Vanilla bicolor Lindl. (Orchidaceae) from the Peruvian Amazon: auto-fertilization in Vanilla and notes on floral phenology

Alex R. Van Dam 0 1 2 3 J. Ethan Householder 0 1 2 3 Pesach Lubinsky 0 1 2 3 0 P. Lubinsky Department of Botany and Plant Sciences, University of California , Riverside, CA 92521-0124, USA 1 J. E. Householder Botanical Research Institute of Texas (BRIT) , 509 Pecan St., Fort Worth, TX 76102, USA 2 A. R. Van Dam Department of Entomology, University of California , Riverside, CA 92521-0124, USA 3 A. R. Van Dam (&) Department of Animal Science, University of California Davis , One Shields Avenue, Davis, CA 95616, USA Minimal documentation exists for natural pollination in wild Vanilla spp., despite the economic importance of this genus, additionally commercial vanilla (V. planifolia Jacks.) is one of very few crops whose production depends entirely on artificial pollination. Flowering and fruiting phenology of Vanilla bicolor Lindl., a close relative of V. planifolia, was documented in a palm swamp in the Peruvian Amazon. V. bicolor was found to autofertilize via bagging experiments. This ecotype had an average fruit set per raceme of 42.50 2.5%. Pollen removal experiments suggest that stigmatic leak may be the mechanism by which auto-pollination occurs in V. bicolor. - out-crossing and asexual propagation, auto-pollination has also been reported in Vanilla (van der Pijl and Dodson 1966). Examples of species that are thought to auto-pollinate, as evidenced by their high fruit sets, atypical in outcrossing species, are V. palmarum (Salzm. ex Lindl.) Lindl., V. savannarum Britton, and V. griffithii Rchb. f. (Cameron and Soto Arenas 2003). The mechanisms and concomitant selective pressures for auto-pollination in Vanilla remain unclear at best. There are three proposed mechanisms for autopollination in Vanilla: (1) stigmatic leak by which stigma lobes release a fluid, that contacts the pollen and induces germination of the pollen tubes (van der Pijl and Dodson 1966), (2) by dehydration or underdevelopment of the rostellum, which could also allow for pollen to contact the stigmatic cavity (Garay and Sweet 1974), (3) a third mechanism of self-fertilization in Vanilla that has remained over looked is agamospermy. In the Neuwiedia veratrifolia Blume (Apostasioideae) the method of selfpollination was determined to be auto-pollination not agamospermy via emasculation experiments (Okada et al. 1996; Kocyan and Endress 2001). Thus, auto-pollination is the method of self-fertilization that occurs at the clade sister to the rest of Orchidaceae. Documentation of any of the above three mechanisms remain absent in Vanilla. Vanilla bicolor Lindl. is a widespread locally common hemi-epiphyte endemic to the Neotropics (Christenson 1995). They are found in open swampy areas along rivers in northern South America and exposed humid thickets along mountain crests in the Carribean (Adams 1972; Werkhoven 1986; Christenson 1995; Borhidi 1996). V. bicolor is a close relative of the widely cultivated V. planifolia Jacks (Cameron and Soto Arenas 2003; Lubinsky et al. 2008a) making it an important wild relative. Unlike the exceedingly sparse density of wild V. planifolia plants (Lubinsky et al. 2006, 2008a, b) V. bicolor occur at high densities in southern Peru and we were curious if higher density resulted in higher pollination rate due to increased reward. In this short communication we provide evidence regarding the floral phenology, pollination syndrome (i.e., outcrossing or self-fertilization) and possible mechanism of pollination in a cleistogamous ecotype of V. bicolor from the Southern Peruvian Amazon. Materials and methods Our study site is located in southeastern Peru in the Madre de Dos basin (Fig. 1). The Madre de Dos River is located in the headwaters of the southwest Amazon, draining a portion of the forelands of the Eastern Cordillera. The Madre de Dos River displays high rates of lateral migration and typical meander scroll morphology. Over 300 hundred wetlands occur along the current Madre de Dios River floodplain, generally hugging the terrace escarpments. Some wetlands in particular, palm swamps, are known locally as aguajales, named after the common name of the monodominant palm Mauritia flexuosa L. Carrera, or aguaje. Aguajales in this region range in size from 1 to 2,000 ha, occupying a significant portion of ecologically important floodplain habitat. Wetland soils are permanently saturated year round, fed by perennial spring inputs (personal obsvervation, Householder 2007). Annual average rainfall in the region where our study occurred is 2,995 455 mm. Rainfall is unevenly distributed throughout the year, with greater than 80% falling between October and April. Average temperature fluctuates minimally between 21 and 26 C, however southerly cold fronts rapidly progress northward from Patagonia, known as friajes and are common in June through August (Amazon Conservation Association, unpublished data). These friajes are known to decrease temperatures to 10 C or less in a matter of minutes. Climate data since 2001 is publicly available provided by the Amazon Conservation Association (ATRIUM 2007). Data was collected on V. bicolor in the Peruvian Amazon near the Los Amigos research station in the center of an aguajale (palm swamp) (S12 33.4530 W70 07.3120 at *330 m). We set up a 50 9 55 m plot in which phenological data (excluding the frequency of bloom and auto-fertilization tests which were recorded in the same aguajal but outside of the plot) for V. bicolor vines were recorded during August 1718, 2005. Most plants had finished blooming during this time of the year in August during the dry season. Fig. 1 Map of Madre de Dios River Peru in grey with aguajales shown in color transposed over rainfall isohyets from Sombroek (2001) Rate of fruit set in V. bicolor Multiple individuals of V. bicolor plants are commonly encountered climbing up sides of palm trees (Mauritia flexuosa). We will refer to multiple V. bicolor vines growing up the sides of a single palm tree as a clump. We counted the number of vines per clump of V. bicolor, the number of individual racemes of V. bicolor per clump, the number of un-pollinated flowers per raceme, the number of pollinated flowers per raceme, and the number of buds or new flowers per raceme. An un-pollinated flower was recognized by the presence of a bract on the raceme, but lacking a flower or a fruit as abscission of flowers occurs. A pollination event was also denoted by the presence of fruit on a raceme or a flower with a drooping ovary. The height at which each raceme occurred was measured. The average fruit set per raceme, number of racemes per clump of V. bicolor, average height of the racemes occurring on the V. bicolor vines, and percent of V. bicolor vines that did not produce racemes were calculated. Only racemes that had no new buds and completely finished blooming were used for calculating the fruit set rate. A total of 131 racemes from 49 clumps of V. bicolor growing up palm tree trunks occurred in our study plot. Fruit se (...truncated)