorphologyoftheasexualmorphdoesnotdistinguish A. acrensis reliably from A. arcoverdensis, A. felis, A. pseudoviridinutans, A. udagawae and A. wyomingensis.
Some of these species can be differentiated each from the otherbytheircharacteristicsexualmorph,buttheproductionof ascomata was not induced in A. acrensis despite our at-tempts, similarly to A. arcoverdensis and A. pseudoviridinutans.Although isolate IFM57290was successfully crossedwithisolates of A. udagawaeIFM46972andCMFISB2190in vitro, both the width and height of ascospores were statistically dif-ferent from A. udagawae.Also,abnormalitiesintheshapeandsuperficialornamentation(Fig.9)werepresentinasignificantnumberofspores(equatorialcrestswereabsentin~50%ofascospores).ReliableidentificationofA. acrensis can currently onlybeachievedbymolecularmethods.
Notes— Horieetal.(1995)designatedthespecimenCBM- FA-0711 as a holotype of A. udagawae, a dried culture with ascomata created by crossing the isolatesCBM-FA-0702(MAT1-1-1)×CBM-FA-0703(MAT1-2-1).Althoughthisspeci-mendemonstratesthesexualandasexualmorphof the lifecycle,itisnotsuitableforthepurposesoftherecenttaxonomyforseveralreasons.Firstofall,itisnotclearwhichofthetwocultures contained within the type should be considered the ex-holotypeculture.Additionally, interspecifichybridscanbeinduced by crossing opposite mating type strains of unrelated species in vitro as shown in this study and some previous studies(seeDiscussion),anddepositionofaresultant‘hybrid’typecouldleadtoambiguities.Althoughthissecondargumentdoes not apply to A. udagawae as both isolates included in the holotype are closely related phylogenetically, we believe that amoreclearlydefinedtypeofthisspecieswillfacilitatefuturetaxonomicwork.Becauseitisnotpossibletorecognizewhichpart of the holotype belongs to particular isolate, lectotype designation(inthiscasepartofholotypespecimen)isdifficult.ForthisreasonwedecidedtoselectanepitypePRM945579derivedfromtheIFM46972(=CBM-FA0702)culture.
DISCUSSION
Changing species concepts in the AVSCTheAVSCmembersshowconsiderablephenotypicvariabilitybutusuallyshareproductionofnoddingheads(somevesiclesborneatanangletothestipe)andrelativelypoorsporulationwithabundantaerialmycelium.Allspecieshaveamaximumgrowthtemperatureof42or45°Candthemacromorphology
anddiameteroftheircoloniesaresimilar,exceptforA. viridi-nutans and A. frankstonensis, which grow more slowly than remainingspecies.Inaddition,themorphologyofconidiophoresand conidia is relatively uniform across species, including the superficialornamentationofconidiaasshownhere(Fig.13).ForthesereasonsheterothallicAVSCmembershaveresistedtaxonomicclassificationandwereonlyidentifiedtoaspeciescomplexlevel,untilrecently.Duetotheabsenceoftaxonomicallyinformativecharacters,mostrecentlydescribedspeciesintheAVSCweredelimitedusingtheGCPSRrules.Usingthisapproach,thespeciesarerecognizedbasedonconcordancebetweensingle-genephy-logeniesandtheabsenceoftreeincongruities.TheGCPSRhasfoundwideapplicationinthetaxonomyoffungi(Dettmanetal.2006,Hubkaetal.2013a,Petersonetal.2015,Visagieetal. 