Temporal Isolation Explains Host-Related Genetic Differentiation

Temporal isolation explains host-related genetic differentiation in a group of widespread mycoparasitic fungi Levente Kiss, Alexandra Pintye, Gabor Kovacs, Tünde Jankovics, Michaël Fontaine, Nick Harvey, Xiangming Xu, Philippe C. Nicot, Marc Bardin, Jacqui Shykoff, et al.

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Levente Kiss, Alexandra Pintye, Gabor Kovacs, Tünde Jankovics, Michaël Fontaine, et al.. Tem- poral isolation explains host-related genetic differentiation in a group of widespread mycoparasitic fungi. Molecular Ecology, Wiley, 2011, 20 (7), pp.1492-1507. 10.1111/j.1365-294X.2011.05007.x. hal-02329042

HAL Id: hal-02329042 https://hal.archives-ouvertes.fr/hal-02329042 Submitted on 29 May 2020

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., Keywords: biotrophicpathogens appears toresult from powdery mildewspecies theywere unlikely to explained bynarrowphysiologicalspecializati in Ampelomyces cannot be differentiation genetic host-related the Thus, occurred. always cross-infections spring, andtostrainsnaturallypresentinother mycohost speciesinautumn, experimentally exposedtoAmpelomyces strain epidemics mainlyinsummer andautumn. epidemics occur before summer, the funga leucotricha startsitslifecycleearlyin powdery mildewspeciesacrossEu Ampelomyces fromother highly differentiated populations foundinappl Internal transcribed spacer sequences andmicr Ampelomyces depends onthelife cyclesof thepowderymildew speciesthey parasitize. powdery mildew fungi,themselves plantpa among sympatricpopulationsof couldberesponsible isolation whether temporal plants andparasites,bu emergence ofnewdiseases via pathogens ondifferenthostsis France Montfavet, UR407,UnitédePathol UK,**INRA, ME19 6BJ, Understanding the mechanisms Abstract Road,BrightonBN13AG,UK, Windlesham UMR80 CNRS; andAgroParisTech, 1117 Budapest, Hungary, ‡Laboratoire Ecologie, Systém Plant Anatomy, EötvösLorándUniversity,Pázmány PéterSétány1 of †Department Hungary, InstituteoftheHungarianAcad *Plant Protection GIRAUD‡ PHILIPPE C.NICOT,**MARCBARDIN JANKOVICS,* MICHAEL C.FONTAINE LEVENTE KISS,*ALEXANDRAPINT fungiwidespread mycoparasitic Temporal isolation explains host-related geneticof group a in differentiation Version définitivedumanuscritpubliédans 1507 differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), , DOI: 10.1111/j.1365-294X.2011.05007.x. Thedefinitive version 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x temporal isolation. Comment citer cedocument: t is probably one of the least studied speciation processes. We studied studied We processes. speciation studied least ofthe one probably is t e powdery mildew ( , isolationintime, speciati / Finalversionofthemanuscriptpublishe of fundamental importance, esp host shifts.Temporal isolati

Ampelomyces, widespread intracellular mycoparasites of of intracellularmycoparasites widespread Ampelomyces, 79, Orsay Cedex F-91405, France, §Genetic Marker Services, 7 Services, Marker §Genetic France, 79, OrsayCedexF-91405, responsiblefor rope, infecting plant hosts other than other hosts plant infecting rope, apple. While P. – East MallingResearch,NewRoad,EastMalling,KentEast spring, and the main apple powdery mildew mildew spring, andthemain applepowdery ,** JACQUIA.S emy of Sciences, P.O. Box 102, H-1525 Budapest, P.O.Box102,H-1525Budapest, emy ofSciences, YE,* GÁBORM.KOVÁCS,*†TÜNDE on, becauseAmpelomyces wereabletoinfect ,‡ NICKHARVEY, osatellite markers show l hostsoftheotherAmpelomyces cause When two powdery mildewWhen powdery two species were differentiation ofgenetic maintenance for the Podosphaera leucotricha thogens. Thetiming oftransmission of have encountered in nature, but instead s naturallyoccurringin is available atwww3.interscience.wiley.com. populations sampled from several other other several from sampled populations ogie Végétale, Domaineogie Végétale, StMaurice,84140 atique et Evolution, Université de Paris-Sud; Paris-Sud; de Université Evolution, et atique on, tri-trophicinteractions divergence andsp on hasbeenrepo d in : Molecular Ecology, 2011, 20:7,149 ecially inthe HYKOFF‡ andTATIANA § XIANGMINGXU, ed that Ampelomyces Ampelomyces ed that P. leucotricha in ) were genetically ) weregenetically ecialization of context of the rted inafew

⁄ C, H- 2- – Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., published online, maintained. are and formed how discrete and distinct clusters have been 2009); (iii)ifthereisnosex, one must explain occurs, identify barriers togene flow, andth (Taylor 1999;Giraudetal.2008a);(ii)ifsex fungi pathogenic in especially information, ofextinctions random processes et despite lackofhomogeni al. 2007), (Fontaneto species discrete form rest ofthegenomewill although selection can maintain differentiation atlociinvolvedinadaptation(Levene1953),the Gi 1996; al. et in sexualorganisms (Johnson adaptations areseldom sufficien allowing exploitationofnovelhosts,butthese adaptations new require often must diseases ofnew emergence major challengeinevolutionarybiologyandisespecially relevant in the context of the a is parasites these of speciation and evolutionary mechanisms responsible for specialization underlying the Understanding 1998). al. et Meeûs (de parasites among phenomenon The existenceofclos Introduction Version définitivedumanuscritpubliédans species and species exis Ampelomycesspecies distinct species worldwide (Falk etal information). life cycle,including short-andlong- distancedi parts of host plantsin thewhite powdery mildewpatches mycelium (Kiss stageofthesefungiremains2008). Thesexual mildewthe powdery conidiophores Am of pycnidia 1998, 2001).Massesofintracellular interaction takes place exclusivelyonaerialplantsurfaces,facilitating direct observation (Kiss dicotyledonous plantspecies,includingimportant crops (Kiss et al. of powderymildew f The genusAmpelo questions. these investigating ofthe theobservation recombination; presence look for footprints to of markers molecular using by regularly, determine occurs whether sex distri continuous than organisms isunderstandin asexual plan todifferenthost clonal lineagesadapted Giraud et al. 2008a). 2005; al. et (Fisher genome cannot break associ recombination same host. Second, in asexual the species, to adapted individuals other with mate ma must parasites these 2008), because Giraud reproduc pleiotropically causing many fungibelongingtotheAsco between developmentonthehostandmating,suchas disperse cannot that parasites sexual cycles thatin allow simpleFirst, wholegenome. overthe adaptation tocausedifferentiation (T emergence of times different with parasites partner choice, host choice for parasites that mateisolationfortemporaland hosts their within such asallopatry flow mustceaseoverlargeparts, gene formation, differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), Mycoparasites, i.e.fungipa The natural occurrence of Ampelomyces was reported in more than 66 powdery mildew mildew powdery 66 than more reported in was ofAmpelomyces occurrence The natural To understandtheformation ofhost-specificpa ⁄ or the host plant species on which they onwhich plantspecies or thehost DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi will cause this, bu this, cause will ungi (theErysiphales) butions ofphenotypes butions Comment citer cedocument: ely related species parasitizing different hosts is a widespread widespread a is hosts ely related species parasitizing different stillhomogenizeunless zing geneflowincludetheexistence of discrete ecological niches and via host shifts. The formation of sibling species on different hosts hosts different on species ofsibling Theformation shifts. host via of intermediate genotypes ofintermediate rasitizing other fungi, pro- vide an excellent model for for model excellent rasitizing an other fungi, pro- vide / Finalversionofthe manuscriptpub . 1995;Kiss1998),but tive isolation(Giraud t to generate reproductive isolation that willallow speciation reproductive generate to t

