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.

High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae).

BACKGROUND: Morphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics. RESULTS: The genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function. CONCLUSIONS: Our two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

A novel nematode species from the Siberian permafrost shares adaptive mechanisms for cryptobiotic survival with C. elegans dauer larva.

Some organisms in nature have developed the ability to enter a state of suspended metabolism called cryptobiosis when environmental conditions are unfavorable. This state-transition requires execution of a combination of genetic and biochemical pathways that enable the organism to survive for prolonged periods. Recently, nematode individuals have been reanimated from Siberian permafrost after remaining in cryptobiosis. Preliminary analysis indicates that these nematodes belong to the genera Panagrolaimus and Plectus. Here, we present precise radiocarbon dating indicating that the Panagrolaimus individuals have remained in cryptobiosis since the late Pleistocene (~46,000 years). Phylogenetic inference based on our genome assembly and a detailed morphological analysis demonstrate that they belong to an undescribed species, which we named Panagrolaimus kolymaensis. Comparative genome analysis revealed that the molecular toolkit for cryptobiosis in P. kolymaensis and in C. elegans is partly orthologous. We show that biochemical mechanisms employed by these two species to survive desiccation and freezing under laboratory conditions are similar. Our experimental evidence also reveals that C. elegans dauer larvae can remain viable for longer periods in suspended animation than previously reported. Altogether, our findings demonstrate that nematodes evolved mechanisms potentially allowing them to suspend life over geological time scales.

Expanded olfactory system in ray-finned fishes capable of terrestrial exploration.

BACKGROUND: Smell abilities differ greatly among vertebrate species due to distinct sensory needs, with exceptional variability reported in the number of olfactory genes and the size of the odour-processing regions of the brain. However, key environmental factors shaping genomic and phenotypic changes linked to the olfactory system remain difficult to identify at macroevolutionary scales. Here, we investigate the association between diverse ecological traits and the number of olfactory chemoreceptors in approximately two hundred ray-finned fishes. RESULTS: We found independent expansions producing large gene repertoires in several lineages of nocturnal amphibious fishes, generally able to perform active terrestrial exploration. We reinforced this finding with on-purpose genomic and transcriptomic analysis of Channallabes apus, a catfish species from a clade with chemosensory-based aerial orientation. Furthermore, we also detected an augmented information-processing capacity in the olfactory bulb of nocturnal amphibious fishes by estimating the number of cells contained in this brain region in twenty-four actinopterygian species. CONCLUSIONS: Overall, we report a convergent genomic and phenotypic magnification of the olfactory system in nocturnal amphibious fishes. This finding suggests the possibility of an analogous evolutionary event in fish-like tetrapod ancestors during the first steps of the water-to-land transition, favouring terrestrial adaptation through enhanced aerial orientation.

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.

Loss of a gluconeogenic muscle enzyme contributed to adaptive metabolic traits in hummingbirds.

Hummingbirds possess distinct metabolic adaptations to fuel their energy-demanding hovering flight, but the underlying genomic changes are largely unknown. Here, we generated a chromosome-level genome assembly of the long-tailed hermit and screened for genes that have been specifically inactivated in the ancestral hummingbird lineage. We discovered that FBP2 (fructose-bisphosphatase 2), which encodes a gluconeogenic muscle enzyme, was lost during a time period when hovering flight evolved. We show that FBP2 knockdown in an avian muscle cell line up-regulates glycolysis and enhances mitochondrial respiration, coincident with an increased mitochondria number. Furthermore, genes involved in mitochondrial respiration and organization have up-regulated expression in hummingbird flight muscle. Together, these results suggest that FBP2 loss was likely a key step in the evolution of metabolic muscle adaptations required for true hovering flight.

Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing.

BACKGROUND: Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. RESULTS: We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20-25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20-25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. CONCLUSION: We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

Longer metaphase and fewer chromosome segregation errors in modern human than Neanderthal brain development.

