Ivo Tsvetkov

Denitsa Pandeva

Vesselka Gyuleva

the driver Bobi Emil Popov

Alexander Alexandrov

François Hausman

Agnès Labarthe

Roberta Maria

Ladislav Roland

Paule

Population genetic structure in common ash : a focus on southeastern european genetic resources (Unpublished doctoral

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Introduction

This thesis is an attempt to understand the factors that shaped the présent day spatial structure of genetic diversity in the common ash. In the following introduction, I will give some backgrounds on the conservation and sustainable use of forest genetic resources and highlight the importance of genetic knowledge for the setting up of conservation strategies. Using examples from the literature, I will présent genetic diversity patterns observed at different spatial scales in European broadleaved forest tree species and show how they hâve been interpreted. Finally, I will introduce the context, the study species and the objectives of the présent work.

1. Conservation and sustainable use of forest genetic resources 1.1. The policy framework of forest genetic resources conservation in Europe In Europe, major threats affecting forests and forest biodiversity were recognised in the I980's: acid rain, uncontrolled forest genetic érosion, forest fires, décliné of mountain forests, lack of research and scientific knowledge on forest tree physiology and forest ecosystems [START_REF] Arbez | Implémentation of the Strasbourg Resolution S2 on the conservation of forest genetic resources in Europe[END_REF]. European countries expressed their will to commonly address the opportunities and threats related to forests and forestry and initiated a forum at the ministerial level in 1990 in order to develop common instruments and measures for the protection and sustainable management of forests. Forty European countries including the European Union participate in this initiative, which opérâtes through the organisation of Ministerial Conférences, expert level meetings, round table meetings and working groups. To date, three ministerial also matters [START_REF] Frankel | The conservation of plant biodiversity[END_REF]. Hence, in order to préparé forest genetic resources for an uncertain future, the objectives of long-term sustainable management must be conservation and even enhancement of biological diversity.

Biological diversity can be viewed in a hierarchical manner focusing from the diversity of communities or ecosystems to the genetic diversity within species [START_REF] Primack | Essentials of conservation biology[END_REF]. The species certainly has a central place in conservation science as it contains a wealth of diversity from genes to populations and provides a measure for the diversity of communities [START_REF] Frankel | The conservation of plant biodiversity[END_REF]. It must nevertheless be stressed that meaningful species conservation in their natural environment (in situ) is impossible without conservation of their ecosystems (Frankel étal. 1995).

The evolutionary potential of forest tree species lies in their within-species genetic diversity.

The level and structure of this diversity can be investigated by analysing patterns of neutral (i.e. non-adaptive) variation at genetic marker loci such as allozymes, random amplified polymorphie DNA (RAPDs), amplified fragment length polymorphisms (AFLPs) or microsatellites (e.g. Milligan et al. 1994). In particular, in a review of allozyme polymorphisms of 322 woody taxa, Hamrick et al. (1992) pointed out that woody species maintain more variation within species and within populations than other life forms but hâve less variation among populations. These diversity patterns can be explained partly by some common features shared by the majority of tree species, such as (i) mainly outbreeding mating Systems and thus high levels of gene flow, (ii) individual longevity and fecundity and (iii) little or no history of domestication (with the exception of several fruit-bearing species, Loveless & Hamrick 1984, Hamrick et al. 1992).

Evolutionary processes

The evolutionary forces that govem genetic diversity act on allelic frequencies in populations. This is why population structure is of chief importance for conservation efforts at the level of individual species. The evolutionary forces are sélection, mutation, gene flow and genetic drift. It is always essential to consider the balance of evolutionary forces in a particular situation.

Evolutionary forces a. Sélection

Sélection is the only force that causes adaptive évolution by acting on the adaptive traits. It arises because different génotypes hâve different rates of reproduction and survival (reproductive fitness) and will transmit their genes accordingly.

Introduction

The adaptive diversity of a species is mostly expressed trough quantitative variation among individuals in characters or traits. Some adaptive characters are monogenic (e.g. those involved in résistance to pathogens), but most traits are govemed by many loci, which contribute small, often additive effects to total variance, and they are strongly affected by the environment (e.g. [START_REF] Frankham | Introduction to conservation genetics[END_REF]. The proportion of the variance of a particular trait in a population due to genetic différences among individuals is called heritability. The heritability of a trait détermines its response to directional sélection (sélection favouring one extreme, Frankfiam et al. 2002) and therefore the evolutionary potential of the population with respect to that trait. The heritability is comprised between 0 in highly inbred populations without genetic variation and 1 in outbred populations where ail genetic variance is additive and there is no environmental variance [START_REF] Frankham | Introduction to conservation genetics[END_REF]. Most quantitative traits that hâve been studied show additive genetic variance (heritability > 0, e.g. [START_REF] Lynch | Evolution and extinction in response to environmental change[END_REF], which means that populations and species hâve the ability to respond to changing environmental conditions.

If the environment relevant to a particular trait is perceived as heterogeneous in the long term, sélection will favour different génotypes in different areas and among-population différentiation will establish. In this case, stabilising sélection occurs in populations within areas with homogeneous sélection pressure regarding the particular trait, and disruptive sélection is experienced among them, i.e. at the species level (Eriksson 1998). Hence, sélection will maintain overall genetic diversity in heterogeneous environments.

b. Mutation

Introduction increase the genetic diversity of a population if it brings in migrants from populations with different alleles and allele frequencies.

d. Genetic drift

Genetic drift désignâtes the random change in allele frequency that occurs because gametes transmitted from one génération to the next carry a biased sample of the alleles présent in the parental population. In a random mating population under the effect of genetic drift, genetic variation is lost within populations at a rate l/2Ne per génération where is the effective population size (see below). As this loss affects genetic variation in a random fashion, différentiation inevitably increases among populations. The expected increase of inbreeding, or mating among relatives, is also equal to l/2Ae per génération. As inbreeding increases levels of homozygosity, deleterious récessive alleles become expressed, which results in a décliné in (reproductive) fitness in inbred individuals, known as inbreeding dépréssion.

Population size

The effective population size Ne is a fondamental parameter for conservation biology as it détermines the relative importance of directional (migration and sélection) and stochastic (genetic drift) genetic factors. The effective population size is the size of an idéal population that would be subjected to the same amount of genetic drift as the actual population. An idéal population corresponds to a closed random mating population with discrète générations, constant population size, equal sex ratio and Poisson variation in family sizes (e.g. [START_REF] Frankham | Introduction to conservation genetics[END_REF]. Real populations rarely fulfil the assumptions of idéal populations. As a conséquence, the effective size Ne is usually smaller than the census size Ne. Correct estimation of the ratio Ne/Nc is a difficult task, but many estimâtes fall within the range 0.1-0.2 [START_REF] Nunney | Assessing minimum viable population size: demography meets population genetics[END_REF] or 0.1 -0.4 [START_REF] Waples | Définition and estimation of effective population size in the conservation of endangered species[END_REF].

In large populations (populations with large effective size), stochastic genetic factors are negligible. Diversity patterns in large random mating populations are therefore essentially determined by migration and sélection, mutations contributing only weakly. Migration brings in new variants that reflect allele types and frequencies from their source population(s) whereas sélection affects bénéficiai and detrimental alleles, but not neutral ones (except under hitchhiking; i.e. when neutral genes are linked to selected ones). Under distinct environmental conditions, natural sélection favours distinct alleles. This is why patterns of among-population différentiation can vary for different traits and particularly between traits and neutral (i.e. nonselected) genetic markers (e.g. [START_REF] Karhu | Do molecular markers reflect patterns of différentiation of adaptive traits in conifers?[END_REF].

In small populations, the effect of stochastic genetic factors is strong. Variation is lost at a rate l/2Ne, regardless whether it is adaptive or not. Hence, sélection is less efficient in small than in large populations. This has two corollaries: small populations adapt less well to changing environmental conditions than large ones, and deleterious alleles are less likely to be Introduction eliminated by sélection in small populations and may even become fixed. A strong réduction in population size is called a population bottleneck. A bottleneck can occur for example during colonisation of a new area (founder effect) or if a population faces threats (section 1.4.). The magnitude and duration of a bottleneck strongly influence the population's genetic features (allelic richness V5. gene diversity, Nei 1975; inbreeding dépréssion v^. genetic load, [START_REF] Kirkpatrick | The effects of a bottleneck on inbreeding dépréssion and the genetic load[END_REF].

Factors causing endangerment

The threats to a population or a species can be divided into deterministic factors on the one hand, and stochastic factors on the other.

Deterministic factors are directional and relatively consistent in magnitude. They are related to human action. Deterministic factors threatening forest tree species include déforestation, exploitation, fragmentation, démographie and habitat alterations, environmental détérioration, translocation and domestication [START_REF] Ledig | Human impacts on genetic diversity in forest ecosystems[END_REF]. Global climate change is a threat of eminent importance belonging to this category (e.g. [START_REF] Mâtyâs | Setting priorities in conservation of genetic diversity -with spécial référencé to widely distributed conifer species. In.-Conservation of forest genetic resources in Europe[END_REF][START_REF] Eriksson | Global warming and gene conservation of noble hardwoods[END_REF].

Stochastic factors are those that affect populations, in addition, when they hâve a small effective size. [START_REF] Shaffer | Minimum population sizes for species conservation[END_REF] has identified four forms of stochasticity or uncertainty in small populations; (i) démographie stochasticity arises from variation in the survival and reproductive success of individuals; (ii) environmental stochasticity refers to unpredictable fluctuations in rainfall, température, density of competitors, pathogens or herbivores that affect birth and death rates; (iii) natural catastrophes include fires, floods and extremes in climate hazards (e.g. drought, frost) and (iv) genetic stochasticity encompasses inbreeding dépréssion, loss of genetic variation and accumulation of deleterious mutations.

As a response to deterministic threats, for instance global climate change, a population can either evolve or migrate; on the contrary in order to face detrimental effects of stochastic factors, only an increase in population size could be useful.

The threats caused by genetic uncertainty hâve not fully been recognized for a long time; for instance [START_REF] Lande | Genetics and demography in biological conservation[END_REF] view was that démographie and environmental stochasticity or natural catastrophes would lead to population extinction before genetic factors could act. The rôle of genetic threats affecting species or population endangerment is however well established now (e.g. [START_REF] Hedrick | Conservation genetics: where are we now?[END_REF][START_REF] Allendorf | The rôle of genetics in population viability analysis[END_REF].

Utility of genetic markers for conservation

We saw that the central objective for conservation of forest genetic resources is to safeguard their adaptive diversity, their evolutionary potential. The question that promptly arises is: does Introduction population genetic structure investigated with neutral molecular markers reflect patterns of adaptive diversity? [START_REF] Hedrick | Conservation genetics: where are we now?[END_REF] inspected this question and recommends caution in inferring biological différences from significant marker différences among groups. His main argument is that highly variable marker loci used in population biology (e.g. [START_REF] Sunnucks | Efficient genetic markers for population biology[END_REF] lead to high statistical power of différentiation tests (Goudet et al. 1996). As a resuit, the différences at marker loci are real, but they do not necessarily reflect biological différences.

An example of the opposite situation is given in [START_REF] Karhu | Do molecular markers reflect patterns of différentiation of adaptive traits in conifers?[END_REF]: non-significant différentiation at allozyme loci reveals the high levels of gene flow among populations in Scots pine, but diversifying sélection for an adaptive trait, bud set, was strong enough to overcome the homogenising effects of gene flow and produce large adaptive genetic différences between populations.

Neutral molecular markers can nevertheless be used to track adaptive variation when they are employed for association mapping with adaptive traits. The linkage of a marker locus to a trait is used for example in marker-assisted sélection in forest tree breeding and allows considérable shortening of génération turnover [START_REF] O'malley | Molecular markers and forest trees[END_REF].

Molecular markers are used to address many other questions conceming species biology relevant to conservation biology (e.g. [START_REF] Hedrick | Conservation genetics: where are we now?[END_REF]):

• Résolve taxonomie uncertainties (phylogenetics) and identify hybridisation

• Mark the boundaries of evolutionary significant units or management units and • Estimate levels of inbreeding

• Study mating Systems Furthermore, molecular markers permit non-intrusive sampling methods in animais, they provide help in choosing populations and suitable sites for reintrodution and they are used for forensics (e.g. find traces of protected species in goods for consomption, Frankham et al.

2002).

Introduction 1.6. Conservation methods

In situ versus ex situ conservation

There are globally two conservation approaches: In situ conservation refers to conservation of a population or species in the wild, in its natural habitat; whereas ex situ conservation désignâtes conservation away from the natural habitat, which for forest trees could be in provenance trials, in seed orchards or clonal archives, or in in vitro-or cryopreservation. In contrast to ex situ methods, in situ conservation is a dynamic means of conservation, genetic resources are continuously exposed to sélection pressures. Another advantage is that in situ conservation mostly allows sustainable use of the material to be conserved. Both in situ and ex situ approaches can complément each other. In order to know which method to use or how to combine them for a particular species, its State of endangerment has to be evaluated, and associated objectives to long-term gene conservation, like for example improvement through breeding, hâve to be identified.

In situ and circa situ conservation are critically important in forest tree species: indeed, only few tree taxa, around 100, are effectively conserved ex situ and there are often technical and économie limitations to the establishment of ex situ programmes [START_REF] Kanowski | Conserving the genetic resources of trees in situ[END_REF].

Regarding in situ conservation for forest trees, it is however well known from provenance tests that the local material is not always the best adapted to the actual site conditions (e.g. [START_REF] Mâtyâs | Climatic adaptation of forest trees: rediscovering provenance test data[END_REF]). This has raised polemics about the utility of promoting natural régénération and using autochthonous reproductive material for long-term conservation against nonautochthonous material for which the genetic adaptedness of the future stand has been evaluated to be higher (e.g. Geburek & Millier 2000).

Importance of population size and structure

The influences of many threats affecting genetic resources of a species reduce essentially to a decrease in the effective population size. There has been much debate on how large a population should be to maintain sufficient genetic variation in order to be able to adapt in the long term [START_REF] Allendorf | The rôle of genetics in population viability analysis[END_REF]. Basically, an isolated population must be large enough for mutations to compensate the loss of variation caused by genetic drift. The suggestions for the effective population sizes needed range from 500-5000, which in many species cannot be accomplished unless the connectivity among populations is increased over a wide géographie area [START_REF] Allendorf | The rôle of genetics in population viability analysis[END_REF]).

It appears thus that population size and among-population structure are central issues for conservation management. In the spécial case of forest tree species, [START_REF] Eriksson | Dynamic gene conservation for uncertain futures[END_REF] described five basic categories of natural population structure ranging from a single large continuons random mating population over several populations interconnected to varions degrees to a continuons population where the relatedness between individuals dépends on Introduction their spatial distance. Extinction and recolonisation dynamics may also influence the structure of populations [START_REF] Wade | Extinction and recolonization: their effects on the genetic différentiation of local populations[END_REF].

Example of a strategy for conservation and management offorest trees: the multiple populations breeding System applied to noble hardwoods in Europe

Noble hardwoods represent a heterogeneous group of species, which includes, among others, maples, hombeam, ashes, chestnut, walnut, lime trees, elms, wild fruit trees and Sorbus species. The commun features of those species are scattered distribution patterns in European mixed forests, high ecological demands and timber of high quality (Turok et al. 1996). Noble hardwoods are rare and some even endangered (Rotach 1999). Many hâve been neglected

until recently, which explains why information on crucial life-history characteristics of these species (e.g. mating System, pollen and seed dispersai) and on their patterns of genetic variation is still scarce. As biodiversity, sustainability and adaptability of ecosystems are being perceived as highly important, and not least because of a large demand of high-quality timber, noble hardwoods are being promoted now. Their effective conservation and sustainable utilisation are supported by the EUFORGEN Noble Hardwoods Network (Turok et al. 1996, Turok et al. 1998, Turok et al. 1999, Turok et al. 2002). Whenever possible, genetic resources of noble hardwoods should be managed and conserved in situ, in the frame of forestry, to allow for joint gene conservation and production of valuable timber (Rotach 1999).

The gene resource conservation strategy that the EUFORGEN Noble Hardwoods network currently recommends for European noble hardwoods is the multiple populations breeding System (MPBS) [START_REF] Eriksson | Conservation of noble hardwoods in Europe[END_REF] and references therein). The MPBS concept was initially proposed for combined breeding and gene conservation by [START_REF] Namkoong | Strategies for gene conservation in forest tree breeding[END_REF]. [START_REF] Eriksson | Dynamic gene conservation for uncertain futures[END_REF] suggested that MPBS could be applied for various management strategies, from very elaborated breeding programs to simple in situ gene resource populations with no or limited silvicultural intervention. The main idea of MPBS is to identify gene resource (sub)populations and to manage them in situ, or, jointly, in situ and ex situ, in order to increase among-population variance. Ideally, the subpopulations are chosen from heterogeneous environments (according to more than one variable) and/or in a way to maximise their initial différentiation among each other. In situ management aims at increasing the adaptation to local conditions while sites for establishment of populations ex situ are chosen with the objective of minimising among-population gene flow. The total gene resource population should contain approximately 20 subpopulations each of which should hâve Ne > 50. In these subpopulations, variability is lost at a rate of 0.01 and inbreeding accumulâtes at the same rate, which is a figure generally accepted for short-term conservation [START_REF] Allendorf | The rôle of genetics in population viability analysis[END_REF]. The strength of MPBS however is the enhanced among-population différentiation, so that the total adaptive diversity is globally increased.

Introduction [START_REF] Eriksson | Dynamic gene conservation for uncertain futures[END_REF] stressed that a proper sampling of gene resource populations is crucial for efficient gene conservation strategies, both in situ and combined in situ and ex situ. If available, genetic information on among-population différentiation for marker loci and/or traits should be accounted for when choosing gene resource populations. Otherwise, the steepness of ecological gradients and biological characteristics of the species could be helpful to produce educated guesses of among-population différentiation for traits and the strength of gene flow (Eriksson 1998).

According to their occurrence, use and susceptibility to devastating diseases, noble hardwoods are divided into four categories for management following MPBS [START_REF] Eriksson | Conservation of noble hardwoods in Europe[END_REF]):

1. Commonly occurring species. This category addresses mainly ash and maple. Gene resource populations should be chosen in situ from heterogeneous environments, guided by genetic information when available, in order to sample a maximum of variation. In some populations, a larger area should be conserved in order to match the objective of conserving associated species.

2. Rarely occurring species. This category concems most noble hardwoods. Gene resource populations should be chosen in situ for joint conservation of several species when possible. A commonly encountered problem is small effective population size in the wild. Increase of the natural population sizes is encouraged through thinning of competing species and through planting of material of autochthonous origin (e.g. Rotach 1999) conserved in seed orchards or clonal archives {ex situ).

3. Multipurpose species, e.g. used for timber and fruits. The long history of domestication of for example nut-bearing trees such as chestnut is likely to hâve strongly différendated cultivated populations from those in the wild. Breeding activities in the cultivated gene pool should be continued and kept as isolated as possible from natural wild populations in order to minimise genetic érosion of the latter by the cultivated gene pool, which has reached a high domestic fitness at the cost of natural fitness.

4. Populations of elms {Ulmus sp.) hâve drastically declined due to Dutch elm disease (DED). For Ulmus minor, which reproduces vegetatively through root suckers, clones from the whole distribution area should be conserved ex situ in clonal archives and partly in cryopreservation. For U. glabra and U. laevis, MPBS should be applied in situ, except when the risk of DED is high. In that case, conservation is recommended in low clonal hedges, which protect elm plants from the DED vector, Scolytus insects.

Some of those recommendations are being set up, for instance in elms, which are clearly endangered [START_REF] Eriksson | Conservation of noble hardwoods in Europe[END_REF]. EUFORGEN facilitâtes the dissémination of information and collaborative initiatives; political commitments are not within its scope.

Box 1 Statistics commonly used in population genetics

Nuclear genetic markers

Measures of polymorphism

A or K Mean number of alleles; noted As when computed within populations and At when computed in the total sample (ail populations considered together). Values are sometimes standardised to a particular population size.

Ho

Observed heterozygosity, or proportion of hétérozygotes (only for codominant markers). Varies from 0 (homozygotes only) to 1 (ail are heterozygous).

He

Gene diversity or heterozygosity expected under random mating; équivalent to the probability of drawing two different alleles when sampling at random (with replacement) in a population. Varies from 0 to ~1. This statistic is noted Hs within populations and Hj in the total sample.

Measure of inbreeding

Fl

Inbreeding coefficient, or fixation index within individuals, computed as (He-Ho)/Hb, noted Fis within populations and Fu in the total sample. Varies from -1 to +1; there is inbreeding if Fi > 0, selfing species hâve Fi = 1.

Measures of among-population différentiation

Fsrc

Fixation index among populations, computed as (^Hj-Hs)IHj, measures the proportion of diversity distributed among populations. Varies from 0 (ail populations equal) to 1 (populations completely different). The statistic was initially developed for loci with 2 alleles.

Rst

Similar to Fst, this statistic was developed to measure différentiation at microsatellite loci, taking into account the différences of allele sizes (~ number of mutations).

Chloroplast genetic markers

Measures of polymorphism

A or K Mean number of alleles.

^sor/iT Haplotypic' diversity within populations or in the total sample. vs or vt Haplotypic diversity within populations or in the total sample considering the phylogenetic différences among haplotypes.

Measures of among-population différentiation

Gst

Similar to Fst, computed as {hT-hs)/hj.

N SJ

Différentiation statistic that takes into account the phylogenetic différences among haplotypes, computed as (vt-vs)/vt.

Population genetic structure in European forest trees

Today, there is a wealth of genetic markers available for the investigation of patterns of genetic variation [START_REF] Parker | What molécules can tell us about populations: choosing and using a molecular marker[END_REF][START_REF] Sunnucks | Efficient genetic markers for population biology[END_REF], Table 1). The patterns of genetic variation in plant species at a large geographical scale, like the European continent, hâve been found to resuit from the combined actions of a multitude of factors. Some of these are intrinsic to the species life history (breeding System, modes of seed and pollen dispersai, life form, gregariousness), while others are perturbations induced by natural processes (ice âges, climatic stochasticity) or human impact (habitat fragmentation, global change).

Tree species, as briefly mentioned before, présent high genetic diversity at nuclear marker loci at the species and at the population level, but very little genetic différentiation among populations, compared to species with other life forms (Hamrick & Godt 1989, Hamrick et al. 1992, Box 1, Table 2). Furthermore, woody species with large geographical ranges, outcrossing breeding Systems and wind or animal-ingested seed dispersai maintain more diversity within species and populations, but less variation among populations than woody species with other combinations of traits (Hamrick et al. 1992). Although life history and ecological traits explained 34% of the variation among species, the authors pointed to the large proportion of variation that remained unexplained and suggested the importance of the spécifie evolutionary history of each species in determining the level and pattern of genetic diversity.

The climatic changes of the Quatemary (the last 1.6 million years) hâve considerably influenced the evolutionary history of temperate tree species by triggering successive contractions and expansions of their distribution ranges, involving population extinctions, size réductions and long-term isolation. In many species, these historical imprints can still be identified today at a large geographical scale (see Cornes and Kadereit 1998, Newton et al. 1999and Hewitt 2000 for reviews). At the régional or local geographical scale, these patterns are usually blurred by the combined effects of sélection, migration and genetic drift (e.g. Pigliucci et al. 1990[START_REF] Leonardi | Spatial structure of genetic variability in natural stands of Fagus sylvatica L. (beech) in Italy[END_REF]. Streiff et al. 1998[START_REF] Lee | Genetic variation at allozyme and RAPD markers in Pinus longaeva (Pinaceae) of the White Mountains, California[END_REF].

Two main methodological approaches hâve been used to investigate the patterns of genetic variation in plant species at different geographical scales with the aim of making inference on their causal factors. Those methods employed either (i) organelle (mainly chloroplast) DNA markers or (ii) nuclear genetic markers ( 'Hamrick & Godt (1989), Hamrick et al (1992), [START_REF] Hamrick | Effects of life history traits on genetic diversity in plant species[END_REF]). ^SanderefaZ. (2000). ^Alleles hâve the same size but are not identical by descent. '*Alleles that do not amplify in the polymerase chain reaction (PCR) because of mutations in the primer annealing sites. Due to the sympatric occurrence of sessile (Quercus petraea) and pedunculate (Quercus robur) oaks, many studies hâve investigated diversity patterns in both species. Levels of within-population genetic diversity in both species are summarised in Table 3.

It appears that pedunculate and sessile oaks are highly polymorphie species, for instance when comparing their mean within-population genetic diversity for allozymes (He ~ 0.25) to that of other long-lived woody species (mean He = 0.148, Table 2). Microsatellites exhibit higher levels of diversity (A ~ 20, He ~ 0.87) than allozymes (A ~ 3, He ~ 0.25), whereas the dominant multilocus methods RAPD and AFLP show the lowest diversity among the markers assessed (He ~ 0.21).

Significant hétérozygote deficiencies, as reflected by comparison of Hq v5. He or by positive values of the fixation index (inbreeding coefficient) Fis, were found in several studies and the trend was stronger for allozymes than for microsatellites. Possible explanations for high Fis values include inbreeding, especially due to non-random spatial genetic structure because of limited seed and/or pollen dispersai within stands [START_REF] Bacilieri | Intraspecific genetic structure in a mixed population of Quercus petraea (Matt.) Liebl. and Q. roburh[END_REF], Streiff et al. 1998);

and a Wahlund effect [START_REF] Degen | Comparative study of genetic variation and différentiation of two pedunculate oak {Quercus robur) stands using microsatellite and allozyme loci[END_REF]) possibly due to phenological différences [START_REF] Bacilieri | Intraspecific genetic structure in a mixed population of Quercus petraea (Matt.) Liebl. and Q. roburh[END_REF]. [START_REF] Degen | Comparative study of genetic variation and différentiation of two pedunculate oak {Quercus robur) stands using microsatellite and allozyme loci[END_REF] attributed markedly lower F,s values in microsatellites than in allozymes to scoring errors of microsatellites, because similar trends for ail biparentally inherited markers should be expected if the mating System were the only process responsible for hetrozygote deficiency. The usual pattern, however, is to find higher Fis values with In tree species, organelle (mainly chloroplast) DNA markers hâve mostly been used to address phylogeographic' issues, i.e. dealing with the spatial distribution of differentiated lineages of a taxon over a large géographie area (e.g. [START_REF] Démesuré | Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe[END_REF], King & Ferris 1998, Dumolin-Lapègue et al. 1997, Petit et al. 2002a,b). In Gymnosperms in particular, organelle markers hâve additionally served for a wider scope of applications, for instance to investigate patterns of gene flow within populations (e.g. Latta et al. 1998), population genetic structure (e.g. [START_REF] Echt | Chloroplast microsatelites reveal population genetic diversity in red pine, Pinus resinosa Ait[END_REF][START_REF] Hayashi | Genetic différentiation of organelle DNA polymorphisms in Saghalin fir from Hokkaido[END_REF][START_REF] Sperisen | Tandem repeats in plant mitochondrial genomes: application to the analysis of population différentiation in the conifer Norway spruce[END_REF], or hybridisation between species (e.g. Bucci et al. 1998).

Several attributes of chloroplast DNA make it especially suitable for phylogeographic studies.

Chloroplast DNA is haploid, non-recombining and predominantly uniparentally inherited [START_REF] Birky | Evolution and variation in plant chloroplast and mitochondrial genomes[END_REF], Clegg 1989). It therefore evolves under a twofold higher pressure of genetic drift than the nuclear genome in hermaphrodite diploid plants [START_REF] Birky | An approach to population and evolutionary genetic theory for genes in mitochondria and chloroplasts, and some results[END_REF]. This allows for a strong différentiation of isolated lineages, for example during climatic cold stages. Further, in most angiosperms, chloroplast DNA is matemally inherited, i.e. transmitted only through seeds (Ennos 1994), and has thus more restricted dispersai than nuclear markers, which are transmitted by both seeds and pollen. This feature and its low mutation rate [START_REF] Clegg | Rates and patterns of chloroplast DNA évolution[END_REF]) allow chloroplast DNA to retain historical imprints in space over long time periods.

The climate of the late Quatemary, which corresponds to approximately the last 700.000 years, was marked by a sériés of cold and dry glacial periods, each lasting approximately 100.000 years, interrupted by shorter periods (10.000-20.000 years) of warmer and moister climate (Webb & Bartlein 1992). During the cold stages, glaciers covered vast areas, for instance the Scandinavian ice sheet covered Britain and most of northem Europe throughout the last glacial cycle (115.000 BP -10.000 BP, [START_REF] Peltier | Ice Age Paleotopography[END_REF]. The distribution ranges of plants and animais were then contracted to more Southern locations (glacial refuges) and expanded northwards again as the climate improved (Cornes & Kadereit 1998, Newton et al. 1999).

Many tree species hâve reached their présent distribution ranges only recently, i.e. a few thousand years ago. Owing to their long life cycles, only 100 to 1000 générations (depending on the species) hâve elapsed since the end of the last ice âge and therefore their extant genetic structure often still contains historical imprints dating back to that period (Kremer 1994).

Chloroplast DNA surveys on a wide geographical scale allow to identify phylogenetic lineages within taxa and to retrace their postglacial recolonisation routes. They can suggest the location of glacial refuges by pinpointing high diversity areas, but cannot precisely locate them without evidence from fossil data, i.e. pollen or macrofossils (Taberlet et al. 1998, Cornes & Kadereit 1999). Fossil material on the other hand can often not provide taxonomie resolution, e.g. palynological data didn't résolve oak species further as 'deciduous Quercus' (Brewer et al. 2002), but the necessary temporal resolution is provided through radiocarbon dating of fossil material. It appears that both methods are complementary, and molecular genetic markers were therefore jointly used with palynological tools to investigate the phylogeography of oak species (Petit et al. 2002a,b, Brewer et al. 2002).

Phylogeography of European white oaks: location of refuges and recolonisation routes

The studies of Petit et al. (2002a,b) The most important results are the following (Figs. 1 and2):

• Existence of three main refuge areas in the three Southern European peninsulas Fossil pollen data from the end of the late glacial maximum 15000 (15 ka) BP identified three régions of Europe acting as full glacial refuges for oaks: (1) the Southern Iberian Peninsula,

(2) Southern Italy and (3) the Southern Balkan Peninsula with one site in Greece and one near the Bulgarian Black Sea border. These refuges were located in or near mountainous areas, supporting the idea that refuges for deciduous trees would hâve been situated at mid-altitude sites where the précipitation would hâve been higher than in the plains during the arid glacial period (see Brewer et al. 2002).

• Proximity of the Italian and Balkan refuges and existence of landbridges Italy and the Balkan Peninsula share haplotypes from lineages A, C and E (Fig. 1). This is mainly because the sea level was about 100 m lower during the last glacial period than at présent [START_REF] Peltier | Ice Age Paleotopography[END_REF], leaving the northem part of the bed of the Adriatic Sea exposed, which resulted in exchanges of haplotypes between the Italian and Balkan refuges (Fig. 2). As a conséquence, phylogenetically divergent haplotypes hâve sometimes followed similar colonisation routes, limiting phylogeographic structure to some extent. The colonisation of Fig. 2 Schematic distribution of primary and secondary oak réfugia and post-glacial movements.

From Petit et al. 2002a.

the British Isles through what is now the Channel is also an example for the existence of a landbridge.

• Existence of primary and secondary refuges

The climate change between the late glacial maximum and the onset of the Quatemary involved an initial warming to a thermal maximum between 13 and 12.5 ka BP. During this warming, oaks spread rapidly northwards from the full glacial or primary refuges. A short climatic détérioration was noted around 12 ka BP (the Older Dryas), followed by higher but declining températures until the severe cooling of the Younger Dryas (approximately 11 to 10 ka BP). Pollen records noted a décliné of oak pollen déposition during this period. Oaks survived the Younger Dryas in secondary refuges located probably in mountainous areas South of a line running from the Pyrenees in the west to the Carpathians in the east (Fig. 2).

• Northward colonisation: the west to east orientation of mountain chains was sometimes a barrier

During the late glacial interstadial (i.e. before the Younger Dryas), haplotypes from Italy and the Balkans had reached the Po-plain, but could not spread farer north because the Alps represented a major obstacle. Instead migration continued westward, circumventing the Alps to reach secondary refuges. Today, oaks do not grow at altitudes higher than 1400 m in the Alps. However, during a climatic optimum at the onset of the Holocene with higher température than at présent, oaks managed to cross the Alps via high altitude passes (more than 2000 m), and pollen records confirm their presence in the northem slopes by 9 ka BP.

From there, Scandinavia was reached within 1000 years. On the other hand, haplotypes from lineage B from Iberia had circumvented the Pyrenees trough the Basque country (Fig. 2) and subsequently colonised their northem slopes (Fig. 1), central and western France, and areas further to the northeast up to Sweden. The south of Ireland and England was reached as early as between 9.5 and 9 ka BP. The présent distribution range of oaks was reached around 6 ka BP.

• Discontinuities in the range of haplotyes and the influence of man An example of range discontinuity is shown by haplotype 2 from lineage C, which occurs in Sicily and north of Rome, but not further south on the Italian peninsula. This suggests that, in this région, populations may hâve been overlooked, that they hâve gone extinct, or that the discovered haplotypes are distinct but could not be resolved with the techniques employed.

Long-distance seed transport might also account for discontinuities in the range of haplotyes.

Although acoms hâve constituted an important nutritional resource for man and his cattle, it is not likely that man was much implicated in the post-glacial recolonisation of oaks [START_REF] Kônig | Chloroplast DNA variation of oaks in western Central Europe and genetic conséquences of human influences[END_REF]. More recent human influence is however confirmed by the identification populations carrying haplotypes of non-autochthonous origin [START_REF] Kônig | Chloroplast DNA variation of oaks in western Central Europe and genetic conséquences of human influences[END_REF].

Population 1

Population 2

Nst « Gst 3 Fig. 3 Illustration of the correspondence between the phylogénies of the haplotypes and their géographie distribution. When there is correspondence (case 1), the différentiation measured by taking into account the similarities between haplotypes (A^st) is larger than the différentiation based only on the frequency of the haplotypes (Gst)-When the haplotypes are equally related, A^st = Gst (case 2). When the most strongly related haplotypes are never together but always found in different populations, 7Vst< Gst (case 3). From Pons and Petit (1996).

Phylogeography ofEuropean white oaks: patterns of diversity and différentiation

Genetic diversity of haplotype data can be evaluated by two methods: (1) by considering allele identity with the statistic h or (2) by taking into account the phylogenetic distance between alleles with the statistic v ( Box 1, Pons and Petit 1996). The analysed samples display a phylogeographic structure when the among-population différentiation based on phylogenetic distance between haplotypes, Nsj, is significantly larger than the différentiation based on haplotype identity, Gst (Pons & Petit 1996). This is the case when phylogenetically divergent haplotypes are preferentially distributed among populations rather than within populations (Fig. 3). However, a différence between A^st and Gst could also be caused by other factors, such as unequal effects of mutation rates or sampling artefacts (Pons and Petit 1996). Central and northem Europe also display high numbers of haplotypes, but this time, vt is higher than hj (although not significantly so), indicating colonisation from different refuges.

Great Britain, on the contrary, présents overall low diversity values reflecting colonisation from a single refuge.

At the level of the whole dataset, the tendencies outlined for Q. petraea become even clearer (Petit et al. 2002b). ( 1) Areas where refuges had been postulated such as the Iberian and Italian peninsulas generally display higher chloroplast diversity.

(2) Superimposed on the 'refuge' effect, the authors found a 'mixing' effect; régions located at the junction of several colonisation routes, i.e. in central and northem Europe also displayed high diversity.

Mode of colonisation

Introduction later migrants would reach the population already at carrying capacity, thus contributing comparatively little to the established diversity pattern.

The sharing of haplotypes among white oak species represents an interesting example for leading edge colonisation. Petit and colleagues (2002a) argue in favour of an early association of haplotypes to particular species (although not exclusively). Leading edge colonisation followed by pollen-mediated gene flow could account for the establishment of a second oak species in the newly colonised area through hybridisation, using the maternai type of the first colonisers and swamping out their nuclear genome through backcrossing (Petit et al. 1997, Petit et al. 2002a).

In contrast to this mode of colonisation, [START_REF] Austerlitz | Effects of colonization processes on genetic diversity: différences between annual plants and tree species[END_REF] hâve presented a model that proposes the arrivai of a relatively high numbers of colonisers owing to the prolonged juvénile phase of tree species. This model can hardly explain the patchy distribution of maternai types observed in oaks, but allows for more realistic seed to pollen dispersai ratios (i.e. higher than 1:200, Ennos et al. 1994) by reducing substantially the founder effects.