ation between the genes under selection and the rest of the the of rest the and ation betweenthegenesunderselection mycota, adaptation canadaptation mycota, act as a ‘magic trait’ (Gavrilets t and, ifso, whethert these ar g how discrete entities that we recognize as species, rather asspecies,rather we recognize that entities discrete how g , are sometimes visible evento , aresometimes visible t socanintrinsicbarriersto ⁄ genotypes, exist. , well-known pathogens of monocotyledonous and ⁄ absence of the sexual fruiting bodies is not reliable absence ofthesexualfruitingbodiesisnotreliable héron & Combes 1995). Some parasiteshavelife Combes & héron ere may bemultiple barriers(de Vienneetal. te on the host where they grew, they can only only te onthehostwheretheygrew,can raud 2006a,b; Giraud et al. 2008b). Indeed, al. et Giraud 2006a,b; raud the lack of dispersal among different hosts and spersal, issummarized inFig. S1(Supporting nitive version is available at that covers the leaves myces consists of intracellular mycoparasites myces consistsofintracellularmycoparasites barriers to gene flow exist. For species species For exist. flow gene to barriers Adaptation can thus cause the formation of of Adaptation can thus cause the formation if not all, of the ge and thehypo ts. The conceptual difficultyposedby ts. Theconceptual pelomyces, mostly produced in the cells of of cells the in produced mostly pelomyces, thogen species, we therefore have to: (i) were found (Park et were et al.2006,2008a, ⁄ phenotypes (Coyne phenotypes itisstillunclearwhether several lished in : Molecular Ecology, 2011, Asexual organisms doappearto theses invokedto e specialized to themycohost geneflow, in the naked eye asbrownish eye naked the www3.interscience.wiley.com. nome. Extrinsic barriers , stems and other aerial , stems andotheraerial 2004; Kiss 2008). This 2004; Kiss2008).This b, 2010; Le Gac & Gac Le b, 2010; &Orr 2004). al.2010). Cross- unknown. Their unknown. Their cluding mating explainthis Article first 2004),

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., structure and host-related differentiation. In parallel, we conducted field experiments to assess assess Inparallel,weconducted fieldexperimentsto differentiation. host-related and structure markers (Harvey to sequences ITS and 2006) are cross-infectio or specificity restriction host-related of gene the flow? in (v) Do the role geneticall a play isolation temporal Can (iv) dispersal at a European scale? genetic in role a (iii) play Can isolation spatial ( locations? inthesame many othermycohosts) mycoparasites isolated in spring (mostly APM isolates)and autumn (isolatescoming from addressed thefollowing (Gavrilets &Losos2009; speciation that remains oneof themost ecological of understanding our in advance important an represents byhostphenology could beisolated elucid Therefore, divergence. allow enough to alone it if can and be, be may strong mechanism this general how 2003), we do not know Montarry etal.2008,2009)andphytophagousinsect 2007; al. et Putten (Stamm (Van 1983; fungi Devaux & pathogenic flow inschistosomes (Théron &Combes 1995),plant plants gene reducing in role a flowering plays in isolation temporal that Lande 2008) and there mechanism is some evidence speciation as a sympatric isolation al et the season(Szentiványi in spring andearlysummer, whilemost otherpowdery isolat bytemporal lineages Ampelomyces other possibility that the APMAmpelomyces lineage isadistinct cryptic species separated from other powderymildew fungithat the APMAmpelomyces extensivel lineage to diverge isolation ofAmpelomyces or on specialization 2005). mildew, caused by Golovinomycesfollowin orontii, Ampelomyces ITShaplotypeinnature particular Curious 2005). al. et (Szentiványi APMstrains inafew onITSdata based this wasonlyshown However, species. host other Ampelomyces infecting APMinnatureisdis 2005; Park etal. 2010). Such high genetic differ strain isolated from been exclusively inAPM,having of the most closely related ITS haplotype. Furthermore, almost found was haplotype this studied APMstrains sequences ofalltheEuropean strains obtained from leucotricha). byPodosphaera mildew caused apple powdery (APM, The mildew ITS powdery the species nor thehost plant species from whichtheycame,with one exception:neither Ampelomycespredicted 1995; Nilssonetal.2008).Ampelomyces ITShaplotypes published online, usually lessthan5–10 f ascomycetous other In 2010). al. et 2007; Park sequence divergencevaluesforthissamegeneticmarker as high 10–15 as show can species mildew isol strains Ampelomyces hand, other On the et hosts indistantpartsoftheglobe(Szentiványi (rDNA) havebeencollectedfrom genotypes for their internal transcribed spacer 2007; Liangetal. al. 2004;Szentiványiet2005; readilycan host infectmildew powdery given other host species (Sztejnberg a et al. 1989;from Kiss et have repeatedlyshownthatAmpelomyces mycoparasites collected experiments inoculation Version définitivedumanuscritpubliédans differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), To test the hypothesis of the existence ofcryptic ofthe existence thehypothesis To test Why shouldtherebean isolatedlineageof % (Kiss & Nakasone 1998; Sullivan & White 2000; Szentiványi etal.2005;Liang & White 2000;Szentiványi Sullivan 1998; Nakasone & (Kiss DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi Podosphaera pannosa Podosphaera pannosa Comment citer cedocument: % Giraud et al. 2010). 2010). al. et Giraud questions: (i) Isther (i) questions: for ITSsequencesalthoughthisvari ns possible? To address thesequestions, weused microsatellite address To possible? ns / Finalversionofthe manuscriptpub . 2005).Althou

share a commona share geographical range? Here, we explore the ly, despitetheapparent detectedonlyinoneEuropeanand Korean non-APM severalpowderymildew species infecting different plant infecting other rosaceous hosts (Szentiványi et (Szentiványi hosts rosaceous other infecting al. Ampelomyces on APM, but but APM, Ampelomyces on y distinctmycoparasites ating whether sympatric ating whethersympatric investigate the mode of reproduction, of population mode the investigate e genetic differentiation between differentiation genetic Ampelomycese other powdery mildew hosts? What has allowed allowed has other powderymildew What hosts? tinct from congenericmy ii) Do these mycoparasites reproduce sexually? sexually? reproduce mycoparasites ii) Dothese gh theoretical studiesha , APM strains readily infectedtobacco, APMstrains powdery (ITS) region of the nuclearribosomal ofthe DNA region (ITS) ated from the samepowdery different or differentiation, i.e. differentiation, are there apparent limits to ion. APM usuallycomple nitive version is available at entiation in natural populations suggests that that suggests populations innatural entiation so farwere identical and divergent from those al. 2005;Lianget20 controversialtopicsin species separated byte species mildewspeciesstart ungi, divergence among sister speciesis ungi, divergenceamong sister Kiss 2008).Infact, strainswith identical g artificialinoculation(Szentiványi et al. s (Berlocher&Feder 2002;Thomas etal. y geneticallydespiteit almost strict host host strict almost lished in : Molecular Ecology, 2011, es amonggroups(Seifert et al. www3.interscience.wiley.com. species ofAmpelomyces show physiologicalhost ve proposed temporal ve proposedtemporal no clear evidence for evidence no clear coparasites infecting studies ofspeciation 07; Park etal.2010). their lifecyclelater mporal isolation,we tes its lifecycle in its tes specificity ofthis s abilitytoinfect Article first

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., phylogenies, maximum parsimony (MP) analys (MP) parsimony maximum phylogenies, in deposited and 2002) (Filatov 2.9 PROSEQ (Corpet 1988). The alignments Multalin using Multiple alignmentsof homologous ITSseque sequences onITS based analysis Phylogenetic 2.9 (Filatov 2002). usingPREGAP4 electropherograms carried outasreportedinSz reaction (PCR) amplification a obtained from appleleaf sample DNA while used todetermineprofiles, both the rDNA ITSsequence and themicrosatellite pycnidia using a DNeasy Plant Mini Kit (Qiagen). DNAextractedfrom cultivatedstrainswas Ampelomyces strain and from 5to10mg drie Total genomic DNAwasextractedfrom 10to15mgfreeze-dried mycelium of each DNA extractionandsequencing Supporting information) for which Ampelomyces c dissecting mycelia mildew Th storage. their before powdery microscope the in pycnidia theAmpelomyces inculture.ThepresenceofAmpelomyces isolating without below, described collected from apple treesacross Europe. These we Additional powderymildew-infected Apple leafsamples below. described as mycoparasitism These leaves were also stored as herbarium materials and used to assess the intensity of from powderymildew-i mycoparasites Ampelomyces using lightmicroscopy if necessary. werepres isolated were strains field. the detected in species isolated Ampelomyces mycoparasites whenever every plant species present inthe surveyed on infections mildew powdery for searched Europe. Duringfieldsurveys,wehavealways Lycium halimifolium, across frommanyand 120strains otherspeciesoftheErysiphales powderym common 264 strainsfroma Kingdom thathadbeensampled foraprevious published online, fromthesame apple trees fro collections new Supporting information). Ofthese, we et al.2005;Liang2007),and645strains et al.1995;Kiss&Naka (Falk studies previous Eighty-nine Ampelomycesstrain Ampelomyces strains Materials andmethods specialization, inthe genus Ampelomyces. APM strains,butnoevidenceofmycohost physiological between recombination of evidence differen genetic high find would we isolation, APM belongedtoacr in strains the degreeofphysiologicalhost Version définitivedumanuscritpubliédans differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), below, outlined experiments field ofthe course the Over we repeatedly isolated DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi Comment citer cedocument: Powdery mildew-infectedleaves of the rDNA ITS region of therDNAITSregion sed, dried and stored as herbarium materials to be re-examined re-examined be to materials sed, driedandstoredasherbarium entiványi etal.(2 / Finalversionofthe manuscriptpub nd sequencingof bot yptic species isolated from cong s wasusedinthemicrosatellite s, isolated from all overfrom obtained were world, the