Since the ancestors of modern humans separated from those of Neanderthals, around 100 amino acid substitutions spread to essentially all modern humans. The biological significance of these changes is largely unknown. Here, we examine all six such amino acid substitutions in three proteins known to have key roles in kinetochore function and chromosome segregation and to be highly expressed in the stem cells of the developing neocortex. When we introduce these modern human-specific substitutions in mice, three substitutions in two of these proteins, KIF18a and KNL1, cause metaphase prolongation and fewer chromosome segregation errors in apical progenitors of the developing neocortex. Conversely, the ancestral substitutions cause shorter metaphase length and more chromosome segregation errors in human brain organoids, similar to what we find in chimpanzee organoids. These results imply that the fidelity of chromosome segregation during neocortex development improved in modern humans after their divergence from Neanderthals.

Emergence and spread of a sub-lineage of SARS-CoV-2 Alpha variant B.1.1.7 in Europe, and with further evolution of spike mutation accumulations shared with the Beta and Gamma variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution plays a significant role in shaping the dynamics of the coronavirus disease 2019 pandemic. To monitor the evolution of SARS-CoV-2 variants, through international collaborations, we performed genomic epidemiology analyses on a weekly basis with SARS-CoV-2 samples collected from a border region between Germany, Poland, and the Czech Republic in a global background. For identified virus mutant variants, active viruses were isolated and functional evaluations were performed to test their replication fitness and neutralization sensitivity against vaccine-elicited serum neutralizing antibodies. Thereby we identified a new B.1.1.7 sub-lineage carrying additional mutations of nucleoprotein G204P and open-reading-frame-8 K68stop. Of note, this B.1.1.7 sub-lineage is the predominant B.1.1.7 variant in several European countries such as Czech Republic, Austria, and Slovakia. The earliest samples belonging to this sub-lineage were detected in November 2020 in a few countries in the European continent, but not in the UK. We have also detected its further evolution with extra spike mutations D138Y and A701V, which are signature mutations shared with the Gamma and Beta variants, respectively. Antibody neutralization assay of virus variant isolations has revealed that the variant with extra spike mutations is 3.2-fold less sensitive to vaccine-elicited antibodies as compared to the other B.1.1.7 variants tested, indicating potential for immune evasion, but it also exhibited reduced replication fitness, suggesting lower transmissibility. The wide spread of this B.1.1.7 sub-lineage was related to the pandemic waves in early 2021 in various European countries. These findings about the emergence, spread, evolution, infection, and transmission abilities of this B.1.1.7 sub-lineage add to our understanding about the pandemic development in Europe and highlight the importance of international collaboration on virus mutant surveillance.

Genome biology of the darkedged splitfin, Girardinichthys multiradiatus, and the evolution of sex chromosomes and placentation.

Viviparity evolved independently about 150 times in vertebrates and more than 20 times in fish. Several lineages added to the protection of the embryo inside the body of the mother, the provisioning of nutrients, and physiological exchange. This often led to the evolution of a placenta. Among fish, one of the most complex systems serving the function of the placenta is the embryonal trophotaenia/ovarian luminal epithelium of the goodeid fishes. For a better understanding of this feature and others of this group of fishes, high-quality genomic resources are essential. We have sequenced the genome of the darkedged splitfin, Girardinichthys multiradiatus The assembly is chromosome level and includes the X and Y Chromosomes. A large male-specific region on the Y was identified covering 80% of Chromosome 20, allowing some first inferences on the recent origin and a candidate male sex determining gene. Genome-wide transcriptomics uncovered sex-specific differences in brain gene expression with an enrichment for neurosteroidogenesis and testis genes in males. The expression signatures of the splitfin embryonal and maternal placenta showed overlap with homologous tissues including human placenta, the ovarian follicle epithelium of matrotrophic poeciliid fish species and the brood pouch epithelium of the seahorse. Our comparative analyses on the evolution of embryonal and maternal placenta indicate that the evolutionary novelty of maternal provisioning development repeatedly made use of genes that already had the same function in other tissues. In this way, preexisting modules are assembled and repurposed to provide the molecular changes for this novel trait.

