# Evolution of pathogenicity traits in the apple scab fungal pathogen in response to the domestication of its host

Amandine Le Van, Pierre Gladieux, Christophe Lemaire, Amandine Cornille, Tatiana Giraud, Charles Eric Durel, Valerie Caffier, Bruno Le Cam

► To cite this version:

Amandine Le Van, Pierre Gladieux, Christophe Lemaire, Amandine Cornille, Tatiana Giraud, et al. Evolution of pathogenicity traits in the apple scab fungal pathogen in response to the domestication of its host. Evolutionary Applications, 2012, 5 (7), pp.694 - 704. 10.1111/j.1752-4571.2012.00246.x. hal-01209918

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ORIGINAL ARTICLE

# Evolution of pathogenicity traits in the apple scab fungal pathogen in response to the domestication of its host

Amandine Lê Van,¹ Pierre Gladieux,²,³ Christophe Lemaire,⁴ Amandine Cornille,²,³ Tatiana Giraud,²,³ Charles-Eric Durel,¹ Valérie Caffier¹ and Bruno Le Cam¹

1 INRA, UMR1345, IRHS (INRA, Agrocampus-Ouest, Université d'Angers), SFR QUASAV, Beaucouzé, France

2 CNRS, UMR 8079, Ecologie, Systématique et Evolution, Univ. Paris-Sud, Orsay, France

3 AgroParisTech, Orsay, France

4 Université d'Angers, UMR1345 IRHS (INRA, Agrocampus-Ouest, Université d'Angers), SFR QUASAV, Angers, France

**Keywords**

apple scab, coevolution, disease emergence, plant-microbe interactions, wild crop relatives.

**Correspondence**

Bruno Le Cam, UMR1345 IRHS (INRA, Agrocampus-Ouest, Université d'Angers), SFR QUASAV, F-49071 Beaucouzé, France.
Tel.: +33 241 225 735;
fax: + 33 241 225 705;
e-mail: bruno.lecam@angers.inra.fr

Received: 23 December 2011

Accepted: 9 January 2012

doi:10.1111/j.1752-4571.2012.00246.x

**Abstract**

Understanding how pathogens emerge is essential to bring disease-causing agents under durable human control. Here, we used cross-pathogenicity tests to investigate the changes in life-history traits of the fungal pathogen *Venturia inaequalis* associated with host-tracking during the domestication of apple and subsequent host-range expansion on the wild European crabapple (*Malus sylvestris*). Pathogenicity of 40 isolates collected in wild and domesticated ecosystems was assessed on the domesticated apple, its Central Asian main progenitor (*M. sieversii*) and *M. sylvestris*. Isolates from wild habitats in the centre of origin of the crop were not pathogenic on the domesticated apple and less aggressive than other isolates on their host of origin. Isolates from the agro-ecosystem in Central Asia infected a higher proportion of plants with higher aggressiveness, on both the domesticated host and its progenitor. Isolates from the European crabapple were still able to cause disease on other species but were less aggressive and less frequently virulent on these hosts than their endemic populations. Our results suggest that the domestication of apple was associated with the acquisition of virulence in the pathogen following host-tracking. The spread of the disease in the agro-ecosystem would also have been accompanied by an increase in overall pathogenicity.

**Introduction**

The domestication of plants, expanding global trade and agriculture are major drivers of plant pathogen emergence (Pysek et al. 2010). Understanding how pathogens emerge on domesticated plants and spread in the agro-ecosystem is essential to bring disease-causing agents under durable human control (Anderson et al. 2004). Population genetic analyses on samples from domesticated and wild hosts have provided important insights into the origins of fun- gal pathogens (Gomez-Alpizar et al. 2007; Frenkel et al. 2010; Stukenbrock et al. 2011; Torriani et al. 2011). The emergence of pathogens on domesticated hosts can result from the colonization of a novel host or from a process of host-tracking where the pathogen simply follows its

current host along the domestication process (e.g. Munk- acsi et al. 2008; Gladieux et al. 2010; Giraud et al. 2010; Hansen 1987; Stukenbrock and McDonald 2008). Coloni- zation of a novel host can be due to host-range expansion (i.e. colonization of a new host species while remaining pathogenic on the ancestral host) or host-shift (i.e. colo- nization of a new host species associated with the loss of the ability to infect the ancestral host). By definition, host-shifts involve very strong (qualitative) host special- ization and can lead to rapid speciation through the evo- lution of an association between genes involved in host adaptation and genes conferring reproductive isolation (Couch et al. 2005; Giraud et al. 2010). In the case of host-range expansion or host-tracking, specialization is not an obligate outcome but quantitative specialization to
the new host can nonetheless be observed, with a lower
performance on the ancestral host of pathogen popula-
tions from the new host compared to native populations
(Fry 2003; Sicard et al. 2007). In fungal plant pathogens,
specialization is favoured by some particular life-history
traits including a large number of spores, mating within
the host and high selection coefficients on a limited num-
ber of genes (Giraud et al. 2010).

In addition to the divergent selective pressures caused
by the genetic differences among ancestral and domesti-
cated hosts, pathogens are also exposed to a novel habi-
tat, the agro-ecosystem, drastically different from natural
ecosystems. The particular features of the human-
engineered environment are thought to further enhance
pathogen specialization to the domesticated hosts
(Stukenbrock and McDonald 2008). Unlike natural plant
populations, the high density and genetic uniformity of
cultivated plant populations are highly conducive to
pathogen transmission between infected and noninfected
hosts, which favours more aggressive pathogens (Ander-
son and May 1982; Hochberg 2000; Thrall et al. 2007).
Moreover, cultivated crops represent large targets for
pathogens shifting from other hosts, while the large and
widely connected pathogen populations associated with
the agro-ecosystem are potential reservoirs for novel epi-
demics on naïve hosts that do not have evolved defence
mechanisms (Couch et al. 2005). The vast-scale and
homogenous availability of nutrients in the agro-ecosys-
tem is also expected to enable the development of
very large populations of pathogens, thereby increasing
the efficiency of selection and accelerating adaptation
(Karasov et al. 2010).

