A genomic basis of vocal rhythm in birds.

Vocal rhythm plays a fundamental role in sexual selection and species recognition in birds, but little is known of its genetic basis due to the confounding effect of vocal learning in model systems. Uncovering its genetic basis could facilitate identifying genes potentially important in speciation. Here we investigate the genomic underpinnings of rhythm in vocal non-learning Pogoniulus tinkerbirds using 135 individual whole genomes distributed across a southern African hybrid zone. We find rhythm speed is associated with two genes that are also known to affect human speech, Neurexin-1 and Coenzyme Q8A. Models leveraging ancestry reveal these candidate loci also impact rhythmic stability, a trait linked with motor performance which is an indicator of quality. Character displacement in rhythmic stability suggests possible reinforcement against hybridization, supported by evidence of asymmetric assortative mating in the species producing faster, more stable rhythms. Because rhythm is omnipresent in animal communication, candidate genes identified here may shape vocal rhythm across birds and other vertebrates.

A region of suppressed recombination misleads neoavian phylogenomics.

Genomes are typically mosaics of regions with different evolutionary histories. When speciation events are closely spaced in time, recombination makes the regions sharing the same history small, and the evolutionary history changes rapidly as we move along the genome. When examining rapid radiations such as the early diversification of Neoaves 66 Mya, typically no consistent history is observed across segments exceeding kilobases of the genome. Here, we report an exception. We found that a 21-Mb region in avian genomes, mapped to chicken chromosome 4, shows an extremely strong and discordance-free signal for a history different from that of the inferred species tree. Such a strong discordance-free signal, indicative of suppressed recombination across many millions of base pairs, is not observed elsewhere in the genome for any deep avian relationships. Although long regions with suppressed recombination have been documented in recently diverged species, our results pertain to relationships dating circa 65 Mya. We provide evidence that this strong signal may be due to an ancient rearrangement that blocked recombination and remained polymorphic for several million years prior to fixation. We show that the presence of this region has misled previous phylogenomic efforts with lower taxon sampling, showing the interplay between taxon and locus sampling. We predict that similar ancient rearrangements may confound phylogenetic analyses in other clades, pointing to a need for new analytical models that incorporate the possibility of such events.

Evolution and genetic architecture of sex-limited polymorphism in cuckoos.

Sex-limited polymorphism has evolved in many species including our own. Yet, we lack a detailed understanding of the underlying genetic variation and evolutionary processes at work. The brood parasitic common cuckoo (Cuculus canorus) is a prime example of female-limited color polymorphism, where adult males are monochromatic gray and females exhibit either gray or rufous plumage. This polymorphism has been hypothesized to be governed by negative frequency-dependent selection whereby the rarer female morph is protected against harassment by males or from mobbing by parasitized host species. Here, we show that female plumage dichromatism maps to the female-restricted genome. We further demonstrate that, consistent with balancing selection, ancestry of the rufous phenotype is shared with the likewise female dichromatic sister species, the oriental cuckoo (Cuculus optatus). This study shows that sex-specific polymorphism in trait variation can be resolved by genetic variation residing on a sex-limited chromosome and be maintained across species boundaries.

Complexity of avian evolution revealed by family-level genomes.

Despite tremendous efforts in the past decades, relationships among main avian lineages remain heavily debated without a clear resolution. Discrepancies have been attributed to diversity of species sampled, phylogenetic method and the choice of genomic regions1-3. Here we address these issues by analysing the genomes of 363 bird species4 (218 taxonomic families, 92% of total). Using intergenic regions and coalescent methods, we present a well-supported tree but also a marked degree of discordance. The tree confirms that Neoaves experienced rapid radiation at or near the Cretaceous-Palaeogene boundary. Sufficient loci rather than extensive taxon sampling were more effective in resolving difficult nodes. Remaining recalcitrant nodes involve species that are a challenge to model due to either extreme DNA composition, variable substitution rates, incomplete lineage sorting or complex evolutionary events such as ancient hybridization. Assessment of the effects of different genomic partitions showed high heterogeneity across the genome. We discovered sharp increases in effective population size, substitution rates and relative brain size following the Cretaceous-Palaeogene extinction event, supporting the hypothesis that emerging ecological opportunities catalysed the diversification of modern birds. The resulting phylogenetic estimate offers fresh insights into the rapid radiation of modern birds and provides a taxon-rich backbone tree for future comparative studies.

