**3.1.1 Self-pollinating species**

A few *Vanilla* species are described as spontaneously self-pollinating (Householder et al., 2010; Soto Arenas & Cameron, 2003; Soto Arenas & Dressler, 2010; Van Dam et al., 2010), as suggested by their abnormally high fruit set (table 1). This is consistent with general data in orchids showing that autogamous species display a much higher fruit set (77%) than cross pollinating species for which the majority show fruit set <20% (Tremblay et al., 2005). Based on high fruit set, these suggested autogamous species are *V. palmarum*, *V. savannarum*, *V. bicolor* (American species of the *V. palmarum* group), *V. guianensis*, *V. martinezii* (American species of the *V. mexicana* group) and *V. griffithii* (an Asian species). Possible self-pollination for *V. inodora* is also reported (Soto Arenas & Dressler, 2010), due to the large fruit set observed in some populations, although others have a fruit set as low as 2.5%.


Table 1. Suggested self-pollinating *Vanilla* species and recorded natural fruit sets.

More precise observations are available for some of these species. *V. guianensis* is supposedly self-pollinated at early anthesis, as it was observed that the stigma and the

Biodiversity and Evolution in the *Vanilla* Genus 7

Spontaneous self-pollination is sometimes described even in classically outcrossing species. In Oaxaca plantations, cases of *V. planifolia* self-pollination are reported (Soto Arenas & Cameron, 2003) with rates reaching 6% of covered flowers giving fruit. Similar rates (6.06%) were reported for bagged *V. chamissonis* flowers in Sao Paulo (Macedo Reis, 2000). Nothing

For the majority of *Vanilla* species, self-pollination does not occur due to an efficient rostellum and sexual reproduction therefore relies on the intervention of pollinators. Consequently, relatively low natural fruit sets are observed in natural conditions ((Bory et al., 2008b), table 2), consistent with the 17% median natural fruit set reported for tropical orchids (Tremblay et al., 2005). Reproductive success in orchids is pollination – rather than by resource - limited and could depend on pollinator effectiveness, abundance and diversity, and pollen quantity and quality (self *versus* allopollen) (Tremblay et al., 2005). This was demonstrated by crossing experiments in temperate and tropical orchids showing that cross hand-pollination shows significantly greater success (80%) than natural open pollination (26.6%) (Tremblay et al., 2005). Further studies are needed in *Vanilla* to determine the highest fruit sets achievable, but results on *V. barbellata, V. claviculata, V.dilloniana*, and *V. poitaei* have showed up to 100% fruit set under hand pollination experiments (Tremblay et al., 2005), and 75.76% in *V. chamissonis* (Macedo Reis, 2000), much

Reference

(Soto Arenas & Cameron, 2003)

0.9% (Householder et al., 2010)

is known about the mechanisms involved in such exceptional cases.

higher values than what can be observed in natural conditions (table 2).

(open pollination)

*V. barbellata* 18.2 % (Tremblay et al., 2005) *V. chamissonis* 15% (Macedo Reis, 2000) *V. claviculata* 17.9 % (Tremblay et al., 2005) *V. crenulata* 0% Johansson 1974, as cited in

*V. cristato-callosa* 6.6% (Householder et al., 2010) *V. dilloniana* 14.5 % (Tremblay et al., 2005) *V. planifolia* 1% to 1‰ (Soto Arenas, 1999) *V. planifolia* 1% (Childers & Cibes, 1948) *V. planifolia* 1% (Tremblay et al., 2005) *V. planifolia* 1 à 3% (Weiss, 2002) *V. poitaei* 6.4 % (Tremblay et al., 2005)

*V. riberoi* 1.1% (Householder et al., 2010)

If the pollinator of *V. planifolia* was long been considered as a social bee from the *Melipona*  genus, as reported by Deltiel (as cited in (Rolfe, 1896)) and then mentioned in (Bouriquet, 1954a, 1954b; Stehlé, 1954), these records are now admitted as doubtful (Soto Arenas & Cameron, 2003; Van Der Cingel, 2001) as the bee is too small to perform the necessary

Table 2. *Vanilla* out-crossing species and natural fruit sets recorded.

