Genetic diversity and population structure of Tasmanian devils, the largest marsupial carnivore

Genetic diversity and population structure were investigated across the core range of Tasmanian devils (Sarcophilus laniarius; Dasyuridae), a wide-ranging marsupial carnivore restricted to the island of Tasmania. Heterozygosity (0.386-0.467) and allelic diversity (2.7-3.3) were low in all subpopulations and allelic size ranges were small and almost continuous, consistent with a founder effect. Island effects and repeated periods of low population density may also have contributed to the low variation. Within continuous habitat, gene flow appears extensive up to 50 km (high assignment rates to source or close neighbour populations; nonsignificant values of pairwise F-ST), in agreement with movement data. At larger scales (150-250 km), gene flow is reduced (significant pairwise F-ST) but there is no evidence for isolation by distance. The most substantial genetic structuring was observed for comparisons spanning unsuitable habitat, implying limited dispersal of devils between the well-connected, eastern populations and a smaller northwestern population. The genetic distinctiveness of the northwestern population was reflected in all analyses: unique alleles; multivariate analyses of gene frequency (multidimensional scaling, minimum spanning tree, nearest neighbour); high self-assignment (95%); two distinct populations for Tasmania were detected in isolation by distance and in Bayesian model-based clustering analyses. Marsupial carnivores appear to have stronger population subdivisions than their placental counterparts.

The effects of habitat fragmentation due to forestry plantation establishment on the demography and genetic variation of a marsupial carnivore, Antechinus agilis

We conducted a demographic and genetic study to investigate the effects of fragmentation due to the establishment of an exotic softwood plantation on populations of a small marsupial carnivore, the agile antechinus (Antechinus agilis), and the factors influencing the persistence of those populations in the fragmented habitat. The first aspect of the study was a descriptive analysis of patch occupancy and population size, in which we found a patch occupancy rate of 70% among 23 sites in the fragmented habitat compared to 100% among 48 sites with the same habitat characteristics in unfragmented habitat. Mark-recapture analyses yielded most-likely population size estimates of between 3 and 85 among the 16 occupied patches in the fragmented habitat. Hierarchical partitioning and model selection were used to identify geographic and habitat-related characteristics that influence patch occupancy and population size. Patch occupancy was primarily influenced by geographic isolation and habitat quality (vegetation basal area). The variance in population size among occupied sites was influenced primarily by forest type (dominant Eucalyptus species) and, to a lesser extent, by patch area and topographic context (gully sites had larger populations). A comparison of the sex ratios between the samples from the two habitat contexts revealed a significant deficiency of males in the fragmented habitat. We hypothesise that this is due to male-biased dispersal in an environment with increased dispersal-associated mortality. The population size and sex ratio data were incorporated into a simulation study to estimate the proportion of genetic diversity that would have been lost over the known timescale since fragmentation if the patch populations had been totally isolated. The observed difference in genetic diversity (gene diversity and allelic richness at microsatellite and mitochondrial markers) between 16 fragmented and 12 unfragmented sites was extremely low and inconsistent with the isolation of the patch populations. Our results show that although the remnant habitat patches comprise approximately 2% of the study area, they can support non-isolated populations. However, the distribution of agile antechinus populations in the fragmented system is dependent on habitat quality and patch connectivity. (C) 2004 Elsevier Ltd. All rights reserved.

An alternative method for predicting body mass: the case of the Pleistocene marsupial lion

Accurate estimates of body mass in fossil taxa are fundamental to paleobiological reconstruction. Predictive equations derived from correlation with craniodental and body mass data in extant taxa are the most commonly used, but they can be unreliable for species whose morphology departs widely from that of living relatives. Estimates based on proximal limb-bone circumference data are more accurate but are inapplicable where postcranial remains are unknown. In this study we assess the efficacy of predicting body mass in Australian fossil marsupials by using an alternative correlate, endocranial volume. Body mass estimates for a species with highly unusual craniodental anatomy, the Pleistocene marsupial lion (Thylacoleo carnifex), fall within the range determined on the basis of proximal limb-bone circumference data, whereas estimates based on dental data are highly dubious. For all marsupial taxa considered, allometric relationships have small confidence intervals, and percent prediction errors are comparable to those of the best predictors using craniodental data. Although application is limited in some respects, this method may provide a useful means of estimating body mass for species with atypical craniodental or postcranial morphologies and taxa unrepresented by postcranial remains. A trend toward increased encephalization may constrain the method's predictive power with respect to many, but not all, placental clades.