In analogy with the common suite of morphological
and physiological traits that distinguish crops from their
wild ancestors (Doebley et al. 2006; Zeder et al. 2006),
the changes in life-history traits of pathogens adapting to
domesticated hosts and to the agro-ecosystem can be
regarded as a ‘domestication syndrome’. Unlike plants,
however, for which the domestication syndrome has been
extensively investigated, the study of the evolutionary
changes in pathogens associated with the domestication
of plants and ecosystems is still in its infancy. This might
be related to the lack of archaeological data on pathogens
and to the difficulty in identifying, getting access and col-
lecting samples in the centre of origins of diseases. Studies
on the rice blast pathogen, *Magnaporthe oryzae*, have
demonstrated differences in pathogenicity traits between
populations infecting domesticated rice and the ancestral
host of the pathogen, *Setaria millet*. Isolates from Setaria
millet were either not virulent on rice or less aggressive
than isolates from rice (Couch et al. 2005). Similarly, the
pathogen *Rhynchosporium* shifted from an unidentified
ancestor to cultivated barley and rye and speciated after

adaptation to its new hosts at the time of domestication
of cereals in the Fertile Crescent (Zaffarano et al. 2008).

The pathosystem *Malus* spp.-*Venturia inaequalis* (apple
scab) is an excellent system to investigate the changes in
life-history traits of pathogens adapting to domesticated
hosts and to the agro-ecosystem. First, the life-history
traits of *V. inaequalis* confer to this ascomycete an impor-
tant evolutionary potential (McDonald and Linde 2002).
The fungus reproduces both asexually during spring and
summer (epidemic phase) and sexually during winter
(saprotrophic phase). Reproduction occurs between
strains of opposite mating types that have infected the
same leaf. This reproductive system, where mating occurs
between individuals that were able to grow on the same
host genotype, is highly conducive to rapid ecological
divergence (Giraud et al. 2010; Gladieux et al. 2011). Sec-
ond, the story of the apple domestication is well docu-
mented. Historical information (Juniper and Mabberley
2006) and (partial) genetic evidence (Harris et al. 2002;
Velasco et al. 2010) suggested that the centre of origin of
the cultivated apple (*M. × domestica*) was Central Asia,
where the wild apple *M. sieversii*, its main progenitor,
forms forests (Harris et al. 2002). From Central Asia, the
domesticated apple was moved westward to Europe and
eastward to China following the Silk Road (Juniper and
Mabberley 2006). During the spread of apple cultivation,
several other *Malus* species may have contributed to the
gene pool of *M. × domestica*. While the domesticated
varieties appear closely related to *M. sieversii* (Velasco
et al. 2010), a possible contribution from the European
crabapple *M. sylvestris* to the domesticated apple gene
pool is still debated (Coart et al. 2006; Harrison and Har-
rison 2011; Micheletti et al. 2011; A Cornille, P. Gladieux,
I. Roldán-Ruiz, F. Laurens, B. Le Cam, M. J. M. Smul-
ders, A. Nersesyan, J. Clavel, M. Olonova, L. Feugey, I.
Gabrielyan, X. G. Zhang, M. I. Tenaillon, T. Giraud,
unpublished manuscript). The speciation between Euro-
pean and Central Asian wild apples likely occurred during
Pleistocene repeated glaciations owing to the retreat and
fragmentation of an ancient corridor of Tertiary temper-
ate forests that ranged from the Atlantic Ocean to Bering
(Juniper and Mabberley 2006). *Malus sylvestris* is now
considered as an endangered tree species in some Euro-
pean regions, with a very scattered distribution (Stephan
et al. 2003; A Cornille, P. Gladieux, I. Roldán-Ruiz, F.
Laurens, B. Le Cam, M. J. M. Smulders, A. Nersesyan, J.
Clavel, M. Olonova, L. Feugey, I. Gabrielyan, X. G.
Zhang, M. I. Tenaillon, T. Giraud, unpublished manu-
script). Third, population genetics studies on *V. inaequal-
is* provided important clues about the evolutionary
history of this pathogen. A previous study on the popula-
tion structure of *V. inaequalis* showed that the pathogen
shared a common origin with its host in Central Asia
(Gladieux et al. 2008). A subsequent study on populations of *V. inaequalis* infecting the wild apples *M. sieversii* and *M. sylvestris* pinpointed *M. sieversii* as the host of origin of the fungus (Gladieux et al. 2010). Results were consistent with a host-tracking scenario in which *V. inaequalis* spread into Europe together with the domesticated apple and subsequently expanded its range to *M. sylvestris*, previously free of apple scab. Population genetic analyses indicated that apple domestication had a strong impact on the population structure of the pathogen: apple domestication was associated with significant changes in the genetic differentiation of *V. inaequalis* populations in their centre of origin but had little impact on historical demography and mating system of the fungus (Gladieux et al. 2010). Three distinct gene pools were indeed identified based on microsatellite data by Gladieux et al. (2010): a population geographically restricted to the south-eastern mountains of Kazakhstan parasitizing *M. sieversii* (CAM population), an Asian population infecting *M. × domestica* and *M. sieversii* in peri-urban or agricultural areas (CAP population) and a European population present on *M. × domestica* and *M. sylvestris* (EU population) (Gladieux et al. 2010). Gladieux et al. (2010) hypothesized that the mountain population (CAM) could represent a relict of the ancestral populations that infected *M. sieversii* before apple domestication and from which the other populations would have diverged following domestication. The CAM population would represent an undisturbed population of the pathogen from natural ecosystems, while the CAP and EU populations would be evolved populations in contact with the agro-ecosystem.

Here, we used cross-inoculation tests to investigate the
changes in pathogenicity traits of the apple scab fungus
*V. inaequalis* associated with the domestication and
spread of its host. Pathogenicity of 40 isolates collected in
wild and domesticated ecosystems was assessed on the
domesticated apple (*Malus* × *domestica*), its Central Asian
main progenitor (*M. sieversii*) and the wild European
crabapple (*M. sylvestris*). Two components of pathogenic-
ity were analysed: virulence, that is, the ability to infect a
given host genotype, and aggressiveness, that is, the sever-
ity of the disease in successful infections. We tested the
hypotheses that agro-ecosystem features such as high host
density favoured pathogen specialization on *M. × domes-
tica* as well as an increase in aggressiveness, while the fea-
tures of the European forest ecosystem with a very
scattered host distribution did not lead to the specializa-
tion on *M. sylvestris*. We tested more specifically the fol-
lowing hypotheses: (i) host-tracking of *V. inaequalis* from
the wild ancestor to the cultivated apple has been associ-
ated with a gain in virulence; evidence would be that iso-
lates from the wild Asian progenitor are unable to cause
disease on *M. × domestica*; (ii) adaptation to the culti-

vated apple has been associated with increased overall
pathogenicity; evidence would be that isolates from
domesticated apple trees are more aggressive or more fre-
quently virulent on the wild Asian progenitor than the
endemic isolates; (iii) the emergence of apple scab on
wild crabapple populations in Europe was due to a host-
range expansion and not a host-shift of *V. inaequalis*
populations from agro-ecosystems; evidence would be
that isolates from crabapple trees are still able to cause
disease on the domesticated trees; iv) the host-range
expansion on *M. sylvestris*, nevertheless, resulted in a cer-
tain degree of specialization; evidence would be that crab-
apple isolates show lower aggressiveness or lower
frequency of virulence on domesticated trees than isolates
from *M. × domestica*.

