Journal of Chemical Ecology, Vot. 8, No. 7, 1982
I R I D O I D G L Y C O S I D E S IN THE N E C T A R OF Catalpa speciosa A R E U N P A L A T A B L E TO N E C T A R THIEVES
ANDREW
G. S T E P H E N S O N
Department of Biology The Pennsylvania State University University Park Pennsylvania 16802 (Received October 6, 1981; revised November 20, 1981) Abstract--The floral nectar of Catalpa speeiosa has a chemical mechanism that limits thievery. A bioassay employing sphingid larvae, Ceratomia catalpae, shows that catalpa iridoid glycosides are present in the floral nectar. When potential nectar thieves are fed nectar, a sucrose solution of identical concentration, or a sucrose solution plus 0.4% catalpol and 0.4% catalposide (catalpa iridoids), the thieves drink significantly more of the pure sucrose solution than either of the other two sugar sources. Those thieves that drink either the nectar or the sucrose solution ptus catalpa iridoids develop behavioral abnormalities including regurgitation and loss of locomotion. The response of the potential nectar thieves to nectar or the sucrose solution plus catalpa iridoids cannot be distinguished by the amount consumed or by their behavior. The legitimate diurnal pollinators of C. speeiosa are not affected by the iridoid glycosides. Key Words--Catalpa speciosa, catalpol, catalposide, iridoid glycosides, nectar thieves, pollination, toxic nectar.
tNTRODU CTION F l o r a l v i s i t o r s w h i c h r e m o v e the n e c t a r b u t fail to effect p o l l i n a t i o n a r e o f t e n c a l l e d n e c t a r thieves. M o s t c o m m o n l y , n e c t a r t h e f t results f r o m a m i s m a t c h b e t w e e n the m o r p h o l o g y o f the t h i e f a n d the m o r p h o l o g y o f the f l o w e r ( I n o u y e , 1980). F o r e x a m p l e , a f l o r a l v i s i t o r (e.g., a n a n t o r s m a l l bee) m a y be t o o s m a l l to c o n t a c t the s t a m e n s a n d s t i g m a u p o n e n t e r i n g o r l e a v i n g a large f l o w e r , It is c o m m o n l y t h o u g h t t h a t closed, z y g o m o r p h i c f l o w e r s , d e n s e hairs t h a t p r e s e n t a p h y s i c a l i m p e d i m e n t to s m a l l f o r a g e r s , sticky e x u d a t e s o n the p e d u n c l e o r c o r o l l a t u b e , a n d the l a c k o f l a n d i n g p l a t f o r m s o n h u m m i n g b i r d pollinated flowers are morphological adaptations that deter nectar thieves 1025 0098-0331/82/0700d025503,00/0 9 t982 Plenum Publishing Corporation
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STEPHENSON
(Faegri and van der Pijl, 1979; Guerrant and Fiedler, 1981; Kerner, 1878). Recently, Baker and Baker (1975) and Janzen (1977) have hypothesized that some floral nectars contain toxic or unpalatable substances that deter nectar thieves. This hypothesis has been strengthened by the mounting evidence which shows that the nectars of many species contain various secondary chemicals (Baker, 1978; Baker and Baker, 1975; Guerrant and Fiedler, 1981). The flowers of Catalpa speciosa (Warder ex Barney) Engel. (Bignoniaceae) are pollinated during the day by bumblebees and carpenter bees and at night by large moths, especially sphingids (Stephenson and Thomas, 1977; Stephenson, personal observation). Although C. speciosa receives frequent visitations from these legitimate pollinators, in seven field seasons studying various aspects of the reproductive biology of catalpa, I have observed very few nectar thieves. The lack of nectar thieves on C. speciosa is surprising because (1) the floral tube is large and unobstructed; (2) the unvisited flowers contain a large nectar reward compared to other beepollinated species (Stephenson and Thomas, 1977); and (3) catalpa produces a large floral display over a short period of time (the floral rewards are clustered in time and space) (Stephenson, 1979). These characteristics should make catalpa very attractive to nectar foragers that are not large enough to effect pollination. Previously, I reported (Stephenson, 198 l) that potential nectar thieves drank less C. speciosa nectar than a sugar solution of identical concentration. In addition, the thieves developed behavioral abnormalities such as erratic movements, regurgitation, loss of balance, or loss of locomotion after drinking the nectar but had none of the abnormalities after drinking the sugar solution. The findings suggested that C. speciosa has a chemical mechanism to limit thievery. In this paper, I present evidence from field and laboratory experiments which shows that the nectar of C. speciosa contains iridoid glycosides, that the iridoid glycosides are unpalatable to potential nectar thieves, and that bumblebees and carpenter bees (legitimate pollinators) are unaffected by these iridoids. METHODS AND MATERIALS
The leaves, fruits, and bark of Catalpa speciosa, along with its close relatives C. bignonioides, C. ovata, and C. bungei, have been shown to contain a mixture of about 15 iridoid glycosides (Bobbitt et al., 1961, 1966, 1967; Lunn et al., 1962; Nayar and Fraenkel, 1963; and references therein). Nayar and Fraenkel (1963) reported that catalpa iridoids are necessary in order to elicit a feeding response from catalpa's host-specific herbivore, the larva of Ceratornia catalpae (Sphingidae). They showed that these larvae
UNPALATABLE
Catalpa
GLYCOSIDES
1027
w o u l d n o t feed on the leaves o f a n y species o t h e r t h a n c a t a l p a (25 species f r o m 12 families were tested), even when faced with starvation. However, the leaves of v a r i o u s plants when s m e a r e d or c o a t e d with a w a t e r e x t r a c t of c a t a l p a leaves were readily c o n s u m e d . In a d d i t i o n , the larvae c o n s u m e d an artificial diet when it c o n t a i n e d either p o w d e r e d c a t a l p a leaves or a s a m p l e of purified c a t a l p a iridoids. In o r d e r to d e t e r m i n e if the n e c t a r of C. speciosa c o n t a i n e d c a t a l p a iridoids, I p e r f o r m e d the following bioassay. I m a d e an artificial diet similar to the one used by N a y a r and F r a e n k e l (1963), which consisted of 21 ml of water, 1 g cellulose p o w d e r , 0.25 g casein, 0.125 g yeast, 0.25 g glucose, a n d either 4 ml o f C. speciosa n e c t a r o r 4 ml of a sucrose s o l u t i o n of identical c o n c e n t r a t i o n to t h a t of the nectar. The n e c t a r of C. speciosa is sucrose d o m i n a n t (H.G. Baker, p e r s o n a l c o m m u n i c a t i o n ) . The c o m p o n e n t s were m i x e d t o g e t h e r in a b e a k e r , h e a t e d to 80~ in a water b a t h , a n d p o u r e d into a Petri dish while hot. A f t e r the diet h a d cooled a n d h a r d e n e d , 1.0-cm-diameter disks were p u n c h e d out with a c o r k borer. Two disks were placed in a Petri dish lined with filter p a p e r . E a c h Petri dish c o n t a i n e d two disks of the s a m e diet (nectar or sucrose solution) a n d two third instar Ceratomia catalpae larvae t h a t had been starved for 3 hr. Sixteen replicates were run for each diet, a n d each test r a n for 24 hr. The diets were e v a l u a t e d on the basis of the n u m b e r of fecal pellets d e p o s i t e d d u r i n g the test period. RESULTS The results of this e x p e r i m e n t are s u m m a r i z e d in Table 1. A f t e r 3 hr there were no significant differences in the n u m b e r of fecal pellets in the Petri dishes with the n e c t a r diet or the sucrose s o l u t i o n diet. However, I noticed t h a t the TABLE 1. FEEDING RESPONSES OF Ceratomia catalpae TO ARTIFICIAL DIET WITH ADDED SUCROSE SOLUTION OR C. speciosa NECTAR a
Artificial diet With sucrose Solution With C. speciosa nectar
Total fecal Mean number of Total fecal Mean number of pellets after fecal Pellets per pellets after fecal pellets per 3 hr replicate after 3 hrb 24 hr replicate after 24 hrC 47
2.9 -+ 1.7
54
3.4 -+ 1.9
41
2.6 -+ 1.9
120
7.5 -+3.3
a Sixteen replicates of two third-instar larvae were offered each type of artificial diet. The larvae were starved for 3 hr prior to the experiment. ONot significant; 0.3 > P > 0.20; t = 0.59; dr= 30. cp < 0.001 ; t = 43.8; df = 30.
