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History of the invasion of the anther smut pathogen on Silene
latifolia in North America
Michael C. Fontaine1,2,3, Pierre Gladieux1,2,4, Michael E. Hood5 and Tatiana Giraud1,2
1Universit�e Paris-Sud, Laboratoire Ecologie, Syst�ematique et Evolution, UMR8079, Orsay Cedex, F-91405, France; 2CNRS, UMR 8079, Orsay Cedex, F-91405, France; 3Department of
Biological Sciences, University of Notre Dame, Notre Dame, IN, USA; 4Plant and Microbial Biology, University of California, Berkeley, CA, USA; 5Department of Biology, Amherst College,
Amherst, MA, USA
Author for correspondence:Michael C. Fontaine
Tel: +1 574 631 3904
Email: [email protected]
Received: 15 October 2012
Accepted: 9 January 2013
New Phytologist (2013)doi: 10.1111/nph.12177
Key words: costructure, mating system,Microbotryum silenes-dioicae,Microbotryum violaceum, Oomycetes, plantpathogen, population genetics, selfing.
Summary
� Understanding the routes of pathogen introduction contributes greatly to efforts to protect
against future disease emergence.� Here, we investigated the history of the invasion in North America by the fungal pathogen
Microbotryum lychnidis-dioicae, which causes the anther smut disease on the white campion
Silene latifolia. This system is a well-studied model in evolutionary biology and ecology of
infectious disease in natural systems.� Analyses based on microsatellite markers show that the introduced American M. lychnidis-
dioicae probably came from Scotland, from a single population, and thus suffered from1 a
drastic bottleneck compared with genetic diversity in the native European range. The pattern
in M. lychnidis-dioicae contrasts with that found by previous studies in its host plant species
S. latifolia, also introduced in North America. In the plant, several European lineages have
been introduced from across Europe. The smaller number of introductions for M. lychnidis-
dioicae probably relates to its life history traits, as it is2 an obligate, specialized pathogen that is
neither transmitted by the seeds nor persistent in the environment.� The results show that even a nonagricultural, biotrophic, and insect-vectored pathogen
suffering from a very strong bottleneck can successfully establish populations on its intro-
duced host.
Introduction
Biological invasions have had dramatic ecological and economicimpacts (Pimentel et al., 2001). In particular, biological invasionsby fungal or fungal-like (i.e. Oomycetes) pathogens have causedmany emerging and devastating diseases (Anderson et al., 2004;Desprez-Loustau et al., 2007). Among plant pathogens, strikingexamples include the introduced diseases that have affected theAmerican chestnut (Castanea dentate), a formerly dominant treethat is now confined to being an understory shrub. The Ink dis-ease caused by the oomycete Phytophthora cinnamomi first causedthe demise of most chestnut trees in the southern part of theirAmerican distribution (Crandall, 1950). The chestnut blight fun-gus (Cryphonectria parasitica) then eliminated nearly all remain-ing native chestnut trees throughout eastern American forestsduring the 20th Century (Anagnostakis, 1987). Another exampleof devastating emerging fungal plant diseases is Dutch elm dis-ease, first caused by Ophiostoma ulmi, which led to the destruc-tion of many American and European elms (Ulmus Americana)(Brasier, 1991). Its sibling species Ophiostoma novo-ulmi, intro-duced in the 1960s and more aggressive than O. ulmi, then elimi-nated most remaining mature elms (Ulmus glabra, Ulmus procera
and Ulmus minor) across North America and Europe (Brasier,1991). Phytophthora cinnamomi is probably the most notoriousinvasive tree pathogen, known to attack and kill c. 5000 woodyplant species in the world, causing highly destructive epidemicsat ecosystem and landscape scales world-wide (Zentmyer, 1980;Shearer & Tippet, 1989; Shearer et al., 2004; Cahill et al., 2008).
Our primary food production is also at risk as a result ofemerging crop diseases caused by fungi or oomycetes (Strange &Scott, 2005), the most dramatic historical example being the IrishPotato Famine caused by Phytophthora infestans on cultivatedpotato (Solanum tuberosum)3 at the beginning of the 1840s (Fry,2008). More recent examples include the blast disease of wheat(Triticum aestivum) 4that appeared in Brazil in the 1980s and thenspread to other South American countries (Urashima et al.,1993), as well as the Ug99 fungal pathotype causing stem rustdisease of wheat, first identified in 1998 in Uganda and nowthreatening North Africa, the Middle East and Asia (Singh et al.,2008).
Retracing the routes of introduction of fungal pathogens andunderstanding the evolutionary processes during introductionand spread can contribute to invasion prevention and manage-ment programs (Facon et al., 2006). It is important to assess, for
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instance, whether multiple introductions are required for success-ful invasions and whether strong bottlenecks reduce the geneticvariability of pathogens in introduced ranges (Dlugosch &Parker, 2008). In the case of an obligate and specialized patho-gen, where invasion can occur only after introduction of the host,another relevant question is whether the pathogen came from thesame geographic areas as its host, either because they took thesame routes of introduction or because of local adaptation (i.e.when sympatric pathogens perform the best on their local hostscompared with other pathogen populations; Kaltz & Shykoff,1998).
Here, we studied the history of the invasion of the anther smutpathogen of the white campion Silene latifolia in North America.Microbotryum violaceum sensu lato is a species complex of basidio-mycete fungi responsible for the sterilizing and persistent anthersmut disease in many species, mostly in the Caryophyllaceae.Infected plants contain fungal teliospores in place of the pollenand female structures do not mature; female plants in dioeciousspecies also develop spore-bearing anthers. Teliospores are trans-mitted from diseased to healthy plants mostly by insects that nor-mally serve as pollinators. Microbotryum fungi are obligatepathogens. The fungus is not transmitted in the seeds (Baker,1947a,b)5 , nor do the spores appear capable of persisting in theenvironment as the disease is restricted to perennial hosts thatallow for transmission between living plants (Hood et al., 2010).Moreover, the sibling species encompassed in M. violaceum sensulato (Kemler et al., 2006; Le Gac et al., 2007; Denchev et al.,2009) show strong host specificity (de Vienne et al., 2009a) andinter-sterility (de Vienne et al., 2009b). The most widely studiedspecies is Microbotryum lychnidis-dioicae (Denchev et al., 2009)(called MvSl in Le Gac et al. (2007)), parasitizing S. latifolia, andthis is a well-studied plant model in evolutionary biology andecology (Bernasconi et al., 2009). Silene latifolia (syn. S. alba) is adioecious herbaceous perennial with a history of human associa-tion, commonly found in disturbed areas such as roadsides,railroad embankments, cultivated fields, and abandoned lots(Baker, 1948). Microbotryum lychnidis-dioicae has a highly selfingmating system (Delmotte et al., 1999; Giraud et al., 2005,2008a; Vercken et al., 2010), with mostly intratetrad matings(Hood & Antonovics, 2000, 2004). Sex is obligate in the lifecycle of Microbotryum, involving the production of dikaryonsthat are the infectious structures.
Both the plant S. latifolia and its anther smut pathogen havebeen introduced in modern times to North America fromEurope, and the invasion of S. latifolia is now well studied(Wolfe, 2002; Taylor & Keller, 2007; Keller et al., 2009,2012). The plant species arrived in North America from Europein the mid-1800s (Baker, 1947a,b) and has become a mildlyproblematic weed of cultivated fields in southern Canada andthe northern USA (McNeil, 1977). An herbarium study sug-gested that the first introduced plant populations occurred prob-ably in the north-eastern USA, from where plants spreadpredominantly southwards and became common only since the1920s (Antonovics et al., 2003). Introduced populations ofS. latifolia have been shown to originate from Europe, withindependent introductions into eastern and western North
America, and with a rather severe but short-lived genetic bottle-neck (Taylor & Keller, 2007; Keller et al., 2012). Lineages ofS. latifolia from both eastern and western Europe have beenintroduced in both eastern and western North America, offeringopportunities for admixture among previously isolated lineages(Taylor & Keller, 2007; Keller et al., 2012). There are geneti-cally based differences in life-history traits between S. latifoliapopulations from the native European range and the introducedNorth American range, in particular increased susceptibility tonatural enemies in North America (Wolfe et al., 2004).
