Journal of Chemical Ecology, Vol. 18, No. 9, 1992
C H E M I C A L D E F E N S E OF Populus balsamifera: A CLARIFICATION
T H O M A S P. C L A U S E N , I ' * P A U L B. R E I C H A R D T , I J O H N P. B R Y A N T , 2 and A . R . E . S I N C L A I R 3
1Department of Chemistry University of Alaska Fairbanks, Alaska 99775-0520 2Institute of Arctic Biology University of Alaska Fairbanks, Alaska 99775-0180 3Department of Zoology University of British Columbia Vancouver, British Columbia, Canada V6T 1Z4 (Received January 27, 1992; accepted April 22, 1992) Abstract--A previous report that the relative palatabilities of juvenile versus mature Populus balsamifera towards browsing snowshoe hares is due to the presence of 2,4,6-trihydroxydihydrochalcone in the juvenile growth form has been shown to be incorrect. Specifically, 2,4,6-trihydroxydihydrochalcone is present in intemodes of Populus balsamifera in exceedingly low concentrations ( 0.1) between 2,4,6-TDC concentrations in juvenile and mature buds from CSG or between the two poplar populations. Mature older growth buds, however, did contain (P < 0.1; paired t test) less 2,4,6-TDC than buds from either of the other growth stages. Levels of 2,4,6-TDC in internodes ranged between 0.25% (in one mature older growth sample) to less than 0.005 % (detection limit o f analyses), and no set of internodes we examined had an average 2,4,6-TDC concentration of greater than 0.06%. Mature CSG intemodes contained on the average about twice as much 2,4,6-TDC as juvenile CSG intemodes, but this difference was not significant (P > 0.1; paired t test). Again, there was no significant difference between the Alaskan and Kluane Lake populations or between growth stages. Bud scars from Kluane samples contained more (P < 0.1; paired t test) 2,4,6-TDC than the other sections o f internodes, but the levels were all less than 0.22%. There was slightly more, but insignificant (P > 0.1; paired t test), 2,4,6-TDC in the juvenile bud scars compared to the mature bud scars. No
TABLE 1. LEVELSOF 2,4,6-TDC (PERCENT WET WEIGHT) IN BALSAMPOPULAR PARTS AND GROWTH STAGESa Alaskan A. Juvenile current season growth Buds 19.5 (3.6) Internodes 0.0034 (0.0022) Bud scars no data B. Mature current season growth Buds 17.0 (3.8) lnternodes 0.0067 (0.0035) Bud scars no data C. Mature older growth Buds no data Intemodes no data ~'Numbers in parentheses represent standard errors (N = 5).
Kluane Lake 20.0 (1.8) 0 (0) 0.168 (0.022) 25.3 (4.5) 0.0160 (0.0104) 0.135 (0.032) 16.0 (1.1) 0.0629 (0.0476)
CHEMICAL DEFENSE OF
Populus
1509
comparison could be made between the two populations because no bud scars were sampled from the Alaskan population. DISCUSSION
The data of Table 1 show that the concentrations of 2,4,6-TDC in parts and growth stages of balsam poplar from both Kluane Lake and interior Alaska are very similar. Thus the differences between the previous conclusions of Jogia et al. (1989) and Reichardt et al. (1990) are probably not due to differences in the secondary chemistry of plants from these two sites (hypothesis 1). The very low levels of 2,4,6-TDC present in balsam poplar CSG interuodes ( < 0 . 0 2 % ) makes it unlikely that it plays a significant role in the defense of balsam poplar internodes unless it is a very active deterrent. The rather remote chance that 2,4,6-TDC is sufficiently active even at such low concentrations (hypothesis 2), however, cannot explain the relative palatabilities of juvenile vs. mature growth stages of balsam poplar to snowshoe hares. Even if 2,4,6TDC was extremely potent as a deterrent, the levels of 2,4,6-TDC would have to be greater in internodes of the juvenile growth stage to explain hares' preference for the mature growth stage, a situation not substantiated by our results in either population. It is also unlikely that the levels of 2,4,6-TDC in the budscars of juvenile twigs can explain hare preference for the mature stage. First, the results of Jogia et al. (1989) and Reichardt et al. (1990) indicate that hares prefer the mature growth stage about 30-to 100-fold. Such a large discrimination between developmental stages of a single species seems to require a relatively large difference in defensive chemistry. This, however, is shown