Anna J. MacDonald

Foxes are now widespread in Tasmania: DNA detection defines the distribution of this rare but invasive carnivore.

Summary 1. Invasive vertebrate species are a world-wide threat to biodiversity and agricultural production. The presence of foxes, one of the most damaging invasive vertebrates introduced to Australia, has now been confirmed in the island state of Tasmania, placing at risk many species of native vertebrates and substantial agricultural industry. 2. Effective eradication of such a rare but elusive carnivore requires robust strategies informed by novel but systematic detection. 3. We combine DNA detection approaches for trace samples with systematic stratified and opportunistic surveys of carnivore scats to estimate the current distribution of foxes in Tasmania. We use that DNA evidence and other hard evidence provided by carcasses and other material to build a predictive model of fox habitat suitability for all of Tasmania. 4. We demonstrate that this destructive species is widespread in northern and eastern Tasmania but has not yet reached the limits of its range. The widespread nature of this distribution reveals that targeting fox activity hotspots only for eradication is unlikely to be successful and that a strategic and statewide approach is required. Our habitat suitability model can provide a basis for prioritizing areas for fox management. 5. Synthesis and applications. Our approach highlights the importance of early and preemptive surveys of recently established, and therefore rare, invasive species and the necessity of providing a sound and defensible approach to determining the distribution of the invasive species. This approach provides a template for the systematic detection of rare cryptic carnivores.

Integrating survey and molecular approaches to better understand wildlife disease ecology.

Infectious wildlife diseases have enormous global impacts, leading to human pandemics, global biodiversity declines and socio-economic hardship. Understanding how infection persists and is transmitted in wildlife is critical for managing diseases, but our understanding is limited. Our study aim was to better understand how infectious disease persists in wildlife populations by integrating genetics, ecology and epidemiology approaches. Specifically, we aimed to determine whether environmental or host factors were stronger drivers of Salmonella persistence or transmission within a remote and isolated wild pig (Sus scrofa) population. We determined the Salmonella infection status of wild pigs. Salmonella isolates were genotyped and a range of data was collected on putative risk factors for Salmonella transmission. We a priori identified several plausible biological hypotheses for Salmonella prevalence (cross sectional study design) versus transmission (molecular case series study design) and fit the data to these models. There were 543 wild pig Salmonella observations, sampled at 93 unique locations. Salmonella prevalence was 41% (95% confidence interval [CI]: 37–45%). The median Salmonella DICE coefficient (or Salmonella genetic similarity) was 52% (interquartile range [IQR]: 42–62%). Using the traditional cross sectional prevalence study design, the only supported model was based on the hypothesis that abundance of available ecological resources determines Salmonella prevalence in wild pigs. In the molecular study design, spatial proximity and herd membership as well as some individual risk factors (sex, condition score and relative density) determined transmission between pigs. Traditional cross sectional surveys and molecular epidemiological approaches are complementary and together can enhance understanding of disease ecology: abundance of ecological resources critical for wildlife influences Salmonella prevalence, whereas Salmonella transmission is driven by local spatial, social, density and individual factors, rather than resources. This enhanced understanding has implications for the control of diseases in wildlife populations. Attempts to manage wildlife disease using simplistic density approaches do not acknowledge the complexity of disease ecology.

Defining specificity in DNA detection of wildlife: Response to Gonçalves et al. “The risks of using “species-specific” PCR assays in wildlife research: The case of red fox (Vulpes vulpes) identification in Tasmania”

Gonçalves et al. [1] report an examination of the PCR-based species identification test used to detect foxes from faecal material in the ongoing fox eradication programme in Tasmania. The authors rightly point out that poor design in screening methods may lead to false negative or false positive results and they sought to ‘‘better evaluate the specificity of the putatively fox-specific pair of PCR primers’’ as reported in Berry et al. [2] and applied by Sarre et al. [3]. We welcome the scrutiny of the test as DNA-based tests are being used more frequently in wildlife detection [4] and yet estimation of test specificity and sensitivity is rarely attempted. Unfortunately, Gonçalves et al. [1] have undertaken an analysis of an incomplete imitation of the test we applied. In doing so, they reach conclusions that do not apply to the test as it is conducted in practice nor do they actually estimate test specificity. Taken at face value, the findings by Gonçalves et al. [1] could lead to erroneous interpretations, as was the case in a recent publication in which their findings were cited as support for claims of a high rate of false positives in the detection of Tasmanian foxes [5]. The test for fox DNA [2,3] uses a sequential two phase approach aimed at determining, with acceptable probability (Ramsey et al. in review), whether a scat contains fox DNA or not. The first phase of the test comprises an initial screening of DNA through amplification with the primers VV-cytb F and VV-cytb R [2]. These primers preferentially amplify fox mitochondrial cytochrome b DNA and are used to identify samples that are likely to contain fox DNA on the basis of the size of the fragment amplified. They are used as part of a multiplex PCR incorporating a second ‘universal’ primer pair targeting mammalian mitochondrial 12s ribosomal DNA as a positive test for PCR amplification. If the ‘universal’ primers amplify a product but the primers targeting fox cytochrome b DNA do not, then the lack of amplification of possible fox DNA cannot be attributed to PCR failure and is most parsimoniously interpreted as meaning that fox DNA has not been detected. The second phase of the fox DNA detection test is conducted only if a PCR product of the size of the target cytochrome b fragment is detected in the multiplex reaction and involves direct sequencing of the PCR product obtained from a second amplification using the VV-cytb F and VV-cytb R primers only. A scat is considered to contain fox DNA only when a direct match is found between the sequence obtained from the fragment amplified and published fox sequences [2,3,6]. This sequential two phase approach, which is described explicitly in Sarre et al. [6], was adopted to make cost effective the screening of predator scats in the large numbers typically required for the

