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Medicinal tea tree (Melaleuca alternifolia) leaves contain large amounts of an essential oil, dominated by monoterpenes. Several enzymes of the chloroplastic methylerythritol phosphate (MEP) pathway are hypothesised to act as bottlenecks to the production of monoterpenes. We investigated, whether transcript abundance of genes encoding for enzymes of the MEP pathway were correlated with foliar terpenes in M. alternifolia using a population of 48 individuals that ranged in their oil concentration from 39 -122 mg x g DM(-1). Our study shows that most genes in the MEP pathway are co-regulated and that the expression of multiple genes within the MEP pathway is correlated with oil yield. Using multiple regression analysis, variation in expression of MEP pathway genes explained 87% of variation in foliar monoterpene concentrations. The data also suggest that sesquiterpenes in M. alternifolia are synthesised, at least in part, from isopentenyl pyrophosphate originating from the plastid via the MEP pathway.

Introduction: Eucalyptus jensenii has not been explored for its phytoconstituents except for its essential oil although a formylated phloroglucinol compound jensenone has been the focus of several ecological studies., Objective: i) To optimise the extraction conditions for preparative scale isolation of jensenone and other secondary metabolites from leaves. (ii) To develop an RP-HPLC method for simultaneous determination of phloroglucinols and other compounds in Eucalyptus leaves., Methodology: Jensenone and other compounds were isolated from acetone extract using VLC over TLC grade silica. HPLC quantitation of isolated compounds was undertaken on a C₁₈-column using acetonitrile-water (2% formic acid) gradient elution., Results: Extraction conditions for isolation of jensenone were optimised and more than 99% pure jensenone was obtained in a yield of 2.1% from the dried leaves. Ten phloroglucinol compounds, including a new nuclear methylated phloroglucinol named 4-O-demethyl miniatone, and two triterpenoids were also isolated. A RP-HPLC method was developed for simultaneous quantitation of phloroglucinols and other compounds in different extracts of E. jensenii leaves. A total of 19 compounds reported from different species of Eucalyptus was separated by this method., Conclusion: The method described for isolation of jensenone is a simple, rapid and low-cost procedure amenable to the preparative scale. A new monomeric phloroglucinol compound was isolated and characterised from the acetone extract of E. jensenii leaves. The HPLC method developed can be applied successfully to different eucalypt matrices for precise and accurate determination of 19 secondary metabolites belonging to different chemical classes., (Copyright © 2012 John Wiley & Sons, Ltd.)

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.

Context: Eucalyptus has been a source of a number of biologically active compounds. The anti-leishmanial activity of terpenoids from Eucalyptus loxophleba (Benth.) ssp. lissophloia (Myrtaceae) has not yet been investigated., Objective: Isolation of the terpenoidal constituents for evaluation of in vitro anti-leishmanial activity against the Leishmania donovani (Dd8 strain) promastigotes., Materials and Methods: The chloroform-methanol (8:2) extract of dried leaves of Eucalyptus loxophleba was used to isolate terpenoidal constituents employing solvent partitioning, column chromatography and preparative high performance liquid chromatography and characterized from spectral data. The anti-leishmanial activity of the isolated compounds was tested in vitro using an Alamar blue assay against a culture of L. donovani (Dd8 strain) promastigotes., Results: Two new naturally occurring triterpenes, named loxanic acid and 3-acetyl loxanic acid together with four known ursane triterpenoids and one bis-monoterpene glycoside, cuniloside B isolated from the leaves showed anti-leishmanial activity (IC(50) 133 to 235 μM) against the promastigotes of the tested strain., Conclusion: The terpenes isolated from the leaves of E. loxophleba showed moderate anti-leishmanial activity.

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.

Two new naturally occurring formylated phloroglucinol compounds (FPCs), a dimer, loxophlebal B (10) and a cyclized FPC, loxophlebene (8) together with eight other formylated phloroglucinols (1-7 and 9) were isolated from the chloroform-methanol (8:2) extract of the leaves of Eucalyptus loxophleba ssp. lissophloia. The structures of new compounds were established by comprehensive spectral analysis and by comparison of their NMR data with those of related compounds in the literature. All the isolated compounds were evaluated for anti-leishmanial activity against promastigotes of Leishmania donovani., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A simple, rapid, accurate and selective 1H NMR spectroscopic method to detect and quantify euglobals in the leaves of Eucalyptus loxophleba ssp. lissophloia has been developed. The method allows for the estimation of total concentration of diformylphloroglucinol-monoterpene adducts, as well as the quantitation of sabinene- and alpha/beta-phellandrene-adducts, separately. The method was validated for accuracy, precision and linearity using as reference standards 2-ethyl phenol and mixtures of jensenone, a monomeric formylated phloroglucinol, and 2-ethyl phenol.

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., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

• 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., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Individual Eucalyptus trees in south-eastern Australia vary considerably in susceptibility to herbivores. On the one hand, studies with insect herbivores have suggested that variation in the concentrations of foliar monoterpenes is related to variation in susceptibility. On the other, studies with marsupial folivores have suggested that variation in the concentrations of sideroxylonals (a group of formylated phloroglucinol compounds) is responsible for variation in susceptibility. We examined relative importance of sideroxylonals and 1,8-cineole (a dominant monoterpene) in host tree selection by Christmas beetles (Anoplognathus species: Coleoptera: Scarabaeidae) by using no-choice experiments, choice/no-choice experiments, and manipulative experiments in which concentrations of sideroxylonals or 1,8-cineole were altered. We used two species of host Eucalyptus, one species of non-host Eucalyptus, and three species of non-host non-Eucalyptus trees. Leaf consumption by Christmas beetles was negatively correlated with the concentrations of sideroxylonals and 1,8-cineole. Artificial increases in the concentration of sideroxylonals or 1,8-cineole reduced leaf consumption by Christmas beetles. An artificial reduction in foliar monoterpenes had no effect on leaf consumption by the beetles when leaves contained high or very low concentrations of sideroxylonals. However, when the concentration of sideroxylonals was moderate, a reduction in the foliar monoterpenes increased leaf consumption by the beetles. Therefore, monoterpenes such as 1,8-cineole may be used as a negative cue by Christmas beetles. The pattern of food consumption on non-host Eucalyptus species and non-host non-Eucalyptus species suggest that both positive and negative cues may be used by Christmas beetles to select host trees.