In contrast to the invasion by S. latifolia, the invasion historyof its associated anther smut pathogen in North America has notbeen studied. The disease introduction is probably very recent, asan herbarium study did not find any diseased S. latifolia in plantcollections from the USA (Antonovics et al., 2003), even thoughdiseased plants are usually represented without much bias in her-baria (Antonovics et al., 2003; Hood et al., 2010). The first pub-lished record, to our knowledge, of the disease on S. latifolia inNorth America describes its occurrence in the central Appala-chian Mountains of Virginia (Alexander & Antonovics, 1988).Sporadic observations have also been made in recent decades inIllinois (E. Garber, pers. comm.), Michigan (J. Antonovics, pers.comm.), Pennsylvania (E. Lyons & A. Jarosz, pers. comm.), andMassachusetts (T. Meagher, pers. comm.). Two specimens inmycological herbaria from the early 1960s place the pathogen onS. latifolia in Bronx and Dutchess counties of New York State (inthe Gray and Kew mycological herbaria, respectively; M. E.Hood, pers. obs. 6). Previous studies on limited numbers of strainsand based on electrophoretic data or internal transcribed spacer(ITS) sequences suggested that North American populations onS. latifolia were genetically more similar to English populationson S. latifolia than to North American populations on otherSilene host species (Antonovics et al., 1996; Freeman et al.,2002), showing that these North American populations origi-nated through an introduction rather than as a host shift from ahost species native to North America. Few sample localities wereincluded in these previous studies, so no comparisons could bemade among European populations to identify the pathogensource(s) more specifically. Also, the possibility of a host shift asthe source of the disease on North American S. latifolia fromanother European host species, particularly Silene dioica, cannotbe excluded. Host shifts indeed occur in Microbotryum betweenclosely related hosts (Antonovics et al., 2002; Lopez-Villavicencioet al., 2005; Refr�egier et al., 2008; Gladieux et al., 2011), and nat-ural and artificial cross-species disease transmission is successfulbetween S. latifolia and S. dioica (Van Putten et al., 2003; deVienne et al., 2009a).
Our aim here was therefore to reconstruct the invasion historyof Microbotryum on S. latifolia by comparing the populationgenetics of North America and European using multilocus micro-satellite genotyping of M. lychnidis-dioicae from S. latifolia andM. silenes-dioicae parasitizing S. dioica (Vercken et al., 2010;Gladieux et al., 2011). Concurrently, the mating system in intro-duced pathogen populations was assessed in comparison to itsnative range, as examples of striking changes in other introducedorganisms include the loss of sexual reproduction in introduced
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fungal pathogens (Ali et al., 2010; Duan et al., 2010; Saleh et al.,2012) and changes in selfing rates in plants (Amsellem et al.,2001; Bossdorf et al., 2005; Colautti et al., 2010). We thereforeanalyzed North American populations of Microbotryum onS. latifolia in North America, representing good coverage of theextant populations, using genetic data to address the followingspecific questions: What are the European sources from whichthe North American populations originated, in terms of bothspecies and populations? Can we detect footprints of a bottle-neck, and if so how strong was it? Were invasive populationsintroduced from multiple sources? If so, did admixture occur?And/or did hybridization occur with native Microbotryum spe-cies? Has the mating system of the fungus changed comparedwith the populations of origin? How similar are the invasion his-tories of the plant S. latifolia and of its pathogen?
Materials and Methods
Teliospore collection and populations
The individuals of Microbotryum analyzed in this study were col-lected as diploid teliospores from 183 localities on Silene latifolia
7 (n = 773) across Europe and eastern North America (Fig. 1) andstored in silica gel (for a detailed description of the sampling seeSupporting Information Table S1 for North America andVercken et al. (2010) for Europe). DNA extracted from teliosp-ores as described in Giraud (2004) from one flower per diseasedplant was used for genetic analyses. Multiple infections bydifferent genotypes are frequent in the Silene–Microbotryum sys-tem, but teliospores within a single flower originate from a singlediploid individual (Lopez-Villavicencio et al., 2007).
Microsatellite genotyping
Populations from Europe had been genotyped and analyzed in aprevious study (Vercken et al., 2010). For populations fromNorth America, teliospores were genotyped using 11 microsatel-lite markers following the protocol of Giraud (2004) (Table 1).Among the 11 microsatellite loci used, E14, E17 and E18 weredescribed in Bucheli et al. (1998), SL8, SL9, SL12, SL19, SVG5,SVG8 and SVG14 were described in Giraud et al. (2008b), andSL5 was described in Refr�egier et al. (2010).
Data analyses
Descriptive statistics Polymorphism at each locus was quanti-fied using number of alleles (A), allelic richness (Ar), allelic rich-ness private to a specific grouping (pAr), unbiased expectedheterozygosity (He), and fixation index (FIS); the four latter statis-tics control for number of samples. These statistics were calcu-lated using FSTAT 2.9.3.2 (Goudet, 2001) and ADZE (Szpiechet al., 2008). Departures from Hardy–Weinberg expectationswere tested using exact tests implemented in GENEPOP 4.0(Raymond & Rousset, 1995; Rousset, 2008).
Population structure We investigated population structureusing two complementary approaches: principal component anal-yses (PCAs) and Bayesian model-based clustering. PCAs displaygenotypes in a multivariate space described by the principal com-ponent (PC) (Patterson et al., 2006; Jombart et al., 2009).Although the method does not rely on any population geneticmodel, it is useful to represent genetic relationships among indi-viduals (McVean, 2009). By contrast, individual-based Bayesian
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Fig. 1 Map of sampled localities for Microbotryum lychnidis-dioicae in the native European range (MvSl, n = 701; left panel) and the introduced area inthe USA (n = 72; right panels). The sizes of the red dots are proportional to the sample size.
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clustering algorithms, such as STRUCTURE, partition multilocusgenotypes into clusters based on allele frequency and linkageequilibrium among loci, and estimate the admixture proportionsto each cluster (Pritchard et al., 2000; Falush et al., 2003; Hubiszet al., 2009).
In order to check that the anther smut fungus infectingS. latifolia in North America originated from European strainsalso infecting S. latifolia, and not from another EuropeanMicrobotryum species such as Microbotryum silenes-dioicae infect-ing Silene dioca9 , we conducted PCAs on the European genotypicdata from Vercken et al. (2010), which included bothMicrobotryum lychnidis-dioicae and M. silenes-dioicae, togetherwith the North American samples genotyped for the presentstudy. We then ran PCA on theM. lychnidis-dioicae data set fromEurope and North America to provide a better resolution of thegenetic structure. PCA was conducted on the microsatellite allelefrequencies using the ADEGENET package (Jombart, 2010) in theR environment (R Development Core Team, 2010).
For the clustering analysis, the STRUCTURE 2.3.3 program wasused (Pritchard et al., 2000; Falush et al., 2003; Hubisz et al.,2009) with a haploid setting because Microbotryum is almostcompletely homozygous (Table 1 and 2). Run conditions forSTRUCTURE analyses were as follows: a series of independent runswere conducted with different proposals for the number of clus-ters (K), testing all values from 1 to 10. Each run used 1 000 000iterations after a burn-in of 100 000 iterations, using a modelallowing for admixture and correlated allele frequencies. Toensure convergence of the MCMC10 , we performed 10 indepen-dent replicates for each value of K and checked the consistency ofresults visually and using the procedure implemented in the pro-gram CLUMPP 1.1.1 (Jakobsson & Rosenberg, 2007). We usedCLUMPP 1.1.1 to account for label switching and to identifypotential distinct solutions among the results of independent rep-licate runs for each K. For that purpose, we computed with theGREEDY algorithm a symmetric similarity coefficient indexbetween pairs of runs (100 random input sequences, G′ statistic).
Differences in allelic frequencies between groups were assessed12using exact tests implemented in GENEPOP 4.0 (Raymond &
Rousset, 1995; Rousset, 2008) and quantified using Weir andCockerham’s FST estimator (Weir & Cockerham, 1984). Differ-ences in genetic polymorphism between groups were assessedusing a Wilcoxon signed-rank test. The selfing rate within theNorth American population was estimated using the INSTRUCT
program (Gao et al., 2007) using the same run settings as in Verc-ken et al. (2010) and we compared selfing rates with values previ-ously estimated for European clusters (Vercken et al., 2010).
Results
Population study
North AmericanMicrobotryum strains originate from EuropeanM. lychnidis-dioicae populations The PCA on M. lychnidis-dioicae and M. silenes-dioicae microsatellite data confirms that allstrains collected in North American are related to EuropeanM. lychnidis-dioicae populations on S. latifolia rather than being ahost-shift lineage 13from the pathogen on S. dioica (Fig. S1). Wefocused thus only on material of M. lychnidis-dioicae forsubsequent analyses.
Low genetic diversity in North American populations Thegenetic diversity in North American populations was much lowerthan those observed in Europe (Table 1). Most loci displayed astrong deficit in heterozygosity, clearly departing from Hardy–Weinberg expectations.