not to be the case in this study. For instance, if one assumes that bud scars comprise about 33 % of a twig (a considerable overestimate), then a simple calculation using the results from the Kluane population (Table 1) shows that the amount of 2,4,6-TDC in the twigs (including bud scars) of the mature and juvenile growth stages are equal. In addition, the difference between concentrations of 2,4,6-TDC in the bud scars of juvenile and mature CSG was not significant (P > 0.1, paired t test). Finally, the results of Reichardt et al. (1990) suggest that the chemical defense relating to the preference of mature over juvenile twigs resides in the internodes themselves; when buds and budscars are removed, hares still prefer mature over juvenile growth stages by roughly the same factor as whole twigs. Because mature and juvenile internodes contain similar levels of 2,4,6-TDC, hares must use some other means to differentiate between growth forms. This refutation of hypothesis 2, however, does not necessarily mean that 2,4,6-TDC has no effect on the overall palatability of balsam poplar relative to other plant species. This may be especially true for those herbivores such as moose that are incapable of selectively feeding on the internode portion of the twigs.
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The elimination of hypotheses 1 and 2 leads us to believe that differences between the results of Jogia et al. (1989) and Reichardt et al. (1990) concerning the defense of winter dormant balsam poplar against snowshoe hares must be due to contamination of the internodes samples of Jogia et al. with bud oil. It is interesting to note that the 1990 paper by Reichardt et al. also dealt with the secondary chemical defense of balsam poplar buds, but failed to report the role of 2,4,6-TDC or even its presence. The results of that study demonstrated that the hares' rejection o f buds could be explained solely on the basis of the deterrent properties o f several volatile bud metabolites. Nonvolatile metabolites were, therefore, never investigated. They did state in this earlier report that other defensive substances could also be present in buds but their presence would not make buds any less palatable in conventional feeding trials. The demonstration by Jogia et al. (1989) of the antifeedant properties of 2,4,6TDC and its concentrations in buds of winter-dormant Alaskan balsam poplar indicate that the statement of Reichardt et al. (1990) about "defensive overkill" in poplar buds is supported in the sense that other chemicals may be active. In a similar vein, hares' preference for mature over juvenile internodes may be caused by aspects of this plant's complex chemistry that have not yet been discovered. For instance, it is not known if the deterrent substances reported by Reichardt et al. (1990) result in physiological stress to browsing hares or whether these substances advertise the presence of other chemicals that are not deterrent, but nonetheless are noxious. Equally intriguing is the unknown role of 2,4,6TDC in the defense of balsam poplar against herbivores. Since it is now clear that 2,4,6-TDC does not affect hares' discrimination between juvenile and mature balsam poplar, the function of the antifeedant properties of this chemical needs to be further investigated. Acknowledgments--This work was supported by the National Science Foundation (grants BSR-
8416461 and BBS-8614587). REFERENCES GREENAWAY,W., MAY, J., and WHATLEY,F.R. 1989. Flavonoid aglycones identified by gas chromatography-mass spectrometry in bud exudate of Populus balsamifera. J. Chromatogr.
29:393-400. ITUFFORD,C.D., and LASSWELL,W.L., JR. 1978. Antimicrobrial activities of constituents of Uvaria chamac. Lloydia 41 : 156-160. JOGIA,M.K., SINCLAIR,A.R.E., and ANDERSEN,R.J. 1989. An antifeedant in balsam poplar inhibits browsing by snowshoe hares. Oecologia 79:189-192. MATTES,B.R., CLAUSEN,T.P., and REICHARDT,P.B. 1987. Volatile constituents of balsam poplar: The phenol glycoside connection. Phytochemistry 26:1361-1366.
REICHARDT,P.B., BRYANT,J.P., MATTES,B.R., CLAUSEN,T.P., CHAPIN,F.S., III, and MEYER, M. 1990. Winter chemical defense of Alaskan balsam poplar against snowshoe hares. J. Chem. Ecol. 16:1941-1959.
