Vanilla

Séverine Bory, Spencer Brown, Marie-France Duval, and Pascale Besse

Introduction

Intraspecific Diversity

The aromatic species Vanilla planifolia G. Jackson, syn. V. fragrans (Salisb.) Ames, the main source of commercial vanilla, was disseminated from its area of origin (Mexico) following the discovery of the Americas by Christopher Columbus. Plantations were easily established by cuttings but pod production was unsuccessful in the absence of natural pollinators in the areas of introduction. In 1841, a simple method to hand-pollinate vanilla was discovered by Edmond Albius, a slave, in Reunion Island, and vanilla cuttings rapidly spread from Reunion Island to the Indian Ocean area and worldwide (Bory et al., 2008c; Kahane et al., 2008; see Chapter 17). As a consequence of this dissemination history, extremely low levels of genetic diversity are observed in vanilla plantations worldwide as shown by recent molecular genetic studies (Besse et al., 2004; Bory et al., 2008b, 2008d; Lubinsky et al., 2008a; Minoo et al., 2007; Sreedhar et al., 2007) suggesting a single clonal origin for the vanilla crop. This clone 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 (Portères, 1954). Cuttings were sent to the botanical gardens of Paris (France) and Antwerp (Belgium) from where these specimens were disseminated worldwide (Bory et al., 2008c; Kahane et al., 2008). It is thus surprising to observe an important morphological diversity in V. planifolia in the areas of introduction such as Reunion Island (Bory et al., 2008b, 2008c, 2008d) for a crop with a clonal origin and vegetatively propagated by cuttings.

All these observations raise important questions regarding the processes that might be involved in the evolution and diversification of vanilla. Some of the key processes that have been identified so far and the explanations that these can provide for the genetic and taxonomic complexity observed in Vanilla are discussed.

FIGURE 2.1 Morphological vegetative traits in Vanilla species from the CIRAD collection in Reunion Island (see Chapter 3): (a) typical leaf specimen for some species; (b) principal component analysis of vegetative traits (leaf and stem) measured in different species showing the importance of intraspecific variations leading to overlapping of species.

Vegetative versus Sexual Reproduction

For most Vanilla species, vegetative growth occurring naturally from stem cuttings (Portères, 1954) is the predominant reproductive mode, and appears as an efficient strategy adopted by the plant to develop settlements (Figure 2.2). Stems running on the ground are frequently observed, giving new roots and creating new individuals when the stem is cut, as reported for species such as V. bahiana Hoehne (Pignal, 1994) and V. chamissonis Klotzsch (Macedo Reis, 2000) in Brazil, V. barbellata Reichenbach f., V. claviculata (W. Wright) Swartz and V. dilloniana Correll (Nielsen and Siegismund, 1999) in Puerto Rico or V. madagascariensis Rolfe in Madagascar (P. Besse, pers. obs.). In Mexico, with reference to V. planifolia, in natural conditions, the same individual can cover up to 0.2 ha (Soto Arenas, 1999).

In Vanilla species, a rostellum membrane separates the female and male parts of the flower, and pollination therefore depends on the intervention of external pollinators. A notable exception is the species V. palmarum (Salzm. ex Lindl.) Lindl., which spontaneously self-pollinates (Bory et al., 2008c; Soto Arenas, 2006). Consequent, due to the need for pollinators, sexual reproduction is rarely observed in natural conditions. For V. planifolia, rates of 1% to 1‰ are reported (Bory et al., 2008c; Soto Arenas, 1999) with possible natural pollinators in America being orchid bees from the Euglossa and perhaps from the Eulaema genera (Lubinsky et al., 2006; Soto Arenas, 2006). Sexual reproduction rates reported for the species V. chamissonis (6% autogamy and 15% allogamy) are also relatively low (Macedo Reis, 2000).

FIGURE 2.2 Typical vegetative growth observed in Vanilla species. Left: V. madagascariensis in Madagascar. Right: V. pompona in Guadeloupe. (Courtesy of P. Besse.)