2017).Hugeprogresshasbeenmade recently in thedevelopment of statistical methods for multilocus species deli-mitation, driven by advances in the multispecies coalescent model (Bouckaert et al. 2014, Flot 2015, Fontaneto et al.2015,Schwarzfeld&Sperling2015, Jones2017).AlthoughtheideologyofMSCdelimitationmethodsisrelativelysimilartoGCPSR,thesemethodsaremorerobustbecausethespeciesaredelimited in threesteps, i.e.,speciesdiscovery,speciestreeconstructionandspeciesvalidation(Carstensetal.2013).The determination of species boundaries is more objective in contrasttoGCPSRrulesthatarebasedonrelativelysubjectiveevaluationandcomparisonofsingle-genetrees.Inaddition,MSCmethodsareabletodealbetterwithphenomenasuchas incomplete lineage sorting, recombination or non-reciprocal monophyly that lead to incongruences between single-gene trees.Comparedtothephylogeneticanalysisofconcatenatedgenedatasets(includingpartitioneddatasets)andinpartalsoGCPSR,theMSCmethodsarelesspronetoover-delimitationofspecies (Degnan&Rosenberg2006,Kubatko&Degnan2007,Heled&Drummond2010,Rosenberg2013),especiallywhen the results of multiple delimitation methods are compared inoneanalysis.TheGCPSRrulestogetherwithevaluationoflimitedphenotypicdata were recently used for description of A. felis, A. arcover-densis and A. frankstonensisintheAVSC(Barrsetal.2013, Matsuzawaetal.2015,Talbotetal.2017).Genealogicalanaly-sisusingfivegenetic lociwascarriedout fordelimitationof A. parafelis, A. pseudofelis and A. pseudoviridinutans, three close relatives of A. felis (Sugui et al. 2014).Although theauthors found no conflict between single-gene phylogenies, only two isolates of each of these four species were used in analysis,andsequencesofA. felis, A. parafelis and A. pseudo-felis strains includedwerealmost invariable.These isolatesdidnotcoversufficientlythegeneticdiversityofthesespeciesasshownhere.Speciesdelimitation resultsbasedonMSCin this study showed that A. parafelis and A. pseudofelis are included in the genetically diverse lineage of A. felis (Fig.3).
TheintraspecificpairwisegeneticdistancesinA. felis(Table6)rangefrom0.6%(RPB2)to4.2%(benA).Similarly,pairwisegenetic distances in A. udagawae(Table6)are1.1%(benA)to4.9%(act).Suchhighintraspecificdiversityinthesegeneticloci is unusual in Aspergillus and it reflects the intense recom-bination.Thus,whenonlylimitednumberofstrainsfromsuchspecies are selected for phylogenetic analysis, the results of speciesdelimitationtechniquesmaybebiasedandpronetooverestimatethenumberofspecies.Aswehaveshownhere,thiswasclearlythecaseinthestudyofSuguietal.(2014).Thisproblemisprobablywidespreadincurrentfungaltaxonomyand limits possibilities of correct species boundaries delimita-tion.Alsointhisstudy,thenumberofstrainsofsomecloselyrelated and phenotypically similar species is underrepresented, e.g.,A. viridinutans and A. frankstonensis.Inthesecases,thespeciesboundariescannotbe reliablydefinedusingneitherGCPSRrulesnorMSC-basedmethodsusedinthisstudy.