261 strains were isolated from APM, some isolated from from isolated some APM, 261strainswereisolatedfrom specificity. Ourexpectati and GAP4 (Staden etal. 20 appleleavescarrying Ampe ildew species,Arthrocladiella m areas, fromspring early tiation betweenAPM andnonAPM strains, and TreeBASE (Study ID:S11058).Forinferring d apple leafsample containing Ampelomyces m collectionsitesin sone 1998;Sullivan&White 2000; Szentiványi overing theseleaves nitive version is available at is yielded353apple le thesewerefoundinanypowdery mildew is was carried out out using the PAUP* 4.0b10 carried was is study (Szentiványi etal microsatellite profiles were analysed. were checked andma checked were 005). The sequenceswerecompiledfrom nces obtained from nces obtainedfrom re dried, stored as and theirDNAextracted nfected apple,tobacco re newly isolated in this work (Data S1, S1, (Data work this in isolated newly re h strandsof ther lished in : Molecular Ecology, 2011, from whichtheAmpelomyces on wasthat,ifAmpelomyces eneric species eneric species work only. Polymerase chain chain workonly.Polymerase 00) andeditedusing PROSEQ lomyces mycoparasites were www3.interscience.wiley.com. Hungary and theUnited was confirmed undera until lateautumn,and DNA ITSregionwere GenBank weremade af samples (DataS1, . 2005).We isolated and cucumber leaves. leaves. and cucumber nually editedwith ougeotii, infecting ougeotii, only bytemporal Article first

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., published online, in combination with primer ITS1-F (Gardes forthe phylogenetic produced using thealignment an ITS-specific primer (5'-TGAGTCCAGAGGCATAG-3') was designedBased ontheidentityofITS haplotype ITS this for Development ofITS-specificprimers fortheAPMstrains ITS haplotypesandmycohost species. probable it is that va bychance,indicatingathe associationsareformed observed our specificity extreme more The parasitized. they between associations among strainsbelong distribution togeneratethepr each random table.Wecompared the for value calculated the sum of the standardized square specie Ampelomyces haplotypesovermycohost theoretical We distributionbypermutation. tested for significance against a Chi-square distribution.appropriate an generated therefore We expected values were <5 andseveral evenwere were isolated.Fromthis with mycohost species, retainingonlythosefrom atleastfive which Ampelomyces of the four ITS haplotypes crossed table contingency two-way a strains constructed We information). inrecognized theclade a to phylogeneticcorresponding analysis (Fig. S2, Supporting particular mycohostspeciesusin Ampelomy particular whetherthe assessed We Permutation testbasedonITSsequencedata MEGA 4.0 program (Tam the of Explorer Phylogenetic respectively. searches, heuristic a 100 with analyses tested byMPbootstrap with ‘MULTREES’ on.Thestatisticalsupportsof other weight.The hadequal characters and all parameters were the default the set in of Gaps theprogram package (Swofford 2003). PAUP* Version définitivedumanuscritpubliédans characterized by the allelic richness (mean number of allele per locus, A) and the expected the and A) locus, per allele of number characterized (mean by the allelic richness samples,leaf apple 353 obtained from 25 localities in Europe (Fig. 1).was diversity Genetic We analysedthemicrosat Analyses ofmicr fluorescent labellingas st Ampelomyces of 404other isolated earlier(twoofthese,B24andB55,having the 261newlyisolated [c la radioactive using strains APM 50 of subset fromAPM.PCRamplificisolated AQmalus10 byHarv developed AQmalus11), and We usedfive polymorphic micr Microsatellite genotyping °C insteadof 52°C. except (2005) al. et inSzentiványi described one the to >100 otherAmpelomyces strains.ThePCRprotocolwasidentical and strains contingency value foreachcell[(Observed)Expected) Chi-square asthesumof differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), 33 P]- ATP, as described in Harvey (2006). Subseq DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x osatellite data 10.1111/j.1365-294X.2011.05007.x. The defi Comment citer cedocument: APM strains, 353 apple leaf samples, and38ofthe40APM leafsamples, 353apple APM strains, strains table of observed data, we calculat we data, observed of table described by Giraud ( Giraud by described ellite genotypes of 652 APM isolates, consisting of of consisting ellite genotypesof652 APMisolates, obability thatourobservedmatrix couldhavearisenby chance osatellite markers (designated AQmalus3, AQmalus4, AQmalus8, AQmalus8, AQmalus4, AQmalus3, osatellite markers (designated / Finalversionofthe manuscriptpub rains from our culture collection. runusing were These g apermutation test. Four IT the standardized squared deviations from the expected expected fromthe deviations squared standardized the sequences of all the Ampelomyces strains isolated the from APM, all of sequences

ing to the various ITS haplot ing tothevariousITS ations and allele detections were first carried outina ura et al. 2007) and a text editor. editor. text a and 2007) al. et ura 2004). trees andeditedbyTree were visualized that the primer annealing temperature was annealingtemperature primer the that 60 & Bruns 1993) was test was 1993) Bruns & nd 1000 replicates using heuristic and fast- and heuristic using replicates 1000 nd d deviations from the from d deviations <1, thisstatisticisin nitive version is available at alignment were treated as afifth character, belling, i.e.forwardpr been lost after the ITS work) as well as a total total a as well as work) ITS the after lost been generated 999 random assignments of 999 generated s, respecting rowa s, respecting ces ITS haplotypes wereassociatedwith analysesdescribedab lue comparedthis with uent microsatellite genotyping includedall value from our observed data settothis ey (2006),forgeno the branches ofthe branches the inferred trees were 2 ⁄ Expected]. However, becausemany lished in : Molecular Ecology, 2011, ed the equivalent of a contingency ed the equivalent ofacontingency S haplotypeswere ypes and the mycohost species mycohostspecies ypes andthe www3.interscience.wiley.com. flated andshouldnot be nd column totals,and ed onallthe261APM imers end-labelledwith expected contingency expected contingency typing Ampelomyces ove. This primer used ove. This primer used distribution, the less distribution,theless 299 strainsand definedeach Article first

) on the logarithm on of geographi (Loiselleetal.1995), indentifiedintheAPM strains based on microsate ULTI with theSpstatisticestimatedas)b lengths. We used M L 01–500, 501–1000, 1001– 1500 and 1501–2000 1501–2000 and 01–500, 501–1000,1001–1500 s which draws diagrams s which for each locus (1000 permutations) using using for eachlocus (1000 permutations) OCUS compares the length of the most parsimonious parsimonious most ofthe length the compares ficient of the firstof ficient distance class (Vekemans & itted to randomized datarandomized to itted sets. Deviation from stance (Cavalli-Sforza nitive version is available at (Feil et al. 2004; Spratt eton al. 2004) E nkage disequilibrium (r nkage disequilibrium F rthermore, assuming gene dispersal in two two in dispersal gene rthermore, assuming ij is computed based onallelicfrequencies computed is 2.1 (Agapow & Burt 2001).Departure Burt & 2.1 (Agapow within each distance classes differed eachdistanceclassesdiffered within ion between haplotypes is allowed and and ion betweenhaplotypesisallowed D at various spatial scales, as a means of of means a as scales, spatial at various = 0) was assessed by permuting bypermuting = 0)wasassessed lished in : Molecular Ecology, 2011, even in haploid species such as so used the phylogenetic testfrom so usedthephylogenetic ULTI cal distanceseparatingthe www3.interscience.wiley.com. L OCUS & Edwards 1967) onthe E linkinghaplotypes; is higher with more more is higherwith D 2.1 (Agapow & & 2.1 (Agapow ) corrected for ⁄ (1)F1), with b (1)F1), with Article first PAUP llite

* Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., published online, both sites. the isolated and genotyped same as described were above. At time, wealso isolated and genotyped Ampelomyces mycoparasites from mycoparasites APM the at found, When mycelia. microscope under a fordissecting the examined were presence leaves their of and intracellular laboratory pycnidia of Ampelomyces inthepowdery the mildew to taken sites, both from experiment. Twoweekslater,al parasitized by Ampelomyces by inspectiona under k was It apart. two localities km about 20 serve as ‘traps’forAmpelomyces mycoparasites na xanthii) inthegreenh with2–4fullyRajnai Fu¨rto¨s, expanded leaves (G. powdery orontii) in with mildew infected artificially expandedleaves plants, cv.Xanthi,with4–6fully on tobaccoandPodosphaeraxanthi 2005, 2006, 2008a Transmission ofAPMAmpelomycestoothe Interspecific transmission of Mont the with scores local and global variance autoco anditsspatial between individuals sPCA ordinatesgenotypesalongacontinuum of regarding apopulationmodel, nor does it Team 2010)toanalysethegeneticstructureof Core Ampelomyces from APM using microsatelliteda Development (R R-environment the 2008)andADE4pack (Jombart package ADEGENET S1 (Supportinginformation). The number ofstrainsobtainedin from applepowderymildew (APM,Podosphaer Fig. 1Distributionofsample sitesfrom which Version définitivedumanuscritpubliédans differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), We furtherconductedaprin DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi nd 2009, we tested for transmission of Ampelomyces from APM Ampelomyces of to G. orontii transmission for tested we nd 2009, Comment citer cedocument: ouse, were hung on twoon hung were appleouse, (Malus domestica) trees, cv. Jonathan, to the greenhouse, and 10 potted cucu APM- and non-APM Ampelomyces APM- andnon-APMAmpelomyces / Finalversionofthe manuscriptpub cipal component analysis (P l the exposed tobacco and cucumber plants were collected collected were plants cucumber l the exposedtobacco and i oncucumber (Fig.2).Tenpotte

differentplacesisalso indi e Carlo testsimplemented inADEgenet. assign discrete to individuals s r powdery mildew species inspring. powdery r nown that APM on those trees was naturally trees those on APM that nown Ampelomyces strainswererepeatedly isolated relatedness takinginto accountboththegenetic nitive version is available at artificially infected with powdery mildew (P. (P. mildew powdery with infected artificially a leucotricha)insp dissecting microscope atthebeginning ofthe ta. This approach makes no assumptions assumptions no ta. Thisapproachmakes rrelation. We tested the significance of the turally occurringturally in APM. This was done at age (Chessel et al. 2004) implemented in in age (Chesseletal.2004)implemented lished in : Molecular Ecology, 2011, mber (Cucumis cv. sativus)plants, under naturalfieldconditions CA) andaspatialPCAusing cated onthemap. SeealsoData d tobacco (Nicotiana tabacum) d tobacco(Nicotianatabacum) www3.interscience.wiley.com. ring from 1995to2008.

ubpopulations. Rather, ubpopulations. Rather, InApril–May Article first

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., published online, of 1998, (Kiss field the in autumn halimifolium apart (Fig. 2). the same waytoAmpelomyces parasitizing Septemberwe 2007,2009, 2008 and Transmission ofnon-APMAmpelomycestoP.xanthiiand G. orontii in autumn Version définitivedumanuscritpubliédans placed outside the greenhouse on the 1st of September 2005 next 1st to 20 potted, 3-year-old the on greenhouse the outside placed infected on19August pl control as used were and until November them whenevernecessary. The re susceptible (cv. Jonathan) applethroughout seedlings ensureTo greenhouse. athe regular in APM s with infected and from removed chamber thegrowth were healthy seedlings borne AP air- from them protect to chamber seedlings, cv.Jonathan,were Ampelomyces isinred. Po xanthii infectingcucumber. Podosphaera in autumn species totwo‘trap’ mycohost leucotricha) Podosphaera (APM, powdery mildew testingtr experiments ofthefield Fig. 2Outline appleson overwinter could plants toapple experimental infect the APM colonies next spring. testedwhether Wefurther (Fig. 3). Ampelo orotherpowderymildew experimentally us by provided APM, susceptible toparasitismautumn-sporulating byAmpelomyces, either from rare, airborne Ampelomyces inocula present intheenvironment todetermine whether itis byAmpelomyces, areactive.Ther parasitized some autumn itcanalsosporulatein (H conditions, overwintering onappletrees. APM- of Transmission mycoparasites from as wellisolatingandsequencingtheITSofAmpelomyces above, as sites both from After a 2-week exposure, we collectedand an differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), To produce youngappleplantswithfreshlyspor To produce A. mougeotii DOI: wherever this solanaceous plant occurs, is heavily parasitized by Ampelomyces in parasitized heavily is occurs, plant solanaceous this wherever 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x Arthrocladiella mougeotii on 10.1111/j.1365-294X.2011.05007.x. The defi A. mougeotii Lycium Comment citer cedocument: 2005, with freshly sporulating APM colonies on their leaves, were were 2005, withfreshly sporulatingAPMcolonies ontheirleaves, and non-APM Ampelomyces to APM in autumn autumn in APM Ampelomycesto and non-APM leaves was previously confirmedunderadissectingmicroscope. 2008). Thepresence / Finalversionofthe manuscriptpub at bothsites. grown from seedinspring AlthoughAPMusuallysporulat maining 15 mildew-free seedlings were kept in the chamber the chamber maining 15mildew-free seedlings werekeptin

upply of fresh sporesfor thisinoculationupply offresh treatment, we grew exposed thesesame two‘trap’powderymildew species in , awidespreadpowderymild A. mougeotii , Golovinomycesorontii myces contracted by myces contracted M infections.Onthe species actively sporulating ontheirhostplants olb 2005) whenolb many other powdery mildews, ansmission of Ampelomyces strains from apple ansmission fromapple ofAmpelomycesstrains wdery mildew is drawn in black while blackwhile drawnin is wdery mildew nitive version is available at alysed all the tobacco and cucumber the plants all alysed of Ampelomyces mycopara ants in this experiment. The 15seedlings, this in ants efore, we exposed autumn-sporulating APM to to APM exposedautumn-sporulating efore, we in spring andfrom Arthrocladiellamugeotii ulating APM colonies in autumn, 30 apple 30apple autumn, in colonies APM ulating the summer, reinoculating batches of 10 of of 10 of thesummer,reinoculating batches on lished in : Molecular Ecology, 2011, L. halimifolium

2005 inaspore-proof growth es in spring, under some some inspring,under es www3.interscience.wiley.com. APM in autumn on our APM inautumn onour 19th ofAugust2005,15 ew speciesthatinfects infecting tobaccoand sites in the mycelium sites inthemycelium attwosites,10km and their . Inmid- Article first

L. Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., chestnut, Norway maple, pedunculate oak andanumber ofotherplantspecies(Fig.3). oak pedunculate maple, Norway chestnut, the in environment sporulated naturally in September–October 2005 on dandelion, horse any other airborne Ampelomyces inoculum were exposedtoanAPMstrainofAmpelomyces pr Thus,thesporulating trees. 3-year-old these young shoots,and in this summer and earlyautumn can sometimes bedetect apples wasalsomaintained artificiallythrougho published online, red. in is whileAmpelomyces black APM parasitizedbya powdery mildew (APM, Podosphaera leucotricha) from variousmycohost species,including Fig. 3Outlineofthefi of haplotype Ampelomyce APM an with infected apple trees,cv.Jonathan,withsporulating Version définitivedumanuscritpubliédans their genotypes. Counts were performed only in areas of mature pycnidia, characterized by by characterized pycnidia, mature of their genotypes. Countswereperformed only inareas from the experiments, usingthedriedleaves area fields on one leaf from each ofthreeto five potted cucumber andtobacco plants from tobaccoplants, respectiv cucumber and Ampelomyces genotypes that had infectede our powd host indifferent sporulate haplotypes Ampelomyces whether determine To species Assessment ofmycopa ITS andmicrosatellites. microscope.dissecting a under When found, mycoparasites were isolatedwere examined daily, and the presence of and genotyped for plus 4hofartificial burst, the30 pottedapple plants thesearound or in buds (Szentiványi & Kiss 2003). In February 2006,bud precocious provoke to greenhouse. APMoverwinters kept mildew-free inisolation,allnow50–75 Ampelomyces sources since the1stofSeptember, differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), After leaffall inNovember

DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi rasitic activity of different Ampelomycesdifferent of activity rasitic strains in different powdery mildew illumination. The development development of leav The illumination. Comment citer cedocument: known Ampelomyces strain,inautum eld experiment testing case, this was stimulated by pruning apart of the older shoots of 2005, the15pottedseedlings infe / Finalversionofthe manuscriptpub wereplacedinaheated,10–15 in apple buds and Ampelomyces mycoparasites overwinter and buds apple in

ery mildew fungi,we countedpycnidia ofthe different ely. We counted all pycnidiaintwo1.5-mm ely. Wecountedall APM mycelia onthe15newly infectedseedlings Ampelomyces in APM mycelia wasexamined APM known, byisolation andgenotyping,tobe APM known, xperimental P.xanthii ut thesummer. Thesporulationof APMinlate transmission ofAmpelo transmission which strains had been isolated todetermine coming that fromspecies mildew powdery s. The sporulationof nitive version is available at differedab their in esent on the 3-year-old 3-year-old onthe esent as well as the 15 healthy control seedlings 15healthycontrolseedlings the as well as cm tall,wereoverwinteredoutsidethe cm ed inthefield(Holb lished in : Molecular Ecology, 2011, es and APM colonies on the shoots onthe colonies and APM es n. Powderymildew isdrawn in cted with APM andexposed to o C greenhouse with natural natural C greenhouse with www3.interscience.wiley.com. APM onthese 3-year-old or G.orontiionpotted myces strains toapple ility to colonize and ility tocolonizeand apple trees but also to alsoto but trees apple 2005), especiallyon

2 Article first surface surface

wdery mildew fungiwereinfected with either the APM Arthrocladiella andstrains infecting infecting e 1 or 3 (Fig. 4), originating from e 1or3(Fig.4),originating nitive version is available at or by Ampelomyces haplotype within the two haplotypewithinthetwo or byAmpelomyces

lished in : Molecular Ecology, 2011, test whether pycnidial density density testwhetherpycnidial www3.interscience.wiley.com. A. mougeotii Article first

on Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., etal.2007). rates(Turner low mutation radial groups. However, the obtainedis pattern relations the inferring for unreliable diagram Recombinationin al. 2007). et linked together, alsoconsistent with recombination oc MLGs all with group straggly large single a S4, Supporting information) showed recombination (Fig. 5),indicating reticulations analyses thusindi sets,respectiv data complete and corrected 5andma of10.2(min: mean Ampelomyces. Among the652APM isolates genotyped suggestthecooccurrenceofclonalSeveral linesofevidence replication and recombination in APM APM Ampelomycesstrainsprac shown). (data not 235 isolatesandonlyone different hostsincludedin from amplific Poor information). Supporting S1, of 299 APM strains ofhavi thetwo 301 studied, fungi,forallth ofhaploid isolates strain single S1,Supportinginformation).unknown (Data were isolatedinsp U1 andGW, P. leucotricha exhibited ITS sequences than identical to those 4andS2,Suppor (Figs ITShaplotype of theAPM other species mildew powdery of thetrees withinreasonable limits. Only th in sequences foronly18APMstrainswereincluded published online, randomized datasetssi microsatellite datawas from Both valuesweresignificantly different the PCRproductsin>100APM primers specificoftheAPMAmpelomyces haplot coming from byamplifications wasshown This hosts. mildew powdery other withthat previousworkdifferent fromalmost alltheITShaplotypes ITS present in Ampelomyces strains ca obtained onesisolatedin2008acrossEurope, isolatedstudy, previous between 1995 and 2002 All the 301 APMAmpelomyces strains studied in mildew fungibasedonITSseque APM Ampelomycesstrains aregenetically differentiated from those isolated from other powdery Results Version définitivedumanuscritpubliédans association, r locality led to adata set localities. Applyinga different or same the at twice least at sampled were 24ofwhich Weidentified240MLGs, (±0.11). differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), showed SplitsTree4, using built However, the tree of the MLGsidentified intheAPMstrains, The five polymorphic microsatellite markers yiel Linkage disequilibrium among microsatellite lo microsatellite among disequilibrium Linkage DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x D , was-0.16forthecomplete datasetand0.044fortheclone-corrected data set. 10.1111/j.1365-294X.2011.05007.x. The defi cated thatclonalityinfluenced allele Comment citer cedocument: composed of mulating free recombination among lo much shorter(265mu to three amplifying microsatellite markers inthe remaining isolates x: mean diversity(H 21)allelesperlocuswitha genetic nce and microsatellite markers nce andmicrosatellitemarkers tice clonal replication / Finalversionofthe manuscriptpub the microsatellite work, with no amplicons for any marker in in marker any for amplicons themicrosatellite work,withno deed biases the eBURST algorithm, rendering the eBURST eBURST the rendering deed biases theeBURST algorithm, clone-correction bykeepingasi ring whiletheexactisolationtimeofthirdone,AqW, is

strains. AlltheseIT 274 genotypes. ation wasobtainedfor hip among MLGs, especially the connection among the especially MLGs, among hip 0 (P<0.001).Themost stronglyindicativeof re ree of 433 Ampelomyces strains isolated from from ree of433Ampelomyces strainsisolated ely, Fig.S3,Supportinginformation). Both e 652APMisolatesusedin among APM strains. The eBURST diagram (Fig. strains. APM among ng beenlost,and353appleleafsamples (Data curring in populations wi inpopulations curring nitive version is available at and regularrecombination ting information). Twoof thesethreestrains, rried the same ITS haplotype identified in in identified haplotype ITS same the rried ype, developed in this work, and sequencing work,and inthis developed ype, (Szentiványi et al. 2005),newly 261 and tational steps) than those obtained from thoseobtained tational steps)than ci, assessed ci, using the multilocus index of ded one allele per locus, as expected for for expected as locus, per allele ded one the phylogenetic analysis to keep the size size the tokeep analysis thephylogenetic associations (Burt et al. 1996). this work, including 40 strains from a at the 5 microsatellite loci, we found a found we at the5micro- loci, satellite S sequences were id S sequenceswere lished in : Molecular Ecology, 2011, ci (325clone- and for steps 500 ngle copy of each MLG per the 404 non-APM strains the 404non-APMstrains www3.interscience.wiley.com. parsimonious tree forthe gular recombinationand th few alleles (Turner thiswork, entical; therefore, therefore, entical; E consisting ) of 0.74 Article first

g countries oforigin,withth g countries ) 5 for both sPCs). forbothsPCs). F ficantly different from 0(b=)0.016,P different ficantly < i1 rrelograms revealed akinship coefficient (F by upto1618km, suggesti PCs for APM Ampelomyces strains explained and the logarithm ofdistance geographical myces strains, the ITS sequences of Ampelomyces of Ampelomyces theITSsequences strains, myces rains were genetically more similarthanmore mycohost or host plant species(Fig. mycohostorhostplant 4 and S2, d byprincipal components (PCs) analysisof nitive version is available at ew species were highly variable and were did not species ew lite multilocus genotypesmultilocus lite identified in lished in : Molecular Ecology, 2011, weak, implying weak, a e first two spatial PCs (sPCs) (sPCs) PCs e firsttwospatial www3.interscience.wiley.com. yces isolates ng thatdispersalover yces strains