Single-cell long-read sequencing-based mapping reveals specialized splicing patterns in developing and adult mouse and human brain.

RNA isoforms influence cell identity and function. However, a comprehensive brain isoform map was lacking. We analyze single-cell RNA isoforms across brain regions, cell subtypes, developmental time points and species. For 72% of genes, full-length isoform expression varies along one or more axes. Splicing, transcription start and polyadenylation sites vary strongly between cell types, influence protein architecture and associate with disease-linked variation. Additionally, neurotransmitter transport and synapse turnover genes harbor cell-type variability across anatomical regions. Regulation of cell-type-specific splicing is pronounced in the postnatal day 21-to-postnatal day 28 adolescent transition. Developmental isoform regulation is stronger than regional regulation for the same cell type. Cell-type-specific isoform regulation in mice is mostly maintained in the human hippocampus, allowing extrapolation to the human brain. Conversely, the human brain harbors additional cell-type specificity, suggesting gain-of-function isoforms. Together, this detailed single-cell atlas of full-length isoform regulation across development, anatomical regions and species reveals an unappreciated degree of isoform variability across multiple axes.

A reference genome for the Andean cavefish Trichomycterus rosablanca (Siluriformes, Trichomycteridae): Building genomic resources to study evolution in cave environments.

Animals living in caves are of broad relevance to evolutionary biologists interested in understanding the mechanisms underpinning convergent evolution. In the Eastern Andes of Colombia, populations from at least two distinct clades of Trichomycterus catfishes (Siluriformes) independently colonized cave environments and converged in phenotype by losing their eyes and pigmentation. We are pursuing several research questions using genomics to understand the evolutionary forces and molecular mechanisms responsible for repeated morphological changes in this system. As a foundation for such studies, here we describe a diploid, chromosome-scale, long-read reference genome for Trichomycterus rosablanca, a blind, depigmented species endemic to the karstic system of the department of Santander. The nuclear genome comprises 1 Gb in 27 chromosomes, with a 40.0× HiFi long-read genome coverage having an N50 scaffold of 40.4 Mb and N50 contig of 13.1 Mb, with 96.9% (Eukaryota) and 95.4% (Actinopterygii) universal single-copy orthologs (BUSCO). This assembly provides the first reference genome for the speciose genus Trichomycterus, serving as a key resource for research on the genomics of phenotypic evolution.

A High-Quality Blue Whale Genome, Segmental Duplications, and Historical Demography.

The blue whale, Balaenoptera musculus, is the largest animal known to have ever existed, making it an important case study in longevity and resistance to cancer. To further this and other blue whale-related research, we report a reference-quality, long-read-based genome assembly of this fascinating species. We assembled the genome from PacBio long reads and utilized Illumina/10×, optical maps, and Hi-C data for scaffolding, polishing, and manual curation. We also provided long read RNA-seq data to facilitate the annotation of the assembly by NCBI and Ensembl. Additionally, we annotated both haplotypes using TOGA and measured the genome size by flow cytometry. We then compared the blue whale genome with other cetaceans and artiodactyls, including vaquita (Phocoena sinus), the world's smallest cetacean, to investigate blue whale's unique biological traits. We found a dramatic amplification of several genes in the blue whale genome resulting from a recent burst in segmental duplications, though the possible connection between this amplification and giant body size requires further study. We also discovered sites in the insulin-like growth factor-1 gene correlated with body size in cetaceans. Finally, using our assembly to examine the heterozygosity and historical demography of Pacific and Atlantic blue whale populations, we found that the genomes of both populations are highly heterozygous and that their genetic isolation dates to the last interglacial period. Taken together, these results indicate how a high-quality, annotated blue whale genome will serve as an important resource for biology, evolution, and conservation research.

Problem-solving skills are predicted by technical innovations in the wild and brain size in passerines.

