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Genetic diversity among and within populations of all species is necessary for people and nature to survive and thrive in a changing world. Over the past three years, commitments for conserving genetic diversity have become more ambitious and specific under the Convention on Biological Diversity's (CBD) draft post-2020 global biodiversity framework (GBF). This Perspective article comments on how goals and targets of the GBF have evolved, the improvements that are still needed, lessons learned from this process, and connections between goals and targets and the actions and reporting that will be needed to maintain, protect, manage and monitor genetic diversity. It is possible and necessary that the GBF strives to maintain genetic diversity within and among populations of all species, to restore genetic connectivity, and to develop national genetic conservation strategies, and to report on these using proposed, feasible indicators. ispartof: CONSERVATION GENETICS vol:24 issue:2 pages:181-191 ispartof: location:Netherlands status: published

Conservation research has historically been conducted at the macro level, focusing on animals and plants and their role in the wider ecosystem. However, there is a growing appreciation of the importance of microbial communities in conservation. Most microbiome research in conservation thus far has used amplicon sequencing methods to assess the taxonomic composition of microbial communities and inferred functional capabilities from these data. However, as manipulation of the microbiome as a conservation tool becomes more and more feasible, there is a growing need to understand the direct functional consequences of shifts in microbiome composition. This review outlines the latest advances in microbiome research from a functional perspective and how these data can be used to inform conservation strategies. This review will also consider some of the challenges faced when studying the microbiomes of wild animals and how they can be overcome by careful study design and sampling methods. Environmental changes brought about by climate change or direct human actions have the potential to alter the taxonomic composition of microbiomes in wild populations. Understanding how taxonomic shifts affect the function of microbial communities is important for identifying species most threatened by potential disruption to their microbiome. Preservation or even restoration of these functions has the potential to be a powerful tool in conservation biology and a shift towards functional characterisation of gut microbiome diversity will be an important first step.

Background While recent advances in genomics has enabled vast improvements in the quantification of genome-wide diversity and the identification of adaptive and deleterious alleles in model species, wildlife and non-model species have largely not reaped the same benefits. This has been attributed to the resources and infrastructure required to develop essential genomic datasets such as reference genomes. In the absence of a high-quality reference genome, cross-species alignments can provide reliable, cost-effective methods for single nucleotide variant (SNV) discovery. Here, we demonstrated the utility of cross-species genome alignment methods in gaining insights into population structure and functional genomic features in cheetah (Acinonyx jubatas), snow leopard (Panthera uncia) and Sumatran tiger (Panthera tigris sumatrae), relative to the domestic cat (Felis catus). Results Alignment of big cats to the domestic cat reference assembly yielded nearly complete sequence coverage of the reference genome. From this, 38,839,061 variants in cheetah, 15,504,143 in snow leopard and 13,414,953 in Sumatran tiger were discovered and annotated. This method was able to delineate population structure but limited in its ability to adequately detect rare variants. Enrichment analysis of fixed and species-specific SNVs revealed insights into adaptive traits, evolutionary history and the pathogenesis of heritable diseases. Conclusions The high degree of synteny among felid genomes enabled the successful application of the domestic cat reference in high-quality SNV detection. The datasets presented here provide a useful resource for future studies into population dynamics, evolutionary history and genetic and disease management of big cats. This cross-species method of variant discovery provides genomic context for identifying annotated gene regions essential to understanding adaptive and deleterious variants that can improve conservation outcomes.

Biodiversity underlies ecosystem resilience, ecosystem function, sustainable economies, and human well-being. Understanding how biodiversity sustains ecosystems under anthropogenic stressors and global environmental change will require new ways of deriving and applying biodiversity data. A major challenge is that biodiversity data and knowledge are scattered, biased, collected with numerous methods, and stored in inconsistent ways. The Group on Earth Observations Biodiversity Observation Network (GEO BON) has developed the Essential Biodiversity Variables (EBVs) as fundamental metrics to help aggregate, harmonize, and interpret biodiversity observation data from diverse sources. Mapping and analyzing EBVs can help to evaluate how aspects of biodiversity are distributed geographically and how they change over time. EBVs are also intended to serve as inputs and validation to forecast the status and trends of biodiversity, and to support policy and decision making. Here, we assess the feasibility of implementing Genetic Composition EBVs (Genetic EBVs), which are metrics of within-species genetic variation. We review and bring together numerous areas of the field of genetics and evaluate how each contributes to global and regional genetic biodiversity monitoring with respect to theory, sampling logistics, metadata, archiving, data aggregation, modeling, and technological advances. We propose four Genetic EBVs: (i) Genetic Diversity; (ii) Genetic Differentiation; (iii) Inbreeding; and (iv) Effective Population Size (Ne ). We rank Genetic EBVs according to their relevance, sensitivity to change, generalizability, scalability, feasibility and data availability. We outline the workflow for generating genetic data underlying the Genetic EBVs, and review advances and needs in archiving genetic composition data and metadata. We discuss how Genetic EBVs can be operationalized by visualizing EBVs in space and time across species and by forecasting Genetic EBVs beyond current observations using various modeling approaches. Our review then explores challenges of aggregation, standardization, and costs of operationalizing the Genetic EBVs, as well as future directions and opportunities to maximize their uptake globally in research and policy. The collection, annotation, and availability of genetic data has made major advances in the past decade, each of which contributes to the practical and standardized framework for large-scale genetic observation reporting. Rapid advances in DNA sequencing technology present new opportunities, but also challenges for operationalizing Genetic EBVs for biodiversity monitoring regionally and globally. With these advances, genetic composition monitoring is starting to be integrated into global conservation policy, which can help support the foundation of all biodiversity and species' long-term persistence in the face of environmental change. We conclude with a summary of concrete steps for researchers and policy makers for advancing operationalization of Genetic EBVs. The technical and analytical foundations of Genetic EBVs are well developed, and conservation practitioners should anticipate their increasing application as efforts emerge to scale up genetic biodiversity monitoring regionally and globally. ispartof: BIOLOGICAL REVIEWS vol:97 issue:4 pages:1511-1538 ispartof: location:England status: published

