Incomplete lineage sorting and phenotypic evolution in marsupials.

Incomplete lineage sorting (ILS) makes ancestral genetic polymorphisms persist during rapid speciation events, inducing incongruences between gene trees and species trees. ILS has complicated phylogenetic inference in many lineages, including hominids. However, we lack empirical evidence that ILS leads to incongruent phenotypic variation. Here, we performed phylogenomic analyses to show that the South American monito del monte is the sister lineage of all Australian marsupials, although over 31% of its genome is closer to the Diprotodontia than to other Australian groups due to ILS during ancient radiation. Pervasive conflicting phylogenetic signals across the whole genome are consistent with some of the morphological variation among extant marsupials. We detected hundreds of genes that experienced stochastic fixation during ILS, encoding the same amino acids in non-sister species. Using functional experiments, we confirm how ILS may have directly contributed to hemiplasy in morphological traits that were established during rapid marsupial speciation ca. 60 mya.

Translating genomic advances into biodiversity conservation.

A key action of the new Global Biodiversity Framework is the maintenance of genetic diversity in all species to safeguard their adaptive potential. To achieve this goal, a translational mindset, which aims to convert results of basic research into direct practical benefits, needs to be applied to biodiversity conservation. Despite much discussion on the value of genomics to conservation, a disconnect between those generating genomic resources and those applying it to biodiversity management remains. As global efforts to generate reference genomes for non-model species increase, investment into practical biodiversity applications is critically important. Applications such as understanding population and multispecies diversity and longitudinal monitoring need support alongside education for policymakers on integrating the data into evidence-based decisions. Without such investment, the opportunity to revolutionize global biodiversity conservation using genomics will not be fully realized.

Threatened Species Initiative: Empowering conservation action using genomic resources.

Globally, 15,521 animal species are listed as threatened by the International Union for the Conservation of Nature, and of these less than 3% have genomic resources that can inform conservation management. To combat this, global genome initiatives are developing genomic resources, yet production of a reference genome alone does not conserve a species. The reference genome allows us to develop a suite of tools to understand both genome-wide and functional diversity within and between species. Conservation practitioners can use these tools to inform their decision-making. But, at present there is an implementation gap between the release of genome information and the use of genomic data in applied conservation by conservation practitioners. In May 2020, we launched the Threatened Species Initiative and brought a consortium of genome biologists, population biologists, bioinformaticians, population geneticists, and ecologists together with conservation agencies across Australia, including government, zoos, and nongovernment organizations. Our objective is to create a foundation of genomic data to advance our understanding of key Australian threatened species, and ultimately empower conservation practitioners to access and apply genomic data to their decision-making processes through a web-based portal. Currently, we are developing genomic resources for 61 threatened species from a range of taxa, across Australia, with more than 130 collaborators from government, academia, and conservation organizations. Developed in direct consultation with government threatened-species managers and other conservation practitioners, herein we present our framework for meeting their needs and our systematic approach to integrating genomics into threatened species recovery.

Genomic insights into the critically endangered King Island scrubtit.

Small, fragmented or isolated populations are at risk of population decline due to fitness costs associated with inbreeding and genetic drift. The King Island scrubtit Acanthornis magna greeniana is a critically endangered subspecies of the nominate Tasmanian scrubtit A. m. magna, with an estimated population of < 100 individuals persisting in three patches of swamp forest. The Tasmanian scrubtit is widespread in wet forests on mainland Tasmania. We sequenced the scrubtit genome using PacBio HiFi and undertook a population genomic study of the King Island and Tasmanian scrubtits using a double-digest restriction site-associated DNA (ddRAD) dataset of 5,239 SNP loci. The genome was 1.48 Gb long, comprising 1,518 contigs with an N50 of 7.715 Mb. King Island scrubtits formed one of four overall genetic clusters, but separated into three distinct subpopulations when analysed independently of the Tasmanian scrubtit. Pairwise FST values were greater among the King Island scrubtit subpopulations than among most Tasmanian scrubtit subpopulations. Genetic diversity was lower and inbreeding coefficients were higher in the King Island scrubtit than all except one of the Tasmanian scrubtit subpopulations. We observed crown baldness in 8/15 King Island scrubtits, but 0/55 Tasmanian scrubtits. Six loci were significantly associated with baldness, including one within the DOCK11 gene which is linked to early feather development. Contemporary gene flow between King Island scrubtit subpopulations is unlikely, with further field monitoring required to quantify the fitness consequences of its small population size, low genetic diversity and high inbreeding. Evidence-based conservation actions can then be implemented before the taxon goes extinct.

A chromosome-level genome assembly for the dugong (Dugong dugon).