Clinically relevant species and their identification in clini-cal settingAlthoughsect.Fumigati harbours many important pathogenic species,membersoftheAVSChavebeenoverlookedbybothcliniciansandmycologistsuntilrecently.Thepresenceofthesesoil-bornespeciesinclinicalmaterialwasfirstreportedbyKatzetal.(2005)whoexaminedphylogeneticpositionsofseveral‘atypical’(poorlysporulating)clinicalisolatesofA. fumigatus.The majority of these strains grouped with, but were not identi-cal to, A. viridinutans and A. aureolusfromtheAVSC.Sincethen many similar epidemiological and clinical studies have reportedthepathogenicroleofAVSCmembersinhumansandanimals,asreviewedbyTalbot&Barrs(2018).Inhumansthemost common manifestation of disease is chronic invasive pul-monaryaspergillosisinimmunocompromisedpatients.AVSCspeciesarealsofrequentlyreportedasacauseofsino-orbitalaspergillosis(SOA)incatsthatischronic,butfrequentlyfatal.In contrast to humans and dogs, the disease usually affects ostensiblyimmunocompetentcats.ThisincreasinglyrecognisedclinicalentityismostfrequentlycausedbytheAVSCspeciesandlessfrequentlybyothercrypticspeciesinsect.Fumigati (Barrsetal.2012,2013,2014).Basedonthespeciesboundariesredefinedinthisstudy,theAVSCencompasses fourspecies thatareconfirmedoppor-tunisticpathogens.Accordingtoanumberofreportedcases,in humans A. udagawae is the most important opportunistic pathogenicfromtheAVSCfollowedbyA. felis and A. pseudo-viridinutans.Incontrast,SOAincatsismostcommonlycausedby A. felis andmuch less frequently byA. udagawae and A. wyomingensis (Barrsetal.2013,2014).MedicallyimportantspeciesfromtheAVSCdemonstrateele-vatedminimuminhibitoryconcentrations(MICs)ofitraconazoleandvoriconazolein vitro, and a variable susceptibility to ampho-tericinB,whileposaconazoleandechinocandinshavepotentin vitroactivities(Lyskovaetal.2018).SincetheintraspecificvariationinMICsofparticularantifungalsisusuallyhigh,theuseofreliablemethodsforMICdeterminationstakesprecedenceovercorrectidentificationtoaspecieslevel.Thelattermaybechallengingorevenimpossibleintheclinicalsetting.However,identificationtothelevelofspeciescomplexanddifferentia-tion from A. fumigatus is important due to strikingly different antifungalsusceptibilitypatterns.In contrast to A. fumigatus, theAVSCspecies donot growat 47and50°C,usually sporulate lessandaproportionoftheirvesiclesareborneatanangletothestipe.Inaddition,someisolatesproduceacidiccompoundsdetectableonCREA(Barrsetal.2013,Novákováetal.2014,Talbot&Barrs2018).DespitethefactthatITSrDNAregionsequencesarenotavail-able forallAVSCmembers, thisuniversalmarker for fungal
speciesidentificationandbarcodingcanbeusedtoachieveidentificationtoaspeciescomplexlevel.Thesequencesfromallsixprotein-codinggenesincludedinthisstudy(Table2)havesufficientdiscriminatorypowerforspecieslevelidentificationofallclinicallyrelevantspecies.Amongthesegenes,sequencesofβ-tubulinandcalmodulinbelongtothemostcommonlyusedintheclinicalpracticewhencorrectidentificationisrequired(epi-demiological studies, outbreak investigations or when dealing withinfectionsrefractorytoantifungaltherapy).However,thediscrimination between A. felis and A. pseudoviridinutans can belimitedwhenusingtheβ-tubulingeneduetotheincompletelineagesortingphenomenondetectedinthisstudy(Fig.3).Additionally,theincreasinglyusedmethodofmatrix-assistedlaser desorption/ionization time-of-flightmass spectrometry(MALDI-TOFMS)givespromisingresultsforrapidandaccuratediscrimination between A. fumigatus and other clinically relevant aspergillifromsect.Fumigati(Alanioetal.2011,Nakamuraetal.2017).Thedevelopmentofmorerobust,curatedandac-cessibleMALDI-TOFspectrumdatabasesshouldenabletheimplementationofMALDI-TOFMSforroutineidentificationoflesscommonaspergilliinfuture.SeveralPCRassaystargetingprotein-coding or microsatellite loci have also been developed andshowgoodefficiencyindiscriminationoflesscommonpath-ogenicspeciesinsect.Fumigati (Araujoetal.2012,Fernandez- Molinaetal.2014,Chongetal.2017).
