New approaches to tannin analysis of leaves can be used to explain in vitro biological activities associated with herbivore defence.

Although tannins have been an important focus of studies of plant-animal interactions, traditional tannin analyses cannot differentiate between the diversity of structures present in plants. This has limited our understanding of how different mixtures of these widespread secondary metabolites contribute to variation in biological activity. We used UPLC-MS/MS to determine the concentration and broad composition of tannins and polyphenols in 628 eucalypt (Eucalyptus, Corymbia and Angophora) samples, and related these to three in vitro functional measures believed to influence herbivore defence: protein precipitation capacity, oxidative activity at high pH and capacity to reduce in vitro nitrogen (N) digestibility. Protein precipitation capacity was most strongly correlated with concentrations of procyanidin subunits in proanthocyanidins (PAs), and late-eluting ellagitannins. Capacity to reduce in vitro N digestibility was affected most by the subunit composition and mean degree of polymerisation (mDP) of PAs. Finally, concentrations of ellagitannins and prodelphinidin subunits of PAs were the strongest determinants of oxidative activity. The results illustrate why measures of total tannins rarely correlate with animal feeding responses. However, they also confirm that the analytical techniques utilised here could allow researchers to understand how variation in tannins influence the ecology of individuals and populations of herbivores, and, ultimately, other ecosystem processes.

Intraspecific Variation in Nutritional Composition Affects the Leaf Age Preferences of a Mammalian Herbivore.

Ecologists have long been interested in how the nutritional composition of leaves changes as they age, and whether this affects herbivore feeding preferences. As a consequence, the literature abounds with reports that younger leaves contain higher concentrations of nitrogen and plant secondary metabolites (PSMs) than do older leaves. Most of these studies, however, base their conclusions on average values that often mean little to herbivores. We examined this issue in the well-studied marsupial-eucalypt system, using Eucalyptus melliodora and captive common brushtail possums (Trichosurus vulpecula) offered branches from individual trees containing both young and mature leaves. Like many plants, the concentrations of N and PSMs differed among individual E. melliodora. Although young leaves were, on average, "better defended" by the PSM sideroxylonal than were mature leaves, some trees produced leaves that were relatively undefended at both ages. In response, possums chose different proportions of young and mature leaves depending on the chemistry of the individual tree. Possums did not always prefer leaves with lower concentrations of sideroxylonal (mature leaves) or those with higher concentrations of available N (young leaves). Instead, the sideroxylonal concentration of young leaves dictated their choice: possums preferred young leaves with low sideroxylonal concentrations, but not with high concentrations. By skewing their feeding toward trees producing young leaves with low concentrations of PSMs, possums may influence plant fitness. Researchers will detect these potentially important interactions only if they are aware that measuring variation among plants discloses more information than do average relationships.

Genus-wide variation in foliar polyphenolics in eucalypts.

Many studies quantify total phenolics or total tannins, but understanding the ecological role of polyphenolic secondary metabolites requires at least an understanding of the diversity of phenolic groups present. We used UPLC-MS/MS to measure concentrations of different polyphenol groups - including the four most common tannin groups, the three most common flavonoid groups, and quinic acid derivatives - in foliage from 628 eucalypts from the genera Eucalyptus, Angophora and Corymbia. We also tested for phylogenetic signal in each of the phenolic groups. Many eucalypts contained high concentrations of polyphenols, particularly ellagitannins, which have been relatively poorly studied, but may possess strong oxidative activity. Because the biosynthetic pathways of many phenolic compounds share either precursors or enzymes, we found negative correlations between the concentrations of several of the constituents that we measured, including proanthocyanidins (PAs) and hydrolysable tannins (HTs), HTs and flavonol derivatives, and HTs and quinic acid derivatives. We observed moderate phylogenetic signal in all polyphenol constituents, apart from the concentration of the prodelphinidin subunit of PAs and the mean degree of polymerisation of PAs. These two traits, which have previously been shown to be important in determining plants' protein precipitation capacity, may have evolved under selection, perhaps in response to climate or herbivore pressure. Hence, the signature of evolutionary history appears to have been erased for these traits. This study is an important step in moving away from analysing "totals" to a better understanding of how phylogenetic effects influence phenolic composition, and how this in turn influences ecological processes.

