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

doi:10.21203/rs.3.rs-3723914/v1

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

https://doi.org/10.21203/rs.3.rs-3723914/v1

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published 01 Jul, 2024

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The Ninu (Greater bilby, Macrotis lagotis) is a desert-dwelling, culturally and ecologically important marsupial. In collaboration with Indigenous rangers and conservation managers, we generated the first Ninu chromosome-level genome assembly (3.66 Gbp) and genome sequences for the extinct Yallara (lesser bilby, Macrotis leucura) to understand their biology and guide Ninu conservation management. Resequenced genomes (temperate Ninu=6; semi-arid Ninu=6; Yallara=4) revealed two major population crashes during global cooling events. Despite their 45-year long captive history, Ninu have fewer long runs of homozygosity than other larger mammals, which may be attributable to their boom-bust life-history. We assessed the beneficial impact of targeted conservation actions, like translocations, in the contemporary metapopulation (N=363 Ninu) and designed a SNP panel used by Indigenous rangers in monitoring remote wild populations. We dig into the unique Ninu biology using 12 tissue transcriptomes revealing expression of all 115 conserved eutherian chorioallantoic placentation genes in the uterus; an XY₁Y₂ sex chromosome system generated by fusion of the X with a large telocentric autosome; genes involved in anatomical and metabolic pathway adaptations to aridity; and expansions in olfactory receptor genes. Together, we demonstrate the holistic value of genomics in addressing key Indigenous and conservation management questions for ongoing conservation efforts.

Biological sciences/Genetics/Genomics/Conservation genomics

Biological sciences/Ecology/Population dynamics

Biological sciences/Evolution/Evolutionary genetics

Macrotis lagotis

Macrotis leucura

Greater bilby

Indigenous knowledge

comparative genomics

conservation genomics

Bilbies are unique marsupials and are the only members of the family Thylacomyidae. They include the extant Greater bilby (Macrotis lagotis; Figure 1A) and the extinct Lesser bilby (Macrotis leucura; Figure 1B). Bilbies are culturally important to Indigenous Australians, with their common name derived from the Yuwaalaraay word, Bilba. The many First Nations across Australia have different names for bilby (Extended Data Table 1), but here we use Ninu for the Greater bilby, as used by the Kiwirrkurra community, and Yallara for the lesser bilby. Ninu were once an important meat source for desert people, and their valuable long black tails with white fluffy ends were used as ornaments associated with their deep symbolism in love and marriage¹. Bilby songlines, ceremonies, and stories exist across Australia, linking sites and people.

Historically Ninu were wide-ranging, distributed across both arid and temperate regions, whilst the Yallara (Lesser bilby) were restricted to the sandy deserts². The declines of both bilby species’ are attributed to the introduction of feral pests into Australia by European settlers, particularly predation by cats (Felis catus) and foxes (Vulpes vulpes) and competition from European rabbits (Oryctolagus cuniculus), as well as changes to cultural fire regimes³. Sadly, the Yallara is now extinct, last reported alive in 1931, although it may have survived in some desert areas until the 1960s³ and was well known to the Indigenous peoples of the central deserts (Figure 1B)⁴. The now-threatened Ninu is believed to exist in only 20% of its former range in the semi-arid regions of north Western Australia (WA), the Northern Territory (NT), and Queensland (QLD; Figure 1A)⁵. Therefore, the conservation of the Ninu is now of critical importance as it is the last remaining extant member of the Thylacomyidae marsupial family.

To ensure the long-term survival of the species, Ninu are managed as a metapopulation consisting of individuals in zoos as well as those in several fenced sanctuaries and on islands. Although Ninu were periodically held in zoos in the early 20^th century, a captive breeding colony was formally established in 1979. The zoo-based populations were managed as two separate evolutionary units (NT/WA and QLD), until 2016 when they were combined into one metapopulation⁶. However, since 1996 Ninu from the zoo population have been released to large, fenced sanctuaries and islands. The genetic consequences of these ongoing translocation events are not known but are explored here. Current methods used to understand the status of wild Ninu populations rely on a combination of track and scat surveys often undertaken by Indigenous rangers⁷, including microsatellite genotyping of scats to estimate abundance⁸. Although these surveys ascertain Ninu presence/absence and breeding (based on expert interpretation of the size of tracks and scats), they provide no insight into sex ratios, relatedness, or gene flow between wild locations. In remote areas of the Ninu range, many Indigenous communities follow traditional practices for actively managing fire regimes and invasive species^9,10. Here we develop genetic tools to support Indigenous communities in conserving not only the Ninu but the cultural practices associated with the species. We also for the first time undertake a genetic comparison between the managed metapopulation and wild individuals to better understand if the metapopulation is representative of wild genetic diversity ensuring we can manage its long-term adaptive potential.

