David H. Siemens

Herbivore-induced ethylene suppresses a direct defense but not a putative indirect defense against an adapted herbivore.

Abstract. Herbivory induces both direct and indirect defenses in plants; however, some combinations of these defenses may not be compatible. The jasmonate signal cascade activated both direct (nicotine accumulations) and indirect (mono- and sesquiterpene emissions) whole-plant defense responses in the native tobacco Nicotiana attenuata Torr. Ex Wats. Nicotine accumulations were proportional to the amount of leaf wounding and the resulting increases in jasmonic acid (JA) concentrations. However, when larvae of the nicotine-tolerant herbivore, Manduca sexta, fed on plants or their oral secretions were applied to leaf punctures, the normal wound response was dramatically altered, as evidenced by large (4- to 10-fold) increases in the release of (i) volatile terpenoids and (ii) ethylene, (iii) increased (4- to 30-fold) accumulations of endogenous JA pools, but (iv) decreased or unchanged nicotine accumulations. The ethylene release, which was insensitive to inhibitors of induced JA accumulation, was sufficient to account for the attenuated nicotine response. Applications of ethylene and ethephon suppressed the induced nicotine response and pre-treatment of plants with a competitive inhibitor of ethylene receptors, 1-methylcyclopropene, restored the full nicotine response. This ethylene burst, however, did not inhibit the release of volatile terpenoids. Because parasitoids of Manduca larvae are sensitive to the dietary intake of nicotine by their hosts, this ethylene-mediated switching from direct to a putative indirect defense may represent an adaptive tailoring of a plants defense response.

COST OF DEFENSE IN THE CONTEXT OF PLANT COMPETITION: BRASSICA RAPA MAY GROW AND DEFEND

Theory on costs of plant defense against herbivory in stressful environments predicts that costs should increase when competition is intense. This amplifies a fundamental dilemma that plants are thought to face: allocate limited resources to grow fast enough to compete, or invest these resources in secondary metabolites to maintain defense. We studied costs associated with genetic and environmental variation in secondary metabolite pro- duction of Brassica rapa in the presence and absence of the generalist competitor Lolium perenne. We used experimental quantitative genetics (artificial selection) to manipulate genetic variation, and herbivore-induction treatments to produce environmental variation in myrosinase and glucosinolate concentrations and resistance. Glucosinolates, and their byproducts after breakdown by myrosinase, are known to affect herbivory on plants in the Brassicaceae family. Defense costs were significant in the absence of competitors, but in contrast to theoretical predictions, costs of constitutive defense (measured as growth rates) were not detectable and the cost of induced defense remained the same in the competitive environment. To understand what factors made constitutive defense costs not detectable under competition we conducted several experiments to assess the effects of limited re- sources and allelopathy on costs and benefits of the defense chemicals. None of the ex- periments involving nutrient supply and weak competition supported the hypothesis that the lack of defense costs in competitive environments was due to limited resources. Instead, the breakdown products of the glucosinolate-myrosinase reaction appeared to function as allelopathic agents, which may benefit B. rapa plants in competition, thereby reducing net costs of chemical defense. We found that: (1) the effects of exogenous glucosinolates on Lolium root length depended on the presence of myrosinase. (2) In the absence of nutrients, Lolium root lengths were shorter when seeds germinated with B. rapa. (3) Genetic increases in glucosinolate concentration negatively affected Lolium seedling growth only when there were simultaneous genetic increases in myrosinase concentration. Activated carbon treat- ments designed to neutralize allelopathic effects and restore costs in the competitive en- vironments were, however, not statistically significant. When plant defenses also function to benefit plants in competitive interactions, plants may evolve to compete and defend.

ECOLOGICAL COSTS OF PLANT RESISTANCE TO HERBIVORES IN THE CURRENCY OF POLLINATION

In this paper, we examine how ecological costs of resistance might be manifested through plant relationships with pollinators. If defensive compounds are incorporated into floral structures or if they are sufficiently costly that fewer rewards are offered to pollinators, pollinators may discriminate against more defended plants. Here we consider whether directional selection for increased resistance to herbivores could be constrained by opposing selection through pollinator discrimination against more defended plants. We used artificial selection to create two populations of Brassica rapa plants that had high and low myrosinase concentrations and, consequently, high and low resistance to flea beetle herbivores. We measured changes in floral characters of plants in both damaged and undamaged states from these populations with different resistances to flea beetle attack. We also measured pollinator visitation to plants, including numbers of pollinators and measures of visit quality (numbers of flowers visited and time spent per flower). Damage from herbivores resulted in reduced petal size, as did selection for high resistance to herbivores later in the plant lifetime. In addition, floral display (number of open flowers) was also altered by an interaction between these two effects. Changes in floral traits translated into overall greater use of low‐resistance, undamaged plants based on total amount of time pollinators spent foraging on plants. Total numbers of pollinators attracted to plants did not differ among treatments; however, pollinators spent significantly more time per flower on plants from the low‐resistance population and tended to visit more flowers on these plants as well. Previous work by other investigators on the same pollinator taxa has shown that longer visit times are associated with greater male and female plant fitness. Because initial numbers of pollinators did not differ between selection regimes, palatability and/or amount of rewards offered by high‐ and low‐resistance populations are likely to be responsible for these patterns. During periods of pollinator limitation, less defended plants may have a selective advantage and pollinator preferences may mediate directional selection imposed by herbivores. In addition, if pollinator preferences limit seed set in highly defended plants, then lower seed set previously attributed to allocation costs of defense may also reflect greater pollinator limitation in these plants relative to less defended plants.

