Journal of Chemical Ecology, Vol. 21. No, 7. 1995
ONTOGENIC DEVELOPMENT OF CHEMICAL DEFENSE BY SEEDLING RESIN BIRCH: ENERGY COST OF DEFENSE PRODUCTION
JOHN P. B R Y A N T I'* a n d R I I T T A J U L K U N E N - T I I T T O
2
~lnstitute of Arctic Biology, Universi 0, of Alaska. Fairbanks Fairbanks, Alaska 99775-7000 'Department of Biology. University of Joensuu Joensuu SF-8010I Finlond (Received February 14. 1995:. accepted March 10, 1995j
Abstract--Whether production of chemical defenses by plants is or is not an energetically costly process is an important, but unresolved, question in chemical ecology. We suggest studies of the ontogenetic development of plant delense systems can help resolve the question. As an example of this approach to the cost question, we explore the problems associated with production of immobile chemical defenses that defend juvenile resin birches against browsing by mammals. From this exploration we draw two conclusions: (1) Shortly after germination, production of chemical defenses by small-seeded species, such as birch, is energetically costly. {2) Opposing selection for defense versus competitive ability in the seedling stage of birch has resulted in a trade-off in allocation of carbon to production of immobile chemical defense versus allocation of carbon to production of storage reserves. We suggest this trade-off results in a large indirect cost of defense because carbon used for production of immobile chemical defenses is unavailable for support of growth in the future, but stored carbon can be used to support future growth. Key Words--Plant chemical defense, storage, seedling, cost, ontogeny.
INTRODUCTION R e s e a r c h o n p l a n t c h e m i c a l d e f e n s e h a s i n c r e a s e d d r a m a t i c a l l y in t h e p a s t t w o d e c a d e s . F r o m t h e e a r l y 1 9 7 0 s to t h e m i d - 1 9 8 0 s t h e n u m b e r o f p r i m a r y p u b l i cations dealing with chemical defense against insect herbivory increased from a b o u t 2 0 p e r y e a r to a b o u t 2 0 0 p e r y e a r ( S c r i b e r a n d A y e r s , 1988), a n d r e c e n t r e v i e w o f t h i s t o p i c ( F e e n y , 1992; R o i t b e r g a n d I s m a n , 1992; B e r n a y s , 1994) *To whom correspondence should be addressed. 883 {lO9S-O33t/95/U71)O0883507.50/U ,,' 19~5 Plenum Publishing Corp~ralkm
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indicate this rate of growth is continuing. Study of chemical defense against vertebrate herbivory in terrestrial ecosystems and chemical defense against herbivory in marine ecosystems started later, but recent reviews of these subjects indicate a similar rate of growth (Hay and Fenical, 1988; Palo and Robbins, 1991: Bryant et al., 1992; Hay and Steinberg, 1992). This research explosion has been fueled by a succession of theoretical models (coevolution, optimal defense, resource availability, carbon/nutrient balance, growth/differentiation balance) that attempt to explain the evolution and ecology of plant antiherbivore defense systems (Ehrlich and Raven, 1964; Feeny, 1976; Rhoades and Cates, 1976; Rhoades, 1979; Bryant et al., 1983a; Coley, 1983; Coley et al., 1985; Lorio, 1986; Herms and Mattson, 1992). Although each model has increased our knowledge of plant antiherbivore defense, empirical research has now identified deficiencies in all models. Thus, plant defense theory is beginning a period of revision. The objective of this symposium was to identify research topics that are likely to provide direction to this revision. The general area of research we have selected for discussion is the early ontogenetic development of plant defense systems. We suggest that research on this subject will further development of plant defense theory for the fullowing reasons. The seedling stage is generally considered to be of paramount importance in understanding plant population dynamics and adaptations (Harper, 1977), and studies of plant population of dynamics have shown the direct negative effect of herbivory on survival of plant genets is generally greatest when herbivory occurs during juvenile stages of development (Watkinson, 1986). Thus, selection for chemical defense by herbivores should be comparatively strong in juvenile developmental stages (Bryant et al., 1983a; Langeheim and Stubblebein, 1983; Kearsely and Whitham, 1989), Yet, in comparison to what we know of chemical defense systems of mature plants, we know virtually nothing about the early ontogenetic development of plant defense systems (Simms, 1992). The specific research problem we focus on is the energy cost paid by juvenile plants to produce chemical defenses. In this paper we suggest three hypotheses that could help resolve this question of cost, which is central to plant defense theory (Feeny, 1976; Rhoades and Cates, 1976; Rhoades, 1979; Bryant et al., 1983a; Coley et al., 1985; Bazzaz et al., 1987; Lorio, 1986; Herms and Mattson, 1992; Zangerl and Bazzaz, 1992): Hypothesis 1. In many species, juvenile developmental stages are more chemically defended than the mature stage. Hypothesis IL In the specific case of species that reproduce by small seeds, the energy cost paid by young seedlings for formation of chemical defense is very high. Thus, production of immobile chemical defenses, sensu Coley et al. (1985), is minimal shortly after seed germination. Hypothesis III. Opposing selection for chemical defense versus competitive ability (Herms and Mattson, 1992) in the seedling stage of small-seeded species
BIRCH CHEMICAL DEFENSE
885
has resulted in a biochemical trade-off in allocation of carbon to formation of immobile antiherbivore defenses versus allocation of carbon to formation of storage reserves sensu Chapin et al. (1990). The outcome of this trade-off is a large indirect cost of defense (Bloom et al., 1985; Gulmon and Mooney, 1986; Herms and Mattson, 1992; Zangerl and Bazzaz, 1992) because carbon used for formation of immobile defenses cannot be used to support growth in the future, but carbon allocated to storage reserves can be used to support growth for defense in the future (Kozlowski and Keller, 1966; Kozlowski, 1971; Chapin et al., 1990). These hypotheses are explored in the following sections.
HYPOTHESIS l
It has long been known that seedlings, saplings, and the mature developmental stages of woody plants differ predictably in one or more distinctive morphologic, anatomic, physiologic, and biochemical traits (Knight, 1795; Schaffatitzky de Muckadell, 1954, 1969; Waring, 1959; Sax, 1962; Kozlowski, 1971; Borchert, 1976). Among traits that change predictably during ontogeny as a result of meristem-based changes in gene expression (Kozlowski, 1971 ) are traits that have been assigned a defensive function. For example, woody species that produce thorns or spines are usually most thorny or most spinescent when juvenile (Schaffalitzky de Muckadell, 1969; Kozlowski, 1971). Similarly, the secondary chemistry of juvenile stages often differs either qualitatively or quantitatively from the secondary chemistry of the conspecific mature stage (Kozlowski, 1971). The best documentation that chemical antiherbivore defenses of woody plants can change dramatically during ontogeny comes from studies of the interaction between woody plants and hares (Lepus) in subarctic forests (Bryant, 1981, 1987; Bryant et al., 1983a,b, 1985a,b, 1989, 1994; Reichardt et al., 1984, 1990; Tahvanainen et al., 1985; Clausen et al., 1986; Sinclair et al., 1988; Swihart et at., 1994). In the taiga and boreal forest of North America and Eurasia, winter-dormant twigs of juvenile trees and juvenile shrubs are less palatable to hares (L. americanus and L. timidus, respectively) than winterdormant twigs of their conspecific mature stage. In every case that has been well-studied chemically, the low palatability of twigs of the juvenile stage has been related to increased production of specific feeding deterrent secondary metabolites (reviewed by Bryant et al., 1991, 1992). For example, Reichardt et al. (1984) found that winter-dormant internodes of juvenile Alaska paper birch (Betula resinifera) are defended against hare browsing by high concentrations of terpene resins such as papyriferic acid that are not even present in internodes of mature B. resinifera.