Classic Mendelian inheritance is the bedrock of population genetics and underpins pedigree-based management of animal populations. However, assumptions of Mendelian inheritance might not be upheld in conservation breeding programmes if early viability selection occurs, even when efforts are made to equalise genetic contributions of breeders. To test this possibility, we investigated deviations from Mendelian proportions in a captive metapopulation of the endangered Tasmanian devil. This marsupial population is ideal for addressing evolutionary questions in conservation due to its large size, range of enclosure types (varying in environmental conditions), good genomic resources (which aid interpretation), and the species' biology. Devil mothers give birth to more offspring than they can nurse in the pouch, providing the potential for intense viability selection amongst embryos. We used data from 140 known sire-dam-offspring triads to isolate within-family selection from population-level mechanisms (such as mate choice or inbreeding), and compared observed offspring genotypes at 123 targeted SNPs to neutral (i.e., Mendelian) expectations. We found lower offspring heterozygosity than expected, and subtle patterns that varied across a gradient of management intensity from zoo-like enclosures to semi-wild environments for some loci. Meiotic drive or maternal-foetal incompatibilities are consistent with our results, although we cannot statistically confirm these mechanisms. We found some evidence that maternal genotype affects annual litter size, suggesting that family-level patterns are driven by differential offspring mortality before birth or during early development. Our results show that deviations from Mendelian inheritance can occur in conservation programmes, despite best-practice management to prevent selection.

Conservation breeding programs such as zoos play a major role in preventing extinction, but their sustainability may be impeded by neutral and adaptive population genetic change. These changes are difficult to detect for a single species or context, and impact global conservation efforts. We analyse pedigree data from 15 vertebrate species – over 30,000 individuals – to examine offspring survival over generations of captive breeding. Even accounting for inbreeding, we find that the impacts of increasing generations in captivity are highly variable across species, with some showing substantial increases or decreases in offspring survival over generations. We find further differences between dam and sire effects in first- versus multi-generational analysis. Crucially, our multispecies analysis reveals that responses to captivity could not be predicted from species’ evolutionary (phylogenetic) relationships. Even under best-practice captive management, generational fitness changes that cannot be explained by known processes (such as inbreeding depression), are occurring., Captive breeding could prevent species extinctions, but selection for captivity may decrease fitness. Here the authors analyse pedigree data on 15 long-running vertebrate breeding programs and find generational fitness changes that processes such as inbreeding depression cannot explain.

Species’ declines around the globe have reached such a rapid rate that without the implementation of extreme conservation methods, we are likely to witness the next mass extinction. Translocation is a popular and growing conservation technique, especially to islands. However, despite the widespread use of this method, the long-term genetic outlook of populations established on islands (namely those with small carrying capacity, and isolated from other populations) is often poorly quantified, and there are conflicting data supporting the relative roles of evolutionary processes such as drift, balancing selection, directional selection and genetic purging. Taking examples from avian translocations, this paper reviews recent data on the genetic dynamics of island populations established for conservation purposes. The goal of conservation genetics is to preserve species’ adaptive potential for the long term. In general, population bottlenecks cause losses of genetic diversity, but there is mixed evidence for the importance of other evolutionary processes, such as maintenance of genetic diversity by balancing selection. There is a clear need for long-term studies that test for associations between genetic change and success of populations established via translocation to conservation islands, and for integration of these observations with ecological and climate data. Combining data types into the future will provide a holistic view on the interactions between the role selection plays, how island populations established by translocations may respond, and how this information can improve conservation strategy.

The increased availability of genomic resources for many species has expanded perspectives on problems in conservation by helping to design management strategies for threatened species. Tasmanian devils (Sarcophilus harrisii) are an iconic and endangered marsupial with an intensively managed breeding program aimed at preventing extinction in the wild caused by devil facial tumour disease. Between 2015 and 2017, 85 devils from this program were released to three sites in Tasmania to support wild populations. Of these, 26 were known to have been killed by vehicles shortly after release. A previous analysis indicated that increased generations in captivity was a positive predictor of vehicle strike, with possible behavioural change hypothesised. Here we use 39 resequenced devil genomes to characterise diversity at 35 behaviour-associated genes, which contained 826 single nucleotide polymorphisms (24 were non-synonymous). We tested for a predictor of survival by examining three genes (AVPR1B, OXT and SLC6A4) in 62 released devils with known fates (survived, N = 39; died, N = 23), and genome-wide associations via reduced-representation sequencing (1727 single nucleotide polymorphisms [SNPs]), in 55 devils with known fates (survived, N = 38; died, N = 17). Overall, there was little evidence of an association between genetic profile and probability of being struck by a vehicle. Despite previous evidence of low genetic diversity in devils, the 35 behaviour-associated genes contained variation that may influence their functions. Our dataset can be used for future research into devil behavioural ecology, and adds to the increasing body of research applying genomics to conservation problems.

Captive breeding for conservation is widely used to prevent extinction, however these programs face many challenges due to small population size, founder effects, lack of migration and potential adaptation to captivity. Together these influence how selection, genetic drift and gene flow shape the genetic makeup of small populations, so examining these in a captive setting is valuable for appropriate biodiversity management. We have quantified the effects of selection, drift and gene flow in 503 individuals across five generations from the Tasmanian devil insurance population. To determine whether different processes were acting in different settings, we separately analysed animals housed under individual-based management, versus those that were released to an island site. We found that a greater proportion of alleles were lost over time in the smaller island population than in captivity and propose that genetic drift is the most likely process influencing this result. We found that the captive population became more heterozygous over time, while the island population stayed constant. Our molecular measure of inbreeding found a decrease over generations in captivity that is not captured by pedigree-based analysis. As management of breeding interactions only takes place in the captive population, our results are consistent with gene flow through managed breeding in captivity minimising inbreeding over time. Gene flow was also evident through changes to population structure in the captive population. Our findings serve as an example of how conservation managers can assess the impact of evolutionary processes on managed populations and adapt management practices if required.

Inter-individual variation at genes known to play a role in reproduction may impact reproductive fitness. The Tasmanian devil is an endangered Australian marsupial with low genetic diversity. Recent work has shown concerning declines in productivity in both wild and captive populations over time. Understanding whether functional diversity exists at reproductive genes in the Tasmanian devil is a key first step in identifying genes that may influence productivity. We characterised single nucleotide polymorphisms (SNPs) at 214 genes involved in reproduction in 37 Tasmanian devils. Twenty genes contained non-synonymous substitutions, with genes involved in embryogenesis, fertilisation and hormonal regulation of reproduction displaying greater numbers of nonsynonymous SNPs than synonymous SNPs. Two genes, ADAMTS9 and NANOG, showed putative signatures of balancing selection indicating that natural selection is maintaining diversity at these genes despite the species exhibiting low overall levels of genetic diversity. We will use this information in future to examine the interplay between reproductive gene variation and reproductive fitness in Tasmanian devil populations.

