Platypus and echidna genomes reveal mammalian biology and evolution.

Egg-laying mammals (monotremes) are the only extant mammalian outgroup to therians (marsupial and eutherian animals) and provide key insights into mammalian evolution1,2. Here we generate and analyse reference genomes of the platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus), which represent the only two extant monotreme lineages. The nearly complete platypus genome assembly has anchored almost the entire genome onto chromosomes, markedly improving the genome continuity and gene annotation. Together with our echidna sequence, the genomes of the two species allow us to detect the ancestral and lineage-specific genomic changes that shape both monotreme and mammalian evolution. We provide evidence that the monotreme sex chromosome complex originated from an ancestral chromosome ring configuration. The formation of such a unique chromosome complex may have been facilitated by the unusually extensive interactions between the multi-X and multi-Y chromosomes that are shared by the autosomal homologues in humans. Further comparative genomic analyses unravel marked differences between monotremes and therians in haptoglobin genes, lactation genes and chemosensory receptor genes for smell and taste that underlie the ecological adaptation of monotremes.

Divergent sensory and immune gene evolution in sea turtles with contrasting demographic and life histories.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

Caecilian Genomes Reveal the Molecular Basis of Adaptation and Convergent Evolution of Limblessness in Snakes and Caecilians.

We present genome sequences for the caecilians Geotrypetes seraphini (3.8 Gb) and Microcaecilia unicolor (4.7 Gb), representatives of a limbless, mostly soil-dwelling amphibian clade with reduced eyes, and unique putatively chemosensory tentacles. More than 69% of both genomes are composed of repeats, with retrotransposons being the most abundant. We identify 1,150 orthogroups that are unique to caecilians and enriched for functions in olfaction and detection of chemical signals. There are 379 orthogroups with signatures of positive selection on caecilian lineages with roles in organ development and morphogenesis, sensory perception, and immunity amongst others. We discover that caecilian genomes are missing the zone of polarizing activity regulatorysequence (ZRS) enhancer of Sonic Hedgehog which is also mutated in snakes. In vivo deletions have shown ZRS is required for limb development in mice, thus, revealing a shared molecular target implicated in the independent evolution of limblessness in snakes and caecilians.

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 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.

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.

Chromosome-scale Genome Assembly of the Rough Periwinkle Littorina saxatilis.

The intertidal gastropod Littorina saxatilis is a model system to study speciation and local adaptation. The repeated occurrence of distinct ecotypes showing different levels of genetic divergence makes L. saxatilis particularly suited to study different stages of the speciation continuum in the same lineage. A major finding is the presence of several large chromosomal inversions associated with the divergence of ecotypes and, specifically, the species offers a system to study the role of inversions in this divergence. The genome of L. saxatilis is 1.35 Gb and composed of 17 chromosomes. The first reference genome of the species was assembled using Illumina data, was highly fragmented (N50 of 44 kb), and was quite incomplete, with a BUSCO completeness of 80.1% on the Metazoan dataset. A linkage map of one full-sibling family enabled the placement of 587 Mbp of the genome into 17 linkage groups corresponding to the haploid number of chromosomes, but the fragmented nature of this reference genome limited the understanding of the interplay between divergent selection and gene flow during ecotype formation. Here, we present a newly generated reference genome that is highly contiguous, with a N50 of 67 Mb and 90.4% of the total assembly length placed in 17 super-scaffolds. It is also highly complete with a BUSCO completeness of 94.1% of the Metazoa dataset. This new reference will allow for investigations into the genomic regions implicated in ecotype formation as well as better characterization of the inversions and their role in speciation.

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.

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.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles gambiae, Giles, 1902, Ifakara strain.

We present a genome assembly from an individual female Anopheles gambiae (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), Ifakara strain. The genome sequence is 264 megabases in span. Most of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

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.

Genomics of cold adaptations in the Antarctic notothenioid fish radiation.

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.

An improved germline genome assembly for the sea lamprey Petromyzon marinus illuminates the evolution of germline-specific chromosomes.

Programmed DNA loss is a gene silencing mechanism that is employed by several vertebrate and nonvertebrate lineages, including all living jawless vertebrates and songbirds. Reconstructing the evolution of somatically eliminated (germline-specific) sequences in these species has proven challenging due to a high content of repeats and gene duplications in eliminated sequences and a corresponding lack of highly accurate and contiguous assemblies for these regions. Here, we present an improved assembly of the sea lamprey (Petromyzon marinus) genome that was generated using recently standardized methods that increase the contiguity and accuracy of vertebrate genome assemblies. This assembly resolves highly contiguous, somatically retained chromosomes and at least one germline-specific chromosome, permitting new analyses that reconstruct the timing, mode, and repercussions of recruitment of genes to the germline-specific fraction. These analyses reveal major roles of interchromosomal segmental duplication, intrachromosomal duplication, and positive selection for germline functions in the long-term evolution of germline-specific chromosomes.

