In this study we conclude that R. tuan-mudae is an endothermic flower, showing a maximum of 6 K of excess tissue temperature in one flower. Endothermy in Rafflesia has been suggested previously (Meeuse, 1978; Beaman et al., 1988) and efforts to detect heating were made (Nais, 1997). Nais’s studies on R. keithii in Sabah, Malaysia, did not reveal any significant excess tissue temperature. There were important differences in the methodology of the two studies. In the present study, we inserted thermocouples inside the tissue and measured every 6 or 20 s and recorded as means of 10 or 15 readings once per 1 or 5 min during the whole period of blooming (5–8 d). We detected 1.5–6.0 K excess temperatures in the internal parts of the flower. In Nais’s study, only the surface temperatures were measured, as spot readings, with a probe that was held against the surface.
During the day, the buds were cooler than the air (Fig. 3b), suggesting that evaporative heat transfer may be significant, and may affect the thermal balance of the buds cooling down the surface when the radiation load from the surroundings is higher and VPD increases. In the present study it was not possible to measure the stomatal conductance or transpiration rate of the floral surface, but stomata are present in the adaxial surface of the perigone lobe (Cammerloher, 1920). Consequently, we have not been able to construct the complete energy budget of the flower.
The role of endothermy in flowers of R. tuan-mudae and R. lowii of the parasitic family Rafflesiaceae
The question posed was: is endothermy part of the mimicry of the flower to attract the pollinating flies? Endothermy was detected in young buds and mature buds of R. lowii and remained during blooming. This flower showed signs of thermoregulation by maintaining fairly constant temperature during and after anthesis (Patiño et al., 2000). Similarly, young buds of R. tuan-mudae showed some endothermy (mature buds were not studied for the reasons stated previously), but the flowers were shown to have only a weak pattern of endothermy and there was no sign of thermoregulation. Therefore, the mechanism for endothermy in these two flowers is present at the early stages of floral development. The photosynthetic products that provide the respiratory substrate for endothermy are available ‘free of charge’ from the vine host. There would, of course, be a certain limit imposed by the productivity of the host and the translocation rates: the vine might not have unlimited photosynthates for its parasites.
It has been demonstrated that many flowers increase their temperature above ambient as a consequence of endothermy, the heat being produced by the cyanide-insensitive respiration (Meeuse & Raskin, 1988; Skubatz et al., 1990). It is possible that endothermy is a facultative characteristic in most plants and perhaps all flowers. This assumption is supported by the fact that most plant mitochondria contain a cyanide and antimycin-insensitive alternative terminal oxidase (Lambers, 1980; Moreau & Romani, 1982). The alternative respiratory pathway has been detected in different tissues of many plants belonging to different taxa, for example ripening fruits (Cruzhernandez & Gomezlim, 1995), roots and cotyledons of soybean, potato, sweet potato and cassava (Day et al., 1994; Millar et al., 1994; Ribascarbo et al., 1995), sugar beet callus (Shugaev et al., 1998), Acer pseudoplatanus (Aubert et al., 1997), rootstocks of pears (Wagner et al., 1992; Tamura et al., 1996), nongreen tissues of Petunia hybrida (Wagner & Wagner, 1995), water-stressed plants of sorghum (Kumar & Sinha, 1994), shoot tips of Douglas fir (Fielder & Owens, 1992), roots of white spruce (Weger & Guy, 1991), Convolvulus (Van der Plas et al., 1977), wheat (Lundegårdh, 2001) and beans (Rychter et al., 1992).
As the tropical environment is rarely cold, it seems likely that endothermy in R. tuan-mudae and R. lowii is present as the result of alternative respiration that is not coupled to energy conservation. High rates of respiration are assumed to occur owing to the nature of the flowers: nonphotosynthetic and parasitic on the roots of the host (where they can easily can obtain the substrate of respiration). Therefore, endothermy may have evolved not merely to maintain the tissue at a high temperature, but to ensure pollination. The suggestion from this present work is that the production of CO2, and possibly other volatiles is important. The flowers in this case are conspicuous by virtue of the CO2 and/or volatiles they produce. The poorly mixed air at the forest floor may contain the olfactory signal to pollinators, but an important question is whether or not CO2 plays a role in the pollination of R. lowii and R. tuan-mudae. Further studies are now needed to prove the relationship between the high respiration rate of parasitic plants and attraction of pollinators.
