Mickael Bourge

Mario Gomez Pacheco

Sandrine Fontaine

Raphaël Cornette

 Nélida

Padilla Garcia

Manuel Cartereau

Bernard Pasquier

Sophie Nadot

Thierry Robert

Marwa Daoud

Sonja Siljak-Yakovlev

Nathalie Machon
email: nathalie.machon@mnhn.fr

Intraspecific DNA Content Variability: Consequences for Conservation

Keywords: chromosome number, evolutionarily significant units, flow cytometry, intraspecific ploidy variation, microsatellite genotyping, monoploid genome size Chromosome number, evolutionarily significant units, flow cytometry, intraspecific ploidy variation, microsatellite genotyping, monoploid genome size Seed germination of Arenaria grandiflora L. species complex (Caryophyllaceae) dormancy, germination protocol, in vitro culture, ploidy levels, scarification, seed provenance Herbarium material, Leaf elongation, Log-Shape Ratio (LSR), Principal Component Analysis (PCA)

Five years ago, I started this project as a thesis project, but it turned out to be long battle against a largeflowered Sandwort from Caryophyllaceae (Arenaria grandiflora L.). This thesis is the report of this long process, but it cannot express the long days spent in both the field and the lab, battling shoulder to shoulder with my fellow scientists. It cannot also express the stress, the sadness and the tiredness because of the war and the victims in my country (Syria). The first year was only just unsuccessful attempts to detect both the nuclear microsatellite markers and the chloroplast markers (we used 4 pairs of universal primers (MatK 390F / 1440R, TrnK-MatK 3914F / 1110R, TrnL-trnF e / f and Trnt-L La / Lb), in dry and old herbarium' specimens (Paris' herbarium, Montpellier' herbarium, Geneva' herbarium and Barcelona' herbarium). A new and different scheme to search was established in the second year.

VIII

Consequently, all these findings are fundamental for the determination of the evolutionarily significant units (ESUs) within A. grandiflora species complex and thus the definition of efficient restoration plans in the future. This study would consider as the preliminary signal for necessary revision for the intraspecific taxonomic keys problematic for this species complex.

Chapter I:

General introduction

Population-level conservation is recognized as an important requirement to prevent biodiversity loss within species [START_REF] Purvis | Getting the measure of well-being[END_REF], but the first necessity is to determine both number and distribution of populations and then differentiation among populations, that is needed to define population-based units for conservation purposes. Presently, there are three population-based approaches for identifying populations to be protected; as the use of evolutionarily significant units (ESUs) advocated by [START_REF] Ryder | Species conservation and systematics: the dilemma of subspecies[END_REF]. This approach was expanded by [START_REF] Moritz | Defining " Evolutionarily Significant Units " for conservation[END_REF] and [START_REF] Waples | Evolutionarily Significant Units, Distinct Population Segments, and the Endangered Species Act: Reply to Pennock and Dimmick[END_REF] to the concepts of management units (MUs) and distinct population segments (DPS), respectively, for species that had undergone distribution range fragmentation. Furthermore, it is a difficult task to identify the ESUs within a species, it requires the use of natural history information on populations, i.e. its ranges and distribution data, morphometric analysis, as well as genetic and cytogenetic analysis… etc. [START_REF] Ryder | Species conservation and systematics: the dilemma of subspecies[END_REF]. Setting conservation priorities within species (i.e. at the population level) may be a challenge in species complex for which ranges become fragmented, such as a case of Arenaria grandiflora species complex.

Little is known about the biology of this widespread species complex through Western Europe and its taxonomic status is even controversial and problematic. Seven or five geographic subspecies, few varieties, and forms have been described until now. While the presence of intraspecific variability was documented within A. grandiflora species complex, no research has addressed how the assessment of differentiation among populations, could be important to take the best conservation measure for preserving this species complex that is distributed in small and disjunctive populations. The genetic variability within A. grandiflora species complex, is still little known, especially, the polyploid nature of this species complex, that has been neglected in a previous conservation program in the Fontainebleau forest in Paris. This manuscript will present in five successive chapters the results of analyses carried out to explore the genetic and phenotypic patterns of variation. Before showing the results of analyses, this manuscript starts with a general introduction about the polyploidy in plants, the history of the evolutionarily significant units (ESUs) and the biology of the genre Arenaria L., with a little available information about our interesting species complex of A. grandiflora. The second chapter describes genetic and cytogenetic studies. For that purpose, we used nuclear microsatellite markers for the study of genetic variability, a classical karyological method for chromosome number count and flow cytometry for assessment of DNA content variability. The third chapter used seed germination protocols to determine the percentage of germination for different populations of A. grandiflora species complex. The fourth chapter presents the morphometric variability observed in the leaf shape among specimens of herbarium' Paris. It is followed by a general discussion and conclusion.

Polyploidy in plants -a brief history

The widespread occurrence of polyploidy in plants is well-known because it has attracted much of scientific studies for almost a century. The polyploids have multiple (more than two) sets of chromosomes in their cells. A series of estimates of the frequency of whole genome duplication (WGD; polyploidy) in angiosperms have been provided during the past years by plant biologists (70% by [START_REF] Masterson | Stomatal Size in Fossil Plants: Evidence for Polyploidy in Majority of Angiosperms[END_REF]; 50 % by [START_REF] Müntzing | The evolutionary significance of autopolyploidy[END_REF]. Many reviews have uncovered that most of the angiosperm Fig. 1 Ancestral polyploidy events in seed plants and angiosperms; from [START_REF] Jiao | Ancestral polyploidy in seed plants and angiosperms[END_REF] have a polyploid origin [START_REF] Leitch | Polyploidy in Angiosperms: Monocotyledons[END_REF]; [START_REF] Soltis | Polyploidy: recurrent formation and genome evolution[END_REF]; [START_REF] Mable | Breaking down taxonomic barriers in polyploidy research[END_REF]; [START_REF] Soltis | Advances in the study of polyploidy since Plant speciation[END_REF]); [START_REF] Soltis | The polyploidy revolution then...and now: Stebbins revisited[END_REF]. Recently, [START_REF] Jiao | Ancestral polyploidy in seed plants and angiosperms[END_REF] have elucidated that there are two WGDs in ancestral lineages before the diversification of extant seed plants and extant angiosperms, (see Fig. 1).

Since its discovery in 1907, polyploidy has been considered as an important dynamic process in the evolutionary success [START_REF] Otto | Polyploid incidence and evolution[END_REF], but the direct effect of polyploidy on the evolutionary trajectory of a species is still unclear (Madlung 2013). The abundance of polyploids refers to multiple sets of genomes and offers an evolutionary advantage, probably through a broader phenotypic and genotypic range than that of their progenitors and because of the increased fitness afforded by fixed heterozygosity [START_REF] Hegarty | Polyploidy: Doubling up for Evolutionary Success[END_REF]; [START_REF] Fawcett | Angiosperm polyploids and their road to evolutionary success[END_REF]. For example, the genetic, epigenetic and transcriptional changes associated with polyploidy in plants, like the greater pool of genes and alleles available for selection, might contribute to the evolutionary success of polyploid plants [START_REF] Hegarty | Genomic Clues to the Evolutionary Success of Polyploid Plants[END_REF], 2007) The whole-genome duplication has provided an important pathway for diversification and speciation in plants [START_REF] Vamosi | Polyploidy and Diversification: A Phylogenetic Investigation in Rosaceae[END_REF]; [START_REF] Rieseberg | Plant Speciation[END_REF]. However, the complexity of understanding the role of genome duplication in species diversification is still disputed. It has been established that 15% of angiosperm speciation events involve a change in genome duplication [START_REF] Wood | The frequency of polyploid speciation in vascular plants[END_REF]). Moreover, [START_REF] Laport | Ecological distributions, phenological isolation, and genetic structure in sympatric and parapatric populations of the Larrea tridentata polyploid complex[END_REF]) reported a part of this complexity in the Larrea tridentata (DC.) Coville polyploid complex, that its cytotypes have different environmental distributions, phenological isolation, and varied genetic structure. In another hand, the polyploidization events are responsible for the increased species richness in Rosaceae [START_REF] Vamosi | Polyploidy and Diversification: A Phylogenetic Investigation in Rosaceae[END_REF]. The complexity of polyploids has been underlined by several different genetic, ecological, physiological and morphological aspects that have been associated with polyploidy. The interactions among these various possible factors can give a large part of the phenotypic, genomic, ecological and evolutionary consequences of polyploidy (reviewed in [START_REF] Balao | Phenotypic consequences of polyploidy and genome size at the microevolutionary scale: A multivariate morphological approach[END_REF]; [START_REF] Yang | Genomic aspects of research involving polyploid plants[END_REF]; [START_REF] Birchler | Genetic Consequences of Polyploidy in Plants James[END_REF]; [START_REF] Moghe | The causes and molecular consequences of polyploidy in flowering plants[END_REF]; [START_REF] Ramsey | Ecological studies of polyploidy in the 100 years following its discovery[END_REF]).

Evolutionarily significant units (ESUs) -a confusing history

The general concept of the evolutionarily significant units (ESUs) has evolved over time. The question is: how precisely identifying this basic unit of conservation? In this question which seems even more complicated is defining the criteria for ESUs recognition within species that composes of many subspecies and ecotypes [START_REF] Ryder | Species conservation and systematics: the dilemma of subspecies[END_REF]. Historically, the ESUs are defined as subsets of the more inclusive entity species, which possess genetic attributes significant for the present and future generations of the species in question [START_REF] Ryder | Species conservation and systematics: the dilemma of subspecies[END_REF]. Then, the ESUs concept was developed by [START_REF] Waples | Evolutionarily Significant Units, Distinct Population Segments, and the Endangered Species Act: Reply to Pennock and Dimmick[END_REF], this novel concept recognizes the ESUs as a population or group of populations that: (i) is substantially reproductively isolated from other conspecific population units; and (ii) represents an important component of the evolutionary legacy of the species.

In the middle of the 1990s, [START_REF] Moritz | Defining " Evolutionarily Significant Units " for conservation[END_REF] focused on the evolutionary past of the taxa and thought that "ESUs should be reciprocally monophyletic for mtDNA alleles (the divergence in mtDNA reflects long-term restriction of gene flow) and show a significant divergence of allele frequencies at nuclear loci". However, [START_REF] Crandall | Considering evolutionary processes in Conservation Biology[END_REF] thought that this concept of ESUs was not adequate for determining appropriate conservation actions. In their paper, they suggested a broader categorization of population distinctiveness based on concepts of ecological and genetic exchangeability. For example, the populations within a species that show adaptive genetic differentiation may justify separate units.

In this thesis, we used different technics to better explore the genetic and phenotypic variation [START_REF]Évolution, Biodiversité (ISYEB[END_REF] to improve the knowledge of the species complex of Arenaria grandiflora (2) to examine if [START_REF] Moritz | Defining " Evolutionarily Significant Units " for conservation[END_REF] and [START_REF] Crandall | Considering evolutionary processes in Conservation Biology[END_REF] points of view are compatible for this case of A. grandiflora species complex.

Arenaria L. -a complex history

The biology of the genus Arenaria has been studied through several points:

1.4. [START_REF]Évolution, Biodiversité (ISYEB[END_REF] The complex taxonomic history

Over the years, the taxonomy of the genus Arenaria and its allied genera has been controversial and considered unsatisfactory. For the first time, Linnaeus (Linnaeus 1737) defined this genus in the thirties of the eighteenth century. Many detailed studies have focused on the circumscription of the generic and infra-generic boundaries in Arenaria, that consistently has a confusing history. Certainly, these studies included two concepts: one held by Fernald, a wide point of view, maintaining the genus Arenaria as a whole unit. Fernald and several American botanists did not consider the different characters (valves of the capsule, foliage, ovary, inflorescence and seeds) sufficiently to demonstrate the segregated genera as distinct from Arenaria sensu stricto [START_REF] Fernald | The specific identity of Arenaria groenlandica and A. glbra[END_REF]. Whereas the other concept divides the group Arenaria into many genera and subgenera, many botanists have adopted this strict generic concept. For example, Bentham and Hooker have recognized 11 subgenera, including the subgenus of Arenaria s.

str., and proposed up to 130 species in the genus Arenaria [START_REF] Bentham | Genera Plantarum[END_REF]. Furthermore, in the first taxonomic revision of Arenaria, Williams defined seven subgenera and 34 sections and subsections, which grouped 168 species [START_REF] Williams | Revision of the genus ARENAIA[END_REF]. Likewise, there are more than 300 species of Arenaria in the world belonging to ten subgenera and 24 sections, according to [START_REF] Zhou | On the geographical distribution of Arenaria L[END_REF]. Recently, in the last thirteen years, several authors defined a new key for the genus Arenaria and new species in Iran and in Turkey (Aytaç & Duman 2004); [START_REF] Fadaie | A new species of the genus Arenaria L. (Caryophylaceae) from Iran[END_REF]; [START_REF] Fadaie | Three new species and a key to the species of the genus Arenaria L. in Iran. Iran[END_REF].

In addition, Fior and Karis have suggested that the seed strophiole should be considered when determining keys to separate Moehringia L. from Arenaria [START_REF] Fior | Phylogeny, evolution and systematics of Moehringia (Caryophylla-ceae) as inferred from molecular and morphological data: a case of homology reassessment[END_REF]. However, McNeill's viewpoint has more clearly outlined by defining the criteria of generic and infra-generic classification of Arenaria. Seven genera, and ten subgenera had been distinguished by McNeill, including the subgenus of Arenaria, that has also subdivided into several sections and series [START_REF] Mcneill | Taxonomic studies in the Alsinoideae: I Generic and Infra-generic groups[END_REF]. Despite the difficulties in selecting the characters to determine the limitations, there are exceptions in a limited area, for example, in Europe [START_REF] Fernald | The specific identity of Arenaria groenlandica and A. glbra[END_REF], where the lines separating the Arenaria genus complex (Arenaria, Moehringia and Minuartia) are well defined by [START_REF] Mcneill | Taxonomic studies in the Alsinoideae: I Generic and Infra-generic groups[END_REF]. The section Grandiflorae is one of 11 sections created by [START_REF] Mcneill | Taxonomic studies in the Alsinoideae: I Generic and Infra-generic groups[END_REF]. It has closely related to section Plinthine. Over and above, it comprises only two species complexes: Arenaria grandiflora and Arenaria valentina Boiss. Members of section Grandiflorae are perennial herbs and native to the Iberian Peninsula, Balearic Islands, Southern, Western and Central Europe and North Africa.

The difficulties in splitting the Arenaria grandiflora species complex (Fig. 2) have led to a confusing intraspecific taxonomic history. The so-called A. grandiflora species complex has explicated by its polymorphous populations and its variation in the level of ploidy. However, a few micro morphological features in the indumentum can use as key characters for some infraspecific taxa [START_REF] Sáez | A new subspecies of Arenaria grandiflora (Caryophyllaceae) from the Rif Mountains (northern Morocco)[END_REF]. Therefore, the species complex A. grandiflora includes only three subspecies as mentioned in Flora Europaea: Arenaria grandiflora ssp. grandiflora; A. grandiflora ssp. bolosii and A. grandiflora ssp. incrassata). Nevertheless, according to the Global Biodiversity Information Facility (GBIF; international open data infrastructure), this species complex involves five accepted subspecies, two accepted varieties and two accepted forms. The five subspecies are:

1) Arenaria grandiflora ssp. grandiflora, that distributes in Baleares (Mallorca), France, Spain, Andorra, Italy, Yugoslavia, Tunisia, Algeria and Morocco.

2) Arenaria grandiflora ssp. bolosii (Cañigueral) Küpfer, it is an endemic restricted in Baleares (Mallorca)

3) Arenaria grandiflora ssp. incrassata (Lange) C. Vicioso ex Rivas Mart, that exists in Baleares and Spain 4) Arenaria grandiflora ssp. valentina (Boiss.) O. Bolòs and Vig, that occurs in SE of Spain 5) Arenaria grandiflora ssp. gomarica L. Sáez. J.M. Monts. and Rosselló, that distributes in Baleares (Mallorca) and Morocco Also, the accepted varieties and forms are as following:

a) Arenaria grandiflora var. stolonifera Ser, its distribution extends along the Pyrenees Chain and Switzerland (Alpine mountains) b) Arenaria grandiflora var. grandiflora c) Arenaria grandiflora f. longifolia Maire d) Arenaria grandiflora f. grandiflora Furthermore, according to other databases such as: The Plant List and International Plant Names Index and NUB Generator, seven subspecies have accepted, they are the first five subspecies plus two subspecies which are:

1) Arenaria grandiflora ssp. glabrescens (Willk.) G. López and Nieto Feliner, that is a Majorcan mountain endemic

2) Arenaria grandiflora ssp pseudoincrassata Malag.

Moreover, the subspecies which have been reported in the cytogenetic studies are: the subspecies Arenaria grandiflora ssp. grandiflora that have a basic chromosome number x=11 including two cytotypes, one diploid (2n= 22) throughout the Pyrenean chain and one tetraploid (2n= 44) in the massif of Nevada in Spain Küpfer (1974). The tetraploid cytotype, detected in the Balearic taxa of this complex, is the endemic Arenaria grandiflora ssp. bolosii that is a tetraploid (2n = 44; [START_REF] Cardona | Nmeros cromosmicos para la flora espaola[END_REF]. The chromosome numbers of Arenaria grandiflora ssp. glabrescens (Willk.)

G. López and Nieto Feliner (2n = 44), indicated that it is tetraploid subspecies [START_REF] Castro | New chromosome numbers for plant taxa endemic to the Balearic Islands[END_REF]. This subspecies Arenaria grandiflora ssp. glabrescens is closely related to the North African A. grandiflora ssp. gomarica, which is also tetraploid [START_REF] Sáez | A new subspecies of Arenaria grandiflora (Caryophyllaceae) from the Rif Mountains (northern Morocco)[END_REF]. The Arenaria grandiflora ssp.

incrassata is another subspecies of this complex but it is diploid. Unfortunately, no chromosome counts are available for neither Arenaria grandiflora ssp. valentina nor Arenaria grandiflora ssp.

pseudoincrassata.

The distribution and ecology

The genus Arenaria is geographically widespread, mainly distributed in arctic regions, such as the Euro-Siberian region. An example is the Arenaria norvegica ssp. anglica Halliday, which is an arctic-montane endemic that occurs in Iceland, Norway, Sweden, Finland, Shetland and North-West Scotland [START_REF] Walker | The distribution, ecology and conservation of Arenaria norvegica subsp. anglica Halliday ( Caryophyllaceae)[END_REF]. The genus also occurs in northern temperate regions (Eurasian Mountains, Andes., Asia Minor).

