Juha-Pekka Salminen

Insect Herbivores Drive Real-Time Ecological and Evolutionary Change in Plant Populations

Plant Anti-Insect Armaments Because individual plants are unable to relocate, they are subject to extreme selection by the insects feeding upon them. One means by which plants suppress herbivory is to produce toxic compounds to deter feeding (see the Perspective by Hare). Agrawal et al. (p. 113) compared pesticide–treated or untreated evening primroses. Over 5 years of pesticide treatment, the production of defensive chemicals in the fruit reduced and flowering times shifted, and the primroses competitive ability against dandelions improved. Züst et al. (p. 116) examined large-scale geographic patterns in a polymorphic chemical defense locus in the model plant Arabidopsis thaliana and found that it is matched by changes in the relative abundance of two specialist aphids. Thus, herbivory has strong and immediate effects on the local genotypic composition of plants and traits associated with herbivore resistance. Protecting the common evening primrose from being eaten by insects alters its phenotype in only five growing seasons. Insect herbivores are hypothesized to be major factors affecting the ecology and evolution of plants. We tested this prediction by suppressing insects in replicated field populations of a native plant, Oenothera biennis, which reduced seed predation, altered interspecific competitive dynamics, and resulted in rapid evolutionary divergence. Comparative genotyping and phenotyping of nearly 12,000 O. biennis individuals revealed that in plots protected from insects, resistance to herbivores declined through time owing to changes in flowering time and lower defensive ellagitannins in fruits, whereas plant competitive ability increased. This independent real-time evolution of plant resistance and competitive ability in the field resulted from the relaxation of direct selective effects of insects on plant defense and through indirect effects due to reduced herbivory on plant competitors.

SEASONAL VARIATION IN THE CONTENT OF HYDROLYZABLE TANNINS, FLAVONOID GLYCOSIDES, AND PROANTHOCYANIDINS IN OAK LEAVES

Oaks have been one of the classic model systems in elucidating the role of polyphenols in plant–herbivore interactions. This study provides a comprehensive description of seasonal variation in the phenolic content of the English oak (Quercus robur). Seven different trees were followed over the full course of the growing season, and their foliage repeatedly sampled for gallic acid, 9 individual hydrolyzable tannins, and 14 flavonoid glycosides, as well as for total phenolics, total proanthocyanidins, carbon, and nitrogen. A rare dimeric ellagitannin, cocciferin D2, was detected for the first time in leaves of Q. robur, and relationships between the chemical structures of individual tannins were used to propose a biosynthetic pathway for its formation. Overall, hydrolyzable tannins were the dominant phenolic group in leaves of all ages. Nevertheless, young oak leaves were much richer in hydrolyzable tannins and flavonoid glycosides than old leaves, whereas the opposite pattern was observed for proanthocyanidins. However, when quantified as individual compounds, hydrolyzable tannins and flavonoid glycosides showed highly variable seasonal patterns. This large variation in temporal trends among compounds, and a generally weak correlation between the concentration of any individual compound and the total concentration of phenolics, as quantified by the Folin–Ciocalteau method, leads us to caution against the uncritical use of summary quantifications of composite phenolic fractions in ecological studies.

Characterisation of hydrolysable tannins from leaves of Betula pubescens by high-performance liquid chromatography–mass spectrometry

A high-performance liquid chromatography-electrospray ionisation mass spectrometry (HPLC-ESI-MS) method, assisted by diode array detection, for the characterisation of individual hydrolysable tannins in birch leaves was developed. With the method, it was found that birch (Betula pubescens) leaves contained an exceptionally complex mixture of hydrolysable tannins; 14 gallotannins and 20 ellagitannins were identified. The developed HPLC-ESI-MS method allows the qualitative and quantitative determination of individual gallotannins and ellagitannins directly from crude birch leaf extract. This is important in studying ecological functions of these phenolic compounds, especially their role in the resistance of birch leaves against insects.