**Materials and methods**

Fungal isolates

This study was based on a total of 40 isolates of *V. inaequalis* (Table 1) sampled on *M. sieversii*, *M. × domestica* and *M. sylvestris*. Three core collections of *V. inaequalis* were previously constructed, one per *Malus* species of origin, as maximizing neutral genetic diversity among isolates genotyped with 12 SSR loci (Lê Van et al. 2011). Each core collection was constituted by 15 isolates except the ‘*M. × domestica* core collection’ constituted by 10 isolates. The isolates originating from CAM, CAP or EU populations (Gladieux et al. 2010) were classified into five pools labelled after their geographic origin (Asia or Europe), the environment of origin (wild or agro-ecosystem) and the host of origin (*M. sieversii*, *M. × domestica* or *M. sylvestris*): ‘WildAsiaSiev’, ‘AgroAsiaSiev’, ‘AgroAsiaDom’, ‘AgroEuDom’ and ‘WildEuSylv’ (Table 1).

Plant material

Two cultivars of *M. × domestica*, three accessions of
*M. sieversii* (GMAL 3619.b, PI 633797.d and PI 633799.e)
and four accessions of *M. sylvestris* (X 9650, X 9651, X
9653 and X 9654) were used in this study. The two culti-
vars, Gala and Top Red Delicious (latter on called ‘Top
Red’), are extensively cultivated worldwide. *Malus sieversii*
accessions were collected in Kazakhstan in two different
localities (Figure S1). GMAL 3619.b and PI 633799.e were
collected from the Tarbagatai mountain range, and PI
633797.d was collected from the Djungarsky mountain
range (Forsline et al. 2010; USDA website: http://
www.ars-grin.gov/cgi-bin/npgs/html/search.pl). *Malus syl-
vestris* accessions were collected in the French forest of
Rambouillet. Budwoods were grafted on ‘MM106’ apple
rootstocks and then maintained in a greenhouse. The
plants of *M. sylvestris* used in pathogenicity tests were
<table><caption>**Table 1.** Description of the *Venturia inaequalis* isolates used in this study.</caption><thead><tr><th>Isolate</th><th>Country of origin</th><th>Sampled year</th><th>Population name</th><th>Malus host (Cultivar)</th></tr></thead><tbody><tr><td>2217</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2219</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2220</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2221</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2222</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2223</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2224</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2225</td><td>Kazakhstan</td><td>2006</td><td>WildAsiaSiev†</td><td>M. sieversii</td></tr><tr><td>2227</td><td>Kazakhstan</td><td>2006</td><td>AgroAsiaSiev‡</td><td>M. sieversii</td></tr><tr><td>2228</td><td>Kazakhstan</td><td>2006</td><td>AgroAsiaSiev‡</td><td>M. sieversii</td></tr><tr><td>2229</td><td>Kazakhstan</td><td>2006</td><td>AgroAsiaSiev‡</td><td>M. sieversii</td></tr><tr><td>2230</td><td>Kazakhstan</td><td>2006</td><td>AgroAsiaSiev‡</td><td>M. sieversii</td></tr><tr><td>2231</td><td>Kazakhstan</td><td>2006</td><td>AgroAsiaSiev‡</td><td>M. sieversii</td></tr><tr><td>2233</td><td>China</td><td>2005</td><td>AgroAsiaSiev‡</td><td>M. sieversii</td></tr><tr><td>2234</td><td>China</td><td>2005</td><td>AgroAsiaSiev‡</td><td>M. sieversii</td></tr><tr><td>2278</td><td>China</td><td>2005</td><td>AgroAsiaDom‡</td><td>M. × domestica (Golden Delicious)</td></tr><tr><td>2279</td><td>China</td><td>2005</td><td>AgroAsiaDom‡</td><td>M. × domestica (Gala)</td></tr><tr><td>2281</td><td>China</td><td>2005</td><td>AgroAsiaDom‡</td><td>M. × domestica (New Century)</td></tr><tr><td>2284*</td><td>China</td><td>2005</td><td>AgroAsiaDom‡</td><td>M. × domestica (New Century)</td></tr><tr><td>2285</td><td>China</td><td>2005</td><td>AgroAsiaDom‡</td><td>M. × domestica (Gala)</td></tr><tr><td>2286</td><td>France</td><td>2005</td><td>AgroEuDom§</td><td>M. × domestica (Mutsu)</td></tr><tr><td>2288</td><td>France</td><td>2005</td><td>AgroEuDom§</td><td>M. × domestica (Mutsu)</td></tr><tr><td>2289</td><td>France</td><td>2005</td><td>AgroEuDom§</td><td>M. × domestica (Mutsu)</td></tr><tr><td>2291</td><td>Spain</td><td>2005</td><td>AgroEuDom§</td><td>M. × domestica (Wellspur)</td></tr><tr><td>EU-D-16</td><td>Germany</td><td>1999</td><td>AgroEuDom§</td><td>M. × domestica (Coop 9)</td></tr><tr><td>2237</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2238*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2239*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2240*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2241*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2245</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2246*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2247</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2248*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2249*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2251*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2252*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2254*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2255*</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr><tr><td>2256</td><td>France</td><td>2005</td><td>WildEuSylv§</td><td>M. sylvestris</td></tr></tbody></table>

genotyped using microsatellite markers, and their assignment to the gene pool of their putative species of origin was checked using a reference data set (Data S1).