C H E M I C A L D E F E N S E OF Populus balsamifera: A CLARIFICATION
T H O M A S P. C L A U S E N , I ' * P A U L B. R E I C H A R D T , I J O H N P. B R Y A N T , 2 and A . R . E . S I N C L A I R 3
1Department of Chemistry University of Alaska Fairbanks, Alaska 99775-0520 2Institute of Arctic Biology University of Alaska Fairbanks, Alaska 99775-0180 3Department of Zoology University of British Columbia Vancouver, British Columbia, Canada V6T 1Z4 (Received January 27, 1992; accepted April 22, 1992) Abstract--A previous report that the relative palatabilities of juvenile versus mature Populus balsamifera towards browsing snowshoe hares is due to the presence of 2,4,6-trihydroxydihydrochalcone in the juvenile growth form has been shown to be incorrect. Specifically, 2,4,6-trihydroxydihydrochalcone is present in intemodes of Populus balsamifera in exceedingly low concentrations ( 0.1) between 2,4,6-TDC concentrations in juvenile and mature buds from CSG or between the two poplar populations. Mature older growth buds, however, did contain (P < 0.1; paired t test) less 2,4,6-TDC than buds from either of the other growth stages. Levels of 2,4,6-TDC in internodes ranged between 0.25% (in one mature older growth sample) to less than 0.005 % (detection limit o f analyses), and no set of internodes we examined had an average 2,4,6-TDC concentration of greater than 0.06%. Mature CSG intemodes contained on the average about twice as much 2,4,6-TDC as juvenile CSG intemodes, but this difference was not significant (P > 0.1; paired t test). Again, there was no significant difference between the Alaskan and Kluane Lake populations or between growth stages. Bud scars from Kluane samples contained more (P < 0.1; paired t test) 2,4,6-TDC than the other sections o f internodes, but the levels were all less than 0.22%. There was slightly more, but insignificant (P > 0.1; paired t test), 2,4,6-TDC in the juvenile bud scars compared to the mature bud scars. No
TABLE 1. LEVELSOF 2,4,6-TDC (PERCENT WET WEIGHT) IN BALSAMPOPULAR PARTS AND GROWTH STAGESa Alaskan A. Juvenile current season growth Buds 19.5 (3.6) Internodes 0.0034 (0.0022) Bud scars no data B. Mature current season growth Buds 17.0 (3.8) lnternodes 0.0067 (0.0035) Bud scars no data C. Mature older growth Buds no data Intemodes no data ~'Numbers in parentheses represent standard errors (N = 5).
Kluane Lake 20.0 (1.8) 0 (0) 0.168 (0.022) 25.3 (4.5) 0.0160 (0.0104) 0.135 (0.032) 16.0 (1.1) 0.0629 (0.0476)
CHEMICAL DEFENSE OF
Populus
1509
comparison could be made between the two populations because no bud scars were sampled from the Alaskan population. DISCUSSION
The data of Table 1 show that the concentrations of 2,4,6-TDC in parts and growth stages of balsam poplar from both Kluane Lake and interior Alaska are very similar. Thus the differences between the previous conclusions of Jogia et al. (1989) and Reichardt et al. (1990) are probably not due to differences in the secondary chemistry of plants from these two sites (hypothesis 1). The very low levels of 2,4,6-TDC present in balsam poplar CSG interuodes ( < 0 . 0 2 % ) makes it unlikely that it plays a significant role in the defense of balsam poplar internodes unless it is a very active deterrent. The rather remote chance that 2,4,6-TDC is sufficiently active even at such low concentrations (hypothesis 2), however, cannot explain the relative palatabilities of juvenile vs. mature growth stages of balsam poplar to snowshoe hares. Even if 2,4,6TDC was extremely potent as a deterrent, the levels of 2,4,6-TDC would have to be greater in internodes of the juvenile growth stage to explain hares' preference for the mature growth stage, a situation not substantiated by our results in either population. It is also unlikely that the levels of 2,4,6-TDC in the budscars of juvenile twigs can explain hare preference for the mature stage. First, the results of Jogia et al. (1989) and Reichardt et al. (1990) indicate that hares prefer the mature growth stage about 30-to 100-fold. Such a large discrimination between developmental stages of a single species seems to require a relatively large difference in defensive chemistry. This, however, is shown not to be the case in this study. For instance, if one assumes that bud scars comprise about 33 % of a twig (a considerable overestimate), then a simple calculation using the results from the Kluane population (Table 1) shows that the amount of 2,4,6-TDC in the twigs (including bud scars) of the mature and juvenile growth stages are equal. In addition, the difference between concentrations of 2,4,6-TDC in the bud scars of juvenile and mature CSG was not significant (P > 0.1, paired t test). Finally, the results of Reichardt et al. (1990) suggest that the chemical defense relating to the preference of mature over juvenile twigs resides in the internodes themselves; when buds and budscars are removed, hares still prefer mature over juvenile growth stages by roughly the same factor as whole twigs. Because mature and juvenile internodes contain similar levels of 2,4,6-TDC, hares must use some other means to differentiate between growth forms. This refutation of hypothesis 2, however, does not necessarily mean that 2,4,6-TDC has no effect on the overall palatability of balsam poplar relative to other plant species. This may be especially true for those herbivores such as moose that are incapable of selectively feeding on the internode portion of the twigs.
