Spatial Genetic Structure and Dispersal of the Cacao Pathogen Moniliophthora Perniciosa in the Brazilian Amazon Artero, A

Aberystwyth University

Spatial genetic structure and dispersal of the cacao pathogen Moniliophthora perniciosa in the Brazilian Amazon Artero, A. S.; Silva, J. Q.; Albuquerque, P. S. B.; Bressan, E. A.; Leal Jr., G. A.; Sebben, A. M.; Griffith, G. W.; Figueira, A.

Published in: Plant Pathology DOI: 10.1111/ppa.12644 Publication date: 2017 Citation for published version (APA): Artero, A. S., Silva, J. Q., Albuquerque, P. S. B., Bressan, E. A., Leal Jr., G. A., Sebben, A. M., Griffith, G. W., & Figueira, A. (2017). Spatial genetic structure and dispersal of the cacao pathogen Moniliophthora perniciosa in the Brazilian Amazon. Plant Pathology, 358-371. https://doi.org/10.1111/ppa.12644

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Brazil. This article isprotected Allrights bycopyright. reserved. doi: 10.1111/ppa.12644 thisversionandthe between lead todifferences been through the copyediting, typesetting, and proofreading which may pagination process, forpublicati accepted This articlehasbeen & #b #a 4 3 2 1 A.S. Artero Brazilian Amazon pathogen cacao ofthe and dispersal structure genetic Spatial Article type : Original Article 18-Oct-2016 : Date Accepted :17-Oct-2016 Date Revised :09-Nov-2015 Date Received Penglais, Ceredigion,Penglais, SYD3 3DD, Wales, United Kingdom; Aberystwyth University, Cledwyn Building, Institute São de Florestal Estação Tupi, ExperimentalPaulo, de Piracicaba, São Brazil; Instituto Paulo, Executiva do Comissão Cacaueira, ERJOH, daLavoura Plano Marituba, Pará, Brazil; Nucl São de deEnergia Paulo, Centro Universidade These authors contributed equally to this to work. authors equally contributed These Current address: Universidade Federal do daBahia, Universidade Recôncavo address: deCiências Federal Centro Current Agrárias, Accepted de deAlagoas, Centro Ciências Federal address: Universidade Agrárias,Current Alagoas,Rio Largo, Article 1,& 4 ; J. Q. Silva ; A. Figueira 1,#a,& 1* ; P.S.B. Albuquerque P.S.B. ; on and undergone full peer review buthasnot review undergone fullpeer on and 2 ; E.A.Bressan ear na Agricultura, Piracicaba, São Paulo, Brazil; Paulo, Piracicaba, São Agricultura, na ear Version of Record. PleaseVersion cite thisarticle as of Biological, Environmental and RuralSciences, 1 Moniliophthora perniciosa perniciosa Moniliophthora ; G.A. Leal Jr. 1,#b ; A.M. Sebbenn A.M. ; in the 3 , genotyped at 11 microsatellite loci found that S- and especially L-biotypes showed a higher genetic genetic ahigher showed S-and locifound that L-biotypes especially at 11 microsatellite genotyped isolates, of Analysis 131 L-biotype. from the cladeseparated formed a well-supported S-biotypes that C-and rRNAITS the based reconstruction diversity. showed regions on the Phylogenetic genetic andtobiotypes, identify for theC-biotype, those lianas (L-biotype). Our objectives were to clarify the genetic relationship between the three on growing endophytically found andthe third (C-andS-biotypes), cacao or Solanaceae species biotypes of This article is protected by copyright. All rights reserved. copyright. This articleis protectedby diversity and Amazonian C-biotype populations, Acre and West Amazon displayed the largest genotypic ‘clonal’, as evidenced by high frequency of regions containing extensive cacao plantings, C-biotype populations were essentially Amazonia for up to 137 km, suggesting isolation by distance mode of dispersal diversity. * population. population. Amazon to from isolates the possibly assigned Amazon, Lower andWest Rondonia through origin highallele richness, invasions, but suggesting multiple genotypic thewith diversity, lowest exhibited Bahia The population cacao isnot endemic. where rest of the valley, the to Amazon eastward direction flow followed river Acre,the of downstream Rondonia from have Amazon and West Moniliophthora perniciosaMoniliophthora Abstract Keywords head: Running e-mail: [email protected] Accepted Article Corresponding author

A significant spatial genetic structure was detected for the C-biotype populations in : Crinipellis M. perniciosa might be part of the center of diversity of the fungus. The pathogen dispersal may dispersal pathogen may fungus.The the part of diversity of the be of center might Moniliophthora perniciosa Moniliophthora ; Marasmius are recognized, differing is the causal agent of witches’ broom in ; Theobroma diversity ; witches’ broom disease. broom ; witches’ repeated multilocus genotypes. Among the regions intheBrazilianAmazon with the greatest in host specificity, with two causing symptoms on

Theobroma cacao (cacao). Three . However, in However, infected pods anddebilitated trees. losses derive from but mainly economic ‘brooms’, are symptoms, buds, called the remarkable most infected of andhyperplastic Hypertrophic growth Schmidt, (Purdy & 1996). development stage of inducingarange pods), anddeveloping cushions, This article is protected by copyright. All rights reserved. copyright. This articleis protectedby and basidiospores is the causal agent of the witches’ broom disease of compatible mating partner. individu fittest the propagate to means rapid strategies, suchas reproductive since s intracontinental regionalor scale.Fungal mating at global, populations between flow gene potential andthe populations between differentiation invasions. harmful The dispersal ability of path reduce strategies avoid to potentially and or their indeveloping inagriculture impact assist manage (Giraud pathogens of plant andpopulation genetics biology evolutionary to the hasbeen given attention Little Introduction Accepted verrucosa Arrabidaea Incoastal Ecuador, and Amazonian 1994b). Hedger, inliving wo found forminghas typically basidiocarps (Bastos & Evans, 1985). The L-biotype hasnot been can it inoculation, artificial symptoms broom cause naturallybiotype infects various weedy solanaceous species (e.g. Strains that infect cacao and relatives are termed C-biotype (Griffith &Hedger, 1994a), whilst the S- symptomssw of development (tissue fungusexhi The (C-, S-,andL-biotypes). biotypes broom symptoms in many of these. tissuesinfecting living plants to diveof belonging from the Malvaceae Article Theobroma Theobroma Griffith &Hedger (1994a) proposed that Moniliophthora infect actively growing meristematic tissues (shoot apices, singleflowers, flower congeners, andspecies from the sister genus et al senso (Bignoniaceae) (Griffith & Hedger, 1994b). (Griffith 1994b). (Bignoniaceae) &Hedger, ., 2008), but establishing but ., 2008),

perniciosa

latu

(formely Sterculiaceae). However, since the 1980s, it has been found hasbeen it 1980s, since the However, Sterculiaceae). (formely It was believed thatoriginally (Marasmiaceae elling) in distinct among cross-inoculations andhosts. biotypes elfing orclonality, may be beneficial for pathogens as a ogens, especially fungi,defines the genetic basic processes inthelife history of parasites may M.perniciosa bits host specificity, with only very limitedhost very specificity,bits withonly als, without requiring the presence of a system plays animportant role in dispersalability rse unrelated hosts, causingdistinctive witches´ of symptoms depending on organ infected and on infected organ depending of symptoms shown to cause witches’ broom symptoms and symptoms broom to shown cause witches’ on cultivated species, suchas andpepper on cultivated tomato ody liana vines andassociated liana ody (Griffith debris vines & the L-biotype was consistently found on vines of of on found vines consistently was the L-biotype sensu stricto sensu Theobroma cacao Theobroma

contains atleast three discrete Herrania Solanum M. perniciosa ; Aime & Phillips-Mora, 2005) (Purdy & Schmidt, 1996), all 1996), & Schmidt, (Purdy

rugosum L (cacao). Its L(cacao). infects onlycacao ). However, upon upon ). However, commercial product during product commercial the 17 al potential socioeconomic with cacao catastrophic production, consequences. world of the 70% over potential about the concerns risk of This article is protected by copyright. All rights reserved. copyright. This articleis protectedby disease reaching over 250,000 tonnes.year broom losses from witches’ cacao estimated producingwith countries, andCaribbean American found in allSouth pathogen. fungusispresently invasionThe an avoided bythe earlier likely controls phytosanitary andsome Figure 1) km; Bahiaand 2,000 Amazon (over region the between in region southern in Bahiastate producing (Andebrhan 1989 (1939),Tobago andGrenada (1948) (Baker &Ho reported inBritish Gu sequentially occurring was commercial cacaoplantati affecting first report broom of witches’ The unwittingly. being Caribbean, transported often and the anddevastated local industries in invaded it cacao-producing across regions South Americavarious Thomas resistancefor inthe Amazon putativel region, formation lead hetero-dikaryons. the to could of brooms, isfusion it that derived hyphae genetically possibly infected basidiospores different from of karyogamy two identicalof nucleiisnot following division and recognize that here.However, we meiotic ‘clonal’ thiswe term also use is strategy the as C-andS-biotypes breeding often1994c). of &Hedger, described (Griffith The the typical dikaryot brooms, with clamped green germinate andinfect hosttissues a forming swolle innature basidiospores However, germination. following aperiod weeks of 1-3 over autodikaryotize germinated to agar basidiospores areable uninucleate on media haploid, fungi whereby an exhibit unusual non-outcrossing and S-biotypes the C- requires Incontrast, compatible first connections, between two monokaryons. mating clamp bearing agaric found dikaryotic among mycelium fertile formation the fungi,suchthat widely of 1994b) showed that the L-biotype is with a multia (Baker & Holliday, 1957; Purdy &Schmidt, 1996) andits host

