Philippine Rafflesia: Exploration and Discovery

Project Description, from National Geographic Society Grant to D. L. Nickrent

Introduction and Progress to Date

Philippine tropical rainforests are among the World’s Biodiversity Hotspots threatened by human activities. They are home to the holoparasitic Rafflesia, famous for possessing the largest single flower in the world (13).  Until 2002, only two species of Philippine Rafflesia were known, but following many scientific investigations (1-8, 10-11), nine or ten endemic species are now recognized: R. baletei, R. leonardi, R. lobata, R. manillana, R. mira, R. philippensis, R. schadenbergiana, and R. speciosa and two additional unnamed potentially new species. A molecular study of Rafflesia (9) included 13 of the 23 species (13-15), but only two are from the Philippines.

Our research on Rafflesia evolution focusses upon the Philippine species. Fieldwork in May 2008 was successful in obtaining data for 6 species. Our preliminary analyses support Barkman et al. (9) in that 1) the Philippine species are monophyletic (i.e. more closely related to each other than to those outside of the Philippines) and 2) genetic variation among species is low (Figure 1). Our data show that R. philippensis (small-flowered) is sister to the remaining Philippine species, not to R. baletei with which it is morphologically most similar. The large flowered R. schadenbergiana occurs at an intermediate position on the tree. This demonstrates that floral size increases and decreases have occurred multiple times independently and that species relationships are indicated best by geographic location, not flower size.

Objectives

Our major objectives are as follows:

1.  Conduct two trips over the course of two field seasons to obtain data from unsampled Rafflesia species/populations: R. baletei, R. mira, R. manillana, and two unnamed species (Rafflesia sp. 1 Luzon, Rafflesia sp. 2 Mindanao).
2. Document pollinator visitations, sex ratios, fruit production, and population sizes.
3. Determine phylogenetic relationships among Philippine Rafflesia and their host plants using DNA sequence data.
4. Obtain seeds and cuttings of host and parasite for ex situ cultivation and experimentation.

Field Methodologies

Fieldwork will be conducted in seven predetermined Rafflesia sites, four in Luzon Island: Mt. Banahaw for R. philippensis, Mt. Isarog for R. baletei, Mt. Natib for R. manillana, and the Quirino Protected Landscape (QPL) for Rafflesia sp. 1, two in Mindanao Island: Mt. Candalaga for R. mira and Mt. Matutum for Rafflesia sp. 2, and one in Samar Island, type locality of R. manillana. All sites in Luzon and Samar are inside Protected Areas whereas those in Mindanao are not. At 206,875 hectares, QPL (Presidential Proclamation 578 dated 2004) is home to rich tropical forests and two indigenous ethnic groups, the Agtas and Bugkalots. Because QPL is experiencing pressures from several factions (see Gorospe-Ibuan 2004), access maybe difficult. We will begin the process of obtaining collecting permit in QPL immediately. Rafflesia sp. 1, found in QPL, is morphologically similar to R. tengku-adlinii from Borneo. However, since molecular data indicate high levels of endemism, Barcelona, et al. (2008, submitted) is describing this as new.

We will conduct non-destructive sampling by removing small portions of the petals without negatively impacting floral function. While in the field, we will determine the number of flowers, buds, and fruits, their sizes, and GPS coordinates and record pollinator behavior (insect samples collected) at all open flowers. We will also search for fruits and collect seeds for ex situ cultivation experiments. 

Ex Situ Cultivation Methodologies

Rafflesia cultivation is reported by Veldkamp in 2007 (15). More recently, Nais (12) reported success with R. keithii in the Kinabalu Park, Sabah, Malaysia. Despite these claims, no published methodologies are available.  Moreover, the host plants used were at locations where Rafflesia already existed, hence the host may have already contained the parasite. The lack of basic life history information currently limits the application of ex situ methods. To date, no observations have been made of Rafflesia seedlings attaching to Tetrastigma (Vitaceae) host tissues.

