Population genomics of a predatory mammal reveals patterns of decline and impacts of exposure to toxic toads.

Mammal declines across northern Australia are one of the major biodiversity loss events occurring globally. There has been no regional assessment of the implications of these species declines for genomic diversity. To address this, we conducted a species-wide assessment of genomic diversity in the northern quoll (Dasyurus hallucatus), an Endangered marsupial carnivore. We used next generation sequencing methods to genotype 10,191 single nucleotide polymorphisms (SNPs) in 352 individuals from across a 3220-km length of the continent, investigating patterns of population genomic structure and diversity, and identifying loci showing signals of putative selection. We found strong heterogeneity in the distribution of genomic diversity across the continent, characterized by (i) biogeographical barriers driving hierarchical population structure through long-term isolation, and (ii) severe reductions in diversity resulting from population declines, exacerbated by the spread of introduced toxic cane toads (Rhinella marina). These results warn of a large ongoing loss of genomic diversity and associated adaptive capacity as mammals decline across northern Australia. Encouragingly, populations of the northern quoll established on toad-free islands by translocations appear to have maintained most of the initial genomic diversity after 16 years. By mapping patterns of genomic diversity within and among populations, and investigating these patterns in the context of population declines, we can provide conservation managers with data critical to informed decision-making. This includes the identification of populations that are candidates for genetic management, the importance of remnant island and insurance/translocated populations for the conservation of genetic diversity, and the characterization of putative evolutionarily significant units.

Population genomics and conservation management of a declining tropical rodent.

Conservation management is improved by incorporating information about the spatial distribution of population genetic diversity into planning strategies. Northern Australia is the location of some of the world's most severe ongoing declines of endemic mammal species, yet we have little genetic information from this regional mammal assemblage to inform a genetic perspective on conservation assessment and planning. We used next-generation sequencing data from remnant populations of the threatened brush-tailed rabbit-rat (Conilurus penicillatus) to compare patterns of genomic diversity and differentiation across the landscape and investigate standardised hierarchical genomic diversity metrics to better understand brush-tailed rabbit-rat population genomic structure. We found strong population structuring, with high levels of differentiation between populations (FST = 0.21-0.78). Two distinct genomic lineages between the Tiwi Islands and mainland are also present. Prioritisation analysis showed that one population in both lineages would need to be conserved to retain at least ~80% of alleles for the species. Analysis of standardised genomic diversity metrics showed that approximately half of the total diversity occurs among lineages (δ = 0.091 from grand total γ = 0.184). We suggest that a focus on conserving remnant island populations may not be appropriate for the preservation of species-level genomic diversity and adaptive potential, as these populations represent a small component of the total diversity and a narrow subset of the environmental conditions in which the species occurs. We also highlight the importance of considering both genomic and ecological differentiation between source and receiving populations when considering translocations for conservation purposes.

Overlapping den tree selection by three declining arboreal mammal species in an Australian tropical savanna.

Tree cavities are important denning sites for many arboreal mammals. Knowledge of cavity requirements of individual species, as well as potential den overlap among species, is integral to their conservation. In Australia's tropical savannas, development of tree cavities is enhanced by high termite activity, and, conversely, reduced by frequent fires. However, it is poorly understood how the availability of tree cavities in the tropical savannas impacts tree cavity use and selection by cavity-dependent fauna. There has been a severe decline among arboreal mammal species in northern Australia over recent decades. Investigation of their cavity requirements may illuminate why these species have declined drastically in some areas but are persisting in others. Here we examined this issue in three species of arboreal mammals (Trichosurus vulpecula, Mesembriomys gouldii, Conilurus penicillatus) on Melville Island, northern Australia. We radiotracked individuals to their den sites to evaluate whether the species differ in their den tree and tree-cavity selection. The strongest influence on den tree selection was the presence of large cavities (> 10 cm entrance diameter), with all three species using larger cavities most frequently. Conilurus penicillatus, the smallest species, differed the most from the other species: it frequently was found in smaller, dead trees and its den sites were closer to the ground, including in hollow logs. The two larger species had broader den tree use, using larger live trees and dens higher up in the canopy. Dens of C. penicillatus are likely to be more susceptible to predation and destruction by high-intensity savanna fires. This may have contributed to this species' rapid decline, both on Melville Island and on the mainland. However, the apparent preference for larger tree cavities by all three arboreal species is concerning due to the limited availability of large trees across Australian savannas, which are subject to frequent, high-intensity fires.

