Description:
Invasive sambar deer (Rusa unicolor) are having significant detrimental impacts on natural environments in south-eastern Australia. Little, however, is known about their ecology, limiting evidence-based management strategies directed at reducing deer impacts. Genetic data, generated from DNA isolated from deer scats, can be used to fill ecological knowledge gaps. This study outlines a non-invasive genetic sampling strategy by which good-quality DNA from a single deer scat can be used to determine (1) species of origin, (2) sex and (3) a unique DNA profile. DNA from deer tissue and sambar deer scat samples were used to develop and optimise molecular methods to collect reliable genetic information. A DNA toolbox is presented that describes how to find, collect and store scat samples, isolate DNA and use molecular markers to generate informative genetic data. Generating genetic data using this approach will support studies aimed at acquiring ecological knowledge about sambar deer. Such knowledge will be critical for developing evidence-based recommendations to improve on-ground management decisions for sambar deer.
Description:
Invasive sambar deer (Rusa unicolor) are having significant detrimental impacts on natural environments in south-eastern Australia. Little, however, is known about their ecology, limiting evidence-based management strategies directed at reducing deer impacts. Genetic data, generated from DNA isolated from deer scats, can be used to fill ecological knowledge gaps. This study outlines a non-invasive genetic sampling strategy by which good-quality DNA from a single deer scat can be used to determine (1) species of origin, (2) sex and (3) a unique DNA profile. DNA from deer tissue and sambar deer scat samples were used to develop and optimise molecular methods to collect reliable genetic information. A DNA toolbox is presented that describes how to find, collect and store scat samples, isolate DNA and use molecular markers to generate informative genetic data. Generating genetic data using this approach will support studies aimed at acquiring ecological knowledge about sambar deer. Such knowledge will be critical for developing evidence-based recommendations to improve on-ground management decisions for sambar deer.
Description:
Conserving biodiversity is of utmost importance on a global scale. Species conservation, however, is a challenging task, which is often compounded by a lack of knowledge of target species. New advances in information technology and molecular techniques, however, are enabling conservation biologists to obtain large amounts of data quickly, which will certainly aid in assigning conservation priorities. This article reviews the use of genetics in conservation biology and highlights, using the Powerful Owl Ninox strenua as an example, how DNA can be a valuable source of data
Delineating genetic management units of sambar deer (Rusa unicolor) in south-eastern Australia, using opportunistic tissue sampling and targeted scat collection
Description:
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.
Description:
The Powerful Owl is an elusive species inhabiting the forests of mainland eastern Australia. Obtaining crucial information on aspects of their breeding behaviour and dispersal has proven extremely difficult, even though other aspects of their ecology are well studied. Here we use molecular methods to investigate the breeding behaviour and dispersal of the Powerful Owl in two different habitats: highly fragmented forest along the urban fringe and continuous forest. DNA profiles of Powerful Owls were obtained predominately from shed feathers collected opportunistically between 1995 and 2006. Seven breeding pairs of Powerful Owls were identified, from which shed feathers were collected during 2003, 2004 and 2005. By comparing DNA profiles, one pair of Owls was found to have occupied the same breeding site for 10 years (1995–2005). The dispersal or movements of five offspring from this pair was also determined to be either of two scenarios: (1) the juvenile moves from the natal territory; however, isn’t breeding; and (2) the juvenile is recovered as part of a breeding pair. Two pairs of Owls breeding in the urban fringe habitat were closely related, but no incidences of extra-pair fertilisation were detected among pairs in either habitat. This study provides new information about the breeding behaviour and dispersal of the Powerful Owl, and shows the potential of using genetic data sourced from shed feathers for studying cryptic, rare or elusive species.
Description:
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.
