**4.3 Conclusions**

16 The Dynamical Processes of Biodiversity – Case Studies of Evolution and Spatial Distribution

hypomethylated at locus Eco-AG/Hpa-AAC/98bp and CR0341 at locus Eco-AC/Hpa-ATT/426bp. Accessions CR0632 and CR0711 were hypermethylated, they presented some bands in their *Msp*I profiles whereas the other accessions presented these bands in both their *Hpa*II and *Msp*I profiles. Accession CR0632 was hypermethylated at locus *Eco*-AG/*Hpa*-ATG/205bp and at locus *Eco*-AG/*Hpa*-AAC/382bp. Finally, accession CR0711 was

These results showed that methylation is present in *V. planifolia* genome, with 7.45% of the fragments revealed being methylated. This value is in accordance with methylation rates reported in banana (7,5%, (Noyer et al., 2005)), but less than what is revealed in other conventionally propagated plant species such as rice (16.3%, (Xiong et al., 1999)), other bananas (18.4%, (Peraza-Echeverria et al., 2001)), apple (25%, (Xu et al., 2000)) and cotton

A limited amount of methylation polymorphism (1%) was detected among 'Classique' and 'Mexique' accessions but the methylation patterns revealed were accession specific. Even for CR0340/0341/0342 which are three clones of the same accession, two different methylation polymorphisms were revealed in CR0340 and CR0341 and none in CR0342, showing that these methylation patterns are either not transmitted trough asexual propagation, or have appeared after clonal propagation. In all cases, no methylation marker was identified which could allow to specifically distinguish the 'Classique' and 'Mexique' morphotypes. A similar conclusion was obtained in studies performed on vegetatively propagated plants such as banana (Baurens et al., 2003; Noyer et al., 2005). Methylation polymorphisms were revealed

We therefore conclude that the 'Mexique' morphotype showing no detectable AFLP or MSAP polymorphism is most probably the result of a limited genetic or epigenetic

The mysterious history of the origin of *V. ×tahitensis*, the so called Tahitian vanilla, has partly been solved. As opposed to its allied species (*V. planifolia*) it cannot be found wild in tropical American forests (Moore, 1993; Portères, 1954; Soto Arenas & Cameron, 2003) but was described from cultivated material found in the Island of Raiatea (Lubinsky et al., 2008b), where it had been introduced via the botanical garden of Papeete from the Philipines in 1848 (Soto Arenas & Dressler, 2010). Molecular sequencing (ITS and cpDNA) have recently shown that *V. ×tahitensis* would be a hybrid, intentional or not between *V. planifolia* and *V. odorata* dating from vanilla exploitation by Mayas in Mesoamerica between years

As much as 18 different morphotypes are described in *V. ×tahitensis* in French Polynesia beside the most widely cultivated type 'Tahiti' (Lepers-Andrzejewski et al., 2011a). These include: 'Haapape' (the second most cultivated type because of its bigger fruits), 'Tahiti Court', 'Tiarei', 'Ofe Ofe', 'Oviri', 'Parahurahu' and 'Sterile'. A study of 16 different accessions using AFLP markers revealed a Dmax value of 0.150, a slightly higher value than what was revealed in *V. planifolia*. All accessions had patterns related to that of 'Tahiti' (either identical or showing missing bands) which led the authors to conclude of a single introduction event in French Polynesia of a 'Tahiti' vine, consistently with the fact that this accession is the oldest one recorded in Polynesia (Lepers-Andrzejewski et al., 2011a). Ten accessions showed more or less the AFLP profile of 'Tahiti'. These included 'Haapape' and 'Tiarei', which were shown to be autotetraploids based on flow cytometry and chromosome

hypermethylated at locus *Eco*-AG/*Hpa*-AAG/393bp.

but could not be correlated to morphological variations.

dominant mutation event with pleiotropic effects.

**4.2** *V. ×tahitensis* **in French Polynesia** 

1359-1500 (Lubinsky et al., 2008b).

(32%, (Keyte et al., 2006)).

These results therefore highlight two different domestication models. In both cases, the genetic base of the cultivated material is very narrow with obviously a single genotype introduced ('Classique' *V. planifolia* in Reunion Island and other cultivation areas; 'Tahiti' *V. ×tahitensis* in French Polynesia). Genetic variation revealed is however slightly higher in *V. ×tahitensis* than in *V. planifolia* because most of *V. ×tahitensis* morphotypes have resulted from selfing of the original 'Tahiti' (with sometimes more than one generation involved) (Lepers-Andrzejewski et al., 2011a). Only one rare case of self-pollination ('Aiguille') was detected in Reunion (Bory et al., 2010). This shows that deliberate or inadvertent seed germination has been strongly involved in the domestication of *V. ×tahitensis* in French Polynesia. In Reunion Island, the limited amount of variation revealed is more related to vegetative propagation and the consecutive accumulation of point mutations.

In both cases however, a noticeable diversification was achieved through polyploidy. Autotetraploidy generated varieties with bigger leaves and fruits, and autotriploidy generated self-sterile individuals. It is noteworthy that self-sterile *V. planifolia* varieties were also described in Mexico ('Oreja de Burro') (Castillo Martinez & Engleman, 1993; Soto Arenas & Dressler, 2010). It is most likely that these have resulted as well from autotriploidy. These results, as well as those that surveyed genome sizes in a wide range of *Vanilla* species (Bory et al., 2010) provide converging evidences for the importance of polyploidy and genome rearrangements during *Vanilla* evolution. Polyploidy can be of major importance in cultivation as well as in natural populations as triploidy and to a certain extent tetraploidy can be responsible for dramatic loss in fruit set. Further work is therefore needed to assess polyploidization consequences on *Vanilla* reproductive biology.