How innovations such as vision, flight and pregnancy evolve is a central question in evolutionary biology. Examination of transitional (intermediate) forms of these traits can help address this question, but these intermediate phenotypes are very rare in extant species. Here we explore the biology and evolution of transitional forms of pregnancy that are midway between the ancestral state of oviparity (egg-laying) and the derived state, viviparity (live birth). Transitional forms of pregnancy occur in only three vertebrates, all of which are lizard species that also display intraspecific variation in reproductive phenotype. In these lizards (Lerista bougainvillii, Saiphos equalis, and Zootoca vivipara), geographic variation of three reproductive forms occurs within a single species: oviparity, viviparity, and a transitional form of pregnancy. This phenomenon offers the valuable prospect of watching ‘evolution in action’. In these species, it is possible to conduct comparative research using different reproductive forms that are not confounded by speciation, and are of relatively recent origin. We identify major proximate and ultimate questions that can be addressed in these species, and the genetic and genomic tools that can help us understand how transitional forms of pregnancy are produced, despite predicted fitness costs. We argue that these taxa represent an excellent prospect for understanding the major evolutionary shift between egg-laying and live birth, which is a fundamental innovation in the history of animals.

The Coalition for Conservation Genetics (CCG) brings together four eminent organizations with the shared goal of improving the integration of genetic information into conservation policy and practice. We provide a historical context of conservation genetics as a field and reflect on current barriers to conserving genetic diversity, highlighting the need for collaboration across traditional divides, international partnerships, and coordinated advocacy. We then introduce the CCG and illustrate through examples how a coalition approach can leverage complementary expertise and improve the organizational impact at multiple levels. The CCG has proven particularly successful at implementing large synthesis-type projects, training early-career scientists, and advising policy makers. Achievements to date highlight the potential for the CCG to make effective contributions to practical conservation policy and management that no one “parent” organization could achieve on its own. Finally, we reflect on the lessons learned through forming the CCG, and our vision for the future.

A correction to this paper has been published: https://doi.org/10.1007/s10592-021-01376-9

Establishing populations of endangered species in captivity is becoming an increasingly common component of species recovery programs for insurance against extinction, and/or for reintroductions. It is important for the success of these efforts that captive populations are genetically representative of wild source populations, and that genetic diversity is maintained over time. Our study presents SNP data from wild and captive populations of the critically endangered Australian orange-bellied parrot (Neophema chrysogaster). We examine the genetic effects of a decision made in the 2010/2011 breeding season to recruit half of the juvenile cohort (n = 21), from a wild population in decline, to supplement an existing captive program. We report that heterozygosity among wild birds decreased in the years after this action. Following multiple releases of captive-reared birds back into the wild (occurring annually since 2013), captive and wild populations have attained similar overall levels of heterozygosity, and genetic differentiation between these populations is low. Parentage analyses confirm that captive-bred released individuals have successfully paired with wild birds and produced offspring. Our study suggests that translocation of wild individuals into captivity, from wild populations in decline, can potentially have deleterious lasting impacts on genetic diversity levels in these populations. However, our data also confirm that in captivity, founder diversity can be successfully preserved over time, and addition of wild founders can improve captive population health. The genetic diversity retained in captive populations can also be reintroduced to wild populations at a later date, provided that captive-release individuals are able to reproductively contribute to their recipient population.

Incorporating mate choice into conservation breeding programs can improve reproduction and the retention of natural behaviors. However, different types of genetic‐based mate choice can have varied consequences for genetic diversity management. As a result, it is important to examine mechanisms of mate choice in captivity to assess its costs and benefits. Most research in this area has focused on experimental pairing trials; however, this resource‐intensive approach is not always feasible in captive settings and can interfere with other management constraints. We used generalized linear mixed models and permutation approaches to investigate overall breeding success in group‐housed Tasmanian devils at three nonmutually exclusive mate choice hypotheses: (a) advantage of heterozygous individuals, (b) advantage of dissimilar mates, and (c) optimum genetic distance, using both 1,948 genome‐wide SNPs and 12 MHC‐linked microsatellites. The managed devil insurance population is the largest such breeding program in Australia and is known to have high variance in reproductive success. We found that nongenetic factors such as age were the best predictors of breeding success in a competitive breeding scenario, with younger females and older males being more successful. We found no evidence of mate choice under the hypotheses tested. Mate choice varies among species and across environments, so we advocate for more studies in realistic captive management contexts as experimental or wild studies may not apply. Conservation managers must weigh up the need to wait for adequate sample sizes to detect mate choice with the risk that genetic changes may occur during this time in captivity. Our study shows that examining and integrating mate choice into the captive management of species housed in realistic, semi‐natural group‐based contexts may be more difficult than previously considered.

Conservation breeding programs, such as fenced reserves, conserve species by removing populations from key threatening processes and providing supplementary resources to support species preservation. This form of management often has the eventual aim to reintroduce individuals back to their native habitat. A key issue with captive management is the small breeding pool, due to generally small population sizes and limited migration, which may lead to inbreeding and consequent inbreeding depression. We investigated inbreeding and its potential fitness consequences in a large, free-ranging, captive population of an Australian marsupial, the long-nosed potoroo (Potorous tridactylus), a species that has been recently impacted by severe bushfire. We used reduced-representation sequencing to genotype single nucleotide polymorphisms and reconstruct a pedigree for the population. We detected low levels of inbreeding in our partial pedigree that incorporated 50.7% (34/67) of sampled individuals. Inbreeding depression was investigated using heterozygosity–fitness correlations of six fitness measurements, but we found no evidence of inbreeding depression in the population despite strong molecular evidence for inter-individual variation in inbreeding coefficient. Our findings suggest that the potoroo population at Tidbinbilla Nature Reserve (TNR) maintains a high level of diversity. As the population is descendant of animals from divergent sources, the TNR population also helps conservationists to learn more about potential consequences of admixture, and plan recovery strategies for the species. For TNR potoroos, we suggest that current management protocols have been effective at maintaining diversity to date. More generally, our results show how modern molecular techniques can efficiently characterise the genetic profile of a free-ranging, captive population to help inform management guidelines for wild recovery.

