A predisposed motor bias shapes individuality in vocal learning.

The development of individuality during learned behavior is a common trait observed across animal species; however, the underlying biological mechanisms remain understood. Similar to human speech, songbirds develop individually unique songs with species-specific traits through vocal learning. In this study, we investigate the developmental and molecular mechanisms underlying individuality in vocal learning by utilizing F1 hybrid songbirds (Taeniopygia guttata cross with Taeniopygia bichenovii), taking an integrating approach combining experimentally controlled systematic song tutoring, unbiased discriminant analysis of song features, and single-cell transcriptomics. When tutoring with songs from both parental species, F1 hybrid individuals exhibit evident diversity in their acquired songs. Approximately 30% of F1 hybrids selectively learn either song of the two parental species, while others develop merged songs that combine traits from both species. Vocal acoustic biases during vocal babbling initially appear as individual differences in songs among F1 juveniles and are maintained through the sensitive period of song vocal learning. These vocal acoustic biases emerge independently of the initial auditory experience of hearing the biological father's and passive tutored songs. We identify individual differences in transcriptional signatures in a subset of cell types, including the glutamatergic neurons projecting from the cortical vocal output nucleus to the hypoglossal nuclei, which are associated with variations of vocal acoustic features. These findings suggest that a genetically predisposed vocal motor bias serves as the initial origin of individual variation in vocal learning, influencing learning constraints and preferences.

Heterotypic macrophages/microglia differentially contribute to retinal ischaemia and neovascularisation.

AIMS/HYPOTHESIS: Diabetic retinopathy is characterised by neuroinflammation that drives neuronal and vascular degenerative pathology, which in many individuals can lead to retinal ischaemia and neovascularisation. Infiltrating macrophages and activated retina-resident microglia have been implicated in the progression of diabetic retinopathy, although the distinct roles of these immune cells remain ill-defined. Our aim was to clarify the distinct roles of macrophages/microglia in the pathogenesis of proliferative ischaemic retinopathies. METHODS: Murine oxygen-induced retinopathy is commonly used as a model of ischaemia-induced proliferative diabetic retinopathy (PDR). We evaluated the phenotype macrophages/microglia by immunostaining, quantitative real-time RT-PCR (qRT-PCR), flow cytometry and scRNA-seq analysis. In clinical imaging studies of diabetic retinopathy, we used optical coherence tomography (OCT) and OCT angiography. RESULTS: Immunostaining, qRT-PCR and flow cytometry showed expression levels of M1-like macrophages/microglia markers (CD80, CD68 and nitric oxide synthase 2) and M2-like macrophages/microglia markers (CD206, CD163 and macrophage scavenger receptor 1) were upregulated in areas of retinal ischaemia and around neo-vessels, respectively. scRNA-seq analysis of the ischaemic retina revealed distinct ischaemia-related clusters of macrophages/microglia that express M1 markers as well as C-C chemokine receptor 2. Inhibition of Rho-kinase (ROCK) suppressed CCL2 expression and reduced CCR2-positive M1-like macrophages/microglia in areas of ischaemia. Furthermore, the area of retinal ischaemia was reduced by suppressing blood macrophage infiltration not only by ROCK inhibitor and monocyte chemoattractant protein-1 antibody but also by GdCl3. Clinical imaging studies of diabetic retinopathy using OCT indicated potential involvement of macrophages/microglia represented by hyperreflective foci in areas of reduced perfusion. CONCLUSIONS/INTERPRETATION: These results collectively indicated that heterotypic macrophages/microglia differentially contribute to retinal ischaemia and neovascularisation in retinal vascular diseases including diabetic retinopathy. This adds important new information that could provide a basis for a more targeted, cell-specific therapeutic approach to prevent progression to sight-threatening PDR.

Ghost introgression in ricefishes of the genus Adrianichthys in an ancient Wallacean lake.