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STEPHENSON
nectar diet 'was partially consumed after 3 hr, whereas the sucrose solution diet did not appear to have been eaten. Consequently, the fecal pellets that appeared during the first 3 hr were probably the result of feedings prior to the initiation of the experiment. The larvae were not starved long enough. After 24 hr, however, there was a mean of 7.5 + 3.3 (SD) pellets per dish with the nectar diet and only 3.4 _+ 1.9 pellets per dish with the sucrose solution diet. These differences are highly significant. Only seven new fecal pellets were deposited in the 16 dishes containing the sucrose solution diet during the final 21 hr of the experiment. In addition, the diets with nectar were either partially or totally consumed after 24hr while the sucrose solution diets were not eaten. Because catalpa iridoids have been shown to be a necessary phagostimulant for Ceratomia catalpae, I conclude from this experiment that catalpa iridoids are present in the nectar of C. speciosa. About nine months after the above bioassay was performed, I received purified samples of the two most common catalpa iridoids (catalpol and catalposide) from Professors J.S.D. Bacon, J.M. Bobbitt, and J.T. Edward. The samples were run on thin-layer chromatography (0.4% of each sample in a 36% sucrose solution) alongside C. speciosa nectar (which had been frozen since extraction) with n-butanol-ethanol-water (40:11:19) as the solvent (Kooiman, 1970). The dried chromatograms were sprayed with p-anisidine phosphate reagent and heated for 1 min at 105~ UV light was used to aid in the detection of the resultant spots. The nectar produced two spots that were identical in color and Rj to the catalposide and catalpol. The nectar also produced a third, unknown spot. These data support the conclusion drawn from the bioassay. I then wanted to determine if catalpa iridoids are responsible for deterring the potential nectar thieves of C. speciosa. That is, do these iridoids limit the amount of nectar consumed by the thieves, and do they cause the behavioral abnormalities that I reported previously (Stephenson, 1981)? In order to answer these questions, I collected approximately 1 ml of nectar using microcapillary tubes in June 1981. The sugar concentration of the pooled nectar was determined to be 36% sucrose-equivalents, using a temperaturecompensated refractometer. It was then necessary to identify potential nectar thieves. I established three criteria: A potential thief must avidly gather nectar; it must be active at the same time and location that Catalpa speciosa flowers; and it must be too small to be a pollinator. The ants that visit the extrafloral nectaries on the leaves of C. speciosa (Stephenson, 1982) and the common skipper butterfly (Poanes hobomok Harris, Hesperiidae), which was actively visiting flowers near the stand of catalpa, fit these criteria. The ants that visit the extrafloral nectaries are the workers of Camponotus noveboracenis (Fitch), C. nearcticus Emery, Prenolepis imparis (Say), Formica lasioides Emery, F. pallidefulva nitidiventris Emery, and Crematogaster cerasi (Fitch), Hymenoptera, Formicidae. No attempt was made to identify the ants as they
UNPALATABLE Catalpa GLYCOSIDES
1029
participated in the experiment. These potential nectar thieves are the same ones that I used previously (Stephenson, 1981). The palatability of catalpa iridoids to ants was determined by placing 20 #1 of nectar, 20tzl of sucrose solution of identical concentration, and 20#1 of the sucrose solution plus 0.4% catalpol and 0.4% catalposide approximately 8 cm apart on the bark of a catalpa tree trunk. This concentration of iridoids was chosen because (1) it had a taste similar to that of nectar and (2) Bobbitt et al., (196 l) reported that the leaves of catalpa contain 0.8% catalpa iridoids. As the ants that were moving up the tree trunk to the extrafloral nectaries discovered one of these sugar sources, I recorded the a m o u n t of time each visitor spent drinking it. Every 30 min I replaced each sugar source with a fresh supply. The palatability of catalpa iridoids to the other potential thief, the skipper P o a n e s h o b o r n o k , was determined by the methods I used previously (Stephenson, 1981), except the butterflies were fed either nectar, sucrose solution or sucrose solution plus 0.4% catalpol and 0.4% catalposide. Basically, the m e t h o d consists of placing a butterfly's proboscis into a calibrated microcapillary tube filled with one of the sugar sources and recording the a m o u n t that the butterfly drinks. The results of these experiments are summarized in Tables 2 and 3. There are no significant differences in the a m o u n t of time the ants spent drinking the nectar and sucrose solution plus catalpa iridoids, whereas the ants spent significantly more time drinking the sucrose solution than either the nectar or the sucrose solution plus catalpa iridoids. In addition, m a n y of the ants that d r a n k the nectar or sucrose solution plus iridoids exhibited behavioral abnormalities: they fell f r o m the tree; vigorously wiped their heads and antennae, or ran in circles. None of the ants that drank the sucrose solution exhibited these abnormalities. TABLE 2. PALATABILITY OF C. s p e c i o s a NECTAR, SUCROSE SOLUTION, AND SUCROSE SOLUTION PLUS CATALPA IRIDIODS (0.4% CATALPOSIDE; 0.4% CATALPOL) TO ANTS (MEAN -+ SD)
N Nectar Sucrose solution Sucrose solution plus iridoids
Time spent d~inking (sec)a
58 38
25 -+41 118 -+73
52
33 +-49
aNectar vs. sucrose solution: t = 7.85;df = 94;P < 0.001. Nectar vs. sucrose solution plus iridoids: t = 0.92; d f = 108; P > 0.10. Sucrose solution vs. sucrose solution plus iridoids: t = 6 . 6 2 ; d f = 88;P < 0.001.
