**3.** *Vanilla* **biodiversity in the wild**

Most *Vanilla* species are hemiepiphytic vines climbing up to 30 meters high (*V. insignis*) (Soto Arenas & Dressler, 2010) and growing in tropical wet forests between 0-1000m (Portères, 1954). Only a few species are adapted to drier conditions (*V. calycullata*, (Soto Arenas & Dressler, 2010)), although extreme xeric adaptation is observed in the 18 leafless species of the genus (Portères, 1954). Vegetative reproduction (by natural stem cuttings) is the predominant reproduction mode adopted by most *Vanilla* species to develop settlements, such as *V. bahiana*, *V. chamissonis*, *V. madagascariensis*, *V. dilloniana*, *V. barbellata*, *V. claviculata* (reviewed in (Bory et al., 2010)). Some vines can grow up to 100 meters long (*V. insignis* (Soto Arenas & Dressler, 2010)) and in *V. planifolia* the same individual can cover up to 0.2ha (Soto Arenas, 1999). However a few species might be strictly sexually reproducing, such as *V. bicolor* and *V. palmarum* which are described as epiphytic on palm trees (Householder et al., 2010; Pignal, 1994), and *V. mexicana* (Bory et al., 2010; Cameron, 2010). Another notable exception is the species *V. dietschiana* which is non lianescent and 40 cm high, and has long been classified for these reasons as a different genus *Dictyophyllaria*  (Pansarin, 2010a, 2010b; Portères, 1954).

of the *Vanilla* genus and concluded that *Vanilla* must be considered as a TCG, a "Taxonomic Complex Group" (Ennos et al., 2005). Indeed, it exhibits (i) an uniparental reproduction mode (vegetative growth) (Portères, 1954) (ii) interspecific hybridization in sympatric areas (Bory et al., 2010; Bory et al., 2008c; Nielsen, 2000; Nielsen & Siegismund, 1999) and (iii) polyploidy (Bory et al., 2010; Bory et al., 2008a; Lepers-Andrzejewski et al., 2011a; Lepers-Andrzejewski et al., 2011b). These mechanisms have profound effects on the organization of the biological diversity and have been described as responsible for the difficulty to define discrete, stable and coherent taxa in such TCGs (Ennos et al., 2005). *Vanilla* is a typical example of a genus for which the barcoding protocols (*mat*K and *rbc*L) as proposed by the CBOL (M.L. Hollingsworth et al., 2009; P.M. Hollingsworth & CBOL Plant Working Group, 2009 ; Ratnasingham & Hebert, 2007), will therefore not be sufficient to revise the species taxonomy. The lack of genetic incompatibility between most *Vanilla* species (Bory et al., 2010) and the proven occurrence of inter-specific hybridizations in the genus (Bory et al., 2010; Bory et al., 2008c; Nielsen, 2000; Nielsen & Siegismund, 1999) will necessitate the obligate survey of nuclear regions in addition to cpDNA markers to resolve introgression patterns and correctly identify *Vanilla* species (Rubinoff, 2006). As an example, the species *V. ×tahitensis* was recently shown to be a *V. planifolia* x *V. odorata* hybrid using a combined ITS and chloroplastic phylogenetic analysis (Lubinsky et al., 2008b), when chloroplastic DNA alone repeatedly identified this species as identical to its maternal donor parent *V. planifolia* (figure 1). Moreover molecular genetic diagnostics can only be useful for barcoding biodiversity when species delimitations are either subtle or cryptic but nonetheless clear-cut. In a TCG, taxon limits are themselves diffuse, therefore genetic analysis alone might fail in the identification of discrete species (Ennos et al., 2005). A typical example of expected difficulties will be within the *V. pompona* species complex which was recently described as containing subspecies *pompona*, *pittieri*, and *grandiflora* based on ITS data, although the latter two are rather paraphyletic (Soto Arenas & Cribb, 2010) . In *Vanilla*, taxonomic revision of species will therefore have to use a combination of taxonomic, morphological, ecological, reproductive biology, cytogenetic (polyploidy

estimates) and genetic (nuclear and chloroplastic) assessments.

Most *Vanilla* species are hemiepiphytic vines climbing up to 30 meters high (*V. insignis*) (Soto Arenas & Dressler, 2010) and growing in tropical wet forests between 0-1000m (Portères, 1954). Only a few species are adapted to drier conditions (*V. calycullata*, (Soto Arenas & Dressler, 2010)), although extreme xeric adaptation is observed in the 18 leafless species of the genus (Portères, 1954). Vegetative reproduction (by natural stem cuttings) is the predominant reproduction mode adopted by most *Vanilla* species to develop settlements, such as *V. bahiana*, *V. chamissonis*, *V. madagascariensis*, *V. dilloniana*, *V. barbellata*, *V. claviculata* (reviewed in (Bory et al., 2010)). Some vines can grow up to 100 meters long (*V. insignis* (Soto Arenas & Dressler, 2010)) and in *V. planifolia* the same individual can cover up to 0.2ha (Soto Arenas, 1999). However a few species might be strictly sexually reproducing, such as *V. bicolor* and *V. palmarum* which are described as epiphytic on palm trees (Householder et al., 2010; Pignal, 1994), and *V. mexicana* (Bory et al., 2010; Cameron, 2010). Another notable exception is the species *V. dietschiana* which is non lianescent and 40 cm high, and has long been classified for these reasons as a different genus *Dictyophyllaria* 

**3.** *Vanilla* **biodiversity in the wild** 

(Pansarin, 2010a, 2010b; Portères, 1954).

In natural conditions, vanilla plant density can be extremely variable from being very high in certain areas (*V. trigonocarpa* (Soto Arenas & Dressler, 2010), *V. pompona* (Householder et al., 2010)) from very low as reported for wild *V. planifolia* in Mexico with less than one plant found per square kilometre (Soto Arenas, 1999). Some species are known to flower very frequently (*V. chamissonis,* (Macedo Reis, 2000)) to very un-frequently (*V. planifolia, V. hartii*, (Schlüter, 2002; Soto Arenas & Dressler, 2010)). A single flower per inflorescence generally opens in *Vanilla*, except 2-3 in some species (*V. odorata, V. martinezii, V. insignis*) and flowers are ephemeral (one day) except for some rare species such as *V. inodora* (2-3 days) (Soto Arenas & Dressler, 2010) or *V. imperialis* for which the flowers can be fertilized 4-5 days after opening (unpublished data). Seedlings can be found very frequently for species such as *V. bicolor* and *V. palmarum* (Householder et al., 2010) or be extremely rare as in *V. pompona* in Madre de Dios (Householder et al., 2010) or *V. planifolia* in Mexico (Schlüter, 2002). All these natural history traits will have deep effects on the levels of *Vanilla* species biodiversity that can be found in the wild. Particularly, the relative balance between vegetative and sexual reproduction and their relative efficiency will be of major importance in shaping populations genetic diversity. Exploring *Vanilla* species reproductive systems is therefore essential in this context.
