**3.4 Conclusions**

Many *Vanilla* species are threatened in the wild. This is particularly the case for *V. planifolia* in Mexico, its centre of origin. Proper conservation strategies need to be developed, but this will require gaining a better knowledge on the reproductive strategies and the derived levels of genetic diversity in these *Vanilla* species. This will include assessing the relative contribution of vegetative *vs* sexual reproduction, self-compatibility (auto *vs* allo fecundation success), pollination syndromes (pollinators, reward/deceit) and seed dispersion systems.

There is a considerable lack of genetic studies of *Vanilla* species biodiversity in the wild. The only published data concern the aphyllous species *V. barbellata*, *V. dilloniana* and *V. claviculata* on the island of Puerto Rico (Nielsen, 2000; Nielsen & Siegismund, 1999) using isozyme markers. Genotypic frequencies were in accordance with Hardy-Weinberg proportions for all species, which could suggest random crosspollination. High differentiation among populations was detected, supposedly attributed to limited seed dispersal by bees. Genetic drift was also demonstrated in some isolated populations (Nielsen & Siegismund, 1999). Soto Arenas also conducted *V. planifolia* population genetic studies in Mexico using isozymes (Soto Arenas, 1999), surprisingly demonstrating homozygous excess corresponding to preferential autogamous reproduction for this species. Development of suitable approaches to the analysis of genetic diversity in a spatial context, where factors such as pollination, seed dispersal, breeding system, habitat heterogeneity and human influence are appropriately integrated in combination with molecular

Biodiversity and Evolution in the *Vanilla* Genus 13

Indies, Mexico) and of different morphotypes (from Reunion 'Classique', 'Mexique', 'Sterile', 'Grosse Vanille' (table 3) and from Mexico 'Mansa', 'Acamaya', 'Mestiza') (Bory et al., 2008c; Lubinsky et al., 2008a) derive from a single introduced genotype. It could correspond to the lectotype that was introduced, early in the nineteenth century, by the Marquis of Blandford into the collection of Charles Greville at Paddington (UK) (Portères, 1954). Cuttings were sent to the botanical gardens of Paris (France) and Antwerp (Belgium) from where these specimens were disseminated to Reunion Island (by the ordinance officer

Consequently, cultivated accessions in Reunion Island exhibit extremely low levels of genetic diversity and have evolved by the accumulation of point mutations through vegetative multiplication (Bory et al., 2008c) (table 3). Maximum genetic distance (Dmax) was 0.106 and the majority of the polymorphic AFLP bands revealed had frequencies in the extreme (0-10% and 90-100%) ranges, therefore corresponding to rare AFLP alleles (presence or absence) a pattern typical of point mutations (Bory et al., 2008c). One peculiar and rare phenotype 'Aiguille' found in Reunion Island was shown to result from sexual reproduction (selfing) (Bory et al., 2008c) (table 3) as its AFLP pattern fell within a group of selfed progeny with Dmax=0.140 and showed a strong pattern of segregation bands. The hypothesis was that it resulted from manual self-pollination and subsequent seed germination from a forgotten pod (Bory et al., 2008c). Flow cytometry, microdensitometry, chromosome counts and stomatal lengths showed that polyploidization has been actively involved in the diversification of *V. planifolia* in Reunion Island (Bory et al., 2008a). Three ploidy levels (2x, 3x, 4x) were revealed that allowed to explain the features of the 'Sterile' type which is autotriploid and of the 'Grosse Vanille' type, auto-tetraploid (Bory et al., 2008a). It was suggested that these resulted from the production of non-reduced gametes during the course of

of Bourbon, Marchant) and then worldwide (Bory et al., 2008b; Kahane et al., 2008).

manual self-pollination performed by growers (Bory et al., 2010; Bory et al., 2008a).

and transmitted as well as by asexual propagation (Peraza-Echevarria et al., 2001).

As the particular phenotype 'Mexique' encountered in Reunion could not be explained by genetic or cytogenetic variations, we tested whether it could have resulted from epigenetic modifications as some studies showed that morphological variations in clonal populations could be explained by a combination of genetic and epigenetic factors (Imazio et al., 2002). Epigenetics corresponds to reversible but heritable modifications of gene expression without changes in the nucleotidic sequence (Mathieu et al., 2007; Wu & Morris, 2001), such as DNA methylation (Finnegan et al., 1998). Epigenetic modifications are heritable (Akimoto et al., 2007; Finnegan et al., 1996; Grant-Downton & Dickinson, 2006; Martienssen & Colot, 2001)

Sometimes, a phenotypic reversion correlated with demethylation of the epi-mutated gene can occur and its expression is restored (Jaligot et al., 2004). These epigenetic mutations have important phenotypic as well as evolutionary consequences, this representing a current field of investigation (Finnegan, 2001; Kalisz & Purugganan, 2004; B. Liu & Wendel, 2003). DNA methylation proceeds by the addition in a newly replicated DNA of a methyl group by a DNA methlytransferase (Finnegan et al., 1998; Martienssen & Colot, 2001). Cytosine is the most frequently methlylated base, resulting in 5-methylcytosine formation (5mC) (Martienssen & Colot, 2001). Plant methylation is restricted to the nuclear genome and is concentrated in repeated sequence regions (Finnegan et al., 1998). Methylation is implied in many biological processes such as 'gene silencing', mobile DNA elements control, DNA replication duration, chromosome structure determination, and mutation frequency increase (Finnegan et al., 1998; Paszkowski & Whitham, 2001). Many spontaneous or induced epimutations are known in maize, Arabidopsis and other plant species and are responsible

population genetic estimates, will be essential (Escuderoa et al., 2003) to provide new insights in the understanding of the mechanisms of maintenance and dynamics of *Vanilla* populations and to provide guidelines for their preservation.