Mating behaviour in the AVSC – heterothallic speciesTheincreasingavailabilityofPCR-basedtoolsforidentificationoffungalgenesresponsibleforsexualandsomaticincompati-bilityhasfacilitatedtheabilitytoinducethesexualmorphinfungi(Dyer&O’Gorman2011).Thecharacterisationofmatingtype(MAT)genesbecameroutinewheninducingthesexualmorph of heterothallic species in vitro.Using thisapproach,thesexualmorphhasbeen inducedrecently inat leastfivemembersofsect.Fumigati (O’Gormanetal.2009,Barrsetal.2013,Swilaimanetal.2013,Novákováetal.2014,Hubkaet al. 2017).Thediscoveryof a sexual cycle in pathogenicandmycotoxigenicfungihasmanyimportantconsequences,becausefungiwithafunctionalsexualcyclehavegreaterpo-tential to increase their virulence and to develop resistance to antifungals,fungicides,etc.(Kwon-Chung&Sugui2009,Leeetal.2010,Swilaimanetal.2013).Here,weinducedthesexualmorphwitharelativelyhighrateofsuccess in A. felis, A. udagawae and A. wyomingensis(Fig.6).We demonstrated that ascospores of these three species have relativelystablemorphology(Fig.7)andthatthesizeoftheirascosporesissignificantlydifferentfromoneanother(Fig.6)andcanbedifferentiatedbyequatorialcrestlength(Table5).However,notalloppositematingtypestrainsofthesamespe-cies are able to produce ascomata in vitro as demonstrated in allthreementionedspecies(Fig.6).Asimilardeclineinmatingcapacity was also demonstrated in previous studies on the AVSC(Suguietal.2010,Novákováetal.2014),butalsoinA. lentulus(Swilaimanetal.2013)andA. fumigatus(O’Gormanetal.2009).Thesespeciesrequirerelativelyrigidconditionstocompletetheirsexualcycleandsomecrossesproducelownumbersoforinfertileascomataordonotmateatall(Balajeeetal.2006,Yaguchietal.2007,Kwon-Chung&Sugui2009,Suguietal.2010,Novákováetal.2014).Forinstance,fertilitybetween two opposite mating-type isolates may be influenced by the vegetative incompatibility genes (Olarteet al. 2015),regulators of cleistothecium development and hyphal fusion (Szewczyk&Krappmann2010).Wewerenotabletoinducethesexualmorphinthreeheterothal-licmembersoftheAVSC,i.e.,A. acrensis, A. arcoverdensis and A. pseudoviridinutans, despite the relatively high number of opposite mating-type strains that was available for the mating
assays (Fig.6).Itisnotclearifthesespeciesrequiredifferentconditionsforsuccessfulmating,ifthereareotherunidentifiedpre-zygoticmating barriers between oppositemating typestrains,oriftheyhavelosttheabilitytocompletetheirsexualcycle.TheevidencethattwoofthesespecieswereabletomatewithdifferentspeciesfromAVSCmakesthelastpossibilityim-probable(Fig.8,10).ThesehybridscanbedifferentiatedfromA. udagawae and A. felis,respectively,bytheirdimensions(Fig.8,10)andsurfaceornamentation(Fig.9,11;Table5).Itdemon-strates that both A. acrensis and A. pseudoviridinutans should be treatedasseparate taxonomicentities from their relatedspecies.Similardeviationsinsizeandsurfaceornamentationofascosporesweredemonstratedinotherinterspecifichybrids(Fig.8–11)whentheywerecomparedtoparentalspecies.