Occupancy of the Invasive Feral Cat Varies with Habitat Complexity.

The domestic cat (Felis catus) is an invasive exotic in many locations around the world and is thought to be a key factor driving recent mammal declines across northern Australia. Many mammal species native to this region now persist only in areas with high topographic complexity, provided by features such as gorges or escarpments. Do mammals persist in these habitats because cats occupy them less, or despite high cat occupancy? We show that occupancy of feral cats was lower in mammal-rich habitats of high topographic complexity. These results support the idea that predation pressure by feral cats is a factor contributing to the collapse of mammal communities across northern Australia. Managing impacts of feral cats is a global conservation challenge. Conservation actions such as choosing sites for small mammal reintroductions may be more successful if variation in cat occupancy with landscape features is taken into account.

Influence of Fire Mosaics, Habitat Characteristics and Cattle Disturbance on Mammals in Fire-Prone Savanna Landscapes of the Northern Kimberley.

Patch mosaic burning, in which fire is used to produce a mosaic of habitat patches representative of a range of fire histories ('pyrodiversity'), has been widely advocated to promote greater biodiversity. However, the details of desired fire mosaics for prescribed burning programs are often unspecified. Threatened small to medium-sized mammals (35 g to 5.5 kg) in the fire-prone tropical savannas of Australia appear to be particularly fire-sensitive. Consequently, a clear understanding of which properties of fire mosaics are most instrumental in influencing savanna mammal populations is critical. Here we use mammal capture data, remotely sensed fire information (i.e. time since last fire, fire frequency, frequency of late dry season fires, diversity of post-fire ages in 3 km radius, and spatial extent of recently burnt, intermediate and long unburnt habitat) and structural habitat attributes (including an index of cattle disturbance) to examine which characteristics of fire mosaics most influence mammals in the north-west Kimberley. We used general linear models to examine the relationship between fire mosaic and habitat attributes on total mammal abundance and richness, and the abundance of the most commonly detected species. Strong negative associations of mammal abundance and richness with frequency of late dry season fires, the spatial extent of recently burnt habitat (post-fire age <1 year within 3 km radius) and level of cattle disturbance were observed. Shrub cover was positively related to both mammal abundance and richness, and availability of rock crevices, ground vegetation cover and spatial extent of ≥4 years unburnt habitat were all positively associated with at least some of the mammal species modelled. We found little support for diversity of post-fire age classes in the models. Our results indicate that both a high frequency of intense late dry season fires and extensive, recently burnt vegetation are likely to be detrimental to mammals in the north Kimberley. A managed fire mosaic that reduces large scale and intense fires, including the retention of ≥4 years unburnt patches, will clearly benefit savanna mammals. We also highlighted the importance of fire mosaics that retain sufficient shelter for mammals. Along with fire, it is clear that grazing by introduced herbivores also needs to be reduced so that habitat quality is maintained.

A synthesis of postfire recovery traits of woody plants in Australian ecosystems.