Accepted., 2006; Motamayor Article The natural distribution distribution natural of The Defining the areas with the largest genetic diversity would help to devise strategies to search to strategies devise to help would diversity genetic largest the with areas the Defining (Griffith Hedger, biotypes & three of the biology the breeding of investigation detailed More et al et ., 2012). The C-biotype is endemic to the Amazon (Purdy &Schmidt, 1996), from where et al ons occurred in ons inoccurred Suriname Afterwards, pathogen (Rorer, the 1895 1913). ., 2008; Thomas Thomas 2008; ., th T. cacao century, cultivation extended eastward to near the to eastward of the mouth cultivation extended century, M. perniciosa clonality -1 inBrazilislimited to the Upper Amazon region (Sereno et al (Bowers y considered the center of origin of both ., 2012), butwhen cacao became a valuable lliday, 1957), reaching the largest Brazilian cacao largest Brazilian reaching the lliday, 1957), ic mycelium only appearingas die the brooms inits strictest sense. Furthermore, in multiply yana (1906), Ecuador (1921), Trinidad (1928), invasion inWest Africa, a region responsible for llelic tetrapolar outcrossing mechanism, asis n and multinucleate ‘biotrophic’n and mycelium within multinucleate breeding strategy found among breeding strategy agaric rarely only et al ., 2001). There are current serious T. cacao

et al ., 1999). The long distance (Motamayor (Motamayor et al M. perniciosa ., 2008; et Ecuador (Figure 1). Infected cacao shoots (‘brooms’) were collected from trees under cultivated from (‘brooms’) cultivated (Figure 1). Infected or cacaoshoots collected trees under Ecuador were naturally, including occurs sites in conducted from infected from infected conducted perniciosaMoniliophthora Materials and methods Transamazon). incacao genotypes (Bahia introduced andto Amazon the producing and origin regions of the verify microsatellite loci, to determine the pathogen gene in regions the BrazilianAmazon with internal based transc ribosomalon gene biotypes populations. pathogen of andstructure these patterns, about cacao-producing isuseful inform programs to major dispersal these breeding regions diversity, 1999; Rincones detected by various consistently (Andebrhan been molecular means have lineages distinct but unknown, least genotypesat remains two clearly source original of the The 1991). (Homewood, (Andebrhan outbreaks local cupuassu local ( from km), (~500 away) km Amazon oraround Belem (~400 along plantations River the old the from M. perniciosa (Godar stands (Bartley, 2005). Since the establishment of parts road theeastern Transamazon of (Central Para cacao state) didnot contain spontaneous with natural incidence of contained cacao spontaneous standswild diversity, (possibly ancient plantings), pre-European from 1970s and 1980s (Godar duringthe 1), aspart road Transamazon (Figure along state ofcolonization projects the Central Para Later, cacao cultivation was encouraged by the Br This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article inthe rubber by late 19 supplanted being until economy basis Amazon original the the of Jesuit particularly Cacao by River, Amazon (Bartley,stimulated missionaries production was 2005). et al et Therefore, the objectives of this study were to describe the relationship between the The spread of the fungus to Southern Bahia intwo Southern fungusto spread apparently occurred primary of the The in 1989, ., 2012), witches’ broom became a major limiting factor for production. But the the factor becameorigin a But of for limiting production. ., broom witches’ major 2012), in the region along the Transamazon road in either as region isunclear, the have it Transamazon derived the along could et al T. grandiflorum T. ., 2003;Santana et al et et al T. cacao M. perniciosa M. perniciosa A sampling: systematic collection of isolates from the C-biotype was ., 1999), possibly by either deliberat ., 2012). The states of Rondonia and Acre, near the putative center center Rondonia the The andAcre, of states near ., 2012). of putative ) stands, or from) stands,elsewhere. or treesin 2004 in areas of the Amazonian region where the fungus the states of Acre, Amazonas, Pará, andRondonia inBrazil,and in the largest genetic diversity of the pathogenic C-biotype using et al et (Bartley, 2005; Thomas Thomas 2005; (Bartley, ., Investigation 2012). of the of theorigin genotypes in ribed sequences the (ITS), defining spacer while azilian government intheazilian government state Rondonia andin of cacao cultivation in the tic population structure, the dispersal pattern in

et al e or accidental intervention by man by man intervention accidental or e ., 2012). On the other hand, the Transamazon road region Transamazon th century. century. et al ., This article is protected by copyright. All rights reserved. copyright. This articleis protectedby conducte was reconstruction Phylogenetic settings). the (v.6.1.6)Geneious within bioinformatics package (default for sequence using MAFFT alignment al wasamplifie region (spanningITS1 andITS2) spacer genetic transcribed the analysis, For internal reconstruction: and analysis sequence phylogenetic ITS Pharmacia Biotech; Buckinghamshire, UK). al and filtration stored at -80º C.DNA was extracted from mycelia following aCTAB protocol (Silva for two temperature rpm) weeks. (100 wasMycelium then agitation room by collected under at (0.17% malt extract, 0.5% yeast extract, 5% glycerol). These cultures were incubated in the dark + G liquid MYE medium of 50 mL used inoculated to were culture the of edge growing the from DNA extraction: Isolates were cultivated on PDA in Petri dishes for two weeks, when hyphae plugs the Brazilian Ministry of Agriculture(‘Parecer Técnico’ DFCP/CGPP 003/2010). permit under the for Cocoa were Studies,Barro transported of Bahia, Brazil.Isolates Preto, Center the Mycology culture collection at Aberystwyth Un isolates from the L-biotypes, originally collected M. perniciosa of isolates Bahia fourteenAtotal C-biotype Itabuna, of (theCEPLAC, 111 state remaining isolates). and (isolates 74, 76, Campinas75, -UNICAMP” de 77), Gramacho Karina andby Peres fromDr. mg L 100 containing (Potato-Dextrose-Agar PDA usingaselective cultivated medium were mycelia where theRecursos at Genéticolaboratory “Estação de and back the fromto taken trees long) were collected cm brooms’, 20-50 (‘green cacao shoots of the ExecutivaAgriculture “Comissão do da Lavoura (CEPLAC).Cacaueira” Plano infected Briefly, Acre, Amazonas, Pará and wereRondonia collected by the staff of the Brazilian Ministry of km between sites, with the geographic positioning recorded (Table 1, Table S1). The isolates from natural conditions along atmain roads the BrazilianAmazonian, located at a minimum distance of 10 isolates from Bahia were kindly provided by Prof. Gonçalo Pereira from the “Universidade Estadual Gonçalo “Universidade from by the Prof. fromPereira provided Bahia kindly isolates were media. sterilization with hypochloride5% sodium solution

Accepted was conducted technology. Sequence management BigDye andsequenced using Sanger ., 1990), fluorimetry using by was determined Quant concentration DyNA (Amersham 2000 DNA ., 2008). Article -1 benzamidazole and L 100 mg benzamidazole C-biotype isolates from Southern Bahia were included to investigate their origins. These These investigate their origins. includedto Southern Bahiawere from isolates C-biotype were analyzed (Table 1, Table S1). Inaddi -1 streptomycin) from small slices shootof tissues after surface s José(ERJOH), Haroldo” Marituba, Para state, in Brazil and Ecuador (Table 1) were sourced in Brazil (Table sourced from andEcuador 1) were . Allisolates were routinely maintained on PDA iversity iversity byor Dr. the AlanPomella from Mars d, using the primer pairs ITS1F and ITS4 (White pairsITS1Fd, andITS4 primer using the d using PhyML and the GTR substitution model. and the GTR dsubstitution usingPhyML tion, 14 isolates from the S-biotype and six

et et estimation of the average co-ancestry coefficient ( the analysis of spatial genetic st region Amazonian from the genotypes ( unique usingonly multilocus investigated analyses structure by genetic Spatial andisolation distance: isolation bydistanceThe (IBD) was 1995). (Goudet, program, using FSTAT 2.9.3.2 version the estimated parameters andanalyses were these each population was estimated by: by: estimated was population each inbreeding. To inbreeding. To visualise the SGS, advantage to not assume Hardy-We individuals. To test whether there was a significant deviation from random spatial genetic spatial random structure; deviation was genetic asignificant from there whether test To individuals. plotted againstdistances. The distance classes were permutation of alleles among individuals and a Bonferroni correction alleles among ( correction andaBonferroni of multiple tests of individuals permutation the frequency the frequency of the This article is protected by copyright. All rights reserved. copyright. This articleis protectedby ( genotypes different multilocus have at individuals taken random that two likelihood The isolates, we used the genetic identity anal among genotypes of presence multilocus identify repeated the To analyses: diversity Genetic USA). MA, Waltham, (Fermentas, inrelation distance a conducted by bp DNA migration to calls were 100 ladder molecular loci ( nine L-biotype (Table Table1, S1) was analyzed usingsp Microsatellite loci analyses: The genetic diversity of the 131 isolates of fixation index ( version 1.3 (Hardy & Vekemans, 2002). The estimates of based on the rarefaction method (El Mousadik & Petit, 1996), expected heterozygosity ( heterozygosity (Elexpected 1996), & rarefaction Petit, on Mousadik the method based locus ( per alleles the average of number parameters: usingconventional determined diversities were genetic ran in Tris-borate buffer at 50 buffer staining at for (Silva silver W 50 h,and ran inTris-borate by 2 visualized indenaturi separated were products Amplification et al ( mMpCena22 mMpCena19, D MsCepec_14

