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Plants that take up relatively large amounts of heavy metals from the soil and sequester them in their tissues are termed metal hyperaccumulators (Brooks et al., 1977). The threshold metal content used to define a hyperaccumulator depends on the particular metal accumulated. For instance, nickel (Ni) hyperaccumulators, which comprise more taxa than any other type of metal hyperaccumulator, contain more than 1000 µg Ni g−1 dry tissue (Brooks et al., 1977). Boyd & Martens (1992) summarized several hypotheses that have been advanced to explain the evolution of hyperaccumulation; however, it is the metal defence hypothesis that has garnered the most supporting evidence (Boyd & Martens, 1998a). Metals in plant tissues deter feeding (Pollard & Baker, 1997; Jhee et al., 1999), delay larval development (Martens & Boyd, 1994; Boyd & Moar, 1999) and are acutely toxic to certain insect herbivores (Boyd & Martens, 1994; Martens & Boyd, 1994; Boyd & Moar, 1999) and plant pathogens (Boyd et al., 1994; Ghaderian et al., 2000).
Because metals are complexed with common metabolic products such as citrate (Sagner et al., 1998), malate (Mathys, 1977; Gabbrielli et al., 1991), amino acids (Krämer et al., 1996), or oxalate (Mathys, 1977), translocation and compartmentalization may represent the only direct metabolic costs for metal-based defences. Thus, the metabolic cost for metal-based defences is presumed to be low compared with the costs of constructing and maintaining carbon-based defences (Martens & Boyd, 1994). For example, Gulmon & Mooney (1986) estimated the cost of synthesis of phenolics to be 2.6 g of assimilated CO2 g−1 of phenolic compound, whereas the cost for leaf production was 1.9–2.7 g CO2 g−1 of leaf tissue, thus the carbon cost of tannin production may rival the cost of producing new leaves. Sequestration of toxic heavy metals may provide defence (benefit) against insects and pathogens and reduce the need (cost) to produce expensive carbon-based defences. Although the ‘basal allocation’ (sensuMcDonald et al., 1999) of metabolic resources toward defence may be genetically fixed, further allocation of resources to defensive chemistry should occur only when the benefits of avoiding herbivory exceed defence costs (Bazzaz et al., 1987). Therefore, we predict that plants defended by metals will invest fewer resources into other types of organic defenses.
Certain elemental defences (e.g. silicification) have been shown to be inducible (McNaughton & Tarrants, 1983). However, since metal hyperaccumulation is a constitutive trait (Boyd & Martens, 1998b) and may have functions other than defence (Boyd & Martens, 1992; Boyd & Martens, 1998a), it is not likely to be affected by the presence or absence of herbivore damage. To date, the inducibility of metal-based defences has only been investigated in one species, the Ni hyperaccumulator, Streptanthuspolygaloides Gray (Davis & Boyd, 2000). In that study, foliar Ni levels of S. polygaloides were unaffected by the presence of either simulated herbivore damage or live herbivores, whereas levels of organic defences (glucosinolates) were increased (Davis & Boyd, 2000).
Leaf maturity also influences leaf defensive characteristics. In tropical forest understories, immature leaves of woody plants experience up to 100 times as much damage from herbivory and disease compared to older leaves (Coley & Aide, 1991). Up to 70% of lifetime damage can occur during leaf expansion (Coley & Kursar, 1996). Young leaves are attractive to herbivores because they are less tough (Nichols-Orians & Schultz, 1990) and have higher nitrogen and water contents than mature leaves (Nichols-Orians & Schultz, 1990; Kursar & Coley, 1991). To reduce damage from herbivory, young leaves of woody tropical plants have higher concentrations of phenolic compounds than mature leaves (Coley & Aide, 1991; Coley & Kursar, 1996). Although Ni levels can be higher in mature leaves than in expanding leaves (Boyd et al., 1999), it is not known if temporal distribution patterns of Ni affect levels of carbon-based defences.
Few studies have examined interactions between defences, including the interaction between organic and elemental chemical defences (Pennings, 1996; Pennings et al., 1998). The purpose of this study was to characterize constitutive and induced levels of a carbon-based defence (phenolics) and a metal-based defence (Ni) in a Ni hyperaccumulator species. Specifically, we wanted to: determine the effects of simulated herbivory on levels of Ni-based defences; characterize developmental patterns of Ni-based defences; contrast the developmental and induced responses of Ni-based and organic defences; and compare these responses with a co-occuring nonhyperaccumulator shrub. Plants that are well defended by foliar metal should invest less carbon towards the construction of organic defences than plants that do not utilize a metal-based defence. Therefore, we expected to find reduced tannin content in the foliage of a hyperaccumulator when compared to leaves of a nonhyperaccumulator species.