Phylogeographic structure differs among species

In analogy to the example of European white oaks, most studies dealing with chloroplast genetic markers in temperate woody species revealed strong différentiation among populations and a relatively concordant geographical distribution of phylogenetically related haplotypes, resulting in a phylogeographic pattern. In Europe, the contribution of the different refuges is species-specific. In beech [START_REF] Demesure | Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe[END_REF] for instance, much of central Europe was colonised from a single refuge located in the Carpathians, and distinctive cpDNA haplotypes restricted to Italy suggest that beech was unable to cross the Alps during postglacial recolonisation. In hazel, on the other hand, most of Europe was colonized from a western refuge (Palmé and Vendramin 2002). Other European woody species where phylogeographic structure was detected include aider (King and Ferris 1998), ivy [START_REF] Grivet | Phylogeography of the common ivy (Hedera sp.) in Europe: genetic différentiation through space and time[END_REF], Scots pine [START_REF] Soranzo | Patterns of variation at a mitochondrial sequence-tagged-site locus provides new insights into the postglacial history of European Pinus sylvestris populations[END_REF] and olive [START_REF] Besnard | Olea europaea (Oleaceae) phylogeography based on chloroplast DNA polymorphism[END_REF]. Some recent surveys of cpDNA over a wide géographie range were however unable to detect phylogeographic patterns. This was the case for sloe. Prunus spinosa [START_REF] Mohanty | Chloroplast DNA diversity within and among populations of the allotetraploid Prunus spinosa L[END_REF], wild cherry. Prunus avium (Mohanty et al. 2001) and for wild service tree, Sorbus torminalis (Oddou-Muratorio et al. 2001). Low différentiation among populations was detected with cpDNA markers in these bird-dispersed early successional species, suggesting that mode of seed dispersai and successional behaviour hâve an impact on historical imprints of cpDNA. Zanetto and Rremer (1995) investigated allozyme diversity in acoms from 81 populations of sessile oak from the whole distribution area. Diversity at the species level was high with an average number of alleles per locus of A = 4.77 and genetic diversity H = 0.265. Genetic différentiation among populations was very low, Fst = 0.025, lower than the average for woody species (Gst=0.084, Table 2). The comparison of Fst for biparentally inherited markers to différentiation for matemally inherited chloroplast markers (Gst = 0.865)

illustrâtes the homogenising power of gene flow through pollen.

Despite the low level of différentiation identified by Zanetto and Kremer (1995), allelic richness and gene diversity varied among populations and variation was organised geographically at the European scale. Populations from the centre of the range (from the Loire to the Rhine river) displayed higher genetic diversity values than those from the edges of the distribution. For allelic richness, the trend was exactly opposite, with populations at the margins of the distribution comprising more alleles than those from the central part (Fig. 4).

This pattern resulted in a positive corrélation with longitude for allelic richness, and a négative one for genetic diversity.

The observed patterns of clinal variation in allele frequencies with longitude were concordant in seven out of eight loci, hence the authors concluded that they were not due to sélection, but rather a conséquence of postglacial recolonisation. To explain the géographie variation of allelic richness, the authors hypothesised loss of alleles in the northwestem part and/or gain of alleles on the edges of the distribution. They excluded the hypothesis of repeated bottlenecks bringing about a loss of alleles during recolonisation, because some northem populations exhibited high numbers of alleles. Sélection against rare alleles on optimum sites (in northem France) was considered a possible explanation for the low allelic richness. A gain of alleles on the (mainly Southern) edges of the distribution was attributed to an increase in the frequency of rare alleles above equilibrium values after recovery from bottlenecks. The antagonistic trends of variation in allelic richness and gene diversity could be explained if the loss of rare alleles was paralleled by an increase in frequency of an allele with intermediate frequency,

and not the most common allele.

In their allozyme data set on 389 European populations of the common beech (Fagus sylvatica), Comps et al. (2001) observed a similar pattern of antagonistic trends in allelic richness and gene diversity over the distribution area as the authors of the sessile oak study. Comps et al. (2001) took up again the hypothesis of successive bottlenecks, producing a graduai loss of allelic richness with post-glacial recolonisation. They proposed that a concomitant increase in gene diversity might be due to combined effects of (i) admixture of A recent study by Kremer et al. (2002) tested the hypothesis that common refuge origin may resuit in a corrélation between cpDNA variation and nuclear variation. The authors performed

Mantel tests to investigate the corrélation between the matrix of chloroplast genetic distances and the matrix of standard genetic distances for either allozymes or RAPDs.

According to Kremer et al. (2002), cytonuclear disequilibria were established through genetic drift during the glacial period. Drift would hâve been strong enough to differentiate populations among refuges for both chloroplast and nuclear DNA, because populations were small and isolated for a long time. During recolonisation, cytonuclear disequilibria would hâve been erased to a large extent by at least 2 forces: (i) gene flow, homogenising nuclear genes more strongly than chloroplast genes, because of more extensive pollen flow, and (ii) sélection, acting on spécifie gene products. Gene flow was likely to be the only force in action for allozymes and RAPDs, as they are supposedly neutral markers. Kremer et al. (2002) found significant cytonuclear disequilibria for both RAPDs and allozymes when not correcting for géographie distance, a resuit approximately équivalent to the détection of allele frequency variation according to geographical position found by Zanetto and Kremer (1995).

After correction for geographical distance, however, the corrélation with chloroplast genetic distances remained significant for RAPDs, but not for allozymes. Hence, Kremer et al. (2002) demonstrated that postglacial gene flow had been able to break up the linkages between chloroplast and nuclear genes in the case of allozymes, and they also revealed some influence of sélection pressures on RAPD markers.

Comparison of chloroplast patterns of variation with nuclear controlled phenotypic traits

In oaks like in most plant species, phenotypic traits exhibit higher différentiation among populations than allozymes; for instance in sessile oak, différentiation coefficients for bud burst and height growth were generally higher than 20% [START_REF] Kremer | Multilocus and multitrait measures of différentiation for gene markers and phenotypic traits[END_REF]) compared to 2.5% for allozymes (Zanetto & Kremer 1995). Kremer et al. (2002) assessed the corrélation with chloroplast lineage in four categories of traits: survival, growth, stem form and phenology. Only six out of 62 trait x provenance trial combinations were correlated with chloroplast genetic distances, and only two of them remained significant after correction for géographie distance. Vice versa, corrélation with géographie distance was significant for 13

Introduction trait combinations, and ail remained significant after correction for chloroplast genetic distance. This indicates that traits were more closely correlated to géographie distance than to chloroplast genetic distance. The 13 significant corrélations ail concemed phenology and none involved growth traits or form. This confirmed earlier results (référencés in Kremer et al. 2002) that phenological traits are under photoperiodic or beat sum control and exhibit géographie patterns at large spatial scales, whereas growth and form, which are influenced by local soil and compétition conditions, show patterns at a microscale level.

According to Kremer et al. (2002), the observed patterns are due to local sélection pressures acting on recolonised populations since their establishment and differentiating them increasingly over time for phenotypic traits. The extant différentiation among populations at phenotypic traits is therefore very different from the one of ice âge refuges, and significant association with maternai lineages is no longer observed.

Materials and Objectives

This thesis was initiated in the framework of the coopération project GENFOR: Genetic Romania and northem Bulgaria (river Danube) and in the Republic of Moldavia (river Dniestr, Fig. 5). As a resuit, the species diversity of forests is high in these régions because they contain different forest ecological zones, comprising broadleaved deciduous forests, mixed ecotonal forests with conifers as well as evergreen mediterranean forests [START_REF] Longauer | Forest resources of the PHARE countries[END_REF]. These ecosystems, which are often less fragmented than in western Europe, provide habitats for a large variety of animal species some of which hâve gone extinct in their western range (i.e. bears in the Pyrenees or lynxes in Germany, [START_REF] Klimo | Forest biodiversity and ecological networks[END_REF].

Another et al. 1964). This high species diversity in the Balkan Peninsula compared to the Italian and Iberian Peninsulas might partly be due to the proximity of Turkey and the near and middle East. These areas may hâve acted as a "buffer zone" during range contractions in the glacial periods of the Quatemary, offering areas of favourable climate for the retraction of species, while species retracting themselves to the other peninsulas may hâve gone extinct because of the presence of the Mediterranean Sea.

The Balkan Peninsula has held refuge areas for many tree species during the glaciations of the Quatemary (Huntley & Birks 1983) and is therefore expected to harbour also high withinspecies diversity (Hewitt 1996). Several southeastem European sites, mainly mountainous ones, are known to hâve acted as glacial refuges from fossil pollen (Huntley & Birks 1983, Brewer 2001), and they hâve been confirmed with molecular data, mostly chloroplast DNA surveys, e.g. the Carpathians for beech (Fagus sylvativa. [START_REF] Démesuré | Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe[END_REF] [START_REF] Longauer | Forest resources of the PHARE countries[END_REF]. Also, in contrast with western Europe where several countries hâve faced severe déforestation at the end of the Middle Ages, forests hâve been continuously présent in most eastem European countries, and large areas of semi-natural forests still predominate especially in the Carpathians and in the Balkans [START_REF] Klimo | Forest biodiversity and ecological networks[END_REF].

In Bulgaria, Romania and the Republic of Moldavia, forests are mainly managed as evenaged communities according to shelterwood Systems, i.e. part of the mature forest is left intact as a light canopy cover after felling of a large area, allowing for natural régénération [START_REF] Longauer | Forest resources of the PHARE countries[END_REF]. Natural régénération from seed is indeed the largely prevailing régénération practice in Romania and Bulgaria (there are no data for the Republic of Moldavia) with over 50% of the total régénération, distinguishing these countries clearly from the near Hungary and Slovakia, where most régénération occurs by planting [START_REF] Longauer | Forest resources of the PHARE countries[END_REF]. It is noteworthy that in Bulgaria, more than 30% of the forest cover is coppice forest, whereas this management form makes up only a few percent in Romania (there are no data for the Republic of Moldavia, Longauer 1999). Further, large forest areas in our focal countries (more than 30%) hâve been designated for spécial fonctions such as water and soil protection or conservation and are therefore managed at low intensity (i.e. the semi-natural forests mentioned before, [START_REF] Klimo | Forest biodiversity and ecological networks[END_REF][START_REF] Longauer | Protective, conservation and spécial forest functions[END_REF].

The previously stated features of southeastem European forests suggest that they are likely to represent a more close-to-nature situation than most western European forests. They should therefore contain genetic resources that are suitable for the application of molecular markers in order to investigate population genetic structure in forest tree species and to infer of relevant biological information conceming for instance tree species life history characteristics (e.g. Milligan et al. 1994).

3.2. Common ash (Fraxinus excelsior L.)

Common ash {Fraxinus excelsior, Oleaceae) is the tallest of four native ash species in Europe (Tutin et al, 1972). Just as F. angustifolia Vahl and F. pallisiae Wilmott, it is wind-pollinated and belongs to section Fraxinus of the genus, whereas the insect-pollinated F. omus belongs to section Omus (Wallander 2001). Common ash is a tree of 25-35 (40) m, with grey bark, black buds and imparipennate leaves with 7-13 leaflets (Fig. 6). Its mean longevity is 150-200 years.

Distribution

The natural distribution of common ash covers large parts of Europe (Fig. 7), ranging from northera Spain to Southern Scandinavia and from the shores of the Atlantic in the west to the Volga river in the east. In southeastem Europe, the southem limit of common ash is in

Ecology

Common ash is a postpioneer species, exhibiting properties intermediate between typical pioneer woody species (birch, willow, poplar) and permanent forest components (oaks, beech, [START_REF] Giertych | Jesion wyniosly -Fraxinus excelsior L[END_REF]. It readily colonises fertile, temporarily forest-absent habitats (forest clearings, roadside and streamside slopes and ravines) but its compétition ability is high only when its ecological requirements are met. Common ash mainly occurs in floodplain forests, where it can represent a high percentage of the canopy, associated with black aider and/or elms [START_REF] Thill | Le frêne et sa culture[END_REF][START_REF] Claessens | Le frêne en Condroz -stations et productivité potentielles[END_REF]. It also occurs in fertile mixed broadleaved forests associated with mainly oak, hombeam and maple [START_REF] Thill | Le frêne et sa culture[END_REF][START_REF] Claessens | Le frêne en Condroz -stations et productivité potentielles[END_REF], but its distribution is discontinuons and scattered and it is not as common as the social species oak and beech [START_REF] Giertych | Jesion wyniosly -Fraxinus excelsior L[END_REF], Pliura 1999). The occurrence of ash in habitats with contrasting features, such as wet lowland sites as opposed to dry chalk-rich sites suggested the existence of differentiated soil-ecotypes, which could however not be confirmed experimentally (Weiser 1995 and references therein). Common ash best grows in fertile, fresh and profound soils, with high organic matter content and good moisture availability.

Mating System

Common ash is a polygamous species; it shows continuons phenotypic sex expression from male via hermaphrodite to female individuals (Picard 1982[START_REF] Lamb | The reproductive biology ofcommon ash (Fraxinus excelsior): a literature review[END_REF][START_REF] Binggeli | Gender variation in ash[END_REF], Wallander 2001). Five basic types of flowers [(i), male with 2 stamens; (ii) male with 2 stamens and a rudimentary pistil; (iii) hermaphrodite; (iv) female with a pistil and mdimentary stamens; and (v) female with a pistil only; [START_REF] Binggeli | Gender variation in ash[END_REF] combine into four types of inflorescences (male; mixed male and hermaphrodite; hermaphrodite; and female; Wallander 2001). Their occurrence in individuals allows to define the three classes of phenotypic gender expression, although some variation was noted between individuals and years (Wallander 2001). Flowering occurs before leafing in spring, as in most wind-pollinated trees. Hermaphrodite flowers are self-compatible, but they may avoid self-pollination by being protogynous (Wallander 2001). For instance, Morand et al. (2002) stated selffertilisation as one of the possible explanations for the high values of Wrights inbreeding coefficient (Fis = 0.16 in secdlings and Fis = 0.29 in adults) that they observed in a microsatellite study of 12 French populations. The fruiting pattern of common ash is irregular and synchrony among individuals indicates that it is probably triggered by some environmental component (for instance early leafing in the preceding year, [START_REF] Tapper | Irregular fruiting in Fraxinus excelsior[END_REF][START_REF] Tapper | Long-term patterns of mast fruiting in Fraxinus excelsior[END_REF]. The syncarpous ovary contains two ovules in each of two locules, but only one develops after fertilisation. The fruit is a one-seeded samara (Fig. 6 b), which is mainly winddispersed, although some authors argue in favour of additional bird-mediated dispersai to Fig. 8 Distribution of Fraxinus excelsior -type pollen deposits 9.000 radiocarbon years ago. From Huntley and Birks (1983).

expiait! features of postglacial rccolonisation [START_REF] Wilkinson | Plant colonization: are wind-dispersed seeds really dispersed by birds at larger spatial and temporal scales[END_REF]) and the strong colonising behaviour of ash [START_REF] Giertych | Jesion wyniosly -Fraxinus excelsior L[END_REF].

Location of glacial refuges and postglacial recolonisation history inferred front fossil pollen evidence

Three species belong to the Fraxinus excelsior-type pollen taxon: Fraxinus excelsior, F.

angustifolia and F. pallisiae (Huntley & Birks 1983). Two subspecies are recognised in F.

excelsior, ssp. excelsior, which occurs throughout the range of the species and ssp. would be associated with woodlands in which ash is dominant or co-dominant. These authors reported the presence of ash pollen during the last glacial cycle (115 ka BP -10 ka BP) in Greece and Italy. In the early Holocene (10 ka BP), they documented expansions of ash from a southeastem European and an Alpine centre. Large amounts of pollen were recorded around 9 ka BP in eastem central Europe (Fig. 8) and interpreted either as a rapid expansion of the Southern centre or, altematively, as an expansion from a putative northeastem European refuge. These centres subsequently merged and expansion continued, reaching England, Denmark, the southem Baltic and western France by 6 ka BP. By this time, a possible fourth centre became apparent in the Pyrenees. Since 5 ka BP, ash became more and more abundant in western Europe, especially in the British Isles, while a retreat was observed in eastem

Europe. The décliné in the east was attributed to forest clearance and/or the immigration of taxa which expanded later (e.g. Carpinus betulus). Another possible reason was a réduction of the precipitation/evaporation ratio in that area, as Fraxinus excelsior favours moist soils (Huntley & Birks, 1983). According to these authors, the highest migration rates during recolonisation were observed in the period up to 6000 BP, with values comprised between 200 and 500 m per year.

A more recent survey based on a greater density of pollen records (Brewer 2001) identified sites with more than 5% ash pollen représentation 12 ka BP at the Bulgarian Black Sea coast and in southem Italy near Naples (Fig. 9). These are probably full glacial refuges. Additional sites with ash pollen représentation above 1% (i.e. local presence of ash sensu Huntley & Birks 1983) were located in central Portugal in the Serra da Estrela, in the French Massif Central, in central Italy in the Apennines, in the eastem Alps at the German-Austrian border.

Fig. 9 Distribution of Fraxinus excelsior -type pollen deposits 12.000 and 8.000 radiocarbon years ago. From Brewer (2001).

in the Greek Pindos mountains, in the Bulgarian Rhodopy mountains, just north of the Carpathians in southeastem Poland, in Scotland at the northem coast of the Firth of Forth and in Southern Sweden near Gôteborg. Based on [START_REF] Peltier | Ice Age Paleotopography[END_REF] maps on déglaciation history, the Scottish and Fennoscandian ice sheets must hâve freed the two latter areas only very recently before pollen déposition. Little change was observed in fossil pollen maps until after 10 ka BP, the end of the Younger Dryas cold period. Ash then spread north and west from the refuges and by 8 ka BP it had reached the Pyrenees, it was abundant at the northem slopes of the Alps and in Poland, and it was présent, but to a lesser extent, in the Baltic States and in Southern Scandinavia (Brewer 2001, Fig. 9). Ash had fully colonised the British Isles around 4,5 ka BP, after which a décliné of ash became évident over large parts of the range, but mainly in the eastem part (Brewer 2001). According to Huntley and Birks' (1983) (Huntley & Birks 1983).

Another recent fossil pollen survey by Gliemeroth (1997) located the Iberian refuge for ash rather in the south of that Peninsula; it confirmed the refuges situated in the Alps and the Balkan Peninsula suggested by the previous authors; and it proposed additional refuges for ash in the Turkish mountains and at the northem Black Sea border.

Summarising the results from these three pollen surveys, it appears that there is strong support for full glacial refuges for ash in the Alps and the Balkan Peninsula, and weaker support for refuges located in Iberia, Turkey and at the northem Black Sea coast. Early occurrence of ash was reported in some other sites in France, Scotland, Sweden and Poland and their rôle as putative primary or secondary refuges deserves further attention. The colonisation of the European continent was first marked by an east-west migration, followed by a south-north trend, and the distribution limits were reached between 6000 and 4000 BP.

Géographie patterns of variation.

Not much information is available on géographie variation in common ash. One study assessed genetic diversity in French populations with nuclear microsatellite markers (Morand et al. 2002); and first surveys of phenotypic markers hâve been reported from two recently established provenance trials describing, respectively, patterns of variation in central Europe [START_REF] Kleinschmit | Erste Ergebnisse des Eschen-Herkunftsversuches von[END_REF] and Scandinavia [START_REF] Baliuckas | Within-population variation in juvénile growth rythm and growth in Fraxinus excelsior L. and Prunus avium L[END_REF]. I will briefly review the results of these works here.

In their study of twelve common ash populations from northeastem France with five microsatellite loci, Morand et al. (2002) found high within-population gene diversity, = 0.900 ± 0.047 (SD), as expected for a wind-pollinated tree species (compare with values for Introduction oak. Table 4, p.l6). A low proportion of the total diversity was distributed among populations, as expected, Fst = 0.043 ±0.017 (SD), and no corrélation was observed between géographie distance and genetic différentiation. [START_REF] Kleinschmit | Erste Ergebnisse des Eschen-Herkunftsversuches von[END_REF] reported results on a total of 52 provenances, from Germany (43), Switzerland (3), Austria (4) and Remania (2), established in a total of 26 test sites in Germany ( 19), The Netherlands (2), Belgium (2), France (2) and Remania (1). Analysed traits included bud burst, bud set and damages due to late frost (recorded in the nursery); as well as losses, height at âge 6 or 7 and shape (recorded on test sites). Ail characters displayed extensive ecotypic variation patterns at local géographie scale, and generally, différences within provenances were as large as among provenances. Some géographie trends of variation were identified, notably for bud burst and bud set (growth cessation), which are important adaptive characters. Dates of bud burst and bud set were however not correlated to each other. Forty percent of the variation in bud burst was explained by longitude, whereas latitude and longitude together explained 27% of the variation in bud set. Romanian provenances flushed early and set buds late, and therefore suffered from early and from late frosts in Germany.

This also resulted in bad stem form because terminal buds were killed by frost. Height was strongly correlated to the date of bud set (i.e. length of the growth period), and decreased with increasing latitude. [START_REF] Baliuckas | Within-population variation in juvénile growth rythm and growth in Fraxinus excelsior L. and Prunus avium L[END_REF] analysed 20 to 29 open-pollinated familles from four Swedish provenances of common ash, sampled along a latitudinal gradient and established in two test sites. They assessed bud burst, growth cessation and height in saplings at âges 3, 4 and 5 years. They found a négative relationship between growth cessation and latitude, confirming the results of [START_REF] Kleinschmit | Erste Ergebnisse des Eschen-Herkunftsversuches von[END_REF]. Large variation was identified among familles within provenances, and the northemmost provenances exhibited as much variation as the others, which conflicts with the expectation that populations at the distribution edges should présent reduced diversity because of the occurrence of bottlenecks and genetic drift (e.g. Hewitt 1996, Soltiseta/. 1997).

Objectives

The central objective of this thesis is to investigate which factors shaped the spatial structure of genetic diversity in the common ash and how strong are their relative contributions. The région of main interest is the Balkan Peninsula, and the results are expected to lead to the formulation of recommendations in the context of management and conservation of common ash populations. The genetic diversity of common ash was analysed at different spatial scales with a sériés of molecular tools. The work was divided into five parts which are briefly introduced here and presented in the form of scientific manuscripts in the following chapters:

1. Assessment of genetic structure within and among Bulgarian populations of the common ash (Fraxinus excelsior L.)

The main objectives of this chapter were to provide a first measure of the level of genetic marker diversity in the species using nuclear microsatellite markers and to investigate the partitioning of diversity at three géographie scales in Bulgaria, (i) within populations, (ii)

among populations within natural régions of Bulgaria, and (iii) among régions. Biological characteristics of the species, such as the level of inbreeding and the variance of gene dispersai distance were inferred.

2. Estimating seed versus pollen dispersai from spatial genetic structure in the common ash This part deals with the estimation of the variance of gene dispersai distance within a large Romanian common ash population. The aim was to gather information on the relative contributions of seed and pollen to total gene dispersai by comparing the decrease of kinship between individuals against distance in the study population with data from simulations on an analogous population, where seed and pollen dispersai were under control.

Nuclear microsatellites reveal contrasting patterns of diversity in western and eastem

European populations of the common ash {Fraxinus excelsior L.)

In this chapter, patterns of genetic diversity were investigated within and among 36

European common ash populations with nuclear microsatellite markers. Data were analysed with classic statistical tools and with recently developed approaches which exploit the high levels of polymorphism characteristic for microsatellites. The objective was to explain the diversity patterns in the context of population demography and postglacial recolonisation history, with particular emphasis on the inference power of the new analysis methods.

4. Different evolutionary history and geographical distribution of even-and odd-sized alleles at a microsatellite locus in Fraxinus excelsior (L.)

In the genetic diversity surveys in Bulgaria and in Europe, even-and odd allele sizes were observed at the microsatellite locus FEMSATL4 in common ash. The purpose of this chapter was to investigate the molecular diversity underlying this electrophoretic variation and to regard it in the context of the evolutionary history of the locus by sizing and sequencing amplification products from four species at increasing phylogenetic distance from common ash.

Incongruence between chloroplast and nuclear marker data among European populations of common ash

Introduction

The aim of this part was to identify patterns of chloroplast genetic diversity at the European scale and compare them to the patterns previously obtained at nuclear markers in the context of the séparation of populations in ice âge refuges and subséquent postglacial recolonisation. The utility of using two differentially inherited types of genetic markers for the inference of population history is assessed.

Introduction

Forest genetic resources are threatened by a broad array of factors such as climate change, environmental pollution, habitat destruction, compétition and pest attacks. The capacity of forest tree species to survive these threats and to persist in spatially and temporarily heterogeneous environments is dépendent on their adaptive potential, which is determined by the within-species genetic diversity. This is why conservation strategies for forest trees should be based on an evolutionary approach and focus on the maintenance of within-species genetic diversity [START_REF] Breitenbach-Dorfer | The contact zone between two migration routes of silver fir, Abies alba (Pinaceae), revealed by allozyme studies[END_REF][START_REF] Eriksson | Global warming and gene conservation of noble hardwoods[END_REF]. Noble hardwood species (e.g. Fraxinus, Acer, Tilia, Castanea, Juglans, Prunus, Almts, Ulmus) hâve a scattered distribution within mixed forests owing to their limited interspecific compétition ability and to their rather narrow ecological plasticity The conservation and promotion of these tree species is most promising in situ, within the frame of forestry management (Rotach 1999), with the aim of combining gene conservation efforts and the production of high quality timber. As conservation programmes of noble hardwood species are built up (Turok étal. 1996(Turok étal. , 1998(Turok étal. ,1999), it appears that information on crucial life-history characteristics of these species (e.g. mating System, pollen and seed dispersai) and on their patterns of genetic variation is still lacking. In this context, the application of molecular genetic techniques to the conservation of noble hardwood species is anticipated to bring valuable data that could be used to cxtract relevant biological information (Milligan et al. 1994), to document hotspots of genetic diversity, and to infer their phylogeography (Newton et al. 1999). Forests from southeastem Europe constitute highly valu able réservoirs of genetic resources of noble hardwood species for two reasons. Firstly, many forest stands hâve been maintained in a seminatural status becauseof the low intensity of économie exploitation (European Commission Phare Programme 1999). Secondly, southeastern Europe has been identified as an important refuge for plant species during the Quatemary glacial periods (for reviews see Cornes &Kadereit 1998 andNewton et al. 1999). For Abies nlba, allozyme (Konnert & Bergmann 1995) and chloroplastic microsatellite (cpSSR) markers ( Vendramin et al. 1999) revealed high-diversity potential refuges in the Southern and southeastern Balkan peninsula and in the Croatian Balkans. Chloroplast DNA restriction fragment analysis revealed that central and western European populations of Fagus sylvatica (Démesuré étal. 1996) and Alnus glutinosa (King & Ferris 1998) may hâve evolved exclusively from the expansion of their respective Carpathian refuges.

Important glacial refuges in southeastern Europe were also documented in Quercus ssp. and Picea abies (Cornes & Kadereit 1998). Huntley & Birks (1983) reported expansion of Fraxinus excelsior in the early Holocene from the northern Apaennine peninsula and from the northem and northwestern Black Sea coasts based on fossil pollen evidence.

The common ash, F. excelsior L-, is a postpioneer heli-ophUous tree species occurring in mixed deciduous forests ail over Europe. It is a protandreous wind-pollinated spiecies with a complex mating System, showing variation in sex expression from male to female with hermaphroditic intermediates (Wardle 1961;Picard 1982). Forestry man agement in Europe has shown increased interest in the common ash in the last 30-40 years, as a conséquence of the récognition of its high économie value (Pliura 1999). However, because of technical difficulties with methods of allozyme electrophoresis, little information is so far available on the patterns of genetic variation in the common ash.

In this study, we assess the level of genetic différentiation in F. excelsior from southeastern Europe at three different geographical scales (among individuals within populations; among populations within natural régions of Bulgaria; and between régions) using six nuclear microsatellite loci, with the aim of inferring biological characteristics useful for conservation management such as the level of inbreeding and the variance of gene dispersai distances.

Materials and methods

Plant material

Samples of Fraxinus excelsior were collected in mixed deciduous forests originating from three distinct régions of Bulgaria. Buds or young leaves from about 30 trees were sampled in each of 10 pxapulafions (Fig. 1): three populations were located in the west, near Sofia; three in central Bulgaria; and the remaining four in the northeastem part of the country. Characteristics of the forest stands are given in Table 1. Elévations vary between 100 m and 1050 m. The origin of the common ash populations is putatively autochthonous. Two stands (1 and 2) resuit from coppice re-sprouting, the others are naturally regenerated from seeds. Within each stand, individual trees were sampled at regular intervals and their p>osition was identified with respect to a reference point. In total, 321 F excelsior trees were sampled. Samples of young leaves were dried and kept at room température prior to DNA extraction. Buds were shipped to the laboratory on their twigs wrapped in Fig- 1 Locations of Fraxinus excelsior ptipulations sampled in Bulgaria. Population numbers arc tire same as in Table 1.

Chapter l: Genetic structure ofcommon ash in Bulgaria assessed with nuclear microsatellites 'Brachet et al. 1999. tLefort et al, 1999. wet paper. They were then dissected oui, separated from their scales, and frozen at -70 °C until DNA extraction.

GENETIC STRUCTURE OF COMMON ASH IN BULGARIA 1617

Microsatellite analysis

Total DNA was extracted from 60 to 90 mg dry leaves or 50-70 mg buds using the QIAGEN Plant mini kit (QIAGEN) with slight modifications, including a 15-min incubation at room température of the ground tissue with API buffer and RNAse. Six primer pairs of microsatellite loci wlaich had previously been shown to display easy to read band patterns and a high degree of polymorphism in F. excelsior were used for polymerase chain reaction (PCR) [START_REF] Brachet | Rapid identification of microsatellite loci using 5' anchored PCR in the common ash Fraxinus excelsior[END_REF]Lefortcf al. 1999; see f>erformed on a Biometra® UNO 11 thermocycler or on a Perkin Elmer GeneAmp® PCR System 2400. Tlie forward sequence of each primer pair was labelled with a fluore scent dye (PE Applied Biosystems) at its 5' end: 6-FAM for FEMSATU and FEMSATL16, HEX for M2-30 and FEMSATL19, and NED for FEMSATLll and FEMSATL12.

This allowed the pooling of three PCR products together with an internai size standard (Genescan-350 with ROX dye) in each well after adjustment of their relative concentrations.

As the size ranges of ail loci are overlapping, it was not possible to load more than three IXIR products in one well.

Electrophoresis and détection of PCR products were carried out on denaturing polyacrylamide gels (4.4% acrylamidebisacrylamide 19:1 from Biorad, 36 cm) using an ABI PRISM® 377 DNA sequencer from Perkin Elmer with filter set D. Gels were run for 2 h at 3000 V in TBE buffer. The resulting electrophoregrams were analysed with software Genotypcr® 2.5 from Perkin Elmer.

Estimation of genetic diversity

The following statistics of genetic variation within popu lations were computed as averages over loci with the software gen-survey (Vekemans & Lefèbvre 1997): mean 1618 M. HEUERTZ ET AL.

number of alleles per locus. A; average observed heterozygosity, Hq; average gene diversity, Hg, computed according to Nei (1978); and Wright's inbreeding coefficient, F[g corrected for small sainple size (Kirby 1975). Déviation of genotypic frequencies from Hardy-Weinberg propor tions was tested with the program genepop version 3.1 d [START_REF] Raymond | GENEPOP (Version 1.2) Population genetics software for exact tests and ecumenicism[END_REF].

Within each population, a test of isolation by distance and an indirect estimation of the variance of gene dispersai distances were performed according to a spatial autocor rélation method [START_REF] Hardy | Isolation by distance in a continuons population: réconciliation between spatial autocorrélation and population genetics models[END_REF]) using the software AUTOCORG ver. 2.0 (ohardy@ulb.ac.be). Kinship coefficients were computed between ail pairs of individuals using the sta tistic of Loiselle et al. (1995). The latter is defined for each aUele k and each pair of individuaLs, i and J, as Fjj = (p, -p) i.Pj-p)/(.pO -p)) + l/(2n -1) where p,, pj are the allele frequencies of ; and j, respectively (taking the following possible values: 0,0.5,1), p is the average allele frequency over the reference population, and n is the sample size used to estimate p. This formula produces kinship coeffi cients that are relative to the reference population.

Average multiaUelic and multilocus estimators were obtained by weighting the F, ^ values paer allele by p(l -p). The matrix of pairwise kinship coefficients was regressed on the matrix of the logarithm of pairwise geographical distances between individuals, giving the slop>e 5,,^. Mantel tests with 10 0(K) permutations were used to check the significance of the corrélation between the two matrices. The extent of gene dispersai was estimated as Nb = -(1 -b|og, where Nb can be interpreted as an estimator of Wright's neighbourhood size defined as Nb = 4nDa^, with D equal to the population density, and equal to the axial variance of gene dispersai distances. For graphical représ entation, average kinship coefficients were also computed for pairs of individuals separated by the following distance classes (0-10,10-20,20-40,40-80, > 80 m).

The population genetic structure of the overall sample from Bulgaria was analysed with hierarchical F-statistics computed with the software arlequin (Schneider étal. 2000). The overall inbreeding coefficient (F,j) was com puted, and the proportion of genetic variance was determined for the following components: among populations in the overall sample (Fgj); among populations within régions (Fg,,); and among régions (Ffjy). Tlie significance of each variance component was tested with permutation tests (Excoffier étal. 1992). Because at each locus many pairs of alleles were found that differ by less than one repeat unit, we did not compute statistics based on the stepwise mutation model such as Rgj statistics [START_REF] Slatkin | A measure of population subdivision based on microsatellite allele frequencies[END_REF].

Isolation by distance among populations was assessed by computing Fgf/ (1 Fgy) ratios for each population pair using GENEPOP version 3.1d (Raymond & Rousset 1995). A Mantel test on the matrix of pairwise Fsr/d-fsr) ratios and that of the logarithm of geograpliical distances (natural logarithm-scale) was performed to test for isolation by distance. An indirect estimate of Nb was computed as Nh = \/ (Rousset 1997), where is the slope from the régression of the pairwise Fgj/(1 values on the loga rithm of the geographical distances.

Results

Allelic diversiti/ of microsatellite loci

The total number of putative alleles at each locus and the size ranges of the PCR products corresponding to these alleles are given as observed in this study, and as reported in the original publications that first described these loci (Table 2). It app>eared that ail six microsatellite loci scored in this study were highly pol)rmorphic, displaying a high number of alleles (from 10 to 59 alleles per locus) and a wide size range of PCR products. We recorded between two to five times more alleles per locus than in the original publications, but the original size ranges are only slightly expanded. The total number of alleles scored in 321 individuals over ail loci was 202. For the computation of further statistics we omitted locus FEMSATL12 because it produced two singular features: (i) the single locus estimate of inbreeding coefficient (Fjg = 0.697) was much higher than for the other loci (F|g ranging from -0.016 to 0.061); and (ii) in most p>opulations a large proportion of individuals (varying from 5% to 35% of the sample size) did not give successful amplification after three separate trials. These features could be related to the presence of null alleles at high frequency or to unreliable amplification reactions. Analyses of offspring from controlled crossings are needed to résolve this issue.

Genetic variation within populations

Statistics of genetic diversity within populations are given in Table 3. High polymorphism was found within popu lations, as on average more than 12 alleles were observed per locus (A = 12.36 + 3.22) and the probability that two randomly sampled alleles in a given population were different was 73% (Hg = 0.731 ). The observed heterozygosity (Hq = 0.720) was slightly lower than the expected hetero zygosity (Hg), causing a low but significantly positive mean inbreeding coefficient (Fjg = 0.014). Five out of 10 populations showed an overall significant departure from Hardy-Weinberg genotypic proportions (showing a déficit and an excess of hétérozygotes in two and three populations, respectively), with a mean inbreeding coefficient compriscd between Fjg = -0.037 and F,g = 0.089. Assuming that the small positive value of the mean inbreeding coefficient was caused by partial selfing, we can compute an expected value of the selfing rate according to s = 2F[g/(l -h F|g) (Hartl & Clark 1989), which gives s = 0.027. In each population, the relationship between the matrix of pairwise kinship coefficients betw'een individuals and that of logarithm of spatial distance was analysed with a Mantel test. Significant tests at the 5% level were obtained in ptopulations 1, 2, 8 and 9 (Table 4). Values of the régres sion slopes (bjug) were négative, indicating that on average, individuals which were spatially close were more likely to be genetically related than individuals which were separated by a larger distance (Table 4). The multilocus estimâtes of neighbourhood size (Nb) based on the values varied from 38 to 66 individuals (Table 4). This means that, on average, 86.5% of matings occur within local neighbourhoods comprising from 38 to 66 individuals. For graphical représentation of isolation by distance in these popula fions, we computed for each of five classes of distance cl between individuals the averages (f^) of the multilocus pairwise kinship coefficients

The average kinship coefficients (fjj) were then plotted against d (Fig. 2). Positive values of were found at short distances, meaning that neighbour individuals had a higher genetic relatedness than random pairs of individuals, whereas négative values of occurrcd (Rousset 1997).

loci Overall -----------------------------------------------------------------------
at larger distances, a pattern indicating isolation by distance within a population [START_REF] Hardy | Isolation by distance in a continuons population: réconciliation between spatial autocorrélation and population genetics models[END_REF]). Population 7 was also included in Fig. 2 because the value at the shortest distance class was significantly higher than zéro (P < 0.05) based on a permutation test.

Genetic différentiation among populations and régions

Total genetic diversity recorded in Bulgarian samples of Fraxinus excelsior was high (Hj = 0.793 ± 0.300) and différ entiation among populations was weak but significant: only 8.7% (F^ = 0.087) of the total diversity was attributable to différentiation among populations (Table 5). Of the total différentiation among populations, 80% was explained by divergence among populations within régions (Fjr = 0.070), whereas only 20% accounted for divergence among régions (Frt = 0.018). Substandal inbreeding in the overall sample was ob.served, as measured by the overall inbreeding coefficient (F^ = 0.104).