Acceptedg Article were obtained using the Multilocus program (Agapow & Burt, 2001). Biotype and population and program & usingthe (Agapow Burt, population obtained Biotype Multilocus 2001). were . (2007). amplification reactions wereThe . (2007). mMpCena3 A ), allele richness estimated for a minimum sample size size for richness), allele a of individuals sample 9 ( estimated or 6 minimum , MsCepec_15 F ). Thestatistical significance of the i , th mMpCena4 multilocus genotype multilocus in each population (Kalinowski , and , MsCepec_16 ructure (SGS). Spatial genetic structure was determined usingthe (SGS).ructure structure was determined Spatial genetic mMpCena26 θ , xy mMpCena8 D inberg equilibrium anditcanbe usedinpopulations with values values were averaged over a set of ten distance classes and g = [ , MsCepec_19 ysis implemented inCervus 3.0 (Kalinowski n /( n ) described by Silva Silva ) described by conducted as previously described described (Silva conducted aspreviously − , mMpCena11 1 )]( ng sequencing gels (7% polyacrylamide, 7 M urea) 7Murea) (7% polyacrylamide, gels ng sequencing θ ecific primers for 14 microsatellite loci, including 1 chosencontain tothe number same pair of of xy F − values was estimated based on was Carlo values estimated Monte ) between pairs of isolates and SPAGeDi and pairs isolates SPAGeDi of ) between  ,

and θ p xy i 2 MsCepec_45 ) based Loiselleon ,

, where is the sample size and sample size isthe , where mMpCena12 et al . (2008), and additional five loci. (2008), and five additional M. perniciosa ) developed by Gramacho by ) developed , et al et mMpCena16 et al et al et ., 2007). Estimates of n ., 2008). Allele = 56) based on 56) = . (1995) have the of C-, S-and of the et al et α R et al H = 0.05). All = 0.05). ) estimated , ., 2007). D e ) and ., 2008). 2008). ., g ) in p i is biotype is distinct from the C-/S- biotypes consiste biotypes C-/S- from isdistinct the biotype Eastern Brazil (Minas Gerais). For the purpose of the present study, these analyses confirm that L- brasiliensis species the new of sequence (AY317137) the contained also clade the ‘Malphigiaceae’ L-biotype statistical Furthermore, good support. separately clustered hosts with liana isidentified as either Bign biotype of Ecuado Amazonian sample from L-biotype the from isolates from coastal Ecuador (112-116) a forming distinct well-supported clade, clearly separated samples, were clearly from with C-/S-biotype separated isolates L-biotype support. statistical poor separated but with only were isolates clade, S-andC-biotype this However, regions. within spacer ITS1/2 found polymorphisms few within the 2) with very variation clade(Figure well-supported a formed C-and that the S-biotype showed inGenBank deposited andseveral study present the from reco Phylogenetic samples sequenced. was biotype and andS-biotype asubset of C- ITS for region genotypes, allL-biotype ‘DNAbarcode’ the three Phylogenetic analysis of the three biotypes: In order to better define the relationship between the Results to belong population. that individual didnot that the indicated replacement. If the observed likelihood of the indivi from runswith based population, onsampling generated 10,000 allele of the frequencies population thelikelihood comparing value of theindividual resulting in afalse positive assignment. Therefore, a measure of confidence was needed that the group for notincludethe true control population of the origin populations might reference set of the However, assigned always inany be set which individuals to to. could reference the population populations approach using leave-one-out likely a (self-assignment). sampled There the was most reference allindividualsBased dataset Efron from self-classified (1983), to 2004). the were on the tested individuals truly belonged to a given population (Halkett population (Halkett agiven to belonged truly individuals tested This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Articlemultilocus approach (Cornuet assignment carried assignment: Individual tests genotype outAnalysis using were of Bayesian the locations. among individuals of permutations 1,000 the 95% confidence interval wascalculated for each observed value and each distance class from M. perniciosa that was reported by Arruda by reported that was from Brazil are deposited on GenBank (Tarnowski & Ploetz, 2008). The host host The 2008). & (Tarnowski Ploetz, on fromGenBank Brazilaredeposited et al oniaceae or Malphigiaceae and isolates obtained from these distinct ., 1999) implemented in the Geneclass2 program (Piry (Piry program inthe Geneclass2 implemented ., 1999) et al et . (2005) forming brooms on test with the likelihood distribution theof nt with its outcrossing breeding strategy (Griffith nstruction based on alignments of of sequences based on alignments nstruction r (117). Several unpublished sequences of the L- sequences unpublished Several r (117). dual test was outside the distribution, then this

et al Crinipellis ., 2010). This was developed by by developed was This 2010). ., Heteropterys acutifolia (= Moniliophthora et al ., ) in Analyses Analyses of microsatellite loci from isolates of the three biotypes: Analysis of the 14 microsatellite (FigureS1). anidentical their confirming identity gave thus pattern PCR-RFLP isolates fragment) and digested by either eight loci ( alleles. two only alleles per locus,rangingfrom the at loci. present A alleles polymorphism was found,5.1 withallelic amean 11 total were of of 56 inall loci isolates131 revealed sizepolymorphism at all butone locus, and these,of unambiguous This article is protected by copyright. All rights reserved. copyright. This articleis protectedby disclosed,alleles were with averageallele 2.63 an average 3.72of alleles per locusandsix private a locus, with eight private alleles (Table S2). For th Brasileia). 70 Thaumaturgo, and 58 Ceplac/ERNEG), (56, Ouro Rondonia 57 biotype isolates were identified heterozygous for were Mocajuba) Ale Uruará, 15 14 Medicilândia, for heterozygous were andAmazonas (27 Trairão) state Pará and (43 Costa44 daConceição, and 46 45 Ceplac/ERNEG) from isolates a Acre (65 C-biotype heterozygous. MsCepec_19 MsCepec_16 confirm their identity (Table 1, TableS1). formation reported for Bignoniaceae theL-biotypeon of specialization & Hedger, 1994a). However, further work isneeded to determine whether there ishost Acceptedthe L-biotype was the only one exhibiting two alleles for loci were disclosedheterozygous individuals only from For another loci ( and S-biotypes. four were observed for Article Most isolates were homozygous were isolates for nearly allthe for loci,Most was detected heterozygosity but Isolates from the L-biotype displayed atotal of fromdisplayed the L-biotype Isolates As allof the C-biotype isolates were obtained from infected tissues, it was necessary to mMpCena3 . For ,

and MsCepec_16 MsCepec_19 mMpCena3 , mMpCena4 mMpCena8 M. brasiliensis , only the L-biotype isolate (Pichilingue,114 Ecuador) was . For . For ) for afew isolates. Heterozygous isolates from all three biotypes nquer, 17 Santarém, 19, 20 and 21 Cametá, 22 Baião, 23 Baião, and 24 and 23 22 Cametá, 20 21 19, Santarém, nquer, 17 from from Pará (26 Trairão), Amazonas (43 Costa daConceição, 45 Msp mMpCena22 mMpCena22 mMpCena19 , , whereas isolates from Pará state (1 Anapu, 7, 8and 9 mMpCena8 mMpCena11 by Arruda I or I or mMpCena4 The ITS1-5.8S-ITS2 region was amplified ( Hinf I (Arruda Preto D’Oeste), and Acre (65 and 66 Mal. and66 andAcre (65 D’Oeste), Preto s per locus and with only three private alleles. alleles. private locus s per three andwith only and Malphigiaceae and also whether the broom andalso the and whether Malphigiaceae locus with atotal of 9 alleles, to , (Table 1, Table S1). For For S1). Table 1, (Table mMpCena11, mMpCena19, mMpCena11, mMpCena19, mMpCena22 , e S-biotype isolates, alleles41 were identified, with nd 66 Mal. Thaumaturgo, 69 and 73Assis Brasil), et al et

, heterozygosity was revealed for isolates from L- mMpCena19 L-biotype.Isolate Ecuador) (Pichilingue, 113 of lleles. For all 111 C-biotype isolates, a total 29 of . (2005) occurs onho 42 alleles, with anaverage of 3.81 alleles per et al ., 2003). All putative mMpCena4

