Ben D. Moore

Tree use by koalas in a chemically complex landscape

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.

Explaining intraspecific diversity in plant secondary metabolites in an ecological context.

Plant secondary metabolites (PSMs) are ubiquitous in plants and play many ecological roles. Each compound can vary in presence and/or quantity, and the composition of the mixture of chemicals can vary, such that chemodiversity can be partitioned within and among individuals. Plant ontogeny and environmental and genetic variation are recognized as sources of chemical variation, but recent advances in understanding the molecular basis of variation may allow the future deployment of isogenic mutants to test the specific adaptive function of variation in PSMs. An important consequence of high intraspecific variation is the capacity to evolve rapidly. It is becoming increasingly clear that trait variance linked to both macro- and micro-environmental variation can also evolve and may respond more strongly to selection than mean trait values. This research, which is in its infancy in plants, highlights what could be a missing piece of the picture of PSM evolution. PSM polymorphisms are probably maintained by multiple selective forces acting across many spatial and temporal scales, but convincing examples that recognize the diversity of plant population structures are rare. We describe how diversity can be inherently beneficial for plants and suggest fruitful avenues for future research to untangle the causes and consequences of intraspecific variation.

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.

Eucalyptus foliar chemistry explains selective feeding by koalas.

The koala is the quintessential specialist herbivore, feeding almost exclusively on Eucalyptus foliage. Consequently, the limitations imposed on the koalas 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 simple, integrative assay to quantify nutritional quality of browses for herbivores

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.

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.

FOLIAR NUTRITION, SITE QUALITY, AND TEMPERATURE INFLUENCE FOLIAR CHEMISTRY OF TALLOWWOOD (EUCALYPTUS MICROCORYS)

The distribution of marsupial folivores in Australian eucalypt forests is extremely patchy and, although distribution patterns are known to be partly determined by nutritional factors, the role of plant secondary metabolites (PSMs) has been little studied. We investigated variation in the foliar concentrations of five constituents: total phenolics (TP); the foliar component bound by polyethylene glycol (PEGBC); the formylphloroglucinol compound, sideroxylonal (a strong deterrent of mammalian herbivory); the terpene, cineole; and nitrogen (N). These constituents were analyzed using near infrared spectroscopy in 501 individual tallowwood (Eucalyptus microcorys) trees at 42 sites across a broad geographic range in eastern Australia. We tested three hypotheses: (1) trees with lower foliar N produce higher PSM concentrations; (2) PSM concentrations are greater at colder sites; and (3) PSM concentrations are higher at sites with low resource availability. Hypothesis 1 was true for all PSMs considered and was the result of variation that occurred within sites. This is consistent with the carbon/nutrient balance hypothesis and with the hypothesis that PSMs protect leaves from photodamage. Hypothesis 2 was upheld for all constituents. This was consistent with both the growth–differentiation balance hypothesis, and with the hypothesis that PSMs represent an evolutionary adaptation to greater risks of photodamage and frost damage in cold environments. Hypothesis 3 was rejected, with one PSM, sideroxylonal, being affected by resource availability in a manner opposite to that predicted. The optimal defense hypothesis offers an explanation for this result (as sideroxylonal concentrations increased along with the apparent nutritional quality of the foliage, possibly to provide defense against higher rates of herbivory). We found that trees associated with koala fecal pellets had lower concentrations of sideroxylonal and cineole. We discuss this finding and further implications for the herbivores of Eucalyptus of the patterns of plant defense that we detected.

Metabolites of dietary 1,8-cineole in the male koala (Phascolarctos cinereus).

