The mycobiome of Australian tree hollows in relation to the Cryptococcus gattii and C. neoformans species complexes

Cryptococcosis is a fungal infection caused by members of the Cryptococcus gattii and C. neoformans species complexes. The C. gattii species complex has a strong environmental association with eucalypt hollows (particularly Eucalyptus camaldulensis), which may present a source of infection. It remains unclear whether a specific mycobiome is required to support its environmental survival and growth. Conventional detection of environmental Cryptococcus spp. involves culture on differential media, such as Guizotia abyssinica seed agar. Next‐generation sequencing (NGS)‐based culture‐independent identification aids in contextualising these species in the environmental mycobiome. Samples from 23 Australian tree hollows were subjected to both culture‐ and amplicon‐based metagenomic analysis to characterize the mycobiome and assess relationships between Cryptococcus spp. and other fungal taxa. The most abundant genera detected were Coniochaeta, Aspergillus, and Penicillium, all being commonly isolated from decaying wood. There was no correlation between the presence of Cryptococcus spp. in a tree hollow and the presence of any other fungal genus. Some differences in the abundance of numerous taxa were noted in a differential heat tree comparing samples with or without Cryptococcus‐NGS reads. The study expanded the known environmental niche of the C. gattii and C. neoformans species complexes in Australia with detections from a further five tree species. Discrepancies between the detection of Cryptococcus spp. using culture or NGS suggest that neither is superior per se and that, rather, these methodologies are complementary. The inherent biases of amplicon‐based metagenomics require cautious interpretation of data through consideration of its biological relevance.     

Thermoregulation by kangaroos from mesic and arid habitats: influence of temperature on routes of heat loss in eastern grey kangaroos (Macropus giganteus) and red kangaroos (Macropus rufus)

We examined thermoregulation in red kangaroos (Macropus rufus) from deserts and in eastern grey kangaroos (Macropus giganteus) from mesic forests/woodlands. Desert kangaroos have complex evaporative heat loss mechanisms, but the relative importance of these mechanisms is unclear. Little is known of the abilities of grey kangaroos. Our detailed study of these kangaroos' thermoregulatory responses at air temperatures (T(a)) from -5 degrees to 45 degrees C showed that, while some differences occur, their abilities are fundamentally similar. Both species show the basic marsupial characteristics of relatively low basal metabolism and body temperature (T(b)). Within the thermoneutral zone, T(b) was 36.3 degrees + or - 0.1 degrees C (X + or - SE) in both species, and except for a small rise at T(a) 45 degrees C, T(b) was stable over a wide range of T(a). Metabolic heat production was 25% higher in red kangaroos at T(a) -5 degrees C. At the highest T(a) (45 degrees C), both species relied on evaporative heat loss (EHL) to maintain T(b); both panting and licking were used. The eastern grey kangaroo utilised panting (76% of EHL) as the principal mode of EHL, and while this was so for red kangaroos, cutaneous evaporative heat loss (CEHL) was significant (40% of EHL). CEHL appeared to be mainly licking, as evidenced from surface temperatures. Both species utilised peripheral vascular adjustments to control heat flow, as indicated by changes in dry conductance (C(dry)). At lower temperatures, C(dry) was minimal, but it increased significantly at T(a) just below T(b) (33 degrees C); in these conditions, the C(dry) of red kangaroos was significantly higher than that of eastern grey kangaroos, indicating a greater reliance on dry heat loss. Under conditions where heat flows into the body from the environment (T(a) 45 degrees C), there was peripheral vasoconstriction to reduce this inflow; C(dry) decreased significantly from the values seen at 33 degrees C in both kangaroos. The results indicated that, while both species have excellent thermoregulatory abilities, the desert red kangaroos may cope better with more extreme temperatures, given that they respond to T(a) 45 degrees C with lower respiratory evaporation than do the eastern grey kangaroos.     