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STEVHENSON
TABLE 3. PALATABILITY OF C. speciosa NECTAR, SUCROSE SOLUTION, AND SUCROSE SOLUTION PLUS CATALPA IRIDOIDS (0.4% CATALPOSIDE; 0.4% CATALPOL) TO Poanes hobomok, THE COMMON SKIPPER (MEAN 4- SD)
Nectar Sucrose solution Sucrose solution plus iridoids
N
A m o u n t consumed (#l)a
Behavioral abnormalities
14 21
3.2 _+ 1.4 9.5 +- 6.3
3 were comatose none
22
3.7 -+ 3.2
7 were comatose
aNectar vs. sucrose solution: t = 3.7; df = 33; p < 0.001. Nectar vs. sucrose solution plus iridoids: t = 0.50; df = 34; 0.40 > P > 0.30. Sucrose solution vs. sucrose solution plus iridoids: t = 3 . 8 ; d r = 4 1 ; P < 0.001.
There are also no significant differences in the amount of nectar and sucrose solution plus catalpa iridoids that the skippers drank (Table 3). However, the skippers drank significantly more sucrose solution than either nectar or sucrose solution plus iridoids. In addition, many of the skippers that drank the nectar or sucrose solution plus iridoids exhibited behavioral abnormalities: most could not reroll their proboscides; others could not walk or fly upon completion of the experiment and appeared to be comatose. All of the skippers that were fed the sucrose solution could reroll their proboscides and could walk and fly upon completion of the experiment. From these data I conclude that catalpa iridoids are the unpalatable substances to the potential nectar thieves of C. speciosa. Because nectar is a primary reward for the legitimate pollinators of C. speciosa, any advantage associated with nectar that is unpalatable to thieves would be negated if the nectar is also unpalatable to the legitimate pollinators. In order to examine the palatability of C. speciosa nectar to bumblebees and carpenter bees, I captured bees (Bombus bimaculatus, B. fervidus, B. impatiens, B. vagans, and Xylocopa sp.) that were foraging on or near C. speciosa, placed them into a screen-covered cage, and starved them for 2 hr. I then permitted the bees to forage on an artificial flower-board. Wells 3 m m deep and 4 m m in diameter were drilled 10 cm apart into a 30-cm • 20-cm • 6-mm clear Plexiglas sheet. This produced two rows of three evenly spaced wells. A blue cardboard square (4 cm • 4 cm) was centered and affixed under each well. The flower-board was then placed over a piece of green cardboard. Next, every well on the board was filled with 20 #1 of either C. speciosa nectar, sucrose solution, or sucrose solution plus catalpa iridoids. All of the wells on any given board were filled with the same sugar source. After filling the wells, the board was slipped into the cage and the foraging behavior of individual bees was monitored. After a bee foraged on the board, the board was removed and the amount of the sugar source
UNPALATABLE
Catalpa
GLYCOSIDES
1031
remaining in the first two wells visited by the bee was determined by using calibrated microcapillary tubes. The bee was observed for 5 min and then removed from the cage. The board was cleaned and filled with a different sugar source, and the procedure was repeated. In most cases, a bee began to forage on the flower-board within 10 min of its placement into the cage. Usually, only one bee at a time foraged on the board and usually the bee removed the sugar source from all six wells (although only the first two wells were analyzed). When two bees foraged simultaneously, the data were counted if the bees visited two wells prior to encountering each other. If, however, the bees attempted to forage at the same well, or if a bee visited a well previously emptied by the other bee, the data were discounted and the bees were removed from the cage. The results of this experiment are summarized in Table 4. Most bees removed all or nearly all of the sugar source in the first two wells. Furthermore, there were no significant differences in the amounts of the three sugar sources the bees removed (see Table 4). Even though many of the bees visited all six wells, none appeared to behave abnormally after consuming any of the sugar sources. From these data, I conclude that all three of these sugar sources are palatable to the legitimate daytime pollinators of C. speciosa. DISCUSSION The floral nectar of Catalpa speciosa is a large and potentially exploitable resource for nectar thieves. However, potential nectar thieves consume only limited amounts of catalpa nectar and appear to become intoxicated after doing so (Stephenson, 1981). The data presented here suggest that the nectar contains iridoid glycosides and that these iridoids are unpalatable to potential thieves but not to bumblebees and carpenter bees, the legitimate diurnal pollinators. The unpalatable nectar of C. speciosa can be viewed as a mechanism that reduces both the amount of resource per flower that can be TABLE 4. PALATABILITYOF C. speciosa NECTAR, SUCROSE SOLUTION,AND SUCROSE SOLUTION PLUS CATALPAIRIDOIDS (0.4% CATALPOSIDEAND 0.4% CATALPOL) TO BUMBLEBEESAND CARPENTER BEES (MEANS + SD)
Reward
C. speciosa nectar Sucrose solution Sucrose solution plus indoids
Number of Wells
Amount consumed per well (M)a
50 54
19.3 -+2.4 19.7 +- 1.4
20
19.5 -+1.4
ap ~ 0.20; H= 0.43; dr= 2; Kruskal-Wallis test.