Seasonal variation in the content of hydrolysable tannins in leaves of Betula pubescens

The contents of 13 hydrolysable tannins in the leaves of white birch (Betula pubescens L.) trees were analysed at twelve stages throughout the growing season. All individual galloylglucoses, from 1-O-galloyl-beta-D-glucopyranose to 1,2,3,4,6-penta-O-galloyl-beta-D-glucopyranose, accumulated in young leaves, while ellagitannins showed significantly variable seasonal trends. The major ellagitannin during the whole growing season was pedunculagin while 2,3-(S)-HHDP-glucopyranose. the end product of the proposed ellagitannin pathway, accumulated in mature leaves. Relationships between the characteristics of seasonal variation in the contents of individual ellagitannins and their chemical structures were used to unravel the biogenesis of ellagitannins in birch leaves. Evidence of degradation of ellagitannins through hydrolysis during leaf growth and development is presented and implications for herbivory are discussed.

Ellagitannins have Greater Oxidative Activities than Condensed Tannins and Galloyl Glucoses at High pH: Potential Impact on Caterpillars

Plants synthesize a diversity of tannin structures but little is known about whether these different types have different oxidative activities in herbivores. Oxidative activities of hydrolyzable and condensed tannins were compared at pH 10 with two methods: EPR spectrometry was used to quantify semiquinone radicals in anoxic conditions and a spectrophotometric assay was used to measure the rate of browning of phenolics oxidized in ambient oxygen conditions. A little-studied group of hydrolyzable tannins (ellagitannins) contained the most active tannins examined, forming high concentrations of semiquinone radicals and browning at the highest rates. On average, galloyl glucoses and high-molecular-weight gallotannins had intermediate to low oxidative activities. Condensed tannins generally formed low levels of semiquinone radicals and browned most slowly. The results suggest that ellagitannin-rich plants have active oxidative defenses against herbivores, such as caterpillars, whereas the opposite may hold true for plants that contain predominantly condensed tannins or high-molecular-weight gallotannins.

Heritability, covariation and natural selection on 24 traits of common evening primrose (Oenothera biennis) from a field experiment

This study explored genetic variation and co‐variation in multiple functional plant traits. Our goal was to characterize selection, heritabilities and genetic correlations among different types of traits to gain insight into the evolutionary ecology of plant populations and their interactions with insect herbivores. In a field experiment, we detected significant heritable variation for each of 24 traits of Oenothera biennis and extensive genetic covariance among traits. Traits with diverse functions formed several distinct groups that exhibited positive genetic covariation with each other. Genetic variation in life‐history traits and secondary chemistry together explained a large proportion of variation in herbivory (r2 = 0.73). At the same time, selection acted on lifetime biomass, life‐history traits and two secondary compounds of O. biennis, explaining over 95% of the variation in relative fitness among genotypes. The combination of genetic covariances and directional selection acting on multiple traits suggests that adaptive evolution of particular traits is constrained, and that correlated evolution of groups of traits will occur, which is expected to drive the evolution of increased herbivore susceptibility. As a whole, our study indicates that an examination of genetic variation and covariation among many different types of traits can provide greater insight into the evolutionary ecology of plant populations and plant–herbivore interactions.

Phylogenetic Trends in Phenolic Metabolism of Milkweeds (Asclepias): Evidence for Escalation

Although plant-defense theory has long predicted patterns of chemical defense across taxa, we know remarkably little about the evolution of defense, especially in the context of directional phylogenetic trends. Here we contrast the production of phenolics and cardenolides in 35 species of milkweeds (Asclepias and Gomphocarpus). Maximum-likelihood analyses of character evolution revealed three major patterns. First, consistent with the defense-escalation hypothesis, the diversification of the milkweeds was associated with a trend for increasing phenolic production; this pattern was reversed (a declining evolutionary trend) for cardenolides, toxins sequestered by specialist herbivores. Second, phylogenetically independent correlations existed among phenolic classes across species. For example, coumaric acid derivatives showed negatively correlated evolution with caffeic acid derivatives, and this was likely driven by the fact that the former are used as precursors for the latter. In contrast, coumaric acid derivatives were positively correlated with flavonoids, consistent with competition for the precursor p-coumaric acid. Finally, of the phenolic classes, only flavonoids showed correlated evolution (positive) with cardenolides, consistent with a physiological and evolutionary link between the two via malonate. Thus, this study presents a rigorous test of the defense-escalation hypothesis and a novel phylogenetic approach to understanding the long-term persistence of physiological constraints on secondary metabolism.