The Fg.p/(1 -Fgj) ratio for pairs of populations increased linearly with the natural logarithm of the geographical distance (Fig. 3; Mantel test: P = 0.0005, R2 = 0.152), showing a typical pattern of isolation by distance (Rousset 1997), although explaining only 15% of the total variance. The reciprocal of the slop>e (B]"g) of the régression of Fst/(1-Fst) on the logarithm of geographical distance provides an estimate of neighbourhood size, Nb = 126 ~ 0.00797), wlaich is higher but within the same order of magnitude as estimâtes obtained within populations.

Discussion

The diversity assessed with microsatellite markers in our sample of populations of Fraxinus excelsior from Bulgaria is high: the total gene diversity is Hj = 0.793 ± 0.300 and the observed number of alleles per locus ranges from 10 to 59. The allelic diversity reported here is two to five times higher than in the original publications describing the screening of microsatellite loci in F. excelsior [START_REF] Brachet | Rapid identification of microsatellite loci using 5' anchored PCR in the common ash Fraxinus excelsior[END_REF]Lefort et al. 1999), while the size range of PCR products is slightly more expanded. The substantia! différence in allelic richness is most probably due to the larger number of individuals scored in this study (n = 321) compared to the original studies [START_REF] Brachet | Rapid identification of microsatellite loci using 5' anchored PCR in the common ash Fraxinus excelsior[END_REF] Overall, populations of F. excelsior from Bulgaria are slightly, but significantly inbred (F^ = 0.014, Fjj. = 0.104).

For microsatellites, the most commonly reported cause of positive values of the inbreeding coefficient, indicating hétérozygote deficiency, is the presence of null alleles [START_REF] Bruford | Characteristics of microsatellites[END_REF]. This is a likely cause of the singular patterns found at locus FEMSATL12 that weomitted from the analyses. Although we cannot exclude this interprétation for additional loci, other possible explanations are selffertilization and biparental inbreeding, i.e. inbreeding caused by matings between related individuals. Evidence for the occurrence of biparental inbreeding cornes from the analysis of genetic structure within populations. Indeed, we detected an association between genetic relatedness and spatial position of individuals, in agreement with the concept of isolation by distance [START_REF] Hardy | Isolation by distance in a continuons population: réconciliation between spatial autocorrélation and population genetics models[END_REF], in four out of 10 populations. Hence, the positive value of F|g may be due in part to biparental inbreeding but know ledge of the dispersai curves of pollen and seeds would be necessary to quantify the contribution of this process to the observed value of Fjg (Fenstcr et al. in préparation). Ignoring the effects of biparental inbreeding we computed a maximum expected value of the selfing rate that gives § = 0.027. Hence, our computation suggests that the selfing rate within Bulgarian populations of the common ash is lower than 3%.

We detected a pattern of genetic structure consistent with the model of isolation by distance at both intrapopulation and interpopulation levels. Estimâtes of the variance of gene dispersai distances obtained from intrapopulation analyses ranged from 38 to 66 individuals, when expressed as Wright's neighbourhood size, and a value of 126 was obtained from the interpopulation analysis. Similar con gruence between estimâtes of gene disp>ersal obtained from analyses within and between populations has been reported in two other plant spodes (on oak, when comparing © 2001 Blackwell Science Ltd, Moltxular Emlogt/, 10,1615-1623 results from Le Corre et al. 1998 andStreiff et al. 1999;on Qiamaecrista fasciculata, Fenster et al. in préparation). In another noble hardwood sjocies, sugar maple, [START_REF] Perry | Spatial genetic structure within three sugarmaple (Acer sacchantm Marsh.) stands[END_REF] also found significant spatial genetic struc ture within three forest stands. From a reanalysis of their data sets with similar procedures as those used in this study, we obtained estimâtes of neighbourhood size close to those in the common ash, ranging from 29 to 59 indi viduals. In contrast, in oak, estimâtes of neighbourhood size ranging from 3000 to 4000 trees, and of neighbourhood area between 12 and 20 ha, hâve been obtained based on analyses of spatial distribution of allozyme frequencies (Le Corre étal. 1998) and on paternity analysis (Streiff étal. 1999). For comparison, the neighbourhood area in the common ash (computed as Nb/D where D is the pxjpulation density) would vary between 0.63 and 0.81 ha based on within-population estimâtes of Nb when an average density of 200 mature trees per hectare in a mixed decidu ous forest with a proportion of F. excelsior ranging from 10 to 100% (Table 1) is assumed. There is thus one order of magnitude différence between estimâtes of gene dispiersal in oak and in the common ash. This could resuit from true différences in dispersai abilities of the two tree sp>ecies. Indeed, very limited pollen dispersai has been reported in F. excelsior (50% and 95% of pollen dispersed at less than 10 m and 50 m, resp>ectively, Altman & Dittmer 1964). Also seed disp>ersal mechanisms differ with heavy winddispiersed seeds in the common ash and a combination of gravity and bird-mediated disp>ersal in oaks. Alterna tively, the large différence in gene flow estimâtes could resuit from différences in sampling (much smaller sampling area in our study as compared to those for oak) and estimation procedures (indirect vs. direct estimation of variance in gene dispiersal distances). In this study, an indirect method based on the effect of restricted gene flow on spatial genetic structure was used. Such methods are derived from par ticularly idealized models of populations from which many assumptions may be violated in nature (e.g. the equilibrium hypothesis, Whitlock & McCauley 1999). Moreover we did not find any indication of spatial genetic structure within five of 10 populations. This could be due to the particular history of these populations, i.e. recent origin or influence of forestry management, or it could resuit from the fact that the sampling area was shorter than the actual neighbourhood area in these forest stands (Rousset 20(K)). In the latter case, ignoring these populations would lead to a downward bias in the estimation of gene dispersai.

As commonly found in tree species, we observed a low overall différentiation among populations (Fgj = 0.087). In comparison, the average estimate of population différ entiation among 322 woody taxa for allozyme lod is Ggj. = 0.084 (Hamrick et al. 1992), whereas lower estimâtes were obtained for oak (Ggj = 0.024 and 0.032 in Quercus petraea and Q. robur, respectively, Zanetto étal. 1994) and beech (in Lagus syhmtica, GgT = 0.054, Comp.s étal. 1990; Fst = 0 .046, [START_REF] Leonardi | Genetic variability of beech (Fagus fylvatica L.) in Italy: the rôle of postglacial recolonization[END_REF]. These comparisons are in agreement with the suggestion that gene flow is more restricted in F. excelsior than in oak and beech, but could aiso be due to différences in processes of postglacial recoloniza tion [START_REF] Austerlitz | Effects of colonization processes on genetic diversity: différences between annual plants and tree species[END_REF]. 11 must be recognized tha t comparisons of values between allozyme and microsatellite data may be misleading because of the very high mutation rates of microsatellite loci tha t can cause a downward bias in estimation of genetic différentiation [START_REF] Nagylaki | Fixation Indices in Subdivided Populations[END_REF]). However, this should not hamper the conclusion that based on our study the différentiation among popula tions is higher in the common ash than in oak and beech. [START_REF] Wright | Isolation by distance[END_REF][START_REF] Epperson | Recent advances in corrélation analysis of spatial patterns of genetic variation[END_REF][START_REF] Doligez | Fine-scale spatial genetic structure with non-uniform distribution of individuals[END_REF]. Gene flow through pollen and seed dispersai is a key déterminant in the establishment of genetic structure: when overall gene flow is restricted, a decrease in genetic relatedness among pairs of individuals is expected with increasing géographie distance between them, in agreement with models of isolation by distance [START_REF] Wright | Isolation by distance[END_REF][START_REF] Malécot | Quelques schémas probabilistes sur la variabilité des populations naturelles[END_REF].

Many studies on forest tree species hâve tested for local spatial genetic structure using genetic markers and the tools of spatial autocorrélation analysis. In most cases, evidence for statistically significant spatial genetic structure was found (e.g. [START_REF] Schnabel | Comparative analysis of population genetic structure of two co-occurring tree species, Madura pomifera (Moraceae) and Gleditsia triacanthos (Leguminosae)[END_REF][START_REF] Sork | Macrogeographic and fine-scale genetic structure in a North American oak species, Quercus rubra L[END_REF][START_REF] Berg | Fine-scale genetic structure of a turkey oak forest[END_REF]Streiff et al. 1998;Chung & Epperson 2000;[START_REF] Takahashi | Différences in genetic structure between two Japanese beech (Fagus crenata Blume) stands[END_REF][START_REF] Ueno | Genetic structure of Camellia japonica L. in an old-growth evergreen forest, Tsushima, Japan[END_REF][START_REF] Dutech | Evidence of low gene flow in a neotropical clustered tree species in two rainforest stands of French Guiana[END_REF]. In some species, however, spatial distribution of génotypes was close to random [START_REF] Epperson | Spatial autocorrélation analysis of the distribution of génotypes within populations of lodge-pole pine[END_REF][START_REF] Knowles | Spatial genetic structure within two natural stands of black spruce {Picea mariana (Mill.) B.S.P.)[END_REF][START_REF] Xie | Spatial genetic substructure within natural populations of jack pine (Pinus banksiana)[END_REF]Chung et al. 2000). The latter situation was observed in tree species where both pollen and seed dispersai are wide-ranging, as for example in wind-pollinated and wind-dispersed [START_REF] Berg | Fine-scale genetic structure of a turkey oak forest[END_REF]Hamrick 1995, Q. robur andQ. petraea, Streiff et al. 1998) was observed at short spatial scale. This pattern was attributed to localised, gravity-mediated seed dispersai, as pollen dispersai by wind is extensive in those species [START_REF] Sork | Macrogeographic and fine-scale genetic structure in a North American oak species, Quercus rubra L[END_REF][START_REF] Berg | Fine-scale genetic structure of a turkey oak forest[END_REF], Streiff et al. 1998). Strong spatial genetic structure was found in tree species featuring substantial restrictions to both pollen and seed dispersai, i.e. pollination by small insects and seed dispersai by gravity (Gleditsia triacanthos, [START_REF] Schnabel | Comparative analysis of population genetic structure of two co-occurring tree species, Madura pomifera (Moraceae) and Gleditsia triacanthos (Leguminosae)[END_REF]; Eurya emarginata, Chung & Epperson 2000).

Relative pollen and seed contributions to gene flow can readily be estimated from genetic structure when it is analysed concomitantly at markers with different modes of inheritance (Latta et al. 1998[START_REF] Latta | Cytonuclear disequilibrium and genetic drift in a natural population of ponderosa pine[END_REF]). However, it remains unclear whether the relative contributions of pollen and seed dispersai could be deduced from the observed patterns of spatial genetic structure at nuclear loci alone. Methods for estimating the extent of local gene flow from the observed spatial genetic structure within populations hâve recently been proposed [START_REF] Hardy | Isolation by distance in a continuons population: réconciliation between spatial autocorrélation and population genetics models[END_REF]Rousset 2000) and applied to several plant species

Materials and Methods

Plant material

The study site is a putatively autochthonous continuons mixed deciduous forest (approximately 710 ha) located in southeastem Romania (44°50'N, 26°04'E) near the city of Ploesti (Fig. 1). Fraxinus excelsior occurs there at high density, on average 200 mature trees (trees with diameter at breast height, d.b.h. >15 cm) per ha, in mixture with Quercus robur, Acer platanoides, A. campestre, A. tataricum, Ulmus minor and other broadleaved tree

species. An average of 30 non-adjacent common ash trees were sampled and mapped in each of five sampling locations, called subpopulations hereafter, covering an area of 0.71 to 3.69 ha (average 1.68 ha). Distances between subpopulations ranged from 514 to 3043 m. In total, 152 trees were sampled. Samples consisted of buds, which were shipped to the laboratory on their twigs wrapped in wet paper. They were then dissected out, separated from their scales and kept at -70°C until DNA extraction.

Microsatellite analysis

Total DNA was extracted with the CTAB procedure of the NucleoSpin Plant kit (Macherey Nagel) from 50 to 70 mg of buds ground in an automatic grinding mill (Retsch MM200).

Microsatellite analysis was performed as described in Heuertz et al. (2001). Five highly polymorphie microsatellite loci were amplified with polymerase chain reaction (PCR)

(Table 1). The quantity of template DNA in the PCR could be increased to 3 to 10 ng compared with the previously published protocol, probably because métabolites susceptible to interfère with PCR were better eliminated with the DNA extraction kit used here. Fluorescent labeling of the forward primers allowed détection of amplification products on an automated DNA sequencer (ABI PRISM® 377 DNA sequencer). Sizing of fragments was performed with the software programs Genescan® 3.1 and Genotyper® 2.5 from Applied Biosystems by comparison with an internai sizing standard (Genescan-350 Rox). Another modification brought to the previously published protocol was that polyacrylamide gels were produced with 10% LongRanger® gel solution (Sanver Tech).

Microsatellite data analysis

The following statistics of genetic variation were computed for each locus and as averages In order to test for isolation by distance, the multilocus kinship coefficient for each pair of individuals was plotted against the logarithm of the géographie distance separating them (kinship-distance plot). The significance of the linear régression line was tested by Mantel tests with 10000 permutations. The extent of gene dispersai was estimated from the observed régression slope bro as

Nb =-H-Fo)/bro (1)
where Fo is the kinship coefficient between adjacent individuals, i.e. in distance class zéro.

Nb can be interpreted as an estimator of Wright's neighbourhood size defined as Nb = AnDeO^, where De is the effective population density, and cr/ the second moment of gene dispersai distance between parents and offspring [START_REF] Hardy | Isolation by distance in a continuons population: réconciliation between spatial autocorrélation and population genetics models[END_REF], Rousset 2000). A linear decrease of pairwise kinship coefficients with the logarithm of distance is expected within a distance range comprised between approximately Gg and 20(7^, and consequently the inference of Nb is most reliable in this range (Rousset 1997(Rousset , 2000)). Ail computations were populations (Kremer 1994) and which allowed to neglect mutation. Seed and pollen dispersai followed an isotropie bivariate normal distribution characterized by parameters Oi and Op, respectively. We ran 100 independent simulation replicates in each of 56 combinations of dispersai parameters, where Oi = 0.75, 1,2,3,4,5,7,10 and Gp = 3,5,7,10,20,50, 100 (in lattice units; these values are to be multiplied by seven to obtain a in meters). After 1000 simulation cycles, equilibrium spatial genetic structure was reached for most parameter combinations, except when both seed and pollen dispersai were very narrow. In each replicate, 152 individuals were then sampled at the grid positions corresponding to those closest to the position of sampled trees in the reference population if the latter had been overlaid by a 7 x 7 m grid. Average multilocus kinship coefficients per distance intervals, Fk, were computed for the following 17 distance classes (upper bound distance in grid units;

times 7 to obtain meters): 1,2,3,4,5,6,7,8,9,10,12,15,20,40,200,300,600. We used short intervals for the first distance classes to get a detailed picture of how kinship varies with distance at a small spatial scale (i.e. within subpopulations, <40 lattice units). At larger spatial scales, wider intervals were used because kinship is then expected to vary little and estimâtes suffer less stochastic variance.

Analysis of simulated data sets

In order to identify trends of variation among the kinship-distance plots, kinship coefficients Assuming that density was constant over time, the effective density De was computed from [START_REF] Crawford | The estimation of neighbourhood parameters for plant populations[END_REF], where D corresponds to the density of individuals, V is the variance of the lifetime reproductive success among individuals, and F\ is Wright's inbreeding coefficient. V and Fj were recorded in the course of the simulations and we found V~5 and F|=0.01 for ail parameter combinations, resulting in De~0.56D. Similarly, as seed and pollen dispersai events occurring in the simulations were recorded, we also computed the realised neighbourhood size and pollen and seed dispersai standard déviations,

Dg = d[4/(2-i-F)][i/(1-i-F/)]
Nbr, Osr and Opr, respectively (cr^ = rf jN , where r, is the distance crossed by the /* dispersai event, N being the total number of dispersai events recorded; 9.5, 14, 22, 27.5, 36.5, 42.5, 49, 56, 63, 71, 84, 105, 140, 280, 1400, 2100, 4200.

Subsequently, the fit of the observed kinship plot was investigated (i) using a statistic describing the global departure between observed and simulated kinship coefficients over ail distance classes, (ii) using two statistics based on the régression of kinship coefficients on the To represent graphically the quality of the fit to the simulation results according to each of the three statistics described above, we attributed P-values for each parameter combination according to the ranking of the statistic obtained for the common ash data with those of the simulation replicates. P-values were plotted on a grid representing ail 56 parameter combinations and contours of P = 0.05, P = 0.2 and P = 0.5 were interpolated automatically with Microsoft Excel. Parameter combinations that had not been rejected in any of the three tests defined the space of parameters compatible with the common ash data.

Results

Microsatellite analysis of the common ash population

Allelic diversity of microsatellite loci. The total number of alleles per microsatellite locus in the total sample of 152 individuals was comprised between 10 and 42, with an overall total of 148 alleles scored over the five loci (Table 1). Gene diversity H ranged from 0.568 to 0.918 per locus, and Wright's inbreeding coefficient F\ from -0.066 to 0.165 (Table 1). The exact test for departure from Hardy-Weinberg genotypic proportions was non-significant for ail loci.

Genetic diversity within subpopulations and the total population. Genetic diversity statistics for subpopulations and the total population are given in Table 2. Average number of alleles per locus A and gene diversity H were similar in ail subpopulations; they ranged respectively from 13.2 to 16.6 and from 0.765 to 0.807. For the total population, A = 29.6 and H = 0.792.

Wright's inbreeding coefficient Pi was comprised between -0.022 and 0.06 within subpopulations and Pi = 0.029 for the total population. No departure from Hardy-Weinberg genotypic proportions was detected.

Spatial genetic structure. In agreement with models of isolation by distance, a significant linear decrease of pairwise kinship coefficients Pÿ with the logarithm of increasing géographie distance was detected in subpopulations 1, 2, and 4 (P < 0.05) and in the total population (P < 0.01) (Table 3), although only few average kinship values per distance class lay outside the 95% confidence interval obtained from 10000 random permutations of locations among individuals (Fig. 2). The neighbourhood size Nb could be estimated from but can become substantially biased under wide-ranging dispersai (tTg>70m, x symbols).

Chapter //; Seed versus pollen disperstil in conimon asii N h = 518.5, an estimate five limes larger than at short spatial scale (Table 3). Inferred neighbourhood sizes within subpopulations are in agreement with those previously obtained at a similar spatial scale in Bulgaria {Nb = 37.9 -66.3, Heuertz et al. 2001). Note that régressions were applied over the whole available distance ranges, which did not necessarily correspond to the adéquate distance range for Nb inference based on équation (1).

Computer simulations

Analysis of simulated data sets. Within the distance range comprised between Og and 20Og, average kinship coefficients decreased linearly with the logarithm of géographie distance (Fig. 3), as verified by the average value of the coefficient of détermination, ^ = 0.968 ± 0.044 (SD), for parameter combinations with ap<140m. For combinations with larger Op, the régression could not be computed because the upper bound, 20Og, lay outside the simulated distance range. Wide-ranging gene dispersai (Og=60m, filled symbols in Fig. 3) produced shallower slopes than narrower dispersai (ag=37m, open symbols). Linear régression over the total distance range resulted in R^= 0.737 ± 0.272 (SD) for ail parameter combinations and = 0.875 ± 0.100 (SD) for those with Op< 140m, showing that déviation from linearity is substantially more pronounced than within the Og to 20Og distance range. Interestingly, déviations from the linear relationship at a distance shorter than Og depended much on the relative contributions of pollen and seed dispersai, as illustrated by three parameter combinations with similar total gene dispersai (üg = 37 m; Fig. 3): narrow dispersai of seeds as compared to pollen produced an upward concave initial curving (squares in Fig. 3); and, as the relative seed dispersai contribution increased, the shape of the curve flattened (circles) before becoming downward concave (triangles). It is noteworthy that, in agreement with theoretical expectations, the three parameter combinations with contrasting <3^ and Op but very similar Og values show the same pattern of decrease of kinship coefficients within the üg -20

Og distance range (Fig. 3).

Estimâtes of standard déviations of gene dispersai distances using slopes br computed over the adéquate distance range (Og to 20 Og, [START_REF] Roussel | Genetic différentiation and estimation of gene flow from F-statistics under isolation by distance[END_REF]Roussel , 2000) ) were close to the expected values (Fig. 4). When the régression was applied on a too short distance range, such as within common ash subpopulations, substantial errors appeared on the estimation of Og (Fig. 4):

dispersai was underestimated when the seed vs. pollen contribution was small (i.e. steep négative slope at short distances, Fig. 3); and it was overestifnated when the relative seed contribution was important (shallower slope at short than at large distance, Fig. 3).

After Slope br. Wide-ranging pollen dispersai and a combination of narrow pollen and narrow seed dispersai were incompatible with the observed data (Fig. 6b). Overall, 31 of the 56 simulated parameter combinations remained compatible at a = 0.04. A zone of relatively good fit (here: P > 0.5) was observed for roughly the same parameter combinations as in the Chi-square-like test (compare Figs. 6a and6b).

Shape d. Generally, narrow pollen dispersai ((Tp<49m) produced shapes of kinship plots differing from the observed data: although only 13 parameter combinations with o), < 49 m could be rejected at a = 0.04 (Fig. 6c), several others were at the verge of significance. Good fit (P > 0.5) was observed for narrow seed dispersai ((7,<14 (21) m) and moderate to wideranging pollen dispersai {Op> (70) 140m).

The direct comparison of the shape parameter in the common ash population, d = -0.084, with the relationship of d to seed vs. pollen dispersai contributions (Fig. 5) produced an estimate of CTj'/fO.ScTp) comprised between approximately 0.002 and 0.04. The resulting ratio of seed vs.

pollen dispersai standard déviations was 7.1 < Op/Gs <31.6.

Discussion

In the Ploesti forest in southeastem Romania, we detected spatial genetic structure in common ash in three of the five subpopulations and over the area of the total population. In general, kinship coefficients among pairs of individuals decreased with increasing logarithm of géographie distance between individuals. However, this decrease was not uniformly linear over the whole range of distances, as reflected by steep régression slopes at short distance, and a shallower slope at large distance. Resulting estimâtes of the neighbourhood size were more than five times larger at the scale of the total population than within subpopulations. In order to find explanations for this observed pattern, possibly in terms of seed vs. pollen dispersai, we thoroughly examined the shape of kinship to distance plots from simulated datasets.

Estimating gene dispersai from spatial genetic structure by the régression approach Simulations hâve shown that linearity of the decrease of kinship coefficients against the logarithm of distance (Rousset 1997(Rousset , 2000) ) is verified when régression is performed over an adéquate distance range (Fig. 3). The resulting estimâtes of gene dispersai are close to their expectations (Fig. 4), confirming the models of [START_REF] Hardy | Isolation by distance in a continuons population: réconciliation between spatial autocorrélation and population genetics models[END_REF] and Rousset (2000).

However, substantial errors occurred on the estimation of gene dispersai when régression was performed over a too short distance range (Fig. 4). Using the proper interval for régression is thus essential for a reliable estimate of Nb. As cr^ is usually unknown, the following approach can be attempted to détermine the right interval: (i) estimating Gg from the first Nb estimate, knowing De-, (ii) estimating Nb again by performing linear régression over an interval of to 20(7^; and (iii) repeating the procedure iteratively until the estimâtes and Nb stabilise (e.g. Fenster et al. in préparation). When this method was initialised on the entire dataset

Chapter II: Seed versus pollen dispersai in common asli using within-subpopulation dispersai estimâtes, successive shifted repeatedly to larger values, suggesting that the within-subpopulation scale is too narrow to estimate Nb. However, even at the level of the whole population, did not stabilise because the a posteriori régression slope became positive for ô^> 53m or Nb> 395 (using De = 0.56D). This was attributed to stochastic variation of the data (see below). Hence, the suggested approach to déterminé the relevant interval for estimating Nb could not be applied to the current data set.

Application to other data sets will be needed to test the usefulness of the approach.

Impact of seed vs. pollen dispersai on the observed spatial genetic structure at nuclear loci The kinship-distance plot in the entire common ash population was characterised by a markedly sharper decrease of kinship at short than at long distances. One possible explanation is restricted seed vs. pollen dispersai as revealed by our simulations (Fig. 3), where we assumed normal dispersai distributions. More generally, simulation results showed that a steep decrease of kinship at short distance occurs when gene dispersai follows a highly leptokurtic distribution (O. J. Hardy, unpublished data). Highly leptokurtic gene dispersai distributions resuit, firstly, from situations where seeds are substantially less dispersed than pollen; i.e. half the genes move over short distances (the maternai ones), and the other half can move over large distances (the patemal ones), producing a leptokurtic composite distribution. If in that case, seed and pollen dispersai distributions are not normal as we assumed, but for example exponential, the adequacy of our method to estimate the ratio of seed V5. pollen dispersai still needs to be tested. Secondly, highly leptokurtic gene dispersai distributions also occur when both seed and pollen dispersai distributions are (i) equal and (ii) highly leptokurtic themselves. In that case, our method would produce a wrong estimate of seed vs. pollen dispersai, as it assumes normality. Wind-dispersed pollen and seed, which are both characteristic for the common ash, may follow leptokurtic dispersai functions, as suggested in the literature. For instance, pollen déposition patterns in anemophilous plants were explained through leptokurtic dispersai functions (e.g. [START_REF] Levin | Gene flow in seed plants[END_REF][START_REF] Ellstrand | Gene flow by pollen: implications for plant conservation genetics[END_REF][START_REF] Richards | Plant Breeding Systems Second édition[END_REF], and patemity analyses hâve often left high proportions of offspring with no potential father within the study population, demonstrating a strong long-distance component in wind-mediated pollen dispersai [START_REF] Burczyk | Male reproductive success in Pseudotsuga menziesii (Mirb.) Franco: the effects of spatial structure and flowering characteristics[END_REF], Streiff et al. 1999[START_REF] Vassiliadis | High male reproductive success of hermaphrodites in the androdioecious Phillyrea angustifolia[END_REF]. Indications for leptokurtic dispersai of wind-bome winged seeds are weaker, referring mainly to an important déposition close to the source detected in direct studies (e.g. [START_REF] Levin | Gene flow in seed plants[END_REF][START_REF] Johnson | Estimating dispersibility of Acer, Fraxinus and Tilia in fragmented landscapes from patterns of seedling establishment[END_REF]). Wind-dispersed seed of tree species are not expected to move as far as pollen, because of their generally much greater mass. More restricted dispersai of wind-bome seed compared to pollen was also confirmed by a stronger spatial genetic structure at matemally than patemally inherited markers in conifer species Fig. 7 Stochasticity in plots of average kinship coefficients Fÿ against the logarithm of distance: observed values in the common ash population (bold line) and four independent simulation replicates for <Ts=7m and CTp=70m (plain lines) (Latta et al. 1998, Liepelt et al. 2002). Hence, although it is likely that dispersai functions of pollen and seed in common ash may contain a leptokurtic component potentially biasing our estimate of relative dispersai levels, we think that most of the leptokurtosis of gene dispersai is due to the relative différence between mean seed and pollen dispersai distances.

Implications of the shape of the kinship-distance plot on the estimate of dispersai

In the case of the common ash data, the steep négative initial slope of the kinship-distance plot and the démonstration that the within-subpopulation scale is inadéquate for the régression approach indicate that we most certainly underestimated Nb at short distance.

In general, departures from linearity of kinship-distance plots, possibly originating from unequal seed vs. pollen dispersai, resuit in a non-random distribution of residuals from linear régression. The latter can be approximated by cubic régression functions whose coefficients, and notably the coefficient d of the term of third power of the logarithm of distance, are strongly correlated to the relative contributions of seed v^. pollen to total dispersai. Hence, in the context of a particular data set, d can provide an estimate of the relative magnitudes of seed and pollen dispersai, independently of the slope of the kinship-distance plot. The effect of seed and pollen on d is especially verified for seed contributions 1000 times smaller up to equalling pollen contributions (Fig. 5). Larger seed than pollen contributions probably do not reflect a biologically realistic situation in wind-pollinated and wind-dispersed species like common ash, and seed contributions more than 1000 times smaller than pollen contributions most likely represent rather isolated cases of long-distance pollination.

Power of methods and stochastic variation

The analysis of the fit of the observed spatial genetic structure to that obtained from simulations revealed that the décomposition of the dispersai fonction into a slope and a shape component allowed to withdraw more information than considering simply the overall departure using a Chi-square statistic; i.e. the combined tests on the slope br and the shape d were able to reject 31 parameter combinations at a = 0.04, compared to the Chi-square-like test, which rejected only 20 at a = 0.05. Notwithstanding, a large range of parameter combinations remained compatible with the observation. Although more simulation replicates (200 or 500 instead of 100) might hâve reduced the number of compatible combinations by rejecting those at the limit of significance, we argue that the main cause for weak rejection power is the stochasticity inhérent to the data. Large fluctuations of kinship values were indeed found in the observed and the simulated data, despite the analysis of 5 highly polymorphie microsatellites in 152 individuals (Fig. 7). Such fluctuations could be related to the sampling scheme, however, simulations with other sampling locations but respecting the same distance classes showed similar variation. The précision that can be reached on an Nb estimate based on spatial genetic structure is actually much dépendent on the real Nb value itself: the stronger the spatial structure (i.e. the smaller the Nb), the smaller the coefficient of variation of the régression slope, hence the more précisé the Nb estimate. The présent study suggests that a larger sample size would hâve been necessary to infer gene dispersai with sufficient précision in common ash.

Estimâtes of dispersai in common ash

For the common ash, results from our investigations suggest a neighbourhood size. Nb= 519, relative contributions of pollen vs. seed dispersai of 7.1 < OpIOs <31.6, and restricted seed and moderate pollen dispersai, with most probable parameters of, respectively, Oi < 14m and 70m

S <Tp < 140m. The latter would translate into a neighbourhood size estimate eomprised between 363 and 1468 individuals when computed as Nb = 47tDg (cr^ + 0.5(7^ ).

These estimâtes of Nb are much higher than our previous results from Bulgaria {Nb= 37.9-66.3, Heuertz et al. 2001); but the latter estimâtes are likely biased downward because the samples were taken at too narrow distance. The Ploesti common ash population is an unusually dense one, as this species mostly occurs scattered in European mixed deciduous forests. The very restricted seed dispersai suggested here probably reflects the high vertical terminal velocities of ash seeds (1.2-1.7 m/s, Johnson 1988, Greene and[START_REF] Greene | Long-distance wind dispersai of tree seeds[END_REF] dispersing under a relatively closed forest canopy where wind velocity and turbulence are reduced [START_REF] Levin | Gene flow in seed plants[END_REF]. Our estimate is smaller than that of Morand-Prieur et al.

(in préparation) who found a mean seed dispersai distance of 125m in a parentage study in a French common ash population (which corresponds to = 99.7 m in case of a normal dispersai fonction, using distance d = ô.yjTr/2 ). Various factors might contribute to explain this discrepancy, for instance a lower density and an elongated shape following a brook, with a preferential direction of seed dispersai in the latter population. Our own estimâtes of fis and

(Tp might also be underestimated if the effective density in the real population was much lower than we assumed (i.e. DJD = 0.5). Our 7 to 32-fold farer pollen than seed dispersai estimate based on standard déviations compares favourably with the estimated ratio of 13 based on mean dispersai distances by Morand-Prieur et al. (in préparation). Estimâtes of pollen dispersai from the présent study are slightly lower, but of the same order of magnitude than in the widely occurring European white oaks, where patemity analysis revealed Gp = 140 m for Q. petraea and Op = 176 m for Q. robur (Streiff et al. 1999); and pollen sédimentation velocities in oaks and in ashes are similar (~ 3 cm/s, Jackson & Lyford 1999).

Introduction

The patterns of genetic variation within plant species over a wide distribution range, like the western European continent, hâve been found to resuit from a combination of factors, some of which are intrinsic to the species life history (breeding System, modes of seed and pollen dispersai, life form, gregariousness), while others are perturbations induced by natural processes (ice âges, climatic stochasticity) or human impact (habitat fragmentation, global change).

For temperate tree species, a common observation is that they présent very high genetic diversity at nuclear loci, but very little genetic différentiation among populations (Hamrick & Godt 1989, Hamrick et al. 1992). Many biological characteristics of temperate tree species hâve been put forward to explain this general pattern: long génération time, woody life form, high fecundity, outcrossing mating System, prédominant wind pollination [START_REF] Hamrick | Influence of microhabitat heterogeneity on gene frequency distribution and gametic phase disequilibrium in Avena barbata[END_REF], Loveless & Hamrick 1984). Indeed, some of these characteristics are expected to influence genetic variation and population genetic stmcture through their effects on the mutation-selection balance [START_REF] Charlesworth | The effect of deleterious mutations on neutral molecular variation[END_REF], effective population size [START_REF] Pollak | On the theory of partially inbreeding finite populations. I. Partial selfing[END_REF][START_REF] Schoen | Intraspecific variation in population gene diversity and effective population size correlates with the mating System in plants[END_REF], and gene flow [START_REF] Levin | Dispersai versus gene flow in plants[END_REF].

Details of historical processes, such as the most recent post-glacial recolonisation évents, hâve also been shown to imprint the genetic stmcture of several temperate tree species (Cornes & Kadereit 1998, Newton et al. 1999, Hewitt 2000). In many species successive founder effects during recolonisation lead to a graduai decrease in genetic diversity from ancient glacial refuges towards recently recolonised areas [START_REF] Tomaru | Genetic diversity in Fagus crenata (Japanese beech): influence of the distributional shift during the late-Quatemary[END_REF], Ally et al. 2000[START_REF] Ledig | Founder effects and the genetic stmcture of Coulter pine[END_REF].

Populations founded by recolonisation from distinct refuges are often highly divergent (Lagercrantz & Ryman 1990, Konnert & Bergmann 1995), and régions where previously separated lineages merge often feature high diversity (Lagercrantz & Ryman 1990, Zanetto & Kremer 1995, Comps et al. 2001). Interactions between these historical events and biological characteristics of temperate tree species, such as potential for long-distance dispersai (Ibrahim et al. 1996, Le Corre & Kremer 1998) and an extended juvénile phase [START_REF] Austerlitz | Effects of colonization processes on genetic diversity: différences between annual plants and tree species[END_REF], hâve probably contributed to a large extent to shaping their current diversity patterns. Human Another approach is to study founder events associated to the recolonisation process by comparing actual gene diversity and gene diversity estimated from the observed number of alleles assuming mutation-drift equilibrium (Comuet & Luikart 1996), knowing that bottlenecks produce transiently a stronger decrease in allelic richness than gene diversity (Nei et al. 1975). Results with this method reach from the détection of significant bottlenecks, i.e.

in 19% of European beech populations, ail located away from refuge areas (Comps et al. 2001); to the opposite pattern, compatible with recent population expansion rather than size réduction, i.e. in mountain hemlock (Ally et al. 2000).

Recently, a new approach has been proposed to identify patterns of genetic diversity: a model-based clustering method that uses multilocus génotypes to infer population stmcture and assign individuals to populations, allowing for population admixture (Pritchard et al. 2000). The method has successfully identified population structure and population admixture in several mammal species including humans [START_REF] Rosenberg | Distinctive genetic signatures in the Libyan Jews[END_REF][START_REF] Wilson | Genetic evidence for different male and female rôles during cultural transitions in the British Isles[END_REF]) and cats [START_REF] Randi | Genetic identification of wild and domestic cats {Felis sylvestris) and their hybrids using Bayesian clustering methods[END_REF] has not yet been applied to large-scale investigations of population structure in tree species. This is possibly because hitherto most large-scale genetic surveys in tree species hâve been conducted with allozyme markers, which hâve limited resolution in identifying multilocus génotypes. Another limitation of allozymes is their low phylogeographic information content, making it difficult to distinguish alternative causes for diversity patterns, i.e. recent gene flow or recolonisation history (Zanetto & Kremer 1995). In this respect, nuclear haplotype data are becoming a promising new tool for evolutionary inference [START_REF] Hare | Prospects for nuclear gene phylogeography[END_REF]. Altematively, when using microsatellite markers, phylogenetic information may be contained in the distribution of allelic sizes. A useful test for detecting such information has been developed (Hardy et al. 2002).

The general objective of our study is to evaluate whether the use of microsatellite markers may substantially improve the identification of the factors responsible for the current genetic patterns in tree species. Microsatellites allow better estimâtes of population genetic statistics because their high polymorphism ensures low standard errors (Goudet et al. 1996) and they enable the application of new approaches of data analysis such as the model-based clustering method and a test of phylogenetic information content of allele sizes. Our model plant is the common ash, Fraxinus excelsior, a temperate tree species occurring in mixed deciduous forests from nearly ail Europe, with the exclusion of the most southem and most northem parts. Common ash exhibits intermediate properties between a pioneer species and a permanent forest component; it features strong colonization capacity, but compétitive ability is high only when ecological requirements are met. Flowers are wind-pollinated and the single-seeded fmits, the samaras, are wind-dispersed. The mating System of common ash is complex and variable: flowers are male, hermaphroditic or female and there is a continuum from pure male to pure female individuals with hermaphroditic intermediates (Wardle 1961, Chapter III: Genetic structure ofcommon asii in Europe assessed with nuclear microsatellites Picard 1982[START_REF] Binggeli | Gender variation in ash[END_REF]. Chloroplast haplotype data (G. G. Vendramin, in préparation) and fossil pollen records (Huntley & Birks 1983, Gliemeroth 1997, Brewer 2001) suggest glacial refuges for ash in the Balkans and Italy and/or the Alps, while refuges in Iberia and north of the Black Sea are less strongly supported. Postglacial recolonisation of continental Europe would hâve occurred from those refuges.