Genetic structure The population structure of M. lychnidis-dioicae in Europe was very strong, as described in Vercken et al.(2010); increasing K consistently identified new distinct clustersin STRUCTURE analyses. The main clusters in Europe at K = 3(Fig. 2) were: the Western group (blue at K = 3), which split athigher K values into north Western (NWest) and south Western(SWest) 14clusters (respectively in blue and yellow at K � 4); theItalian cluster (in green); and the Eastern cluster (in red at K = 3),which split at higher Ks into the Balkan and Eastern clusters(respectively in purple and red at K � 5). The American popula-tions remained together with the NWest cluster regardless of thenumber of clusters implemented (see K = 3–7; higher K valuesnot shown). This indicates that American populations are closelyrelated to the populations from northwestern Europe 15, and in par-ticular to the populations from the UK. This was also indicatedby the PCA (Fig. 3a), on which American genotypes clusteredtogether with northwestern European populations; the three first
16PCs accounted for 35% of the total variance.Even with high K values we could not obtain finer resolution
for the origin of the North American populations using the fulldata set (Fig. 2a). However, the clustering analyses on a reduceddata set (n = 149) focusing on American samples and the Euro-pean samples that belonged to the same cluster at K = 7 showedthat all strains from North America clustered with a few strainscollected in Scotland (Fig. 4). Estimations of the number of clus-ters for this reduced data set, based on either the posterior
Table 1 Genetic polymorphism at each locus in European and Americansamples8
Locus
Europe USA
N A He FIS N A He FIS
E14 670 11 0.71 0.96** 70 2 0.07 �0.03 ns
E17 649 15 0.85 0.97** 61 1 0.00 –
E18 535 20 0.90 0.99** 69 1 0.00 –
SL5 678 12 0.80 1.00** 72 1 0.00 –
SL8 534 10 0.60 0.98** 52 2 0.04 1.00**SL9 683 12 0.81 0.98** 70 2 0.47 1.00**SL12 680 7 0.62 0.91** 68 2 0.04 �0.02 ns
SL19 662 9 0.84 0.80** 61 1 0.00 –
SVG5 695 7 0.72 0.89** 72 2 0.44 0.97**SVG8 679 15 0.87 0.97** 69 3 0.04 0.67*SVG14 683 7 0.31 0.90** 67 2 0.03 �0.01 ns
Mean 649.8 11.4 0.73 0.94** 66.5 1.7 0.10 0.84**� SD 58.3 4.1 0.17 0.06 6.1 0.6 0.17 0.51
N, sample size; A, number of alleles; He, expected heterozygosity; FISvalue: ns, not statistically different from 0; *, P < 0.05; **, P < 0.01.
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probability calculation (P(K|X), where X is the data) (Pritchardet al., 2000) or the method advocated by (Evanno et al., 2005),indicated that K = 3 was the most likely solution (P(K = 3|X)
= 0.99 and maximum DeltaK 18obtained for K = 3; Fig. S2).Indeed, the Scottish samples were most closely related to Ameri-can samples with a multi-locus genotype almost identical to some
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Fig. 2 Genetic clustering analyses of Microbotryum lychnidis-dioicaemicrosatellite genotypes. Barplots display estimated individual admixture proportionfor the global data set (n = 773), including samples from both Europe and USA, assuming K = 3 to K = 7 clusters (a). Each individual is represented by a thinvertical line divided into K colored segments that represent the individual’s estimated admixture fractions in the putative number of K clusters assumed.Black lines separate individuals from different geographic areas. In each case, only the most frequent solution among the 10 replicates is displayed. (b, c)Maps show the admixture proportions estimated from the STRUCTURE analysis at K = 7 averaged over localities.
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Fig. 3 Principal component analysis (PCA)on Microbotryum lychnidis-dioicae
microsatellite genotypes. (a) Genotypesfrom Europe and the USA are plotted in thethree-dimensional space defined by the firstthree principal components (PCs). Eachgenotype has been colored according to theassignment values based on the clusteringanalysis in Vercken et al. (2010). (b) PCAconducted on all genotypes that displayedscore values for the three first17 PCsoverlapping with those from the USA.Countries in which these genotypesoccurred are coded with different colors andsymbols. The dotted lines split individualgenotypes into three distinct groupsconsistent with the STRUCTURE clusteringanalysis.
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strains found in North America, differing only at one locus, forwhich the North American population had an allele at the mostpolymorphic locus E18 that was not shared by any Europeanpopulation ofM. lychnidis-dioicae19 .
Providing results consistent with the clustering analyses, PCAon genotypes that displayed score values for the first three PCs ofthe global analyses overlapping with the score values of the NorthAmerican samples, these introduced genotypes appeared againclosely related to the Scottish samples20 (Figs 3b, 4, S3).
The level of divergence in allelic frequencies (FST) betweenNorth American populations and the five European clusters iden-tified in Vercken et al. (2010) was significant for all pair-wisecomparisons and ranged between 0.31 and 0.64 (Table 2). Thelowest value was observed between the North American popula-tions and the NWest cluster, in agreement with the close geneticrelationship estimated from the PCA and STRUCTURE analysis.The North American populations displayed a significantly lowerlevel of genetic diversity than the European NWest cluster(Table 3), as estimated either using the allelic richness (Ar) or thegenetic diversity (He) (Wilcoxon signed rank test, P < 0.001 forboth Ar and He statistics). North American populations also hada very low private allelic richness (pAr) compared with the Euro-pean values, meaning that all the alleles found in the NorthAmerican samples were also detected in European populations.The selfing rate within the North American population estimatedusing the INSTRUCT program was high, as expected given theknown mating system in Microbotryum species. The estimated
selfing rate was significantly lower than in European populations(Tables 1, 3), but the difference was small and the variance in FISvalues across loci was large (Table 1).
Discussion
This study confirms that the introduced M. lychnidis-dioicae pop-ulations on S. latifolia in North America originated from anintroduction of European populations of M. lychnidis-dioicaespecialized on S. latifolia, and did not result from a host shift.Yet, among the documented cases of recent disease emergence asa result of introductions of fungal or oomycete pathogens intonew continents, infection of novel hosts (i.e. host-shifts, host-range expansion, or pathogen spillover) is often involved (Parker& Gilbert, 2004; Slippers et al., 2005). Examples include theintroduction of C. parasitica in the USA, the most probablesource of which was Japanese chestnut trees (Castanea crenata)that were imported and planted throughout the country(Milgroom et al., 1996; Anagnostakis, 2001).
The strong genetic structure of M. lychnidis-dioicae in Europeallowed us to determine that Scotland is the most probable sourceof introduction for the North American populations. No foot-print of admixture or hybridization with native Microbotryumspecies was found, which probably relates to the high selfing ratein M. lychnidis-dioicae (> 85%; Hood & Antonovics, 2004;Giraud et al., 2005; Gladieux et al., 2011), as well as to the stronghost specificity and post-zygotic isolation among Microbotryumspecies (de Vienne et al., 2009a,b). The high level of homozygos-ity found in the North American populations also indicated ahigh selfing rate of the introduced populations. The fungus prob-ably has thus kept the same mating system as in its native area.This is in contrast to other fungal or oomycete pathogens that donot undergo sexual reproduction in their introduced ranges, suchas the rice blast fungus Magnaporthe oryzae (Saleh et al., 2012),the yellow rust fungus Puccinia striiformis f.sp tritici (Ali et al.,2010; Duan et al., 2010), or the potato late blight oomyceteP. infestans (Day et al., 2004). In the latter case, only one matingtype – A1 – had long been present in the introduced populations
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(b)
(c)
USAUK
Longitude
Latitu
de
K3
K2
Fig. 4 Genetic clustering analyses ofMicrob-
otryum lychnidis-dioicaemicrosatellitegenotypes on a reduced data set (n = 147)consisting of USA samples and Europeansamples which grouped with the USAsamples at K = 7 in the global populationstructure analysis. STRUCTURE barplots for K = 2to K = 4 and maps displaying results at K = 4in the native and introduced areas areprovided in (a), (b) and (c), respectively.
Table 2 Pairwise differentiation among clusters expressed as FST values(P < 0.001 for all values)11
FST Balkan NWest Italian SWest Eastern
NWest 0.44Italian 0.35 0.31SWest 0.47 0.24 0.34Eastern 0.45 0.43 0.36 0.44US 0.62 0.32 0.47 0.44 0.64
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in Europe, preventing the occurrence of sex (Day et al., 2004).The introduction of the A2 mating type from Mexico in the1970s, c. 130 yr after the A1 mating type, caused dramaticchanges in the population structure and aggressiveness of theEuropean P. infestans population as a consequence of23 the result-ing sexual recombination (Goodwin, 1997; Cooke et al., 2012).Similarly, Phytophthora ramorum24 and P. cinnamomi reproduceonly clonally in their world-wide introduced ranges, as only theirrespective A2 mating types occur in their invasive natural popula-tions (Zentmyer, 1980; Old et al., 1984; Arentz & Simpson,1986; Shearer & Tippet, 1989; Gr€unwald et al., 2012). InMicrobotryum, the failure to evolve asexual reproduction is notsurprising, as the formation of a dikaryon by sexual fusion ofhaploid cells is required for plant infection. The lack of change inthe mating system also contrasts to some invasive plants, whichhave evolved higher selfing rates (Amsellem et al., 2001; Bossdorfet al., 2005; Colautti et al., 2010); however, this may not be sur-prising either given the already very high selfing rates ofMicrobotryum in its native range (Vercken et al., 2010).
