Mitochondrial sequences have integrated into the nuclear genome since the origin of eukaryotes. Recent insertions that retain homology to extant mitochondrial DNA (mtDNA), termed NUMTs, confound mtDNA sequence analysis. Here, we use great ape Telomere-to-Telomere (T2T) genomes to study NUMTs in bonobo, chimpanzee, human, gorilla, and Bornean and Sumatran orangutans. A phylogeny based on shared and lineage-specific NUMTs accurately recapitulates the great ape species tree topology. NUMTs are enriched at nonfunctional nonrepetitive regions of the nuclear genome, and depleted within enhancers and coding sequences, suggesting negative selection. We validate the presence of a 76-kilobase-long heterozygous NUMT in chimpanzee, which is larger than any other NUMT observed in great apes, and find that dozens of NUMTs on the Pan Y Chromosome expanded together with palindromes. Finally, by analyzing intraspecific variation, we confirm that the vast majority of species-specific NUMTs identified in T2T assemblies are fixed or present at high frequencies in each species. Our study highlights NUMTs as a dynamic evolutionary force contributing to shaping ape genomes, and is valuable for characterizing mtDNA in great apes.

Abstract Developing an effective DNA extraction method that meets requirements for long-read sequencing of poorly preserved samples, such as museum specimens or ancient material, offers new opportunities for genomic analysis of endangered or extinct species for which samples are rare. However, these samples often yield degraded and highly fragmented DNA, rendering long-read sequencing infeasible for many specimens residing in museum collections. Herein, we demonstrate a protocol for successfully extracting DNA of sufficient quality for sequencing on the Oxford Nanopore Technologies long-read sequencing PromethION platform from a desiccated, museum-grade blue carpenter bee specimen ( Xylocopa caerulea ). We find the protocol is reproducible across specimens and yields high levels of long, endogenous X. caerulea -derived DNA, highlighting the utility of our method for enabling genomic studies of historical collections. From a single flow cell, we assembled the full-length mitochondrial genome and used this assembly to perform a phylogenetic analysis, accurately placing our X. caerulea specimen among related Xylocopa species, thus demonstrating the phylogenetic utility of long-read museomics. Using these long-read data, we analyzed native CpG methylation, finding endogenous methylation signals that correlate with genic and exonic sequences. This method expands the feasibility of genomic and epigenomic analyses from challenging samples, enhancing our ability to investigate the genomes of endangered and extinct species through archival resources.

Subspecies represent evolutionarily distinct lineages that preserve patterns of adaptive diversity and ecological specialization within species. Genomic tools can assist with subspecies delineation, and provide support for classifications based solely on morphological, geographic, or ecological traits. The American mink (Neogale vison), as semi-aquatic mustelid carnivore with a broad ecological range across North America, includes several putative subspecies of conservation concern. To investigate their evolutionary history and adaptive signatures, chromosome-scale genome assemblies were generated for six individuals representing three subspecies: N. vison evergladensis, N. vison vulgivaga, and N. vison lutensis. Genomes were assembled using Illumina short reads, scaffolded with Oxford Nanopore long reads, and aligned to the phased N. vison reference genome. The reference-free pangenome revealed an open architecture and presence/absence analysis was consistent with distinct adaptive profiles. Neogale vison evergladensis showed enrichment in traits related to reproduction and sensory function, N.v. vulgivaga in cytoskeletal remodeling and oxidative stress, and N. v. lutensis in neuronal development and synaptic plasticity. Mitochondrial analyses resolved N.v. lutensis as a distinct lineage, while nuclear data lacked fine-scale resolution. Our findings support N.v. evergladensis as a distinct subspecies, supported by both the mitogenome phylogeny and functional differentiation. Neogale vison evergladensis also exhibited multiple indicators of small population size, including high inbreeding estimates, and evidence of sustained population decline. As the first pangenome for Mustelidae, this study demonstrates the power of natural history specimens to detect signatures of adaptation and inform the management of threatened populations.