The Relative Concentrations of Nutrients and Toxins Dictate Feeding by a Vertebrate Browser, the Greater Glider Petauroides volans.

Although ecologists believe that vertebrate herbivores must select a diet that allows them to meet their nutritional requirements, while avoiding intoxication by plant secondary metabolites, this is remarkably difficult to show. A long series of field and laboratory experiments means that we have a good understanding of the factors that affect feeding by leaf-eating marsupials. This knowledge and the natural intraspecific variation in Eucalyptus chemistry allowed us to test the hypothesis that the feeding decisions of greater gliders (Petauroides volans) depend on the concentrations of available nitrogen (incorporating total nitrogen, dry matter digestibility and tannins) and of formylated phloroglucinol compounds (FPCs), potent antifeedants unique to Eucalyptus. We offered captive greater gliders foliage from two species of Eucalyptus, E. viminalis and E. melliodora, which vary naturally in their concentrations of available nitrogen and FPCs. We then measured the amount of foliage eaten by each glider and compared this with our laboratory analyses of foliar total nitrogen, available nitrogen and FPCs for each tree offered. The concentration of FPCs was the main factor that determined how much gliders ate of E. viminalis and E. melliodora, but in gliders fed E. viminalis the concentration of available nitrogen was also a significant influence. In other words, greater gliders ate E. viminalis leaves with a particular combination of FPCs and available nitrogen that maximised the nutritional gain but minimised their ingestion of toxins. In contrast, the concentration of total nitrogen was not correlated with feeding. This study is among the first to empirically show that browsing herbivores select a diet that balances the potential gain (available nutrients) and the potential costs (plant secondary chemicals) of eating leaves. The major implication of the study is that it is essential to identify the limiting nutrients and relevant toxins in a system in order to understand feeding behaviour.

Nutritional correlates of koala persistence in a low-density population.

It is widely postulated that nutritional factors drive bottom-up, resource-based patterns in herbivore ecology and distribution. There is, however, much controversy over the roles of different plant constituents and how these influence individual herbivores and herbivore populations. The density of koala (Phascolarctos cinereus) populations varies widely and many attribute population trends to variation in the nutritional quality of the eucalypt leaves of their diet, but there is little evidence to support this hypothesis. We used a nested design that involved sampling of trees at two spatial scales to investigate how leaf chemistry influences free-living koalas from a low-density population in south east New South Wales, Australia. Using koala faecal pellets as a proxy for koala visitation to trees, we found an interaction between toxins and nutrients in leaves at a small spatial scale, whereby koalas preferred trees with leaves of higher concentrations of available nitrogen but lower concentrations of sideroxylonals (secondary metabolites found exclusively in eucalypts) compared to neighbouring trees of the same species. We argue that taxonomic and phenotypic diversity is likely to be important when foraging in habitats of low nutritional quality in providing diet choice to tradeoff nutrients and toxins and minimise movement costs. Our findings suggest that immediate nutritional concerns are an important priority of folivores in low-quality habitats and imply that nutritional limitations play an important role in constraining folivore populations. We show that, with a careful experimental design, it is possible to make inferences about populations of herbivores that exist at extremely low densities and thus achieve a better understanding about how plant composition influences herbivore ecology and persistence.

Four species of arboreal folivore show differential tolerance to a secondary metabolite.

