Evergrowing incisors of diprotodont marsupials record age and life history.

OBJECTIVE: Tooth growth and wear are commonly used tools for determining the age of mammals. The most speciose order of marsupials, Diprotodontia, is characterised by a pair of procumbent incisors within the lower jaw. This study examines the growth and wear of these incisors to understand their relationship with age and sex. DESIGN: Measurements of mandibular incisor crown and root length were made for two sister species of macropodid (kangaroos and wallabies); Macropus giganteus and Macropus fuliginosus. Histological analysis examined patterns of dentine and cementum deposition within these teeth. Broader generalisability within Diprotodontia was tested using dentally reduced Tarsipes rostratus - a species disparate in body size and incisor function to the studied macropodids. RESULTS: In the macropodid sample it is demonstrated that the hypsodont nature of these incisors makes measurements of their growth (root length) and wear (crown length) accurate indicators of age and sex. Model fitting finds that root growth proceeds according to a logarithmic function across the lifespan, while crown wear follows a pattern of exponential reduction for both macropodid species. Histological results find that secondary dentine deposition and cementum layering are further indicators of age. Incisor measurements are shown to correlate with age in the sample of T. rostratus. CONCLUSIONS: The diprotodontian incisor is a useful tool for examining chronological age and sex, both morphologically and microstructurally. This finding has implications for population ecology, palaeontology and marsupial evolution.

Emx2 underlies the development and evolution of marsupial gliding membranes.

Phenotypic variation among species is a product of evolutionary changes to developmental programs1,2. However, how these changes generate novel morphological traits remains largely unclear. Here we studied the genomic and developmental basis of the mammalian gliding membrane, or patagium-an adaptative trait that has repeatedly evolved in different lineages, including in closely related marsupial species. Through comparative genomic analysis of 15 marsupial genomes, both from gliding and non-gliding species, we find that the Emx2 locus experienced lineage-specific patterns of accelerated cis-regulatory evolution in gliding species. By combining epigenomics, transcriptomics and in-pouch marsupial transgenics, we show that Emx2 is a critical upstream regulator of patagium development. Moreover, we identify different cis-regulatory elements that may be responsible for driving increased Emx2 expression levels in gliding species. Lastly, using mouse functional experiments, we find evidence that Emx2 expression patterns in gliders may have been modified from a pre-existing program found in all mammals. Together, our results suggest that patagia repeatedly originated through a process of convergent genomic evolution, whereby regulation of Emx2 was altered by distinct cis-regulatory elements in independently evolved species. Thus, different regulatory elements targeting the same key developmental gene may constitute an effective strategy by which natural selection has harnessed regulatory evolution in marsupial genomes to generate phenotypic novelty.

The people behind the paper - Peter Kozulin, Rodrigo Suárez, Qiong-Yi Zhao, Linda Richards and Laura Fenlon.

The neocortex is unique to mammals and so, for evolutionary studies, researchers have compared eutherians and marsupials. A new paper in Development uncovers key differences in the timing of gene expression changes in the cortical development of the mouse and the similarly sized marsupial, the fat-tailed dunnart. We caught up with the authors from The University of Queensland, Australia, to find out more about their research and their future plans.

Divergent evolution of developmental timing in the neocortex revealed by marsupial and eutherian transcriptomes.

Only mammals evolved a neocortex, which integrates sensory-motor and cognitive functions. Significant diversifications in the cellular composition and connectivity of the neocortex occurred between the two main therian groups: marsupials and eutherians. However, the developmental mechanisms underlying these diversifications are largely unknown. Here, we compared the neocortical transcriptomes of Sminthopsis crassicaudata, a mouse-sized marsupial, with those of eutherian mice at two developmentally equivalent time points corresponding to deeper and upper layer neuron generation. Enrichment analyses revealed more mature gene networks in marsupials at the early stage, which reverted at the later stage, suggesting a more precocious but protracted neuronal maturation program relative to birth timing of cortical layers. We ranked genes expressed in different species and identified important differences in gene expression rankings between species. For example, genes known to be enriched in upper-layer cortical projection neuron subtypes, such as Cux1, Lhx2 and Satb2, likely relate to corpus callosum emergence in eutherians. These results show molecular heterochronies of neocortical development in Theria, and highlight changes in gene expression and cell type composition that may underlie neocortical evolution and diversification. This article has an associated 'The people behind the papers' interview.