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.

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.

Loxophlebal A, a new antibacterial formylated phloroglucinol was isolated from the mother liquor obtained after separation of sideroxylonals from the chloroform-methanol extract of leaves of Eucalyptus loxophleba ssp lissophloia. The structure of loxophlebal A was determined to be 3-desformyl sideroxylonal A by spectroscopic methods including 1D- and 2D-NMR. The stereochemistry of loxophlebal A was determined by chemical correlation with sideroxylonal A. This article also reports an efficient, simple and economic method for large scale isolation of sideroxylonals in a purity of >90% from the leaves of Eucalyptus loxophleba ssp lissophloia., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Melaleuca quinquenervia (Cav) S.T. Blake (broadleaf paperbark) is an Australian tree that has become a serious weed in many places around the world. Two insects Oxyops vitiosa (the melaleuca weevil), and Boreioglycaspis melaleucae (the melaleuca psyllid), which were introduced to Florida as part of a biological control programme, have been very effective in reducing survival and reproduction of this weed. There are two terpene chemotypes of M. quinquenervia; one rich in the sesquiterpene E-nerolidol whereas the other is rich in viridiflorol. Viridiflorol is a strong feeding deterrent for the melaleuca weevil and retards larval development. The larvae therefore avoid the viridiflorol-rich chemotype, in contrast, female melaleuca psyllids prefer to oviposit on these leaves. To identify the molecular basis of these preferences, we isolated and characterised two terpene synthases from the viridiflorol-rich chemotype, both of which utilise farnesyl pyrophosphate and have the same product profile. Chemotypic variation in terpenes in M. quinquenervia is under strong genetic control and the reproductive potential of each chemotype is limited by a different insect. These insects could, therefore, be selective agents for the maintenance of chemotypic variation in M. quinquenervia., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Different folivorous marsupials select their food from different subgenera of Eucalyptus, but the choices cannot be explained by known antifeedants, such as formylated phloroglucinol compounds or tannins, or by nutritional quality. Eucalypts contain a wide variety of plant secondary metabolites so it is difficult to use traditional methods to identify the chemicals that determine food selection. Therefore, we used a metabolomic approach in which we employed (1)H nuclear magnetic resonance spectroscopy to compare chemical structures of representatives from the two subgenera and to identify chemicals that consistently differ between them. We found that dichloromethane extracts of leaves from most species in the subgenus Eucalyptus differ from those in Symphyomyrtus by the presence of free flavanones, having no substitution in Ring B. Although flavanoids are known to deter feeding by certain insects, their effects on marsupials have not been established and must be tested with controlled feeding studies.

Myrtaceae is one of the chemically most variable and most significant essential oil yielding plant families. Despite an abundance of chemical information, very little work has focussed on the biochemistry of terpene production in these plants. We describe 70 unique partial terpene synthase transcripts and eight full-length cDNA clones from 21 myrtaceous species, and compare phylogenetic relationships and leaf oil composition to reveal clades defined by common function. We provide further support for the correlation between function and phylogenetic relationships by the first functional characterisation of terpene synthases from Myrtaceae: a 1,8-cineole synthase from Eucalyptus sideroxylon and a caryophyllene synthase from Eucalyptusdives., (Copyright 2010 Elsevier Ltd. All rights reserved.)

The variation of foliar monoterpenes in the Australian Tea Tree (Melaleuca alternifolia) has been of significant interest both to the essential oil industry as well as to ecologists. The majority of studies on leaf chemistry have been aimed directly towards obtaining oil of higher quality or quantity. In the current study, we aimed to understand how molecular mechanisms contribute to the chemical variability of this species, based on chemical analysis of the leaf oils from a biochemical perspective. Correlations between monoterpenes across the species as well as within chemotypes show strong, persistent patterns, which enable us to establish groups based on possible common biosynthetic origins. We found that three distinct enzymes corresponding to these groups: a sabinene-hydrate synthase, a 1,8-cineole synthase, and a terpinolene synthase may be sufficient to explain all six chemotypes in M. alternifolia.

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.

This review covers the synthetic aspects of various naturally occurring phloroglucinols, describing syntheses of 42 compounds and citing 137 references.

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.

Background: There is little information about the DNA sequence variation within and between closely related plant species. The combination of re-sequencing technologies, large-scale DNA pools and availability of reference gene sequences allowed the extensive characterisation of single nucleotide polymorphisms (SNPs) in genes of four biosynthetic pathways leading to the formation of ecologically relevant secondary metabolites in Eucalyptus. With this approach the occurrence and patterns of SNP variation for a set of genes can be compared across different species from the same genus., Results: In a single GS-FLX run, we sequenced over 103 Mbp and assembled them to approximately 50 kbp of reference sequences. An average sequencing depth of 315 reads per nucleotide site was achieved for all four eucalypt species, Eucalyptus globulus, E. nitens, E. camaldulensis and E. loxophleba. We sequenced 23 genes from 1,764 individuals and discovered 8,631 SNPs across the species, with about 1.5 times as many SNPs per kbp in the introns compared to exons. The exons of the two closely related species (E. globulus and E. nitens) had similar numbers of SNPs at synonymous and non-synonymous sites. These species also had similar levels of SNP diversity, whereas E. camaldulensis and E. loxophleba had much higher SNP diversity. Neither the pathway nor the position in the pathway influenced gene diversity. The four species share between 20 and 43% of the SNPs in these genes., Conclusion: By using conservative statistical detection methods, we were confident about the validity of each SNP. With numerous individuals sampled over the geographical range of each species, we discovered one SNP in every 33 bp for E. nitens and one in every 31 bp in E. globulus. In contrast, the more distantly related species contained more SNPs: one in every 16 bp for E. camaldulensis and one in 17 bp for E. loxophleba, which is, to the best of our knowledge, the highest frequency of SNPs described in woody plant species.