Primordial germ cells (PGCs) are critical tools for genome engineering and conservation in birds. Although culture systems for chicken PGCs have been well established for nearly two decades, efforts to propagate PGCs from other avian species have proved exceptionally challenging, limiting the broader application of artificial reproductive technologies in birds. Here we report the first successful derivation and long-term culture of PGCs from the rock dove, or common pigeon (Columba livia). Guided by transcriptomic profiling of PGCs, we developed a species-specific medium that supports PGC maintenance and expansion. We identify insulin signaling as a requirement for survival under the conditions tested, and demonstrate that inhibition of the retinoic acid receptor, in the presence of vitamin A, supports propagation. Supplementation with bone morphogenetic protein 4, leukemia inhibitory factor (LIF), glial cell line-derived neurotrophic factor, and pleiotrophin further enhances PGC proliferation. Cultured cells express canonical germline markers and migrate to the gonads following injection into both rock dove and chicken embryos, confirming functional migratory competency. These findings establish a platform for germline manipulation and biobanking in Columbidae, broadening the potential applicability of reproductive technologies to conservation efforts.

ABSTRACT Butternut ( Juglans cinerea ) is a North American hardwood in rapid decline, driven largely by butternut canker disease (BCD). Within the genus, Juglans ailantifolia (Japanese walnut) is comparatively resistant and hybridizes readily with J. cinerea , yet the genomic basis of the difference is unresolved. The first chromosome-scale reference genome for J. ailantifolia is presented here, together with a second J. cinerea accession from the species’ primary range, with independent scaffolding and uniform annotation throughout. Gene family evolution was assessed across twelve Juglandaceae genomes, and a four accession pangenome contrasted the BCD resistant section Cardiocaryon ( J. ailantifolia , J. mandshurica ) with the BCD susceptible section Trachycaryon ( J. cinerea , Iowa and New Brunswick accessions). Across sections separated by approximately 30 My, most defense gene families differ in presence and absence rather than copy number; five surface receptor and protease families are overrepresented in the lineage specific complement of both sections, indicating rapid bidirectional turnover. Five families differ between sections in copy number while remaining stable between the conspecific accessions, all favoring the susceptible section. The smaller set includes chitinases and NB-LRR receptors biased toward the resistant section, and dehydrins, pectin-modifying enzymes, pathogenesis-related proteins and the CBF regulon toward the susceptible one. The largest copy number difference separates the two conspecific accessions at a senescence associated cysteine protease. The northern New Brunswick accession also retains lower heterozygosity, a distinct demographic trajectory, and unique gene content enriched for calcium transport, salt stress regulation and raffinose family oligosaccharide biosynthesis, consistent with freezing tolerance. Significance Statement Juglans cinerea (butternut), a threatened North American walnut tree, is being lost to butternut canker disease, while Juglans ailantifolia (Japanese walnut) is resistant to the fungal pathogen. Chromosome-scale genomes and a pangenome place defense differences between resistant and susceptible lineages primarily in gene presence and absence. The pangenome and demographic modeling distinguish the northern J. cinerea accession by unique gene content consistent with cold tolerance and a distinct population history.

Centromere identity is specified by CENP-A, a histone H3 variant that epigenetically defines centromere position. How CENP-A is maintained at one location in rapidly evolving centromeric DNA is unknown. Using single-cell-derived clones of human cell lines, we demonstrate heterogeneity in CENP-A position within cell populations at neocentromeres and a native centromere. CENP-A heterogeneity is accompanied by heterogeneous DNA methylation patterns, with DNA methylation shifting according to CENP-A position. We demonstrate centromere epigenetic plasticity over extended proliferation, with native centromeres maintaining stable DNA methylation boundaries, but neocentromeres exhibiting DNA methylation instability, boundary loss, and increased missegregation. Finally, we show that neocentromeres are more sensitive to DNA methylation inhibition than native centromeres, and that this inhibition is accompanied by expanded CENP-A-enriched domains and increased missegregation. This study supports a role for DNA methylation boundaries in maintaining centromere position, stability, and function and highlights the intrinsic instability of DNA methylation at neocentromeres.

Cancer is ubiquitous in multicellular life, yet susceptibility varies significantly between species. Previous studies have shown a genetic basis for cancer resistance in many species, but few studies have investigated the inverse: why some species are particularly susceptible to cancer. The Dasyuridae are a family of carnivorous marsupials that are frequently reported as having high rates of cancer prevalence. We hypothesized that this high susceptibility also has a genetic basis. To investigate this, we generated reference genomes for the kowari (Dasyuroides byrnei), a dasyurid species with one of the highest rates of reported cancer prevalence among mammals, and a non-dasyurid marsupial, the eastern barred bandicoot (Perameles gunnii). We used these to perform a comparative genomics analysis alongside nine previously assembled reference genomes: four dasyurid species and five non-dasyurid marsupial species. Genomes were annotated using FGENESH++ and assigned to orthogroups for input to computational analysis of gene family evolution (CAFE) to identify gene families that had undergone significant expansions or contractions in each lineage. In the dasyurids, we identified large expansions in Ras genes, a family of oncogenes. Interestingly, a similar expansion of Ras genes was also identified in the bandicoot and bilby. These genes were primarily expressed in tissues such as testes, ovaries, and yolk sac, so we hypothesize they serve a reproductive role. Future work is required to identify the potential roles of oncogene expansions in cancer susceptibility in these marsupial species.