Because speciation might have been promoted by ancient introgression from an extinct lineage, it is important to detect the existence of 'ghost introgression' in focal taxa and examine its contribution to their diversification. In this study, we examined possible ghost introgression and its contributions to the diversification of ricefishes of the genus Adrianichthys in Lake Poso, an ancient lake on Sulawesi Island, in which some extinctions are known to have occurred. Population-genomic analysis revealed that two extant Adrianichthys species, A. oophorus and A. poptae are reproductively isolated from each other. Comparisons of demographic models demonstrated that introgression from a ghost population, which diverged from the common ancestor of A. oophorus and A. poptae, is essential for reconstructing the demographic history of Adrianichthys. The best model estimated that the divergence of the ghost population greatly predated the divergence between A. oophorus and A. poptae, and that the ghost population secondarily contacted the two extant species within Lake Poso more recently. Genome scans and simulations detected a greatly divergent locus, which cannot be explained without ghost introgression. This locus was also completely segregated between A. oophorus and A. poptae. These findings suggest that variants that came from a ghost population have contributed to the divergence between A. oophorus and A. poptae, but the large time-lag between their divergence and ghost introgression indicates that the contribution of introgression may be restricted.

Single-cell RNA sequencing of intestinal immune cells in neonatal necrotizing enterocolitis.

PURPOSE: Necrotizing enterocolitis (NEC) causes fatal intestinal necrosis in neonates, but its etiology is unknown. We analyzed the intestinal immune response to NEC. METHODS: Using single-cell RNA sequencing (scRNA-seq), we analyzed the gene expression profiles of intestinal immune cells from four neonates with intestinal perforation (two with NEC and two without NEC). Target mononuclear cells were extracted from the lamina propria of the resected intestines. RESULTS: In all four cases, major immune cells, such as T cells (15.1-47.7%), B cells (3.1-19.0%), monocytes (16.5-31.2%), macrophages (1.6-17.4%), dendritic cells (2.4-12.2%), and natural killer cells (7.5-12.8%), were present in similar proportions to those in the neonatal cord blood. Gene set enrichment analysis showed that the MTOR, TNF-α, and MYC signaling pathways were enriched in T cells of the NEC patients, suggesting upregulated immune responses related to inflammation and cell proliferation. In addition, all four cases exhibited a bias toward cell-mediated inflammation, based on the predominance of T helper 1 cells. CONCLUSION: Intestinal immunity in NEC subjects exhibited stronger inflammatory responses compared to non-NEC subjects. Further scRNA-seq and cellular analysis may improve our understanding of the pathogenesis of NEC.

Human-specific features of spatial gene expression and regulation in eight brain regions.

Molecular maps of the human brain alone do not inform us of the features unique to humans. Yet, the identification of these features is important for understanding both the evolution and nature of human cognition. Here, we approached this question by analyzing gene expression and H3K27ac chromatin modification data collected in eight brain regions of humans, chimpanzees, gorillas, a gibbon, and macaques. An analysis of spatial transcriptome trajectories across eight brain regions in four primate species revealed 1851 genes showing human-specific transcriptome differences in one or multiple brain regions, in contrast to 240 chimpanzee-specific differences. More than half of these human-specific differences represented elevated expression of genes enriched in neuronal and astrocytic markers in the human hippocampus, whereas the rest were enriched in microglial markers and displayed human-specific expression in several frontal cortical regions and the cerebellum. An analysis of the predicted regulatory interactions driving these differences revealed the role of transcription factors in species-specific transcriptome changes, and epigenetic modifications were linked to spatial expression differences conserved across species.

Population history and genomic admixture of sea snakes of the genus Laticauda in the West Pacific.

Speciation in the open ocean has long been studied, but it remains largely elusive what factors promote or inhibit speciation in such an open environment. Marine amniotes, which evolved from terrestrial ancestors, provide valuable opportunities for studying speciation in the ocean because of their evident aquatic origins. Sea snakes are phylogenetically related to terrestrial elapid snakes and consist of two monophyletic groups (Hydrophiini and Laticaudini). These two groups migrated from land to water almost at the same time, but species diversities are remarkably different: there are approx. 60 species in 16 genera described for hydrophiins, whereas only eight species in the genus Laticauda are described for laticaudins. Here, we provide a high-quality reference genome assembly of a laticaudin L. colubrina with a scaffold N50 value of 40 Mbp, and focused on laticaudins to consider why they have seldom speciated. We performed whole-genome shotgun sequencing of several species of laticaudins sampled in their southmost (Vanuatu) and northmost (Ryukyu) habitats. Demographic histories of Vanuatu and Ryukyu populations suggest that populations of broadly distributed major species are geographically structured. Each species is genetically clearly distinguished, but there is a considerable amount of gene flow between two sibling species distributed sympatrically in Vanuatu. In addition, inter-species genomic admixture is ubiquitously observed among laticaudins even between phylogenetically distant species. Broad distribution of major species combined with such genetic mixability might have prevented laticaudins from genetic isolation and speciation.