Plant secondary metabolites (PSMs) strongly influence diet selection by mammalian herbivores. Concentrations of PSMs vary within and among plant species, and across landscapes. Therefore, local adaptations may cause different populations of herbivores to differ in their ability to tolerate PSMs. Here, we tested the food intake responses of three populations of a marsupial folivore, the common brushtail possum (Trichosurus vulpecula Kerr), from different latitudes and habitat types, to four types of PSMs. We found clear variation in the responses of northern and southern Australian possums to PSMs. Brushtail possums from southern Australia showed marked decreases in food intake in response to all four PSMs, while the two populations from northern Australia were not as sensitive and their responses did not differ from one another. These results were unexpected, based on our understanding of the experiences of these populations with PSMs in the wild. Our results suggest that geographically separated populations of possums may have evolved differing abilities to cope with PSMs, as a result of local adaptation to their natural environments. Our results provide the basis for future studies to investigate the mechanisms by which populations of mammalian species differ in their ability to tolerate PSMs.

A central goal in understanding the ecology and evolution of animals is to identify factors that constrain or expand breadth of diet. Selection of diet in many animals is often constrained by chemical deterrents (i.e., secondary metabolites) in available food items. The integration of chemistry and ecology has led to a significant understanding of the chemical complexity of prey (e.g., animals, plants, and algae) and the resultant foraging behavior of consumers. However, most of the literature on chemical defenses of marine and terrestrial prey lacks a mechanistic understanding of how consumers tolerate, or avoid, chemically-defended foods. In order to understand ecological patterns of foraging and co-evolutionary relationships between prey and consumers, we must advance our understanding of the physiological mechanisms responsible for chemical interactions. Such mechanistic studies require the integration of the discipline of pharmacology with ecology, which we call "PharmEcology." Pharmacology provides the tools and insight to investigate the fate (what the body does to a chemical) and action (what a chemical does to the body) of chemicals in living organisms, whereas ecology provides the insight into the interactions between organisms (e.g., herbivores) and their environment (e.g., plants). Although, the general concepts of pharmacology were introduced to ecologists studying plant-herbivore interactions over 30 years ago, the empirical use of pharmacology to understand mechanisms of chemical interactions has remained limited. Moreover, many of the recent biochemical, molecular and technical advances in pharmacology have yet to be utilized by ecologists. The PharmEcology symposium held at a meeting of the Society for Integrative and Comparative Biology in January of 2009 was developed to define novel research directions at the interface of pharmacology and ecology.

Background: Phloroglucinol compounds, both synthetic as well as natural, have shown a vast array of biological activities. There are a wide range of applications of phloroglucinol compounds in pharmaceuticals, cosmetics, textiles, paints and dyeing industries. Although many of the phloroglucinols have shown promising results in various biological assays, very few have reached clinics., Objective: To compile the patented information on various therapeutically active phloroglucinol molecules, so that technologies used in isolation and activity assessment of these compounds could be unearthed and the compiled information be utilized for further development of these molecules., Methods: The European Patent Office database (official website: espacenet.com) was searched with a keyword "phloroglucinol". In addition, patents were searched using names of compounds listed in our previous review., Conclusions: This class holds potential for development of molecules in various therapeutic areas. There exist a number of patents on preparations that have phloroglucinol compounds as active ingredient(s). Many such preparations have been tested in vitro and/or in vivo for their efficacy and proven to be active and non-toxic. Commercialization of existing technology on phloroglucinol molecules can yield fruitful results.

Plants contain a variety of chemical defenses that strongly affect feeding rates in captive mammals, but their effects on the fitness of wild herbivores are largely unknown. This is because the complexity of defensive compounds, and herbivores' counteradaptations to them, make their effects in the wild difficult to measure. We show how tannins interact with protein to produce spatial variation in the nutritional quality of eucalypt foliage, which is related to demography in a wild population of a marsupial folivore, the common brushtail possum (Trichosurus vulpecula Kerr). Tannins reduced the digestibility of nitrogen (N) in vitro, creating variation in available N concentrations among the home ranges of individual possums in an otherwise homogeneous habitat. This was strongly correlated with reproductive success: females with better quality trees in their home range reproduced more often and had faster-growing offspring. These results demonstrate a powerful mechanism by which spatial variation in plant chemistry may control herbivore population dynamics in nature.