## Cross-pathogenicity tests

The 40 isolates were inoculated onto all host genotypes during three rounds of experiments per host species. For each experiment, one to six isolates of each fungal popu-

lation were inoculated onto one *Malus* species. For inoculations onto *M. sieversii*, 28 of 40 isolates were inoculated because of limited plant material available (Table 1). Only actively growing plants with uniform growth were chosen and transferred to a quarantine-controlled climate chamber for subsequent inoculations. We used a quarantine room because of the exotic origin of numerous isolates whose unknown virulence may present a risk. Inocula were obtained by growing monoconidial isolates of the

<footnote>*These isolates were inoculated onto *M. × domestica* and *M. sylvestris* but not onto *M. sieversii*.</footnote>

<footnote>†This population belongs to the previously identified CAM (Central Asian Mountains) population (Gladieux et al. 2010).</footnote>

<footnote>‡This population belongs to the previously identified CAP (Central Asian Plains) population.</footnote>

<footnote>§This population belongs to the previously identified EU (European) population.</footnote>
fungus on cellophane sheets deposited onto malt agar medium (Bus et al. 2005). Monoconidial suspensions of each isolate were adjusted to a concentration of $1.5 \times 10^5$ conidia/mL. Germination rates were assessed for each monoconidial suspension on malt agar plates to check for the viability of conidia. Germination rates ranged from 34% to 95% depending on the isolate and the experiment, with more than 64% reached in 75% of cases. Each isolate was sprayed using an air pressure hand-sprayer on four to five replicates of each host genotype. All leaves were inoculated. For the first 48 h after inoculation, the plants were kept in darkness with humidity maintained at 100% and temperature at 18°C to allow conidia germination and fungal infection. Humidity was then reduced to 80% with 16-h light per day. Percentage of each leaf showing sporulation was scored visually at 14, 21 and 28 days after inoculation (dai) on an ordinal scale, ranging from 0 (no sporulation) to 8 (100% of leaf area showing sporulation) (Lê Van et al. 2011). For virulence, each replicate was either scored as infected or not (no visible sporulating symptoms at 28 days).

## Data analyses

### Analyses of virulence

Virulence was defined as the ability of an isolate to produce sporulating symptoms on a host genotype. A Pearson's chi-squared test was performed on contingency tables to test for independence between virulence and population of origin. Because of expected cell count below five, P-values were computed for a Monte Carlo test (Hope 1968) with $1 \times 10^5$ replicates. When virulence and the population of origin were not independent, multiple comparisons were made using Pearson's chi-squared tests on two-by-two contingency tables using the Bonferroni correction. A Pearson's chi-squared test was also conducted to test for independence between virulence and *Malus* species tested.

### Analyses of aggressiveness

Aggressiveness was measured as the area under the disease progress curve (AUDPC) calculated on the sporulation percentage of the most diseased leaf of each replicate. The 'AUDPC' variable was analysed using a linear mixed-effect model (LME). The 'isolate' was treated as random factor and nested in the population of origin. The cultivars were treated as fixed factors. A variance function was used for modelling the within-group heteroscedasticity. Each factor (isolate, population of origin, *Malus* species of origin, tested cultivar and round of experiment) was included in the model based on an ascendant selection using BIC (Bayesian Information Criterion) to select the best model (Pinheiro and Bates 2000). The model was fit-

ted by maximizing the log-likelihood. All statistical analyses (virulence and aggressiveness) were performed using the 'nlme' package (Pinheiro et al., 2008) in R version 2.10.1 (R-Development-Core-team, 2008).

## Results

### Virulence

*Malus sieversii* plants were significantly more often infected by isolates from Central Asia sampled in the agro-ecosystem area, either from *M. sieversii* (AgroAsia-Siev population) or from *M. × domestica* (AgroAsiaDom population), than by isolates sampled in the wild area in Central Asia (WildAsiaSiev population) or isolates from Europe, either from wild or from agro-ecosystem (Pearson's chi-squared tests on two-by-two contingency tables; Fig. 1). Isolates belonging to the Asian population from the agro-ecosystem were able to infect up to three *M. sieversii* accessions, whereas isolates from Europe were able to infect up to two accessions. Only one of the three accessions was infected by isolates from the WildAsiaSiev population (Table 2). The higher frequency of virulence on *M. sieversii* in fungal populations from Asian agro-ecosystems suggests that the spread of the disease on the domesticated apple has been associated with an increase in pathogenicity of *V. inaequalis*.

None of the *M. × domestica* cultivars was infected by the WildAsiaSiev population. *Malus × domestica* cultivars were significantly less frequently infected by the WildAsia-Siev population than by other pathogen populations (Pearson's chi-squared tests on two-by-two contingency tables; Fig. 1). All other populations were able to infect both *M. × domestica* cultivars (Fig. 1). The lack of pathogenicity of isolates from wild habitats of the centre of origin of the crop when inoculated on the domesticated apple indicates that the host-tracking of *V. inaequalis* from the wild ancestor to the cultivated apple has demanded acquisition of new virulence.

Although isolates from both *M. × domestica* and *M. sylvestris* were able to infect *M. × domestica* cultivars, the number of infected plants was significantly lower when challenged with isolates from *M. sylvestris* (WildAsia-Sylv population) than with isolates from *M. × domestica* either collected in Asia or in Europe ($\chi^2 = 9.44$; *P* < 0.01 and $\chi^2 = 6.82$; *P* < 0.01, respectively). This suggests a certain degree of specialization by isolates parasitizing the European crabapple. No significant differences were observed between isolates from *M. × domestica* from either Asia or Europe (*P* = 1) (Fig. 1).

*Malus sylvestris* plants were significantly more frequently diseased after inoculation by isolates from *M. sylvestris* than by isolates from other *Malus* species (Pearson's chi-squared tests on two-by-two contingency
<figure><img src="image_2.png" /><figcaption>**Figure 1** Percentage of plants of three *Malus* species infected by five *Venturia inaequalis* populations. Note that an empty place indicates zero infected plant for the corresponding pathogen population. Virulence was measured on three accessions for *M. sieversii*, two cultivars for *M. × domestica* and four accessions for *M. sylvestris*. Different letters indicate significant differences between populations (*P* < 0.05) (Pearson's chi-squared tests with Bonferroni correction on two-by-two contingency tables).</figcaption></figure>

