The Anther Oil of Symphonia Globulifera L.F. (Clusiaceae)

Biochemical Systematics and Ecology 49 (2013) 131–134

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Biochemical Systematics and Ecology

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The anther oil of Symphonia globulifera L.f. (Clusiaceae)

Volker Bittrich a,*, José E. do Nascimento-Junior b, Maria do Carmo E. Amaral b, Paulo Cesar de Lima Nogueira c a R. Mario de Nucci, 500, 13083-290 Campinas, Brazil b Department of Plant Biology, Institute of Biology, CP 6109, University of Campinas – UNICAMP, 13083-970 Campinas, SP, Brazil c Department of Chemistry, CCET, Federal University of Sergipe, Av. Marechal Rondon s/n, Jd. Rosa Elze, 49100-000 São Cristóvão, SE, Brazil article info abstract

Article history: The chemical composition of the oil on the anthers of Symphonia globulifera L. f., in which Received 20 November 2012 the pollen grains are completely immersed, was investigated. An unsaturated fatty acid Accepted 9 March 2013 methyl ester (methyl nervonate), very rare in plants, was found to be the only component Available online 15 April 2013 of the oil. The possible ecological implications of the presence of this oil on the anthers of the ornithophilous ﬂowers of Symphonia are discussed. Keywords: Ó 2013 Elsevier Ltd. All rights reserved. Clusiaceae Symphonia Floral oils Ornithophily Pollenkitt Methyl nervonate

1. Introduction

Angiosperm flowers use various chemical devices to ensure the success in an often complex pollination process: flower colors and fragrances to attract, sometimes deceive, animal pollinators; various rewards, like pollen, nectar, oils, resins, among others, pollenkitt to glue the pollen grains to each other and to the pollinator’s body, and various kinds of secretions on the stigma surface. Guttiferae (today divided into Clusiaceae s.s., Hypericaceae and Calophyllaceae) seem to be genetically especially preadapted for the synthesis of floral oils and resins involved in the pollination process (e.g., Bittrich et al., 2006; Nogueira et al., 1998; Porto et al., 2000). Especially the androecium, morphologically of a dazzling variety probably unknown of other angiosperm families, shows an impressing capacity to produce oils and resins. Oils can be fragrant, then normally probably serving mainly for attracting pollinators (e.g., Caraipa spp. of Calophyllaceae), but also sometimes functioning additionally as reward collected by male euglossine bees (Tovomita spp. of Clusiaceae s.s.; Nogueira et al., 1998). Sometimes the secreted oils are neither aromatic nor functioning as a reward for animal flower visitors, however, but rather guarantee adherence to and a safe pollen transport on the body of the pollinators. This was first observed in Clusia spp. of sect. Chla- mydoclusia (e.g., C. grandiflora Splitg.), where the elongated anther connectives bear tiny droplets of a clear oil of still unknown chemical composition (Bittrich and Amaral, 1997). The flower morphology guarantees that this oil is transferred together with the dry pollen to the body of visiting bees. Another but rather different case occurs in the tribe Symphonieae of Clusiaceae, and is especially evident in S. globulifera L.f. (Bittrich and Amaral, 1996). This species occurs in Africa and the