large extentof Article first

ij ) Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., while therestofstrainsfell intoclade1. haplotype APM the of that to strain (GW) ofthose25isolated non-APM One sporulate andspread ontheir information). Incontrastto 25 strains fr were isolated strainsintheEurope. Inspring,wefoundnon-APM field only once, in southern France, where from the different sequences Forthosecollected forthis studies. previous Glawe 2008). represent species complexes thatincludecrypticspecieswithdifferent host plant ranges (e.g., binomials, suchas because speciesdelimitation isnot always the most regardless of the binomials used forthese plant powdery mildew fungiin character,and treatedasthefifth ITS sequenceswere the of alignment the in gaps way, the localities of origin.Toensurethatthisan China, Israel,SouthAfrica andtheUSA;thus, 91 strains carrying identical ITS sequences were isolatedindifferent Europeancountries, that differed inone orafewnucleotides from eachother (Fig. S2, Supporting information). The sequences and grouped intoclade1togetherwith anumber ofotherstrains with ITS sequences Phyllactinia mildew fungi belong species. For example, 91strain mildew powdery information). from detected evenonthesame In information). to 2008(Data S1, Supporting these strains were isolated inHungary, thus in arelatively small geographical area, from 1990among them (seestrainsingreenFig. 4andS2, S strains ofthe267,andtherewereconsiderabledifferences 65 in determined were sequences ITS The summer. before mostly epidemics causes and spring its early life cycle in (Kiss1998),incontrasttoAPM thatstarts September to August from starting areas sampling common a powdery mildewfungusrepresents strains, atotalof267,was Ampelomyces overrepresented in were foundalmost exclusivelyin that wasevidentformycohostsother than just APM. Ampelomyces strains belonging to clade 4 Ampelomyces ITShaplotypeswithmycohostsp Supporting information). Noneth 1andthef belonged toclade inFig.S2,Supportinginforma colour blue in pannosa infecting Rosa,fourofthem belongingto markedly different in were Ampelomyces strains isolated from P. sequences ITS the example, published online, powdery mildew samehost the species infecting same the from isolated were powdery different mildew and species, different ITS haplotypes Version définitivedumanuscritpubliédans differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), The only powdery mildewspecies powdery The only Unfortunately, collectiondate,andhenceseason, Reciprocally, Ampelomyces strainswithidenti A. mougeotii and DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi Sawadaea , JY3 and HMLAC202, also belonged to different clades (Fig. S2, Supporting Supporting S2, (Fig. clades , JY3andHMLAC202,alsobelongedtodifferent A. mougeotii ing tosixgenera, P. xanthii,fusca Comment citer cedocument: fecting different host plant generaplant host different fecting were treated as different taxa , andcollectedfrom21hostplant ge L. halimifolium APM, speciesareknowntoactively allthesefivemycohost . om fivepowderymildew species(Data S1, Supporting ourth one to clade 2 (see strain 2(see clade to one ourth APM haplotypewerecollectedinSeptember-October across / Finalversionofthe manuscriptpub respective host plants during

P. fusca eless, there wasthere eless, an overall significant association of other than APM fromwhic APM than other Podosphaera, Erysiphe,Golovinomyces, Arthrocladiella, A. mougeotii and bush inHungary,andthetwoChinesestrains study, the 383 non-APM strains that exhibited ITS ITS exhibited that study, the383non-APMstrains and alysis was carried out in the most conservative conservative alysis wascarriedoutinthemost tion). Similarly, three strains from E. sordida sordida E. from tion). Similarly, threestrains there was noindicationof was there P. xanthii pathogens. Thislatter criterion was necessary cal ITS foundindifferent sequences were nitive version is available at in spring exhibited an ITS sequence identical identical sequence in springexhibitedanITS ecies (P = 0.001 from the permutation test) test) permutation ecies (P=0.001from the some cases,different ITShaplotypes were s isolated from different species of powdery powdery of species fromdifferent s isolated species that becomes widespread in our in widespread becomes that species clade1andthefifth toclade3(seestrains G. cichoracearum plant species,evenin is unknown for most ofthestrainsusedin is unknownformost accurate within the Er infecting upporting information) , andthosefrom cl lished in : Molecular Ecology, 2011, nera, all exhibited identical ITS ITS exhibitedidentical all nera, the whole vegetation period. the wholevegetationperiod. s inbrowncolourFig. S2, L. halimifolium h wehadalargenumber of www3.interscience.wiley.com. , mentioned inthiswork, any association with anyassociation thesame area.For ysiphales, and some ysiphales, andsome ades 1and2were . Allbuttwoof bushes.This Article first

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., the overwintered APM,15were the overwintered 15experimentalin APMoffivethe i.e. present, were plants, but none of the apple 15 controls. mycoparasites plantsAmpelomyces experimental 15 all on developed APM environment, andmildew powdery Ampelomycesnatural any before inoculum appeared in the (Fig. 3). After beingoutdoor overwintered from Ampelomyces non-APM to and Ampelomyces of haplotype APM the with infected be potted to apple trees bearing sporulating APM known 3-year-old from Ampelomyces APM to exposed were after exposure.Theappleseedlings weeks 3 infection were placedoutdoors inSeptember2005showedAmpelomyces that seedlings autumn-sporulating APMboth by infection Ampelomycesto susceptible strains and Ampelomyces strains that infect APM usually sporulates onlyinspring,when APM canbeinfectedby autumn. haplotypes ofAmpe successfully parasitized byboth 1 or3(Fig. 4).Thus,thetwopowdery mildew species each andonestrainfrom powdery mildews, ofthe22stra The ITShaplotypes years). these powdery mildew onlyin2008and cucumber andtobaccopowderymildew 2 week both of mycelia Ampelomyces (Fig.2).Themycoparasitesappearedinthe harboured infected withitspowdery mildew species, plant the when placednear andtobacco cucumber on species mildew powdery powdery mildew species. other infect Ampelomyces strains infecting the powdery mildew mildew species powdery provided these were available in their closetime. that at vicinities colonies soon after bud break in spring (Szentiványi & Kiss 2003),couldeasilyspreadtoother confirmed thatthemycoparasite ofAmpelomyces, APMhaplotypes as the did th two powdery mildew species afte The ITSsequencesandmicrosatellite profiles of the 27 Ampelomyces strains isolated from these tob of mildew werefoundin3of4yearsand AmpelomycesAmpelomy Successful infections. harbour Ampelomyces-infected APM(Fig. 2), xanthii and tobacco infected experimentally plants by ca Spring APMAmpelomycesstrains parasitized naturally sources mildew powdery infected near Ampelomyces. hosts potential providing published online, Lycium halmifolium A. mougeo parasitizing found often 1, clade to belonging non-APM Ampelomyces strains to those of theidentical surroun plant species were able toinfect alternat parasitizing APMinspringand result from physiological other powderyde fungi, mildew The highgeneticdifferentiationof fieldconditions natural APM andnon-APMAmpelomycesstra Version définitivedumanuscritpubliédans differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), and G. orontii, DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi and several other powdery mildew speci mildew other powdery several and lomyces coming from coming lomyces powdery mildew spec were suspended, after bud burst in spring, on apple trees known to trees apple on spring, in burst bud after were suspended, Comment citer cedocument: hostspecificity. We tested whether Ampelomyces strains naturally APM andnon-APMAmpelom ding infected apple trees, andtw appletrees, ding infected s that overwintered onappletrees / Finalversionofthe manuscriptpub those parasitizing other powdery those parasitizingother APM strains of Ampelomyces in APM strainsofAmpelomyces spite long-dista r 2weeksexposureonin of the APMand haplotype ITS Ampelomyces in strains found

2009 (the experimentalcucumber ins areeasilytransmitted to different mycohost species under n infectotherpowdery mildews. We obtained successful infection of these same two two same these We obtainedsuccessfulinfectionof s and having bud burst forced in the gree s andhavingbudburstforcedinthe A. mougeotii ive powderymildew speci L. halimifolium with their respective powdery mildew species, respective their with acco powderymildew all4yearsofthisstudy. ies. Sporulating APMon15pottedapple had successfully overwintered emerged, and species Arthrocladiella nce dispersalabilityand recombination, could nitive version is available at manipulated tosporul . Of the 17 Ampelomyces strains isolated from from . Ofthe17Ampelomyces strainsisolated e strains isolated from ces infections of the cucumber powdery powdery ces infectionsofthecucumber ins isolated from the cucumberortobacco from the ins isolated A. mougeotii these powderymildew contracted species s after exposure in 2007 and of tobacco 2007andoftobacco in exposure after s several other plant species in the vicinity naturallyinfecting fected apple trees all corresponded to correspondedto fected appletreesall es (Fig. S2, Supporting information). lished in : Molecular Ecology, 2011, bush, all belonged to either clade bush,allbelongedtoeitherclade P. xanthii P.xanthii yces strains inautumn. o hadITSsequencesidentical to , in autumn, whenthislatter APM from those isolated from from isolated those from APM mildew species on other host host other on species mildew , present inthe primary APM springandtwo different ITS had microsatellite genotypes had microsatellite genotypes plants diedprematurely in www3.interscience.wiley.com. mougeotii inautumncan When potted cucumber Whenpottedcucumber ate inautumn, itwas es by experimentally es byexperimentally the apple This trees. and L. halimifolium G. orontii L. halimifolium L. halimifolium Although Article first nhouse tii from tii from were

in P. Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., F mycohost species. host, andthemycopa autumn. This suggests thatsporulationdensity although thestrainswere published online, difference indensityofpowderym pycnidia Ampelomyces densitywasa tobaccoplants,each and exposed cucumber species. The density ofwas pycnidia Ampelomyces Mycoparasitic activityof genetical species was available in theautumn andoverwinteronapple. Therefore, bothnon-APMandAPMhaplotypesof Version définitivedumanuscritpubliédans Ampelomyces strainson thesame theseby production mycoparasites. However, we found no significant heterogeneity am differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), 1,40 = 299.33(P<0.0001)inbothspringand autumn experiments. This directly reflects the ⁄ cm 2 mycelium) as in DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi Comment citer cedocument: rasitic activityofgeneti genetically diversecoming from APM in spring and P. xanthii bout twiceasgreatin / Finalversionofthe manuscriptpub ly differentAmpelomy