Postfire resprouting and recruitment from seed are key plant life-history traits that influence population dynamics, community composition and ecosystem function. Species can have one or both of these mechanisms. They confer resilience, which may determine community composition through differential species persistence after fire. To predict ecosystem level responses to changes in climate and fire conditions, we examined the proportions of these plant fire-adaptive traits among woody growth forms of 2880 taxa, in eight fire-prone ecosystems comprising ~87% of Australia's land area. Shrubs comprised 64% of the taxa. More tree (>84%) than shrub (~50%) taxa resprouted. Basal, epicormic and apical resprouting occurred in 71%, 22% and 3% of the taxa, respectively. Most rainforest taxa (91%) were basal resprouters. Many trees (59%) in frequently-burnt eucalypt forest and savanna resprouted epicormically. Although crown fire killed many mallee (62%) and heathland (48%) taxa, fire-cued seeding was common in these systems. Postfire seeding was uncommon in rainforest and in arid Acacia communities that burnt infrequently at low intensity. Resprouting was positively associated with ecosystem productivity, but resprouting type (e.g. basal or epicormic) was associated with local scale fire activity, especially fire frequency. Although rainforest trees can resprout they cannot recruit after intense fires and may decline under future fires. Semi-arid Acacia communities would be susceptible to increasing fire frequencies because they contain few postfire seeders. Ecosystems dominated by obligate seeders (mallee, heath) are also susceptible because predicted shorter inter-fire intervals will prevent seed bank accumulation. Savanna may be resilient to future fires because of the adaptive advantage of epicormic resprouting among the eucalypts. The substantial non-resprouting shrub component of shrublands may decline, but resilient Eucalyptus spp. will continue to dominate under future fire regimes. These patterns of resprouting and postfire seeding provide new insights to ecosystem assembly, resilience and vulnerability to changing fire regimes on this fire-prone continent.

Mammals of Australia's tropical savannas: a conceptual model of assemblage structure and regulatory factors in the Kimberley region.

We construct a state-and-transition model for mammals in tropical savannas in northern Australia to synthesize ecological knowledge and understand mammalian declines. We aimed to validate the existence of alternative mammal assemblage states similar to those in arid Australian grasslands, and to speculate on transition triggers. Based on the arid grassland model, we hypothesized that assemblages are partitioned across rainfall gradients and between substrates. We also predicted that assemblages typical of arid regions in boom periods would be prevalent in savannas with higher and more regular rainfall. Data from eight mammal surveys from the Kimberley region, Western Australia (1994 to 2011) were collated. Survey sites were partitioned across rainfall zones and habitats. Data allowed us to identify three assemblage states: State 0:--low numbers of mammals, State II:--dominated by omnivorous rodents and State III:--dominated by rodents and larger marsupials. Unlike arid grasslands, assemblage dominance by insectivorous dasyurids (State I) did not occur in savannas. Mammal assemblages were partitioned across rainfall zones and between substrates as predicted, but-unlike arid regions-were not related strongly to yearly rainfall. Mammal assemblage composition showed high regional stability, probably related to high annual rainfall and predictable wet season resource pulses. As a consequence, we speculate that perpetually booming assemblages in savannas allow top-down control of the ecosystem, with suppression of introduced cats by the dingo, the region's top predator. Under conditions of low or erratic productivity, imposed increasingly by intense fire regimes and introduced herbivore grazing, dingoes may not limit impacts of cats on native mammals. These interacting factors may explain contemporary declines of savanna mammals as well as historical declines in arid Australia. The cat-ecosystem productivity hypothesis raised here differs from the already-articulated cat-habitat structure hypothesis for mammal declines, and we suggest approaches for explicit testing of transition triggers for competing hypotheses.

Protection by methylproamine of irradiated human keratinocytes correlates with reduction of DNA damage.

PURPOSE: The therapeutic ratio for ionising radiation treatment of tumour is a trade-off between normal tissue side-effects and tumour control. Application of a radioprotector to normal tissue can reduce side-effects. Here we study the effects of a new radioprotector on the cellular response to radiation. Methylproamine is a DNA-binding radioprotector which, on the basis of published pulse radiolysis studies, acts by repair of transient radiation-induced oxidative species on DNA. To substantiate this hypothesis, we studied protection by methylproamine at both clonogenic survival and radiation-induced DNA damage, assessed by γH2AX (histone 2AX phosphorylation at serine 139) focus formation endpoints. MATERIALS AND METHODS: The human keratinocyte cell line FEP1811 was used to study clonogenic survival and yield of γH2AX foci following irradiation (¹³7Cs γ-rays) of cells exposed to various concentrations of methylproamine. Uptake of methylproamine into cell nuclei was measured in parallel. RESULTS: The extent of radioprotection at the clonogenic survival endpoint increased with methylproamine concentration up to a maximum dose modification factor (DMF) of 2.0 at 10 µM. At least 0.1 fmole/nucleus of methylproamine is required to achieve a substantial level of radioprotection (DMF of 1.3) with maximum protection (DMF of 2.0) achieved at 0.23 fmole/nucleus. The γH2AX focus yield per cell nucleus 45 min after irradiation decreased with drug concentration with a DMF of 2.5 at 10 µM. CONCLUSIONS: These results are consistent with the hypothesis that radioprotection by methylproamine is mediated by attenuation of the extent of initial DNA damage.