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Serpentine soils are typically characterized by a paucity of certain nutrients (e.g. Ca, K, P) and relatively high levels of Ni, Mg, Co, and Cr (Brooks, 1987). These harsh conditions often result in serpentine floras that are specifically adapted to ultramafic soils, although it is unclear if nutrient limitations (Kruckeberg, 1984; Nagy & Proctor, 1997) or other factors such as drought and soil depth (Kruckeberg, 1984) are responsible for these unique communities. Jaffré (1980) estimated that 60% of the 1500 plant species found on New Caledonian serpentine soils are endemic to their ultramafic substrates. Within the serpentine flora (but not always endemic to serpentine soils) is an even smaller ‘flora’, the metal hyperaccumulators. This distinct trait has been the focus of many studies, but the metal-based plant defence hypothesis has received the most experimental support among the current hypotheses seeking to explain the evolution of hyperaccumulation (Boyd & Martens, 1998a).
Martens & Boyd (1994 ) suggested that one benefit of a metal-based defence is the decreased need to invest resources (i.e. C and N) into organic defences. Thus, species that are putatively protected by metals should not invest as much carbon into organic defences as nonhyperaccumulators. Our data did not support this prediction; the Ni-hyperaccumulator, P . douarrei , had 12% more total phenolics, 49% more condensed tannins, and 73% higher protein precipitation ability than the nonhyperaccumulator, F . webbiana ( Table 1 ). Since total carbon content (%) did not differ between species, it appears that Psychotria invests more of its leaf carbon budget toward organic defences than does Ficus .
Why would Psychotria divert cellular resources into synthesis of organic defences if its foliage is already defended by Ni? Both Ni and tannins may have multiple functions, including defence. Nickel hyperaccumulation may be involved in drought or metal tolerance (Boyd & Martens, 1992) and may not have evolved solely as an herbivore defence for Psychotria. Likewise, Bernays (1981) has suggested that tannins have cellular and ecological functions that do not involve defence. For example, tannins may simply be repositories for excess carbon when plant growth occurs under nutrient-limited conditions (Bryant, 1987). For hyperaccumulators, tannins may function as metal-binding compounds. Tannins have many hydroxyl radicals which endow them with a strong affinity for many cations, including metals. Tannins are used industrially as metal chelators to remove Fe ions from water used in food industries (Matsuo et al., 1995) and extract Cu, Pb, Cd, Cr, and Hg from industrial waste waters (Randall et al., 1974). Neumann et al. (1995) found that much of the Cu in the Cu tolerant plant, Armeriamaritima (Mill.) Willd. ssp. halleri A. and D. Love, was localized within the vacuoles of tannin idioblasts. We suggest that the high levels of tannins in Psychotria leaves may function in the chelation or detoxification of Ni, in addition to providing defensive benefits.
Leaf age can affect levels of foliar organic defence compounds. In tropical understoreys, young foliage of woody shrubs generally has higher levels of organic defences than mature foliage (Coley & Aide, 1991; Coley & Kursar, 1996) since expanding leaves are more palatable to herbivores (i.e. young leaves are less tough and have lower C : N ratios than mature leaves) (Kursar & Coley, 1991). Our data support this prediction for both Psychotria and Ficus, with Psychotria leaves exhibiting the largest contrast. Young Ficus leaves had 16% more total phenolics, 13% more condensed tannins, and 14% higher protein precipitation ability than mature leaves, while young Psychotria leaves exhibited 30%, 35%, and 27% higher values than mature leaves for the same parameters.
