Inbreeding depression in one of the last DFTD-free wild populations of Tasmanian devils.

BACKGROUND: Vulnerable species experiencing inbreeding depression are prone to localised extinctions because of their reduced fitness. For Tasmanian devils, the rapid spread of devil facial tumour disease (DFTD) has led to population declines and fragmentation across the species' range. Here we show that one of the few remaining DFTD-free populations of Tasmanian devils is experiencing inbreeding depression. Moreover, this population has experienced a significant reduction in reproductive success over recent years. METHODS: We used 32 microsatellite loci to examine changes in genetic diversity and inbreeding in the wild population at Woolnorth, alongside field data on breeding success from females to test for inbreeding depression. RESULTS: Wefound that maternal internal relatedness has a negative impact on litter sizes. The results of this study imply that this population may be entering an extinction vortex and that to protect the population genetic rescue should be considered. This study provides conservation managers with useful information for managing wild devils and provides support for the "Wild Devil Recovery Program", which is currently augmenting small, isolated populations.

Fecal Viral Diversity of Captive and Wild Tasmanian Devils Characterized Using Virion-Enriched Metagenomics and Metatranscriptomics.

The Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumor disease (DFTD). While research on DFTD has been extensive, little is known about viruses in devils and whether any are of potential conservation relevance for this endangered species. Using both metagenomics based on virion enrichment and sequence-independent amplification (virion-enriched metagenomics) and metatranscriptomics based on bulk RNA sequencing, we characterized and compared the fecal viromes of captive and wild devils. A total of 54 fecal samples collected from two captive and four wild populations were processed for virome characterization using both approaches. In total, 24 novel marsupial-related viruses, comprising a sapelovirus, astroviruses, rotaviruses, picobirnaviruses, parvoviruses, papillomaviruses, polyomaviruses, and a gammaherpesvirus, were identified, as well as known mammalian pathogens such as rabbit hemorrhagic disease virus 2. Captive devils showed significantly lower viral diversity than wild devils. Comparison of the two virus discovery approaches revealed substantial differences in the number and types of viruses detected, with metatranscriptomics better suited for RNA viruses and virion-enriched metagenomics largely identifying more DNA viruses. Thus, the viral communities revealed by virion-enriched metagenomics and metatranscriptomics were not interchangeable and neither approach was able to detect all viruses present. An integrated approach using both virion-enriched metagenomics and metatranscriptomics constitutes a powerful tool for obtaining a complete overview of both the taxonomic and functional profiles of viral communities within a sample.IMPORTANCE The Tasmanian devil is an iconic Australian marsupial that has suffered an 80% population decline due to a contagious cancer, devil facial tumor disease, along with other threats. Until now, viral discovery in this species has been confined to one gammaherpesvirus (dasyurid herpesvirus 2 [DaHV-2]), for which captivity was identified as a significant risk factor. Our discovery of 24 novel marsupial-associated RNA and DNA viruses, and that viral diversity is lower in captive than in wild devils, has greatly expanded our knowledge of gut-associated viruses in devils and provides important baseline information that will contribute to the conservation and captive management of this endangered species. Our results also revealed that a combination of virion-enriched metagenomics and metatranscriptomics may be a more comprehensive approach for virome characterization than either method alone. Our results thus provide a springboard for continuous improvements in the way we study complex viral communities.

Complex problems need detailed solutions: Harnessing multiple data types to inform genetic management in the wild.

For bottlenecked populations of threatened species, supplementation often leads to improved population metrics (genetic rescue), provided that guidelines can be followed to avoid negative outcomes. In cases where no "ideal" source populations exist, or there are other complicating factors such as prevailing disease, the benefit of supplementation becomes uncertain. Bringing multiple data and analysis types together to plan genetic management activities can help. Here, we consider three populations of Tasmanian devil, Sarcophilus harrisii, as candidates for genetic rescue. Since 1996, devil populations have been severely impacted by devil facial tumour disease (DFTD), causing significant population decline and fragmentation. Like many threatened species, the key threatening process for devils cannot currently be fully mitigated, so species management requires a multifaceted approach. We examined diversity of 31 putatively neutral and 11 MHC-linked microsatellite loci of three remnant wild devil populations (one sampled at two time-points), alongside computational diversity projections, parameterized by field data from DFTD-present and DFTD-absent sites. Results showed that populations had low diversity, connectivity was poor, and diversity has likely decreased over the last decade. Stochastic simulations projected further diversity losses. For a given population size, the effects of DFTD on population demography (including earlier age at death and increased female productivity) did not impact diversity retention, which was largely driven by final population size. Population sizes ≥500 (depending on the number of founders) were necessary for maintaining diversity in otherwise unmanaged populations, even if DFTD is present. Models indicated that smaller populations could maintain diversity with ongoing immigration. Taken together, our results illustrate how multiple analysis types can be combined to address complex population genetic challenges.

No evidence of inbreeding depression in a Tasmanian devil insurance population despite significant variation in inbreeding.

