Genome sharing in gonochorous species is expected to result in intraspecific conflicts due to intersexual competition. The emergence of sexual dimorphism is thus connected to the evolution of mechanisms that, starting from a similar genomic background, produce sufficiently disparate phenotypes to attenuate sexually antagonistic selection. From a molecular perspective it can be achieved through sex-specific differences in gene expression, splicing, non-coding regulation or epigenetic marks. The tawny owl (Strix aluco) is a sexually dimorphic species where females and males evolved distinct body sizes (smaller males), which results in sex-specific roles and therefore is a robust example of resolved sexual conflict. Here, we explore transcriptional variation among 32 juvenile tawny owls with the objective of investigating molecular signatures of resolved sexual conflict. Our results show substantial sex-specific variation in terms of differentially expressed genes, single nucleotide polymorphisms and alternative exon usage in genes involved in life history traits (ZGRF1, VLDLR), behaviour (GSK3B, SLC12A) and aspects of growth (GHR, EGF, EPS8L2). Exploring sex-specific DEG revealed enrichment for biological functions associated with melanogenesis and pigment granulation in males, which together with the identification of a single up-regulated autosomal gene involved in melanogenesis (RAB38) in brown males strongly suggests different timings for the onset of pigmentation between sexes. Overall, our results reveal some of the sex-specific molecular signatures expected to be observed in the context of a resolved sexual conflict.

Feral pigs (Sus scrofa) in Hawai'i pose a persistent threat to native biodiversity, endemic species, and culturally important resources. Polynesian pigs, or pua'a, were brought to the Hawaiian Islands with Polynesian settlement in the mid-1200s and represent part of the cultural legacy of Hawai'i. Since the introduction of European pigs in 1778 and onward, the ancestral composition of contemporary animals has been debated, and conservation efforts for island endemic species have been challenged by tension between ecological destruction caused by contemporary feral pigs and the cultural importance of this animal. To inform this complex issue, our objective was to evaluate the genetic ancestry of contemporary feral pig populations across Hawai'i to elucidate genetic remnants of past introductions. We used a high-resolution single nucleotide polymorphism (SNP) array, providing a survey of the entire genome, to characterize ancestry, including hybridization of Polynesian pigs with global Asian and European lineages. We assembled a comprehensive reference set-representing the S. scrofa wild-domestic species complex-from which we queried 608 Hawaiian feral pig samples to quantify ancestral composition. Our results demonstrate that contemporary Hawaiian feral pigs have admixed ancestry influenced by European Heritage breeds and animals of Asian origin-potentially descending from initial Polynesian introductions. Importantly, we establish that European domestic lineages represent the dominant ancestral influence among contemporary feral pigs in Hawai'i, which challenges previous claims of genetic uniqueness of these populations within the broader S. scrofa wild-domestic species complex.

Sexual dimorphism is a defining vertebrate feature, yet its sex-specific molecular architecture remains poorly understood. Here we established a sex-balanced, uniformly reared chicken cohort to map this landscape, integrating individual whole-genome sequencing with 7,969 bulk and 779,380 single nucleus transcriptomes across 32 tissues from 280 birds. We identified 495,098 independent expression quantitative trait loci for 20,194 genes, including 10,937 loci modulated by cell-type composition. Notably, 340 genes were regulated by 449 loci in a sex-dependent manner, significantly enrichment in endocrine tissues like adipose and the adrenal gland. Furthermore, we fine-mapped 1,219 structural variants, demonstrating their unique roles to tissue- and sex-specific expression beyond SNPs. Ultimately, we showed the utility of these regulatory effects in elucidating the molecular basis of metabolism and complex traits in both chickens and humans. This comprehensive atlas of regulatory effects provides profound insights into the genomic and molecular basis of sexual dimorphism in vertebrates.