We sampled thirty-six populations of common ash in Europe and analysed them at five nuclear microsatellite loci with the objectives of ( 1) identifying geographical patterns of within-and among-population diversity, (2) clarifying genetic and phylogenetic relationships between régions using distance or model-based clustering methods and analyses of allelic size distributions, and (3) attempting to interpret the observed patterns in ternis of originating evolutionary events with particular emphasis on discussing whether the properties of microsatellite markers added additional power in the evolutionary inference.

Materials and Methods

Plant material

Samples of Fraxinus excelsior were collected in 36 putatively autochthonous forests in Europe (Table 1, Figure 3). Samples consisting of buds or leaves were taken from an average of 30 non-adjacent trees within each population. Buds were shipped to the laboratory on their twigs, wrapped in wet paper. They were dissected out, separated from their scales and showed less thcn half the polymorphism of other loci. Little variation in Fst (CV = 25.9%) but strong variation in F (CV = 168.8%) and /?st (CV = 93.5%) were observed among loci (Table 2). In a previous publication conceming only 321 individuals from ten populations of Bulgaria (Heuertz et al. 2001), we observed an overall total of 184 alleles for the same set of loci. Total allelic richness standardised to the size of the Bulgarian data set is higher for the présent study (At= 226), indicating that the higher allelic diversity detected is not only due to a larger sample size, but also to the larger géographie area investigated. In a study of 240 ash seedlings from 12 French populations, Morand et al. (2002) found 169 alleles for four of the loci that we used (ail except FEMSATL16); omitting this locus, our présent data set featured 259, and the Bulgarian set 174 distinct alleles.

Genetic variation within populations

Statistics of genetic variation are given as population averages for the twelve local régions and the four zones (Table 3). Corrélation coefficients between geographical variables and within-population statistics are given in Table 4.

The mean allelic richness (As_36) within individual populations varied from 6.14 to 14.90 (standardised samples of 36 gene copies) with an overall mean of 11.33. Nei's gene diversity (He) varied from 0.650 to 0.853, with a mean value of 0.770. An overall significant departure from Hardy-Weinberg genotypic proportions (Fisher's exact test, P < 0.05) was observed in 15 out of 36 populations, two of which showed an excess and 13 a déficit of hétérozygotes.

Statistically significant values of Wright's inbreeding coefficient (F) were comprised between -0.028 and 0.157, and the overall mean value was 0.033, indicating a low overall déficit in hétérozygotes within populations. Averages of the three statistics varied significantly among local régions (As, P < 0.001; He, P < 0.01 and F, P < 0.001), based on 1000 permutations of populations among régions. Among zones, variation was significant for He (F < 0.05) and marginally significant for F {P = 0.051). Gene diversity was significantly positively correlated to both As and F (Table 4). No corrélation was found between latitude and any of the withinpopulation statistics. In contrast, we observed a négative association between longitude and values of He (r = -0.345) and F (r = -0.425) but the corrélation was non-significant after application of the sequential Bonferroni correction. Accordingly, high gene diversity was found in populations from Ireland and France, whereas several régions from eastem Europe showed lower values of He (Bulgaria and Romania). Values of Wright's inbreeding coefficient F were also found to be the highest in western populations (Ireland and France), whereas the lowest values occurred in southeastem Europe. Values of allelic richness did not show the same pattern as the highest values occurred in populations from Lithuania and the Republic of Moldova. However, average values for ail three statistics (As, He and F) at the zonal level showed a similar overall pattern of a decrease from western to eastem Europe (Table 3). This pattern hides some local peculiarities, for instance very high genetic variation and high inbreeding coefficients were found in populations from Lithuania.

Linkage disequilibrium was detected (a = 0.05) for one pair of loci in seven populations, two pairs in five populations, three pairs in three populations, four pairs in two populations, five pairs in four populations and ail ten pairs in one population (12, GerFr from Germany). No corrélation between the number of pairs of loci in linkage disequilibrium and withinpopulation diversity statistics or geographical location was identified. For a given pair of loci, the number of populations with significant linkage disequilibrium was comprised between two and ten.

The two bottleneck statistics T2 lAM and Tj SMM were strongly positively correlated (r = 0.935, P < 0.001, Table 4) but the values of T2 SMM were consistently lower than those of T2 lAM. The lowest values for both statistics were observed in Romania (mean T2IAM = -1.332, mean T2 SMM = -8.892) and Sweden (mean T2 LAM = -1.397, mean T2 SMM = -8.553), whereas the highest were recorded in France (mean T2IAM = 1.167, mean T2 SMM = -1.350) and Germany (mean T2 lAM = 0.858, mean T2 SMM = -2.686)(Table 3). Similarly to the other intrapopulation statistics, T2 decreased from western to eastem Europe. For T2 lAM, a négative association with longitude was observed (r = -0.455, P < 0.08): the western zone featured a mean T2 lAM = 0.9, the eastem zones' mean T2 LAM was = -0.9 in the northeast and == -0.5 in the southeast. Hence, western populations show an excess of heterozygosity relative to that expected from the observed number of alleles, which is consistent with a recent bottleneck. Conversely eastem populations show a déficit of heterozygosity explicable by population expansion. Wilcoxon's signed rank test (a = 0.05) applied to T2 lAM identified significant signatures of bottlenecks in seven populations (IreAv,2 and LreCa,3 from Ireland,FraGo,5 and FraLr,6 from France,GerTi,10 from Germany and BulLj,20 and BulMo,22 from western Bulgaria) and of expansion in two populations (RomTu, 23 from Romania and BulVi, 21 from western Bulgaria). For T2 SMM, the négative association with longitude was less marked (r = -0.404). Ail populations but one showed négative T2 SMM values and consequently Wilcoxon's signed rank test identified no significant signal of bottlenecks, but 28 populations showed a significant signal of démographie expansion. Summarizing these results, it can be said that populations from western and central Europe showed a stronger tendency towards an excess heterozygosity than populations from eastem Europe.

Total diversity and population genetic structure

Statistics of total diversity and différentiation among populations were computed for each of the twelve local régions, for each of the four zones and at the European scale (Table 5).

Corrélation coefficients between total diversity and population structure statistics and both geographical variables and within-population statistics are presented in Table 6.

Total diversity in the overall data set was very high with an average of 16 different alleles per locus (At_36 = 16.13), when considering a standardised overall sample of 36 gene copies, and total gene diversity {Hj) equal to 0.832. At the régional level, total allelic richness (At) and total gene diversity (Hj) showed a geographical pattern similar to that described for the corresponding within-population statistics (As and respectively) to which they were significantly positively correlated. Accordingly, the highest values for Aj were observed in the Republic of Moldova and Lithuania, whereas the lowest values were found in Sweden and western Bulgaria. The latter two régions were also characterised by the lowest values for total gene diversity Hr, while the highest values of Hj occurred in western European populations and in Lithuania. At the zonal level, however, the patterns of variation of Aj and Hj were distinct from the longitudinal gradient described for within-population statistics, mainly because the southeastem zone exhibited low within-population diversity but very high total diversity.

The overall population différentiation measured as Fst was 0.076, indicating that more than 92% of the genetic variation at nuclear microsatellite loci occurs within populations, whereas the overall allelic différentiation statistic, Ast, showed a four-fold higher value (A$t = 0.318).

The population différentiation statistics Ast, ^st and Fst varied widely at the régional level, and showed a pattern of variation very different from either the within-population or the total diversity statistics. Indeed, each of these différentiation statistics was significantly negatively correlated to both As and The observation that Dst showed a very similar pattern to Fst indicates that the négative corrélation between Fst and the within-population statistics was not an artifact due to the computation of Fst as a ratio statistic. Régions with low genetic variation, i.e. Sweden and western Bulgaria, thus exhibited strong population genetic structure (Fst > 0.1). In contrast, régions with high genetic variation like the Republic of Moldova, Lithuania and France showed low différentiation among populations (Fst < 0.01). At the zonal level, ail différentiation statistics displayed high values for the two eastem European zones, whereas populations from western and central Europe showed very little population genetic structure.

The différentiation statistic based on the distributions of allele size (Fst = 0.173) was about two times higher than that based on allele identities (Fst = 0.076) on the overall data set (Table 5). The différence between the two statistics was highly significant (F < 0.001), when permutations of allele sizes among alleles were performed. At the régional level, Fst was found to be significantly positively correlated to Fst (r = 0.615, P < 0.05), with values higher than Fst in seven out of the twelve régions, but a significant différence between Fst and Fst occurred only in one région (Romania, P < 0.01). In Romania, the Fst statistic was more than three-fold higher than the Fst. In contrast, in Sweden, the Fst statistic was half the value of Fst-At the zonal level, values of the Fst were higher than the Fst in three out of four cases. but a significant différence between F$t and R$t (P<0.001) occurred only in the southeastem group.

-------21 BulVi "31 BulDu -33 BulZd -----36 MolHa --------34 MolHr -------35 MolHi -----------32 BulKo -------------

Patterns of différentiation among populations

Strong géographie différentiation was apparent from the midpoint-rooted neighbor joining tree based on Cavalli-Sforza and Edwards' (1967) chord distance (Figure 1). One major cluster contained ail populations from the western and the central zones. It was close to populations from Sweden, Lithuania, Slovakia and Hungary and was inserted among clusters with populations from eastem Europe. Among eastem populations, those from the Danube and Dniestr plains, i.e. eastem Bulgaria, the Republic of Moldova and eastem Romania, were grouped together. A strikingly long internai branch separated the three populations from eastem Romania (24,RomBaa;25,RomBab and 26,RomBac) from the rest of the tree.

Because of the very limited number of loci, only 126 bootstraps could be performed. Very low support for internai branches was observed, with the séparation of the western and central ■y European group being given a score of 66% of bootstraps. The tree based on the (ôp) distance also isolated the three populations of eastem Romania trough a long internai branch, supported by 71% of the bootstraps (Figure 2). No clear geographical structure was found among the other populations.

Inference of population stmeture was obtained from an analysis of multilocus génotypes using the model-based clustering method of Pritchard et al. (2000) allowing for population admixture. The highest likelihood of the observed genotypic data X conditional to a given number K of clusters or gene pools was obtained for K=6, ln[Pr(A| K=6)] = -27583, and the posterior probability Pr(A'=6| X) was very close to one. These six identified gene pools showed a strong géographie structure. A first gene pool was prédominant in ail populations from the western and central zones where 54 to 68% of the genes from each population were assigned to it (Figure 3). It was also présent in Slovakia, Hungary and Lithuania. A second gene pool was mainly présent in populations from Slovakia, Lithuania and two Swedish populations (SweRoe,13 and SweAk,15), whereas a third gene pool occurred in populations from Hungary, central Romania, population BulMo ( 22) from western Bulgaria and populations SweRoe (13) and SweEhd ( 14) from Sweden. A fourth gene pool was primarily found in ail populations from western and central Bulgaria except in population BulMo (22).

Populations from the Danube and Dniestr plains were assigned to two separate gene pools, the fifth gene pool comprising populations from eastem Romania and the sixth gene pool prevailing in those from the Republic of Moldova and eastem Bulgaria. Estimâtes of population admixture varied greatly among populations. On average the proportion of genes assigned to the best scoring gene pool within a population was Pbest = 0.66 ±0.15 (S.D.).

Lower than average population admixture was found within some géographie groups in 6) appeared to be the most common gene pool in distinct populations from those régions. The highest population admixture was found in populations from Lithuania, Slovakia and Hungary (Pbest = 0.42) with on average 19%, 39% and 21% of genes assigned to the first, second and third gene pools, respectively. High population admixture was also found in ail populations from the western and central zones (Pbest = 0.62), which showed a strikingly homogeneous pattern with the first gene pool predominating, and, on average 17% of genes assigned to the second gene pool and 11% of genes assigned to the third gene pool. The situation in Sweden was singular as two populations show little evidence for population admixture, but with distinct prédominant gene pools that were found in admixture in the third Swedish population.

Isolation by distance tested at the European level by a permutation procedure was rejected both for Fst and R$t computed as multilocus statistics. However, a significant pattern of isolation by distance (P<0.001) was detected with the Fst/(1-Fst) statistic but not with /?st/(1-Rst) in a subset of the data, comprising populations from Scotland, Ireland, France, Germany, Slovakia, Hungary and Lithuania (i.e. from the régions in which admixture of gene pools 1 to 3 was found from the model-based cluster analysis). In this subset, the Fst/(1-Fst) ratio increased with the natural logarithm of geographical distance (Blog = 0.00491, R^ = 0.190; Figure 4).

Discussion

The overall pattern of genetic variation at microsatellite loci that we observed for the common ash is typical for a long-lived outerossing species with high génie diversity and a low level of inbreeding within populations (He = 0.77 and F = 0.03), and low différentiation among populations (Fst = 0.08). Nevertheless, over the distribution range, we observed two contrasting patterns in terms of level of within and among-population diversity: (1) régions with high allelic and génie diversity within populations, but very low différentiation among populations; and (2) régions with relatively low allelic and génie diversity within, but relatively strong genetic divergence among populations. The former pattern occurs mainly in the western and central European zones, although it also occurs in Lithuania, whereas the latter is found only in Sweden and in the southeastem zone. These patterns were also associated to striking différences in levels of the inbreeding coefficient, that was positively correlated to the within population genetic variation, and the T2 statistics of Comuet and Luikart (1996), which indicated a tendency to excess of heterozygosity with respect to that expected from the observed number of alleles in régions with high within-population genetic variation, and the opposite pattern in régions of high genetic différentiation among populations. Finally, the southeastem région, which has high genetic différentiation in terms of Fst, also shows a large and significant excess of Rsj over the Fst-Although our sampling scheme is too limited to make full inference of historical processes, we will discuss below possible causes of such clear contrasting patterns.

Patterns of genetic diversity and postglacial recolonisation processes

Palynological data since the last ice âge suggest the occurrence of glacial refuges for ash in the eastem Alps and in the Southern Balkans, as well as, but with lower support, northeast of the Black Sea and in the Pyrenees (Huntley & Birks 1983). From interprétation of Brewer's palynological maps (Brewer 2001), additional refuges were located within the Italian peninsula and at the Bulgarian Black Sea coast; the western refuge is weakly supported and may rather be situated within the Iberian peninsula, and the refuge north of the Black sea is not confirmed. The analysis of pollen diagrams by Gliemeroth (1997) supported the occurrence of refuges in the Iberian peninsula and at the northem Black Sea coast, whereas refuges in the three peninsulas and in the Alps are supported by chloroplast haplotype data (G. G. Vendramin, in préparation). Recolonisation from the refuges started northward about 12000 years BP and was followed by a westward migration, which began roughly 9000 years BP (from Brewer 2001). In several tree species from North America (Tsuga mertensiana, Ally et al. 2000;Pinus coulteri, Ledig 2000) and Japan (Fagus crenata, [START_REF] Tomaru | Genetic diversity in Fagus crenata (Japanese beech): influence of the distributional shift during the late-Quatemary[END_REF]) a decrease of both allelic richness and gene diversity within populations has been reported from Southern to northem areas of the distribution, and interpreted as a conséquence of founder effects associated to postglacial recolonisation. In the common ash, allelic richness and gene diversity also varied concomitantly with geography but genetic variation was highest in régions that hâve been colonized more recently, i.e. France and the British Isles. Thus in the common ash, we found no evidence for genetic impoverishment due to founder effects during the recolonisation process. The relationship between allelic richness and gene diversity is condensed in Comuet and Luikart's (1996) bottleneck statistic T2 for which we recorded low values close to refuges, i.e. a relative excess of allelic richness, but high values in recently recolonised areas, i.e. a relative excess of gene diversity. Similar variation of the bottleneck statistic with geography was observed by Comps et al. (2001) in European Fagus sylvatica.

However, these authors, as well as Zanetto and Kremer (1995) in European Quercus petraea, found a négative corrélation between régional averages of allelic richness and gene diversity, with higher allelic richness in régions corresponding to the glacial refuges but higher gene diversity in recently colonized régions. Comps et al. (2001) interpreted these observations as a conséquence of post-glacial recolonisation from multiple refuges simultaneously, in analogy with the "migrant pool" model of metapopulation dynamics (Wade & MeCauley 1988).

Hence, the drift effect associated to founder events could hâve been counteracted by admixture of previously differentiated gene pools and they presented simple calculations illustrating that this process may act differentially on allelic richness and gene diversity Chapter lll: Genetic structure ofcommon ash in Europe assessed with nuclear microsatellites (Comps et al. 2001). We propose that admixture of differentiated gene pools originating from distinct refuges may also explain the diversity patterns found in the common ash. This hypothesis is reinforced by the results from the model-based cluster analysis, which identified higher levels of population genetic admixture in recently colonized areas (i.e., western and central zones) than in régions closest to glacial refuges (southeastem zone). The topography of Europe allowing recolonising lineages to merge, and the evidence of a westward colonization of ash after migration from the refuges (from S. Brewer, 2001) also support this proposition. The lack of a decrease in allelic richness from refuges to recolonised areas in the common ash may also be attributable to the genetic markers employed in this study: microsatellites hâve indeed a higher mutation rate than allozymes (Jame & Lagoda, 1996) and may thus hâve recovered alleles more quickly after founder events.

Géographie variation in within-population level of inbreeding

We observed a low positive but significant average inbreeding coefficient within European populations of the common ash (F = 0.033) but among-population variability for F was high (CV = 158%). Moreover a longitudinal gradient of decreasing F from west to east was observed. This could be explained by variation across Europe either in the mating System, in the level of biparental inbreeding (mating among relatives), or in the frequency of null alleles. The latter explanation is a general feature of microsatellite loci, the higher the frequency of null alleles, the higher the value of the observed inbreeding coefficient [START_REF] Bruford | Characteristics of microsatellites[END_REF]).

Although we cannot rule out this hypothesis, we did not observe unreliable amplification reactions at any analysed locus. In a previous paper on populations from Bulgaria, we studied the association between the spatial position of individual trees within stands and their genetic relatedness (Heuertz et al. 2001). Our results indicated the presence of spatially-determined biparental inbreeding in several populations. A comparison of the average values of the inbreeding coefficient (F = 0.014) with that of the kinship coefficient between neighbor plants (^short distance ciass = 0.021) indicated that most of the observed inbreeding seemed to be caused by biparental inbreeding. In contrast, within a large French population, M.-E. Morand (in préparation) observed a much higher value of the inbreeding coefficient (F = 0.122) than kinship coefficients between neighbour plants (Fshon distance ciass = 0.026). Their results suggest that in this French population most inbreeding is due either to self-fertilization or to nonspatially determined biparental inbreeding, as for instance assortative mating. We thus suggest that in the common ash a gradient of increasing selfing may occur from eastem to western Europe, with an inferred average selfing rate in the western zone as high as 17%

[computed as s=2F/(l+F)]. Altematively, following the hypothesis of admixture of differentiated gene pools at the time of recolonisation in western Europe, biparental inbreeding could hâve been enforced in the descendant populations through the occurrence of some reproductive barriers between individuals from the co-occurring gene pools. Data on variation in morphology, phenology and sexual phenotype within populations, as well as direct assessments of outcrossing rates would be necessary to distinguish between these two hypothèses. For instance in Fraxinus lanuginosa, an androdioecious ash species from Japan, increasing levels of inbreeding attributed to self-fertilization were observed in seeds as the frequency of male individuals decreased within populations [START_REF] Ishida | Mating System and population genetic structure of an androdioecious tree, Fraxinus lanuginosa Koidz. (Oleaceae) in northem Japan[END_REF].

Patterns of divergence among populations

Results from the distance-based and the model-based cluster analyses clearly showed that populations from the western and central zones, as well as populations from Slovakia, Hungary and Lithuania but to a lower extent, hâve a very similar genetic composition (Fst < 0.02, Rst < 0.03). This suggests either a common history of recolonisation, probably occurring mainly westwards in agreement with fossil pollen maps, or several events of recolonisation followed by homogénisation through extensive gene flow. A higher than average level of population admixture in these populations suggests that intermixing of génotypes derived from several refuges did occur, and the observed pattern of isolation by distance supports some common migration history. Chloroplast haplotype data are necessary to clarify the proposed hypothèses. In contrast, in the southeastem zone, we found evidence for the co-occurrence of several well differentiated gene pools (Fst = 0.09, Rst = 0.22) at relatively short géographie distance. This observation would agréé with restricted inter regional gene flow, and accordingly, in a previous investigation on spatial patterns of genetic différentiation within and among populations from Bulgaria, we found indirect evidence for relatively limited levels of pollen and seed effective dispersai in the common ash (Heuertz et al. 2001). Hence, the observed strong genetic différentiation in the southeastem zone could be explained by long-term coexistence of several gene pools under a stable démographie history and restricted gene flow. This pattern is not at odds with the observed low levels of différentiation among populations in western and central Europe; it has been shown that during recolonisation similar levels of dispersai of seed or pollen into low-density stands could resuit in higher effective gene flow than under stable démographie conditions [START_REF] Austerlitz | Effects of colonization processes on genetic diversity: différences between annual plants and tree species[END_REF]. Finally, another striking pattern was the very large divergence between populations 24 to 26 from Romania and ail other samples. This pattern was even more pronounced when the variances in microsatellite allelic sizes were taken into account (Rst » Rst; see also Figure 2). Although this pattern could be partly explained by évolution from separate glacial refuges, we suggest that additional processes such as interspecific hybridisation could be responsible for it, as other species of Fraxinus, i.e. F. angustifolia, F. pallisiae and F. omus are common in this région (Huntley & Birks 1983; M. Heuertz, Personal observation).

Population admixture

Our results suggested that areas most recently colonized showed evidence for higher population admixture than those from areas doser to the glacial refuges. It is striking that most genes from the populations with higher than average admixture were assigned with different proportions to a combination of mainly three gene pools using the model-based clustering approach: gene pools 1 to 3, which were prédominant in a very large géographie area ranging from the British Isles to eastem Romania and eastem Bulgaria, including northem populations from Sweden and Lithuania. Hence these gene pools could originale from several identified potential refuges, the Iberian Peninsula, Italy and/or the Alps, or the Balkans. These results should be taken with caution, however, because the model-based clustering method has not yet been applied to large datasets on plant species, and evolutionary processes such as pollen gene flow, isolation by distance, founder effects may hâve confounding effects on the observed patterns. When the method was applied to populations from the western and central zones alone, the signal of population admixture disappeared. This suggests either that the détection of admixture in the overall analysis is an artifact, or that the events leading to population admixture occurred a long time ago and most linkage disequilibrium within populations has already decayed. The géographie cohérence of the distribution of the putative gene pools suggests however that the signal of admixture has a historical foundation, with for instance, the occurrence of a suture zone between the western group and the southeastem group in Slovakia and Hungary where the highest levels of admixture were recorded.

A putative northern cryptic refuge

In Sweden, the patterns of genetic variation were very similar to those observed in the southeastem zone, although Swedish populations are thought to hâve a history of postglacial recolonisation: low allelic richness and gene diversity; low inbreeding coefficient; low values of the T2 statistics; lower than average population admixture. A careful investigation of fossil pollen maps (from Brewer et al. 2001) confirms that pollen records are known from southem Sweden as early as 12500 years BP. We thus suggest that our data might confirai a putative cryptic northem glacial refuge for common ash in southem Sweden. Such cryptic northem refuges hâve recently been identified in other plant and animal species (Stewart & Lister 2001). Moreover, they could hâve played a rôle in the postglacial recolonisation process, because one of the six identified gene pools (number 2) is prédominant in one Swedish population but is found in admixture across the western and central zones as well as in Lithuania.

Microsatellite versus aliozyme patterns of variation

Up to now, most large-scale studies of patterns of genetic variation in tree species were performed with aliozyme markers. Although no data on allozymes are available in Fraxinus excelsior, our results on relative statistics such as F and Fst are comparable to those obtained on species with similar life historiés but with allozymes (Hamrick & Godt 1989, Hamrick et al. 1992). Like these other studies, we also observed géographie patterns of variation in statistics describing within-and among-population genetic variation. However, data from five highly variable microsatellite loci allowed us to identify several additional patterns that may hâve been missed with aliozyme markers. First we observed a subtle pattern of géographie variation in F and in the statistic Tz, that was détectable thanks to the comparatively lower variance associated to microsatellite markers (Goudet et al. 1996). Second, a comparison between Fst and Fst values allowed us to identify a région with potential long-term genetic différentiation and/or interspecific hybridisation. Third, the application of the model-based cluster analysis of Pritchard et al. (2000) on multilocus génotypes allowed to suggest the occurrence of population admixture associated to recolonisation of northwestem Europe.

Concluding remarks

We suggest that the refuge areas analysed, i.e. southeastem Europe, contain differentiated genetic resources of the common ash, which may hâve been coexisting for a long time without substantial genetic exchanges. These areas thus constitute important source populations for the conservation of genetic resources in the common ash. In contrast, central and western European populations harbor much more homogeneous resources which would resuit from the processes of recolonisation and admixture of previously differentiated gene pools.

Référencés

Aldrich PR, Hamrick JL, Chavarriaga P and Kochert G (1998) Microsatellite analysis of démographie genetic structure in fragmented populations of the tropical tree Symphonia globulifera. Molecular Ecology, 7, 933-944.

Ally D, El-Kassaby YA and Ritland K (2000) Genetic diversity, différentiation and mating System in mountain hemlock {Tsuga mertensiana) across British Columbia. Forest Genetics, 7, 97-108.

Austerlitz F, Mariette S, [START_REF] Austerlitz | Effects of colonization processes on genetic diversity: différences between annual plants and tree species[END_REF] Effects of colonization processes on genetic diversity: différences between annual plants and tree species. Genetics, 154, 1309-1321. [START_REF] Binggeli | Gender variation in ash[END_REF] Gender variation in ash. Available from http://members.tripod. co.ukAVoodyPlantEcology/species/ash.htm Introduction Microsatellites or simple sequence repeats (SSRs) are tandemly repeated DNA motifs, one to six nucléotides long [START_REF] Queller | Microsatellites and kinship[END_REF]. They are highly polymorphie mendelian markers, extensively used for mapping and for population genetic and démographie issues (e.g. Jame & Lagoda 1996[START_REF] Estoup | Microsatellites and minisatellites for molecular ecology: theoretical and empirical considérations[END_REF].

Mutation rates at microsatellite loci are generally high; on the order of 10' to 10' per locus per génération for animais (Jame & Lagoda 1996[START_REF] Estoup | Microsatellites and minisatellites for molecular ecology: theoretical and empirical considérations[END_REF]. In plants, estimâtes ranged from 5.1 x 10'^ to 1.1 x 10'^ in maize [START_REF] Vigouroux | Rateand pattern of mutation at microstellite loci in maize[END_REF] and from 3.9 x 10'^ to 10'^ in chickpea [START_REF] Udupa | High mutation rate and mutational bias at (TAA)(n) microsatellite loci in chickpea {Cicer arietinum L.)[END_REF]. The favoured mutation mechanism for microsatellites is slip-strand mispairing, i.e. relative slippage of the nascent and the template strands during DNA réplication (Levinson & Gutman 1997, Primmer et al. 1996a), increasing or decreasing the allele size by one repeat motif. More complicated mutation processes may occur, involving multi-repeat mutations (e.g. [START_REF] Karhu | Rapid extension of microsatellite sequences in pines[END_REF][START_REF] Vigouroux | Rateand pattern of mutation at microstellite loci in maize[END_REF], single nucléotide substitutions or indels (e.g. [START_REF] Primmer | Patterns of évolution in avian microsatellites[END_REF][START_REF] Van Oppen | Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in lake Malawi Cichlids[END_REF][START_REF] Barrier | Interspecific évolution in plant microsatellite structure[END_REF] or large indels [START_REF] Van Oppen | Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in lake Malawi Cichlids[END_REF][START_REF] Colson | Evidence for complex mutations at microsatellite loci in Drosophila[END_REF].

Mutations in Banking régions are also common [START_REF] Brohede | Microsatellite évolution: polarity of substitutions within repeats and neutrality of flanking sequences[END_REF][START_REF] Colson | Evidence for complex mutations at microsatellite loci in Drosophila[END_REF], and sometimes linked to certain allele types [START_REF] Blankenship | Evolution of a perfect simple sequence repeat locus in the context of its flanking sequence[END_REF].

Frequent and variable mutations lead to size homoplasy, i.e. alleles hâve the same electrophoretic size but they are not identical by descent (reviewed in [START_REF] Estoup | Homoplasy and mutation model at microsatellite loci and their conséquences for population genetics analysis[END_REF].

Homoplasious alleles can hâve the same sequence or not. In the latter case, size homoplasy is referred to as "molecularly accessible" [START_REF] Estoup | Homoplasy and mutation model at microsatellite loci and their conséquences for population genetics analysis[END_REF], and it was observed among alleles from the same species (e.g. [START_REF] Culver | Patterns of size homoplasy at 10 microsatellite loci in pumas {Puma concolor)[END_REF], or from different species [START_REF] Estoup | Size homoplasy and mutational processes of inteirupted microsatellites in two bee species. Apis mellifera and Bombus terrestris (Apidae)[END_REF][START_REF] Primmer | Patterns of évolution in avian microsatellites[END_REF][START_REF] Markova | Evolution of microsatellite alleles in four species of mice (genus Apodemus)[END_REF][START_REF] Van Oppen | Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in lake Malawi Cichlids[END_REF].

Although there is substantial interlocus variation of mutation rates, empirical studies hâve revealed associations between mutations and features of the microsatellite (revieved in Estoup &[START_REF] Estoup | Microsatellites and minisatellites for molecular ecology: theoretical and empirical considérations[END_REF][START_REF] Eisen | Mechanistic basis for microsatellite instability[END_REF]): (i) mutation rates are on average inversely proportional to the size of the repeat unit; (ii) the likelihood for slip-strand mispairing, and a fortiori the mutation rate, increase with repeat count; (iii) tracts with multiple repeats are more subjected to large, multi-repeat délétions than short tracts; and (iv) the presence of interruptions or variant repeats stabilises the locus by reducing the opportunity for slip-strand mispairing.

For adéquate inference of population genetics parameters from microsatellite data, statistics hâve been developed based on models which take into account the peculiarities of microsatellite mutations [START_REF] Slatkin | A measure of population subdivision based on microsatellite allele frequencies[END_REF], Goldstein et al. 1995). The simples! model of microsatellite évolution, the stepwise mutation model (SMM, [START_REF] Ohta | A model of mutation appropriate to estimate the number of electrophoretically détectable alleles in a finite population[END_REF] assumes évolution by addition or délétion of one repeat motif. Recent models additionally allow for more complex mutation events, involving several repeat motifs (the two-phase model, TPM, Di [START_REF] Rienzo | Mutational Processes of simple sequence repeats in human populations[END_REF] or point mutations (e.g. [START_REF] Kruglyak | Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations[END_REF]. However, statistics Fig. 1 Geographical distribution of odd-and even-sized alleles at microsatellite locus FEMSATL4 in 36 European Fraxinus excelsior populations. based on allele size variation typically suffer higher sampling variances than their counterparts based on allele identity information, as demonstrated for instance for the estimators of among-population différentiation i?sx and Fst [START_REF] Slatkin | A measure of population subdivision based on microsatellite allele frequencies[END_REF][START_REF] Balloux | Statistical properties of population différentiation estimators under stepwise mutation in a finite island model[END_REF]. In this context, a useful test was recently developed which assesses whether step-wise like mutations significantly contributed to population différentiation (Hardy et al. 2002).

The mutation process at microsatellite loci can be investigated directly from observation of mutations in the germline, for example in humans [START_REF] Amos | Microsatellites show mutational bias and hétérozygote instability[END_REF] or bam swallows (Primmer et al. 1996a). Altematively, sequence variation accumulated over evolutionary time can be studied by using the same primers in related taxa [START_REF] Primmer | Patterns of évolution in avian microsatellites[END_REF][START_REF] Van Oppen | Extensive homoplasy, nonstepwise mutations, and shared ancestral polymorphism at a complex microsatellite locus in lake Malawi Cichlids[END_REF][START_REF] Karhu | Rapid extension of microsatellite sequences in pines[END_REF]. This is the approach we used for the compound dinucleotide repeat locus FEMSATL 4, originally isolated as (CA)2(AG)24 in common ash, Fraxinus excelsior, a European forest tree species (Lefort et al. 1999). Priming sites of this locus hâve been conserved in many ash species and in three additional généra (Ligustrum, Olea and Phillyrea, Lefort et al. 1999) from the tribe Oleeae, in the relatively young family Oleaceae (most recent common ancestor ~ 37 MYBP, E. Wallander personal communication). Our study was motivated by the finding of alleles of even and odd electrophoretic sizes with different géographie distribution in a population genetic study of common ash at the European scale (Heuertz et al. unpublished). Our objectives were (i) to investigate the molecular diversity underlying the electrophoretic variation in Fraxinus excelsior and (ii) to regard it in the context of the evolutionary history of the locus by analysing amplification products from four species with increasing phylogenetic distance to F. excelsior. F. omus\ {Olea europaea and Phillyrea angustifolia), and Ligustrum vulgare.

Materials and methods

Materials

The studied species include common ash {Fraxinus excelsior), a large European forest tree; flowering ash {F. omus), a small south European tree; Ligustrum vulgare, a European bush, sometimes planted in hedges; olive tree {Olea europaea), a largely cultivated tree species with a Mediterranean distribution; and Phillyrea angustifolia, a Mediterranean bush. Samples were collected in order to represent a large geographical range: leaf or bud samples from a total of 1050 F. excelsior trees from 36 European populations (Heuertz et al. unpublished,Fig. 1); leaf or DNA samples from a total of 21 F. omus trees from two Bulgarian and one Italian population; leaf samples from a total of 36 Ligustmm vulgare bushes from one Dutch, one Belgian, two French and two Moldavian populations; DNA samples from a total of 69 olive trees belonging to 26 cultivars from different south-European, north African and near Eastem countries or to five natural populations from south Italy, leaf samples from three omamental Values followed by the same letter are not significantly different at the 5% probability level (T-tests).

' Diversity statistics were not computed because the sample size was too small.

Cloning and sequencing of PCR products

For cloning, we selected individuals carrying alleles from throughout the observed size range (Table 1). In order to minimize the number of cloning reactions, heterozygous individuals were preferred. FEMSATL4 was amplified with non-fluorescent primers in a total volume of 25 |il; 4 pi were run on 1% agarose gels to check the quality of PCR products, the rest was purified with the QIAQuick PCR purification kit (QIAGEN). Purified DNA was cloned using the TOPO TA® cloning kit (Invitrogen). For each cloning reaction, ten bacterial colonies were screened for the presence of an insert of suitable size as follows. The colonies were suspended in 25 pl stérile water, boiled for 5 min and spun for 1 min at 12000 G. Three pl of the supematant were used as a PCR template for Ml3 forward and reverse primers annealing to the cloning vector. The PCR reactions were performed in a mix (25pl) containing 2.5 mm MgCh, 0.5 U per reaction of Taq polymerase (APBiotech) in APBiotech PCR buffer, 0.5 pM More precisely, in O. europaea, as much as 83% of the recovered sequences corresponded to alleles; and the species also featured a very low percentage of stutter bands (5%). In ail other species the percentage recovery of fragments of expected size was worse, notably in P.

angustifolia and in F. excelsior where only a quarter of the sequences corresponded to alleles and half of them were unreadable or contained no microsatellite. The bad resuit for F.

excelsior could however be linked to experimental conditions (see discussion).

Sequence variation in the microsatellite repeat région and in flanking régions Microsatellite repeat région. Sequence variation at the microsatellite locus FEMSATL4 is summarized in Fig. 2. Ail species carried a (GA)n motif in their repeat région (box in Fig. 2), European populations (Fig. 1). Odd and even allele sizes are represented by white and grey bars, respectively. Two alleles larger than 251 nucléotides were found in low frequency and are not shown. " When five loci were analysed, multilocus statistics were computed. Values are followed by the significance level for the tests with HO: Fst = 0, and HO:

-0, respectively, assessed with 1000 permutations: ns, non significant; ***, P<0.001.

""Data from Heuertz et al. (unpublished).

Nine south-eastem European populations with more than 25 % even-sized alleles at locus FEMSATL4.

Discussion

Allelic diversity

Among the five Oleaceae species screened at microsatellite locus FEMSATL4, allelic diversity was highest in Fraxinus excelsior. This is in agreement with Primmer et al.'s (1996b) finding that microsatellite loci tend to be more polymorphie in the species in which they were initially isolated than in related species. Additionally, cross-species priming may produce a higher amount of null alleles in related species, because of mutations in the priming sites (e.g. [START_REF] Estoup | Microsatellites and minisatellites for molecular ecology: theoretical and empirical considérations[END_REF]. Our data set bears no evidence for this, since inbreeding coefficients were not higher in related species than in F. excelsior. It has been argued that microsatellites were longer in the focal species, due to preferential sélection of loci with a large number of repeats during the isolation process (ascertainment bias, e.g. [START_REF] Ellegren | Microsatellite évolution: a reciprocal study of repeat lengths at homologous loci in cattle and sheep[END_REF], but see Crawford et al. 1998). We found that F. excelsior alleles were indeed longer than those found in L. vulgare or in O. europaea, but shorter than those identified in F. omus.