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STEPHENSON
taken and the number of flowers that can be visited per unit time by the thieves. This would effectively shrink the size of the resource from the perspective of the thieves (but not the pollinators). Consequently, alternative nectar sources, such as other flowering species and extrafloral nectaries, may then be more attractive to thieves than the floral nectar of C. speciosa. Secondary chemicals have been reported in the floral nectars of many species (Baker, 1978; Baker and Baker, 1975; Clinch et al., 1972; Deinzer et al., 1977; Guerrant and Fiedler, 1981; Pryce-Jones, 1944). In some cases, these chemicals in the nectar have been shown to adversely affect various aspects of commercial honey production or pose potential health hazards to humans who consume honey made from these nectars (Clinch et al., 1972; Deinzer et al., 1977; Pryce-Jones, 1944). Ecologists have only begun to explore the role of toxic nectars in the pollination biology of plants (Feinsinger and Swarm, 1978; Guerrant and Fiedler, 1981). To my knowledge, this is the first report of iridoid glycosides in nectar. The toxic or unpalatable effect of iridoids on insects is well known. They are often referred to as bitter substances (Thomas, 1961; Hegnauer, 1966). Hagnauer (1973) reported that iridoids are toxic to generalist insects and that some plant species with iridoids (including species containing catalpol) have been used as insecticides. Iridoids have also been reported in the defensive secretions of many arthropods (see Cavill, 1969; Harborne, 1977; Roth and Eisner, 1962). In addition, some iridoids are antimalarial and antimicrobial (Merck Index, 1976). Finally, iridoids have been implicated in the unpalatability defenses of the checkerspot butterfly, Euphydryas spp. (Bowers, 1981), which suggests that they may also affect vertebrates. This study makes no attempt to understand the mechanisms by which the bumblebees and carpenter bees circumvent the toxic effects of the catalpa iridoids. Given the rather large percentage of floral nectars that contain secondary chemicals (Baker, 1978; Baker and Baker, 1975; Guerrant and Fiedler, 1981), it is reasonable to assume that bees frequently encounter these chemicals and must have well-developed mechanisms for dealing with them (Rhoades and Bergdahl, 1981). It should also be noted that some of the bees in this study consumed up to 120 #1 of nectar or sucrose solution plus catalpa iridoids over a very short period of time without showing any adverse effects; this is probably very close to the maximum amount that a large bee will take on any one foraging trip. Real (1981) noted that bumblebees usually consume only 60-80 #1 of nectar per trip when foraging on an artificial flower-board. This study did not examine the effects of catalpa iridoids on the nocturnal pollinators of C. speciosa. However, one of the principal nocturnal pollinators is the sphingid, Ceratomia catalpae, whose larvae eat the leaves of catalpa. It is reasonable to speculate that the adult moth has retained the capacity to detoxify catalpa iridoids. From the data presented here, it appears that the extrafloral nectar of C. speciosa differs from the floral nectar because the same ants that avidly
UNPALATABLE Catalpa GLYCOSIDES
1033
gather extrafloral nectar (Stephenson, 1982) find the floral nectar to be unpalatable. This suggests that the extrafloral nectar either lacks or has very low concentrations of iridoid glycosides. In other species, it has been shown that floral and extrafloral nectar from the same plant differ in sugar concentration (Vansell, 1939), in the complement of sugars (Elias et al., 1975; Elias and Gelband, 1975; Keeler, 1977) and in the amount and types of amino acids (Baker et al., 1978). Baker et al., (1978) suggest that these differences occur because the two types of nectaries function to attract different insects with different nutritional requirements and preferences. In conclusion, it is thought that floral morphology functions to both attract some floral visitors and exclude others (Faegri and van der Pijl, 1979). Recent studies suggest that the concentration and amount of lipids, amino acids, and sugars in floral nectar also have adaptive significance in attracting and excluding floral visitors (see Baker and Baker, 1975; Heinrich, 1975; Heinrich and Raven, 1972). This study provides evidence that secondary chemicals in floral nectars may also have adaptive significance in excluding some floral visitors, Acknowledgments--I thank B.H. Allen and S.T. Stephenson for their help in the field and J.L. Burris for technical advice. Professors J.S.D. Bacon, J.M. Bobbitt, and J. M. Edward kindly provided catalposide, and J.S.D. Bacon and J.M. Bobbitt also provided catalpol. I thank D.R. Smith for identifying the ants, F.C. Evans for identifying the bees, and W.S. Benninghoff for use of the facilities at the Matthaei Botanical Gardens of the University of Michigan. This research was supported by NSF grant DEB-7905508. To all I am grateful.
REFERENCES BAKER, H.G. 1978. Chemical aspects of the pollination of woody plants in the tropics, pp. 57-82, in P.B. Tomlinson, and M. Zimmerman (eds.). Tropical Trees as Living Systems. Cambridge University Press, New York. BAKER, H.G., and BAKER, I. 1975. Studies of nectar-constituents and pollinator plant coevolution, pp. 100-140, in L.E. Gilbert and P.H. Raven (eds.). Coevolution of Plants and Animals. University of Texas Press, Austin. BAKER, H.G., OPLER, P.A., and BAKER,l. 1978. A comparison of the amino acid complements of floral and extrafloral nectars. Bot. Gaz. 139:322-332. BOBBITT, J.M., SCHMID, H., and AFRICA, T.B. 1961. Catalpa glycosides. I. The characterization of catalposide. J. Org. Chem. 26:3090-3094. BOBBITT, J.M., SPIGGLE, D.W., MAHBOOB, S., SCHMID, H., and VON PHILIPSBORN, W. 1966. Catalpa glycosides. Ill. The structure of catalposide. 3. Org. Chem. 31:500-506. BOBBITT, J.M., KIELY, D.E., LAM, A.Y., and SNYDER, E.I. 1967. Catalpa glycosides. IV. The sterochemistry of catalposide. J. Org. Chem. 32:1459-1461. BOWERS, M.D. 1981. Unpalatability as a defense strategy of western checkerspot butterflies (Euphydryas Scudder, Nymphalidae). Evolution 35:367-375. CAVILL, G.W.K. 1969. Insect terpenoids and nepetalactone, pp. 203-238, in W.I. Taylor, and A.R. Battersby (eds.). Cyclopentanoid Terpene Derivatives. Marcel Dekker, Inc., New York. CLINCH, P.G., PALMER-JONES,T., and FORSTER, I.W. 1972. Effect on honeybees of nectar from yellow Kowhai (Sophora microphylla Ait.). N.Z.J. Agric. Res. 15:194-201.
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DEINZER, M,L., THOMSON,P.A., BURGETT,D.M., and ISAACSON,D.L. 1977. Pyrrolizidine alkaloids: Their occurrence in honey from tansy ragwort (Senecio jacobaea L.). Science 195:497-499.