EVOLUTION OF PEST-INDUCED DEFENSES IN BRASSICA PLANTS: TESTS OF THEORY

Theory on the evolution of pest-induced defenses in plants predicts (1) a negative genetic correlation between induced and constitutive (basal) levels of secondary metabolites, and (2) costs of maintaining high constitutive levels of secondary metabolites. We tested these predictions with genetically diverged populations created by artificial selection on myrosinase and glucosinolate levels in Brassica rapa. Glucosinolates and their breakdown products from the action of the enzyme myrosinase are putative defensive compounds in brassicas. Theory also suggests that effects of genetic changes in secondary metabolites may depend on resource availability, so nitrogen, a main constituent of glucosinolates and myrosinase was added in fertilizer treatments to assess costs. We used the fungal pathogen Leptosphaeria maculans and diamondback moth larvae Plutella xylostella as induction agents in comparisons of the diverged myrosinase populations. We found pleiotropic effects among constitutive myrosinase levels and pathogen-induced levels of myrosinase, glucosinolates, and resistance to diamondback moth larvae. In field experiments, genetic increases in myrosinase production were associated with significant decreases in estimated seed production, despite potential benefits from increased resistance to flea beetles Phylotreta cruciferae. No genotype-by-nitrogen interaction was found. Although costs were detected, our results indicate, in contrast to theory, mainly positive pleiotropic effects between constitutive levels of secondary metabolites and induced responses.

Cost of resistance and tolerance under competition: the defense-stress benefit hypothesis

Defense costs provide a major explanation for why plants in nature have not evolved to be better defended against pathogens and herbivores; however, evidence for defense costs is often lacking. Plants defend by deploying resistance traits that reduce damage, and tolerance traits that reduce the fitness effects of damage. We first tested the defense-stress cost (DSC) hypothesis that costs of defenses increase and become important under competitive stress. In a greenhouse experiment, uniparental maternal families of the host plant Arabis perennans were grown in the presence and absence of the bunch grass Bouteloua gracilis and the herbivore Plutella xylostella. Costs of resistance and tolerance manifest as reduced growth in the absence of herbivory were significant when A. perennans grew alone, but not in the competitive environment, in contrast to the DSC hypothesis. We then tested the defense-stress benefit (DSB) hypothesis that plant defenses may benefit plants in competitive situations thereby reducing net costs. For example, chemical resistance agents and tolerance may also have functions in competitive interactions. To test the DSB hypothesis, we compared differentially competitive populations for defense costs, assuming that poorer competitors from less dense habitats were less likely to have evolved defenses that also function in competition. Without competitive benefits of defenses, poorer competitors were expected to have higher net costs of defenses under competition in accordance with DSB. Populations of A. perennans and A. drummondii that differed dramatically in competitiveness were compared for costs, and as the DSB hypothesis predicts, only the poor competitor population showed costs of resistance under competition. However, cost of tolerance under competition did not differ among populations, suggesting that the poor competitors might have evolved a general stress tolerance. Although the DSC hypothesis may explain cases where defense costs increase under stress, the DSB hypothesis may explain some cases where costs decrease under competitive stress.

Alternative Seed Defense Mechanisms in Congeneric Plants

Plants in the same genus often differ by certain putative defense compounds even though they are attacked by the same or similar herbivores. We compared two such congeners in their interactions with the same seed predators. In the Sonoran Desert, seeds of the palo verdes Cercidium floridum and C. microphyllum (Leguminosae) were fed upon by the bruchid beetles Mimosestes amicus, M. ulkei, and Stator limbatus, several species of imperfect soil fungi, and at least one species of termite. Seed suitability was compared in field and laboratory experiments. Seeds of C. floridum were significantly more resistant except to a putative subpopulation of S. limbatus that normally feeds on C. floridum. Cercidium floridum seed resistance was located in the seed coat and was chemically based, as determined by a seed coat removal experiment and experiments in which we transferred extracts from resistant seeds to susceptible seeds. The resistance chemical(s) was not iden- tified, though tests for tannins were negative. Alternatively, C. microphyllum abscised pods early, which significantly reduced attack by the seed predators. Early pod abscission was more effective at reducing seed predation than were resistant seed coats. We suggest that very different plant defense mechanisms may evolve under similar selection pressures within the same lineage, contrary to conventional wisdom.

