Phytochemistry, Vol. 21, No.5, pp. 1035-1037, 1982. Printed in Great Britain. 0031-9422/82/051035-03$03.00/0 © 1982 Pergamon Press Ltd. THE INDOLE ALKALOID, HYPAPHORINE AND PTEROCARPUS SEED PROTECTION DANIEL H. JANZEN, DAVID G. LYNN,* LINDA E. FELLoWSt and WINIFRED HALLWACHS* Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.; *Department of Chemistry, University of Virginia, Charlottesville, VA 22901, U.S.A.; tJodrell Laboratory, Royal Botanical Gardens, Kew, TW9 3DS, U.K.; tEcology and Systematics, Cornell University, Ithaca, NY 14853, U.S.A. (Received 18 August 1981) Key Word Index-Pterocarpus officinalis; Leguminosae; L-hypaphorine; indole alkaloid; Liomys salvini; rodent; feeding deterrence. Abstract-L-Hypaphorine has been isolated as a major constituent of the seeds of Pterocarpus officinalis, seeds which are rejected as a food source by a wide variety of seed-eating rodents dwelling in the same habitat. Incorporation of the isolated hypaphorine into artificial diets of a small seed predator, Liomys salvini (a rodent) supports its role as a feeding deterrent. INTRODUCTION The large tree, Pterocarpus officinalis (Leguminosae), grows in natural pure stands of as much as 1 ha adjacent to the species-rich rainforests [1] of the Corcovado National Park in Costa Rica, a rainforest rich in seed-eating mammals (e.g. Dasyprocta punc- tata, Aguti paca, Proechimys cayennensis, Tayassu tajacu, Tapirus bairdii, Heteromys desmarestianus, Oryzomys caliginosus, Sciurus granatensis, Micro- sciurus allari and several species of climbing rats). On approximately alternating years these stands of P. officinalis produce so many fruits that densities of l00/m 2 on the ground are commonplace. Each of the soft, indehiscent and quasi-wind-dispersed 4 cm diameter fruits contains a soft seed weighing 1-3 g (ca 60% dry wt). Despite this enormous abundance of readily available seeds, there is no sign whatsoever of seed predation by mammals. We have investigated these seeds and found that they contain ca 5% dry wt of the alkaloid L-hypaphorine (1). This alkaloid has now been shown to function as an effective mammalian feeding deterrent for the seed predator Liomys salvini (Heteromyidae) by incorporating L-hypaphorine into artificial diets. RESULTS AND DISCUSSION The water-soluble crystalline material isolated from the seeds of P. officinalis showed a strong UV max- imum at 281 nm with shoulders at 289 and 274 nm, and the EI-MS (70 e V, 200°) gave an apparent mole- cular ion at m/z 187. The CI-MS (CH4) positive ion spectra supported the molecular ion assignment with m/z 188.0712 (C lI H ION02, calc. for 188.0727) and the negative ion spectra (CH4 /N20) showed evidence for chlorine attachment, m/ z 222. Conflicting evidence appeared in the IH NMR (360 MHz) giving integrated intensities for at least 17 protons. The 9H singlet at 8 3.20 taken together with the broadened carbon signal at 8 50.1 and the intense, sharp IR (KBr) band at 1350 cm- I , strongly supported the presence of a methylated quaternary amine [2], which is lost on volatilization in EI and CI mass spectrometry. Thermal fragmentation occurs to give a neutral fragment which undergoes protonation or chlorine attachment under chemical ionization conditions. Further assignments of the NMR data, taken together with optical rotation measurements, + 102° (c = 0.05 m) have allowed the assignment of structure 1, L-hypaphorine. Hypaphorine was first isolated from seeds of Erythrina hypaphorus [3] and has been detected in several other papilionoid genera. High concentrations of alkaloids and non-protein amino acids are known to deter insect feeding [4-9] and it is interesting in this context that the seeds of the Costa Rican Ptero- carpus rohrii, which lacks L-hypaphorine or other alkaloids, are attacked by the larvae of a weevil and a bruchid beetle [10]. Less is known about the ecologi- cal role these substances playas regards seed-eating mammals. Liomys salvini, or spiny pocket mouse, was the only wild tropical rodent available for testing hypaphorine's role as a mammalian feeding deterrent (Table 1). L. salvini is a professional seed predator [11,12] and consumes many species of tropical seeds during the year. It is the Costa Rican dry forest analogue to Heteromys desmarestianus [12, 13], the heteromyid seed-eating rodent that lives in the rain- forest containing the stand of Pterocarpus officinalis. Five adult female wild-caught L. salvini weighing 39-50 g (x = 44.8 g; s.d. = 5.3) were placed on a diet of reconstituted laboratory rat chow (ground, adul- terated, slightly moistened, pressed into balls, air 1035