Sylvie Oddou-Muratorio

Etienne K Klein

Giovanni G Vendramin

Bruno Fady

Etienne K Klein §

Giovanni G Vendramin #

Spatial vs. temporal effects on demographic and genetic structures: the roles of dispersal, masting and differential mortality on patterns of recruitment in Fagus sylvatica

Keywords: contemporary gene flow, spatial genetic structure, microsatellite, tree, spatially explicit mating model

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Understanding the effects of demographic and genetic processes on the amount and spatial distribution of genetic variation in natural populations is one of the main objectives of modern evolutionary ecology. These issues have gained a renewed interest in the context of increased habitat fragmentation and rapid environmental change, which stress the need to understand and predict evolutionary trajectories of populations at ecological time scale. At short time and spatial scales, demographic (survival, growth, competition) and evolutionary forces (selection, gene flow and genetic drift) tightly interact to shape spatial patterns of allelic frequencies across life stages or generations. In return, genetic variation affects population dynamics by determining individual capacities of survival, growth, and reproduction [START_REF] Lande | A quantitative genetic theory of life history evolution[END_REF]. The study of this interplay between demographic and short-term evolutionary processes is sometimes referred to as demo-genetics and builds on the theoretical framework mainly developed by [START_REF] Lande | A quantitative genetic theory of life history evolution[END_REF] and recently adapted by [START_REF] Coulson | Putting evolutionary biology back in the ecological theatre: a demographic framework mapping genes to communities[END_REF].

In trees, the period spanning from seed dispersal to early seedling recruitment is thought to be a major transition step where important demo-genetic interactions take place and have pervasive consequences on the structure and dynamics of tree populations [START_REF] Petit | Some Evolutionary Consequences of Being a Tree[END_REF]. This step is characterised in particular by a massive mortality of seeds/seedlings produced during an individual life time, with typically only one seed in a million surviving as a reproductive adult [START_REF] Petit | Some Evolutionary Consequences of Being a Tree[END_REF]. Moreover, recruitment studies highlight the role of various demographic and genetic processes and of their variation on patterns of regeneration; major processes are the spatial distribution and density of reproductive plants, their seed outputs, the shape and form of their dispersal kernel and the spatial patterns of microsites favorable for seedling establishment [START_REF] Clark | Are plant populations seed limited? A critique and meta-analysis of seed addition experiments[END_REF][START_REF] Clark | Interpreting recruitment limitation in forests[END_REF][START_REF] Nathan | Spatial patterns of seed dispersal, their determinants and consequences for recruitment[END_REF]. In the following section, we will focus in particular on: (1) dispersal limitation, which is a major factor shaping variation of recruitment through space; (2) mast seeding (i.e. synchronous intermittent production of large seed crops) which is an important factor shaping the variation of recruitment through time particularly in temperate forests [START_REF] Kelly | Mast seeding in perennial plants: Why, How, Where?[END_REF][START_REF] Piovesan | Masting behaviour in beech: linking reproduction and climatic variation[END_REF]; (3) mortality at early stages of the tree life cycle that causes major variation in the demographic and genetic structure across life stages.

The combined role of propagule (seed and pollen) production and dispersal on patterns of recruitment is widely acknowledged and studied in population dynamics and genetics. Seed dispersal and individual seed production shape both the initial spatial pattern of seedling abundance [START_REF] Clark | Are plant populations seed limited? A critique and meta-analysis of seed addition experiments[END_REF][START_REF] Clark | Interpreting recruitment limitation in forests[END_REF][START_REF] Nathan | Spatial patterns of seed dispersal, their determinants and consequences for recruitment[END_REF] and genetic relatedness among established individuals (Wright, 1943). Unless pollen production is strongly limited or population density is very low (Sagnard et al., 2011), pollen dispersal is usually considered as driving mainly patterns of genetic relatedness. By contrast, the temporal component of the regeneration pattern due to inter-annual variation in seed production has received less attention, in particular from a population genetics perspective. It has been suggested that the genetic consequences of high variation in seed production among individuals are reduced over time due to the fact that seed production ranking among trees varies strongly between years [START_REF] Krouchi | Year and tree effect on reproductive organisation of Cedrus atlantica in a natural forest[END_REF]. This phenomenon tends to increase the effective population size and therefore decrease the spatial variation in genetic relatedness over the whole regeneration phase. From a population dynamics perspective, the few studies focusing on recruitment patterns across space and time show contrasting results. Some studies have found across-year consistency in seed-fall and seedling distribution with a strong site effect (Wright et al., 2005), while in others, across-year variation was higher than variation across sampling sites [START_REF] Beckage | Survival of tree seedlings across space and time: estimates from long-term count data[END_REF]. It thus remains largely unknown how spatial and temporal recruitment dynamics interact across heterogeneous landscapes, which of these components has a greater effect on patterns of regeneration in long-lived plants, and under 1982;Janzen, 1970;[START_REF] Nathan | A simple mechanistic model of seed dispersal, predation and plant establishment: Janzen-Connell and beyond[END_REF] can result in higher average distances between mothers and successfully established offspring than those expected from seed dispersal alone. The existence of Janzen-Connell effects in forest trees is supported by different studies across different forest systems including temperate deciduous forest [START_REF] Ris Lambers | Effects of dispersal, shrubs, and density-dependent mortality on seed and seedling distributions in temperate forests[END_REF]. Janzen-Connell effects are expected to result in a decrease in structure and relatedness from the initial seed rain to recruited seedlings (Trapnell et al., 2008). Alternatively, an increase in genetic structure and relatedness from the initial seed rain to recruited seedlings can be expected when mortality is driven by microsite heterogeneity and genotype-microsite interactions, especially in spatially variable environments (Sagnard et al. 2010). Moreover, because the genetic load of trees is high [START_REF] Petit | Some Evolutionary Consequences of Being a Tree[END_REF], the purging of inbred individuals may contribute to seedling mortality [START_REF] Ferriol | Variation of early stage inbreeding depression and outbreeding depression within an admixed Cedrus population[END_REF].

However, such processes are notoriously difficult to demonstrate in natural populations, and require monitoring the demo-genetics of naturally established seedlings through time [START_REF] Kalisz | Spatial population genetic structure in Trilium grandiflorum: the roles of dispersal, mating, history and selection[END_REF]. Most frequently, genetic structure studies taking a life-stage approach compare very distant cohorts, typically seeds, seedlings and adults [START_REF] Alvarez-Buylla | Demographic genetics of a pioneer tropical tree species: patch dynamics, seed dispersal and seed banks[END_REF][START_REF] Jones | Demographic spatial genetic structure of the Neotropical tree, Jacaranda copaia[END_REF]) .

Here we used a demo-genetic approach to investigate the consequences of the spatiotemporal patterns of regeneration on the demo-genetic structure of European beech (Fagus sylvatica L.). This wind-pollinated species is both gravity-and animal-dispersed [START_REF] Jensen | Seed-seed predator interactions of European beech, Fagus silvatica and forest rodents, Clethrionomys glareolus and Apodemus flavicollis[END_REF] and produces beech nuts in irregular mast years. Previous demographic estimates of et al., 2007). The European beech is described as a shade tolerant species able to survive under 1-2% of full above-canopy light, but showing optimal growth potential at 30-40% of abovecanopy light [START_REF] Kunstler | Fagus sylvatica L. recruitment across a fragmented Mediterranean Landscape, importance of long distance effective dispersal, abiotic conditions and biotic interactions[END_REF].

The originality of this study lies in the fact that we investigated the role of various genetic and demographic processes (inter-annual variation in seed production, dispersal, and mortality) on the distribution pattern of genetic diversity in three regeneration plots with different canopy closure in a beech stand where seedling recruitment was monitored from 2004 to 2006, and extrapolated back to 1993 by estimating seedling age. Because it is difficult to disentangle the different ecological factors shaping genetic structure, we also directly investigated the seed and pollen dispersal processes using parentage/paternity analyses. This approach allowed us to better test the consequences of a process (dispersal) and its variation in space (with stand density) or time (among year).

Based on this data set, the following specific questions were addressed: (1) how do patterns of seedling density and survival correlate in space and time? (2) How spatially restricted are contemporary pollen and seed dispersal? (3) Which spatial or temporal processes are the major factors shaping seedling genetic structure? (4) Does mortality affect the genetic structure of the seedlings?

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Version définitive du manuscrit publié dans / Final version of the manuscript published in : Molecular Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011

Material and Methods

Study species and sampling design:

The European beech, Fagus sylvatica L. (Fagaceae), is a monoecious diploid (2n = 24) late-successional forest tree. It is a highly outcrossing windpollinated species (selfing <10% ; [START_REF] Merzeau | Estimation of Fagus sylvatica L mating system parameters in natural populations[END_REF]. Reproductive trees (typically older than 60-80 years in dense stands) produce beech nuts in irregular mast years, with an intermast interval of at least 2 years [START_REF] Nilsson | Seed predation and cross-pollination in mast-seeding beech (Fagus sylvatica) patches[END_REF]Teissier du Cros, 1981). Seeds are primarily dispersed by gravity, and secondarily dispersed by rodents (Apodemus flavicollis, Clethrionomys glareolus) and birds (Glandus glandularius) that scatter hoard them [START_REF] Jensen | Seed-seed predator interactions of European beech, Fagus silvatica and forest rodents, Clethrionomys glareolus and Apodemus flavicollis[END_REF].