However, even rare sexual reproduction events can generate an important genetic diversification because a single sexual reproduction event is able to generate numerous genotypes that can vegetatively propagate rapidly. Heterozygosity observed in V. planifolia was reported to be 0–0.078 using isozymes (Soto Arenas, 1999), 0.154 using SSR markers (Bory et al., 2008b) and 0.293 using AFLPs (Bory et al., 2008d). Given these heterozygosity levels, even selfing can generate genetic diversity, as demonstrated through manual self-pollination experiments (Bory et al., 2008d) leading to increased diversity estimates (D_max from 0.106 to 0.140) through novel allelic combinations (Figure 2.3). This is well illustrated in the case of V. planifolia in areas of introduction, where natural pollinators are absent. In these areas, such as in Reunion Island, traditional cultivation practices involve vine propagation by cuttings, and manual self-pollination to produce pods. This resulted in the appearance of novel vanilla varieties such as the “Aiguille” type observed in Reunion Island, which most likely resulted from accidental seed germination in the field from a forgotten pod, and subsequent vegetative propagation of the individual (Bory et al., 2008d) (Figure 2.3). Such a novel type can rapidly spread in plantations given the vegetative propagation used to multiply vines. This must also happen in the wild. A combination of sexual and vegetative reproduction, where one creates diversity and the other helps settlement, has already been suggested for the species V. pompona Schiede and V. bahiana in tropical America based on AFLP patterns (Bory et al., 2008d). Sexual reproduction is therefore a key evolutionary process for most species of the genus despite its low rates and because of their major vegetative reproduction. A few species of Vanilla appear to rely solely on sexual reproduction for propagation. This is the case for V. palmarum, which is entirely epiphytic on a palm tree with a short lifecycle (Pignal, 1994) and for V. mexicana Mill. (syn V. inodora Shiede) in which even artificial vegetative propagation is unsuccessful (P. Feldmann, pers. com.) (Figure 2.4).

FIGURE 2.3 Factorial analysis from AFLP markers on different American Vanilla species illustrating the increased diversity for V. planifolia selfed progenies.

FIGURE 2.4 Exclusive sexually reproducing species. Left: V. palmarum in the CIRAD collection. Fruits were spontaneously obtained in an insect proof quarantine glasshouse. (Courtesy of M. Grisoni.) Right: V. mexicana in Guadeloupe. (Courtesy of P. Besse and P. Feldmann.)

Interspecific Hybridization

The main factors preventing interspecific hybridization in the Orchidaceae family are pre-pollination mechanisms such as pollinator specificity, flowering phenologies, or mechanical barriers in flowers (Dressler, 1981; Gill, 1989; Grant, 1994; Paulus and Gack, 1990; Van Der Pijl and Dodson, 1966). On the contrary, genetic incompatibility between closely related species is rarely observed (Dressler, 1993; Johansen, 1990; Sanford, 1964, 1967). This is also the case for Vanilla. Indeed, most inter specific artificial crosses attempted to date in Vanilla have been successful showing the lack of genetic incompatibility between the species involved. Interspecific hybrids were successfully obtained between closely related American species (V. planifolia × V. tahitensis J.W. Moore—accession Hy0003 in Figures 2.1 and 2.3, V. planifolia × V. pompona) in breeding programs in Madagascar (Bory et al., 2008c), and even between distantly related species such as the Indian V. aphylla Blume and the American V. planifolia in breeding programs in India (Minoo et al., 2006).

There is a growing evidence for the occurrence of natural interspecific hybridization in Vanilla. A study on three native species of Vanilla, V. claviculata, V. barbellata, and V. dilloniana in the western part of the island of Puerto Rico, showed the possibility of interspecific hybridization between V. claviculata and V. barbellata in sympatric areas (Nielsen, 2000; Nielsen and Siegismund, 1999). This was demonstrated by using isozyme markers, and floral morphological observations confirmed the hybrid status of sympatric populations. On the other hand, V. dilloniana, showing a different phenology, did not hybridize with the other two species. Recent work using AFLP and SSR markers also suggested the possibility of interspecific hybrid formation in tropical America, involving species such as V. bahiana, V. planifolia, or V. pompona (Bory, 2007; Bory et al., 2008d) (Figure 2.5). The species V. tahit-ensis was also recently shown using nuclear ITS and cp DNA sequences to result from intentional or inadvertent hybridization between the species V. planifolia and V. odorata C. Presl that could have happened during the Late Postclassic (1350– 1500) in Mesoamerica (Lubinsky et al., 2008b).