Determinants of host range in bruchid beetles

We asked what factors keep the seed beetles Stator limbatus and S. pruininus (Bruchidae) restricted to different species of Acacia (Leguminosae) in sympatry. Factors assessed were plant microenvironment, interspecific competition, suitability of seeds for food, and enemy impact. Females selectively oviposited on seeds of their normal host when given a choice of acacia seeds in their normal host microenvironment. This result occurred whether seeds of the other acacia were offered with or without potential com- petitors in them. Host suitability was determined by comparing full-siblings on both acacias. Three performance variables were measured: (1) emergence per egg laid, (2) time to emer- gence, and (3) size of females. Stator limbatus performed best on its normal host in all three variables measured, while S. pruininus performed best on its normal host in emergence time but showed no difference in percent emergence or female size. We also assessed the impact of the egg parasite Uscana semifumipennis (Trichogrammatidae) on the two species of bruchids when the bruchids occurred in their normal hosts. Uscana attacked both bruchids at the same level (20% of eggs parasitized). Thus, the proximate determinant of locality restricted use of hosts is oviposition behavior, which we suggest relates best to differences in the suitability of hosts for larvae.

Physiology and costs of resistance to herbivory and disease in Brassica

We used artificial selection experiments to study genetic allocation costs and physiological mechanisms of resistance to herbivory and fungal disease. Genetic costs to resistance were present in some instances and absent in others. Genetic resistance to the fungal pathogen, Leptosphaeria maculans was cost‐free, while resistance to Peronospora parasitica showed a negative genetic correlation between disease resistance and growth rate. Leptosphaeria resistant genotypes had 13% higher chitinase activity. Genetic increases in myrosinase activity were correlated with increased resistance to flea beetles (Phyllotreta cruciferae), but resulted in lower plant fecundity, presumably due to production costs of myrosinase. Genetic costs of resistance may maintain genetic variation in natural plant populations. These studies demonstrate the predictive and explanatory power of a functional approach to plant‐herbivore and plantpathogen interactions.

Bruchid Oviposition Patterns Beneath Guanacaste Trees (Mimosaceae) in Venezuela: Probable Consequences of Extinct Seed Dispersers

Guanacaste seeds in Venezuela accumulate beneath parent trees presumably because of extinct seed dispersers. We asked if the number of eggs laid per seed by the bruchid beetle Stator generalis and factors affecting this oviposition pattern are consequences of seed accumulation. We compared patterns of oviposition to theoretical expectations and to another study on a congeneric bruchid which faced limited seed availability. Theory predicts bruchids should lay one egg per seed when many seeds are readily available and when there are only negative effects to multiple eggs on seeds. From collections of hundreds of seeds from beneath three trees, we found the median number of eggs per seed to range from 4 to 8.5, even though many seeds (33%) had zero eggs. These patterns of oviposition were nonrandom. Theory also predicts that when seeds are limited, more eggs per seed should be laid. Thus, the relatively high numbers of eggs per seed on guanacaste seeds could be an anachronism more appropriate for limited seed availability. Alternatively, other factors associated with seed accumulation may cause increased egg densities. Indirect evidence suggests multiple oviposition may be more frequent, and that density dependent egg parasitism has changed from positive to negative, which selects for increased egg densities.

Observations on Host Plant and Biogeographical Differences in Bruchid Beetles in Southern Ecuador

An abrupt change of bruchid taxa, distribution, and host range is evident in central and southwestern Ecuador. There are several hypotheses supported by data. One is expanded host range of the genus Sennius, which is very specific to Cassia (s.l.) north of Colombia, but feeds in seeds of other genera and subfamilies of Leguminosae in South America. Stator testudinarius was found also to feed in Pithecellobium saman. Acanthoscelides isla has a disjunct distribution due to past geologic changes. It feeds in seeds of Rhynchosia spp. only in Panama, Costa Rica, and Ecuador. Endemism in a biogeographically isolated area is exemplified by Acanthoscelides siemensi which feeds only in seeds of Duranta dombeyana in one locality in Ecuador. Guilds of bruchids are discussed because Stator testudinarius and Pseudopachymerina spinipes replace other bruchids in the same seed guilds there. Based on our data, we hypothesize that the distribution of bruchids in Ecuador is due to geologic events and present floral composition.