1510
CLAUSEN ET AL.
The elimination of hypotheses 1 and 2 leads us to believe that differences between the results of Jogia et al. (1989) and Reichardt et al. (1990) concerning the defense of winter dormant balsam poplar against snowshoe hares must be due to contamination of the internodes samples of Jogia et al. with bud oil. It is interesting to note that the 1990 paper by Reichardt et al. also dealt with the secondary chemical defense of balsam poplar buds, but failed to report the role of 2,4,6-TDC or even its presence. The results of that study demonstrated that the hares' rejection o f buds could be explained solely on the basis of the deterrent properties o f several volatile bud metabolites. Nonvolatile metabolites were, therefore, never investigated. They did state in this earlier report that other defensive substances could also be present in buds but their presence would not make buds any less palatable in conventional feeding trials. The demonstration by Jogia et al. (1989) of the antifeedant properties of 2,4,6TDC and its concentrations in buds of winter-dormant Alaskan balsam poplar indicate that the statement of Reichardt et al. (1990) about "defensive overkill" in poplar buds is supported in the sense that other chemicals may be active. In a similar vein, hares' preference for mature over juvenile internodes may be caused by aspects of this plant's complex chemistry that have not yet been discovered. For instance, it is not known if the deterrent substances reported by Reichardt et al. (1990) result in physiological stress to browsing hares or whether these substances advertise the presence of other chemicals that are not deterrent, but nonetheless are noxious. Equally intriguing is the unknown role of 2,4,6TDC in the defense of balsam poplar against herbivores. Since it is now clear that 2,4,6-TDC does not affect hares' discrimination between juvenile and mature balsam poplar, the function of the antifeedant properties of this chemical needs to be further investigated. Acknowledgments--This work was supported by the National Science Foundation (grants BSR-
8416461 and BBS-8614587). REFERENCES GREENAWAY,W., MAY, J., and WHATLEY,F.R. 1989. Flavonoid aglycones identified by gas chromatography-mass spectrometry in bud exudate of Populus balsamifera. J. Chromatogr.
29:393-400. ITUFFORD,C.D., and LASSWELL,W.L., JR. 1978. Antimicrobrial activities of constituents of Uvaria chamac. Lloydia 41 : 156-160. JOGIA,M.K., SINCLAIR,A.R.E., and ANDERSEN,R.J. 1989. An antifeedant in balsam poplar inhibits browsing by snowshoe hares. Oecologia 79:189-192. MATTES,B.R., CLAUSEN,T.P., and REICHARDT,P.B. 1987. Volatile constituents of balsam poplar: The phenol glycoside connection. Phytochemistry 26:1361-1366.
REICHARDT,P.B., BRYANT,J.P., MATTES,B.R., CLAUSEN,T.P., CHAPIN,F.S., III, and MEYER, M. 1990. Winter chemical defense of Alaskan balsam poplar against snowshoe hares. J. Chem. Ecol. 16:1941-1959.