Domestication involves huge phenotypic shifts via strong directional selection. The resulting changes, often termed the Domestication Syndrome, typically encompass numerous traits; however, the most universal of these are changes in reduced fear of humans (tameness) and brain composition. To assess how early domestication selection may have focused on tameness and its interaction with brain composition, a Red Junglefowl (Gallus gallus) population (the wild progenitor of the domestic chicken) was used to create two lines bidirectionally selected for fear of humans over eight generations of selection. These selection lines were then used to make an intercross population. Using a combination of genome-wide mapping in the intercross and between-line analysis of the selection lines, we show that the genetic loci for tameness co-localise with genetic loci for brain composition and anxiety behaviour. Furthermore, the detected loci for brain composition also co-localise with brain composition loci identified in a separate wild × domestic intercross. These results indicate that tameness and brain composition are either pleiotropic or genetically linked, and that tameness selection appears to recapitulate the same loci that have been selected by domestication itself. Therefore, selection for increased tameness could be the initial selection pressure driving the core of the domestication syndrome.

The crowing of the male chicken is a charismatic example of vocal display in a bird. It is regarded as the main territorial announcement of the ancestral red junglefowl. The call has been preserved throughout domestication, although several of its elements have been altered. To assess these alterations, we assayed crowing spectrograms from wild and captive-held red junglefowl populations from India, along with two red junglefowl populations held in long-term captivity in Sweden, and a domestic white Leghorn breed. We find consistent differences between the different Indian red junglefowl and the domestic white Leghorn for a range of characteristics, including the duration of the last syllable and the number of formants and their frequency in the last and second-to-last syllable. To analyse the genetic architecture of crowing vocalization, we performed a quantitative trait loci (QTL) experiment using a wild × domestic advanced intercross to identify QTL that explained a large percentage of the variation present for the duration of the last syllable and the number of formants in the second to last syllable. With this study we thus demonstrate consistent differences in red junglefowl and white Leghorn chickens and identify a relatively simple genetic architecture for some of these traits.This article is part of the theme issue 'Unravelling domestication: multi-disciplinary perspectives on human and non-human relationships in the past, present and future'.

A social network is a group of individuals forming a non-random social structure where the number of interactions systematically vary among individuals. The Red Junglefowl, the ancestor of all domesticated chickens, has been shown to form social structures within a larger group; however, how poultry in egg production are socially organised is poorly understood. Egg layers in groups of thousands of individuals are suggested to either, just as Red Junglefowl, form subgroups or to lose social affiliations and replace these with a system of social tolerance. In this case study, a stable group of 20 laying hens with known genetic relationships housed in an outdoor aviary were observed for 3 h per day, during 20 days, in order to determine the social structure in the group. Social affiliations and centralisation measurements were assessed by proximity. The results show that some individuals were consistently more centralised compared to others, which indicates a stable individual variation in sociality. Furthermore, no individual spent more than 10% of her total number of interactions with a particular individual, i.e., little consistency in social affiliations could be found. The results of this case study support the theory of a higher social tolerance rather than a consistent social organisation in domesticated egg layers.

Across species, prenatal maternal stress has been shown to create heterogeneity in behavioural phenotypes. Research has recently highlighted that individuals vary in how predictable they are in their behavioural responses. This within-individual variation in behaviour is likely to be of biological importance, since individuals interact with the world not only through their mean behavioural phenotype, but also through their full range of behavioural variation. Yet, the underlying mechanisms that create and constrain between-individual variation in behavioural predictability remain largely unexplored. Here, we estimate whether experimental elevation of maternal corticosterone during egg laying (to model prenatal maternal stress) can cause variation in behavioural predictability in a population of chickens. Offspring’s behavioural predictability was quantified by testing them repeatedly (16 times) in a standard anxiety test (open-field test). Elevated maternal corticosterone resulted in less anxious and more predictable offspring compared to control offspring. These findings provide the first evidence that maternal corticosterone levels, via prenatal pathways, may influence multi-hierarchical behavioural plasticity by affecting both the magnitude and the predictability of behavioural responses. These results not only expand our current knowledge about the ways maternal stress can affect offspring’s behavioural phenotypes but also suggest a possible proximate mechanism underlying within-population variation in individual behavioural predictability.