Food for folivores: nutritional explanations linking diets to population density

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

Extensive Acclimation in Ectotherms Conceals Interspecific Variation in Thermal Tolerance Limits

Species’ tolerance limits determine their capacity to tolerate climatic extremes and limit their potential distributions. Interspecific variation in thermal tolerances is often proposed to indicate climatic vulnerability and is, therefore, the subject of many recent meta-studies on differential capacities of species from climatically different habitats to deal with climate change. Most studies on thermal tolerances do not acclimate animals or use inconsistent, and insufficient, acclimation times, limiting our knowledge of the shape, duration and extent of acclimation responses. Consequently patterns in thermal tolerances observed in meta-analyses, based on data from the literature are based on inconsistent, partial acclimation and true trends may be obscured. In this study we describe time-course of complete acclimation of critical thermal minima in the tropical ectotherm Carlia longipes and compare it to the average acclimation response of other reptiles, estimated from published data, to assess how much acclimation time may contribute to observed differences in thermal limits. Carlia longipes decreased their lower critical thermal limits by 2.4°C and completed 95% of acclimation in 17 weeks. Wild populations did not mirror this acclimation process over the winter. Other reptiles appear to decrease cold tolerance more quickly (95% in 7 weeks) and to a greater extent, with an estimated average acclimation response of 6.1°C. However, without data on tolerances after longer acclimation times available, our capacity to estimate final acclimation state is very limited. Based on the subset of data available for meta-analysis, much of the variation in cold tolerance observed in the literature can be attributed to acclimation time. Our results indicate that (i) acclimation responses can be slow and substantial, even in tropical species, and (ii) interspecific differences in acclimation speed and extent may obscure trends assessed in some meta-studies. Cold tolerances of wild animals are representative of cumulative responses to recent environments, while lengthy acclimation is necessary for controlled comparisons of physiological tolerances. Measures of inconsistent, intermediate acclimation states, as reported by many studies, represent neither the realised nor the potential tolerance in that population, are very likely underestimates of species’ physiological capacities and may consequently be of limited value.     

Seedling Mortality Due to Drought and Fire Associated with the 2002 El Niño Event in a Tropical Rain Forest in North‐East Queensland,Australia1

We monitored survival of seedlings in 216 1‐m² quadrats in lowland rain forest in tropical north Queensland between December 2001 and December 2002. During this time, the region experienced severe drought associated with an El Nińo Southern Oscillation event. The 2001 census recorded 124 species and 2912 individuals. In late November 2002 (2 wk prior to the second census), a low intensity fire passed through approximately half of the study site removing all evidence of seedlings from 110 plots. Only 482 (17%) individuals and 64 (52%) species recorded in 2001 survived the 12‐mo period. In 96 quadrats not affected by fire, mortality was high, but considerably variable between species. Six of the 20 most abundant species in 2001 experienced mortality rates higher than the community average and two of the most abundant species showed rates lower than average. Overall, conditions experienced during 2002 caused significant changes in the rank abundances of species between censuses. Mortality due to fire was less severe and mortality more uniform across species, resulting in significant concordance between pre and postfire rankings, once the effects of drought had been considered. Our results provide the first indication of how differences in survival after a perturbation predicted to become more frequent in future global climates may alter the size and species composition of the seedling bank in Australian tropical rain forests.     

Metabolites released by Cryptococcus neoformans var. neoformans and var. gattii differentially affect human neutrophil function