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Other studies have provided evidence that chemical defense of the juvenile stage against browsing is widespread among both mammals and biomes. Basey et al. (1988) found juvenile quaking aspen (Populus tremuloids) in the Great Basin of North America is more defended against beaver by phenolics than is mature quaking aspen. Circumstantial evidence for this hypothesis comes from studies of willow (Salix spp.) and microtine rodents (Danell et al., 1987) and those of birch and moose (Alces alces) (Danell et al., 1990) in the boreal forest of Sweden, Monterey pine (Pinus radiata) and jackrabbits (Lepus) in California Mediterranean woodlands (Libby and Hood, 1976), Douglas fir (Pseudotsuga menzesii) and black-tailed deer (Odocoileus hemionus columbianus) and snowshoe hare in the coastal evergreen forest of northwestern North America (Dimock, 1974: Dimock et al., 1976), several birches and aspens and snowshoe hare in the temperate deciduous forests of eastern North America (Swihart et al., 1994), and several trees and elephant (Loxodonta africana africana) in the miombo woodland of Africa (Jachman, 1989). Juvenile woody plants and mature woody plants can also differ in chemical defense against insect herbivory. Langenheim and her students have demonstrated seedlings of some Brazilian trees have better constitutive defenses (terpene resins) than the conspecific mature stage (Langenheim and Stubblebine, 1983; Macedo and Langenheim, 1989), and Wagner (1988) found the shortterm inducible defense of Ponderosa pine (Pinus ponderosa) to be restricted to saplings. Results of Kearsely and Whitham (1989) suggest chemical defenses of Populus angustifolia change during ontogenetic development, but defenses of different developmental stages have different efficacies against different insects. In comparison to woody species, virtually nothing is known about the ontogenetic development of chemical defense of herbaceous species, but what is known indicates chemical defenses of herbs also change during ontogeny. For example, in a particularly elegant study of Plantago lanceolata, Bowers and Stamp (1993) found young plants to be more defended by iridoid glycosides against both generalist insects and specialists insects than the mature stage, and plant age explained about twice as much of the variation in leaf iridoid glycoside concentration as did plant genotype. Gershenzon (unpublished data) has found young mint (Mentha) contain less monoterpene than does older mint. These data suggest two generalizations. First, chemical defenses change during plant ontogenetic development. Second, in many cases, juvenile stages of development are more defended than the conspecific mature stage. They further suggest enhanced chemical defense of the juvenile stage may be more consistent in the case of mammalian herbivory than in the case of insect herbivory. We suggest comparative studies of effects of mammalian herbivory versus insect herbivory on plant recruitment in pristine ecosystems will be needed to determine the relative contributions of these two groups of herbivores to the evolution of chemical defenses of juvenile plants.
BIRCH CHEMICAL DEFENSE
887
HYPOTHESIS II
The energy cost of producing antiherbivore defenses should be highest in periods of the life cycle when competition for carbon between chemical defense and other plant functions is intense. It is generally recognized that the onset of reproduction may be such a period (Bazzaz et al., 1987). In the case of smallseeded plants, it is likely that another such period is when energy reserves of the seed have been exhausted, and the plant first has to rely totally on photosynthesis for growth (Bryant et al., 1991, 1992). In these plants, growth of the radicle depletes energy reserves of the seed to such an extent that when the epicotyl emerges above the soil further growth is almost entirely dependent on the seedling's rate of carbon acquisition (Ingestad, 1962: Kozlowski, 1971). Thus, at this stage of seedling development, allocation of photosynthate to substances such as immobile chemical defenses that do not immediately support growth would be selectively disadvantageous, because it would greatly limit seedling establishment (Bryant et al., 1991, 1992). This is the reason we have predicted production of immobile chemical defenses by plants that grow from small seeds will be minimal when the epicotyl emerges from the soil. We are testing this hypothesis by studying the ontogeny of chemical defense against browsing by mammals in birch. The birches we are focusing on are B. resinifera and B. pendula family E1970 × E1980 (Finnish Foundation for Tree Breeding, Helsinki). By the sapling stage, B. resinifera is more defended by terpene resin than is B. pendula (Bryant et al., 1989), and the resin concentration of juveniles of B. pendula family E1970 × E1980 is the lowest yet reported for a B. pendula family (Rousi et al., 1991, 1993). We are studying these birches and their chemical defenses against browsing in winter for the following reasons: (1) Mammal browsing in winter is a major cause of death of recruitment of these birches (Rousi, 1990; Bryant, unpublished data), so juveniles of these birches should have evolved defenses against winter browsing. (2) Birch produces small seeds (mass about 0.6 mg), so energy limits the growth of young birch seedlings (Ingestad, 1962, 1970, 1971, 1979; Ingestad and Lund, 1979: Ingestad and McDonald, 1989) for reasons given above. (3) B. resinifera and B. pendula use triterpenes such as papyriferic acid and sesquiterpenes to deter browsing in winter (Reichardt, 1981; Reichardt et al., 1984; Bryant et al., 1989; Lapinjoki et al., 1991; Vainiotalo et al., 1991; Rousi et al., 1991, 1993; Taipale and Lapinjoki, 1992). These defensive substances are energetically expensive to produce for two reasons. First, the mass of glucose (Glc) used to produce a gram of defense is comparatively high for secondary metabolites: In the case of papyriferic acid it is 2.72 g Glc/g product, and in the case of sesquiterpenes it ranges from 3.34 to 3.54 g Glc/g product. By comparison, the glucose cost of producing tannin ranges from 1.28 g Glc/g product to 2.08 g Glc/g product (Gershenzon, 1994). Second, the resin glands
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that secrete these defensive substances onto the epidermis of birch seedlings (Lapinjoki et al., 1991) are also energetically costly to produce (Gershenzon, 1994). Moreover, because these substances can occur in high concentrations in internodes of juvenile birch, their cost of production per gram tissue can be very high (Table 1). For example, Gershenzon (1994) has calculated B. r e s i n i f e r a saplings use 307 g Glc/g tissue to defend current-year-growth intemodes with papyriferic acid, and papyriferic acid is only one third of the defensive resin found in current-year-growth internodes of sapling B. resinifera (Reichardt et al., 1984). (4) These substances appear to have no other function than defense against browsing by mammals in winter (Bryant et al., 1992), so the energy cost of producing them is not offset by use for other functions (Seigler and Price, 1976; Simms, 1992), (5) Finally, these defenses are immobile, sensu Coley et al. (1985), because they are deposited by resin glands on the surface of the epidermis (Reichardt et al., 1984; Lapinjoki et al., 1991). In our experiments, seedlings were grown in pots in a mix of vermiculite and perlite (1 : 2 v/v) in a glasshouse. Light and temperature were characteristic of the environments in which these birches grow, which are essentially the same and seedlings were fed a nutrient solution developed by Ingestad specifically for growing birch seedlings in pots (Ingestad, 1962). This solution provided nitrogen at an optimum level (140 ppm N as NH4NO3). Other macronutrients and micronutrients were adjusted to N as recommended by Ingestad. Pots were sufficiently large to prevent root binding, and pots were drenched daily with nutrient solution to prevent desiccation and salt accumulation.