Convergent and lineage-specific genomic differences in limb regulatory elements in limbless reptile lineages.

Loss of limbs evolved many times in squamate reptiles. Here we investigated the genomic basis of convergent limb loss in reptiles. We sequenced the genomes of a closely related pair of limbless-limbed gymnophthalmid lizards and performed a comparative genomic analysis including five snakes and the limbless glass lizard. Our analysis of these three independent limbless lineages revealed that signatures of shared sequence or transcription factor binding site divergence in individual limb regulatory elements are generally rare. Instead, shared divergence occurs more often at the level of signaling pathways, involving different regulatory elements associated with the same limb genes (such as Hand2 or Hox) and/or patterning mechanisms (such as Shh signaling). Interestingly, although snakes are known to have mutations in the Shh ZRS limb enhancer, this enhancer lacks relevant mutations in limbless lizards. Thus, different mechanisms could contribute to limb loss, and there are likely multiple evolutionary paths to limblessness in reptiles.

Deletion of lrrk2 causes early developmental abnormalities and age-dependent increase of monoamine catabolism in the zebrafish brain.

LRRK2 gain-of-function is considered a major cause of Parkinson's disease (PD) in humans. However, pathogenicity of LRRK2 loss-of-function in animal models is controversial. Here we show that deletion of the entire zebrafish lrrk2 locus elicits a pleomorphic transient brain phenotype in maternal-zygotic mutant embryos (mzLrrk2). In contrast to lrrk2, the paralog gene lrrk1 is virtually not expressed in the brain of both wild-type and mzLrrk2 fish at different developmental stages. Notably, we found reduced catecholaminergic neurons, the main target of PD, in specific cell populations in the brains of mzLrrk2 larvae, but not adult fish. Strikingly, age-dependent accumulation of monoamine oxidase (MAO)-dependent catabolic signatures within mzLrrk2 brains revealed a previously undescribed interaction between LRRK2 and MAO biological activities. Our results highlight mzLrrk2 zebrafish as a tractable tool to study LRRK2 loss-of-function in vivo, and suggest a link between LRRK2 and MAO, potentially of relevance in the prodromic stages of PD.

Phylogenetic analysis of SARS-CoV-2 lineage development across the first and second waves in Eastern Germany in 2020: insights into the cause of the second wave.

In Germany, Eastern regions had a mild first wave of coronavirus disease 2019 (COVID-19) from March to May 2020, but were badly hit by a second wave later in autumn and winter. It is unknown how the second wave was initiated and developed in Eastern Germany where the number of COVID-19 cases was close to zero in June and July 2020. We used genomic epidemiology to investigate the dynamic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineage development across the first and second waves in Eastern Germany. With detailed phylogenetic analyses we could show that SARS-CoV-2 lineages prevalent in the first and second waves in Eastern Germany were different, with several new variants including four predominant lineages in the second wave, having been introduced into Eastern Germany between August and October 2020. The results indicate that the major driving force behind the second wave was the introduction of new variants.

Low Threshold for Cutaneous Allergen Sensitization but No Spontaneous Dermatitis or Atopy in FLG-Deficient Mice.