Towards HCP-Style macaque connectomes: 24-Channel 3T multi-array coil, MRI sequences and preprocessing.

Macaque monkeys are an important animal model where invasive investigations can lead to a better understanding of the cortical organization of primates including humans. However, the tools and methods for noninvasive image acquisition (e.g. MRI RF coils and pulse sequence protocols) and image data preprocessing have lagged behind those developed for humans. To resolve the structural and functional characteristics of the smaller macaque brain, high spatial, temporal, and angular resolutions combined with high signal-to-noise ratio are required to ensure good image quality. To address these challenges, we developed a macaque 24-channel receive coil for 3-T MRI with parallel imaging capabilities. This coil enables adaptation of the Human Connectome Project (HCP) image acquisition protocols to the in-vivo macaque brain. In addition, we adapted HCP preprocessing methods to the macaque brain, including spatial minimal preprocessing of structural, functional MRI (fMRI), and diffusion MRI (dMRI). The coil provides the necessary high signal-to-noise ratio and high efficiency in data acquisition, allowing four- and five-fold accelerations for dMRI and fMRI. Automated FreeSurfer segmentation of cortex, reconstruction of cortical surface, removal of artefacts and nuisance signals in fMRI, and distortion correction of dMRI all performed well, and the overall quality of basic neurobiological measures was comparable with those for the HCP. Analyses of functional connectivity in fMRI revealed high sensitivity as compared with those from publicly shared datasets. Tractography-based connectivity estimates correlated with tracer connectivity similarly to that achieved using ex-vivo dMRI. The resulting HCP-style in vivo macaque MRI data show considerable promise for analyzing cortical architecture and functional and structural connectivity using advanced methods that have previously only been available in studies of the human brain.

Evolution of the sperm methylome of primates is associated with retrotransposon insertions and genome instability.

Changes in gene expression resulting from epigenetic and/or genetic changes play an important role in the evolutionary divergence of phenotypes. To explore how epigenetic and genetic changes are linked during primate evolution, we have compared the genome-wide DNA methylation profiles (methylomes) of humans and chimpanzees, which have a 1.2% DNA sequence divergence, of sperm, the frontal cortices, B cells, and neutrophils. We revealed that species-specific differentially methylated regions (S-DMRs), ranging from several hundred base pairs (bp) to several kilo base pairs (kb), were frequently associated with sequence changes in transcription factor-binding sites and insertions of Alu and SVA retrotransposons. We then generated a reference macaque sperm methylome map and revealed, in sperm, that both human and chimpanzee S-DMRs arose more frequently owing to methylation loss rather than gain. Moreover, we observed that the sperm methylomes contained many more hypomethylated domains (HMDs), ranging from 20 to 500 kb, than did the somatic methylomes. Interestingly, the sperm HMDs changed rapidly during primate evolution; hundreds of sperm HMDs were specific to humans, whereas most somatic HMDs were highly conserved between humans and chimpanzees. Notably, these human-specific sperm HMDs frequently occurred in regions exhibiting copy number variations. Our findings indicate that primate evolution, particularly in the germline, is significantly impacted by reciprocal changes in the genome and epigenome.

The life history of retrocopies illuminates the evolution of new mammalian genes.