TABLE l. HIGHESTRAW MATERIALS(GLucosE) COSTSESTIMATEDBYGERSHENZON (1994) FOR PRODUCTIONOF CLASSESOF DEFENSECOMPOUNDSa
Chemical class Terpenoids Phenolics Alkaloids Cyanogenic glycosides Glucosinolates Nonprotein amino acids Protinase inhibitors
Plant species
Defense compound
Cost per gram of plant tissue (mg glucose/g)
Betula resinifera Isocoma acradenia Camellia sinensis
Papyriferic acid Apigenin Caftine
307 103 72
Amelanchier alnifolia Cleome serrula
Prunasin Methyl glucosinolate
7.8 20
Leucaena leucocephala
Mimosine
42
Lycopersicon esculanturn
Inhibitor1 from tomato
0.27
OEstimates take into accountall the starting materialsand cofactors needed for biosynthesis.
BIRCH CHEMICAL DEFENSE
889
For seven weeks after the epicotyl emerged above the soil, we followed production of resin glands in the upper 2 cm of internodes for 50 seedlings of each birch. These measurements were made for the first 2 cm below the apical meristem, because in these birches intemode resin glands differentiate from the primary epidermis just below the apical meristem (Lapinjoki et al., 1991). To avoid destructive sampling before final harvest, we made rank estimates of the density of resin glands in weeks 1, 2, 3, 5, and 7. A rank of zero indicated no observable resin glands, and each higher rank indicated an increase of about 25-30 glands per square centimeter of intemode epidermis. In week 7 we measured the seedlings' net photosynthetic rates, and then harvested them. After harvest, for each seedling we measured its height, its total leaf area, and the dry masses of its leaves, shoot, and roots. We also used the photographic procedure of Rousi et al. (199t, 1993) to count the number of resin glands in the top 2 cm of intemode epidermis. The epicotyls of both birches emerged above the soil at the same time, about one week after planting. At this time, neither birch produced intemode resin glands (Figure IA). B. resinifera began producing resin glands in week 2, and B. pendula began producing resin glands three weeks later. For both birches, resin gland production increased as the birches increased in size and their leaf area increased. This result was expected, because as the leaf area of a seedling birch increases, so does its ability to acquire carbon (Ingestad, 1962; Ingestad and Lund, 1979; Ingestad and McDonald, 1989). Thus, production of resin glands by both birches was minimal shortly after germination (Figure 1A). Furthermore, B. resinifera seedlings always produced more resin glands than did B. pendula seedlings (Figure tA and B; P < 0.0001). Similar results have been obtained for other birches and other small-seeded species. In a set of comparisons of eight birch species based on 12 secondary metabolites (96 total comparisons), R. Julkunen-Tiitto (unpublished data) found the concentrations of the 12 metabolites in CAG internodes of seedlings were less than those found in CAG intemodes of saplings in 94 of the 96 comparisons. Reichardt and Chapin (unpublished data) found production of condensed tannin by CAG internodes of Salix alaxensis seedlings, production of pinosylvin and pinosylvin methyl ether by CAG intemodes of Alnus crispa seedlings, and production of monoterpenes by CAG internodes of Picea glauca seedlings was tow in comparison to production of the same substances by CAG internodes of conspecific saplings, respectively. In the case of herbaceous species, D. Bowers (personal communication) has found production of iridoid glycosides by Plantago lanceolata is minimal shortly after seed germination, and J. Gershenzon (personal communication) has obtained the same result for production of monoterpenes by mint (Mentha). Our interpretation of these results is that the direct energetic cost of producing defensive resins is high enough that seedlings of small-seeded plants
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INDEX OF RESIN GLAND DENSITY
4l A L~ CI Z
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3 0 - - o B . resinilero // t ~--~B pendula / 2 P I o.oool ~¢."
z Ld II
O0
#' •
2
/
./.i . J
4
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6
8
WEEKS AFTER EPICOTYL EMERGENCE WEEK 7 RESIN GLAND COUNT EJ B res~nifero B pendulo u~
I00
7s so 25 c~
0
FIG. 1. Production of resin glands by seedlings of two birches, B. resinifera and B. pendula family E1970 x Et980. Mean +_ 1 SE presented. N = 50 seedlings for each birch. such as birch are unable to produce much of these substances. This conclusion contrasts with the suggestion of Rousi et al. (1991, 1993) that in the case of seedling resin birch, the direct energetic cost of producing terpene resins is negligible. Since this conclusion, and recent similar conclusions by others (e,g., Simms and Rausher, 1987, 1989; Simms, 1992; Karban, 1993), challenge the widely accepted hypothesis that production of chemical defense has an energetic cost, it is useful to examine the basis for the conclusion of Rousi et al. (1991, 1993). In their experiments, Rousi et al. (1991, 1993) correlated the density of resin glands on intemodes of 1-year-old birch seedlings with two measures of seedling growth, height (Rousi et al., 1991) and biomass accretion (Rousi et al., 1993). In both studies they are unable to demonstrate a negative correlation between defense (density of resin glands on intemodes) and seedling growth. Similarly, in our experiments with birch seedlings, we also failed to find a negative correlation between resin gland density (Figure 1) and these two measures of growth (Table 2). Such failures to find negative correlations between simple measures of plant growth such as plant size or biomass accretion have often been interpreted to mean that production of chemical defense has no energy cost (Simms, 1992). Thus, it is not surprising that Rousi et al. (1991, 1993) reached their conclusion.
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BIRCH CHEMICAL DEFENSE
TABLE 2, SEEDLING HEIGHT, TOTAL LEAF AREA, DRY BIOMASS, AND NET PHOTOSYNTHETIC RATE a
Biomass
Birch
B. resinifera B. pendula
Height (cm)
Leaf area (cm 2)
Leaf (g)
Shoot (g)
Root (g)
Total (g)
Photosynthesis (#rnol/m" sec)
26 + 0.9 27 + 0. t
433 ± 23 414 ± 23
1.3 + 0,1 1.4 i 0. l
0.4 + 0 . | 0.5 ± 0.1
1.1 ± 0.1 1.1 +_ 0.1
2.8 + 0.1 2.9 ± 0.1
18.8 +_ 0.5 18.6 _+ 0.5
"Mean + 1 SE presented.
We suggest conclusions drawn from this approach to testing the "energy cost of defense hypothesis" should be viewed with caution for three reasons. These simple estimates of growth do not take into account: (1) differences in the ability to acquire carbon, (2) differences in maintenance respiration, or (3) biochemical trade-offs in partitioning of carbon between chemical defense and competing plant functions. Yet these differences are the physiological and biochemical basis for the energetic cost of producing chemical defense (Gulmon and Mooney, 1986; Chapin et al., 1990; Bryant et al., 1991, 1992; Herms and Mattson, 1992; Zangerl and Bazzaz, 1992). Our experiment with B. resinifera and B. pendula indicates a difference in the ability to acquire carbon is unlikely to explain why birch seedlings that differ in defense can accrete biomass at the same rate. We found that seedlings of B. resinifera and seedlings of B. pendula that differed in production of resin glands (Figure 1A and B; P < 0.001) and that had the same biomass after seven weeks of growth (Table 2), also had the same ability to acquire carbon (Table 2). We further suggest a difference in maintenance respiration is unlikely to explain why birch seedlings that differ in defense can accrete biomass at the same rate, because maintenance respiration has a minimal effect on patterns of carbon allocation by birch seedlings (Ingestad and McDonald, 1989; Ingestad and Agren, 1991).
HYPOTHESIS III
These conclusions suggest that if birch seedlings pay an energy cost to defend themselves against browsing in winter, this cost is an indirect cost resulting from trade-off in allocation of carbon between defense and storage. Two observations from common garden experiments support this hypothesis: (1) In contrast to 1-year-old B. pendula seedlings, when Rousi et al. (1991) examined
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BRYANT AND JULKUNEN-TIITTO
8-year-old B. pendula saplings they found a negative correlation between defense (resin gland density) and growth (sapling height). This result is expected if there is a trade-off between defense and storage in juvenile stages of B. pendula, and juvenile B. pendula is polymorphic for this tradeoff, (2) After two to three years of growth in common gardens in Finland (M. Rousi, personal communication) and Michigan (M. Herms, personal communication) the comparatively heavily defended saplings of B. resinifera are growing at less than half the rate of saplings of the less-defended B. pendula.