The marsupials that eat Eucalyptus in south-eastern Australia provide an example of animals with similar niche requirements occurring sympatrically. They certainly differ in size, ranging from about 1 kg in the greater glider (Petauroides volans) and the closely related common ringtail possum (Pseudocheirus peregrinus), to 4 kg (common brushtail possum, Trichosurus vulpecula) and up to 15 kg in the koala (Phascolarctos cinereus). All species, however, may eat considerable amounts of eucalypt foliage, often favouring the same species, and thus appear to compete for food. In order to better understand the degree of competition for food, we measured feeding by the greater glider in response to increasing concentrations of a specific group of eucalypt plant secondary metabolites (PSM), the sideroxylonals, and then compared it to results published for the other species. The greater glider was more resilient than the other species to increasing concentrations of sideroxylonals. We suggest this allows gliders to feed on leaves from the eucalypt subgenus, Symphyomyrtus, while its small size and gliding ability allow it to feed where koalas cannot, on the young leaves on top of the canopy. In contrast, the common ringtail possum is well adapted to feeding from species of the subgenus Eucalyptus, which do not produce sideroxylonals but contain less available nitrogen (AvailN) than do the symphyomyrtles. These 'nutritional niches' segregate the forest and along with other factors, such as generalist and specialist feeding strategies and differences in body size and requirements for shelter, presumably minimise competition between the marsupial species.

Feeding rates of a mammalian browser confirm the predictions of a 'foodscape' model of its habitat.

Adequate nutrition is a fundamental requirement for the maintenance and growth of populations, but complex interactions between nutrients and plant toxins make it difficult to link variation in plant quality to the ecology of wild herbivores. We asked whether a 'foodscape' model of habitat that uses near-infrared spectroscopy to describe the palatability of individual trees in the landscape, predicted the foraging decisions of a mammalian browser, the koala (Phascolarctos cinereus). Specifically, we considered four behavioural decision points at which nutritional quality may influence an animal's decision. These were: which tree to enter, whether to feed from that tree, when to stop eating, and how long to remain in that tree. There were trends for koalas to feed in eucalypt trees that were more palatable than unvisited neighbouring conspecific trees, and than trees that they visited but did not eat. Koalas ate longer meals in more palatable trees, and stayed longer and spent more time feeding per visit to these trees. Using more traditional chemical analyses, we identified that an interaction between the concentrations of formylated phloroglucinol compounds (a group of plant secondary metabolites) and available N (an integrated measure of tannins, digestibility and N) influenced feeding. The study shows that foodscape models that combine spatial information with integrated measures of food quality are a powerful tool to predict the feeding behaviour of herbivores in a landscape.

Digestive strategies and food choice in mantled howler monkeys Alouatta palliata mexicana: bases of their dietary flexibility.

Mantled howler monkeys (Alouatta palliata) occupy a wide variety of tropical habitats and are the most folivorous of New World primates. However, their diet may include fruits, buds, petioles, and flowers, as well as leaves, suggesting they must cope with variations in the nutrient composition of their food. We studied the physiological basis of the dietary flexibility of these monkeys by comparing food choice, digestive performance and patterns of digesta flow in six adults, fed diets of either leaves or a mixture of fruit and leaves. Although monkeys ate similar amounts of the two diets, they ingested more digestible protein when offered the leaf diet, on which they lost body mass, but they ingested much more soluble sugars when offered fruit and leaves on which they gained mass. Digestibilities of dry matter, fat, energy and fibre did not differ between diets, but those of crude protein, soluble sugars and minerals were higher on the fruit-leaf diet. Mean retention times in the gut of solute (Co-EDTA) and particulate markers (Cr-mordanted cell walls) did not differ between diets, but on both diets the monkeys retained the particulate marker (mean retention time ca 55 h) for longer than they did the solute marker (MRT ca 50 h). A lack of selective retention of solutes and small particles in the gastro-intestinal tract of howler monkeys probably restricts them to mixed diets but their digestive strategy is sufficiently flexible to allow them to feed on a diet of leaves when fruit is unavailable.

Whole-body protein turnover reveals the cost of detoxification of secondary metabolites in a vertebrate browser.