Likewise, according to Flora Iranica [START_REF] Rechinger | Flora Iranica: Caryophyllaceae II-Alsinoideae and Silenoideeae[END_REF]). Arenaria genus is present in the Irano-Turanian The species complex of A. grandiflora has a distribution stretching from France, Spain, Switzerland, Andorra, Italy and ex-Yugoslavia, to Tunisia, Morocco and Algeria (Fig. 3); where it occurs in fragmented areas (disjunctive distributions). However, most members of the Arenaria grandiflora species complex are orophilous plants, aside from those in the lowlands, where it very rarely occurs, such as in the Indre-et-Loire (Les Sablons et Beaumont-en-Véron) and in Seine-et-Marne (Fontainebleau) (Chater & Halliday 1964;[START_REF] Bottin | Re-establishment trials in endangered plants: a review and the example of Arenaria grandiflora, a species on the brink of extinction in the Parisian region (France)[END_REF] as well as in the Pavlov Hills in Czech Republic [START_REF] Danihelka | Pavlov Hills: botanical excursion guide[END_REF], also, in the low hills of Rocamadour (300m) and the Dordogne Valley (Galinat 1938). Therefore, in Switzerland, its distribution is limited to the alpine summits in the Jura region, in rocks or rock debris and in full sun exposure in very well-drained soil with low humidity (Mt Chasseron, Mt Suchet and Mt Chasseral) (Favarger 1959;[START_REF] Hess | Vitro Culture Introduction for Exsitu Conservation of Some Rare Plant Species[END_REF]).

Indeed, the species complex of A. grandiflora has a wide range of distribution in Spain and it has represented by several subspecies, as like as A. grandiflora ssp. incrassata, A. grandiflora ssp.

glabrescens and A. grandiflora ssp. grandiflora, these subspecies have been featured by several studies in many provinces (Huesca, Alicante, Galician, Balearic Islands, Soria, Castellón, Sierra Tramuntana of Mallorca and Sierra Pozo in Jaén, Gerona, Lleida, Andalusia, Tarragona and Valencia). However, the A. grandiflora ssp grandiflora is a widespread subspecies, shows a restricted geographical distribution in dry rocky and stony places in the southern Provence of France such as in the summits and crests above 900 m in Sainte Baume [START_REF] Youssef | Factors underlying the narrow distribution of the Mediterranean annual plant Arenaria provincialis (Caryophyllaceae)[END_REF]. In addition, it is restricted to the highest summits (1600-2150 m) of the mountains of the western Rif in Morocco [START_REF] Sáez | A new subspecies of Arenaria grandiflora (Caryophyllaceae) from the Rif Mountains (northern Morocco)[END_REF].

1.4.3

The life forms and cycles

As we mentioned above, the geographical distributions differ extremely in genus Arenaria. Therefore, the members of this genus can be grouped into different life-forms and cycles. In general, the species of genus Arenaria are either annual or perennial herbs, sometimes biennial. However, very few detailed studies have focused on plant growth forms for this genus, McNeill has worded its life form spectrum in a way, which was categorized as plant habit. Some of McNeill's categories are small annuals, xerophytes of Mediterranean, densely pulvinate alpines and spiny sub-fruticose (i.e., buds that are protected by dead plant remains) perennials [START_REF] Mcneill | Taxonomic studies in the Alsinoideae: I Generic and Infra-generic groups[END_REF]. [START_REF] Raunkiaer | The life forms of plants and statistical geography[END_REF] has classified plants according to the place where the growth point is located during the less favorable seasons. The buds of Chamaephytes are on persistent shoots near the ground and the Hemicryptophytes have their dormant buds in the upper crust of the soil, just below the surface [START_REF] Raunkiaer | The life forms of plants and statistical geography[END_REF]. The Chamaephytes reach their highest density rates within 1400-2000m [START_REF] Giménez | Endemic ora biodiversity in the south of the Iberian Peninsula: altitudinal distribution, life forms and dispersal modes[END_REF]), thus, we can understand that the Arenaria tomentosa Willk. (endemic species in the south of the Iberian Peninsula) is chamaephytes (Fig. 4). Furthermore, Arenaria polytrichoides Edgew is a long-lived perennial herb that forms cushion chamaephytes [START_REF] Yang | Positive associations between the cushion plant Arenaria polytrichoides (Caryophyllaceae) and other alpine plant species increase with altitude in the Sino-Himalayas[END_REF]. A cushion chamaephyte is a life form in harmony with the alpine conditions. Therefore, the winter annual Arenaria serpyllifolia L. is a chamaephyte or therophyte herb (Fig. 5b).

However, there are no studies that explain clearly the life forms in Arenaria grandiflora species complex, it is a chamaephyte or a hemicryptophyte (Fig. 5a). Under less favorable conditions, the chamaephyte form, as well as the hemicryptophyte form, have been found [START_REF] Raunkiaer | The life forms of plants and statistical geography[END_REF]. Dispersal of seeds is an important process within the plant's life cycle. Up to now, dispersal modes at the level of Arenaria species have not been studied in detail. However, [START_REF] Youssef | Factors underlying the narrow distribution of the Mediterranean annual plant Arenaria provincialis (Caryophyllaceae)[END_REF] have reported the mode of dispersal occurring in some species of Arenaria in southeastern France. The dispersal modes in Arenaria grandiflora, A. aggregata, A. cinerea and A. provincialis has been reported to be anemochory (dispersal by wind) or barochory (dispersal by means of gravitation) i.e. without clear morphological adaptations for seed dispersal [START_REF] Youssef | Factors underlying the narrow distribution of the Mediterranean annual plant Arenaria provincialis (Caryophyllaceae)[END_REF]. Indeed, a species has sometimes dispersed by more than one process. Such cases have called diplo-or polychory, like as Arenaria lanuginose (Michaux) [START_REF] Frantzen | Dispersal and growth form patterns of some zonal páramo vegetation types[END_REF]).

The morphological description

The morphological characteristics of Arenaria genus have described in many Floras in the world as Flora of China, Flora Europaea, Flora of North America, Flora Coste, etc. Therefore, the descriptive details have been defined in different artificial keys to subgenera, sections, and species by [START_REF] Mcneill | Taxonomic studies in the Alsinoideae: I Generic and Infra-generic groups[END_REF], [START_REF] Zhou | On the geographical distribution of Arenaria L[END_REF] and [START_REF] Williams | Revision of the genus ARENAIA[END_REF]. Here, we exhibit a few of characteristics of the genus

Arenaria. livermorensis) and its ovary are with numerous ovules. Styles are often 2 or 3(-5).

Our interesting species complex of Arenaria grandiflora has outlined the morphological characteristics in the France Flora (Coste 1937), Flora of the Jurassic Chains [START_REF] Grenier | Flora de la Chains Jurassique[END_REF] and Flora Helvetica (Switzerland) etc. The stem is 5-15 cm tall, branched, ascending and sometimes hairless. The rootstock is woody and many-headed. The simple leaves are opposite and sessile; they are lanceolate or linear-lanceolate, acuminate, thickened at the margin. Actinomorphic flowers with long petals characterize the large flower sandwort, these petals are clearly exceeding the calyx (Figure 2. The sepals are ovate or glandular-pubescent. The petals are oblong-obovate and they are twice as long of the sepals.

Inflorescence 1-3(-6)-flowered, with large white flowers in spring. The flowers are with pedicels that measure about 2-6 times as long as sepals and these pedicels are glandular-pubescent. The stamens are 10 and the styles are usually 3. The plants of the species complex of A. grandiflora have a short capsule, opening by valves in several equal or doubles that of styles. The fruit is an ovoid capsule (Coste 1937).

Very few of studies reported the diagnostic features of the infraspecific taxa of A. grandiflora species complex. For instance, the subspecies of A. grandiflora species complex can be differentiated mainly on the base of the leaf shape, pedicels, calyx, seeds and the pubescence on the pedicels and calyx of plants [START_REF] Sáez | A new subspecies of Arenaria grandiflora (Caryophyllaceae) from the Rif Mountains (northern Morocco)[END_REF].

The seeds

Seed characteristics generally have great contributions in taxonomic studies. In the genus Arenaria and its allied genera, they are significant enough to allow taxonomic delimitation of the family Caryophyllaceae at various levels [START_REF] Justice | Principles and Practices of Seed Storage[END_REF]) like the five types of strophioles which have been recognized in Moehringia species by [START_REF] Minuto | Seed morphology in Moehringia L. and its taxonomic significance in comparative studies within the Caryophyllaceae[END_REF]). Furthermore, the micromorphology studies of the seeds showed a significant variation at the species level in several species of Minuartia L. [START_REF] Mostafavi | Seed micromorphological survey of the Minuartia species (Caryophyllaceae) in Iran[END_REF]) and suggested a variation in the exomorphology of the seeds of Stellaria L.

species [START_REF] Mahdavi | The systematic significance of seed micro-morphology in Stellaria L. (Caryophyllaceae) and its closest relatives in Iran[END_REF]. Interspecific variation in seed morphology of Arenaria was noted by [START_REF] Fernald | The specific identity of Arenaria groenlandica and A. glbra[END_REF] and [START_REF] Sadeghian | Systematic implication of seed micromorphology in Arenaria (Caryophyllaceae) and allied genera. Flora: Morphology, Distribution[END_REF]) which found a significant polymorphism in seed coat morphology in certain of Arenaria species. Furthermore, an intraspecific variation in seed morphology was analyzed in populations of A. uniflora (Walt.) Muhl. by [START_REF] Wyatt | Intraspecific Variation in Seed Morphology of Arenaria uniflora (Caryophyllaceae)[END_REF].

According to Central and East-European flora [START_REF] Bojnanský | Atlas of seeds and fruits of central and east-european flora the carpathian mountains region[END_REF], seeds of A.

grandiflora species are reniform and laterally compressed and the margin is dense, the surface is tuberculate, it is ranged from brown to reddish-brown. The seed traits (such as mass and size of seeds, number of seeds) are vital components for distribution of A. grandiflora which inhabit dry and rocky habitats because of the correlations between habitats, seed traits and root development [START_REF] Youssef | Factors Underlying the Narrow Distribution of the Mediterranean Annual Plant Arenaria provincialis (Caryophyllaceae)[END_REF].

In some species of Arenaria, the seed dormancy has been rather questionable. In their studies of A. glabra Michx and A. fontinalis (Short and Peter) Shinners, Baskin andBaskin (1982, 1987) mentioned that most of the freshly-matured seeds were innately dormant. A physiological dormancy was prevalent in Arenaria ciliata L., according to [START_REF] Schwienbacher | Seed dormancy in alpine species[END_REF]).

The threat and protection

The European Red List of Vascular Plants defined the status of many species of Arenaria such as A.

nevadensis, which is Critically Endangered and A. humifusa, which is Near Threatened [START_REF] Bilz | European Red List of Vascular Plants[END_REF] (see Figure 6) Additionally, the status of Arenaria cinerea DC., is determined as vulnerable (VU) in Provence-Alps-Côte d'Azur (France) as mentioned in the Red Book of endangered flora in France [START_REF] Olivier | Livre Rouge de la flore menacée de France. Tome I: Espèces prioritaires[END_REF]. There are also others national sources, such as the Red List of endangered species in Switzerland (ferns and flowering plants), that mentioned the status of two species of Arenaria, one of which is Near Threatened (A. bernensis) and the other is Regionally Extinct in Switzerland (OFEV 2011). Although IUCN is the best scientific tool, there are some cases with increasing knowledge of taxa, where the re-evaluation of its status is necessary, such as Arenaria provincialis Chater and

Hallyday, which has reevaluated by [START_REF] Véla | Exemple d' une nouvelle évaluation du statut de menace suivant les cri-tères de l"UICN version 3.1. : le cas de l"endémique provençale Arenaria provincialis Chater & Halliday (Caryophyllaceae)[END_REF]. Its new status is "Near Threatened" (NT), instead of the ancient category "Rare" (R).

Fig. 6: IUCN Red List Categories at regional scale [START_REF] Bilz | European Red List of Vascular Plants[END_REF] Globally, the Red List of the International Union for the Conservation of Nature (IUCN) is an important scientific tool for making decisions for evaluating the species and their conservation. In 1997, Arenaria grandiflora was as a vulnerable (VU) in Jura, according to IUCN (list of threatened species) and it has been completely protected since 2005 in Vaud (Switzerland). Furthermore, in Austria, A. grandiflora has been characterized as "Rare" and is potentially endangered. Else, this taxon occurs only at a few localities in the Czech Republic are considered as rare species and critically threatened taxa (CR) [START_REF] Grulich | Red List of vascular plants of the Czech Republic: 3rd edition[END_REF], because of its occurring at 1-5 localities and one out of these five populations is known to have become extinct. In France, according to the Regional Red List of the vascular flora of Ile-de-France, Arenaria grandiflora species has nationally characterized as Critically Endangered in areas of occupancy and in areas of occurrence. Its decline is continuing in the quality of habitat, the number of locations or subpopulations, or in the number of mature individuals (Auvert et al. 2011).

In 1991 and 1993, this species has protected by regional texts in Ile de France and Centre. In 1999, a restoration plan was initiated in Fontainebleau Forest in Paris region (Machon et al. 1999). It consisted of founding 3 populations of the same mixture of plants of Fontainebleau (2/3 of local plants)

and plants from the Chinon region (exogenous contribution for 1/3). Seventeen years later, two populations still exist. Transplanted individuals have virtually all disappeared but have been replaced by abundant offspring. The dataset consists of the follow-up of each individual transplanted or born in the reintroduction sites since the implementation of the plan (about 17 000 data), as well as its size and the number of flowers it produced each year.

1.4.6

The phylogeny

The phylogeny has been illustrated Arenaria sensu [START_REF] Mcneill | Taxonomic studies in the Alsinoideae: I Generic and Infra-generic groups[END_REF] by Nepokroeff et al (2001) conducting a DNA survey of the Caryophyllaceae using sequence data from matK and the rps16 intron.

This phylogeny mentioned to Arenaria is polyphyletic, with two major distinct clades. Recently, the molecular studies have referred to three subfamilies of Caryophyllaceae [Alsinoideae (Burnett.)

Caryophylloideae (Arn.) and Paronychioideae (A. St)], which are polyphyletic (Fior et al. 2006). It has revealed by using ITS sequence data in combination with the chloroplast marker matK.

Thereafter, Harbaugh et al. (2010) found that the traditional three-subfamily subdivision of the Caryophyllaceae has not reflected and the morphological characters used to delimit them are unreliable due to the extensive convergent evolution. According to their study, large genera, such as Arenaria are polyphyletic and their species have scattered among three separated clades corresponding to several subgenera in [START_REF] Mcneill | Taxonomic studies in the Alsinoideae: I Generic and Infra-generic groups[END_REF] Finally, in 2007, 13 microsatellite markers were developed for the species A. grandiflora [START_REF] Zavodna | Development and characterization of microsatellite markers for Arenaria grandiflora L. (Caryophyllaceae)[END_REF]. They allowed to genotype the founding individuals of the populations (which always existed in ex-situ collection) and to determine the gene's share from Chinon and Fontainebleau in the progenies that lived in 2007 and 2010.

Conclusion of the introduction

It is apparent from this introduction that there is generally high complexity in the genus Arenaria, but for this thesis, we are interested in the species complex of Arenaria grandiflora, currently composed of two cytotypes (diploid and tetraploid), a few subspecies (3, 5 or 7 accepted subspecies according different authors) and varieties (2 accepted varieties), especially, the subspecies A. grandiflora ssp.

grandiflora composed also of two cytotypes (diploid and tetraploid) and many widespread populations that show disjunctive and declining distributions and encountered in many different types of habitats in Western Europe. Disjunctive populations are often particularly important to study, because most of them are small (less than 100 individuals) and thus threatened and because they also often contain original traits. We use the term of species complex to reflect the complicated nomenclatural and taxonomical history of this taxon. Despite of the previous studies on A. grandiflora, there are still major gaps in the knowledge of this species complex. To implement coherent and effective conservation plans, it is necessary to clearly identify the protected species. Especially, it is necessary to well delineate evolutionary significant units (ESUs) for its conservation. In this thesis, we used the opportunity of the work on both the natural population and the herbarium' specimens of Arenaria grandiflora species complex to bring new elements to the definition of ESUs for the future conservation plans. Thus, the scientific question of this work is: on what should we base to define conservation units?

2. Chapter II: Genetic variation within the species complex of Arenaria grandiflora L.

Introduction to Chapter II

As mentioned earlier in the preface, there had been difficulties to reveal nuclear microsatellite loci on ancient (e.g. >200 years) herbarium' specimens within the Arenaria section Grandiflorae, collected from different herbaria (Montpellier, Geneva, Barcelona and Paris herbaria). The DNA was perhaps too degraded. That is why we used the same technic but with fresh specimens of the species complex of Arenaria grandiflora. Thus, in the next year, samples of natural populations have been collected from its disjunctive populations in Western Europe. These fresh specimens have been studied to reveal the nuclear microsatellite loci at the intraspecific level at the Molecular Systematics Service (SSM) in the French National Museum of Natural History. Because we found for some specimens (in some populations) more than two alleles per locus, we supposed that there could be tetraploids within our fresh samples which were collected from the natural populations?

To confirm this result, cytogenetic analyses had been carried out at the Ecology and Systematics Evolution (ESE) in University of Paris-Sud Saclay to detect the chromosome numbers and flow cytometry approach had been used to reveal the intraspecific genome size variability and thus better define the ploidy levels within this species complex.

INTRODUCTION

The doubling of complete chromosome sets (polyploidization) occurs in angiosperms at several levels (Madlung 2013;[START_REF] Weiss-Schneeweiss | Evolutionary consequences. constraints and potential of polyploidy in plants[END_REF], including the intraspecific level (Schönswetter et al. 2007;[START_REF] Trávnícek | Remarkable coexistence of multiple cytotypes of the Gymnadenia conopsea aggregate (the fragrant orchid): evidence from flow cytometry[END_REF]). In the context of the implementation of management's plans for threatened species, and the definition of evolutionarily significant units (ESUs: a group of organisms that has been isolated from other groups within the same species for a sufficient period of time to exhibit significant genetic divergence [START_REF] Ryder | Species conservation and systematics: the dilemma of subspecies[END_REF][START_REF] Moritz | Defining " Evolutionarily Significant Units " for conservation[END_REF])), the phenomenon of intraspecific polyploidy must be taken into account (Brown and Young 2000;[START_REF] Severns | Intraspecific chromosome number variation: A neglected threat to the conservation of rare plants[END_REF]. Indeed, because crosses between some populations with different ploidy levels may be impossible or lead to sterile offspring, it is necessary to document the spatial distribution of such populations to define the best management plans. In this framework, the natural populations of Arenaria grandiflora species complex from the carnation family (Caryophyllaceae) in Western Europe represent an interesting study case.