The up-regulation of secondary metabolic pathways following herbivore attack and the subsequent reduction in herbivore performance have been identified in numerous woody plant species. Eucalypts constitutively express many secondary metabolites in the leaves, including terpenes and formylated phloroglucinol compounds (FPCs). We used clonal ramets from six clones of Eucalyptus grandis and two clones of E. grandis x camaldulensis to determine if methyl jasmonate (MeJA) treatment could induce changes in the foliar concentrations of either of these groups of compounds. We also used bioassays to determine if any changes in the performance of larvae of Paropsis atomaria, a chrysomelid leaf beetle, could be detected in treated ramets versus the untreated controls, thus indicating whether MeJA induced the up-regulation of defences other than terpenes or FPCs. We found no significant effects of MeJA treatment on either the foliar concentrations of terpenes and FPCs or on herbivore performance. We did, however, detect dramatic differences in larval performance between Eucalyptus clones, thereby demonstrating large variations in the levels of constitutive defence. Larval feeding on clones resistant to P. atomaria resulted in high first instar mortality and disruption of normal gregarious feeding behaviour in surviving larvae. Histological examination of larvae feeding on a resistant clone revealed damage to the midgut consistent with the action of a toxin. These findings concur with mounting evidence that most evergreen perennial plants lack foliar-induced defences and suggest that constitutively expressed secondary metabolites other than those commonly examined in studies of interactions between insect herbivores and Eucalyptus may be important in plant defence.

Many regard the concentrations of nitrogen (N), tannins and plant cell wall constituents (fibre) as key indicators of food quality and habitat suitability for browsing herbivores; yet there is no method for measuring their combined effects. We have developed a simple in vitro assay for measuring the effects of tannins and fibre on N availability in browse. We determined the effects of tannins by measuring the polyethylene glycol (PEG)-binding capacity (PEG-BC) of Eucalyptus leaf samples, followed by a two-stage in vitro digestion with pepsin and cellulase to determine the digestibility of dry matter and N. There was a significant relationship between concentrations of digestible N and the PEG-BC of the leaves. Furthermore, adding PEG significantly improved the digestibility of N. Our results concur with in vivo observations from several mammalian species. This suggests that our method is effective for measuring the nutritional quality of browse and the benefits of adding PEG, providing some index of the detrimental effects of tannins. We further simplified the assay by removing the PEG step, allowing us to quickly analyse samples in bulk. Nevertheless, this simplified method is still not practical for analysing the many samples necessary to compare the nutritional values of different tracts of forest. We used near-infrared reflectance spectroscopy to produce calibration equations and predicted total and digestible N in 322 trees at eleven sites. Both within and between sites, we found a wide variation in concentrations of digestible N but a much lower variation in total N, with either no relationship or poor relationships between the two measures. This confirms the variability in the nutritional quality of eucalypt forests, which may explain the distribution and abundance of mammalian herbivores. Thus, our assay provides a useful tool for understanding how food resources influence herbivore populations at different scales.

In a given period of time, herbivores often eat less as dietary plant secondary metabolite (PSM) concentrations increase. This reduction in total food intake is interpreted as a need for the herbivore to regulate PSM ingestion in order to avoid toxication. However, regulation of PSM ingestion involves more than the reduction of total intake; it involves an alteration of meal patterns, through a reduction in the number and/or the size of the meals eaten. Despite this, studies of how herbivores alter their meal patterns when offered varying concentrations of PSMs are rare. We investigated whether koalas adjust the number and/or the size of their meals when offered eucalypt foliage varying naturally in concentrations of formylated phloroglucinol compounds (FPCs), a group of PSMs that have previously been shown to inhibit total food intake. High FPC concentrations caused koalas to eat more slowly, eat shorter meals and eat less per meal, which resulted in a reduced total intake. Because increasing FPC concentrations did not cause koalas to alter the number of meals that they ate, clear individual differences between koalas were observed, where some consistently ate fewer larger meals and others ate many smaller meals. Thus, different feeding strategies may still achieve the same outcome of a regulated intake of PSMs. The changes observed match the meal patterns of other herbivores ingesting PSMs known to stimulate nausea and emetic pathways, supporting the idea that feedback signals from nausea are an important way that koalas avoid toxication when eating eucalypt foliage.

Plants encounter a broad range of natural enemies and defend themselves in diverse ways. The cost of defense can be reduced if a plant secondary metabolite confers resistance to multiple herbivores. However, there are few examples of positively correlated defenses in plants against herbivores of different types. We present evidence that a genetically variable chemical trait that acts as a strong antifeedant to mammalian herbivores of Eucalyptus also deters insect herbivores, suggesting a possible mechanism for cross-resistance. We provide field confirmation that sideroxylonal, an important antifeedant for mammalian herbivores, also determines patterns of damage by Christmas beetles, a specialist insect herbivore of Eucalyptus. In a genetic progeny trial of Eucalyptus tricarpa, we found significant heritabilities of sideroxylonal concentration (0.60), overall insect damage (0.34), and growth traits (0.30-0.53). Population of origin also had a strong effect on each trait. Negative phenotypic correlations were observed between sideroxylonal and damage, and between damage and growth. No relationship was observed between sideroxylonal concentration and any growth trait. Our results suggest that potential for evolution by natural selection of sideroxylonal concentrations is not strongly constrained by growth costs and that both growth and defense traits can be successfully incorporated into breeding programs for plantation trees.

Exploring the spatial distribution of variation in plant secondary metabolites is critical for understanding the evolutionary ecology of biochemical diversity in wild organisms. In the present study, concentrations of foliar sideroxylonal, an important and highly heritable defense chemical of Eucalyptus melliodora, displayed strong, fine-scale spatial autocorrelation. The spatial patterns observed could promote associational effects on herbivore foraging decisions, which may influence the selection pressures exerted on sideroxylonal content. Multiple chemical traits have roles in certain eucalypt-herbivore interactions, and the spatial characteristics of the herbivore foraging environment are therefore determined by these different factors. We used a model of E. melliodora intake by common brushtail possums (Trichosurus vulpecula), based on the combined effects of two chemical traits, to explore this idea and found that the spatial patterns were different to those of sideroxylonal alone. Spatial genetic autocorrelation, examined using microsatellites, was strong and occurred at a fine scale, implying that restricted gene flow might allow genetic patches to respond to selection relatively independently. Local two-dimensional genetic autocorrelation, explored using a new heuristic method, was highly congruent with the pattern of local phenotypic variation observed for sideroxylonal, suggesting that the genetic variance underlying the sideroxylonal variation is similarly structured. Our results suggest that the spatial distribution of genetic and phenotypic variation could influence both the selective pressure imposed by herbivores on eucalypt defenses and the potential of populations to respond to natural selection. Spatial context should be considered in future studies of plant-herbivore interactions.

