Fiona Hogan

Optimizing the use of shed feathers for genetic analysis

Shed feathers obtained by noninvasive genetic sampling (NGS) are a valuable source of DNA for genetic studies of birds. They can be collected across a large geographical range and facilitate research on species that would otherwise be extremely difficult to study. A limitation of this approach is uncertainty concerning the quality of the extracted DNA. Here we investigate the relationship between feather type, feather condition and DNA quality (amplification success) in order to provide a simple, cost‐effective method for screening samples prior to genetic analysis. We obtained 637 shed feathers of the powerful owl (Ninox strenua) from across its range in southeastern Australia. The extracted DNA was amplified using polymerase chain reaction for a range of markers including mitochondrial DNA, ND3 and nuclear DNA, a simple sequence repeat (Nst02) and a portion of the CHD‐1 gene (P2/P8). We found that feather condition significantly influenced the amplification success of all three loci, with feathers characterized as ‘good’ having greater success. Feather type was found to be of lower importance, with good quality feathers of all types consistently producing high success for all three loci. We also found that the successful amplification of multilocus genotypes was dependant on the condition of the starting material and was highly correlated with successful amplification of the sex‐linked CHD‐1 locus. Samples with low DNA quality have a higher probability of amplification failure and are more likely to produce incorrect genotypes; therefore, identifying samples with high DNA quality can save substantial time and cost associated with the genetic analysis of NGS. As a result, we propose a method for screening shed feathers in order to provide a subset of samples which will have a greater probability of containing high quality DNA suitable for the amplification of multilocus genotypes.

The diet of powerful owls (Ninox strenua) and prey availability in a continuum of habitats from disturbed urban fringe to protected forest environments in south-eastern Australia

This study investigates the diet of six breeding pairs of powerful owls in the Yarra Valley Corridor in Victoria, Australia, and compares prey consumption with prey availability. The six sites represent a continuum of habitats, ranging from urban Melbourne, through the urban fringe interface to a more forested landscape. We found that powerful owls in the Yarra Valley Corridor are reliant almost exclusively on arboreal marsupial prey as their preferred diet, with 99% of their overall diet comprising four arboreal marsupial species. These four species (the common ringtail possum, common brushtail possum, sugar glider and greater glider) were also the most abundant species observed while spotlighting; however, their abundance varied along the continuum. There was a strong positive relationship with the presence of these species in the diet and their site-specific availability, indicating that the powerful owl is a generalist hunter, preying on the most available prey at a given site and in a given season. This study suggests that food resources are high in these disturbed urban fringe sites and it is unlikely that food availability in urban environments will limit the potential survival of urban powerful owls.

Reliable genotyping of the koala (Phascolarctos cinereus) using DNA isolated from a single faecal pellet.

The koala, an Australian icon, has been added to the threatened species list. Rationale for the listing includes proposed declines in population size, threats to populations (e.g. disease) and loss and fragmentation of habitat. There is now an urgent need to obtain accurate data to assess the status of koala populations in Australia, to ensure the long‐term viability of this species. Advances in genetic techniques have enabled DNA analysis to study and inform the management of wild populations; however, sampling of individual koalas is difficult in tall, often remote, eucalypt forest. The collection of faecal pellets (scats) from the forest floor presents an opportunistic sampling strategy, where DNA can be collected without capturing or even sighting an individual. Obtaining DNA via noninvasive sampling can be used to rapidly sample a large proportion of a population; however, DNA from noninvasively collected samples is often degraded. Factors influencing DNA quality and quantity include environmental exposure, diet and methods of sample collection, storage and DNA isolation. Reduced DNA quality and quantity can introduce genotyping errors and provide inaccurate DNA profiles, reducing confidence in the ability of such data to inform management/conservation strategies. Here, we present a protocol that produces a reliable individual koala genotype from a single faecal pellet and highlight the importance of optimizing DNA isolation and analysis for the species of interest. This method could readily be adapted for genetic studies of mammals other than koalas, particularly those whose diet contains high proportions of volatile materials that are likely to induce DNA damage.