RECOMMENDATIONS FOR FUTURE RESEARCH
Two further experiments are needed to verify that seedlings of small-seeded plants subject to differences in the intensity of selection for defense and the intensity of selection for competitive ability are polymorphic for partitioning of carbon between immobile chemical defense and storage reserves. In the one experiment, mass balance measurements should be used to document biochemical carbon partitioning by seedlings of congeners or conspecifics grown in a common environment. In this experiment, masses of carbon allocated to production of immobile defenses and to production of storage compounds (e.g., starch, storage protein, fatty acids) as well as masses of carbon allocated to growth and maintenance (e.g., sugars, amino acids, enzymes), and to structure (e.g., cellulose, lignin) should be determined using the biochemical approach suggested by Chapin et al. (1990). Assuming no differences in carbon acquisition, maintenance respiration, and allocation of carbon to other functions, evidence for a trade-off between defense and storage would be a negative correlation between the amount of glucose used to produce immobile chemical defense and the amount of glucose used to produce storage substances. Furthermore, this trade-off should become more noticeable in later stages of seedling ontogeny, because according to our second hypothesis, shortly after germination allocation of carbon to any function that competes with immediate growth should be minimal. Thus, for later stages of seedling ontogeny we expect "defense-selected" seedlings will have comparatively small storage reserves, and "'competitionselected" seedlings will have comparatively poor chemical defenses. In the other experiment, Jacob. pulse labeling should be used to measure the flow of carbon competing plant functions within a few hours of acquisition. In this experiment, evidence for a "defense-storage" polymorphism would be provided by the following two observations: (1) After production of defenses and storage reserves begins, the short-term flow of carbon into immobile chemical defense will be greatest for "defense-selected seedlings." (2) Conversely, the short-term flow of carbon into storage reserves wilt be greatest for "competition-selected" seedlings.
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CONCLUSIONS C o m p e t i t i o n and herbivory are two o f the greatest challenges faced by j u v e n i l e plants (Harper, 1977; T i l m a n , 1982; W a t k i n s o n , 1986; Huntly, 1991). Increased ability to deter herbivory is often achieved by increased allocation o f resources to chemical defense, and increased competitive ability is generally associated with rapid growth. Thus, in the j u v e n i l e stage, plants that have evolved in e n v i r o n m e n t s with varying levels o f herbivory and competition should be polymorphic for partitioning of carbon b e t w e e n defense and growth (Herms and Mattson, 1992). In this p a p e r we have suggested that studies o f the early ontogenetic d e v e l o p m e n t o f defense systems by plants are needed to test this hypothesis. W e have further suggested the focus o f this research should be a search for a trade-off in allocation of carbon to i m m o b i l e defense versus formation of storage reserves in the seedling stage o f species that grow from small seeds. This research will help resolve a central question o f current plant defense theory: when is production of chemical defense energetically costly?
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T. 1989. Biogeographic evidence for the evolution of chemical defense by boreal birch and willow against mammalian browsing. Am. Nat. 134:20-34.
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HUNTLY, N. 1991. Herbivores and the dynamics of communities and ecosystems. Annu. Rev. Ecol. Syst. 22:477-503. INGESTAD T. 1962. Macro element nutrition of pine, spruce and birch seedlings in nutrient solutions. Medd. Statens Skogsf-lnst. 51:1-150. INGESTAD T, 1970. A definition of optimum nutrient requirements in birch seedlings. 1. Physiol, Plant. 23:1127-1128. INGESTAD T. 1971. A definition of optimum nutrient requirements in birch seedlings. II. Physiol. Plant. 24:118-125. 1NGESTAD T, 1979, Nitrogen stress in birch seedlings II: N, K, P, Ca, and Mg nutrition. Physiol. Plant. 45:149-157. INGESTAD T., and AGREN, G.T. 1991. The influence of plant nutrition on biomass allocation. Ecol. Appl. 2:168-174, INGESTAD T,, and LUND, A. 1979. Nitrogen stress in birch seedlings: I. Growth technique and growth. Physiol. Plant. 45:137-148, INGESTAD T,, and MCDONALD, A.J.S. 1989. The influence of photon flux density on nutrition and growth of birch seedlings at different relative nitrogen addition rates. PhysioL Plant. 45:137148. JACHMANN, H. 1989. Food selection by elephants in the "Miombo'" biome in relation to leaf chemistry. Biochem. Syst. Ecol, 17:15-24. KARBAN, R. 1993. Costs and benefits of induced resistance and plant density. Ecology 74:9-19. KEARSELY, M.J.C., and WHtTHAM, T.G, 1989. Developmental changes in resistance in herbivory: implications for individuals and populations. Ecology 70: 1040-1047. KNIGHT, T.A. 1795. Phil. Trans. 85:290-295, KOZt,OWSKL T.T. 1971. Growth and Development of Trees, Vol. 1. Academic Press, New York. KozLowsKI, T.T., and KELLEa, T. 1966, Food relations of woody plants. Bot. Rev. 22:293382. LANGENHEIM, J.H., and STUBBLEBINE, W H . 1983. Variation in leaf resin composition between parent tree and progeny in Hymenaea: Implications for herbivory in humid tropics, Biochem, Syst. EcoL 11:97-106. LAPlNJOKI, S.P., ELO, H.A., and TA]PALE, H.T. 1991. Development and structure of resin glands on tissues of Betula pendula Roth. during growth. New Phytol. 117:219-223. LIBBY, W.L, and HOOD, J.V. 1976. Juvenility in hedged radiata pine. Acata Hortie. 56:91-98. LOR10, P.L, 1986. Growth-differentiation balance: a basis for understanding southern pine beetletree interactions. For. Ecol. Manage, 14:259-273. MACEDO,C.A., and LANGENHEIM,J.H. 1989. Microlepidopteran herbivory in relation to leaf sesquiterpenes in Copifera langsdorfii adult trees and their seedling progeny in a Brazilian woodland. Biochem. Syst. Ecol. 17:217-224. PALO, R.T., and RoBBms, C.T. 1991, Plant Defense against Mammalian Herbivory. CRC Press. Boca Raton, Florida. REICHARDT, P.B. 1981. Papyriferic acid: A triterpenoid from Alaskan paper birch. J. O~g, Chem. 46:1576-1578. REtCHAROT, P.B., BRYANT, J.P,, CLAUSEN, T.P., and WIEt,AND. G. 1984, Defense of winterdormant Alaska paper birch against Snowshoe hare. Oecologia (Berlin) 65:58-59. REJCHARDT, P.B., BaYANT, J.P., MATTES, B.R,, CLAUSEN, T.P., C,APIN, F.S.. III, and MEYEa, M. 1990. The winter chemical defense of balsam poplar against snowshoe hares, J. Chem. Ecol. 16:1941-1959. RI-IOADES,D.F. 1979. Evolution of plant chemical defense against herbivores, pp. 4-48, in G.A. Rosenthal and D.H, Janzen (eds.), Herbivores: Their Interactions with Secondary Plant MetaboIites. Academic Press, New York. RHOADES, D.F., and CATES, R.G. 1976. Toward a general theory of plant antiherbivore chemistry, pp. 168-2t3, in J,W, Wallace and R.L. Mansell (eds.). Biochemical Interactions between Plants and Insects, Plenum, New York.