We propose beginning a research program to systematically study the life history of Rafflesia, particularly the seedling phase. Funding to the Philippine Native Plant Conservation Society, Inc. (PNPCSI) from the German government has been approved to construct a botanical garden at the DENR campus in Quezon City that includes cultivation of Tetrastigma hosts for possible Rafflesia infection. In collaboration with DENR and PNPCSI, we will conduct detailed studies of the early infection and establishment process. Molecular methods will be used to 1) genotype the host plants used for inoculation experiments and 2) determine whether the host tissue contains Rafflesia.  The latter is feasible because DNA profiles for host and parasite are very different and the PCR technique is extremely specific and sensitive, thus allowing tiny samples to be assayed. Most Filipinos have never seen Rafflesia, thus having this plant in an urban botanical garden would increase public awareness of their country’s natural heritage and strengthen efforts to conserve threatened natural ecosystems. If perfected, such cultivation techniques would allow botanical gardens around the world to grow this amazing host-parasite combination.

Molecular Methodologies

Standard procedures that have already been used for extracting DNA, PCR, and sequencing will be continued. Given the low amount of genetic diversity between species, additional molecular markers will be sought to increase resolution of phylogenetic relationships. The molecular tools are essential to all phases of this project because they allow us to 1) assess interspecific relationships, 2) determine whether potential new species are genetically distinct, 3) provide data useful in studies of the biogeography and floral size evolution of Rafflesia, and 4) provide genetic markers that can be applied to ex situ cultivation experiments.  For the latter, both host and parasite can be “fingerprinted”, thus guiding inoculation experiments. The taxonomy of Tetrastigma is problematic because plants often present only vegetative features (not flowers), thus these molecular studies will help with species identifications. A better understanding of species relationships in Rafflesia also has implication for local politics, ecotourism, management and conservation. Additional projects that will develop as this research program matures involve monitoring Tetrastigma host vine populations for Rafflesia. Given the specificity and sensitivity of PCR, populations will be surveyed to determine incidence of infection by Rafflesia, even in the absence of external flowers or buds.  This type of study would provide the first data useful in determining sizes of the parasite populations.

Time Frame Justification

Our request for two years of support is made with the understanding that such awards are rare. We believe that our project is exceptional for the following reasons. Only certain times are available to conduct fieldwork owing to weather conditions and the PI’s academic schedule (December through April). The time frame for one collection trip is generally four weeks, however, not all of the proposed sites can be visited in that time period.  In some cases (e.g. Rafflesia manillana, R. philippensis, R. baletei) the populations are readily accessible and the likelihood of finding flowers is high. In other cases (R. mira, Rafflesia sp. 1, Rafflesia sp. 2) the populations are remote and finding flowers may require more time.  Moreover, if flowers of these rarely seen species are found, several days of fieldwork will be expended to properly document their populations.  Since security is an issue in Mindanao and Samar Islands, we need more time to carefully assess the situation by seeking assistance of local officials, DENR, and NGO to access the sites without jeopardizing our safety.

Permits

We have strictly followed Philippine regulations in conducting research. By virtue of a Memorandum of Agreement (Feb. 14, 2008) between the Philippine DENR and SIUC entitled “Systematics of Parasitic Plants of the Philippines,” a Wildlife Gratuitous Permit (no. 175) was issued May 5, 2008 to collect specimens for scientific research purposes. Local Transport Permits and Municipal Clearances were obtained for all sites from the local DENR Office. We also obtained Phytosanitation Certificates from the Bureau of Plant Industry, Philippine Dept. Agriculture that allowed export of samples. These guidelines will be followed for all future fieldwork.