Sex-chromosome dosage poses a challenge for heterogametic species in maintaining the proper balance of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage asymmetries in avian cells. Through comprehensive allele-resolved multiome analyses, we examine dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Our data reveal that females upregulate their single Z chromosome through increased transcriptional burst frequency, mirroring mammalian X upregulation. Z-protein levels are further balanced in females through enhanced translation efficiency. Additionally, we present a global analysis of promoter elements regulating transcriptional burst kinetics in birds, revealing evolutionary conservation of the genomic encoding of burst kinetics between birds and mammals. Our study provides insights into the regulation of avian dosage compensation, and when considering all regulatory layers collectively, an unexpected similarity between avian and mammalian dosage compensation becomes apparent.

Introduction: Domestication is the process of modifying animals for human benefit through selective breeding in captivity. One of the traits that often diverges is the size of the brain and its constituent regions; almost all domesticated species have relatively smaller brains and brain regions than their wild ancestors. Although the effects of domestication on the brain have been investigated across a range of both mammal and bird species, almost nothing is known about the neuroanatomical effects of domestication on the world’s most common bird: the chicken (Gallus gallus). Methods: We compared the quantitative neuroanatomy of the telencephalon of white leghorn chickens with red junglefowl, their wild counterpart, and several wild galliform species. We focused specifically on the telencephalon because telencephalic regions typically exhibit the biggest differences in size in domesticate-wild comparisons. Results: Relative telencephalon size was larger in chickens than in junglefowl and ruffed grouse (Bonasa umbellus). The relative size of telencephalic regions did not differ between chickens and junglefowl, but did differ in comparison with ruffed grouse. Ruffed grouse had larger hyperpallia and smaller entopallial, nidopallial, and striatal volumes than chickens and junglefowl. Multivariate analyses that included an additional three wild grouse species corroborated these findings: chicken and junglefowl have relatively larger nidopallial and striatal volumes than grouse. Conversely, the mesopallial and hyperpallial volumes tended to be relatively smaller in chickens and junglefowl. Conclusion: From this suite of comparisons, we conclude that chickens do not follow a pattern of widespread decreases in telencephalic region sizes that is often viewed as typical of domestication. Instead, chickens have undergone a mosaic of changes with some regions increasing and others decreasing in size, and there are few differences between chickens and junglefowl.

Despite a long presence in the contiguous United States (US), the distribution of invasive wild pigs (Sus scrofa × domesticus) has expanded rapidly since the 1980s, suggesting a more recent evolutionary shift towards greater invasiveness. Contemporary populations of wild pigs represent exoferal hybrid descendants of domestic pigs and European wild boar, with such hybridization expected to enrich genetic diversity and increase the adaptive potential of populations. Our objective was to characterize how genetic enrichment through hybridization increases the invasiveness of populations by identifying signals of selection and the ancestral origins of selected loci. Our study focused on invasive wild pigs within Great Smoky Mountains National Park, which represents a hybrid population descendent from the admixture of established populations of feral pigs and an introduction of European wild boar to North America. Accordingly, we genotyped 881 wild pigs with multiple high-density single-nucleotide polymorphism (SNP) arrays. We found 233 markers under putative selection spread over 79 regions across 16 out of 18 autosomes, which contained genes involved in traits affecting feralization. Among these, genes were found to be related to skull formation and neurogenesis, with two genes, TYRP1 and TYR, also encoding for crucial melanogenesis enzymes. The most common haplotypes associated with regions under selection for the Great Smoky Mountains population were also common among other populations throughout the region, indicating a key role of putatively selective variants in the fitness of invasive populations. Interestingly, many of these haplotypes were absent among European wild boar reference genotypes, indicating feralization through genetic adaptation.