The distribution of the A. grandiflora species complex stretches from France, Spain, Switzerland, Andorra, Italy and Yugoslavia to Tunisia, Algeria and Morocco, with populations occurring in fragmented areas; [START_REF] Sáez | A new subspecies of Arenaria grandiflora (Caryophyllaceae) from the Rif Mountains (northern Morocco)[END_REF]Stanisci et al. 2005;[START_REF] Gavilán | An approach towards the knowledge of Iberian high-mountain calcareous grasslands[END_REF]. All members of the A. grandiflora species complex, are orophilous plants but, they can grow in lowlands, though this habitat is rare. In France, they have been found in the Loire region near the city of Chinon (Chater and Halliday 1964), in the low hills (300 m) of Rocamadour (Lot) in the Dordogne Valley (Galinat 1938) and in the Fontainebleau forest 50 km south of Paris (Arnal 1999) and in the Pavlov Hills in the Czech Republic [START_REF] Danihelka | Pavlov Hills: botanical excursion guide[END_REF]). Moreover, this species complex of A. grandiflora grows in small isolated populations almost everywhere it is found (Bottin et al.2007;obs. perso.)According to several regional and national Red Lists, the species complex of A. grandiflora is considered as a threatened taxon in certain locations in Europe, such as in the Czech Republic, it is classified as a critically threatened taxon, while it is vulnerable and protected in Switzerland, and it is defined as a rare taxon in Austria. In addition, A. grandiflora species complex is currently extinct in two regions of France: on the French side of Mt Jura and in the Fontainebleau forest (Prunier 2001 and personal observation). Furthermore, it is now generally a rare species in southeastern France [START_REF] Youssef | Factors underlying the narrow distribution of the Mediterranean annual plant Arenaria provincialis (Caryophyllaceae)[END_REF]. This species complex is only abundant in the Pyrenees Mountains and in Spain at present (Machon et al. 1991). In the particular case of the Fontainebleau forest, the A. grandiflora populations [Mont Merle and Mt Chauvet] rapidly declined during the last century and probably became extinct in 2014 [START_REF] Bottin | Re-establishment trials in endangered plants: a review and the example of Arenaria grandiflora, a species on the brink of extinction in the Parisian region (France)[END_REF] and personal observation). In 1999, before the complete extinction of A. grandiflora, a restoration programme was undertaken at three natural sites in the Fontainebleau forest (Maurice et al. 2013) through the creation of three populations, with 450 clones of both local (Fontainebleau) and non-local (Chinon) individuals being mixed in each population [START_REF] Zavodna | ) differed significantly from others diploid populations as such as Mt Rié population. In addition, the GP of some seed provenances of A. grandiflora species complex varied significantly among the tetraploids ones, like seeds of Mt Cheval Blanc population geminated differently and significantly from seeds of Mt Raton population. Also, in vitro seed germination percentages were the lowest for the diploid populations like Rocamadour, Chinon and Mt Merle populations and differed significantly from all other seed provenances that germinated very well in vitro like the tetraploid populations (Mt Chaudière, Ceresa, Mt Lure, Mt Raton and Mt Cheval Blanc) and the diploid ones (Plan and Mt Sagra). In addition, the GP of seeds from the tetraploid population of Mt Cheval Blanc differed significantly from the GP of seeds of the diploid population of Mt Sagra. The provenance effect on seed germination is a very well-known phenomenon. For example; it was shown for six provenances of Faidherbia albida (Delile) A. Chev in tropical Africa[END_REF]. The programme was implemented as an emergency measure, ignoring the potential intraspecific variation of polyploidy in the A. grandiflora species complex. However, the restoration project was relatively successful [START_REF] Zavodna | ) differed significantly from others diploid populations as such as Mt Rié population. In addition, the GP of some seed provenances of A. grandiflora species complex varied significantly among the tetraploids ones, like seeds of Mt Cheval Blanc population geminated differently and significantly from seeds of Mt Raton population. Also, in vitro seed germination percentages were the lowest for the diploid populations like Rocamadour, Chinon and Mt Merle populations and differed significantly from all other seed provenances that germinated very well in vitro like the tetraploid populations (Mt Chaudière, Ceresa, Mt Lure, Mt Raton and Mt Cheval Blanc) and the diploid ones (Plan and Mt Sagra). In addition, the GP of seeds from the tetraploid population of Mt Cheval Blanc differed significantly from the GP of seeds of the diploid population of Mt Sagra. The provenance effect on seed germination is a very well-known phenomenon. For example; it was shown for six provenances of Faidherbia albida (Delile) A. Chev in tropical Africa[END_REF]: two of the three populations still exist 15 years after their creation, although a slow decline in the size of the remaining populations has been observed since 1999 (personal observation in 2016).

Based on the necessity of documenting the evolutionarily significant units (ESUs) within A.

grandiflora species complex to better define future management plans, we first assessed the neutral genetic variation in their natural populations using nuclear microsatellite (SSR) loci. Then, we counted the chromosome numbers of some of these natural populations using cytogenetic approaches and quantified the genome size of individuals from the same natural populations using flow cytometry (FCM). According to the available literature, the only previous study reporting the genetic diversity of A. grandiflora species complex based on microsatellite markers [START_REF] Zavodna | ) differed significantly from others diploid populations as such as Mt Rié population. In addition, the GP of some seed provenances of A. grandiflora species complex varied significantly among the tetraploids ones, like seeds of Mt Cheval Blanc population geminated differently and significantly from seeds of Mt Raton population. Also, in vitro seed germination percentages were the lowest for the diploid populations like Rocamadour, Chinon and Mt Merle populations and differed significantly from all other seed provenances that germinated very well in vitro like the tetraploid populations (Mt Chaudière, Ceresa, Mt Lure, Mt Raton and Mt Cheval Blanc) and the diploid ones (Plan and Mt Sagra). In addition, the GP of seeds from the tetraploid population of Mt Cheval Blanc differed significantly from the GP of seeds of the diploid population of Mt Sagra. The provenance effect on seed germination is a very well-known phenomenon. For example; it was shown for six provenances of Faidherbia albida (Delile) A. Chev in tropical Africa[END_REF], conducted in two populations (Chinon and Fontainebleau) and their hybrids, considered the plants to be diploids. The most frequent cytotypes within the A. grandiflora species complex in Europe are 2n=20; 2n=22 and 2n=44 (Favarger 1959). Despite the increasing use of flow cytometry techniques to estimate genome size and ploidy levels in plants due to the rapidity and sensitivity of these methods, genome size has been studied in only three species of Arenaria L.

(A. serpyllifolia L.; A. gracilis Waldst. and Kit. and A. deflexa Dec.) [START_REF] Siljak-Yakovlev | Towards a genome size and chromosome number database of balkan flora: C-values in 343 taxa with novel values for 242[END_REF]Bou Dagher-Kharrat et al. 2013).

The present study assessed a combination of cytogenetic and multi-allelic molecular markers (microsatellites) in many wild populations to characterize the genomes of the A. grandiflora species complex. The main objectives of this study were to a) perform molecular profiling using microsatellite markers; b) determine the chromosome number in each population c) assess genome size; d) detect any cytogenetic differences between the investigated populations; and e) define potential the evolutionarily significant units (ESUs) for restoration plans.

MATERIALS AND METHODS

Plant material

The large-flowered sandwort (A. grandiflora) is a perennial plant that is cushion-shaped with woody and ascending stems that can reach a height of 15 centimetres. This small plant of Caryophyllaceae is a Pyrenean-Alpine species that grows on calcareous and sedimentary rocks, xerophytic and oro-basophils lawns and schist screes (Küpfer 1974). It blooms from May to August, A. grandiflora is insect pollinated but is also able to self-reproduce in the absence of pollinators (Machon et al. 1999[START_REF] Machon | Comment sauver Arenaria grandiflora L. de la depression?[END_REF]). The mating system of A. grandiflora is probably predominantly outcrossing, although vegetative reproduction is also possible [START_REF] Bottin | Re-establishment trials in endangered plants: a review and the example of Arenaria grandiflora, a species on the brink of extinction in the Parisian region (France)[END_REF]).

Seed and leaf samples were collected from natural populations in different eco-geographic sites in Western Europe (table 1). Some of the collected seeds were dried and stored in paper or plastic bags under ambient laboratory conditions (approximately 20°C) and some of the collected leaves were stored in silica gel. The geographical origins of the investigated populations are indicated in table 1. The leaves were generally collected from natural populations, but for BBV (the population from Chinon), the leaves were obtained from the clones cultivated in vitro by the National Botanical Conservatory for the Parisian Basin (CBNBP). The root meristem was also sampled from in vitro plants. A protocol was implemented to sterilize the seeds and to cultivate them in vitro, as reported in another article (M. Daoud et al., National Museum of Natural History, France, unpubl. res.).

Microsatellite genotyping

DNA extraction and PCR conditions--Leaf samples were stored in silica gel and were subsequently used to identify SSR loci. To purify A. grandiflora DNA, the leaves were processed using the DNeasy Plant Mini Kit (QIAGEN). Firstly, 20 mg of dry leaves from each individual was weighed and then homogenized using a TissueRuptor homogenizer to obtain a fine powder. The cells were then lysed via the addition of lysis buffer, followed by incubation. After the addition of buffer P3 and incubation of the finale lysate on ice, the proteins and polysaccharides were precipitated. Finally, the lysate was cleared to promote binding of the DNA to the DNeasy membrane. Optimal results were obtained by eluting the samples twice, the elution volume was reduced to 2 x 50 µL.

The samples were genotyped using the ten specific microsatellite markers for A. grandiflora [START_REF] Zavodna | Development and characterization of microsatellite markers for Arenaria grandiflora L. (Caryophyllaceae)[END_REF]). All reactions were carried out in a 10 µL final volume containing 2 ng/µL of template DNA; 0.20 µ/L Taq pol; 0.30 mM dNTPs; 1.00 mM MgCL²; 0.25 µM Primer R; 0.10 µM Primer fluo F; 5.5 µL H2O and 1.00 µL of 10X TP. Optimization of the PCR conditions was performed for each PCR simplex. After this optimization step, all the loci that exhibited non-overlapping size ranges, similar primer annealing temperatures and similar cycles in the same reaction were co-amplified. Finally, all the microsatellite loci were combined into three multiplexes for simultaneous amplification and analysis using the Qiagen Multiplex PCR Kit. The composition of the multiplexes was as follows: AGA3--98; AGA5--123 and AGA2--74 in Multiplex 1; AG9--49; AG3--36 and AGA5--109 in Multiplex 2 and AG3--2; AGA1--150; AGC5--93 and AGA2--73 in Multiplex 3.

The cycling conditions were as follows: 94°C for 5 min; 30 or 32 cycles of 94°C for 1 min (30 for Multiplexes: 1 and 3; and 32 for Multiplex 2), the specific annealing temperature (55°C for Multiplex 1;

50°C for Multiplex 2; and 53°C for Multiplex 3) for 1 min and 72°C for 1 min; with a final extension step at 72°C for 10 minutes. Then, 1 µL of the PCR product was added to 8.8 µL of formamide and 0.20 µL of the marker standard, followed by evaluation via electrophoresis on an automated sequencer (ABI Genetic Analyzer). Gene Mapper software (Applied Biosystems) was used to determine the size of alleles. In addition, the size of the amplification product was followed through visual verification. For each locus, the alleles were recorded based on their size in base pairs and scored, after which a matrix of allelic phenotypes was constructed.

Determination of chromosome numbers

Root tips obtained from seedlings germinated in in vitro culture were pre-treated with 0.002 M 8hydroxyquinoline (Merck. Prolabo), for 3 h at 16°C or in a 0.05% solution of colchicine (Sigma), and for 45 minutes at room temperature. Fixation was performed in 3:1 (v/v) ethanol-acetic acid at 4°C for at least 24-48 h. The fixed root tips were stored for several days in the first fixative or for several months in ethanol (70%) at 4°C. Root tip meristems were then hydrolysed in 1 N HCl for 5 minutes at 60°C or in 5 N HCL for 6 minutes at room temperature and stained with acetic orceine. Squashing was performed using a drop of acetic carmine. Chromosome numbers were determined from at least ten individuals per population and from several well spread metaphases per root tip. Chromosome plates were observed under a Ziess Axiophot microscope and analysed using a CCD camera (RETIGA 2000R. Princeton Instruments Evry. France) in association with an image analyser (Metavue; version 7).

Genome size estimation via flow cytometry and statistical analysis

Cotyledons, fresh leaves, stored seeds and dried leaves (stored in silica gel) were used to obtain nuclear suspensions for genome size evaluation. The quantity of DNA (2C DNA value) was determined via flow cytometry following the technique of (Marie & Brown 1993). Leaves of the investigated Arenaria Sigma P6755), 5 mM sodium meta-bisulfite and 10 µg/ml RNase; Sigma Aldrich, Saint Quentin, France).

The nuclear suspension was then filtered through nylon mesh (pore size 30 µm) and kept at 4°C. The nuclei were stained with a specific DNA-intercalating fluorochrome dye (30 µg/ml propidium iodide; Sigma Chemical Co. St. Louis, USA) after RNase treatment (2.5 units/ml; Roche). In total, 58 samples were analysed using a flow cytometer (CyFlow SL3, Partec, Munster, Germany). At least 2--5 individuals per population and 5.000 to 10.000 nuclei per individual were analysed to obtain the mean DNA content. The 2C DNA value was calculated based on the linear relationship between the fluorescent signals from the stained nuclei of A. grandiflora specimens and the internal standard.

ANOVA with the Welch test for unequal sampled population sizes was performed, followed by a Games--Howell test using IBM--SPSS--statistics version 24--win32 (Field 2013). ANOVA with the Welch test was employed to determine whether there were any significant differences between the mean monoploid genome sizes. A multiple comparisons test (Games-Howell test) was conducted to demonstrate all pairwise comparisons. Because of the unequal number of samples, a nonparametric measure (Spearman's Correlation Coefficient) was employed to investigate the relationships between altitude and both 2C DNA values and the 1Cx monoploid genome size.

RESULTS

The main results of the molecular assessment using microsatellite markers are summarized in table 2; those of the cytogenetic analysis (chromosome counts and ploidy levels) in table 3; and those for the intrapopulation genome size variability over all spatial scales in fig. 1.

Nuclear microsatellite analysis

Ten microsatellite loci were amplified in seven populations. The allelic phenotypes of the analysed samples are presented in table 2. The ploidy level was deduced from the allelic phenotypes at the ten microsatellite loci, and a total of 258 alleles were revealed for the 10 loci (between 4 and 15 alleles per locus over all seven investigated populations). A maximum of four alleles per locus per individual were detected at most loci in the individuals from Mt Chasseral population in the Swiss Jura, Mt Lure population in the Alps and from Gabas population in the Spanish Pyrenees (fig. 2). Individuals from the populations in Andalusia (Mt Sagra), Rocamadour, Chinon (Bauemont en Veron) and the French Pyrenees (Gèdre) displayed a maximum of two alleles per locus per individual that are sometimes recognizable heterozygosity (table 2). 3). The metaphase chromosomes were relatively small (1 µm to 2 µm), morphologically similar and difficult to spread. Diploid populations were frequent in populations of Chinon (Bauemont en Veron), the Spanish Pyrenees (Plan) and Andalusia (Mt Sagra), whereas the tetraploid populations were found in both alpine and pre-alpine populations (Mt Lure, Mt Raton) also in and the Spanish Pyrenees (Ceresa).

Chromosome number and ploidy level

Genome size data

The holoploid genome size (2C value) ranged from (2.08 pg) to (2.98 pg) for diploid populations and from 4.28 pg to 5.62 pg for tetraploid populations. The monoploid (1Cx) genome size ranged from 1.015.85 to 1.455.58 Mbp. The average quantity of nuclear DNA for each population is given in table 3. Based on the flow cytometric analysis of isolated nuclei, histograms of the relative nuclear DNA content were obtained, which showed two dominant peaks corresponding to both the internal standard and the species complex of A. grandiflora for all studied diploid and tetraploid populations (figs 4 a & b).

Statistical analyses (ANOVA) of genome size--ANOVA with the Welch test for monoploid genome sizes (1Cx) revealed significant differences between the studied populations (F=32.394. p=0.000). To indicate pairwise differences in the populations, we applied Games-Howell analysis (see Supplementary 1). There was an interesting significant difference between the diploid population of Sainte Baume in the southeast of France (Pas de l'Ai) and all other diploid populations (fig. 5), except for the population of the Vilas del Turbon and Mt Sagra in Spain (Supplementary 1). Another interesting difference was found between diploid populations one from the Spanish Pyrenees (Vilas del turbon) and others from the French Pyrenees (Lake Gloriettes, Coumely Plateau and Saint Sauveur) (Supplementary 1). Additionally, there was a significant difference among the alpine population of Mt Cheval Blanc and the two pre-alpine populations of the Chaudière and Mt Raton (fig. 5). Indeed, our results also showed a significant difference (p=0.03) between the tetraploid populations in the Spanish Pyrenees (Ceresa and Gabas). Furthermore, a significant difference was detected between the tetraploid population of Mt Chasseral in Swiss Jura and the tetraploid pre-alpine populations of the Chaudière and Mt Raton.

In general, a strong positive correlation was found between altitude and the 2C DNA value (r=0.69. P=0.000). A strong positive correlation was also detected between altitude and the 1Cx monoploid genome size (r=0.58. P=.000). In the diploid populations, a strong positive correlation (r=0.59; P=0.000) was detected between altitude and both the 2C DNA value and 1Cx monoploid genome size, while in the tetraploid populations, there was no statistically significant correlation (r=-0.24. P=0.224)

DISCUSSION

Microsatellite genotyping

The analysis of 10 polymorphic microsatellite loci detected 258 alleles in natural populations of A.

grandiflora species complex and the number of alleles per sample (2-4) corresponded to the intraspecific variation in ploidy observed via flow cytometry or chromosome counting. For the individuals from Swiss Jura (Mt Chasseral) and the Spanish Pyrenees (Gabas) that displayed up to four alleles per locus (fig. 2), so, the number of alleles could be an indicator of the ploidy level. Here, the observed microsatellite patterns suggested that these populations are tetraploid and the count chromosomes affirmed these findings.

Furthermore, because all ten loci that we examined using microsatellite markers were the same for the diploid and tetraploid plants and tetraploids exhibited up to 4 alleles, we can conclude that the tetraploids that occurred in some of the A. grandiflora populations could have resulted from spontaneous genome duplication or from the joining of unreduced gametes. Polyploids that form via genome doubling within a species have been well described by [START_REF] Soltis | Autopolyploidy in angiosperms: have we grossly underestimated the number of species?[END_REF] in many species, such as Tolmiea menziesii (Pursh)

Torr. & Gray., Galax urceolata (Poir.) Brummit., and Chamerion angustifolium (L.) Holub.

Chromosome number

Our results indicated a basic chromosome number of x=11, including two cytotypes, one diploid (2n=22)

and one tetraploid (2n=44). These results basically agreed with those reported by Küpfer (1974) but differed from the x=10 chromosome number observed for A. grandiflora subsp. grandiflora in the Sierra de Baza in the Province of Granada (Luque & Lifante 1991), which we were unable to find during our collection session. We did not detect any cases of mixed ploidy, as found in other species complex (e.g., Festuca pallens [START_REF] Smarda | Intraspecific DNA content variability in Festuca pallens on different geographical scales and ploidy levels[END_REF]).