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ROITBERG, B.D., and ISMAN, M.B. 1992. Insect Chemical Ecology: An Evolutionary Approach. Chapman & Hall, New York. ROUSl, M. 1990. Breeding forest trees for resistance to mammalian herbivores--a study based on European white birch. Acta For. Fenn. 210:1-20. ROUSl, M., TAHVANAINEN,J.. and UOTILA, 1. 1991. Mechanism of resistance to hare browsing in winter-dormant silver birch (Betula pendula). Am. Nat. 137:64-82, RousI, M., TAHVANAINEN,J., HEN't~ONEN. H,. and UOTILA, I. 1993. Effects of shading and fertilization on resistance of winter-dormant birch (Betula pendula) to voles and hares. Ecology 74:30-38. Snx, K. 1962. Aspects of aging in plants. Annu. Rev. Plant. Physiol. 13:489-506. SCHAFFALITZKYDE MUCKADELL, M. 1954, Juvenile stages in woody plants. Physiol. Plant. 7:782796. SCHAFFALITZKYDE MUCKADELL, M. 1969. Environmental factors in developmental stages of trees, pp. 289-298, in T,T. Kozlowski (ed.). Tree Growth. Ronald Press, New York. SCRIBER, J.M., and AVERS. M.P. 1988. Leaf chemistry as a defense against insects. Atlas of science: Plants" and Animals. 1:117-123. SE]GLER, D,, and PRICE, P.W. 1976. Secondary compounds in plants: Primary functions. Am. Nat. 110:101-105. SIMMS, E.L. 1992. Costs of plant resistance to herbivory, pp. 392-425, in R.S. Fritz and E.L, Simms (eds.). Plant Resistance to Herbivores and Pathogens: Ecology, Evolution, and Genetics. University of Chicago Press, Chicago. SIMMS, E,L., and RAUSHER, M.D. 1987. Costs and benefits of plant defense to herbivory. Am. Nat. 130:570-581. SIMMS, E,L., and RAUSHER, M.D. 1989. The evolution of resistance to herbivory in lpomoea purpurea. II. Natural selection by insects and costs of resistance. Evolution 43:573-585, SINCLAm, A.R.E., JOGJA, M.K., and ANDERSON,R.J. 1988. Camphor from juvenile white spruce as an antifeedent for snowshoe hares, J. Chem, Ecol. 14:1505-1514. SW;HART, R,K., BRYAN'r.J.P., and NEWTON,L. 1994. Latitudinal patterns in consumption of woody plants by snowshoe hares in the eastern United States. Oikos 70:427~-34. TAHVANAINEN,J.. HELLE, E.. JULKUNEN-TIITTO,R., and LAVOLA, A. 1985. Phenolic compounds of willow bark as deterrents against feeding by mountain hare, Oecologia (Berlin) 65:319323. TAtPALE, H,T., and LAPINJOKI,S.P. 1992. Use of evaporative light scattering mass detection in high performance liquid chromatography of triterpenes in the bark resin of Betula species. Phytochem. Anal. 2:84-86. TILMAN, D. 1982. Resource Competition and Community Structure. Princeton University Press, Princeton. VAINIOTALO,P., JULKUNEN-Tu'I-rO,R., JUNTHEIKKI,M.R.. REICHARDT, P., and AURIOLA,S. 1991. Chemical characteristics of herbivore defenses in Betula pendula winter-dormant young stems. J. Chromatogr. 547:367-376. WAGNER, M.R. 1988, Induced defenses in ponderosa pine against defoliating insects, pp. 141-156, in W.J. Mattson, J. Levieux, and C. Bemard-Dagen (eds.). Mechanisms of Woody Plant Defenses Against Insects; Search for a Pattern. Springer-Verlag, New York. WARING, P.F. 1959, Problems of juvenility and flowering in trees. J. Linn. Soc. London, Bot. 56:282-289. WAT~dNSON, A.R. 1986. Plant population dynamics, pp. 137-184, in M.J. Crawley (ed.). Plant Ecology. Blackwell Scientific, Oxford, England, ZANGERI, A.R.. and BAZZAZ, F.A. 1992. Theory and pattern in plant defense allocation, pp. 363392, in R.S. Fritz and E.L. Simms (ed.). Plant Resistance to Herbivores and Pathogens: Ecology. Evolution, and Genetics. University of Chicago Press, Chicago.
ONTOGENIC DEVELOPMENT OF CHEMICAL DEFENSE BY SEEDLING RESIN BIRCH: ENERGY COST OF DEFENSE PRODUCTION
JOHN P. B R Y A N T I'* a n d R I I T T A J U L K U N E N - T I I T T O
2
~lnstitute of Arctic Biology, Universi 0, of Alaska. Fairbanks Fairbanks, Alaska 99775-7000 'Department of Biology. University of Joensuu Joensuu SF-8010I Finlond (Received February 14. 1995:. accepted March 10, 1995j
Abstract--Whether production of chemical defenses by plants is or is not an energetically costly process is an important, but unresolved, question in chemical ecology. We suggest studies of the ontogenetic development of plant delense systems can help resolve the question. As an example of this approach to the cost question, we explore the problems associated with production of immobile chemical defenses that defend juvenile resin birches against browsing by mammals. From this exploration we draw two conclusions: (1) Shortly after germination, production of chemical defenses by small-seeded species, such as birch, is energetically costly. {2) Opposing selection for defense versus competitive ability in the seedling stage of birch has resulted in a trade-off in allocation of carbon to production of immobile chemical defense versus allocation of carbon to production of storage reserves. We suggest this trade-off results in a large indirect cost of defense because carbon used for production of immobile chemical defenses is unavailable for support of growth in the future, but stored carbon can be used to support future growth. Key Words--Plant chemical defense, storage, seedling, cost, ontogeny.