New genes contribute substantially to adaptive evolutionary innovation, but the functional evolution of new mammalian genes has been little explored at a broad scale. Previous work established mRNA-derived gene duplicates, known as retrocopies, as models for the study of new gene origination. Here we combine mammalian transcriptomic and epigenomic data to unveil the processes underlying the evolution of stripped-down retrocopies into complex new genes. We show that although some robustly expressed retrocopies are transcribed from preexisting promoters, most evolved new promoters from scratch or recruited proto-promoters in their genomic vicinity. In particular, many retrocopy promoters emerged from ancestral enhancers (or bivalent regulatory elements) or are located in CpG islands not associated with other genes. We detected 88-280 selectively preserved retrocopies per mammalian species, illustrating that these mechanisms facilitated the birth of many functional retrogenes during mammalian evolution. The regulatory evolution of originally monoexonic retrocopies was frequently accompanied by exon gain, which facilitated co-option of distant promoters and allowed expression of alternative isoforms. While young retrogenes are often initially expressed in the testis, increased regulatory and structural complexities allowed retrogenes to functionally diversify and evolve somatic organ functions, sometimes as complex as those of their parents. Thus, some retrogenes evolved the capacity to temporarily substitute for their parents during the process of male meiotic X inactivation, while others rendered parental functions superfluous, allowing for parental gene loss. Overall, our reconstruction of the "life history" of mammalian retrogenes highlights retroposition as a general model for understanding new gene birth and functional evolution.

Loss of olfaction in sea snakes provides new perspectives on the aquatic adaptation of amniotes.

Marine amniotes, a polyphyletic group, provide an excellent opportunity for studying convergent evolution. Their sense of smell tends to degenerate, but this process has not been explored by comparing fully aquatic species with their amphibious relatives in an evolutionary context. Here, we sequenced the genomes of fully aquatic and amphibious sea snakes and identified repertoires of chemosensory receptor genes involved in olfaction. Snakes possess large numbers of the olfactory receptor (OR) genes and the type-2 vomeronasal receptor (V2R) genes, and expression profiling in the olfactory tissues suggests that snakes use the ORs in the main olfactory system (MOS) and the V2Rs in the vomeronasal system (VNS). The number of OR genes has decreased in sea snakes, and fully aquatic species lost MOS which is responsible for detecting airborne odours. By contrast, sea snakes including fully aquatic species retain a number of V2R genes and a well-developed VNS for smelling underwater. This study suggests that the sense of smell also degenerated in sea snakes, particularly in fully aquatic species, but their residual olfactory capability is distinct from that of other fully aquatic amniotes. Amphibious species show an intermediate status between terrestrial and fully aquatic snakes, implying their importance in understanding the process of aquatic adaptation.

Considerable Synteny and Sequence Similarity of Primate Chromosomal Region VIIq31.

Human chromosome 7 has been the focus of many behavioral, genetic, and medical studies because it carries genes related to cancer and neurodevelopment. We examined the evolution of the chromosome 7 homologs, and the 7q31 region in particular, using chromosome painting analyses and 3 paint probes derived from (i) the whole of chimpanzee chromosome VII (wcVII), (ii) human 7q31 (h7q31), and (iii) the chimpanzee homolog VIIq31 (cVIIq31). The wcVII probe was used instead of the whole human chromosome 7 because the chimpanzee contains additional C-bands and revealed large areas of synteny conservation as well as fragmentation across 20 primate species. Analyses focusing specifically on the 7q31 homolog and vicinity revealed considerable conservation across lineages with 2 exceptions. First, the probes verified an insertion of repetitive sequence at VIIq22 in chimpanzees and bonobos and also detected the sequence in most subtelomeres of the African apes. Second, a paracentric inversion with a breakpoint in the cVIIq31 block was found in the common marmoset, confirming earlier studies. Subsequent in silico comparative genome analysis of 17 primate species revealed that VIIq31.1 is more significantly conserved at the sequence level than other regions of chromosome VII, which indicates that its components are likely responsible for critical shared traits across the order, including conditions necessary for proper human development and wellbeing.

Frequent expansions of the bitter taste receptor gene repertoire during evolution of mammals in the Euarchontoglires clade.