Leaves of eucalypt species contain a variety of plant secondary metabolites, including terpenoids and formylated phloroglucinol compounds (FPCs). Both terpene and FPC concentrations are quantitative traits that can show large variation within a population and have been shown to be heritable. The molecular genetic basis of this variation is currently unknown. Progeny from a field trial of a three-generation mapping pedigree of Eucalyptus nitens were assayed for terpenes and FPCs. Quantitative trait loci (QTL) analyses were conducted using a map constructed from 296 markers to locate regions of the genome influencing foliar concentrations of these plant secondary compounds. A large number of significant QTL for 14 traits were located across nine linkage groups, with significant clustering of QTL on linkage groups 7, 8 and 9. As expected, QTL for biosynthetically related compounds commonly colocated, but QTL for unrelated monterpenes and FPCs also mapped closely together. Colocation of these QTL with mapped candidate genes from the various biosynthetic pathways, and subsequent use of these genes in association mapping, will assist in determining the causes of variation in plant secondary metabolites in eucalypts.

Grazing by dugongs and cropping by green turtles have the capacity to alter the subsequent nutritional quality of seagrass regrowth. We examined the effects of simulated light and intensive grazing by dugongs and cropping by turtles on eight nutritionally relevant measures of seagrass chemical composition over two regrowth periods (short-term, 1-4 months; long-term, 11-13 months) at two seagrass communities (a mixed species community with Zostera capricorni, Halophila ovalis, Halodule uninervis, Cymodocea rotundata and C. serrulate; and a monospecific bed of Halodule uninervis) in tropical Queensland, Australia. The concentrations of organic matter, total nitrogen, total water-soluble carbohydrates, total starch, neutral detergent fiber, acid detergent fiber, acid lignin, as well as the in vitro dry matter digestibility (IVDMD) were measured in the leaves and below-ground parts of each species using near-infrared reflectance spectroscopy (NIRS). Regrowth of preferred species such as H. ovalis and H. uninervis from simulated intensive dugong grazing after a year exhibited increased (by 35 and 25%, respectively, relative to controls) whole-plant N concentrations. Similarly, regrowth of H. ovalis from simulated turtle cropping showed an increase in the leaf N concentration of 30% after a year. However, these gains are tempered by reductions in starch concentrations and increases in fiber. In the short-term, the N concentrations increased while the fiber concentrations decreased. These data provide experimental support for a grazing optimization view of herbivory in the tropical seagrass system, but with feedback in a different manner. Furthermore, we suggest that in areas where grazing is the only major source of natural disturbance, it is likely that there are potential ecosystem level effects if and when numbers of dugongs and turtles are reduced.

Most herbivores eat more and survive better when they have access to a variety of foods. One explanation involves the detoxification of plant secondary metabolites (PSMs). By feeding from a variety of plants that contain different classes of PSMs, animals can use multiple detoxification pathways and presumably consume more food. Although popular, this theory is difficult to test because it requires knowledge of the detoxification pathways of each PSM in the diet. We established that common brushtail possums (Trichosurus vulpecula) use various combinations of oxidation, hydrolysis, and conjugation with glucuronic acid (GA) or glycine to detoxify six PSMs. Compared to their ingestion of a single PSM, possums ate more when offered a choice between two diets containing PSMs that require apparently independent detoxification pathways (benzoate and 1,8-cineole, benzoate and p-cymene, benzoate and orcinol, benzoate and salicin, or orcinol and 1,8-cineole). However, possums still did not eat as much of these diets as they did of a basal diet free of PSMs. This suggests that detoxification pathways are never independent, but are separated instead by degrees. In contrast, possums offered a choice of two PSMs that require competing detoxification pathways (1,8-cineole and p-cymene, 1,8-cineole and salicin, or orcinol and salicin) ate no more than when offered diets containing one of the compounds. There was an exception: even though both rutin and orcinol are detoxified via conjugation with GA, the feeding behavior of possums did not suggest competition for detoxification pathways. This implies that the supply of GA is not limiting. This study provides the first convincing evidence that herbivorous mammals can eat more by selecting mixed diets with a diversity of PSMs that make full use of their detoxification potential. It also emphasizes that other behavioral and physiological factors, such as transient food aversions, influence feeding behavior.

The detoxification limitation hypothesis is firmly entrenched in the literature to explain various aspects of the interaction between herbivores and plant toxins. These include explanations for the existence of specialist and generalist herbivores and for the prevalence of each of these. The hypothesis suggests that the ability of mammalian herbivores to eliminate plant secondary metabolites (PSMs) largely determines which plants, and how much, they can eat. The value of the hypothesis is that it provides a clear framework for understanding how plant toxins might limit diet breadth. Thus, it is surprising, given its popularity, that there are few studies that provide experimental support either for or against the detoxification limitation hypothesis. There are two likely reasons for this. First, Freeland and Janzen did not formally propose the hypothesis, although it is implicit in their paper. Second, it is a difficult hypothesis to test, requiring an understanding of the metabolic pathways that lead to toxin elimination. Recent attempts to test the hypothesis appear promising. Results suggest that herbivores can recognize mounting saturation of a detoxification pathway and adjust their feeding accordingly to avoid intoxication. One strategy they use is to ingest a food containing a toxin that is metabolized by a different pathway. This demonstrates that careful selection of food plants is a key to existing in a chemically complex environment. As more studies characterize the detoxification products of PSMs, we will better understand how widespread this phenomenon is.