The study site is a mixed beech-oak stand located within a large forest dominated by beech (Haye forest, North-Eastern France, Longitude: 06° 06' 36' ' E;Latitude: 48° 38' 23''N) .

Within the site (~7.8 ha), all 342 adult beech trees were mapped using a Rangemaster 900 Scan telemeter and a compass, with a precision of more than 5 meters (Figure 1). All were sampled for genetic analyses. Adult density varied across the site from <30 stem/ha to >90 stem/ha as a result of the 1999 Lothar storm.

[Figure 1 around here] Young seedlings were sub-sampled in 3 plots (A: 9 m², B: 5 m² and C: 8 m² in area) corresponding to a gradient in the levels of photosynthetically active radiation (PAR) measured in micromol/m²/s with a SunScan ® system. Within each plot 5-6 measurements were averaged and converted into percentage of transmitted PAR ( PAR Incident

PAR

Transmited

) by reference to the average measure of 157.9 micromol/m²/s obtained in the open (=incident PAR). Light availability under the canopy strongly decreased from 55% of transmitted light (plot A) to 31% of incident light (plot B) and finally to 6% of incident light (plot C), in relation to the variation in adult tree density in a 50 meter radius around the plots (A: 56 trees ha -1 ; B: 72 trees ha -1 ; C: 92 trees ha -1 ).

In total, 462 young seedlings were exhaustively mapped, among which 254 where sampled for genetic analyses (Table 1). Among the 462 seedlings, 371 were found in the first year of Genotyping: DNA was isolated from buds (adult trees and seedlings) and embryos (seeds) using the Qiagen DNeasy Plant kit. Individuals were genotyped using 4 nuclear microsatellite markers (FS1-03, FS1-25, FS3-04, FS4-46) developed for Fagus sylvatica [START_REF] Pastorelli | Characterization of microsatellite markers in Fagus sylvatica L. and Fagus orientalis Lipsky[END_REF] and 2 nuclear microsatellite markers (FCM5 and SFC-0161) developed for Fagus crenata [START_REF] Asuka | Development and characterization of microsatellite markers for Fagus crenata Blume[END_REF]Tanaka et al., 1999) (Table 1), following PCR conditions given by the authors. Adult and seed PCR products were separated using an automated 96-capillary MegaBACE™ 1000 sequencer (GE Healthcare). Genotypes were sized using the internal size standards ET400 and the MegaBACE™ Fragment Profiler ver. 1.2 software (GE Healthcare).

Seedling PCR products were separated using a LICOR automated gel-sequencer (some adults were also genotyped on LICOR sequencer for homogenous sizing).

Null alleles and quality of the marker set: Null allele frequencies (NAF) were first estimated by direct counting in maternal progeny arrays (for details see Oddou-Muratorio et al., 2009). Additionally, we estimated NAF in adult and seedling cohorts using the maximumlikelihood method implemented in ML-NullFreq software and accounting for genotyping error [START_REF] Kalinowski | Maximum likelihood estimation of the frequency of null alleles at microsatellite loci[END_REF].

The non-negligible prevalence of null alleles in European beech was confirmed, with four loci out of six affected (Table A1). NAF were > 10% in 2 locus/cohort combinations (adults at locus FS1-25 and FS4-46) and >5% in 9 locus/cohort combinations out of 18. Adults tended to have higher NAF than seedlings at loci FS1-25 and FS4-46, but the opposite trend was observed at locus FS1-03. The difference in NAF between adult and seedling cohorts was highest (~0.13) at locus FS 4-46, despite low genetic differentiation among adult and seedling cohorts (F ST averaged across loci = 0.54%).

When high frequencies of null alleles were found (p>0.05), we evaluated their impact on consanguinity and differentiation estimates using the ENA method proposed by [START_REF] Chapuis | Microsatellite Null Alleles and Estimation of Population Differentiation[END_REF]. This method consists (1) in correcting the original data set by statistical adjustment of genotype frequencies based on estimated null allele frequencies and then (2) in re-estimating F IS or F ST based on visible alleles only. It is designed to yield unbiased F ST values, but not to yield unbiased F IS -values, which are likely to be underestimated.

Significantly positive F IS values were estimated at the four loci affected by null alleles (Table A1). However after correcting the data set for null alleles using the ENA method proposed by [START_REF] Chapuis | Microsatellite Null Alleles and Estimation of Population Differentiation[END_REF], no significant heterozygote deficiency could be detected in either adults or seedlings (results not shown). By contrast, F ST values were of the same order of magnitude in the raw and corrected datasets, showing that genetic differentiation between groups of seedlings was not affected by null alleles.

According to these results, we later considered in our analyses that: (1) null alleles occurred at non-negligible frequencies in the data set, frequencies that were not estimated with precision as depicted by their variation across cohorts at the same locus;

(2) some of the observed positive F IS -values were probably partly affected by null alleles, but also by the mating system; F IS -values were thus assumed to be relevant in a comparative context; (3) genetic differentiation as estimated by F ST was not affected by null alleles.

Genetic diversity:

Genotyping problems occurred at locus FCM5, and particularly for seedlings (81% missing data). Given that missing data affect the estimation of genetic differentiation, all analyses were run with only 5 loci, excluding FCM5. Expected heterozygosity (He), allelic richness (A) and heterozygote deficiency (as measured by F IS ) were estimated using the Fstat software [START_REF] Goudet | FSTAT, a program to estimate and test gene diversities and fixation indices (version 2.9.1). updated from Goudet[END_REF]. Allelic richness was rarefied to a minimum sample size of 10 individuals. Significance of F IS -values was assessed at the 5% confidence level after Bonferonni correction.

Spatio-temporal genetic structure:

To investigate the spatial and temporal components of genetic structure in the seedlings, we used a hierarchical AMOVA design (Excoffier et al., 1992). We analyzed the respective effects of spatial (plot) versus temporal (year of germination) processes, by testing successively two two-level nested models (year within plot and plot within year) for the genotypic frequency vector G ijk of individual k germinated in year j and located in plot i:

G ijk =µ+f i +t j(i) +w ijk (1)
and G ijk =µ+f' j +s i(j) +w ijk (2)

where f i is the average effect of plot i, f' j the average effect of year j, t j(i) is the average effect of year j nested within plot i, s i(j) is the average effect of plot i nested within year j and w ijk is the replication error associated with the k th individual from the i th plot germinated in the j th year. For microsatellites assumed to follow a stepwise mutation model, Slatkin (1995) recommends measuring variation in allelic frequencies and genetic differentiation with alleles ordered according to their size rather than with unordered alleles (identity in state).

Accordingly, we used both F st and R st as estimators of genetic differentiation. Significance of F-statistics was assessed by means of 5000 permutations. All computations were done using the Arlequin software (Schneider et al., 2000). Detailed AMOVA design is described in online appendix A1.

Fine-scale spatial genetic structure (SGS) within plots / cohorts: The classical analysis of SGS consists in plotting the variation in average genetic relatedness among individuals against distance (or logarithm of distance in a two-dimensional space). Under isolation by and low in the other years).

All kinship analyses were performed using Spagedi 1.2 [START_REF] Hardy | SPAGeDi: a versatile compute program to analyse spatial genetic structure at the individual or population levels[END_REF], which makes it possible to specify adult-seedling pairs to be compared. To measure genetic relatedness, we used the kinship coefficient (F ij ) of Loiselle et al. (1995). Fij-values were estimated using 5 loci (excluding FCM5), and assumed not to be affected by null alleles, similarly to F ST (Rousset 2000). The allele frequencies from the whole population (i.e.

grouping adults and seedlings) were used as a reference sample. 1) and ( 2), replacing the average effect of year by that of status (dead/alive) in f' j , t j(i) and s i(j) and considering that w ijk was the replication error associated with the k th individual from the i th plot in state j.

Then, fine-scale patterns of between-generation SGS were investigated as detailed above to test whether gene dispersal patterns from adults to seedlings differed among dead and alive seedlings. mother tree located within the study site (with probability (1m s )). In the latter case, the model considers that offspring i may be the result either of self-pollination (with probability s), pollen flow from outside the neighbourhood (with probability m p ), or pollen from a sampled male (with probability 1-s-m p ). The genotypes of seedlings and candidate mothers/fathers are used to define the compatible offspring-parent triplet, and to compute transition probabilities in a fractional parentage analyses design. The contribution of a sampled and genetically compatible mother tree j to the seedling rain at the location of seedling i is modelled as the product of the probability of a seed to disperse from j to i (the seed dispersal kernel), and of mother intrinsic fecundity. Similarly, the contribution of each sampled father tree k to the pollen cloud above mother tree j at is modelled as the product of the probability of a pollen grain to disperse from k to j (the seed dispersal kernel), and of the father intrinsic fecundity. Here, we used the exponential power function to model the seed and pollen dispersal kernels:

Estimation

              - b a d b) Γ( a b = d) b; p(a, exp / 2 ² 2π ( 3
)
where site and of the 221 seedlings were used for these analyses (33 seedlings were eliminated because they could not be genotyped at more than 2 loci and/or because of missing spatial position data). Note that locus FCM5 was included in these analyses, as missing data do not bias this estimation procedure. Background allele frequencies were assumed to be similar to that of the local population.