International Cooperation

This project provides a unique opportunity to develop a truly international cooperative effort to better understand and conserve Rafflesia. The planned Tetrastigma plots at the Quezon City botanical garden will provide a controlled environment to conduct detailed studies of Rafflesia parasitism. Dr. Antonio Manila (DENR, PNPCSI) has requested our technical assistance in this endeavor. Following a seminar at the University of the Philippines (May 12, 2008), several students expressed interest in Rafflesia.  Perla Visorro, President/CEO, Cagayan Valley Partners in People Development (CAVAPPED) is actively seeking Protective status for the habitats of Rafflesia manillana and R. leonardi in Cagayan Province. It was here that Sumper Aresta (Agta-People’s Organization member) discovered R. leonardi on April 13, 2005. In May 2008, we participated in a conservation management workshop for R. schadenbergiana in Baungon, Bukidnon Province, Mindanao. This meeting demonstrated the commitment of local people to preserve Rafflesia and their understanding of the complexities of conservation biology when weighed against other issues, e.g. subsistence agriculture and ecotourism.

Literature Cited

1. Barcelona, J. F., and E. S. Fernando. 2002. A new species of Rafflesia (Rafflesiaceae) from Panay Island, Philippines. Kew Bulletin 57: 647-651.
2. Barcelona, J. F., M. A. O. Cajano, and A. S. Hadsall. 2006. Rafflesia baletei, another new Rafflesia (Rafflesiaceae) from the Philippines. Kew Bulletin 61: 231-237.
3. Barcelona, J. F., P. B. Pelser, and M. A. O. Cajano. 2007. Rafflesia banahaw (Rafflesiaceae), a new species from Luzon, Philippines. Bumea 52: 345-350.
4. Barcelona, J. F., P. B. Pelser, E. Cabutaje, and N. A. Bartolome. 2008. Another new species of Rafflesia (Rafflesiaceae) from Luzon, Philippines: R. leonardi. Blumea 53: 223-228.
5. Barcelona, J. F., P. B. Pelser, D. S. Balete, and L. L. Co. In press-a. Taxonomy, ecology, and conservation status of Philippine Rafflesia (Rafflesiaceae). Blumea.
6. Barcelona, J. F., P. B. Pelser, A. M. Tagtag, R. G. Dahonog, and A. P. Lilangan. In press-b. The rediscovery of Rafflesia schadenbergiana Göpp. ex Hieron. (Rafflesiaceae). Flora Malesiana Bulletin.
7.  Barcelona, J. F. 2007-onwards. http://picasaweb.google.com/barceljf/PhilippineRafflesia#
8.  Barcelona, J. F., L. L. Co, D. S. Balete, and N. A. Bartolome. submitted. Rafflesia aurantia (Rafflesiaceae): a new species from northern Luzon,  Philippines. Gardens’ Bulletin Singapore.
9. Barkman, T. J., M. Bendiksby, S.-H. Lim, K. Mat Salleh, J. Nais, D. Madulid, and T. Schumacher. 2008. Accelerated rates of floral evolution at the upper size limit for flowers. Current Biology 18: 1508–1513.
10. Fernando, E. S., and P. S. Ong. 2005. The genus Rafflesia R. Br. (Rafflesiaceae) in the Philippines. Asia Life Sciences 14: 263-270. [R. mira]
11. Galang, R., and D. A. Madulid. 2006. A second new species of Rafflesia (Rafflesiaceae) from Panay Island, Philippines Rafflesia. Folia Malaysiana 7: 1-8. [R. lobata]
12. Madulid, D. A., I. E. Buot, and E. M. G. Agoo. 2007. Rafflesia panchoana (Rafflesiaceae), a new species from Luzon Island, Philippines. Acta Manilana 55: 43-47.
13. Nais, J. 2001. Rafflesia of the World. Sabah Parks, Kota Kinabalu. 243 pp.
14. Nickrent, D. L. 1997 onwards.  The Parasitic Plant Connection. College of Science, SIUC. http://www.parasiticplants.siu.edu/ [see Rafflesiaceae]
15.  Philippine Native Plant Society, Inc. Manila. 2008 onwards. [see Rafflesia] http://philippinenativeplants.org/site/
16.  Veldkamp, J. F. 2007. Some notes on the cultivation of Rafflesia. Flora Malesiana Bulletin 14 (1 & 2): 50-3.

SIUC / College of Science / Philippine Rafflesia / Project
URL: http://parasiticplants.siu.edu/PhilRaff
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