‘trap’ powdery mildew species(F ‘trap’ powderymildew ildew conidiophores available for intr for ildew conidiophores available oncucumber (2714.28±115.4pycnidia is determined entirely by the powdery mildew mildew powdery the is determined entirelyby nitive version is available at infected with itspowderymildew fungus. cally distinct strains Ampelomyces could infect APM when this this when APM infect could Ampelomyces counted on the leaves of the experimentally counted ontheleavesofexperimentally ces strainsindiffere G. orontii lished in : Molecular Ecology, 2011, on tobacco(5227±93.3 www3.interscience.wiley.com. 4,40 is similarinagiven acellular pycnidial nt powdery mildew nt powderymildew = 1.37, P = 0.26) 0.26) = 1.37,P ⁄ cm A. mougeotii 2

mycelium), Article first ong

in Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., published online, Discussion Whittlesford (1). WH: (23); Velem Stansted VL: (1); (6); TY: Teynham GT: Gotheron (56); KZ: Keszú (27); OR:Orsay(5 East Mall EM: (1); Dresden DR: (8); Canterbury Version définitivedumanuscritpubliédans ( eigenvalues areshown inthe inserts. The crosse (botto andspatialPCA Fig. 6Centred-PCA(top) differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), n = 274) are as follows: AR: Ahrensburg (1); BP: Budapest (32); CB: BP: Cambridge (8); CT: (1); Ahrensburg AR: = follows: 274) are as DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi Comment citer cedocument: / Finalversionofthe manuscriptpub

nitive version is available at s arethe localitycentroids. The 17locations ing (9); EP: Eperjeske (23); GD: Gödölló(18); m) ontheclone-corrected data set. The 7); OX: Oxford(1); OX: 7); lished in : Molecular Ecology, 2011, www3.interscience.wiley.com. PR: Prague(1);ST:

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Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., Ampelomyces from P. xanthii Ampelomyces mycoparasites asrevealedbyourfieldexperiments. The powdery mildew species asexuality of the former.Ampelomyces inAPMarenotisolated strains Physiological hostspecificity isnotstricteither inthese reproduction, butalsowiththeo APMwithin pattern genetic Ampelomycesrevealed strains consistent with frequent asexual the of distinct maintenance APM Ampelomyces lineage. Analyses usingmarkers microsatellite mildews. from species,distinct acryptic represents APM Ampelomyces strains. Altogether, thissuggeststhatAmpelomyces infectingAPM Ampelomyces non-APM in strains yielded bands with allele sizes different from those carried by cies (Dutechetal.2007),becauseofmutationsin strains, asisfrequentwhenmicrosatellite mark Ampelomyces strain Ampelomyces inmost toamplify non-APM 2006)failed (Harvey 1995;Nilssonet al. et between APM- andnon-APM Ampelomyces corres AP than other species from powderymildew ITS haplotype, divergentfrom ITShaplotypes f mildew onotherplantspecies.Indeed,APMAmpelomyces strains all carrieddistinct from Ampelomycesmyco one identical we reportthatAmpelomyces etal.2005;SzentiványiLiang2000; Nischwitz et al. 2007; Park et al. published online, betw differentiation high the explaining a host. nonhabitual Thus, intrinsic infecting when even pycnidia of numbers large produced successfully strains Ampelomyces infecting sporulating, become infected by the found in recogni been have lineages genetic distinct considered to be a generalistintracellularpara quisqualis, A. species, Ampelomyces mycoparasites currentlybelongtoasinglenominal Version définitivedumanuscritpubliédans autumn. Wedidfindthree exceptions to the APM Ampelomyces Ampelomyces from causing epidemics mostly inautumn (Szentivá summer while most other powdery Indeed isolation. temporal from differentiation. thegenetic explaining specificthat suggesting experiment, overwintering requirements are also insufficient for our in APM with Ampelomyces werecapableofoverwintering inapple buds inassociation non-APM Ampelomyces. However, and non-APM APM- between isolation genetic to contribute therefore that particular adapta site overwintering this APM Ampelomyces shares mating between Ampelomycesstrainsinfectin from dispersal both prevent that exist must nisms because our genetic analyses revealedmecha- Other events. dispersal long-distance frequent differ geography genetic explain thestrong apple and the Ampelomyces my differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), We thenattempted differenthypotheses totest We suggest that genetic isolation between AP that geneticisolationbetween We suggest b inapple The APMoverwinters A. mougeotii L. halimifolium onand cucumber DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi P. leucotricha infecting al.2008).Furthermor Comment citer cedocument: andmildew severalpowdery other foundinAPMcausedby tion overwintering behaviourmay associated with thisparticular L. halimifolium , the life cycle of APM is mostly completed in spring and early early and spring in , the life cycle ofAPM ismostly completed / Finalversionofthe manuscriptpub G. orontii parasites infecting other myc other infecting parasites

coparasites of other powdery and Ampelomyces strainsoftw and uds, a particular ecological s ecological particular a uds, ccurrence of regularof ccurrence recombination. This suggests that the of other powdery mildew hosts that cause epidemics in in epidemics cause that of other powdery mildew hosts mildew species star species mildew physiological barriers to infection areinfection to notbarriers physiological adequate for een theAmpelomyces mycoparasites of Ampelomyces normally associated with on tobacco became infectedwith both APM g APMandthosefound e, microsatellitemarker . We also found thatAPMcould,ifexposedwhile ers from one fungal species ers fromonefungal zed (Kiss&Nakasone zed Ampelomyces species in Ampelomyces species site ofpowderymildews; however, a number of entiation betweenAPMandnon-APM strains M- and non-APM Ampelomyces results mainly M- andnon-APMAmpelomyces resultsmainly M. ThedifferentiationintheITSsequence nyi et al. 2005), thereby isolating the APM theAPM therebyisolating nyi etal.2005), from non-APM Ampelomyces strainsbystrict Ampelomyces non-APM from nitive version is available at about the barriers to gene flow allowing the allowing flow gene to barriers about the APM to other mycohosts APM theflanking regions. The rareamplifications ound inallbutthree other strains collected specificity of the divergent of haplotype ITS (Szentiványi & Kiss Kiss & (Szentiványi ponds to species-level differences (Seifert ponds tospecies-leveldifferences P. leucotricha t theirlife cyclelaterinthe season, lished in : Molecular Ecology, 2011, ohosts responsible for powdery forpowdery responsible ohosts ituation, anditwasshownthat s. Furthermore, allof these o distinctITShaplotypesoften mildew species. Nor can in othermycohosts. s developed from an APM an APM from s developed www3.interscience.wiley.com. 1998; Sullivan & White 1998; Sullivan&White innature aregenetically fecting other powdery powdery other fecting are used for other spe- other for used are 2003). Itis , andviceversa, P. leucotricha A. mougeotii 2010). Here, Article first possible