FIGURE 2.5 Flowers, fruits, and leaves from accession CR0068 (a) from Costa Rica, putatively identified as a hybrid species derived from a maternal V. planifolia donor species (b) based on AFLP, SSR, and cp DNA markers data. (Courtesy of M. Grisoni.)

Polyploidization

We recently demonstrated the occurrence of recent polyploidization events (in less than 200 years as V. planifolia was introduced in 1822) in Reunion Island (Bory et al., 2008a). Congruent evidences (AFLP markers, genome size, chromosome counts, and stomatal length) showed the formation of auto-triploid self-sterile types (“Stérile”), as well as auto-tetraploid types (“Grosse Vanille”), with genome sizes of 2C = 7.5 and 10 pg, respectively, as opposed to 5 pg for conventional “Classique” varieties (Bory et al., 2008a) (Table 2.1). The most likely formation of these types was suggested to be through manual self-pollination accompanied by the formation of unreduced 2n gametes (Bretagnolle and Thompson, 1995), seed germination from a forgotten pod, followed by vegetative multiplication of the individuals (Bory et al., 2008a) (Figure 2.6). Polyploidy was also reported for the cultivated species of V. tahitensis in Polynesia with the existence of diploid and tetraploid (i.e., “Haapape”) types (Duval et al., 2006; see Chapter 13) and this species might have resulted from both polyploidy and sexual regeneration following its V. planifolia × V. odorata origin (Lubinsky et al., 2008b).

FIGURE 2.6 Schematic representation of the possible formation of autotriploid and autotetra-ploid V. planifolia types in Reunion Island.

Polyploidization could therefore be a major phenomenon in the evolution of Vanilla. In order to put this hypothesis to test, we conducted a preliminary survey on genome size variation in different Vanilla species. Thirty-eight accessions were analyzed by flow cytometry according to the protocol detailed by Bory et al. (2008a) using wheat as an internal standard: Triticum aestivum L. cv. Chinese Spring, 2C = 30.9 pg, 43.7% GC (Marie and Brown, 1993). These accessions belong to 17 different Vanilla species and also included 3 artificial hybrids (V. planifolia × V. planifolia, V. planifo-lia × V. tahitensis, V. planifolia × V. phaeantha Rchb. f.). The entire leaf samples were collected from vines maintained in the Vanilla genetic resources collection of CIRAD in Reunion Island (see Chapter 3 and Grisoni et al., 2007). Details for each accession (species, putative continent of origin, place of sampling, and genome size) are presented in Table 2.1. For each species, fluorescence histograms revealed five endoreplicated peaks and the marginal replication ratio was still irregular (from 1.5 to 1.8 instead of 2), as encountered in V. planifolia (Bory et al., 2008a).

2C nuclear DNA content varied from 4.72 (±0.05) pg (V. tahitensis) to 12.00 (±0.11) pg (V. phalaenopsis Reichb. f. ex Van Houtte) for 34 wild accessions. One accession CR0067 (Vanilla sp.) had an extreme value at 22.31 pg (one measure with wheat standard) (Table 2.1), which was confirmed by using another standard (pea, data not shown). Intra-accession variation coefficients did not exceed 5%.

These results indicate that genome size variations exist in Vanilla, which could suggest the occurrence of polyploid species, based on what was detected for V. planifolia (Bory et al., 2008a). In particular, African accessions displayed bigger genome sizes than American accessions, with 2C DNA content ranging from 6.93 to 22.31 pg and 4.72 to 9.23 pg, respectively. African species may, therefore, have been subjected to more dramatic genomic rearrangements and polyploidization events than their American or Asian counterparts. Finally, as it was observed in V. planifolia in Reunion Island (Bory et al., 2008a), intraspecific genome size variations were revealed in some species (V. imperialis Kraenzl., V. albida Blume), which may reflect the occurrence of different ploidy levels even within species. These results need to be explored further by chromosome counts for each species, but they already strongly suggest that polyploidy might be a major phenomenon in the evolution of Vanilla.

In the first three lines, are given results for V. planifolia according to the study of Bory et al. (2008a).

^a According to Portères (1954).

^b From the study of Bory et al. (2008a).