, MsCepec_16 mMpCena3 , mMpCena19 sts inBignoniaecae. and MsCepec_15 M. perniciosa MsCepec_19 circa , heterozygous C- , heterozygous and 750 bp 750 ,

with

), and S-biotype displayed significantly significantly displayed and S-biotype (Central20 (Acre) to Pará), while ranging 10 of genotypes adistinct members, from number contained of multilocus populations biotypes and between within diversity Genetic among the six L-biotype isolates (Table 1, Table S3). (isolates and both120 121, andno from repeated Manaus), detected genotypes was multilocus Amazon) (Table S3). For the S-biotype, only one pa (West from AM Tabatinga, 92 isolate to identical Amazon) were (Lower from Alenquer and109 107 (Acre) was identical to isolates from Central (6 Pará Medicilandia and25 Trairão), whereas isolates Trairão, PA (Central Pará) and 73from Assis Br Ceplac-ERNEG,and 46 Conceição an AM)displayed S3 (Table populationlocations theoriginal outside genotypes identified for the C-biotype, only in three cases identical genotypes were identified in (>40 genotypes multilocus number repeated of largest populations, Central East seven Amazon, the ones Pará, and Bahiawere with these the Rondonia, Solimões Acre, Colombia-Peru at(Upper Amazon Rondonia, andBahia the border), Among (Table 1). Amazon East (around river Amazon Lower (surrounding Amazon the mouth), or Manaus), West This article is protected by copyright. All rights reserved. copyright. This articleis protectedby isolates from the C-biotype distances, longer for inatypical pattern isolation by negative values distance. of significantly a SGS zero up to significant (Figure Amazonrevealed or km from for the 137 3), tending to genotypes one isolate without proper spatial position, the genotypesmultilocus from Bahia (Table 1), where (SGS) Excluding by structure isolates: distance seven genetic andisolation Spatial C-biotype the of identical allele composition with one or isolatesmore (Table 1, Table S3). shared remaining 48 genotypes, with the multilocus unique to corresponded 83 only isolates, multilocus genotypes (‘clonal’, with exact allelic match). Our results indicated that from the 131 for the investigated loci. Therefore, Cervus 3.0 was used to verify and identify isolates with identical indicated that some isolates from the same region repeatedof Identification mult largest genotypic diversity genotypic diversity ( largest Bahia On by andAcre followed (0.32) hand, (0.31). population the held (0.39), other Acre the Pará Acceptedbiotype isolates (0.69/0.56 vs. 0.29; Table 2). Within the C-biotype, the highest Article The SGS analysis allowed us to arbitrarily define seven geographic populations with the populations with the define seven geographic arbitrarily usto SGSanalysis allowed The

of D g M ilocus genotypes within The populations: microsatellite analyses = 0.96), followed by the West Amazon ( . perniciosa there were only six L-biotype isolates (Table 2). Isolates from the L- higher expected heterozygosity ( heterozygosity expected higher (Figure 1): Central Pará region),(Transamazon road Pará asil (Acre). Isolate 64 fro and populations: The seven arbitrary C-biotype C-biotype arbitrary seven The and populations: analysis of the remaining 56 uniquemultilocus ). Isolates from the East Amazon (44 Costa da M. perniciosa ir of repeated multilocus genotypes was identified was genotypes repeated multilocus ir of of collection shared the same allelic composition identical matc multilocus %) (Table From 1). the 47 repeated multilocus

was only recently introduced, and introduced, was recently only H m Marechal Thaumaturgo D e g ) compared to the C- = 0.95), whileBahia = h with isolate 27 from h withisolatefrom 27 H e was observed for wasobserved (Table Allele2). richness ( This article is protected by copyright. All rights reserved. copyright. This articleis protectedby lowest the displayed the S-biotype isolate 131 from Manaus. Aurelino Leal, BA, respectively, Table 3). The group of L-biotype isolates matched, with 100% score, and 117) were assigned at a high score (>92%) the isolate 116 (Janauche) assigned with a high score (> 99%) to the S-biotype isolate 131 (Manaus, AM, Brazil), together with were from 3), allgenotypes thethree (Table remaining against S-andC-biotypes assignment the fro 117 isolate L-biotype the to assigned se the Asagroup (<97.0%). (containing Manaus isolate S-biotype from were assigned genotypes S4).131 (Table five to The remaining genotype from San Carlos, while two (32 and 93) showed lower score (between 90.0 to 93.4%) to the same the L- and S-biotypes (Table S4), 57 were assigned at a high score (>98%) to L-biotype isolate 117 from San Ecuador. Carlos, 117 isolate biotype (Tablebiotype 3). Asagroup, theS-biotype isolat RO), and 119 showed alower score (50.1%) to geno a displayed highassi 124 the genotypes, S-biotype Ofthe two other genotype. the same to (<92%) alower score and displayed 123 129 isolate whereas from inAmazonian SanCarlos isolate 3) L-biotype Table Ecuador, the (>99%, high score 117 to of analyzed were genotypes S-biotype 13 When the populations. genotypes reference to the individual possible multilocus exclude origin of or assign to a assignment test genetic populations: to We undertook genotypes assignment of Genetic Rondonia (0.37). Accepted Articlethat the highest were analyzed genotypes the unique only multilocus When respectively). and (3.56 3.82, higher values displayed richness ( (includingidentica allgenotypes considering when Conversely, when allsix genotypes theof L-biotype from Ecuador were analyzed for from against those were tested C-biotype from the unique genotypes all multilocus When 64 R ) ranged from 2.14 (Bahia) to 1.71 (West

( n H =6, Table 2), the genetic parameters the Table =6, genetic asimilar2), showed trend for cases,most except e was observed for the Bahia population (0.45), followed by followed and by (0.45), (0.40) Pará Bahiawas for population the observed D g (0.77). All C-biotype isolates exhibited a high and significant fixation index isolates ahighandsignificant index AllC-biotype exhibited fixation (0.77). R , but at a lower score (51.6%). The two other L-biotype genotypes (115 (115 genotypes two L-biotype The score, other (51.6%). alower at but ) wasstandardized a for sample size( against all the remaining genoty m San m Carlos, atEcuador a 100% score. to C-biotype genotypes (63 Ji-Paraná, RO or 91 ven populations), the C-biotype genotypes were genotypes C-biotype the populations), ven es were assigned ata 100% probability to the L- gnment score (>99%) to genotype (Ji-Paraná, genotype 63 to (>99%) score gnment l genotypes).multilocus For the C-biotype, allele type 91 (Aurelino Leal, BA), both from the C- Amazonas), whereas the S- and L-biotype

n ) (Table genotypes of 2) nine pes, nine were assignedat a originally neither endemicoriginally itshostnor This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted genotypemultilocus (isolate 109) fromthe same border the between Brazil,at anidentical (>99%) ahighscore displayed and Peru, Colombia with Solimões) from Amazon West (Upper genotypes (score 11 Allthe Ceplac/ERNEG multilocus 76.1%). shared scores between 92.6 to 97.8% to genotype 63 Ji-Paraná, RO, and one to genotype 45 genotypes from Acre, four shared scores between (> Ceplac/ERNEGassignment 99%) 45 genotype to (TableAmazonas S5). Of the genotypes seven from Rondonia, two exhibited high probability of probability.100% 98.2%. As a group, the isolates from Bahia were a 87 and 90) were assigned to genotype 63 from Ji-Paraná, Rondonia, with scores ranging from 71.0 to an origin from Tabatinga, AM (92) with scores ranging from 53.4 to 100.0%, while another three (81, genotype 63 from Ji-Paraná,Rondonia. from derive to certainty a 100% shared population Amazon Lower the group, As respectively. at 81.2 and 91.6% probability to isolate 63 fr Article with from isolate score 110 AguaBlanca,Ecuador (Table genotypes 3). assignedThe were two other genotype 73 from Assis Brasil, Acre (score ranging from 90.7 to 97.7%), whereas four had a 100% Ceplac/ERNEG. genotype 45 the to (99.1%) showed ahighscore Pará Central The population (Table 3). East Amazon, Ceplac/ERNEG which from wa 45 genotype assigned to genotype 73 from AssisBrasil, Acre, tw Assis Brasil,Acre. The 12 genotypes from Central Pa the group, East Amazon C-biotype isolates were fromgenotypes 92 Tabatinga, AM, 63 Ji-Paraná, from genotypes and Bahia).TheAmazon, East 10 Am where C-biotype of the population individual from each arbitrary genotype The same analyses were conducted for the populations from Rondonia, Acreand Rondonia, populations from West for the were conducted same analyses The to and assigned 84 were 91) (74, 77, Bahia, four genotypes genotypes from seven the Among For the nine genotypes from the Lower Amazon population, three were assigned to foreach assignment genetic then putative conducted were origin, the analyses determine To T. cacao om Ji-Paraná,om ROand 45 Ceplac/ERNEG, AM, was endemic (East Amazon, Central Pará, Lower assigned at probability 94.2% to isolate from 73 RO, and from Assis 73 Brasil,AC (Table Asa3). population (isolate S5).population (Table Tabatinga) 92 ssigned to genotype 92 from Tabatinga, AM at from AM Tabatinga, 92 ssigned genotype to , and one to 73 Assis Brasil, Acre. Ofthe eight 80.7to 98.3% genotype to 73Brasil, Assis three o to genotype 63 Ji-Paraná, Rondonia, and four to andfour to Ji-Paraná, Rondonia, 63 genotype o to rá were analyzed in the same way, and six were azon were assigned with a score below 96% to s assigned to genotype 73 Assis Brasil,Acre

M. perniciosa was 42), includingeightprivate 42), allele amplification analyses suggested that the S-biotyp any mating event). (i.e. ability meristem infection a by singlespore the S-andC-biotypes, selection possibly by for homothallism, linked to a more competitive dispersal inbreeding (Griffithoutcrossing mechansism bifactorial in 1994b). Biparental &Hedger, occur might populations) was PacificandAmazonian and (both its to distinct variable more due biotype variability, consistent with their primary homotha genetic (C- and related beclosely andS-)to broom-forming little biotypes exhibting pathogenic Hedger, the (Griffith & analyses found 1994a). Our phylogenetic L-biotype non-pathogenic endophytic apparently the to S-biotype, pathogenic occasionally the cacao, on C-biotype pathogenic in comparisonto to the S- or L-biotypes. It isrecogn al (Arruda sequences nucleotide fromITS Evidence leve alower exhibiting while to similar each other, Arruda meristematic flushing by the disturbed condition ( condition the disturbed flushingby meristematic forest areas, either as weedy invader ( This article is protected by copyright. All rights reserved. copyright. This articleis protectedby by evolved have ( diversity genotypic The Discussion from a more ancestral non-pathogenic form, likely the L-biotype. S-biotype and C-biotype might andC-biotype might S-biotype form, the likely L-biotype. ancestral a non-pathogenic from more (C- AM. results both pathogenic These andS-) Manaus, bioypes that suggested evolved have might Ecuador, while the remaining fiveisolates shared higher score against S-biotype isolate 131 from multilocus genotypes (92%, 59 out of 64) were assigned to L-biotype isolate 117 from San Carlos, isolate (117) could indicate asharedancestry andHedger (1994a). byGriffith suggested arisen in bottleneck have could &Bataillon, Suchagenetic ingenetic 2010). (Stukenbrock variation reduction cases inthe host, even reproducing pathogen, sexually of effect afounder imposes with important