Bilbies also have several unique biological features that could be better understood using comparative genomics. Characterised by their large ears, bilbies are burrowing, nocturnal omnivores that have strong forelimbs with long claws for digging and finding food¹ (Figure 1A).

Bilbies are physiologically well adapted to arid environments, having low metabolic rates and low water turnover¹. Bilbies do not drink free water but are able to obtain sufficient moisture from their food¹¹, which consists of insects, insect larvae, termites, seeds, bulbs, and fungi^12,13. Although not the largest of marsupials, Ninu males (50-84 cm; 0.66-2.5 kg) and females (49-68 cm; 0.66-1.1 kg)³, bilbies are proportionately bigger at birth than other species, which has been attributed to their complex placenta and broad milk composition¹². Bilbies, along with bandicoots, belong to the order Peramelemorphia, and unlike other marsupials have a short-lived chorioallantoic placentas as well as a choriovitelline placenta¹⁴. Like other digging marsupials, female Ninu have a backward-facing pouch and have one or two (rarely three) offspring per breeding event, (average 1.48 - 1.94)¹⁵. Ninu are polyoestrous with an oestrous cycle of 12-37 days (20.6 ± 7.3 days; N = 14) and an oestrous duration of 2-11 days (4.3 ± 2.1 days)¹⁴. Gestation is for 14 ± 1.4 days (N = 4) and offspring exit the pouch at around 80 ± 2 days (N = 6) with a lactation period of 90 days¹². Females are sexually mature from five months of age, and males from seven months¹⁵. Owing to the extreme Australian climate, characterised by periods of significant drought followed by flooding rains, bilbies are known as a boom-bust species¹². That is, their breeding seasons depend on food availability and rainfall meaning population numbers have the capacity to expand and contract rapidly in relation to climatic patterns. In times of high resource availability they can breed every three months, producing up to seven offspring in a 12-month period¹⁴; fecundity reduces in times of low resource availability.

Here, we present the first chromosome-length reference genome assembly for this threatened elusive species, the Ninu. Using this reference, in conjunction with resequenced genomes from both the Ninu and Yallara, we greatly increase our understanding of their biology and demographic histories, in addition to understanding the current genetic status of the Ninu metapopulation. We have developed and tested an innovative scat genotyping tool to inform current and future conservation actions and undertake ecological assessments.

The Ninu reference genome is now one of the highest quality marsupial genomes to date, comparable to that of the koala¹⁶, offering insights into biology, evolution, and contemporary population dynamics. The female Ninu reference genome was generated using a combination of long-read sequencing (HiFi), HiC (Omni-C) scaffolding and short-read (Illumina) polishing. The assembly is 3.66 Gb in size, which is larger than other marsupial genomes (Table 1), with 95.6% assigned to nine nuclear chromosome scaffolds and the mitochondrial genome (scaffold N50, 343.85 Mb; 0.34% gaps; 93.5% complete mammalian BUSCOs; Table 1; Extended Data Figure 1). Chromosome 1 is extremely large at 934,426,298 bp. The global transcriptome (including non-coding transcripts) contains 39,106 genes, with an average transcript length of 6,833 bp and an N50 of 13.4 kb (Extended Data Table 2). For all protein-coding transcripts, the longest open reading frame had an average transcript length of 1,010bp and N50 of 1,620bp. 53.99% of the genome is masked as repetitive (Extended Data Table 2). The Yallara genome assembly is 3.50 Gb in size (6,329,012 contigs; 19.74% gaps; 85.5% complete mammalian BUSCOs; Table 1).

Table 1: Genome statistics of the current Ninu genome compared with recently published marsupial genomes.

Species	Ninu (Greater bilby, Macrotis lagotis) This study	Yallara (Lesser bilby, Macrotis leucura) This study	Tasmanian devil (Purinina, Sarcophilus harrisii)¹⁷	Koala (Guba, Phascolarctos cinereus)¹⁶	Woylie (Brush-tailed bettong; Bettongia penicillata ogilbyi)¹⁸	Short-tailed opossum (Monodelphis domestica)¹⁹
Genome assembly version	v1.9	v1.0	mSarHar1.11	phaCin_unsw_v4.1	mBetpen1.pri.20210916	mMonDom1.pri
Data type	PacBio HiFi, Dovetail HiC, Illumina, 10x	Illumina	ONT 10x ULR, 10x Chromium, BioNano, Dovetail HiC	PacBio RSII, Illumina, BioNano	PacBio HiFi, Illumina	PacBio HiFi, BioNano, Arima HiC
Genome size (Gbp)	3.66	3.50	3.1	3.19	3.39	3.6
GC (%)	36.61	36.12	36	39.05	38.64	37.5
No. scaffolds	10	10	105	1,318	1,116	13
Scaffold N50 (Mbp)	343.85	343.85	611.3	480.11	6.94	538.3
Chromosome 1 (bp)	934,426,298	NA	716,413,629	N/A	N/A	760,810,273
No. contigs	4,429	6,329,012	444	1,935	3,019	2,268
Contig N50 (Mbp)	1.29	851 (bp)	62.3	11.4	1.995	3.9
Complete mammalian BUSCO (v5.3.2)	93.5%	85.5%	94.5% (BUSCO v4.0.2)	94.1%	94.1%
% Gaps	0.34	19.74	3.6	0.01	0.403