Postnatal development in a marsupial model, the fat-tailed dunnart (Sminthopsis crassicaudata; Dasyuromorphia: Dasyuridae).

Marsupials exhibit unique biological features that provide fascinating insights into many aspects of mammalian development. These include their distinctive mode of reproduction, altricial stage at birth, and the associated heterochrony that is required for their crawl to the pouch and teat attachment. Marsupials are also an invaluable resource for mammalian comparative biology, forming a distinct lineage from the extant placental and egg-laying monotreme mammals. Despite their unique biology, marsupial resources are lagging behind those available for placentals. The fat-tailed dunnart (Sminthopsis crassicaudata) is a laboratory based marsupial model, with simple and robust husbandry requirements and a short reproductive cycle making it amenable to experimental manipulations. Here we present a detailed staging series for the fat-tailed dunnart, focusing on their accelerated development of the forelimbs and jaws. This study provides the first skeletal developmental series on S. crassicaudata and provides a fundamental resource for future studies exploring mammalian diversification, development and evolution.

Ontogenetic origins of cranial convergence between the extinct marsupial thylacine and placental gray wolf.

Phenotypic convergence, describing the independent evolution of similar characteristics, offers unique insights into how natural selection influences developmental and molecular processes to generate shared adaptations. The extinct marsupial thylacine and placental gray wolf represent one of the most extraordinary cases of convergent evolution in mammals, sharing striking cranial similarities despite 160 million years of independent evolution. We digitally reconstructed their cranial ontogeny from birth to adulthood to examine how and when convergence arises through patterns of allometry, mosaicism, modularity, and integration. We find the thylacine and wolf crania develop along nearly parallel growth trajectories, despite lineage-specific constraints and heterochrony in timing of ossification. These constraints were found to enforce distinct cranial modularity and integration patterns during development, which were unable to explain their adult convergence. Instead, we identify a developmental origin for their convergent cranial morphologies through patterns of mosaic evolution, occurring within bone groups sharing conserved embryonic tissue origins. Interestingly, these patterns are accompanied by homoplasy in gene regulatory networks associated with neural crest cells, critical for skull patterning. Together, our findings establish empirical links between adaptive phenotypic and genotypic convergence and provides a digital resource for further investigations into the developmental basis of mammalian evolution.

Development of the skin in the eastern quoll (Dasyurus viverrinus) with focus on cutaneous gas exchange in the early postnatal period.

A morphological and morphometric study of the skin development in the eastern quoll (Dasyurus viverrinus) was conducted to follow the transition from cutaneous to pulmonary gas exchange in this extremely immature marsupial species. Additionally, the development of the cardiac and respiratory system was followed, to evaluate the systemic prerequisites allowing for cutaneous respiration. The skin in the newborn D. viverrinus was very thin (36 ± 3 µm) and undifferentiated (no hair follicles, no sebaceous and perspiratory glands). Numerous superficial cutaneous capillaries were encountered, closely associated with the epidermis, allowing for gaseous exchange. The capillary volume density was highest in the neonate (0.33 ± 0.04) and decreased markedly during the first 4 days (0.06 ± 0.01). In the same time period, the skin diffusion barrier increased from 9 ± 1 µm to 44 ± 6 µm. From this age on the skin development was characterized by thickening of the different cutaneous layers, formation of hair follicles (day 55) and the occurrence of subcutaneous fat (day 19). The heart of the neonate D. viverrinus had incomplete interatrial, inter-ventricular, and aortico-pulmonary septa, allowing for the possibility that oxygenated blood from the skin mixes with that of the systemic circulation. The fast-structural changes in the systemic circulations (closing all shunts) in the early postnatal period (3 days) necessitate the transition from cutaneous to pulmonary respiration despite the immaturity of the lungs. At this time, the lung was still at the canalicular stage of lung development, but had to be mature enough to meet the respiratory needs of the growing organism. The morphometric results for the skin development of D. viverrinus suggest that cutaneous respiration is most pronounced in neonates and decreases rapidly during the first 3 days of postnatal life. After this time a functional transition of the skin from cutaneous respiration to insulation and protection of the body takes place.

Developmental Constraints Do Not Influence Long-Term Phenotypic Evolution of Marsupial Forelimbs as Revealed by Interspecific Disparity and Integration Patterns.