Loss of FLG causes ichthyosis vulgaris. Reduced FLG expression compromises epidermal barrier function and is associated with atopic dermatitis, allergy, and asthma. The flaky tail mouse harbors two mutations that affect the skin barrier, Flgft, resulting in hypomorphic FLG expression, and Tmem79ma, inactivating TMEM79. Mice defective only for TMEM79 featured dermatitis and systemic atopy, but also Flgft/ft BALB/c congenic mice developed eczema, high IgE, and spontaneous asthma, suggesting that FLG protects from atopy. In contrast, a targeted Flg-knockout mutation backcrossed to BALB/c did not result in dermatitis or atopy. To resolve this discrepancy, we generated FLG-deficient mice on pure BALB/c background by inactivating Flg in BALB/c embryos. These mice feature an ichthyosis phenotype, barrier defect, and facilitated percutaneous sensitization. However, they do not develop dermatitis or atopy. Whole-genome sequencing of the atopic Flgft BALB/c congenics revealed that they were homozygous for the atopy-causing Tmem79matted mutation. In summary, we show that FLG deficiency does not cause atopy in mice, in line with lack of atopic disease in a fraction of patients with ichthyosis vulgaris carrying two Flg null alleles. However, the absence of FLG likely promotes and modulates dermatitis caused by other genetic barrier defects.

Complete vertebrate mitogenomes reveal widespread repeats and gene duplications.

BACKGROUND: Modern sequencing technologies should make the assembly of the relatively small mitochondrial genomes an easy undertaking. However, few tools exist that address mitochondrial assembly directly. RESULTS: As part of the Vertebrate Genomes Project (VGP) we develop mitoVGP, a fully automated pipeline for similarity-based identification of mitochondrial reads and de novo assembly of mitochondrial genomes that incorporates both long (> 10 kbp, PacBio or Nanopore) and short (100-300 bp, Illumina) reads. Our pipeline leads to successful complete mitogenome assemblies of 100 vertebrate species of the VGP. We observe that tissue type and library size selection have considerable impact on mitogenome sequencing and assembly. Comparing our assemblies to purportedly complete reference mitogenomes based on short-read sequencing, we identify errors, missing sequences, and incomplete genes in those references, particularly in repetitive regions. Our assemblies also identify novel gene region duplications. The presence of repeats and duplications in over half of the species herein assembled indicates that their occurrence is a principle of mitochondrial structure rather than an exception, shedding new light on mitochondrial genome evolution and organization. CONCLUSIONS: Our results indicate that even in the "simple" case of vertebrate mitogenomes the completeness of many currently available reference sequences can be further improved, and caution should be exercised before claiming the complete assembly of a mitogenome, particularly from short reads alone.

Towards complete and error-free genome assemblies of all vertebrate species.

High-quality and complete reference genome assemblies are fundamental for the application of genomics to biology, disease, and biodiversity conservation. However, such assemblies are available for only a few non-microbial species1-4. To address this issue, the international Genome 10K (G10K) consortium5,6 has worked over a five-year period to evaluate and develop cost-effective methods for assembling highly accurate and nearly complete reference genomes. Here we present lessons learned from generating assemblies for 16 species that represent six major vertebrate lineages. We confirm that long-read sequencing technologies are essential for maximizing genome quality, and that unresolved complex repeats and haplotype heterozygosity are major sources of assembly error when not handled correctly. Our assemblies correct substantial errors, add missing sequence in some of the best historical reference genomes, and reveal biological discoveries. These include the identification of many false gene duplications, increases in gene sizes, chromosome rearrangements that are specific to lineages, a repeated independent chromosome breakpoint in bat genomes, and a canonical GC-rich pattern in protein-coding genes and their regulatory regions. Adopting these lessons, we have embarked on the Vertebrate Genomes Project (VGP), an international effort to generate high-quality, complete reference genomes for all of the roughly 70,000 extant vertebrate species and to help to enable a new era of discovery across the life sciences.

Mutations in the splicing regulator Prp31 lead to retinal degeneration in Drosophila.