Behavioural innovations can provide key advantages for animals in the wild, especially when ecological conditions change rapidly and unexpectedly. Innovation rates can be compared across taxa by compiling field reports of novel behaviours. Large-scale analyses have shown that innovativeness reduces extinction risk, increases colonization success and is associated with increased brain size and pallial neuron numbers. However, appropriate laboratory measurements of innovativeness, necessary to conduct targeted experimental studies, have not been clearly established, despite decades of speculation on the most suitable assay. Here we implemented a battery of cognitive tasks on 203 birds of 15 passerine species and tested for relationships at the interspecific and intraspecific levels with ecological metrics of innovation and brain size. We found that species better at solving extractive foraging problems had higher technical innovation rates in the wild and larger brains. By contrast, performance on other cognitive tasks often subsumed under the term behavioural flexibility, namely, associative and reversal learning, as well as self-control, were not related to problem-solving, innovation in the wild or brain size. Our study yields robust support for problem-solving as an accurate experimental proxy of innovation and suggests that novel motor solutions are more important than self-control or learning of modified cues in generating technical innovations in the wild.

Chromosome level genome assembly of the Etruscan shrew Suncus etruscus.

Suncus etruscus is one of the world's smallest mammals, with an average body mass of about 2 grams. The Etruscan shrew's small body is accompanied by a very high energy demand and numerous metabolic adaptations. Here we report a chromosome-level genome assembly using PacBio long read sequencing, 10X Genomics linked short reads, optical mapping, and Hi-C linked reads. The assembly is partially phased, with the 2.472 Gbp primary pseudohaplotype and 1.515 Gbp alternate. We manually curated the primary assembly and identified 22 chromosomes, including X and Y sex chromosomes. The NCBI genome annotation pipeline identified 39,091 genes, 19,819 of them protein-coding. We also identified segmental duplications, inferred GO term annotations, and computed orthologs of human and mouse genes. This reference-quality genome will be an important resource for research on mammalian development, metabolism, and body size control.

A chromosome-level genome assembly for the dugong (Dugong dugon).

The dugong (Dugong dugon) is a marine mammal widely distributed throughout the Indo-Pacific and the Red Sea, with a Vulnerable conservation status, and little is known about many of the more peripheral populations, some of which are thought to be close to extinction. We present a de novo high-quality genome assembly for the dugong from an individual belonging to the well-monitored Moreton Bay population in Queensland, Australia. Our assembly uses long-read PacBio HiFi sequencing and Omni-C data following the Vertebrate Genome Project pipeline to reach chromosome-level contiguity (24 chromosome-level scaffolds; 3.16 Gbp) and high completeness (97.9% complete BUSCOs). We observed relatively high genome-wide heterozygosity, which likely reflects historical population abundance before the last interglacial period, approximately 125,000 yr ago. Demographic inference suggests that dugong populations began declining as sea levels fell after the last interglacial period, likely a result of population fragmentation and habitat loss due to the exposure of seagrass meadows. We find no evidence for ongoing recent inbreeding in this individual. However, runs of homozygosity indicate some past inbreeding. Our draft genome assembly will enable range-wide assessments of genetic diversity and adaptation, facilitate effective management of dugong populations, and allow comparative genomics analyses including with other sirenians, the oldest marine mammal lineage.

Chromosome-level genome assembly of chub mackerel (Scomber japonicus) from the Indo-Pacific Ocean.

Chub mackerels (Scomber japonicus) are a migratory marine fish widely distributed in the Indo-Pacific Ocean. They are globally consumed for their high Omega-3 content, but their population is declining due to global warming. Here, we generated the first chromosome-level genome assembly of chub mackerel (fScoJap1) using the Vertebrate Genomes Project assembly pipeline with PacBio HiFi genomic sequencing and Arima Hi-C chromosome contact data. The final assembly is 828.68 Mb with 24 chromosomes, nearly all containing telomeric repeats at their ends. We annotated 31,656 genes and discovered that approximately 2.19% of the genome contained DNA transposon elements repressed within duplicated genes. Analyzing 5-methylcytosine (5mC) modifications using HiFi reads, we observed open/close chromatin patterns at gene promoters, including the FADS2 gene involved in Omega-3 production. This chromosome-level reference genome provides unprecedented opportunities for advancing our knowledge of chub mackerels in biology, industry, and conservation.

A pangenome graph reference of 30 chicken genomes allows genotyping of large and complex structural variants.