The introduction of M. lychnidis-dioicae on S. latifolia inNorth America appears to have been more recent, and with amuch stronger bottleneck, than the introduction of the host plantspecies (Taylor & Keller, 2007). Substantially fewer propaguleswere successfully introduced for the fungus than for the plant,and only in a small range on the eastern coast of North America,while several introductions occurred for S. latifolia, and indepen-dently on the west and east coasts (Taylor & Keller, 2007). Evenin the area where M. lychnidis-dioicae is present in North Amer-ica, several European lineages of S. latifolia have been detected,originating from different European regions (Taylor & Keller,2007). Local adaptation is therefore not likely to be involved inthe low number of M. lychnidis-dioicae populations introduced.However, we have not been able to integrate samples of the path-ogen from the northeastern USA where the fungus has also beenobserved in a few places (J. Antonovics, pers. comm.), and thepossibility remains that additional allelic variation may exist inM. lychnidis-dioicae in North America that has not been capturedby the current study. The situation in M. lychnidis-dioicae andS. latifolia thus contrasts with plant pathogens such asMycosphaerella graminicola (Banke & McDonald, 2005;Stukenbrock et al., 2007), Ustilago scitaminea (Raboin et al.,2007), Magnaporthe oryza (Couch et al., 2005a), Venturiainaequalis (Gladieux et al., 2008) and P. infestans (Gomez-Alpizaret al., 2007), which seem to share the same geographic origins as
their respective host plant, that is, wheat, sugarcane (Saccharumofficinarum), rice (Oryza sativa) 25; 26, apple 25; 26and potato, respectively.This does not seem to be the case, however, for the barley (Horde-um vulgare) 27pathogen Rhynchosporium secalis (Brunner et al.,2007; Zaffarano et al., 2008) or for the colza 28pathogenLeptosphaeria maculans (Dilmaghani et al., 2012).
It is quite remarkable that there has been trans-continentalintroduction of the anther smut disease at all, as there is no seedtransmission, dispersal of Microbotryum occurs by insects, andthe fungus appears unable to persist as an environmental contam-inant (Hood et al., 2010). One may speculate that, as probablyoccurred for the seeds of the host, diseased flowers of S. latifoliacould have been transported along with forage materials aboardships, as this plant is common in hay fields. Entry of the patho-gen may have occurred through seedlings germinating among thespores where the forage material was off-cast upon arrival toNorth America. Such a scenario may be consistent with thesource populations of introduced populations being from theUK, as human and commercial exchange are known to be espe-cially frequent between these countries, and especially with theeast coast. The successful invasion by an insect-borne biotrophicfungus shows that even pathogens presenting life history traitsthat are a priori less favorable for invasion (Philibert et al., 2011)can still cause biological invasions. An alternative explanation,that a living, diseased plant was transported from Europe toNorth America, seems particularly unlikely as S. latifolia is ashort-lived perennial, easily propagated by seeds, and not of par-ticular interest among gardeners. However, it should be notedthat such an event is not entirely unprecedented, as the gardenfavorite moss campion, Silene acaulis, appears to have been intro-duced along with its anther smut to Hawaii (Farr et al., 1989),where this extremely long-lived species is more likely to be col-lected and transported as living specimens. Also, the anther smutdisease of Dianthus caryophyllus was introduced by unknownmeans to Massachusetts in 1948, but the disease did not becomeestablished in this nonnative host and was present only in agricul-tural settings 29(Spencer & White, 1951). Another example of apriori unlikely invasion that nevertheless occurred is the case ofthe forest pathogen Heterobasidion annosum introduced to Italyby US troops during World War II (Gonthier et al., 2007).
The low genetic diversity in introduced populations is animportant factor that is expected to limit invasion success.Multiple introductions can increase genetic diversity in intro-duced ranges (Genton et al., 2005), which has been argued to
Table 3 Descriptive statistics within each European cluster and in the USA 21
NWest SWest Italian Balkan Eastern US
N 131 159 143 93 84 72Ar 4.14� 0.63 4.44� 0.67 5.67� 0.56 3.70� 0.52 4.95� 0.63 1.65� 0.16pAr 0.44� 0.22 0.74� 0.21 1.14� 0.29 0.46� 0.19 0.84� 0.38 0.03� 0.03He 0.46� 0.26 0.46� 0.33 0.65� 0.12 0.38� 0.27 0.43� 0.28 0.10� 0.17FIS 0.85 0.91 0.95 0.93 0.96 0.84S 0.93 0.95 0.94 0.91 0.92 0.88
N, within-cluster sample size; Ar, allelic richness; pAr, private allelic richness; He, expected heterozygosity; FIS value; S, selfing rate inferred from INSTRUCT
program 22.
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facilitate biological invasions. Here again, however, theM. lychnidis-dioicae case shows that, with only a few genotypes, apopulation of a fungal plant pathogen can become established ina new geographic range.
Biological invasions of whole continents by one or a fewclonal lineages in fact appear to be frequent in fungal andoomycete pathogens (Goodwin et al., 1994; Couch et al., 2005b;Enjalbert et al., 2005; Raboin et al., 2007; Singh et al., 2008;Fisher et al., 2009; James et al., 2009; Mboup et al., 2009), suchas P. infestans on potato (Goodwin et al., 1994), P. cinnamomion trees (Old et al., 1984; Arentz & Simpson, 1986; Dobrowol-ski et al., 2003), U. scitaminea on sugarcane (Raboin et al.,2007), Batrachochytrium dendrobatidis on amphibians (Jameset al., 2009), Seiridium cardinale on cypress30 (Della Rocca et al.,2011), or Puccinia striiformis on wheat (Mboup et al., 2009).Other invasions nevertheless resulted from introductions of a fewdivergent lineages from distinct sources, such as Plasmoparahalstedii on sunflower31 in France (Delmotte et al., 2008; Ahmedet al., 2012), C. parasitica on chestnut in France (Dutech et al.,2010), the grape (Vitis vinfera)32 powdery mildew fungus Erysiphenecator (Brewer & Milgroom, 2010), the apple scab fungusVenturia inaequalis (Gladieux et al., 2008), or the oilseed rape(Brassica napus)33 pathogen Leptosphaeria maculans (Dilmaghaniet al., 2012). Admixture might then occur between the lineagesand influence their evolutionary dynamics. Little admixture has,however, been found between divergent introduced lineages inC. parasitica (Dutech et al., 2010, 2012) and P. ramorum (Ivorset al., 2006; Mascheretti et al., 2008, 2009), probably because ofthe predominantly asexual mode of reproduction of those patho-gens. By contrast, it has been posited that hybridization betweendistant lineages created the hypervirulent strain in the chytridfungus threatening amphibians world-wide (Farrer et al., 2011)and similarly led to new pathogenic abilities on resistant cultivarsin the downy mildew pathogen of sunflowers (Ahmed et al.,2012).
The most devastating epidemics on plants caused by invasivefungal and oomycete pathogens most often involve novel hostspecies that are closely related to the host in the pathogens’ areaof origin. The present study nevertheless adds to a number ofcases of invasions by pathogenic fungi in plants following theintroduction of its host plant and shows that, even where life-his-tory traits are unfavorable for invasions (Philibert et al., 2011),such invasions may occur. Investigations of the evolution ofintroduced populations are also interesting for the understandingand management of biological invasions. The elucidation of theorigin of introduced M. lychnidis-dioicae populations onS. latifolia in North America will make it possible to performmore directed cross-inoculations to investigate coevolution withthe North American plant genotypes, and whether there has beenlocal adaptation, as shown in European S. latifolia for resistanceagainst M. lychnidis-dioicae (Kaltz et al., 1999).
Acknowledgements
T.G. acknowledges receipt of grant ANR 07-BDIV-003 (Emerf-undis project) and M.C.F. a postdoctoral grant from the Ile de
France R�egion. M.E.H. acknowledges receipt of grants NSF-DEB 0747222 and 1115765. We thank Janis Antonovics forhelping with collections and comments on the manuscript, andDoug Taylor for samples.
References
Ahmed S, de LabrouheD, Delmotte F. 2012. Emerging virulence arising from
hybridisation facilitated by multiple introductions of the sunflower downy
mildew pathogen Plasmopara halstedii. Fungal Genetics and Biology 49: 847–855.Alexander HM, Antonovics J. 1988. Disease spread and population dynamics of
anther-smut fungus of Silene alba caused by the fungus Ustilago violacea.Journal of Ecology 76: 91–104.
Ali S, Leconte M, Walker A-S, Enjalbert J, de Vallavieille-Pope C. 2010.
Reduction in the sex ability of worldwide clonal populations of Pucciniastriiformis f.sp tritici. Fungal Genetics and Biology 47: 828–838.
