The piRNA Response to Retroviral Invasion of the Koala Genome.

Antisense Piwi-interacting RNAs (piRNAs) guide silencing of established transposons during germline development, and sense piRNAs drive ping-pong amplification of the antisense pool, but how the germline responds to genome invasion is not understood. The KoRV-A gammaretrovirus infects the soma and germline and is sweeping through wild koalas by a combination of horizontal and vertical transfer, allowing direct analysis of retroviral invasion of the germline genome. Gammaretroviruses produce spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome, but KoRV-A piRNAs are almost exclusively derived from unspliced genomic transcripts and are strongly sense-strand biased. Significantly, selective piRNA processing of unspliced proviral transcripts is conserved from insects to placental mammals. We speculate that bypassed splicing generates a conserved molecular pattern that directs proviral genomic transcripts to the piRNA biogenesis machinery and that this "innate" piRNA response suppresses transposition until antisense piRNAs are produced, establishing sequence-specific adaptive immunity.

Expansion of a retrovirus lineage in the koala genome.

Retroviruses have left their legacy in host genomes over millions of years as endogenous retroviruses (ERVs), and their structure, diversity, and prevalence provide insights into the historical dynamics of retrovirus-host interactions. In bioinformatic analyses of koala (Phascolarctos cinereus) whole-genome sequences, we identify a recently expanded ERV lineage (phaCin-ß) that is related to the New World squirrel monkey retrovirus. This ERV expansion shares many parallels with the ongoing koala retrovirus (KoRV) invasion of the koala genome, including highly similar and mostly intact sequences, and polymorphic ERV loci in the sampled koala population. The recent phaCin-ß ERV colonization of the koala genome appears to predate the current KoRV invasion, but polymorphic ERVs and divergence comparisons between these two lineages predict a currently uncharacterized, possibly still extant, phaCin-ß retrovirus. The genomics approach to ERV-guided discovery of novel retroviruses in host species provides a strong incentive to search for phaCin-ß retroviruses in the Australasian fauna.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry.

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. Our study shed more lights into the genetic determinants of ACE2 as the functional receptor of SARS-CoV-2, which facilitates our understanding of viral entry.

Evaluating the genetic consequences of population subdivision as it unfolds and how to best mitigate them: A rare story about koalas.

The genetic consequences of the subdivision of populations are regarded as significant to long-term evolution, and research has shown that the scale and speed at which this is now occurring is critically reducing the adaptive potential of most species which inhabit human-impacted landscapes. Here, we provide a rare and, to our knowledge, the first analysis of this process while it is happening and demonstrate a method of evaluating the effect of mitigation measures such as fauna crossings. We did this by using an extensive genetic data set collected from a koala population which was intensely monitored during the construction of linear transport infrastructure which resulted in the subdivision of their population. First, we found that both allelic richness and effective population size decreased through the process of population subdivision. Second, we predicted the extent to which genetic drift could impact genetic diversity over time and showed that after only 10 generations the resulting two subdivided populations could experience between 12% and 69% loss in genetic diversity. Lastly, using forward simulations we estimated that a minimum of eight koalas would need to disperse from each side of the subdivision per generation to maintain genetic connectivity close to zero but that 16 koalas would ensure that both genetic connectivity and diversity remained unchanged. These results have important consequences for the genetic management of species in human-impacted landscapes by showing which genetic metrics are best to identify immediate loss in genetic diversity and how to evaluate the effectiveness of any mitigation measures.

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.

Susceptibility to a sexually transmitted disease in a wild koala population shows heritable genetic variance but no inbreeding depression.

The koala, one of the most iconic Australian wildlife species, is facing several concomitant threats that are driving population declines. Some threats are well known and have clear methods of prevention (e.g., habitat loss can be reduced with stronger land-clearing control), whereas others are less easily addressed. One of the major current threats to koalas is chlamydial disease, which can have major impacts on individual survival and reproduction rates and can translate into population declines. Effective management strategies for the disease in the wild are currently lacking, and, to date, we know little about the determinants of individual susceptibility to disease. Here, we investigated the genetic basis of variation in susceptibility to chlamydia using one of the most intensively studied wild koala populations. We combined data from veterinary examinations, chlamydia testing, genetic sampling and movement monitoring. Out of our sample of 342 wild koalas, 60 were found to have chlamydia. Using genotype information on 5007 SNPs to investigate the role of genetic variation in determining disease status, we found no evidence of inbreeding depression, but a heritability of 0.11 (95% CI: 0.06-0.23) for the probability that koalas had chlamydia. Heritability of susceptibility to chlamydia could be relevant for future disease management, as it suggests adaptive potential for the population.