ELIAS, T.S., and GELBAND, H. 1975. Nectar: Its production and functions in trumpet creeper. Science 189:289-291. ELIAS, T.S., ROZlCH, W.R., and NEWCOMBE,L. 1975. The foliar and floral nectaries of Turnera ulmifolia L. Am. J. Bot. 62:570-576. FAEGRI, K., and VAN DER PIJL, L. 1979. The Principles of Pollination Ecology, 3rd revised ed. Pergamon Press, New York. FEINSINGER, P., and SWARM,L.A. 1978. How common are ant-repellent nectars? Biotropica 10:238-239. GUERRANT,E.O., JR., and FIEDLER,P.L. 1981, Flower defenses against nectar-pilferage by ants. Biolropica 13(Suppl.):25-33. HARBORNE, J.B. 1977. Introduction to Ecological Biochemistry. Academic Press, New York. HEGNAUER, R. 1966. Aucubin-like glucosides: Distribution and significance as a systematic criterion. Pharm. Acta Helv. 11:577-587. HEGNAUER,R. 1973. Chemotaxonomie der Pflanzen, Vol. 6. Berkhauser Verlag, Basel. HEINRICrt, B, 1975. Energetics of pollination. Annu. Rev. Ecol. Syst. 6:139-170. HEINRICH, B., and RAVEN,P.H. 1972. Energetics and pollination ecology. Science 176:597-602. INOUYE, D.W. 1980. The terminology of floral larceny. Ecology 61:1251-1253. JANZEN, D.H. 1977. Why ants don's visit flowers. Biotropica 9:252. KEELER,K.H. 1977. The extrafloral nectaries of Ipomoea carnea (Convolvulaceae). Am. J. Bot. 64:1 t82-1188. KERNER VON MARILAUN, A. 1978. Flowers and Their Unbidden Guests. Kega Paul, London. KOOIMAN,P. 1970. The occurrence ofiridoid glycosides in the Scrophulariaceae. Acta Bot. Neerl. 19:329-340. LUNN, W.H., EDWARD, D.W., and EDWARD,J.T. 1962. Studies on the structure of catalposide. Can. J. Chem. 40:104-110. Merck Index. 1976. 9th ed. Merck, Rahway, New Jersey. NAYAR, J.K., and FRAENKEL,C. 1963. The chemical basis of the host selection in the catalpa sphinx, Ceratomia catalpae (Lepidoptera, Sphingidae). Am. Entomol. Soc. Am. 56:119-122, PRYCE-JONES, J. 1944. Some problems associated with nectar, pollen and honey. Proc. Linn. Soc. London 1944:129-174. REAL, L.A. 1981. Uncertainty and pollinator-plant interactions: The foraging behavior of bees and wasps on artificial flowers. Ecology 62:20-26. RHOADES, D.F., and BERGDAHL, F.C. 1981. Adaptive significance of toxic nectar. Am. Nat. 117:798-803. ROTH, L.M., and EISNER, T. 1962. Chemical defenses of arthropods. Annu. Rev. Entomol. 7:107-136. STEVHENSON, A.G, 1979. An evolutionary examination of the floral display of Catalpa speciosa (Bignoniaceae). Evolution 33:1200-1209. SXEPHENSON,A.G. 1981. Toxic nectar deters nectar thieves of Catalpa speciosa. Am. Midl. Nat. 105:381-383. STEP~ENSON, A.G. 1982. The role of the extrafloral nectaries of Catalpa speciosa in limiting herbivory and increasing fruit production. Ecology. STEPHENSON, A.G., and THOMAS,W.W. 1977. Diurnal and nocturnal pollination of Catalpa speciosa (Bignoniaceae). Syst. Bot. 2:191-198. THOMAS,R. 1961. A possible biosynthetic relationship between the cyclopentanoid monoterpenes and the indole alkaloids. Tetrahedron Lett. 16:544-553. VANSELL,G.H. 1939. The sugar concentration of western nectars. J. Econ. Entomol. 35:321-323.
I R I D O I D G L Y C O S I D E S IN THE N E C T A R OF Catalpa speciosa A R E U N P A L A T A B L E TO N E C T A R THIEVES
ANDREW
G. S T E P H E N S O N
Department of Biology The Pennsylvania State University University Park Pennsylvania 16802 (Received October 6, 1981; revised November 20, 1981) Abstract--The floral nectar of Catalpa speeiosa has a chemical mechanism that limits thievery. A bioassay employing sphingid larvae, Ceratomia catalpae, shows that catalpa iridoid glycosides are present in the floral nectar. When potential nectar thieves are fed nectar, a sucrose solution of identical concentration, or a sucrose solution plus 0.4% catalpol and 0.4% catalposide (catalpa iridoids), the thieves drink significantly more of the pure sucrose solution than either of the other two sugar sources. Those thieves that drink either the nectar or the sucrose solution ptus catalpa iridoids develop behavioral abnormalities including regurgitation and loss of locomotion. The response of the potential nectar thieves to nectar or the sucrose solution plus catalpa iridoids cannot be distinguished by the amount consumed or by their behavior. The legitimate diurnal pollinators of C. speeiosa are not affected by the iridoid glycosides. Key Words--Catalpa speciosa, catalpol, catalposide, iridoid glycosides, nectar thieves, pollination, toxic nectar.
tNTRODU CTION F l o r a l v i s i t o r s w h i c h r e m o v e the n e c t a r b u t fail to effect p o l l i n a t i o n a r e o f t e n c a l l e d n e c t a r thieves. M o s t c o m m o n l y , n e c t a r t h e f t results f r o m a m i s m a t c h b e t w e e n the m o r p h o l o g y o f the t h i e f a n d the m o r p h o l o g y o f the f l o w e r ( I n o u y e , 1980). F o r e x a m p l e , a f l o r a l v i s i t o r (e.g., a n a n t o r s m a l l bee) m a y be t o o s m a l l to c o n t a c t the s t a m e n s a n d s t i g m a u p o n e n t e r i n g o r l e a v i n g a large f l o w e r , It is c o m m o n l y t h o u g h t t h a t closed, z y g o m o r p h i c f l o w e r s , d e n s e hairs t h a t p r e s e n t a p h y s i c a l i m p e d i m e n t to s m a l l f o r a g e r s , sticky e x u d a t e s o n the p e d u n c l e o r c o r o l l a t u b e , a n d the l a c k o f l a n d i n g p l a t f o r m s o n h u m m i n g b i r d pollinated flowers are morphological adaptations that deter nectar thieves 1025 0098-0331/82/0700d025503,00/0 9 t982 Plenum Publishing Corporation
1026
STEPHENSON
