Extant and extinct bilby genomes combined with Indigenous knowledge improve conservation of a unique Australian marsupial

Bilbies (*Macrotis* spp.), the only members of the family Thylacomyidae, are culturally and ecologically important Australian marsupials. This study focuses on the greater bilby (*Macrotis lagotis*, referred to as Ninu by the Kiwirrkurra community) and the extinct lesser bilby (*Macrotis leucura*, Yallara). Bilbies once had a wide range but are now threatened due to introduced predators (cats, foxes), competition (rabbits), and altered fire regimes. Indigenous Australians have a deep cultural connection to bilbies, reflected in songlines, ceremonies, and stories, and their knowledge is crucial for conservation. Ninu are currently managed as a metapopulation (zoos, fenced sanctuaries, islands), with translocations occurring since 1996. Existing monitoring methods (track and scat surveys, microsatellite genotyping) lack information on sex ratios, relatedness, and gene flow. This research aims to develop genetic tools to support Indigenous communities in bilby conservation and to understand the genetic status of the Ninu metapopulation compared to wild individuals, ensuring long-term adaptive potential. Bilbies possess unique biological features: large ears, burrowing behavior, nocturnal activity, arid-adapted physiology (low metabolic rate, water turnover), and a boom-bust life history tied to rainfall and food availability. Comparative genomics can enhance understanding of these features.

Existing literature highlights the decline of bilby populations due to the impacts of European settlement. Studies on bilby biology detail their unique physiological adaptations to arid environments and their reproductive strategies. Previous genetic work has used microsatellite genotyping for population assessments but lacked the resolution of whole genome sequencing. Indigenous knowledge about bilby distribution, habitat, and traditional management practices has been documented but not fully integrated into genetic conservation efforts. Comparative genomic studies of marsupials have provided insights into their evolution and adaptation but there was a lack of a high-quality genome for the bilby. This gap has limited the ability to understand the uniqueness of bilby biology at the molecular level and the development of robust conservation tools.

This study employed a multi-faceted approach combining genomics, population genetics, and Indigenous knowledge. A high-quality chromosome-level genome assembly for Ninu (3.66 Gbp) was created using a hybrid approach of long-read (PacBio HiFi), linked-read (10x Genomics), and Hi-C (Dovetail Genomics) sequencing technologies, along with Illumina short-read polishing. The extinct Yallara genome was assembled from a museum specimen using Illumina sequencing. Whole-genome resequencing (WGR) was performed on 12 Ninu (6 temperate, 6 semi-arid) and 4 Yallara samples. Phylogenetic relationships were confirmed using mitogenomes and whole-genome data. Demographic history was inferred using pairwise and multiple sequentially Markovian coalescent (PSMC, MSMC) analyses. Runs of homozygosity (ROH) were analyzed to assess inbreeding. Transcriptomes from 12 Ninu tissues were sequenced to explore gene expression. A scat single nucleotide polymorphism (SNP) panel (35 autosomal, 4 sex-linked markers) was developed using reduced-representation sequencing (RRS) data from 363 Ninu from the metapopulation and tested on scat samples collected by Indigenous rangers. Genome-wide association studies (GWAS) were conducted to identify allele frequency differences between semi-arid and temperate Ninu populations. Comparative genomics analysis was performed to explore the unique biological features of Ninu. This involved gene family analysis, using CAFE, to look at evolutionary rates, as well as analysis of olfactory receptor genes and reproductive and immune system genes. The study also analyzed the sex chromosome system by read depth analysis and immunofluorescence of spermatocytes.

The Ninu reference genome is one of the highest-quality marsupial genomes available, comparable to the koala genome. Phylogenetic and PCA analyses confirmed the divergence of Ninu and Yallara. PSMC and MSMC analyses revealed two major population crashes in both species coinciding with global cooling events. Ninu from the temperate island population showed lower heterozygosity and higher ROH compared to semi-arid individuals, likely due to a small founder effect. The semi-arid Ninu showed variable ROH levels, with both short and long ROHs suggesting past and recent inbreeding. Despite their captive history, Ninu had fewer long ROHs than other larger mammals, potentially due to their boom-bust life history. The metapopulation expansion between 2016 and 2021 increased heterozygosity in translocated populations, demonstrating the success of genetically informed management. The scat SNP panel successfully genotyped wild Ninu, revealing comparable genetic diversity to other wild populations. GWAS identified genes involved in anatomical structure, metabolic pathways, and stress response that differed between semi-arid and temperate Ninu. Ninu showed the highest number of annotated olfactory receptor genes among the ten species compared in CAFE analysis. Association analysis revealed fixed differences in four testis genes between semi-arid and temperate male Ninu. All 115 conserved eutherian chorioallantoic placentation genes were expressed in the Ninu uterus. The immune gene repertoire of Ninu was similar to other marsupials, with marsupial-specific genes and eutherian orthologues identified. The analysis of the XY1Y2 sex chromosome system revealed a large pseudoautosomal region (PAR) in the compound X chromosome that pairs with the Y2 chromosome during meiosis.

This study demonstrates the power of integrating genomics with Indigenous knowledge and conservation management. The high-quality Ninu genome provides a valuable resource for understanding bilby biology and evolution. The findings highlight the impact of past climate change and the effectiveness of genetically informed metapopulation management. The development of a reliable scat genotyping method is a significant breakthrough for monitoring remote populations. The unique biological characteristics revealed through transcriptomics and comparative genomics offer insights into bilby adaptations. The identification of genes involved in key pathways like metabolism and reproduction can inform future conservation strategies. The collaborative nature of this research, involving Indigenous rangers, conservation managers, and scientists, is a model for future conservation genetics projects.

This study provides the first chromosome-level genome assembly for the greater bilby and the first genome sequence for the extinct lesser bilby. The findings demonstrate the value of integrating genomics with Indigenous knowledge and conservation management for the long-term survival of the bilby. Future research should investigate the functional consequences of the identified genetic variations, expand scat sampling across the country, and continue to integrate Indigenous knowledge into conservation planning.

The small sample size for Yallara limited the power of the association studies. The use of museum specimens may lead to DNA degradation and biases in the Yallara genome assembly. The scat genotyping panel may have limited sensitivity in areas with low bilby densities or high scat degradation. Further research is needed to understand the effects of various environmental factors on gene expression and to validate the use of scat sampling methods.