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Neotropics, which it colonized in the Tertiary probably independently three times (Dick et al., 2003). In the flowers of S. globulifera an oily substance, probably of tapetal origin, is covering the anthers at anthesis. The pollen grains are suspended in this oily substance that contaminates the upper side of the beaks of visiting nectar collecting birds (humming-birds and perching birds: Bittrich and Amaral, 1996; Gill et al., 1998; Lasprilla and Sazima, 2004). When the birds are visiting another flower, the oil with the pollen is transferred to a pore-like stigma and enters by capillarity into the hollow space behind the pore. Normal pollenkitt covering the pollen grains (see review of Pacini and Hesse, 2005) to make them stickier is a very common phenomenon in flowers pollinated by animals and can be considered a synapomorphy of the angiosperms (Hesse, 1980). Normal pollenkitt is produced by the tapetum inside the anthers. Oily substances produced outside the anthers, as on the elongated connective in certain Clusia spp. mentioned above, are sometimes termed “accessory pollenkitt” (Vogel, 1984) as they have a similar function as the typical pollenkitt produced by the anther tapetum. Pollination involving an abundant oily fluid in the anthers with pollen grains immersed in it, is a very uncommon phenomenon (e.g. Machado and Lopes, 2000; for Souroubea guianensis Aubl., Marcgraviaceae). In some species of Clusia, however, pollen is immersed in a rather fluid resin, a mixture of benzophenones and free fatty acids (Porto et al., 2000). This mixture is collected by small bees for nest construction (Bittrich and Amaral, 1997). Wang et al. (2004) in Caulokaempferia coenobialis (Hance) K. Larsen (Zingiberaceae) discovered a new self-pollination mechanism, in which an oily fluid with immersed pollen grains slides down from the anthers to the stigma. Highly interesting is the report of Houston et al. (1993) about the flowers of Verticordia (Myrtaceae) that offer both, nectar and anther oil for pollinating Euryglossa (Colletidae) bees, a singular case in angiosperms. Due to technical problems, the chemical composition of normal pollenkitt, present only in very tiny amounts on the pollen grains, is poorly investigated (e.g., Dobson, 1988; Piskorski et al., 2011), but it contains mainly lipids. Houston et al. (1993) discovered various saturated fatty acid esters in the anther oil of Verticordia. The chemical composition of the oily fluids of the anthers in Symphonia and related genera and Souroubea, however, was up to now never studied.

2. Material and methods

Flowers of S. globulifera were collected in the coastal restinga vegetation of Sergipe state (NE Brazil) north of the capital Aracaju in December 2011. Voucher material (J.E. Nascimento-Jr et al. 1121, ASE 22947) was deposited in the herbarium of the Federal University of Sergipe in São Cristóvão, Sergipe (acronym: ASE). The oily fluid was collected from 3 different trees of the same population north of Aracaju. 3–4 flowers were sampled of each tree and the substance kept in different vials, one for each tree. The fluid was carefully scraped with a spatula from the anthers directly after collection of the flowers in the field and transferred to the solvent (CH2Cl2 HPLC grade, Tedia, Brazil) and stored in an amber bottle in a freezer until chemical analysis was performed. Thus any degradation of the substance after removal from the anthers was avoided. Although dif- ferences in the chemical composition of the anther oil of the sampled trees were considered improbable, material from the three different samples was at first examined by silica gel thin layer chromatography (TLC)(Merck, Germany) eluted with hexane-EtOAc (90:10, v/v). As the chromatograms were found to be identical, one sample was further analyzed by GC/MS. Before injection into the GC/MS, the solution was submitted to filtration through a cellulose membrane (pore diameter 0.45 mm, Sartorius).

2.1. GC/MS analysis

The analysis of the anther oil of S. globulifera was performed on a Shimadzu QP5050A GC/MS system equipped with an AOC-20i auto-injector. A J&W Scientific DB-5MS (coated with 5%-phenyl-95%-dimethylpolysiloxane) fused capillary column (30 m 0.25 mm 0.25 mm film thickness) was used as the stationary phase. Helium was the carrier gas at 1.0 mL/min flow rate. Initially, we ran the sample on GC/MS under the conventional conditions for the analysis of volatile compounds: 40 C (hold for 4 min) to 220 C at a rate of 4 C/min. As no volatile component was detected, column temperatures were programmed from 180 C (hold for 1 min) to 300 C at a rate of 20 C/min (hold for 5 min), then raised from 300 C to 320 Cata rate of 5 C/min (hold for 10 min). The injector and detector temperatures were 250 C and 280 C, respectively. MS were taken at 70 eV with a scan interval of 0.5 s and fragments from 40 to 600 Da. The retention index (Van den Dool and Kratz, 1963) was obtained by co-injecting the oil sample with a C9–C40 linear hydrocarbon mixture, and identification was made on the basis of standard compound co-injection and comparison of retention index and MS with those in the literature (Tretyakov, 2007).