Twelve Ninu genomes were whole genome resequenced (WGR) using Illumina Novaseq with an average coverage of 23.9 ± 4.0´ (SD) (range: 13.8-29.6´; Table S1). Six individuals (3 males and 3 females) were from a temperate island (35°S, 136°E) and the other six (3 males and 3 females) were from a semi-arid region (26°S, 146°E; Figure 1A). DNA was extracted from four male and one female Yallara samples collected between 1895 and 1931 (Table S1). Four of these were resequenced using Illumina Novaseq, with 5.9 ± 2.6´ mean coverage (range: 0.73–12.82´). The phylogenetic relationship among the Ninu (N = 6) and Yallara (N = 2) individuals were inferred using the mitochondrial genomes extracted from the final BAM files (Figure 1C). For the high coverage dataset (total 2,146 variants), PC1 splits the Yallara and Ninu explaining 61.6% of the variance (Figure 1D). PC2 splits the Ninu samples into semi-arid and temperate samples, explaining a further 10.9% of the variance observed (Figure 1D).

Our analyses of the Ninu and Yallara effective population sizes, using sequentially Markovian coalescent analyses (PSMC and MSMC), revealed initial declines at 500,000–8,000,000 years ago and 300,000–4,000,000 years ago, respectively (Figure 2A). Both contractions coincide with the cooling of the global surface temperature prior to the last glacial period. The Ninu population expanded 100,000–500,000 years ago, followed by a possible decline in the last 100,000 years. However, this pattern is not well resolved as the bootstrap replicates lacked a signal for a population contraction and the pattern lies at the limits of SMC estimation. There is no clear separation between the semi-arid and temperate populations during the timeframe of inference (Figure 2B). The effective population size of Yallara may be underestimated due to the low coverage (mean 7.18×), falling below previously recommended thresholds for SMC analyses^20,21. Similarly, the flattened peaks and slight offset between the two MSMC estimations of temperate and semi-arid Ninu may be an outcome of differences in the mean sequencing coverage, where semi-arid individuals generally had higher genome coverage (Table S1).

Investigation of the twelve resequenced Ninu genomes reveals differences in heterozygosity and runs of homozygosity (ROH). The six Ninu from the temperate island population had lower heterozygosity and generally higher ROH-based inbreeding coefficients (F_ROH) than those from the semi-arid population (Figure 2C; Table S2). These results are likely due to the relatively small founder size that started the island population in the temperate region (N = 21) and limited gene flow with other populations for ~35-40 generations (Supplementary Note 2.3). F_ROH values of semi-arid Ninu were variable, but generally comprised fewer short ROHs than the temperate individuals (Figure 2C; Supplementary Note 2.2).

The semi-arid Ninu population was originally sourced from the last remaining Ninu population at Astrebla Downs, Queensland (24.20°S, 140.55°E) and have only spent ~1-7 generations in captivity. The number of short ROHs in some of the semi-arid individuals suggests past inbreeding in the wild populations, likely caused by declining population sizes. The long ROHs in these individuals suggest recent inbreeding, potentially within the captive population, indicating management strategies that avoid inbreeding and high population relatedness in the metapopulation should continue. In general, Ninu have relatively few long ROH compared with other threatened mammals^22-24. The fewer long ROHs in Ninu, despite their history of small founder sizes and captive breeding, could be partially attributed to their shorter generation time (hence higher recombination) and potentially higher substitution rate than those of larger mammals²⁵.