BACKGROUND: The red junglefowl, the wild outgroup of domestic chickens, has historically served as a reference for genomic studies of domestic chickens. These studies have provided insight into the etiology of traits of commercial importance. However, the use of a single reference genome does not capture diversity present among modern breeds, many of which have accumulated molecular changes due to drift and selection. While reference-based resequencing is well-suited to cataloging simple variants such as single-nucleotide changes and short insertions and deletions, it is mostly inadequate to discover more complex structural variation in the genome. METHODS: We present a pangenome for the domestic chicken consisting of thirty assemblies of chickens from different breeds and research lines. RESULTS: We demonstrate how this pangenome can be used to catalog structural variants present in modern breeds and untangle complex nested variation. We show that alignment of short reads from 100 diverse wild and domestic chickens to this pangenome reduces reference bias by 38%, which affects downstream genotyping results. This approach also allows for the accurate genotyping of a large and complex pair of structural variants at the K feathering locus using short reads, which would not be possible using a linear reference. CONCLUSIONS: We expect that this new paradigm of genomic reference will allow better pinpointing of exact mutations responsible for specific phenotypes, which will in turn be necessary for breeding chickens that meet new sustainability criteria and are resilient to quickly evolving pathogen threats.

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.

Low mutation rate in epaulette sharks is consistent with a slow rate of evolution in sharks.

Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often acting as apex predators. They are considered a slow-evolving lineage and have been suggested to exhibit exceptionally low cancer rates. These two features could be explained by a low nuclear mutation rate. Here, we provide a direct estimate of the nuclear mutation rate in the epaulette shark (Hemiscyllium ocellatum). We generate a high-quality reference genome, and resequence the whole genomes of parents and nine offspring to detect de novo mutations. Using stringent criteria, we estimate a mutation rate of 7×10-10 per base pair, per generation. This represents one of the lowest directly estimated mutation rates for any vertebrate clade, indicating that this basal vertebrate group is indeed a slowly evolving lineage whose ability to restore genetic diversity following a sustained population bottleneck may be hampered by a low mutation rate.

Prioritizing Endangered Species in Genome Sequencing: Conservation Genomics in Action with the First Platinum-Standard Reference-Quality Genome of the Critically Endangered European Mink Mustela lutreola L., 1761.

The European mink Mustela lutreola (Mustelidae) ranks among the most endangered mammalian species globally, experiencing a rapid and severe decline in population size, density, and distribution. Given the critical need for effective conservation strategies, understanding its genomic characteristics becomes paramount. To address this challenge, the platinum-quality, chromosome-level reference genome assembly for the European mink was successfully generated under the project of the European Mink Centre consortium. Leveraging PacBio HiFi long reads, we obtained a 2586.3 Mbp genome comprising 25 scaffolds, with an N50 length of 154.1 Mbp. Through Hi-C data, we clustered and ordered the majority of the assembly (>99.9%) into 20 chromosomal pseudomolecules, including heterosomes, ranging from 6.8 to 290.1 Mbp. The newly sequenced genome displays a GC base content of 41.9%. Additionally, we successfully assembled the complete mitochondrial genome, spanning 16.6 kbp in length. The assembly achieved a BUSCO (Benchmarking Universal Single-Copy Orthologs) completeness score of 98.2%. This high-quality reference genome serves as a valuable genomic resource for future population genomics studies concerning the European mink and related taxa. Furthermore, the newly assembled genome holds significant potential in addressing key conservation challenges faced by M. lutreola. Its applications encompass potential revision of management units, assessment of captive breeding impacts, resolution of phylogeographic questions, and facilitation of monitoring and evaluating the efficiency and effectiveness of dedicated conservation strategies for the European mink. This species serves as an example that highlights the paramount importance of prioritizing endangered species in genome sequencing projects due to the race against time, which necessitates the comprehensive exploration and characterization of their genomic resources before their populations face extinction.

Songbird species that display more-complex vocal learning are better problem-solvers and have larger brains.