3. Results

According to our analysis the anther oil of S. globulifera is composed of a single component, an unsaturated fatty acid ester, methyl nervonate (15-Tetracosenoic acid methyl ester). The identiﬁcation of the component of the anther oil of S. globulifera was based on the characteristic mass spectrum fragmentation pattern and by comparison of retention time and mass þ spectrum using an authentic sample. The EI spectrum [m/z: 380(M ), 348(26), 83(51), 74(42), 69(64), 55(100), 43(73), 41(74)] was identical with that of methyl nervonate [(15Z)-tetracosenoic acid methyl ester] (Fig. 1). Also, the retention index (RI ¼ 2698) generated from a standard solution of n-alkanes (C9–C40) on DB5-MS column by applying equation of Van den Dool and Kratz (1963) is in agreement with literature (Tretyakov, 2007). V. Bittrich et al. / Biochemical Systematics and Ecology 49 (2013) 131–134 133

(x1,000,000) 4.0 TIC

3.5 A

3.0

2.5

2.0

1.5

1.0

0.5

5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

% 100.0 55 B

75.0 41

69 O

50.0 O

348 25.0 111

125 152 661 194 208 250 264 872 306 236 330 355 380 0.0 50 100 150 200 250 300 350 400

Fig. 1. (A) Total ion chromatogram of anther oil from S. globulifera. (B) Mass spectrum and chemical structure of methyl nervonate.

4. Discussion

To the best of our knowledge, methyl nervonate is very rare as a natural product from plants and has been previously identified only in two Asteraceae (roots of Echinacea angustifolia DC., Dahui et al., 2011; and Senecio stabianus Lacaita, Tundis et al., 2012) and in Durio zibethinus Rumph. ex Murray (Bombacaceae; Jaswir et al., 2008). Methyl nervonate is an apolar viscous substance, fluid at room temperature. Thus it is well-suited for serving as a carrier material for the pollen grains. Pacini and Hesse (2005) and Hesse (2010) discuss numerous possible functions of pollenkitt and several of these may also apply for the oily fluid in the anthers of S. globulifera. It seems, however, to have no function in attracting pollinators. In Central Amazonia, Bittrich and Amaral (1996) observed that the open flowers and even mature buds of S. globulifera were frequently damaged by nectar-robbing Trigona bees. Their tongues are too short to reach the nectar within the tube of an undamaged flower. The oil may have a protective function to avoid pollen loss to these visitors. Bees of the tribe Trigonini are well-known as pollen thieves and robbers (e.g., Renner, 1983; Hargreaves et al., 2009). On flowers of Platonia insignis Mart., Maués and Venturieri (1996) observed Trigona bees to pilfer nectar and to also provoke small injuries in the petal surface to collect later the exuded latex, which they use for nest-construction. Apparently, the anther oil is un- suitable for this purpose as it was not reported to be collected by the bees. The unusual pore-like stigma of Symphonieae permits that the oil with the pollen immersed in it is immediately sucked up into the cavity behind the pore after left on the stigma tip by the beak of a visiting bird. This may serve to avoid that the oil is scraped away during the visit of another bird. Two species belonging to the tribe Symphonieae of Clusiaceae s.s., Moronobea coccinea Aubl. and Platonia insignis are reported to be pollinated by parakeets (Vicentini and Fischer, 1999; Maués and Venturieri, 1996). These birds take nectar and also scratch the pollen-anther-oil mixture with the beak. This indicates that the ingestion of the oily substance of the anthers of these genera is at least not toxic for these pollinators. Its chemical composition is yet unknown. Neither parakeets were observed visiting flowers of S. globulifera nor other birds ever observed to consume the oil-pollen mixture, nor exist reports that the pollen/oil mixture was collected by bees. The nectar in the flowers of Symphonia cannot be removed by parakeets as the petals are firmly pressed against each other. Also different to Platonia and Moronobea, the anthers in Symphonia flowers are attached with their whole length to the fleshy triangular lobes of the androecial tube. The removal of the oil by parakeet beaks would therefore probably damage these lobes and liberate the unpalatable latex. It is less clear why no bees are interested in the oil-pollen mixture. Collection of similar anther oils by bees was, however, only once reported in the literature, on flowers of the genus Verticordia (Myrtaceae; Houston et al., 1993). The anthers of these flowers have pollen suspended in an oil in a similar way as in Symphonia. The oil of Verticordia anthers is a 134 V. Bittrich et al. / Biochemical Systematics and Ecology 49 (2013) 131–134 mixture of various saturated fatty acid esters (Houston et al., 1993). While such an oil-pollen mixture represents an extremely rare floral reward for pollinating bees, floral oil without pollen suspended in it, is more commonly offered by flowers and collected regularly by bees of various tribes. The use of oils as a floral reward for pollinating bees represents a nice example for parallel evolution in various families of flowering plants (see Machado, 2004; Renner and Schaefer, 2010). Reportedly the oil is used by the bees for lining the cells of the nest to make them waterproof and mixed with pollen as larval food. The lipids of the oils were found to contain saturated fatty acid ester, mono-, di-, and triglycerides and free fatty acids (e.g., Renner and Schaefer, 2010). Why the anther oil of Symphonia is not collected by bees remains unclear.