Effîciency of cloning and sequencing reactions

The number of cloned alleles recovered in sequencing reactions was generally low in our study; we re-identified on average only one allele from each cloning reaction, despite choosing as much as eight colonies for sequencing. In some species, we found many stutter bands, 2 to 4 nucléotides shorter, or sometimes longer, than the allele length determined in the sizing experiment. One possible cause for the multiplicity of stutter bands may lie in the experimental protocol: instead of using DNA from a miniprep for sequencing, we used a purified PCR product, generated by amplification with Ml3 primers, hence adding one step of putative polymerase slippage. The use of a proofreading Taq polymerase instead of a conventional one might also hâve improved the situation. In F. excelsior, the low recovery of alleles is probably due to the fact that we failed to clone and sequence several long alleles in this species (repeat région of up to 190 bases). The cloning and sequencing of long alleles is complicated in two respects: (i) if they occur in heterozygous individuals together with a short allele, they amplify more weakly because of compétition in the PCR reaction (short allele dominance, Wattier et al 1998); hence there is a lower concentration of the longer allele in the PCR product for cloning; and (ii) they display more stutter bands than short alleles as there is more opportunity for slippage in a long repeat région than in a short one (in agreement with higher mutation rates in long microsatellites, e.g. [START_REF] Estoup | Microsatellites and minisatellites for molecular ecology: theoretical and empirical considérations[END_REF]. In heterozygous individuals where allele lengths differ by more than 20 nucléotides, we were indeed unable to recover the longer allele. Furthermore, we found sequences carrying very long AG repeats (>60 motifs), but the part the sequence downstream of the microstallite was degenerated, probably because of slippage that had occurred in the pre-sequencing PCR and during cycle sequencing. In O. europaea on the contrary, very few stutter bands were observed, and most alleles differed by 6 bases. Together with the relatively low polymorphism in O. europaea, this indicates that there might be less slippage, i.e. less potential for mutation, at hexanucleotide than at dinucleotide repeats, in agreement with Chakraborty et al.'s (1997) resuit that mutation rates are on average inversely proportional to the size of the repeat unit.

In L. vulgare, F. omus and P. angustifolia, we sequenced numerous copies of a product of 157 bases that did not contain a microsatellite and showed 100% similarity among these species. A gene duplication can be excluded because the fragment shows no homology with the flanking régions of the microsatellite. A BLAST search to déterminé its origin did not reveal any conclusive results. Hence, it is probably a locus with priming sites similar to those of FEMSATL4, which does not amplify in F. excelsior, because primers are more spécifie in the species they hâve been designed for; although it cannot be excluded that this fragment corresponds to a contamination. In our category "unexpected sequences with microsatellite", we grouped two types of products: (i) products where repeat and flanking régions were those of the analysed species, but the product was of unexpected length, and (ii) products with repeat and/or flanking régions atypical for the species. The first type was mainly found in F.

excelsior and O. europaea, and the second in the other species. In P. angustifolia, we identified sequence products with flanking and repeat régions typical for O. europaea in three out of four cloned individuals. In F. omus, similar O. europaea products were found in 3 sequences from one cloned individual; and in L. vulgare, two sequences corresponded to the odd-sized allele type of F. excelsior. The simplest explanation for the détection of these sequences is contamination, which is perhaps more likely to occur in related species where primer annealing may not be optimal. However, one F. omus sequence carried a hexanucleotide repeat typical for O. europaea, but corresponded entirely to the F. omus allele type otherwise. Hence, the hypothesis of a gene duplication and independent évolution to that type of hexanucleotide repeat might deserve further attention in F. omus. A larger sample size should help résolve whether we deal with an artefact here, or not.

Molecular diversity at locus FEMSATL4 in Fraxinus excelsior: origin and geographical distribution In F. excelsior, the ancestral State of the microsatellite locus probably corresponds to the oddsized allele type. It is indeed more abundant, more widespread geographically and notably, it can be identified in the phylogenetically close F. omus, which is not the case for the evensized allele type. The narrower size range of even-sized alleles also points to a shorter evolutionary history of this type. We were however unable to détermine the origin of the oddsized allele, because too few Oleaceae species were studied. The even-sized allele type has probably evolved from the odd-sized type since the F. excelsior -F. omus split, by the délétion of the motif AAA(CA)n. Its geographical distribution suggests two possible evolutionary scénarios, involving hybridisation with another Fraxinus species, or not.

Firstly, the délétion would hâve taken place in F. excelsior during the last glacial cold stage (115.000 -10.000 radiocarbon years BP) in the Balkan area, and possibly near the Black Sea coast, where fossil pollen data provide evidence for a glacial refuge for ash (Brewer 2001).

During post-glacial recolonisation (since 15.000 BP), the new allele type has colonised areas towards the north, but it could not spread ail over Europe because of compétition with northward migrating ashes from other glacial refuges. Therefore, its occurrence remained skewed towards southeastem Europe. This scénario can only be explained if the locus exhibits a very high mutation rate, for example on the order of 10'^ per génération, because 23 new alleles hâve been evolved since the délétion. The expounded mutation rate is possibly a realistic figure, regarding the tremendous polymorphism of the locus. A mutation rate at least one of magnitude lower would place the délétion back to at least 700.000 BP, approximately, the time before the European continent experienced repeated glacial periods (Webb and Bartlein 1992). During the latter, récurrent range contractions and expansions would hâve swept the phylogeographical pattern that is now observed among odd-and even-sized alleles.

Secondly, an alternative explanation for the observed phylogeographic pattern would be regular hybridisation of F. excelsior with another Fraxinus species, possibly F. angustifolia, which would carry the even-sized allele type at high frequency. Four ash species occur sympatrically in the Balkan area: F. excelsior, F. angustifolia ssp. oxycarpa, F. omus and F.

pallisiae (Tutin et al. 1972, and personal observation). Fraxinus excelsior and F. angustifolia ssp. oxycarpa hâve been reported to hybridise naturally in southeastem France (Rameau et al. 1989), and morphologically intermediate individuals are encountered in Romania (personal observation). According to Wallander (2001), Fraxinus pallisiae is a synonym for F.

angustifolia ssp. oxycarpa-, these species should hybridise more easily to F. excelsior than F.

omus, because the former three belong to section Fraxinus of the genus, whereas F. omus belongs to section Omus. Under the hybridisation scénario, the motif AAA(CA)" would hâve been lost in F. angustifolia s.l. after the F. excelsior -F. angustifolia divide. In that case, the presence of even-sized alleles in F. excelsior would be an indication for hybridisation with F.

angustifolia s.l. The relative restriction of even-sized alleles to the Balkan peninsula can be explained by (i) the distribution of F. angustifolia, which is restricted to southem and eastem central Europe (Tutin et al. 1972), (ii) by the fact that we did not analyse F. excelsior populations from another région where both species co-occur (such as Italy or southem France), and (iii) by a stable démographie situation in the Balkan area, with little gene flow among régions (see Heuertz et al. unpublished). Under this scénario, the délétion would be •2 much more ancient than under the first scénario and a lower mutation rate (<10' per génération) for locus FEMSATL4 is therefore suggested. The hybridisation scénario is supported by the fact that a Romanian floodplain population of uncertain taxonomie status (F.

excelsior -F. angustifolia) carried 60% of even-sized alleles.

Further, we found that step-wise like mutations did not significantly contribute to population différentiation at locus FEMSATL4, in neither allele-size type. This could be due to a low mutation rate and a relatively strong impact of the gene flow / genetic drift equilibrium to generate population différentiation. This resuit seems to weaken our first evolutionary scénario, where the even-sized allele type evolved in F. excelsior without hybridisation.

Evolution of microsatellite locus FEMSATL4 in the Oleaceae family

Our results reveal that the compound dinucleotide repeat locus originally isolated from F.

excelsior bears a more complicated repeat structure when related species are analysed. The only éléments common to ail species are a (CA)n and a (GA)n repeat région, and might therefore hâve been contained in the repeat type ancestral to these five species. The phylogenetic proximity of F. excelsior and F. omus on the one hand, and of O. europaea and P. angustifolia on the other (Fig. 3b) can be recognized by comparing their respective repeat régions (Fig. 2). Both species pairs differ from each other by a 16 nucléotides insertion/deletion of the stretch CACG(CA)3TGA4. After the split of F. excelsior and F.

omus, a transversion G C has probably occurred in the F. omus lineage, transforming a perfect (GA) into an interrupted sequence, which then continued to evolve by slippage as a hexanucleotide repeat (GAGACA). In O. europaea, the hexanucleotide repeat (GAGTGA) has probably evolved similarly, through an A ^ T transversion. It is recognized that microsatellites evolve by a combination of slippage events and base pair substitutions, however, the mutated nucléotide mostly seems to hâve the fonction of a repeat interruption, stabilising the microsatellite by reducing the possibility for further slip-strand mispairing (e.g. [START_REF] Angers | Complex Evolution of a Salmonid Microsatellite locus and its conséquences in inferring allelic divergence from size information[END_REF][START_REF] Kruglyak | Equilibrium distributions of microsatellite repeat length resulting from a balance between slippage events and point mutations[END_REF][START_REF] Eisen | Mechanistic basis for microsatellite instability[END_REF][START_REF] Estoup | Microsatellite évolution: inferences from population data[END_REF]. Our results suggest that base substitutions in the microsatellite région produced an elongated repeat motif, but did not inhibit further évolution by slippage. The study of [START_REF] Primmer | Patterns of évolution in avian microsatellites[END_REF] also describes length variation of the repeat unit in a microsatellite locus (in birds), but that situation is more complicated than ours, and it has been interpreted by a combination of substitutions and indels.

Mutations in régions immediately flanking the microsatellites repeat hâve been reported to be more common than in sequences further away [START_REF] Brohede | Microsatellite évolution: polarity of substitutions within repeats and neutrality of flanking sequences[END_REF]. Our results agréé with this, notably because the sequence immediately adjacent to the left side of the (GA)" repeat in L. vulgare shows much less homology to the corresponding sequence in both

Fraxinus species than the 25 sequenced bases located upstream. More generally, the left side of our microsatellite locus seems to be more variable overall than the right side, showing more substitutions in the flanking régions, an indel of the sequence flanking immediately the repeat, and point mutations in this sequence when it has been conserved.

Introduction

Two main methodological approaches hâve been used to characterise patterns of genetic variation over a wide distribution range, with the aim of making inference on the main causal factors: (1) phylogeographic studies based on organelle DNA (mainly chloroplast) patterns of variation;

(2) population genetic structure studies based on analyses of nuclear genetic marker diversity.

In flowering plants, where the chloroplast genome is usually matemally transmitted [START_REF] Birky | Evolution and variation in plant chloroplast and mitochondrial genomes[END_REF], much stronger population genetic structure is generally found for chloroplast than nuclear markers (Ennos 1994, Le Cotre et al. 1997[START_REF] Raspé | Chloroplast DNA haplotype variation and population différentiation in Sorbus aucuparia L. (Rosaceae: Maloideae)[END_REF]. This genetic structure often présents a clear phylogeographic pattern, which means that there is concordance of phylogenetic relationships and spatial distribution of génotypes (e.g. King & Ferris 1998, Petit et al. 2002a, Palmé & Vendramin 2002;see Taberlet et al. 1998, Cornes & Kadereit 1998, Newton et al. 1999 for reviews). Several factors may account for these observations. In angiosperms, the chloroplast genome is generally subjected to a lower level of gene flow than nuclear genes because it is dispersed only in the seed, whereas nuclear genes are transmitted by both seed and pollen. Further, as the chloroplast genome is haploid and uniparentally inherited [START_REF] Birky | Evolution and variation in plant chloroplast and mitochondrial genomes[END_REF], Clegg 1989), it evolves in hermaphrodite diploid plants under a twofold higher pressure of genetic drift than the nuclear genome [START_REF] Birky | An approach to population and evolutionary genetic theory for genes in mitochondria and chloroplasts, and some results[END_REF]. Therefore, when populations are relatively small and isolated, substantial genetic impoverishment may occur within, and large genetic divergence between them at chloroplast DNA markers. In many species, this was for instance the case for populations surviving in separate refuges during the glaciations of the Quatemary (Hewitt 2000). The comparison of phylogenetic relationships among organelle haplotypes with their current geographical occurrence allows to make inference on postglacial recolonisation routes [START_REF] Ferris | Chloroplast DNA recognizes three réfugiai sources of European oaks and suggests independent eastem and western immigrations to Finland[END_REF], Petit et al. 2002b), to propose high diversity areas as putative glacial refuges (King & Ferris 1998[START_REF] Huang | Spatial patterns of chloroplast DNA variation of Cyclobalanopsis glauca in Taiwan and East Asia[END_REF], Petit et al. 2002b) and to corroborate the location of refuges suggested on the basis of palynological data [START_REF] Demesure | Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe[END_REF], Petit et al. 2002b). Uniparentally inherited organelle markers are also used to study the directionality of gene flow in species complexes (Bucci et al. 1998[START_REF] Dumolin-Lapègue | Are chloroplast and mitochondrial DNA variation species independent in oaks?[END_REF] and to identify populations of non-local origin [START_REF] Demesure | Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe[END_REF], Sperisen et al. 1999, Vendramin et al. in préparation).

However, some important evolutionary processes such as recent démographie changes, or interspecific hybridisation events may remain undetected with organelle markers because they often display low polymorphism and hâve uniparental transmission. Moreover, each organelle genome represents only one occurrence of a genealogical process, which obviously does not keep trace of ail major evolutionary events in the species history [START_REF] Rare | Prospects for nuclear gene phylogeography[END_REF].

Nuclear markers are highly suited to study large-scale géographie variation in levels of within-population diversity, and by this means to identify imprints of founder effects resulting Nuclear microsatellites revealed strongly differentiated gene pools for common ash in southeastem Europe, whereas the partitioning of diversity in central and western Europe is much more uniform (Heuertz et al. unpublished,chapter 3). Also, we observed an increase of genetic diversity from populations close to refuges towards recently recolonised areas, which we attributed to admixture events of distinct genepools during recolonisation (Heuertz et al. unpublished,chapter 3).

In this study we assessed genetic diversity at six chloroplast microsatellite loci in 42 European populations of common ash, including the 36 populations previously analysed with nuclear microsatellite markers. Our objectives were (i) to identify geographical patterns of withinand among-population diversity at chloroplast markers, (ii) to compare them to the structure of genetic variation at nuclear markers, and (iii) to explain the current diversity patterns at both types of markers in terms of population demography and postglacial recolonisation dynamics during the current glacial interstadial. with Hl: r > 0, P = 0.000 for the two-tailed Spearman rank corrélation test), indicating that high différentiation at nSSRs occurred concomitantly with low différentiation at cpSSRs, and reciprocally. It appeared that this négative corrélation was essentially due to the comparison of pairs of populations within each of the groups defined from diversity patterns at nuclear loci. The western European group showed low différentiation among populations at nuclear markers, but high divergence among populations at chloroplast markers (Fig. 4). Conversely, the eastem European group displayed relatively high among-population différentiation at nuclear microsatellites, but very weak différentiation at chloroplast markers, as haplotype Hl largely prevails in that région (Fig. 4, compare also Figs. 1 and2). The comparison of différentiation between the two groups did not show a spécifie pattern (results not shown); différentiation at chloroplast markers varied from slightly négative values to one, whereas différentiation at nuclear markers took low to intermediate values.

Materials and Methods

Material

Discussion

Properties of chloroplast microsatellites

The haploid, non-recombinant and predominantly uniparentally inherited nature of chloroplast DNA makes it a useful tool for evolutionary studies (e.g. [START_REF] Olmstead | Chloroplast DNA systematics: a review of methods and data analysis[END_REF][START_REF] Provan | Chloroplast microsatellites: new tools for studies in plant ecology and évolution[END_REF]. Some limitations may however occur, especially in phylogenetic applications, when using chloroplast microsatellites as genetic markers. Like their nuclear counterparts, chloroplast microsatellites are prone to homoplasy, which means that variants at cpSSR loci can be identical in State without being identical by descent [START_REF] Provan | Chloroplast microsatellites: new tools for studies in plant ecology and évolution[END_REF][START_REF] Chapter | Launching microsatellites: a review of mutation processes and methods of phylogenetic inference[END_REF]. Consequently, phylogenetic distances between chloroplast haplotypes should be interpreted with caution, especially when using samples that are likely to be highly divergent, such as in broad-scale phylogeographic studies. In this latter case. Provan et al. opted for cpSSRs. We can assume that the mutation rate in our loci is not very high, as we detected only little polymorphism (8 haplotypes in 323 individuals) and because these markers are highly conserved across angiosperm species [START_REF] Weising | A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chlorolplast genomes of dicotyledonous angiosperms[END_REF].

Nevertheless, homoplasy is likely, and it would be useful to test the relationships we found among cpSSR haplotypes by comparing them with PCR-RFLP markers, even if the latter provided only a low resolution. Because of the little overall number of mutations separating haplotypes (Fig. 3), we were unable to identify strongly divergent haplotype lineages. For this reason, we chose not to présent our data according to groups of populations carrying similar haplotypes.

Géographie structure of chloroplast microsatellite variation Chloroplast haplotype variation in common ash was highly structurée! in Europe (G$t = 0.88).

Similar levels of among-population différentiation were observed for instance in black aider, Alnus glutinosa (Gst = 0.87, King & Ferris 1998), another mid-successional colonising species with wind-dispersed pollen and wind-disseminated seed, and in the more widely occurring European white oaks {Quercus robur, Gst = 0.78; Quercus petraea, Gst = 0.86, Petit et al. 2002a). In those species, a phylogeographic pattern of chloroplast DNA polymorphisms was observed, which means that phylogenetically close haplotypes were found together in the same population more often than randomly chosen different haplotypes.

In common ash however, we did not observe phylogenetic structure {Nsj was not different from Gst) and we attribute this to the overall low haplotypic variation, and especially to the low number of mutational steps separating haplotypes (Fig. 3). A géographie organisation of chloroplast haplotype variation in common ash was observed (Fig. 1) and confirmed by a significant pattern of isolation by distance among populations.

Isolation by distance at matemally inherited markers at a large geographical scale can be due Even though our previous results suggested restrictions to pollen flow (Heuertz et al. unpublished, chapter 2), we argue that wide-ranging gene flow through pollen was possible during colonisation. Colonising populations typically feature low densities, and therefore there is little compétition against immigrating pollen, allowing for high levels of effective gene flow. Theoretical results of [START_REF] Austerlitz | Effects of colonization processes on genetic diversity: différences between annual plants and tree species[END_REF] also predicted stronger gene flow under colonisation than under stable démographie situations. Additional support for such a scénario cornes from silver fir, for which Liepelt et al. (2002) revealed a range-wide cline for a patemally inherited chloroplast marker, demonstrating extensive postglacial pollen flow in a species with much larger pollen grains (Jackson & Lyford 1999) and a longer lifespan than ash. In oaks, the lack of an association between chloroplast and nuclear marker diversity was also mainly attributed to high levels of pollen flow during colonisation (Kremer et al. 2002).

Eastern Europe

In eastern Europe, the strong genetic structure we observed at nuclear loci in the common ash was rather unexpected, especially because chloroplast patterns of diversity did not allow to discem different glacial origins of populations in this area. The nuclear allozyme study in beech (Comps et al. 2001) showed some similarities with our results: among-population différentiation in the postulated refuge area in the Carpathians was higher than average, and the bottleneck statistic was overall low in southeastem Europe, as in our study. In oaks, for which at least two refuges were suggested in the Balkan Peninsula (Brewer et al. 2001), sampling efforts for genetic data were too limited in this area to permit any comparisons.

In common ash, fossil pollen data hâve identified one glacial refuge at the Bulgarian Black Sea coast, one in the Greek Pindus mountains and one in the Bulgarian Rhodopy mountains.

It is likely that, during glacial times, populations in these refuges were différendated at chloroplast markers and at nuclear markers (e.g. Kremer et al. 2002). In this context, the large prevalence of haplotype HT in the Balkan Peninsula suggests that either (i) refuge populations were differentiated at chloroplast markers and postglacial recolonisation occurred from a single refuge, or (ii) refuge populations were not differentiated and haplotype H1 had been fixed in the Balkan area already before the last ice âge (before 115000 BP).

The examination of diversity patterns at nuclear microsatellites within the Balkan Peninsula revealed high divergence among populations and some congruence according to geography, with higher levels of diversity in plain populations east of the Carpathians and Balkans than in populations within these mountains or west of them (Table 5), and a different gene pool composition of both groups of populations (Fig. 2). Three non exclusive hypothèses can be proposed to explain the observed patterns. ( 1) Gene pools might reflect differentiated lineages Contrasting démographie features of refuge populations could contribute to explain the higher levels of within-population diversity in the Danube and Dniestr plains than in mountainous areas. For instance, floodplain refuge populations near the Black Sea, which probably provided the colonisers to the Danube and Dniestr plains, could hâve remained relatively large throughout cold periods, conserving high levels of diversity, whereas mountainous populations could hâve been smaller and more scattered, and therefore subjected to stronger genetic drift. Moisture availability was a key factor for the persistence of refuge populations in the arid glacial periods, and for this reason, many refuges were located in mid-altitude sites (Brewer et al. 2002 and references therein). In sheltered floodplains, like in the Kamcija plain near Varna at the Bulgaria Black Sea coast, we suggest that sufficient moisture was available to sustain relatively large refuge populations. A larger pollen représentation for ash near the Black Sea than in the mountainous area 12000 years ago (Brewer 2001) A particular diversity pattern in Sweden

Populations from Sweden displayed diversity patterns at nuclear loci that were strikingly different from those we observed in other populations from western and central Europe. In Sweden, within-population gene diversity and allelic richness were low, the bottleneck statistic was négative, indicating population expansion without immigration, and populations were in Hardy-Weinberg equilibrium and strongly differentiated from each other (Heuertz et al. unpublished,chapter 3). These features are very similar to those we observed in southeastem European populations. Interestingly, in oak, populations from Denmark and Norway also showed higher allelic richness and lower gene diversity compared to western European populations (Zanetto & Kremer 1995); and in beech, populations from southem Sweden were more differentiated (Fst) and displayed lower values of the inbreeding coefficient (Fj) than populations from Germany or France (Comps et al. 2001). Comps et al. (2001) attributed this to quicker dynamics of the fixation indices compared to gene diversity measures in recently colonised populations. However, these populations also showed négative bottleneck statistics, indicating population expansion, and higher than average total allelic richness (Comps 2001). Populations from the British Isles could be interesting to confirm such diversity patterns as they should also hâve been recolonised most recently, but such populations were not analysed in beech. In oaks, diversity patterns in the British Isles were similar to those in western France, and did not show the particularities of Danish and Norwegian populations.

These unusual diversity patterns in Southern Scandinavian populations of ash, but also of oak and beech brings about the question whether glacial refuges for ash may hâve existed in northem central Europe. Palynological data indeed identified proportions of ash pollen above one percent in southem Sweden as early as 12000 BP (Brewer 2001), which is an indication for local presence of ash according to Huntley and Birks (1983). The inference of glacial refuges on the basis of pollen data alone is however increasingly questioned (e.g. Brewer et al. 2002), as pollen could hâve been transported over long distances. In this context, the importance of macrofossils is more and more stressed, as they represent the only method to provide unequivocal evidence for the presence of a taxon in a particular site (e.g. [START_REF] Birks | Future uses of pollen analysis must include plant macrofossils[END_REF]. Today, increasing evidence in many thermophilous plant and animal species suggests that southem European glacial refuges were supplemented by small "cryptic" refuges in northem Europe, usually located in areas of sheltered topography (Stewart & Lister 2001).

In the particular case of Sweden, macrofossils of boréal tree species like Norway spruce, Scots pine or birch discovered in the Scandes mountains of central Sweden hâve been dated to the late glacial period (the Younger Dryas, 11000 -10000 BP), suggesting colonisation of early deglaciated mountain peaks (nunataks) before complété déglaciation of the massif by 9200 BP [START_REF] Kullman | A new approach to postglacial forest history of northem Scandinavia. Review of megafossil and macrofossil evidence[END_REF]. It was suggested that these species immigrated into the Scandes during the Younger Dryas cold period from a Coastal refuge in the west, where they had persisted as particularly cold-adapted ecotypes (krummholz, i.e. végétative shmb-like growth forms) (Kullmann 2001). For ash, colonisation of Sweden from the west is not very likely not only because krummholz variants are not known, but also as our populations carried the eastem haplotype Hl. Early colonisation from the east is also improbable because the area was glaciated [START_REF] Kullman | A new approach to postglacial forest history of northem Scandinavia. Review of megafossil and macrofossil evidence[END_REF]). In the Swedish Scandes, macrofossils indicate that thermophilous trees such as oaks, elms, black aider, hazel and lime tree had appeared as early as between 8500 and 8000 BP, suggesting that colonisation probably occurred from small stands close to the ice sheets rather than from the southem refuges (Stewart & Lister 2001).

During the late glacial (around 12000 BP), the northem limit of pine forest was in northem Germany and southem Sweden, as inferred from palynological data (Stewart & Lister 2001).

Further, [START_REF] Willis | The full glacial forests of central and southeastem Europe[END_REF] provided evidence that thermophilous trees survived in microenvironmentally favourable pockets within refuges for more cold-adapted species in Hungary. Hence, a possible explanation for an early occurrence of ash in southem Sweden could be the existence of cryptic glacial refuges for ash in this pine forest, close to the Fennoscandian ice sheet. This hypothesis is not at odds with the Swedish populations carrying haplotype Hl, if the latter had been fixed before glacial times, regarding that H1 is found ail over eastem Europe.

In ash, the nuclear gene pool 2 inferred with the method of Pritchard (2000) (Fig. 2) is dominant in a population from Southern Sweden only, and it is présent in admixture in ail western and central European populations. Considering this together with the particular diversity patterns identified in oak and beech, the existence of cryptic refuges for thermophilous tree species in northem central Europe and their contribution to colonisation of western and central Europe might deserve more attention in the future.

Concluding remarks

Our study showed that the combined analysis of chloroplast and nuclear markers provided interesting insights into postglacial history of common ash. Nuclear microsatellites unravelled diversity patterns at high resolution, largely owing to an efficient model-based clustering method, suggesting that they might constitute more valuable tools for the investigation of population history than previously thought. High différentiation and spécifie gene pool compositions were detected among populations from southeastem Europe and from Sweden with nuclear markers, whereas chloroplast markers revealed only very little divergence. These results identified southeastem Europe and Sweden as important centres for the conservation of genetic resources in ash. This study demonstrated that the investigation of population history with only one genetic marker approach may lead to overlook essential diversity patterns in a taxon; for example nuclear markers did not discriminate the different maternai lineages in western and central Europe and chloroplast markers ignored strong différentiation for nuclear markers in the Balkan Peninsula. Therefore, we suggest that strategies for the management and conservation of genetic resources should be based on genetic markers with different modes of inheritance, especially when maternai markers display low polymorphism.

Référencés

Austerlitz F, Mariette S, Machon N, Gouyon PH, Godelle B (2000) Effects of colonization processes on genetic diversity: différences between annual plants and tree species. Genetics, 154, 1309-1321.

Bingelli P, Power J (1999) http://members.tripod.co.uk/WoodyPlantEcology/species/ash.htm [START_REF] Birky | Evolution and variation in plant chloroplast and mitochondrial genomes[END_REF] Evolution and variation in plant chloroplast and mitochondrial genomes. In: Plant evolutionary biology (eds. Gottlieb LD, Jain SK), p. 23-55 Chapman and Hall, London.

Birky CW Jr., [START_REF] Birky | An approach to population and evolutionary genetic theory for genes in mitochondria and chloroplasts, and some results[END_REF] An approach to population and evolutionary genetic theory for genes in mitochondria and chloroplasts, and some results. Genetics, 103, 513-527.

populations are not readily distinguishable by obvious adaptive différences under présent conditions.

In order to capture actual patterns of adaptive variation and enhance them, the EUFORGEN technical guidelines (unpublished) for common ash (see also Pliura 1999) foresee that in each country, eco-geographic or provenance régions should be delimited according to climatic variation, topography, soil and végétation. Then, two complementary networks of gene conservation populations should be established: (i) a network of 20-30 in situ populations selected throughout provenance régions, and (ii) a network of ex situ populations (progeny trials, provenance trials, collections). When possible, in situ conservation activities should be jointly performed for other noble hardwoods.

If common ash occurs in large populations within a country, in situ conservation is sufficient according to EUFORGEN technical guidelines (unpublished), and 1-3 stands of sizes 5-15 ha (or at least 100 flowering trees) should be selected in each région of provenance. In these stands, promoting natural régénération of the target species, monitoring health condition and protection against damage are necessary. Management should aim at creating multi-age structure and rapid génération turnover. For populations that are marginal, isolated, endangered, growing under spécial ecological conditions or carrying rare features, in situ conservation should be complemented by ex situ activities, which is most effective in the form of progeny trials, as it permits joint gene conservation and breeding.

Some additional recommendations for conservation of common ash genetic resources can be formulated on the basis of the molecular marker analyses in this thesis.

The high différentiation among populations detected with nuclear markers at the level of southeastem European countries most probably has a historical origin, regarding the proximity of different glacial refuges inferred from fossil pollen data. Hence, we suggest that southeastem European common ash populations contain differentiated genetic resources, which préparé them well for future uncertainties. In order to value the distinctiveness of genetic resources for common ash in this région, we suggest that a higher density of in situ gene conservation populations should be established in southeastem Europe, especially in Bulgaria and Romania, which were probably colonised from different glacial refuges.

Similarly, in Sweden, genetic resources in ash are highly differentiatied, although the historical origin of this différentiation still has to be verified. Additionally, the adaptation to photoperiod was experimentally confirmed [START_REF] Baliuckas | Within-population variation in juvénile growth rythm and growth in Fraxinus excelsior L. and Prunus avium L[END_REF] and signs for population bottlenecks were not identified neither in that study, nor in the présent one. We therefore also recommend particular conservation efforts in northem central Europe.

In western and central Europe, as well as Lithuania, our study revealed an important homogeneity of the nuclear gene pool and no particular patterns of divergence despite the presence of different maternai lineages. Hence in these régions, it is probably better to look at Further, it is also important to conserve populations close to glacial refuges or even the refuge populations themselves because of their significant rôle in the evolutionary history of the species in the past and potentially also in the future (e.g. Tzedakis et al. 2002); for instance we identified private chloroplast DNA haplotypes in populations close to refuges indicating peculiarities in their genetic composition.

Perspectives

Several perspectives for future research can be identified in the light of the results of this thesis.

A major subject with potentially serions financial implications is certainly the hybridisation between common ash and narrow-leaved ash. Narrow-leaved ash (Fraxinus angustifolia) is a termophilous tree species mainly occurring in Southern Europe (Tutin et al. 1972). It is a smaller tree (up to 25 m high) than F. excelsior (up to 40 m), and its wood is of lower quality [START_REF] Picard | A propos du frêne oxyphylle Fraxinus angustifolia[END_REF]. Common ash can be distinguished from narrow-leaved ash by its black buds and its leaves which show more serrations than latéral veins; in narrow-leaved ash, buds are brown and the leaves show as many serrations as latéral veins (Tutin et al. 1972). These two ash species hâve been reported to commonly hybridise in southeastem France (Rameau et al. 1989) and hybrid individuals were also found in nurseries among seedlings of F. excelsior [START_REF] Picard | A propos du frêne oxyphylle Fraxinus angustifolia[END_REF]. We observed many ash populations featuring a continuum from light brown to black buds in Romania, in the floodplain of the river Mures, west of the Carpathians, and close to the rivers Oit and Jiu east of the Carpathians. Because we were not sure of the taxonomie status, these samples were not included in our survey. Alternativement, ce patron pourrait aussi être expliqué par des caractéristiques contrastées des populations de refuges glaciaires, avec par exemple des populations fragmentées dans les montagnes du sud-est de l'Europe qui auraient subi une dérivé génétique plus importante qu'une large population dans un refuge de plaine alluviale près de la Mer Noire. Nous avons estimé un flux de graines très restreint et un flux de pollen modéré dans une population roumaine de frêne commun en comparant la structure de la diversité génétique dans la population avec des résultats de simulations où nous avions contrôlé la dispersion de graines et de pollen.

Nous nous sommes également intéressés à l'histoire évolutive d'un locus microsatellite nucléaire et nous avons identifié un déterminisme moléculaire distinct et des distributions géographiques distinctes pour des allèles de taille paire ou impaire. Deux scénarios évolutifs ont été proposés pour expliquer ces patrons, impliquant ou non l'hybridation avec une autre espèce de frêne, peut-être le frêne à feuilles étroites. Au sein de la famille des oléacées, le locus microsatellite montre une variabilité importante de la région répétée. Dans l'ensemble, ce travail a identifié le sud-est de l'Europe et le nord de l'Europe centrale comme des centres importants pour la conservation des ressources génétiques chez le frêne.

Summary: This thesis is an attempt to understand the factors that shaped the présent day spatial structure of genetic diversity in the common ash. It focuses on the structure of genetic diversity in southeastem Europe and compares it to diversity patterns in other European régions in the context of population demography and postglacial recolonisation dynamics during the current glacial interstadial.

Chloroplast microsatellite analysis allowed us to corroborate glacial refuges for ash situated in the Alps and in the Balkan Peninsula, as suggested from fossil pollen data, and our results are in agreement with other proposed refuges in Italy and the Iberian Peninsula. Nuclear microsatellite analysis revealed distinct diversity patterns, and notably contrasting situations in western and central Europe on the one hand, and in southeastem Europe and Sweden on the other. In western and central Europe, slight imprints from bottlenecks, high genetic diversity and a homogeneous gene pool over the entire area were interpreted as the resuit of extensive pollen flow, homogenising the nuclear gene pools of colonisers from differentiated refuges during the process of postglacial recolonisation. In southeastem Europe, high among-population différentiation as well as relatively low levels of genetic diversity and no signs from bottlenecks were attributed to the expansion from different glacial refuges with little among-population gene flow, hypothesising that a demographically stable condition with high density of ash was rapidly reached in the area. Higher diversity in the eastem than in the western part of the Balkan Peninsula was tentatively explained by hybridisation with other sympatric ash species in floodplain populations in the east. Altematively, this pattern could also be due to contrasting démographie features of glacial refuge populations, with for instance scattered and fragmented refuge populations in the southeastem European mountains that would hâve been subjected to stronger genetic drift than a large floodplain refuge population near the Black Sea. In Sweden, diversity patterns were similar to southeastem Europe, which lead us to suspect the existence of cryptic full glacial refuges in northem central Europe. Further, very narrow seed dispersai and moderate pollen dispersai were estimated in a Romanian common ash population by comparing the spatial genetic stmeture in the population with results from computer simulations where seed and pollen dispersai were under control. We also investigated the evolutionary history of a nuclear microsatellite locus and identified a different molecular determinism and different geographical distributions of odd-and even-sized alleles in common ash. Two evolutionary scénarios were proposed to explain the observed patterns, involving or not the hybridisation with another ash species, possibly narrow-leaved ash. Within the olive family, the microsatellite locus shows a high variability of the repeat région.

Overall, this work identified southeastem Europe and northem central Europe as important centres for the conservation of genetic resources in common ash.

•

  Assess total gene flow at different scales and relative contributions of pollen and seed to gene flow • Identify founder contributions • Investigate historical gene flow and locate colonisation routes (phylogeography)

Fig. 1

 1 Fig. 1 Map of cpDNA lineages in 8 European oak species. The géographie distribution of the six cpDNA lineages identified (A-F) is provided. From Petit et al. 2002b.

  describe phylogeographic patterns in European white oaks (eight species) based on chloroplast DNA (cpDNA) variation in over 2600 oak populations from ail over Europe. Six haplotype lineages, noted from A to F were identified with the PCR-RFLP (polymerase chain reaction -restriction fragment length polymorphism) technique, the géographie distribution of which is shown in Fig 1. Chloroplast data hâve been interpreted in comparison with Brewer et ai' (2002) data on 'deciduous Quercus' (22 species).

  Fig.4Geographical distribution of mean number of alleles per population of Quercus petraea. Cutoff point was chosen a priori for illustration purposes. FromZanetto and Kremer (1995).

Introduction

  populations colonising from different refuges, (ii) sélection against less viable, inbred individuals during population establishment and (iii) increased pollen flow during colonisation.2.4. Comparison of chloroplast and nuclear patterns of variation at genetic marker loci

Fig. 6

 6 Fig. 6 (a) Flowering common ash trees in the Marner river floodplain forest (Luxembourg); (b) imparipennate leaves and samaras; (c) flushing juvénile, note the black bud scales; (d) opening male inflorescences.