A chromosomal reference genome sequence for the malaria mosquito, Anopheles moucheti, Evans, 1925.

We present a genome assembly from an individual male Anopheles moucheti (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae), from a wild population in Cameroon. The genome sequence is 271 megabases in span. The majority of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.5 kilobases in length.

Chromosome-level Genome Assembly of Acanthopagrus latus Provides Insights into Salinity Stress Adaptation of Sparidae.

The yellowfin seabream, Acanthopagrus latus, is widely distributed throughout the Indo-West Pacific. This species, as a euryhaline Sparidae fish, inhabits in coastal environments with large and frequent salinity fluctuation. So the A. latus can be considered as an ideal species for elucidating the evolutionary mechanism of salinity stress adaption on teleost fish species. Here, a chromosome-scale assembly of A. latus was obtained with PacBio and Hi-C hybrid sequencing strategy. The final assembly genome of A. latus is 685.14 Mbp. The values of contig N50 and scaffold N50 are 14.88 Mbp and 30.72 Mbp, respectively. 29,227 genes were successfully predicted for A. latus in total. Then, the comparative genomics and phylogenetic analysis were employed for investigating the different osmoregulation strategies of salinity stress adaption on multiple whole genome scale of Sparidae species. The highly accurate chromosomal information provides the important genome resources for understanding the osmoregulation evolutionary pattern of the euryhaline Sparidae species.

The genome sequence of the malaria mosquito, Anopheles funestus, Giles, 1900.

We present a genome assembly from an individual female Anopheles funestus (the malaria mosquito; Arthropoda; Insecta; Diptera; Culicidae). The genome sequence is 251 megabases in span. The majority of the assembly is scaffolded into three chromosomal pseudomolecules with the X sex chromosome assembled. The complete mitochondrial genome was also assembled and is 15.4 kilobases in length.

Significantly improving the quality of genome assemblies through curation.

Genome sequence assemblies provide the basis for our understanding of biology. Generating error-free assemblies is therefore the ultimate, but sadly still unachieved goal of a multitude of research projects. Despite the ever-advancing improvements in data generation, assembly algorithms and pipelines, no automated approach has so far reliably generated near error-free genome assemblies for eukaryotes. Whilst working towards improved datasets and fully automated pipelines, assembly evaluation and curation is actively used to bridge this shortcoming and significantly reduce the number of assembly errors. In addition to this increase in product value, the insights gained from assembly curation are fed back into the automated assembly strategy and contribute to notable improvements in genome assembly quality. We describe our tried and tested approach for assembly curation using gEVAL, the genome evaluation browser. We outline the procedures applied to genome curation using gEVAL and also our recommendations for assembly curation in a gEVAL-independent context to facilitate the uptake of genome curation in the wider community.

A high-quality, chromosome-level genome assembly of the Black Soldier Fly (Hermetia illucens L.).

Hermetia illucens L. (Diptera: Stratiomyidae), the Black Soldier Fly (BSF) is an increasingly important species for bioconversion of organic material into animal feed. We generated a high-quality chromosome-scale genome assembly of the BSF using Pacific Bioscience, 10X Genomics linked read and high-throughput chromosome conformation capture sequencing technology. Scaffolding the final assembly with Hi-C data produced a highly contiguous 1.01 Gb genome with 99.75% of scaffolds assembled into pseudochromosomes representing seven chromosomes with 16.01 Mb contig and 180.46 Mb scaffold N50 values. The highly complete genome obtained a Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness of 98.6%. We masked 67.32% of the genome as repetitive sequences and annotated a total of 16,478 protein-coding genes using the BRAKER2 pipeline. We analyzed an established lab population to investigate the genomic variation and architecture of the BSF revealing six autosomes and an X chromosome. Additionally, we estimated the inbreeding coefficient (1.9%) of the lab population by assessing runs of homozygosity. This provided evidence for inbreeding events including long runs of homozygosity on chromosome 5. The release of this novel chromosome-scale BSF genome assembly will provide an improved resource for further genomic studies, functional characterization of genes of interest and genetic modification of this economically important species.

The complete genome sequence of Eimeria tenella (Tyzzer 1929), a common gut parasite of chickens.

We present a genome assembly from a clonal population of Eimeria tenella Houghton parasites (Apicomplexa; Conoidasida; Eucoccidiorida; Eimeriidae). The genome sequence is 53.25 megabases in span. The entire assembly is scaffolded into 15 chromosomal pseudomolecules, with complete mitochondrion and apicoplast organellar genomes also present.

The genome sequence of the European water vole, Arvicola amphibius Linnaeus 1758.

We present a genome assembly from an individual male Arvicola amphibius (the European water vole; Chordata; Mammalia; Rodentia; Cricetidae). The genome sequence is 2.30 gigabases in span. The majority of the assembly is scaffolded into 18 chromosomal pseudomolecules, including the X sex chromosome. Gene annotation of this assembly on Ensembl has identified 21,394 protein coding genes.