Estimation of the pollen dispersal kernel based on maternal progenies: SEMM was also used to estimate the shape and the range of the pollen dispersal kernel from genotypes and positions of maternal trees, progeny arrays, and potential fathers (online Appendix A3 supplementary material). The model is simpler than the seedling model above (Oddou- et al., 2005) and considers that a given seed i collected on mother-tree j may be the result of self-pollination (with probability s), pollen flow from outside the neighbourhood N (with probability m p ), or pollen from a sampled male (with probability 1-s-m p ).

Muratorio
The genotypes and spatial positions of all 342 adult trees as well as the genotypes of the 372 seeds collected on 29 fruiting trees were used for these analyses.

Results

Demographic dynamics in seedlings plots

Effective recruitment patterns were highly variable among years, with on average 41.3% of seedlings germinated in 2002, and several years without significant recruitment (Table 1). For seedlings germinated before 2004, these variations can result either from low seed production and/or germination, or from high seedling mortality between the germination year and 2004.

These effective recruitment patterns are consistent with the expected effect of the Lothar storm in 1999, as most of the seedlings observed alive between 2004 and 2005 germinated after the stand canopy was significantly opened by the storm. Overall, seedling density observed in 2004 was lower in plot A (12.7 m-²) than in plot B (31 m-²) or plot C (24 m-²)

(Figure 2).

Genetic diversity within seedling and adult cohorts

Levels of diversity did not differ among adult and seedling cohorts, among seedling plots, or among dead and alive seedlings (Table 2). Nei's genetic diversity was high both in seedlings and adults (H e = 0.71 and 0.72, respectively), and allelic richness was also comparable in seedlings and adults (A = 5.71 and 5.94, respectively). By contrast, the within-individual structure of genetic diversity differed between adult and seedling cohorts, with a higher heterozygote deficiency in the adults (F IS = 0.131**) than in the seedlings (F IS = 0.069**).

However, when the three loci affected by null alleles (FS1-25, FS4-46, FS1-03) were removed, F IS -values did not differ significantly from 0.

[Table 2 around here]

Spatio-temporal genetic structure of seedlings

Spatial (among plots) versus temporal (among year classes) components of seedling genetic structure were first investigated using two different two-level AMOVA models: (Model1) years nested within plots (Table 3) and (Model2) plots nested within years. The main effect for years was not significant (Model2 see Table A2), while the main effect for plots was significant (Model1). Detailed analyses of Model1 (Table 3) showed that the "among-plot" component of genetic variation was not negligible (F RT = 2.6% of the total variation) considering the small spatial scale investigated (average distance among plots ~100 m). By contrast, year-to-year variation in a given plot was not significant (F SR = 0.5%) but contributed to overall differentiation (F ST = 3.1%). Differentiation estimates using R-statistics were similar to these values.

[Table 3 around here]

To investigate whether the lack of a significant among year/plot effect could be due to an over-representation of seedlings germinated in year 2002 in the data set (68% of the 234 seedlings), we rarefied the sample to balance sample size within year (so that year 2002 represented 47% of 139 seedlings) and ran the AMOVA analyses again (Table 3). Results

were consistent with those obtained with the complete data set, with a significant main spatial effect (F RT = 3.2%).

Pairwise F ST among seedling plots (Table A3) showed that only plot C was significantly differentiated from plots B and A (F ST = 1.3% in both cases). All seedling plot genetic frequencies significantly differed from that of the adult populations (F ST ranging from 0.9% for B plot to 2.3% for A plot).

Fine-scale spatial genetic structure (SGS) within plots and within cohorts

The [Figure 3 around here] 'Between-generation' SGS varied among plots (Fig 3B). However, trends were not significant due to the high standard errors of F ij and b estimates (Table A4). There was also no clear trend of increasing'between-generation' SGS during years of low recruitment, as could have been expected with a reduced contribution of adults to reproduction in non-masting years. Year 2001 and 2004 (low recruitment) showed higher and lower 'between-generation' SGS, respectively, whereas year 2002 showed intermediate SGS (Fig 3C).

Direct gene flow estimates

Maximum-likelihood estimates of seed and pollen dispersal as well as mating system parameters were obtained using SEMM (Table 4). Both for pollen and seed dispersal, the exponential kernel (i.e. fixing b p = 1 and b s = 1 and estimating solely a p and a s ) provided a better fit than the Gaussian kernel (i.e. fixing b p = 2 and b s = 2). The exponential power kernel with joint estimates of b and a parameters did not improve the fit (results not shown). We estimated a larger mean distance for pollen dispersal ( p δ ˆ≈57 m; CI = 30.6 -123.4 m) than for seed dispersal ( s δ ˆ≈11 m ;CI = 9.4 -12.9 m). Also, the pollen immigration rate ( p m ˆ=71.6%; CI = 60.2 -85%) was significantly higher than the seed immigration rate ( s m ˆ=20.5%; CI = 13.5 -27.1%). The selfing rate was significantly positive (s= 3.5%).

[Table 4 around here] Independent estimates of pollen dispersal and selfing rate based on maternal progenies fell within the same range of that of estimates based on established seedlings. The best fit for

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Version définitive du manuscrit publié dans / Final version of the manuscript published in : Molecular Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011

Impact of mortality on genetic structure

Because there was strong among-plot genetic structure, the relationship between survival and genetic differentiation was tested using a two-level AMOVA design with status (alive/dead) nested within plot. Neither the main effect nor the interaction effect of status was significant (result not shown), showing that dead and alive seedling were not genetically differentiated within plot.

The fine-scale variations of 'between-generation' SGS were analyzed by plotting genetic relatedness among seedling-adult pairs against distance for dead and alive seedlings (Fig 3D).

The 'between-generation' SGS of dead and alive seedlings was very similar, with a marked peak in the first distance interval (>10 m), followed by a notable decrease between 20 and 40 m.

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Version définitive du manuscrit publié dans / Final version of the manuscript published in : Molecular Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011 However, recruitment patterns between years 1999 and 2004 are consistent with a massive seedling germination event in 2002 and with the assumption that seed production is a limiting factor for recruitment in beech [START_REF] Piovesan | Masting behaviour in beech: linking reproduction and climatic variation[END_REF].

Demographic and genetic structure show opposite spatio-temporal effects

By contrast, spatial heterogeneity in seedling density was weak, with a trend of lower initial density in the plot with open canopy (PAR= 55%, 12.7 seedlings.m -² in 2004), compared to

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Version définitive du manuscrit publié dans / Final version of the manuscript published in : Molecular Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111 (PAR=6%, 24 seedlings.m -²). However, our experimental design with only three plots was not conceived to address the impact of canopy closure on initial seedling density or to separate this effect from that of seed-tree density. Still, our results show that despite variable seed-tree density across the plot (from <30 to >90 trees per ha -1 ), there was a high density of seedlings even under unusually high canopy openness (>9,700 ha -1 in 2006, which is several times higher than recommended for afforestation rates). This is consistent with other studies, which have found that seedling germination is almost independent of light availability (Szwagrzyk et al., 2001), contrary to subsequent growth and long-term survival [START_REF] Kunstler | Growth, mortality, and morphological response of European beech and downy oak along a light gradient in sub-Mediterranean forest[END_REF]Szwagrzyk et al., 2001).

In contrast to their density, the genetic structure of recruited seedlings was significantly shaped by spatial processes and poorly affected by temporal heterogeneity of the seed rain.

Here, the stand-level spatio-temporal genetic structure was investigated by testing successively the main and nested effects of spatial location and year of germination on genetic differentiation among seedlings as measured by F ST . The main effect of spatial location was strongly significant, and translated into a significant level of differentiation between plots of F ST = 2.6%. By contrast, genetic differentiation among temporal cohorts within plots was not significant. Moreover, genetic frequencies significantly differed between plot C and the two other plots (F ST C-A = F ST C-B = 1.3%) and between each seedling plot and adults (0.9<F ST Adult- seedling <2.3%; Table A2). These F ST -values may look weak, but by comparison, genetic differentiation at allozyme loci measured over 389 populations across Europe were not larger than 5.9% [START_REF] Comps | Diverging trends between heterozygosity and allelic richness during postglacial colonization in the european Beech[END_REF]. Using SSR markers to measure genetic differentiation among 10 populations across Europe, [START_REF] Buiteveld | Genetic diversity and differentiation in European beech (Fagus sylvatica L.) stands varying in management history[END_REF] reported pairwise F ST -values ranging between 0.8% and 5.3%, with an overall F ST of 5.3%. The major role of spatial versus temporal processes in shaping plant genetic structure has been acknowledged in previous studies, usually by comparing genetic structure across different life-stages [START_REF] Alvarez-Buylla | Demographic genetics of a pioneer tropical tree species: patch dynamics, seed dispersal and seed banks[END_REF][START_REF] Chung | Genetic structure of age class in Camellia japonica (Theaceae)[END_REF][START_REF] Jones | Demographic spatial genetic structure of the Neotropical tree, Jacaranda copaia[END_REF].

