Pouch appearance is a reliable indicator of the reproductive status in the Tasmanian devil and the spotted-tailed quoll

Female Tasmanian devils (TDs) Sarcophilus harrisii and spotted-tailed quolls (STQs) Dasyurus maculatus were monitored to assess changes in plasma progesterone and faecal oestrogens/progestagens, vaginal smears and qualitative changes in pouch appearance during the oestrous cycle. Pouch condition was characterized based on size, colour and secretions, and was found to accurately reflect reproductive status, being significantly correlated with changes in both sex steroids and vaginal cytology. During the follicular phase, pouch redness and secretions were maximal, and associated with increased sex steroid concentrations, a karyopyknotic index of >90% and the onset of copulation. Post-ovulation, pouches became wet and deep and developed a glandular appearance; plasma progesterone/faecal progestagen concentrations remained high and sustained throughout the luteal phase. These features were identical during the pregnant and non-pregnant oestrous cycle. This study demonstrated that pouch appearance is a reliable physical indicator of the stage of oestrous in the TD and STQ, and provides an alternative non-invasive method for evaluating the ovarian cycle of these threatened species. This technique can be readily applied to monitor individuals in free-ranging or captive populations, and will aid as a practical tool for improved breeding management.     

Isolation of 21 polymorphic microsatellite markers for the Virginia opossum (Didelphis virginiana)

Twenty-one polymorphic microsatellite markers were developed for the Virginia opossum (Didelphis virginiana). The number of alleles ranged from two to 13 and observed heterozygosities ranged from 0.464 to 0.964. Significant heterozygote deficiencies were observed at three loci and null alleles were detected at five loci. Evidence for gametic disequilibrium was observed between three sets of paired loci after a sequential Bonferroni correction was applied. These markers will enable us to investigate the mating tactics, movement behaviour and social structure of Virginia opossum populations inhabiting fragmented agricultural landscapes.     

Leader of the pack: faecal pellet deposition order impacts PCR amplification in wombats

DNA sourced from faeces is notoriously less reliable than that from tissue. Hence, understanding whether faecal pellet quality varies within faecal piles may be important for sample selection. We hypothesized that the order in which faecal pellets are deposited may influence microsatellite polymerase chain reaction (PCR) amplification success from sampled faeces, more specifically, that first pellets deposited will have signatures of greater success than later ones. In a first test of the hypothesis, first and later-deposited pellets, as determined from the direction of footprints, were collected from fresh (overnight) faecal piles of northern hairy-nosed wombats (Lasiorhinus krefftii). DNA extracts were typed for seven microsatellite loci. We found that faecal deposition order significantly affected optical density of bands on autoradiographs (a measure of PCR amplification success) when the first faecal pellet was compared with the last one, but not when the first pellet was only distinguishable from later ones. The absence of a difference in amplification rate between first and later pellets is likely a reflection of the overall high amplification success in this study. That first pellets deposited yield more product suggests they contain more intestinal cells. Although further comparisons are needed, these results may inform sample selection in species for which success of microsatellite PCR amplification of faecal DNA is low. Deposition order may have more of an impact on amplification success and genotyping errors as faecal age increases.     

Spatial ecology of the mulgara in arid Australia: impact of fire history on home range size and burrow use

Knowledge about the spatial ecology of small mammals in relation to fire history in arid zones in general and Australia in particular is limited. Here, we report data on the spatial ecology of the brush-tailed mulgara Dasycercus blythi in the hummock grasslands of Ulu r u – Kata Tju t a National Park during winter 2006, the beginning of the breeding season for this species. About 73% of the study area had been burnt in 2002 and spinifex cover was sparse. Mulgaras Marsupialia: Dasyuridae (six males and three females) were implanted with radio-transmitters and monitored daily for between 6 and 55 days. All mulgaras appeared to use defined home ranges, which overlapped extensively with those of several neighbours. Spatial overlap occurred between as well as within sexes. On average, males (25.5 ha) occupied significantly larger home ranges than females (10.8 ha). Mulgaras used a number of burrows within home ranges and several were used by more than one individual. Moreover, occasionally, two individuals used the same burrow simultaneously. Home ranges and burrows encompassed both mature spinifex Triodia basedowii and open regrowth areas and mulgaras did not exhibit a significant preference for either habitat type. However, three males were killed by introduced-predators and they all lived predominantly in the open regrowth area. We conclude that mulgaras do not select the dense cover of mature spinifex habitat, and might be subjected to increased risk from introduced predators, especially following fire.     