Conclusions

There is, therefore, growing evidence that demonstrate the complexity of the processes involved in the evolution and diversification of Vanilla. As for many species for which vegetative reproduction is predominant, we observed phenotypic diversity at the intraspecific level in V. planifolia, which was not congruent with the observed low genetic diversity of this clonal crop. We demonstrated that this discrepancy was in part due to the occurrence of rare sexual reproduction events, as well as to the occurrence of polyploidization in this species. Given that these variations have happened in Reunion Island in less than 200 years; there is little doubt that such intraspecific variations exist in other species of the genus found in the wild, and might be responsible for the difficulty to correctly identify species solely based on morphological observations. This is exacerbated by the occurrence of interspecific hybridizations in the genus, which makes clear taxonomic designation is even more delicate.

Vanilla can therefore be considered as a TCG, a “Taxonomic Complex Group” sensu Ennos et al. (Ennos et al., 2005). Indeed, it exhibits (1) a uniparental reproduction mode (vegetative reproduction), (2) interspecific hybridization in sympatric areas, and (3) polyploidy. These mechanisms have profound effects on the organization of the biological diversity and have been described as being responsible for the difficulty to define discrete, stable and coherent taxa in such TCGs (Ennos et al., 2005). TCGs are widespread in plants and uniparental reproduction can produce a complex mixture of sexual outcrossing and uniparental lineages that can be at different ploidy levels and the whole complex can be involved in reticulate evolution generating novel uniparental lineages by hybridization (Ennos et al., 2005). This has great implications on the way conservation programs should be conducted. In such TCGs, as it is often not possible to classify biodiversity into discrete and unambiguous species, traditional species-based conservation programs are not appropriate. As recommended, in situ conservation should focus on the evolutionary processes that generate taxonomic diversity rather than on the poorly defined taxa resulting from this evolution (Ennos et al., 2005). This includes concentrating on species that might be widespread (and thus not concerned by classical conservation efforts) but responsible for the generation of taxonomic diversity (through hybridization, introgression, or polyploidization).

Therefore, not only the mechanisms described in this chapter provide a better understanding of the Vanilla genus evolution, but they also are of major importance with regard to future genetic resources management and conservation (Crandall et al., 2000; Moritz, 2002).

These mechanisms are also of major interest with regard to the future improvement of V. planifolia. Interspecific hybridizations between V. planifolia and other aromatic species have already proved successful. In Madagascar, the production of V. planifolia × V. tahitensis hybrid variety “Manitra ampotony” led to an increased vanillin content (6.7% vanillin versus 2.5% in V. planifolia) and the (V. planifolia × V. pompona) × V. planifolia “Tsy taitry” shows increased resistance to different diseases (Dequaire, 1976; FOFIFA, 1990; Nany, 1996). In India, V. planifolia × V. aphylla hybrids were produced to increase Fusarium resistance (Minoo et al., 2006). At the intraspecific level, self-pollination could also be used to increase diversity in this genetically uniform crop (Bory et al., 2008d; Minoo et al., 2006). Furthermore, the agronomic characterization (vigor, resistance, vanillin production) of autotetraploid types is currently being performed in Reunion Island as a first step for testing for the potentialities of polyploidy breeding strategies in V. planifolia.

Unraveling the evolution and acquisition of traits of agronomic interest in the genus will also be of major importance. These traits include fragrance of fruits, which despite its considerable breeding interest has received limited attention particularly with regards to its evolution. Fragrant fruit species are almost exclusively restricted to America (Soto Arenas, 2003), and this character could have been selected as favoring fruit dispersion by bats (Soto Arenas, 1999) or sticky-seed dispersion by bees through fruit fragrance collection as observed in V. grandifl ora (Lubinsky et al., 2006). This matter was recently addressed by surveying intron length variations in the COMT gene family (Besse et al., 2009), encoding key enzymes in the phenylpropanoid pathway putatively involved in the biosynthesis of vanillin. Further work is also needed to understand the evolution, mechanisms and genetic determinism of spontaneous self-pollination in the genus (V. palmarum) — a highly desirable character that would considerably reduce bean production costs. Finally, elucidating how the aphyllous species of the genus have evolved and differentiated might be of great interest in our understanding of adaptation to dry conditions, given the predicted future of great climatic changes.

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