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., latter. With this defin poses theoreticaldifficultiestoexplaindifferentiation,in contrastcase former to thethe as factors control theprobabilityof matingbetween Mina (1986)whosuggestedthatsympatry requires that only the genotypes and no extrinsic operational and useful very definitionA question. of sympatry is provided by Kondrashov& considered sympatric andbecause their mycohosts. mycoparasites withwidehostrangeswhosephenol differentiation. Here,weprovideacompelling et al.2008,2009)areal plants 2006) al. (Savolainen et flowering 1995), Combes & (Théron parasites Devaux &Lande2008).Observationsfromanimal that increasing phenological divergenceeven can reproduction hasbeenre that aresporulatingoverthewhole other by Ampelomycesinfection strains. Ampelomyces spreadbecomes dormant after itsspringoutbreaks,li betweenspecies mildew powdery Ampelomyces detectedinthesemycohosts. Furthermore, APM of haplotypes non-APM of than APMthatareactivelygrowin other species mildew powdery of number low the chances forsucheventsarelimited, inpartbecauseof establ become principle, in could, and season other than APM.Thismeans thatsuch haplotypes arealso mycohosts present in early in the and another onefromKorea(Parketal. 2007) al. et (Liang China from reported Another strainwith APMITShaplotypewas APM host, it appears to occurin predominantly APM strains. APMITS althoughthis Therefore, known, werecollectedinspring published online, Ampelomyces inoursample.Twoof Version définitivedumanuscritpubliédans without selection for ec occurred in eff divergence that suggest may hostuse are not strict. vice and hosts Genotype, mildew powdery other to transmit therefore, does not determine host usesuch as and ability externalby governed speciation barriers to gene flow as in allopatry. scenario,a such it would be by gradual divergence thereby resemble allopatry(Giraud2006a,b).Indeed, if speciation were to occur under barrier to gene flowand genes, its by wouldcaused not i.e. beAmpelomyces, to extrinsic s would environment placed inthat particular determined bytheplant and th particular hostwithaphenology. In mainly inspring,then 2005). Ifonlyparticul variation, sogenotypedirectly animals, and plants most genetic control.In temporal isolationcanbeconsideredsympatri evidence that APM Ampelomyces is a cryptic Ampelomyces APM that evidence and divergentmicrosatellite loci differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), Whether geneticisolation,when geneticdifferentia Increasing also were haplotypesofAmpelomyces Non-APM We haveshownthatAPMAmpelomyces strainsform DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi ar Ampelomyces genotypes are able to exploit APM, which sporulates sporulates which APM, exploit to ar able Ampelomyces genotypes are ition, genetic isolation that is initiated and initiated is that ition, genetic isolation Comment citer cedocument: ological divergence. divergence. ological so consistent with the the with consistent so temporal isolationiscausedby ported innumerous organisms (Hendry &Day e powdery mildew phenologies, andanyAmpelomyces genotype phenologies, mildew powdery e influences which mating partnersareavailable (Hendry & Day / Finalversionofthe manuscriptpub and alleles from therestof and plantpathog tion with increasing divergence in theseasonal timing of increasing with tion

the vicinity of apple trees in trees ofapple vicinity the of ecological factors or because of allopatry is not a trivial trivial a not is of ecologicalfactorsorbecauseallopatry vegetation periodappearsmore likely. 2010), bothfromrosa it occurswithinarestricted geographical area, should be these strains, these thos thereisagenetic component for phenological idea that temporal isolation can allow ecological ecological canallow isolation temporal idea that porulate and transmit exclusively in spring, the transmit and porulate c if the timing of reproduction islargelyunder ofreproduction timing c ifthe species.Theabilityfo spring, at least in the sampled European region. region. spring, atleastinthesampledEuropean ished inAPM.However, g and sporulating in spring and the low number lownumber the springand g andsporulatingin miting windowavailablefor temporal the two individuals. The distinction is important important is distinction The two individuals. nitive version is available at ective allopatryviatemporal isolation,i.e. leadto sympatric sp versa suggests thatgeneticdeterminants of ogy is largely controlled by the phenology of of phenology the by controlled ogy islargely enic fungi (Van Putten case that such a mechanism operates in in operates mechanism a such that case contrast,if temporal of a subset of genoty haplotype is not entirelynot is haplotype restricted to the a distinct clade with detected, albeit infrequently, inspring infrequently, detected,albeit e forwhichthedate ceous hostplants. lished in : Molecular Ecology, 2011, genotype viagenotype its adaptation to a the Ampelomyces. This is strong strong is This the Ampelomyces. fected withAPM ⁄ or maintained because of www3.interscience.wiley.com. r APMAmpelomycesto eciation (Stamm 1983; 1983; (Stamm eciation asingleITShaplotype pes inisolat isolationisentirely et al.2007;Montarry itappears that the 2005). Modelsshow of collectionwas parasitized by ion, i.e. a ion, i.e.a Article first

Version postprint Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Manuscrit d’auteur / Author manuscript Bardin, M., Shykoff,J., Giraud,T.(2011). Temporal isolationexplains host-related genetic Kiss, L., Pintye,A.,Kovacs, G.,Jankovics, T.,Fontaine, M.,Harvey, N.,Xu,X.,Nicot, P., Giraud T(2006b)Speciation in immigrant inviability barrier in the pathogens: migrant against Selection (2006a) T Giraud Giraud T(2004)Patterns of Gavrilets S, Losos JB(2009)Adaptiveradiation: Or andthe Gavrilets S(2004)FitnessLandscapes Gardes M, Bruns TD (1993) Independently (2007) al. Cet EA,Boschetti D,Herniou Fontaneto evolving species in WP,deHoogSetal.(2005) Fisher MC,Hanage forpreparationandevolutionary asoftware Filatov DA(2002)ProSeq: analysis of WP,Sp DM,Hanage Feil EJ,LiBC,Aanensen Falk SP, Gadoury DM,Co microsatellite isolation of in fungi. Challenges (2007) al. et E Fournier J, Enjalbert C, Dutech published online, Devaux C,LandeR(2008)Incipien S Associates, Sinauer OrrHA(2004)Speciation. Coyne JA, clustering. Nucleic Acids hierarchical with alignment sequence Multiple (1988) F Corpet Chessel D,DufourAB,ThiouluoseJ(2004)Theade4package–I: one-table methods. R Cavalli-Sforza LL,Edward Burt A,CarterDA,KoenigGL,White TJ,Taylor JW (1996)Molecular markers reveal cryptic phytophagous insects: moving in speciation Sympatric (2002) JL Feder SH, Berlocher Indicesofmu P-M,BurtA(2001) Agapow References project. EU grant an (FP 7-SME-2007-1-222045- by acknowledges afellowshipoftheFrenchgove andOd Mayer EvaArpadne thank authors The Acknowledgements Version définitivedumanuscritpubliédans differentiation ina group of widespread mycoparasitic fungi. Molecular Ecology, 20(7), allopatry. Trends in plant Silene the latifolia. Heredity, 95, 559–565. parasitising violaceum Microbotryum of mycorrhi identification the to application pathogens. Heredity,97,316–318.pathogens. 732–737. Princeton, NJ. 5(4),e87. PLoS Biology, rotifers. bdelloid asexual Pathogens, 1(2),e20. in heterogeneous landscapes by theendemic pathogenPenicillium marneffei. PLoS Ecology Notes, 2, 621–624. Molecular sets. data sequence sequence typingdata.Journal mu from genotypes bacterial related of evolutionary descent among clusters Ampelomyces quisqualis. Phytopathology, 85, 794–800. mycoparasite the by ascomata Fungal Genetics&Biology,44,933–949. mutation, andgeneticdrift.ProceedingsoftheRoyalSociety time, B, London, Series flowering by mating Research, 16,10881–10890. 4,5–10. News, Human Genetics, of 19, 233–257. Journal American procedures. USA,93,770–773. ofthe Sciences Coccidioides immitis pathogen human the in sex beyond controversy.AnnualReview of Entomology, 47, 773–815. Ecology Notes,1,101–102. DOI: 1492-1507. DOI : 10.1111/j.1365-294X.2011.05007.x 10.1111/j.1365-294X.2011.05007.x. The defi 275,2723–2732. Parasito Comment citer cedocument: rtesi P,Pearson RC, Seem RC(1995) s AWF (1967)Phylogenetican logy, 22,151–152. of Bacteriol / Finalversionofthe manuscriptpub ITS primers withenhanced specificity for basidiomycetes- within populationdispersion andmating of the fungus

parasites:hostswitching doesnot automatically lead to t allochronicsp ogy, 186,1518–1530. zae andrusts.Molecu BCA_grape) and an INRAHAS collaborative collaborative andanINRAHAS BCA_grape) ratt BG (2004) eBURST: inferring patterns of of patterns inferring ratt BG(2004)eBURST: Low effective dispersal of asexual genotypes genotypes asexual of dispersal Loweffective nitive version is available at igin of Species. Princeton University Press, Press, University igin ofSpecies.Princeton rnment. This study was supported in part rnment. supportedinpart This studywas ltilocus linkagedisequilibrium. Molecular contrasting theorycontrasting with data. Science, 323, eciation due to eciation dueto . Proceedingsof theNationalAcademyof ile Jonot for technicalassistance.AP lished in : Molecular Ecology, 2011, alysis: models and estimation estimation and alysis: models underland, MA. Parasitism ofUncinulanecator lar Ecology, 2,113–118. non-selective assortative www3.interscience.wiley.com. Article first ltilocus DNA

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