Clustered DNA lesion sites as a source of mutations during human colorectal tumourigenesis.

The role of gene mutations in tumourigenesis is well understood, however, the mechanism(s) by which they arise are less clear. Indeed, the common assumption that tumourigenic mutations are the result of DNA replication errors is apparently contradicted by the very low division frequency of the cells from which tumours are thought to arise (i.e. deep stem cells). As a potential solution to this paradox, we tested a model whereby clustered DNA lesion sites (CLS) (where several lesions occur within a few base pairs of each other on opposing strands) could give rise to mutations in quiescent cells. We used statistical analyses to search for sets of dinucleotide sequences (designated target sequences) that are present at and in close proximity to mutation sites in four genes associated with human colorectal tumourigenesis (adenomatosis polyposis coli (APC), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA), and tumour protein p53 (TP53)). The dinucleotides CG, AC-GT, TG, and GC were identified as target sequences in at least three of the genes analysed. Consistent with their designation as target sequences, these dinucleotides have all been identified as high probability sites of oxidative damage formation in in vitro studies. Our results strongly suggest a statistical association between the presence of multiple, clustered target sequences and mutational events. We propose that CLS are a major source of mutations during human tumourigenesis.

Uncoupling of telomere length and radiosensitivity in mouse lymphoma cell lines of similar genetic background.

PURPOSE: To investigate the link between radiosensitivity and telomere length in murine lymphoid cell line stocks that have similar genetic backgrounds but different radiosensitivities. MATERIALS AND METHODS: We used two stocks from both the parental L5,178Y-R cell line and the repair-deficient radiosensitive subline, L5,178Y-S, to assess telomere length. We used terminal restriction fragment analysis and flow-fluorescence in situ hybridization (FISH) telomere length assessment to determine telomere lengths in the related radiosensitive and non-radiosensitive cell lines. Each cell line was further tested for retention of its original radiation response phenotype using cell growth assays after treatment with ionizing radiation. RESULTS: One stock of L5,178Y-R cells had long telomeres, whereas the other stock had short telomeres. Likewise, one stock of L5,178Y-S cells had long telomeres, whereas the other stock had short telomeres. Telomere lengths in these cell lines were relatively stable for over 80 divisions in culture. Each cell line was confirmed to have retained its original radiosensitivity phenotype. CONCLUSION: We conclude that radiosensitivity is independent of telomere length in these genetically similar cell lines.

Ionizing radiation-induced chromosomal rearrangements occur in transcriptionally active regions of the genome.

PURPOSE: The mechanism by which ionizing radiation induces chromosomal rearrangements in mammalian cells has for long been a subject of debate. In order to dissect these events at a molecular level, we have studied the sequences involved in gamma irradiation-induced rearrangements. MATERIALS AND METHODS: An inverse polymerase chain reaction (PCR)-based methodology was used to amplify rearrangements that had occurred between one of four target regions (in or neighbouring the avian myelocytomatosis viral oncogene homologue (c-MYC), cyclin-dependent kinase inhibitor 1A (CDKN1A), fibroblast growth factor receptor 2 (FGFR2), or retinoblastoma 1 (RB1) genes) and sequences elsewhere in the genome, following gamma irradiation and subsequent incubation at 37 degrees C of normal human IMR-90 fibroblasts. RESULTS: The sequences of 90 such rearrangements, including both inter- and intra-chromosomal events, have been analysed. Sequence motifs (including DNA topoisomerase recognition sites) were not found to be consistently present either at or near rearrangement breakpoint sites. Statistical analysis suggested that there was significantly more homology between the sites of DNA rearrangement breakpoints than would be expected to occur by chance, however, most DNA rearrangements showed little or no homology between the interacting sequences. The rearrangements were shown to predominantly involve transcriptionally active sequences, a finding that may have significant implications for our understanding of radiation-induced carcinogenesis. CONCLUSION: The results obtained are difficult to reconcile with most models for ionizing radiation-induced chromosomal aberration formation, but are consistent with the Transcription-Based model.