INTRODUCTION R e s e a r c h o n p l a n t c h e m i c a l d e f e n s e h a s i n c r e a s e d d r a m a t i c a l l y in t h e p a s t t w o d e c a d e s . F r o m t h e e a r l y 1 9 7 0 s to t h e m i d - 1 9 8 0 s t h e n u m b e r o f p r i m a r y p u b l i cations dealing with chemical defense against insect herbivory increased from a b o u t 2 0 p e r y e a r to a b o u t 2 0 0 p e r y e a r ( S c r i b e r a n d A y e r s , 1988), a n d r e c e n t r e v i e w o f t h i s t o p i c ( F e e n y , 1992; R o i t b e r g a n d I s m a n , 1992; B e r n a y s , 1994) *To whom correspondence should be addressed. 883 {lO9S-O33t/95/U71)O0883507.50/U ,,' 19~5 Plenum Publishing Corp~ralkm
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indicate this rate of growth is continuing. Study of chemical defense against vertebrate herbivory in terrestrial ecosystems and chemical defense against herbivory in marine ecosystems started later, but recent reviews of these subjects indicate a similar rate of growth (Hay and Fenical, 1988; Palo and Robbins, 1991: Bryant et al., 1992; Hay and Steinberg, 1992). This research explosion has been fueled by a succession of theoretical models (coevolution, optimal defense, resource availability, carbon/nutrient balance, growth/differentiation balance) that attempt to explain the evolution and ecology of plant antiherbivore defense systems (Ehrlich and Raven, 1964; Feeny, 1976; Rhoades and Cates, 1976; Rhoades, 1979; Bryant et al., 1983a; Coley, 1983; Coley et al., 1985; Lorio, 1986; Herms and Mattson, 1992). Although each model has increased our knowledge of plant antiherbivore defense, empirical research has now identified deficiencies in all models. Thus, plant defense theory is beginning a period of revision. The objective of this symposium was to identify research topics that are likely to provide direction to this revision. The general area of research we have selected for discussion is the early ontogenetic development of plant defense systems. We suggest that research on this subject will further development of plant defense theory for the fullowing reasons. The seedling stage is generally considered to be of paramount importance in understanding plant population dynamics and adaptations (Harper, 1977), and studies of plant population of dynamics have shown the direct negative effect of herbivory on survival of plant genets is generally greatest when herbivory occurs during juvenile stages of development (Watkinson, 1986). Thus, selection for chemical defense by herbivores should be comparatively strong in juvenile developmental stages (Bryant et al., 1983a; Langeheim and Stubblebein, 1983; Kearsely and Whitham, 1989), Yet, in comparison to what we know of chemical defense systems of mature plants, we know virtually nothing about the early ontogenetic development of plant defense systems (Simms, 1992). The specific research problem we focus on is the energy cost paid by juvenile plants to produce chemical defenses. In this paper we suggest three hypotheses that could help resolve this question of cost, which is central to plant defense theory (Feeny, 1976; Rhoades and Cates, 1976; Rhoades, 1979; Bryant et al., 1983a; Coley et al., 1985; Bazzaz et al., 1987; Lorio, 1986; Herms and Mattson, 1992; Zangerl and Bazzaz, 1992): Hypothesis 1. In many species, juvenile developmental stages are more chemically defended than the mature stage. Hypothesis IL In the specific case of species that reproduce by small seeds, the energy cost paid by young seedlings for formation of chemical defense is very high. Thus, production of immobile chemical defenses, sensu Coley et al. (1985), is minimal shortly after seed germination. Hypothesis III. Opposing selection for chemical defense versus competitive ability (Herms and Mattson, 1992) in the seedling stage of small-seeded species
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has resulted in a biochemical trade-off in allocation of carbon to formation of immobile antiherbivore defenses versus allocation of carbon to formation of storage reserves sensu Chapin et al. (1990). The outcome of this trade-off is a large indirect cost of defense (Bloom et al., 1985; Gulmon and Mooney, 1986; Herms and Mattson, 1992; Zangerl and Bazzaz, 1992) because carbon used for formation of immobile defenses cannot be used to support growth in the future, but carbon allocated to storage reserves can be used to support growth for defense in the future (Kozlowski and Keller, 1966; Kozlowski, 1971; Chapin et al., 1990). These hypotheses are explored in the following sections.
HYPOTHESIS l
It has long been known that seedlings, saplings, and the mature developmental stages of woody plants differ predictably in one or more distinctive morphologic, anatomic, physiologic, and biochemical traits (Knight, 1795; Schaffatitzky de Muckadell, 1954, 1969; Waring, 1959; Sax, 1962; Kozlowski, 1971; Borchert, 1976). Among traits that change predictably during ontogeny as a result of meristem-based changes in gene expression (Kozlowski, 1971 ) are traits that have been assigned a defensive function. For example, woody species that produce thorns or spines are usually most thorny or most spinescent when juvenile (Schaffalitzky de Muckadell, 1969; Kozlowski, 1971). Similarly, the secondary chemistry of juvenile stages often differs either qualitatively or quantitatively from the secondary chemistry of the conspecific mature stage (Kozlowski, 1971). The best documentation that chemical antiherbivore defenses of woody plants can change dramatically during ontogeny comes from studies of the interaction between woody plants and hares (Lepus) in subarctic forests (Bryant, 1981, 1987; Bryant et al., 1983a,b, 1985a,b, 1989, 1994; Reichardt et al., 1984, 1990; Tahvanainen et al., 1985; Clausen et al., 1986; Sinclair et al., 1988; Swihart et at., 1994). In the taiga and boreal forest of North America and Eurasia, winter-dormant twigs of juvenile trees and juvenile shrubs are less palatable to hares (L. americanus and L. timidus, respectively) than winterdormant twigs of their conspecific mature stage. In every case that has been well-studied chemically, the low palatability of twigs of the juvenile stage has been related to increased production of specific feeding deterrent secondary metabolites (reviewed by Bryant et al., 1991, 1992). For example, Reichardt et al. (1984) found that winter-dormant internodes of juvenile Alaska paper birch (Betula resinifera) are defended against hare browsing by high concentrations of terpene resins such as papyriferic acid that are not even present in internodes of mature B. resinifera.
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Other studies have provided evidence that chemical defense of the juvenile stage against browsing is widespread among both mammals and biomes. Basey et al. (1988) found juvenile quaking aspen (Populus tremuloids) in the Great Basin of North America is more defended against beaver by phenolics than is mature quaking aspen. Circumstantial evidence for this hypothesis comes from studies of willow (Salix spp.) and microtine rodents (Danell et al., 1987) and those of birch and moose (Alces alces) (Danell et al., 1990) in the boreal forest of Sweden, Monterey pine (Pinus radiata) and jackrabbits (Lepus) in California Mediterranean woodlands (Libby and Hood, 1976), Douglas fir (Pseudotsuga menzesii) and black-tailed deer (Odocoileus hemionus columbianus) and snowshoe hare in the coastal evergreen forest of northwestern North America (Dimock, 1974: Dimock et al., 1976), several birches and aspens and snowshoe hare in the temperate deciduous forests of eastern North America (Swihart et al., 1994), and several trees and elephant (Loxodonta africana africana) in the miombo woodland of Africa (Jachman, 1989). Juvenile woody plants and mature woody plants can also differ in chemical defense against insect herbivory. Langenheim and her students have demonstrated seedlings of some Brazilian trees have better constitutive defenses (terpene resins) than the conspecific mature stage (Langenheim and Stubblebine, 1983; Macedo and Langenheim, 1989), and Wagner (1988) found the shortterm inducible defense of Ponderosa pine (Pinus ponderosa) to be restricted to saplings. Results of Kearsely and Whitham (1989) suggest chemical defenses of Populus angustifolia change during ontogenetic development, but defenses of different developmental stages have different efficacies against different insects. In comparison to woody species, virtually nothing is known about the ontogenetic development of chemical defense of herbaceous species, but what is known indicates chemical defenses of herbs also change during ontogeny. For example, in a particularly elegant study of Plantago lanceolata, Bowers and Stamp (1993) found young plants to be more defended by iridoid glycosides against both generalist insects and specialists insects than the mature stage, and plant age explained about twice as much of the variation in leaf iridoid glycoside concentration as did plant genotype. Gershenzon (unpublished data) has found young mint (Mentha) contain less monoterpene than does older mint. These data suggest two generalizations. First, chemical defenses change during plant ontogenetic development. Second, in many cases, juvenile stages of development are more defended than the conspecific mature stage. They further suggest enhanced chemical defense of the juvenile stage may be more consistent in the case of mammalian herbivory than in the case of insect herbivory. We suggest comparative studies of effects of mammalian herbivory versus insect herbivory on plant recruitment in pristine ecosystems will be needed to determine the relative contributions of these two groups of herbivores to the evolution of chemical defenses of juvenile plants.