Recent work indicates that feralisation is not a simple reversal of domestication, and therefore raises questions about the predictability of evolution across replicated feral populations. In the present study we compare genes and traits of two independently established feral populations of chickens (Gallus gallus) that inhabit archipelagos within the Pacific and Atlantic regions to test for evolutionary parallelism and/or divergence. We find that feral populations from each region are genetically closer to one another than other domestic breeds, despite their geographical isolation and divergent colonisation histories. Next, we used genome scans to identify genomic regions selected during feralisation (selective sweeps) in two independently feral populations from Bermuda and Hawaii. Three selective sweep regions (each identified by multiple detection methods) were shared between feral populations, and this overlap is inconsistent with a null model in which selection targets are randomly distributed throughout the genome. In the case of the Bermudian population, many of the genes present within the selective sweeps were either not annotated or of unknown function. Of the nine genes that were identifiable, five were related to behaviour, with the remaining genes involved in bone metabolism, eye development and the immune system. Our findings suggest that a subset of feralisation loci (i.e. genomic targets of recent selection in feral populations) are shared across independently established populations, raising the possibility that feralisation involves some degree of parallelism or convergence and the potential for a shared feralisation 'syndrome'.

Feathers comprise a series of evolutionary innovations but also harbor colour, a biological trait with immense selective value and known to co-vary with life history or complex traits. Such an intricate web of relationships is particularly true in melanin-based pigmentation species, mainly due to known pleiotropic effects of the melanocortin pathway – originating so-called melanin-phenotypes. Here we explored the molecular basis of melanin coloration and expected co-variation at molecular level in a melanin-based, colour polymorphic benchmark system, the tawny owl. An extensive body of literature has revealed grey and brown tawny owl color morphs differ in a series of life history and behavioral traits. We assembled the first draft genome of the species against which we mapped ddRADseq reads from 220 grey and 150 brown morphs - representing 10 years of pedigree data from a population in Southern Finland - and explored genome-wide associations with colour phenotype. Our results revealed molecular signatures of cold adaptation strongly associated with grey coloration, namely a non-synonymous substitution in MCHR1 detected when comparing genomes, plus 2 substitutions in non-coding regions of FTCD and FAM135A whose genotype combinations obtained a predictive power of up to 100% (predicting grey colour). All these genes have functions related to energy homeostasis, fat deposition and control of starvation response and indicate the molecular basis of some cold environment adaptations predicted to be grey-morph specific. our results unveil part of the molecular machinery of melanin-phenotypes and shed light on the maintenance and evolution of colour polymorphism in melanin-based pigmented species.

DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. To investigate how variation in DNA methylation is regulated on the Z chromosome we utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals. The relatively large numbers of individuals allowed us to identify additional genomic MHM regions on the Z chromosome that were significantly differentially methylated between the sexes. These regions appear to down-regulate local gene expression in males, but not remove it entirely (unlike the lncRNAs identified in the initial MHM regions). These MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. In addition, quantitative trait loci (QTL) that regulate variation in methylation on the Z chromosome, and those loci that regulate methylation on the autosomes that derive from the Z chromosome were mapped. Trans-effect hotspots were also identified that were based on the autosomes but affected the Z, and also one that was based on the Z chromosome but that affected both autosomal and sex chromosome DNA methylation regulation. We show that both cis and trans loci that originate from the Z chromosome never exhibit an interaction with sex, whereas trans loci originating from the autosomes but affecting the Z chromosome always display such an interaction. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes.

Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency similar to mammalian X-upregulation, and that Z-protein levels are further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species. ### Competing Interest Statement The authors have declared no competing interest.

Mito-nuclear insertions, or NUMTs, relate to genetic material of mitochondrial origin that have been transferred to the nuclear DNA molecule. The increasing amounts of genomic data currently being produced presents an opportunity to investigate this type of patterns in genome evolution of non-model organisms. Identifying NUMTs across a range of closely related taxa allows one to generalize patterns of insertion and maintenance in autosomes, which is ultimately relevant to the understanding of genome biology and evolution. Here we collected existing pairwise genome-mitogenome data of the order Strigiformes, a group that includes all the nocturnal bird predators. We identified NUMTs by applying percent similarity thresholds after blasting mitochondrial genomes against nuclear genome assemblies. We identified NUMTs in all genomes with numbers ranging from 4 in Bubo bubo to 24 in Ciccaba nigrolineata. Statistical analyses revealed NUMT size to negatively correlate with NUMT's sequence similarity to with original mtDNA region. Lastly, characterizing these nuclear insertions of mitochondrial origin in a comparative genomics framework produced variable phylogenetic patterns, suggesting in some cases that insertions might pre-date speciation events within Strigiformes.