The detoxification limitation hypothesis predicts that the metabolism and biotransformation of plant secondary metabolites (PSMs) elicit a cost to herbivores. There have been many attempts to estimate these costs to mammalian herbivores in terms of energy, but this ignores what may be a more important cost-increases in protein turnover and concomitant losses of amino acids. We measured the effect of varying dietary protein concentrations on the ingestion of two PSMs (1,8 cineole-a monoterpene, and benzoic acid-an aromatic carboxylic acid) by common brushtail possums (Trichosurus vulpecula). The dietary protein concentration had a small effect on how much cineole possums ingested. In contrast, protein had a large effect on how much benzoate they ingested, especially at high dietary concentrations of benzoate. This prompted us to measure the effects of dietary protein and benzoate on whole-body protein turnover using the end-product method following an oral dose of [(15)N] glycine. Increasing the concentration of dietary protein in diets without PSMs improved N balance but did not influence whole-body protein turnover. In contrast, feeding benzoate in a low-protein diet pushed animals into negative N balance. The concomitant increases in the rates of whole-body protein turnover in possums eating diets with more benzoate were indicative of a protein cost of detoxification. This was about 30 % of the dietary N intake and highlights the significant effects that PSMs can have on nutrient metabolism and retention.

A faecal index of diet quality that predicts reproductive success in a marsupial folivore.

Estimating the nutritional value of a herbivore's diet is difficult because it requires knowing what the animal eats, the relative quality of each component and how these components interact in relation to animal physiology. Current methods are cumbersome and rely on many assumptions that are hard to evaluate. We describe a new method for estimating relative diet quality directly from faeces that avoids the problems inherent in other methods. We combine this method with near infrared reflectance spectroscopy (NIRS) to analyse many samples and thus provide a technique with immense value in ecological studies. The method stems from the correlation between the concentrations of dietary and faecal nitrogen in herbivores eating a tannin-free diet, but a weaker relationship in browsers that ingest substantial amounts of tannins, which form complexes with proteins. These complexes reduce the availability of nitrogen and may increase faecal nitrogen concentrations. Using the tannin-binding compound, polyethylene glycol, we showed that tannin-bound nitrogen is a significant and variable part of faecal nitrogen in wild common brushtail possums (Trichosurus vulpecula). We developed a technique to measure faecal available nitrogen and found that it predicted the reproductive success of female brushtail possums in northern Australia. Faecal available nitrogen combined with NIRS provides a powerful tool for estimating the relative nutritional value of the diets of browsing herbivores in many ecological systems. It is a better indicator of diet quality than other commonly used single-nutrient measures such as faecal nitrogen and foliage analysis paired with observed feeding behaviour.

Correlations between physical and chemical defences in plants: tradeoffs, syndromes, or just many different ways to skin a herbivorous cat?

Most plant species have a range of traits that deter herbivores. However, understanding of how different defences are related to one another is surprisingly weak. Many authors argue that defence traits trade off against one another, while others argue that they form coordinated defence syndromes. We collected a dataset of unprecedented taxonomic and geographic scope (261 species spanning 80 families, from 75 sites across the globe) to investigate relationships among four chemical and six physical defences. Five of the 45 pairwise correlations between defence traits were significant and three of these were tradeoffs. The relationship between species' overall chemical and physical defence levels was marginally nonsignificant (P = 0.08), and remained nonsignificant after accounting for phylogeny, growth form and abundance. Neither categorical principal component analysis (PCA) nor hierarchical cluster analysis supported the idea that species displayed defence syndromes. Our results do not support arguments for tradeoffs or for coordinated defence syndromes. Rather, plants display a range of combinations of defence traits. We suggest this lack of consistent defence syndromes may be adaptive, resulting from selective pressure to deploy a different combination of defences to coexisting species.

Mosaic eucalypt trees suggest genetic control at a point that influences several metabolic pathways.