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."

Novel phylogenetic methods are needed for understanding gene function in the era of mega-scale genome sequencing.

Ongoing large-scale genome sequencing projects are forecasting a data deluge that will almost certainly overwhelm current analytical capabilities of evolutionary genomics. In contrast to population genomics, there are no standardized methods in evolutionary genomics for extracting evolutionary and functional (e.g. gene-trait association) signal from genomic data. Here, we examine how current practices of multi-species comparative genomics perform in this aspect and point out that many genomic datasets are under-utilized due to the lack of powerful methodologies. As a result, many current analyses emphasize gene families for which some functional data is already available, resulting in a growing gap between functionally well-characterized genes/organisms and the universe of unknowns. This leaves unknown genes on the 'dark side' of genomes, a problem that will not be mitigated by sequencing more and more genomes, unless we develop tools to infer functional hypotheses for unknown genes in a systematic manner. We provide an inventory of recently developed methods capable of predicting gene-gene and gene-trait associations based on comparative data, then argue that realizing the full potential of whole genome datasets requires the integration of phylogenetic comparative methods into genomics, a rich but underutilized toolbox for looking into the past.

Koala methylomes reveal divergent and conserved DNA methylation signatures of X chromosome regulation.

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is an iconic example of epigenetic regulation. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here, we examine genome-wide signatures of DNA methylation across fives tissues from a male and female koala (Phascolarctos cinereus), and present the first whole-genome, multi-tissue marsupial 'methylome atlas'. Using these novel data, we elucidate divergent versus common features of representative marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in koalas primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx, the primary lncRNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-specific (and presumably inactive X chromosome-specific) expression. Finally, we use the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5%) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.

Degradation and remobilization of endogenous retroviruses by recombination during the earliest stages of a germ-line invasion.

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded.

High-throughput immunogenetic typing of koalas suggests possible link between MHC alleles and cancers.

Characterizing the allelic diversity within major histocompatibility complex (MHC) genes is an important way of determining the potential genetic resilience of a population to infectious and ecological pressures. For the koala (Phascolarctos cinereus), endemic diseases, anthropogenic factors and climate change are all placing increased pressure on this vulnerable marsupial. To increase the ability of researchers to study MHC genetics in koalas, this study developed and tested a high-throughput immunogenetic profiling methodology for targeting MHC class I UA and UC genes and MHC class II DAB, DBB, DCB and DMB genes in a population of 82 captive koalas. This approach was validated by comparing the determined allelic profiles from 36 koala family units (18 dam-sire-joey units and 18 parent-joey pairs), finding 96% overall congruence within family profiles. Cancers are a significant cause of morbidity in koalas and the risk factors remain undetermined. Our analysis of this captive population revealed several novel MHC alleles, including a potential link between the DBB*03 allele and a risk of developing cancer. This method offers a reliable, high-throughput protocol for expanded study into koala immunogenetics.

Inbreeding and disease avoidance in a free-ranging koala population.

Habitat destruction and fragmentation are increasing globally, forcing surviving species into small, isolated populations. Isolated populations typically experience heightened inbreeding risk and associated inbreeding depression and population decline; although individuals in these populations may mitigate these risks through inbreeding avoidance strategies. For koalas, as dietary specialists already under threat in the northern parts of their range, increased habitat fragmentation and associated inbreeding costs are of great conservation concern. Koalas are known to display passive inbreeding avoidance through sex-biased dispersal, although population isolation will reduce dispersal pathways. We tested whether free-ranging koalas display active inbreeding avoidance behaviours. We used VHF tracking data, parentage reconstruction, and veterinary examination results to test whether free-ranging female koalas avoid mating with (a) more closely related males; and (b) males infected with sexually transmitted Chlamydia pecorum. We found no evidence that female koalas avoid mating with relatively more related available mates. In fact, as the relatedness of potential mates increases, so did inbreeding events. We also found no evidence that female koalas can avoid mating with males infected with C. pecorum. The absence of active inbreeding avoidance mechanisms in koalas is concerning from a conservation perspective, as small, isolated populations may be at even higher risk of inbreeding depression than expected. At risk koala populations may require urgent conservation interventions to augment gene flow and reduce inbreeding risks. Similarly, if koalas are not avoiding mating with individuals with chlamydial disease, populations may be at higher risk from disease than anticipated, further impacting population viability.