Acknowledgments

The authors are indebted to CNPq (grants to PCLN and MCEA) and to FAPESP (studenschip to JENJr) for ﬁnancial support. The authors thank Dr. Gizelle Cristina Bedendo (SINC/Brazil) for kindly donating a sample of methyl nervonate and Dr. Marcelo da Rosa Alexandre (Department of Chemistry, Federal University of Sergipe, Brazil) who kindly provided a sample of n-alkanes for the calculation of the retention index.

References

Bittrich, V., Amaral, M.C.E., 1996. Pollination biology of Symphonia globulifera L.f. (Clusiaceae). Plant Syst. Evol. 200, 101–110. Bittrich, V., Amaral, M.C.E., 1997. Flower biology of some Clusia species from Central Amazonia. Kew Bulletin 52, 617–635. Bittrich, V., Amaral, M.C.E., Machado, S.M.F., Zacharias, M.E., Marsaioli, A.J., 2006. Oils, resins and the pollination biology of the Clusiaceae. In: Texeira da Silvam, J.A. (Ed.), 2006. Floriculture, Ornamental and Plant Biotechnology, Vol. 4. Global Science Books, Middlesex (UK), pp. 387–394. Dahui, L., Zaigui, W., Yunhua, Z., 2011. Antifungal activity of extracts by supercritical carbon dioxide extraction from roots of Echinacea angustifolia and analysis of their constituents using gas chromatography-mass spectrometry (GC-MS). J. Med. Plants Res. 5, 5605–5610. Dick, C.W., Abdul-Salim, K., Bermingham, E., 2003. Molecular systematics reveals cryptic Tertiary diversification of a widespread tropical rainforest tree. Amer. Naturalist 162, 691–703. Dobson, H.E.M., 1988. Survey of pollen and pollenkitt lipids – chemical cues to flower visitors? Amer. J. Bot. 75, 170–182. Gill, G.E., Fowler, R.T., Mori, S.A., 1998. Pollination biology of Symphonia globulifera (Clusiaceae) in central French Guiana. Biotropica 30, 139–144. Hargreaves, A.L., Harder, L.D., Johnson, S.D., 2009. Consumptive emasculation: the ecological and evolutionary consequences of pollen theft. Biol. Rev. 84, 259–276. Hesse, M., 1980. Pollenkitt is lacking in Gnetum gnemon (Gnetaceae). P1. Syst. Evol. 136, 41–46. Hesse, M., 2010. Bonding single pollen grains together: how and why? In: von Byern, J., Grunwald, I. (Eds.), Biological Adhesive Systems. Springer Verlag, Wien, pp. 3–14. Houston, T.F., Lamot, B.B., Radford, S., Errington, S.G., 1993. Apparent mutualism between Verticordia nitens and V. aurea (Myrtaceae) and their oil-ingesting bee pollinators (Hymenoptera, Colletidae). Austr. J. Bot. 41, 369–380. Jaswir, I., Man, Y.B.C., Selamat, J., Ahmad, F., Sugisawa, H., 2008. Retention of volatile components of durian fruit leather during processing and storage. J. Food Process. Preserv. 