In support of the role of CO2 as an insect attractant, it has been shown, for example, that CO2 is the only attractant volatile of the larvae of western corn rootworm (Diabrotica virgifera virgifera) to corn roots (Bernklau & Bjostad, 1998). The detection of CO2 by identified peripheral sensory organs of some terrestrial arthropods (e.g. nematodes, larva and adult beetles, centipedes, ants, termites, fig pollinators, honey bees, mosquitoes, flies, bugs, ticks and moths) is now well established, and the resulting coordinated behavioural responses at concentrations that occur naturally in the habitats of these organism has been well documented (Stange & Wong, 1993; Stange, 1996). Furthermore, there is evidence that the blowfly has CO2-specific sensory receptors (Stange, 1975). It has also been demonstrated that CO2 has an anaesthetic effect in blowflies and that they are considerably more sensitive to CO2 than to other anaesthetics (Diesendorf, 1975). It is therefore possible that the CO2 produced by the flowers of R. tuan-mudae and R. lowii plays a role on the pollination by the blowflies.
The signals emitted by R. tuan-mudae and R. lowii may be different. Despite Rhizanthes and Rafflesia attracting blowflies of the same genera Lucilia, Chrysomya and Hypopygiopsis (Beaman et al., 1988; Bänziger, 1991, 1996; Hidayati et al., 2000), it has been observed that R. lowii stimulated oviposition in the flies, suggesting that R. lowii is releasing specific volatiles that trick female flies. Oviposition by the blowflies on R. tuan-mudae was not observed in this study and has not been observed in other Rafflesia species (Bänziger, 1991; Beaman et al., 1988; Bänziger, 1996).
Further work is required to determine whether the olfactory signal is more complex than simply CO2. The volatiles identified in the headspace of R. lowii– 3-hydroxy-2-butanone, 2-ethyl-1-hexanol and N,N-diethyl-3-methyl-benzamide (S. Patiño, A. A. Edwards and J. Grace, unpublished) – have been found in other flowers (Knudsen et al., 1993). The volatiles identified in R. tuan-mudae– dimethyl disulphide and dimethyl trisulphide (S. Patiño, A. A. Edwards and J. Grace, unpublished) – have been found in various flowers from the family Araceae such as Amorphophallus, Pseudodracontium (Stransky & Valterova, 1999) and Hydrosme (Kite & Hetterschieid, 1997), in one Hydnoraceae (Hydnora) (Burger & Munro, 1988) and in bat-pollinated flowers (Knudsen & Tollsten, 1995), with dimethyl disulphide being the component preferred by the bats (von Helversen et al., 2000). Dimethyl disulphide and dimethyl trisulphide are compounds commonly related to bacterial growth on meat (Senter et al., 2000) and dimethyl disulphide is the compound that gives the characteristic taste to Camembert cheese (Demarigny et al., 2000). It has been found that the antennae of the female blowfly have a specific receptor neurone tuned to dimethyl disulphide (Park & Cork, 1999) and it has been suggested that dimethyl trisulphide may be one of the major cues for host location by calliphorid flies (Nilssen et al., 1996). Location of the flower by the flies may follow the classical downwind model (Stange, 1996) and once the flies have landed on the flower, mechanical and contact chemical inputs may guide them into the diaphragm and to the gynoecium. Although, Rafflesia is not a ‘trap’ flower, as defined by Dafni (1984), it has been observed that a few flies remain inside the R. tuan-mudae diaphragm for some hours (pers. obs.), perhaps because of the CO2 anaesthetic effect, as suggested by Dafni (1984).
Further work is now needed on the behaviour of the pollinating flies and identification of the species visiting the flowers.
A further step in elucidating the role of carbon dioxide in the pollination syndrome of Rafflesia tuan-mudae and Rhizanthes lowii would be to perform direct measurements of gas exchange, and careful identification of the volatiles that compose the odour, with bioassays of the substances using the pollinating flies and other species of Rafflesia and Rhizanthes. Experiments with model flowers, in which the odours are experimentally emitted, would enable identification of the role of specific gases as well as the importance of size, shape and colour of the flower.