The Australian continent exhibits complex biogeographic patterns but studies of the impacts of Pleistocene climatic oscillation on the mesic environments of the Southern Hemisphere are limited. The koala (Phascolarctos cinereus), one of Australia’s most iconic species, was historically widely distributed throughout much of eastern Australia but currently represents a complex conservation challenge. To better understand the challenges to koala genetic health, we assessed the phylogeographic history of the koala. Variation in the maternally inherited mitochondrial DNA (mtDNA) Control Region (CR) was examined in 662 koalas sampled throughout their distribution. In addition, koala CR haplotypes accessioned to Genbank were evaluated and consolidated. A total of 53 unique CR haplotypes have been isolated from koalas to date (including 15 haplotypes novel to this study). The relationships among koala CR haplotypes were indicative of a single Evolutionary Significant Unit and do not support the recognition of subspecies, but were separated into four weakly differentiated lineages which correspond to three geographic clusters: a central lineage, a southern lineage and two northern lineages co-occurring north of Brisbane. The three geographic clusters were separated by known Pleistocene biogeographic barriers: the Brisbane River Valley and Clarence River Valley, although there was evidence of mixing amongst clusters. While there is evidence for historical connectivity, current koala populations exhibit greater structure, suggesting habitat fragmentation may have restricted female-mediated gene flow. Since mtDNA data informs conservation planning, we provide a summary of existing CR haplotypes, standardise nomenclature and make recommendations for future studies to harmonise existing datasets. This holistic approach is critical to ensuring management is effective and small scale local population studies can be integrated into a wider species context.
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.
Koala populations are in catastrophic decline in certain eastern Australian regions. Spanning from 1997-2013, a database derived from wildlife hospitals in southeast Queensland with N = 20,250 entries was classified by causes of morbidity and mortality. A total of 11 aetiologies were identified, with chlamydiosis, trauma, and wasting being most common. The clinical diagnosis at submission varied significantly over the observation period. Combinations of aetiologies were observed in 39% of koalas submitted, with chlamydiosis frequently co-occurring. Urogenital (cystitis 26.8%, bursitis 13.5%) and ocular (conjunctivitis 17.2%) chlamydiosis were the most frequently diagnosed representations of the infection. Approximately 26% of submissions comprised koalas involved in vehicle accidents that were otherwise healthy. Age and sex of the koala as well as season and submission period were compared for the case outcomes of ‘dead on arrival’, ‘euthanized’, or ‘released’ for the four most common clinical diagnoses using multinomial logistic regression models. Exploratory space-time permutation scans were performed and overlapping space-time clusters for chlamydiosis, motor vehicle traumas and wasting unveiled high risk areas for koala disease and injury. Our results suggest that these aetiologies are acting jointly as multifactorial determinants for the continuing decline of koalas.
Koala (Phascolarctos cinereus) populations in the wild are in sharp decline in Australia due to deforestation, road accidents, dog attacks, and disease from infection with sexually transmitted Chlamydia spp. Severely diseased koalas that are captured are euthanized for humane reasons because antibiotics are not effective. Paradoxically, we propose that euthanizing more koalas could help to increase koala population numbers. We investigated the potential impact of systematically euthanizing diseased koalas. Using data from a well-studied koala population, and an individual-based computer simulation model, we predict that such a program would result in a larger population of koalas after 7 yr than would exist without the program. If terminally diseased and sterile koalas are euthanized and other infected captured koalas are given antibiotics, chlamydial infection could be eliminated and population growth observed after 4 yr. The practical implementation of such a program would be facilitated with further development of tools to diagnose infection and internal disease in the field.
Although endogenous retroviruses are common across vertebrate genomes, the koala retrovirus (KoRV) is the only retrovirus known to be currently invading the germ line of its host. KoRV is believed to have first infected koalas in northern Australia less than two centuries ago. We examined KoRV in 28 koala museum skins collected in the late 19th and 20th centuries and deep sequenced the complete proviral envelope region from five northern Australian specimens. Strikingly, KoRV env sequences were conserved among koalas collected over the span of a century, and two functional motifs that affect viral infectivity were fixed across the museum koala specimens. We detected only 20 env polymorphisms among the koalas, likely representing derived mutations subject to purifying selection. Among northern Australian koalas, KoRV was already ubiquitous by the late 19th century, suggesting that KoRV evolved and spread among koala populations more slowly than previously believed. Given that museum and modern koalas share nearly identical KoRV sequences, it is likely that koala populations, for more than a century, have experienced increased susceptibility to diseases caused by viral pathogenesis.
Endogenous retroviruses (ERVs) comprise 8% of the human genome and are common in all vertebrate genomes. The only retrovirus known to be currently transitioning from exogenous to endogenous form is the koala retrovirus (KoRV), making koalas (Phascolarctos cinereus) ideal for examining the early stages of retroviral endogenization. To distinguish endogenous from exogenous KoRV proviruses, we isolated koala genomic regions flanking KoRV integration sites. In 3 wild southern Australian koalas, there were fewer KoRV loci than in 3 captive Queensland koalas, consistent with reports that southern Australian koalas carry fewer KoRVs. Of 39 distinct KoRV proviral loci examined in a sire-dam-progeny triad, all proved to be vertically transmitted and endogenous; none were exogenous. Of the 39 endogenous KoRVs (enKoRVs), only 1 was present in the genomes of both the sire and the dam, suggesting that, at this early stage in the retroviral invasion of a host germ line, very large numbers of ERVs have proliferated at very low frequencies in the koala population. Sequence divergence between the 5' and 3' long terminal repeats (LTRs) of a provirus can be used as a molecular clock. Within each of 10 enKoRVs, the 5' LTR sequence was identical to the 3' LTR sequence, suggesting a maximum age for enKoRV invasion of the koala germ line of ca. 22,200 to 49,900 years ago, although a much younger age is possible. Across the 10 proviruses, 7 LTR haplotypes were detected, indicating that at least 7 different retroviral sequences had entered the koala the germ line.