Efforts to improve Ninu genetic diversity through genetically driven population management actions were successful. The expansion of the managed metapopulation occurred between 2016 to 2021, where existing zoo-based and fenced sanctuary populations were used as source populations for new fenced sanctuaries (Figure 3A; Supplementary Note 2.3 & 2.4). We used over 9,000 SNPs called from reduced representation sequencing data (RRS; Table 2) of 363 individuals aligned to the reference genome to inform translocations and understand the genetic outcomes of our management recommendations (Supplementary Note 2.6). Observed heterozygosity across these source populations ranged from 0.1005 to 0.1839 (Figure 3B; Table 2). As expected, the mixed translocated populations had higher observed heterozygosity ranging from 0.1470 to 0.1916, and this flowed through to the two offspring populations, assessed as part of this study, with 0.1888 and 0.1913 (Figure 3B; Table 2). Most populations exhibited lower observed heterozygosity than expected under Hardy Weinberg equilibrium (Table 2). In concordance with the observed excess of homozygosity, mean inbreeding (F_IS) of the source populations was statistically significant for the Pilbara, Scotia, Thistle Island, and the zoo-based (ZAA) populations (Table 2). High allelic richness was observed across the translocated populations, likely due to these sites being recently established by genetically differentiated source populations (Figure 3B). Mean kinship (relatedness) was highest in some of the isolated source populations (Venus Bay, Yooka1 and Yooka2). Effective population size estimates were low across the populations and were generally estimated with poor precision (Table 2). It is important to note that sample size can affect the accuracy of estimating such population genetic statistics, so results from populations with low sample sizes (e.g., fewer than six individuals) should be treated with caution²⁹.

A toolkit for measuring the success of conservation efforts has been developed and tested. Using RRS SNPs aligned to the reference genome, a MassARRAY panel of 35 autosomal and four sex-linked markers was developed (Supplementary Note 2.5). This was used to genotype 195 scats collected by Indigenous rangers from the Kiwirrkurra Community across two locations, south and northeast of the community, in 2021 and 2022. Traditional hunting of feral cats and regular fire management is implemented in areas south of the Kiwirrkurra Community to reduce predation pressure but not in the north-eastern Ninu colonies. Community Rangers were interested in determining baseline data on the abundance of Ninu in the two areas before predator baiting is instigated around the northern sites, and whether these two colonies have become genetically isolated. Whilst survey of the north-eastern Ninu colonies was not as comprehensive as the southern colonies, we detected more Ninu (N = 16) in the area where traditional cat hunting occurs than in the north-eastern area (N = 9; Figure 3C; Table S3). Cumulatively, based on the 35 autosomal SNPs from the MassARRAY panel, the genetic diversity of the wild Ninu population at Kiwirrkurra was comparable to other wild populations in the Pilbara and Kimberley, (Ho = 0.34, 0.29 and 0.37, respectively; Extended Data Table 3). The north-eastern and southern Ninu colonies are located approximately 70 km apart but appear to be connected with detection of several half-sib and higher relationships amongst individuals located in the northeastern MU colony and southern colonies, and little genetic structuring observed in PCoA (Figure S1).

Managing metapopulations is complex but has been assisted greatly in recent years with genetic data³⁰. There is evidence of population stratification based on the demographic and translocation history of the Ninu populations (Figure 3B; Figure S2)³¹. Throughout this study a combination of the known translocation histories and genetic data were used to develop translocation recommendations for the National Bilby Recovery Team (Supplementary Note 2.6). These resulted in the movement of 225 individuals between 2016 and 2021 to establish five new populations within sanctuary areas (Figure 3B; Supplementary Note 2.3). Offspring sampled at two of these locations show the benefits of genetic mixing within the metapopulation (Figure 3B; Table 2), suggesting that this practice should continue in order to maintain Ninu genetic diversity in a protected environment. Based on the success of the scat case study, Indigenous rangers and other conservation agencies can now use this method to undertake a whole country survey for the species to better understand its distribution and movement between isolated populations, both in the wild and sanctuary locations, and estimate census population size³².

Table 2. Population genetic statistics, excluding Birdsville (QLD) and Currawinya (wild) samples (n = 1 for each population). SE is calculated as SD/sqrt(N), where N is the number of genotyped loci for each population. The full set of 9,906 SNPs was used. N_E was calculated on randomly selected subset of 5,000 loci and reported as the estimated N_E (no singletons), jackknifed 95% CIs and harmonic mean sample size. As Group 2 represents the founding animals that were sourced from Group 1 populations, F_IS and N_E were not calculated as the Wahlund effect would likely influence results due to the mixing of diverse source populations at translocated sites³³.