Amsellem L, Noyer JL, Hossaert-McKey M. 2001. Evidence for a switch in the
reproductive biology of Rubus alceifolius (Rosaceae) towards apomixis, between
its native range and its area of introduction. American Journal of Botany 88:2243–2251.
Anagnostakis. 1987. Chestnut blight: the classical problem of an introduced
pathogen.Mycologia 79: 23–37. 34
Anagnostakis S. 2001. The effect of multiple importations of pests and pathogens
on a native tree. Biological Invasions 3: 245–254.Anderson PK, Cunningham AA, Patel NG, Morales FJ, Epstein PR, Daszak P.
2004. Emerging infectious diseases of plants: pathogen pollution, climate
change and agrotechnology drivers. Trends in Ecology & Evolution 19: 535–544.Antonovics J, Hood ME, Partain J, Heuhsen AM. 2002. The ecology and
genetics of a host-shift:Microbotryum as a model system. American Naturalist160: S40–S53.
Antonovics J, Hood ME, Thrall PH, Abrams JY, Duthie GM. 2003.Herbarium
studies on the distribution of anther-smut fungus (Microbotryum violaceum)and Silene species (Caryophyllaceae) in the eastern United States. AmericanJournal of Botany 90: 1522–1531.
Antonovics J, Stratton D, Thrall PH, Jarosz AM. 1996. An anther-smut disease
(Ustilago violacea) of fire-pink (Silene virginica): its biology and relationship tothe anther-smut disease of white campion (Silene alba). American MidlandNaturalist 135: 130–143.
Arentz F, Simpson JA. 1986. Distribution of Phytophthora cinnamomi in Papua
New Guinea and notes on its origin. Transactions of the British MycologicalSociety 87: 289–295.
Baker HG. 1947a. Biological flora of the British Isles:Melandrium (Roehling
em.) Fries (Lychnis L. (1753) partim.). Journal of Ecology 35: 271–292.Baker HG. 1947b. Infection of species of Melandrium by Ustilago violacea(Pers.) Fuckel and the transmission of the resultant disease. Annals of Botany11: 333–348.
Baker HG. 1948. Stages in invasion and replacement demonstrated by species of
Melandrium. Journal of Ecology 36: 96–119.Banke S, McDonald BA. 2005.Migration patterns among global populations of
the pathogenic fungusMycosphaerella graminicola.Molecular Ecology 14: 1881–1896.
Bernasconi G, Antonovics J, Biere A, Charlesworth D, Delph LF, Filatov D,
Giraud T, Hood M, Marais G, McCauley D et al. 2009. Re-emergence of
Silene as a classic model system in ecology and evolution. Heredity 103: 5–14.Bossdorf O, Auge H, Lafuma L, Rogers WE, Siemann E, Prati D. 2005.
Phenotypic and genetic differentiation between native and introduced plant
populations. Oecologia 144: 1–11.Brasier CM. 1991. Ophiostoma novo-ulmi sp. nov., causative agent of currentDutch elm disease pandemics.Mycopathologia 115: 151–161.
Brewer MT, Milgroom MG. 2010. Phylogeography and population structure of
the grape powdery mildew fungus, Erysiphe necator, from diverse Vitis species.BMC Evolutionary Biology 10: 268.
Brunner PC, Schurch S, McDonald BA. 2007. The origin and colonization
history of the barley scald pathogen Rhynchosporium secalis. Journal ofEvolutionary Biology 20: 1311–1321.
New Phytologist (2013) � 2013 The Authors
New Phytologist� 2013 New Phytologist Trustwww.newphytologist.com
Research
NewPhytologist8
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Bucheli E, Gautschi B, Shykoff JA. 1998. Isolation and characterization of
microsatellite loci in the anther smut fungusMicrobotryum violaceum.Molecular Ecology 7: 665–666.
Cahill DM, Rookes JE, Wilson BA, Gibson L, McDougall KL. 2008.
Phytophthora cinnamomi and Australia’s biodiversity: impacts, predictions and
progress towards control. Australian Journal of Botany 56: 279–310.Colautti RI, White NA, Barrett SCH. 2010. Variation of self-incompatibility
within invasive populations of purple loosestrife (Lythrum salicaria L.) fromEastern Noth America. International Journal of Plant Sciences 171: 158–166.
Cooke DE, Cano LM, Raffaele S, Bain RA, Cooke LR, Etherington GJ, Deahl
KL, Farrer RA, Gilroy EM, Goss EM, et al. 2012. Genome analyses of an
aggressive and invasive lineage of the irish potato famine pathogen. PLoSPathogens 8: e1002940.
Couch BC, Fudal I, Lebrun MH, Tharreau D, Valent B, van Kim P,
Notteghem JL, Kohn LM. 2005b.Origins of host-specific populations of the
blast pathogenMagnaporthe oryzae in crop domestication with subsequent
expansion of pandemic clones on rice and weeds of rice. Genetics 170:613–630.
Couch BC, Fudal I, Lebrun M-H, Tharreau D, Valent B, van Kim P,
Nott�eghem J-L, Kohn LM. 2005a. Origins of host-specific populations of the
blast pathogen,Magnaporthe oryzae, in crop domestication with subsequent
expansion of pandemic clones on rice and weeds of rice. Genetics 70: 613–630.Crandall BS. 1950. The distribution and significance of the chestnut root rot
Phytophthoras, P. cinnamomi and P. cambivora. Plant Disease Reporter 34:194–196.
Day JP, Wattier RAM, Shaw DS, Shattock RC. 2004. Phenotypic and genotypic
diversity in Phytophthora infestans on potato in Great Britain, 1995–98. PlantPathology 53: 303–315.
Della Rocca G, Eyre CA, Danti R, Garbelotto M. 2011. Sequence and simple-
sequence repeat analyses of the fungal pathogen Seiridium cardinale indicateCalifornia is the most likely source of the cypress canker epidemic for the
mediterranean region. Phytopathology 101: 1408–1417.Delmotte F, Bucheli E, Shykoff JA. 1999.Host and parasite population structure
in a natural plant-pathogen system. Heredity 82: 300–308.Delmotte F, Giresse X, Richard-Cervera S, M’Baya J, Vear F, Tourvieille J,
Walser P, Tourvieille de Labrouhe D. 2008. Single nucleotide polymorphisms
reveal multiple introductions into France of Plasmopara halstedii, the plantpathogen causing sunflower downy mildew. Infection, Genetics and Evolution 8:534–540.
Denchev C, Giraud T, Hood M. 2009. Three new species of anthericolous smut
fungi on Caryophyllaceae.Mycologia Balcanica 6: 79–84.Desprez-Loustau MR, C, Buee M, Courtecuisse R, Garbaye J, Suffert F, Sache
I, Rizzo D. 2007. The fungal dimension of biological invasions. Trends inEcology and Evolution 22: 472–480.35
Dilmaghani A, Gladieux P, Gout L, Giraud T, Brunner PC, Stachowiak A,
Balesdent M-H, Rouxel T. 2012.Migration patterns and changes in
population biology associated with the worldwide spread of the oilseed rape
pathogen Leptosphaeria maculans.Molecular Ecology 21: 2519–2533.Dlugosch K, Parker I. 2008. Founding events in species invasions: genetic
variation, adaptive evolution, and the role of multiple introductions.MolecularEcology 17: 431–449.
Dobrowolski MP, Tommerup IC, Shearer BL, O’Brien PA. 2003. Three clonal
lineages of Phytophthora cinnamomi in Australia revealed by microsatellites.
Phytopathology 93: 695–704.Duan X, Tellier A, Wan A, Leconte M, de Vallavielle-Pope C, Enjalbert J. 2010.
Puccinia striiformis f.sp tritici presents high diversity and recombination in the
over-summering zone of Gansu, China.Mycologia 102: 44–53.Dutech C, Barres B, Bridier J, Robin C, MilgroomMG, Ravigne V. 2012. The
chestnut blight fungus world tour: successive introduction events from diverse
origins in an invasive plant fungal pathogen.Molecular Ecology 21: 3931–3946.Dutech C, Fabreguettes O, Capdevielle X, Robin C. 2010.Multiple
introductions of divergent genetic lineages in an invasive fungal pathogen,
Cryphonectria parasitica, in France. Heredity 105: 220–228.Enjalbert J, Duan X, Leconte M, Hovmoller M, De Vallavieille-Pope C. 2005.
Genetic evidence of local adaptation of wheat yellow rust (Puccinia striiformis f.sp tritici) within France.Molecular Ecology 14: 2065–2073.
Evanno G, Regnaut S, Goudet J. 2005. Detecting the number of clusters of
individuals using the software STRUCTURE: a simulation study.MolecularEcology 14: 2611–2620.
Facon B, Genton BJ, Shykoff JA, Jarne P, Estoup A, David P. 2006. A general
eco-evolutionary framework for understanding bioinvasions. Trends in Ecologyand Evolution 21: 130–135.