Mating behaviour in the AVSC – homothallic speciesAlthoughhomothallicspeciesprevailoverheterothallicinsect.Fumigati (Fig. 1), only twohomothallic species are presentin theAVSC. It is supposed that heterothallism is ancestraltohomothallisminfungi(Nauta&Hoekstra1992), includingAspergillus (Rydholmetal.2007,Leeetal.2010).Itisobviousfrom phylogenetic studies across different subgenera of Asper-gillus, that reproductive strategy is evolutionary conservative andhomothallicaswellasheterothallic(orasexual)speciesaretypicallyclusteredincladeswithauniformreproductivestrategy.For instance insubg.Aspergillus, the31currentlyacceptedspeciesof sect.Aspergillus areall homothallic (Chenet al.2016a)whilesistersect.Restricti encompasses20asexualandonly one distantly related homothallic species, A. halophilicus (Sklenářetal.2017).Similarly,subg.Polypaecilum harbours onlyasexualspecies(Martinellietal.2017,Tanneyetal.2017).Asexualspeciesalsopredominateinsubg.Circumdati(Jurjevićetal.2015)althoughmost,ifnotall,probablyhaveacrypticsexualcycleashighlightedbysexualmorph induction inA. flavus, A. nomius, A. parasiticus, A. terreus and A. tubingen-sis (Hornetal.2009a,b,2011,2013,Arabatzis&Velegraki2013).Astrikinglydifferentsituationispresent insubgeneraNidulantes(Chenetal.2016a,Hubkaetal.2016a),Fumigati (Fig.1)andCremei (Hubkaetal.2016b) where heterothallic and homothallic species interchange like a mosaic along the phylogenetictree.Common genetic distances between closely related sister spe-ciesacrossaspergilliusuallyrangebetween2–4%inbenA and CaMlociand1–2%inRPB2locus;thesituationinAVSCisverysimilar(Table7).Interestingly,thereareonlyfewexamplesofcloselyrelatedhomothallicandheterothallic/asexualspeciesin Aspergillusdespite their commonorigin.Geneticsimilari-ties between related couples of homothallic and heterothallic/asexualexceeding95%arerare,withonlytwoexamplesinsubg.Circumdati andoneinsubg.Cremei(Table8).SectionFumigatiisexceptionalbecauseitcontainsatleastfivepairsofhighlyrelatedhomothallicandheterothallicspecies(Table8;Fig.1).Aspergillus acrensis, described here, and A. aureolus represent the most closely related pair across genus Aspergil-lus(Table8)andthuscouldbeanidealmodelforstudyingtheevolutionofreproductivemodes.Ifweacceptthehypothesisabout the derived origin of homothallic species, it is probable that A. aureolus evolved in the lineage of A. acrensis rela-tivelyrecently,duetotheextremelylowgeneticdistancesofbothspecies.Thisisalsolikelythereasonwhythemultilocus speciesdelimitationmethodSTACEYandalsosomesingle-locus methods failed to segregate A. acrensis from A. aureolus (Fig.2)inthisstudy.
Interspecific hybridization in fungi and its consequencesInterspecifichybridizationisanimportantprocessaffectingspe-ciation and adaptation of micro- and macroorganisms, however, S
relativelylittleisstillknownaboutthefrequencyofhybridiza-tioninfungianditsroleinevolutionoffungalspecies.Fungalhybridsmayformeitherbyapartialorcompletesexualcycleorbyaparasexualprocess.Matingbetweentwospeciesmaybepreventedbypre-zygoticbarriers(e.g.,gameterecognition)and variouspost-zygotic barriers (developmental problems,hybridviabilityandabilitytoreproduce,etc.).Thedisagreementbetween phylogenetic/morphological species concepts and bio-logical species compatibilities has been repeatedly described infungi.Phylogeneticdivergenceinsomefungalgroupsmighthave preceded development of reproductive barriers as shown byinterspecifichybridsinduced in vitro between primary hu-man and animal pathogenic Trichophytonspecies(Kawasakietal.2009,2010,Anzawaetal.2010,Kawasaki2011),op-portunistic pathogenic Candida albicans and C. dubliniensis (Pujol et al. 2004),members ofAspergillus sect.Fumigati (Suguietal.2014,Talbotetal.2017),mycotoxigenicA. flavus and A. parasiticus(Olarteetal.2015),A. flavus and A. mini-sclerotigenes(Damann&DeRobertis2013),phytopathogenicspecies from the Fusarium graminearumcomplex(Bowden&Leslie1999)andspeciesofNeurospora(Dettmanetal.2003).Naturalinterspecifichybridsresultingfromrecombinationbe-tweenspeciesorparasexualreproductionaremostcommonlyreported andhavebeenextensively studied in saprophyticyeasts(Gonzálezetal.2008,Sipiczki2008,Nakaoetal.2009,Louisetal.2012), theplantendophyteEpichloë (Coxetal.2014,Charltonetal.2014,Shymanovichetal.2017)andinvarious plant pathogenic fungi including species of Fusarium graminearumcomplex(O’Donnelletal.2004,Starkeyetal.2007),Ophiostoma (Brasier et al. 1998,Solla et al. 2008),Microbotryum (Gladieuxetal.2010),Melampsora (Spiers&Hopcroft1994,Newcombeetal.2000),Botrytis (Staatsetal.2005),Verticillium(Inderbitzinetal.2011)andHeterobasidion (Gonthieretal.2007,Lockmanetal.2014).