Accepted from isolates C-biotype the a for the diversity indicated lower genetic results Indeed, our ., 2006). Article three et al L-biotype isolates exhibited thegreatest level heterozygosity,of possesing more alleles ( The convergence of most S- and C-biotypes multilocus genotypes to the same L-biotype the same to L-biotype genotypes S-multilocus most of andC-biotypes convergence The M. perniciosa recognized ., 2003) have previously suggested that the broom-forming C- and S-biotypes were more more C-andS-biotypes were the broom-forming suggested that previously have ., 2003) disruptive selection by infection on distinct host species D g ). Somatic and compatibility molecular by a to as breeding switch from strategy, outcrossing non-outcrossing M. perniciosa s, the largest allele richess ( biotypes exhibit contrasting life styles, ranging from the highly ranging highly the styles, life from contrasting exhibit biotypes eg . Solanaceae induced or asa species) with host susceptible of both biotypes. Most of the C-biotype unique unique C-biotype the of Most both biotypes. of could leadcould to later basidiocarpformation without et al et llic (homomictic) breedingstrategy, whereas the L- Theobroma e is more diverse than the C-biotype (Rincones l incomparisonof diversity, to the L-biotype. ized that apathogen transi ., 2005), karyotype and microsatellite telomeric R ), expected heterozygosity ( heterozygosity ), expected analyses (Griffith & Hedger, 1994a; and

Herrania ). indisturbed occurring tion an to agricultural H e ) and A et = This article is protected by copyright. All rights reserved. copyright. This articleis protectedby adapted mating prior to sexual reproduction, leading to the spread of geographically C-biotype mycelia ishomomictic, dispersal ca East Amazonas,Pará, Rondonia andBahia as largestands of cacao, commercial such in regions containing especially region, genotypes (47 in 111 isolates) were detected among C-biotype isolates from the same of ‘isolation by distance’ (IBD) modeof dispersal. A large number of repeated multilocus Amazon, with significant co-ancestry detected for up to 137 km, suggesting the occurrence significant spatial genetic stru and potential between distant flow the populations. gene populations mechanism for longer distance dispersal. infected plant material under unwitting human intervention might predominate as a favor spread over long distances upon the appropriate conditions. However, movement of monocyclic within ayear (Purdy & Schimdt, regimes do not typically limit infection cycles over the year, but infection tends to be been reported to up to 50-70 Kmin Ecuador (Wheeler & Soarez, 1993). Tropical rainfall losing viability within hours, but long distance dispersal, most likely during the night, has from basidiocarps (Purdy & Schimdt, 1996). Basi basidiospores, the only recognized infective structure of the C-biotype, following meiosis appeared to be broom (‘ be to witches’ symptoms appeared described what in region this cacao plants 1785-1787 Ferreira, exhibiting visited who Rodrigues Schmidt, (Purdy species & cacao andrelated 1996) Accepted its distribution. original natural the area, beyond spread throughout have period, of cacao cultivation had 100-year Amazon modern inthe expanded and the might pathogen plantations was documented since the first outbreak introduction of the Subsequently, disease. of the report Article The C-biotype of The dispersal ability pathogens,of especially f of dispersal The

lineages (Griffith & Hedger, 1994b), similar to ‘clones’ (Stukenbrock & Bataillon, 2010). M. perniciosa M. perniciosa cture (SGS)for isolates of the C-biotype in the Brazilian isendemic to the Upper Amazon valley, where it infects depends on the release of the haploid uninucleate . C-biotype isolates were mostly homozygous. lagarto 1996), and continuous inoculum release may n be achieved without the requirement for . The travel report of the naturalist Alexandre ’ [= Silva, lizard]; 1987), representing the earliest diopores are highly sensitive to desiccation, in 1895 in Suriname (Rorer, 1913). Within this ungi, defines thegenetic differentiation among M. perniciosa

into commercial cacao Here, we detected a we Here, detected extensive Central Central A s the s the

individual multilocus genotypes from the East fromAmazon, the genotypes Amazon, andBahia Central Lower Pará, multilocus individual ofgene center the bepart of and might therefore these two populations appeared to contain the largest genetic diversity of Acre, or from the Santarém region, a traditional region of cacao cultivation, where the West Amazon from and directly Rondonia introduced was either pathogen suggesting the Amazon, West not that introduc from derived exclusively have to appeared population Pará Central agoThe (Bartley, 2005). years around 300 cultivation started modern where West Amazon eastwards to the rest of the Amazon valley to regions where cacao is not native, and downstream from river and flow the direction Rondonia of Acre, followed have pathogen might the of region. Amazon The movement the Upper didnot species occur naturally outside host the since the diversity pathogen. limitedof the have genetic which were collected, might isolates important cacao producing region since the 1970s, with large areas of cultivation, from where the derivedRondonia Acre from (73 Assis Brasil) or This article is protected by copyright. All rights reserved. copyright. This articleis protectedby number ofheterozygous isolates for traced back to isolate 63 from But Ji-Paraná. Amazon populations corresponded geographically to the regions that presumably genotypes from Rondonia also pointed to a West Amazon origin East Amazon, Lower Amazon and Bahia populations was from the West Amazon. Some from the Amazon. genotypes multilocus part be of the origin Acremight of many that from genotypes to assigned Acre,be or alesser to extent, Rondonia to corroborating (isolate 63), the least repeated multilocus genotypes. but the diversity, different spores. The West Amazon population also presented an elevated heterozygosity could have arisen following mult heterozygosity (at two loci) was detected within the C-biotype populations. Such Brazil. Ofthese, the Acre population was the more diverse and was the only area where fungusin diversity part center of the be the of might andboth of within the C-biotype, diversity Acceptedthe center of diversity of Article The Rondonia population as population animportant isolates, appeared have dispersal to Rondonia inthe role of The The spread of the pathogen elsewhere occurred pr The genetic assignment test indicated that the putative origin of isolates from the The Acre and West Amazon populations lowest T. cacao allele richness (Sereno , and together with the Acre population, both contained tic diversity and diversity fungus. of the tic dispersal m MpCena3 et al Rondonia showed no private alleles and alimited Most of the multilocus genotypes from Acre tended to from genotypes Acretended of the multilocus Most ., 2006; Motamayor Motamayor 2006; ., West Amazon (92 Tabatinga). Rondonia became became an Rondonia Tabatinga). West (92 Amazon tions of genotypes from Acre and Rondonia, but but from Acretions genotypes andRondonia, of . According to GeneClass, the genotypes from . According GeneClass, to the genotypes iple infections of meristems bygenetically appeared to contain the greatest genetic obably inassociation with cacao dispersal,

et al et . The Acre and West ., 2008;Thomas genotypic genotypic M. perniciosa et al encompass ., 2012), 2012), ., , Cocoa Research funding GWG appreciated. Ltd. acknowledges isgreatly from UK Pimentel recipients Brazilianof National Research Council (CNP for technica and CEPLAC 07/07175-0) (03/11483-0, authors wishThe to thank the “São Research (FAPESP) Foundation” Paulo for financial support Acknowledgments pathogen.destructive light locations could shed on from collections diverse the more origins of this sampled, additional under- are currently populations S-biotype since these hosts. However, solanaceous infecting population from an ancestral evolved but have it may uncertain C-biotype remain of the origins the man.However, the and pathogen by of known incidences translocation of suspected consistent with (clonal) spread. The presence of genetically very genetic withinvariability populations C-biotype of Bahia. Southern with isolates from the Lower Amazon population (Pará), an Ploetz Amazon). BahiaintheCacao 18 inSouthern wasAmazon). introduced two origins, withancestry to genotype 63 from Ji-Paraná (Rondonia) and 92 from Tabatinga (West Andebrhan Andebrhan presence of two distinct lineages of inSouthern introduced genotypes clearly Bahia.The indicated originsfortwo the results major at Uruçuca reported were and Camacan free of This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Article invasion of documented suggesting multiple introductions. The multiloc inthat population. alsodetected origins were Amazon Amazon likely as Lower was from the less West primarily Introduction detected. not were genotypes et al et M. perniciosa The microsatellite analysis deployed in the present study has demonstrated only limited study hasdemonstrated present analysis inthe deployed The microsatellite The Bahia population exhibited the lowest genotypic diversity, but high allele richness, but highallele diversity, genotypic lowest the Bahiapopulation exhibited The ., 2005; Santana Santana 2005; ., et al . (1999) and later confirmed by many other genetic analyses (Rincones (Rincones genetic analyses by confirmed other and later many . (1999) for over 200 years. In 1989, two independent foci of foci of independent two In1989, years. 200 over for M. perniciosa et al ., 2012). Thus,it ispossible that this invasion occurred based on M. perniciosa (Andebrhan (Andebrhan municipalities, 120 municipalities, apart km 120 (Andebrhan us genotypes from Bahia,us genotypes arecent and well- similar strainsingeographically distant areas is isolates inBahia was originally demonstrated by M. perniciosa l support. ASA,PSBA,l support. GAL,AMS, andAF were et al et q) fellowships.Technical assistance by Edivaldo areawith an easier and more intense contact ., 1999), displayed the clear migration from the clear migration displayed ., 1999), th century, and the region managed to be be to managed and region the century,