Plant secondary metabolites (PSMs) offer plants chemical defences against herbivores, and are known to influence intake and diet choice in both insect and mammalian herbivores. However, there is limited knowledge regarding how PSMs influence herbivore foraging decisions. Herbivore foraging decisions, in turn, directly impact on which individual plants, and plant species, are selected for consumption. We took advantage of the natural variation in sideroxylonal concentrations in the foliage of Eucalyptus melliodora (Cunn. ex Schauer) to investigate feeding patterns of a marsupial folivore, the common ringtail possum, Pseudocheirus peregrinus (Boddaert 1785). Foliage, collected from six trees, contained between 0.32 and 12.97 mg g-DM(-1) sideroxylonal. With increasing sideroxylonal concentrations, possums decreased their total intake, rate of intake and intake per feeding bout, and increased their cumulative time spent feeding. Possums did not alter their total feeding time, number of feeding bouts or time per feeding bout in response to increasing sideroxylonal concentrations. Results demonstrate important behavioural changes in foraging patterns in response to sideroxylonal. These behavioural changes have important implications, in relation to altered foraging efficiency and potential predation risk, for herbivores foraging in the field. As a result, the spatial distribution of dietary PSMs across a landscape may directly influence herbivore fitness, and ultimately habitat selection of mammalian herbivores.

Marker-based methods for estimating heritability and genetic correlation in the wild have attracted interest because traditional methods may be impractical or introduce bias via G x E effects, mating system variation, and sampling effects. However, they have not been widely used, especially in plants. A regression-based approach, which uses a continuous measure of genetic relatedness, promises to be particularly appropriate for use in plants with mixed-mating systems and overlapping generations. Using this method, we found significant narrow-sense heritability of foliar defense chemicals in a natural population of Eucalyptus melliodora. We also demonstrated a genetic basis for the phenotypic correlation underlying an ecological example of conditioned flavor aversion involving different biosynthetic pathways. Our results revealed that heritability estimates depend on the spatial scale of the analysis in a way that offers insight into the distribution of genetic and environmental variance. This study is the first to successfully use a marker-based method to measure quantitative genetic parameters in a tree. We suggest that this method will prove to be a useful tool in other studies and offer some recommendations for future applications of the method.

Plant secondary metabolites can constrain the diet of vertebrates and these effects can flow through to community dynamics. Recent studies have moved beyond attempting to correlate diet choice with secondary metabolite profiles and instead focus on mechanisms that animals use to detect toxins and to regulate their intake and absorption. These include molecularly determined taste specificity, serotonin-mediated learning and the control of toxin absorption by permeability-glycoproteins. Focus on the detoxification pathways employed by specialist and generalist herbivores has facilitated explicit tests of the long-standing hypothesis that detoxification rates limit feeding. Understanding the molecular basis of differences amongst species in their tolerance of plant secondary metabolites opens many opportunities for understanding the evolutionary history of interactions between vertebrates and their food plants.

Although defence against herbivores is often argued to be the main action of plant secondary metabolites (PSMs), very few examples have demonstrated that intraspecific variation in PSM concentrations influences foraging by wild vertebrate herbivores. Experiments with captive animals often indicate that PSM concentrations influence how much herbivores eat from individual plants, but these experiments do not replicate the subtle trade-offs in diet selection faced by wild animals, which must avoid predators and extremes of weather, interact with conspecifics, and achieve a balanced, nutritious diet, while avoiding intoxication by PSMs. We characterized the foliar chemistry of every tree from two Eucalyptus species available to a population of koalas (Phascolarctos cinereus) and considered rates of tree visitation over a ten-year period. We show that visitation rate was most strongly influenced by tree size, but that koalas also visited trees less frequently if the foliage contained either high concentrations of deterrent PSMs known as formylated phloroglucinol compounds, or low concentrations of nitrogen. Consequently, plant chemistry restricts the use of trees by this herbivore, and thus limits the food available to koalas and potentially influences koala populations.

The koala is the quintessential specialist herbivore, feeding almost exclusively on Eucalyptus foliage. Consequently, the limitations imposed on the koala's diet by plant defences indicate the extent to which evolutionary adaptations allow mammalian herbivores to circumvent such defences. We tested whether a recently discovered group of plant secondary metabolites, the formylated phloroglucinol compounds (FPCs), deters koalas from feeding on some eucalypt foliage. We found that captive koalas ate less foliage in a single night from trees with high FPC concentrations. Individual trees also differ in the types of FPC they possess, but for a given eucalypt species, most FPCs were similarly effective deterrents. Two closely related and sympatric eucalypt species could be clearly separated by the amounts that koalas ate from each; however, this difference could not be explained by total FPC concentrations alone. We suggest, that in this case, the presence of a distinct type of FPC deters koala herbivory on the less palatable species, and may have facilitated the evolutionary divergence of these species. We conclude that plant defences probably play an important role in determining the distribution and abundance of koalas.

A rapid method is described for the quantification of sideroxylonals, a group of formylated phloroglucinol compounds found in some eucalypts. Samples of dry, ground foliage were extracted by sonication with 20% methanol in acetonitrile, 7% water in acetonitrile or 40% water in acetonitrile and the extracts analysed by reversed phase HPLC. The extracts from the two water-acetonitrile extractions were stable for at least 48 h. All three sonication methods recovered more sideroxylonals than did the Soxhlet extraction with petroleum spirit and acetone. Adding 0.1% trifluoracetic acid to the water-acetonitrile extraction solvents led to even higher recoveries of sideroxylonals. Soaking the sample in extracting solvent for 5 min recovered 70% of the sideroxylonals, whilst sonicating the suspension for 1 min recovered the remainder. The developed method involving sonication of the sample for 5 min in 7% water in acetonitrile with 0.1% trifluoroacetic acid is fast and requires minimal equipment and solvents compared with the traditional methods. With an autosampler it is possible to prepare and run 100 samples a day. More importantly, the technique is ideal for the analysis of small samples, e.g. individual leaves, which is essential when studying the evolutionary ecology of eucalypts.