Considering that in vitro induction of hybrids is relatively suc-cessful, it is surprising that reports on the isolation of naturally occurringhybridsareinfrequentinhumanandanimalpatho-genicfungi.Itsuggeststhatpost-zygoticmatingbarriersplayafundamentalroleinthemaintenanceofspeciesboundaries.Naturally occurring hybrids have been detected in yeasts and dimorphic fungi, including between Candidaspp.(Schröderetal.2016),Malassezia spp.(Wuetal.2015),Cryptococcus neo-formans and C. gattii (Boversetal.2006,2008,Kwon-Chung&Varma2006,Aminnejadetal.2012)andCoccidioides immitis and C. posadasii (Johnsonet al. 2015).However, to date,reportsonthesehybridsinfilamentousfungiarerestrictedtothe Neocosmospora solanicomplex(Shortetal.2013,2014).Speciesdefinitionhasbecomeacontroversialissueinsomeof thesespeciescomplexeswithnaturallyoccurringhybridsbecause of differing opinions on species concepts among taxonomists (Kwon-Chung&Varma2006,Kawasaki 2011,Kwon-Chungetal.2017).Evenincaseswhereinterspecifichybridswithhighfitnessandfertility can be demonstrated, the intensity of gene flow between naturalpopulationsmustbesufficienttoopposegeneticdriftinordertohaveasignificantimpactongeneticisolationofspecies.In fungi, these processes cannot be evaluated rigorously by in vitromatingassays,asthesecannotbeextrapolatedfullytoanaturalsetting(Starkeyetal.2007,Suguietal.2014,Hubkaetal.2015a).Indeed,naturalinterspecifichybridshaveneverbeenreported for the majority of species that readily produce hybrids in vitro, including Aspergillus, dermatophytes and Neurospora.TheMSCandGCPSRapproachesprovidepracticaltoolsfor evaluatingthesignificanceofgeneflowbetweennaturalpopu-lations and for assessing species limits.The interpretationof in vitro mating assays without a robust phylogeny is thus controversial, because a number of clearly phylogenetically, morphologically and ecologically distinct species lack effective
subg.Aspergillus A. halophilicus(Restricti)–anyspecies ≤89 A. montevidensis(Aspergillus)–anyspecies ≤88
subg.Circumdati A. alliaceus(Flavi)–A. lanosus 96.4/95.7/99.1 A. muricatus(Circumdati)–A. ochraceus ≤91 A. neoflavipes(Flavipedes)–A. micronesiensis 94.8/91.9/97.5 A. neoniveus(Terrei)–anyspecies ≤90
subg.Cremei A. chrysellus (Cremei)–A. wentii 97.1/97.2/97.7 A. cremeus (Cremei) – any species ≤91 A. stromatoides (Cremei) – any species ≤93
subg.Fumigati A. acanthosporus(Clavati)–A. clavatus ≤93 A. aureolus(Fumigati)–A. acrensis 99.6/98.8/99.0 A. cejpii(Clavati)–anyspecies ≤88 A. fischeri(Fumigati)–A. fumigatus 94.3/94.5/97.9 A. posadasensis(Clavati)–A. clavatus 95.1/92.6/93.5 A. quadricinctus (Fumigati)–A. duricaulis 92.6/95.0/99.1 A. siamensis(Fumigati)–A. wyomingensis 97.1/96.5/98.9 A. waksmanii (Fumigati)–A. nishimurae 97.8/98.4/96.6
subg.Nidulantes A. discophorus (Nidulantes, A. aeneus clade)–A. karnatakaensis ≤92 A. falconensis (Nidulantes, A. nidulans clade)–A. recurvatus ≤93 A. monodii (Usti)–anyspecies ≤90 A. nidulans (Nidulantes, A. nidulans clade)–anyspecies ≤92 A. pluriseminatus (Nidulantes, A. multicolor clade)–anyspecies ≤92 A. purpureus (Nidulantes, A. spelunceus clade)–anyspecies ≤90 A. undulatus (Nidulantes, A. stellatus clade)–anyspecies ≤891Ifnoneofthreegeneticsimilaritiesexceed95%,thevaluesarereplacedbyonlyonehighestvalue(usuallyRPB2locus).