Recent advances in molecular genetics have enabled a great deal of information about species to be obtained from analysis of non-invasively collected samples such as scat. Scat provides genetic information via residual host DNA on the outside of the scat, via characterising the genetic makeup of intestinal microbes that are present in the scat, or by examining the DNA remnants of prey items that have passed through the animal’s digestive tract. In this review, we provide a case study to demonstrate how these approaches are being used to better understand the threatened Tasmanian devil in the landscape, and to support the species’ conservation. Scat analysis enables us to quantify the genetic diversity of remote populations, where trapping is logistically challenging. We are beginning to learn how conservation management impacts the microbiome of threatened species, and investigate how various management strategies may be impacting the diverse array of bacteria and viruses that devils, like all animal species, are host to. We are using scat samples to better understand the interaction between devils and other animals in their environment by learning more about what they eat. We explore the strengths and challenges of these approaches by comparing our work to that conducted in other species. Finally, we provide specific examples of how our results are being integrated into conservation strategy for the devil.

Top carnivores are essential for maintaining ecosystem stability and biodiversity. Yet, carnivores are declining globally and current

Quantifying fitness of wild organisms from genomic data alone is a challenging frontier

The major histocompatibility complex (MHC) plays a critical role in the vertebrate immune system. Accurate MHC typing is critical to understanding not only host fitness and disease susceptibility, but also the mechanisms underlying host-pathogen co-evolution. However, due to the high degree of gene duplication and diversification of MHC genes, it is often technically challenging to accurately characterise MHC genetic diversity in non-model species. Here we conducted a systematic review to identify common issues associated with current widely used MHC typing approaches. Then to overcome these challenges, we developed a long-read based MHC typing method along with a new analysis pipeline. Our approach enables the sequencing of fully phased MHC alleles spanning all key functional domains and the separation of highly similar alleles as well as the removal of technical artefacts such as PCR heteroduplexes and chimeras. Using this approach, we performed population-scale MHC typing in the Tasmanian devil (Sarcophilus harrisii), revealing previously undiscovered MHC functional diversity in this endangered species. Our new method provides a better solution for addressing research questions that require high MHC typing accuracy. Since the method is not limited by species or the number of genes analysed, it will be applicable for studying not only the MHC but also other complex gene families.

ispartof: CONSERVATION GENETICS vol:22 issue:4 pages:533-536 status: published

Reduced-representation sequencing methods have wide utility in conservation genetics of non-model species. Several methods are now available that reduce genome complexity to examine a wide range of markers in a large number of individuals. We produced two datasets collected using different laboratory techniques, comprising a common set of samples from the greater bilby (Macrotis lagotis). We examined the impact of differing data filtering thresholds on downstream population inferences. We found that choice of restriction enzyme and data filtering thresholds, especially the rate of allowable missing data, impacted our ability to detect population structure. Estimates of FST were robust to alterations in laboratory and bioinformatic protocols while principal coordinates and STRUCTURE analyses showed variation according to the number of loci and percent missing data. We advise researchers using reduced-representation sequencing in conservation projects to examine a range of data thresholds, and follow these through to downstream population inferences. Multiple measures of population differentiation should be used in order to fully understand how data filtering thresholds influence the final dataset, paying particular attention to the impact of allowable missing data. Our results indicate that failure to follow these checks could impact conclusions drawn, and conservation management decisions made.

Mate choice contributes to driving evolutionary processes when animals choose breeding partners that confer genetic advantages to offspring, such as increased immunocompetence. The major histocompatibility complex (MHC) is an important group of immunological molecules, as MHC antigens bind and present foreign peptides to T-cells. Recent studies suggest that mates may be selected based on their MHC profile, leading to an association between an individual’s MHC diversity and their breeding success. In conservation, it may be important to consider mate choice in captive breeding programs, as this mechanism may improve reproductive rates. We investigated the reproductive success of Tasmanian devils in a group housing facility to determine whether increased MHC-based heterozygosity led individuals to secure more mating partners and produce more offspring. We also compared the breeding success of captive females to a wild devil population. MHC diversity was quantified using 12 MHC-linked microsatellite markers, including 11 previously characterized markers and one newly identified marker. Our analyses revealed that there was no relationship between MHC-linked heterozygosity and reproductive success either in captivity or the wild. The results of this study suggest that, for Tasmanian devils, MHC-based heterozygosity does not produce greater breeding success and that no specific changes to current captive management strategies are required with respect to preserving MHC diversity.

Captive animals are commonly housed in groups to make efficient use of limited resources and allow for natural social behaviour. Captive management relies on accurate pedigrees to estimate various population genetic parameters, such as genetic contributions of breeders, but pedigrees of group-housed offspring can be uncertain. Pedigree analysis software incorporates genetic information from multiple putative parents (“MULT”). Molecular pedigree reconstruction to resolve pedigree uncertainties can be costly. We quantify the need for molecular parentage assignment by comparing predicted offspring contributions (based on uncertain “MULT” pedigrees) to contributions obtained from a molecular genetic pedigree reconstruction. Parentage of 81 insurance population Tasmanian devils (Sarcophilus harrisii) born in free-range enclosures from 2011 to 2017 was resolved using 891 single nucleotide polymorphisms. We observed large discrepancies between the MULT pedigree and molecular pedigree data, revealing both overestimates and underestimates of genetic contributions of individuals, and different pedigree-based effective population sizes (102 vs. 158 respectively). The molecular data revealed that reproductive skew (proportion of adults that failed to breed) was high for both sexes. Over half of the wild-born individuals in our dataset were found to have not bred. If undetected, variation in breeding success undermines the utility of pedigree management and may threaten the success of captive breeding. Molecular techniques are increasingly cost-effective, and our data demonstrate that they are critical to devil management. Where feasible, we recommend molecular management of group-housed species in captivity to avoid inaccurate estimates of genetic diversity and to identify non-breeding individuals, in particular founders, for targeted breeding.