Marsupials show a smaller range of forelimb ecomorphologies than placental mammals, and it is hypothesized that this results from macroevolutionary constraints imposed by the specialized reproductive biology of marsupials. Specifically, the accelerated development of the marsupial forelimb allows neonates to crawl to the mother's pouch but may constrain adult morphology. This hypothesis makes three main predictions: (i) that marsupial forelimbs should show less interspecific disparity than their hindlimbs, (ii) that morphological integration within the marsupial forelimb is stronger than integration between limbs, and (iii) that these patterns should be strongest in diprotodontians, which undergo the most rigorous crawls as neonates. We use a three-dimensional geometric morphometric data set of limb bones for 51 marsupial species to test these predictions. We find that (i) marsupial forelimbs and hindlimbs show similar disparities, (ii) no clear differences in integration exist either within or between limbs, and (iii) the same patterns occur in diprotodontians as in other marsupials, even correcting for lineage age. Therefore, there is currently little evidence that the developmental biology of marsupials has constrained their macroevolutionary patterns. It is possible that functional selection can overcome the effects of developmental constraint on macroevolutionary timescales. Our findings suggest that the role of developmental constraints in explaining the limited phenotypic variability of marsupials (compared with that of placentals) should be reconsidered.

Quantitative Analysis of the Timing of Development of the Cerebellum and Precerebellar Nuclei in Monotremes, Metatherians, Rodents, and Humans.

We have used a quantitative statistical approach to compare the pace of development in the cerebellum and precerebellar systems relative to body size in monotremes and metatherians with that in eutherians (rodents and humans). Embryos, fetuses, and early postnatal mammals were scored on whether key structural events had been reached in the development of the cerebellum itself (CC-corpus cerebelli; 10 milestones), or the pontine and inferior olivary precerebellar nuclear groups (PC; 4 milestones). We found that many early cerebellar and precerebellar milestones (e.g., formation of Purkinje cell layer and deep cerebellar nuclei) were reached at a smaller absolute body length in both metatherians and eutherians together, compared to monotremes. Some later milestones (e.g., formation of the external granular layer and primary fissuration) were reached at a smaller body length in metatherians than eutherians. When the analysis was performed with proportional body length expressed as a natural log-transformed ratio of length at birth, milestones were reached at a much smaller proportional body length in rodents and humans than in the metatherians or monotremes. The findings are consistent with the slower pace of metabolic activity and embryonic development in monotremes. They also indicate slightly advanced maturation of some early features of the cerebellum in some metatherians (i.e., early cerebellar development in dasyurids relative to body size), but do not support the notion of an accelerated development of the cerebellum to cope with the demands of early birth. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1998-2013, 2020. © 2019 American Association for Anatomy.

Growth and development of the Mahogany Glider (Petaurus gracilis).

The growth and development of the endangered Mahogany Glider (Petaurus gracilis) was monitored in a captive population at Burleigh Heads, Queensland, Australia. Video surveillance confirmed that the gestation period for this species was 16 days. Morphometric data and developmental milestones were recorded from 10 Mahogany Gliders from birth to weaning. Growth curves were developed for head length, ulna length, tail length, and body weight. Weekly inspections of female pouches revealed the young's eyelid margins were visible by Day 21, the first hair erupted on the bridge of the nose at Day 30, pigmentation of the body developed at Day 63, and they started detaching from the teat intermittently, and the body was covered in short fur by Day 70. The young were left in the nest alone from Days 84 to 87, their eyes opened between Days 84 and 94, and there was a rapid increase in length and density of fur from Day 98 onwards. At Days 101 to 105 of age the young left the nest box with its mother as back young. Weaning occurred from 184 to 187 days. Typically, the reproductive rate was two young per annum per pair, but one pair produced five young in 19 months. Females produced young from 12 months to 7 years of age, males up to 9.4 years of age. The average longevity of Mahogany Gliders in the studbook in 2018 was 11.6 years. This study provides data on the reproductive biology of the Mahogany Glider that will assist in its captive breeding, management, and conservation.

Mechanical similarity across ontogeny of digging muscles in an Australian marsupial (Isoodon fusciventer).