  Chapter I: Genetic structure ofcommon ash in Bulgaria assessed with nuclear microsatelUtes GENETIC STRUCTURE OF COMMON ASH IN BULGARIA 1619

Fig. 1

 1 Fig. 1 Sampling locations of common ash trees in the Ploesti forest.

  over loci for each of the five subpopulations and for the entire population with the program GEN-SURVEY(Vekemans and Lefèbvre 1997):(1) the average number of observed alleles A, (2) the average expected heterozygosity or gene diversity H corrected for small sample size(Nei 1978) and (3) Wright's inbreeding coefficient F corrected for small sample size(Kirby 1975). Déviation of genotypic frequencies from Hardy-Weinberg proportions was tested with exact tests with the program GENEPOP version 3.3 d (Raymond and Rousset 1995). Spatial genetic structure was analysed using kinship coefficients. The a priori kinship coefficient is the probability that a gene drawn at random from individual i and a gene drawn at random from individual j are identical by descent (IBD). From a sample of genotyped individuals, only relative kinship coefficients, Fy = -o)/(l -o), can actually be estimated, where O is the mean Oÿ over ail pairs of sampled individuals. Kinship coefficients were computed for ail pairs of individuals in each subpopulation and in the entire population using the statistic of Loiselle et al. (1995), which is defined for each allele k and each pair of individuals, i and j, as Fy = (pi -pk)iPj -pùK (1-PÙ) + l/(2n-l) where pi, pj are the frequencies of allele k in individuals i and j (taking the values 0, 0.5 or 1) and pk is the average allele frequency of allele k in the reference population with sample size n. Average multiallelic (multilocus) estimâtes were computed by weighting the Fÿ for each allele k by its polymorphism index p^fl-pk).

  www.ulb.ac.be/sciences/lagev). For graphical représentation of kinship in the total population, average kinship coefficients were computed for 17 distance classes, équivalent to those used for the analysis of simulated data (see below).Computer simulation procedureIn order to détermine whether inference on the relative contributions of pollen and seeds to total gene flow could be drawn from spatial genetic structure, we compared the kinship-distance plot of the common ash population to kinship-distance plots from simulated data sets produced with various combinations of pollen and seed dispersai parameters. Computer simulations were carried out on a theoretical population that closely matched features of the real population. In the real population, the density was on average 200 mature trees/ha and the maximum distances between samples were 3.43 km and 1.75 km on the abscissa and the ordinate of an orthogonal grid. Hence, the population could be represented by 490 x 250 trees positioned on a rectangular grid where adjacent trees were separated by 7 meters. To minimise edge effects, a larger theoretical population of 600 x 300 regularly spaced mature hermaphrodite diploid individuals was simulated considering overlapping générations.Simulations proceeded by the following steps: (1) an initial population is created by defining individual génotypes, choosing alleles stochastically at five loci according to allele numbers and frequencies observed in the common ash population; (2) each individual on the grid is given a 80% chance of surviving (assuming one simulation cycle corresponds to 1/5 of average lifetime); (3) each empty cell (death event) is replaced by an offspring whose maternai parent is chosen stochastically around the empty cell according to a pre-defined seed dispersai curve, and the patemal parent is chosen stochastically around the maternai parent according to a pre-defined pollen dispersai curve (individuals présent at the previous step were considered when selecting parents); (4) the génotype of each new offspring is defined by choosing, at each locus, one allele of each parent at random; (5) once ail individuals hâve been considered, a new population is defined by keeping previously surviving individuals and replacing dead ones by the offspring. Stages 2 to 5 were repeated 1000 times. This corresponds to 200 générations, which is a realistic figure for postglacial recolonised

Fk

  in each distance class k were averaged over the 100 simulation replicates, yielding for each class k in each parameter combination. First, to verify the expected linear decrease of kinship-distance plots, we plotted against the logarithm of distance (measured in m), and computed the slope br of the linear régression (i) over ail distances and (ii) over distances ranging from Og to 20 cr^, with cr/ = cr/ + where Oi, Op are the standard déviations of seed and pollen dispersai distances, respectively, entered as parameters. To compute br, F^ values were weighted by the number of pairs of individuals belonging to class k, nk'. -^iFk -f) k where Xk is the average of /«(distance between individuals), with distance in m, for distance class k; x is the weighted average of Xk, x -/'^^k i andF is the weighted average k / k kinship coefficient F-^n^F,^ j^rii^. Equation (2) ensures that br is essentially k ! k independent of distance class désignations because it is nearly équivalent to regressing ail pairwise Fÿ values on the logarithm of distance. The fit of the linear régression for each parameter combination was evaluated with the coefficient of détermination R , which expresses the proportion of variation in F^ values explained by the linear régression model. The extent of local gene flow, Og, was estimated from the régression slope br using the équation ô^ = -JNbl(47iD^), with the estimated neighbourhood size Nb= -(l-Fk=j)/br.

  ------------------). Realised values were slightly smaller than their /2 P'-\2 + V)(1 + F,) corresponding parameter values because of edge effects and the use of a lattice rather than a continuons space. The estimâtes were compared to their expected values computed as f^gexp=V^'r+0-5< •The shape of dispersai curves was further investigated by examining plots of residuals dk = F,-Fke (3) with Fke being the kinship coefficient for class k expected from linear régression over the entire distance range. Residuals dk were divided by Sy[ï^, (4) to obtain plots in standard déviation scale, where s = j{n-2) + riixi^-xYj^n^^ixf.-xy'iYic leverage coefficient of distance class k, as recommended bySokal and Rohlf (1995, p. 531), weighted by the number of pairs of individuals in class k. The total number of classes K was 17. The visual appearance of plots of standardised residuals against distance suggested that they might be explained by polynomial régression. We fitted polynomial fonctions up to the third degree of the logarithm of distance to the observed standardised residuals plots followingSokal and Rohlf (1995, p.616) and weighting each distance class by its respective number of pairs. Coefficients of these fonctions were compared to the ratio of seed V5. pollen contributions to total dispersai. Fitting the observed spatial genetic structure to simulation results The direct comparison of kinship values observed in the common ash population with those from simulated datasets required équivalent définitions of distance classes in the real and theoretical populations. Upper bounds of classes in the real population were determined by (i) multiplying those of the theoretical population by 7 m and (ii) adding or subtracting a maximum of 1.5 m to correct for rounding errors from positioning of the sample points in the theoretical population. The resulting 17 distance classes were (upper bound distances in m):

Fig. 2

 2 Fig. 2 Average kinship coefficients Fÿ between pairs of individuals plotted against the logarithm of geographical distance in the whole population. Dashed Unes represent 95% confidence intervals for Fij under the null hypothesis that génotypes are randomly distributed.

équation ( 1 )Fig. 3 Fig. 4

 134 Fig. 3 Average kinship coefficients Fÿ over 100 independent replicates of four simulated parameter combinations, plotted against the logarithm of distance. For squares, Oi=5 m, Op=49 m; for circles, o;v=14 m, Op=49 m; for triangles, (Ts=28 m, fTp=35 m; for diamonds, Oi=49 m, (7^=49 m. Open symbols stand for parameter combinations with (7g ~ 35-37m, whereas filled symbols feature C7g ~ 60m. Arrows represent the distance range between trand 20(7, where a linear decrease of Fjj is expected, the dashed arrow corresponding to (7g=31 m, the solid arrow to cTj=60 m.

Fig. 5

 5 Fig. 5 Shape parameter d of the kinship-distance plots according to the ratio of seed vs. pollen contributions to total gene dispersai, o] j{0.50^^). The quantity d corresponds to the coefficient of the term of third power of the polynomial fonctions (y=a+bx+cx +dx ) that were fitted to the standardized residuals of kinship coefficients (see text for details). The arrow stands for the observed d value in the common ash population and the corresponding range of estimâtes of ct,7(0.5<t;).

Fig. 6

 6 Fig.6Fit of kinship-distance plots from simulated data sets to the observed kinship-distance plot for three statistics: a, Chi-square; b, slope, br statistic; c, shape, d statistic. The axes represent the seed and pollen dispersai standard déviations used for simulations (scales not linear). Parameter combinations in white areas were rejected; darker shades represent an increasingly better fit of simulations with the observed data (see legend). The contours of shaded areas were interpolated. Lines of equal realised neighbourhood size Nbr were also plotted in (a).

  impact, through past and présent management, i.e. multiplication of interesting phenotypes (in chestnut,[START_REF] Fineschi | Chloroplast DNA reveals little geographical structure in Castanea sativa Mill. Fagaceae throughout southem European countries[END_REF], harvesting within stands (i.e.[START_REF] Rajora | Microsatellite DNA analysis of genetic effects of harvesting in old-growth eastem white pine (Pinus strobus) in Ontario, Canada[END_REF] and forest fragmentation (i.e.Aldrich et al. 1998), has also strongly influenced the patterns of genetic diversity of tree species.Large-scale patterns of nuclear genetic diversity in tree species hâve generally been investigated by analyzing the variation of population genetics statistics such as allelic richness, gene diversity and Wright's Fst as a function of geographical variables like latitude, longitude and altitude. Such analyses revealed either similar (mountain hemlock, Ally et al. 2000) or contrasting (sessile oak, Zanetto & Kremer 1995; beech, Comps et al. 2001) trends of variation in allelic richness and gene diversity over the investigated distribution area.

  conserved at -70° prior to DNA extraction. Leaves were dried between paper sheets or in plastic bags in the presence of silica gel. They were kept at room température until DNA extraction. DNA extraction Total DNA was extracted from 60 to 90 mg of dry leaves or from 50 to 70 mg fresh weight of buds. The plant material was ground by hand or in the automatic grinding mill MM200 (Retsch) for extraction with the DNeasy Plant mini kit (Qiagen) or the CTAB procedure of the NucleoSpin Plant kit (Macherey Nagel). Altematively, high throughput DNA extraction was performed by grinding simultaneously 192 samples of about 20 mg of dry leaves in the mill MM300 (Retsch) and subsequently extracting the DNA with the DNeasy 96 Plant Kit (Qiagen). Overall, the DNA extracts of ash performed with the NucleoSpin plant kit had to be less diluted for polymerase chain reaction (PCR) than the ones performed with the DNeasy kits, probably because of a better élimination with the former kit of métabolites susceptible to interfère with PCR.

Fig 2 -

 2 Fig 2 -Midpoint-rooted neighbour joining tree computed from Goldstein et al.'s (1995) (ô|i)^ distance among pairs of populations. The bootstrap support of the branches is given in bold numbers as the percentage out of 126 possible bootstraps.

  Fig 4 -Plot of pairwise Fst/(1-Fst) ratios against the logarithm of distances (in km) between populations. Under isolation by distance, values of the ratios are expected to increase linearly with the logarithm of distance (ROUSSET 1997).

  Chapter IV: Evolution ofa microsatellite locus in Fraxinus excelsior olive trees from Portugal; and DNA samples from four Phillyrea bushes from Southern France, the Balearic Islands and Sicily.Screening for allelic diversityTotal DNA was extracted from 60 to 90 mg of dry leaves or from 50 to 70 mg fresh weight of buds. The plant material was ground by hand or in the automatic grinding mill MM200 (Retsch) and extracted with the DNeasy Plant mini kit (Qiagen) or the CT AB procedure of the NucleoSpin Plant kit (Macherey Nagel). Polymerase chain reaction (PCR) was performed with FEMSATL4 primers(Lefort et al 1999) as described inHeuertz et al. (2001) andHeuertz et al. (unpublished). Difficult templates were PCR amplified with LATaq polymerase (TaKaRa). Fluorescent labelling of the forward primer allowed détection of amplification Products on an ABI PRISM® 377 DNA sequencer. Sizing of fragments was performed with the software programs Genescan® 3.1 and Genotyper® 2.5 from Applied Biosystems by comparison with an internai sizing standard (Genescan-350 Rox), and alleles were called by eye. Différences in mean allele sizes among pairs of species were tested with T-tests. For the two Faxinus species, for Olea and for Ligustrum, the following genetic diversity statistics were computed for the overall species samples with the program FSTAT v. 2.9.3.2(Goudet 2001): the allelic richness A, which quantifies allelic diversity with the property of being independent of sample size variation, for a standardised sample size of n=21 diploid individuals (corresponding to the smallest sample size, observed in F. omus) according to El Mousadik andPetit (1996); the total gene diversity H\ and the overall inbreeding coefficient F\. Déviation from Hardy-Weinberg genotypic proportions was tested with exact tests with the program GENEPOP v. 3.3(Raymond & Rousset 1995).

F

  ____ T.................................................................................................................................... T ........................................... G T............................................. G O. europaea 1 .........................................................................................................................................G O. europaea 2 : ..........................................................................................................................................G O. europaea 3 : .................................... G................................................................................................ G O. europaea 4 = .........................................................................................................................................G P. angustlfolia 1 : .......................................... T.............................................................................................. P. angustlfolia 2 = .......................................... T..............................................................................................

Fig. 2

 2 Fig.2Alignment of nucléotide sequences between five Oleaceae species for the microsatellite locus of the study. Primers are excluded from the sequences. Dots (.) show identical nucléotides. Gaps (-) hâve been placed to increase the similarity. The box contains what is defined as the microsatellite repeat région. Numbers next to aligned sequences indicate nucléotide positions when the repeat région is excluded. Species names in bold correspond to haplotypes defined by variation in the flanking régions, and the corresponding microsatellite repeat sequences. Plain sequence names indicate sequences with additional variation in flanking or repeat régions, which were occasionally observed (at least two observations for each sequence).

Fig. 3

 3 Fig. 3 Agreement of phylogenetic relationships among five species of the tribe Oleae, based on (a) number of mutations in flanking régions of microsatellite FEMSATL4 (rectangular cladogram of midpoint-rooted neighbour joining tree) and (b) on sequence variation at two non-coding chloroplast loci, the rpsl6 intron and the tmL-F région (the species of interest are indicated with arrows; part of Fig.l extracted from[START_REF] Wallander | Phylogeny and classification of Oeaceae based on rpsl6 and tmL-F sequence data[END_REF]. The original figure in this reference shows a strict consensus tree of the most parsimonious trees from the combined data set at the two loci, with indels coded as separate characters, for 76 Oleaceae species. Jackknife support values over 50% are shown above the branches.

Fig. 1

 1 Fig. 1 Géographie distribution of the haplotypes identified with chloroplast microsatellites.

  Samples of Fraxinus excelsior were collected in 42 putatively autochthonous forests in Europe, comprising the 36 populations previously analysed at nuclear microsatellite loci(Heuertz et al. unpublished, chapter 3; Figs. 1 and 2) and six additional populations from Poland (number 37, Choszno, 53°10'40"N, 15°18'09"E; 38, Jawor, 50°58'58"N, 16°12'23"E; 39, Jamy-Chelmo, 53°20'59"N, 18°23'52"E; 40, Jamy-Bialochowo, 53°32'59"N, 15°18'09"E; 41, Nowogard, 53°38'27"N, 15°05'27"E; 42, Szczecinek, 53°44'35"N, 16°42'28"E; Fig.1).Samples consisted of buds or leaves. Buds were brought to the laboratory on their twigs, wrapped in wet paper. They were dissected out, separated from their scales and conserved at -70°C prior to DNA extraction. Leaves were dried between paper sheets or in plastic bags in the presence of silica gel. They were kept at room température until DNA extraction. Total DNA was extracted from 60 to 90 mg of dry leaves or from 50-70 mg fresh weight of buds with the DNeasy plant mini kit (Qiagen) or the CTAB procedure of the NucleoSpin Plant kit (Macherey Nagel).Chloroplast microsatellite analysisA total of 323 trees were analysed at chloroplast microsatellite (cpSSR) loci, which corresponds to an average of 7.7 ± 3.2 (SD) trees per population. Chloroplast microsatellites corresponding to poly(A) or poly (T) repeats were polymerase chain reaction (PCR) amplified with six universal primer pairs for angiosperms (ccmp2, ccmp3, ccpm4, ccmp6, ccmp7, and ccmplO from Weising & Gardner 1999) in a total reaction volume of 25 pi with the following

(

  2001) recommended to preferentially use PCR-restriction fragment length polymorphism (PCR-RFLP) markers, which are based on nucléotide substitution polymorphisms, if these display enough polymorphism. In our study organism, PCR-RFLP markers show very little polymorphism (G.G. Vendramin & D. Salvini, personal communication), which is why we

  Chapter V: Genetic structure ofcotnmon ash in Europe assessed with chloroplast and nuclear microsatellites of common ash, for example ecotypes adapted to mountainous or floodplain habitats, and each lineage would display characteristic diversity patterns. (2) Some gene pools might reflect hybridisation with other ash species, like F. angustifolia or F. pallisiae, occurring in the Balkan Peninsula(Tutin et al. 1972). As F. excelsior and F. angustifolia hâve been reported to hybridise in southeastem France(Rameau et al. 1989), hybridisation might account for some of the différentiation between F. excelsior populations, for instance between hybridising and non-hybridising ones. (3) Gene pools might reflect glacial origin. The strong différentiation and the very heterogeneous gene pool composition among populations in the Balkan Peninsula suggest that postglacial recolonisation most probably occurred from multiple refuges, in agreement with fossil pollen data(Huntley & Birks 1983, Gliemeroth 1997, Brewer 2001). Under this hypothesis, haplotype H1 must hâve been fixed in the Balkan area before the last glacial period. A rapid expansion in the early Holocene from several refuges in the Southern Balkan Peninsula as suggested byHuntley and Birks (1983) is very compatible with our results and evokes the following steps. (1) As the climate improved, multiple populations differentiated for their nuclear gene pools expanded mostly at their edges. (2) Rare long-distance colonisation events would not hâve been very successful because of the proximity of the refuges and the rapidly achieved high density of ash in the area. (3) Similarly, because of high density, high compétition would hâve taken place against incoming pollen, and therefore, among-population pollen flow would hâve rarely resulted in effective gene flow. (4) Additionally, the complex topography might hâve made gene flow even more difficult, keeping the gene pools differentiated. A scénario of expansion of multiple differentiated gene pools with little immigration is also in agreement with low values of the bottleneck statistic in the Balkan Peninsula.

  provides additional support to this matter. The slightly larger différentiation values in the western part of the Balkan Peninsula could also reflect a stronger fragmentation of populations in mountain refuges. Further, hybridisation is probably a stronger issue for floodplain than for mountain populations, as F. angustifolia and F. pallisiae preferentially occur in floodplain habitats(Tutin et al. 1972). Therefore, hybridisation might also contribute to explain the higher levels of diversity found in F. excelsior populations from the Danube and Dniestr floodplains compared to those from the mountains, and/or the strong divergence of populations 23-25 from populations 30-36 in that area.Eastern European contact zoneA zone of admixture of haplotype H1 of eastern European origin and haplotype H2 from the Alps was observed in Germany and Poland. This confirms an early northeastward colonisation form an Alpine refuge, joining the westward expansion observed in Poland as suggested from fossil pollen data (around 8000 BP,Huntley & Birks 1983). Whether haplotype H1 in the contact zone originates from the southem Balkan Peninsula, or possibly from a northem Carpathian refuge in southeastem Poland(Huntley & Birks 1983, Brewer 2001) is yet unclear. In case such a refuge contributed to postglacial recolonisation, it is most likely that it contained haplotype Hl, providing another argument for an ancient fixation of this haplotype in eastern Europe. At the level of the nuclear gene pool, the strong admixture revealed in populations 18 and 19 from Slovakia and Hungary by the method ofPritchard et al. (2000) (Fig.2) suggests contact of central and eastern European gene pools. The advance of a central European maternai lineage up to northem Poland can also partially explain the high levels of admixture discovered in Lithuania (Fig.2).

  présent patterns of adaptation according to ecological zones, and to identify particular features. For instance, an important dieback of young ash populations due to a yet unidentified pest is actually reported in Poland and Lithuania (J. Nowakowska and A. Pliura, Personal communication), and résistant populations should benefit from complementary ex situ conservation. Despite little différentiation at nuclear markers in our study, we identified gradients of increasing levels of inbreeding from southeastem Europe towards the west and the north, with the exception of Sweden. If this pattern originated from increased selfing ability (which we stated as one possible explanation,Heuertz et al. unpublished, chapter 3), it could be meaningful in the context of adaptation, at least during colonisation, where longdistance colonisers with a potential for self-fertilisation would hâve had a sélective advantage over colonisers lacking such a mechanism. In order to capture such large-scale trends of potentially adaptive variation, we propose that in large geographical régions with similar ecological features, several geographically distant populations should be chosen for in situ gene conservation.

Table

Table

  Frequently used molecular markers in population genetics

		Markers that address variation in nuclear DNA		Chloroplast DNA markers
		Isozymes /	Nuclear	Random	Amplifled	Polymerase	Chloroplast
		allozymes:	microsatellites	amplified	fragment	Chain reaction	microsatellites
			/ simple	polymorphie	length	-Restriction
			sequence	DNA (RAPD)	polymorphism	fragment
			repeats (SSRs)		(AFLP)	length
						polymorphism
						(PCR-RFLP)
	Inheritance	Codominant	Codominant	Dominant	Dominant	Uniparental	Uniparental
	Polymorphism	Modest	High	High	High	Modest	Moderate
	Main use	Genetic	Genetic	Genetic	Genetic	Phylo-	Phylo-
		diversity and	structure at	diversity and	diversity and	geography	geography
		structure	local scale	structure	structure
	Advantage	Many studies	Application in	Multilocus	Multilocus		Higher
		available for	related species	technique	technique, high		polymorphism
		comparison'	sometimes		reproducibility		than RCR-
			possible				RFLP
	Disadvantage	Sélection	omoplasy^,	Low	Scoring is		HomoplasyĈ
		cannot be	null alleles"*,	reproducibility	difficult
		excluded^H scoring
			problems
	ost	Moderate	High,	Low	High	Moderate	Moderate
			especially the
			development

Table 2

 2 Oaks are abondant and widely spread in Europe and play important rôles from both economical and ecological points of view. Their morphology and molecular genetics hâve been thoroughly studied for practical reasons, for example in the view of developing methods for tracing the géographie origin of seed lots, identifying the source of plantations, checking the autochthonous status of oak woods or forests for conservation purposes or even tracing the origin of wood products(Petit et al. 2002a). Biologically, oaks represent a highly challenging study subject, as many of them undergo extensive introgressive hybridisation, forming a species complex. Sessile oak (Quercus petraea) is a commercially important widespread species covering most parts of Europe from Spain to Russia and Scotland to Turkey. It is

	He	Gst

Levels of allozyme diversity within and among populations of species belonging to different life forms. He, genetic diversity within populations; Gst, proportion of the total diversity among populations. Means followed by the same letter are not significantly different at the 5% probability level. From

Hamrick et al. (1992)

. complex. These examples are meant to introduce the reader to population genetic structure of European forest trees and will serve for comparison in the discussion of the genetic structure patterns of common ash presented in this thesis. sympatric with pedunculate oak (Quercus robur). Both species often occur in the same stands and occupy different but proximal ecological niches.

2.1. Genetic diversity within populations of sessile and pedunculate oak analysed with nuclear DNA markers.

Table 3

 3 Levels of genetic diversity within populations of Quercus robur and Q. petraea: A, mean number of alleles per population; Ho, observed heterozygosity; He, gene diversity; Fis, fixation index.

			No.		No.
	Marker Species	pops No. trees/pop	loci A	Ho He ^IS Référencé
	allozymes	Q. robur	7	120	13	2.703 0.184 0.252	Zanello et al. 1994
	allozymes	Q. robur	1 mixed	217	7			0.244 Bacilieri et al. 1994
	allozymes	Q. robur	2	156	7	3.065 0.298	0.072 Degenefa/. 1999
	allozymes	Q. petraea	7	120	13	2.725 0.222 0.245	Zanettoera/. 1994
	allozymes	Q. petraea	1 mixed	190	7			0.304 Bacilieri ef a/. 1994
	allozymes	Q. petraea	81	120	8	2.629 0.228 0.257	Zanetto, Kremer 1995
	allozymes	Q. robur & Q. petraea 1 mixed 183 2-r. \66Q.p.	4	4.3	0.25	0.34	0.16 Streiff et al. 1998
	SSR	Q. robur	2	156	7	17.88 0.896	-0.022 Degen et al. 1999
	SSR	Q. robur	7 mixed	174	6	18.86 0.792 0.868 0.083 Mariette et al. 2002
	SSR	Q. petraea	7 mixed	168	6	20.22 0.831 0.878 0.047 Mariette et al. 2002
					6	21.7	0.81	0.87	0.07 Streiff et al. 1998
	RAPD	Q. petraea	21	23	31			0.233	Le Cotre et aZ. 1997
	AFLP	Q. robur	7 mixed	174	155			0.195	Mariette et al. 2002

SSR

Q. robur & Q. petraea 1 mixed 183 2r. 166 2-P-microsatellite markers than allozymes, because of the frequent occurrence of null alleles at microsatellite loci (e.g.

[START_REF] Callen | Incidence and origin of 'null' alleles in the (AC)n microsatellite markers[END_REF]

.

Table 4

 4

		No.	No.	hs	vs	hf	Vt	Gst	Nst
		Pops. Haplo
			types
	Alpine région	121	6 0.062 0.075 ns	0.574 0.642 ns	0.892 0.884 ns
	France	170	6 0.118 0.061 ns	0.712 0.539 ns	0.835 0.886 ns
	Central Europe	169	9 0.154 0.174 ns	0.765 0.857 ns	0.799 0.797 ns
	Great Britain	84	4 0.094 0.034 ns	0.605 0.239 ns	0.845 0.857 ns
	North Balkans	97	11 0.139 0.070 **	0.792 0.593 ns	0.825 0.883 **
	Northern Europe	27	8 0.107	0.068 ns	0.842 0.892 ns	0.873 0.924
	Q. petraea	650	17 0.122 0.091 ns	0.847 0.835 ns	0.856 0.891

Levels of diversity and différentiation in Quercus peîraea.

From Petit et al. (2002b)

.

Table 4

 4

summarises diversity and différentiation levels observed in Quercus petraea. It is immediately obvions that within-population diversity is low (mean hs = 0.122, mean vs = 0.091), but total diversity is high, resulting in strong différentiation among populations (typically Gst > 0.8). Overall, phylogeographic structure is observed, as Nst is significantly larger than Gst-A refuge effect can be observed in the Northern Balkans. This région is characterised by a high number of haplotypes (

11

) and relatively high diversity hs and hj, but low phlyogenetic information content (vs and vt smaller than the corresponding h values).

  Rhodopy mountains in southem Bulgaria, the Dinaric Alps in the area of former Yugoslavia and in Albania and the Pindos in Greece; interspersed with large plains in Hungary and the north of former Yugoslavia (rivers Danube, Save and Tisa), in southem

	Bulgaria, the
	iTISLAVA
	BUDAPEST	.iAlKi	ÀHULno»^'i !. ^-yv':-
	ZAGREB
			BLACK BEA
	;rade		BUCHAREST
	>N MARINO
			VarnaÂ
	DRIATIC^d
	i Varsno
			Oh
	■Vbunt ^meo
	H EN I AN SEA
	Resources of Broadleaved Forest Tree Species in Southeastem Europe, financed by the
	Luxembourgish Ministry of Finance and coordinated by the International Plant Genetic
	Resources Institute (IPGRI). The financial support to this project by the Luxembourgish fatermo
	Government can be seen as an important contribution to the implémentation of Strasbourg
	resolution S2 on forest genetic resources. Partners in the first phase of the project were the
	Forest Research Institute in Sofia, Bulgaria; the Institute of Botany in Chisinau, the Republic
	of Moldavia; the ICAS Forestry Administration, Bucharest, Romania; and the Public
	Research Centre -Gabriel Lippmann, Luxembourg. The main objectives of the project were Fig. 5 Topographie map of the Balkan Peninsula
	to carry out new forest inventories in southeastem European countries to investigate
	distribution and health status of forest tree species and to organise exchanges of researches for
	training and common research activities in the fields of in vitro propagation, adaptation to
	physiological stresses and population genetics of forest tree species. The présent work was
	initiated from the latter subject and focuses on the géographie structure of genetic diversity in
	the common ash {Fraxinus excelsior L.), mainly in southeastem Europe, but also in the
	context of diversity patterns in other European régions.
	3.1. Forest resources in southeastem Europe
	Southeastem Europe is a rich area with respect to forest genetic resources because it contains
	a variety of ecological régions. The climate in the Balkan Peninsula is mainly continental, but

mediterranean on the west coast, and the topography is highly variable with the mountain chains of the Carpathians reaching from Slovakia to Romania, the Balkans in central

  Greece and in northem Turkey; it is présent around the Black Sea and as far east as the Caspian Sea and northem Iran.

	M				KNometers
	0 150 300	600	900	1,200	1.500
	Fig. 7 Fraxinus excelsior distribution map (from EUFORGEN Noble Hardwoods
	Network).

northem

  coriariifolia, which is distributed in southeastem Europe and Iran(Tutin et al. 1972).

	Fraxinus angustifolia occurs in Southern and south-central Europe; it also contains two
	subspecies, with non-overlapping ranges, ssp. angustifolia, spread in the western
	mediterranean région and Portugal, and ssp. oxycarpa, which occurs in eastem-central Europe
	and in Southern Europe, from northeastem Spain eastwards (Tutin et al. 1972). Fraxinus
	pallisiae is distributed in southeastem Europe, from Turkey to Moldavia.
	As F. excelsior has a poor pollen représentation, Huntley and Birks (1983) suggested that
	values above 1% in pollen cores would indicate local presence, whereas values above 5%

Table 1

 1 Characteristics of common ash populations sampled from Bulgaria

					Elévation		Proportion	Basic
	Population		Stand type	(m above sea level)	Location	(%)'	substrate
	1. Za dol		coppioe		100		43°50'N, 27°03'E	30	limestone
	2. Kodga ormani		coppice		200		43°54' N, 26°54' E	40	loess
	3. Iri hissar		mature light	150		43°51'N,26°46'E	10	loess
	4. Dulovo		mature dense	200		43°54'N, 26"54'E	30	loess
	5. Bukatchov chukar	mature high	850		42°50'N, 26°02'E	20	mari
	6. Golyamoto ravnishte	young dense	800		42°50'N,26"02'E	100	mari
	7. Elena		young dense	750		42°50'N,26°03'E	30	mari
	8. Kokalyane monastir	mature high	1000		42''33'N,23°26'E	30	schist
	9. Ljulin monastir		mature high	1050		42°39'N, 23''11'E	30	conglomérâtes
	10. Vitosha		mature high	900		42°38'N,23°14'E	30	andésite
	'Proportion of common ash in the canopy.
							Table 2 AUelic diversity of the microsatellite
				Original publications	This study	scored in the common ash
		Repeat		No.of	Range of	No.of	Range of
	Locus	motif		alleles	sizes(bp)	alleles	sizes (bp)
	M2-.30*	(TG),5 (AG)23		18	182-248	59	182-294
	FEMSATL4t	(CA)j (AG)24		9	164-228	50	158-251
	FEMSATLll+	(GA>2o (TA)4		11	180-226	32	176-266
	FEMSATL12t	(GA)^CA(GA)"	9	180-262	18	181-264
	FEMSATL16t	(CA), CG (CA),0	4	180-200	10	176-204
	FEMSATL19t	(CA),, CGGC (CA), ,	12	174-214	33	142-229

Table 2

 2

	). The PCR reactions
	were performed in a mix containing 2.5 mM MgClj, 0.2 unit
	per reaction of Taq polymerase (Gibco BRL) in Gibco PCR
	buffer, 0.4 pM of each primer, 0.2 mM t)f each dNTP and
	approximately 30 pg of template DNA (1 pL of extract
	diluted 100 times) in a total reaction volume of 10 pL. After
	an initial denaturing step for 4 min at 95 °C, amplification
	comprised 35 cycles of 30 s at 94 °C, 45 s at either 52 °C

(FEMSATL4, FEMSATLll, FEMSATL12, FEMSATL16, FEMSATL19)

or 56 °C (M2-30) and 1 min at 72 °C. Final élongation was for 5 min at 72 °C. PCR reactions were ©2CX)1 Blackwell Science Ltd, Molecular Ecologi/, 10,1615-1623

Table 3

 3 Statistics of genetic variation within populations at five microsateUite loci

	Population	tî	A	«0	He	^is'*'
	l.Zlidol	36 17.4	0.720 0.751	0.041*
	2. Kodga ormani	36 14.6	0.756 0.738 -0.024
	3. Iri hissar	20 12.4	0.740 0.716 -0.034
	4. Dulovo	33 13.8	0.691 0.758	0.089***
	5. Bukatchov chukar	30 11.2	0.641 0.687	0.066
	6. Golyamoto ravnishte	37 15.4	0.762 0.807	0.055
	7. Elena	30 12.8	0.780 0.779 -0.001
	8. Kokalyane monastery 37	9.0	0.735 0.727 -0.012**
	9. Ljulin monastery	30	6.4	0.661 0.638 -0.037*
	10. Vitosha	32 10.6	0.709 0.713 -0.004***
	Overall mean		12.36 0.720 0.731	0.014***
	■Standard déviation		3.22 0.045 0.048	0.045

fl, number of trees sampled; A, average nuniber of alleles per locus; Hçy average proportion of hétérozygotes; Hg, average gene diversity; F,5, average inbreeding coefficient. tExact test of departure front Hardy-Weinberg genotypic proportions: *P < 0.05; **P < 0.01 ; ***P < 0.001 ; ail other values, not significant.

Table 5

 5 Hierarchical f-statistics at the fivc Différentiation atnong: microsatellite

  Degen étal. (1999) analysed four of the mentioned lod in two populations of Q. robur and found an average Hg = 0.88 and between 14 and 33 alleles p>er locus.

	GENETIC STRUCTURE OF COMMON ASH IN BULGARIA 1621
	1999: n = 16). However, this observation could be an
	indication of a particularly high genetic diversity in
	Bulgarian populations of F. excelsior, in agreement with the
	hypothesis based on palynological data that this area of the
	Balkans acted as a glacial refuge for the spocies (Huntley &
	Birks 1983). Clearly, studies on microsatellite variation in
	common ash populations from other régions of Europ>e are
	needed to confirm this hypothesis. In studies of other
	deciduous temjaerate tree sp>ecies using microsatellite
	markers, similarly high polymorphism has been reported:
	Isagi et al. (2000) found Hg = 0.87 ± 0.08 and 7-40 alleles at
	eight microsatellite loci in Magnolia obovata; Streiff étal.
	(1998) reported mean gene diversity Hg = 0.87 and observed
	number of alleles comprised between 16 and 32 for six
	microsatellite loci in a mixed stand of Quercus robur and
	Quercus petraea-

: n = 50,

Lefort et al.

  When compared to other noble hardwood species studied at a similar geographical scale, our results of population différentiation in the common ash fall within the reported François Hausman is senior researcher at CRP-Gabriel Lippmann, working on the physiological response of woody species to environmental constraints. Nathalie Frascaria-Lacoste is a lecturer in forest genetics at the ENGREF Institution (PARIS). Ivaylo Tsvetkov is senior researcher at Forest Research Institue, Sofia, woking on micro propagation and régénération of tree species. Xavier Vekemans is senior researcher at ULB working in plant population genetics. Introduction In natural plant populations, spatial genetic structure of sexually reproducing individuals is determined by the combined effects of gene flow, local genetic drift, varions forais of natural sélection and the spatial arrangement of individuals

range:, e.g. Jugtans regia, ^ST within different European countries ranging from 0.018 to 0.071 (Fomari étal. 1999); Castanea sativa from Italy, Fg.,. = 0.10 (Pigliucci étal. 1990), from Turkey, Fgj = 0.184

(Villani étal. 1999)

; Ulmus minor from France, Fgy = 0.178

(Machon étal. 1997)

. Further investigations on pollen and seed dispersai with direct methods in noble hardwood species are needed to test whether différences in levels of genetic différentiation can be mainly explained by variaHon in gene dispersai.

M5rriam Heuertz is a PhD student at CRP-Gabriel Lippmann and ULB working under the supervision of Jean-François Hausman and Xavier Vekemans on the conservation of genetic resources in the common ash from Eastern Europe. Jean-

Table 1

 1 Statistics of genetic diversity of microsatellite loci in the overall sample. , total number of alleles; H, gene diversity; F], Wright's inbreeding coefficient."Exact tests for departure from Hardy-Weinberg genotypic proportions were non-significant for ail loci.

	Locus	K	H
	M2-30	42	0.918	0.043
	FEMSATL4	37	0.880	0.007
	FEMSATLll	32	0.918	0.003
	FEMSATL16	10	0.663	0.165
	FEMSATL19	27	0.568	-0.066

K

Table 2

 2 Genetic diversity statistics within subpopulations and the total population.

	Population	n	A	H	Fi"
	Subpopulation 1	30	13.2	0.799	-0.011
	Subpopulation 2	30	14.8	0.765	0.044
	Subpopulation 3	30	13.8	0.773	0.045
	Subpopulation 4	32	16.6	0.807	0.060
	Subpopulation 5	30	14.8	0.794	-0.022
	Mean subpopulations	30.4	14.6	0.788	0.023
	Total population	152	29.6	0.792	0.029

n, sample size; A, average number of alleles per locus; H, gene diversity; Fj, Wright's inbreeding coefficient.

" Exact tests for departure from Hardy-Weinberg genotypic proportions were non-significant for ail subpopulations and for the total population.