Population	Samples	N loci genotyped	H_O(SE)	H_E (SE)	AR^* (SE)	F_IS (95% CI)	MK (SE)	N_E (95% CI)	Harmonic mean N
GROUP 1 – SOURCE POPULATIONS
Arid Recovery	16	9,855	0.1700 (0.002071)	0.1795 (0.001928)	1.179 (0.001930)	0.0369 (-0.0011; 0.0667)	0.0850 (0.0023)	207.1 (77.1; INF)	12.7
Kimberley (wild)	5	9,835	0.1451 (0.002331)	0.1570 (0.002159)	1.156 (0.002160)	-0.0082 (-0.1168; 0.0660)	0.1358 (0.0125)	INF (10.4; INF)	4.4
Pilbara (wild)	5	9,749	0.1005 (0.002079)	0.1705 (0.002602)	1.156 (0.002392)	0.3007 (0.0840; 0.8603)	0.0747 (0.0067)	INF (INF; INF)	3.0
Scotia	53	9,905	0.1499 (0.001855)	0.1690 (0.001818)	1.169 (0.001814)	0.0981 (0.0442; 0.1393)	0.1268 (0.0020)	5.1 (3.3; 6.6)	29.0
Thistle Island	89	9,905	0.1761 (0.001909)	0.1835 (0.001828)	1.183 (0.001827)	0.0344 (0.0229; 0.0462)	0.0626 (0.0005)	83.8 (63.8; 106.7)	72.3
Venus Bay	5	9,807	0.1319 (0.002460)	0.1317 (0.002090)	1.131 (0.002098)	-0.0374 (-0.2736; 0.1793)	0.2443 (0.0312)	4.7 (0.6; INF)	4.7
Yookamurra 1	19	9,621	0.1245 (0.002433)	0.1062 (0.001889)	1.113 (0.002074)	0.0599 (-0.0060; 0.1527)	0.2644 (0.0052)	8.7 (2.8; 34.7)	8.5
Yookamurra 2	3	9,792	0.1546 (0.002727)	0.1554 (0.002370)	1.155 (0.002359)	-0.1151 (-0.1773; -0.0386)	0.1681 (0.0127)	INF (0.2; INF)	2.9
ZAA	78	9,906	0.1839 (0.001707)	0.1964 (0.001612)	1.196 (0.001611)	0.0556 (0.0352; 0.0752)	0.0376 (0.0010)	20.8 (17.4; 25.1)	58.4
GROUP 2 – TRANSLOCATED POPULATION FOUNDERS
Currawinya	35	9,906	0.1837 (0.001774)	0.1978 (0.001672)	1.198 (0.001669)	-	0.0421 (0.0025)	-	-
Dubbo	18	9,906	0.1916 (0.001878)	0.2024 (0.001730)	1.202 (0.001722)	-	0.0355 (0.0044)	-	-
Mallee Cliffs	50	9,904	0.1709 (0.001651)	0.1981 (0.001661)	1.198 (0.001658)	-	0.0377 (0.0014)	-	-
Mt Gibson	26	9,904	0.1470 (0.001871)	0.1710 (0.001846)	1.170 (0.001836)	-	0.0948 (0.0047)	-	-
Pilliga	36	9,900	0.1752 (0.001842)	0.1917 (0.001756)	1.191 (0.001755)	-	0.0527 (0.0018)	-	-
GROUP 3 – OFFSPRING POPULATIONS
Currawinya	35	9,906	0.1913 (0.001927)	0.1925 (0.001724)	1.192 (0.001724)	-0.0127 (-0.0391; 0.0141)	0.0275 (0.0022)	16.5 (13.4; 26.6)	25.7
Dubbo	46	9,905	0.1888 (0.001972)	0.1873 (0.001743)	1.187 (0.001744)	-0.0228 (-0.0478; -0.0029)	0.0342 (0.0019)	13.7 (10.7; 18.5)	33.3

*Rarefied to 2

A total of 3,858 SNPs that met our criteria were common across all three association analyses (Chi-square association, Fisher’s test, F_ST outlier test). We identified 339 unique genes between semi-arid and temperate individuals (Figure 4A; a full list of GO terms and associated genes is given in Table S4). As only two individuals had high sequencing coverage for the Yallara (Table S1), association analyses could not be undertaken.

Metabolism and olfactory receptors

Ninu have the lowest standard metabolic rate and the largest olfactory bulbs of any marsupial, which is reflected in their genome. The top 10 GO terms between temperate and semi-arid Ninu are associated with genes involved in anatomical structure (including SYNE1 and FMR1 involved in brain development), metabolic and cellular pathways (including BBOX1 and ACSBG1 involved in fatty acid metabolism), and response to stress (including GRM7 involved in neurotransmission in mammalian central nervous systems; Figure 4A; Table S4). It is not surprising that seven of the top ten GO terms are involved in cell differentiation, transport, and metabolic pathways as Ninu are known to have a low standard metabolic rate (58% of eutherian standard) compared with other marsupials (70% of eutherian standard)¹², as well as consuming very small quantities of water (56-68 mL/day) compared with bandicoots (46-341 mL/day), koala (296-414 mL/day) and possums (114-140 mL/day)³⁴.