Falush D, Stephens M, Pritchard J. 2003. Inference of population structure
using multilocus genotype data: linked loci and correlated allele frequencies.
Genetics 164: 1567–1587.Farr DF, Bills GF, Chamuris GP, Rossman AY. 1989. Fungi on plants and plantproducts in the United States. VIII, 1252 S. St. Paul, MN, USA: The American
Phytopathological Society (APS) Press.
Farrer RA, Weinert LA, Bielby J, Garner TWJ, Francois Balloux FC, Bosch J,
Cunningham AA, Weldon C, Preez LHd, Anderson L et al. 2011.Multiple
emergences of genetically diverse amphibianinfecting chytrids include a
globalized hypervirulent recombinant lineage. Proceedings of the NationalAcademy of Sciences, USA 108: 18732–18736.
Fisher MC, Garner TWJ, Walker SF. 2009. Global emergence of
Batrachochytrium dendrobatidis and amphibian Chytridiomycosis in space,
time, and host. Annual Review of Microbiology 63: 291–310.Freeman AB, Kellye K, Shi T-L, Hughes CF, Perlin MH. 2002. Isolates of
Microbotryum violaceum from North American host species are phylogenetically
distinct from their European host-derived counterparts.Molecular Phylogeneticsand Evolution 23: 158–170.
Fry W. 2008. Phytophthora infestans: the plant (and R gene) destroyer.MolecularPlant Pathology 9: 385–402.
Gao H, Williamson S, Bustamante CD. 2007. A Markov Chain Monte Carlo
approach for joint inference of population structure and inbreeding rates from
multilocus genotype data. Genetics 176: 1635–1651.Genton B, Shykoff JA, Giraud T. 2005.High genetic diversity in French invasive
populations of common ragweed Ambrosia artemisiifolia as a consequence ofmultiple sources of introduction.Molecular Ecology 14: 4275–4285.
Giraud T. 2004. Patterns of within population dispersion and mating of the
fungusMicrobotryum violaceum parasitising the plant Silene latifolia. Heredity93: 559–565.
Giraud T, Jonot O, Shykoff JA. 2005. Selfing propensity under choice
conditions in a parasitic fungus,Microbotryum violaceum, and parameters
influencing infection success in artificial inoculations. International Journal ofPlant Sciences 166: 649–657.
Giraud T, Yockteng R, Lopez-Villavicencio M, Refregier G, Hood ME. 2008a.
The mating system of the anther smut fungus,Microbotryum violaceum: selfingunder heterothallism. Eukaryotic Cell 7: 765–775.
Giraud T, Yockteng R, Marthey S, Chiapello H, Jonot O, Lopez-Villavicencio
M, de Vienne DM, Hood ME, Rrefr�egier G, Gendrault-Jacquemard A et al.
2008b. Isolation of 60 polymorphic microsatellite loci in EST libraries of four
sibling species of the phytopathogenic fungal complexMicrobotryum.MolecularEcology Resources 8: 387–392.
Gladieux P, Vercken E, Fontaine MC, Hood ME, Jonot O, Couloux A, Giraud
T. 2011.Maintenance of fungal pathogen species that are specialized to
different hosts: allopatric divergence and introgression through secondary
contact.Molecular Biology and Evolution 28: 459–471.Gladieux P, Zhang X-G, Afoufa-Bastien D, Sanhueza R-MV, Sbaghi M, Cam
BL. 2008.On the origin and spread of the scab disease of apple: out of central
Asia. PLoS ONE 3: e1455.
Gomez-Alpizar L, Carbone I, Ristaino JB. 2007.AnAndean origin of Phytophthorainfestans inferred frommitochondrial and nuclear gene genealogies. Proceedings ofthe National Academy of Sciences, USA 104: 3306–3311.
Gonthier P, Nicolotti G, Linzer R, Guglielmo F, Garbelotto M. 2007.
Invasion of European pine stands by a North American forest pathogen and
its hybridization with a native interfertile taxon. Molecular Ecology 16: 1389–1400.
Goodwin SB. 1997. The population genetics of Phytophthora. Phytopathology 87:462–473.
Goodwin SB, Cohen BA, Fry WE. 1994. Panglobal distribution of a single clonal
lineage of the Irish potato famine fungus. Proceedings of the National Academy ofSciences, USA 91: 11591–11595.
� 2013 The Authors
New Phytologist� 2013 New Phytologist TrustNew Phytologist (2013)
www.newphytologist.com
NewPhytologist Research 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Goudet J. 2001. FSTAT, a program to estimate and test gene diversities and fixationindices (version 2.9.3). [WWW document] URL http://www2.unil.ch/popgen/
softwares/fstat.htm.36
Gr€unwald NJ, Garbelotto M, Goss EM, Heungens K, Prospero S. 2012.
Emergence of the sudden oak death pathogen Phytophthora ramorum. Trends inMicrobiology 20: 131–138.
Hood M, Mena-Al�ı J, Gibson A, Oxelman B, Giraud T, Yockteng R, Arroyo M,
Conti F, Pedersen A, Gladieux P et al. 2010. Distribution of the anther-smut
pathogenMicrobotryum on species of the Caryophyllaceae assessed from natural
history collections. New Phytologist 187: 217–229.Hood ME, Antonovics J. 2000. Intratetrad mating, heterozygosity, and the
maintenance of deleterious alleles inMicrobotryum violaceum (=Ustilagoviolacea). Heredity 85: 231–241.
Hood ME, Antonovics JA. 2004.Mating within the meiotic tetrad and the
maintenance of genomic heterozygosity. Genetics 166: 1751–1759.Hubisz MJ, Falush D, Stephens M, Pritchards JK. 2009. Inferring weak
population structure with the assistance of sample group information.
Molecular Ecology Resources 9: 1322–1332.Ivors K, Garbelotto M, Vries ID, Ruyter-Spira C, Te Hekkert B, Rosenzweig N,
Bonants P. 2006.Microsatellite markers identify three lineages of Phytophthoraramorum in US nurseries, yet single lineages in US forest and European nursery
populations.Molecular Ecology 15: 1493–1505.Jakobsson M, Rosenberg N. 2007. CLUMPP: a cluster matching and
permutation program for dealing with label switching and multimodality in
analysis of population structure. Bioinformatics 23: 1801–1806.James TY, Litvintseva AP, Vilgalys R, Morgan JAT, Taylor JW, Fisher MC,
Berger L, Weldon C, du Preez L, Longcore JE. 2009. Rapid global expansion
of the fungal disease Chytridiomycosis into declining and healthy amphibian
populations. PLoS Pathogens 5: e1000458.Jombart T. 2010. Package ‘adegenet’. a R package for the multivariate analysis of
genetic markers. [WWW document] URL http://cran.r-project.org/web/
packages/adegenet/index.html.37
Jombart T, Pontier D, Dufour AB. 2009. Genetic markers in the playground of
multivariate analysis. Heredity 102: 330–341.Kaltz O, Gandon S, Michalakis Y, Shykoff J. 1999. Local maladaptation of the
plant pathogenMicrobotryum violaceum to its host Silene latifolia: evidencefrom a cross-inoculation experiment. Evolution 53: 395–407.
Kaltz O, Shykoff JA. 1998. Local adaptation in host-parasite systems. Heredity81: 361–370.
Keller SR, Gilbert KJ, Fields PD, Taylor DR. 2012. Bayesian inference of a
complex invasion history revealed by nuclear and chloroplast genetic diversity
in the colonizing plant, Silene latifolia.Molecular Ecology 21: 4721–4734.Keller SR, Sowell DR, Neiman M, Wolfe LM, Taylor DR. 2009. Adaptation
and colonization history affect the evolution of clines in two introduced species.
New Phytologist 183: 678–690.Kemler M, Goker M, Oberwinkler F, Begerow D. 2006. Implications of
molecular characters for the phylogeny of the Microbotryaceae (Basidiomycota:
Urediniomycetes). BMC Evolutionary Biology 6: 35.Le Gac M, Hood ME, Fournier E, Giraud T. 2007. Phylogenetic evidence
of host-specific cryptic species in the anther smut fungus. Evolution 61:15–26.
Lopez-Villavicencio M, Enjalbert J, Hood ME, Shykoff JA, Raquin C, Giraud
T. 2005. The anther smut disease on Gypsophila repens: a case of parasite sub-optimal performance following a recent host shift? Journal of EvolutionaryBiology 18: 1293–1303.
Lopez-Villavicencio M, Jonot O, Coantic A, Hood M, Enjalbert J, Giraud T.
2007.Multiple infections by the anther smut pathogen are frequent and
involve related strains. PloS Pathogens 3: e176.Mascheretti S, Croucher PJ, Kozanitas M, Baker L, Garbelotto M. 2009.
Genetic epidemiology of the sudden oak death pathogen Phytophthoraramorum in California.Molecular Ecology 18: 4577–4590.