The prion protein gene: Identifying regulatory signals using marsupial sequence

The function of the prion protein gene (PRNP) and its normal product PrPC is elusive. We used comparative genomics as a strategy to understand the normal function of PRNP. As the reliability of comparisons increases with the number of species and increased evolutionary distance, we isolated and sequenced a 66.5 kb BAC containing the PRNP gene from a distantly related mammal, the model Australian marsupial Macropus eugenii (tammar wallaby). Marsupials are separated from eutherians such as human and mouse by roughly 180 million years of independent evolution. We found that tammar PRNP, like human PRNP, has two exons. Prion proteins encoded by the tammar wallaby and a distantly related marsupial, Monodelphis domestica (Brazilian opossum) PRNP contain proximal PrP repeats with a distinct, marsupial-specific composition and a variable number. Comparisons of tammar wallaby PRNP with PRNPs from human, mouse, bovine and ovine allowed us to identify non-coding gene regions conserved across the marsupial-eutherian evolutionary distance, which are candidates for regulatory regions. In the PRNP 3' UTR we found a conserved signal for nuclear-specific polyadenylation and the putative cytoplasmic polyadenylation element (CPE), indicating that post-transcriptional control of PRNP mRNA activity is important. Phylogenetic footprinting revealed conserved potential binding sites for the MZF-1 transcription factor in both upstream promoter and intron/intron 1, and for the MEF2, MyTI, Oct-1 and NFAT transcription factors in the intron(s). The presence of a conserved NFAT-binding site and CPE indicates involvement of PrPC in signal transduction and synaptic plasticity. (c) 2004 Elsevier B.V. All rights reserved.

Restricted mating dispersal and strong breeding group structure in a mid-sized marsupial mammal (Petrogale penicillata)

Ecological genetic studies have demonstrated that spatial patterns of mating dispersal, the dispersal of gametes through mating behaviour, can facilitate inbreeding avoidance and strongly influence the structure of populations, particularly in highly philopatric species. Elements of breeding group dynamics, such as strong structuring and sex-biased dispersal among groups, can also minimize inbreeding and positively influence levels of genetic diversity within populations. Rock-wallabies are highly philopatric mid-sized mammals whose strong dependence on rocky terrain has resulted in series of discreet, small colonies in the landscape. Populations show no signs of inbreeding and maintain high levels of genetic diversity despite strong patterns of limited gene flow within and among colonies. We used this species to investigate the importance of mating dispersal and breeding group structure to inbreeding avoidance within a 'small' population. We examined the spatial patterns of mating dispersal, the extent of kinship within breeding groups, and the degree of relatedness among brush-tailed rock-wallaby breeding pairs within a colony in southeast Queensland. Parentage data revealed remarkably restricted mating dispersal and strong breeding group structuring for a mid-sized mammal. Breeding groups showed significant levels of female kinship with evidence of male dispersal among groups. We found no evidence for inbreeding avoidance through mate choice; however, anecdotal data suggest the importance of life history traits to inbreeding avoidance between first-degree relatives. We suggest that the restricted pattern of mating dispersal and strong breeding group structuring facilitates inbreeding avoidance within colonies. These results provide insight into the population structure and maintenance of genetic diversity within colonies of the threatened brush-tailed rock-wallaby.