(Faegri and van der Pijl, 1979; Guerrant and Fiedler, 1981; Kerner, 1878). Recently, Baker and Baker (1975) and Janzen (1977) have hypothesized that some floral nectars contain toxic or unpalatable substances that deter nectar thieves. This hypothesis has been strengthened by the mounting evidence which shows that the nectars of many species contain various secondary chemicals (Baker, 1978; Baker and Baker, 1975; Guerrant and Fiedler, 1981). The flowers of Catalpa speciosa (Warder ex Barney) Engel. (Bignoniaceae) are pollinated during the day by bumblebees and carpenter bees and at night by large moths, especially sphingids (Stephenson and Thomas, 1977; Stephenson, personal observation). Although C. speciosa receives frequent visitations from these legitimate pollinators, in seven field seasons studying various aspects of the reproductive biology of catalpa, I have observed very few nectar thieves. The lack of nectar thieves on C. speciosa is surprising because (1) the floral tube is large and unobstructed; (2) the unvisited flowers contain a large nectar reward compared to other beepollinated species (Stephenson and Thomas, 1977); and (3) catalpa produces a large floral display over a short period of time (the floral rewards are clustered in time and space) (Stephenson, 1979). These characteristics should make catalpa very attractive to nectar foragers that are not large enough to effect pollination. Previously, I reported (Stephenson, 198 l) that potential nectar thieves drank less C. speciosa nectar than a sugar solution of identical concentration. In addition, the thieves developed behavioral abnormalities such as erratic movements, regurgitation, loss of balance, or loss of locomotion after drinking the nectar but had none of the abnormalities after drinking the sugar solution. The findings suggested that C. speciosa has a chemical mechanism to limit thievery. In this paper, I present evidence from field and laboratory experiments which shows that the nectar of C. speciosa contains iridoid glycosides, that the iridoid glycosides are unpalatable to potential nectar thieves, and that bumblebees and carpenter bees (legitimate pollinators) are unaffected by these iridoids. METHODS AND MATERIALS
The leaves, fruits, and bark of Catalpa speciosa, along with its close relatives C. bignonioides, C. ovata, and C. bungei, have been shown to contain a mixture of about 15 iridoid glycosides (Bobbitt et al., 1961, 1966, 1967; Lunn et al., 1962; Nayar and Fraenkel, 1963; and references therein). Nayar and Fraenkel (1963) reported that catalpa iridoids are necessary in order to elicit a feeding response from catalpa's host-specific herbivore, the larva of Ceratornia catalpae (Sphingidae). They showed that these larvae
UNPALATABLE
Catalpa
GLYCOSIDES
1027
w o u l d n o t feed on the leaves o f a n y species o t h e r t h a n c a t a l p a (25 species f r o m 12 families were tested), even when faced with starvation. However, the leaves of v a r i o u s plants when s m e a r e d or c o a t e d with a w a t e r e x t r a c t of c a t a l p a leaves were readily c o n s u m e d . In a d d i t i o n , the larvae c o n s u m e d an artificial diet when it c o n t a i n e d either p o w d e r e d c a t a l p a leaves or a s a m p l e of purified c a t a l p a iridoids. In o r d e r to d e t e r m i n e if the n e c t a r of C. speciosa c o n t a i n e d c a t a l p a iridoids, I p e r f o r m e d the following bioassay. I m a d e an artificial diet similar to the one used by N a y a r and F r a e n k e l (1963), which consisted of 21 ml of water, 1 g cellulose p o w d e r , 0.25 g casein, 0.125 g yeast, 0.25 g glucose, a n d either 4 ml o f C. speciosa n e c t a r o r 4 ml of a sucrose s o l u t i o n of identical c o n c e n t r a t i o n to t h a t of the nectar. The n e c t a r of C. speciosa is sucrose d o m i n a n t (H.G. Baker, p e r s o n a l c o m m u n i c a t i o n ) . The c o m p o n e n t s were m i x e d t o g e t h e r in a b e a k e r , h e a t e d to 80~ in a water b a t h , a n d p o u r e d into a Petri dish while hot. A f t e r the diet h a d cooled a n d h a r d e n e d , 1.0-cm-diameter disks were p u n c h e d out with a c o r k borer. Two disks were placed in a Petri dish lined with filter p a p e r . E a c h Petri dish c o n t a i n e d two disks of the s a m e diet (nectar or sucrose solution) a n d two third instar Ceratomia catalpae larvae t h a t had been starved for 3 hr. Sixteen replicates were run for each diet, a n d each test r a n for 24 hr. The diets were e v a l u a t e d on the basis of the n u m b e r of fecal pellets d e p o s i t e d d u r i n g the test period. RESULTS The results of this e x p e r i m e n t are s u m m a r i z e d in Table 1. A f t e r 3 hr there were no significant differences in the n u m b e r of fecal pellets in the Petri dishes with the n e c t a r diet or the sucrose s o l u t i o n diet. However, I noticed t h a t the TABLE 1. FEEDING RESPONSES OF Ceratomia catalpae TO ARTIFICIAL DIET WITH ADDED SUCROSE SOLUTION OR C. speciosa NECTAR a
Artificial diet With sucrose Solution With C. speciosa nectar
Total fecal Mean number of Total fecal Mean number of pellets after fecal Pellets per pellets after fecal pellets per 3 hr replicate after 3 hrb 24 hr replicate after 24 hrC 47
2.9 -+ 1.7
54
3.4 -+ 1.9
41
2.6 -+ 1.9
120
7.5 -+3.3
a Sixteen replicates of two third-instar larvae were offered each type of artificial diet. The larvae were starved for 3 hr prior to the experiment. ONot significant; 0.3 > P > 0.20; t = 0.59; dr= 30. cp < 0.001 ; t = 43.8; df = 30.