Microscale methods for cell-based assays typically rely on macroscopic reagent handling and fluidic loading protocols that are technically challenging and do not scale with the number of assays favorably. Here, we demonstrate a microfluidic platform technology called “Kit-On-A-Lid-Assay” (KOALA), that enables the creation of self-contained microfluidic cell-based assays, integrating all the steps required to perform cell-based assays. The KOALA platform allows the pre-packaging of reagents, cryopreservation of cell suspensions, thawing of cell suspensions, culture of cells, and operation of whole cell-based assays. The operation of the KOALA platform is user-friendly and consists of bringing together a lid containing the microchannels, and a base containing the pre-packaged reagents, thereby causing fluidic exchange in all the channels simultaneously. We demonstrate that the KOALA cell-based assays can be simply operated from start to finish without any external laboratory equipment.
Despite being a charismatic and well-known species, the social system of the koala (Phascolarctos cinereus-the only extant member of the family Phascolarctidae) is poorly known and much of the koala’s sociality and mating behaviors remain un-quantified. We evaluated these using proximity logging-GPS enabled tracking collars on wild koalas and discuss their implications for the mating system of this species. The frequency and duration of male-female encounters increased during the breeding season, with male-male encounters quite uncommon, suggesting little direct mating competition. By comparison, female-female interactions were very common across both seasons. Body mass of males was not correlated with their interactions with females during the breeding season, although male size is associated with a variety of acoustic parameters indicating individuality. We hypothesise that vocal advertising reduces the likelihood of male-male encounters in the breeding season while increasing the rate of male-female encounters. We suggest that male mating-season bellows function to reduce physical confrontations with other males allowing them to space themselves apart, while, at the same time, attracting females. We conclude that indirect male-male competition, female mate choice, and possibly female competition, mediate sexual selection in koalas.
The marsupial family Diprotodontidae (Diprotodontia, Vombatiformes) is a group of extinct large-bodied (60-2500 kg) wombat-like herbivores that were common and geographically widespread in Cenozoic fossil deposits of Australia and New Guinea. Typically they are regarded to be gregarious, terrestrial quadrupeds and have been likened in body form among placental groups to sheep, rhinoceros and hippopotami. Arguably, one of the best represented species is the zygomaturine diprotodontid Nimbadon lavarackorum which is known from exceptionally well-preserved cranial and postcranial material from the middle Miocene cave deposit AL90, in the Riversleigh World Heritage Area, northwestern Queensland. Here we describe and functionally analyse the appendicular skeleton of Nimbadon lavarackorum and reveal a far more unique lifestyle for this plesiomorphic and smallest of diprotodontids. Striking similarities are evident between the skeleton of Nimbadon and that of the extant arboreal koala Phascolarctos cinereus, including the powerfully built forelimbs, highly mobile shoulder and elbow joints, proportionately large manus and pes (both with a semi-opposable digit I) and exceedingly large, recurved and laterally compressed claws. Combined with the unique (among australidelphians) proportionately shortened hindlimbs of Nimbadon, these features suggest adept climbing ability, probable suspensory behaviour, and an arboreal lifestyle. At approximately 70 kg, Nimbadon is the largest herbivorous mammal to have occupied the forest canopies of Australia - an ecological niche that is no longer occupied in any Australian ecosystem and one that further expands the already significant niche diversity displayed by marsupials during the Cenozoic.
Koala retroviruses (KoRV) have been isolated from wild and captive koalas in Australia as well as from koala populations held in zoos in other countries. They are members of the genus Gammaretrovirus, are most closely related to gibbon ape leukemia virus (GaLV), feline leukemia virus (FeLV) and porcine endogenous retrovirus (PERV) and are likely the result of a relatively recent trans-species transmission from rodents or bats. The first KoRV to be isolated, KoRV-A, is widely distributed in the koala population in both integrated endogenous and infectious exogenous forms with evidence from museum specimens older than 150 years, indicating a relatively long engagement with the koala population. More recently, additional subtypes of KoRV that are not endogenized have been identified based on sequence differences and host cell receptor specificity (KoRV-B and KoRV-J). A specific association with fatal lymphoma and leukemia has been recently suggested for KoRV-B. In addition, it has been proposed that the high viral loads found in many animals may lead to immunomodulation resulting in a higher incidence of diseases such as chlamydiosis. Although the molecular basis of this immunomodulation is still unclear, purified KoRV particles and a peptide corresponding to a highly conserved domain in the envelope protein have been shown to modulate cytokine expression in vitro, similar to that induced by other gammaretroviruses. While much is still to be learned, KoRV induced lymphoma/leukemia and opportunistic disease arising as a consequence of immunomodulation are likely to play an important role in the stability of koala populations both in the wild and in captivity.