To our knowledge and according to the available literature, our identification of the chromosome number (2n=22) is novel for the population of Chinon from the Loire Valley. Furthermore, a 2n=22 chromosome number was also observed in population from the Spanish Pyrenees (Huesca. Plan), which is in agreement with the chromosome counts previously reported in populations of A. grandiflora subsp.

grandiflora throughout the Pyrenean chain (Küpfer 1974). While our results indicated that the population from Mt Sagra in Andalusia was a diploid population (2n=22), Küpfer observed two populations on the other massif (Sierra Nevada), among which one was described as tetraploid (Küpfer 1974). On the other hand, this result agrees with the counts previously detected in east-central Spain (Teruel) and the Province of Cordoue in Andalusia (Peniarroya) (Küpfer 1974).

Our chromosome count of 2n=44 for populations from the Spanish Pyrenees (Huesca; Ceresa altitude: 1711 m) also strongly supported an earlier report on A. grandiflora ssp grandiflora (Favarger 1959(Favarger . 1962) in the Spanish Pyrenees, also located in Huesca, at an altitude of 1.600 m. Furthermore, the population from both the Alps and the pre-Alps (Mt Lure, Mt Raton) showed 2n=44 as well, which is in accordance with another tetraploid population from Mont Ventoux in the same massif (Monts de Vaucluse -Alps) (Küpfer 1974). Our identification of the chromosome number (2n=44) is unprecedented for all the investigated populations from both the Alps and the pre-Alps.

Because most tetraploid individuals that we identified grew at altitude, we can assume that polyploidy has occurred and spread in regions with extreme climatic conditions, such as alpine zones, as polyploidy may increase adaptability to unfavourable habitats (Flovik 1940).

In accordance with an earlier report on the chromosome counts (2n=44) and sizes of Majorcan mountain-endemic A. grandiflora subsp. glabrescens in the Balearic Islands [START_REF] Castro | New chromosome numbers for plant taxa endemic to the Balearic Islands[END_REF]. The metaphase chromosomes of the populations investigated in the present study were relatively small (1-2 µm) and morphologically similar.

Intraspecific variation in genome size

To our knowledge and according to the available databases, our results regarding 2C DNA values are novel for the A. grandiflora species complex in Western Europe. This study identified intraspecific variation in genome size among 21 populations of the species complex of A. grandiflora from its natural distribution ranges for the first time. Results of ANOVA--In general, the variation of 1Cx values may be explained by the potential for adaptation to different environmental conditions, including environmental extremes at high latitudes and altitudes and harsh climatic conditions (Comai 2005;[START_REF] Ramsey | Polyploidy and ecological adaptation in wild yarrow[END_REF][START_REF] Brochmann | Polyploidy in arctic plants[END_REF]). In the samples of the species complex of A. grandiflora, the higher ploidy levels showed a positive correlation with altitude. This correlation was noted previously by [START_REF] Löve | The significance of differences in the distribution of diploids and polyploids[END_REF] and Sharma & Sharma (1958). The findings showed that the genome size of the Mt Cheval Blanc population (the highest population) was significantly different from those found at Beaumont-en-Véron near the city of Chinon (the lowest population, at 35 m)

and Mt Raton (an intermediate population, at 1.473 m). These results suggested that local adaptation may be related to genome enlargement and duplication [START_REF] Ramsey | Polyploidy and ecological adaptation in wild yarrow[END_REF][START_REF] García-Fernández | Ploidy level and genome size of locally adapted populations of Silene ciliata across an altitudinal gradient[END_REF]). However, genome downsizing is a widespread phenomenon in polyploids [START_REF] Leitch | The ups and downs of genome size evolution in polyploid species of nicotiana (Solanaceae)[END_REF]) and our findings highlighted that this phenomenon is not observed in the tetraploid populations in the Pre-Alps (Chaudière 1.560 m and Mt Raton 1.473 m) or Spanish Pyrenees (Gabas 1830 m). The largest monoploid genome size was found in the diploid population from the southeast of France (Pas de l'Ai) growing at 1.034 m.

According to [START_REF] Youssef | Factors underlying the narrow distribution of the Mediterranean annual plant Arenaria provincialis (Caryophyllaceae)[END_REF], this population exhibits a narrower and more marginal niche than other species of Arenaria. Moreover, the Sainte Baume mountain chain has a Mediterranean climate. In this case, it might be assumed that the increased monoploid genome size could be correlated with the climatic conditions of Mediterranean mountains, which often present stressful environments (high temperatures or serious drought conditions in summer (Grime & Mowforth 1982;Giménez-Benavides et al. 2008;2007)).

However, two diploid populations were also located at high altitudes of the Mt Sagra in Andalusia ( 2279m) and the Spanish Pyrenees (the Vilas del Turbon 1720 m) and displayed larger monoploid genome sizes compared with other diploid populations. It was also found that the population from Plan in the Spanish Pyrenees (1720 m) presented a large monoploid genome size compared with other diploid populations from plains and small hills. At the genomic level, the intraspecific variation in genome size is usually caused by differences in the content of noncoding repetitive DNA, such as transposable elements (e.g., via the activity of retrotransposons) [START_REF] Smarda | Intrapopulation genome size dynamics in Festuca pallens[END_REF], satellite DNA, introns, and pseudogenes (Bennett & Leitch 2005;

Smarda & Bures 2010)

Intraspecific polyploidy and conservation

The study of intraspecific genome size variation can reveal inter-and even intra-populational variations in ploidy [START_REF] Siljak-Yakovlev | Towards a genome size and chromosome number database of balkan flora: C-values in 343 taxa with novel values for 242[END_REF]. The phenomenon of intraspecific ploidy variation is prevalent (Keeler 2004;[START_REF] Suda | Genome size variation and species relationships in hieracium sub-genus Pilosella (Asteraceae) as inferred by flow cytometry[END_REF]Duchoslav et al. 2010;[START_REF] Realini | Intraspecific variation of genome size in maize: cytological and phenotypic correlates[END_REF], and its frequency has recently been estimated to be 12-13% for angiospermes [START_REF] Wood | The frequency of polyploid speciation in vascular plants[END_REF]). However, less attention has been paid to this phenomenon when conservation priorities are being set and it remains unaddressed in plant conservation

CONCLUSION

In this report, we show that different techniques can be used to estimate ploidy levels in threatened and rare species complex of A. grandiflora and can contribute to improved definition of restoration plans. Among these techniques, flow cytometry can be a useful tool. Small amounts of leaf tissue, cotyledons of germinated seedlings, dried seeds, or stored leaves in silica gel are sufficient for the estimation of ploidy level and nuclear genome size. Because of the difficulty of applying classical karyological techniques in the A.

grandiflora species complex due to seed dormancy, crystals of oxalate of calcium that occur in the roots or the presence of endophytic fungi in seeds, the technique of flow cytometry was found to be very helpful for the rapid estimation of ploidy levels in this study and may be a powerful alternative karyological approach for threatened species. The use of microsatellite markers provided an additional opportunity to obtain information on genetic diversity, which can also aid in the definition of conservation priorities (Loeschcke et al. 1994).

The co-occurrence of diploid-tetraploid populations of A. grandiflora species complex in the Spanish Pyrenees leads to many questions regarding the origins of polyploid populations. The most interesting finding emerging from this study was the presence of populations with different ploidy levels, even within the same subspecies of the species complex of A. grandiflora. Favarger (1959Favarger ( . 1962)), Küpfer (1974); N -number of investigated individuals 1 pg=978 Mbp according (Doležel et al. 2003)

ACKNOWLEDEMENT

Chapter III:

Germination patterns within the species complex of Arenaria grandiflora L.

Introduction to Chapter III

As reported in the first paper, we had many difficulties in seed germination of Arenaria grandiflora species complex certainly because of seed dormancy and presence of endophytic fungi in the used seeds.

In addition, according to the literature, there was a lack of knowledge on seed germination and no adequate or valid protocol to germinate the seeds of A. grandiflora complex existed. Furthermore, while implementing our different studies (such as intraspecific DNA content variation) at ESE in University of Paris-Sud Saclay, we had frequently needed young roots and seedlings of A. grandiflora species complex. Consequently, there was a challenge to find the most appropriate protocol to overcome difficulties of seed germination of A. grandiflora species complex. Therefore, we reviewed many currently available data about seed germination for other species of the genus of Arenaria and its close genera as Minuartia and Moehringia and then, we tried different conditions of storage and germination protocols, on different genotypes collected in natural populations.

Because the results varied greatly from one population to the other but also with the protocols, we took advantage to these experiments to determine the ecological and genetic factors that could have influenced the germination capacity of the seeds.

Introduction

Ex-situ seed storage is one of the best procedures to maintain large pools of plant genetic resources (Satterthwaite et al. 2007;Coppi et al. 2015). Defining the storage conditions and periods is essential to preserve the seed viability during long periods (Leino & Edqvist 2010) (Onyekwelu et al. 2007).

Actually, the seeds of many plant species are extremely tolerant to harsh environmental conditions provided they are in a state of desiccation (Hay and Probert 2013). In this dry state, their metabolic activity is drastically reduced to a very low level (quiescence) while retaining their ability to germinate for considerable periods; e.g. 2000 years for date seeds (Phoenix dactylifera L.) recovered from a Herodian fortress near the Dead Sea (Rajjou & Debeaujon 2008) and 151 years for Acacia Mill. spp.

seeds from Egypt stored in Swedish museums (Leino and Edqvist 2010).

The reinforcement of wild populations using stored seed material may be a valuable tool for conserving endemic and threatened species (Giménez-Benavides et al. 2005;Coppi et al. 2015).

Likewise, the study of seeds germination of protected flora species is vital for the success of ecological restoration projects (Bischoff et al. 2006). For twenty years, the preservation of a species very rare and threatened in lowlands (for Arenaria grandiflora in Fontainebleau forest) was conducted. This plant is more common but progressively disappearing in western mountains of Europe [START_REF] Bottin | Re-establishment trials in endangered plants: a review and the example of Arenaria grandiflora, a species on the brink of extinction in the Parisian region (France)[END_REF]).

Concerning the germination of this species complex, information is lacking and up to now, no studies were conducted on the seed germination of A. grandiflora. For different studies, such as the impact of climatic changes (Fontaine et al., submitted) and intraspecific DNA content variability in species complex of A. grandiflora (Daoud et al., submitted), young roots and seedlings are needed. Therefore, while carrying out these studies in our laboratory, we have made many attempts relying on many and previous protocols in the germination of seeds for other species of the genus of Arenaria and its close genres as Minuartia and Moehringia. For example, the seeds of some species of Arenaria do not have a light requirement for germination like as A. meyeri (Boiss) Edgew and Hook f and A. serpyllifolia L., according to King (1975) and Ronnenberg et al. (2007). On the other hand, the germination of other species such as A. glabra Michx and A. fontinalis (Short and Peter) is controlled by both the temperature and availability of water (Baskin & Baskin 1982, 1987). Several authors showed the impact that period of seed storage had on the germination percentage; for example, Ronnenberg et al. (2007) found that the germination of fresh seeds was significantly higher in A. meyeri. Also, (Baskin & Baskin 1987) revealed that seeds of A. fontinalis did not germinate after ripening when stored in dry at ambient laboratory conditions.

It is also well-known that dormancy-breaking and germination requirements are specific for each species and depend on geographic distribution, habitat and life cycle (Vandelook et al. 2009;Copete et al. 2011;Zharare 2012). In general, studies can show variation in germination responses between populations of one single species [START_REF] Lord | Variation in Festuca-Novae-Zelandiae (Hack) Cockayne Germination Behavior with Altitude of Seed Source[END_REF]Baskin et al. 1998;Hernández Verdugo et al. 2001;Cochrane et al. 2014). The altitude and latitude of the seed source, population size and abiotic factors like substrate type or climate are generally regarded as important factors that affect seed germination percentages (Menges 1991;[START_REF] Lord | Variation in Festuca-Novae-Zelandiae (Hack) Cockayne Germination Behavior with Altitude of Seed Source[END_REF]Keller & Kollmann 1999;Vandelook et al. 2009;Wang et al. 2012;Rosbakh & Poschlod 2015).

The species complex of A. grandiflora occurs as diploid and tetraploid cytotypes in its native range in Western Europe. The case of polyploidy is also interesting because it can induce the changes in seed characteristics and may have important consequences on their germination response as mentioned by (Tyler et al. 1978;Thomas et al. 1994;Hoya et al. 2007;Hahn et al. 2013).

In this paper, we report germination experiments executed on seeds collected in Spanish and

French natural populations of the species complex of A. grandiflora. The purposes of this study were:

(a) to define the methodology which allows A. grandiflora seed germination; b) to assess, if it exists, a variation of germination rates of seeds according to their geographical origin (altitude, latitude and longitude), period and condition (temperature) of seed storage and their ploidy levels.

Material and Methods:

Study Species

The large-flowered sandwort (A. grandiflora) is a perennial plant that is cushion-shaped with woody and ascending stems that can reach a height of 15 centimeters. This small Caryophyllaceae is a Pyrenean-Alpine species that grows on calcareous and sedimentary rocks, xerophytic and oro-basophils lawns, and schist screes (Küpfer 1974). In general, it blooms from May to August. The species complex of A.

grandiflora is insect pollinated but is also able to self-reproduce in the absence of pollinators (Machon et al. 1999[START_REF] Machon | Comment sauver Arenaria grandiflora L. de la depression?[END_REF]). The mating system of A. grandiflora is probably predominantly outcrossing, although vegetative reproduction is also possible [START_REF] Bottin | Re-establishment trials in endangered plants: a review and the example of Arenaria grandiflora, a species on the brink of extinction in the Parisian region (France)[END_REF]). The lowland populations of A. grandiflora in the Puys of Chinonais and in the Fontainebleau forest have been protected since 1991 when a restoration program was conducted by the Botanical Conservatory in Ile-de-France. According to field observations, the natural populations of the species complex of A. grandiflora in the Fontainebleau forest got probably extinct (fully) in 2014.

Origin of material

The seeds used for the experiments of germination were collected from eleven populations of A.

grandiflora complex in Western Europe. These populations are representing the natural distribution of the species in Western Europe (Mt Merle, Chinon, Rocamadour, Mt Lure, Mt Raton, Mt Chaudière, Mt Cheval Blanc, Mt Rié, Mt Sagra, Plan and Ceresa). Table [START_REF]Évolution, Biodiversité (ISYEB[END_REF] shows each studied population with indications of altitude, latitude, longitude, collection year and site, both condition and period of seeds storage and ploidy levels. The size of the collected seed samples was variable depending on the population size Table 2.

Conditions (Temperature) and periods of seed storage

Most of the seed samples were kept in paper bags and stored in a dry place at room temperature, during three months for the populations of Chinon, Plan and Ceresa. However, the seeds from Mt Raton and Mt Chaudière populations that were stored during 84 and 96 months, respectively.

Except for the Mt Merle population, the seeds were kept in paper bags at room temperature (for 2 years), then they were conserved in a refrigerator at (-4 °C) during seven years in CNPMAI (Conservatoire National des Plantes à Parfum, Médicinales, aromatiques et Industrielles). In addition, for the seeds of Mt Cheval Blanc population were kept in paper bags and stored in a cold room at + 4 °C at 40% of humidity in CBNA (Conservatoire botanique national alpin) during 300 months (see Table 1).

Test of germination methods

Three germination methods were used: (i) seed germination in pots, (ii) seed germination on filter paper and (iii) in vitro culture with scarified seeds. These three methods are detailed below.

1) Test of germination in pots

The experiment of germination on potting soil was preliminary attempt to germinate the seeds of A.

grandiflora, according to a protocol which was inspirited of Meta-analysis for the genre Arenaria and its allied genera Minuartia and Moehringia which were already reported in the literature and in previous studies. Considering the results of this Meta-analysis, we started these experiments with four French populations of A. grandiflora (Table 1): Mt Raton, Mt Chaudière, Mt Rié, and Chinon.

Seeds were planted on potting soil in terrine at a depth of 0.5cm with a spacing of 2 cm between each seed, 150 seeds per terrine. The number of seeds tested could vary according to the quantity of seed available. The seeds were kept on the moist soil, continuously. The terrines were placed in a seedbed under controlled conditions of temperature and light: 22°C (light 14h) + 15°C (dark 10h). The temperature was measured using Tinytag Temperature Data Logger. Data were recorded every 10 min.

Three steps were performed:

(i) Media preparation

We prepared four culture media: two of these media were GMC6 and GMC610 containing the macronutrients of Estelle and Somerville (1987) modified as follow 2.5 mMol KH2PO4 were replaced by 2 mM KH2PO4 and 0.5 mM K2HPO4 4H2O. So, the medium was directly adjusted to pH=6. The mineral nutrient solution contained per liter 5mM KNO3; 1.85 mM MgSO4 7H2O; 1mM Ca(NO3)2 4H2O; 0.1 mM Fe-EDTA; 1mM NaCl Half concentration of the mineral micronutrients of Murasnige and Skoog (1962) was added 2g/l gelrite and 4g/l of agar was used as gelling agents before autoclaving for 30 minutes at 115°C. 10 g/l of sucrose (about 30mM) was only added to the GMC610 medium. The author two media were prepared one with sugar (GH0) and one without sugar (GH10), contained the macronutrients (MCC2) modified according to (Guillon and Raquin 2002). The modification of the MS micronutrient solution just consisted in the addition of 0.01mM of Na3VO4 (sodium vanadate). The composition of the solution of macronutrients (MCC2) was as the following per liter: 40 mM NH4NO3; 100 mM KNO3; 30 mM MgSO4; 10 mM Mg(NO3)2; 30mM Ca(NO3)2. The four media were placed in 55mm diameter Petri dishes and they were left to dry a few hours. They were then stored at room temperature in closed boxes to limit evaporation of the medium. To detect the possible problem of contaminations we waited a few days before using them.

(ii) Surface sterilization

Because there was a possibility of infections caused by fungus or bacteria present over the coating of the collected seeds from the field. The seeds were treated with a solution containing calcium hypochlorite (Ca (ClO)2 1 2% w/v) and sodium hydroxide (NaOH 0 4% w/v). About 15-20 small seeds were put in each Eppendorf 2ml with about 1.7 ml of the solution of sterilization. They were first centrifuged for 1 minute to eliminate air bubbles on the surface of the seeds then they were kept 20 min in the solution at room temperature. The hypochlorite was removed by a transfer pipette. Because the seeds were fragile they were rinsed with the same solution diluted 1/10. The disinfected seeds were placed on germination media in Petri dishes and were left during six days to ensure that there were no contaminations observed.

(iii) Scarification and seeds germination Mechanical scarification was done by using a scarifier to cut the seed coat of each seed under the Enlarger. The surface-scarified seeds were placed on germination media in Petri dishes and incubated in a growth chamber of 23°C± 2. According to the availability of the seeds (Table 2), 12-15 Petri dishes containing different types of agar media, for each population, each one including the seeds (7-20 per dish) were used in the seed germination experiments. The seeds were considered germinated when the root tip was visible (protruded 1 mm above the seed coat). In each run, the number of germinated seeds was recorded during one month (30 days), because almost no further germination occurred afterwards.