Human centromeres are large, complex chromosomal loci that serve as the foundation for kinetochore assembly, contribute to chromosome architecture and sister chromatid cohesion, and participate in chromosome separation during cell division. Encoded by thousands to millions of base pairs of repetitive DNA, these regions were previously represented as gaps in the human genome assembly due to limitations in sequencing technologies and computational tools that could accurately distinguish and anchor the highly similar repeats within a linear genome assembly. Substantial advances in long-read sequencing over the past 5 years have permitted these large human centromere regions to be spanned, revealing new genomic and epigenomic information and the structural organization of these essential regions. Here, we review these discoveries and discuss knowledge gaps that have been filled and emerging functional questions. The complex and repetitive nature of centromeres has historically posed challenges to their genomic assembly and functional understanding, which are now being overcome with long-read sequencing. This Review discusses recent genomic and epigenomic insights into human centromere biology that have positioned the field for future studies of centromere structure in chromosome biology and human disease.

Human genome resequencing typically involves mapping reads to a reference genome to call variants; however, this approach suffers from both technical and reference biases, leaving many duplicated and structurally polymorphic regions of the genome unmapped. Consequently, existing variant benchmarks, generated by the same methods, fail to assess these complex regions. To address this limitation, we present a telomere-to-telomere genome benchmark that achieves near-perfect accuracy (i.e. no detectable errors) across 99.4% of the complete, diploid HG002 genome. This benchmark adds 701.4 Mb of autosomal sequence and both sex chromosomes (216.8 Mb), totaling 15.3% of the genome that was absent from prior benchmarks. We also provide a diploid annotation of genes, transposable elements, segmental duplications, and satellite repeats, including 39,144 protein-coding genes across both haplotypes. To facilitate application of the benchmark, we developed tools for measuring the accuracy of sequencing reads, phased variant call sets, and genome assemblies against a diploid reference. Genome-wide analyses show that state-of-the-art de novo assembly methods resolve 2-7% more sequence and outperform variant calling accuracy by an order of magnitude, yielding just one error per 100 kb across 99.9% of the benchmark regions. Adoption of genome-based benchmarking is expected to accelerate the development of cost-effective methods for complete genome sequencing, expanding the reach of genomic medicine to the entire genome and enabling a new era of personalized genomics.

Abstract Small marsupials in the family Dasyuridae are a key component of Australia’s arid and semi-arid fauna, whose high species richness is proposed to reflect an opportunity-driven adaptive radiation. Despite growing interest in this group from both ecological and evolutionary perspectives, genomic data for most species is non-existent, or limited to a few marker loci. Here, we generated a chromosome-level reference genome and a de novo mitochondrial genome for the desert-dwelling Wongai ningaui ( Ningaui ridei ). The nuclear genome assembly is highly contiguous, with a scaffold N50 of 594.5 MB and high BUSCO gene recovery (93.8%). Additionally, we produced a draft assembly for the related, semi-arid slender-tailed dunnart (Sminthopsis murina ). We then used these assemblies to explore the demographic histories of these species. We find evidence for contrasting patterns of population growth during the late Pleistocene and early Holocene, corresponding with differences in local climate, potentially consistent with differences in optimal habitat. The new genomic resources and demographic findings presented here provide a foundation for future studies on adaptive specialisation in this group of Australian marsupials. Significance Statement Dasyurid marsupials are the primary carnivorous and insectivorous mammals in Australia. This family includes species such as the endangered Tasmanian devil ( Sarcophilus harrisii ) and quolls (Genus Dasyurus ), as well as an emerging model species, the fat-tailed dunnart ( Sminthopsis crassicaudata ). Despite the species richness within dasyurids, most species remain under-studied. This is particularly true of arid and semi-arid zone species, who are often small in size, live in remote habitats and are cryptic by nature. By creating genome assemblies for two dasyurid species, this study provides resources to support phylogenetic, comparative and conservation research in arid zone marsupials. Importantly, the study’s finding that arid and semi-arid species show distinct trajectories of demographic change in response to historical climate may have implications for the resilience of locally-adapted dasyurid species to ongoing climate change.