Genetic structure revealed low levels of genetic drift despite restricted dispersal

In their pioneer demo-genetic study in the tropical tree Cecropia obtusifolia, [START_REF] Alvarez-Buylla | Demographic genetics of a pioneer tropical tree species: patch dynamics, seed dispersal and seed banks[END_REF] showed that patchy recruitment in gaps markedly affect the genetic composition of the seed rain, with higher differentiation among gaps than among-life-stages. More recently, [START_REF] Jacquemyn | Multigenerational analysis of spatial structure in the terrestrial, food-deceptive orchid Orchis mascula[END_REF] used multi-stage spatial genetic structure analyses combined with parentage analyses in the perennial Orchis mascula to show that patterns of SGS were mostly shaped by pollen and seed-mediated gene dispersal, and were consistent across life-stage.

By contrast however, we focussed here on a more fine-scale temporal structure. We

Seed and pollen dispersal direct estimates

Contemporary estimates of seed and pollen dispersal based on parentage/paternity analyses shed light on dispersal processes and their ecological determinants in European beech.

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Version définitive du manuscrit publié dans / Final version of the manuscript published in : Molecular Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011 They are also consistent with life history traits of the beech dispersers. The rodents involved in secondary dispersal of beech seeds have been shown to remove seeds a few meters away from the source tree (4.1 m on average, [START_REF] Jensen | Seed-seed predator interactions of European beech, Fagus silvatica and forest rodents, Clethrionomys glareolus and Apodemus flavicollis[END_REF], whereas frequent 1 km-dispersal events have been reported for jays [START_REF] Nilsson | Ecological and Evolutionary Interactions between Reproduction of Beech Fagus silvatica and Seed Eating Animals[END_REF].

Our results suggest greater dispersal abilities for pollen than for seeds, with both a higher mean dispersal distance (δ p =43.7 -57.2 m versus δ s =10.9 m), and a higher immigration rate (m p = 71.6% to 77.9% versus m s = 20.5%). The results based on established seedling or openpollinated progeny were highly consistent (Table 4). This supports the accuracy of the SEMM. Moreover, it shows that early selective processes acting between seed release and seedling establishment may not be driven by the genetic origin of the pollen grain (no outbreeding or inbreeding depression).

From direct estimates of seed and pollen dispersal, we can estimate real-time, total gene flow estimates (σ rt ), as detailed in [START_REF] Oddou-Muratorio | Comparing direct vs. indirect estimates of gene flow within a population of a scattered tree species[END_REF]. In a two dimensional space, for hermaphrodite, outcrossing species:σ rt 2 = ½σ p-rt 2 + σ s-rt 2 , where σ² p-rt and σ² s-rt are the respective second moments of the pollen and seed dispersal kernels. In our case, σ rt = 51.35 m (CI = 29-108m); this is consistent with the shape of the between-generation auto-correlograms (Figure 3) which show significant SGS up to ~50m.

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Version définitive du manuscrit publié dans / Final version of the manuscript published in : Molecular Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111 We reasonably assumed that differentiation (F ST ) and SGS estimates were not biased by the frequencies of null alleles estimated in F. sylvatica (between 0 and 14% depending on cohort and method of estimation, with all but two estimates <10%; see Table A1). However, attention should be put in evaluating the potential impact of null alleles on direct estimates of seed and pollen dispersal. Somehow reassuringly, [START_REF] Dakin | Microsatellite null alleles in parentage analysis[END_REF] showed using simulations that the range of null allele frequency observed in this study (NAF<10%) equates to a less than 5% risk of falsely excluding an actual parent of a heterozygous offspring in parentage/paternity analyses. Additionally, we also estimated NAF using our mating model parameters, using a modified version of the SEMM and adult and seedlings genetic and spatial data as input (Chybicki & Burczyk, 2010). Interestingly, "direct" NAF estimates were lower (2% on average, see Table A1) than those obtained by traditional methods (between 4.3 and 6.7%). The reason for this discrepancy may be that "direct" NAF estimates account for the SGS present in the population. By contrast, in the case of significant SGS and preferential local mating, biparental inbreeding can result in a deficit of heterozygotes (similar to a Wahlund effect) that traditional methods could misinterpret as a signature of null alleles

Evolutionary and ecological drivers of mortality

The overall mortality rate over the two years of the survey (from 2004 to 2006) To investigate evolutionary drivers of mortality, we first estimated genetic differentiation between dead and alive seedlings (as measured by hierarchical F-statistics within plots). We did not observe any significant differences between the two groups. Moreover, levels of heterozygote deficiency were consistent among groups (F IS = 0.073* in alive seedlings versus 0.058 NS in dead seedlings, Table 2). Although the level of inbreeding estimated by F IS may be affected by null alleles, F IS -values can still be used to compare dead and alive seedlings because of the absence of genetic differentiation between these groups. Thus, there was no evidence that mortality is driven by the purging of selfed individuals in the studied beech stand. Finally, between-generation patterns of spatial genetic structure (SGS) were also consistent for dead and alive seedlings (Fig. 3), indicating that levels of genetic relatedness within the stand did not significantly contribute to mortality. Overall, we did not find any evidence that mortality is driven by inbreeding or lack of local adaptation.

Interestingly, patterns of inbreeding and relatedness coefficients were actually stronger for adults compared to seedlings (F IS = 0.131 in adults versus 0.069 in seedlings). This indicates that massive recruitment during a single mast year does not reduce effective population size as could have been expected.

Perspectives

This study highlights different magnitudes of temporal versus spatial effects on demographic and genetic patterns of early recruitment. The high heterogeneity among year classes in recruited seedling density revealed a major effect of mast seeding on demographic patterns of recruitment. By contrast, the low genetic differentiation among seedlings recruited in different years indicates balanced contribution of adult trees to reproduction within year. The significant spatial genetic structure was consistent with the strong spatial limitation of both seed and pollen dispersal detected using parentage analyses and neighborhood mating models.

As a direct consequence for forest managers, our results highlight that genetic diversity within beech stands is mostly shaped by gene dispersal and adult tree density. Consequently, high levels of genetic diversity can be maintained within stand even if young seedlings are recruited in a reduced number of mast years. , 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011 1993 1995 1997 1999 2000 2001 2002 2003 2004 2005

Figure legend

  the survey (2004) while 91 new seedlings germinated in spring 2005 and were mapped in summer 2005. Survival of all seedlings was recorded from 2004 to 2006. Germination year was estimated retrospectively by counting node scars and ranged from 1993 to 2005. Finally, 372 seeds were collected in the autumn of 2004 in the crown of 29 fruiting trees (5-16 seeds/tree, mean = 12.8, S.D. = 2.98).

  of the seed and pollen dispersal kernel based on established seedlings: the spatially explicit mating model (SEMM) developed by Burczyk et al. (2006) and Oddou-Muratorio & Klein (2008) was used to estimate the shape and range of seed and pollen dispersal kernels from genotypes and positions of established seedlings and their potential parents. The model considers that each seedling i can be mothered either (1) by a mother tree located outside the study site due to seed immigration (with probability m s ) or (2) by a local

  d is the distance of interest (mother-seedling or father-mother) and Γ is the classically defined gamma function. The parameter b is the shape parameter affecting the tail of the dispersal function and a is a scale parameter homogeneous to distance. When b=1 this model simplify to an exponential; when b<1 the kernel is fat-tailed and when b>1 the kernel is thintailed. Through parameter b, this model thus allows to estimate whether long-distance dispersal events are respectively more or less important as compared to the exponential kernel. The model allows for a simultaneous estimation of seed and pollen immigration levels (m s and m p ), selfing rate (s) along seed and pollen dispersal kernels parameters (a s , b s , a p , b p ), as detailed in Oddou-Muratorio & Klein (2008) and in online Appendix A2 (supplementary material). SEMM requires as input the mapped locations of all sampled seedlings and all potential reproductive adult males and females within a local population, the multilocus genotypes of seedlings and adults, and allele frequencies of the same species in surrounding (background) populations. The genotypes and spatial positions of all the 342 adult trees found within the Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 14

  fine-scale variations of 'between-generation' SGS were analyzed by plotting genetic relatedness among all seedling-adult pairs against distance (Fig 3A). Then, similar plots were obtained by computing genetic relatedness among seedling-adult pairs for seedlings belonging to the same plot (Fig 3B) or the same year class (Fig 3C). Patterns of SGS were always markedly significant, with regression slopes of F ij on log(distance) different from zero in all cases (p<0.001). Overall, the 'between-generation' SGS was strong and decreased rapidly with distance, with a marked peak of SGS in the first distance interval (F 10m = 0.039) followed by a notable decrease between 20 and 40 m and no more significant trend after 40 m (Fig 3A). The adult SGS tended to be even stronger (F 10m = 0.065; Fig 3A).