Many mammals dig, either during foraging to access subsurface food resources, or in creating burrows for shelter. Digging requires large forces produced by muscles and transmitted to the soil via the skeletal system; thus fossorial mammals tend to have characteristic modifications of the musculoskeletal system that reflect their digging ability. Bandicoots (Marsupialia: Peramelidae) scratch-dig mainly to source food, searching for subterranean food items including invertebrates, seeds, and fungi. They have musculoskeletal features for digging, including shortened, robust forelimb bones, large muscles, and enlarged muscle attachment areas. Here, we compared changes in the ontogenetic development of muscles associated with digging in the Quenda (Isoodon fusciventer). We measured muscle mass (m m ), pennation angle, and fiber length (FL) to calculate physiological cross-sectional area (PCSA; a proxy of maximum isometric force) as well as estimate the maximum isometric force (Fmax) for 34 individuals ranging in body size from 124 to 2,390 g. Males grow larger than females in this bandicoot species, however, we found negligible sex differences in mass-specific m m , PCSA or FL for our sample. Majority of the forelimb muscles PCSA showed a positive allometric relationship with total body mass, while m m and FL in the majority of forelimb muscles showed isometry. Mechanical similarity was tested, and two thirds of forelimb muscles maximum isometric forces (Fmax) scaled with isometry; therefore the forelimb is primarily mechanical similar throughout ontogeny. PCSA showed a significant difference between scaling slopes between main movers in the power stroke, and main movers of the recovery stroke of scratch-digging. This suggests that some forelimb muscles grow with positive allometry, specially these associated with the power stroke of digging. Intraspecific variation in PCSA is rarely considered in the literature, and thus this is an important study quantifying changes in muscle architectural properties with growth in a mammalian model of scratch-digging.

Too much of a good thing? Finding the most informative genetic data set to answer conservation questions.

Molecular markers are a useful tool allowing conservation and population managers to shed light on genetic processes affecting threatened populations. However, as technological advancements in molecular techniques continue to evolve, conservationists are frequently faced with new genetic markers, each with nuanced variation in their characteristics as well as advantages and disadvantages for informing various questions. We used a well-studied population of Tasmanian devils (Sarcophilus harrisii) from Maria Island, Tasmania, to illustrate the issues associated with combining multiple genetic data sets and to help answer a question posed by many population managers: which data set will provide the most precise and accurate estimates of the population processes we are trying to measure? We analysed individual heterozygosity (as internal relatedness, IR) of 96 individuals, calculated using four genetic marker types (putatively neutral microsatellites, major histocompatibility complex-linked microsatellites, reduced representation sequencing, and candidate region resequencing). We found no correlation in IR values across marker types, suggesting that various genetic markers reflect different aspects of genomic diversity. In addition, some marker types were more informative than others for conservation decision-making. Reduced representation sequencing provided the highest precision (lowest error) for estimating population-level genetic diversity, and most closely reflected genome-wide heterozygosity both theoretically and empirically. Within the conservation context, our results highlight important considerations when choosing a molecular technique for wildlife genetics.

Molecular identification and gene expression profiles of the T cell receptors and co-receptors in developing red-tailed phascogale (Phascogale calura) pouch young.

Expressed coding sequences were identified for the T cell receptors TCRα, TCRß, TCRγ, TCRδ, TCRµ, and co-receptors CD3ε, CD4, and CD8α in the red-tailed phascogale (Phascogale calura). Expression of these genes was examined in the developing thymus and spleen of pouch young and juvenile individuals to detect any changes in expressionrelative to developmental stages. Transcripts of CD3ε, CD4, TCRδ and TCRµ were first detected in the thorax at 1 day postpartum (dpp), and transcripts of all target genes were detected in the thymus from 3 dpp, and the spleen from 17 dpp. The relative expression of all target genes changed significantly over the course of pouch life and was associated with histological milestones of both the thymus and the spleen. Peak gene expression was observed early in pouch life in the thymus, with a decrease in expression associated with timing of thymic involution, while expression peaked later in the spleen and was associated with the initial differentiation of the red and white pulp and a population of T cells forming the periarterial lymphatic sheath (PALS). Active expression of these target genes within the thymus and spleen indicates that the young marsupials are actively developing immunocompetence during pouch life.

Transcriptomic changes in the pre-implantation uterus highlight histotrophic nutrition of the developing marsupial embryo.