Genome studies of mammals in the superorder Euarchontoglires (a clade that comprises the orders Primates, Dermoptera, Scandentia, Rodentia, and Lagomorpha) are important for understanding the biological features of humans, particularly studies of medical model animals such as macaques and mice. Furthermore, the dynamic ecoevolutionary signatures of Euarchontoglires genomes may be discovered because many species in this clade are characterized by their successful adaptive radiation to various ecological niches. In this study, we investigated the evolutionary trajectory of bitter taste receptor genes (TAS2Rs) in 28 Euarchontoglires species based on homology searches of 39 whole-genome assemblies. The Euarchontoglires species possessed variable numbers of intact TAS2Rs, which ranged from 16 to 40, and their last common ancestor had at least 26 intact TAS2Rs. The gene tree showed that there have been at least seven lineage-specific events involving massive gene duplications. Gene duplications were particularly evident in the ancestral branches of anthropoids (the anthropoid cluster), which may have promoted the adaptive evolution of anthropoid characteristics, such as a trade-off between olfaction and other senses and the development of herbivorous characteristics. Subsequent whole-gene deletions of anthropoid cluster TAS2Rs in hominoid species suggest ongoing ectopic homologous recombination in the anthropoid cluster. These findings provide insights into the roles of adaptive sensory evolution in various ecological niches and important clues related to the molecular mechanisms that underlie taste diversity in Euarchontoglires mammalian species, including humans.

Bidirectional promoters are the major source of gene activation-associated non-coding RNAs in mammals.

BACKGROUND: The majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples. RESULTS: We found that there is only about 1% of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs. CONCLUSIONS: We propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression.

Expansion of learning capacity elicited by interspecific hybridization.

Learned behavior, a fundamental adaptive trait in fluctuating environments, is shaped by species-specific constraints. This phenomenon is evident in songbirds, which acquire their species-specific songs through vocal learning. To explore the neurogenetic mechanisms underlying species-specific song learning, we generated F1 hybrid songbirds by crossing Taeniopygia guttata with Aidemosyne modesta. These F1 hybrids demonstrate expanded learning capacities, adeptly mimicking songs from both parental species and other heterospecific songs more extensively than their parental counterparts. Despite the conserved size of brain regions and neuron numbers in the neural circuits for song learning and production, single-cell transcriptomics reveals distinctive transcriptional characteristics in the F1 hybrids, especially in vocal-motor projection neurons. These neurons exhibit enrichment for nonadditively expressed genes, particularly those related to ion channel activity and cell adhesion, which are associated with the degree of song learning among F1 individuals. Our findings provide insights into the emergence of altered learning capabilities through hybridization, linked to cell type-specific transcriptional changes.

A method to analyze gene expression profiles from hippocampal neurons electrophysiologically recorded in vivo.

Hippocampal pyramidal neurons exhibit diverse spike patterns and gene expression profiles. However, their relationships with single neurons are not fully understood. In this study, we designed an electrophysiology-based experimental procedure to identify gene expression profiles using RNA sequencing of single hippocampal pyramidal neurons whose spike patterns were recorded in living mice. This technique involves a sequence of experiments consisting of in vivo juxtacellular recording and labeling, brain slicing, cell collection, and transcriptome analysis. We demonstrated that the expression levels of a subset of genes in individual hippocampal pyramidal neurons were significantly correlated with their spike burstiness, submillisecond-level spike rise times or spike rates, directly measured by in vivo electrophysiological recordings. Because this methodological approach can be applied across a wide range of brain regions, it is expected to contribute to studies on various neuronal heterogeneities to understand how physiological spike patterns are associated with gene expression profiles.

Neuronal DSCAM regulates the peri-synaptic localization of GLAST in Bergmann glia for functional synapse formation.

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

Regional DNA methylation differences between humans and chimpanzees are associated with genetic changes, transcriptional divergence and disease genes.

Changes in gene expression have been proposed to have an important role in the evolutionary changes in phenotypes. Interspecific changes in gene expression can result not only from genetic changes in regulatory regions but also from epigenetic changes in such regions. Here we report the identification of genomic regions showing differences in DNA methylation between humans and chimpanzees (termed S-DMRs for species-specific differentially methylated regions) on chromosomes 21 and 22. These regional methylation differences are frequently associated with genes, including those relevant to a disease, such as Alzheimer's disease, diabetes mellitus or cancer. Methylation differences are often correlated with changes in promoter activity or alternative splicing. Comparative studies including other great ape species provide evidence for the contribution of genetic changes to some of these S-DMRs. Genetic changes responsible for the S-DMRs include gain or loss of CTCF-binding site and changes in CpG density in microsatellite repeats. Our results suggest that DNA methylation changes, often caused by small sequence changes, contribute to transcriptional and phenotypic diversification in hominid evolution.

