A DNA toolbox for non-invasive genetic studies of sambar deer (Rusa unicolor)

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

Delineating genetic management units of sambar deer (Rusa unicolor) in south-eastern Australia, using opportunistic tissue sampling and targeted scat collection

Context: Invasive species are major drivers of biodiversity loss, requiring management to reduce their ecological impacts. Population genetics can be applied to delineate management units, providing information that can help plan and improve control strategies. Aim: The present study aims to use a genetic approach to test the existence of three previously proposed sambar deer populations in south-eastern Australia. In doing so, the study aims to delineate management units of sambar deer in south-eastern Australia. Methods: Sambar deer DNA was sourced opportunistically from tissue samples and targeted scat collection. Samples were collected from three areas in Victoria, south-eastern Australia: Mt Cole (MC), French Island (FI) and eastern Victoria (EV). Contemporary population structure was assessed using a suite of 11 polymorphic microsatellite markers. The number of maternal sambar deer lineages in south-eastern Australia was investigated through sequencing of the mitochondrial (mt)DNA control region. Key results: Three distinct genetic clusters were identified. Differentiation among inferred clusters was found to be high, with FST ranging from 0.24 between EV and FI clusters and 0.48 between MC and FI clusters. Two mtDNA haplotypes were identified; R.u1 was found throughout EV and FI, and R.u2 was unique to MC. DNA isolated from scats provided reliable data and proved critical for sampling areas where hunting and culling of deer are not generally undertaken. Conclusions: Three genetically distinct sambar deer management units in south-eastern Australia are defined-MC, FI and EV. Sambar deer control strategies should be applied to each management unit independently. This may be difficult or infeasible for the EV management unit, which is large and geographically complex. Further research may help identify additional fine-scale genetic structure in EV, allowing smaller, more practicable management units to be identified. Implications: Genetic data can be used to identify management units for invasive species, which will be critical for the development of future management strategies and improving control operations. The approach outlined here could also be applied to improve the management of other introduced deer species in south-eastern Australia. © 2022 CSIRO Open Access

Validating the use of non-invasively sourced DNA for population genetic studies using pedigree data

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. © Author(s) 2017. CC Attribution 3.0 License

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

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

Hybridisation rates, population structure, and dispersal of sambar deer (Cervus unicolor) and rusa deer (Cervus timorensis) in south-eastern Australia

Context. Introduced populations of sambar deer (Cervus unicolor) and rusa deer (Cervus timorensis) are present across south-eastern Australia and are subject to local population control to alleviate their negative impacts. For management to be effective, identification of dispersal capability and management units is necessary. These species also readily hybridise, so additional investigation of hybridisation rates across their distributions is necessary to understand the interactions between the two species. Aims. Measure the hybridisation rate of sambar and rusa deer, assess broad-scale population structure present within both species and identify distinct management units for future population control, and measure the likely dispersal capability of both species. Methods. In total, 198 sambar deer, 189 rusa deer, and three suspected hybrid samples were collected across Victoria and New South Wales (NSW). After sequencing and filtering, 14 099 polymorphic single-nucleotide polymorphism (SNP) markers were retained for analysis. Hybridisation rates were assessed before the data were split by species to identify population structure, diversity indices, and dispersal distances. Key results. Across the entire dataset, 17 hybrids were detected. Broad-scale population structure was evident in sambar deer, but not among the sites where rusa deer were sampled. Analysis of dispersal ability showed that a majority of deer movement occurred within 20 km in both species, suggesting limited dispersal. Conclusions. Distinct management units of sambar deer can be identified from the dataset, allowing independent population control. Although broad-scale population structure was not evident in the rusa deer populations, dispersal limits identified suggest that rusa deer sites sampled in this study could be managed separately. Sambar × rusa deer hybrids are present in both Victoria and NSW and can be difficult to detect on the basis of morphology alone. Implications. Genetic analysis can identify broad-scale management units necessary for population control, and estimates of dispersal capability can assist in delineating management units where broad-scale population structure may not be apparent. The negative impacts associated with hybridisation require further investigation to determine whether removal of hybrids should be considered a priority management aim. © 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Genetic data from koala scats: method development and investigation of a unique population in South Gippsland

Koala populations are being impacted by threats including habitat loss, urban development, disease, dog attacks, road trauma and climatic events such as drought and bush fire. Due to a lack of genetic diversity, Victorian koalas are also vulnerable to future environmental change. This research developed molecular methods which used DNA isolated from koala scats to determine gender, unequivocally identify individuals and detect pathogens affecting koala health. Developed methods were used to investigate koalas in South Gippsland, Victoria, which were found to be genetically more diverse than other Victorian populations, warranting targeted conservation strategies

Genetic data from koala scats: method development and investigation of a unique population in South Gippsland

Koala populations are being impacted by threats including habitat loss, urban development, disease, dog attacks, road trauma and climatic events such as drought and bush fire. Due to a lack of genetic diversity, Victorian koalas are also vulnerable to future environmental change. This research developed molecular methods which used DNA isolated from koala scats to determine gender, unequivocally identify individuals and detect pathogens affecting koala health. Developed methods were used to investigate koalas in South Gippsland, Victoria, which were found to be genetically more diverse than other Victorian populations, warranting targeted conservation strategies

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

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

Data from: 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