<table><caption>**Table 2.** Number of virulent isolates of *Venturia inaequalis* out of the number of isolates tested for each genotype of three *Malus* species.</caption><thead><tr><th rowspan="2">Pathogen population</th><th colspan="3">M. sieversii accessions</th><th colspan="2">M. × domestica cultivars</th><th colspan="4">M. sylvestris accessions</th></tr><tr><th>GMAL 3619.b</th><th>PI 633 797.d</th><th>PI 633 799.e</th><th>Gala</th><th>Top Red</th><th>X9650</th><th>X9651</th><th>X9653</th><th>X9654</th></tr></thead><tbody><tr><td>WildAsiaSiev</td><td>8/8</td><td>0/8</td><td>0/7</td><td>0/8</td><td>0/7</td><td>0/8</td><td>0/8</td><td>0/8</td><td>0/8</td></tr><tr><td>AgroAsiaSiev</td><td>7/7</td><td>3/7</td><td>5/7</td><td>6/7</td><td>7/7</td><td>0/7</td><td>0/7</td><td>2/7</td><td>0/6</td></tr><tr><td>AgroAsiaDom</td><td>4/4</td><td>3/4</td><td>3/4</td><td>5/5</td><td>5/5</td><td>0/5</td><td>0/5</td><td>1/4</td><td>0/4</td></tr><tr><td>AgroEuDom</td><td>5/5</td><td>1/5</td><td>1/5</td><td>5/5</td><td>5/5</td><td>0/5</td><td>0/5</td><td>0/5</td><td>1/5</td></tr><tr><td>WildEuSylv</td><td>4/4</td><td>0/4</td><td>1/4</td><td>13/15</td><td>12/15</td><td>1/14</td><td>2/15</td><td>4/12</td><td>6/14</td></tr></tbody></table>

tables; Fig. 1), except for AgroAsiaDom. Moreover, some isolates from *M. sylvestris* were able to infect up to three different *M. sylvestris* accessions, whereas isolates from other populations were able to infect a single accession. This suggests that the colonization of *M. sylvestris* has demanded a certain degree of adaptation.

The number of diseased plants was not significantly different when challenged with isolates from the agro-ecosystem in Asia sampled on *M. × domestica* or on *M. sieversii* ($\chi^2$ = 0.03; *P* = 1). Similar to the pattern of virulence observed on *M. × domestica*, the eight isolates from WildAsiaSiev were not able to infect *M. sylvestris* accessions (Fig. 1). *Malus sylvestris* accessions were resistant to a significantly higher number of isolates than *M. × domestica* or *M. sieversii* accessions ($\chi^2$ = 356.28; *P* < 0.0001).

## Aggressiveness

Data of all experiments conducted on the same *Malus* species were pooled. The experiment factor had no significant effect and did not improve LME models. As a consequence, it was not eventually included in the models. The within-group heteroscedasticity was modelled as a power function of mean fitted values.

Because a single *M. sieversii* accession was susceptible to all pathogen populations, statistical analyses were conducted only for this accession (GMAL 3619.b). The factor ‘population of origin’ explained the area under the disease progress curve (AUDPC) variance (BIC = 3158) and significantly improved the null model (BIC = 3176; *P* < 0.0001). Isolates from Asia collected in the agro-ecosystem, either from *M. sieversii* or from *M. × domestica*, were significantly more aggressive than isolates from other populations on the *M. sieversii* accession (Fig. 2). The AgroAsiaSiev and the AgroAsiaDom populations were on average fivefold and fourfold, respectively, more aggressive than the WildAsiaSiev population. This higher aggressiveness of populations from agro-ecosystems on *M. sieversii* is another evidence indicating an increase in overall pathogenicity following the spread of the pathogen on the domesticated apple.

Aggressiveness of isolates from WildAsiaSiev was significantly lower than aggressiveness of isolates from AgroAsiaSiev, but not significantly different from aggressiveness of European isolates, regardless of their host of origin (*M. × domestica* or *M. sylvestris*). The response of isolates from *M. × domestica* was different across populations. Isolates from *M. × domestica* collected in Asia (AgroAsiaDom) were significantly more aggressive
than isolates from Europe (AgroEuDom) on *M. sieversii*. The lower aggressiveness and the lower frequency of virulent isolates in European population from the agro-ecosystem support the view that, unlike in Central Asia, pathogen populations in Europe evolved a quantitative specialization to the apple-based agro-ecosystem.

For tests on *M. × domestica* accessions, the best model included the factor 'Malus* species* of origin' (BIC = 3709), significantly improving the null model (BIC = 3715; *P* < 0.001). There was no significant effect of the cultivar, cv. Gala and cv. Top Red having similar responses. Thus, adding this trait to the model did not significantly improve the BIC score (BIC = 3721). The most aggressive isolates were those from *M. × domestica* regardless of their geographic origin (Asia or Europe) (Fig. 2). However, isolates from *M. × domestica* were not significantly more aggressive than isolates from AgroAsia-Siev (P = 0.81). WildEuSylv was the least aggressive population, suggesting that the emergence of the disease on *M. sylvestris* was followed by the evolution of quantitative specialization in pathogen populations.

The number of isolates virulent on *M. sylvestris* accessions was low (Table 2). Furthermore, the severity of the disease caused by virulent isolates was weak (Fig. 2). As a consequence, the statistical analysis of aggressiveness had too low a power to infer any reliable conclusion and is therefore not presented.

Discussion

Previous studies exploiting population genetic inference revealed a marked impact of domestication on fungal pathogen population structure, leading in some cases to the emergence of novel pathogen species (Couch et al. 2005; Munkacsi et al. 2007; Stukenbrock et al. 2007).

However, the impact of domestication on pathogenicity traits has rarely been investigated, despite being of major importance to understand the consequences of modern human activities on disease emergence. We used cross-pathogenicity tests in controlled quarantine conditions to investigate the changes in pathogenicity traits of the apple scab fungus, *V. inaequalis*, associated with the domestication and spread of its host. Our main findings were that host-tracking was associated with a change in virulence and an increase in aggressiveness of pathogen populations from the agro-ecosystem. Our results suggested that the transition from wild to apple-based agro-ecosystem did not promote the evolution of specialized populations of *V. inaequalis*, as they were still able to infect the ancestral host plant. In contrast, host-range expansion from the domesticated apple to the European wild apple was associated with a certain degree of host specialization, as populations of *V. inaequalis* from *M. sylvestris* caused a less severe disease on *M. × domestica*.