Table 3

 3 Spatial genetic structure and estimâtes of gene dispersai.

		Pairs of					sampling
	Population	individuals	bro	P-value	Fo	Nb	area (ha)
	Subpopulation 1	435	-0.0177	0.0201	-0.012	57.3	0.83
	Subpopulation 2	435	-0.0165	0.0217	0.054	57.3	1.38
	Subpopulation 3	435	-0.0039	0.2475	0.047	ne	3.69
	Subpopulation 4	496	-0.0170	0.0241	0.064	55.1	1.76
	Subpopulation 5	435	0.0005	0.5001	-0.022	ne	0.71
	Mean subpopulations	2236	-0.0106	0.0019	0.027	92.2	1.68
	Total population	11476	-0.0019	0.0038	0.031	518.5	-710

bro, slope of the régression of pairwise kinship coefficients on the logarithm of géographie distance; P-value of the onesided Mantel test with HO; observed bn > 0; Fo, average kinship coefficient between adjacent individuals; Nb, estimate of neighbourhood size; ne, not computed because the Mantel test was not significant distance (m)

Table 1

 1 Sampling locations and sample sizes for common ash.

		Population	Acronym	Latitude	Longitude n	Région"	Zone"
	1	Camolin	IreCa	52=3674	6=28'W 30	Ircland	West
	2	Avova	IreAv	52°54'N	6=1 l'W 29	Ireland	West
	3	Kilmacurra	IreKi	53°00'N	6=10'W 30	Ireland	West
	4	Loch Tay	ScotTa	56=3574	4=03 AV 30	Scotland	West
	5	Saint Gobain	FraGo	49=3574	3=22'E 20	France	West
	6	La Romagne	FraLr	49=4074	4=19'E 20	France	West
	7	Bremgarten	SwiB	47=2074	8=18'E 30	Switzerland	Center
	8	Ehrendingen	SwiG	47=2974	8=2 l'E 30	Switzerland	Center
	9	Eglisau	SwiE	47=3574	8=3 l'E 30	Switzerland	Center
	10	Tiroler Ache	GerTi	47=5074	12=3l'E 30	Germany	Center
	11	Chiemsee	GerCh	47=4874	12=3 l'E 29	Germany	Center
	12	Freilassing	GerFr	47=5074	12°59'E 30	Germany	Center
	13	ROdjan	SweRoe	57=1974	13=59'E 30	Sweden	Northeast
	14	Ehd	SweEhd	57=1474	13=59'E 30	Sweden	Northeast
	15	Akerâs	SweAk	57=1774	14=04'E 29	Sweden	Northeast
	16	Zeimelis	LitZei	56=1674	24=03'E 30	Lithuania	Northeast
	17	Kaisiadorys	LitKai	54=5374	24=22'E 30	Lithuania	Northeast
	18	Slovakia	SloPa	48=3474	19°08'E 26	Slovakia & Hungary	Southeast
	19	Lillafüred Lusta-Vôlgy	HunLue	48=0874	20=40'E 28	Slovakia & Hungary	Southeast
	20	Ljulin monastir	BulLj	42=39'N	23=1l'E 29	Western Bulgaria	Southeast
	21	Vitosha	BulVi	42=3874	23=14'E 32	Western Bulgaria	Southeast
	22	Kokalyane monastir	BulMo	42=3374	23=26'E 37	Western Bulgaria	Southeast
	23	Tutuleac	RomTu	46=13'N	24=48'E 31	Romania	Southeast
	24	Balota 1	RomBaa	44=5074	26=04'E 30	Romania	Southeast
	25	Balota 2	RomBab	44=5074	26=04'E 30	Romania	Southeast
	26	Balota 3	RomBac	44=5074	26=03'E 30	Romania	Southeast
	27	Golyamoto ravnishte	BulGr	42=5074	26=03'E 36	Central Bulgaria	Southeast
	28	Bukatchov chukar	BulBc	42=5074	26=03'E 30	Central Bulgaria	Southeast
	29	Elena	BulEl	42=5074	26=04'E 30	Central Bulgaria	Southeast
	30	Iri hissar	Bullh	43=5174	26=46'E 20	Eastem Bulgaria	Southeast
	31	Dulovo	BulDu	43=5474	26=54'E 32	Eastem Bulgaria	Southeast
	32	Kodga ormani	BulKo	43=5474	26=54'E 36	Eastem Bulgaria	Southeast
	33	Zli dol	BulZd	43=5074	27=03'E 35	Eastem Bulgaria	Southeast
	34	Hrjauca 1	MolHr	47=1874	28=12'E 31	Republic of Moldova	Southeast
	35	Hijauca2	MolHi	47=1774	28=15'E 27	Republic of Moldova	Southeast
	36	Hrjauca 3	MolHa	47=2074	28=17'E 32	Republic of Moldova	Southeast

n, sample size per population. " For the définition of régions and zones, see Material and Methods.

Table 2

 2 Allelic diversity of the nuclear microsatellite loci scored in the common ash. Ti, bottleneck statistic from Cornuet and Luikart (1996) computed on the basis of the infinité alleles model lAM) and the stepwise mutation model (SMM); S.D. (populations), standard déviation over populations. " For the définition of groups, see Material and Methods Chapter III: Genetic structure ofcoiiimon ash in Europe assessed witli nuclear microsatellites

	Locus	Repeat motif	(bp) Range of sizes	K	Ht	F	fsT	Rst
	M2-30	(TG),5(AG)23"	176-294	83	0.979 0.018 0.059 0.150
	FEMSATL4	(CA)2 (AG)24"	155-298	71	0.898 0.062 0.066 0.066
	FEMSATLll (GA)2o(TA)4''	176-249	48	0.904 -0.018 0.067 0.034
	FEMSATL16 (CA)3 CG (CA)io''	170-210	16	0.433 0.203 0.093 0.079
	FEMSATL19 (CA)ô CGGC (CA),3''	142-230	57	0.949 -0.001 0.106 0.350
	K, total number of alleles; Hr, total gene diversity; F, Wright's inbreeding coefficient; Fsr. relative différentiation based
	on allele identity; Rsv, relative différentiation based on allele size.
	* from Brachet et al. 1999
	** from Lefort et al. 1999

Diversity statistics were computed as multilocus statistics averaged over populations from the same région or zone. N^, number of populations included in each group; n, average sample size per population; As_36, allelic richness for standardized samples of 36 gene copies (standard déviation over loci); gene diversity (standard déviation over loci);

F, Wright's inbreeding coefficient (standard déviation over loci) and exact test of departure from Hardy-Weinberg genotypic proportions: *, P<0.05; **, P<0.01; ***, P<0.(X)1; ns, not significant;

Table 4

 4 Ti lAM, bottleneck statistic based on the infinité alleles model; T2 SMM, bottleneck statistic based on the stepwise mutation model. Significance levels of corrélation coefficients are given after sequential Bonferroni correction as *, P<0.05; **, P<0.01; ***, P<0.001.

		Corrélation matrix between within-population diversity statistics and geographical
	variables.
		Lat	Long	As_36	He	F	T2IAM
	Long	-0.569 **
	As_36	0.204	-0.122
	He	0.268	-0.345	0.823 ***
	F	0.290	-0.425	0.431	0.545 *
	T2IAM	0.052	-0.455	0.164	0.523 *	0.346
	72 SMM	0.015	-0.404	0.234	0.503 *	0.309	0.929 ***
	Lat, latitude; Long, longitude; As_36, allelic richness for standardised samples of 36 gene copies;	gene diversity; F,
	Wright's inbreeding coefficient;

Table 5

 5 Statistics of régional population genetic structure.

	Group	Populations At_36	Ht	■4st	Dst	Fst	Rst	P(2-sided)
	Ireland	3	12.79 (5.65)	0.841 (0.140)	0.074 0.009 0.015 0.039	ns
	France	2	12.84 (6.22)	0.845 (0.151)	0.054 0.002 0.004 -0.015	ns
	Switzerland	3	13.09 (6.92)	0.771 (0.312)	0.040 0.004 0.008 0.003	ns
	Germany	3	12.37 (6.33)	0.802 (0.207)	0.076 0.007 0.013 0.018	ns
	Sweden	3	10.45 (5.37)	0.766 (0.182)	0.191 0.081 0.153 0.077	ns
	Slovakia & Hungary	2	12.84 (5.21)	0.840 (0.165)	0.088 0.014 0.033 0.059	ns
	Western Bulgaria	3	10.94 (4.60)	0.752 (0.282)	0.362 0.068 0.126 0.138	ns
	Lithuania	2	14.19(6.60)	0.856 (0.132)	0.001 0.004 0.010 -0.003	ns
	Romani a	4	12.97 (4.95)	0.808 (0.160)	0.167 0.055 0.089 0.328	**
	Central Bulgaria	3	12.18(5.64)	0.782 (0.275)	0.159 0.034 0.064 0.097	ns
	Eastem Bulgaria	4	13.81 (6.63)	0.765 (0.328)	0.191 0.030 0.048 0.060	ns
	Republic of Moldova	3	14.48 (6.39)	0.801 (0.268)	0.044 0.002 0.003 0.000	ns
	West	6	13.63 (6.40)	0.844(0.154)	0.111 0.011 0.016 0.021	ns
	Center	6	13.18 (6.87)	0.790 (0.258)	0.092 0.010 0.015 0.030	ns
	Northeast	5	12.53 (6.05)	0.811 (0.162)	0.144 0.059 0.090 0.045	ns
	Southeast	19	15.94 (7.17)	0.822 (0.255)	0.335 0.070 0.088 0.216	***
	Overall	36	16.13 (7.66)	0.832 (0.266)	0.318 0.062 0.076 0.173	***

At_36, total allelic richness for standardized samples of 36 gene copies (standard déviation over loci); f/j, total gene diversity (standard déviation over loci); Ast, différentiation computed from allelic richness; Dsr, absolute différentiation; Fst, relative différentiation based on allele identity; /?st. relative différentiation based on allele size; P(2sided), test of information content of allele sizes from

Hardy et al. (2002)

: ns, not significant; **, P<0.01; ***, P<0.001.

Table 6

 6 Corrélation matrix between total diversity and genetic structure statistics and geographical variables and within-population diversity statistics Différentiation statistics were computed among populations from twelve local régions (see Materials and Methods), and diversity statistics as averages over populations from each of those régions. At_36, total allelic richness for standardized samples of 36 gene copies; Hj, total gene diversity; Ast. différentiation computed from allelic richness; Dst, absolute différentiation; Fyr, relative différentiation based on allele identity; Lat, latitude; Long, longitude; As_36, allelic richness for standardized samples of 36 gene copies; gene diversity; F, Wright's inbreeding coefficient; Ti lAM, bottleneck statistic based on the infinité alleles model; Ti SMM; bottleneck statistic based on the stepwise mutation model. Significance levels of corrélation coefficients are given after sequential Bonferroni correction as *, P<0.05; **, P<0.01;***,P<0.001.

		At_36	Hr	Ast	Dÿj	Fst
	Lat	-0.059	0.462	-0.466	-0.076	-0.041
	Long	0.208	-0.329	0.319	0.296	0.278
	As_36	0.904 **	0.650	-0.918 ***	-0.876 **	-0.887 **
	He	0.662	0 923 ***	-0.847 *	-0.872 **	-0.862 **
	F	0.417	0.753	-0.748	-0.677	-0.669
	T2IAM	0.025	0.449	-0.285	-0.553	-0.514
	TjSMM	0.111	0.382	-0.326	-0.652	-0.608

  Fig 1 -Midpoint-rooted neighbour joining tree computed from Cavalli-Sforza and Edwards' (1967) chord distance among pairs of populations. The bootstrap support of the branches is given in bold numbers as the percentage out of 126 possible bootstraps.

	21 BuIVi
		71	26 RomBac
			56	r	24 RomBaa
				25 RomBab
	50	20 BuILj	22 BuIMo
			52 I-	• 32 BulKo
			' 30 Bullh
			33 BulZd
				31BulDu
				23 RomTu
				34 MolHr
				36 MolHa
				35 MolHi
				29 BuLEl
				9 SwiE
				I 2 IreAv
				^ 12 GerFr
				11 GerCh
				4	10 GerTi -28 BulBc
				13 SweRoe
				30 Bullh 6 FraLr
				26 RomBac _T 14 SweEhd
				' 8 SwiG	24 RomBaa
				1 IreCa	25 RomBab
				■ 24 RomTu 15 SweAk -22 BulMo 16 LitZei ■ 28 BulBc 5 FraGo	20 BulLj
				27 BulGr 17 LitKai
				-------29 BulEl -7 SwiB
		0.1		4 ScotTa 19 HunLue H'
				3IreKi
				18 SloPa
				27 BulGr

Table 1

 1 Statistics of genetic diversity of the microsatellite locus FEMSATL4 in five Oleaceae species. Wright's inbreeding coefficient; allele range, range of allele sizes in nucléotides; mean allele size, mean size of alleles in nucléotides and standard déviation in parenthèses.

						allele	mean allele
	Species	n	K	A	H	range	size
	Fraxinus excelsior	1050	71	19.2 0.897 0.123 **	155-298	175.7 (17.4)'=
	Fraxinus omus	21	11	11.0 0.885 0.085 ns	179-213	189.8(10.7)''
	Ligustrum vulgare	37	13	10.2 0.786 0.002 ns	152-210	169.8 (9.3)"
	Olea europaea	72	10	7.5	0.764 0.091 *** 159-201	170.7 (15.9)*'
	Phillyrea angustifolia^4	8			140-198	170.0 (20.5)*="

n, sample size; K, total number of alleles; A, allelic richness standardised to a sample size of 21 diploid individuals; H, total gene diversity; F\, "Exact test of departure from Hardy-Weinberg genotypic proportions: ns, non significant; **, P<0.01; ***, P<0.001.

Table 2

 2 Sample sizes and efficiency of cloning and sequencing reactions. (ô|i,)^ distance was constructed with PHYLEP v. 3.57c(Felsenstein 1993) in order to clarify géographie patterns of variation.

		Fraxinus	Fraxinus	Ligustrum	Olea	Phillyrea
	Sample sizes	excelsior	omus	vulgare	europaea	angustifolia
	Cloning reactions	14	5	9	8	4
	Alleles chosen for cloning	26	9	9	8	8
	Alleles recovered from sequencing
	reactions	14	6	6	8	4
	Sequencing products (number)	>100	40	84	58	27
	of which alleles (%)	25	50	35	83	26
	shorter stutter bands (%)	23	13	19	5	4
	longer stutter bands (%)	6	0	9	0	0
	unexpected sequences
	with microsatellite (%)	2	13	7	7	22
	no microsatellites or bad
	sequences (%)	50	25	30	5	48

Table 1 .

 1 After standardising for equal sample sizes, allelic richness was highest in F. excelsior {A = 19.2) and lowest in O. europaea (A = 7.5). In P. angustifolia, a standardised allelic richness could not be computed, because we analysed only four individuals, but ail of them were heterozygous and ail detected alleles were different {K = 8); for comparison, in O. europaea, 72 samples were analysed and only two more alleles were identified (K = 10). Size ranges of observed alleles were overlapping in ail species, but comparatively large alleles were found in F. excelsior. Mean allele size was largest in F. omus (189.8 ± 10.7 nucléotides), followed by F. excelsior (175.7 ± 17.4 nucléotides). Gene diversity was high in ail species, ranging from H = 0.764 in O. europaea io H = 0.897 in F.excelsior. Significant inbreeding in the overall species samples was found in F. excelsior and O. europaea. The experiment performed slightly better in F. omus and L. vulgare, and it was truly successful in O. europaea, where ail eight cloned alleles were recovered in sequencing.

	Efficiency of cloning and sequencing reactions
	Table 2 summarises the number of cloning reactions performed in each species, the number of
	alleles involved, and the assignment of sequencing products to different categories. It appears
	that for F. excelsior, only 14 of the 26 alleles implicated in cloning were recovered in
	sequencing products. Similarly, only four out of eight alleles were recovered in P.
	angustifolia.

  The box contains what is defined as the microsatellite repeat région. Numbers next to aligned sequences indicate nucléotide positions when the repeat région is excluded. Species names in bold correspond to haplotypes defined by variation in the flanking régions, and the corresponding microsatellite repeat sequences. Plain sequence names indicate sequences with additional variation in flanking or repeat régions, which were occasionally observed (at least two observations for each sequence).

	a	Ligustrum vulgare
		Fraxinus excelsior
		Fraxinus ornus
		Olea europaea 1
		O/ea europaea 2
		Phillyrea angustifolia
		Syringa pakintnsfs
		Syringa raticutata Syringa vulg^ris
		Syrirtga yunmrtensis Uguszrum vt^gara	-^
		ügustrum samjparvirans
		Ugustrum oyarifolium Uguatrum sintnsa
		Ccmorarkhus madagascarians.
		Comora/khus minor
		Schrabara aiata
		Sehrabara mazoansis
		Fraxinus amaricara
		Fraxinm dipatala
		Fraxima artomala
		Fraxinus quadrangulata
		Fraxinus cusfxdata Fraxinus gragai	.
		Fraxinus axcaisior	^-----------
		Fraxinus xanthoxyloidas
		Fraxinus chinarais	Fraxinus
		ornus	Ĉhionamftus
		filiformis
		Ohonanthus virginicus Forastiara aggarsiana
		Forastiara sag-agata
		Hasparalaaa paimeri
		NoronNa amar^nata
		OIaa panicutata
		Osmanthus amarieanus
		Priogymnanthus apartus
		Prio^mnanthus hassierianus
		Forastiara aeuminata
		Forastiara naomaxicana
		Haanianthus irKrassatus
		Haananthus salicifotius OIaa capensis	.
		Oiaa auropaea	^--------------
		ChionantfHJS ramiflorus
		CfUonanthus ratusus ' Olea brachiata
		Nastagis sandyricansis
		' Osmanthus fraçyans
		Osmanthus insularis • Phillyraa angustifolia ^
		Phillyraa latifolia Phillyraa madla
		Piceonia axcalsa
		Osmanthus haterophyllus Osmamhus rigidus
		Nastagis lancaoéata Nastagis apatala
		Nastagis cimninghamii
		Notalaaa longifôJia
		Notalaaa microcarpa Notalaaa purKtata

Table 3

 3 Structure of genetic diversity among populations based on allele identity (Fst) or allele sizes (Rst).

	Populations	Locus	Fst	Rst^P (l-sided)
	36 European''	5 loci	0.076***	0.173***
	36 European''	M2-30	0.059 ***	0.150***	**
	36 European''	FEMSATL 11	0.067***	0.034***	ns
	36 European''	FEMSATL 16	0.093***	0.073***	ns
	36 European''	FEMSATL 19	0.106***	0.350***
	36 European	FEMSATL 4 ail alleles	0.066***	0.066***	ns
	36 European	FEMSATL 4 odd alleles	0.071***	0.064***	ns
	9 SE European*'''	5 loci	0.066***	Q 2^-77***	**
	9 SE European	FEMSATL 4 ail alleles	0.042***	0.009 ns	ns
	9 SE European	FEMSATL 4 odd alleles	0.053***	0.023 ns	ns
	9 SE European	FEMSATL 4 even alleles	0.053***	-0.025 ns	ns
	P (l-sided), test of information content of allele sizes (Hardy et al 2002): ns, non significant; **, P<0.01; ***, P<0.(X)1.

Table 1

 1 Characteristics of the cpDNA haplotypes of the common ash identified from six microsatellite loci. Codes of loci are as in[START_REF] Weising | A set of conserved PCR primers for the analysis of simple sequence repeat polymorphisms in chlorolplast genomes of dicotyledonous angiosperms[END_REF] and codes of haplotypes as inVendramin et al. (in préparation).

	Haplotypes	number of individuals	size of amplified fragment (nucléotides) ccmp2 ccmp3 ccmp4 ccmp6	ccmp7	ccmplO
	H1	207	194	97	140	97	118	103
	H2	39	194	97	140	99	117	104
	H3	16	194	97	140	99	117	103
	H4	47	194	97	140	98	118	104
	H5	7	194	97	140	98	117	103
	H8	5	194	97	140	97	118	104
	H13	1	194	97	140	97	118	106
	H14	1	194	96	140	97	118	103

Table 2

 2 Chloroplast marker diversity statistics fromPons and Petit (1996) from 42 common ash populations in Europe. Standard errors are given between brackets.

	Unordered alleles	Ordered alleles
	hs	0.066 (0.0212)	Vs	0.060 (0.0243)
	hj	0.556 (0.0762)	Vj	0.591 (0.0786)
	Gst	0.881 (0.0389)	N ST	0.898 (0.0410)

Table 3

 3 Statistics of chloroplast genetic diversity in polymorphie populations of the common ash: n, number of individual trees analysed; K, number of haplotypes identified; hs and vs, genetic diversity based on unordered and ordered alleles, respectively. Ail 33 remaining populations were monomorphic. Haplotype network constructed from variation at the chloroplast microsatellite loci. The circles represent putative haplotypes that were not detected in our dataset.

	Population	Country	n	K	hs	Vs
	1 Camolin	Ireland	8	2	0.250	0.087
	2 Kilmacurra	Ireland	12	2	0.167	0.058
	10 Tiroler Ache	Germany	8	2	0.571	0.792
	11 Chiemsee	Germany	8	2	0.250	0.170
	20 Ljulin Monastery	Bulgaria	8	2	0.250	0.087
	23 Tutuleac	Romania	14	2	0.363	0.126
	30 Iri Hissar	Bulgaria	8	2	0.250	0.250
	37 Choszno	Poland	6	2	0.333	0.462
	41 Nowogard	Poland	6	2	0.333	0.462

Table 4

 4 Pearson moment corrélation coefficients among within-population diversity statistics computed from chloroplast microsatellite data and geographical coordinates or within-population diversity statistics computed from nuclear microsatellite data. Ail corrélations were non significant.

		Diversity at chloroplast microsatellites
		K	hs	vs
	Latitude	0.011	-0.000	0.050
	Longitude	-0.209	-0.140	-0.084
	Diversity at nuclear microsatellites
	As	-0.239	-0.158	0.017
	He	-0.167	-0.109	0.035
	Fl	-0.118	-0.217	-0.244

Table 5

 5 Statistics of genetic diversity and différentiation at nuclear microsatellite markers between groups of common ash populations. Compare population numbers with Fig.2. As, allelic richness for population samples of standardised size 18 diploid individuals; gene diversity; T2 lAM and T2 SMM, bottleneck statistics based on the infinité alleles model and the step-wise mutation model, respectively; F\, Wright's inbreeding coefficient; Fst, relative among-population différentiation based on allele identity; Test, significance level (P) of tests with 10000 permutations for As, //e, P\ and Fst, and results of the analysis of variance for the bottleneck statistics T2 lAM and T2 SMM, where F is the ratio of mean squares to compare with the F-distribution, and P the significance level obtained. Patterns of différentiation among populations. Fsj statistics were computed between pairs of populations and values at nuclear loci were plotted against those at chloroplast loci. The plot represents values computed among populations within each of two groups defined on the basis of polymorphism patterns at nuclear loci (see materials and methods). Squares represent différentiation values among eastem European populations; triangles stand for différentiation among western European populations. Between-group comparisons are not shown.

	European scale
	Populations from western and	Populations from Sweden and
	central Europe and Lithuania	southeastem Europe (numbers	Test
	(numbers 1-12, 16-19)	13-15, 20-36)
	As = 12.355	As = 10.508	F = 0.0048
	He = 0.814	He = 0.729	F = 0.0001
	T2 lAM = 0.648	F2lAM =-0.711	F= 17.22 F = 0.0002
	T2 SMM = -3.055	T2 SMM = -6.438	F= 17.27 F = 0.0002
	Fi = 0.064	II	0 0 0	F = 0.0008
	Fst -0.027	Fst -0.105	F = 0.0001
	Southeastem European scale
	Populations from within or west of	Populations from east of the
	the Carpathians and Balkans	Carpathians and Balkans (numbers	Test
	(numbers 20-23, 27-29)	24-26, 30-36)
	As = 8.941	As = 12.162	F = 0.0013
	He = 0.714	He = 0.769	F = 0.0261
	T2 lAM =-0.045	F2 lAM =-0.970	F =2.58 F = 0.1292
	T2 SMM =-4.942	T2 SMM =-6.850	F= 1.68 F = 0.2143
	Fi = 0.004	Fi = 0.017	F = 0.5709
	Fst = 0.101	Fst -0.067	F = 0.2310

  Allele frequency distributions in four microsatelite loci over a mean of 1069 common ash trees Cette thèse s'intéresse aux facteurs responsables de la structure de la diversité génétique chez le frêne commun. L'accent est mis sur les patrons de diversité génétique au sud-est de l'Europe, qui sont comparés à la structure génétique dans d'autres régions d'Europe dans le contexte de la démographie des populations et de la recolonisation postglaciaire du frêne. L'analyse de microsatellites chloroplastiques nous a permis de confirmer la localisation de refuges glaciaires pour le frêne dans les Alpes et dans la Péninsule Balkanique, suggérée par des données de pollen fossile. Nos résultats sont aussi en accord avec d'autres refuges proposés en Italie et dans la Péninsule Ibérique. Les microsatellites nucléaires ont révélé des patrons de diversité différents, avec notamment des situations contrastées à l'ouest et au centre de l'Europe d'un côté, et au sud-est de l'Europe et en Suède de l'autre. A l'ouest et au centre de l'Europe, une diversité élevée, de légères empreintes de goulots d'étranglement et un pool génique homogène sur toute la région ont été interprétés comme résultant d'un flux de pollen considérable au cours de la recolonisation postglaciaire, homogénéisant les pools géniques de colonisateurs issus de refuges différenciés. Au sud-est de l'Europe, une différenciation importante ainsi qu'une diversité relativement faible et l'absence de signes de goulots d'étranglement ont été attribuées à une expansion depuis plusieurs refuges avec peu de flux de gènes entre populations, sous l'hypothèse qu'une situation démographique stable avec une densité élevée en frêne aurait été rapidement atteinte dans la région. Une diversité plus élevée à l'est qu'à l'ouest de la Péninsule Balkanique pourrait être due à de l'hybridation avec d'autres espèces de frênes sympatriques dans des populations proches des grandes rivières à l'est.

	Résumé:								Appendices Appendices
	Appendice III: Statistics of genetic diversity in 36 European pqiulations of
	common ash
					FEMSATL19
		0.1 -
			Population	As 36	//e	FEMSATL 11 F,s		Ti lAM	T2 SMM
		1	IreCa	11.475	0.8368		0.0916		1.467	-1.698
		0.18 -	-	-		-	-
		2	IreAv	12.684	0.8465		0.0574	ns	1.149	-2.585
		3	IreKi	11.5984	0.8108		0.1109	♦ **	-0.174	-5.501
		4	ScotTa	13.214	0.8191		0.0389	***	0.776	-3.262
	trequency	5 6 7	FraGo FraLr SwiB	12.9946 11.4212 12.7026	0.8423 0.8376 0.7644		0.1566 0.0845 0.0478	** ** *	1.289 1.045 0.334	-1.235 -1.465 -3.079
		8	SwiG	12.502	0.7429		0.0396	ns	-0.361	-4.885
		9	SwiE	12.5984	0.7899		0.0922	*	1.048	-1.936
		10	GerTi	12.7518	0.8291			-0.0235	ns	1.215	-2.144
		11	GerCh	10.3068	0.7365		0.0378	ns	0.397	-3.440
		12	GerFr	11.4668	0.8193		0.0268	**	0.963	-2.473
		13	SweRoe	10.0816	0.7434	0.0191	ns	-0.427	-6.046
		14 15	SweEhd SweAk	8.5356 7.3316	0.6609 0.6505	-0.0393 0.0159	ns ns	-1.96 -1.805	-9.199 -10.415	248 ]
		16	LitZei	13.4694	0.8475		0.103	***	0.24	-4.857
		17	LitKai	14.9014	0.8528	M230 0.0773	***	-0.415	-4.295
		0.06 ^ -18	SloPa	11.8542	0.8305		0.0844	**	0.847	-2.331
		19	HunLue	11.742	0.8195	FEMSATL16 0.0015	ns	0.555	-3.699
	frequency	0.05 20 21 22 23 24 25 26 27 28 29 30 31 32 33 0.8 ^allele size BulLj 6.1466 0.6499 -0.0217 BulVi 7.962 0.6793 -0.1085 BulMo 7.906 0.7239 -0.0018 RomTu 9.3636 0.6761 -0.0197 RomBaa 11.1366 0.7994 -0.0111 RomBab 11.803 0.7648 0.0438 RomBac 11.57 0.7731 0.0449 BulGr 11.334 0.7938 0.0192 BulBc 8.4638 0.6661 0.093 BulEl 11.4122 0.7823 0.0551 Bullh 10.8918 0.7076 -0.029 BulDu 11.1984 0.7435 0.065 BulKo 11.4784 0.7416 -0.0164 BulZd 11.8866 0.7456 0.0221 34 MolHr 14.3858 0.8019 -0.0052	* ns *** ns ns ns ns ns ns ns ns ** ns ns ns	1.974 -1.769 1.663 -1.771 -0.377 -2.317 -0.861 0.418 -1.288 0.457 -1.854 -1.019 -0.131 -1.902 -0.657	0.008 -8.778 -0.747 -9.311 -6.797 -12.011 -7.449 -4.295 -7.583 -3.889 -6.317 -8.325 -5.386 -8.242 -5.092	294
		35	MolHi	13.6782	0.7832	0.0161	ns	-0.24	-3.743
		36	MolHa	13.5956	0.8138	0.0255	**	-0.345	-5.135
										132 133

The development of frequency frequency Appendices Appendice II:

© 2001 Blackwell Science Ltd, Motecular Ecologt/, 10,1615-1623

© 2001 Blackwell Science Ltd, Molecular Eoelogy, 10,1615-1623

© 2001 Blackwell Science Ltd, Mokcuhr Ecologx/, 10,1615-1623

© 2001 Blackwell Science Ltd, Molecular Ecology, 10,1615-1623

Acknowledgements

Acknowledgements

This paper represents a portion of the doctoral research of Myriam Heuertz who received a scholarsltip (rcf. BFR 99/046) from MCESR (Ministry of Culture, of Higher Education and of Research). This work was funded by IPGRI (International Plant Genetic Resources Institute) (project GENFOR). We are gralefvil to G. Muller from Laboratoire National de Santé, Department of Immunology, for letting us use the automated sequencer. We wish to thank M. E. Morand and O. Hardy for collaboration and stimulating discus sions and to L. Hoffmann and two anonymous référées for helpful comments on an earlier draft of the manuscripL Référencés Altman PL, Dittmer DS (1964) Biology Data Book, pp. 428-431. Fédération of American Societies for Experimental Biology, Washington, DC. cited by: Richards AD (1997) Plant Breeding Systems 2nd edn, p. 183. Chapman & Hall. London. Austerlitz F, Mariette S, Machon N, Gouyon PH, Godelle B (2000) Effects of cokmization processes on genetic diversity: différ ences between annual plants and tree species. Genetics, 154, 1309-1321. Brachet S, Jubier MF, Richard M, Jung-Muller B, Frascaria-Lacoste N (1999) Rapid identification of microsatellite loci using 5' anchored PCR in the common ash Fraxinus excelsior. Mükcular Ecology, 8,160-163. Bruford MW, Ciofi C, Funk SM (1998) Qiaracteristics of microsatelllites. In: Molecular Tools for Screening Biodiversity (eds Karp A, Isaac PG, Ingram DS), pp. 202-205. Chapman & Hall, London. Cornes HP, Kadereit JW (1998) The effect of Quatemary climatic changes on plant distribution and évolution. Trends in Plant Science, 3, 432-438. Comps B, Thiébaut B, Paule L, Merzeau D, Letouzey J (1990) Allozymic variabUity in beechwood (Fagus sylvatica L.) over Central Europe: spatial différentiation among and within populations. Hercdity, 65, 407-417. Degen B, Streiff R, Ziegenhagen B (1999) Comparative study of genetic variation and différentiation of two pedunculate oak (.Quercus robur) stands using microsatellite and allozyme loci. Heredity, 83, 597-603. Demesure B, Comps B, Petit R (1996) CMoroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe. Evolutüm, 50, 2515-2520. Eriksson G (1999) Global warming and gene conservation of Noble Hardwcxxis. In: 'Nohte Hardwoods Network. Report of the Third Meeting, 13-16 ]une 1998, Sagadi, Estonia (compilées Turok J, Jensen J,Palmberg-LercheC et al.), pp. 98-112. International Plant Genetic Resources Institute, Rome. European Commission Phare Programme (1999) Conservation and Sustainabte Management ofForests in Central and Eastern European Countries. Phare Environment Consortiiun, Brussels. Excoffier L, Smouse P, Quattro J (1992) Analysis of molecular variance inferred from metric distances among DNA haplo types: application to human mitochondrial DNA restriction data. Genetics, 131,479-491. Fomari B, Cannata F, Spada M, Malvolti ME (1999) AUozynne analysis of genetic diversity and différentiation in European and Asiatic walnut ( Jugions regia L.) populations. Forest Genetics, 6,115-127. Geburek T (1997) Isozymes and DNA markers in gene conserva tion of forest trees. Biodiversity and Conservation, 6,1639-1654. Hamrick JL, Godt MJW, Sherman-Broyles SL (1992) Factors influencing levels of genetic diversity in woody plant species. New Forests, 6,95-124. Hardy OJ, Vekemans X (1999) Isolation by d istance in a continuons population: réconciliation between spatial autocorrélation and population genetics models. Heredity, 83,145-154. Hartl DL, Clark AG (1989) Principics of population genetics, 2nd edn, p. 682. Sinauer Associates, Sunderland, MA. Huntley B, Birks HJB (1983) An Atias ofPast atui Présent Pollen Maps for Europe: 0-13 000 YearsAgo,pp. 209-223. Cambridge University Press, Cambridge. Isagi Y, Kanazashi T, Suzuki W, Tanaka H, Abe T (2000) Micro satellite analysis of the régénération process of Magnolia obovata Thunb. Heredity, 84,143-151. King RA, Ferris C (1998) Chloroplast DNA phylogeography of AInus glutinosn (L.) Gaertn. Molecular Ecology, 7,1151-1161. Kirby GC (1975) Hétérozygote frequencies in small subpopula tions. Theoretiael Population Biology, 8,31-48. Konnert M, Bergmann F (1995) The geographical distribution of genetic variation in silver fir (Abies alba, Pinaceae) in relation to its migration history. Plant Systematics and Evolution, 196, 19-30. Le Corre V, Roussel G, Zanetto A, Kremer A (1998) Geographical structure of gene diversity in Quercus petraea (Matt.) Liebl. III. Patterns of variation identified by geostatistical analyses. Heredity, 80,464-473. Lefort F, Brachet S, Frascaria-Lacoste N, Edwards KJ, Douglas GC (1999) Identification and characterisation of microsatellite Icxd in ash (Fraxinus excelsior L.) and their conservation in the olive family (Oleaceae). Molecular Ecology, 8,1088-1091.

 ------------------------------------------------------English Version -------------------------------------------------, and helped printing! Thanks! Hello also to my brother Claude, who I see more rarely, but we are a good team! I acknowledge financial support from the Public Research Centre-Gabriel Lippmann CRP-GL; the Luxembourgish Ministry of Culture, Higher Education and Research MCESR; the International Plant Genetic Resources Institue IPGRI; the Free University of Brussels ULB; the Luxembourgish National Research Fund FNR; the Belgian National Fund for Scientific Research FNRS; the Centre de Documentation et d'information sur les Etudes Supérieures in Luxembourg CEDIES; the Rotary Club section Ettelbruck-Diekirch in Luxembourg and the European Science Foundation ESF. Thanks to you ail, and to ail those I forgot! And finally a little hello and a message for those who work in the European Union-funded ash projects RAP and FRAXIGEN: There is still a lot more to do; go ahead!

indicated above must always be used to provide access to the

Chapter II: Seed versus pollen dispersai in common ash (Heuertz et al. 2001;[START_REF] Dutech | Evidence of low gene flow in a neotropical clustered tree species in two rainforest stands of French Guiana[END_REF]Fenster et al. in préparation). These methods are based on the rate of decrease of genetic relatedness between individuals with géographie distance [START_REF] Hardy | Isolation by distance in a continuons population: réconciliation between spatial autocorrélation and population genetics models[END_REF]Rousset 2000). Theoretical models of isolation by distance in a two-dimensional space predict that relatedness decreases linearly with the logarithm of the distance at a rate inversely proportional to the product of the effective population density, D, and half the mean squared gene dispersai distance, CTg , within an adéquate distance range (ca. Og to 20(7^, Rousset 1997Rousset , 2000)). Gene dispersai is contributed by pollen and seed dispersai {Og = Oi + cr^ /2, where Os , <7p are half the mean squared dispersai distances of seed and pollen, respectively; [START_REF] Crawford | The estimation of neighbourhood parameters for plant populations[END_REF]), but the relative contribution of each component to total gene dispersai cannot be inferred using this approach.

However, since this method focuses on the rate of decrease of relatedness within a limited distance range only, it does not exploit the whole information available from the plot of relatedness against distance. In this study, we attempted to gather information on the relative contributions of seed and pollen dispersai by inspecting in detail how relatedness between individuals, as assessed by biparentally inherited markers, decreases over an unrestricted range of spatial distances.

Common ash (Fraxinus excelsior L., Oleaceae) is a temperate tree species which occurs in mixed deciduous forests nearly ail over Europe. It is wind-pollinated and its single-seeded fruits, the winged samaras, are wind-dispersed. Its mating System is polygamous: there is a continuum from pure male to pure female individuals with hermaphroditic intermediates (Picard 1982;A Lamb and D Boshier unpublished;Wallander 2001). In a previous study, we hâve identified strong genetic structure at short géographie scale (less than 1 ha) in four out of 10 common ash populations from Bulgaria (Heuertz et al. 2001). This pattern was tentatively attributed to restricted seed dispersai (heavy wind-dispersed seed) and possibly restricted pollen dispersai. Our objective in the présent study was to investigate whether a pattern of isolation by distance could be observed at larger geographical scale in a common ash population from southeastem Romania and whether some inference could be drawn on the relative contributions of pollen and seed to total gene flow from the observed spatial genetic structure. For this purpose we used intensive computer simulations of a theoretical population that closely matched features of the study population. We simulated different combinations of pollen and seed dispersai parameters and examined the resulting plots of relatedness against distance in order to (1) investigate how the relative contributions of seed vs. pollen dispersai affect the patterns of decrease of relatedness with distance and (2) infer levels of pollen and seed dispersai in the study population by comparing observed and simulated data.