Formylated phloroglucinol compounds (FPCs) are the single most important factor determining the amount of foliage that marsupial folivores eat from individual Eucalyptus trees. Folivores need to recognize which trees contain FPCs if they are to avoid them and forage efficiently, they are challenged by great diversity in the types and quantities of FPCs present, even within eucalypt species. We investigated the relationship between FPCs and terpenoids in species with both simple and complex FPC profiles and found strong positive correlations between terpenes generally, and several monoterpenes in particular, and FPCs. Terpene cues also indicated qualitative differences in trees' FPC profiles. We describe significant qualitative and quantitative variation in FPCs in several species that are important food sources for marsupial folivores. New discoveries include the fact that macrocarpals occur as two major, distinct groups and several new dimeric acylphloroglucinols from Eucalyptus strzeleckii. These patterns add to the chemical complexity of the foraging environment for folivores.

The resistance of Eucalyptus to browsing mammals has been related to the level and type of formylated phloroglucinol compounds (FPCs) present in the leaf. The antifeedant activity of FPCs appears to depend on their aldehyde groups, but little else is known of their mode of action. We have sought to elucidate this further by examining the biological reactivity and disposition of jensenone, a model FPC. Neither jensenone nor any metabolites were detected in urine or feces of marsupial brushtail or ringtail possums that had ingested up to 725 mg x kg(-0.75). When jensenone was incubated in rat gastrointestinal segments in vitro, it rapidly disappeared. Jensenone also reacted rapidly with glutathione, cysteine, glycine, ethanolamine, and trypsin, and more slowly with acetylcysteine and albumin. Sideroxylonal, a more complex FPC, exhibited the same reactivity. Torquatone, a related compound that lacks both aldehyde groups and antifeedant activity, was unreactive. Mass spectroscopic analysis indicated that the adducts were Schiff bases formed between the aldehyde groups of FPCs and amine groups of the conjugating molecules. Successive adducts were formed with the two aldehyde groups of jensenone, and the four groups of sideroxylonal. The jensenone bis-glutathione adduct appeared to cyclize to the disulfide form. These findings suggest that the antifeedant effects of FPCs are due to their facile binding to amine groups on critical molecules in the gastrointestinal tract, leading to a loss of metabolic function. The consequent toxic reaction, probably involving chemical mediators such as 5-hydroxytryptamine (5HT), may cause colic, nausea, and a general malaise, resulting in anorexia.

This paper describes the extraction and quantification of sideroxylonals, a group of formylated phloroglucinol compounds found in the foliage of some eucalypt species. Samples of dry, ground foliage were Soxhlet-extracted with light petroleum spirit:acetone (4:1) and the resultant extract analysed (in the presence of internal standard) by reversed-phase HPLC without further purification. The yield of sideroxylonals was exponential with time and showed an inflection at ca. 4 h of extraction. It is recommended that samples be extracted for 6 h, giving a 92% recovery of the sideroxylonals. The title compounds deteriorate under various conditions, e.g. 10% are lost when foliage is oven-dried at 40 degrees C compared to freeze-drying. Storing samples in mobile phase led to a slow deterioration of sideroxylonals with a 7% loss after 4 days, while 22% of these compounds were lost from dry, ground eucalypt leaf stored at room temperature for 20 months.

Glucuronuria is normal in marsupial folivores such as the koala (Phascolarrctos cinereus), which excretes 2-3 g glucuronic acid daily. Although this has long been attributed to the metabolites of Eucalyptus terpenes, we have found that these are mostly excreted in the unconjugated form. We now report on the aglycones that account for most of the glucuronic acid in koala urine. Urine (24 hr) was collected from six male koalas (8.8 +/- 0.4 kg, mean +/- SE) that were maintained on E. cephalocarpa foliage. Urine samples were analyzed by liquid and gas chromatography (LC and GC) coupled with mass spectrometry (MS). Glucuronides were readily identified by LC-MS/MS, which generated characteristic product ions at m/z 113 and 175. From the corresponding parent glucuronide ions, the masses of the aglycones were calculated. Confirmation of identity was by GC-MS after hydrolysis with beta-glucuronidase and comparison with standard compounds. Quantitation was by GC. The major non-terpene aglycones were 4-methylcatechol, resorcinol, salicyl alcohol, and two unidentified C7H8O2 phenols. Smaller amounts of benzoic acid, benzyl alcohol, orcinol, p-cresol, phenol, and phloroglucinol were detected. We have previously reported that terpene metabolites account for about 10% urinary glucuronides in the same koalas fed E. cephalocarpa. The present study found that an additional 60% urinary glucuronic acid is conjugated with non-terpene, mainly phenolic, aglycones. It seems likely that these phenolic compounds are present in leaves as glycosides and are chiefly responsible for the glucuronuria in koalas.