Here, we tested the influences of three factors on germination percentages: (a) the seed source (provenances of seeds), (b) the four media of culture in vitro and (c) the ploidy levels.

Statistical analyses

 In general, for each trial

The final germination percentage (GP) of sown seeds was defined as the proportion of germinated seeds on the total number. Germination percentage is the fraction of seeds germinated in each Petri-dish after 30 to 50 days of incubation. To test the impact of the different methodologies used on the GP, we used the non-parametric test of Kruskal-Wallis coupled to Wilcoxon tests to explore the differences of germination rates among germination methods.

 For the germination experiments in pots

All statistical tests were performed using R version 2 software (R Development Core Team 2016).

Germination percentage (GP) in each Petri-dish was calculated after 50 days of incubation. To test the impacts of both the seed sources (seed provenances) and the seed type ("Normal" vs "Deformed") on GP, we used Chi 2 Pearson test. A Tukey's honest significant test (HSD), for equal sampled population sizes, was used to explore the differences of germination rates among seed sources (provenances of seeds) with mixed seeds ("Normal" and "Deformed"). The significance level for all statistical tests was p-value <0.05.

 For the germination experiments in filter paper

No statistical tests were performed because all the results of the germination percentages were zero.

 For the germination experiments, in vitro culture (with scarification)

All statistical tests were performed using SPSS (IBM SPSS Statistics version 24). Germination percentage (GP) in each Petri-dish was calculated after 30 days of incubation. To test the effects of each one of these following factors on the germination percentage: the seed sources (seed provenances), their four media of culture (GH0, GH10, GMC6 and GMC610) in vitro and their ploidy levels, we used oneway ANOVAs.

The significant variance in germination percentages among the seed sources (provenances of seeds) was investigated by an ANOVA with Welch test, for unequal sampled population sizes. Also, a test of multiple comparisons (Games-Howell test), for unequal sampled population sizes, was conducted to demonstrate all pairwise comparisons of the means for the seed sources (provenances of seeds). The significance level for all statistical tests was p-value <0.05.

To characterize, as much as possible, the optimal germination conditions, we excluded all GP < 10% from the whole data, because of the probability that the seeds were dead or immature was too high.

Then we applied statistical analyses on the new data, like following:

 For germinated experiments in all trials (GP>10 %)

 Impact of some parameters on GP For the populations having significantly germinated (GP> 10 %) in all the trials, we tested the significant effects of each one of the following factors: the geographical parameters (altitude, latitude and longitude) of the collected seeds provenances, their ploidy levels and the period and temperature of seed storage on the germination percentage, by using an ANOVA with Welch test. To demonstrate all pairwise comparisons of the means of GP, a test of Multiple Comparisons (Games-Howell test) was conducted. The significance level for all statistical tests was p-value <0.05.

 Modeling (linear regression)

To model the relationships among six explanatory variables (predictors) and a response variable of germination (different values of GP), we used two methods: simple and multiple linear regression analyses. The six predictors were latitude, longitude, altitude, ploidy levels and both period and condition of seeds storage. The multiple linear regression analysis was fitted by a linear equation y = a + b X [START_REF]Évolution, Biodiversité (ISYEB[END_REF](2)(3)(4)(5)(6). To compare the models, a standard error of estimation and both associated r and r 2 and a P value were given for each suggested model. All regressions were considered significant at P < 0.05. A regression analysis was performed also by SPSS (IBM SPSS Statistics version 24).

Results

Methodology

The three methods under which seeds were sown had a significant impact on GP (Kruskal-Wallis test; p < 0.0001). For the method of seed germination on filter papers, no germination was obtained and fungal contamination was observed for all the seed provenances. Both the method of seed germination in potting soil and the method of seed germination in scarification in vitro culture (with pretreatment by scarification) were significantly better than the method of seed germination in filter papers (Wicoxon tests p < 0.0001). Even if, the GP greatly varied according to the tested populations, from 0.4% to 72 % (for method of potting soil) and from 0% to 83 % (for method of in vitro culture) (see Table 1). For populations whose seeds were not contaminated with fungi, the method of in vitro culture was the one that yielded the best percentages. So, the two populations located in the mountains, Mt Raton and the Mt Chaudière, had have a very low germination rates (1.1% to 4%) with the method of potting soil, but had have a good GP with the method of in vitro culture.

Germination in potting soil

In our experiments, the seed GP varied considerably from 0% to around 74 % (see Fig. 1A). The seed provenances had a significant impact on the percentage of germination (Chi 2 Pearson test. P = 0.023).

On the contrary, seed type ("Normal" vs "Deformed") had no influence on GP (Chi 2 Pearson test. p > 0 05) for the four studied sites (Fig. 1A). So, we used mixed seeds for the rest of the analyses. There was a variation among the seed provenances (with mixed seeds) (F = 278.234; P = 0.000). In other words, the seeds harvested from the two locations of the alpine mountains, Mt Raton and the Mt Chaudière showed a very low germination rate (1.1% to 4%). Moreover, seeds from Mt Rié (French Pyrenees) had a good germination percentage. 50.3%; but these germination percentages were lower than that of the seeds from the Puy Chinonnais (plains) (71.58%) (see Fig. 1B). The Chinon population differed significantly in the germination percentage compared with both Mt Raton and Mt Chaudière population and Mt Rié population (P = 0.000). There were no significant differences between alpine populations (Mt Raton and Mt Chaudière) (P > 0.05).

Germination percentage in all trials (GP > 10 %)

 Impacts of intrinsic and environmental parameters

The factors like the geographical parameters (altitude, latitude and longitude) (F=9.306; P= 0.000 for each one out three of these factors) induced significant differences among the populations, as well as the polyploidy seemed to have a significant impact on the germination rate (F=12.434; P=0.001) (see Fig. 3).  Impacts of conditions (temperature) and periods of seed storage

The periods and conditions of seed storage had a significant impact on the GP (F=4.129, P=0.004; F=11.743, P=0.001 respectively). Furthermore, the seeds from Mt Cheval Blanc population which were stored for 300 months germinated differently and significantly compared with each of Mt Raton and Mt

Chaudière populations (that were stored for 84 and 96 months respectively). In addition, all the seeds of populations, that were stored three months (Chinon, Plan and Ceresa) germinated differently and significantly compared to seeds of Mt Cheval Blanc population.

 Modeling

We tested six predictor variables in the regression analysis. Every value of each one of the independent variables x (1-6) is associated with a value of the dependent variable (GP) y. So, the value of the GP changed when each of the predictor variables (altitude, longitude, latitude, ploidy levels, both period and condition of seed storage) varied, while the others were fixed. We defined the regression line for 6 explanatory variables x [START_REF]Évolution, Biodiversité (ISYEB[END_REF](2)(3)(4)(5)(6) to be y= (-328.238) + (-4.355) *ploidy levels + (0.041) *altitude + (8.913) *latitude + (-4.431) *longitude+ (0.117) *period of seeds storage + (-32.758) *condition of seeds storage. The best model among all models which had the upper value of r = 0.633 and the lowest value of the standard error of estimation =16.43872, this model that was more explicit by its six predictors (altitude, longitude and latitude, ploidy levels, both period and condition of seed storage) (see Table 3).

There was a good significant impact of both period and condition of seeds storage on the final germination percentage (r = 0.350, P = 0.000; r = 0.326, P = 0.000 respectively). Furthermore, the germination percentages of seeds had been equally and well affected by ploidy levels and altitude (r = 0.334, P = 0.000), the tetraploids germinated better than diploids. However, both altitude and longitude of seed provenances seemed to have influences on the final germination percentage (r = 0.334, P = 0.000; r = 0.208, P = 0.018 respectively), the latitude had no effect on the GP (r = 0.165. p >0.05).

Discussion

Methodology of seed germination of Arenaria grandiflora

While seeds of some species of Arenaria germinate easily without any special treatment, others are more difficult. For example, seeds of A. meyeri Fenzl and A. serpyllifolia L., according to (King 1975) and Ronnenberg et al. (2007) do not need light for germination. Reliable technics for achieving in vitro seed germination with pre-treatment (scarification) in the complex of Arenaria grandiflora is documented in the present study. Our results showed that a scarification is required for a successful in vitro seed germination of the tetraploids of A. grandiflora species complex. The mechanical scarification allowed water to penetrate the tough outer coating to start the germination process. Indeed, we found that the mechanical scarification produced higher germination percentage for most of the tetraploid populations.

Thus, we could consider that the scarification of the seeds and then the use of in vitro culture technics could be a great method for both overcoming the inhibition of germination caused by seedcovering and multiplication the tetraploids of Arenaria grandiflora complex. For other species, these technics initiated from seeds of orchids has been widely used at Kew (Muir 1989). Moreover, many carnivorous plants have been propagated in vitro from seeds (Vasile et al. 2011;Fay 1992;[START_REF] Holobiuc | in Vitro Culture Introduction for Exsitu Conservation of Some Rare Plant Species[END_REF]. Furthermore, Ghosh and Maiti (2014) indicated that both of the in vitro germination of seeds and the shortening of their dormancy period for the Abrus precatorius L. species are possible with the different method of seeds scarification treatment. However, we recommend these technics for the tetraploids of A. grandiflora species complex. The diploid populations could better germinate with another method, clearly simple.

Environmental effects and germination

In the present study, the seed collection sites differed widely in elevation (from 87 to 2280 m) and in longitude (from 0.12 to 6.20 E°) and the germination percentage correlated with the altitude and longitude of the seed sources. These findings agreed with the altitudinal and interpopulation variability in high mountain Mediterranean species that had reported by (Giménez-Benavides et al. 2005). These factors are likely to be associated with differentiation among populations because they represent the different environments under which the seeds were produced and matured (Cavieres and Arroyo 2000).

The seed germination of populations from different environments may have conducted different responses as it was found for Banksia species that were collected from a longitudinal climate gradient in Western Australia (Cochrane et al. 2014). Thus, the differences could arise from inherited environmental effects, "environmental preconditioning" or "maternal environmental effect" (Luzuriaga et al. 2005;Hermesh and Acharya 1992;Galloway 2001). These sources of variation are widespread in plants and could be the product of both the parental genes and the parental environment, or their interaction, and can include contributions that reflect the abiotic, nutritional and other ecological features of a parental environment (He et al. 2014;Rossiter 1996). The findings suggest that variability in germination percentages could reflect a local adaptive strategy to unpredictable environmental conditions (Gutterman 1994).

However, seeds collected at higher altitudes reached higher germination percentage than those from lower elevations (Giménez-Benavides et al. 2005), the results present that the GP of seeds from both similar and higher altitudinal populations significantly differed among themselves. For instance, the GP of seeds from the Mediterranean mountains (Mt Sagra at 2279 m) differed significantly from seeds of the alpine mountains (Mt Cheval Blanc at 2280 m). Furthermore, the type of climate, especially seasonal changes, could be the most obvious pre-harvest factor affecting seed viability and storability, because the different pre-harvest conditions will have caused different amounts of deterioration by the time (Justice and Bass 1978). Thus, we could interpret these differences by the type of climate in which each population grows. According to Köppen classification based on precipitation and temperatures, the climate of Mt Cheval Blanc population (Cfb) is temperate and humid, the precipitation occurs every month of the year, there is no dry season and the summer is temperate with warmest month temperature <22° C, when the climate of Mt Sagra (Csa) is temperate, but the summer is hot and dry with low precipitation (<40 mm) and the mean temperature of the warmest month is > 22 °C.

The genetic variation and the germination percentage

Another possible source of differentiation among populations is the ploidy level of the individuals. As found in another study (e.g. Daoud et al. submitted), Arenaria grandiflora can have tetraploid or diploid populations. Our results showed that the germination rate was generally higher for the tetraploids than for the diploids (at the exception of the population of Plan). A similar phenomenon occurs in Matricaria perforata Mérat (Thomas et al. 1994). The whole genome duplication events may provide a subfunctionalization of duplicate genes. So, it has been hypothesized that polyploidy could induce small changes in the characteristics of seeds that may induce a variation in germination ability. Thus, the differences in germination rate (Fig. 2) among populations may have been also attributed to genotypic differences among these populations.

Years' seed and conditions storage effects

Our findings demonstrated that there were also significant effects of storage conditions (papers bags in room temperature and +4°C at 40% humidity) and different periods of seed storage on the germination rates. These findings agree with the high effects of the storage conditions (room temperature, +4°C, and -15°C) on seed germination percentage in Swertia chirata by (Pradhan & Badola 2012). Our study indicated that the storage at +4°C temperature and 40% humidity (Mt Cheval Blanc population) can retain seed viability for the longer period than the storage at -4°C temperature (Mt Merle population). Also, our results are in agreement with mentioned results by (Andersson and Milberg 1998) which showed that the germination of four species (Sinapis arvensis L., Silene noctiflora L., Spergula arvensis L. and Thlaspi arvense L.) differed between seeds collected in different years.

However, these results did not agree with results of germination of the perennial widespread Arenaria meyeri (Ronnenberg et al. 2007) in the mountain steppes of southern Mongolia where the germination of fresh seeds (100%) was significantly higher than dry seeds (91.5%). In addition, Baskin and Baskin (1987) revealed that seeds did not germinate after ripening when stored in dry at ambient laboratory conditions. Other alpine species of Minuartia Loefl which is a genus closed to Arenaria. as e.g. M.

obtusiloba (Rydb.) House and M. rubella (Wahlenb.) Some of the alpine species exhibited a general tendency toward improved germinability with years' seed storage according to (Bonde 1965).

Seed germination and dormancy

Generally, differential seed dormancy mechanisms arise among plant provenances (Andersson and Milberg 1998) due to adaptations to local environmental (Zharare 2012;Huang et al. 2015). Our results could suggest that the seeds of A. grandiflora of the mountain populations (alpines and sup-alpines populations) have a physical dormancy. So, the seed coat inhibition is probably the most common cause of alpine seed dormancy as mentioned by (Amen 1966). Moreover, other species of Arenaria showed some degrees of dormancy like (Arenaria serpyllifolia L.) (Pons 1991) and Arenaria nevadensis Boiss.

& Reuter (Lorite et al. 2007). The range of suitable conditions for the germination of the dormant seeds is restricted. Seed dormancy in A. grandiflora had been a major problem for our previous cytogenetic research. The results of this study showed that the mechanical scarification could remove the physical barriers due to water-impermeable seed coat (Baskin & Baskin 2004;Chaves et al. 2017;Amen 1966) and thus, highlight the benefit of in vitro culture technics for species with dormant seeds (Fay 1992).

Moreover, the mechanical scarification which induced the highest percentage of germination in our

Arenarias is known to break physical dormancy of hard seed coats especially in Acacia polyacantha

Willd (Missanjo et al. 2014) and in Faidherbia albida (Fredrick et al. 2016).

Conclusions

Our results revealed that seed dormancy in some populations of A. grandiflora complex is mainly due to the hard seed coat, which can be broken effectively by physical scarification. This study of seed germination of an endangered flora species (A. grandiflora) is crucial and vital for its future conservation strategies and provides an overall view and basic information needed for genetic resources conservation.

The present study stressed 1) the variability in seed germination in eleven populations collected from different altitudes in Western Europe that were stored in two diverse conditions for various periods.

2) the high seed germination percentages found for the tetraploids of A. grandiflora complex pretreated with mechanical scarification and cultivated in vitro.

Considering the significant differences in germination rates observed among populations, the question of the provenance of seeds to be reintroduced in situ should be resolved in choosing the seeds with the best GP. However, the local seed provenance is often used and recommended in restoration because seeds are expected to be better adapted to local habitat conditions, and no effect of outbreeding depression is expected. The use of non-local seed provenance might be also interesting when the aim is to introduce both diversity and seeds with better germination capacities. For example, introducing Chinon' individuals whose seeds well germinate, in the Fontainebleau sites where plants hardly germinated was certainly one of the factors that led to the success of the restoration plan (Maurice et al. 2013).

gynandra and two Amaranthus species. African Journal of Agricultural Reseearch, 7(36), pp.5049-5059. [START_REF] Mitteroecker | A brief review of shape, form, and allometry in geometric morphometrics, with applications to human facial morphology[END_REF], "the shape of an object is the geometric properties that are invariant to translation, rotation, and scaling". Studies on plant species identification using morphometrics (Conesa et al. 2012;[START_REF] Cope | Plant species identification using digital morphometrics: A review[END_REF]Viscosi and Cardini 2011;[START_REF] Viscosi | Leaf Shape and Size Differentiation in White Oaks: Assessment of Allometric Relationships among Three Sympatric Species and Their Hybrids[END_REF] have shown that, among other traits, leaf shape is of great significance to help distinguish between different taxa. Both leaf shape and leaf dimensions differ within species as in Atriplex canescens (Pursh)

Nutt (Dunford 1985) and in Buddleja macrostachya Wallich ex Benth (Chen et al. 2009). In addition, polyploidy could be one of the features that influenced the most vegetative morphological features [START_REF] Chansler | The role of polyploidy in shaping morphological diversity in natural populations of Phlox amabilis[END_REF][START_REF] Padoan | Ploidy levels in Citrus clementine affects leaf morphology , stomatal density and water content[END_REF]. For example, the polyploidization is sometimes associated with differences in both leaf shape and elongation (Van Laere et al. 2011;[START_REF] Sun | Variation of phenotype, ploidy level, and organogenic potential of in vitro regenerated polyploids of Pyrus communis[END_REF]. As a further matter, the leaf shape depends on the interaction between genetic features of the individuals and the environments in which they grow (Dkhar and Pareek 2014). Nevertheless, because the influence of environment could disturb the genotype/phenotype relationships, we wonder how is leaf shape variation distributed in a species complex comprising plants from different ploidy levels and growing in different habitats.

We performed a morphometric study on the Arenaria grandiflora species complex that is characterized by two cytotypes. Individuals can be diploid or tetraploid, with a basic number x = 11 (see more Daoud et al. submitted;Favarger 1959;Küpfer 1974) and grow at different altitudes in western Europe.

According to the Flora of France (Coste 1937), plants in the A. grandiflora species complex are organized into perennial caespitose tuft with woody rootstocks, but the stems are herbaceous, short, erected or ascendant, and usually about ten cm high. The leaves are coriaceous (stiff and tough, but somewhat flexible), opposite and sessile, glabrous or glabrescent, with an entire and thick margin. They are lanceolate-linear, oblong-lanceolate or ovate-orbicular. The leaf apex is often subulate (e.g. more than 3:1 L/w), usually prolonged, straight and stiff. The leaf is characterized by a very prominent middle rib.

The populations of plants belonging to the A. grandiflora species complex occur in disjunctive areas of Europe. In addition, the splitting of this species complex into discrete entities at the sub-specific level is controversial, because of the different numbers of subspecies that were mentioned in different databases. For example, three, five or seven accepted subspecies according to Flora Europaea, the Global Biodiversity Information Facility (GBIF; international open data infrastructure) and to the International Plant Names Index, respectively. As demonstrated by Daoud et al. (submitted) the patterns of genetic variation among populations of different ploidy levels discriminate among the studied populations as presented in Fig. 1. For instance, the populations from the plains are predominantly diploids, and the populations of high altitude are mostly tetraploids.