Dire wolves (Aenocyon dirus) are extinct predators of Pleistocene North America. Although phenotypically similar to living wolves (Canis lupus), dire wolves have yet to be placed confidently in the canid family tree. We generated 3.4× and 12.8× paleogenomes from two well-preserved dire wolves dating to >13,000 and >72,000 years ago and estimated consensus species trees for these and 10 canid species. Our results revealed that ∼2/3 of dire wolf ancestry is most likely derived from a lineage sister to the clade comprising the gray wolf, coyote, and dhole and the remaining ∼1/3 from a lineage near the base of Canini diversity. Our results underscore the power of paleogenomes to resolve long-standing taxonomic questions and contribute to growing evidence of the role of post-speciation gene flow as an evolutionary force.

The evolutionary plasticity of the three-dimensional (3D) genome organization in vertebrates, and its transmission through the germ line, is central to understanding genome function and evolution. Yet, the mechanisms regulating these processes remain poorly characterized across lineages. Here, we integrate fluorescence-activated cell sorting, in situ chromosome capture conformation (Hi-C), and single-cell RNA sequencing to investigate germ line genome architecture in eutherians, marsupials, and reptiles, lineages that last shared a common ancestor ~350 million years ago. We uncover lineage-specific chromatin folding patterns in germ cells, shaped by chromosome morphology and genome size, which constrain DNA loop formation and inter-chromosomal interactions during meiosis. We also explore the relationship between 3D genome remodeling and gene regulation in the context of meiotic sex chromosome inactivation (MSCI). In the tammar wallaby, we identify regions of the X that escape MSCI checkpoint, suggesting incomplete silencing in marsupials. These findings provide high-resolution insights into the evolution of germ line chromatin architecture and the co-evolution of genome structure and function across vertebrates.

The marsupial moles are arguably Australia’s most enigmatic marsupials. Almost indistinguishable from placental (eutherian) moles, they provide a striking example of convergent evolution. Exploring the genome of the southern marsupial mole, we provide insights into its unusual biology. We show definitively by retrophylogenomic analysis that marsupial moles are most closely related to bandicoots and bilbies (order Peramelemorphia). We find evidence of a marked decline in marsupial mole effective population size, most likely preceding the arrival of humans in regions near its range, and potentially corresponding to periods of climatic change. Our analysis of loss of eye function—an adaptation to subterranean life—reveals a structured order of loss of gene function associated first with the lens, then cone, and finally rod cells. Last, we identify genetic changes suggestive of adaptation to an oxygen-poor environment and of its evolution of partially descended testes.

The attachment of the kinetochore to the centromere is essential for genome maintenance, yet the highly repetitive nature of satellite regional centromeres limits our understanding of their chromatin organization. We demonstrate that single-molecule chromatin fiber sequencing (Fiber-seq) can uniquely co-resolve kinetochore and surrounding chromatin architectures along point centromeres, revealing largely homogeneous single-molecule kinetochore occupancy. In contrast, the application of Fiber-seq to regional centromeres exposed marked per-molecule heterogeneity in their chromatin organization. Regional centromere cores uniquely contain a dichotomous chromatin organization (dichromatin) composed of compacted nucleosome arrays punctuated with highly accessible chromatin patches. CENP-B occupancy phases dichromatin to the underlying alpha-satellite repeat within centromere cores but is not necessary for dichromatin formation. Centromere core dichromatin is conserved between humans and primates, including along regional centromeres lacking satellite repeats. Overall, the chromatin organization of regional centromeres is defined by marked per-molecule heterogeneity, buffering kinetochore attachment against sequence and structural variability within regional centromeres.

Human neocentromeres are functional centromeres demarcated by CENP-A nucleosomes that form ectopically at alpha satellite-free loci. How neocentromeres reshape local chromatin and which features of native centromeric chromatin are preserved are unknown. We generated gapless, haplotype-resolved assemblies of native and neocentromeres from three patient-derived cell lines. Integrating CpG methylation, CENP-A profiling, and single-molecule chromatin fiber sequencing, we reveal chromatin features that define the essential centromeric architecture reconstituted during neocentromere establishment. We find that a deletion within the satellite array encompassing the hypo-CpG methylation centromere dip regions (CDRs) led to native centromere inactivation, that neocentromeres harbor CDRs and a dichromatin architecture, recapitulating features of alpha-satellite centromeres, and that LINEs demarcate neocentromere boundaries, implicating transposable elements in restricting CENP-A domain spreading. Moreover, neocentromeric chromatin is incompatible with promoter-like chromatin states, redefining the regulatory landscape within genic regions. Finally, using haplotype-specific chromatin footprinting, we resolve CENP-A nucleosome chromatin architecture of active centromeres.