Genomic comparisons reveal biogeographic and anthropogenic impacts in the koala (Phascolarctos cinereus): a dietary-specialist species distributed across heterogeneous environments.

The Australian koala is an iconic marsupial with highly specific dietary requirements distributed across heterogeneous environments, over a large geographic range. The distribution and genetic structure of koala populations has been heavily influenced by human actions, specifically habitat modification, hunting and translocation of koalas. There is currently limited information on population diversity and gene flow at a species-wide scale, or with consideration to the potential impacts of local adaptation. Using species-wide sampling across heterogeneous environments, and high-density genome-wide markers (SNPs and PAVs), we show that most koala populations display levels of diversity comparable to other outbred species, except for those populations impacted by population reductions. Genetic clustering analysis and phylogenetic reconstruction reveals a lack of support for current taxonomic classification of three koala subspecies, with only a single evolutionary significant unit supported. Furthermore, ~70% of genetic variance is accounted for at the individual level. The Sydney Basin region is highlighted as a unique reservoir of genetic diversity, having higher diversity levels (i.e., Blue Mountains region; AvHecorr=0.20, PL% = 68.6). Broad-scale population differentiation is primarily driven by an isolation by distance genetic structure model (49% of genetic variance), with clinal local adaptation corresponding to habitat bioregions. Signatures of selection were detected between bioregions, with no single region returning evidence of strong selection. The results of this study show that although the koala is widely considered to be a dietary-specialist species, this apparent specialisation has not limited the koala's ability to maintain gene flow and adapt across divergent environments as long as the required food source is available.

A novel Australian flying-fox retrovirus shares an evolutionary ancestor with Koala, Gibbon and Melomys gamma-retroviruses.

A novel gamma-retroviral sequence (7912 bp), inclusive of both partial 5' and 3' long terminal repeat regions, was identified from the brain of a black flying-fox (Pteropus alecto), Queensland, Australia. The sequence was distinct from other retroviral sequences identified in bats and showed greater identity to Koala, Gibbon ape leukaemia, Melomys burtoni and Woolly monkey retroviruses, forming their own phylogenetic clade. This finding suggests that these retroviruses may have an unknown common ancestor and that further investigation into the diversity of gamma-retroviruses in Australian Pteropus species may elucidate their evolutionary origins.

Characterisation of MHC class I genes in the koala.

Koala (Phascolarctos cinereus) populations are on the decline across the majority of Australia's mainland. Two major diseases threatening the long-term survival of affected koala populations are caused by obligate intracellular pathogens: Chlamydia and koala retrovirus (KoRV). To improve our understanding of the koala immune system, we characterised their major histocompatibility complex (MHC) class I genes, which are centrally involved in presenting foreign peptides derived from intracellular pathogens to cytotoxic T cells. A total of 11 class I genes were identified in the koala genome. Three genes, Phci-UA, UB and UC, showed relatively high genetic variability and were expressed in all 12 examined tissues, whereas the other eight genes had tissue-specific expression and limited polymorphism. Evidence of diversifying selection was detected in Phci-UA and UC, while gene conversion may have played a role in creating new alleles at Phci-UB. We propose that Phci-UA, UB and UC are likely classical MHC genes of koalas, and further research is needed to understand their role in koala chlamydial and KoRV infections.

Demographic, environmental and genetic determinants of mating success in captive koalas (Phascolarctos cinereus).