Background Recent advances in genomics have greatly increased research opportunities for non-model species. For wildlife, a growing availability of reference genomes means that population genetics is no longer restricted to a small set of anonymous loci. When used in conjunction with a reference genome, reduced-representation sequencing (RRS) provides a cost-effective method for obtaining reliable diversity information for population genetics. Many software tools have been developed to process RRS data, though few studies of non-model species incorporate genome alignment in calling loci. A commonly-used RRS analysis pipeline, Stacks, has this capacity and so it is timely to compare its utility with existing software originally designed for alignment and analysis of whole genome sequencing data. Here we examine population genetic inferences from two species for which reference-aligned reduced-representation data have been collected. Our two study species are a threatened Australian marsupial (Tasmanian devil Sarcophilus harrisii; declining population) and an Arctic-circle migrant bird (pink-footed goose Anser brachyrhynchus; expanding population). Analyses of these data are compared using Stacks versus two widely-used genomics packages, SAMtools and GATK. We also introduce a custom R script to improve the reliability of single nucleotide polymorphism (SNP) calls in all pipelines and conduct population genetic inferences for non-model species with reference genomes. Results Although we identified orders of magnitude fewer SNPs in our devil dataset than for goose, we found remarkable symmetry between the two species in our assessment of software performance. For both datasets, all three methods were able to delineate population structure, even with varying numbers of loci. For both species, population structure inferences were influenced by the percent of missing data. Conclusions For studies of non-model species with a reference genome, we recommend combining Stacks output with further filtering (as included in our R pipeline) for population genetic studies, paying particular attention to potential impact of missing data thresholds. We recognise SAMtools as a viable alternative for researchers more familiar with this software. We caution against the use of GATK in studies with limited computational resources or time. Electronic supplementary material The online version of this article (10.1186/s12864-019-5806-y) contains supplementary material, which is available to authorized users.

Molecular markers are a useful tool allowing conservation and population managers to shed light on genetic processes affecting threatened populations. However, as technological advancements in molecular techniques continue to evolve, conservationists are frequently faced with new genetic markers, each with nuanced variation in their characteristics as well as advantages and disadvantages for informing various questions. We used a well-studied population of Tasmanian devils (Sarcophilus harrisii) from Maria Island, Tasmania, to illustrate the issues associated with combining multiple genetic data sets and to help answer a question posed by many population managers: which data set will provide the most precise and accurate estimates of the population processes we are trying to measure? We analysed individual heterozygosity (as internal relatedness, IR) of 96 individuals, calculated using four genetic marker types (putatively neutral microsatellites, major histocompatibility complex-linked microsatellites, reduced representation sequencing, and candidate region resequencing). We found no correlation in IR values across marker types, suggesting that various genetic markers reflect different aspects of genomic diversity. In addition, some marker types were more informative than others for conservation decision-making. Reduced representation sequencing provided the highest precision (lowest error) for estimating population-level genetic diversity, and most closely reflected genome-wide heterozygosity both theoretically and empirically. Within the conservation context, our results highlight important considerations when choosing a molecular technique for wildlife genetics.

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.

Background The Tasmanian devil (Sarcophilus harrisii) is the world's largest extant marsupial carnivore. Since the emergence of devil facial tumour disease in 1996, the species has undergone a severe population decline. The insurance population (IP) was established in 2006 to build a disease-free captive population to maintain 95% of the wild Tasmanian devil genetic diversity for 50 years. Captive and semi-wild Tasmanian devils are fed with possum and wallaby meat provided by local hunters, who use lead ammunition. Lead ingestion can cause acute toxicity, including ataxia, coma and death, or chronic subclinical deleterious effects including decreased fertility. Methods We determined blood lead concentrations in 26 captive and 133 wild Tasmanian devils from various sites across Tasmania. Results Captive Tasmanian devils showed significantly higher blood lead concentrations than their conspecifics in the wild. In captivity, older animals had higher blood lead concentrations than young animals, which suggested regular exposure, as lead can accumulate in a living organism in the blood, soft tissues and bones. After a response measure was implemented by removing the heads and wounds containing lead from the diet, blood concentrations significantly decreased in animals at one of the captive study sites, supporting the suspicion of food as the source of lead. Conclusion This study highlights the need to ensure meat fed to captive carnivores is not contaminated by lead, especially in the context of a conservation program breeding individuals in captivity, as for Tasmanian devils.

Conservation introductions to islands and fenced enclosures are increasing as in situ mitigations fail to keep pace with population declines. Few studies consider the potential loss of genetic diversity and increased inbreeding if released individuals breed disproportionately. As funding is limited and post-release monitoring expensive for conservation programs, understanding how sampling effort influences estimates of reproductive variance is useful. To investigate this relationship, we used a well-studied population of Tasmanian devils (Sarcophilus harrisii) introduced to Maria Island, Tasmania, Australia. Pedigree reconstruction based on molecular data revealed high variance in number of offspring per breeder and high proportions of unsuccessful individuals. Computational subsampling of 20%, 40%, 60%, and 80% of observed offspring resulted in inaccurate estimates of reproductive variance compared to the pedigree reconstructed with all sampled individuals. With decreased sampling effort, the proportion of inferred unsuccessful individuals was overestimated and the variance in number of offspring per breeder was underestimated. To accurately estimate reproductive variance, we recommend sampling as many individuals as logistically possible during the early stages of population establishment. Further, we recommend careful selection of colonizing individuals as they may be disproportionately represented in subsequent generations. Within the conservation management context, our results highlight important considerations for sample collection and post-release monitoring during population establishment.

Extinction risk is increasing for a range of species due to a variety of threats, including disease. Emerging infectious diseases can cause severe declines in wild animal populations, increasing population fragmentation and reducing gene flow. Small, isolated, host populations may lose adaptive potential and become more susceptible to extinction due to other threats. Management of the genetic consequences of disease-induced population decline is often necessary. Whilst disease threats need to be addressed, they can be difficult to mitigate. Actions implemented to conserve the Tasmanian devil (Sarcophilus harrisii), which has suffered decline to the deadly devil facial tumour disease (DFTD), exemplify how genetic management can be used to reduce extinction risk in populations threatened by disease. Supplementation is an emerging conservation technique that may benefit populations threatened by disease by enabling gene flow and conserving their adaptive potential through genetic restoration. Other candidate species may benefit from genetic management via supplementation but concerns regarding outbreeding depression may prevent widespread incorporation of this technique into wildlife disease management. However, existing knowledge can be used to identify populations that would benefit from supplementation where risk of outbreeding depression is low. For populations threatened by disease and, in situations where disease eradication is not an option, wildlife managers should consider genetic management to buffer the host species against inbreeding and loss of genetic diversity.