Retinitis pigmentosa (RP) is a clinically heterogeneous disease affecting 1.6 million people worldwide. The second-largest group of genes causing autosomal dominant RP in human encodes regulators of the splicing machinery. Yet, how defects in splicing factor genes are linked to the aetiology of the disease remains largely elusive. To explore possible mechanisms underlying retinal degeneration caused by mutations in regulators of the splicing machinery, we induced mutations in Drosophila Prp31, the orthologue of human PRPF31, mutations in which are associated with RP11. Flies heterozygous mutant for Prp31 are viable and develop normal eyes and retina. However, photoreceptors degenerate under light stress, thus resembling the human disease phenotype. Degeneration is associated with increased accumulation of the visual pigment rhodopsin 1 and increased mRNA levels of twinfilin, a gene associated with rhodopsin trafficking. Reducing rhodopsin levels by raising animals in a carotenoid-free medium not only attenuates rhodopsin accumulation, but also retinal degeneration. Given a similar importance of proper rhodopsin trafficking for photoreceptor homeostasis in human, results obtained in flies presented here will also contribute to further unravel molecular mechanisms underlying the human disease.This paper has an associated First Person interview with the co-first authors of the article.

Six reference-quality genomes reveal evolution of bat adaptations.

Bats possess extraordinary adaptations, including flight, echolocation, extreme longevity and unique immunity. High-quality genomes are crucial for understanding the molecular basis and evolution of these traits. Here we incorporated long-read sequencing and state-of-the-art scaffolding protocols1 to generate, to our knowledge, the first reference-quality genomes of six bat species (Rhinolophus ferrumequinum, Rousettus aegyptiacus, Phyllostomus discolor, Myotis myotis, Pipistrellus kuhlii and Molossus molossus). We integrated gene projections from our 'Tool to infer Orthologs from Genome Alignments' (TOGA) software with de novo and homology gene predictions as well as short- and long-read transcriptomics to generate highly complete gene annotations. To resolve the phylogenetic position of bats within Laurasiatheria, we applied several phylogenetic methods to comprehensive sets of orthologous protein-coding and noncoding regions of the genome, and identified a basal origin for bats within Scrotifera. Our genome-wide screens revealed positive selection on hearing-related genes in the ancestral branch of bats, which is indicative of laryngeal echolocation being an ancestral trait in this clade. We found selection and loss of immunity-related genes (including pro-inflammatory NF-κB regulators) and expansions of anti-viral APOBEC3 genes, which highlights molecular mechanisms that may contribute to the exceptional immunity of bats. Genomic integrations of diverse viruses provide a genomic record of historical tolerance to viral infection in bats. Finally, we found and experimentally validated bat-specific variation in microRNAs, which may regulate bat-specific gene-expression programs. Our reference-quality bat genomes provide the resources required to uncover and validate the genomic basis of adaptations of bats, and stimulate new avenues of research that are directly relevant to human health and disease1.

The round goby genome provides insights into mechanisms that may facilitate biological invasions.

BACKGROUND: The invasive benthic round goby (Neogobius melanostomus) is the most successful temperate invasive fish and has spread in aquatic ecosystems on both sides of the Atlantic. Invasive species constitute powerful in situ experimental systems to study fast adaptation and directional selection on short ecological timescales and present promising case studies to understand factors involved the impressive ability of some species to colonize novel environments. We seize the unique opportunity presented by the round goby invasion to study genomic substrates potentially involved in colonization success. RESULTS: We report a highly contiguous long-read-based genome and analyze gene families that we hypothesize to relate to the ability of these fish to deal with novel environments. The analyses provide novel insights from the large evolutionary scale to the small species-specific scale. We describe expansions in specific cytochrome P450 enzymes, a remarkably diverse innate immune system, an ancient duplication in red light vision accompanied by red skin fluorescence, evolutionary patterns of epigenetic regulators, and the presence of osmoregulatory genes that may have contributed to the round goby's capacity to invade cold and salty waters. A recurring theme across all analyzed gene families is gene expansions. CONCLUSIONS: The expanded innate immune system of round goby may potentially contribute to its ability to colonize novel areas. Since other gene families also feature copy number expansions in the round goby, and since other Gobiidae also feature fascinating environmental adaptations and are excellent colonizers, further long-read genome approaches across the goby family may reveal whether gene copy number expansions are more generally related to the ability to conquer new habitats in Gobiidae or in fish.