32, 740–750. Lasprilla, L.R., Sazima, M., 2004. Interacciones planta-colibrí en tres comunidades vegetales de la parte suroriental del Parque Nacional Natural Chiribiquete, Colombia. Ornitol Neotrop 15 (Suppl), 183–190. Machado, I.C., 2004. Oil-collecting bees and related plants: a review of the studies in the last twenty years and case histories of plants occurring in NE Brazil. In: Freitas, B.M., Pereira, J.O.P. (Eds.), Solitary Bees, Conservation, Rearing and Management for Pollination. Federal University of Ceará, Ceará, Brazil, pp. 225–280. Machado, I.C., Lopes, A.V., 2000. Souroubea guianensis Aubl.: quest for its legitimate pollinator and the first record of tapetal oil in the Marcgraviaceae. Ann. Bot. 85, 705–711. Maués, M.M., Venturieri, G.C., 1996. Ecologia da polinizacao do bacurizeiro (Platonia insignis Mart.). Clusiaceae. Embrapa-CPATU, Boletim de Pesquisa 170, 24. Belém do Pará, Brasil. Nogueira, P.C.D., Marsaioli, A.J., Oliveira, M.C. de, Bittrich, V., 1998. The fragrant floral oils of Tovomita species. Phytochemistry 49, 1009–1012. Piskorski, R., Kroder, S., Dorn, S., 2011. Can pollen headspace volatiles and pollenkitt lipids serve as reliable chemical cues for bee pollinators? Chem. Biodivers. 8, 577–586. Pacini, E., Hesse, M., 2005. Pollenkitt: its composition, forms and function. Flora 200, 399–415. Porto, A.L.M., Machado, S.M.F., Oliviera, C.M.A. de, Bittrich, V., Amaral, M.C.E., Marsaioli, A.J., 2000. Polyisoprenylated benzophenones from Clusia floral resins. Phytochemistry 55, 755–768. Renner, S., 1983. The widespread occurrence of anther destruction by Trigona bees in Melastomataceae. Biotropica 15, 251–256. Renner, S.S., Schaefer, H., 2010. The evolution and loss of oil offering flowers: new insights from dated phylogenies for angiosperms and bees. Philos. Trans. Roy. Soc. B 265, 423–435. Tretyakov, K.V., 2007. Retention data. In: Linstrom, P.J., Mallard, W.G. (Eds.), NIST Chemistry Webbook, NIST Standard Reference Database Number 69. National Institute of Standards and Technology, Gaithersburg, MD. http://webbook.nist.gov/chemistry/ (accessed October, 2012). Tundis, R., Menichini, F., Loizzo, M.R., Bonesi, M., Solimene, U., Menichini, F., 2012. Studies on the potential antioxidant properties of Senecio stabianus Lacaita (Asteraceae) and its inhibitory activity against carbohydrate-hydrolysing enzymes. Nat. Prod. Res. 26, 393–404. Van den Dool, H., Kratz, P.D., 1963. A generalization of the retention index system including linear temperature programmed gas-liquid partition chromatography. J. Chromatogr. 11, 463–471. Vicentini, A., Fischer, E., 1999. Pollination of Moronobea coccinea (Clusiaceae) by the golden winged parakeet in the Central Amazon. Biotropica 31, 692–696. Vogel, S., 1984. Blütensekrete als akzessorischer Pollenkitt. In: Ehren-dorfer, F. (Ed.), Mitteilungsband Botanikertagung in Wien. Institut für Botanik, Wien, p. 123. (Abstract). Wang, Y., Zhang, D., Renner, S.S., Chen, Z., 2004. A new self-pollination mechanism. Nature 431, 39–40.