Complex vocal learning, a critical component of human spoken language, has been assumed to be associated with more-advanced cognitive abilities. Tests of this hypothesis between individuals within a species have been inconclusive and have not been done across species. In this work, we measured an array of cognitive skills-namely, problem-solving, associative and reversal learning, and self-control-across 214 individuals of 23 bird species, including 19 wild-caught songbird species, two domesticated songbird species, and two wild-caught vocal nonlearning species. We found that the greater the vocal learning abilities of a species, the better their problem-solving skills and the relatively larger their brains. These conclusions held when controlling for noncognitive variables and phylogeny. Our results support a hypothesis of shared genetic and cognitive mechanisms between vocal learning, problem-solving, and bigger brains in songbirds.

A Chromosome-Level Reference Genome for the Black-Legged Kittiwake (Rissa tridactyla), a Declining Circumpolar Seabird.

Amidst the current biodiversity crisis, the availability of genomic resources for declining species can provide important insights into the factors driving population decline. In the early 1990s, the black-legged kittiwake (Rissa tridactyla), a pelagic gull widely distributed across the arctic, subarctic, and temperate zones, suffered a steep population decline following an abrupt warming of sea surface temperature across its distribution range and is currently listed as Vulnerable by the International Union for the Conservation of Nature. Kittiwakes have long been the focus for field studies of physiology, ecology, and ecotoxicology and are primary indicators of fluctuating ecological conditions in arctic and subarctic marine ecosystems. We present a high-quality chromosome-level reference genome and annotation for the black-legged kittiwake using a combination of Pacific Biosciences HiFi sequencing, Bionano optical maps, Hi-C reads, and RNA-Seq data. The final assembly spans 1.35 Gb across 32 chromosomes, with a scaffold N50 of 88.21 Mb and a BUSCO completeness of 97.4%. This genome assembly substantially improves the quality of a previous draft genome, showing an approximately 5× increase in contiguity and a more complete annotation. Using this new chromosome-level reference genome and three more chromosome-level assemblies of Charadriiformes, we uncover several lineage-specific chromosome fusions and fissions, but find no shared rearrangements, suggesting that interchromosomal rearrangements have been commonplace throughout the diversification of Charadriiformes. This new high-quality genome assembly will enable population genomic, transcriptomic, and phenotype-genotype association studies in a widely studied sentinel species, which may provide important insights into the impacts of global change on marine systems.

Species-wide genomics of kakapo provides tools to accelerate recovery.

The kakapo is a critically endangered, intensively managed, long-lived nocturnal parrot endemic to Aotearoa New Zealand. We generated and analysed whole-genome sequence data for nearly all individuals living in early 2018 (169 individuals) to generate a high-quality species-wide genetic variant callset. We leverage extensive long-term metadata to quantify genome-wide diversity of the species over time and present new approaches using probabilistic programming, combined with a phenotype dataset spanning five decades, to disentangle phenotypic variance into environmental and genetic effects while quantifying uncertainty in small populations. We find associations for growth, disease susceptibility, clutch size and egg fertility within genic regions previously shown to influence these traits in other species. Finally, we generate breeding values to predict phenotype and illustrate that active management over the past 45 years has maintained both genome-wide diversity and diversity in breeding values and, hence, evolutionary potential. We provide new pathways for informing future conservation management decisions for kakapo, including prioritizing individuals for translocation and monitoring individuals with poor growth or high disease risk. Overall, by explicitly addressing the challenge of the small sample size, we provide a template for the inclusion of genomic data that will be transformational for species recovery efforts around the globe.

Scalable, accessible, and reproducible reference genome assembly and evaluation in Galaxy.

Improvements in genome sequencing and assembly are enabling high-quality reference genomes for all species. However, the assembly process is still laborious, computationally and technically demanding, lacks standards for reproducibility, and is not readily scalable. Here we present the latest Vertebrate Genomes Project assembly pipeline and demonstrate that it delivers high-quality reference genomes at scale across a set of vertebrate species arising over the last ~500 million years. The pipeline is versatile and combines PacBio HiFi long-reads and Hi-C-based haplotype phasing in a new graph-based paradigm. Standardized quality control is performed automatically to troubleshoot assembly issues and assess biological complexities. We make the pipeline freely accessible through Galaxy, accommodating researchers even without local computational resources and enhanced reproducibility by democratizing the training and assembly process. We demonstrate the flexibility and reliability of the pipeline by assembling reference genomes for 51 vertebrate species from major taxonomic groups (fish, amphibians, reptiles, birds, and mammals).