A non-invasive tool for assessing pathogen prevalence in koala (Phascolarctos cinereus) populations: detection of Chlamydia pecorum and koala retrovirus (KoRV) DNA in genetic material sourced from scats

Pathogenic diseases may threaten the viability of wild animal populations, especially when already vulnerable. The mitigation of risks associated with pathogenic infections in populations is an important factor in conservation strategies. Koalas are of conservation concern across the north of their range and are affected by two main pathogens; Chlamydia pecorum and the koala retrovirus (KoRV). This study tested whether DNA from C. pecorum and KoRV could be detected in genetic material isolated from koala scats. Detection of C. pecorum in scat isolated DNA samples was compared with results obtained from urogenital swabs collected from the same individuals as part of an independent study. The ability to detect KoRV in scats from both northern and southern regions of the koala’s range was also assessed. There was a high level of concordance (5/6) between the detection of C. pecorum in DNA isolated from scats and urogenital swabs from the same individual. In positive samples, C. pecorumompA genotypes were identical between DNA from scats and urogenital swabs in two out of three cases. In samples from the south of the koala’s range, KoRV copy number was higher in DNA isolated from scats compared to DNA isolated from ear tissue, potentially indicating the detection of horizontally acquired infections. Our results demonstrate the ability to detect C. pecorum and KoRV in DNA isolated from koala scats. This method will be useful for studying the prevalence, transmission and impact of these pathogens in wild populations which may subsequently inform conservation management strategies.

A morphological model for sexing nestling peregrine falcons (Falco peregrinus macropus) verified through genetic analysis

Adult peregrine falcons (Falco peregrinus macropus) have monotypic plumage and display strong reversed sexual dimorphism, with females significantly larger than males. Reversed sexual dimorphism is measurable among nestlings in the latter stages of their development and can therefore be used to differentiate between sexes. In the early stages of development, however, nestlings cannot be sexed with any degree of certainty because morphological differentiation between the sexes is not well developed. During this study we developed a model for sexing younger nestlings based on genetic analysis and morphometric data collected as part of a long-term banding study of this species. A discriminant function model based on morphological characteristics was developed for determining the sex of nestlings (n = 150) in the field and was shown to be 96.0% accurate. This predictive model was further tested against an independent morphometric dataset taken from a second group of nestlings (n = 131). The model correctly allocated sex to 96.2% of this second group of nestlings. Sex can reliably be determined (98.6% accurate) for nestlings that have a wing length of at least 9 cm using this model. Application of this model, therefore, allows the banding of younger nestlings and, as such, significantly increases the period of time over which banding can occur. Another important implication of this model is that by banding nestlings earlier, they are less likely to jump from the nest, therefore reducing the risk of injury to both the brood and the bander.

Genetic structure and diversity of the koala population in South Gippsland, Victoria: a remnant population of high conservation significance

In the Australian state of Victoria, the history of koalas and their management has resulted in the homogenisation and reduction of genetic diversity in many contemporary populations. Decreased genetic diversity may reduce a species’ ability to adapt to future environmental pressures such as climate change or disease. The South Gippsland koala population is considered to be unique in Victoria, as it is believed to be a remnant population, not originating from managed populations that have low genetic variation. This study investigated genetic structure and diversity of koalas in South Gippsland, with comparison to other populations in Victoria (French Island/Cape Otway, FI and Raymond Island, RI), New South Wales and south east Queensland. Population analyses were undertaken using both microsatellite genotype and mitochondrial DNA sequence data. Non-invasive sampling of koala scats was used to source koala DNA, allowing 222 South Gippsland koalas to be genotyped. Using nuclear data the South Gippsland koala population was found to be significantly differentiated (Djost 95% CI SG–RI = 0.03–0.06 and SG–FI = 0.08–012) and more diverse (AR 95% CI SG = 4.7–5.6, RI = 3.1–3.3, FI = 3.0–3.3; p = 0.001) than other Victorian koala populations, supporting the premise that koalas in the South Gippsland region are part of a remnant population, not derived from translocated island stock. These results were also supported by mitochondrial data where eight haplotypes (Pc4, Pc17, Pc26, Pc27, and Pc56–Pc59) were identified in South Gippsland while a single haplotype (Pc27) was found in all island koalas tested. Compared to other Victorian koala populations, greater genetic diversity found in South Gippsland koalas, may provide this population with a greater chance of survival in the face of future environmental pressures. The South Gippsland koala population is, therefore, of high conservation significance, warranting the implementation of strategies to conserve this population and its diversity into the future.

Genetic structure and sex-biased dispersal of a declining cooperative-breeder, the Grey-crowned Babbler, Pomatostomus temporalis, at the southern edge of its range

Abstract Loss and fragmentation of habitat can disrupt genetic exchange between populations, which is reflected in changes to the genetic structure of populations. The Grey-crowned Babbler (Pomatostomus temporalis) is a cooperatively breeding woodland bird, once common and widespread in south-eastern Australia. The species has suffered population declines of >90% across its southern distribution as a result of loss and fragmentation of habitat. We investigated patterns of genetic diversity and population structure of Grey-crowned Babblers in fragmented habitats at the southernmost extent of its range. We sampled blood from 135 individual Babblers from 39 groups stratified into six subpopulations in three regions. Genotypic data were used to estimate genetic diversity, population substructure, local relatedness and dispersal patterns. Individuals showed high heterozygosity within regions, and varying numbers of private alleles among regions suggested differences in levels of connectivity between regions. Four genetic clusters revealed population substructure consistent with treeless landscapes acting as strong barriers to gene flow. In contrast to previous studies, we identified a male-biased dispersal pattern and significant isolation-by-distance patterns for females at fine spatial scales. We recommend that conservation plans for this species incorporate opportunities to increase and enhance corridor areas to facilitate genetic exchange among subpopulations.