Mosaic trees contain more than one phenotype. The two Eucalyptus mosaic trees studied here (E. melliodora and E. sideroxylon) are predominantly susceptible to insect herbivory, with the leaves on a single large branch on each tree resisting herbivory. We used gas chromatography-mass spectrometry and high-pressure liquid chromatography to analyze the chemical profile of leaves of the mosaic trees, as well as leaves of adjacent non-mosaic con-specifics. We show that the leaves of the two phenotypes are distinctly different. Compared to the susceptible (S) leaves on the same tree, the resistant (R) leaves on the mosaic trees had low concentrations of sesquiterpenes (E. melliodora: 2 vs. 24 mg·g(-1) dry matter; E. sideroxylon: 8 vs. 22 mg·g(-1) dry matter), high concentrations of FPCs (E. melliodora: 5.4 vs. 0.3 mg·g(-1) dry matter; E. sideroxylon: 9.8 vs. 0.2 mg·g(-1) dry matter) but similar concentrations of nitrogen (E. melliodora: 15.4 vs. 16.8 mg·g(-1) dry matter; E. sideroxylon: 13.1 vs. 14.0 mg·g(-1) dry matter). The only difference between the two mosaic trees was in the levels of monoterpenes. The R leaves from the mosaic E. melliodora contained higher concentrations of monoterpenes compared to the S leaves (12 vs. 6 mg·g(-1) dry matter). In contrast, the leaves from the E. sideroxylon mosaic contained much higher concentrations of monoterpenes with a reversed pattern (R: 26 vs. S: 45 mg·g(-1) dry matter). There were qualitative differences too on the mosaic trees. The R leaves of both species contained much higher concentrations of the monoterpene, 1,8-cineole, whereas the S chemotype of E. sideroxylon only had high concentrations of phellandrenes. Furthermore, the chemical differences between leaves on the R and S branches of the mosaic trees resembled those between the leaves of R and S con-specific trees in the same population. We use these data and knowledge of secondary metabolite biosynthesis to propose that high-level transcriptional differences may control the profile of specialized metabolites in eucalypts.

Nitrogen content, amino acid composition and digestibility of fungi from a nutritional perspective in animal mycophagy.

Fungi comprise a major part of the diet of many animals. Even so, the nutritional value of fungi has been much debated, with some arguing that fungi are nutritionally poor. However, the chemical composition of fungi and of the biology of the animals that eat them are not well understood, particularly in reference to amino acid (AA) composition of fungi and digestibility of fungal protein. We analysed fibre, total nitrogen (N), available N, and AA contents and measured in vitro digestibility of a wide range of epigeous and hypogeous fungi collected in Australia and the USA to test three hypotheses: (i) fungi are nutritionally poor because they contain few nutrients or are otherwise of low digestibility, (ii) fungi vary substantially in their nutritional composition; and (iii) animals can counter this variable quality by eating diverse taxa. Resultant data indicate many fungi are a reasonable source of AAs and digestible nitrogen. However, they vary highly between species in AA content, and the protein has a poor balance of digestible AAs. This helps explain why many mycophagous animals eat a wide array of fungi and often have digestive strategies to cope with fungi, such as foregut fermentation. Another common strategy is to supplement the diet with high quality protein, such as insect protein. Accordingly, evaluating nutritional value of fungi requires consideration of physiology of the animal species and their whole diet.

Food for folivores: nutritional explanations linking diets to population density.

Ecologists want to explain why populations of animals are not evenly distributed across landscapes and often turn to nutritional explanations. In seeking to link population attributes with food quality, they often contrast nutritionally positive traits, such as the concentration of nitrogen, against negative ones, such as fibre concentration, by using a ratio of these traits. This specific ratio has attracted attention because it sometimes correlates with the biomass of colobine primates across sites in Asia and Africa. Although empirically successful, we have identified problems with the ratio that may explain why it fails under some conditions to predict colobine biomass. First, available nitrogen, rather than total nitrogen, is nutritionally important, while the presence of tannins is the major factor reducing the availability of nitrogen in browse plant species. Second, tannin complexes inflate measures of fibre. Finally, simple ratios may be unsound statistically because they implicitly assume isometric relationships between variables. We used data on the chemical composition of plants from three continents to examine the relationships between the concentrations of nitrogen, available nitrogen, fibre and tannins in foliage and to evaluate the nitrogen to fibre ratio. Our results suggest that the ratio of the concentration of nitrogen to fibre in leaves does sometimes closely correlate with the concentration of available nitrogen. However, the ratio may give misleading results when leaves contain high concentrations of tannins. The concentration of available nitrogen, which incorporates measures of total nitrogen, dry matter digestibility and tannins, should give a better indication of the nutritional value of leaves for herbivorous mammals that can readily be extrapolated to habitats.