  Our results first indicate strong temporal and low spatial heterogeneity in recruited seedling density, contrasting with the strong spatial and low temporal heterogeneity in their genetic structure. Beginning with seedling density, we observed a strong temporal heterogeneity on early-recruitment patterns in European beech, with 41.3% of seedlings germinated in 2002, and several years out of the 13 under study (from 1993 to 2005) without significant recruitment. These variations can result from low seed production, low germination, high mortality, or a combination of these factors. Many sites favorable for seedling establishment were opened by the 1999 Lothar storm, explaining the lack of recruitment before 1999.

  investigated the genetic structure among seedlings recruited in consecutive years, and thus belonging to a single life-stage from the point of view of most previous studies. Because beech trees produce nuts in irregular mast years, we expected significant genetic differentiation among year-classes due to inter-annual variance in reproductive success. Such fine-scale temporal differentiation has been reported in marine perennial organisms (e.g.(Planes & Lenfant, 2002). In our case, the across-year genetic homogeneity of the seed rain suggest low levels of genetic drift, i.e. high effective population size and/or relatively even contributions of all adult trees to reproduction either as male or female, even in non-masting years. Fine-scale patterns of between-generation SGS confirmed (1) the across-year genetic homogeneity of the seed rain (Fig 3C) and(2) the major role of gene dispersal and effective population size on spatial patterns of allelic frequencies.

Figure 1 .Figure 2 .

 12 Figure 1. Haye forest study plot, with exhaustive mapping of adult trees (plotted as circles: ○

Figure 3 .

 3 Figure 3. Between-generation patterns of spatial genetic structure : A. for all seedling-adult

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  seed dispersal obtained through seed trapping and inverse modeling methods showed restricted dispersal abilities, with a median distance of seed dispersal of ~6.50 m (Sagnard

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  relationship is expected to be linear in a part of the distance range, and shows a decay rate proportional to 1/d e σ² e , d e being the effective population density, and σ² e the axial variance in gene dispersal distance(Rousset, 2000). Here, our sampling design with only three seedling plots was not conceived to investigate the variation of genetic relatedness among pairs of seedlings over a large range of distances. Instead, we focused on the "between-generation" component of SGS, by computing coefficients of genetic relatedness (F ij ) among all pairs of individuals that involved one seedling (i = 1 to N S , where N S = total number of seedlings) and one adult (j= 1 to N A where N A = total number of adults). Computed in this way, F ij coefficients reflect the parent-offspring component of genetic structure, with expected values equal to 0.25 when i and j are related (parent-offspring) or 0 when i and

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Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011.

05039.x Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript 11 distance, this j are unrelated. As proposed by Hampe et al. (2010), we analyzed between-generation SGS for different group of seedlings, i.e. seedlings grouped by spatial plots (A, B and C) or by germination year. This allowed us to investigate the variation in SGS with spatial adult tree density (which decreased from plot C to A) and temporal adult tree density (high in the masting year 2002

  The genetic differentiation between dead and alive seedlings in year 2006 was first investigated using the hierarchical AMOVA design described above by equations (

To visualize SGS, F ij -values were averaged over a set of distance classes (d) (with a minimum number of 80 pairs of individuals per distance class) and plotted against distance. To test SGS, F ij values were regressed on ln(d ij ), where d ij is the spatial distance between individuals i and j, to provide the Impact of mortality on genetic structure:

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		[Table 1 around here]
		[Figure 2 around here]
	Manuscrit d'auteur / Author manuscript	The average mortality rate was 22.7% between 2004 and 2005, and 8.2% between 2005 and 2006. From year 2004 to 2005, there was a trend of higher mortality under low light conditions (high canopy closure), with mortality rate increasing from 15% in plot A to 21 % in plot B and finally to 27% in plot C (χ² test: p-value = 0.10). By contrast, from year 2005 to year 2006, the mortality rate was lower in plot C (0.3%) than in plots A (9%) and B (12%) (χ² test: p-value = 0.01). Overall, mortality from 2004 to 2006 (average mortality rate = 29%) tended to reduce variation in seedling density among plots (as measured by the coeeficient of
		variation (CV) of seedling density, CV 2004 = 0.36 versus CV 2006 =0.31), with final seedling
		density in 2006 ranging from 9.8 m
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-² (plot A) to 16.9 m -² on plot C and to 21.2 m -² on plot B.

  .05039.x ˆ value ≈ 44 m. The selfing rate was significantly positive (s= 2.1%).

	pollen dispersal was also obtained with the exponential kernel (fixing b p ) with an estimated
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p δ

  .05039.x Our results highlight some major spatial and temporal characteristics in the development of a demographic and genetic structure within beech populations. These results were based on a more than 10 year survey, with seedling establishment monitored from 2004 to 2006, and extrapolated back to 1993 by estimating seedling age. This 10 year time scale is relevant considering that in most forests across Europe, the management strategy of beech is high forest (even-aged stands resulting from natural regeneration). In this type of silviculture, stands are regenerated over a period spanning 10 to 20 years. At the beginning of the regeneration phase, adult trees are selectively logged to leave approx. 100-150 mature adult trees/ha. Because of masting and strong seedling vigor, all beech seedlings that will effectively contribute to the new reproductive stands are often recruited in less than 20 years.

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	plots with intermediate canopy closure (PAR= 31%, 31 seedlings.m -²) or high canopy closure
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		Considering first seed dispersal patterns as estimated from established seedlings, our results
		reflected preferential short distance dispersal as depicted by the low average distance of seed
	Manuscrit d'auteur / Author manuscript	dispersal ( s δ ˆ≈11 m). However, events of long distance dispersal appeared not negligible and may contribute to high effective population size in beech, as depicted by the seed immigration rate ( m s =20.5%) and the exponential-shaped dispersal kernel. These estimates convert into a median dispersal distance of 7.6 m and are consistent with previous demographic estimates of seed dispersal in beech (median dispersal distance of 6.49 m in Sagnard et al., 2007), or with recent genetic estimates in beech species across different sites (Oddou-Muratorio et al 2010).
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Table 1 .

 1 .05039.x Effective recruitment patterns of beech seedlings in the three studied regeneration 730 plots. Total count of seedlings in year 2004 (NS) and the number of seedlings used for 731 genetic analyses (NG) per plot and per year of germination. 732

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Table 3 .

 3 Nested analysis of molecular variation for genetic variation among 3 beech seedlings plots, each sampled in 3 successive years. 234 seedlings were used for these analyses.Version définitive du manuscrit publié dans / Final version of the manuscript published in : MolecularEcology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011.05039.x

		234 individuals			135 individuals
	Source of	df *	Sum of	Est.	% total	df *	Sum of	Est.	% total
	variation		squares	Var.	variance		squares	Var.	variance
	Among
		2	17.07	0.05	2.59	2	13.77	0.06	3.21
	subplots
	Among
		7	14.03	0.01	0.50	7	11.78	0.01	0.44
	Years/subplots
	Within years	352-				225 -
			709.60	1.75	96.91		433.56	1.75	96.36
		452				269
	Total		740.69	1.80			459.11	1.82

* df = degree of freedom for the within year component. df varied among loci due to missing data.

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Table 4 :

 4 Direct estimates of selfing (s), pollen and seed migration rates (resp. m p and m s ) and pollen and seed dispersal distance (δ p and δ s ) through spatially explicit mating models (SEMM) applied either on seedlings (e.g.[START_REF] Oddou-Muratorio | Comparing direct vs. indirect estimates of gene flow within a population of a scattered tree species[END_REF] or seeds (e.g.Oddou-Muratorio et al 2005).

		Seedlings SEMM	Seeds SEMM
	Parameter Estimate	Confidence	Estimate	Confidence
			Interval		Interval
	s	3.5%	4.2-23.8%	2.1%	0.4-3.8%
	m p	71.6%	60.2-85.0 %	78.0%	72.1-83.0 %
	δ p (m)	57.25	30.6-123.4	43.5	25.4-61.7
	m s	20.5%	13.5-27.1 %	-	-
	δ s (m)	10.92	9.4-12.9	-	-

Version définitive du manuscrit publié dans / Final version of the manuscript published in : MolecularEcology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011.05039.x

Acknowledgments

We thank A. Amm, H. Davi, C. Garcia, T Lander, F. Lefèvre and C. Pichot for revision of previous version of this manuscript. We also thank Alex Widmer, Dana Berens and two other anonymous reviewers for their detailed comments on previous version of this manuscript. E.

Teissier du Cros initiated this project. We are indebted to B. Jouaud (INRA Avignon) for sampling, genotyping and managing the lab work, as well as to T. Paul, P. Legroux, A.

Nassau, F. Bonne, R Herbeck et J.P. Lemaire (UEFL, Nancy) for field work (mapping and in situ observations). This study was funded by the French Bureau des Ressources Génétiques (BRG grant AIP 223 N° 88 -2003-2004) and the EC-supported Network of Excellence Evoltree (GOCE-016322).

Author information box

Sylvie Oddou-Muratorio is interested in estimating tree species abilities of pollen and seed dispersal and their role in adaptation in the context of climate change. Etienne Klein is interested in modelling and estimating long-distance dispersal through pollen and seed.

Giovanni G. Vendramin is a population geneticist involved in the application of molecular markers to population genetics and genomics of forest tree species. Bruno Fady is a researcher studying patterns of genetic diversity in forest trees in Mediterranean regions.

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Version définitive du manuscrit publié dans / Final version of the manuscript published in : Molecular Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011 Ecology, 2011, Vol.20, no.9, 1997-2010, DOI: 10.1111/j.1365-294X.2011.