Gallic acid and hydrolysable tannins are formed in birch leaves from an intermediate compound of the shikimate pathway

Gallic acid is the starting material for hydrolysable tannin synthesis, but the mechanism of its formation in higher plants has not been known. To elucidate the pathway of gallic acid synthesis in the leaves of mountain birch (Betula pubescens ssp. czerepanovii), we studied the effects of glyphosate (N-[phosphonomethyl]-glycine) on the levels of individual hydrolysable tannins. Glyphosate is known to block 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, an enzyme of the shikimate pathway, thus reducing the synthesis of aromatic amino acids and phenolics derived from phenylalanine. However, when birch leaves were treated with glyphosate (1 mM, 72 h), the contents of individual hydrolysable tannins increased about twofold compared to control leaves. Our findings indicate that the immediate precursor of gallic acid may be an intermediate compound of the shikimate pathway before EPSP, most probably 3-dehydroshikimic acid. Consistently with this assumption, a highly active enzyme dehydroshikimate dehydrogenase (DSDG), which catalyses the direct conversion of 3-dehydroshikimic acid into gallic acid, was found in birch leaves. This new enzyme was partially purified to study its properties. It was found that DSDG is an NADP-dependent enzyme with a pH optimum at about 10.0. The enzyme showed an affinity for NADP+ approximately 60-fold (Km=0.008 mM) that for dehydroshikimate (Km=0.49 mM). The intracellular organisation of the shikimate pathway and the hydrolysable tannin pathway are discussed.

Geographical variation in egg mass and egg content in a passerine bird.

Reproductive, phenotypic and life-history traits in many animal and plant taxa show geographic variation, indicating spatial variation in selection regimes. Maternal deposition to avian eggs, such as hormones, antibodies and antioxidants, critically affect development of the offspring, with long-lasting effects on the phenotype and fitness. Little is however known about large-scale geographical patterns of variation in maternal deposition to eggs. We studied geographical variation in egg components of a passerine bird, the pied flycatcher (Ficedula hypoleuca), by collecting samples from 16 populations and measuring egg and yolk mass, albumen lysozyme activity, yolk immunoglobulins, yolk androgens and yolk total carotenoids. We found significant variation among populations in most egg components, but ca. 90% of the variation was among individuals within populations. Population however explained 40% of the variation in carotenoid levels. In contrast to our hypothesis, we found geographical trends only in carotenoids, but not in any of the other egg components. Our results thus suggest high within-population variation and leave little scope for local adaptation and genetic differentiation in deposition of different egg components. The role of these maternally-derived resources in evolutionary change should be further investigated.

Phylogenetic ecology of leaf surface traits in the milkweeds (Asclepias spp.): chemistry, ecophysiology, and insect behavior.

The leaf surface is the contact point between plants and the environment and plays a crucial role in mediating biotic and abiotic interactions. Here, we took a phylogenetic approach to investigate the function, trade-offs, and evolution of leaf surface traits in the milkweeds (Asclepias). Across 47 species, we found trichome densities of up to 3000 trichomes cm(-2) and epicuticular wax crystals (glaucousness) on 10 species. Glaucous species had a characteristic wax composition dominated by very-long-chain aldehydes. The ancestor of the milkweeds was probably a glaucous species, from which there have been several independent origins of glabrous and pubescent types. Trichomes and wax crystals showed negatively correlated evolution, with both surface types showing an affinity for arid habitats. Pubescent and glaucous milkweeds had a higher maximum photosynthetic rate and lower stomatal density than glabrous species. Pubescent and glaucous leaf surfaces impeded settling behavior of monarch caterpillars and aphids compared with glabrous species, although surface types did not show consistent differentiation in secondary chemistry. We hypothesize that pubescence and glaucousness have evolved as alternative mechanisms with similar functions. The glaucous type, however, appears to be ancestral, lost repeatedly, and never regained; we propose that trichomes are a more evolutionarily titratable strategy.