reproductivebarriers.Inaddition,theevaluationofbiologicalspecieslimitsusingmatingassaysrequiresdeterminationofthefitnessandfertilityofprogeny,whichisdemandinginbothtimeandcost.In general, mating success between different species under laboratoryconditionsismuchlowercomparedtointraspecificmating, suggesting strong reproductive isolation between speciesandadherencetothebiologicalspeciesconcept.Inagreementwiththis,onlyalimitednumberofstrainswithex-ceptionalmatingcapacityareusuallycapableofinterspecifichybridizationwithstrainsofdifferentspecies,e.g.,A. udagawae strainIFM46972(Fig.8)orA. pseudoviridinutansstrainIFM59502(Fig.10).Severalstudiesdemonstratedthatinterspecifichybridsexpressgeneticabnormalitiesorhavedecreasedfertilityandviability.GeneticanalysisoftheprogenyofacrossbetweenF. asiaticum × F. graminearum detected multiple abnormalities that were absent in intraspecific crossesofF. graminearum, i.e., pro-nounced segregation distortion, chromosomal inversions, and recombinationinseveralstudiedlinkagegroups(Jurgensonetal.2002,Galeetal.2005).MatingsbetweenC. neoformans × C. gattiiproducedonlyalowpercentageofviableprogeny.Ithas been suggested that C. neoformans and C. gattii produce onlystablediploidhybrids,butnottruerecombinants(Kwon‐Chung&Varma2006).AlthoughOlarteetal.(2015)obtainedhybrid progeny of A. flavus and A. parasiticus, fertile crosses were rare and involved only one parental strain of A. flavus.Viableascosporeswereextremelyrare,suggestingextensivegeneticincompatibilityandpost-zygoticincompatibilitymecha-nisms.Morphologically, the progeny differed fromparentalstrains in growth rate, sclerotium production, stipe length, co-nidialheadseriationandconidialfeatures(Olarteetal.2015).Decreasedviabilityofhybridascosporeswasalsodetectedamong Neurospora spp.(Dettmanetal.2003)andinAspergil-lussect.Fumigati, in addition to abnormalities in their surface ornamentationvisualisedbySEM(Suguietal.2014),whichisinagreementwiththepresentstudy(Fig.9,11).Apartfromas-cosporeornamentation,wealsofoundsignificantdifferencesinhybridascosporedimensionsfromparentalspecies(Fig.8,10).Therelativelyrecentglobalizationoftradeinhorticulturalandagricultural plants, and introduction of non-native plant species has resulted in the inadvertent introduction of alien plant patho-gens into non-endemic areas, contributing to the emergence of somedevastatingplantdiseases(Brasier2001,Mehrabietal.2011,Dickieetal.2017).Anthropogenicactivitiesorchangesinthedistributionoffungi(e.g.,inresponsetoclimatechanges)may bring together related, previously allopatric pathogenic species.Subsequentinterspecifichybridizationcouldgiveriseto pathogens with new features, including adaptation to new niches and host species, and varying degrees of virulence, as evidenced in Verticillium longisporum, Zymoseptoria pseudo-tritici, Blumeria graminis f.sp. triticale, and hybrids between Ophiostoma novo-ulmi and O. ulmi(Brasier2001,Schardl&Craven2003,Depotteretal.2016).As far as we know, the occurrence of Aspergillusinterspecifichybrids in nature has not been proven despite successful hybri-dizationofsomespeciesin vitro.However,thereisnoreasontoassumethatthisphenomenondoesnotoccuroccasionally.Geneticrecombinationsimilartothatfoundinintraspecificmat-ing occurred in half of the progeny produced by mating A. fumi-gatus with A. felis, while the other half were probably diploids or aneuploids(Suguietal.2014).Progenyresultingfrommatingbetween A. flavus and A. minisclerotigenes was fertile when crossedwithparentalstrainsandthefrequencyofsuccessfulmatings was similar to that within pairs of A. flavus and A. mini- sclerotigenes strains, respectively (Damann&DeRobertis