Many factors have been shown to affect mating behavior. For instance, genes of the major histocompatibility complex (MHC) are known to influence mate choice in a wide variety of vertebrate species. The genetic management of captive populations can be confounded if intrinsic mate choice reduces or eliminates reproductive success between carefully chosen breeding pairs. For example, the San Diego Zoo koala colony only has a 45% copulation rate for matched individuals. Herein, we investigated determinants of koala mating success using breeding records (1984-2010) and genotypes for 52 individuals at four MHC markers. We quantified MHC diversity according to functional amino acids, heterozygosity, and the probability of producing a heterozygous offspring. We then used categorical analysis and logistic regression to investigate both copulation and parturition success. In addition, we also examined age, day length, and average pairwise kinship. Our post-hoc power analysis indicates that at a power level of 1-ß = 0.8, we should have been able to detect strong MHC preferences. However, we did not find a significant MHC effect on either copulation or parturition success with one exception: pairs with lower or no production of a joey had significantly lower MHC functional amino acid diversity in the categorical analysis. In contrast, day length and dam age (or age difference of the pair) consistently had an effect on mating success. These findings may be leveraged to improve the success of attempted pairs, conserve resources, and facilitate genetic management.

Characterization of the antimicrobial peptide family defensins in the Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus), and tammar wallaby (Macropus eugenii).

Defensins comprise a family of cysteine-rich antimicrobial peptides with important roles in innate and adaptive immune defense in vertebrates. We characterized alpha and beta defensin genes in three Australian marsupials: the Tasmanian devil (Sarcophilus harrisii), koala (Phascolarctos cinereus), and tammar wallaby (Macropus eugenii) and identified 48, 34, and 39 defensins, respectively. One hundred and twelve have the classical antimicrobial peptides characteristics required for pathogen membrane targeting, including cationic charge (between 1+ and 15+) and a high proportion of hydrophobic residues (>30%). Phylogenetic analysis shows that gene duplication has driven unique and species-specific expansions of devil, koala, and tammar wallaby beta defensins and devil alpha defensins. Defensin genes are arranged in three genomic clusters in marsupials, whereas further duplications and translocations have occurred in eutherians resulting in four and five gene clusters in mice and humans, respectively. Marsupial defensins are generally under purifying selection, particularly residues essential for defensin structural stability. Certain hydrophobic or positively charged sites, predominantly found in the defensin loop, are positively selected, which may have functional significance in defensin-target interaction and membrane insertion.

HapFlow: visualizing haplotypes in sequencing data.

SUMMARY: HapFlow is a python application for visualizing haplotypes present in sequencing data. It identifies variant profiles present and reads and creates an abstract visual representation of these profiles to make haplotypes easier to identify. AVAILABILITY AND IMPLEMENTATION: HapFlow is freely available (under a GPL license) for download (for Mac OS X, Unix and Microsoft Windows) from github (http://mjsull.github.io/HapFlow). CONTACT: apolking@usc.edu.au.

Proliferation of endogenous retroviruses in the early stages of a host germ line invasion.

Endogenous retroviruses (ERVs) comprise 8% of the human genome and are common in all vertebrate genomes. The only retrovirus known to be currently transitioning from exogenous to endogenous form is the koala retrovirus (KoRV), making koalas (Phascolarctos cinereus) ideal for examining the early stages of retroviral endogenization. To distinguish endogenous from exogenous KoRV proviruses, we isolated koala genomic regions flanking KoRV integration sites. In three wild southern Australian koalas, there were fewer KoRV loci than in three captive Queensland koalas, consistent with reports that southern Australian koalas carry fewer KoRVs. Of 39 distinct KoRV proviral loci examined in a sire-dam-progeny triad, all proved to be vertically transmitted and endogenous; none was exogenous. Of the 39 endogenous KoRVs (enKoRVs), only one was present in the genomes of both the sire and the dam, suggesting that, at this early stage in the retroviral invasion of a host germ line, very large numbers of ERVs have proliferated at very low frequencies in the koala population. Sequence divergence between the 5'- and 3'-long terminal repeats (LTRs) of a provirus can be used as a molecular clock. Within each of ten enKoRVs, the 5'-LTR sequence was identical to the 3'-LTR sequence, suggesting a maximum age for enKoRV invasion of the koala germ line of approximately 22,200-49,900 years ago, although a much younger age is possible. Across the ten proviruses, seven LTR haplotypes were detected, indicating that at least seven different retroviral sequences had entered the koala germ line.