Kathrine A. Handasyde

Intraspecific variation in Eucalyptus secondary metabolites determines food intake by folivorous marsupials

Abstract Traditional approaches to the question of the effects of plant secondary metabolites on the feeding choices of folivores of Eucalyptus have focused on the tree species level, although numerous field studies of foraging behaviour have identified selection at the level of the individual trees. Attempts to relate these decisions to deterrency resulting from secondary leaf chemistry have been inconclusive because assays used have focused on broad groups of compounds such as “total” phenolics. In this study we have conducted no-choice feeding trials with two arboreal mammalian folivores, the common ringtail possum (Pseudocheirus peregrinus) and the koala (Phascolarctos cinereus), to measure deterrency of individual trees of two species of Eucalyptus, E. ovata and E. viminalis. Average daily intakes of E. ovata foliage by common ringtail possums ranged from 2.5 to 50 g kg−0.75 body mass. Koala intakes of foliage from the same individual trees ranged from 22.4 to 36.3 g kg−0.75 body mass. When fed foliage from different individual E. viminalis trees, common ringtail possums ate between 1.26 and 6.28 g kg−0.75 body mass while koalas ate from 14.3 to 45.9 g kg−0.75 body mass. Correlative analyses showed no relationships between feeding and several measures of nutritional quality, nor with total phenolics or condensed tannins. They did, however, identify two groups of plant secondary metabolites that may cause deterrency: terpenes, and a defined group of phenolic compounds, the diformylphloroglucinols (DFPs). Further bioassay experiments with common ringtail possums showed that only the DFPs could cause the effects seen with the foliage experiments at concentrations similar to those found in the leaves. We argue that, when in sufficiently high concentrations, DFPs determine the level of food intake by these animals irrespective of other questions of nutritional quality of the leaves.

A Major Role for Mammals in the Ecology of Mycobacterium ulcerans

Background Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a destructive skin disease found predominantly in sub-Saharan Africa and south-eastern Australia. The precise mode(s) of transmission and environmental reservoir(s) remain unknown, but several studies have explored the role of aquatic invertebrate species. The purpose of this study was to investigate the environmental distribution of M. ulcerans in south-eastern Australia. Methodology/Principal Findings A range of environmental samples was collected from Point Lonsdale (a small coastal town southwest of Melbourne, Australia, endemic for BU) and from areas with fewer or no reported incident cases of BU. Mycobacterium ulcerans DNA was detected at low levels by real-time PCR in soil, sediment, water residue, aquatic plant biofilm and terrestrial vegetation collected in Point Lonsdale. Higher levels of M. ulcerans DNA were detected in the faeces of common ringtail (Pseudocheirus peregrinus) and common brushtail (Trichosurus vulpecula) possums. Systematic testing of possum faeces revealed that M. ulcerans DNA could be detected in 41% of faecal samples collected in Point Lonsdale compared with less than 1% of faecal samples collected from non-endemic areas (p<0.0001). Capture and clinical examination of live possums in Point Lonsdale validated the accuracy of the predictive value of the faecal surveys by revealing that 38% of ringtail possums and 24% of brushtail possums had laboratory-confirmed M. ulcerans skin lesions and/or M. ulcerans PCR positive faeces. Whole genome sequencing revealed an extremely close genetic relationship between human and possum M. ulcerans isolates. Conclusions/Significance The prevailing wisdom is that M. ulcerans is an aquatic pathogen and that BU is acquired by contact with certain aquatic environments (swamps, slow-flowing water). Now, after 70 years of research, we propose a transmission model for BU in which terrestrial mammals are implicated as reservoirs for M. ulcerans.

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.

Detecting bottlenecks using BOTTLENECK 1.2.02 in wild populations: the importance of the microsatellite structure

Reduced, or bottlenecked, populations are more prone to adverse events. Thus, the detection of genetic bottleneck signatures in wildlife is an important issue for conservation. BOTTLENECK 1.2.02 is a software commonly used for detecting genetic characteristics of past bottlenecks. Here we test the efficiency with which this software detects bottlenecks in two koala populations of known history. The sign test performed well for both populations, particularly under the infinite alleles model for mutation. This suggests this model could be the more realistic for marsupial microsatellites than other mutation models. Under the allele frequency distribution test, the two populations falsely appeared to be at mutation/drift equilibrium. However, this test could detect the bottleneck when only imperfect repeat microsatellites were included in the analysis. We thus recommend further investigation of imperfect repeat microsatellites, which could be more powerful for bottleneck detection. These results underline the cautious approach researchers and conservationists should take when studying the past of unknown populations.