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Ben Mekki

Vincent Khalifa

Hatem Simon

Benoît Fakhfakh

Moury

M Ben Khalifa

V Simon

H Fakhfakh

Benoît Moury
email: benoit.moury@avignon.inra.fr

Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions

Keywords: Capsicum annuum, eukaryotic initiation factor 4E (eIF4E), Potyvirus, recessive resistance, matching allele model, resistance durability

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Potato virus Y (PVY; genus Potyvirus; family Potyviridae) is an important pathogen in solanaceous crops including potato, pepper, tomato and tobacco. PVY isolates have been classified into three main phylogenetic groups, named groups O, N and C, which possess particular host range properties. Almost all pepper isolates of PVY belong to the C group, while almost all potato isolates belong to the O or N groups [START_REF] Blanco-Urgoiti | PVY C isolates are a homogeneous pathotype but two different genetic strains[END_REF]. Several recombinant isolates have also been characterized, mainly between the N and O groups [START_REF] Revers | Frequent occurrence of recombinant potyvirus isolates[END_REF][START_REF] Glais | Genetic variability in Potato potyvirus Y (PVY): evidence that PVYNW and PVYNTN variants are single to multiple recombinants between PVYO and PVYN isolates[END_REF][START_REF] Ogawa | Genetic structure of a population of Potato virus Y inducing potato tuber necrotic ringspot disease in Japan; comparison with North American and European populations[END_REF]. Two recessive resistance alleles at the pvr2 locus have been used extensively for more than 50 years to control PVY in pepper crops. The pvr2 2 allele is highly durable since no resistance breakdown has been observed in the field and very few isolates have been described that are able to infect plants carrying that resistance (Gébré- [START_REF] Sélassié | Variabilité naturelle des souches du virus Y de la pomme de terre dans les cultures de piment du sud-est de la France. Caractérisation et classification en pathotypes[END_REF][START_REF] Arteaga | Presence of PVY1-2 pathotype in pepper crops in Spain[END_REF]). The pvr2 1 allele is less durable since it has been broken down in some regions. However, Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H., Moury, B. (2012) [START_REF] Arteaga | Biological characterization of PVY as isolated from pepper in Spain. VI Meeting on Capsicum and eggplant[END_REF]. Seven additional pvr2 alleles confer particular resistance specificity towards PVY isolates and correspond to particular amino acid substitutions in the pvr2-encoded product, i.e. the eukaryotic translation initiation factor 4E (eIF4E) [START_REF] Charron | Natural variation and functionnal analyses provide evidence for coevolution between plant eIF4E and potyviral VPg[END_REF]). These alleles have not been used in the breeding of elite F 1 hybrids but are present in local pepper populations. Interaction of PVY isolates with the four pvr2 alleles pvr2 + (the susceptibility allele), pvr2 1 , pvr2 2 and pvr2 3 is controlled by a 23-amino-acid-long region in the viral genome-linked protein (VPg) [START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF][START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF]. Amino acid substitutions in VPg and eIF4E can modify their binding properties and are responsible for the pepper resistance specificity and PVY pathogenicity. When this interaction is impaired, the virus fails to accumulate in inoculated tissues and to complete its infection cycle [START_REF] Charron | Natural variation and functionnal analyses provide evidence for coevolution between plant eIF4E and potyviral VPg[END_REF]. Genetic diversity of PVY populations infecting pepper in northern Tunisia was analysed by RFLP haplotypes of three genome regions, including the VPg-coding region (Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF].

The study revealed a high genetic diversity and a strong genetic differentiation between the PVY populations due to sampling locality. The sampled plants were either susceptible to PVY (they carried the susceptibility allele pvr2 + ) or homozygous for the pvr2 1 allele and hence potentially resistant to some PVY isolates. Surprisingly, the polymorphism at the pvr2 locus of the sampled plants did not contribute significantly to the genetic differentiation of PVY. The aim of this study was consequently to characterize the pathogenicity of some of these Tunisian PVY isolates towards the pepper resistances at the pvr2 locus.

MATERIALS AND METHODS

PVY isolates

Details of all PVY isolates used in this study, their locality of origin, collection year, original host plant and, for some of them, the pvr2 genotype of these plants together with GenBank accession numbers are shown in table 1. Tunisian PVY isolates used in this study were obtained from fieldinfected Capsicum annuum plants in northern Tunisia in 2005 or 2006 or in 1994 in the centre of Tunisia (region of Kairouan) or in northern Tunisia (Borj el Amri). Isolates from 2006 were chosen to represent different haplotypes previously described by Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF] and to avoid plants infected simultaneously by several virus variants. Other isolates were chosen randomly.

Since their isolation, they were propagated once onto the laboratory host Nicotiana tabacum cv.

Xanthi-nc and were stored as dehydrated infected leaves.

cDNA synthesis and sequencing of the PVY VPg cistron

Total RNAs were purified from a 0•5 g piece of flesh of PVY-infected pepper fruits with the Tri-Reagent kit (Molecular Research Center Inc.), and were used as template for RT-PCR [START_REF] Moury | Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum[END_REF]. Part of the VPg cistron was amplified with primers VPg-F and VPg-R (Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF] designed to be polyvalent for all PVY groups and amplified 505 nucleotides of the VPg cistron. Sequencing reactions were performed directly on RT-PCR products with primer VPg-F by Genome Express (Grenoble, France).

Phylogenetic analyses

Using the CLUSTAL W program (Thomson et al., 1994), we obtained a 462-nucleotide-long alignment of the PVY VPg coding region, corresponding to the region from position 5796 to position 6257 of the genome of isolate SON41p (accession number AJ439544). Phylogeny construction and evaluation was done using the neighbor-joining (NJ) method implemented in the MEGA software [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF] and the robustness of the tree topology was evaluated with 1,000 bootstrap resampling. The codeml program of the software PAML version 4.2 [START_REF] Yang | PAML 4: Phylogenetic analysis by maximum likelihood[END_REF]) was used to estimate the selection intensity in the VPg central region, which is involved in PVY pathogenicity towards the pvr2 resistances (codon positions 101 to 123), along the different branches of the tree using the majority-rule consensus tree topology obtained above. For this, estimates of the numbers of synonymous (s) and nonsynonymous (n) nucleotide substitutions were obtained for each branch of A test of positive selection and tests of episodic evolution along a specific branch of the tree were performed as in [START_REF] Zhang | Small-sample tests of epidemic adaptative evolution: a case study of primate lysozymes[END_REF] (see also Table 2). For the test of positive selection, the numbers of synonymous and nonsynonymous substitutions estimated to have occurred along that particular branch were compared to the expected numbers under the null hypothesis of neutral evolution. For the tests of episodic evolution, the numbers of synonymous and nonsynonymous substitutions estimated to have occurred along that particular branch were compared to the sum of the numbers of synonymous and nonsynonymous substitutions estimated to have occurred (i) in all branches corresponding to the descendant lineages or (ii) in all branches corresponding to the lineages of other clades. Fisher exact tests of homogeneity were performed for all these comparisons.

Biological characterization of PVY pathotypes

The C. annuum inbred lines used for characterization of the pathogenicity properties of PVY isolates were Yolo Wonder (pvr2 + /pvr2 + ), Yolo Y (pvr2 1 /pvr2 1 ), Florida VR2 (pvr2 2 /pvr2 2 ) and HD285 (pvr2 3 /pvr2 3 ). Virus isolates kept as dehydrated material were first multiplied on Xanthi-nc plants and PVY-infected leaves of these plants were used to prepare inocula for pepper genotypes.

For each PVY isolate and each pepper genotype, 20 seedlings at the two-cotyledon stage (two to three weeks after sowing) were inoculated on the two cotyledons as in [START_REF] Moury | Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum[END_REF]. One month post inoculation, PVY infection of the pepper plants was assessed by DAS-ELISA [START_REF] Moury | Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum[END_REF].

RESULTS

Diversity of the VPg pathogenicity factor of Tunisian PVY isolates from pepper

About 100 PVY isolates have been isolated from pepper plants in Northern Tunisia in 2006 and previously subjected to an RFLP analysis covering three genome regions (Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF]). This study revealed a high level of genetic variability and a large number of plants infected by mixtures of different PVY haplotypes. On this basis, we chose 13 isolates that belonged to different haplotypes and that did not show evidence of mixed infection. We also analysed nine isolates collected in 2005 and chosen randomly among a collection of 50 isolates, and five isolates collected in 1994 in Northern or central Tunisia (Table 1). A 462 nucleotide long sequence of the VPg coding region (from position 5796 to position 6257 of the reference genome of isolate SON41p; accession number AJ439544) was obtained for all these isolates (GenBank accession numbers JF824713 to JF824736). There were very few ambiguous nucleotides (double peaks) in the sequence chromatograms and in these cases there was usually a marked difference in the peak heights. At these positions, only the predominant nucleotide in the population was considered in further analyses. Phylogenetic analysis of the sequenced region revealed three separate groups of isolates supported by high bootstrap values (>99%; Fig. 1). Compared to the overall PVY diversity, these isolates belonged to the C1 subgroup of clade C, as do most pepper isolates (96% bootstrap support; Blanco-Urgoiti et al. 1998 and data not shown). These three groups possess particular amino acids in the central part of the VPg (amino acid positions 101 to 123) which was shown to determine the pathogenicity properties of PVY towards the recessive resistances at the pvr2 locus in pepper [START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF][START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF] (Fig. 2). In this VPg region, all group 1 isolates possess identical amino acids with the exception of isolate K1694 which is composed of a mixture of viruses possessing either a valine or a leucine at position 118 (Fig. 2). Similarly, all group 3 isolates possess identical amino acids in this region. Finally, group 2 isolates show two different amino acid sequences that differ at position 115 only (Fig. 2). The fact that the central part of PVY VPg is subjected to positive selection [START_REF] Moury | Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum[END_REF]) could induce a biased image of the phylogenetic relationships between PVY isolates. Consequently, we also performed the phylogenetic analysis after excluding codon positions 101 to 123 from the sequence alignment. This second analysis revealed the same tree topology as before, the three major PVY groups showing high bootstrap support (>90%; data not shown). The tree topology was consequently There was no link between the PVY groups and the geographic or cultivar origin of the isolates (Table 1). The genotype at the pvr2 locus of the sampled plants was determined previously for the isolates collected in 2006. Two groups of plants can be distinguished: susceptible plants that carry the pvr2 + allele and pvr2 1 /pvr2 1 plants that are potentially resistant to some PVY isolates (Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF]). All of four isolates collected on pvr2 1 /pvr2 1 plants belonged to group 1 of isolates, while the nine isolates collected on pvr2 + /-plants were shared between the three PVY groups. Despite the small number of isolates, this discrepancy suggested some correspondence between the phylogeny and pathogenicity properties of the isolates.