1028
STEPHENSON
nectar diet 'was partially consumed after 3 hr, whereas the sucrose solution diet did not appear to have been eaten. Consequently, the fecal pellets that appeared during the first 3 hr were probably the result of feedings prior to the initiation of the experiment. The larvae were not starved long enough. After 24 hr, however, there was a mean of 7.5 + 3.3 (SD) pellets per dish with the nectar diet and only 3.4 _+ 1.9 pellets per dish with the sucrose solution diet. These differences are highly significant. Only seven new fecal pellets were deposited in the 16 dishes containing the sucrose solution diet during the final 21 hr of the experiment. In addition, the diets with nectar were either partially or totally consumed after 24hr while the sucrose solution diets were not eaten. Because catalpa iridoids have been shown to be a necessary phagostimulant for Ceratomia catalpae, I conclude from this experiment that catalpa iridoids are present in the nectar of C. speciosa. About nine months after the above bioassay was performed, I received purified samples of the two most common catalpa iridoids (catalpol and catalposide) from Professors J.S.D. Bacon, J.M. Bobbitt, and J.T. Edward. The samples were run on thin-layer chromatography (0.4% of each sample in a 36% sucrose solution) alongside C. speciosa nectar (which had been frozen since extraction) with n-butanol-ethanol-water (40:11:19) as the solvent (Kooiman, 1970). The dried chromatograms were sprayed with p-anisidine phosphate reagent and heated for 1 min at 105~ UV light was used to aid in the detection of the resultant spots. The nectar produced two spots that were identical in color and Rj to the catalposide and catalpol. The nectar also produced a third, unknown spot. These data support the conclusion drawn from the bioassay. I then wanted to determine if catalpa iridoids are responsible for deterring the potential nectar thieves of C. speciosa. That is, do these iridoids limit the amount of nectar consumed by the thieves, and do they cause the behavioral abnormalities that I reported previously (Stephenson, 1981)? In order to answer these questions, I collected approximately 1 ml of nectar using microcapillary tubes in June 1981. The sugar concentration of the pooled nectar was determined to be 36% sucrose-equivalents, using a temperaturecompensated refractometer. It was then necessary to identify potential nectar thieves. I established three criteria: A potential thief must avidly gather nectar; it must be active at the same time and location that Catalpa speciosa flowers; and it must be too small to be a pollinator. The ants that visit the extrafloral nectaries on the leaves of C. speciosa (Stephenson, 1982) and the common skipper butterfly (Poanes hobomok Harris, Hesperiidae), which was actively visiting flowers near the stand of catalpa, fit these criteria. The ants that visit the extrafloral nectaries are the workers of Camponotus noveboracenis (Fitch), C. nearcticus Emery, Prenolepis imparis (Say), Formica lasioides Emery, F. pallidefulva nitidiventris Emery, and Crematogaster cerasi (Fitch), Hymenoptera, Formicidae. No attempt was made to identify the ants as they
UNPALATABLE Catalpa GLYCOSIDES
1029
participated in the experiment. These potential nectar thieves are the same ones that I used previously (Stephenson, 1981). The palatability of catalpa iridoids to ants was determined by placing 20 #1 of nectar, 20tzl of sucrose solution of identical concentration, and 20#1 of the sucrose solution plus 0.4% catalpol and 0.4% catalposide approximately 8 cm apart on the bark of a catalpa tree trunk. This concentration of iridoids was chosen because (1) it had a taste similar to that of nectar and (2) Bobbitt et al., (196 l) reported that the leaves of catalpa contain 0.8% catalpa iridoids. As the ants that were moving up the tree trunk to the extrafloral nectaries discovered one of these sugar sources, I recorded the a m o u n t of time each visitor spent drinking it. Every 30 min I replaced each sugar source with a fresh supply. The palatability of catalpa iridoids to the other potential thief, the skipper P o a n e s h o b o r n o k , was determined by the methods I used previously (Stephenson, 1981), except the butterflies were fed either nectar, sucrose solution or sucrose solution plus 0.4% catalpol and 0.4% catalposide. Basically, the m e t h o d consists of placing a butterfly's proboscis into a calibrated microcapillary tube filled with one of the sugar sources and recording the a m o u n t that the butterfly drinks. The results of these experiments are summarized in Tables 2 and 3. There are no significant differences in the a m o u n t of time the ants spent drinking the nectar and sucrose solution plus catalpa iridoids, whereas the ants spent significantly more time drinking the sucrose solution than either the nectar or the sucrose solution plus catalpa iridoids. In addition, m a n y of the ants that d r a n k the nectar or sucrose solution plus iridoids exhibited behavioral abnormalities: they fell f r o m the tree; vigorously wiped their heads and antennae, or ran in circles. None of the ants that drank the sucrose solution exhibited these abnormalities. TABLE 2. PALATABILITY OF C. s p e c i o s a NECTAR, SUCROSE SOLUTION, AND SUCROSE SOLUTION PLUS CATALPA IRIDIODS (0.4% CATALPOSIDE; 0.4% CATALPOL) TO ANTS (MEAN -+ SD)
N Nectar Sucrose solution Sucrose solution plus iridoids
Time spent d~inking (sec)a
58 38
25 -+41 118 -+73
52
33 +-49
aNectar vs. sucrose solution: t = 7.85;df = 94;P < 0.001. Nectar vs. sucrose solution plus iridoids: t = 0.92; d f = 108; P > 0.10. Sucrose solution vs. sucrose solution plus iridoids: t = 6 . 6 2 ; d f = 88;P < 0.001.
1030
STEVHENSON
TABLE 3. PALATABILITY OF C. speciosa NECTAR, SUCROSE SOLUTION, AND SUCROSE SOLUTION PLUS CATALPA IRIDOIDS (0.4% CATALPOSIDE; 0.4% CATALPOL) TO Poanes hobomok, THE COMMON SKIPPER (MEAN 4- SD)
Nectar Sucrose solution Sucrose solution plus iridoids
N
A m o u n t consumed (#l)a
Behavioral abnormalities
14 21
3.2 _+ 1.4 9.5 +- 6.3
3 were comatose none
22
3.7 -+ 3.2
7 were comatose
aNectar vs. sucrose solution: t = 3.7; df = 33; p < 0.001. Nectar vs. sucrose solution plus iridoids: t = 0.50; df = 34; 0.40 > P > 0.30. Sucrose solution vs. sucrose solution plus iridoids: t = 3 . 8 ; d r = 4 1 ; P < 0.001.
There are also no significant differences in the amount of nectar and sucrose solution plus catalpa iridoids that the skippers drank (Table 3). However, the skippers drank significantly more sucrose solution than either nectar or sucrose solution plus iridoids. In addition, many of the skippers that drank the nectar or sucrose solution plus iridoids exhibited behavioral abnormalities: most could not reroll their proboscides; others could not walk or fly upon completion of the experiment and appeared to be comatose. All of the skippers that were fed the sucrose solution could reroll their proboscides and could walk and fly upon completion of the experiment. From these data I conclude that catalpa iridoids are the unpalatable substances to the potential nectar thieves of C. speciosa. Because nectar is a primary reward for the legitimate pollinators of C. speciosa, any advantage associated with nectar that is unpalatable to thieves would be negated if the nectar is also unpalatable to the legitimate pollinators. In order to examine the palatability of C. speciosa nectar to bumblebees and carpenter bees, I captured bees (Bombus bimaculatus, B. fervidus, B. impatiens, B. vagans, and Xylocopa sp.) that were foraging on or near C. speciosa, placed them into a screen-covered cage, and starved them for 2 hr. I then permitted the bees to forage on an artificial flower-board. Wells 3 m m deep and 4 m m in diameter were drilled 10 cm apart into a 30-cm • 20-cm • 6-mm clear Plexiglas sheet. This produced two rows of three evenly spaced wells. A blue cardboard square (4 cm • 4 cm) was centered and affixed under each well. The flower-board was then placed over a piece of green cardboard. Next, every well on the board was filled with 20 #1 of either C. speciosa nectar, sucrose solution, or sucrose solution plus catalpa iridoids. All of the wells on any given board were filled with the same sugar source. After filling the wells, the board was slipped into the cage and the foraging behavior of individual bees was monitored. After a bee foraged on the board, the board was removed and the amount of the sugar source