Mascheretti S, Croucher PJ, Vettraino A, Prospero S, Garbelotto M. 2008.
Reconstruction of the Sudden Oak Death epidemic in California through
microsatellite analysis of the pathogen Phytophthora ramorum.MolecularEcology 17: 2755–2768.
Mboup M, Leconte M, Gautier A, Wan A, Chen W, de Vallavieille-Pope C,
Enjalbert J. 2009. Evidence of genetic recombination in wheat yellow rust
populations of a Chinese oversummering area. Fungal Genetics and Biology 46:299–307.
McNeil J. 1977. The biology of Canadean weeds. Canadian Journal of Botany 57:1103–1114.
McVean G. 2009. A Genealogical Interpretation of Principal Components
Analysis. Plos Genetics 5: e1000686.Milgroom MG, Wang K, Zhou Y, Lipari SE, Kaneko S. 1996. Intercontinental
population structure of the chestnut blight fungus, Cryphonectria parasitica.Mycologia 88: 179–190.
Old KM, Moran GF, Bell JC. 1984. Isozyme variability among isolates of
Phytophthora cinnamomi from Australia and Papua-New-Guinea. CanadianJournal of Botany-Revue Canadienne De Botanique 62: 2016–2022.
Parker IM, Gilbert GS. 2004. The evolutionary ecology of novel plant-pathogen
interactions. Annual Review of Ecology Evolution and Systematics 35: 675–700.Patterson N, Price AL, Reich D. 2006. Population structure and eigenanalysis.
Plos Genetics 2: e190.Philibert A, Desprez-Loustau M-L, Fabre B, Frey P, Halkett F, Husson C,
Lung-Escarmant B, Marcais B, Robin C, Vacher C et al. 2011. Predicting
invasion success of forest pathogenic fungi from species traits. Journal of AppliedEcology 48: 1381–1390.
Pimentel D, McNair S, Janecka J, Wightman J, Simmonds C, O’Connell C,
Wong E, Russel L, Zern J, Aquino T et al. 2001. Economic and
environmental threats of alien plant, animal, and microbe invasions. AgricultureEcosystems & Environment 84: 1–20.
Pritchard J, Stephens M, Donnelly P. 2000. Inference of population structure
using multilocus genotype data. Genetics 155: 945–959.R Development Core Team. 2010. R: a language and environment for statisticalcomputing. 38
Raboin LM, Selvi A, Oliveira KM, Paulet F, Calatayud C, Zapater MF, Brottier
P, Luzaran R, Garsmeur O, Carlier J et al. 2007. Evidence for the dispersal of
a unique lineage from Asia to America and Africa in the sugarcane fungal
pathogen Ustilago scitaminea. Fungal Genetics and Biology 44: 64–76.Raymond M, Rousset F. 1995. GENEPOP (Version 1.2): population genetics
software for exact tests and eucumenism. Journal of Heredity 86: 248–249.Refr�egier G, Hood ME, Giraud T. 2010. No evidence of reproductive character
displacement between two sister fungal species causing anther smut disease in
Silene. International Journal of Plant Sciences 171: 847–859.Refr�egier G, Le Gac M, Jabbour F, Widmer A, Hood M, Yockteng R, Shykoff J,
Giraud T. 2008. Cophylogeny of the anther smut fungi and their
Caryophyllaceous hosts: prevalence of host shifts and Importance of delimiting
parasite species. BMC Evolutionary Biology 8: 100.Rousset F. 2008. genepop’007: a complete re-implementation of the Genepop
software for Windows and Linux.Molecular Ecology Resources 8: 103–106.Saleh D, Xu P, Shen Y, Li C, Adreit H, Milazzo J, Ravigne V, Bazin E,
Notteghem J-L, Fournier E et al. 2012. Sex at the origin: an Asian population
of the rice blast fungusMagnaporthe oryzae reproduces sexually.MolecularEcology 21: 1330–1344.
Shearer BL, Crane CE, Cochrane A. 2004.Quantification of the susceptibility of
the native flora of the South-West Botanical Province, Western Australia, to
Phytophthora cinnamomi. Australian Journal of Botany 52: 435–443.Shearer BL, Tippet JT. 1989. Jarrah dieback: the dynamics and management of
Phytophthora cinnamomi in the jarrah (Eucalyptus marginata) forest of
south-western Australia. [Research Bulletin No.3.] Department of
Conservation and Land Management, Como, West Australia.
Singh RP, Hodson DP, Huerta-Espino J, Jin Y, Njau P, Wanyera R, Herrera-
Foessel SA, Ward RW. 2008.Will stem rust destroy the world’s wheat crop?
Advanced Agronomy 98: 271–309.Slippers B, Stenlid J, Wingfield MJ. 2005. Emerging pathogens: fungal host
jumps following anthropogenic introduction. Trends in Ecology and Evolution20: 420–421.
Spencer JL, White HE. 1951. Anther smut of carnation. Phytopathology 41: 291–299.
Strange R, Scott P. 2005. Plant disease: a threat to global food security. AnnualReview of Phytopathology 43: 83–116.
Stukenbrock EH, Banke S, Javan-Nikkhah M, McDonald BA. 2007.Origin and
domestication of the fungal wheat pathogenMycosphaerella graminicola viasympatric speciation.Molecular Biology and Evolution 24: 398–411.
New Phytologist (2013) � 2013 The Authors
New Phytologist� 2013 New Phytologist Trustwww.newphytologist.com
Research
NewPhytologist10
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
Szpiech Z, Jakobsson M, Rosenberg N. 2008. ADZE: a rarefaction approach for
counting alleles private to combinations of populations. Bioinformatics 24:2498–2504.
Taylor DR, Keller SR. 2007.Historical range expansion determines the
phylogenetic diversity introduced during contemporary species invasion.
Evolution 61: 334–345.Urashima AS, Igarashi S, Kato H. 1993.Host range, mating-type, and fertility of
Pyricularia grisea from wheat in Brazil. Plant Disease 77: 1211–1216.Van Putten WF, Biere A, Van Damme JMM. 2003. Intraspecific competition
and mating between fungal strains of the anther smutMicrobotryum violaceumfrom the host plants Silene latifolia and S. dioica. Evolution 57: 766–776.
Vercken E, Fontaine MC, Gladieux P, Hood ME, Jonot O, Giraud T. 2010.
Glacial refugia in pathogens: European genetic structure of anther smut
pathogens on Silene latifolia and Silene dioica. PloS Pathogens 6: e1001229.de Vienne DM, Hood ME, Giraud T. 2009a. Phylogenetic determinants of
potential host shifts in fungal pathogens. Journal of Evolutionary Biology 22:2532–2541.
de Vienne DM, Refr�egier G, Hood M, Guigue A, Devier B, Vercken E, Smadja
C, Deseille A, Giraud T. 2009b.Hybrid sterility and inviability in the parasitic
fungal species complexMicrobotryum. Journal of Evolutionary Biology 22: 683–698.
Weir B, Cockerham C. 1984. Estimating F-statistics for the analysis of
population structure. Evolution 38: 1358–1370.39
Wolfe LM. 2002.Why aliens invaders succeed: support for the escape-ennemy
hypothesis. The American Naturalist 160: 705–711.Wolfe LM, Elzinga JA, Biere A. 2004. Increased susceptibility to enemies
following introduction in the invasive plant Silene latifolia. Ecology Letters 7:813–820.
Zaffarano PL, McDonald BA, Linde CC. 2008. Phylogeographical analyses
reveal global migration patterns of the barley scald pathogen Rhynchosporiumsecalis.Molecular Ecology 18: 279–293.
Zentmyer GA. 1980. Phytophthora cinnamomi and the diseases it causes. St Paul,MN, USA: The American Phytopathological Society.
Supporting Information
Additional supporting information may be found in the onlineversion of this article.
Fig. S1 Principal component analysis (PCA) on Microbotryumlychnidis-dioicae and Microbotryum silenes-dioicae microsatellitegenotypes.
Fig. S2 Population structure estimated from STRUCTURE analysesconducted on a reduced data set (n = 149) considering only Ame-rican and European samples grouping together at K = 7 in theglobal analyses.
Fig. S3 Principal component analysis (PCA) on Microbotryumlychnidis-dioicae strains whose genotypes overlap with thosefound in the USA for the three first 40principal components (PCs)on the PCA on the total data set.
Table S1 Number of genotypes and GPS coordinates of samplescollected in North America
Please note: Wiley-Blackwell are not responsible for the contentor functionality of any supporting information supplied by theauthors. Any queries (other than missing material) should bedirected to the New Phytologist Central Office.