Evidence for habitat fragmentation altering within-population processes in wombats

Habitat fragmentation and associated reduced dispersal of wildlife can lead to an accumulation of related individuals in fragments. The altered kin interactions and amplified chance of inbred matings has profound implications for mating and social systems, and ultimately population persistence. Nonetheless, within-fragment population processes are rarely studied. With this aim, we examined relatedness structure in two candidate isolated populations (Kulpara and Scrubby Peak) of southern hairy-nosed wombats ( Lasiorhinus latifrons ). Wombats were sampled by remote hair-trapping for genotyping at 14 microsatellite loci, enabling individual identification and estimation of space-use and associative behaviour with respect to relatedness. Genetic data indicated that Scrubby Peak was not strongly isolated, against predictions from landscape structure and history. In isolated Kulpara, inhibited female dispersal (normally the dispersing sex) was associated with high population density and altered kin relationships. First, female relatives preferentially coexisted, a radical departure from the previously reported active avoidance of female relatives in continuous habitat. This is consistent with females in altered habitat interacting with more- rather than less-related females to minimize the cost:benefit ratio of proximity to other wombats. Second, inbreeding avoidance appeared to be stronger at Kulpara than in conspecific populations with natural population structures. Although these adaptive behaviours may have contributed to persistence of the Kulpara population in the short term, they are unlikely to ensure its long-term viability in the face of ongoing isolation because they can act only to slow the rate of inbreeding and mitigate some of its negative impacts.     

Does soil type drive social organization in southern hairy‐nosed wombats?

Spatiotemporal distributions of key resources are hypothesized to underpin sociobiological patterns. Burrow availability and quality is of paramount importance to fossorial animals. The southern hairy-nosed wombat (Lasiorhinus latifrons) burrows in both hard and friable soils. Theoretical and empirical studies suggest that the harder substrate should promote closer geographical clustering of burrows than in softer soils. Clustered burrows are expected to be associated with larger group sizes. If sociality is driven by constraint rather than advantage, patterns of spatial and temporal distribution of animals within and among groups may show indications of avoidance or even antagonism, and 'making the best of a bad job' via positive kin associations to offset the disadvantages of high-density living. To test these ideas, we compared warren relatedness and social structure of L. latifrons on friable soils (Nullarbor Plain) and hard calcrete (Brookfield Conservation Park, BCP). Individuals were sampled by noninvasive collection of hairs for genotyping to identify individuals and to estimate their space-use and associative behaviour with respect to relatedness. Burrows in calcrete were indeed more clumped, and warren and group size larger. Differences in spatiotemporal organization and relatedness structure between sites were in the expected direction: (i) Nullarbor males associated and shared warrens less than at BCP; and (ii) Nullarbor spatial relatedness patterning data were not consistent with proposed female breeding dispersal, in contrast to those at BCP. Under Nullarbor (low density) conditions, cooperation or tolerance between males may be less advantageous, and accessing or digging burrows should be less of a constraint for juvenile females.     

Isolation and characterization of 10 MHC Class I-associated microsatellite loci in tammar wallaby (Macropus eugenii)

The major histocompatibility complex (MHC) contain genes which play a key role in immune response and mate choice, and are therefore of functional importance to molecular ecologists. Here we describe the design of 10 MHC Class I-associated microsatellite loci from the tammar wallaby. All 10 loci are highly polymorphic, with the expected heterozygosity ranging from 0.547 to 0.919. Six loci successfully cross-amplify in other macropodid species. These microsatellites will serve as useful tools for studying the level of MHC diversity, the impact of selection on genetic variation and the unique structure of the tammar wallaby MHC.     

Genetic variation of microsatellite loci in a bottlenecked species: the northern hairy-nosed wombat Lasiorhinus krefftii

We investigate the utility of hypervariable microsatellite loci to measure genetic variability remaining in the northern hairy-nosed wombat, one of Australia's rarest mammals. This species suffered a dramatic range and population reduction over the past 120 years and now exists as a single colony of about 70 individuals at Epping Forest National Park, central Queensland. Because our preliminary research on mitochondrial DNA and multilocus DNA fingerprints did not reveal informative variation in this population, we chose to examine variation in microsatellite repeats, a class of loci known to be highly polymorphic in mammals. To assess the suitability of various wombat populations as a reference for comparisons of genetic variability and subdivision we further analysed mitochondrial DNA cytochrome b sequence, using phylogenetic methods. Our results show that appreciable levels of variation still exist in the Epping Forest colony although it has only 41% of the heterozygosity shown in a population of a closely-related species. From museum specimens collected in 1884, we also assessed microsatellite variation in an extinct population of the northern hairy-nosed wombat, from Deniliquin, New South Wales, 2000 km to the south of the extant population. The apparent loss of variation in the Epping Forest colony is consistent with an extremely small effective population size throughout its 120-year decline.     