Description:
We isolated 24 novel polymorphic microsatellite markers from the tawny frogmouth, a nocturnal bird endemic to Australia, which has successfully adapted to urban environments. Initially, 454 shotgun sequencing was used to identify 733 loci with primers designed. Of these, we trialled 30 in the target species of which all amplified a product of expected size. Subsequently, all 30 of these loci were screened for variation in 25 individuals, from a single population in Melbourne, Victoria, Australia. Twenty-eight loci were polymorphic with observed heterozygosity ranging from 0.03 to 0.96 (mean 0.58) and the number of alleles per locus ranged from 2 to 18 (average of 6.5); we confirmed that 24 loci conformed to Hardy–Weinberg expectations. The 24 loci identified here will be sufficient to unequivocally identify individuals and will be useful in understanding the reproductive ecology, population genetics and the gene flow amongst localities in urban environments where this bird thrives.
Description:
Masked owls, reputedly all of the Tasmanian race (Tyto novaehollandiae castanops) were introduced onto Lord Howe Island (LHI) in the 1920s in an attempt to control the black rat (Rattus rattus). This attempt, however, has been unsuccessful and a co-eradication of the rats and masked owls has been planned to reduce the threat to endemic species and breeding seabirds on the island. As the Tasmanian masked owl is considered endangered, translocation of LHI masked owls to Tasmania has been suggested. Before translocation is considered the ancestry of the LHI masked owl needs to be confirmed, as LHI masked owls are typically smaller and paler than individuals occurring in Tasmania. Here we sequenced three sections of mitochondrial gene regions: cytochrome b, ATP6 and ND3 to assess the provenance of the LHI masked owl and screened a suite of microsatellite loci isolated from the barn owl (Tyto alba) to assess contemporary divergence. Phylogenetic analysis revealed two clades, one exhibited by individuals from LHI and south-eastern mainland Australia and the second by those from Tasmania. Cross species amplification of microsatellite loci was successful, with 18 loci polymorphic. Genotypic data revealed significant sub-structuring between LHI, south-eastern mainland Australia and Tasmania. Data presented here indicate that the south-eastern mainland masked owl was introduced to LHI and subsequently reproduced. The genetic integrity of the LHI masked owl population is therefore questionable and as such LHI individuals may not be suitable for translocation to Tasmania.
Description:
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.
Description:
This research was supported by an Australian Government Research Training Program (RTP) scholarship and Federation University Australia’s School of Health and Life Science.
Description:
Urban expansion is a principal process threatening biodiversity globally. It is predicted that over half of the world's population will reside in urban centres by 2010. If we are to conserve biodiversity, a shift in perspective from traditional ecological studies based in natural environments, to studies based in less natural environments is paramount. To effectively conserve species which occur in urban environments, comprehensive analysis is necessary to determine the processes that are driving this urban usage. Geographical Information Systems (GIS) technology provides a valuable tool for efficient spatial analysis and predictive mapping of species distributions. This study used GIS to analyze current breeding sites for the powerful owl, a vulnerable top order predator in urban Melbourne, Australia. GIS analysis suggests that a number of ecological attributes were influencing powerful owl usage of urban environments. Using these ecological attributes, predictive mapping was undertaken, which identified a number of potential breeding sites for powerful owls within urbanized Melbourne. Urban environments are traditionally perceived as “the wastelands” of natural environments, however, this study demonstrates that they have the potential to support apex predators, an important finding for the management of rare and threatened species.
Description:
Abstract 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.
Description:
A rich source of markers may be overlooked by screening for polymorphism in the source species only. We screened 129 microsatellite loci isolated from the powerful owl (Ninox strenua) against two closely related species; Ninox connivens and Ninox novaeseelandiae. From the screening effort 20 polymorphic markers were isolated, including six loci which were originally discarded as they were monomorphic in the source species. Further cross-species amplification of all 20 loci across species from two families, Strigidae and Tytonidae, revealed unusually high levels of polymorphism within closely related species, and limited success within phylogenetically distant species. Routine screening of multiple species during the marker development phase can yield a wider range of polymorphic markers which can subsequently enhance cross-species amplification attempts
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
Description:
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.