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Author Query Form
Journal: NPH
Article: 12177/2012-14489
Dear Author,During the copy-editing of your paper, the following queries arose. Please respond to these by marking up your proofs with thenecessary changes/additions. Please write your answers on the query sheet if there is insufficient space on the page proofs. Please writeclearly and follow the conventions shown on the attached corrections sheet. If returning the proof by fax do not write too close to thepaper’s edge. Please remember that illegible mark-ups may delay publication.
Many thanks for your assistance.
Query reference Query Remarks
1 AUTHOR: and thus suffered from - change OK?
2 AUTHOR: as it is - change OK?
3 AUTHOR: potato (Solanum tuberosum) - binomial added OK?
4 AUTHOR: wheat (Triticum aestivum) - binomial added OK?
5 AUTHOR: Baker, 1947 has been changed to Baker, 1947a, 1947b so that thiscitation matches the Reference List. Please check whether Baker, 1947 refers to1947a or 1947b, or both.
6 AUTHOR: Please check inserted initials ‘M. E.’ for Hood are correct.
7 AUTHOR: Silene latifolia - please give the authority
8 AUTHOR: To fit journal style, please insert the species under investigationinto the table legend.
9 AUTHOR: Silene dioca - genus added OK?
10 AUTHOR: MCMC - please define
11 AUTHOR: To fit journal style, please insert the species under investigationinto the table legend.
12 AUTHOR: assessed - change OK here and below to avoid ‘tested using… tests’?
13 AUTHOR: rather than being a host-shift lineage - change OK?
14 AUTHOR: north Western (NWest) and south Western (SWest) - definitionsadded OK?
15 AUTHOR: northwestern Europe - OK?
16 AUTHOR: the three first - should this be ‘the first three’?
17 AUTHOR: the three first - should this be ‘the first three’?
18 AUTHOR: DeltaK - OK or should this be DK?
19 AUTHOR: Indeed, the Scottish samples… any European population ofM. lychnidis-dioicae - please clarify this sentence. Does ‘with a multi-locusgenotype’ refer to the Scottish samples? If so, please add a comma before ‘with’.Do ‘one locus’ and ‘the most polymorphic locus’ refer to the same locus? If so,please reword as e.g. ‘differing only at one locus, E18, which was the most poly-morphic locus, and for which the North American population had an allelethat was not shared…’
20 AUTHOR: Providing results consistent with the clustering analyses, PCA ongenotypes that displayed score values for the first three PCs of the global analy-ses overlapping with the score values of the North American samples, theseintroduced genotypes appeared again closely related to the Scottish samples -please also reword this sentence to clarify meaning.
21 AUTHOR: To fit journal style, please insert the species under investigationinto the table legend.
22 AUTHOR: For Ar, pAr, and He, please indicate whether the values are� SD orSE
23 AUTHOR: as a consequence of - change OK?
24 AUTHOR: Phytophthora ramorum - genus added OK?
25 AUTHOR: sugarcane (Saccharum officinarum), rice (Oryza sativa) - binomialsadded OK?
26 AUTHOR: apple - please give the Latin binomial
27 AUTHOR: barley (Hordeum vulgare) - binomial OK?
28 AUTHOR: colza - please give the Latin binomial
29 AUTHOR: and was present only in agricultural settings - change OK?
30 AUTHOR: cypress - please give the Latin binomial
31 AUTHOR: sunflower - please give the Latin binomial
32 AUTHOR: grape (Vitis vinfera) - binomial added OK?
33 AUTHOR: oilseed rape (Brassica napus) - binomial OK?
34 AUTHOR: Please provide the givenNames/initials for the author Anagnostakisfor reference Anagnostakis (1987).
35 AUTHOR: Please provide the familyname for the author C for reference Des-prez-Loustau et al. (2007).
36 AUTHOR: Please provide Accessed date, month and year for reference Goudet(2001).
37 AUTHOR: Please provide Accessed date, month and year for reference Jombart(2010).
38 AUTHOR: Please insert additional information for this citation for reference RDevelopment Core Team (2010).
39 AUTHOR: Please check the page range for reference Weir and Cockerham(1984).
40 AUTHOR: the three first - should this be ‘the first three’?
O n c e y o u h a v e A c r o b a t R e a d e r o p e n o n y o u r c o m p u t e r , c l i c k o n t h e C o m m e n t t a b a t t h e r i g h t o f t h e t o o l b a r :
S t r i k e s a l i n e t h r o u g h t e x t a n d o p e n s u p a t e x tb o x w h e r e r e p l a c e m e n t t e x t c a n b e e n t e r e d .‚ H i g h l i g h t a w o r d o r s e n t e n c e .‚ C l i c k o n t h e R e p l a c e ( I n s ) i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ T y p e t h e r e p l a c e m e n t t e x t i n t o t h e b l u e b o x t h a ta p p e a r s .
T h i s w i l l o p e n u p a p a n e l d o w n t h e r i g h t s i d e o f t h e d o c u m e n t . T h e m a j o r i t y o ft o o l s y o u w i l l u s e f o r a n n o t a t i n g y o u r p r o o f w i l l b e i n t h e A n n o t a t i o n s s e c t i o n ,p i c t u r e d o p p o s i t e . W e ’ v e p i c k e d o u t s o m e o f t h e s e t o o l s b e l o w :S t r i k e s a r e d l i n e t h r o u g h t e x t t h a t i s t o b ed e l e t e d .
‚ H i g h l i g h t a w o r d o r s e n t e n c e .‚ C l i c k o n t h e S t r i k e t h r o u g h ( D e l ) i c o n i n t h eA n n o t a t i o n s s e c t i o n .
H i g h l i g h t s t e x t i n y e l l o w a n d o p e n s u p a t e x tb o x w h e r e c o m m e n t s c a n b e e n t e r e d .‚ H i g h l i g h t t h e r e l e v a n t s e c t i o n o f t e x t .‚ C l i c k o n t h e A d d n o t e t o t e x t i c o n i n t h eA n n o t a t i o n s s e c t i o n .‚ T y p e i n s t r u c t i o n o n w h a t s h o u l d b e c h a n g e dr e g a r d i n g t h e t e x t i n t o t h e y e l l o w b o x t h a ta p p e a r s .
M a r k s a p o i n t i n t h e p r o o f w h e r e a c o m m e n tn e e d s t o b e h i g h l i g h t e d .‚ C l i c k o n t h e A d d s t i c k y n o t e i c o n i n t h eA n n o t a t i o n s s e c t i o n .‚ C l i c k a t t h e p o i n t i n t h e p r o o f w h e r e t h e c o m m e n ts h o u l d b e i n s e r t e d .‚ T y p e t h e c o m m e n t i n t o t h e y e l l o w b o x t h a ta p p e a r s .
I n s e r t s a n i c o n l i n k i n g t o t h e a t t a c h e d f i l e i n t h ea p p r o p r i a t e p a c e i n t h e t e x t .‚ C l i c k o n t h e A t t a c h F i l e i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ C l i c k o n t h e p r o o f t o w h e r e y o u ’ d l i k e t h e a t t a c h e df i l e t o b e l i n k e d .‚ S e l e c t t h e f i l e t o b e a t t a c h e d f r o m y o u r c o m p u t e ro r n e t w o r k .‚ S e l e c t t h e c o l o u r a n d t y p e o f i c o n t h a t w i l l a p p e a ri n t h e p r o o f . C l i c k O K .
I n s e r t s a s e l e c t e d s t a m p o n t o a n a p p r o p r i a t ep l a c e i n t h e p r o o f .‚ C l i c k o n t h e A d d s t a m p i c o n i n t h e A n n o t a t i o n ss e c t i o n .‚ S e l e c t t h e s t a m p y o u w a n t t o u s e . ( T h e A p p r o v e ds t a m p i s u s u a l l y a v a i l a b l e d i r e c t l y i n t h e m e n u t h a ta p p e a r s ) .‚ C l i c k o n t h e p r o o f w h e r e y o u ’ d l i k e t h e s t a m p t oa p p e a r . ( W h e r e a p r o o f i s t o b e a p p r o v e d a s i t i s ,t h i s w o u l d n o r m a l l y b e o n t h e f i r s t p a g e ) .
A l l o w s s h a p e s , l i n e s a n d f r e e f o r m a n n o t a t i o n s t o b e d r a w n o n p r o o f s a n d f o rc o m m e n t t o b e m a d e o n t h e s e m a r k s . .‚ C l i c k o n o n e o f t h e s h a p e s i n t h e D r a w i n gM a r k u p s s e c t i o n .‚ C l i c k o n t h e p r o o f a t t h e r e l e v a n t p o i n t a n dd r a w t h e s e l e c t e d s h a p e w i t h t h e c u r s o r .‚
T o a d d a c o m m e n t t o t h e d r a w n s h a p e ,m o v e t h e c u r s o r o v e r t h e s h a p e u n t i l a na r r o w h e a d a p p e a r s .‚
D o u b l e c l i c k o n t h e s h a p e a n d t y p e a n yt e x t i n t h e r e d b o x t h a t a p p e a r s .