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HYPOTHESIS II
The energy cost of producing antiherbivore defenses should be highest in periods of the life cycle when competition for carbon between chemical defense and other plant functions is intense. It is generally recognized that the onset of reproduction may be such a period (Bazzaz et al., 1987). In the case of smallseeded plants, it is likely that another such period is when energy reserves of the seed have been exhausted, and the plant first has to rely totally on photosynthesis for growth (Bryant et al., 1991, 1992). In these plants, growth of the radicle depletes energy reserves of the seed to such an extent that when the epicotyl emerges above the soil further growth is almost entirely dependent on the seedling's rate of carbon acquisition (Ingestad, 1962: Kozlowski, 1971). Thus, at this stage of seedling development, allocation of photosynthate to substances such as immobile chemical defenses that do not immediately support growth would be selectively disadvantageous, because it would greatly limit seedling establishment (Bryant et al., 1991, 1992). This is the reason we have predicted production of immobile chemical defenses by plants that grow from small seeds will be minimal when the epicotyl emerges from the soil. We are testing this hypothesis by studying the ontogeny of chemical defense against browsing by mammals in birch. The birches we are focusing on are B. resinifera and B. pendula family E1970 × E1980 (Finnish Foundation for Tree Breeding, Helsinki). By the sapling stage, B. resinifera is more defended by terpene resin than is B. pendula (Bryant et al., 1989), and the resin concentration of juveniles of B. pendula family E1970 × E1980 is the lowest yet reported for a B. pendula family (Rousi et al., 1991, 1993). We are studying these birches and their chemical defenses against browsing in winter for the following reasons: (1) Mammal browsing in winter is a major cause of death of recruitment of these birches (Rousi, 1990; Bryant, unpublished data), so juveniles of these birches should have evolved defenses against winter browsing. (2) Birch produces small seeds (mass about 0.6 mg), so energy limits the growth of young birch seedlings (Ingestad, 1962, 1970, 1971, 1979; Ingestad and Lund, 1979: Ingestad and McDonald, 1989) for reasons given above. (3) B. resinifera and B. pendula use triterpenes such as papyriferic acid and sesquiterpenes to deter browsing in winter (Reichardt, 1981; Reichardt et al., 1984; Bryant et al., 1989; Lapinjoki et al., 1991; Vainiotalo et al., 1991; Rousi et al., 1991, 1993; Taipale and Lapinjoki, 1992). These defensive substances are energetically expensive to produce for two reasons. First, the mass of glucose (Glc) used to produce a gram of defense is comparatively high for secondary metabolites: In the case of papyriferic acid it is 2.72 g Glc/g product, and in the case of sesquiterpenes it ranges from 3.34 to 3.54 g Glc/g product. By comparison, the glucose cost of producing tannin ranges from 1.28 g Glc/g product to 2.08 g Glc/g product (Gershenzon, 1994). Second, the resin glands
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that secrete these defensive substances onto the epidermis of birch seedlings (Lapinjoki et al., 1991) are also energetically costly to produce (Gershenzon, 1994). Moreover, because these substances can occur in high concentrations in internodes of juvenile birch, their cost of production per gram tissue can be very high (Table 1). For example, Gershenzon (1994) has calculated B. r e s i n i f e r a saplings use 307 g Glc/g tissue to defend current-year-growth intemodes with papyriferic acid, and papyriferic acid is only one third of the defensive resin found in current-year-growth internodes of sapling B. resinifera (Reichardt et al., 1984). (4) These substances appear to have no other function than defense against browsing by mammals in winter (Bryant et al., 1992), so the energy cost of producing them is not offset by use for other functions (Seigler and Price, 1976; Simms, 1992), (5) Finally, these defenses are immobile, sensu Coley et al. (1985), because they are deposited by resin glands on the surface of the epidermis (Reichardt et al., 1984; Lapinjoki et al., 1991). In our experiments, seedlings were grown in pots in a mix of vermiculite and perlite (1 : 2 v/v) in a glasshouse. Light and temperature were characteristic of the environments in which these birches grow, which are essentially the same and seedlings were fed a nutrient solution developed by Ingestad specifically for growing birch seedlings in pots (Ingestad, 1962). This solution provided nitrogen at an optimum level (140 ppm N as NH4NO3). Other macronutrients and micronutrients were adjusted to N as recommended by Ingestad. Pots were sufficiently large to prevent root binding, and pots were drenched daily with nutrient solution to prevent desiccation and salt accumulation.
TABLE l. HIGHESTRAW MATERIALS(GLucosE) COSTSESTIMATEDBYGERSHENZON (1994) FOR PRODUCTIONOF CLASSESOF DEFENSECOMPOUNDSa
Chemical class Terpenoids Phenolics Alkaloids Cyanogenic glycosides Glucosinolates Nonprotein amino acids Protinase inhibitors
Plant species
Defense compound
Cost per gram of plant tissue (mg glucose/g)
Betula resinifera Isocoma acradenia Camellia sinensis
Papyriferic acid Apigenin Caftine
307 103 72
Amelanchier alnifolia Cleome serrula
Prunasin Methyl glucosinolate
7.8 20
Leucaena leucocephala
Mimosine
42
Lycopersicon esculanturn
Inhibitor1 from tomato
0.27
OEstimates take into accountall the starting materialsand cofactors needed for biosynthesis.
BIRCH CHEMICAL DEFENSE
889
For seven weeks after the epicotyl emerged above the soil, we followed production of resin glands in the upper 2 cm of internodes for 50 seedlings of each birch. These measurements were made for the first 2 cm below the apical meristem, because in these birches intemode resin glands differentiate from the primary epidermis just below the apical meristem (Lapinjoki et al., 1991). To avoid destructive sampling before final harvest, we made rank estimates of the density of resin glands in weeks 1, 2, 3, 5, and 7. A rank of zero indicated no observable resin glands, and each higher rank indicated an increase of about 25-30 glands per square centimeter of intemode epidermis. In week 7 we measured the seedlings' net photosynthetic rates, and then harvested them. After harvest, for each seedling we measured its height, its total leaf area, and the dry masses of its leaves, shoot, and roots. We also used the photographic procedure of Rousi et al. (199t, 1993) to count the number of resin glands in the top 2 cm of intemode epidermis. The epicotyls of both birches emerged above the soil at the same time, about one week after planting. At this time, neither birch produced intemode resin glands (Figure IA). B. resinifera began producing resin glands in week 2, and B. pendula began producing resin glands three weeks later. For both birches, resin gland production increased as the birches increased in size and their leaf area increased. This result was expected, because as the leaf area of a seedling birch increases, so does its ability to acquire carbon (Ingestad, 1962; Ingestad and Lund, 1979; Ingestad and McDonald, 1989). Thus, production of resin glands by both birches was minimal shortly after germination (Figure 1A). Furthermore, B. resinifera seedlings always produced more resin glands than did B. pendula seedlings (Figure tA and B; P < 0.0001). Similar results have been obtained for other birches and other small-seeded species. In a set of comparisons of eight birch species based on 12 secondary metabolites (96 total comparisons), R. Julkunen-Tiitto (unpublished data) found the concentrations of the 12 metabolites in CAG internodes of seedlings were less than those found in CAG intemodes of saplings in 94 of the 96 comparisons. Reichardt and Chapin (unpublished data) found production of condensed tannin by CAG internodes of Salix alaxensis seedlings, production of pinosylvin and pinosylvin methyl ether by CAG intemodes of Alnus crispa seedlings, and production of monoterpenes by CAG internodes of Picea glauca seedlings was tow in comparison to production of the same substances by CAG internodes of conspecific saplings, respectively. In the case of herbaceous species, D. Bowers (personal communication) has found production of iridoid glycosides by Plantago lanceolata is minimal shortly after seed germination, and J. Gershenzon (personal communication) has obtained the same result for production of monoterpenes by mint (Mentha). Our interpretation of these results is that the direct energetic cost of producing defensive resins is high enough that seedlings of small-seeded plants
890
BRYANT AND JULKUNEN-TIITTO
INDEX OF RESIN GLAND DENSITY
4l A L~ CI Z
~.//
3 0 - - o B . resinilero // t ~--~B pendula / 2 P I o.oool ~¢."