Powerful Owls: Possum Assassins Move into Town

Once thought to live only in large forested areas, the powerful owl (Ninox strenua), Australia’s largest and most iconic of owls (figure 11.1), surprisingly is now turning up frequently in the cities of eastern Australia. Powerful owls require ample prey and large tree cavities for nest sites; how this top-order predator is able to survive in human-dominated landscapes is an important question for conservation and the focus of ongoing research. The powerful owl is endemic to Australia, resident in the three eastern mainland states and the Australian Capital Territory, and classified nationally as “rare.”2,3 First described by Gould in 1838, powerful owls are an unusual raptor in that they do not exhibit reversed sexual size dimorphism, the prevalent trait among raptors in which females are larger than males. For reasons still not understood, male powerful owls grow to a height of 65 cm and weigh up to 1,700 g, compared to females, which grow to a height of 54 cm and weigh up to 1,308 g.1

Isolating DNA sourced non-invasively from koala scats: a comparison of four commercial DNA stool kits

Genetic sampling from faeces is a useful method for obtaining DNA samples non-invasively. The quantity and quality of DNA isolated from faecal samples is, however, an important factor affecting the success of downstream analyses. Commercial DNA isolation kits offer an efficient and convenient means for recovering DNA, but the kit methodology can influence the quantity and quality of DNA obtained. Comparisons of kit performance for the isolation of DNA from non-invasive sources for ecological studies based on genetic analysis are uncommon in the literature. This study compared the quantity and quality of DNA isolated from surface washings of fresh koala (Phascolarctos cinereus) faecal pellets (scats) using four commercial DNA isolation kits: Axygen® AxyPrep™ MAG Soil, Stool, and Water DNA Kit (AX), Bioline ISOLATE Fecal DNA Kit (BL), Qiagen QIAamp® Fast DNA Stool Mini Kit (QFS), and Qiagen QIAamp® DNA Stool Mini Kit (QS). DNA quantitation, standard PCR and electrophoresis, real time PCR and replicate genotyping using capillary electrophoresis were used to compare the performance of resultant DNA isolates. The performance of DNA isolated from koala scats varied substantially with the DNA kit utilised. All kits provided accurate genotypes but with differing amounts of missing data. Overall, kit AX performed best, providing DNA isolates of higher quantity and quality compared to kit QS, which has previously been thoroughly assessed for genotyping reliability using DNA from koala scats. Given the high variability noted, assessing kit performance is an important way to maximise data quality from non-invasively sourced DNA.

Using non-invasive sampling methods to determine the prevalence and distribution of Chlamydia pecorum and koala retrovirus in a remnant koala population with conservation importance

Abstract Context. Pathogenic infections are an important consideration for the conservation of native species, but obtaining such data from wild populations can be expensive and difficult. Two pathogens have been implicated in the decline of some koala (Phascolarctos cinereus) populations: urogenital infection with Chlamydia pecorum and koala retrovirus subgroup A (KoRV-A). Pathogen data for a wild koala population of conservation importance in South Gippsland, Victoria are essentially absent. Aims. This study uses non-invasive sampling of koala scats to provide prevalence and genotype data for C. pecorum and KoRV-A in the South Gippsland koala population, and compares pathogen prevalence between wild koalas and koalas in rescue shelters. Methods. C. pecorum and KoRV-A provirus were detected by PCR of DNA isolated from scats collected in the field. Pathogen genetic variation was investigated using DNA sequencing of the C. pecorum ompA and KoRV-A env genes. Key results. C. pecorum and KoRV-A were detected in 61% and 27% of wild South Gippsland individuals tested, respectively. KoRV-A infection tended to be higher in shelter koalas compared with wild koalas. In contrast with other Victorian koala populations sampled, greater pathogen diversity was present in South Gippsland. Conclusions. In the South Gippsland koala population, C. pecorum is widespread and common whereas KoRV appears less prevalent than previously thought. Further work exploring the dynamics of these pathogens in South Gippsland koalas is warranted and may help inform future conservation strategies for this important population. Implications. Non-invasive genetic sampling from scats is a powerful method for obtaining data regarding pathogen prevalence and diversity in wildlife. The use of non-invasive methods for the study of pathogens may help fill research gaps in a way that would be difficult or expensive to achieve using traditional methods.