Expression of taste signal transduction molecules in the caecum of common marmosets.

The extraoral presence of taste signal transduction proteins has recently been reported in rodents and humans. Here, we report for the first time the presence of these signal transduction proteins in the caecum of a non-human primate, the common marmoset. Quantitative RT-PCR data on the gene expression of taste signal transduction molecules (gustducin and TRPM5) in common marmosets suggested high expression in the caecum, which was not observed in other non-human primates. Immunohistochemical analysis confirmed the specific presence of gustducin and taste receptors in marmoset caecal cells. These results may relate to the specific feeding behaviour of marmosets, which consume plant exudates, primarily gums.

Gene expression profiling of the masticatory muscle tendons and Achilles tendons under tensile strain in the Japanese macaque Macaca fuscata.

Both Achilles and masticatory muscle tendons are large load-bearing structures, and excessive mechanical loading leads to hypertrophic changes in these tendons. In the maxillofacial region, hyperplasia of the masticatory muscle tendons and aponeurosis affect muscle extensibility resulting in limited mouth opening. Although gene expression profiles of Achilles and patellar tendons under mechanical strain are well investigated in rodents, the gene expression profile of the masticatory muscle tendons remains unexplored. Herein, we examined the gene expression pattern of masticatory muscle tendons and compared it with that of Achilles tendons under tensile strain conditions in the Japanese macaque Macaca fuscata. Primary tenocytes isolated from the masticatory muscle tendons (temporal tendon and masseter aponeurosis) and Achilles tendons were mechanically loaded using the tensile force and gene expression was analyzed using the next-generation sequencing. In tendons exposed to tensile strain, we identified 1076 differentially expressed genes with a false discovery rate (FDR) < 10-10. To identify genes that are differentially expressed in temporal tendon and masseter aponeurosis, an FDR of < 10-10 was used, whereas the FDR for Achilles tendons was set at > 0.05. Results showed that 147 genes are differentially expressed between temporal tendons and masseter aponeurosis, out of which, 125 human orthologs were identified using the Ensemble database. Eight of these orthologs were related to tendons and among them the expression of the glycoprotein nmb and sphingosine kinase 1 was increased in temporal tendons and masseter aponeurosis following exposure to tensile strain. Moreover, the expression of tubulin beta 3 class III, which promotes cell cycle progression, and septin 9, which promotes cytoskeletal rearrangements, were decreased in stretched Achilles tendon cells and their expression was increased in stretched masseter aponeurosis and temporal tendon cells. In conclusion, cyclic strain differentially affects gene expression in Achilles tendons and tendons of the masticatory muscles.

The admixed brushtail possum genome reveals invasion history in New Zealand and novel imprinted genes.

Combining genome assembly with population and functional genomics can provide valuable insights to development and evolution, as well as tools for species management. Here, we present a chromosome-level genome assembly of the common brushtail possum (Trichosurus vulpecula), a model marsupial threatened in parts of their native range in Australia, but also a major introduced pest in New Zealand. Functional genomics reveals post-natal activation of chemosensory and metabolic genes, reflecting unique adaptations to altricial birth and delayed weaning, a hallmark of marsupial development. Nuclear and mitochondrial analyses trace New Zealand possums to distinct Australian subspecies, which have subsequently hybridised. This admixture allowed phasing of parental alleles genome-wide, ultimately revealing at least four genes with imprinted, parent-specific expression not yet detected in other species (MLH1, EPM2AIP1, UBP1 and GPX7). We find that reprogramming of possum germline imprints, and the wider epigenome, is similar to eutherian mammals except onset occurs after birth. Together, this work is useful for genetic-based control and conservation of possums, and contributes to understanding of the evolution of novel mammalian epigenetic traits.