Morphometric methods in both plants and animals are powerful tools to reveal differences in organismal shapes. We used the collection of A. grandiflora species complex from the French National Herbarium (acronym P), consisting of 610 specimens, some being more than 200 years old. We described the variation in leaf shape using a morphometric approach. The main objective was to identify both the leaf shape and elongation that could discriminate the sampled specimens belonging to different cytotypes and collected from different habitats.

Materials and Methods

Sampling and data collection

Forty-two herbarium specimens from the A. grandiflora species complex from Western Europe were used. Seventeen and 25 specimens were collected from populations that had been formerly described as diploid or tetraploid (see Daoud et al. submitted;Favarger 1959 andKüpfer 1974, for details). A map of the collection sites was drawn using ArcGIS (Fig. 1). For each specimen, we sampled five leaves from the central part of the individual. Measurements were hence made on a total of 210 leaves. We used simmering water to hydrate the dry leaves. We glued them on microscope slides (10 leaves [i.e.

two specimens] on each slide) with white glue (Fig. 2). The microscope slides were numbered and the correspondence between these numbers and the specimen barcodes is given in Table 1. We put all the microscope slides on an inverted scanner (HerbScan) and then took a digital 1200 DPI image of the set of leaves (Fig. 2a). Morphological linear measurements of the sampled leaves were made on the resulting picture using tpsDig version 2.26 (Rohlf 2015).

Leaf measurements

The following leaf measurements were performed in microns (Fig. 2b): the overall leaf length (L) and the leaf width at three levels dividing the leaf into three equally long segments (Fig. 2b). Thus, the raw linear measurements of the leaves composed the raw dataset.

Shape parameters: Log-Shape Ratio (LSR)

LSR (Mosimann 1970) is one of the methods which is used to compare shape parameters amonggeographically distributed populations while removing size by partitioning the form into size and shape and to provide study differences in shape proportions. First, the isometric size was computed as a geometric mean of all logarithmic measurements by specimen for each leaf, then each measurement was divided by isometric size to obtain the LSR.

Leaf elongation

Based on the relation with leaf elongation (leaf length to maximal leaf width (L / W2)), three leaf types were defined: acicular: L / W2 > 12:1; linear: ratio between 12:1 and 6:1; and lanceolate: ratio between 6:1 and 3:1.

Sampling approach with a priori grouping A sampling approach with a priori grouping was performed on the leaf shape dataset by using each one of the factors as a priori grouping. The factors were as follows: [START_REF]Évolution, Biodiversité (ISYEB[END_REF] The ploidy level (diploids and tetraploids) of our studied populations that were recovered from previous studies (see more Daoud et al. submitted;Favarger 1959;Küpfer 1974); ( 2) Their habitats which were classified according to the altitude (alt.> 900m: mountains; alt. < 250m: plains; 250 < alt. < 500m: hills); (3) The century of the collection (19 th century or 20 th century); (4) The flowering time that was extrapolated from the collection month of our selected specimens (May, June, July, August, and October).

Data analysis

The statistical analyses were done using the Rmorph statistical software packages (R Core Team, 2016).

A Principal Component Analysis (PCA) was performed on the leaf shape dataset, to visualize the structure of leaf shape variation within the species complex of A. grandiflora. The results were then presented as scatter plots. A multivariate analysis of variance (MANOVA) was run on the principal component scores, to reveal significant statistical differences among populations and to test the significance of the following factors: the ploidy levels, the habitats, the century of collection and the flowering time.

Results

Leaf shape discriminates between two cytotypes

The ploidy level separates well the populations according to their leaf shape. The results showed that the polyploidy was discriminant in the species complex of A. grandiflora and we could distinguish evidently the data of leaf shape into the diploids and the tetraploids (Fig. 3a-b). A MANOVA of significance performed on the PCA of leaf shape data showed that there were significant differences among the specimens of different ploidy levels (F=97.467, P = 0.000).

parnassifolius L. [START_REF] Cires | Intraspecific genome size variation and morphological differentiation of Ranunculus parnassifolius (Ranunculaceae), an Alpine-Pyrenean-Cantabrian polyploid group[END_REF], and a negative relationship with leaf mass per unit area (LMA) in angiosperms by (Beaulieu et al. 2007). However, a considerable variation in genome size was detected among populations of diploid plants. For example, the two populations Mt Sagra and Ste Baume had a genome size of about 2.44 pg and 2.70 pg respectively (Daoud et al. submitted), when the others had about 2.11 pg. Thus, we could suppose that this high genome size for both the Mt Sagra and Ste Baume in the Mt Mediterranean, could explain their distinguishable leaves from the other diploid ones growing on the plains or in the Pyrenees chain.

Nevertheless, the morphological diversity in leaf shape could be a response to abiotic stress in their natural habitat (see Hovenden and Vander Schoor 2006;[START_REF] Xu | Habitat effects on leaf morphological plasticity[END_REF][START_REF] Royer | Phenotypic plasticity of leaf shape along a temperature gradient in Acer rubrum[END_REF]). For instance, the simple leaves of Quercus acutissima become narrower in the xeric environments [START_REF] Xu | Leaf morphology correlates with water and light availability: What consequences for simple and compound leaves[END_REF]). Thus, it seems that leaf shape is sensitive to climate change. For example, [START_REF] Royer | Phenotypic plasticity of leaf shape along a temperature gradient in Acer rubrum[END_REF] highlighted the leaf-climate relationships in Acer rubrum. The leaf shape of native European hardwoods correlates strongly with temperature [START_REF] Traiser | Environmental signals from leaves--a physiognomic analysis of European vegetation[END_REF], and it can also change according to salinity in Vitis vinifera L. (Sinclair and Hoffmann 2003). Our results showed that the habitats are not sufficient to separate clearly the leaf shape dataset into distinct groups, although they suggest a significant statistical variation. We observed that leaf shape of A. grandiflora species complex could be linked with the habitats in which they grow. Indeed, A. grandiflora species complex occurs in the dry mountains of North Africa, the Mediterranean islands and the Pyrenees in Spain and France where this species complex survives the harshest conditions. When growing in dry rocks or screes, in the alpine mountains, they produce small, thin and pointed leaves which may help them to bear extreme frost and dryness (such as lanceolate leaves of the tetraploids). Furthermore, as expected, the leaves of plants have grown in sunny environments were generally smaller than leaves of plants of shaded environments. This result might be due to the shade avoidance response as explained in [START_REF] Franklin | Shade avoidance[END_REF] or Tsukaya (2005), which is expressed by the populations exposed to the weak light of the Fontainebleau forest (compared to alpine ones) resulting in different leaf elongation (acicular and linear leaves).

Conclusion

Our results showed a limited effect of habitats on leaf shape variation in the species complex of Arenaria grandiflora. This result is probably due to genetic founding and drift effects. Because this species complex is so sparsely distributed, gene flow among populations is limited and thus leads to differentiated genomes. This phenomenon could explain a large part of the differences observed among the leaves. In this study, we assume that the respective influences of many factors, that likely drive the leaf shape variation in the Arenaria grandiflora species complex, are difficult to assess because some of them are confounding (ploidy levels, habitats, flowering time). This study would be interesting to replicate in the future on plants from current natural populations to estimate, for such a trait, the speed of the drift and the impact of climate change.

Figure Captions

Fig. 1 Distribution map of the herbarium specimens sampled from the collection of Arenaria grandiflora species complex, with their associated ploidy levels as mentioned in the literature (see Daoud et al. submitted, Favarger 1959, Küpfer 1974)

Chapter V:

General Discussion be frequently surveyed by microsatellite markers, for example; a genetic differentiation among local populations of Magnolia stellata (Siebold & Zucc.) Maxim., was estimated using the microsatellite markers [START_REF] Setsuko | Population differentiation and gene flow within a metapopulation of a threatened tree, Magnolia stellata (magnoliaceae)[END_REF]) and [START_REF] Walser | Microsatellites reveal regional population differentiation and isolation in Lobaria pulmonaria, an epiphytic lichen[END_REF]) addressed a genetic differentiation among lichen populations using the microsatellite markers. In this study, we used the microsatellite markers in a different way a little bit, it has been tested whether such markers are used as a considerable indication to the ploidy levels, for the first time. Here, the microsatellites markers reveal a remarkable differentiation among populations at the ploidy levels within the species complex of A. grandiflora, using the allelic phenotypes at the ten microsatellite DNA loci that were amplified in some of the investigated populations. Some of the microsatellite loci in some of the populations were characterized by one or two alleles per locus per individual and made recognizable, in some of the cases, diploid heterozygosity. However, in populations of both Mt Jura and Mt Alps, the same loci displayed more than two alleles (up to four alleles) per individual, signal of tetraploid heterozygotes.

Furthermore, a classical karyological approach using conventional staining techniques was another proof demonstrating that explicit differentiation among populations exists within the A. grandiflora species complex. In this species complex, the occurrence of intraspecific chromosome number variation has been reported by two basic chromosome numbers of x=11,10 (Küpfer 1974;Luque & Lifante 1991, respictively). However, our results showed one basic chromosome numbers of x=11.Two chromosome numbers corresponding to two different ploidy levels were detected, diploid (2n=2x=22) and tetraploid (2n=4x=44), both with small chromosomes and characterized by the basic chromosome number of x = 11. Because of many obstacles like seed dormancy, crystals of oxalate of calcium that occurred in the roots and the presence of endophytic fungi in seeds, it has been very difficult to apply this classical karyological technique in A. grandiflora species complex. Thus, we used the technics of flow cytometry that can be a powerful tool for rapid and detailed analysis of populations of threatened species by using various materials such as leaf tissue (small amount), stored leaves in silica gel, cotyledons of germinated seedlings or dried seeds, which are adequate to estimate the variability among populations in genome size of the species complex of A. grandiflora. As well as a range of uses in plant researchers, flow cytometry offers a useful approach for monitoring the stability or the rapid evolution of genome size within an individual, likewise within a species, the genome size is usually constant within a species such as some of species of the Mediterranean genus Cistus L., [START_REF] Ellul | Intra-and interspecific variation in DNA content in Cistus (Cistaceae)[END_REF], and [START_REF] Lysak | Limited Genome Size Variation in Sesleria albicans[END_REF] demonstrated that Sesleria albicans Deyl., belongs to the plant taxa with a highly stable genome size, although there are notable exceptions, where a rapid evolution of genome size causes remarkable variation such as intraspecific differences in nuclear DNA content were found among populations of Koeleria macrantha (Ledeb.) Schult., [START_REF] Pecinka | Nuclear DNA content variation among central European Koeleria taxa[END_REF], also an intraspecific genome size variation was observed in Camellia sinensis (L.) Kuntze [START_REF] Huang | Genome Size Variation among and within Camellia Species by Using Flow Cytometric Analysis[END_REF]). In addition, a significance of intraspecific genome size variation was found in quinoa (Chenopodium quinoa Willd.) using flow cytometry technic, with propidium iodide as the DNA stain [START_REF] Kolano | Genome size variation in Chenopodium quinoa (Chenopodiaceae)[END_REF]. The present study suggests that the species complex of A. grandiflora has no stable genome size, because of a considerable intraspecific variation in genome size among its natural and fragmented populations (21 populations) was identified.

Furthermore, [START_REF] Severns | Intraspecific chromosome number variation: A neglected threat to the conservation of rare plants[END_REF] uncovered large gaps in the current state of cytological knowledge for threatened and endangered plants and a lack of attention given to the topic of intraspecific chromosome number variation and plant conservation. Thus, our assessment of the distribution of this cytogenetic variability present within the species complex of A. grandiflora with its fragmented distribution in Western Europe was critically and important requirement for the success of any genetic conservation plan in the future.

Nevertheless, if the phenomenon of differentiation among populations in seed germination rates is well known (Pérez-García et al. 2003) (Ginwal et al. 2005) [START_REF] Bahru | Effect of provenances on seed germination, early survival and growth performance of Tamarindus indica L. in Ethiopia : a key multipurpose species[END_REF]) (Fredrick et al. 2016) (Xu et al. 2016), the seed germination response variability in the investigated populations of A. grandiflora species complex was described for the first time. The seed provenances were significantly differentiated by the seed germination percentages. In spite of the different designs in the experiments, such as potting soil and agar substrates, the findings were convergent. The germination percentages (GP) greatly varied from 0% to 80 % within the species complex of A. grandiflora. Thus, we suppose that this high intraspecific variation in germination rate of seeds could be interpreted by various factors like the maternal environmental effect of seed provenances (both altitude and longitude of seed provenances impacted the final germination percentage), the ploidy levels of individuals (the tetraploids germinated better than the diploid ones) and both period and condition of seeds storage (seed viability for the longer period is retained at + 4 °C temperature and 40% humidity). Our results in seed germination trials provide valuable and baseline information of populations of A. grandiflora species complex to address conservation concerns for this endangered species.

Finally, the phenotypic patterns of differentiation among populations have been detected in herbarium' specimens belonging to some of populations of A. grandiflora species complex, that were stored in the French National Herbarium (in Paris). The distinct patterns of both shape and elongation variation have been exhibited in the leaves of A. grandiflora species complex by using the morphometric approaches. This variation in both of leaf shape and elongation has been distinguishable between the two cytotypes (diploids and tetraploids) of the species complex of A. grandiflora. In other words, we found that the leaves of diploids were typically acicular or linear and the leaves of tetraploids were typically lanceolate. An interesting leaf elongation variation within the diploids of A. grandiflora species complex has been found. Both the acicular and the linear leaves were shown, the leaves of specimens from Mt Sagra and Ste Baume populations are acicular, different from the linear leaves of specimens which are included in other populations as like as Fontainebleau, Chinon, Rocamadour and Gèdre.

As mentioned by [START_REF] Otto | Polyploid incidence and evolution[END_REF] and [START_REF] Levin | The role of chromosomal change in plant evolution[END_REF], the process of polyploidization is often accompanied by changes in morphology, physiology and associated with some geographic locations, types of environment as well as the life history of populations. Here, we observe an impressive incidence of polyploidy among the natural and fragmented populations of A. grandiflora complex over its spatial scale in Western Europe. Moreover, this incidence of polyploidy seemed to influence the tested characters such as 1) molecular genotypes (variant number of alleles at some of the microsatellite loci), 2) seed germination responses (GP variation) and 3) both leaf shape and elongation variability.

The results on ploidy levels within perennial herbaceous of A. grandiflora species complex agree with the current evidences which suggest that the frequency of polyploidization events differ among different life forms. For example, [START_REF] Stebbins | Cytological Characteristics Associated with the Different Growth Habits in the Dicotyledons[END_REF] and [START_REF] Otto | Polyploid incidence and evolution[END_REF] reported that the high proportion of polyploids is generally among perennial herbaceous plants, compared to annuals species.

Furthermore, the polyploids frequently differ in geographic ranges from diploid relatives, and often have larger geographic distribution and occupy more extreme environments such as high altitudes than diploids [START_REF] Otto | Polyploid incidence and evolution[END_REF][START_REF] Levin | The role of chromosomal change in plant evolution[END_REF]). These differences in geographic ranges have been explained through different aptitudes of polyploids such as best ecological tolerance (plasticity), due to higher heterozygosity and allelic diversity [START_REF] Otto | Polyploid incidence and evolution[END_REF].

Protocol for seed germination and influential factors

To understand the effects of many factors on the germination percentage, we validated a linear model of regression with the 6 explanatory variables: altitude, longitude, latitude, ploidy levels and both period and condition of seed storage.

As well known, the reinforcement with new individuals is often recommended to preserve some populations for extinction [START_REF] Bottin | Re-establishment trials in endangered plants: a review and the example of Arenaria grandiflora, a species on the brink of extinction in the Parisian region (France)[END_REF]). Nevertheless, producing the individuals from seeds can be challenging because little is known about the germination requirements of many endangered species.

Here, we established for the first time, a reliable protocol of seed germination that achieved a higher germination rate for most of the tetraploid populations of A. grandiflora species complex. As mentioned above, the seed coat dormancy is one of the obstacles that have been found in many populations of A.

grandiflora species complex (especially tetraploid ones) and have been sideswiped by using in vitro seed germination with mechanical scarification. In vitro culture after mechanical scarification has been applied to seeds which have been collected from different altitudes and have been stored in two types of conditions for different periods. Four culture media with and without sugar have been prepared to germinate the seeds. Furthermore, the protocol of the surface sterilization has been used for pretreatment the seeds to avoid the contamination. According to our novel protocol of seed germination, the propagation from seeds will be an efficient method for the in-situ restoration of these tetraploid populations.

Diploid cytotypes

Here, we present and discuss some of the diploid populations that we consider particularly remarkable for the future conservation plans for A. grandiflora species complex.

One of the most interesting populations is Mt Sagra population in the Mediterranean mountains that occurs in Granada (Andalusia, Spain) at high elevation (2279 m). According to [START_REF] Ryder | Species conservation and systematics: the dilemma of subspecies[END_REF] who identified the populations as ESUs, the Mt Sagra population is considered by this study as distinct ESU, presenting a particular monoploid genome size which has been detected for the first time. Our findings exhibited that the Mt Sagra population had a maximum of two alleles per locus by using the microsatellite markers and showed a mean 2C value in pg ±SD (2.44±0.96) detected by flow cytometry approach, Küpfer (1974) reported that one diploid population occurred in the Sierra Nevada (another massif in Granada-Andalusia, Spain). Thus, the population of Mt Sagra is worthy of follow-up, especially because it presents a good seed germination percentage (52,86± 22,14) in in vitro culture (with mechanical scarifying) and a significant difference from the GP for seeds of tetraploid population of the alpine mountains (Mt Cheval Blanc at 2280 m, in the same altitude).

So, the different seed germination responses we observed could be explained by the different types of climate i.e. the Mediterranean mountain and the alpine mountain ones, marked especially by seasonal changes that are affecting seed viability and storability. Moreover, the leaf shape of herbarium specimens of Mt Sagra population is another argument for which this population could be considered as an interesting distinct population among other diploids ones. Its leaves are acicular and seem different from the linear ones of the other diploid populations. The phenotypic findings on leaves could be explained by a large monoploid genome size that has been found and already counted as a distinct cytogenetic character in this population. Consequently, the Mt Sagra population revealed a distinguished pattern of genetic, cytogenetic and phenotypic variation and thus could be considered as a unit for population-level conservation to stop the biodiversity loss within a species complex such as A.

grandiflora [START_REF] Purvis | Getting the measure of well-being[END_REF].