**3.1.2 Outcrossing species and pollinators** 

Species Natural fruit set

*V. pompona subsp. grandiflora* 

anther grew to contact one another; and no pollinators were observed despite the high fruit set recorded in Peru (Householder et al., 2010). The lack of observed local pollinators and the high fruit set also suggested that *V. bicolor* and *V. palmarum* were self-pollinating species in Peru (Householder et al., 2010).

Two mechanisms were proposed to account for self-pollination in *Vanilla* species (Van Dam et al., 2010): true self-pollination occurring by either stigmatic leak and/or the presence of a dehydrated or reduced rostellum, or agamospermy. In *V. bicolor*, pollen removal experiments showed that agamospermy was not the mechanism in play (Van Dam et al., 2010). Also all fertilized flowers showed fully developed rostellum. This suggested that a stigmatic leak, where stigma lobes release a fluid that contacts the pollen and induces germination of the pollen tubes (Van Der Pijl & Dodson, 1966) was the more likely explanation for self-pollination in this species (Van Dam et al., 2010). The observation of the occurrence of a thick rostellum in *V. palmarum* led to the suggestion of an identical mechanism (Householder et al., 2010). Our own observations on *V. palmarum* reveal selfpollination most likely due to a rostellum reduced in width, allowing pollinaria to get in contact with the stigmata on both sides of the rostellum (figure 2). A similar situation is found for the self-fertile species *V. lindmaniana* (data not shown).

Fig. 2. Detailed structure of the pollinaria, rostellum and stigmata in the species *V. palmarum:* (a) and (b) accession CR0891, (c) accession CR0083, maintained in BRC Vatel (Reunion Island).

anther grew to contact one another; and no pollinators were observed despite the high fruit set recorded in Peru (Householder et al., 2010). The lack of observed local pollinators and the high fruit set also suggested that *V. bicolor* and *V. palmarum* were self-pollinating species

Two mechanisms were proposed to account for self-pollination in *Vanilla* species (Van Dam et al., 2010): true self-pollination occurring by either stigmatic leak and/or the presence of a dehydrated or reduced rostellum, or agamospermy. In *V. bicolor*, pollen removal experiments showed that agamospermy was not the mechanism in play (Van Dam et al., 2010). Also all fertilized flowers showed fully developed rostellum. This suggested that a stigmatic leak, where stigma lobes release a fluid that contacts the pollen and induces germination of the pollen tubes (Van Der Pijl & Dodson, 1966) was the more likely explanation for self-pollination in this species (Van Dam et al., 2010). The observation of the occurrence of a thick rostellum in *V. palmarum* led to the suggestion of an identical mechanism (Householder et al., 2010). Our own observations on *V. palmarum* reveal selfpollination most likely due to a rostellum reduced in width, allowing pollinaria to get in contact with the stigmata on both sides of the rostellum (figure 2). A similar situation is

found for the self-fertile species *V. lindmaniana* (data not shown).

Fig. 2. Detailed structure of the pollinaria, rostellum and stigmata in the species *V. palmarum:* (a) and (b) accession CR0891, (c) accession CR0083, maintained in BRC Vatel

in Peru (Householder et al., 2010).

(Reunion Island).

Spontaneous self-pollination is sometimes described even in classically outcrossing species. In Oaxaca plantations, cases of *V. planifolia* self-pollination are reported (Soto Arenas & Cameron, 2003) with rates reaching 6% of covered flowers giving fruit. Similar rates (6.06%) were reported for bagged *V. chamissonis* flowers in Sao Paulo (Macedo Reis, 2000). Nothing is known about the mechanisms involved in such exceptional cases.