Interestingly, the gene families involved in anatomical structure development, a range of metabolic processes and response to stress were also identified as fast evolving using a Computational Analysis of gene Family Evolution (CAFE) analysis of Ninu compared to ten other species’ genomes (Figure 4B & C; Table S5; Supplementary Note 3), including six marsupials across the marsupial lineage (antechinus, Tasmanian devil, koala, wombat, tammar wallaby, and opossum), one monotreme (platypus) and three eutherian mammals (human, mouse, and cow). We also show Ninu to have the highest number of annotated olfactory receptor genes (OR1D2 and OR1D5) across these 10 species (Figure 4C; Supplementary Note 3.7). This is unsurprising as bilbies rely on olfactory cues for locating food, leaving scent markings for male-male signalling³⁵, and avoiding predators^36,37. Further, larger olfactory bulbs have been observed in this species and are likely linked to their reliance on olfactory cues³⁸.

Genomics of the reproductive and immune systems

The association analyses of the male Ninu from the temperate and semi-arid populations revealed four genes expressed in the testis (SPEF2, TBC1D21, SYNE1, and NME8) that are involved in spermatogenesis, with each population having private and fixed alleles for all four genes. SPEF2 is critical in sperm tail development and head shape³⁹; TBC1D21 is similarly essential for sperm tail function⁴⁰. NME8 is involved in sperm tail maturation⁴¹, while SYNE1 (KASH1) is involved in sperm head formation⁴². It is tempting to speculate on the functional effect of these fixed differences in genes essential for male fertility between these two populations. However, the small testis size of Ninu relative to body mass⁴³ and the fact that only litters with single paternity have been observed (B. Coulter, unpublished data), suggests that they do not have a multi-male mating system that might produce differential rates of sperm competition between different populations. Instead, these sequence differences may be due to either population genetic differences caused by drift, or possibly higher mutation rates in animals living in warmer climates. Future studies in this area should examine mating structures, differential testis gene expression, sperm function and dominance in male breeding activity across Ninu populations.

The Peramelemorphia (bilbies and bandicoots) standout amongst marsupials due to their invasive chorioallantoic placenta, while most other marsupials rely on a yolk sac (choriovitelline) placenta⁴⁴. All 115 of the genes that show conserved chorioallantoic expression across all eutherians⁴⁵ are expressed in the Ninu uterus (TPM > 2). During formation of the peramelemorphian chorioallantoic placenta, uterine epithelia and trophoblast cells fuse together to form a heterokaryotic syncytium⁴⁶. Syncytia in both eutherians and marsupials have evolved through the incorporation of fusogenic retro-viral envelope (env) genes referred to as syncytins^47,48. All marsupials examined have at least one incorporated syncytin (Env2)⁴⁸. Since a chorioallantoic syncytium is unique to Peramelemorphia, we might expect that incorporation of multiple syncytins has occurred in this group. The Ninu genome has a diversity of encoded retro-elements, including more than 45,000 LTR retrotransposons, from which further envelope genes could have been co-opted. This genome provides the foundation for future studies of the fusion of maternal and fetal cells in the unique peramelemorphian placenta, if placental tissues can be obtained.

The immune gene repertoire of the Ninu is similar to those of other marsupials^49,50, with marsupial-specific genes and eutherian orthologs identified. Immune genes were annotated in the Ninu genome and transcriptome using similarity-based search methods such as BLAST⁵¹ and HMMER⁵² with known marsupial immune gene sequences as queries. This resulted in the manual characterisation of over 562 immune genes, from six immune gene families or groups (Table 3): cytokines, toll-like receptors (TLR), the major histocompatibility complex (MHC-I, MHC-II & MHC-III), natural killer cell (NK) receptors, immunoglobulins (IG), and T cell receptors (TCR). Relatively conserved immune genes, such as TLRs and constant regions of TCR and IG, were identified in addition to those immune genes unique to the marsupial lineage; including TLR1/6, TCRμ, MHC-I (-UM) and MHC-II (-DA, -DB, -DC) genes (Figure S3). Large marsupial-specific gene expansions within the LRC NK receptors were characterised, as well as reduced gene content within the NKC cluster of NK receptors (Table 3). Consistent with other marsupials investigated to date Igδ was not found⁵³. The organisation of the MHC region in the Ninu is similar to those of other marsupials in that the MHC-I and -II genes are interspersed, there is a MHC-III region and framework region, and the core MHC cluster is flanked by extended MHC genes (Figure 4D)⁵⁴. However, a few key distinct differences exist, with the four DAB genes positioned 8.7 Mb from the flanking extended region, and the translocation of four MHC-I genes onto scaffold 1 (Figure 4D). The mean sequence similarity between MHC-I genes was 76.4% in coding sequences, and 63.6% in the translated amino acids (Table S6). The class I genes that translocated onto scaffold 1 show very high sequence similarities (e.g., 99.3% between -UA and -UB, and 99% between -UC and -UD) and strong bootstrap support (100%).