, consistent with non-outcrossing with, consistent non-outcrossing M. perniciosa et al ., 1999). Our et al occurrence ., 2003; 2003; ., Arruda MCC,Sepulveda GFC, Miller RNG,Ferreir Cornuet JM, S,Luikart Cornuet Piry A, SolignacG, Estoup multilocus New methods employing M, 1999. JH,Bailey BA, HebbarBowers S, Sanogo PK, impactLumsden RD, of The plant 2001. diseases on Bastos CN, Evans HC, 1985. A new pathotype of Bartley BGD, 2005. The genetic diversity cacaoof a Baker RED, P,1957. Holliday Witches’ broom disease cacaoof ( This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted of Morocco. argan of the tree phylogeography Chloroplast DNA A,RJ, 1996. Petit El Mousadik cross-validation. on rate the aprediction Estimating error B,of rule-improvement Efron 1983. ArticleArruda MCC,Ferreira MASV, Miller RNG,Resende ML A, Figueira Yamada Furtek MM, T, Andebrhan agent The causal 2005. W, MC,Phillips-Mora Aime Agapow BurtA,PM, 2001. Indices of multilocus linkage disequilibrium. References data. data. 2005. MSS, Felipe Phytopathological papersno. 2. The Commonwealth Mycological Institute, Kew, 42 p. Research genotypes to select or exclude populations as origins of individuals. as individuals. origins to excludeof populations select or genotypes chocolate production. world solanaceous hosts. Molecular Ecology Molecular Association Statistical American in variability rDNA Brazil. ( disease broom outbreaks of witches' primary (chocolate, 101-2. Mycologia European J Plant Pathology J European Plant

107 Theobroma , 25–37.

97 Crinipellis brasiliensis

Crinipellis perniciosa 5 , 1348-61. Plant Pathology Plant , 547–55.

cacao Plant Health Plant ) form anew lineage of Marasmiaceae. 78 105 , 316-31.

34 , 167-75. , 306–12. from different geographic origins and hosts. andhosts. from different origins geographic , a new species based on morphological and molecular and molecular on, a morphological based species new doi: 10.1094/PHP-2001-0709-01-RV. DB, 1999. Molecular fingerprinting suggeststwo Crinipellis perniciosa a MASV, Santiago DVR, Resende DVR, JC, a MASV,Santiago MLV,Dianese Crinipellis nd its utilization. CABI Oxforshire, UK.Publishing, of withces’ andfrosty broom rot cocoapod of V, Felipe MSS, 2003. Nuclear andmitochondrial

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Marasmius (witches’ broom disease) on ) of Genetics Molecular Ecology Mycologia Theobroma cacao

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153

97 , 1989-2000. Stahel). Stahel). , 1012-22. 1012-22. , Mycological Mycological

Notes inBahia, J

1 , Gramacho KP, Risterucci AM, Lanaud C,Lima LS, Lo to program acalculate computer 1.2: FSTAT version J, 1995. Goudet This article is protected by copyright. All rights reserved. copyright. This articleis protectedby biology breeding JN, The GW,1994a. Griffith Hedger is respon Who B,2012. EJ, J, Pokorny Godar Tizado Giraud T,Refrégier G, Le Gac M, de Vienne DM, Hood ME, 2008. Speciation in fungi. Loiselle BA, VL, Sork NasonLoiselle Spatial structure of atropicalJ, Graham C, 1995. understory genetic Kalinowski ST, Taper ML, ST, ML, Taper how computer Marshall TC, Revising Kalinowski program 2007. the CERVUS spatial genetic analyse program to a versatile computer 1.2: SPAGeDi O,Vekemans X, 2002. Hardy Halkett F,Rivas CD, Platero GG,Zapater MF, Abadie Griffith GW, Hedger JN, 1994c. Dual culture of distribution Spatial JN, of GW,Griffith Hedger 1994b. AcceptedHomewood B, Fungus1991. threat to Brazil’s 300 million cocoa trees. Article in the fungal plant pathogen pathogen fungalplant in the 6. http://www.unil.ch/popge 73. explicit analysis along the Transamazon in Highway Brazil. Biology cocoa, 16, shrub, accommodates genotyping error increases success in paternity assignment. success assignment. increases in error genotyping paternity accommodates 16. levels. at population the individual or structure fijiensis Mycosphaerella pathogenic the plant populations fungus of established inrecently discontinuities genetic Pathology ofPlant Journal perniciosa Crinipellis http://www.ulb.ac.be/sciences/lagev/software.html. http://www.ulb.ac.be/sciences/lagev/software.html. 1099-106. 1099-106. Psychotria officinalis Psychotria Crinipellis perniciosa

45 , 791–802. , 791–802. (Stahel) Singerintropical forest. . Molecular Ecology Molecular

100, (Rubiaceae). . Heredity Crinipellis perniciosa Crinipellis n/softwares/fstat.htm. n/softwares/fstat.htm. 371-9.

72 , 278-89. 278-89. , Amercan JBotanyAmercan

Crinipellis perniciosa 19 , 3909-23. Molecular EcologyNotes Molecular sible for deforestation intheAmazon? Aspatially pes UV, Characterization2007. of microsatellites C, J, by Carlier Isolation 2010. and distance . of biotypes of the witches’ broom pathogen of mycelia theof liana(L-) biotype of the agarica Molecular EcologyNotes Molecular New Phytologist Forest Ecology Management Ecology Forest

82 ,1420-5. and potato andpotato callus. F -statistics. New Scientist

127

2 , 618-20. , 243-59.

7 Molecular Ecology , 153-5. , 153-5. J Heredity

1778 Fungal Genetics Fungal Genetics European European (July 1991), 1991), (July

267 86, 485– , 58–

Silva JRQ,FigueiraSilva A, Pereira GAG,Albuquerque Cacau por elagartão Alexandreregistros feitos P,1987, Rodrigues e Silva vassoura-de-bruxa: This article is protected by copyright. All rights reserved. copyright. This articleis protectedby and diversity natural Genetic A, Figueira 2006. PSB, R, Vencovsky Albuquerque ML, Sereno Araújo EF, ESG, MF, Santana Souza Queiroz Mizubuti JT, MV, Development of 2012. molecular Tobago. disease & Agriculture. of cacao. Surinam Trinidad Board of The JB,1913. witch-broom Rorer Rincones J, Meinhardt LW, Vidal BC, Pereira GAG, 2003. Electrophoretic karyotype analysis of JS,Schnell JC, JWS, R, Geographic Brown RJ, DN, Loor P, Mota Lachenaud Kuhn 2008. Motamayor AcceptedRincones J,Mazotti GD, Griffith GW, Pomela A, Article and epidemiology, Status of cacao witches biology, RA, Schmidt LH, broom: 1996. Purdy Ploetz RC, SchnellRJ, YingZ, S, A, Baudouin a GENECLASS Alapetite for A, L,Estoup JM, Cornuet D, software Piry Paetkau 2: 2004. Molecular EcologyResourcesMolecular from na Amazonia. e1787 em1785 Ferreira microsatellite markers. population structure of cacao ( of structure cacao population perniciosa markers based retrotransposons on for the analysis of genetic in variability pp. 13 Circular no. (July1913) 10 America. perniciosa Crinipellis Plos One ( tree Amazonian chocolate the differentiation of population and genetic polymorphisms of the witches’ broom pathogen pathogen broom witches’ the of polymorphisms Azevedo RA, Pereira GA, Meinhardt LW, 2006. Genetic andchromosome-length variability Research Mycological management. 317-32. diversity in molecular detection. assignment andfirst-generation migrant genetic Moniliophthora perniciosa

3, Mycological ResearchMycological . e3311. European J European Pathology Plant Annual Review Phytopathology , the causal agent of witches’ broom disease disease of broom causal, the agent of witches’

Crinipellis perniciosa 107 Conservation Genetics Zheng Q, Olano CT, Motamayor JC, Johnson ES, 2005. Analysis of , 452-8.

8 , 783-5. Theobroma cacao

110 , causal agent of the witches’ broom disease disease , causalagent of broom the of witches’ , 821-32.

Boletim técnico CEPEC 134 with AFLP markers. with , 497-507. Figueira A,Leal Jr.GA, Queiroz MV, Pereira JF, PSB, 2008. Development ofPSB, microsatellites novel 2008.

34, 7 , 13-24. , 13-24. L.)from the Brazilian Amazon evaluated by Crinipellis perniciosa 573-94.

J Heredity (Itabuna, Brazil) (Itabuna, European JPlant Pathology Theobroma cacao from various hosts in South inSouth from hosts various

95, Theobroma cacao Theobroma 536-9. 536-9. Moniliophthora Moniliophthora 146 Theobroma cacao Theobroma , 21 p., 21 .