Changes in pathogenicity traits associated with
host-tracking

The pathogenicity of isolates collected in natural ecosystems on the wild apple *M. sieversii* (WildAsiaSiev) can be compared to more recently founded pathogen populations from the agro-ecosystem to draw inferences on the evolutionary changes associated with the emergence and spread of *V. inaequalis* on the domesticated apple. The lack of pathogenicity of isolates from the WildAsiaSiev population when inoculated onto the domesticated apple suggests a strong ecological differentiation between the WildAsiaSiev population and the populations from the agro-ecosystem. A similar pattern of pathogenicity was

<figure><img src="image_3.png" /><figcaption>**Figure 2** Mean area under the disease progress curve (AUDPC) (+SE) of five *Venturia inaequalis* populations inoculated onto three *Malus* species. AUDPC was measured on one accession for *Malus sieversii*, two cultivars for *M. × domestica* and two accessions for *M. sylvestris*. Different letters indicate significant differences between populations (*P* < 0.05), parameters being estimated by the maximum-likelihood algorithm in the linear mixed-effect model.</figcaption></figure>
observed in a study on the rice blast fungus where isolates of *M. oryzae* from ancestral undomesticated hosts (Setaria millet) were not able to infect or were less aggressive on domesticated rice (Couch et al. 2005). Unlike *M. oryzae*, however, *V. inaequalis* did not emerge on the domesticated crop following a host-shift but through a more continuous process of host-tracking. It could be hypothesized that the disruptive change during domestication corresponded to input of new resistance genes in the domesticated apple species. Indeed, along the process of domestication, *M. × domestica* might have hybridized with several wild species of *Malus* such as *M. sieversii*, *M. baccata*, *M. kirghisorumi*, *M. orientalis* (Coart et al. 2006; Forsline et al. 2010; A Cornille, P. Gladieux, I. Roldán-Ruiz, F. Laurens, B. Le Cam, M. J. M. Smulders, A. Nersesyan, J. Clavel, M. Olonova, L. Feugey, I. Gabrielyan, X. G. Zhang, M. I. Tenaillon, T. Giraud, unpublished manuscript) from which new resistant genes could have been introgressed. Resistance genes from *M. sieversii* might also be unconsciously selected during the course of domestication. A recent study also suggests that novel resistant alleles may also have been created in crops during domestication (Zhai et al. 2011).

Additional insights into the changes in pathogenicity associated with the apple domestication can be gained by comparing the frequency of virulence and aggressiveness of isolates collected in populations from natural and agro-ecosystems. The different populations could only be compared on *M. sieversii*, as isolates from the WildAsia-Siev population were avirulent on *M. × domestica*. Isolates from the WildAsiaSiev population were less aggressive and less frequently virulent on *M. sieversii* than isolates from the population representing the Central Asian agro-ecosystem (AgroAsiaSiev and AgroAsiaDom populations), suggesting that spread of the disease on the domesticated apple may have been associated with a quantitative increase in pathogenicity. These differences in overall pathogenicity (both virulence and aggressiveness) may be related to the contrasted ecological properties of the two environments. Higher density and homogeneity of the agro-ecosystem would have promoted higher pathogenicity of populations infecting domesticated hosts, while the patchy and geographically structured populations of *M. sieversii* (Richards et al. 2009) would have impeded the evolution of high pathogenicity in populations from natural ecosystems. The lower overall pathogenicity of WildAsiaSiev could also be explained by differences in resistance to *V. inaequalis* between *M. sieversii* populations from Central Asian mountains and plains. A study of local adaptation using *M. sieversii* genotypes from which WildAsiaSiev and AgroAsiaSiev populations were sampled would be interesting for assessing to what extent WildAsiaSiev and AgroAsiaSiev populations are adapted

to their host, taking into account the potential diversity in resistance to *V. inaequalis* existing within *M. sieversii*.

## Apple-based agro-ecosystem did not promote the evolution of specialized populations of *V. inaequalis*

The higher level of environmental homogeneity of the agro-ecosystem is thought to promote the ecological specialization of pathogens associated with cultivated species (Stukenbrock and McDonald 2008). Following host-tracking, *V. inaequalis* populations on *M. × domestica* did not loose their capacity to infect their wild native host *M. sieversii*. In Central Asia, specialization of the *V. inaequalis* population from *M. × domestica* agro-ecosystem might have been impeded by recurrent gene flow between populations infecting domesticated apples and neighbouring populations from *M. sieversii* in human-managed habitats. In Europe, pathogenicity experiments supported quantitative specialization of the *V. inaequalis* population from the agro-ecosystem. This population was less frequently virulent and less aggressive than Asian populations from the agro-ecosystem on *M. sieversii*. Allelic combinations providing higher pathogenic fitness on *M. sieversii* would have been unnecessary in European populations and would thus have been progressively lost through genetic drift. The observed quantitative specialization in Europe could therefore be due to geographic distance rather than to environmental differences between wild and agricultural ecosystems.

## The emergence of apple scab on the European wild apple resulted from a host-range expansion associated with quantitative host specialization

The pathogenicity of isolates from European crabapple on the domesticated trees indicated that the emergence of apple scab on *M. sylvestris* resulted from a host-range expansion and not a host-shift. Analyses further revealed that the host-range expansion from *M. × domestica* to *M. sylvestris*, nevertheless, led to a quantitative specialization with a lower frequency of virulence and lower aggressiveness of populations from *M. sylvestris* on the native host *M. × domestica*, suggesting a trade-off between adaptations onto these two hosts. The lower phylogenetic distance between *M. sieversii* and *M. × domestica* (Velasco et al. 2010) compared with that between *M. × domestica* and *M. sylvestris* could explain the less pronounced trade-off between adaptations to these two former hosts. Stronger trade-offs are indeed expected for host-range expansion involving phylogenetically distant hosts than for host-tracking. Isolates from *M. sylvestris* were not more aggressive on *M. sylvestris* than isolates sampled on other hosts. The highly scattered distribution of *M. sylvestris*
across Europe and within forests may not promote evolution towards higher aggressiveness (Gilbert 2002). Environmental context could thus be of primary importance for the evolution of pathogenicity and aggressiveness.

## Concluding remarks

We have investigated the changes in pathogenicity in populations of the pathogen *V. inaequalis*, associated with the domestication of their host. The emergence of *V. inaequalis* on domesticated hosts was associated with a gain in virulence and a subsequent increase in aggressiveness on *M. sieversii* trees in contact with the agro-ecosystem. These results are expected characteristics of the domestication syndrome in pathogens. We hypothesize that pathogen populations did not specialize on the domesticated host because of the close relatedness between their original wild host and *M. × domestica* and the persistence of gene flow between pathogen populations from *M. sieversii* and *M. × domestica* in Central Asia. A decrease in the levels of gene flow in Europe would have led to a decrease in aggressiveness of the European population from *M. × domestica* but without a loss of its capacity to infect its original wild host. The introduction of *V. inaequalis* in Europe was followed by a host-range expansion from *M. × domestica* to the more phylogenetically distant *M. sylvestris*. The existence of efficient resistance traits in this wild species, in association with its very low density in forests, may have limited the increase in aggressiveness of the corresponding pathogen population.