Pathogenicity of Tunisian PVY isolates against pepper genotypes carrying different pvr2 alleles

The pathogenicity of nine isolates belonging to the different PVY groups was evaluated against a series of reference pepper genotypes homozygous for the pvr2 + , pvr2 1 , pvr2 2 or pvr2 3 alleles (Table 1). Pathogenicity could not be established by the occurrence of symptoms on inoculated plants because all isolates were in mixture with Cucumber mosaic virus (CMV; genus Cucumovirus, family Bromoviridae). Consequently, DAS-ELISA detection of PVY was performed one month post-inoculation on apical leaves. On this basis, two categories of PVY isolates were observed: isolates that infected all plants from the reference susceptible genotype (pvr2 + /pvr2 + ) only and isolates that infected all plants except those from the pvr2 2 /pvr2 2 genotype (Table 1). According to the nomenclature for PVY pathotypes (for example [START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF], the former isolates belonged to pathotype (0) and the latter to pathotype (0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF]. The absence of infection of pepper plants carrying the pvr2 1 , pvr2 2 or pvr2 3 resistances by a large number of PVY isolates indicated the absence of synergism between CMV and PVY in regard with these eIF4E-mediated resistances and validated the analysis of the pathogenicity of the PVY isolates. There was a perfect correspondence between the pathogenicity properties and phylogeny. PVY isolates from group 1 belonged to pathotype (0,1,3) while isolates from groups 2 and 3 belonged to pathotype (0). This was logical considering that the phylogeny itself corresponded to the polymorphism observed in the central part of the VPg.

Phylogenetic analysis of selection intensity in PVY VPg

Given the correspondence between phylogeny and pathogenicity properties of Tunisian PVY isolates, we asked whether the diversification into three major groups could have been the result of the selection imposed by the host plant resistance. For this, the PAML software was used to estimate the selection intensity within the region involved in pathogenicity (i.e. codon positions 101 to 123) and along the different branches of the phylogenetic tree obtained previously (Fig. 1). A single branch showed an apparent excess of nonsynonymous substitutions: the branch linking PVY group 1, corresponding to pathotype (0,1,3), to the rest of the tree. Along that branch, 8.1 nonsynonymous substitutions and zero synonymous substitutions were estimated to have occurred (Fig. 1). A formal test of positive selection comparing observed and expected synonymous and nonsynonymous changes along that particular branch [START_REF] Zhang | Small-sample tests of epidemic adaptative evolution: a case study of primate lysozymes[END_REF]) was however only marginally significant (P-value=0.10, Fisher exact test; Table 2A), but the small number of substitutions does not provide a high statistical power. Tests of episodic evolution, comparing the selection intensity along that particular branch and either (i) along the set of branches linking the descendant lineages in group 1 or (ii) along the set of branches corresponding to groups 2 and 3 were highly significant (P-values=0.0018 and 0.001, respectively; Fisher exact tests; Tables 2B and2C).

DISCUSSION

Evolution towards pathogenicity against the pvr2 1 resistance allele in Tunisian PVY isolates

The two isolates from group 1 whose pathogenicity properties were evaluated against the pvr2 alleles (isolates AOUA2 and GHD4) belonged to pathotype (0,1,3). Given that all isolates from group 1 share identical amino acid residues at VPg positions 101 to 123 and that this region is the [START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF][START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF], we inferred that all group 1 isolates belonged to pathotype (0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF]. For similar we considered that all isolates from groups 2 and 3 belonged to pathotype (0). As a consequence, the most parsimonious scenario for acquisition of pathogenicity toward pvr2 1 and pvr2 3 corresponds to a single event (along branch (a); Fig. 1). This event was probably the result of the selection imposed by resistant pepper plants, though the positive selection test along that particular branch of the phylogenetic tree was only marginally significant (Table 2A). Indeed, a significant acceleration of amino acid substitutions in the VPg region determining pathogenicity properties was observed along that branch compared to the rest of the tree (Tables 2B and2C). Since about eight amino acid substitutions were estimated to have occurred along that branch, it is probable that several of them were required for resistance breakdown. Each of two amino acid substitutions observed in the VPg of isolates from group 1, the serine to glycine substitution at position 101 and the threonine to arginine substitution at position 115, have been described as sufficient for breakdown of the pvr2 3 resistance [START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF]). The other amino acid substitutions are specific to this group of Tunisian isolates and have not been shown to alter PVY pathogenicity before.

If several amino acid substitutions were required for acquisition of resistance breaking properties by group 1 isolates, this could explain why such event was rare during PVY history and why isolates from the two other groups did not succeed to break the pvr2 1 resistance of Tunisian pepper cultivars down. This event is not very recent since isolate K1694, collected in 1994, belongs also to group 1. It is possible that emergence of group 1 occurred locally. No other PVY isolate close to group 1 is present in sequence databanks, while isolate LYE84.2 from the Canary Islands (accession number AJ439545) belongs to group 2 and French isolates To72 and SON41p (accession numbers EU334782 and AJ439544, respectively) are close to group 3 (Fig. 1).

Choosing the most efficient pvr2 resistance allele to control PVY in pepper in Tunisia

Quite a large number of amino acid substitutions in PVY VPg have been identified that confer particular pathogenicity properties towards the pvr2 resistance alleles of pepper [START_REF] Ayme | Different mutations in the genome-linked protein VPg of Potato virus Y confer virulence on the pvr2 3 resistance in pepper[END_REF][START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF]. These data can be used to choose the most appropriate resistance allele in order to control PVY in a given geographical region. The most widespread resistance allele in the sampled Tunisian pepper cultivars is pvr2 1 (Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF]) and pvr2 1 -resistance breaking isolates are widespread in these crops (Table 1, Fig. 2). Although pvr2 3 is not present in the sampled Tunisian pepper cultivars, isolates from group 1 are already able to break this resistance down. Consequently, the pvr2 3 resistance will probably be useless to control PVY in these crops. The resistance conferred by pvr2 2 would certainly be more appropriate. This resistance has been used worldwide for ca. 50 years and has not been broken down. Only two PVY isolates able to infect pvr2 2 /pvr2 2 plants have been described. The SON41p isolate has been selected from a pathotype (0,1) isolate by serial passages in pvr2 2 /pvr2 2 pepper plants (Gebre Selassie et al. 1985). The second case is a field isolate collected in the region of Málaga, Spain (Luis [START_REF] Arteaga | Presence of PVY1-2 pathotype in pepper crops in Spain[END_REF]) that was not mentioned afterwards. This latter isolate was not characterized at the molecular level. Isolates that are closest to SON41p at the critical residues in the centre of the VPg that control PVY pathogenicity properties towards the pvr2 alleles differ by two amino acids (corresponding to two nucleotide substitutions in the coding region) and are similar to isolate To72 (accession number EU334782; Figs. 2 and3) which belongs to pathotype (0) [START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF]). The combination of these two substitutions is required for infection of pvr2 2 /pvr2 2 plants [START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF]. Tunisian isolates from group 3 also differ by two amino acids from SON41p and evolution towards a VPg identical to that of SON41p would require three nucleotide substitutions (and three amino acid substitutions) (Figs. 2 and3), which is less likely than for To72. Isolates from other groups are more distant from SON41p and consequently much less likely to break the pvr2 2 resistance down. Consequently, the pvr2 2 resistance would certainly confer an efficient and durable resistance towards the Tunisian PVY pepper isolates.