Interestingly, there were fewer MHC-I and IG variable genes in the Ninu, Tasmanian devil, and opossum than in the other marsupials (Table 3). The loss of MHC-I and IG variable genes in the Ninu may be because of its invasive placenta, placing embryonic tissues in closer proximity to maternal tissues compared with epitheliochorial placentation. In the opossum, the brief phase of placental attachment is accompanied by inflammatory signalling⁵⁵, although there is little evidence for maternal recognition in marsupials outside of the macropods⁵⁶. Changes in vertebrate immunity have been noted in other species where pregnancy has evolved including the losses/modifications to the MHC-II pathway and expansion of the MHC-I repertoire in seahorses and pipefish⁵⁷. However, without a Ninu pregnant uterus, or placenta, we can only speculate on the meaning of the loss of MHC-I and IG variable genes in this species relative to the presence of 115 eutherian conserved chorioallantoic genes.

Bilby chromosomes

As with several other marsupial species, Ninu chromosomes have a number of rearrangements (Figure 5A-B; Extended Data Figure 2). The Ninu genome provides insights into chromosome evolution showing the Ninu XY₁Y₂ system was generated by fusion of the X with a telocentric autosome. Early work on marsupial karyotypes described a 2n=18 complement with 9 chromosome pairs in the Ninu including a large submetacentric X in females, and a 2n=19 complement in males with a single X, and two male-specific Y chromosomes⁵⁸. The Y₁ chromosome is very small, as is the case with the Y in most marsupial species, and represents the ancestral Y. In contrast, the Y₂ is a long telocentric chromosome, with a size and morphology like that of the long arm (Xq) of the Ninu X⁵⁸. However, alignment of the Illumina male genome sequence to the female Ninu reference genome now reveals more detail of this XY₁Y₂ system. Read depth analysis shows that the compound X is demarcated into an X-specific region (Xp) with half read depth, and a large pseudoautosomal region (Xq) with full read depth in males that pairs with Y₂ at male meiosis (Figure 5C). Additionally, reduced read depth extends into the fused autosome, so represents new X-specific material. Interestingly, this region does not pair with Y₂ during male meiotic prophase I (Figure 5D). Future work on bilby Y₂ chromosomes will yield information about sex chromosome differentiation and future comparisons between male and female transcriptomes will inform our understanding of meiotic sex chromosome inactivation in marsupials.

Table 3: Comparison of the immune gene repertoire of the Ninu with two marsupial species with partially invasive placentae (Tasmanian devil and opossum) and non-invasive placentae (koala and woylie). Only species with chromosome length genome assemblies were selected because genome quality influences the ability to characterise immune genes⁵⁹.

Immune Gene Family	Ninu (Greater bilby)	Tasmanian devil (Purinina)¹⁷	Short-tailed opossum¹⁹	Koala (Guba)¹⁶	Woylie¹⁸
Cytokines	84	72	76	82	77
TLR	10	10	10	10	10
MHC-I	6	6	6	19	17
MHC-II	12	8	12	16	23
MHC-III	38	36	33	39	37
Ext. MHC & framework genes	29	32	28	27	31
NKC	12	16	15	17	17
LRC (IG domains)	92	92	123	51	60
Extended LRC	22	16	9	6	22
IG constant	14	11	13	15	20
IG variable	116	61	89	289	226
TCR constant	9	13	14	10	12
TCR variable	118	82	67	103	122
Total	562	385	495	658	674

Rapid advances in genome sequencing technology have allowed us to sequence genomes for both the extant Ninu and the extinct Yallara leading to advances in our understanding of their unique biology and a toolkit for measuring the success of conservation efforts. Ninu are the only surviving species in their marsupial family, are known ecosystem engineers, and have significant cultural value to Indigenous Australians. Here we showcase Ninu cultural value and their importance by recognising the many First Nations names for the bilbies and describing their cultural meaning, in addition to their unusual biology.

Although once wide-ranging across the continent the long-term survival of Ninu in the wild is hampered by the presence of invasive pest species and altered fire regimes. Management of wild populations to date has been hindered by their cryptic, nocturnal nature making our scat genotyping array panel a critical component for understanding wild populations in the future. For the first time, we provide an understanding of the remaining wild Ninu genetic diversity relative to the managed semi-wild populations that are currently a conservation reservoir for the species. Throughout our project, we have worked closely with the conservation managers and Indigenous rangers, so our latest genetic research data has informed their management actions in real-time. Notably we have provided a worked example of the value of a high-quality reference genome, through gene discovery and interpretation, to downstream applied conservation actions. Our approach showcases what can be achieved when academics partner with Indigenous communities to understand culturally and ecologically significant species and is relevant to genome biologists, evolutionary and conservation geneticists, and conservation managers. This is not just another genome project but rather represents the holistic value of reference genomes to answer key Indigenous and conservation end-user management questions and understand the evolution and fundamental biology of a unique Australian species.