Our findings have important implications regarding the assessment of risk for the emergence of highly aggressive pathogens in wild and agricultural ecosystems. We show here that regulation agencies, policy makers, as well as plant breeders, should consider very carefully the risk of host-tracking by pathogens onto domesticated species. The favourable environment provided by the agro-ecosystem can foster the emergence of new pathogens with increased virulence and aggressiveness. While many plant and animal species are still under domestication (Diamond 2002), our results point to the considerable risk that potentially unnoticed pathogens can adapt via host-tracking and subsequently spread across continents. Moreover, the finding that a pathogen having emerged in the agro-ecosystem has increased its aggressiveness without loosening its ability to infect its original host also suggests that such pathogens can subsequently pose serious threats to wild crop relatives. The policy regarding cultivated areas should therefore take into account the surrounding wild ecosystem to prevent a 'boomerang' effect, that is, the return of more aggressive pathogens back on wild original hosts.

## Acknowledgements

We are greatly indebted to all the people that helped with sample collection: Catherine Peix, Xiu Guo Zhang, François Laurens, Laurent Brun, Marta Pujol, Brigitte Musch, Laurent Levèque, M. Rigoleur, M. Arigoni, M. Ihl and M. Le Valégan. We are also grateful to Pascal Heitzler for providing *M. sieversii* budwoods and Yves Le Vallegan and M. Arigoni for providing *M. sylvestris* budwoods. We thank Pauline Lasserre-Zuber and Caroline Denancé for complementary *Malus* genotyping, Pascale Expert for her help in pathological tests and Frédérique Didelot and Frédéric Fabre for advices on statistical analyses. Amandine Lê Van was supported by a fellowship from INRA, Department SPE and GAP and the Region Pays de La Loire. This work was funded by an Agence Nationale de la Recherche grant ANR 07-BDIV-003 and by the CO-SAVE program (Région Pays de La Loire).

## Data archiving statement

Data deposited in the Dryad repository: doi:10.5061/dryad.6bp470fn

## Literature cited

- Anderson, R. M., and R. M. May 1982. Coevolution of hosts and parasites. *Parasitology* **85**:411–426.

- Anderson, P. K., A. A. Cunningham, N. G. Patel, F. J. Morales, P. R. Epstein, and P. Daszak 2004. Emerging infectious diseases of plants: pathogen pollution, climate change and agrotechnology drivers. *Trends in Ecology & Evolution* **19**:535–544.

- Bus, V. G. M., F. N. D. Laurens, W. E. van de Weg, R. L. Rusholme, E. H. A. Rikkerink, S. E. Gardiner, H. C. M. Bassett *et al*. 2005. The *Vh8* locus of a new gene-for-gene interaction between *Venturia inaequalis* and the wild apple *Malus sieversii* is closely linked to the *Vh2* locus in *Malus pumila* R12740-7A. *New Phytologist* **166**:1035–1049.

- Coart, E., S. Van Glabeke, M. De Loose, A. S. Larsen, and I. Roldan-Ruiz 2006. Chloroplast diversity in the genus *Malus*: new insights into the relationship between the European wild apple (*Malus sylvestris* (L.) Mill.) and the domesticated apple (*Malus domestica* Borkh.). *Molecular Ecology* **15**:2171–2182.

- Couch, B. C., I. Fudal, M. H. Lebrun, D. Tharreau, B. Valent, P. van Kim, J. L. Notteghem *et al*. 2005. 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* **170**:613–630.

- Diamond, J. 2002. Evolution, consequences and future of plant and animal domestication. *Nature* **418**:700–707.

- Doebley, J. F., B. S. Gaut, and B. D. Smith 2006. The molecular genetics of crop domestication. *Cell* **127**:1309–1321.

- Forsline, P. L., H. S. Aldwinckle, E. E. Dickson, J. J. Luby, and S. C. Hokanson 2010. Collection, maintenance, characterization, and utilization of wild apples of Central Asia. In: J. Janick, ed. *Horticultural Reviews: Wild Apple and Fruit Trees of Central Asia*, Vol. 29, pp. 1–61. John Wiley & Sons, Inc, Oxford, UK.
Frenkel, O., T. L. Peever, M. I. Chilvers, H. Ozkilinc, C. Can, S. Abbo, D. Shtienberg et al. 2010. Ecological genetic divergence of the fungal pathogen *Didymella rabiei* on sympatric wild and domesticated *Cicer* spp (Chickpea). Applied and Environmental Microbiology **76**:30–39.

Fry, J. D. 2003. Detecting ecological trade-offs using selection experiments. Ecology **84**:1672–1678.

Gilbert, G. S. 2002. Evolutionary ecology of plant diseases in natural ecosystems. Annual Review of Phytopathology **40**:13–43.

Giraud, T., P. Gladieux, and S. Gavrilets 2010. Linking the emergence of fungal plant diseases with ecological speciation. *Trends in Ecology & Evolution* **25**:387–395.

Gladieux, P., X. G. Zhang, D. Afoufa-Bastien, R. M. V. Sanhueza, M. Sbaghi, and B. Le Cam 2008. On the origin and spread of the scab disease of apple: out of Central Asia. PLoS One **3**:e1455.

Gladieux, P., X. G. Zhang, I. Roldan-Ruiz, V. Caffier, T. Leroy, M. Devaux, S. Van Glabeke et al. 2010. Evolution of the population structure of *Venturia inaequalis*, the apple scab fungus, associated with the domestication of its host. Molecular Ecology **19**:658–674.

Gladieux, P., F. Guérin, T. Giraud, V. Caffier, L. Parisi, F. Didelot, C. Lemaire et al. 2011. Emergence of novel fungal pathogens by ecological speciation: importance of the reduced viability of immigrants. Molecular Ecology **20**:4521–4532.

Gomez-Alpizar, L., I. Carbone, and J. B. Ristaino 2007. An Andean origin of *Phytophthora infestans* inferred from mitochondrial and nuclear gene genealogies. Proceedings of the National Academy of Sciences of the United States of America **104**:3306–3311.

Hansen, E. M. 1987. Speciation in plant pathogenic fungi: the influence of agricultural practice. Canadian Journal of Plant Pathology **9**:403–410.