The dugong (Dugong dugon) is a marine mammal widely distributed throughout the Indo-Pacific and the Red Sea, with a Vulnerable conservation status, and little is known about many of the more peripheral populations, some of which are thought to be close to extinction. We present a de novo high-quality genome assembly for the dugong from an individual belonging to the well-monitored Moreton Bay population in Queensland, Australia. Our assembly uses long-read PacBio HiFi sequencing and Omni-C data following the Vertebrate Genome Project pipeline to reach chromosome-level contiguity (24 chromosome-level scaffolds; 3.16 Gbp) and high completeness (97.9% complete BUSCOs). We observed relatively high genome-wide heterozygosity, which likely reflects historical population abundance before the last interglacial period, approximately 125,000 yr ago. Demographic inference suggests that dugong populations began declining as sea levels fell after the last interglacial period, likely a result of population fragmentation and habitat loss due to the exposure of seagrass meadows. We find no evidence for ongoing recent inbreeding in this individual. However, runs of homozygosity indicate some past inbreeding. Our draft genome assembly will enable range-wide assessments of genetic diversity and adaptation, facilitate effective management of dugong populations, and allow comparative genomics analyses including with other sirenians, the oldest marine mammal lineage.

A targeted approach to investigating immune genes of an iconic Australian marsupial.

Disease is a contributing factor to the decline of wildlife populations across the globe. Koalas, iconic yet declining Australian marsupials, are predominantly impacted by two pathogens, Chlamydia and koala retrovirus. Chlamydia is an obligate intracellular bacterium and one of the most widespread sexually transmitted infections in humans worldwide. In koalas, Chlamydia infections can present as asymptomatic or can cause a range of ocular and urogenital disease signs, such as conjunctivitis, cystitis and infertility. In this study, we looked at differences in response to Chlamydia in two northern populations of koalas using a targeted gene sequencing of 1209 immune genes in addition to genome-wide reduced representation data. We identified two MHC Class I genes associated with Chlamydia disease progression as well as 25 single nucleotide polymorphisms across 17 genes that were associated with resolution of Chlamydia infection. These genes are involved in the innate immune response (TLR5) and defence (TLR5, IFNγ, SERPINE1, STAT2 and STX4). This study deepens our understanding of the role that genetics plays in disease progression in koalas and leads into future work that will use whole genome resequencing of a larger sample set to investigate in greater detail regions identified in this study. Elucidation of the role of host genetics in disease progression and resolution in koalas will directly contribute to better design of Chlamydia vaccines and management of koala populations which have recently been listed as "endangered."

Future-proofing the koala: Synergising genomic and environmental data for effective species management.

Climatic and evolutionary processes are inextricably linked to conservation. Avoiding extinction in rapidly changing environments often depends upon a species' capacity to adapt in the face of extreme selective pressures. Here, we employed exon capture and high-throughput next-generation sequencing to investigate the mechanisms underlying population structure and adaptive genetic variation in the koala (Phascolarctos cinereus), an iconic Australian marsupial that represents a unique conservation challenge because it is not uniformly threatened across its range. An examination of 250 specimens representing 91 wild source locations revealed that five major genetic clusters currently exist on a continental scale. The initial divergence of these clusters appears to have been concordant with the Mid-Brunhes Transition (~430 to 300 kya), a major climatic reorganisation that increased the amplitude of Pleistocene glacial-interglacial cycles. While signatures of polygenic selection and environmental adaptation were detected, strong evidence for repeated, climate-associated range contractions and demographic bottleneck events suggests that geographically isolated refugia may have played a more significant role in the survival of the koala through the Pleistocene glaciation than in situ adaptation. Consequently, the conservation of genome-wide genetic variation must be aligned with the protection of core koala habitat to increase the resilience of vulnerable populations to accelerating anthropogenic threats. Finally, we propose that the five major genetic clusters identified in this study should be accounted for in future koala conservation efforts (e.g., guiding translocations), as existing management divisions in the states of Queensland and New South Wales do not reflect historic or contemporary population structure.

The relevance of pedigrees in the conservation genomics era.

Over the past 50 years conservation genetics has developed a substantive toolbox to inform species management. One of the most long-standing tools available to manage genetics-the pedigree-has been widely used to characterize diversity and maximize evolutionary potential in threatened populations. Now, with the ability to use high throughput sequencing to estimate relatedness, inbreeding, and genome-wide functional diversity, some have asked whether it is warranted for conservation biologists to continue collecting and collating pedigrees for species management. In this perspective, we argue that pedigrees remain a relevant tool, and when combined with genomic data, create an invaluable resource for conservation genomic management. Genomic data can address pedigree pitfalls (e.g., founder relatedness, missing data, uncertainty), and in return robust pedigrees allow for more nuanced research design, including well-informed sampling strategies and quantitative analyses (e.g., heritability, linkage) to better inform genomic inquiry. We further contend that building and maintaining pedigrees provides an opportunity to strengthen trusted relationships among conservation researchers, practitioners, Indigenous Peoples, and Local Communities.

How much is enough? Sampling intensity influences estimates of reproductive variance in an introduced population.

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.

Annotation of immune genes in the extinct thylacine (Thylacinus cynocephalus).