ispartof: BIOLOGICAL CONSERVATION vol:253 status: published

Understanding how inbreeding affects endangered species in conservation breeding programs is essential for their recovery. The Hawaiian Crow ('Alalā) (Corvus hawaiiensis) is one of the world's most endangered birds. It went extinct in the wild in 2002, and, until recent release efforts starting in 2016, nearly all of the population remained under human care for conservation breeding. Using pedigree inbreeding coefficients (F), we evaluated the effects of inbreeding on Hawaiian Crow offspring survival and reproductive success. We used regression tree analysis to identify the level of inbreeding (i.e., inbreeding threshold) that explains a substantial decrease in 'Alalā offspring survival to recruitment. Similar to a previous study of inbreeding in 'Alalā, we found that inbreeding had a negative impact on offspring survival but that parental (vs. artificial) egg incubation improved offspring survival to recruitment. Furthermore, we found that inbreeding did not substantially affect offspring reproductive success, based on the assumption that offspring that survive to adulthood breed with distantly related mates. Our novel application of regression tree analysis showed that offspring with inbreeding levels exceeding F = 0.098 were 69% less likely to survive to recruitment than more outbred offspring, providing a specific threshold value for ongoing population management. Our results emphasize the importance of assessing inbreeding depression across all life history stages, confirm the importance of prioritizing parental over artificial egg incubation in avian conservation breeding programs, and demonstrate the utility of regression tree analysis as a tool for identifying inbreeding thresholds, if present, in any pedigree-managed population.Moverse de las Tendencias a los Referentes Mediante el Análisis de Árbol de Regresión para Encontrar los Umbrales de Endogamia de una Ave en Peligro Crítico Resumen Es necesario entender cómo afecta la endogamia a las especies en peligro que se encuentran en programas de reproducción para la conservación para la recuperación exitosa de estas especies. El cuervo hawaiano (‘Alalā) (Corvus hawaiiensis) es una de las especies de ave que se encuentra en mayor peligro de extinción en todo el mundo. Esta especie se extinguió en vida libre en el 2002 y no fue hasta los recientes esfuerzos de liberación que iniciaron en 2016 que casi toda la población permaneció bajo cuidado humano en programas de reproducción para la conservación. Usamos los coeficientes de linaje endogámico (F) para evaluar los efectos de la endogamia sobre el éxito reproductivo y de supervivencia de la descendencia de los cuervos hawaianos. Usamos un análisis de árbol de regresión para identificar el nivel de endogamia (es decir, el umbral de endogamia) que explicara una disminución sustancial en la supervivencia de la descendencia de ‘Alalā hasta el reclutamiento. Similar a un estudio previo de la endogamia en los ‘Alalā, descubrimos que la endogamia tuvo un impacto negativo sobre la supervivencia de la descendencia pero que la incubación parental (vs. la artificial) de huevos mejoró la supervivencia de la descendencia hasta el reclutamiento. Además, encontramos que la endogamia no afectó de manera importante al éxito reproductivo de la descendencia, esto basado en la suposición de que los descendientes que sobreviven hasta la edad adulta se reproducen con parejas cuya relación familiar es lejana. Nuestra aplicación novedosa del análisis de árbol de regresión mostró que la descendencia con niveles de endogamia que exceden F = 0.098 tuvieron 69% menos de probabilidad de sobrevivir hasta el reclutamiento que aquella progenie con menor endogamia, lo que proporciona un valor específico de umbral para el manejo continuo de la población. Nuestros resultados resaltan la importancia de la evaluación de la depresión endogámica en todas las etapas de la historia de vida, confirman la importancia de priorizar la incubación parental de huevos sobre la artificial en los programas de conservación de aves y demuestran la utilidad del análisis de árbol de regresión como herramienta para identificar los umbrales de endogamia, cuando presentes, en cualquier población manejada por linaje.

The 196 parties to the Convention on Biological Diversity (CBD) will soon agree to a post-2020 global framework for conserving the three elements of biodiversity (genetic, species, and ecosystem diversity) while ensuring sustainable development and benefit sharing. As the most significant global conservation policy mechanism, the new CBD framework has far-reaching consequences- it will guide conservation actions and reporting for each member country until 2050. In previous CBD strategies, as well as other major conservation policy mechanisms, targets and indicators for genetic diversity (variation at the DNA level within species, which facilitates species adaptation and ecosystem function) were undeveloped and focused on species of agricultural relevance. We assert that, to meet global conservation goals, genetic diversity within all species, not just domesticated species and their wild relatives, must be conserved and monitored using appropriate metrics. Building on suggestions in a recent Letter in Science (Laikre et al., 2020) we expand argumentation for three new, pragmatic genetic indicators and modifications to two current indicators for maintaining genetic diversity and adaptive capacity of all species, and provide guidance on their practical use. The indicators are: 1) the number of populations with effective population size above versus below 500, 2) the proportion of populations maintained within species, 3) the number of species and populations in which genetic diversity is monitored using DNA-based methods. We also present and discuss Goals and Action Targets for post-2020 biodiversity conservation which are connected to these indicators and underlying data. These pragmatic indicators and goals have utility beyond the CBD; they should benefit conservation and monitoring of genetic diversity via national and global policy for decades to come.\ud\ud Previous article in issue

Reintroducing captive giant pandas (Ailuropoda melanoleuca) to the wild is the ultimate goal of their ex situ conservation. Choosing higher fitness candidates to train prior to release is the first step in the giant panda reintroduction program. Disease resistance is one important index of individual fitness and presumed to be related to variation at major histocompatibility complex genes (MHC). Here, we used seven polymorphic functional MHC genes (Aime-C, Aime-I, Aime-L, Aime-DQA1, Aime-DQA2, Aime-DQB1 and Aime-DRB3) and estimate their relationship with Baylisascaris schroederi (Ascarididae) infection in giant panda. We found that DQA1 heterozygous pandas were less frequently infected than homozygotes. The presence of one MHC genotype and one MHC allele were also associated with B. schroederi infection: Aime-C*0203 and Aime-L*08 were both associated with B. schroederi resistance. Our results indicate that both heterozygosity and certain MHC variants are important for panda disease resistance, and should therefore be considered in future reintroduction programs for this species alongside conventional selection criteria (such as physical condition and pedigree-based information)., Graphical abstract Image 1, Highlights • MHC heterozygous pandas were less frequently infected by Baylisascaris schroederi than homozygotes. • Presence of Aime-C*0203 and Aime-L*08 are associated with Baylisascaris schroederi resistance. • MHC types are important for panda parasite resistance.