The effect of two plant secondary metabolites, tannins and formylated phloroglucinol compounds (FPCs), on the intake of Eucalyptus foliage by common ringtail (Pseudocheirus peregrinus) and common brushtail possums (Trichosurus vulpecula) was studied. We manipulated the amount of tannin that was free to bind with protein by coating foliage with polyethylene glycol 4000 (PEG) and relied on natural intraspecific variation in FPC concentrations. In contrast to ringtail possums, brushtail possums showed a greater tolerance to FPCs and ate more foliage when it was coated with PEG, suggesting that tannins limited their food intake. Brushtails detected the effects of tannins through immediate oral sensations rather than through systemic effects. Ringtail possums appeared highly tolerant of foliar tannins yet susceptible to low concentrations of FPCs. We could not detect any interaction between tannins and FPCs that affected the intake of Eucalyptus foliage by either species of possum. Although ringtail and brushtail possums are widely regarded as specialist and generalist folivores, respectively, their differential susceptibility to co-occurring secondary metabolites suggests greater complexity. Each possum species appears to be a specialist in its own right, which leads to a partitioning of available foliage. Brushtails avoid tannins and ringtails avoid FPCs.

In this study of the amino acid nutrition of a marsupial we tested three hypotheses: (a) that brushtail possums eat less when diets contain excesses or deficiencies in essential amino acids, (b) that brushtail possums choose diets that do not contain amino acid excesses, and (c) that amino acid consumption is mediated partly by the 5HT3 receptor. Possums ate less when 0.2-1.0% methionine (wet matter) was added to the diet, but similar concentrations of lysine and threonine had little effect. However, when given a choice, possums always selected the basal ration over one with added lysine, methionine or threonine at concentrations between 0.05% and 0.9%. In contrast to the experiments with excess amino acids, possums did not eat less of a diet almost devoid of an essential amino acid. Instead, the possums ate less when their diets contained synthetic amino acids rather than similar amounts and proportions of amino acids as casein. Contrary to the third hypothesis, the 5HT3 receptor antagonist, ondansetron, did not affect feeding by possums given a diet containing 0.8% methionine, suggesting that post-ingestive feedback, via the 5HT3 receptor, does not regulate amino acid intake when diets contain amino acid excesses.

We investigated the utility of near-infrared reflectance spectroscopy (NIRS) as a means of rapidly assaying chemical constituents of Eucalyptus leaves and of directly predicting the intake of foliage from individual trees by greater gliders (Petauroides volans) and common ringtail possums (Pseudocheirus peregrinus). The concentrations of total nitrogen, neutral detergent fiber, condensed tannins and total phenolics could be predicted accurately by partial least squares regression models relating the near-infrared reflectance spectra of foliage samples to analyses performed using standard laboratory procedures. Coefficients of determination (r 2 ) for all four constituents ranged between 0.88 and 0.98, and standard errors of prediction between 0.80 mg g -1 dry matter (DM) for total nitrogen and 5.14 quebracho equivalents g -1 DM for condensed tannins. Near-infrared spectral-based models of food intake had r 2 values of 0.90 and 0.95 with a standard error of prediction of 3.4 and 8.3 g DM kg -0.75  day -1 for greater gliders and common ringtail possums respectively. We used the predictive model of food intake for greater gliders to examine the relationship between leaf palatability and documented food preferences of animals in the wild. Ranked differences in leaf palatability across four Eucalyptus species were consistent with documented food preferences of greater gliders in the wild. We conclude that NIRS provides a powerful tool to predict foraging behaviour of herbivores where forage choices are determined by compositional attributes of food.

Salicin was administered orally to six brushtail possums by incorporation in food for six days at three dose levels (0.05, 0.5, and 1.5% wet weight), giving mean +/- SD daily intakes of 0.31 +/- 0.09, 2.76 +/- 0.75, and 6.04 +/- 1.12 mmol salicin. Metabolites were identified by mass spectrometry and assayed by HPLC. Salicyl alcohol glucuronide accounted for 56-64% of urinary metabolites over the three doses, salicyluric acid 15-26%, salicin 10-18%, and there were smaller amounts of free (2-4%) and conjugated (0-6%) salicylic acid. beta,2-Dihydroxyphenylpropionic acid was a minor metabolite. The hydrolysis of dietary salicin enabled reconjugation of its aglycone, salicyl alcohol, with a more polar sugar, glucuronic acid, thus enhancing its renal excretion and resulting in little net loss of substrates for conjugation and a low measurable metabolic cost of excretion.

Formylated phloroglucinol compounds (FPCs) in Eucalyptus leaves are important determinants of feeding in marsupial folivores and have a wide range of other biological actions. We conducted a survey of the occurrence of formylated phloroglucinol compounds (euglobals, macrocarpals and sideroxylonals) in acetone-petrol extracts of 41 species of Eucalyptus from among seven informal subgenera growing on the East Coast of Australia. We used electrospray ionisation, Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTMS) to search crude extracts of eucalypt leaves for molecular weights characteristic of FPC compounds. We found masses characteristic of reported FPCs in 27 of the 41 species examined. The most frequently identified group of compounds was the sideroxylonals. Notable was the lack of known FPCs in the informal subgenus Monocalyptus.

The use of fruit-based diets by a small arboreal marsupial, the woolly opossum (Caluromys philander) was studied to elucidate the mechanisms used to compensate for a low dietary protein concentration. The passage of a liquid phase digesta marker (Cr-EDTA) through the gut was significantly faster when animals were fed a diet containing 0.45% N compared to that measured on a diet containing 0.90% N. The size of the gut of the two groups was similar except that the caecum of animals fed 0.45% N was significantly larger than in those animals fed 0.90% N. Animals fed a diet of 0.45% N ate significantly more food than those fed higher levels of nitrogen but there was no significant difference in the dry matter digestibility of the diet. The maintenance nitrogen requirement of the animals was 176 mg dietary N or 146 mg truly digestible nitrogen per kg metabolic body mass, with low losses of non-dietary faecal nitrogen being notable. There was no significant difference between diets in any measured parameter of urea metabolism and all animals recycled between 60% and 80% of the endogenously synthesised urea.