An examination of the accuracy of a sequential PCR and sequencing test used to detect the incursion of an invasive species: the case of the red fox in Tasmania.

Summary Polymerase chain reaction (PCR) diagnostic tests are increasingly applied to the identification of wildlife. Yet rigorous verification is rare and the estimation of test accuracy (the probability that true positive and true negative samples are correctly identified – test sensitivity and specificity, respectively), particularly in combination with sequencing, is uncommon. This is important because PCR-based tests are prone to contamination in sampling and the laboratory. Here, we use an experimental case–control approach to estimate the sensitivity and specificity of a sequential PCR-based wildlife detection test used to identify incursions of red foxes into Tasmania from predator faeces (scats). Our results show that the sensitivity of the fox test is high (∼94%) for the PCR-based test on its own, but this decreases to ∼84% when combined with the DNA sequencing step. In contrast, the specificity increases from ∼96% in the PCR-only test to ˜99·6% after inclusion of the DNA sequencing step. The intense public scrutiny of the fox eradication programme in Tasmania has undoubtedly influenced the application of a sequential PCR test that maximizes specificity at the expense of sensitivity and so increases the risk that scats containing fox DNA would not be detected. This could lead to the establishment of foxes in Tasmania as a consequence. Synthesis and applications. Importantly, the estimation of the sensitivity and specificity of sequential tests enables decisions about the risk associated with mistaken identification (i.e. false negatives vs. false positives) to be quantified for decision-makers. The cost of false-negative errors should be balanced against the costs of false-positive errors, which could include the expenditure incurred in the application of unnecessary management actions were foxes not in fact present. Understanding the risks and costs associated with both false-negative and false-positive errors is therefore a key component to the decision-making process for the management of the Tasmanian fox incursion.

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.

Primers for detection of Macquarie perch from environmental and trace DNA samples

Predation by invasive salmonids threatens the conservation of the endangered Australian freshwater fish Macquarie perch (Macquaria australasica). Predation is difficult to detect visually because larval fish are rapidly digested. Here, we present PCR primers that will enable the detection of M. australasica from trace samples, removing the need to detect undigested larval fish to confirm predation. We tested these primers on DNA from all fish species present in an upstream section of the Cotter River, a key locality for the species. We demonstrate the primers to be species-specific, amplifying DNA from M. australasica only.

A framework for developing and validating taxon-specific primers for specimen identification from environmental DNA

Taxon‐specific DNA tests are applied to many ecological and management questions, increasingly using environmental DNA (eDNA). eDNA facilitates noninvasive ecological studies but introduces additional risks of bias and error. For effective application, PCR primers must be developed for each taxon and validated in each system. We outline a nine step framework for the development and validation of taxon‐specific primers for eDNA analysis in ecological studies, involving reference database construction, phylogenetic evaluation of the target gene, primer design, primer evaluation in silico, and laboratory evaluation of primer specificity, sensitivity and utility. Our framework makes possible a rigorous evaluation of likely sources of error. The first five steps can be conducted relatively rapidly and (where reference DNA sequences are available) require minimal laboratory resources, enabling assessment of primer suitability before investing in further work. Steps six to eight require more costly laboratory analyses but are essential to evaluate risks of false‐positive and false‐negative results, while step 9 relates to field implementation. As an example, we have developed and evaluated primers to specifically amplify part of the mitochondrial ND2 gene from Australian bandicoots. If adopted during the early stages of primer development, our framework will facilitate large‐scale implementation of well‐designed DNA tests to detect specific wildlife from eDNA samples. This will provide researchers and managers with an understanding of the strengths and limitations of their data and the conclusions that can be drawn from them.