Another interesting diploid population that could be considered as a good ESUs, is the Ste Baume population. This diploid population of the species complex of A. grandiflora exhibits both in a restricted geographical distribution (1034 m) and a narrower and more marginal niche than other species of Arenaria, in dry rocky in the southern Provence of France [START_REF] Youssef | Factors underlying the narrow distribution of the Mediterranean annual plant Arenaria provincialis (Caryophyllaceae)[END_REF]. In addition, it is a very small population (< 25 individual, obs. perso.2016). Here, the cytogenetic results show that Ste Baume population has the largest monoploid genome size with an interesting significant difference with other diploid ones. Furthermore, the leaves of herbarium specimens of Ste Baume population are acicular and look distinguishable from the linear leaves of the other diploid ones in the plains or in the Pyrenees chain. Thus, we have a distinct unit for population-level conservation in Mediterranean mountains that has presented a restricted geographical pattern of both cytogenetic and phenotypic variation. Because of all above, we suggest a serious and effective interest in the future for the Ste Baume population.

both leaf sampling and seed harvesting had been very difficult. The chromosome number of individuals from Mt Chasseral population were detected by Favarger (1959Favarger ( , 1962) ) and Küpfer (1974) (2n =4x = 44). Here, we reveal, for the first time for this population, both the number of alleles for microsatellite loci and the mean of 2C DNA content (4.39±1.54 pg.).

The presence of populations with different ploidy levels in the same mountain chain such as in the Spanish Pyrenees (Huesca), e.g. two tetraploid populations (Ceresa and Gabas) co-occur with two diploid ones (The Vilas del Turbon and Plan), arises new questions about the origins of polyploid populations.

General Conclusion

Taxonomic revision_ point of view

The Large Flowered Sandwort (A. grandiflora) is a widely distributed species complex with disjunctive populations in Western Europe but has an uncertain taxonomic status. Seven, sometimes five or three geographic subspecies, two varieties and two forms had sometimes been described. In this thesis, A.

grandiflora had been considered as species complex, because of the existence of numerous synonyms and intraspecific karyotypic variability, whereas, two cytotypes (diploid and tetraploid) have been detected among its fragmented populations. As demonstrated, the thesis has shown the patterns of genetic and phenotypic variation in A. grandiflora species complex. A correlation was found between polyploidy and phenotypic variation, there were differences between the two cytotypes (diploid and tetraploid). We found polymorphism for all the characters we studied, such as the genotyping of microsatellite loci, the chromosome number, the 2C-values of nuclear DNA content, the seed germination percentage and the leaf shape. The recent approach of flow cytometry has allowed more accurate and rapid estimates of the genome size and its variation within the species complex of A.

grandiflora, including the correlations with the geographic range of its distribution and morphometric traits of its leaves. The mean 2C DNA value within the species complex of A. grandiflora ranges from 2.11±0.74 pg to 2.70±0.11 pg for the diploids and from 4.30±1.51 pg to 5.27±0.14 pg for the tetraploid ones. These results substantially support that both the genome size and the polyploidy are probably major causes of diversity within this species complex. However, as known, the plant genome sizes range it is not surprising that some of these changes should be fixed evolutionarily (Kellogg and Bennetzen 2004). Thus, a taxonomic revision of this species complex A. grandiflora is a critical step in the future, based on the observations of wild populations and the extensive herbarium material. Furthermore, the ploidy levels, the genome size, the seed germination percentage and the leaf shape could be used with a degree of confidence to discriminate among the disjunctive populations of studied species complex.

Consequently, these patterns of genetic, cytogenetic and phenotypic variation could suggest a development of the intraspecific taxonomic keys problematic for this species complex and describe the taxonomic subgroups in ways that are useful to systematists, field botanists, and plant conservation biologists.

Evolutionarily significant units (ESUs) _ points of view

Over the past three decades, there has been an intense debate about the concept of the evolutionarily significant units (ESUs) [START_REF] Ryder | Species conservation and systematics: the dilemma of subspecies[END_REF][START_REF] Moritz | Defining " Evolutionarily Significant Units " for conservation[END_REF][START_REF] Crandall | Considering evolutionary processes in Conservation Biology[END_REF]. Moritz (1994a) proposed a definition based on neutral genetic divergence at microsatellite loci. In contrast, [START_REF] Crandall | Considering evolutionary processes in Conservation Biology[END_REF] argued that the degree, to which populations are adapted to the same ecological niche and are thus exchangeable, must be considered. In this thesis, we found significant differences both genetically and morphologically among and within populations of A. grandiflora species complex by combining the genetic, phenotypic (seed germination rates and morphometric) technics. In our study, the combination of genetic and morphological differences indicated that diploid and tetraploid populations could likely be reproductively isolated. This strongly suggests that these populations should represent different ESUs (see fig. 7). Most of the habitats of diploids and tetraploids differ considerably. Thus, we argue that the identification of conservation units, could base on both species' genetics and ecology. It means that both [START_REF] Moritz | Defining " Evolutionarily Significant Units " for conservation[END_REF] and [START_REF] Crandall | Considering evolutionary processes in Conservation Biology[END_REF] concepts are compatible for this species complex because genetics and ecology are obviously closely linked.

Fig. 2 :

 2 Fig.2: Illustration of Arenaria grandiflora from Tela Botanica

  region (N. W. NW. C of Iran). The Subgenus of Arenaria is centered in Asia and in the Mediterranean region, where the section Plinthine McNeill extended to the Maritime Alps (S.E. of France and N.W. of Italy). Other sections of the Arenaria Subgenus are distributed in Greenland, America (Texas and New Mexico) and Northeastern Africa.

Fig. 3

 3 Fig.3Distribution of some subspecies of Arenaria grandiflora species complex in Europe according to

Fig. 4

 4 Fig. 4 Chamaephytes (buds near soil) according to Raunkiaer's classification (Beentje and Williamson

Fig. 5

 5 Fig.5 (a-b) 5a: Hemicryptophytes: buds in the upper crust of the soil, 5b: Therophytes: the growing

  specimens and an internal standard [Petunia hybrida (Hook) Vilm. cv.' PxPC6', 2.85/2C pg or Solanum lycopersicum L. cv. Montfavet 63-5. 2C=1.99 pg (Lepers-Andrzejewski et al. 2011)] were chopped together with a razor blade in 600 µL of cold Gif Nuclear Buffer (45 mM MgCl2, 30 mM sodium citrate, 60 mM 4morpholine propane sulfonate pH 7. 0.1% (w/v) Triton X--100. 1% polyvinylpyrrolidone (~10.000 Mr.

  Two chromosome numbers corresponding to two ploidy levels, diploid (2n=2x=22; figs 3 a & b) and tetraploid (2n=4x=44; figs 3c & d), were detected in the A. grandiflora species complex (table

  programmes. For example, two-thirds of threatened or endangered plants in the USA are known to exhibit intraspecific chromosome number variation. yet this has been taken into account for very few species in conservation plans to our knowledge[START_REF] Severns | Intraspecific chromosome number variation: A neglected threat to the conservation of rare plants[END_REF] Delaney & Baack 2012). To avoid the risk of omitting information on ploidy levels in restoration measures, it would be desirable to first determine the existence of potential cytotypes. Our study identified different ploidy levels in native populations of threatened and endangered species and highlighted the importance of this form of genetic variation in plant conservation. In programmes aimed at reinforcement of populations, mixing of individuals with different ploidy levels should be stringently avoided. Here, we might consider all the populations with different ploidy levels, such as the alpine and pre-alpine populations (Mt Lure, Mt Cheval Blanc, the Mt Chaudière and Mt Raton) also, the Mt Chasseral population in the Swiss Jura, as distinct ESUs. Moreover, we could define the diploid populations presenting intraspecific variation in monoploid genome size from Pas de l'Ai in the Mt Sainte Baume chain in France and the Mt Sagra in Andalusia as distinct ESUs.

Figure 1 -

 1 Figure Captions

Figure 2 -

 2 Figure 2--The four alleles of three individuals from two tetraploid populations of Arenaria grandiflora

Figures

  Figures 3A, B, C & D--Metaphase chromosome plates of Arenaria grandiflora species complex, that were

Figure 5 -

 5 Figure 5--Histogram showing the genome size (2C in pg) for each population of Arenaria grandiflora

Figure 1 -

 1 Figure 1--Map of the distribution of the sampled populations from Western Europe, indicating ploidy

Figure 2 -

 2 Figure 2--The four alleles of three individuals from two tetraploid populations: Gabas in the Spanish

Figures

  Figures 3A, B, C & D--Metaphase chromosome plates of Arenaria grandiflora species complex, that were

  Fig. Captions

Fig. 1 Fig. 2

 12 Fig.1The seed germination percentages in the potting experiments for four populations of Arenaria

Fig. 3

 3 Fig. 3 Histogram shows the means of germination percentage (GP) for good germinated populations of

Fig. 1 (Fig. 2

 12 Fig.1 (A and B): The seed germination percentages in the potting soil experiments for four populations of Arenaria grandiflora species complex in function of

Fig. 3

 3 Fig.3Histogram shows the means of germination percentage (GP) for good germinated populations of A. grandiflora species complex in all trials (without GP<

Fig. 2 a

 2 Fig.2 a: Set of microscope slides on which leaves sampled from herbarium specimens identified as

Fig. 3

 3 Fig.3Plots of Principal Component Analysis (PCA) of leaf shape variation within the species complex

Fig. 4

 4 Fig.4A scatter plot of Principal Component Analysis (PCA) reveals the structure leaf shape variation

Fig. 5

 5 Fig. 5 Plot of Principal Component Analysis (PCA) of the leaf shape variation among the diploid

Fig. 1

 1 Fig.1 Distribution map of the herbarium specimens sampled from the collection of Arenaria grandiflora

Fig. 3

 3 Fig.3Plots of Principal Component Analysis (PCA) of leaf shape variation within the species complex of Arenaria grandiflora: a: plot of the diploids and the

Fig. 4

 4 Fig. 4 A scatter plot of Principal Component Analysis (PCA) reveals the structure leaf shape variation

Fig. 5

 5 Fig. 5 Plot of Principal Component Analysis (PCA) of the leaf shape variation among the diploid

  from the smallest (1C = 0•0648 pg in Genlisea margaretae Hutch,Bennett and Leitch 2011) to the largest (1C = 152•23 pg in Paris japonica Franch.(Pellicer et al. 2010), there is no evidence supporting a link between overall genome size and diversification. Nevertheless, the rate of genome size evolution enhances the acquisition of the novel traits, the reproductive barriers and the movement into new niches, which have aided the diversification of angiosperms(Puttick et al. 2015). Moreover, when the changes in the overall genome size, the ploidy level and the chromosome number occur continuously in plants,

Fig. 7

 7 Fig.7A map presents some of proposed ESUs within the species complex of Arenaria grandiflora in

  The stems are ascending, simple or branched, often caespitose or pulvinate. Leaves are opposite, mostly sessile, linear or linear-lanceolate, usually elliptic to ovate or rarely orbicular,

sometimes sub-succulent or succulent, subulate, setaceous and apex blunt or obtuse to acute, acuminate, margin entire and usually flat. Inflorescences are solitary flowers. Stamens 10 (2-5 or 8 in A.

  system and have described as a new tribe; Eremogoneae Rabeler and W. Brot. and A. hispida has obtained by[START_REF] Youssef | Factors underlying the narrow distribution of the Mediterranean annual plant Arenaria provincialis (Caryophyllaceae)[END_REF]. The molecular studies, specifically of the genus Arenaria s.str., are limited to the findings ofSheidai et al. (2011).

	L. Wagner (Harbaugh et al. 2010). With only a few exceptions, these results of tribal classification have
	supported by Greenberg et al (2011) via inferring a larger, more equally sampled, phylogenetic tree for
	Caryophyllaceae to more obviously resolve relationships within this clade.
	Nevertheless, the phylogenetic analysis of the western Mediterranean Arenaria section Plinthine
	(Valcárcel et al. 2006) distinguishes between genus Moehringia and Arenaria. and has not been without
	controversy. Moehringia is paraphyletic to Arenaria with Iberian taxa, according to the phylogeny of
	the genus Moehringia, that was investigated by Fior and Karis (2007) by analyzing nuclear (ITS) and
	chloroplast (matK) sequence data in combination with morphological characters. However, the
	phylogenetic knowledge of the species Arenaria in southern Provence of France, A. provincialis, A.
	modesta, A. serpyllifolia, A. aggregata, A. grandiflora, A. cinerea DC., A. ermarginata, A.

conimbricensis

Table 1 -

 1 -Geographical origins of the studied materials.

	No	Population	Latitude	Longitude	Altitude	Type of Tissue/Date	Region
					(m)
	1	Baumont-en-Véron*	47°11'42.93"N 0°11'9.87"E	35	leaves/2015	Loire Valley
	2	Rocamadour.Casetl	44°47'53.03"N 1°36'53.69"E	331	leaves /2014	Languedoc-Roussillon Midi Pyrenees
	3	Rocamadour.roadside	44°47'53.46"N 1°37'5.25"E	260	leaves /2014	Languedoc-Roussillon Midi Pyrenees
	4	Lake Gloriettes	42°45'24.29"N 0° 3'19.89"E	1690	leaves /2013	Gèdre-Hautes-Pyrenees
	5	Coumely Plateau	42°43'0.54"N 0° 1'17.42"W	1600	leaves /2013	Gavarnie-Hautes-Pyrenees
	6	Plan	42°33'13.5''N 0°26'9.80"E	1720	seeds + leaves /2015	Huesca-Pyrenees
	7	Mt Sagra	37°57'13.20"N 2°33'34.90"O	2279	leaves/2014 + seeds /2015 Granada-Andalusia
	8	Queue de vache East** 48°23'17.21"N 2°36'24.81"E	131	leaves /2016	Fontainebleau forest
	9	Queue de vache West** 48°23'11.12"N 2°36'17.75"E	129	leaves /2016	Fontainebleau forest
	10	Mont Chauvet	48°25'43.36"N 2°40'59.83"E	87	leaves /2013	Fontainebleau forest
	11	Mt Lure	44° 7'26.93"N 5°46'23.16"E	1718	seeds /2013	Monts de Vaucluse-Alps
	12	Mt Chasseral	47° 7'50.43"N 7° 3'42.98"E	1 502	leaves /2013	Mt Jura -Vaud
	13	Ceresa	42°29'24.90"N 0°12'34.60"E	1711	seeds + leaves /2015	Huesca-Pyrenees
	14	Gabas	42°27'48.9''N 0°28'00.5''E	1830	leaves/2014	Huesca-Pyrenees
	15	Mt Cheval Blanc	44° 07′ 39″ N 6° 25′ 26″ E	2300	seeds /1990	Alps de Haute Provence
	16	Pas de l'Ai	43° 34′ 180″ N 5° 81′ 854″ E	1034	leaves/2016	Sainte Baume-Provence
	17	Mt Raton	44°23'53.71"N 5°27'47.25"E	1473	seeds /2008	Prealps
	18	Saint Sauveur	42°51'35.1"N 0°00'44.0"O	899	leaves/2016	Languedoc-Roussillon Midi Pyrenees
	19	Butte de Sers	42°53'16.0"N 0°02'28.7"O	1185	leaves/2016	Languedoc-Roussillon Midi Pyrenees

Table 2 -

 2 -The allelic phenotypes at the ten microsatellite loci for some analysed individuals. Allele size is given in bp. Number of alleles per individual for each locus: (Aind); number of alleles per locus for each individual: (ALoc). N° of alleles per individual. ALoc: N° of alleles per locus. Ind: individus Sagra: Mt Sagra; B. en V. : Bauemont en Veron; Lure: Mt Lure; Lake Glo.: Lake Gloriettes; Roca: Rocamadour.Casetl; Chass.: Mt Chasseral.

	Individu s	Sag ra	Sa gr a	B. en V.	B. en V.	Lu re	Ga bas	Ga bas	Lake Glo.	Lake Glo.	Ro ca.	Ro ca.	Ch ass.	Ch ass.	Ch ass .	= 14 Ind
	Aind	15 15 14	15	23 26 24 16	17	16 13 24 20 = 258
	Ploidy Levels	2x 2x 2x	2x	4x 4x 4x 2x	2x	2 x 2 x 4x 4x 4x	ALoc
	Loci / Allele	184	17 8	181 181	18 1	178 181 178	178	196 181 184 184
	AGA3-98	184	17 8	181 181	18 1	181 184 184	184	196 181 184 184		4
							184 190
							190 190
		148	14 3	177 161	14 6	146 146 173	167	148 140 155 155
	AG3-2	179	16 7	179 179	14 6	146 146 173	170	152 181 155 181		14
						15 9							159 181
						16 3							181 181
		138	13 8	150 150 -	136 136 162	148	160 170 128 138
	AG3-36 142	13 8	170 150 -	136 136 170	148	182 182 130 144		15
						-	138 138					138 148
						-	160 160					154 154
		267	26 5	287 263	25 9	259 281 265	263	263 263 263 267
	AG9-49 267	26 7	287 263	25 9	259 287 281	267	273 263 263 267		9
						25 9	267						265 267
						26 5	271						267 277
		209	20 9	209 209	20 3	209 209 215	209	215 209 209 209
	AGA2-74	209	20 9	215 209	20 9	215 215 221	209	215 215 209 209		5
							227 227
							227 227
		147	14 5	147 143 -	143 145 145	145	153 153 149 149
	AGC5-93	151	15 1	147 145 -	145 145 151	147	159 153 149 151		8
						-	147 149					153 155

Table 3 -

 3 -The results of cytogenetic analyses including: Chromosome number (2n), ploidy level and genome size for the investigated Arenaria grandiflora populations.

	Population	2n Ploidy level Mean 2C value 1Cx	Number of studied
			in pg ±SD	in Mpb individuals/population
	Bauemont en Veron	22 2x	2.13±0.78	1039.17
	Plan	22 2x	2.19±0.77	1071.89
	Mt Sagra	22 2x	2.44±0.96	1195.12
	Mont Chauvet	2x	2.11±0.74	1033.01
	Queue de vache East	2x	2.11±0.74	1033.9
	Queue de vache West	2x	2.13±0.74	1039.8
	Rocamadour. roadside	2x	2.17±0.76	1064.85
	Rocamadour. Castle	2x	2.17±0.76	1062.04
	Gloriettes Lack	2x	2.17±0.76	1059.37
	Coumely Plateau	2x	2.16±0.76	1054.77
	Mt Lure	44 4x	4.64±1.63	1134.11
	Ceresa	44 4x	4.30±1.51	1051.47
	Mt Chasseral	44* 4x	4.39±1.54	1072.7
	Gabas	4x	4.81±0.12	1 177.03
	Mt Cheval Blanc	4x	4.56±0.03	1 115.40
	Pas de l'Ai	2x	2.70±0.11	1 321.90
	Mt Raton	44 4x	4.98±0.05	1 218.32
	Saint Sauveur	2x	2.21±0.09	1 079.09
	Butte de Sers	2x	2.23±0.03	1 090.08
	The Vilas del Turbon	2x	2.39±0.01	1147.10
	The Chaudière	4x	5.27±0.14	1 288.75
	* From

  Significant differences were reported among Rocamadour population that occurs at (331 m; 44.47 N° and 1.36 E°) and all other germinated populations (GP >10 %) as well as Chinon population at (90 m; 47.19 N° and 0.24 E°) and Mt Cheval Blanc population at (2280 m; 44.08 N° and 6.20 E°) but not for Mt Rié population at (900 m; 42.54 N° and 0.40 E°). Moreover, Mt Cheval Blanc population at (2280 m; 44.08 N° and 6.20 E°) differed significantly from Mt Raton population at (1450 m; 44.25 N° and 5.25 E°), on one side and Mt Sagra population on the other side (2279 m; 37.57 N° and 2.33 E°).