111 L). , . This article is protected by copyright. All rights reserved. copyright. This articleis protectedby 2. Figure 1. Figure Figure Legends White TJ, BrunsT, Lee S, Taylor J, 1990. Amplific Wheeler BJE, Soarez C, TarnowskiTL, Ploetz RC, 2008.A multilocus phylogeny ofbiotypes the of and on the emergence perspective genomics Apopulation EH, 2010. Bataillon T, Stukenbrock Thomas E, van Zonneveld M, Loo J, Hodgkin T, Gall J,Hodgkin Loo M, E, van Zonneveld Thomas Accepted 3. Figure Article SPAGeDi 1. The solid line represents the average from the class distance region, exclusively Amazon using each within populations estimated lack spatial (one the for to was of the due excluded genotypes position) C-biotype multilocus 56 and and L-biotypes. andapplications methods guide to In: for phylogenetics. InnisMA,genes Gelfand Sninsky JJ,White DH, eds. TJ, broom witches’ of epidemiology Comparative Rudgard SA, Maddison AC, Andebrhan T, eds. witches’ broom on cacao.cause of doi:10.1371/journal.ppat.1002893. adaptation of new plant pathogens in agro-ecosystems. multilocus genotypes. Co-ancestry coefficient ( refugia followed by human-influenced by followed dispersal. refugia patterns of shown at salient nodes.shown Scalebar site. indicates substitutions per unknown al. (2005). (2005). M Approximate location of collection of the Maximum likelihood tree of 33 Correlogram pairwiseof co-ancestry coefficient ( indicates host in indicates Malphigiaceae. Moniliophthora Theobroma cacao 1993. Disease gradients of of gradients Disease 1993. species (MCA2500). Bootstrap values (% from 1000 replicates) are L.in the Neotropics reflect genetic differentiation in Pleistocene . New York, USA:Academic Press, 315-22. Phytopathology M. perniciosa M* was named was ation and direct sequencing of fungal ribosomal RNA ofation sequencing fungalribosomal and direct θ uzzzi G, van Etten J, 2012. Present spatial diversity spatial diversity J,Present 2012. Etten uzzzi G,van Moniliophthora perniciosa xy Plos One θ . London, UK:. London, Chapman &Hall, 157–64. Crinipellis perniciosa ) was estimated as Loiselle Disease Management in Cocoa: inCocoa: Management Disease xy

sequences (704 bp alignment), rooted with an rooted with bpalignment), (704 sequences 98 value, whereas the dashed lines represent :S155. θ Moniliophthora brasiliensisMoniliophthora PLoS Pathogen xy

) among the unique 56 C-biotype 56C-biotype unique the ) among 7

, e47676. B indicates host Bignoniaceae Moniliophthora

8 oncocoa seedlings. In: (9), e1002893. e1002893. (9), isolates from the C-,S- PCR Protocols:a et al . (1995) for the by Arruda by

perniciosa et et , biotypes. and incomparison Acre; Amazon with eight fromWest from the population; 11 other Rondonia of C-biotype the genotypes from unique multilocus seven assignment each the test Results of the of of Table S5 test or as a group, in comparison with the S- and L-biotypes. individual from Results the C-biotype. from genotypes unique 64 multilocus ofthe test Assignment Table S4 Multilocus genotype with Table S3 allelesPrivate identified from microsatellite loci for isolates (coded as Table S1) Table S2 and site. bygeographical coordination ofthe and country state followed year collection; of isolate identification, locale collection host adopted andof with code previous (ID), biotype, the Figure S1 Figure Information Supporting This article is protected by copyright. All rights reserved. copyright. This articleis protectedby Accepted Articleisolates of Description Table S1 regionITS2 with rDNA ITS1-5.8S-ITS2regionamplified with the rDNA ITS1-5.8S-ITS2 region of 61 C-biotype isolates of identity of of Analysis permutations of individuals among of spatial distance. of spatial individuals among of permutations the 95% (two-tailed) confidence interval of the average HinfI .

Monilihtora perniciosa Moniliophthora perniciosa the respective identical a individual Msp isolates by rDNA-ITS region. A by isolates rDNA-ITS region. andB. Amplification of I. E and F. Digestion of the amplified rDNA ITS1-5.8S- theI. E rDNA amplified of and F.Digestion C-, S- and L- biotypes analyzed. Information about analyzed. Information C-, biotypes S-andL- M. perniciosa θ xy nd original locationnd original collection. of

distribution calculated by 1,000 distribution . C and D. Digestion . Cand the of D. Digestion .

Central Pará Central Pará C-Biotype S-Biotype L-Biotype Accepted populations C-biotype Article individual for each multilocus genotype using the program Cervus using the 3.0. program for each genotype multilocus individual id number isolate localewith of collection original isolates; of Biotypes/ x

Anapú (1); Altamira (2, 3); (22); Baião Brasil Novo (4, Medicilândia5); (6, 7, 8, Pichilingue (112,Pichilingue 113, 114, Jauneche115), (116), San Carlos, Napo (117), Gandu, BA (126), Gandu, BA (126), Manaus, AM Pomba, MG Locale collectionof (number of isolate identification) y (125), Viçosa, MG Moniliophthora perniciosa z Ecuador (119, 120, 121, 128, 129, 130, 131),Rio y (118, 123, 124, 127), unknown (122) (122) unknown 127), 124, 123, (118,

-- entificationnu S1); (see Table x

(L-, S-, and C-biotypes, with seven geographic populations), (L-, S-, andtotal seven geographic of number with C-biotypes, with mber of of multilocus genotypes multilocus of of mber Total number number Total of isolates 111 2 14 6 0 Multilocus genotypes genotypes 12 64 13 6 ; and number of identical NumberIdentical of Individuals (clones)Individuals 47 8 1 0 This article is protected by copyright. All rights reserved. This article is protected bycopyright.

AcceptedAcre Rondonia Solimões) (Upper West Amazonas ArticleEast Amazonas Amazon) (Lower Pará region)(Transamazon Assis Brasil (68, 69, 72, 73); Brasiléia (70, 71); Marechal Thaumaturgo (64, 65, Ceplac/ERNEG (45, 46); Costa (45, 46); Ceplac/ERNEG da Concei (102, 103);Tabatinga (92,94, 93, 95, Ariquemes (47, 48, 49, 50, 51); Cacaulândia (52, 53, 54); Cacoal (61); Jaru Atalaia do NorteAtalaia do (105); Benjamim Constant (104); São dePaulo Olivença 9, 10, 11); Mocajuba (23, 24); Trairão (25, 26, 27); Uruará (12, 13, 14) Alenquer (15, 16, 107, 108, 109); Cametá (19, 20, 21); Óbidos (106); (106); 20, Óbidos (19, 21); Cametá 109); 108, 107, (15, 16, Alenquer AugustoMontenegro (33,36, 34, 35,38, 37, 39); Boa(32); Vista (55); Ji-Paraná (60, 62, 63); Ouro Preto D’Oeste (56, 57, 58, 59) Ecuador x (110); San Carlos, (110); Ecuador Uricurituba (40, 42) (40, 41, Uricurituba Santarém (17, 18)

96, 97, 98, 99, 100, 101); Agua Blanca, ção (43, 44); Manaus29,30,(28, 31); x (111) (111) 1 1 16 19 11 0 7 11 1 8 7 9 0 1 2 5 9 2 0 This article is protected by copyright. All rights reserved. This article is protected bycopyright. y x Total Overall z – AM = Brazilian state of Amazonas. – isolates collected originally in Ecuador; – MG = Brazilian state of Minas Gerais; Accepted ArticleBahia Aurelino Leal (91); Camacan (79, 87); Ca 87); (79, Camacan (91); Leal Aurelino Azul (85, 86); Gandú (84); Ilhéus (76); It Ilhéus (76); Gandú (84); 86); Azul (85, Teolândia Uruçuca Una (80); (81, (89); Teolândia

66); Plácido de Castro (67)

abuna (75); Itajuípe (74); Itapebi (77); (77); Itapebi (74); Itajuípe abuna (75); mamú (90); Canavieiras (78); Floresta 82, 83); Wenceslau Guimarães (88) Wenceslau Guimarães 83); 82, 131 18 83 7 48 11 This article is protected by copyright. All rights reserved. This article is protected bycopyright. about Information 2. ( sample size Table Accepted 111 C-Biotype S-Biotype 14 L-Biotype 6 Article multilocus genotypes,multilocus respectively, were used in perniciosa Moniliophthora alleles ( (Transamazon region)(Transamazon Population (C) ( C A ) ), and fixation index (

Central Pará

Pará (Lower Amazon) Amazon)

20 11 n , as well as for unique multilocus genotype. The parameter The genotype. multilocus unique , as for as well

F ) for the L-, S- and C-biotypes 1.80 1.90 3.56 3.82 1.91 R n ±SE ± ± ± ± ± ), with results allele of richess ( 0.26 0.12 0.29 0.90 0.56 0.05 0.69 0.41 All genotypes

the analyses. SE is the standard error. * 0.26 0.39 H

e ± ± ± ± ± ±SE 0.04 0.04 0.15 0.06 0.09 0.15

(all genotypes) and

.913 0.99 6 1.00 .412 0.94 0.93 D -64 R g ); expected heterozigosity ( heterozigosity ); expected

n 9

the seven geographic populations of isolates from the C-biotype of of fromC-biotype isolates the populations of geographic seven the R 3.73 3.82 1.91 2.73 1.91 P was estimated for nine or six individuals, when all or unique unique all or when individuals, six or for nine estimated was < 0.05. A ±SE ± ± ± ± ±

0.59 3.43 0.50 3.82 0.21 0.24 2.37 0.21 Only unique multilocus genotypes unique multilocus Only

H e );genotypic diversity ( diversity );genotypic 1.81 1.91 R ±SE ± ± ± ± ± 0.48 0.57 0.50 0.66 0.16 0.43 0.18 0.21