z Ld II
O0
#' •
2
/
./.i . J
4
•/
//
6
8
WEEKS AFTER EPICOTYL EMERGENCE WEEK 7 RESIN GLAND COUNT EJ B res~nifero B pendulo u~
I00
7s so 25 c~
0
FIG. 1. Production of resin glands by seedlings of two birches, B. resinifera and B. pendula family E1970 x Et980. Mean +_ 1 SE presented. N = 50 seedlings for each birch. such as birch are unable to produce much of these substances. This conclusion contrasts with the suggestion of Rousi et al. (1991, 1993) that in the case of seedling resin birch, the direct energetic cost of producing terpene resins is negligible. Since this conclusion, and recent similar conclusions by others (e,g., Simms and Rausher, 1987, 1989; Simms, 1992; Karban, 1993), challenge the widely accepted hypothesis that production of chemical defense has an energetic cost, it is useful to examine the basis for the conclusion of Rousi et al. (1991, 1993). In their experiments, Rousi et al. (1991, 1993) correlated the density of resin glands on intemodes of 1-year-old birch seedlings with two measures of seedling growth, height (Rousi et al., 1991) and biomass accretion (Rousi et al., 1993). In both studies they are unable to demonstrate a negative correlation between defense (density of resin glands on intemodes) and seedling growth. Similarly, in our experiments with birch seedlings, we also failed to find a negative correlation between resin gland density (Figure 1) and these two measures of growth (Table 2). Such failures to find negative correlations between simple measures of plant growth such as plant size or biomass accretion have often been interpreted to mean that production of chemical defense has no energy cost (Simms, 1992). Thus, it is not surprising that Rousi et al. (1991, 1993) reached their conclusion.
891
BIRCH CHEMICAL DEFENSE
TABLE 2, SEEDLING HEIGHT, TOTAL LEAF AREA, DRY BIOMASS, AND NET PHOTOSYNTHETIC RATE a
Biomass
Birch
B. resinifera B. pendula
Height (cm)
Leaf area (cm 2)
Leaf (g)
Shoot (g)
Root (g)
Total (g)
Photosynthesis (#rnol/m" sec)
26 + 0.9 27 + 0. t
433 ± 23 414 ± 23
1.3 + 0,1 1.4 i 0. l
0.4 + 0 . | 0.5 ± 0.1
1.1 ± 0.1 1.1 +_ 0.1
2.8 + 0.1 2.9 ± 0.1
18.8 +_ 0.5 18.6 _+ 0.5
"Mean + 1 SE presented.
We suggest conclusions drawn from this approach to testing the "energy cost of defense hypothesis" should be viewed with caution for three reasons. These simple estimates of growth do not take into account: (1) differences in the ability to acquire carbon, (2) differences in maintenance respiration, or (3) biochemical trade-offs in partitioning of carbon between chemical defense and competing plant functions. Yet these differences are the physiological and biochemical basis for the energetic cost of producing chemical defense (Gulmon and Mooney, 1986; Chapin et al., 1990; Bryant et al., 1991, 1992; Herms and Mattson, 1992; Zangerl and Bazzaz, 1992). Our experiment with B. resinifera and B. pendula indicates a difference in the ability to acquire carbon is unlikely to explain why birch seedlings that differ in defense can accrete biomass at the same rate. We found that seedlings of B. resinifera and seedlings of B. pendula that differed in production of resin glands (Figure 1A and B; P < 0.001) and that had the same biomass after seven weeks of growth (Table 2), also had the same ability to acquire carbon (Table 2). We further suggest a difference in maintenance respiration is unlikely to explain why birch seedlings that differ in defense can accrete biomass at the same rate, because maintenance respiration has a minimal effect on patterns of carbon allocation by birch seedlings (Ingestad and McDonald, 1989; Ingestad and Agren, 1991).
HYPOTHESIS III
These conclusions suggest that if birch seedlings pay an energy cost to defend themselves against browsing in winter, this cost is an indirect cost resulting from trade-off in allocation of carbon between defense and storage. Two observations from common garden experiments support this hypothesis: (1) In contrast to 1-year-old B. pendula seedlings, when Rousi et al. (1991) examined
892
BRYANT AND JULKUNEN-TIITTO
8-year-old B. pendula saplings they found a negative correlation between defense (resin gland density) and growth (sapling height). This result is expected if there is a trade-off between defense and storage in juvenile stages of B. pendula, and juvenile B. pendula is polymorphic for this tradeoff, (2) After two to three years of growth in common gardens in Finland (M. Rousi, personal communication) and Michigan (M. Herms, personal communication) the comparatively heavily defended saplings of B. resinifera are growing at less than half the rate of saplings of the less-defended B. pendula.
RECOMMENDATIONS FOR FUTURE RESEARCH
Two further experiments are needed to verify that seedlings of small-seeded plants subject to differences in the intensity of selection for defense and the intensity of selection for competitive ability are polymorphic for partitioning of carbon between immobile chemical defense and storage reserves. In the one experiment, mass balance measurements should be used to document biochemical carbon partitioning by seedlings of congeners or conspecifics grown in a common environment. In this experiment, masses of carbon allocated to production of immobile defenses and to production of storage compounds (e.g., starch, storage protein, fatty acids) as well as masses of carbon allocated to growth and maintenance (e.g., sugars, amino acids, enzymes), and to structure (e.g., cellulose, lignin) should be determined using the biochemical approach suggested by Chapin et al. (1990). Assuming no differences in carbon acquisition, maintenance respiration, and allocation of carbon to other functions, evidence for a trade-off between defense and storage would be a negative correlation between the amount of glucose used to produce immobile chemical defense and the amount of glucose used to produce storage substances. Furthermore, this trade-off should become more noticeable in later stages of seedling ontogeny, because according to our second hypothesis, shortly after germination allocation of carbon to any function that competes with immediate growth should be minimal. Thus, for later stages of seedling ontogeny we expect "defense-selected" seedlings will have comparatively small storage reserves, and "'competitionselected" seedlings will have comparatively poor chemical defenses. In the other experiment, Jacob. pulse labeling should be used to measure the flow of carbon competing plant functions within a few hours of acquisition. In this experiment, evidence for a "defense-storage" polymorphism would be provided by the following two observations: (1) After production of defenses and storage reserves begins, the short-term flow of carbon into immobile chemical defense will be greatest for "defense-selected seedlings." (2) Conversely, the short-term flow of carbon into storage reserves wilt be greatest for "competition-selected" seedlings.
893
BIRCH CHEMICAL DEFENSE
CONCLUSIONS C o m p e t i t i o n and herbivory are two o f the greatest challenges faced by j u v e n i l e plants (Harper, 1977; T i l m a n , 1982; W a t k i n s o n , 1986; Huntly, 1991). Increased ability to deter herbivory is often achieved by increased allocation o f resources to chemical defense, and increased competitive ability is generally associated with rapid growth. Thus, in the j u v e n i l e stage, plants that have evolved in e n v i r o n m e n t s with varying levels o f herbivory and competition should be polymorphic for partitioning of carbon b e t w e e n defense and growth (Herms and Mattson, 1992). In this p a p e r we have suggested that studies o f the early ontogenetic d e v e l o p m e n t o f defense systems by plants are needed to test this hypothesis. W e have further suggested the focus o f this research should be a search for a trade-off in allocation of carbon to i m m o b i l e defense versus formation of storage reserves in the seedling stage o f species that grow from small seeds. This research will help resolve a central question o f current plant defense theory: when is production of chemical defense energetically costly?
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