Advances in genome sequencing technology have enabled genomes of extinct species to be sequenced. However, given the fragmented nature of these genome assemblies, it is not clear whether it is possible to comprehensively annotate highly variable and repetitive genes such as those involved in immunity. As such, immune genes have only been investigated in a handful of extinct genomes, mainly in human lineages. In 2018 the genome of the thylacine (Thylacinus cynocephalus), a carnivorous marsupial from Tasmania that went extinct in 1936, was sequenced. Here we attempt to characterise the immune repertoire of the thylacine and determine similarity to its closest relative with a genome available, the Tasmanian devil (Sarcophilus harrisii), as well as other marsupials. Members from all major immune gene families were identified. However, variable regions could not be characterised, and complex families such as the major histocompatibility complex (MHC) were highly fragmented and located across multiple small scaffolds. As such, at a gene level we were unable to reconstruct full-length coding sequences for the majority of thylacine immune genes. Despite this, we identified genes encoding functionally important receptors and immune effector molecules, which suggests the functional capacity of the thylacine immune system was similar to other mammals. However, the high number of partial immune gene sequences identified limits our ability to reconstruct an accurate picture of the thylacine immune repertoire.

First evidence of deviation from Mendelian proportions in a conservation programme.

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.

Expanding the conservation genomics toolbox: Incorporating structural variants to enhance genomic studies for species of conservation concern.

Structural variants (SVs) are large rearrangements (>50 bp) within the genome that impact gene function and the content and structure of chromosomes. As a result, SVs are a significant source of functional genomic variation, that is, variation at genomic regions underpinning phenotype differences, that can have large effects on individual and population fitness. While there are increasing opportunities to investigate functional genomic variation in threatened species via single nucleotide polymorphism (SNP) data sets, SVs remain understudied despite their potential influence on fitness traits of conservation interest. In this future-focused Opinion, we contend that characterizing SVs offers the conservation genomics community an exciting opportunity to complement SNP-based approaches to enhance species recovery. We also leverage the existing literature-predominantly in human health, agriculture and ecoevolutionary biology-to identify approaches for readily characterizing SVs and consider how integrating these into the conservation genomics toolbox may transform the way we manage some of the world's most threatened species.

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.

From reference genomes to population genomics: comparing three reference-aligned reduced-representation sequencing pipelines in two wildlife species.

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.

Contraceptive efficacy and dose-response effects of the gonadotrophin-releasing hormone (GnRH) agonist deslorelin in Tasmanian devils (Sarcophilus harrisii).

Contraception is increasingly used to manage breeding opportunities in conservation-dependent species. This study aimed to determine the efficacy, duration of effect, optimal dose and potential side effects of Suprelorin contraceptive implants in Tasmanian devils, for use in the conservation breeding program. In our pilot study, Suprelorin was found to effectively suppress oestrous cycles in female devils, yet caused a paradoxical increase in testosterone in males. Therefore, we focussed on females in further trials. Females received one (n=5), two (n=5) or no (n=5) Suprelorin implants, with quarterly gonadotrophin-releasing hormone (GnRH) challenges used to test pituitary responsiveness over two breeding seasons. Both Suprelorin doses suppressed pituitary responsiveness for at least one breeding season, with a reduced effect in the second. There was a dose-response effect on duration rather than magnitude of effect, with high-dose devils remaining suppressed for longer than low-dose animals. There were no apparent negative effects on general health, yet captivity and contraception together may cause weight gain. Suprelorin contraceptive implants are now routinely used in the Save the Tasmanian Devil Program insurance metapopulation to meet the aims of maintaining genetic and behavioural integrity by controlling individual reproductive contributions in group housing situations.

A role for selective contraception of individuals in conservation.

Contraception has an established role in managing overabundant populations and preventing undesirable breeding in zoos. We propose that it can also be used strategically and selectively in conservation to increase the genetic and behavioral quality of the animals. In captive breeding programs, it is becoming increasingly important to maximize the retention of genetic diversity by managing the reproductive contribution of each individual and preventing genetically suboptimal breeding through the use of selective contraception. Reproductive suppression of selected individuals in conservation programs has further benefits of allowing animals to be housed as a group in extensive enclosures without interfering with breeding recommendations, which reduces adaptation to captivity and facilitates the expression of wild behaviors and social structures. Before selective contraception can be incorporated into a breeding program, the most suitable method of fertility control must be selected, and this can be influenced by factors such as species life history, age, ease of treatment, potential for reversibility, and desired management outcome for the individual or population. Contraception should then be implemented in the population following a step-by-step process. In this way, it can provide crucial, flexible control over breeding to promote the physical and genetic health and sustainability of a conservation dependent species held in captivity. For Tasmanian devils (Sarcophilus harrisii), black-flanked rock wallabies (Petrogale lateralis), and burrowing bettongs (Bettongia lesueur), contraception can benefit their conservation by maximizing genetic diversity and behavioral integrity in the captive breeding program, or, in the case of the wallabies and bettongs, by reducing populations to a sustainable size when they become locally overabundant. In these examples, contraceptive duration relative to reproductive life, reversibility, and predictability of the contraceptive agent being used are important to ensure the potential for individuals to reproduce following cessation of contraception, as exemplified by the wallabies when their population crashed and needed females to resume breeding.