UNPALATABLE
Catalpa
GLYCOSIDES
1031
remaining in the first two wells visited by the bee was determined by using calibrated microcapillary tubes. The bee was observed for 5 min and then removed from the cage. The board was cleaned and filled with a different sugar source, and the procedure was repeated. In most cases, a bee began to forage on the flower-board within 10 min of its placement into the cage. Usually, only one bee at a time foraged on the board and usually the bee removed the sugar source from all six wells (although only the first two wells were analyzed). When two bees foraged simultaneously, the data were counted if the bees visited two wells prior to encountering each other. If, however, the bees attempted to forage at the same well, or if a bee visited a well previously emptied by the other bee, the data were discounted and the bees were removed from the cage. The results of this experiment are summarized in Table 4. Most bees removed all or nearly all of the sugar source in the first two wells. Furthermore, there were no significant differences in the amounts of the three sugar sources the bees removed (see Table 4). Even though many of the bees visited all six wells, none appeared to behave abnormally after consuming any of the sugar sources. From these data, I conclude that all three of these sugar sources are palatable to the legitimate daytime pollinators of C. speciosa. DISCUSSION The floral nectar of Catalpa speciosa is a large and potentially exploitable resource for nectar thieves. However, potential nectar thieves consume only limited amounts of catalpa nectar and appear to become intoxicated after doing so (Stephenson, 1981). The data presented here suggest that the nectar contains iridoid glycosides and that these iridoids are unpalatable to potential thieves but not to bumblebees and carpenter bees, the legitimate diurnal pollinators. The unpalatable nectar of C. speciosa can be viewed as a mechanism that reduces both the amount of resource per flower that can be TABLE 4. PALATABILITYOF C. speciosa NECTAR, SUCROSE SOLUTION,AND SUCROSE SOLUTION PLUS CATALPAIRIDOIDS (0.4% CATALPOSIDEAND 0.4% CATALPOL) TO BUMBLEBEESAND CARPENTER BEES (MEANS + SD)
Reward
C. speciosa nectar Sucrose solution Sucrose solution plus indoids
Number of Wells
Amount consumed per well (M)a
50 54
19.3 -+2.4 19.7 +- 1.4
20
19.5 -+1.4
ap ~ 0.20; H= 0.43; dr= 2; Kruskal-Wallis test.
1032
STEPHENSON
taken and the number of flowers that can be visited per unit time by the thieves. This would effectively shrink the size of the resource from the perspective of the thieves (but not the pollinators). Consequently, alternative nectar sources, such as other flowering species and extrafloral nectaries, may then be more attractive to thieves than the floral nectar of C. speciosa. Secondary chemicals have been reported in the floral nectars of many species (Baker, 1978; Baker and Baker, 1975; Clinch et al., 1972; Deinzer et al., 1977; Guerrant and Fiedler, 1981; Pryce-Jones, 1944). In some cases, these chemicals in the nectar have been shown to adversely affect various aspects of commercial honey production or pose potential health hazards to humans who consume honey made from these nectars (Clinch et al., 1972; Deinzer et al., 1977; Pryce-Jones, 1944). Ecologists have only begun to explore the role of toxic nectars in the pollination biology of plants (Feinsinger and Swarm, 1978; Guerrant and Fiedler, 1981). To my knowledge, this is the first report of iridoid glycosides in nectar. The toxic or unpalatable effect of iridoids on insects is well known. They are often referred to as bitter substances (Thomas, 1961; Hegnauer, 1966). Hagnauer (1973) reported that iridoids are toxic to generalist insects and that some plant species with iridoids (including species containing catalpol) have been used as insecticides. Iridoids have also been reported in the defensive secretions of many arthropods (see Cavill, 1969; Harborne, 1977; Roth and Eisner, 1962). In addition, some iridoids are antimalarial and antimicrobial (Merck Index, 1976). Finally, iridoids have been implicated in the unpalatability defenses of the checkerspot butterfly, Euphydryas spp. (Bowers, 1981), which suggests that they may also affect vertebrates. This study makes no attempt to understand the mechanisms by which the bumblebees and carpenter bees circumvent the toxic effects of the catalpa iridoids. Given the rather large percentage of floral nectars that contain secondary chemicals (Baker, 1978; Baker and Baker, 1975; Guerrant and Fiedler, 1981), it is reasonable to assume that bees frequently encounter these chemicals and must have well-developed mechanisms for dealing with them (Rhoades and Bergdahl, 1981). It should also be noted that some of the bees in this study consumed up to 120 #1 of nectar or sucrose solution plus catalpa iridoids over a very short period of time without showing any adverse effects; this is probably very close to the maximum amount that a large bee will take on any one foraging trip. Real (1981) noted that bumblebees usually consume only 60-80 #1 of nectar per trip when foraging on an artificial flower-board. This study did not examine the effects of catalpa iridoids on the nocturnal pollinators of C. speciosa. However, one of the principal nocturnal pollinators is the sphingid, Ceratomia catalpae, whose larvae eat the leaves of catalpa. It is reasonable to speculate that the adult moth has retained the capacity to detoxify catalpa iridoids. From the data presented here, it appears that the extrafloral nectar of C. speciosa differs from the floral nectar because the same ants that avidly
UNPALATABLE Catalpa GLYCOSIDES
1033
gather extrafloral nectar (Stephenson, 1982) find the floral nectar to be unpalatable. This suggests that the extrafloral nectar either lacks or has very low concentrations of iridoid glycosides. In other species, it has been shown that floral and extrafloral nectar from the same plant differ in sugar concentration (Vansell, 1939), in the complement of sugars (Elias et al., 1975; Elias and Gelband, 1975; Keeler, 1977) and in the amount and types of amino acids (Baker et al., 1978). Baker et al., (1978) suggest that these differences occur because the two types of nectaries function to attract different insects with different nutritional requirements and preferences. In conclusion, it is thought that floral morphology functions to both attract some floral visitors and exclude others (Faegri and van der Pijl, 1979). Recent studies suggest that the concentration and amount of lipids, amino acids, and sugars in floral nectar also have adaptive significance in attracting and excluding floral visitors (see Baker and Baker, 1975; Heinrich, 1975; Heinrich and Raven, 1972). This study provides evidence that secondary chemicals in floral nectars may also have adaptive significance in excluding some floral visitors, Acknowledgments--I thank B.H. Allen and S.T. Stephenson for their help in the field and J.L. Burris for technical advice. Professors J.S.D. Bacon, J.M. Bobbitt, and J. M. Edward kindly provided catalposide, and J.S.D. Bacon and J.M. Bobbitt also provided catalpol. I thank D.R. Smith for identifying the ants, F.C. Evans for identifying the bees, and W.S. Benninghoff for use of the facilities at the Matthaei Botanical Gardens of the University of Michigan. This research was supported by NSF grant DEB-7905508. To all I am grateful.
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