Chapter U: Seed versus pollen dispersai in common asli logarithm of distances, one being the régression slope, the other being a shape index based on the residuals. These statistics are defined as follows.

Chi-square-like test. For each independent simulation replicate in each parameter combination, we computed where Fk is the average multilocus kinship coefficient in distance class k, and and var(F<:) are, respectively, the average and variance of Fk over the 100 independent simulation replicates of the investigated parameter combination. The numerator of équation (4) measures the relative squared error of a single replicate from the average over replicates, and the divisor weights for sample size in the corresponding distance class (large numbers of observations, i.e. pairs of individuals, produce a small variance, and reciprocally). For each parameter combination, the observed was obtained by replacing Fk values in équation (5) by their respective values observed in the real population. The smaller , the better the simulations fitted the observation. Parameter combinations for which X^ was larger than the 95% lowest X values from independent replicates were considered incompatible with the common ash data, and therefore rejected (one-sided test).

Linear régression. For each independent simulation replicate in each parameter combination and for the real population, kinship coefficients Fk in each distance class k were plotted against the logarithm of the distance (measured in m). The slope br of the linear régression was computed following équation (2), with Fk instead of F^ (the whole distance range was considered). The slope bro of the observed kinship plot was compared to the 100 ranked br values from independent simulation replicates for each parameter combination. A parameter combination was considered incompatible with the observation when bro was smaller than the third or larger than the 98'*' ranked br value; i.e. lying outside of a 96% confidence interval (two-sided test).

Polynomial fit of residuals. Residuals from the linear régression described above were computed for each independent simulation replicate in each parameter combination and for the real population as in équation (3) with F* replacing F^. After standardising the scale following équation (4), a polynomial fonction involving the third power of the logarithm of distance, y=a+bx-¥cx +dx , was fitted to the plot of residuals against distance, weighting each distance class by its number of observations. As we observed that the relative contribution of seed v^. pollen dispersai was closely related to the coefficient d of the polynomial (see results), the coefficient do of the term in x^ in the real population was compared to the 100 ranked values of d of independent replicates in each parameter combination. A parameter

(5)

Microsatellite analysis

Microsatellite analysis was performed as previously described (Heuertz et al. 2001): Five primer pairs of highly polymorphie microsatellite loci (table 2) were chosen on the basis of the quality of their banding pattern and used for PCR. Fluorescent labeling of the forward primers allowed détection of amplification products on an automated DNA sequencer (ABI PRISM® 377 DNA sequencer). Sizing of fragments was performed with the software programs Genescan® 3.1 and Genotyper® 2.5 from Applied Biosystems by comparison with an internai sizing standard (Genescan-350 Rox). The following modifications were applied to the previously published protocol: (1) when DNA was extracted with the NucleoSpin Plant kit (Macherey-Nagel), the quantity of template DNA in the PCR was increased to 3-10 ng, (2) samples which produced bad amplifications were reamplified with LA Taq polymerase (TaKaRa) and (3) polyacrylamide gels were produced with 10% LongRanger® gel solution (Sanver Tech).

Data analysis

Estimation of genetic variation within populations. The within-population statistics are presented as average values over populations grouped into twelve régions defined on the basis of géographie proximity and on an additional criterion of inclusion of between two to four populations per région ( The following statistics of genetic variation within populations were computed as averages over loci: (1) The allelic richness A quantifies allelic diversity with the property of being independent of sample size variation. It estimâtes the expected number of alleles A_n in a subsample of n genes, given that N genes hâve been sampled (N > n), according to the raréfaction method [START_REF] Hurlbert | The nonconcept of species diversity: a critique and alternative parameters[END_REF] adapted by El Mousadik and Petit (1996). The allelic richness within populations was computed with the software FSTAT version 2.9.3 (J. Goudet 2001, available from http://www.unil.ch/izea/softwares/fstat.html) as As_36 for a sub-sample of n = 36 gene copies or 18 diploid individual trees, which corresponds to the smallest sample size with a complété génotype at ail five loci in a population (in population 30, Bullh). ( 2) The average expected heterozygosity or gene diversity He corrected for small sample size (Nei 1978) and

(3) Wright's inbreeding coefficient F corrected for small sample size (Kirby 1975) were

Chapter III: Genetic structure ofcommon ash in Europe assessed with nuclear microsatellites computed with the program GEN-SURVEY (Vekemans & Lefèbvre 1997). Déviation of genotypic frequencies from Hardy-Weinberg proportions was tested with Fisher's exact test with the program GENEPOP version 3.3 (Raymond & Rousset 1995). Heterogeneity among local régions or zones in mean values of these statistics was tested with a permutation procedure with ESTAT version 2.9.3 (J. Goudet 2001). ( 4) The "bottleneck" statistic T2 from Comuet and Luikart (1996) measures the déviation in observed gene diversity (He) from the expected equilibrium value computed from the observed number of alleles (Ha), assuming mutation-drift equilibrium. The principle of the statistic is derived from the realization that a population suffering from a recent bottleneck expériences a larger réduction of its number of alleles at selectively neutral loci than of its gene diversity (Nei et al. 1975). Hence, positive values of T2 indicate an excess in observed heterozygosity, consistent with a recent population bottleneck, whereas négative values are consistent with recent population expansion without immigration. T2 was computed using the software BOTTLENECK version 1.2.02 (Comuet & Luikart 1996) according to two mutation models, the Infinité Alleles Model {T2 lAM), and the

Stepwise Mutation Model (T2 SMM), which represent two extreme models as our loci do not follow a strict SMM (i.e. we observe many alleles differing by one base pair, which is less than one repeat unit). We also report populations with significant déviation from equilibrium heterozygosity Ha as determined with the Wilcoxon signed rank test, which is the most appropriate test when only few polymorphie loci are analysed [START_REF] Piry | BOTTLENECK: a computer program for detecting recent réductions in the effective population size using allele frequency data[END_REF].

Analysis oflinkage disequilibrium. Linkage disequilibrium was analysed for ail pairs of loci in each population with GENEPOP version 3.3.

Estimation of total diversity and population genetic structure. Statistics of total diversity are given for the overall sample and for each of the twelve previously defined régions and the four zones. Total allelic richness At was computed standardised to a sample size of 36 gene copies (At_36), and relative différentiation among populations on the basis of allelic richness was determined as Ast= 1-( As_36-1)/(At_36-1) as suggested by Comps et al. (2001). Total gene diversity T/xcorrected for small sample size [START_REF] Nei | Estimation of fixation indices and gene diversities[END_REF] and average absolute gene diversity between populations Dst = Hj -He were computed with GEN-SURVEY.

Relative différentiation values among populations, Fst based on allele identity and /?st based on allele size, were computed following an ANOVA approach (Weir & Cockerham 1984, for Fst;Michalakis & Excoffier 1996, for Fsx) with the program SPAGeDi (Hardy & Vekemans 2002). This program also permitted to test for significance of the information content of allele sizes, i.e. whether Fst was significantly different from Fst, by means of a permutation procedure where allele sizes were permuted among alleles within populations (Hardy et al. 2002) Corrélation analysis between geographical variables and diversity statistics. The Pearson moment corrélation coefficient was computed between the population latitude and longitude and the genetic diversity statistics presented above, as well as among diversity statistics.

Significance levels are given after sequential Bonferroni correction (Rice 1989).

Patterns of différentiation among populations. Patterns of among-population différentiation

were investigated with distance-based methods ( 1) and with a model-based clustering method

(2). ( 1 (Pritchard et al. 2000). We ran the program for 100000 itérations after a bum-in period of 100000 with a number of clusters varying from one to eight, allowing for population admixture. For each individual, the fraction of ancestry from each of the clusters was estimated. The partition of the samples into clusters was computed as the averages over individuals belonging to a sample of the fractions of ancestry from each cluster.

Isolation by distance. Isolation by distance was investigated by computing the slope Blog of the linear régression of the Fst/(1-Est) and /?st/(1-Rst) ratios among pairs of populations on the natural logarithms of their géographie distances (Rousset 1997) with SPAGeDi version 0.0. The significance of the isolation by distance pattern was tested by comparing the observed slope with values obtained after 1000 random permutations of population locations.

The analysis was performed for the whole data set as well as for a subset of populations found to hâve a similar composition of clusters as determined with STRUCTURE, comprising the populations from Ireland, Scotland, France, Switzerland, Germany, Hungary, Slovakia and Lithuania.

Results

Allelic variation at microsatellite loci

The total number of putative alleles per locus in the overall sample of 1069 individuals ranged from 16 to 83, with an overall total of 275 alleles scored over the five loci (Table 2). The size ranges of the PCR products corresponding to these alleles are given in Table 2. Overall gene diversities (Hj) were very similar for each locus with the exception of FEMSATL16 that

Chapter IV : Different evolutionary history and geographical distribution of even-and odd-sized alleles at a microsatellite locus in Fraxinus excelsior (L.)

Abstract

The evolutionary history of the molecular determinism of even-and odd-sized alleles in the compound dinucleotide microsatellite locus FEMSATL4 in Fraxinus excelsior was investigated by sequencing a total of 38 alleles in five species of the olive family (Oleaceae).

In F. excelsior, the even-sized allele type was mainly observed in southeastem Europe and differed from the more common odd-sized allele type by the absence of the stretch AAA(CA)n. The odd-sized allele type was also found in the closely related F. omus, suggesting that it corresponds to the ancestral allele type in F. excelsior, but its origin could not be traced further back into the past. Two evolutionary scénarios are proposed to account for the current phylogeographic pattern in F. excelsior, involving (i) a délétion of the motif within F. excelsior during the last glacial period, or (ii) hybridisation with a closely related ash species occurring in southeastem Europe, probably F. angustifolia, which would carry the odd-sized allele type at high frequency. Further, our results suggest that two hexanucleotide repeats hâve evolved independently in F. omus and Olea europaea, probably trough a substitution within a perfect (GA)n repeat, followed by slippage affecting six nucléotides. This finding challenges the view that base substitutions occurring within microsatellites usually stabilise the locus by preventing further slippage events. ), the direct sequence information could not be used.

Instead, we used a distance matrix based on the number of nucléotide différences between ail pairs of flanking région haplotypes to compute a neighbour joining cladogram with the software package PHYLIP v. 3.57c (Felsenstein 1993). In F. excelsior, alleles of even and odd size corresponded to two different types of microsatellite repeat sequences (see results).

Their respective frequencies and average sizes were investigated based on allele sizing data (Heuertz et al. unpublished) from 36 European populations. The corrélation of withinpopulation allele type frequency with géographie coordinates was tested with the Pearson moment corrélation coefficient. Geographical trends in the other loci of that study (Heuertz et al. unpublished) were investigated by testing the corrélation of mean allele size with geographical position. Genetic différentiation was analysed at locus FEMSATL4 among the 36 populations and among 9 southeastem European populations containing more than 25% of even-sized alleles (i) based on ail alleles, and (ii) according to allelic type. Estimators based on allele identity (Fst) or allele size (Fst) were computed and the contribution of step-wise mutations to population différentiation was tested (Hardy et al. 2002). Results were compared to single locus estimâtes for four other loci in the overall dataset, and to multilocus estimâtes for five microsatellite loci in both datasets (data from Heuertz et al. unpublished). Ail computations were performed with the program SPAGeDi (Hardy & Vekemans, 2002). When

Fst was significantly larger than Fst, a neighbour joining tree based on Goldstein et al.'s but in addition to that, there was substantial variation, especially among species. In F.

excelsior, two different patterns of variation were identified in the repeat région and unambiguously assigned to odd and even-sized alleles respectively. In odd-sized alleles, the (GA)n repeat was preceded by the stretch AAA(CA)n (sequence types F. excelsior odd I and 2 in Fig. 2; detected in alleles of length 163, 165, 167, 169, 171, 175, 183, 187, 195, 219 and 227 nucléotides). This motif was missing in even-sized alleles (sequence types F. excelsior even 1 and 2 in Fig. 2; verified in alleles of length 160, 166 and 188, and in stutter bands of length 162, 170, 172 and 178 nucléotides). In F. omus, the motif characteristic for odd allele size in F. excelsior appeared in ail sequences and it was mostly followed by a hexanucleotide repeat (GAGACA)n. Some more variation in the repeat région (sequence types F. omus 2-4 in Fig. 2) was observed in few sequences (at least 2 sequences for each type), but not unambiguously confirmed (e.g. observed in sequences cloned from only one individual and therefore possibly due to a réplication error of the Taq polymerase, and/or in sequences with inferior resolution). The repeat région in L. vulgare was a perfect dinucleotide repeat (GA)n, but the first nucléotide positions immediately flanking the repeat showed little homology to the Fraxinus species. The variable sequence in O. europaea was a compound dinucleotide / hexanucleotide repeat (CA)n(GAGTGA)n, sometimes interrupted by a single GA motif; additional variation in the repeat région (sequence type O. europaea 3) was not unequivocally confirmed. Three of the four detected P. angustifolia alleles carried a perfect (GA)" repeat, in the fourth allele, it was preceded by a (CA)" motif.

Flanking régions. The overall beginning of the repeat région was defined at position 26 (box in Fig. 2), where it is located in O. europaea and P. angustifolia. According to this définition, eleven sites in the sequenced flanking régions were polymorphie among species, comprising one insertion/deletion and ten substitutions (compare sequence types in bold in Fig. 2). The variation at these sites defined six haplotypes (sequence types in bold, Fig. 2); one in each species except in O. europaea, where there were two due to a single polymorphie site (position 21). In the other species, no variation in flanking régions was confirmed, but there were five candidate substitutions (in non-bold sequence types in Fig. 2, e.g. F. omus 2, position 16: G) and one délétion (in O. europaea 4). Cladistic methods could not be applied to sequence variation in flanking régions among haplotypes because there was only one phylogenetically informative site (position 13), which allowed to group O. europaea and P.

angustifolia against the other species. The number of mutations between pairs of species (with the indel coded as a separate character) was comprised between two (F. excelsior -F.

omus) and eight (L. vulgare -P. angustifolia), and phylogenetic relationships between species based on the number of mutations in flanking régions of locus FEMSATL4 were in agreement with the phylogeny of the tribe Oleae established by [START_REF] Wallander | Phylogeny and classification of Oeaceae based on rpsl6 and tmL-F sequence data[END_REF] from sequence data at two non-coding chloroplast loci (Fig. 3, compare a and b).

Concluding remarks

The results of this study unravelled the molecular diversity accounting for the appearance of odd-and even-sized allele types at locus FEMSATL4 in Fraxinus excelsior and suggested two evolutionary scénarios to explain the current géographie distribution of these allele types.

Further, support for the évolution of a hexanucleotide repeat through a base substitution and subséquent slippage was given on two occasions. In contrast, our results were unable to détermine (i) the origin of the stretch AAA(CA)n, which distinguishes both F. excelsior allele types, and (ii) the time of its délétion, i.e. discriminate the two evolutionary scénarios. The sequencing of the microsatellite locus in other closely related généra (Cormoranthus, Schrebera and from the subtribe Oleinae, see [START_REF] Wallander | Phylogeny and classification of Oeaceae based on rpsl6 and tmL-F sequence data[END_REF] and in other Fraxinus species, especially in F. angustifolia, is expected to résolve these issues. Chapter V: Genctic structure ofcommon ush in Europe assessed witli chloroplast and nuclear microsatellites from long-distance dispersai during postglacial recolonisation (Lagercrantz & Ryman 1990, Zanetto & Kremer 1995, Comps et al. 2001). Géographie variation in patterns of genetic structure can also be studied with nuclear markers at high resolution (Machon et al. 1997[START_REF] Murillo | Genetic diversity in natural populations of Alnus acuminata ssp. Arguta (Schlechtendal) Furlow in Costa Rica and Panama[END_REF], and hybridisation events between closely related species or between lineages at the intraspecific level can be identified [START_REF] Marchelli | Genetic diversity and différentiation in a Southern beech subjected to introgressive hybridization[END_REF][START_REF] Breitenbach-Dorfer | The contact zone between two migration routes of silver fir, Abies alba (Pinaceae), revealed by allozyme studies[END_REF]. However, a limitation with nuclear markers in tree species is that most variation is found within populations so that their phylogeographic information content is low. As a conséquence, alternative causes of the observed patterns of variation, i.e.

recent gene flow or recolonisation history, cannot be easily distinguished (Zanetto & Kremer 1995). In this respect, nuclear haplotype data are becoming a promising new tool for evolutionary inference [START_REF] Hare | Prospects for nuclear gene phylogeography[END_REF]. Another strategy is to assess simultaneously organelle and nuclear DNA markers on the same set of populations. The combination of both markers may lead to a better understanding of the relative rôles of different evolutionary factors, i.e.

gene flow, postglacial history, and démographie events [START_REF] Latta | A comparison of population différentiation across four classes of gene markers in Limber pine (Pinus flexilis James)[END_REF], Le Corre et al. 1997[START_REF] Terry | Genetic variation in chloroplast and nuclear ribosomal DNA in Utah juniper (Juniperus osteosperma, Cupressaceae): evidence for interspecific gene flow[END_REF]. Moreover, when no phylogeographic structure occurs, this allows to détermine the relative contributions of seed and pollen to gene flow (Ennos 1994).

The general objective of this work was to combine both approaches on the same population samples to better characterise diversity patterns and to test whether the combination provides a clearer identification of the factors shaping these patterns. Our study plant is the common ash, Fraxinus excelsior, a temperate tree species occurring in floodplain forests and in mixed deciduous forests nearly ail over Europe. Common ash exhibits intermediate properties between a pioneer species and a permanent forest component; it features strong colonisation capacity, but compétitive ability is high only when ecological requirements are met. Flowers are wind-pollinated and single-seeded fruits, the samaras, are wind-dispersed. The mating System of common ash is polygamous and highly variable: flowers are male, hermaphroditic or female and there is a continuum from pure male to pure female individuals with hermaphroditic intermediates (Picard 1982, A Lamb & D Boshier unpublishe,;Bingelli & Power 1999, Wallander 2001). Palynological data strongly support full glacial refuges for ash in the eastem Alps and in the Balkan Peninsula (Huntley & Birks, 1983;Gliemeroth 1997;Brewer 2001), with up to three sites in the latter (Brewer 2001). Fossil pollen evidence is weaker for putative refuges in the Iberian Peninsula, in the Italian Peninsula, in Turkey and at the northem Black Sea coast (Huntley & Birks, 1983, Gliemeroth 1997, Brewer 2001).

Further, the most detailed survey (Brewer 2001) identified sites compatible with early occurrence of ash in the French Central Massif, in southeastem Poland, in Scotland at the northem coast of the Firth of Forth and in southem Sweden near Gôteborg. The rôle of these sites in postglacial history of ash still needs to be clarified. Postglacial colonisation was marked by an east-west migration in the early Holocene (after 10.000 BP), followed by a south-north trend.

Data analysis

A chloroplast haplotype network was constructed based on the minimum number of length différences of PCR-amplified fragments separating the haplotypes, using TCS 1.13 [START_REF] Clement | TCS: a computer program to estimate gene généalogies[END_REF]. The distance matrix among haplotypes was computed defining the distance between two haplotypes as the sum over the 6 loci of the absolute number of nucléotides distinguishing the haplotypes, assuming that a one-nucleotide différence corresponded to a single mutation, i.e. a slippage event at a microsatellite locus involving a single nucléotide.

Within populations, we determined the number of alleles K and the haplotypic diversity based on haplotype identity (/zs) or on the minimum number of microsatellite length mutations separating haplotypes (vs) following Pons and Petit (1996). In order to allow for straightforward comparison of within-population haplotypic diversity statistics, the weights for v-type statistics were divided by a correction factor computed from the overall matrix of différences among haplotypes (the matrix that we also used to build the haplotype network, see above) according to Petit et al. (2002a). Géographie variation of within-population diversity was investigated by computing the Pearson product moment corrélation between diversity statistics (number of alleles, hs, vs) and geographical coordinates.

In the overall sample, total haplotypic diversity statistics based on ordered or unordered alleles {hj and vj, respectively) were calculated following Pons and Petit (1996).

Différentiation among populations was computed from ordered and from unordered alleles (VsT and Gst, respectively) and a permutation procedure was carried out to test for the presence of phylogeographic structure (i.e. whether Nsj > Gst) following Burban et al.

(1999), using the program PERMUT 2 (Pons & Petit 1996). An analysis of isolation by distance was performed using the matrix of F$t values among pairs of populations computed with ARLEQUIN 2.0 [START_REF] Schneider | Arlequin v. 2.000: A software for population genetics data analysis[END_REF] and the matrix of the logarithms of géographie distances among pairs of populations computed with SPAGeDi 1.0 (Hardy & Vekemans 2002). Values of Fst were regressed on the logarithm of distance and a Mantel test between both matrixes was computed with the program CADM [START_REF] Legendre | CADM[END_REF].

Chapter V: Genetic structure ofcommon ash in Europe assessed with chloroplast and nuclear microsatellites A previous analysis of 36 common ash populations with five nuclear microsatellite (nSSR) loci has revealed distinct patterns of genetic diversity among eastem and western European populations (Heuertz et al. unpublished,chapter 3). More precisely, our data hâve suggested congruent patterns of variation in western and central Europe and in Lithuania on the one hand (hereafter referred to as "western group"), and in southeastem Europe and Sweden on the other (hereafter referred to as "eastem group"). A synthetic visualisation of the patterns was obtained with the model-based clustering method by Pritchard et al. (2000) (Fig. 2). In this study, we tested the discrepancy between the two suggested groups of populations at the European scale, as well as between populations located east or west of the Carpathian and

Balkan mountain range at the scale of southeastem Europe. We performed permutation tests with ESTAT 2.3.5 (Goudet 2001) for the following statistics in each group: the withinpopulation allelic richness, As, standardised to a sample size of 18 diploid individuals (which corresponds to the smallest sample size among the 36 populations) according to El Mousadik and Petit (1996), the within-population genetic diversity (//e), the within-population inbreeding coefficient (F\), and the statistic of among-population différentiation, Fst-We also tested with an analysis of variance whether there were différences among groups in the mean value of the bottleneck statistic Tz (Comuet & Luikart 1996). This statistic is based on the principle that a drastic size réduction in a population, i.e. a bottleneck, transiently produces a stronger decrease of the number of alleles than of gene diversity He (Nei et al. 1995). The Tj Chloroplast and nuclear patters of variation were compared by means of the Pearson product moment corrélation coefficient between within-population diversity statistics at both types of markers, including (i) the number of alleles (K) and haplotypic diversity based on ordered (vs)

or unordered (hs) alleles for chloroplast microsatellites; and (ii) allelic richness (As), gene diversity (He) and the inbreeding coefficient (F\) for nuclear microsatellites (Heuertz et al. unpublished,chapter 3). As we tested multiple corrélations, a sequential Bonferroni correction (Rice 1989) was applied to significance levels in order to exclude corrélations that might hâve appeared by chance. We also compared pairwise population différentiation (Fst) matrixes for both types of markers with a Mantel test using CADM [START_REF] Legendre | CADM[END_REF]). These comparisons were performed over ail 36 populations and within each of the two groups of populations for which nuclear markers had suggested contrasting diversity patterns.

Results

Variation at chloroplast microsatellites Two of the six cpSSR loci analysed were monomorphic (ccmp2 and ccmp4, Table 1). The other loci showed low levels of polymorphism. Two distinct size variants separated by one nucléotide were observed at both ccmp3 and ccmp7 (Table 1). Ccmpô and ccmplO displayed three size variants each; with amplification fragment sizes of 96, 97 and 98 nucléotides, and 103, 104 and 106 nucléotides, respectively (Table 1). These size variants combined into a total of 8 haplotypes (Table 1). In the overall sample of 323 individuals, two haplotypes (H13 and H14) were encountered in only one individual; the frequencies of the others ranged from 5 individuals (1.5 %, H8, only observed in population 11 from Germany) to 207 individuals (64.1 %, Hl, Table 1). The minimum numbers of evolutionary events separating the haplotypes is visualised in the haplotype network in Fig. 3. Most haplotypes are related to 1 to 3 others by one single mutational step. Three putative haplotypes corresponding to intermediate evolutionary steps were not detected in our dataset (circles in Fig. 3).

Geographical distribution of cpSSR polymorphism

Within-population variation at chloroplast microsatellites was low, with an average number of AT= 1.21 ±0.41 (standard déviation) haplotypes per population and average gene diversity values of /is=0.066 and vs=0.060 based on unordered or ordered alleles, respectively (Table 2). Accordingly, most populations carried a single haplotype, and only nine population samples displayed two distinct haplotypes (Fig. 1). The diversity measure based on ordered alleles (vs) was higher than that based on allele identity (hs) in polymorphie populations from Germany and Poland; in the other polymorphie populations, vs was smaller than h$ (Table 3).

No corrélation was observed between within-population diversity statistics and geographical coordinates (Table 4); hence our dataset bears no evidence for graduai variation of diversity according to latitude or longitude.

Haplotypic diversity in the overall sample was much higher than within populations, hj = 0.556, vt = 0.591, and consequently, différentiation among populations was important: Gst = 0.881 and Nsj = 0.898 (Table 2). The permutation procedure to test for phylogeographic structure revealed that Nsj was not significantly larger than Gst, indicating that on average, phylogenetically close haplotypes were not found together in the same population more often than different haplotypes which would hâve been chosen randomly. A geographical organisation of genetic diversity is nevertheless évident from Fig. 1, with haplotype Hl being prédominant in eastem and northem Europe, haplotypes H2, H3 and H5 occurring in central and western Europe, and haplotype H4 being found only in the British Isles. Also, the test of isolation by distance showed that among-population différentiation increased significantly with the logarithm of geographical distance (Mantel test: P < 0.001, linear régression: = 0.222).

Geographical distribution of nSSR polymorphism

The contrasting patterns in the geographical distribution of genetic diversity at nuclear microsatellite markers at the European scale suggested in our previous study (Heuertz et al. unpublished, chapter 3) were confirmed (Table 5): western and central European, as well as

Lithuanian populations (i.e. the western group) displayed highly significantly larger allelic richness, gene diversity and inbreeding coefficients, but lower levels of among-population différentiation than populations from southeastem Europe and Sweden (i.e. the eastem group). The différence in gene diversity was more marked (P = 0.0001) than that in allelic richness (P = 0.0048). As a conséquence, we observed a gene diversity excess with respect to the one expected from the number of alleles in the western group {Ti lAM = 0.648), and a gene diversity deficiency in the eastem group {Ti lAM = -0.711). Assuming a stepwise mutation model, ail values for the bottleneck statistic T2 SMM were négative, but still higher in the western than in the eastem group. Hence, western European populations carry stronger imprints from recent population bottlenecks than eastem European populations.

Within the Balkan Peninsula, we were able to identify two groups of populations separated by the physical barrier of the Carpathian and Balkan mountains. Populations at the foothills of the mountains or west of them harboured significantly lower allelic richness and gene diversity than populations from the Danube and Dniestr plains in the east (Table 5). The model-based clustering method by Pritchard et al. (2000) also assigned different gene pools to these groups of populations, gene pools 3 and 4 in the west, and gene pools 5 and 6 in the east (Fig. 2).

Comparison of chloroplast and nuclear patterns of variation

The comparison between chloroplast and nuclear within-population diversity statistics over ail 36 populations revealed low and non-significant corrélation coefficients (Table 4). When populations were split into a western and an eastem group, as suggested by patterns of nuclear microsatellite diversity, a weak négative association was observed in the western group between the phylogenetic diversity of haplotypes, vs, and the inbreeding coefficient F\ (Pearson corrélation coefficient r = -0.517), but it was non-significant after application of a sequential Bonferroni correction. The Mantel test identified a négative corrélation between the matrixes of pairwise Fsr-values among populations at chloroplast and nuclear markers (Spearman rank corrélation coefficient r = -0.263, P = 0.993 for the one-tailed Mantel test

Chapter V: Genetic structure ofcommon ash in Europe assessed with chloroplast and nuclear microsatellites haplotypes H2, H3 and H5 suggests that they originate from one or, more probably, a few refuges in the Alps and/or Italy. In line with the now broadly accepted view that refuge populations were able to accumulate high diversity and/or unique haplotypes owing to their persistence and relative stability over glacial cycles (Hewitt 1996, Newton et al. 1999, Tzedakis et al. 2002), the occurrence of private haplotypes in populations from the Alps and the Balkan Peninsula in our dataset (H5 in population 11 in the Alps, and H13 and H14 in populations 20 and 30 from Bulgaria, respectively) provides additional support for the location of refuges in these areas. A suture zone between haplotypes from distinct refuges (H1 and H2, Fig. 1) occurs in Germany and Poland, explaining the relatively high values of phylogenetic diversity within populations, vs, in this area (Table 3). The fact that we identified haplotype H4 exclusively in the British Isles evokes two scénarios: either a colonisation from another distinct refuge, possibly in the Iberian Peninsula, or, but less likely, fixation of a mutant haplotype during colonisation from, potentially, the Balkan Peninsula (H8 or H4, differing from the Eastem European haplotype H1 by only one or two steps, respectively, Fig. 3) and extinction of geographically intermediate populations. The occurrence of H8 in population 23 from Romania as well as in populations 1 and 3 from Ireland weakly supports the latter scénario. Yet, in Vendramin et al.'s (unpublished) chloroplast microsatellite survey at the European scale in common ash, haplotype H4 was also found in northem Spain, which confirms the existence of an Iberian refuge, as suggested from palynological data (Gliemeroth 1997, Brewer 2001).

Joint interprétation of chloroplast and nuclear patterns of genetic diversity

From the previous paragraph it appears that the four major chloroplast haplotypes detected in European populations of the common ash présent a clear geographical structure (Fig. 1), which was most likely shaped during postglacial recolonisation. The géographie structure of genetic diversity at nuclear markers showed a completely different pattern. In western and central Europe, différentiation for cpSSRs was high, as ail four major chloroplast haplotypes occurred there, but very little différentiation among populations was observed at nSSRs.

Conversely, in eastem Europe, différentiation for chloroplast markers was very weak because of the prédominance of haplotype Hl, but most surprisingly, strong différentiation among populations was identified at nuclear markers (compare Figs. 1 and 2, Fig. 4).

Western and central Europe

In western and central Europe, we found a very homogeneous distribution of nuclear marker diversity with Fst= 0.027 and a most likely number of gene pools of one, using the method of Pritchard et al. (2000), i.e. when the method was applied to populations from the western group only, the admixture of gene pools 1, 2 and 3 (Fig. 2) basically reduced to a single gene pool (Heuertz et al. unpublished,chapter 3). Comparée! to southeastem Europe and Sweden, this région was also characterised by comparatively high gene diversity and high values of the inbreeding coefficient, and it showed slight imprints from recent population bottlenecks.

Little structure among populations at nuclear loci, despi te potentially high divergence for chloroplast markers, as well as relatively high gene diversity, seem to be general observations in late-successional broadleaved tree species in western and central Europe. Sessile oak, which has recolonised continental Europe from the three Southern peninsulas (Dumolin-Lapègue et al. 1997, Petit et al. 2002b) et al. 1996) and différentiation for nuclear allozymes in western and central

Europe was also low (Fst= 0.04, Comps et al. 2001). Both of these studies showed a decrease of within-population allelic richness and a concomitant increase of gene diversity following the direction of postglacial recolonisation, which can be understood as a conséquence of population bottlenecks during recolonisation. Hence there seem to be some congruent patterns in the recolonisation of western and central Europe by ash, oak and beech.

In the light of our results, we propose the following scénario for postglacial recolonisation of western and central Europe in common ash. ( 1) During glacial times, populations from different refuges were differentiated at chloroplast and at nuclear markers (e.g. Kremer et al. 2002).

(2) As climatic conditions improved and suitable areas became available in the beginning of the Holocene (10000 BP), differentiated maternai lineages from different refuges started colonising northward. Pollen data suggest that colonisation from Spain started about 1000 years later than from refuges in Italy, the Alps and the Balkan area, which resulted in an east to West trend of migration (Huntley & Birks 1983, Gliemeroth 1997, Brewer 2001) (3)

Pollen flow established contact between the colonising lineages, homogenising progressively the nuclear gene pool of migrating populations. The resulting nuclear gene pool featured high diversity, because it resulted from the admixture of differentiated gene pools, which had evolved in separate glacial refuges. As seed flow was much more limited in general, only little mixing of chloroplast haplotypes occurred. (4) Maternai lineages expanded and continued colonising northward and westward, where new land became available.

Colonisation occurred through leading edge dispersai, i.e. successive but rare events of longdistance dispersai of seed followed by a more compact wave of advance (e.g. Hewitt 1996, Petit et al. 1997). Long-distance dispersai resulted in founder events (or population bottlenecks), of which we still detect some imprints in the nuclear genome today. Multiple founders carrying different alleles helped to maintain overall diversity in the colonising gene pool (Ibrahim et al. 1996, Le Corre & Kremer 1998). ( 5) Extensive gene flow through pollen between the core and the newly colonised populations moderated the conséquences of founder

General discussion

General discussion

A synthetic view of the results of this work is given in Fig. 1. Chloroplast microsatellite analysis allowed us to corroborate glacial refuges for ash situated in the Alps and and the Balkan Peninsula, as suggested from fossil pollen data (Huntley & Birks 1983, Gliemeroth 1997, Brewer 2001)

Recommendations for management and conservation of genetic resources in common ash

The central aim in forest genetic resources conservation is to safeguard a species' adaptive potential (Eriksson et al. 1992[START_REF] Namkoong | Conservation of forest genetic resources in Europe[END_REF]. As the real adaptive potential of populations is generally not known, one possibility is to concentrate on existing patterns of adaptation. This is the scope of the multiple populations breeding System (MPBS) recommended as a conservation strategy for noble hardwoods [START_REF] Eriksson | Conservation of noble hardwoods in Europe[END_REF]) by the EUFORGEN Noble Hardwoods network. The concept of MPBS is to enhance amongpopulation différentiation by promoting natural sélection within populations with the aim to increase the overall adaptive diversity. There is a broad agreement among conservation geneticists that, together with adaptive divergence, historical isolation is an important concept for the délimitation of significant units for conservation (reviewed by [START_REF] Fraser | Adaptive evolutionary conservation: towards a unified concept for defining conservation units[END_REF]. This is because historically isolated populations hâve most certainly accumulated different mutations that préparé them differentially for future challenges, even if these

General discussion

molecular markers for the identification of hybrids is strongly needed in forestry, and some studies hâve already focused on this theme using RAPD [START_REF] Jeandroz | Molecular récognition of the closely related F. excelsior and F. oxyphylla (Oleaceae) by RAPD markers[END_REF] or chloroplast microsatellite markers (Morand-Prieur et al. 2002). Our results (chapter 4) suggest that progress in this field could potentially be made through the sequencing of nuclear microsatellite loci.

In our nuclear microsatellite diversity survey of common ash at the European scale (chapters 3 and5 A technical problem we encountered during this work was the difficulty of correctly scoring the hypervariable microsatellite loci, especially when incorporating many populations over a large géographie area like Europe (chapter 3). Our observed diversity patterns are concordant, which is why we think that we made few scoring mistakes, and that we revealed the true organisation of neutral genetic variation at nuclear markers. If nuclear genetic diversity patterns of ash hâve to be studied at a large scale within a research project involving many laboratories, the reproducibility of scoring among laboratories could however be a serious issue (as it is with microsatellite markers in oaks, [START_REF] Mariette | Comparison of levels of genetic diversity detected with AFLP and microsatellite markers within and among mixed Quercus petraea (Matt.) Liebl. and Q. robur stands[END_REF]. Therefore, it would be interesting to develop a set of less polymorphie nuclear markers. This would also allow the vérification of the diversity patterns obtained in our study.

General discussion

At the within-population level, we obtained some estimâtes for the variance of pollen and seed dispersai distances from spatial genetic structure (chapter 2). Our estimâtes suggested a much lower seed flow in our Romanian population than preliminary results in a French population where a parentage study is in progress (Morand-Prieur et ai, in préparation). It would be interesting to verify our results by a direct method, similarly to Morand-Prieur et al, in order to corroborate or invalidate our estimâtes from spatial genetic structure. Several local-scale studies in selected populations from ail over Europe might also clarify the issue of increasing values of the inbreeding coefficient from southeastem Europe towards the west and towards the north (with the exception of Sweden).

Appendices

Appendice I: Sizing of microsatellite loci with Genescan and Genotyper where the number of observations was small. In each population, I checked if alleles of similar size were put into the same class. Approximately one percent of alleles had to be reclassified with this method, so it seems to be quite robust.

Appendices

y4s_36, allelic richness for standardised samples of 36 gene copies; gene diversity; F, Wright's inbreeding coefficient and exact test of departure from Hardy-Weinberg genotypic proportions: *, P<0.05; **, P<0.01; ***, P<0.001; ns, not significant; T2, bottleneck statistic ffom Comuet and Luikart (1996) computed on the basis of the infinité alleles model {Ti lAM) and the stepwise mutation model (SMM); S.D. (populations), standard déviation over populations.

Samples ffom Bulgaria were rescored between chapters 1 and 111 ffom the thesis, which explains the slight différences between the values.