Chickens are believed to have inhabited the Hawaiian island of Kauai since the first human migrations around 1200AD, but numbers have peaked since the tropical storms Iniki and Iwa in the 1980s and 1990s that destroyed almost all the chicken coops on the island and released large numbers of domestic chickens into the wild. Previous studies have shown these now feral chickens are an admixed population between Red Junglefowl (RJF) and domestic chickens. Here, using genetic haplotypic data, we estimate the time of the admixture event between the feral population on the island and the RJF to 1981 (1976–1995), coinciding with the timings of storm Iwa and Iniki. Analysis of genetic structure reveals a greater similarity between individuals inhabiting the northern and western part of the island to RJF than individuals from the eastern part of the island. These results point to the possibility of introgression events between feral chickens and the wild chickens in areas surrounding the Koke’e State Park and the Alaka’i plateau, posited as two of the major RJF reservoirs in the island. Furthermore, we have inferred haplotype blocks from pooled data to determine the most plausible source of the feral population. We identify a clear contribution from RJF and layer chickens of the White Leghorn (WL) breed. This work provides independent confirmation of the traditional hypothesis surrounding the origin of the feral populations and draws attention to the possibility of introgression of domestic alleles into the wild reservoir.

The allometric scaling of the brain size and neuron number across species has been extensively studied in recent years. With the exception of primates, parrots, and songbirds, larger brains have more neurons but relatively lower neuronal densities than smaller brains. Conversely, when considering within-population variability, it has been shown that mice with larger brains do not necessarily have more neurons but rather more neurons in the brain reflect higher neuronal density. To what extent this intraspecific allometric scaling pattern of the brain applies to individuals from other species remains to be explored. Here, we investigate the allometric relationships among the sizes of the body, brain, telencephalon, cerebellum, and optic tectum, and the numbers of neurons and non-neuronal cells of the telencephalon, cerebellum, and optic tectum across 66 individuals originated from an intercross between wild and domestic chickens. Our intercross of chickens generates a population with high variation in brain size, making it an excellent model to determine the allometric scaling of the brain within population. Our results show that larger chickens have larger brains with moderately more neurons and non-neuronal cells. Yet, absolute number of neurons and non-neuronal cells correlated strongly and positively with the density of neurons and non-neuronal cells, respectively. As previously shown in mice, this scaling pattern is in stark contrast with what has been found across different species. Our findings suggest that neuronal scaling rules across species are not a simple extension of the neuronal scaling rules that apply within a species, with important implications for the evolutionary developmental origins of brain diversity.

The cerebellum has a highly conserved neural structure across species but varies widely in size. The wide variation in cerebellar size (both absolute and in proportion to the rest of the brain) among species and populations suggests that functional specialization is linked to its size. There is increasing recognition that the cerebellum contributes to cognitive processing and emotional control in addition to its role in motor coordination. However, to what extent cerebellum size reflects variation in these behavioral processes within species remains largely unknown. By using a unique intercross chicken population based on parental lines with high divergence in cerebellum size, we compared the behavior of individuals repeatedly exposed to the same fear test (emergence test) early in life and after sexual maturity (eight trials per age group) with proportional cerebellum size and cerebellum neural density. While proportional cerebellum size did not predict the initial fear response of the individuals (trial 1), it did increasingly predict adult individuals response as the trials progressed. Our results suggest that proportional cerebellum size does not necessarily predict an individual’s fear response, but rather the habituation process to a fearful stimulus. Cerebellum neuronal density did not predict fear behavior in the individuals which suggests that these effects do not result from changes in neuronal density but due to other variables linked to proportional cerebellum size which might underlie fear habituation.