Inbreeding and testicular abnormalities in a bottlenecked population of koalas (Phascolarctos cinereus)

Habitat destruction and fragmentation, interactions with introduced species or the relocation of animals to form new populations for conservation purposes may result in a multiplication of population bottlenecks. Examples are the translocations of koalas to French Island and its derivative Kangaroo Island population, with both populations established as insurance policies against koala extinction. In terms of population size, these conservation programs were success stories. However, the genetic story could be different. We conducted a genetic investigation of French and Kangaroo Island koalas by using 15 microsatellite markers, 11 of which are described here for the first time. The results confirm very low genetic diversity. French Island koalas have 3.8 alleles per locus and Kangaroo Island koalas 2.4. The present study found a 19% incidence of testicular abnormality in kangaroo Island animals. Internal relatedness, an individual inbreeding coefficient, was not significantly different in koalas with testicular abnormalities from that in other males, suggesting the condition is not related to recent inbreeding. It could instead result from an unfortunate selection of founder individuals carrying alleles for testicular abnormalities, followed by a subsequent increase in these alleles’ frequencies through genetic drift and small population-related inefficiency of selection. Given the low diversity and possible high prevalence of deleterious alleles, the genetic viability of the population remains uncertain, despite its exponential growth so far. This stands as a warning to other introductions for conservation reasons.

Variation in the use of corridors by introduced and native rodents in south-eastern Australia.

We examined the use of roadside corridors as habitat by introduced and Australian native rodents using a replicated sampling program that compared their abundance in remnant forest, two types of roadside corridor and pasture. Introduced rodents were most abundant in either the corridors (black rat Rattus rattus) or in the corridors and pastures (house mouse Mus musculus), whereas forests were favoured by the native bush rat Rattus fuscipes. For all species, the density of different age-classes varied between the habitats. Additionally, there was interspecific and intraspecific (age-class) variation in the number of males to females in each habitat, and adult female bush rats in corridors weighed less than those in forests. These data indicate that some introduced species may be more prominent in corridors than native taxa, and highlight the importance of considering intraspecific as well as interspecific variation in corridor use when evaluating the role of corridors.

Tree-hugging koalas demonstrate a novel thermoregulatory mechanism for arboreal mammals

How climate impacts organisms depends not only on their physiology, but also whether they can buffer themselves against climate variability via their behaviour. One of the way species can withstand hot temperatures is by seeking out cool microclimates, but only if their habitat provides such refugia. Here, we describe a novel thermoregulatory strategy in an arboreal mammal, the koala Phascolarctos cinereus. During hot weather, koalas enhanced conductive heat loss by seeking out and resting against tree trunks that were substantially cooler than ambient air temperature. Using a biophysical model of heat exchange, we show that this behaviour greatly reduces the amount of heat that must be lost via evaporative cooling, potentially increasing koala survival during extreme heat events. While it has long been known that internal temperatures of trees differ from ambient air temperatures, the relevance of this for arboreal and semi-arboreal mammals has not previously been explored. Our results highlight the important role of tree trunks as aboveground ‘heat sinks’, providing cool local microenvironments not only for koalas, but also for all tree-dwelling species.

Burrow use and ranging behaviour of the southern hairy-nosed wombat ( Lasiorhinus latifrons ) in the Murraylands, South Australia

This study investigated burrow use and ranging behaviour in the southern hairy-nosed wombat Lasiorhinus latifrons in semi-arid South Australia. Sixteen adult wombats were fitted with radio transmitters and monitored monthly from July 2001 to February 2002. Wombats generally used between one and five warrens, preferred large warrens with a greater number of entrances and showed a preference for one or two warrens. Across the study period there was no apparent change in burrows used within warrens. Radio-tracking indicated that animals spent very little time above ground (26% of 1115 night-time fixes), centred their activity around their preferred warrens, and moved, on average, 99 m/h and 221 m/night. Mean home-range size, estimated using minimum convex polygons and the harmonic mean method from location data, obtained through triangulation, and daytime warren fixes, ranged from 1.3 to 4.8 ha. Home-range size was similar between males and females and home ranges overlapped substantially. The data highlight the importance of burrows to southern hairy-nosed wombats in shaping their home ranges. It seems likely that the use of burrows and a specialized diet are important energy saving strategies for this species in such unpredictable regions of South Australia.

Long-term pair-bonds without mating fidelity in a mammal

Most mammals are polygynous and are characterised by male-biased sexual size dimorphism. One hallmark of mammalian monogamy is the lack of such dimorphism. Bobucks, or mountain brushtail possums, Trichosurus cunninghami, lack sexual size dimorphism; however, few behavioural data exist for this species. We studied the mating system of a bobuck population in south-eastern Australia. Adult bobucks were strongly paired: pair-members had exclusive access to a suite of den-trees and overlapped in home range on average by 70%. Pair-members rested together in the same tree-hollow on approximately 70% of days during the breeding season and 47% of days during the non-breeding season. While active, pair-members remained within approximately 8 m of one another during the breeding season and within 31 m of one another during the non-breeding season. Females established pair-bonds at 2-5 years of age; pair-bonds ended only as a result of the death of one pair member. However, molecular paternity analysis established that 35% of young were the result of extra-pair copulations (EPCs). This is the first study to provide strong evidence of long-term pair-bonds in a marsupial and raises questions about the relative benefits of pairing to males and females in this population.

Potential wildlife sentinels for monitoring the endemic spread of human buruli ulcer in South-East australia.

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥106 organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.