2013).Ultimately,theviablehybridmustpresentsomecharac-teristicsthatpromotesitssurvival(Turneretal.2010,Mixão&Gabaldón2018).ForinstanceOlarteatal.(2015)showedthatsome F1 progeny of A. flavus × A. parasiticus produced higher aflatoxinconcentrationscomparedtomidpointparentaflatoxinlevels,andsomehybridssynthesizedGaflatoxinsthatwerenotproducedbytheparents.Thissuggestedthathybridizationisanimportantdiversifyingforcegeneratingnoveltoxinprofiles(Olarteetal.2015).Althoughinterspecifichybridizationinas-pergilli is a relatively newly discovered phenomenon, it is likely tohaveplayedanimportantroleintheevolutionofthegenus.Therelationshipbetweenhybridizationandchangesinvirulencepotential is not well understood in human and animal fungal pathogens but its role in the emergence of novel plant fungal pathogensiswelldocumented,asdiscussed.Theevidenceofbiological compatibility between major pathogens in Aspergillus sect.Fumigatishedsnewlightonpossibleinterspecifictransferof virulence genes, genes responsible for antifungal resist-ance, and other genes influencing adaptation of these fungi to achangingenvironment.Furtherstudiesshouldelucidatetowhatextentinterspecifichybridizationshapedtheevolutionoftheseopportunisticpathogens.
CONCLUSIONS
Based on consensus results of species delimitation methods and after evaluation of mating assay results and phenotypic data, wenowrecognise10specieswithintheAVSC.Thisnumbercomprises nine previously recognised and one new species proposedhere.Aspergillus pseudofelis and A. parafelis are placed in synonymy with A. felis.AllfourgeneticlociusedforphylogeneticanalysisacrosstheAVSChavesufficientvariabilityforreliablespeciesidentificationandcanbeusedasDNAbar-codes.Thoughmorelaborious,theMSCareasuitabletoolfordelimitation of genetically diverse cryptic species in cases where classical phylogenetic, morphological and mating compatibility datadonotyieldsatisfactoryresults.
Acknowledgements This research was supported by the project of the CharlesUniversityGrantAgency(GAUK1434217),CzechScienceFoun-dation (No. 17-20286S), CharlesUniversityResearchCentre programNo.20406,theprojectBIOCEV(CZ.1.05/1.1.00/02.0109)providedbytheMinistryofEducation,YouthandSportsofCRandERDF,andbyaThomp-sonResearchFellowshipfromtheUniversityofSydney.WethankMiladaChudíčkováandAlenaGabrielová for their invaluable assistance in thelaboratory,CCFcollectionstaff(IvanaKelnarováandAdélaKovaříčková)forlyophilizationofthecultures,MiroslavHylišforassistancewithscanningelectronmicroscopy,StephenW.Peterson,KyungJ.Kwon-Chung,AdrianM.Zelazny,MariaDoloresPinheiroandDirkStubbeforprovidingimportantculturesforthisstudy.VitHubkaisgratefulforsupportfromtheCzechoslovakMicroscopySociety(CSMSscholarship2016).
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