Acknowledgements

We acknowledge the traditional custodians of the land upon which bilbies have been, and are still, found and pay respects to their elders past and present. There are a massive number of individuals to acknowledge as part of this project. We thank Perth Zoo and Taronga Zoo for the provision of the individuals (female and male respectively) for the reference genome and transcriptomes. Thanks also to A. Sayer for assistance in acquiring the genome sample from Perth Zoo. We thank staff and teams at the Australian Wildlife Conservancy, Arid Recovery, Department for Environment and Water (South Australia), Save the Bilby Fund and the Zoo and Aquarium Association (ZAA) member institutions holding bilbies for their assistance with sample collection from the bilby metapopulation. We also thank the Kiwirrkurra community rangers and high school students for their collection of scat samples. The lesser bilby samples were provided by the Natural History Museum, London (NHMUK), South Australian Museum (SAMA) and Museum Victoria (NMV). Technical support for the contemporary tissue extractions were provided by K. Heasman and L. Alexander (University of Sydney), for the original lesser bilby extractions by A. MacDonald (ANU), the scat samples by S. McArthur and M. Millar (DBCA), and T. Bertozzi for the Illumina library prep kits for the lesser bilby samples. We also thank M. Dziminski and F. Carpenter (DBCA) for their collection and extraction of the Pilbara tissue samples. The authors acknowledge the technical support provided by the Sydney Informatics Hub, a Core Research Facility of the University of Sydney, particularly T. Chew, C. Willet, and R. Sadsad for the transcriptome pipeline, the earlier WGS NCI Gadi pipeline, and 10x assembly. The WGR alignment and variant calling was performed on the Illumina DRAGEN pipeline, we thank the DRAGEN development team for their assistance. Cloud support was provided by Amazon Web Services, Queensland's Research Computing Centre (RCC) FlashLite facility, and the Pawsey Supercomputing Centre for genome assembly and annotation, and RRS data analysis. We thank the Vertebrate Genomes Project (VGP) members for early pre-publication access to several genomes, in particular G. Myers, E. Jarvis, R. Nespolo and colleagues, and for Trichosurus vulpecula from E. Jarvis, N. Gemmell, T. Hore, M. Laird and colleagues.

Conflict of Interest

The authors declare they have no conflict of interest.

Ethics approval statement

Tissue samples for the reference individual, and the male testis, were collected opportunistically when individuals were euthanised for medical purposes. Ear biopsies are collected as part of the metapopulation routine monitoring programs, or during targeted trapping and capture events, that they conducted in accordance with the standard operating procedures for each organization. These management samples were shared with us as part of a study plan approved by representatives from the participating ZAA facilities, the AWC, the Australian Museum, the University of Sydney, and the Greater Bilby National Recovery Team Metapopulation Committee.

Data Availability

Raw and processed data for the reference genome, transcriptomes, resequenced genomes and DARTseq data is available via NCBI for the Ninu (PRJNA1049866) and Yallara (PRJNA1049868); in addition to the Australasian Genomes website (https://awgg-lab.github.io/australasiangenomes/genomes.html).

Funding information

The 10x Genomics sequencing was provided as part of the Oz Mammals Genomics (OMG), and bioinformatic support was provided as part of the Threatened Species Initiative (TSI), both supported by funding from the Australian Government National Collaborative Research Infrastructure Strategy (NCRIS) Bioplatforms Australia. The PacBio HiFi sequencing and HiC was supported by the University of Sydney through research grants to KB (DP180102465) and CJH (Toledo Zoo and Aquarium). KF, CJH and KB (CE200100012), CMW (DP180103370), and FRJ and SYWH (FT160100167) received funding from the Australian Research Council, KT was supported by an Australian Biological Resources Study (ABRS) grant, and MRW and the Ramaciotti Centre for Genomics acknowledge funding from the Australian Government NCRIS program and Bioplatforms Australia. ARH received funding from the Spanish Ministry of Science and Innovation (PID2020–112557GGB-I00) and the Agència de Gestió d’Ajuts Universitaris i de Recerca, AGAUR (2021 SGR 122). LM-G was supported by the Ministry of Science, Innovation and University (FPU18/03867 and EST22/00661). PDW is supported by the Australian Research Council (DP170101147, DP180100931, DP210103512, and DP220101429) and NHMRC Ideas Grant (2021172). RJE was supported by the Australian Research Council (LP18010072).

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

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