The molecular basis of quantitative variation in foliar secondary metabolites in Eucalyptus globulus.

Eucalyptus is characterized by high foliar concentrations of plant secondary metabolites with marked qualitative and quantitative variation within a single species. Secondary metabolites in eucalypts are important mediators of a diverse community of herbivores. We used a candidate gene approach to investigate genetic associations between 195 single nucleotide polymorphisms (SNPs) from 24 candidate genes and 33 traits related to secondary metabolites in the Tasmanian Blue Gum (Eucalyptus globulus). We discovered 37 significant associations (false discovery rate (FDR) Q < 0.05) across 11 candidate genes and 19 traits. The effects of SNPs on phenotypic variation were within the expected range (0.018 < r(2) < 0.061) for forest trees. Whereas most marker effects were nonadditive, two alleles from two consecutive genes in the methylerythritol phosphate pathway (MEP) showed additive effects. This study successfully links allelic variants to ecologically important phenotypes which can have a large impact on the entire community. It is one of very few studies to identify the genetic variants of a foundation tree that influences ecosystem function.

Putting plant resistance traits on the map: a test of the idea that plants are better defended at lower latitudes.

• It has long been believed that plant species from the tropics have higher levels of traits associated with resistance to herbivores than do species from higher latitudes. A meta-analysis recently showed that the published literature does not support this theory. However, the idea has never been tested using data gathered with consistent methods from a wide range of latitudes. • We quantified the relationship between latitude and a broad range of chemical and physical traits across 301 species from 75 sites world-wide. • Six putative resistance traits, including tannins, the concentration of lipids (an indicator of oils, waxes and resins), and leaf toughness were greater in high-latitude species. Six traits, including cyanide production and the presence of spines, were unrelated to latitude. Only ash content (an indicator of inorganic substances such as calcium oxalates and phytoliths) and the properties of species with delayed greening were higher in the tropics. • Our results do not support the hypothesis that tropical plants have higher levels of resistance traits than do plants from higher latitudes. If anything, plants have higher resistance toward the poles. The greater resistance traits of high-latitude species might be explained by the greater cost of losing a given amount of leaf tissue in low-productivity environments.

Foliage chemistry influences tree choice and landscape use of a gliding marsupial folivore.

The chemical quality of forage may determine landscape use and habitat quality for some herbivorous species. However, studies that investigate the relationship between foliar chemistry and foraging choices in wild vertebrates are rare. Petauroides volans (the greater glider) is unique among Australian marsupial folivores because it glides. It also frequently consumes foliage from both major Eucalyptus subgenera, Eucalyptus (common name "monocalypt") and Symphyomyrtus (common name "symphyomyrtle"), which differ markedly in their foliar chemistry. Such differences are thought to be a product of co-evolution that also led to guild-specific plant secondary metabolite (PSM) specialization among other marsupial eucalypt folivores. To explore whether foliar chemistry influences tree use, we analyzed foliage from eucalypt trees in which we observed P. volans during a radio tracking study and from eucalypt trees in which animals were never observed. We used a combination of chemical assays and near infrared spectrophotometry (NIRS) to determine concentrations of nitrogen (N), in vitro available nitrogen (AvailN), and in vitro digestible dry matter (DDM) from foliage sampled from the monocalypt and symphyomyrtle species, and total formylated phloroglucinol compounds (FPCs) and sideroxylonals (a class of FPCs) from the symphyomyrtle species (FPCs do not occur in monocalypts). Tree size and spatially-dependent, intraspecific variations in sideroxylonals and DDM concentrations in the symphyomyrtle foliage and of N, AvailN, and DDM in the monocalypt species were important indicators of tree use and habitat suitability for P. volans. The results i) demonstrate that guild-specific PSMs do not always lead to guild-specific foraging; ii) provide a compelling co-evolutionary case for the development of gliding in P. volans; and iii) have implications for the management and conservation of this and other folivorous species.