Significant patterns of population genetic structure and limited gene flow in a threatened macropodid marsupial despite continuous habitat in southeast Queensland, Australia

Many endangered species worldwide are found in remnant populations, often within fragmented landscapes. However, when possible, an understanding of the natural extent of population structure and dispersal behaviour of threatened species would assist in their conservation and management. The brush-tailed rock-wallaby (Petrogale penicillata), a once abundant and widespread rock-wallaby species across southeastern Australia, has become nearly extinct across much of the southern part of its range. However, the northern part of the species' range still sustains many small colonies closely distributed across suitable habitat, providing a rare opportunity to investigate the natural population dynamics of a listed threatened species. We used 12 microsatellite markers to investigate genetic diversity, population structure and gene flow among brush-tailed rock-wallaby colonies within and among two valley regions with continuous habitat in southeast Queensland. We documented high and signifcant levels of population genetic structure between rock-wallaby colonies embedded in continuous escarpment habitat and forest. We found a strong and significant pattern of isolation-by-distance among colonies indicating restricted gene flow over a small geographic scale (< 10 km) and conclude that gene flow is more likely limited by intrinsic factors rather than environmental factors. In addition, we provide evidence that genetic diversity was significantly lower in colonies located in a more isolated valley region compared to colonies located in a valley region surrounded by continuous habitat. These findings shed light on the processes that have resulted in the endangered status of rock-wallaby species in Australia and they have strong implications for the conservation and management of both the remaining 'connected' brush-tailed rock-wallaby colonies in the northern parts of the species' range and the remnant endangered populations in the south.

Visual thalamocortical projections in the flying fox: Parallel pathways to striate and extrastriate areas

We studied thalamic projections to the visual cortex in flying foxes, animals that share neural features believed to resemble those present in the brains of early primates. Neurones labeled by injections of fluorescent tracers in striate and extrastriate cortices were charted relative to the architectural boundaries of thalamic nuclei. Three main findings are reported: First, there are parallel lateral geniculate nucleus (LGN) projections to striate and extrastriate cortices. Second, the pulvinar complex is expansive, and contains multiple subdivisions. Third, across the visual thalamus, the location of cells labeled after visual cortex injections changes systematically, with caudal visual areas receiving their strongest projections from the most lateral thalamic nuclei, and rostral areas receiving strong projections from medial nuclei. We identified three architectural layers in the LGN, and three subdivisions of the pulvinar complex. The outer LGN layer contained the largest cells, and had strong projections to the areas V1, V2 and V3. Neurones in the intermediate LGN layer were intermediate in size, and projected to V1 and, less densely, to V2. The layer nearest to the origin of the optic radiation contained the smallest cells, and projected not only to V1, V2 and V3, but also, weakly, to the occipitotemporal area (OT, which is similar to primate middle temporal area) and the occipitoparietal area (OP, a third tier area located near the dorsal midline). V1, V2 and V3 received strong projections from the lateral and intermediate subdivisions of the pulvinar complex, while OP and OT received their main thalamic input from the intermediate and medial subdivisions of the pulvinar complex. These results suggest parallels with the carnivore visual system, and indicate that the restriction of the projections of the large- and intermediatesized LGN layers to V1, observed in present-day primates, evolved from a more generalized mammalian condition. (C) 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

Bi-sensory, striped representations: comparative insights from owl and platypus

Bi-sensory striped arrays are described in owl and platypus that share some similarities with the other variant of bi-sensory striped array found in primate and carnivore striate cortex: ocular dominance columns. Like ocular dominance columns, the owl and platypus striped systems each involve two different topographic arrays that are cut into parallel stripes, and interdigitated, so that higher-order neurons can integrate across both arrays. Unlike ocular dominance stripes, which have a separate array for each eye, the striped array in the middle third of the owl tectum has a separate array for each cerebral hemisphere. Binocular neurons send outputs from both hemispheres to the striped array where they are segregated into parallel stripes according to hemisphere of origin. In platypus primary somatosensory cortex (SI), the two arrays of interdigitated stripes are derived from separate sensory systems in the bill, 40,000 electroreceptors and 60,000 mechanoreceptors. The stripes in platypus SI cortex produce bimodal electrosensory-mechanosensory neurons with specificity for the time-of-arrival difference between the two systems. This thunder-and-lightning system would allow the platypus to estimate the distance of the prey using time disparities generated at the bill between the earlier electrical wave and the later mechanical wave caused by the motion of benthic prey. The functional significance of parallel, striped arrays is not clear, even for the highly-studied ocular dominance system, but a general strategy is proposed here that is based on the detection of temporal disparities between the two arrays that can be used to estimate distance. (C) 2004 Elsevier Ltd. All rights reserved.

