Journal of Chemical Ecology, VoL 7, No. 6, 1981 SEASONAL AND INTRAPLANT VARIATION OF CARDENOLIDE CONTENT IN THE CALIFORNIA MILKWEED, Asclepias eriocarpa, AND IMPLICATIONS FOR PLANT DEFENSE 1 C.J. NELSONY J.N. SEIBER, 2 and L.P. BROWER 3,1 2Department of Environmental Toxicology University of California, Davis, California 95616 3Department of Zoology, University of Florida, Gainesville, Florida 32611 (Received October 31, 1980; revised January 19, 1981) Abstract--Root, stem, leaf, and latex samples of Asclepias eriocarpa collected from three plots in one population at 12 monthly intervals were assayed for total cardenolide content by spectroassay and for individual cardenolides by thin-layer chromatography. From May to September mean milligram equivalents of digitoxin per gram of dried plant were: latices, 56.8 >~ stems, 6.12 ~ leaves, 4.0 > roots, 2.5. With the exception of the roots, significant changes in gross cardenolide content occurred for each sample type with time of collection during the growing season, whereas variation within this population was found to be small. Labriformin, a nitrogen- containing cardenolide of low polarity, predominated in the latices. Leaf samples contained labriformin, labriformidin, desglucosyrioside, and other unidentified cardenolides. In addition to most of the same cardenolides as the leaves, the stems also contained uzarigenin. The roots contained desglucosyrioside and several polar cardenolides. The results are compared with those for other cardenolide-containingplants, and discussed in relation to anti-herbivore defense based on plant cardcnolide content. Arguments are advanced for a central role of the latex in cardenolide storage and deployment which maximizes the defensive qualities of the cardenolides while preventing toxicity to the plant. Key Words- Asclepias eriocarpa, Asclepiadaceae, milkweed, cardenolides, chemical defense, chemical ecology, labriformin, labriformidin, desgluco- syrioside, uzarigenin, variation, season, plant part, defense, root, stem, leaf, latex. ~Research supported by National Science Foundation Grants DEB 75-14266 and DEB 78-15419 (U.C. Davis) and DEB 75-14265, DEB 78-10658, and DEB 80-40388 (U.F.) 4present address: Department of Pharmacy, University of Sydney, N.S.W., Australia 2006. 981 0098-0331 / 81 / I 100-0981 $03.00/0 1981 Plenum Publishing Corporation 982 N~LSON ET AL. INTRODUCTION There is much current interest in plant secondary chemicals and the possible ecological functions they fulfill (Levin, 1976; Cates and Rhoades, 1977; Seigler, 1977; Swain, 1977). Many of these compounds are believed to have evolved in response to plant-herbivore, plant-pathogen, and plant-plant pressures while others may play a role in metabolic regulation or storage within plants. Secondary compounds thought to be antiherbivore defense agents have been divided into two groups. The first characteristically occur in relatively low concentrations (usually less than 2% by dry weight), have pronounced physiological effects, and include alkaloids, cyanogenic glyco- sides, nonprotein amino acids, cardiac glycosides, and many other toxic substances. They are generally found in ephemeral tissues of herbaceous and woody plants. The compounds in the second group occur in much higher concentrations and include digestibility-reducing agents such as tannins and phenolic resins. These occur primarily in mature tissues and organs of woody perennials and have been found in concentrations up to 60% by dry weight (Feeny, 1975; Rhoades and Cates, 1976). Cardenolides (cardiac glycosides) are produced by plants belonging to several families of angiosperms. Their biological properties which might be involved in herbivore defense include bitterness, emeticity, cardiotonic activity, Na+K+-ATPase inhibition, and cytotoxicity (references in Roeske et al., 1976). Many insect species feed on cardenolide-containing plants, and several sequester and store cardenolides for their own defense against vertebrate predators (Brower, 1970; references in Rothschild and Reichstein, 1976). Indications of toxic effects of cardenolides on insects have been indirectly gained from studies on regulation, sequestration, and detoxification of cardenolides by specialist feeders on plants containing these poisons (Duffey et al., 1978; Rafaeli-Bernstein and Mordue, 1978; Seiber et al., 1980). Other investigations both on adapted and nonadapted insects have compared the responses of isolated tissues or organs to purified cardenolides (Vaughan and Jungreis, 1977; Jungreis and Vaughan, 1977; Rafaeli-Bernstein and Mordue, 1978). The kinds, amounts, and distribution of cardenotides within plants should be major factors in governing the degree of protection conferred by these chemicals to any given plant and