Description:
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.
Description:
Non-invasive genetic sampling has provided valuable ecological data for many species - data which may have been unobtainable using invasive sampling methods. However, DNA obtained non-invasively may be prone to increased levels of amplification failure and genotyping error.
Utilizing genotype data from 32 pedigreed koalas, this study aimed to validate the reliability of final consensus genotypes obtained using DNA isolated from koala scats. Pedigree analysis, duplicate genotyping, analysis of mismatched loci and tests for null alleles were used to look for evidence of errors.
All genetically confirmed parent-offspring relationships were found to follow Mendelian rules of inheritance. Duplicate genotypes matched in all cases and there was no evidence of null alleles. Related individuals always had different 12-marker genotypes having a minimum of three unique loci (in one full sibling pair), a mode of seven unique loci and a maximum of 11 unique loci.
This study demonstrates the capacity of DNA recovered from koala scats to provide reliable genotypes that can unequivocally discriminate individuals and infer parentage, provided data are missing from no more than two loci. Validating data obtained using non-invasive sampling is an important step, allowing potential problems to be identified at an early stage.
Description:
Non-invasive genetic sampling has provided valuable ecological data for many species - data which may have been unobtainable using invasive sampling methods. However, DNA obtained non-invasively may be prone to increased levels of amplification failure and genotyping error.
Utilizing genotype data from 32 pedigreed koalas, this study aimed to validate the reliability of final consensus genotypes obtained using DNA isolated from koala scats. Pedigree analysis, duplicate genotyping, analysis of mismatched loci and tests for null alleles were used to look for evidence of errors.
All genetically confirmed parent-offspring relationships were found to follow Mendelian rules of inheritance. Duplicate genotypes matched in all cases and there was no evidence of null alleles. Related individuals always had different 12-marker genotypes having a minimum of three unique loci (in one full sibling pair), a mode of seven unique loci and a maximum of 11 unique loci.
This study demonstrates the capacity of DNA recovered from koala scats to provide reliable genotypes that can unequivocally discriminate individuals and infer parentage, provided data are missing from no more than two loci. Validating data obtained using non-invasive sampling is an important step, allowing potential problems to be identified at an early stage.
Description:
Non-invasive genetic sampling has provided valuable ecological data for many species - data which may have been unobtainable using invasive sampling methods. However, DNA obtained non-invasively may be prone to increased levels of amplification failure and genotyping error.
Utilizing genotype data from 32 pedigreed koalas, this study aimed to validate the reliability of final consensus genotypes obtained using DNA isolated from koala scats. Pedigree analysis, duplicate genotyping, analysis of mismatched loci and tests for null alleles were used to look for evidence of errors.
All genetically confirmed parent-offspring relationships were found to follow Mendelian rules of inheritance. Duplicate genotypes matched in all cases and there was no evidence of null alleles. Related individuals always had different 12-marker genotypes having a minimum of three unique loci (in one full sibling pair), a mode of seven unique loci and a maximum of 11 unique loci.
Description:
Non-invasive genetic sampling has provided valuable ecological data for many species - data which may have been unobtainable using invasive sampling methods. However, DNA obtained non-invasively may be prone to increased levels of amplification failure and genotyping error.
Utilizing genotype data from 32 pedigreed koalas, this study aimed to validate the reliability of final consensus genotypes obtained using DNA isolated from koala scats. Pedigree analysis, duplicate genotyping, analysis of mismatched loci and tests for null alleles were used to look for evidence of errors.
All genetically confirmed parent-offspring relationships were found to follow Mendelian rules of inheritance. Duplicate genotypes matched in all cases and there was no evidence of null alleles. Related individuals always had different 12-marker genotypes having a minimum of three unique loci (in one full sibling pair), a mode of seven unique loci and a maximum of 11 unique loci.