0.27 0.40 H e ± ± ± ± ± ±SE 0.09 0.73 0.04 0.74 0.06 0.92 0.07 0.08 D

), effective ofnumber 0.86 0.92 F ±SE ± ± ± ± ± 0.17 0.17 0.12 0.05 0.05 0.08 0.08

1 ihlnu L 11Mnu,A S 9. 1 ioa G 117 Sa All rights reserved. This article is protected bycopyright. 119 99.8 MG 118 Viçosa, 99.5 AM (S) 131 Manaus, AM (S) 131 Manaus, (L) 113 Pichilingue (L) 112 Pichilingue C-biotype of East S-biotype, 3.fromL-biotype, individual the genotype Results assignment of the each test Table multilocus of 1 ihlnu L 11Mnu,A S 100. AM (S) 131 Manaus, (L) 114 Pichilingue 115 Pichilingue (L) 63 Ji-Paraná, RO (C) RO 63 Ji-Paraná, (L) 115 Pichilingue AcceptedAssigned L-biotype Article biotypes or populations (forC-biotype). the populations or biotypes and from Bahia of Amazon lower Pará of region, Transamazon (C) (C) (Upper Solimões) Solimões) (Upper West Amazonas atAaoa 19 East Amazonas (C) Rondonia 17 Rondonia (C) (C) Bahia 18 Acre 10 1 st rank 16

1.79 2.14 1.71 2.09 1.85 ± ± ± ± ± 0.30 0.38 0.37 0.43 0.24 (%) Score

94122 MG 99.4 0 2 aas M 117 San Carlos (L) 121 Manaus, AM Assigned S-biotype s 0.27 0.32 0.24 0.31 0.26 aas M 1Arln el A() 50.1 BA (C) Leal, 91 Aurelino AM Manaus,

± ± ± ± ± 0.10 0.12 0.12 0.13 0.13 0.09 Moniliophthora perniciosa Moniliophthora

.710 0.87 0.77 11 0.95 0.96 0.81 117 San Carlos (L) (L) 117 San Carlos 1 st rank n Carlos (L) (L) n Carlos 7 8 7 1.91 1.73 2.27 2.09 1.91 and as a group in comparison with genotypes comparison the other from in and as a group ± ± ± ± ± 0.25 0.24 0.27 0.21 0.16

100.0 31 Manaus, AM AM 31 Manaus, 100.0 100.0 28 Manaus, AM AM 28 Manaus, 100.0 0. 30 Manaus, AM 100.0 Score Score (%) y 29 Manaus, AM 1.82 1.70 2.27 2.09 1.91 Assigned East Amazonas (C) ± ± ± ± ± 0.24 0.23 0.27 0.21 0.12

0.28 0.25 0.45 0.35 0.37 ± ± ± ± ± Amazonas Central, Pará of 0.08 0.08 0.09 0.07 0.07

0.87 1.00 1.00 0.77 0.93 2Tbtna M 71.6 AM 92 Tabatinga, 1 3J-aaá O 88.0 63 Ji-Paraná, RO 2Tbtna A 95.8 PA 92 Tabatinga, 3J-aaá O 89.7 63 Ji-Paraná, RO st rank ± ± ± ± ± 0.09 0.00 0.00 0.13 0.06

Score Score (%)

rslNv,P 7 si rsl C 50.9 Brasil, AC 73 Assis PA 4 Brasil Novo,

3 Altamira, PA 51.6 131 Manaus, AM (S) 116 Jauneche (L) eiiâda A 5Cpa/RE,A 8. 16Óio,P 10A lna aai C 0. 90 Camacan 100.0 110 A. Blanca, Manabi, EC All rights reserved. This article is protected bycopyright. 57.6 106 Óbidos, PA 84.2 45 Ceplac/ERNEG, AM 45 Ceplac/ERNEG, AM 10 Medicilândia, PA 7 Medicilândia, PA 2 Altamira, PA 1 Anapu, PA (C) Assigned Central Pará 63 100. MG 124 Viçosa, 92.7 131 Manaus, AM (S) 91 Aurelino BA Leal, (C) isolates L-Biotype 117 San Carlos (L) eiiâda A 3AssBai,A 69.6 Brasil, AC 73 Assis 6 Medicilândia, PA

Accepted Article 3AssBai,A 8. 1 lnur A 73 16 Alenquer, PA 94.6 15 Alenquer, PA 81.0 Brasil, AC 73 Assis Brasil, AC 73 Assis

1 63 Ji-Paraná, RO st rank -itp slts 117 San (L) Carlos S-Biotype isolates 3 aas M 117 San Carlos (L) 131 Manaus, AM 2 aas M 117 San (L) Carlos 117 San (L) Carlos 129 Manaus, AM 117 San (L) Carlos 128 Manaus, AM MG 127 Viçosa, 3 aas M 117 San Carlos (L) 130 Manaus, AM (L) 117 San Carlos BA 126 Gandu, Score Score 37 17 Santarém, PA 93.7 (%) s t v 19 Cametá, PA 73 Assis Brasil, AC Brasil, AC 73 Assis 19 Cametá, PA 107 Alenquer, PA 110 A. Blanca, Manabi, EC 100.0 91 Aurelino Leal 91 Aurelino Leal 100.0 110 A. Blanca, Manabi, EC 107 Alenquer, PA 8Snaé,P 4 ELCEng M 16 84 Gandu 91.6 Erneg, 45 CEPLAC AM 18 Santarém, PA 0 z Amazon (C) (C) Amazon Assigned Lower 2 i ob,B 117 San (L) Carlos Pomba, 125 Rio BA 117 San Carlos (L) MG123 Viçosa,

1 63 Ji-Paraná, RO 73 Assis Brasil, AC Brasil, AC 73 Assis st rank Assis Brasil, AC Brasil, AC Assis Ji-Paraná, RO (C) 0. 4 ot aCneço M 3AssBai,A 93.4 Brasil, AC 73 Assis AM da Conceição, 44 Costa 100.0 0. 3 .Mneer,A 6 iPrn,R 45.3 63 Ji-Paraná, RO AM 39 A. Montenegro, 100.0 0. 100.0 0. 100.0 48.2 AC Brasil, 73 Assis AM da Conceição, 43 Costa 100.0 0. 100.0 Score Score 51.2 99.9 33 A. Montenegro, AM 73 Assis Brasil, 73 Assis AM 33 A. Montenegro, 99.9 11 atAaoa slts 3AssBai,A 94.2 Brasil, AC 73 Assis isolates Amazonas East 91.1 97 5Cpa/RE,A 7 si rsl C 89.5 Brasil, AC 73 Assis 45 Ceplac/ERNEG, AM 99.7 12 81 Uruçuca 81.2 95.6 77 Itapebi 77 Itapebi 95.6 07 74 Itajuípe 90.7 77 87 Camacan 97.7 (%) w 32 Boa Vista, AM Vista, 32 Boa Assigned Bahia (C) Assigned(C) Bahia 3AssBai,A 65.8 Brasil, AC 73 Assis

1 2Tbtna M 100.0 92 Tabatinga, AM 3J-aaá O 71.0 63 Ji-Paraná, RO 2Tbtna M 53.4 99.2 92 Tabatinga, AM 92 Tabatinga, AM 3J-aaá O 99.2 63 Ji-Paraná, RO 2Tbtna M 86.0 92 Tabatinga, AM 3J-aaá O 78.5 63 Ji-Paraná, RO st rank AC 81.0 AC

Score Score (%) p u c x

1Mdclni,P 63 Ji-Paraná, RO 11 Medicilândia, PA This article is protected by copyright. All rights reserved. This article is protected bycopyright. p q r s t v u x w y 99.1 z 45 Ceplac/ERNEG, AM Central Pará group PA26 Trairão, 14 Uruará, PA 12 Uruará, PA 27 Trairão, PA27 Trairão, 2 2 2 2 2 2 2 2 2 2 2 nd nd nd nd nd nd nd nd nd nd nd score = 22.9% for 63 Ji-Paraná, RO; score Ji-Paraná, = for 22.9% 63

Accepted AM; Tabatinga, for 92 = 20.0% score Article score = 21.7% for 92 Tabatinga, AM;score = Tabatinga, for 21.7% 92 score = 43.1% for 63 Ji-Paraná, RO (C); RO (C); for 63 Ji-Paraná, = 43.1% score score = 43.4% for 73 Assis Brasil, AC; Brasil, AC; for 73 Assis = 43.4% score score = 41.1% for 45 Ceplac/ERNEG, AM; AM; for 45 Ceplac/ERNEG, = 41.1% score Carlos (L); for 117 San = 49.1% score score = 45.9% for 73 Assis Brasil, AC. Brasil, AC. for 73 Assis = 45.9% score AM; for 45 Ceplac/ERNEG, = 31.4% score Brasil, AC; for 73 Assis = 31.8% score score = 40.1% for 73 Assis Brasil, AC; AC; Brasil, 73 Assis for = 40.1% score 3AssBai,A 8. 84.5 50.8 Brasil, AC 73 Assis Brasil, AC 73 Assis 5Cpa/RE,A 9. 97.9 45 Ceplac/ERNEG, AM 64.9 45 Ceplac/ERNEG, AM 39 0 lnur A 110 A. 108 Alenquer, PA 73.9 q r 0 uaAeqe,P 10A lna aai C 0. 100.0 110 A. Blanca, Manabi, EC 109 Duca Alenquer, PA oe mzngop 63 Ji-Paraná, RO Amazon group Lower

Bac,Mnb,E 100 Bahia group 100.0 Blanca, Manabi, EC

0. 100.0 2Tbtna M 100.0 92 Tabatinga, AM