the means for bypassing and/or utilizing such chemicals available to adapted herbivores. By analogy with other secondary chemicals, cardenolide content is expected to be influenced by the plant's genetics, development, biochemistry, and physiology and its interaction with and response to biotic and abiotic environments (Levin, 1971; Jones, 1972; McKey, 1974; McKey, 1979; Brower et al., 1981). Biochemical and physiological sources of variation include site of cardenolide synthesis, means of translocation, stabilization and storage, mechanisms for avoiding CARDENOLIDE CONTENT OF Asclepias eriocarpa 983 self-toxicity, and the presence and distribution of other phytochemical or physical defenses. Ecological factors which might affect cardenolide concentration and distribution include herbivores, pathogens, and competing plants as well as day length, temperature, moisture, and nutrients. Assuming a plant has evolved chemical defensive capabilities in response to this host of environ- mental factors, then it could also have evolved the biochemical and physiological capacity to control chemical production and distribution in a manner which optimizes chemical protection throughout life while producing no detrimental effect to the plant in the process. Several milkweeds of the widely distributed North American genus Asclepias (Asclepiadaceae) contain varying amounts of cardenolides (Roeske et al., 1976). Of particular interest are the western A. eriocarpa Benth. and A. labriformis Jones. Both of these species are extremely toxic to livestock due principally to their cardenolides (Seiber et al., 1978; Benson et al., 1978, 1979). Structures for three of the major cardenolides from these two milkweeds [labriformin (1), labriformidin (II), and desglucosyrioside (III), Figure 1], were recently elucidated (Brown et al., 1979; Cheung et al., 1980). Another, uzarigenin (V), is a structurally simple cardenolide which is widely distributed in Asclepias spp. and plants of other families (references in Roeske et al., 1976). Asclepias plants are host to a number of different insect herbivores, some feeding preferentially on particular plant parts and some sequestering and storing cardenolide in the process (Brower, 1969; Isman et al., 1977a,b; Price and Willson, 1979; Vaughan, 1979). In this study we examined the distribution of cardenolides within various organs and latices of A. eriocarpa during an entire year. We then compared these findings to other studies of cardenolide variation in Asclepias and non-Asclepias plants and related them to potential lines of defense these plants may possess by virtue of their cardenolide content. METHODS AND MATERIALS Sampling of Plants. The A. eriocarpa plants were collected at monthly intervals during 1976 from a natural population growing in an occasionally cultivated wheat field located within a 10-hectare rolling hill site along Rd. 19, Woodland (Yolo County), California. Identification was verified by the staff of the Botany Department Herbarium, University of California, Davis (voucher specimen 71895). In August 1975 the field was divided into three plots separated by at least 115 m. Within these plots, five areas (9 X 20 m) were marked where plant growth was especially dense (ca. 100 plants) to aid in the location of roots during the winter months. Beginning in January 1976, samples were taken in each of the plots toward the middle of each month. 984 NELSON ET AL. ~ O HO IJ 18 120 22 "" OH 1 7 RO~~" CH H H H labrifornlin (I) uzarigenin (V, R= H) desglucouzarin (Va, R = 13-D-glucosyl-) uzarin (Vb, R = !~-D-glucosyl-12) 0 0 0 O HO. O~ O oH OH H H labriformidin Ill) ~ HO H o jyo s yriogenin (VI) HO. OH "'" "' OH ..'" O H3C H H 0 0 desgtucosyrioside (111) OH HO. Q O OH "'" H o ----~ '" OH ..- . ", ..-" 0 H H syriobioside (VII) : H: ~ OH H . 3c..-.L.o~o H ~ s yrios ide (IV) FIG. 1. Structures of some cardenolides occurring in Asclepias eriocarpa and/or A. syriaca. Each sample consisted of a composite group of ten plants and each was divided into component parts as follows: Jan.-Feb.--roots only. March--roots; shoots (white, 1-2 cm in length). April--roots; shoots (white and green, up to 16 cm). May--roots; stems (from root juncture to plant apex); latices (collected CARDENOLIDE CONTENT OF Asclepias eriocarpa 985 as drops from leaf and stem junctures and from stems when flower buds were removed); lower leaves (from lowest two whorls); middle leaves; upper leaves (from two whorls just below the plant apex); flower buds (unopened); and shoots (12-20 cm in length). June--same as May, plus flowers, but no shoots. July-Aug.--same as June, but no flowers or buds were available; unripe seed pods were also taken. Sept.--same as July-Aug., except lateral shoots (growth off the lower nodes of the stem) were