Early pregnancy is a critical time for successful reproduction; up to half of human pregnancies fail before the development of the definitive chorioallantoic placenta. Unlike the situation in eutherian mammals, marsupial pregnancy is characterised by a long pre-implantation period prior to the development of the short-lived placenta, making them ideal models for study of the uterine environment promoting embryonic survival pre-implantation. Here we present a transcriptomic study of pre-implantation marsupial pregnancy, and identify differentially expressed genes in the Sminthopsis crassicaudata uterus involved in metabolism and biosynthesis, transport, immunity, tissue remodelling, and uterine receptivity. Interestingly, almost one quarter of the top 50 genes that are differentially upregulated in early pregnancy are putatively involved in histotrophy, highlighting the importance of nutrient transport to the conceptus prior to the development of the placenta. This work furthers our understanding of the mechanisms underlying survival of pre-implantation embryos in the earliest live bearing ancestors of mammals.

Multiple events of gene manipulation via in pouch electroporation in a marsupial model of mammalian forebrain development.

BACKGROUND: The technique of in utero electroporation has been widely used in eutherians, such as mice and rats, to investigate brain development by selectively manipulating gene expression in specific neuronal populations. A major challenge, however, is that surgery is required to access the embryos, affecting animal survival and limiting the number of times it can be performed within the same litter. NEW METHOD: Marsupials are born at an early stage of brain development as compared to eutherians. Forebrain neurogenesis occurs mostly postnatally, allowing electroporation to be performed while joeys develop attached to the teat. Here we describe the method of in pouch electroporation using the Australian marsupial fat-tailed dunnart (Sminthopsis crassicaudata, Dasyuridae). RESULTS: In pouch electroporation is minimally invasive, quick, successful and anatomically precise. Moreover, as no surgery is required, it can be performed several times in the same individual, and littermates can undergo independent treatments. COMPARISON WITH EXISTING METHOD: As compared to in utero electroporation in rodents, in pouch electroporation in marsupials offers unprecedented opportunities to study brain development in a minimally invasive manner. Continuous access to developing joeys during a protracted period of cortical development allows multiple and independent genetic manipulations to study the interaction of different systems during brain development. CONCLUSIONS: In pouch electroporation in marsupials offers an excellent in vivo assay to study forebrain development and evolution. By combining developmental, functional and comparative approaches, this system offers new avenues to investigate questions of biological and medical relevance, such as the precise mechanisms of brain wiring and the organismic and environmental influences on neural circuit formation.

Development of body, head and brain features in the Australian fat-tailed dunnart (Sminthopsis crassicaudata; Marsupialia: Dasyuridae); A postnatal model of forebrain formation.

Most of our understanding of forebrain development comes from research of eutherian mammals, such as rodents, primates, and carnivores. However, as the cerebral cortex forms largely prenatally, observation and manipulation of its development has required invasive and/or ex vivo procedures. Marsupials, on the other hand, are born at comparatively earlier stages of development and most events of forebrain formation occur once attached to the teat, thereby permitting continuous and non-invasive experimental access. Here, we take advantage of this aspect of marsupial biology to establish and characterise a resourceful laboratory model of forebrain development: the fat-tailed dunnart (Sminthopsis crassicaudata), a mouse-sized carnivorous Australian marsupial. We present an anatomical description of the postnatal development of the body, head and brain in dunnarts, and provide a staging system compatible with human and mouse developmental stages. As compared to eutherians, the orofacial region develops earlier in dunnarts, while forebrain development is largely protracted, extending for more than 40 days versus ca. 15 days in mice. We discuss the benefits of fat-tailed dunnarts as laboratory animals in studies of developmental biology, with an emphasis on how their accessibility in the pouch can help address new experimental questions, especially regarding mechanisms of brain development and evolution.

The role of basking in the development of endothermy and torpor in a marsupial.

Marsupials have a slow rate of development and this allows a detailed examination of thermoregulatory developmental changes and stages. We quantified the cooling rates of marsupial dunnarts (Sminthopsis crassicaudata) at 40-56 days (d) old, and torpor and basking behaviour in animals given the option to bask in four age groups from 60 to 150 d. The development of thermoregulation was a continuum, but was characterised by three major thermoregulatory stages: (1) at 40 d, animals were unable to maintain a constant high body temperature during short-term cold exposure; (2) at 60 d, animals could maintain a high T b for the first part of the night at an ambient temperature of 15.0 ± 0.7 °C; later in the night, they entered an apparent torpor bout but could only rewarm passively when basking under a heat lamp; (3) from ~90 d, they expressed prolonged torpor bouts and were able to rewarm endogenously. Young newly weaned 60 d animals were able to avoid hypothermia by basking. In this case, basking was not an optional behavioural method of reducing the cost of rewarming from torpor, but was essential for thermoregulation independent of the nest temperature. Results from our study suggest that basking is a crucial behavioural trait that permits young marsupials and perhaps other juvenile altricial mammals to overcome the developmental stage between poikilothermy early in development and full endothermy later in life.