Imatinib. Novartis.

Novartis has launched imatinib, an inhibitor of tyrosine kinases, including Bcr-Abl, for the treatment of chronic myeloid leukemia (CML). Imatinib selectively inhibits activation of target proteins involved in cellular proliferation. It also inhibits c-KIT tyrosine kinase activity and is equally effective against both wild-type and constitutively active enzyme. Close correlation between in vitro responses to IFNalpha and imatinib suggested that it may be an alternative to IFNalpha therapy for chronic-phase CML, and the compound has the advantage that it can be administered orally. Futhermore, Bcr-Abl-expressing cells treated with imatinib undergo apoptosis. Imatinib also has potential for the treatment of other cancers that express these kinases, including acute lymphocytic leukemia and certain solid tumors. In February 2002, the FDA approved imatinib for the treatment of inoperable and/or metastatic malignant gastrointestinal stromal tumors (GIST); in September 2001, launch for the indication was expected in 2002. In November 2000, imatinib was granted Orphan Drug status in Japan for the target indication of Philadelphia chromosome-positive leukemia. By May 2001, imatinib had entered phase II trials for small cell lung cancer, prostate cancer and glioma. Imatinib has been launched in more than 35 countries, including the US, Brazil, Switzerland, Australia and the UK. By December 2001, the drug had also been launched in Japan. The drug is marketed as Gleevec (imatinib mesilate) in the US, and Glivec (imatinib) outside the US. In August 2001, Deutsche Bank estimated sales of SFr 233 million in 2001, rising to SFr 850 million in 2005; while Bear Stearns & Co predicted sales of SFr 250 million in 2001, rising to SFr 800 million in 2005.

Invasiveness and comparative life-history traits of exotic and indigenous Senecio species in Australia.

A comparative ecological study of closely related invasive and non-invasive species, Senecio madagascariensis and S. lautus (Asteraceae), investigated what traits might confer invasive ability in very similar species. Life-history attributes of the weed S. madagascariensis were compared to five habitat-specific subspecies of S. lautus: S. l. alpinus, S. l. dissectifolius, S. l. lanceolatus and two forms of S. l. maritimus. Field populations of each taxon were monitored to compare their population ecology. Relative rates of phenological development were compared at a single location. Seed germination was studied in a laboratory experiment. Transplant experiments were conducted in a range of S. madagascariensis and S. lautus habitats to compare performance in different environments. In monitored field populations S. madagascariensis produced seedlings and reproductive cohorts more frequently, flowered for longer periods, produced more seeds and had fewer germinable achenes in the soil compared to S. lautus taxa. S. madagascariensis achenes had higher rates of germination than S. lautus in both light and dark conditions. S. madagascariensis was found to have higher rates of survival than S. lautus taxa in a range of habitats and to be faster to flower in both transplant and standard glasshouse environments. Overall S. madagascariensis performed better than S. lautus ecotypes in terms of seedling, growth and fecundity measurements and second best for achenes. Despite relatively good performance in terms of life-history traits there is no evidence that S. madagascariensis is invading S. lautus habitats. We speculate that physiological and morphological adaptations to specialised environments are a better explanation for success of Senecio taxa/ecotypes than generalised life-history trait performance. We suggest that invasiveness is essentially unpredictable, due to habitat/plant specific interactions between invader and area of introduction. In the absence of predictive theory, quarantine authorities should use a combination of methods to assess invasive potential including a database of known weeds, performance comparisons between congeneric natives and exotics in a range of habitats at proposed point of introduction and monitoring of introduced species to determine if they spread.