Palatability mapping: a koala's eye view of spatial variation in habitat quality.

Ecologists trying to understand the value of habitat to animals must first describe the value of resources contained in the habitat to animals and, second, they must describe spatial variation in resource quality at a resolution relevant to individual animal foraging. We addressed these issues in a study of koalas (Phascolarctos cinereus) in a Eucalyptus woodland. We measured beneficial and deterrent chemical characteristics as well as the palatability of trees using a near-infrared spectroscopic model based on direct feeding experiments. Tree use by koalas was influenced by tree size and foliar quality but was also context-dependent: trees were more likely to be visited if they were surrounded by small, unpalatable trees or by large, palatable trees. Spatial autocorrelation analysis and several mapping approaches demonstrated that foliar quality is spatially structured in the woodland at a scale relevant to foraging decisions by koalas and that the spatial structure is an important component of habitat quality.

Genetic and environmental contributions to variation and population divergence in a broad-spectrum foliar defence of Eucalyptus tricarpa.

BACKGROUND AND AIMS: Both environmental and genetic effects contribute to phenotypic variation within and among populations. Genetic differentiation of quantitative traits among populations has been shown in many species, yet it can also be accompanied by other genetic changes, such as divergence in phenotypic plasticity and in genetic variance. Sideroxylonal (a formylated phloroglucinol compound or FPC) is an important chemical defence in eucalypts. The effect of environmental variation on its production is a critical gap in our understanding of its genetics and evolution. METHODS: The stability of genetic variation in sideroxylonal was assessed within and among populations of Eucalyptus tricarpa in three replicated provenance/progeny trials. The covariance structure of the data was also modelled to test whether genetic variances were consistent among populations and Fain's test was applied for major gene effects. KEY RESULTS: A significant genotype x environment interaction occurred at the level of population, and was related to temperature range and seasonality in source populations. Within-population genetic variation was not affected by genotype x environment effects or different sampling years. However, within-population genetic variance for sideroxylonal concentration differed significantly among source populations. Regression of family variance on family mean suggested that this trait is subject to major gene effects, which could explain the observed differences in genetic variances among populations. CONCLUSIONS: These results highlight the importance of replicated common-garden experiments for understanding the genetic basis of population differences. Genotype x environment interactions are unlikely to impede evolution or responses to artificial selection on sideroxylonal, but the lack of genetic variation in some populations may be a constraint. The results are broadly consistent with localized selection on foliar defence and illustrate that differentiation in population means, whether due to selection or to drift, can be accompanied by changes in other characteristics, such as plasticity and genetic variance.

Acid loads induced by the detoxification of plant secondary metabolites do not limit feeding by common brushtail possums (Trichosurus vulpecula).

We fed common brushtail possums artificial diets containing a buffer and the plant secondary metabolite (PSM), orcinol, to test the hypothesis that organic acids, common products of PSM metabolism, limit feeding by common brushtail possums (Trichosurus vulpecula). We introduced several diets containing orcinol and a buffer (urinary alkalising agent) over a course of three experiments. A diet containing 2% orcinol (wet matter) caused possums to reduce their food intake immediately, but feeding returned to normal 1-2 days later. Even though possums excreted strongly acidic urine (pH 5.1) and had perturbed nitrogen metabolism, they maintained their food intake and body mass until the experiment terminated 9 days after the introduction of orcinol. Possums ate 52% less when the basal diet contained 4% orcinol. As expected, the acid loads caused a change in the composition of urinary nitrogen with possums excreting more ammonium than urea and a large amount of unidentified nitrogenous material. Supplementing the diet containing orcinol with buffer neutralised the metabolic acid load and partly restored normal nitrogen metabolism, but did not restore feeding. Also, animals eating orcinol excreted normal amounts of 3-methylhistidine, indicating no increase in muscle protein catabolism. This suggests that a limitation to the rate of detoxification or toxicosis, rather than acid loads, limits the ingestion of acid-inducing PSMs.