Immunity genes are proposed to be informative about evolutionary processes acting upon introduced populations (e.g., showing signatures of selection). This is because immunity genes are expected to be under pathogen-mediated selection, and this type of selection can be more pronounced when individuals are exposed to new environmental conditions. Here we assessed innate immune genetic diversity, via Toll-like receptors (TLRs), to quantify genetic differentiation between a deliberately introduced population of dunnocks (Prunella modularis) in New Zealand and its source population in the United Kingdom. We also asked whether the introduced population shows signatures of intergenerational and current selection in TLR. We expect intergenerational and current selection patterns because New Zealand dunnocks have been exposed to new environmental conditions following their introduction around the late 1800s, which may have driven pathogen-mediated selection. Counter to our expectation, we found only weak and non-significant genetic differentiation between the introduced and source populations. Further, the levels of genetic differentiation (G ST ′ ) found in TLRs were similar to those found in microsatellites across the populations. Dunnocks, in general, have been under strong purifying selection over evolutionary time, but we found little evidence to support signatures of contemporary selection in TLR in the introduced population. Notably, however, we found a statistically significant heterozygosity advantage for males in TLR3, which lends support to possible current selection acting upon the introduced population. Overall, it appears that New Zealand dunnocks have retained a high proportion of the immunogenetic diversity of the source population, and that such diversity has probably been sufficient to defend against the potential pathogens found in New Zealand. Our results may explain—at least in part—why the introduction of dunnocks has been so successful; dunnocks have become one of the most common birds in New Zealand.

In order to project and plan for the future of threatened species, conservationists require good quality estimates of basic population parameters, such as population sex ratios and breeding rates. These data can be difficult to obtain in many threatened species where pervasive threats perturb population dynamics. For Tasmanian devils, previous studies at several sites across Tasmania revealed demographic conse-quences of the fatal and transmissible devil facial tumour disease (DFTD). In the current analysis, we take advantage of broad-scale survey data across the state, since 2003, to examine the differences between DFTD-present and DFTD-absent populations at the landscape level. Consistent with expectations based on previous studies, we found that devils trapped in DFTD-present sites were significantly younger (based on tooth-wear age estimates) than those in DFTD-absent sites. Interestingly, we also found that females in DFTD-present sites were more likely to show evidence of breeding than females in DFTD-absent sites. This difference could not be attributed to differences in age or body condition. Devil populations are declining, so increased female breeding rate is unlikely to be successfully compensating for decreased lifespan as a result of DFTD at the population level. These data can be used to inform stochastic and evolu-tionary models of population dynamics for devils, and inform the assessment of conservation strategies for the species in the presence of disease.

Successfully establishing captive breeding programs is a priority across diverse industries to address food security, demand for ethical laboratory research animals, and prevent extinction. Differences in reproductive success due to birth origin may threaten the long-term sustainability of captive breeding. Our meta-analysis examining 115 effect sizes from 44 species of invertebrates, fish, birds, and mammals shows that, overall, captive-born animals have a 42% decreased odds of reproductive success in captivity compared to their wild-born counterparts. The largest effects are seen in commercial aquaculture, relative to conservation or laboratory settings, and offspring survival and offspring quality were the most sensitive traits. Although a somewhat weaker trend, reproductive success in conservation and laboratory research breeding programs is also in a negative direction for captive-born animals. Our study provides the foundation for future investigation of non-genetic and genetic drivers of change in captivity, and reveals areas for the urgent improvement of captive breeding., Aquaculture, conservation, and biological research are reliant on the successful breeding of animals in captivity. Here, Farquharson et al. report that, in captivity, captive-born animals have decreased reproductive success compared to wild-born individuals, across diverse species and contexts.

Diet and immunity are both highly complex processes through which organisms interact with their environment and adapt to variable conditions. Parents that are able to transmit information to their offspring about prevailing environmental conditions have a selective advantage by ‘priming’ the physiology of their offspring. We used a meta-analytic approach to test the effect of parental diet on offspring immune responses. Using the geometric framework for nutrition (a method for analysing diet compositions wherein food nutrient components are expressed as axes in a Cartesian coordinate space) to define dietary manipulations in terms of their energy and macronutrient compositions, we compiled the results of 226 experiments from 38 published papers on the intergenerational effects of diet on immunity, across a range of study species and immunological responses. We observed intergenerational impacts of parental nutrition on a number of offspring immunological processes, including expression of pro-inflammatory biomarkers as well as decreases in anti-inflammatory markers in response to certain parental diets. For example, across our data set as a whole (encompassing several types of dietary manipulation), dietary stress in parents was seen to significantly increase pro-inflammatory cytokine levels measured in offspring (overall d = 0.575). All studies included in our analysis were from experiments in which the offspring were raised on a normal or control diet, so our findings suggest that a nutrition-dependent immune state can be inherited, and that this immune state is maintained in the short term, despite offspring returning to an ‘optimal’ diet. We demonstrate how the geometric framework for nutrition can be used to disentangle the role that different forms of dietary manipulation can have on intergenerational immunity. For example, offspring B-cell responses were significantly decreased when parents were raised on a range of different diets. Similarly, our approach allowed us to show that a parental diet elevated in protein (regardless of energy composition and relative to a control diet) can increase expression of inflammatory markers while decreasing B-cell-associated markers. By conducting a systematic review of the literature, we have identified important gaps that impair our understanding of the intergenerational effects of diet, such as a paucity of experimental studies involving increased protein and decreased energy, and a lack of studies directed at the whole-organism consequences of these processes, such as immune resilience to infection. The results of our analyses inform our understanding of the effects of diet on physiological state across diverse biological fields, including biomedical sciences, maintenance of agricultural breed stock and conservation breeding programs, among others.

In the original publication of the article Evol Ecol 34, 803–820 (2020) we suggested that “Historical records from the Otago Acclimatisation Society indicate that in the Otago region…a total of 185 individuals…were introduced from United Kingdom between 1865 and 1907 (Santos 2012; Santos and Nakagawa 2013)”.