Inbreeding depression occurs when inbred individuals experience reduced fitness as a result of reduced genome-wide heterozygosity. The Tasmanian devil faces extinction due to a contagious cancer, devil facial tumour disease (DFTD). An insurance metapopulation was established in 2006 to ensure the survival of the species and to be used as a source population for re-wilding and genetic rescue. The emergence of DFTD and the rapid decline of wild devil populations have rendered the species at risk of inbreeding depression. We used 33 microsatellite loci to (1) reconstruct a pedigree for the insurance population and (2) estimate genome-wide heterozygosity for 200 individuals. Using heterozygosity-fitness correlations, we investigated the effect of heterozygosity on six diverse fitness measures (ulna length, asymmetry, weight-at-weaning, testes volume, reproductive success and survival). Despite statistically significant evidence of variation in individual inbreeding in this population, we found no associations between inbreeding and any of our six fitness measurements. We propose that the benign environment in captivity may decrease the intensity of inbreeding depression, relative to the stressful conditions in the wild. Future work will need to measure fitness of released animals to facilitate translation of this data to the broader conservation management of the species in its native range.

Development of a SNP-based assay for measuring genetic diversity in the Tasmanian devil insurance population.

BACKGROUND: The Tasmanian devil (Sarcophilus harrisii) has undergone a recent, drastic population decline due to the highly contagious devil facial tumor disease. The tumor is one of only two naturally occurring transmissible cancers and is almost inevitably fatal. In 2006 a disease-free insurance population was established to ensure that the Tasmanian devil is protected from extinction. The insurance program is dependent upon preserving as much wild genetic diversity as possible to maximize the success of subsequent reintroductions to the wild. Accurate genotypic data is vital to the success of the program to ensure that loss of genetic diversity does not occur in captivity. Until recently, microsatellite markers have been used to study devil population genetics, however as genetic diversity is low in the devil and potentially decreasing in the captive population, a more sensitive genotyping assay is required. METHODS: Utilising the devil reference genome and whole genome re-sequencing data, we have identified polymorphic regions for use in a custom genotyping assay. These regions were amplified using PCR and sequenced on the Illumina MiSeq platform to refine a set a markers to genotype the Tasmanian devil insurance population. RESULTS: We have developed a set of single nucleotide polymorphic (SNP) markers, assayed by amplicon sequencing, that provide a high-throughput method for monitoring genetic diversity and assessing familial relationships among devils. To date we have used a total of 267 unique SNPs within both putatively neutral and functional loci to genotype 305 individuals in the Tasmanian devil insurance population. We have used these data to assess genetic diversity in the population as well as resolve the parentage of 21 offspring. CONCLUSIONS: Our molecular data has been incorporated with studbook management practices to provide more accurate pedigree information and to inform breeding recommendations. The assay will continue to be used to monitor the genetic diversity of the insurance population of Tasmanian devils with the aim of reducing inbreeding and maximizing success of reintroductions to the wild.

Genomic insights into a contagious cancer in Tasmanian devils.

The Tasmanian devil faces extinction due to a contagious cancer. Genetic and genomic technologies revealed that the disease arose in a Schwann cell of a female devil. Instead of dying with the original host, the tumour was passed from animal to animal, slipping under the radar of the immune system. Studying the genomes of the devil and the cancer has driven our understanding of this unique disease. From characterising immune genes and immune responses to studying tumour evolution, we have begun to uncover how a cancer can be 'caught' and are using genomic data to manage an insurance population of disease-free devils for the long-term survival of the species.

Lack of genetic diversity across diverse immune genes in an endangered mammal, the Tasmanian devil (Sarcophilus harrisii).

The Tasmanian devil (Sarcophilus harrisii) is threatened with extinction due to the spread of devil facial tumour disease. Polymorphisms in immune genes can provide adaptive potential to resist diseases. Previous studies in diversity at immune loci in wild species have almost exclusively focused on genes of the major histocompatibility complex (MHC); however, these genes only account for a fraction of immune gene diversity. Devils lack diversity at functionally important immunity loci, including MHC and Toll-like receptor genes. Whether there are polymorphisms at devil immune genes outside these two families is unknown. Here, we identify polymorphisms in a wide range of key immune genes, and develop assays to type single nucleotide polymorphisms (SNPs) within a subset of these genes. A total of 167 immune genes were examined, including cytokines, chemokines and natural killer cell receptors. Using genome-level data from ten devils, SNPs within coding regions, introns and 10 kb flanking genes of interest were identified. We found low polymorphism across 167 immune genes examined bioinformatically using whole-genome data. From this data, we developed long amplicon assays to target nine genes. These amplicons were sequenced in 29-220 devils and found to contain 78 SNPs, including eight SNPS within exons. Despite the extreme paucity of genetic diversity within these genes, signatures of balancing selection were exhibited by one chemokine gene, suggesting that remaining diversity may hold adaptive potential. The low functional diversity may leave devils highly vulnerable to infectious disease, and therefore, monitoring and preserving remaining diversity will be critical for the long-term management of this species. Examining genetic variation in diverse immune genes should be a priority for threatened wildlife species. This study can act as a model for broad-scale immunogenetic diversity analysis in threatened species.