Relatedness structure detected by microsatellite analysis and attempted pedigree reconstruction in an endangered marsupial, the northern hairy-nosed wombat Lasiorhinus krefftii

The northern hairy-nosed (NHN) wombat is perhaps Australia's most endangered mammal. Being fossorial and nocturnal as well as rare, NHN wombats are difficult to observe in the wild. Hence little is known of their social biology, such as their mating and dispersal systems. A hypothesis has been advanced that adult females of the species disperse post-breeding, leaving their young to inhabit the natal burrow. Female-biased dispersal is expected to result in higher relatedness amongst males in a burrow cluster than amongst females in a burrow cluster. The usefulness of a panel of microsatellite markers in estimating the relatedness structure, and in reconstructing pedigrees for, the sole known population of NHN wombats was assessed. Microsatellite genotypes at eight or nine loci were obtained from 58 of the 85 known individuals, and used to estimate pairwise individual relatedness using Queller & Goodnight's (1989) RELATEDNESS 4.2. Our analysis gave the unexpected result that both males and females were significantly more closely related to their same-sex burrow cluster mates than random, while opposite-sex animals sharing burrows were only slightly (nonsignificantly) more related than random. This raises the possibility of dispersal patterns which lead to association of same-sex relatives. The observed relatedness structure is not expected to make likely a high incidence of inbred matings, as close relatives of the opposite sex are not significantly associated in space. Parentage analysis was attempted using genetic exclusion and LOD likelihood ratios, but proved difficult because of low genetic variation, incomplete sampling of potential parents, and paucity of ecological data such as known mother/offspring pairs and ages of individuals.     

Sex-linked and autosomal microsatellites provide new insights into island populations of the tammar wallaby

The emerging availability of microsatellite markers from mammalian sex chromosomes provides opportunities to investigate both male- and female-mediated gene flow in wild populations, identifying patterns not apparent from the analysis of autosomal markers alone. Tammar wallabies (Macropus eugenii), once spread over the southern mainland, have been isolated on several islands off the Western Australian and South Australian coastlines for between 10 000 and 13 000 years. Here, we combine analyses of autosomal, Y-linked and X-linked microsatellite loci to investigate genetic variation in populations of this species on two islands (Kangaroo Island, South Australia and Garden Island, Western Australia). All measures of diversity were higher for the larger Kangaroo Island population, in which genetic variation was lowest at Y-linked markers and highest at autosomal markers (θ=3.291, 1.208 and 0.627 for autosomal, X-linked and Y-linked data, respectively). Greater relatedness among females than males provides evidence for male-biased dispersal in this population, while sex-linked markers identified genetic lineages not apparent from autosomal data alone. Overall genetic diversity in the Garden Island population was low, especially on the Y chromosome where most males shared a common haplotype, and we observed high levels of inbreeding and relatedness among individuals. Our findings highlight the utility of this approach for management actions, such as the selection of animals for translocation or captive breeding, and the ecological insights that may be gained by combining analyses of microsatellite markers on sex chromosomes with those derived from autosomes.     

Pedigrees and microsatellites among endangered ungulates: what do they tell us?

Relationships between pedigree coefficients of inbreeding and molecular metrics are generally weak, suggesting that measures of heterozygosity estimated using microsatellites may be poor surrogates of genome-wide inbreeding. We compare three endangered species of gazelles ( Gazella ) with different degrees of threat in their natural habitats, for which captive breeding programmes exist. For G. dorcas, the species with the largest founding population, the highest and most recent number of founding events, the correlation between pedigree coefficient of inbreeding and molecular metrics was higher than for outbred populations of mammals, probably because it has both higher mean f and variance. For the two species with smaller founding populations, conventional assumptions about founders, i.e. outbred and unrelated, are unrealistic. When realistic assumptions about the founders were made, clear relationships between pedigree coefficients of inbreeding and molecular metrics were revealed for G. cuvieri. This population had a small founding population, but it did experience admixture years later; thus, the relationship between inbreeding and molecular metrics in G. cuvieri is very similar to the expected values but lower than in G. dorcas . In contrast, no relationship was found for G. dama mhorr which had a much smaller founding population than had been previously assumed, which probably had high levels of inbreeding and low levels of genetic variability, and no admixture. In conclusion, the strength of the association between pedigree coefficient of inbreeding and molecular metrics among endangered species depends on the level of inbreeding and genetic variability present in the founding population, its size and its history.     

Development of tetranucleotide microsatellite markers for the cushion star, Asterina gibbosa, and cross-species amplification

We describe nine polymorphic tetranucleotide microsatellite loci from the starfish, Asterina gibbosa. Loci were isolated from a partial genomic library that had been enriched for AAAC repeat sequences. Number of alleles per locus ranged from two to 14 in a sample of 85 individuals from three populations (two from Spain and one from the UK). Observed and expected heterozygosities per population ranged from 0.000 to 0.400 and from 0.040 to 0.784, respectively. All loci presented significant heterozygote deficits in one or more populations. Eight of these loci were amplified and variable in A. pancerii and A. phylactica. These loci will be used to study population structure in A. gibbosa.