Tasmanian devils have experienced an 85% population decline since the emergence of an infectious cancer. In response, a captive insurance population was established in 2006 with a subpopulation later introduced onto Maria Island, Tasmania. We aimed to (1) examine the genetic parameters of the Maria Island population as a stand-alone site and within its broader metapopulation context, (2) assess the efficacy of assisted colonisations, and (3) inform future translocations. This study reconstructs the pedigree of 86 island-born devils using 31 polymorphic microsatellite loci. Combined molecular and pedigree analysis was used to monitor change in population genetic parameters in 4 years since colonisation. Molecular analysis alone revealed no significant change in genetic diversity, while DNA-reconstructed pedigree analysis revealed a statistically significant increase in inbreeding due to skewed founder representation. Pedigree modelling predicted that gene diversity would only be maintained above the threshold of 95% for a further 2 years, dropping to 77.1% after 40 years. Modelling alternative supplementation strategies revealed introducing eight new founders every 3 years will enable the population to retain 95% gene diversity until 2056, provided the translocated animals breed; to ensure this we recommend introducing ten new females every 3 years. We highlight the value of combining pedigree analyses with molecular data, from both a single-site and metapopulation viewpoint, for analysing changes in genetic parameters within populations of conservation concern. The importance of post-release genetic monitoring in an established population is emphasised, given how quickly inbreeding can accumulate and gene diversity be lost.

Background Captivity presents extreme lifestyle changes relative to the wild, and evidence of microbiome dysbiosis in captive animals is growing. The gut microbiome plays a crucial role in host health. Whilst captive breeding and subsequent reintroduction to the wild is important for conservation, such efforts often have limited success. Post-release monitoring is essential for assessing translocation success, but changes to the microbiome of released individuals are poorly understood. The Tasmanian devil was previously shown to exhibit loss of microbiome diversity as a result of intense captive management. This current study examines changes in the devil gut microbiome in response to translocation and aims to determine if perturbations from captivity are permanent or reversible. Methods Using 16S rRNA amplicon sequencing, we conducted temporal monitoring of the gut microbiome of released devils during two translocation events, captive-to-wild and wild-to-wild. To investigate whether the microbiome of the released devils changed following translocation, we characterized their microbiome at multiple time points during the translocation process over the course of 6–12 months and compared them to the microbiome of wild incumbent devils (resident wild-born devils at the respective release sites). Results We showed that the pre-release microbiome was significantly different to the microbiome of wild incumbent animals, but that the microbiomes of animals post-release (as early as 3 to 4 weeks post-release) were similar to wild incumbents. The gut microbiome of released animals showed significant compositional shifts toward the wild incumbent microbiome of both translocation events. Conclusion Our results suggest that the devil gut microbiome is dynamic and that loss of microbiome diversity in captivity can be restored following release to the wild. We recommend the broader application of microbiome monitoring in wildlife translocation programs to assess the impacts of translocation on animal microbiomes.

As species extinction rates increase, genomics provides a powerful tool to support intensive management of threatened species. We use the Tasmanian devil (Sarcophilus harrisii) to demonstrate how conservation genomics can be implemented in threatened species management. We conducted whole genome sequencing (WGS) of 25 individuals from the captive breeding programme and reduced-representation sequencing (RRS) of 98 founders of the same programme. A subset of the WGS samples was also sequenced by RRS, allowing us to directly compare genome-wide heterozygosity with estimates from RRS data. We found good congruence in interindividual variation and gene-ontology classifications between the two data sets, indicating that our RRS data reflect the genome well. We also attempted genome-wide association studies with both data sets (regarding breeding success), but the genomic data suffered from small sample size, while the RRS data suffered from lack of precision, highlighting a key trade-off in the design of conservation genomic research. Nevertheless, we identified a number of candidate genes that may be associated with variation in breeding success. Individual heterozygosity, as measured by WGS or RRS, was not associated with breeding success in captivity but was negatively associated with litter sizes of breeding females in the RRS data set. Our findings enable conservation managers to have confidence in RRS data while understanding its limitations, and provide avenues for further investigation into which processes underlie variation in breeding success in captive Tasmanian devils. We caution, however, that deep functional insights using RRS may be impaired by a lack of precision, especially when marker density is low.

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., 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.

Outbreaks of disease in threatened species can increase the likelihood of extinction by reducing population size. Low immunogenetic diversity can increase susceptibility to, and severity of, disease outbreaks. Our study measured genetic diversity at innate immune genes the Toll-like receptors (TLRs) in a critically endangered Australian bird, the orange-bellied parrot (Neophema chrysogaster). Conservation of this species has recently been challenged by several disease outbreaks. We found low diversity at six TLR genes in both captive and wild orange-bellied parrot populations, similar to results seen in other threatened bird species. Three loci were found to be monomorphic across all samples, including birds which were recorded to have been involved in recent disease events. Phylogenetic analysis of TLR sequences from Neophema chrysogaster as well as representatives of all other species within parrot genera Neophema and Neopsephotus revealed a small number of sites under pervasive and/or episodic positive selection, and a number of sites under negative selection. This study is the first to investigate functional genetic diversity in the orange-bellied parrot.

BackgroundThe Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumour disease (DFTD). While research on DFTD has been extensive, little is known about the viruses present in devils, and whether any of these are of potential conservation relevance for this endangered species.MethodsUsing both metagenomics based on virus-like particle (VLP) enrichment and sequence-independent amplification (VLP metagenomics), and meta-transcriptomics based on bulk RNA sequencing, we characterised and compared the faecal viromes of captive and wild Tasmanian devils.ResultsA total of 54 devil faecal samples collected from captive (n = 2) and wild (n = 4) populations were processed for virome characterisation using both approaches. We detected many novel, highly divergent viruses, including vertebrate viruses, bacteriophage and other dietary associated plant and insect viruses. In total, 18 new vertebrate viruses, including novel sapelovirus, astroviruses, bocaviruses, papillomaviruses and gammaherpesvirus were identified, as well as known mammalian pathogens including rabbit haemorrhagic disease virus 2 (RHDV2). Captive devils showed significantly lower levels of viral diversity than wild devils. Comparison of the two methodological approaches revealed substantial differences in the number and types of viruses detected, with meta-transcriptomics mainly identifying RNA viruses, and VLP metagenomics largely identifying DNA viruses.ConclusionThis study has greatly expanded our knowledge of eukaryotic viruses in the Tasmanian devil and provides important baseline information that will contribute to the conservation and captive management of this endangered species. In addition, our results showed that a combination of VLP metagenomics and meta-transcriptomics may be a more comprehensive approach to virome characterisation than either method alone.

We apply a recently established nutritional framework for defining dietary generalism to global populations of wild boar (Sus scrofa). Across its range, wild boar consume a diversity of foods that vary in nutritional composition. The macronutrient (carbohydrate, protein and fat) composition of the diets composed from those foods also varies substantially between countries, particularly in terms of proportion of energy from protein. These results suggest that as a species wild boar have a wide fundamental macronutrient niche, which likely contributes to the success of the species as an invader of novel environments.