Haplotype-Resolved Genome of the Critically Endangered, Paleo-endemic Tree, Eidothea hardeniana

A fully phased, chromosome-level reference genome assembly for an IUCN-declared critically endangered and paleo-endemic tree, Eidothea hardeniana (Nightcap Oak) was assembled de novo using long-read PacBio HiFi, Oxford Nanopore (ONT) and Hi-C sequencing technologies. Consensus assembly represented high contiguity (N50 ∼ 49 Mb), (BUSCO 99.2%), assembly size in accordance with flow cytometry derived genome size estimate, and the presence of telomeric and centromeric repeats on pseudochromosomes. Haplotype 1 (598 Mb) achieved an N50 of 45.8 Mb and 99.1% BUSCO completeness, while Haplotype 2 (596 Mb) showed similar quality (N50: 47.3 Mb; BUSCO: 98.4%). Repeat content comprised approximately 54% of the genome. Gene annotation confirmed approximately 30120 protein coding genes with BUSCO completeness of 99.5%. Comparative orthology with other assembled Proteaceae genomes identified ∼13,000 shared ortholog clusters and 227 Eidothea-specific orthologs clusters. Heterozygosity in E. hardeniana was estimated at 1.17 SNVs per kilobase (0.12%), which is approximately one-third of the level reported for the other sequenced critically endangered Proteaceae species, Macadamia jansenii. This genome represents the first assembled genome from the Australian lineage of the Proteoideae subfamily within Proteaceae. As a high-quality reference for conservation genomics, it provides a foundation for SNP genotyping, population structure analyses, and the development of informed genetic rescue strategies for E. hardeniana and related species.