Ni-based defences also can be influenced by the stage of leaf development. Ni concentrations were higher in the older foliage of both Psychotria and Ficus in this study. Greater Ni concentrations in older leaves indicate that Ni is an immobile element that accumulates in tissues over time. Since Ni levels were lowest in the young, palatable leaves for both species, it appears that young leaves of Psychotria are less defended by Ni. However, the mean Ni level in young Psychotria leaves observed in this study (13 700 µg g−1) is likely to be acutely toxic to generalist folivores. For example, Martens & Boyd (1994) demonstrated that a leaf Ni concentration of 3000 µg g−1 was toxic to a generalist folivore (Pierisrapae L.) feeding on the Ni hyperaccumulator, Thlaspimontanum var. montanum P. Holmgr. (Martens & Boyd, 1994). In fact, further experimentation with artificial diet studies showed that Ni concentrations of only 1000 µg g−1 were fatal to Pierisrapae larvae (Martens & Boyd, 1994). Prior studies of Psychotriadouarrei have shown that leaf Ni levels vary greatly: 13 400 (Lee et al., 1977), 14 900–27 700 (Boyd et al., 1999), 19 900 (Kelly et al., 1975), and 47 000 µg g−1 (Jaffré & Schmid, 1974) have all been reported. Since these values range from 13 to 47 times greater than the amount of Ni necessary to be acutely toxic to an unadapted herbivore (Martens & Boyd, 1994), defence is a likely function of Ni in Psychotria leaves at all developmental stages.
Plant defences are not always static; in fact, many defences are induced by herbivory. Although tannins are generally not considered to be inducible (but see Baldwin & Schultz, 1983; Stock et al., 1993), little is known about the inducibility of metal-based defences (Davis & Boyd, 2000). In the current study, neither tannin nor Ni concentrations were increased by simulated herbivore damage, implying noninducibility. There were, however, significant reductions for some measures of organic defence compounds. Total carbon content was less in severely damaged leaves than in moderately damaged leaves for both species (with the exception of mature Ficus leaves), indicating that carbon export increased with increased severity of damage. Lower levels of total phenolics and condensed tannins in severely damaged leaves of Ficus, and condensed tannins in severely damaged, young Psychotria leaves, could be due to mobilization of carbon to other plant organs or failure to reinvest carbon to replace compounds lost from the normal turnover costs of cellular metabolism. It should be noted that the measure of ecological activity of tannins, or protein precipitation ability, was not affected by damage for either species. These patterns in response to damage for both tannins and Ni accumulation indicate that neither defence is induced by simulated herbivory in either Psychotria or in Ficus. This evidence supports both the widely accepted concept that tannins are constitutive defences (Feeny, 1970) and the more recent idea that Ni hyperaccumulation is also a constitutive trait (Boyd & Martens, 1998b).
Metal hyperaccumulators may have better metal uptake abilities than nonhyperaccumulator plants. Obviously the Ni-hyperaccumulator Psychotria should contain more Ni than the nonhyperaccumulator Ficus, however, the difference in Ni levels between the two species is staggering; mean Ni content for Psychotria leaves was 16 400 µg g−1 d. wt vs 90 µg g−1 d. wt for Ficus leaves. In addition, Zn, Cr, Co, and Pb concentrations were 2.5x, 1.5x, 13.0x, and 6.5x higher in Psychotria leaves (Table 1). Furthermore, elevated levels of Zn, Cr, Pb, and Co suggest that Psychotria has a more efficient metal uptake system (or a less efficient system of exclusion) than Ficus. The ‘inadvertent uptake’ hypothesis, reviewed by Boyd & Martens (1992) and Boyd & Martens (1998), suggests that the evolution of metal hyperaccumulation resulted from a preadapted, enhanced nutrient uptake system, and may not have originated solely in response to herbivore selection pressures. Our data provide support for this hypothesis.
Few studies have attempted to describe multiple defence mechanisms within a species (Pennings, 1996). Unfortunately, chemical (and physical) defences may have subtle interactive effects that can be overlooked by conventional studies focusing on single defensive characteristics. Synergism among defences should increase their effectiveness for overall herbivore protection. It is possible, however, that chemical defences may have antagonistic effects. Goldstein & Spencer (1985) showed that tannins inhibit cyanogenesis in Caricapapaya L. Similarly, the possibility exists for an antagonism between tannins and Ni. Pizarro et al. (1994)) showed that herbal infusions that are high in tannins may inhibit gastrointestinal absorption of Fe, Zn and Cu. A similar interaction may occur between Ni and tannins within the gut of herbivores, thereby limiting the toxicity of Ni. However, preliminary artificial diet studies have shown that Ni and tannic acid produce a synergistic, negative effect on the growth and mortality of Spodopteraexigua Hübner larvae (data not shown). Undoubtedly, complete comprehension of the evolution of plant defences of hyperaccumulators cannot occur without consideration of all possible defences within the ‘green arsenal.’ As our understanding of chemical defences increases, it becomes more apparent that chemical defences work in concert rather than in absence of each other.