Differences in the ability of Cryptococcus neoformans var. neoformans (CNVN) and var. gattii (CNVG) to establish localized lesions in the lungs of healthy humans remain unexplained. In this study, CNVG infection in a rat model was characterized by early neutrophil invasion into lung tissue, but phagocytosis of cryptococci was not observed. The chemical composition of non-enzymic components secreted by one strain of each variety (heat-inactivated supernatants from CNVN and CNVG, termed vns and vgs, respectively) were compared, using magnetic resonance spectroscopy. Effects on human neutrophil viability and functions at both pH 5.5 and 7.0 were investigated, as the pH of cryptococcomas was found to be 5.4-5.6 in vivo. The supernatants were similar in composition, although metabolites in vns were generally present in higher concentrations. In addition, vgs contained two novel metabolites-acetoin and dihydroxyacetone. Polyphosphate was observed in cells from both varieties and may be a source of extracellular inorganic phosphate. Superoxide production in the presence of phorbol ester was enhanced by treatment with vns and decreased by vgs. At pH 5.5, vns caused high levels of necrosis in neutrophils, as well as increased adhesion/migration through A549 lung epithelial cell monolayers. Individual supernatant components such as polyols, acetoin, dihydroxyacetone, and gamma-aminobutyric acid exhibited both pro- and anti-inflammatory properties. Overall, we found that vgs was potentially less pro-inflammatory than vns. Inhibition of neutrophil function by products of CNVG may promote survival of extracellular organisms, and local multiplication to form cryptococcomas.     

Evidence of recombination in mixed-mating-type and alpha-only populations of Cryptococcus gattii sourced from single eucalyptus tree hollows

Disease caused by the pathogenic yeast Cryptococcus gattii begins with the inhalation of an infectious propagule. As C. gattii is heavily encapsulated, this propagule is most likely to be a basidiospore. However, most C. gattii strains are infertile in laboratory crosses, and population studies indicate that recombination and dispersal are very restricted. In addition, strains of the alpha mating type predominate, which would not be expected in a mating population. C. gattii comprises four genetically distinct molecular genotypes, designated VGI to VGIV. C. gattii molecular type VGI has a strong association with Eucalyptus camaldulensis and can be found in high numbers in E. camaldulensis hollows. Previous work on isolates obtained from E. camaldulensis suggested that environmental populations of C. gattii are highly fragmented, have limited ability to disperse, and are confined to individual tree hollows. In the current study, we examined large numbers of isolates from three separate hollows for evidence of recombination. In two hollows, the alpha and a mating types were present in approximately equal numbers. The third hollow had alpha cells only and was from a region where a isolates have never been found. Statistical analysis of multilocus genotypes revealed recombining subpopulations in the three Eucalyptus hollows. Recombination was equally present in the alpha-a and alpha-only populations. This is consistent with recent studies that have found evidence suggestive of alpha-alpha mating in C. gattii and Cryptococcus neoformans and raises the possibility this may be a widespread phenomenon, allowing these fungi to recombine despite a paucity of a mating partners.     

The Distribution and Abundance of an Island Population of Koalas (Phascolarctos cinereus) in the Far North of Their Geographic Range

Koalas are an iconic species of charismatic megafauna, of substantial social and conservation significance. They are widely distributed, often at low densities, and individuals can be difficult to detect, making population surveys challenging and costly. Consequently, koala population estimates have been limited and the results inconsistent. The aims of this study were to estimate the distribution, relative abundance and population size of the koalas on Magnetic Island, far north Queensland. Population densities were estimated in 18 different vegetation types present on the island using a Fecal Standing Crop Method. Koala density ranged from 0.404 ha⁻¹, recorded in forest red gum and bloodwood woodland, to absence from eight of the vegetation types surveyed. The second highest density of 0.297 koalas ha⁻¹ was recorded in mixed eucalypt woodland, which covers 45% of the island. The total abundance of koalas on Magnetic Island, not including those present in urban areas, was estimated at 825±175 (SEM). The large variation in koala density across vegetation types reinforces the need for sampling stratification when calculating abundance over large areas, as uniformity of habitat quality cannot be assumed. In this context, koala populations also occur in low densities in areas generally regarded as poor quality koala habitat. These results highlight the importance of protecting vegetation communities not traditionally considered to have high conservation value to koalas, as these habitats may be essential for maintaining viable, widespread, low-density populations. The results from this study provide a baseline to assess future trends in koala distribution, density and abundance on Magnetic Island.     

Inter-population differences in the tolerance of a marsupial folivore to plant secondary metabolites

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.