Table 1 :

 1 Seed provenances, ploidy levels, year of seed collection, both period and temperature of seeds storage and geographical parameters (latitude, longitude,

		and altitude) of investigated populations
	No	Population	Latitude (°N)	Longitude	Altitude	Year	Period of	Temperature of seed	Ploidy	Collection site
				(°E)	(m)	collection	seed storage	storage	levels	(Provenance)
							(month)
		Mt Raton	44.25	5.25	1450	2007/2008	3/84/96	room temperature	4	The pre-Alps
		Mt Sagra	37.57	2.33	2279	2015	3	room temperature	2	Mt Mediterranean
		Plan	42.33	0.26	1720	2015	3	room temperature	2	Spanish Pyrenees
		Ceresa	42.29	0.12	1711	2015	3	room temperature	4	Spanish Pyrenees
		Rocamadour	44.47	1.36	331	2015	3	room temperature	2	French Pyrenees
		Mt Lure	44.7	5.46	1718	2013	24	room temperature	4	The pre-Alps
		Mt Rié	42.54	0.40	900	2007	3	room temperature	2	French Pyrenees
		Mt Chaudière	44.37	5.13	1560	2007	96	room temperature	4	The pre-Alps
		Chinon	47.19	0.24	90	2007/2008	3	room temperature	2	Loire Valley
		Mt Merle	48.25	2.40	87	2006	108	room temperature	2	Fontainebleau Forest
								(for 2 years) then at (-4 °C)
								for 7 years
		Mt Cheval Blanc	44.08	6.20	2280	1990	300	+ 4 °C and 40% of Humidity	4	The Alps

Table 3 :

 3 Summary of models and ANOVA for the dependent variable (GP %) germination percentage and the linear regression's F-test, P values, R, R square and Std, the error of the estimate; a. predictors: (constant), period of seeds storage (month); b. predictors: (constant), period of seeds storage (month), altitude;

	c. predictors: (constant), period of seeds storage (month), altitude, latitude; d. predictors: (constant), altitude, latitude; e. predictors: (constant), altitude, latitude,
	longitude; f. predictors: (constant), condition of seeds storage, latitude, ploidy levels, longitude, altitude, period of seeds storage (month)
	Models	R	R square Std. the error of the estimate F	P values
	period of seeds storage	.350 a	0.122	19.37623	13.811	.000 a
	period of seeds storage, altitude	.409 b	0.168	18.9675	9.863	.000 b
	period of seeds storage, altitude, latitude	.558 c	0.312	17.33442	14.651	.000 c
	altitude, latitude	.551 d	0.304	17.34666	21.377	.000 d
	altitude, latitude, longitude	.616 e	0.379	16.46044	19.772	.000 e
	condition and period of seeds storage, latitude, ploidy levels, longitude, altitude	.633 f	0.4	16.43872	10.455	.000 f

Table 1

 1 List of herbarium specimens sampled from the collection of Arenaria grandiflora species complex, corresponding microscope slides and collection year. High-resolution images of the herbarium

	specimens	can	be	accessed	using	the	following	link:
	https://science.mnhn.fr/institution/mnhn/collection/p/item/search/form
	N° studied sample	N° microscope slide	Specimen barcode	Collection year
	1		2		P04933087
	2		3		P04936269
	3		4		P04936299
	4		5		P04936290
	5		7		P04936267
	6		8		P04933084
	7		9		P04936139
	8		10		P05162981
	9		17		P04933086
	10		21		P04936144
	11		22		P04936143
	12		26		P04933116
	13		27		P04933065
	14		28		P04933061
	15		29		P04933064
	16		30		P04932285
	17		38		P05160377
	18		44		P05162986
	19		45		P05109875
	20		50		P05161419
	21		52		P05167087
	22		53		P05109867

Acknowledgements Acknowledgements

The study was funded by the institutional resources of the two laboratories CESCO (MNHN) and ESE (University of Paris-Sud, Saclay). The team of this framework thanks Nélida PADILLA GARCIA.

Bernard PASQUIER from CNPMAI (Conservatoire National des Plantes à Parfum. Médicinales, aromatiques et Industrielles), Frédéric MARQUIS from CBNA (Conservatoire botanique national alpin) and Lyon Botanical Garden for their assistance in collection of the materials. This study was partly funded by the Labex BCDIV.

Acknowledgement

We thank Myriam GAUDEUL from Paris' herbarium in the French National Museum of Natural History, Paris, France. The authors also thank. We are grateful to Sophie NADOT from Paris-Sud University for her constructive advice. This study was partly funded by the Labex BCDIV.

This work is to the best of my knowledge original except where references are made to previous work. This thesis has been writing based on experiments conducted in the scientific group of both two laboratories Center for Ecology and Conservation Sciences (CESCO; MNHN) and

Table 2 The mean values ± standard deviation of germination percentages for all the trials with the substrate of the germination and number of the cultivated seeds for each trial Populations Means of GP (± s.d.) Means of GP (± s.d.) Means of GP (± s.d.) Nb cultivated seeds In Filtre Papers In Potting Soil In vitro with scarification

Paper 2

Seed germination of Arenaria grandiflora L. species complex (Caryophyllaceae) This study was conducted with Sandrine Fontaine and Christian Raquin from University of Paris-

Saclay

This study is still preliminary to be submitted in Seed science research

The terrines were examined at 1-day intervals and during each examination, the number of germinated seeds was recorded. Protrusion of the cotyledons was the criterion of germination. The seed germination was observed and recorded every day during 50 days' germination experiments in the pots. We subdivided seeds into two categories: Normal (N) and Deformed (D), because of the number of seeds in A. grandiflora capsule ranged from one to eleven but the size of the capsules was roughly the same. In a capsule containing a few seeds, the seeds had more space to grow and therefore grew into a rounded shape. Contrariwise, the capsules with many seeds did not have ample space for the seeds to grow and consequently grow deformed.

We tested the impacts of two seed parameters on germination percentage: (a) the seed sources (provenances of seeds), and (b) the seed type ("Normal" vs "Deformed"). Experiments were carried out in autumn 2007 or 2008.

In view of this first attempt, we tried again by using another methodology, but with less seeds than for the first attempt because of the low available quantity remaining.

2) Test of germination on filter paper

The material which was used for these experiments were seeds of A. grandiflora species complex from Mt Merle, Rocamadour, Mt Lure, Mt Cheval Blanc, Mt Raton, Mt Chaudière, Mt Sagra, Plan and Ceresa populations. Three Petri dishes of 9 cm in diameter were used for the seeds of each population under ambient lab conditions (approximately 20 °C). Each dish contained (10-18) seeds. As required, the filter paper was moistened with distilled water at the beginning of the experiment. The seed germination was observed every day. The germination percentage is the fraction of seeds germinated in each Petri-dish after 45 days of incubation. These experiments of germination on filter paper were carried out in autumn 2015.

Therefore, in view of the above (two attempts and their results), we decided to perform the 3rd test, by in vitro culture.

3) In vitro germination (with scarification) experiments

Ten out of the eleven populations were sown in vitro experiments, except Mt Rié population (French Pyrenees), because of lack of seeds. The experiments of in vitro culture were carried out in autumn 2008 or 2016.

Germination in filter papers

In our preliminary experiments in filter paper, no germination was obtained and fungal contamination was observed.

Germination of scarified seeds in vitro

Means of seed germination percentage (GP) in vitro for scarified seeds greatly varied from 0% to 80 % (see Fig. 2). However, both their seed provenances and their ploidy levels affected the percentage of germination (ANOVA: P = 0.021; P = 0.000, respectively), but there was no impact of the four media (GH0, GH10, GMC6 and GMC610) of in vitro culture (ANOVA: p > 0.05). Thus, ANOVA with Welch test was conducted only for the seed provenances and the ploidy levels. Significant differences among both the seed provenances and their ploidy levels were observed (F=51.232, p = 0.000; F=71.349, p = 0.000 respectively). The variation in GP among the seed provenances in function of their ploidy levels was showed in Fig. (2).

The pairwise differences of the seed provenances were demonstrated by using Games-Howell analysis (see Supplementary1). We observed that the lowest GP of seeds from Rocamadour populations (GP ± s.d. = 10.76 ± 12.37 %), Chinon and Mt Merle populations (GP = 0.00 %) differed significantly from all other seed provenances that very well germinated in vitro as well as Mt Chaudière (GP ± s.d. = 69.57± 11.31%), Ceresa (GP ± s.d. = 68.63± 20.54%), Mt Lure (GP ± s.d. = 68.92± 17.59 %), Mt Raton (GP ± s.d. = 62.03± 14.29 %), Mt Cheval Blanc (GP ± s.d. = 82.85± 7.76%), Plan (GP ± s.d. = 72.48± 16.24 %) and Mt Sagra (GP ± s.d. = 52.86± 22.14 %) (P < 0.05). Furthermore, a significant difference in the germination percentage was reported among the Mt Cheval Blanc population (GP ± s.d. = 82.85± 7.76%), in one side, and both Mt Raton population (GP ± s.d. = 62.03± 14.29 %) and Mt Sagra population (GP ± s.d. = 52.86± 22.14 %) on the other side.

Moreover, the seeds from Chinon population (the Puy du Chinonnais) and Mt Merle population in the Fontainebleau forest had not germinated after the scarification. The culture dishes were infected by fungi that are probably of endogenous origin (endo-fungal) in the seeds. Generally, we could affirm that differences in GP among the seed provenances were highly significant for all the germinated seed provenances, in all our experiments, except for the experiments in filter papers where the GP = 0 % (see

Leaf elongation

The leaf shape variation within the species complex of A. grandiflora could be also explained in relation with leaf elongation, that included some of information about the leaf types and separated the leaves sampled into three groups (acicular leaves, linear leaves, and lanceolate leaves). The specimens (Fig. 3b)

were separated according to the variables of PC1, both the leaf length (L) and the leaf width (W2).

Along the PC1, in the diploids, leaf length increases while leaf width is decreased, thus we found two of leaf types: acicular and linear leaves. In contrast, in the tetraploids, leaf length decreases while the leaf width is increased (one of leaf types: lanceolate leaves).

Leaf shape data analysis

The scatter plot of Principal Component Analysis (PCA) showed the structure of leaf shape variation in the species complex of A. grandiflora (Fig. 4). The first principal component (PC1) axis accounted for 66.04 % of the total leaf shape variation while the second principal component (PC2) accounted for 21.47 % and PC3 12.49 % (see Fig. 4). The first principal component is clearly congruent with the separation of the tested specimens; it opposes leaf length with its maximal width W2. The second principal component axis contrasts leaf width at the highest point (W1) to leaf width at the basal point (W3).

Leaf shape discriminates within the diploids

We found that leaf shape varied within the diploids of A. grandiflora species complex: acicular and linear leaves were defined with significant variation (F= 41.173; P = 0.000) (see Fig. 3b). We could distinguish the leaf shape of the specimens that are belonging to the diploid population from Sainte Baume from the leaves of specimens which are belonged to other diploid ones (Fontainebleau, Chinon, Rocamadour, and Gèdre) (see Fig. 5). In addition, the leaves of plants harvested from the diploid specimens of Mt Sagra population seem different from the other ones that were collected from the diploid populations of Fontainebleau, Chinon, Rocamadour, and Gèdre. Moreover, the leaves of the diploid populations of Fontainebleau, Chinon, Rocamadour, and Gèdre are not distinguishable.

Significant effects for both flowering time and habitat

The results of the PCA was performed on the leaf shape dataset using a sampling approach with three out of four priori groupings. Two out them (flowering time, habitats) had a significant variation (P < 0.05), but the century of collection, the fourth priori grouping, had no statistical significance effects. The MANOVA of significance performed on PCA of the leaf shape data showed significant differences among the studied populations for the flowering time, their habitats (F = 2.974, P = 0.000; F = 13.707, P = 0.000 respectively).

Discussion

Intraspecific leaf shape variation

Leaves of individuals from the species complex of A. grandiflora showed distinct patterns of shape and elongation variation, that were well related to the two cytotypes (diploids and tetraploids). Moreover, the leaf elongation in the A. grandiflora species complex ranged from acicular or linear to lanceolate, with an aristate apex, perfectly continuous margins, glabrous and entire. Leaves of tetraploids were typically lanceolate and the leaves of diploids were typically acicular or linear. These results agree partially with the general morphological description of the lanceolate or linear-lanceolate leaves of our model A. grandiflora in the French Flora (Coste 1937) and the Flora of the Jurassic Chains [START_REF] Grenier | Flora de la Chains Jurassique[END_REF]. Furthermore, the tetraploid specimens that were investigated in this study were restricted to the high-altitude areas in both the Swiss Jura mountains and the alpine mountains.

Why does leaf shape differ in the A. grandiflora species complex?

Many hypotheses have been proposed to explain the leaf shape variation. In fact, leaf shape reflects complex assemblages of shape-determining elements: genetic control, environmental factors (Dkhar and Pareek 2014); reviewed in a model plant, Arabidopsis thaliana by (Tsukaya 2005), and hormonal control. The gibberellin (GA) reduce marginal elaborations of tomato leaves [START_REF] Hay | The gibberellin pathway mediates KNOTTED1-type homeobox function in plants with different body plans[END_REF][START_REF] Jasinski | PROCERA encodes a DELLA protein that mediates control of dissected leaf form in tomato[END_REF]. Moreover, many genes have been described that control leaf development and shape (reviewed by Byrne 2005;[START_REF] Li | A brassica rapa linkage map of EST-based SNP markers for identification of candidate genes controlling flowering time and leaf morphological traits[END_REF]Tsukaya 2005). Several studies reported important relationships between the DNA amount and many phenotypic characteristics such as leaf and flower characters (both discrete and continuous quantitative characters) in the polyploid group of Ranunculus

The investigation within a widespread species of plants at large spatial scales in nature exhibits opportunities to characterize the importance of differentiation in many domains of biological research.

The general objective of this doctoral thesis was to give a faithful portrait about the variation within the species complex of Arenaria grandiflora, with a view to providing complementary information useful for improving the management decisions for the future population-level conservation plans.

Differentiations among populations

Understanding the genetic or phenotypic patterns of differentiation among natural populations is one of the central concerns of many types of research such as population genetics, ecology, taxonomy, and conservation research. Moreover, the plant population is one of the most basic levels of plant studies, and because of naturally fragmented distributions of plant populations, an important genetic differentiation should be found among these naturally fragmented populations. For example, the isolated populations of a rare alpine species Campanula thyrsoides L. show a high genetic diversity and a considerable population differentiation [START_REF] Aaegisdóttir | Isolated populations of a rare alpine plant show high genetic diversity and considerable population differentiation[END_REF]). In the case of the Arenaria grandiflora species complex, within its range of the widespread distribution, there were isolated, small populations (< 20 individuals; in Mt Chasseral and Ste Baume (obs. perso.)), and often threatened (or extinct; on the French side of Mt Jura and in the Fontainebleau forest [START_REF] Prunier | Données nouvelles sur quelques éléments remarquables de la Réserve Naturelle de la Haute-Chaîne du Jura[END_REF]) and perso. obs.), and genetically distinct population (Favarger 1959(Favarger , 1962;;Küpfer 1974). Thus, the present project provides a synthesis of results from studies on the natural populations of this species complex (in total 27 population) from

Western Europe. This study has also proven interesting to ongoing research into patterns of genetic, cytogenetic and phenotypic variation within A. grandiflora species complex. The variation patterns of A. grandiflora populations seemed to be characterized by their ploidy levels i.e. chromosome numbers as well as their genome size, likewise, their seed germination percentage and leaf shape. As far as we know, this is the first integrative study of genetic and phenotypic diversities of A. grandiflora species complex on a large geographical scale across Western Europe, for which, we applied different approaches.

Firstly, the microsatellite analysis was one of the most important indications that revealed a differentiation among populations. Though microsatellite markers are routinely used to investigate the genetic structuring of natural populations, reliable estimates of differentiation among populations could

Tetraploid cytotypes

However, all the investigated populations (except Mt Chasseral population in Swiss Jura, already protected since 2005) with high genome size (from 4.30 to 5.27 pg.) have been considered, in this project, as distinct ESUs. The tetraploidy of all the following populations: the alpine and pre-alpine populations (Mt Lure, Mt Cheval Blanc, the Chaudière and Mt Raton), the Swiss Jura population (Mt Chasseral) and the Spanish Pyrenees populations (Ceresa and Gabas) has also been detected for the first time. The present results have showed that the seeds of all investigated tetraploids were physically dormant (seed coat dormancy). Though all those populations have been defined as ESUs, the Mt Cheval Blanc population, the highest population (i.e. situated at 2300 m) deserves to be interesting issue in the future studies. It differed significantly in genome size (4.56±0.03 pg.) from other both alpine (Mt Lure)

and pre-alpine populations (Chaudière and Mt Raton). In addition, the seeds from Mt Cheval Blanc population could keep its viability and germinability (GP ± s.d. = 82.85± 7.76 %) for a longer period (until 300 months) with storage at the +4°C temperature and 40% humidity. Moreover, the germination percentage for the Mt Cheval Blanc population showed a significant difference from both pre-alpine populations (Mt Raton; GP ± s.d. = 62.03± 14.29 %; and Mt Chaudière; GP ± s.d. = 69.57± 11.31) that were stored during 84 and 96 months respectively.

As observed in the French National Herbarium (in Paris), the specimens from the tetraploid alpine and pre-alpine populations (Mt Lure, Mt Cheval Blanc, the Chaudière and Mt Raton) have not been harvested, except tetraploid specimens from the Swiss Jura populations (Mt Chasseron, Mt Suchet, and Mt Chasseral) and from the alpine population of Mont Ventoux. Moreover, the leaves of tetraploids (from the Swiss Jura populations and the alpine population of Mont Ventoux) were typically lanceolate and differ significantly from both the acicular and linear ones in the diploid populations. Thus, it will be very interesting to study the morphometric traits of leaves of the alpine and pre-alpine populations from its natural distributed range (Mt Lure, Mt Cheval Blanc, Chaudière and Mt Raton) in the future researches about this species complex of A. grandiflora.

Furthermore, one of the most interesting populations, that is already protected in Switzerland, is the Mt Chasseral population, that grows in rock debris and at a full sun exposure in very well-drained soil (Favarger 1959;[START_REF] Hess | Vitro Culture Introduction for Exsitu Conservation of Some Rare Plant Species[END_REF]. This population has only 8 individuals (obs. perso., 2013), so,