Creating new evolutionary pathways through bioinvasion: the population genetics of brushtail possums in New Zealand.

Rapid increases in global trade and human movement have created novel mixtures of organisms bringing with them the potential to rapidly accelerate the evolution of new forms. The common brushtail possum (Trichosurus vulpecula), introduced into New Zealand from Australia in the 19th century, is one such species having been sourced from multiple populations in its native range. Here, we combine microsatellite DNA‐ and GIS‐based spatial data to show that T. vulpecula originating from at least two different Australian locations exhibit a population structure that is commensurate with their introduction history and which cannot be explained by landscape features alone. Most importantly, we identify a hybrid zone between the two subspecies which appears to function as a barrier to dispersal. When combined with previous genetic, morphological and captive studies, our data suggest that assortative mating between the two subspecies may operate at a behavioural or species recognition level rather than through fertilization, genetic incompatibility or developmental inhibition. Nevertheless, hybridization between the two subspecies of possum clearly occurs, creating the opportunity for novel genetic combinations that would not occur in their natural ranges and which is especially likely given that multiple contact zones occur in New Zealand. This discovery has implications for wildlife management in New Zealand because multiple contact zones are likely to influence the dispersal patterns of possums and because differential susceptibility to baiting with sodium fluoroacetate between possums of different origins may promote novel genetic forms.

Species assignment from trace DNA sequences: an in silico assessment of the test used to survey for foxes in Tasmania

Summary Diagnostic DNA tests have become important for species detection from environmental samples and are increasingly applied to the analysis of ecological systems and in wildlife management. The availability of reference DNA sequences from many taxa enables the development of diagnostic PCR primers. Where there is a high risk of false-positive PCR amplification, or where even a low rate of false positives has serious management implications, DNA sequencing is crucial for accurate specimen identification. The ability of DNA sequencing to discriminate among target and non-target species must be explored for each system. The red fox Vulpes vulpes is an invasive pest in Australia. A fox-specific PCR and sequencing test has been applied to a systematic survey of scats collected in Tasmania, where the management of a recent fox incursion remains controversial. We investigated the risk that DNA sequences obtained using this test might be mistakenly assigned to fox in cases of non-specific amplification, or mistakenly assigned to another species when fox DNA was correctly amplified. We conducted an analysis of barcoding efficacy using cytochrome b sequences from 74 vertebrates. In our analysis, no non-fox sequences were identified as fox (false positives) and no genuine fox sequences were misidentified (false negatives). This two-stage DNA test, including PCR screening and sequencing steps, can reliably discriminate fox DNA from that of other Australian species. Synthesis and applications. DNA tests are attractive to wildlife managers interested in detecting species that are cryptic or difficult to identify. When DNA data directly influence management decisions, it is important to understand the limitations of the genetic markers and the likely causes of failed identifications or erroneous species assignments. We show that short cytochrome b sequences can provide high specificity for vertebrate species assignment. We highlight the importance of developing appropriate reference sequence data bases for each study system, and of evaluating the potential for misidentification of different taxa.

Detecting rare carnivores using scats: Implications for monitoring a fox incursion into Tasmania

Abstract The ability to detect the incursion of an invasive species or destroy the last individuals during an eradication program are some of the most difficult aspects of invasive species management. The presence of foxes in Tasmania is a contentious issue with recent structured monitoring efforts, involving collection of carnivore scats and testing for fox DNA, failing to detect any evidence of foxes. Understanding the likelihood that monitoring efforts would detect fox presence, given at least one is present, is therefore critical for understanding the role of scat monitoring for informing the response to an incursion. We undertook trials to estimate the probability of fox scat detection through monitoring by scat‐detector dogs and person searches and used this information to critically evaluate the power of scat monitoring efforts for detecting foxes in the Tasmanian landscape. The probability of detecting a single scat present in a 1‐km2 survey unit was highest for scat‐detector dogs searches (0.053) compared with person searches (x¯≅0.015) for each 10 km of search effort. Simulation of the power of recent scat monitoring efforts undertaken in Tasmania from 2011 to 2015 suggested that single foxes would have to be present in at least 20 different locations or fox breeding groups present in at least six different locations, in order to be detected with a high level of confidence (>0.80). We have shown that highly structured detection trials can provide managers with the quantitative tools needed to make judgments about the power of large‐scale scat monitoring programs. Results suggest that a fox population, if present in Tasmania, could remain undetected by a large‐scale, structured scat monitoring program. Therefore, it is likely that other forms of surveillance, in conjunction with scat monitoring, will be necessary to demonstrate that foxes are absent from Tasmania with high confidence.