Sperm membrane fatty acid composition in the Eastern grey kangaroo (Macropus giganteus), koala (Phascolarctos cinereus), and common wombat (Vombatus ursinus) and its relationship to cold shock injury and cryopreservation success

Marsupial spermatozoa tolerate cold shock well, but differ in cryopreservation tolerance. In an attempt to explain these phenomena, the fatty acid composition of the sperm membrane from caput and cauda epididymides of the Eastern grey kangaroo, koala, and common wombat was measured and membrane sterol levels were measured in cauda epididymidal spermatozoa. While species-related differences in the levels of linolenic acid (18:3, n-6) and arachidonic acid (20:4, n-6) were observed in caput epididymal spermatozoa, these differences failed to significantly alter the ratio of unsaturated/saturated membrane fatty acids. However in cauda epididymidal spermatozoa, the ratio of unsaturated/saturated membrane fatty acids in koala and kangaroo spermatozoa was approximately 7.6 and 5.2, respectively; substantially higher than any other mammalian species so far described. Koala spermatozoal membranes had a higher ratio of unsaturated/saturated membrane fatty acids than that of wombat spermatozoa (t = 3.81; df = 4; p less than or equal to 0.02); however, there was no significant difference between wombat and kangaroo spermatozoa. The highest proportions of DHA (22:6, n-3), the predominant membrane fatty acid in cauda epididymidal spermatozoa, were found in wombat and koala spermatozoa. While species-related differences in membrane sterol levels (cholesterol and desmosterol) were observed in cauda epididymidal spermatozoa, marsupial membrane sterol levels are very low. Marsupial spermatozoal membrane analyses do not support the hypothesis that a high ratio of saturated/unsaturated membrane fatty acids and low membrane sterol levels predisposes spermatozoa to cold shock damage. Instead, cryogenic tolerance appears related to DHA levels. (C) 2004 Elsevier Inc. All rights reserved.

Cone topography and spectral sensitivity in two potentially trichromatic marsupials, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus)

The potential for trichromacy in mammals, thought to be unique to primates, was recently discovered in two Australian marsupials. Whether the presence of three cone types, sensitive to short- (SWS), medium-(MWS) and long-(LWS) wavelengths, occurs across all marsupials remains unknown. Here, we have investigated the presence, distribution and spectral sensitivity of cone types in two further species, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus). Immunohistochemistry revealed that SWS cones in the quokka are concentrated in dorso-temporal retina, while in the quenda, two peaks were identified in naso-ventral and dorso-temporal retina. In both species, MWS/LWS cone spatial distributions matched those of retinal ganglion cells. Microspectrophotometry (MSP) confirmed that MWS and LWS cones are spectrally distinct, with mean wavelengths of maximum absorbance at 502 and 538 nm in the quokka, and at 509 and 551 nm, in the quenda. Although small SWS cone outer segments precluded MSP measurements, molecular analysis identified substitutions at key sites, accounting for a spectral shift from ultraviolet in the quenda to violet in the quokka. The presence of three cone types, along with previous findings in the fat-tailed dunnart and honey possum, suggests that three spectrally distinct cone types are a feature spanning the marsupials.