Harris, S. A., J. P. Robinson, and B. E. Juniper 2002. Genetic clues to the origin of the apple. Trends in Genetics **18**:426–430.

Harrison, N., and R. J. Harrison 2011. On the evolutionary history of the domesticated apple. Nature Genetics **43**:1043–1044.

Hochberg, M. E. 2000. Evidence that specialists are special. Trends in Ecology & Evolution **15**:490.

Hope, A. C. A. 1968. A simplified Monte Carlo significance test procedure. Journal of the Royal Statistical Society **30**:582–598.

Juniper, B. E., and D. J. Mabberley 2006. The Story of the Apple. Timber Pressm, Portland.

Karasov, T., P. W. Messer, and D. A. Petrov 2010. Evidence that adaptation in Drosophila is not limited by mutation at single sites. Plos Genetics **6**:e1000924.

Lê Van, A., C. E. Durel, B. Le Cam, and V. Caffier 2011. The threat of wild habitat to scab resistant apple cultivars. Plant Pathology **60**:621–630.

McDonald, B. A., and C. Linde 2002. Pathogen population genetics, evolutionary potential, and durable resistance. Annual Review of Phytopathology **40**:349–379.

Micheletti, D., M. Troggio, F. Salamini, R. Viola, R. Velasco, and S. Salvi 2011. On the evolutionary history of the domesticated apple. Nature Genetics **43**:1044–1045.

Munkacsi, A. B., S. Stoxen, and G. May 2007. Domestication of maize, sorghum, and sugarcane did not drive the divergence of their smut pathogens. Evolution **61**:388–403.

Munkacsi, A. B., S. Stoxen, and G. May 2008. *Ustilago maydis* populations tracked maize through domestication and cultivation in the Americas. Proceedings of the Royal Society B-Biological Sciences **275**:1037–1046.

Pinheiro, J. C., and D. M. Bates 2000. Mixed-Effects Models in S and S-PLUS, Statistics and Computing. Springer-Verlag, New York, USA.

Pinheiro, J. C., D. M. Bates, S. DebRoy, and D. Sarkar; R-Core-team 2008. Nlme: Linear and Nonlinear Mixed Effects Models. R Package (version 3). http://www.R-project.org (accessed on 1 December 2008).

Pysek, P., V. Jarosik, P. E. Hulme, I. Kuhn, J. Wild, M. Arianoutsou, S. Bacher et al. 2010. Disentangling the role of environmental and human pressures on biological invasions across Europe. Proceedings of the National Academy of Sciences of the United States of America **107**:12157–12162.

R-Development-Core-team 2008. R: A Language and Environment for Statistical Computing. Vol. ISBN 3-900051-07-0. R foundation for Statistical Computing, Vienna, Austria [http://www.R-project.org] (accessed on 14 December 2009).

Richards, C. M., G. M. Volk, A. A. Reilley, A. D. Henk, D. R. Lockwood, P. A. Reeves, and P. L. Forsline 2009. Genetic diversity and population structure in *Malus sieversii*, a wild progenitor species of domesticated apple. Tree Genetics & Genomes **5**:339–347.

Sicard, D., P. S. Pennings, C. Grandclement, J. Acosta, O. Kaltz, and J. A. Shykoff 2007. Specialization and local adaptation of a fungal parasite on two host plant species as revealed by two fitness traits. Evolution **61**:27–41.

Stephan, B. R., I. Wagner, and J. Kleinschmit 2003. EUFORGEN Technical Guidelines for Genetic Conservation and Use for Wild Apple and Pear (*Malus sylvestris* and *Pyrus pyraster*). International Plant Genetic Resources Institute, Rome, Italy, 6.

Stukenbrock, E. H., and B. A. McDonald 2008. The origins of plant pathogens in agro-ecosystems. Annual Review of Phytopathology **46**:75–100.

Stukenbrock, E. H., S. Banke, M. Javan-Nikkhah, and B. A. McDonald 2007. Origin and domestication of the fungal wheat pathogen *Mycosphaerella graminicola* via sympatric speciation. Molecular Biology and Evolution **24**:398–411.

Stukenbrock, E. H., T. Bataillon, J. Y. Dutheil, T. T. Hansen, R. Li, M. Zala, B. A. McDonald et al. 2011. The making of a new pathogen: insights from comparative population genomics of the domesticated wheat pathogen *Mycosphaerella graminicola* and its wild sister species. Genome Research **21**:2157–2166.

Thrall, P. H., M. E. Hochberg, J. J. Burdon, and J. D. Bever 2007. Coevolution of symbiotic mutualists and parasites in a community context. Trends in Ecology & Evolution **22**:120–126.

Torriani, S. F. F., P. C. Brunner, and B. A. McDonald 2011. Evolutionary history of the mitochondrial genome in *Mycosphaerella* populations infecting bread wheat, durum wheat and wild grasses. Molecular Phylogenetics and Evolution **58**:192–197.

Velasco, R., A. Zharkikh, J. Affourtit, A. Dhingra, A. Cestaro, A. Kalyanaraman, P. Fontana et al. 2010. The genome of the domesticated apple (*Malus × domestica* Borkh.). Nature Genetics **42**:833–839.

Zaffarano, P. L., B. A. McDonald, and C. C. Linde 2008. Rapid speciation following recent host shifts in the plant pathogenic fungus *Rhynchosporium*. Evolution **62**:1418–1436.

Zeder, M. A., E. Emshwiller, B. D. Smith, and D. G. Bradley 2006. Documenting domestication: the intersection of genetics and archaeology. Trends in Genetics **22**:139–155.

Zhai, C., F. Lin, Z. Dong, X. He, B. Yuan, X. Zeng, L. Wang et al. 2011. The isolation and characterization of *Pik*, a rice blast resistance gene which emerged after rice domestication. New Phytologist **189**:321–334.
**Supporting Information**

Additional Supporting Information may be found in the online version of this article:

**Figure S1.** Geographic location of strains (circles) and hosts (squares) sampling sites. Populations of strains are identified by different colours: WildAsiaSiev (red), AgroAsiaSiev (orange), AgroAsiaDom (purple), AgroEuDom (yellow) and WildEuSylv (green). Red squares represent the three accessions of *M. sieversii* from Kazakhstan. Accessions of *M. sylvestris* are located in France in the Rambouillet forest (next to Paris). Maps are provided by Google Earth®.

**Data S1.** *M. sylvestris* accessions were genotyped using microsatellite markers, and their assignment to the gene pool of their putative species of origin was checked using a reference data set.

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