Patterns in the bony skull development of marsupials: high variation in onset of ossification and conserved regions of bone contact.

Development in marsupials is specialized towards an extremely short gestation and highly altricial newborns. As a result, marsupial neonates display morphological adaptations at birth related to functional constraints. However, little is known about the variability of marsupial skull development and its relation to morphological diversity. We studied bony skull development in five marsupial species. The relative timing of the onset of ossification was compared to literature data and the ossification sequence of the marsupial ancestor was reconstructed using squared-change parsimony. The high range of variation in the onset of ossification meant that no patterns could be observed that differentiate species. This finding challenges traditional studies concentrating on the onset of ossification as a marker for phylogeny or as a functional proxy. Our study presents observations on the developmental timing of cranial bone-to-bone contacts and their evolutionary implications. Although certain bone contacts display high levels of variation, connections of early and late development are quite conserved and informative. Bones that surround the oral cavity are generally the first to connect and the bones of the occipital region are among the last. We conclude that bone contact is preferable over onset of ossification for studying cranial bone development.

Leptin levels, seasonality and thermal acclimation in the Microbiotherid marsupial Dromiciops gliroides: Does photoperiod play a role?

Mammals of the Neotropics are characterized by a marked annual cycle of activity, which is accompanied by several physiological changes at the levels of the whole organism, organs and tissues. The physiological characterization of these cycles is important, as it gives insight on the mechanisms by which animals adjust adaptively to seasonality. Here we studied the seasonal changes in blood biochemical parameters in the relict South American marsupial Dromiciops gliroides ("monito del monte" or "little mountain monkey"), under semi-natural conditions. We manipulated thermal conditions in order to characterize the effects of temperature and season on a battery of biochemical parameters, body mass and adiposity. Our results indicate that monitos experience an annual cycle in body mass and adiposity (measured as leptin levels), reaching a maximum in winter and a minimum in summer. Blood biochemistry confirms that the nutritional condition of animals is reduced in summer instead of winter (as generally reported). This was coincident with a reduction of several biochemical parameters in summer, such as betahydroxybutyrate, cholesterol, total protein concentration and globulins. Monitos seem to initiate winter preparation during autumn and reach maximum body reserves in winter. Hibernation lasts until spring, at which time they use fat reserves and become reproductively active. Sexual maturation during summer would be the strongest energetic bottleneck, which explains the reductions in body mass and other parameters in this season. Overall, this study suggests that monitos anticipate the cold season by a complex interaction of photoperiodic and thermal cues.

Temporal Patterns in the Abundance of a Critically Endangered Marsupial Relates to Disturbance by Roads and Agriculture.

The aim of this study was to investigate how landscape disturbance associated with roads, agriculture and forestry influenced temporal patterns in woylie (Bettongia penicillata) abundance before, during and after periods of rapid population change. Data were collected from an area of approximately 140,000 ha of forest within the Upper Warren region in south-western Australia. Woylie abundance was measured using cage trapping at 22 grid and five transect locations with varying degrees of landscape disturbance between 1994 and 2012. We found evidence that the distribution and abundance of woylies over time appears to be related to the degree of fragmentation by roads and proximity to agriculture. Sites furthest from agriculture supported a greater abundance of woylies and had slower rates of population decline. Sites with fewer roads had a greater abundance of woylies generally and a greater rate of increase in abundance after the implementation of invasive predator control. The results of this study suggest that landscape disturbance is less important at peak population densities, but during times of environmental and population change, sites less dissected by roads and agriculture better support woylie populations. This may be due to the role these factors play in increasing the vulnerability of woylies to introduced predators, population fragmentation, weed species invasion, mortality from road collisions or a reduction in available habitat. Strategies that reduce the impact of disturbance on woylie populations could include the rationalisation of forest tracks and consolidation of contiguous habitat through the acquisition of private property. Reducing the impact of disturbance in the Upper Warren region could improve the resilience of this critically important woylie population during future environmental change.