The novel long non-coding RNA (lncRNA) Leat1 is extraordinarily conserved in both its location (syntenic with EfnB2, an essential gene in anogenital patterning) and sequence. Here we show that Leat1 is upregulated following the production of testosterone from the developing testis in mice and interacts with EfnB2, positively regulating its expression. Leat1 expression is suppressed by estrogen, which in turn suppresses the expression of EfnB2. Moreover, the loss of Leat1 leads to reduced EfnB2, resulting in a severe hypospadias phenotype. The human LEAT1 gene is also co-expressed with EFNB2 in the developing human penis, suggesting a conserved function for this gene in urethral closure. Together our data identify Leat1 as a novel molecular regulator of urethral closure and implicate it as a target of endocrine disruption in the etiology of hypospadias.

Horseshoe crabs, considered living fossils with a stable morphotype spanning similar to 445 million years, are evolutionarily, ecologically, and biomedically important species experiencing rapid population decline. Of the four extant species of horseshoe crabs, the Atlantic horseshoe crab, Limulus polyphemus, has become an essential component of the modern medicine toolkit. Here, we present the first chromosome-level genome assembly, and the most contiguous and complete assembly to date, for L. polyphemus using nanopore long-read sequencing and chromatin conformation analysis. We find support for three horseshoe crab-specific whole-genome duplications, but none shared with Arachnopulmonata (spiders and scorpions). Moreover, we discovered tandem duplicates of endotoxin detection pathway components Factors C and G, identify candidate centromeres consisting of Gypsy retroelements, and classify the ZW sex chromosome system for this species and a sister taxon, Carcinoscorpius rotundicauda. Finally, we revealed this species has been experiencing a steep population decline over the last 5 million years, highlighting the need for international conservation interventions and fisheries-based management for this critical species.

Centromere location is specified by CENP-A, a centromere-specific histone that epigenetically defines centromere identity. How CENP-A is maintained at one location in rapidly evolving centromeric DNA is unknown. Using single-cell-derived clones of human cell lines, we demonstrate single-cell heterogeneity in CENP-A position within cell populations at neocentromeres and a native centromere. CENP-A heterogeneity is accompanied by unique DNA methylation and H3K9me3 patterns, with DNA methylation shifting according to CENP-A position. We further demonstrate centromere epigenetic evolution over prolonged proliferation, with native centromeres maintaining stable heterochromatin boundaries, but neocentromeres exhibiting DNA methylation instability, H3K9me3 gain, boundary loss and fragility. Lastly, prolonged CENP-A and HJURP overexpression leads to centromere and neocentromere expansion, gradual CENP-A depletion, neocentromere destabilization and CENP-A re-localization that is accompanied by local heterochromatin remodeling. This study reveals the naturally evolving epigenetic plasticity of human centromeres and neocentromeres and highlights the importance of repressive chromatin boundaries in maintaining centromere stability.

The most dynamic and repetitive regions of great ape genomes have traditionally been excluded from comparative studies 1–3 . Consequently, our understanding of the evolution of our species is incomplete. Here we present haplotype-resolved reference genomes and comparative analyses of six ape species: chimpanzee, bonobo, gorilla, Bornean orangutan, Sumatran orangutan and siamang. We achieve chromosome-level contiguity with substantial sequence accuracy (<1 error in 2.7 megabases) and completely sequence 215 gapless chromosomes telomere-to-telomere. We resolve challenging regions, such as the major histocompatibility complex and immunoglobulin loci, to provide in-depth evolutionary insights. Comparative analyses enabled investigations of the evolution and diversity of regions previously uncharacterized or incompletely studied without bias from mapping to the human reference genome. Such regions include newly minted gene families in lineage-specific segmental duplications, centromeric DNA, acrocentric chromosomes and subterminal heterochromatin. This resource serves as a comprehensive baseline for future evolutionary studies of humans and our closest living ape relatives.