Plant-based immunocontraceptive control of wildlife - "potentials, limitations, and possums"

Possums (Trichosurus vulpecula), originally introduced from Australia, are spread over 90% of New Zealand and cause major economic and environmental damage. Immunocontraception has been suggested as a humane means to control them. Marsupial-specific reproductive antigens expressed at high levels in edible transgenic plant tissue might provide a safe, effective, and cheap oral delivery bait for immuno-contraceptive control. As proof of concept, female possums vaccinated with immunocontraceptive antigens showed reduced fertility, and possums fed with potato-expressed heat labile toxin-B (LT-B) had mucosal and systemic immune responses to the antigen. This demonstrated that immunocontraception was effective in possums and that oral delivery in edible plant material might be possible. Nuclear transformation with reporter genes showed that transgenic carrot roots accumulate high levels of foreign protein in edible tissues, indicating their potential as a delivery vector. However, prior to attempts at large scale production, more effective immunocontraceptive antigen-adjuvant formulations are probably required before plant-based immunocontraception can become a major tool for immunocontraceptive control of overabundant vertebrate pests. (c) 2004 Elsevier Ltd. All rights reserved.

Papillomavirus in healthy skin of Australian animals

Papillomaviruses are a group of ubiquitous viruses that are often found in normal skin of humans, as well as a range of different vertebrates. In this study, swab samples collected from the healthy skin of 225 Australian animals from 54 species were analysed for the presence of papillomavirus DNA with the general skin papillomavirus primer pair FAP59/FAP64. A total of five putative and potential new animal papillomavirus types were identified from three different animal species. The papillomaviruses were detected in one monotreme and two marsupial species: three from koalas, and one each from an Eastern grey kangaroo and an echidna. The papillomavirus prevalence in the three species was 14% (10/72) in koalas, 20% (1/5) in echidnas and 4% (1/23) in Eastern grey kangaroos. Phylogenetic analysis was performed on the putative koala papillomavirus type that could be cloned and it appears in the phylogenetic tree as a novel putative papillomavirus; genus. The data extend the range of species infected by papillomaviruses to the most primitive mammals: the monotremes and the marsupials.

An investigation into the similarities and differences governing the cryopreservation success of koala (Phascolarctos cinereus: goldfuss) and common wombat (Vombatus ursinus: shaw) spermatozoa

The aim of this study was to determine the relative cryopreservation success of koala and wombat spermatozoa and to investigate reasons for their respective post-thaw survival by examining the sperm's response to a range of osmotic media and determining the presence and distribution of F-actin. An hypothesis was proposed that F-actin may be imparting a degree of structural inflexibility to the koala sperm plasma membrane; hence, exposure of spermatozoa to cytochalasin D (5 mu M), a F-actin depolymerisation agent, should result in increased plasticisation of the membrane and greater tolerance of cell volume changes that typically occur during cryopreservation. In experiment 1, koala (n = 4) and wombat (n = 4) spermatozoa packaged in 0.25 mL straws were cryopreserved using two freezing rates (fast-3 cm above liquid N2 interface; slow-6 degrees C/min in a freezing chamber) and two glycerol concentrations (8 and 14% v/v) in a tris-citrate glucose buffer with 15% (v/v) egg yolk. Wombat spermatozoa showed better (P < 0.01) post-thaw survival (% motile, % intact plasma membranes, % decondensed sperm heads) than koala spermatozoa. When exposed to media of varying osmolality, koala spermatozoa were less tolerant (% intact plasma membrane) of hyper-osmotic conditions (920 and 1410mOsmol/kg) than wombat spermatozoa. F-actin was localised using a monoclonal antibody but only found in the wombat sperm head. When koala and wombat spermatozoa were exposed to media of varying osmolality, cytochalasin D had no beneficial effect on sperm survival (% intact plasma membranes). This study has demonstrated that wombat spermatozoa are highly tolerant of cryopreservation when compared to koala sperm but that spermatozoa from both species show greatest post-thaw survival when frozen slowly in 14% glycerol. Koala sperm are also particularly susceptible to hyper-osmotic environments but lack of detectable F-actin in the koala spermatozoan suggests that poor cryopreservation success in this species is unlikely to be associated with F-actin induced plasma membrane inflexibility. (c) 2006 Elsevier Inc. All rights reserved.