The in vivo metabolic fate of 1,8-cineole was investigated in six male koalas. Koalas were fed ad lib a diet of Eucalyptus cephalocarpa leaf with a 1,8-cineole concentration of 2.53+/-0.70% dry mass of leaf, corresponding to a 1,8-cineole intake of 2.4+/-1.1 mmol/kg (3.1+/-1.3 g). Urine and faeces were collected for 24 h and metabolites identified by GC-MS and LC-MS. Metabolites were quantified before and after hydrolysis with beta-glucuronidase to give free and total levels, respectively. Fractional recovery of ingested 1,8-cineole was 1.3+/-0.4 and 1.4+/-0.4 (mean+/-S.D.) for free and total measurements, respectively. Seven metabolites were identified and quantified: 9- and 7-hydroxycineole, 9- and 7-cineolic acid, 7-hydroxy-9-cineolic acid, 9-hydroxy-7-cineolic acid and 7,9-dicineolic acid. The hydroxycineolic acids dominated the metabolite profile (85%). 7,9-Dicineolic acid, a novel metabolite of 1,8-cineole, accounted for almost 10% of the recovered dose making it the second most abundant metabolite after 7-hydroxy-9-cineolic acid (77%). Together, the less oxidised metabolites, the hydroxycineoles and cineolic acid, accounted for only 5% of the cineole consumed. Significant conjugation only occurred with four minor, less oxidised, alcohol and carboxylic acid metabolites. We have shown that the koala detoxifies and eliminates 1,8-cineole primarily by extensive oxidation without utilising conjugation pathways.

FATE OF THE DIETARY TERPENE, p-CYMENE, IN THE MALE KOALA

The fate of the dietary terpene, p-cymene, is reported in a specialist Eucalyptus leaf folivore, the koala (Phascolarctos cinereus). Six male koalas were given two single oral doses of p-cymene (0.37 mmol/kg and 1.49 mmol/kg). Urine and feces were collected for two 24-hr periods after each dose and analyzed for metabolites by extraction, gas chromatography, and mass spectrometry. A total of six metabolites were detected in the urine. A novel, extensively oxidized metabolite, 4-(1,2-dihydroxy-1-methylethyl)-benzoic acid, was identified and its structure elucidated by high resolution and chemical ionization mass spectrometry. Minor amounts of two glycine conjugated metabolites were also detected. Five metabolites were present in sufficient amounts to quantify, using isolated urinary metabolites as reference standards. The mean fractional recovery of administered p-cymene was 0.77 ± 0.09 and 0.84 ± 0.12 for the low and high dose, respectively. The major metabolite excreted was the novel carboxy diol compound. No fecal metabolites were found. Thus, the koala employs a strategy in the metabolism of p-cymene that promotes the production of extensively oxidized metabolites that consequently have increased polarity and enhanced renal excretion. This strategy is compared with that employed by other Eucalyptus leaf folivores.

Do multiple herbivores maintain chemical diversity of Scots pine monoterpenes

A central issue in our understanding of the evolution of the diversity of plant secondary metabolites (PSMs) is whether or not compounds are functional, conferring an advantage to the plant, or non-functional. We examine the hypothesis that the diversity of monoterpene PSMs within a plant species (Scots pine Pinus sylvestris) may be explained by different compounds acting as defences against high-impact herbivores operating at different life stages. We also hypothesize that pairwise coevolution, with uncorrelated interactions, is more likely to result in greater PSM diversity, than diffuse coevolution. We tested whether up to 13 different monoterpenes in Scots pine were inhibitory to herbivory by slugs (Arion ater), bank voles (Clethrionomys glareolus), red deer (Cervus elaphus) and capercaillie (Tetrao urogallus), each of which attack trees at a different life stage. Plants containing more α-pinene were avoided by both slugs and capercaillie, which may act as reinforcing selective agents for this dominant defensive compound. Herbivory by red deer and capercaillie were, respectively, weakly negatively associated with δ3-carene, and strongly negatively correlated with the minor compound β-ocimene. Three of the four herbivores are probably contributory selective agents on some of the terpenes, and thus maintain some, but by no means all, of the phytochemical diversity in the species. The correlated defensive function of α-pinene against slugs and capercaillie is consistent with diffuse coevolutionary processes.