The apparent lack of fitness cost associated to pathogenicity against the pvr2 resistance alleles supports the matching allele model for plant-virus interactions

Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H., Moury, B. (2012) [START_REF] Sacristan | The evolution of virulence and pathogenicity in plant pathogen populations[END_REF] proposed that the interaction and coevolution between plant recessive resistances and viruses correspond to the "matching allele" model. Under that model, "infection of plants requires a specific match between host and parasite genes" (Sacristán and García-Arenal 2008). For the pepper-PVY interaction, this was established by [START_REF] Charron | Natural variation and functionnal analyses provide evidence for coevolution between plant eIF4E and potyviral VPg[END_REF] who showed that infection depended on the direct binding between the plant eIF4E and virus VPg, and that amino acid substitutions in pepper eIF4E or PVY VPg that disrupt binding conferred an absence of infection of the plant. At the population scale, the matching allele model predicts (i) that no parasite genotype evolves a general pathogenicity on all host genotypes and (ii) that there is no fitness cost associated to pathogenicity [START_REF] Agrawal | Infections genetics: gene-for-gene versus matching-alleles models and all points in between[END_REF]. The first prediction was partly validated in the PVY-pepper interaction since a large number of PVY mutants that gained pathogenicity toward a particular pvr2 allele simultaneously lost their pathogenicity towards other alleles [START_REF] Ayme | Molecular dissection of the Potato virus Y VPg virulence factor reveals complex adaptations to the pvr2 resistance allelic series in pepper[END_REF]). Concerning the second prediction, no exhaustive fitness comparisons have been made in controlled laboratory conditions between PVY isolates with different pathogenicity properties. However, PVY epidemiological data in Tunisia suggest lack of, or low fitness costs associated to pathogenicity toward the pvr2 resistances. First, there was no effect of the pvr2 1 resistance in the sampled plants on the genetic structure of PVY populations (Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF]. This was probably due to the fact that a large number of pathotype (0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF] isolates are present in pvr2 + /pvr2 + plants. In the sampled areas, an average of 23% of plants possess a pvr2 1 /pvr2 1 genotype (Ben [START_REF] Khalifa | Contribution of host plant resistance and geographic distance to the structure of Potato virus Y (PVY) populations in pepper in Northern Tunisia[END_REF]). If pathotype (0,1,3) isolates were as fit as pathotype (0) isolates in susceptible pvr2 + /-plants, their expected frequency would be 61.5% on average (the 23% of pvr2 1 /pvr2 1 plants plus half of the 77% of pvr2 + /pvr2 + plants would be infected by pathotype (0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF] isolates while half of the 77% of pvr2 + /pvr2 + plants would be infected by pathotype (0) isolates; 1×0.23+0.5×0.77=0.615). Twelve of 22 PVY isolates (54.5%) belong to pathotype (0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF] in our set of isolates either chosen randomly (those from 2005) or at random among RFLP haplotypes (those from 2006) (Table 1). This observed value is not significantly different from expectations under an equal-fitness model (the 95% confidence interval is [8,18] for pathotype (0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF] isolates under this model; Monte Carlo simulations with the R software). Consequently, there is no apparent fitness cost of pathotype (0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF] isolates in susceptible pvr2 + /pepper plants in the Tunisian epidemiological conditions. Isolates from group 1 are not only infectious in pvr2 1 /pvr2 1 plants but they can also infect pvr2 3 /pvr2 3 plants, although this genotype is absent or very rare in Tunisia. Consequently, pathogenicity towards pvr2 3 also does not seem to be costly for group 1 isolates. It is plausible that pathogenicity towards pvr2 3 is a byproduct of pathogenicity selected by the pvr2 1 resistance due to the fact that the three-dimensional structure of the VPg of group 1 isolates was by chance able to bind the eIF4Es encoded by both pvr2 1 and pvr2 3 . Then, these isolates were maintained in PVY populations by the selective pressure of pvr2 1 /pvr2 1 plants and the absence of (or low) counter selection in pvr2 + /-plants.

Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H., Moury, B. (2012)

Figure 1 :Figure 2 :Figure 3 :

 123 Figure 1: Neighbor joining phylogenetic tree of part of the VPg coding region of Tunisian PVY isolates and reference isolates (identified by their accession numbers) that cluster with them. Bootstrap percentages above 70% are shown. The scale bar indicates branch lengths in substitutions per nucleotide. The numbers of nonsynonymous and synonymous nucleotide substitutions for internal branches are indicated as n/s above these branches as estimated by the codeml program of the software PAML (Yang 1997).

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : PlantPathology, 2012, 61, 441-447; DOI: 10.1111 DOI: 10. /j.1365 DOI: 10. -3059.2011.02540..02540.x. The original publication is available at http://onlinelibrary.wiley.com/doi/10.1111/j.1365- 3059.2011.02540.x/abstract. -breaking PVY isolates are usually less frequent than avirulent ones (Luis

	pvr2 1
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. Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : PlantPathology, 2012, 61, 441-447; DOI: 10.1111 DOI: 10. /j.1365 DOI: 10. -3059.2011.02540..02540.x. The original publication is available at http://onlinelibrary.wiley.com/doi/10.1111/j.1365- 3059.2011.02540.x/abstract.

Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H.,

Moury, B. (2012)

. Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript Manuscrit d'auteur / Author manuscript reliable and was not mainly influenced by a small number of positively-selected amino acid sites.

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : PlantPathology, 2012, 61, 441-447; DOI: 10.1111 DOI: 10. /j.1365 DOI: 10. -3059.2011.02540..02540.x. The original publication is available at http://onlinelibrary.wiley.com/doi/10.1111/j.1365- 3059.2011.02540.x/abstract.

	sole pathogenicity determinant towards the pvr2 resistance alleles in pepper
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Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H.,

Moury, B. (2012)

. Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x

  Version définitive du manuscrit publié dans / Final version of the manuscript published in : PlantPathology, 2012, 61, 441-447; DOI: 10.1111 DOI: 10. /j.1365 DOI: 10. -3059.2011.02540..02540.x. The original publication is available at http://onlinelibrary.wiley.com/doi/10.1111/j.1365- 3059.2011.02540.x/abstract.

	Sacristán and García
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	Manuscrit d'auteur / Author manuscript
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. Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x

Table 1 :

 1 . Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H., Moury, B. (2012). Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x Version définitive du manuscrit publié dans / Final version of the manuscript published in : Plant Pathology, 2012, 61, 441-447; DOI: 10.1111/j.1365-3059.2011.02540.x. The original publication is available at http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3059.2011.02540.x/abstract. Potato virus Y isolates used in this study. Version définitive du manuscrit publié dans / Final version of the manuscript published in : Plant Pathology, 2012, 61, 441-447; DOI: 10.1111/j.1365-3059.2011.02540.x. The original publication is available at http://onlinelibrary.wiley.com/doi/10.1111/j.1365-3059.2011.02540.x/abstract.

	Isolate	Pepper cultivar (pvr2	Locality	Year	Accession	Pathotype
	Manuscrit d'auteur / Author manuscript	genotype) or host			number
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a Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H., Moury, B. (2012). Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x

Table 2 :

 2 Tests of positive selection and episodic evolution in the region spanning codon positions 101 to 123 of the VPg cistron along branch (a) of the phylogenetic tree (Fig.1) linking group 1 of pathotype(0,1,[START_REF] Marchoux | Since the isolate stored as dehydrated material was not infectious, its pathogenicity against pvr2 3 could not be evaluated[END_REF] isolates to the rest of the tree (seeZhang et al. 1998 for details). s: number of synonymous nucleotide substitutions for branch (a); n: number of nonsynonymous nucleotide substitutions for branch (a); S: number of synonymous sites in the alignment; N: number of nonsynonymous sites in the alignment. These numbers were estimated by the codeml program of the software PAML(Yang 1997).Version définitive du manuscrit publié dans / Final version of the manuscript published in : PlantPathology, 2012, 61, 441-447; DOI: 10.1111 DOI: 10. /j.1365 DOI: 10. -3059.2011.02540..02540.x. The original publication is available at http://onlinelibrary.wiley.com/doi/10.1111/j.1365- 3059.2011.02540.x/abstract.

	A	Test of positive selection along branch (a)
		Nonsynonymous	Synonymous	Probability a
	Changes	8.1 (n)	0.0 (s)
	No changes	44.4 (N-n)	16.5 (S-s)	0.10
	B	Test 1 of episodic evolution
		Nonsynonymous	Synonymous	Probability a
	Along branch (a)	8.1	0.0
	Descendant lineages 2.0	7.2	0.0018
	C	Test 2 of episodic evolution
		Nonsynonymous	Synonymous	Probability a
	Along branch (a)	8.1	0.0
	Lineages of groups 2	7.4	14.8	0.0010
	and 3

a Tail probability in Fisher's exact test of homogeneity. Comment citer ce document : Ben Khalifa, M., Simon, V., Fakhfakh, H., Moury, B. (2012). Tunisian Potato virus Y isolates with unnecessary pathogenicity towards pepper : Support for the matching allele model in eIF4E resistance -potyvirus interactions. Plant Pathology, 0032-0862, 61 (3), 441-447. DOI : 10.1111/j.1365-3059.2011.02540.x

Acknowledgements

This study was supported by a Franco-Tunisian CORUS program from the French Ministère des Affaires Etrangères, the Tunisian Ministère de l'Education Supérieure, de la Recherche Scientifique et de la Technologie, a program 'Ecologie pour la gestion des Ecosystèmes et de leurs Ressources' and a program 'Agriculture et Développement Durable' from the Agence Nationale de la Recherche. We thank Julie Quenouille for interesting comments on an earlier version of the manuscript.