The tuatara genome reveals ancient features of amniote evolution.

The tuatara (Sphenodon punctatus)-the only living member of the reptilian order Rhynchocephalia (Sphenodontia), once widespread across Gondwana1,2-is an iconic species that is endemic to New Zealand2,3. A key link to the now-extinct stem reptiles (from which dinosaurs, modern reptiles, birds and mammals evolved), the tuatara provides key insights into the ancestral amniotes2,4. Here we analyse the genome of the tuatara, which-at approximately 5 Gb-is among the largest of the vertebrate genomes yet assembled. Our analyses of this genome, along with comparisons with other vertebrate genomes, reinforce the uniqueness of the tuatara. Phylogenetic analyses indicate that the tuatara lineage diverged from that of snakes and lizards around 250 million years ago. This lineage also shows moderate rates of molecular evolution, with instances of punctuated evolution. Our genome sequence analysis identifies expansions of proteins, non-protein-coding RNA families and repeat elements, the latter of which show an amalgam of reptilian and mammalian features. The sequencing of the tuatara genome provides a valuable resource for deep comparative analyses of tetrapods, as well as for tuatara biology and conservation. Our study also provides important insights into both the technical challenges and the cultural obligations that are associated with genome sequencing.

Publisher Correction: The tuatara genome reveals ancient features of amniote evolution.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Genomic Perspective on the Evolutionary Diversification of Turtles.

To examine phylogenetic heterogeneity in turtle evolution, we collected thousands of high-confidence single-copy orthologs from 19 genome assemblies representative of extant turtle diversity and estimated a phylogeny with multispecies coalescent and concatenated partitioned methods. We also collected next-generation sequences from 26 turtle species and assembled millions of biallelic markers to reconstruct phylogenies based on annotated regions from the western painted turtle (Chrysemys picta bellii) genome (coding regions, introns, untranslated regions, intergenic, and others). We then measured gene tree-species tree discordance, as well as gene and site heterogeneity at each node in the inferred trees, and tested for temporal patterns in phylogenomic conflict across turtle evolution. We found strong and consistent support for all bifurcations in the inferred turtle species phylogenies. However, a number of genes, sites, and genomic features supported alternate relationships between turtle taxa. Our results suggest that gene tree-species tree discordance in these data sets is likely driven by population-level processes such as incomplete lineage sorting. We found very little effect of substitutional saturation on species tree topologies, and no clear phylogenetic patterns in codon usage bias and compositional heterogeneity. There was no correlation between gene and site concordance, node age, and DNA substitution rate across most annotated genomic regions. Our study demonstrates that heterogeneity is to be expected even in well-resolved clades such as turtles, and that future phylogenomic studies should aim to sample as much of the genome as possible in order to obtain accurate phylogenies for assessing conservation priorities in turtles. [Discordance; genomes; phylogeny; turtles.].

Elephant Genomes Reveal Accelerated Evolution in Mechanisms Underlying Disease Defenses.

Disease susceptibility and resistance are important factors for the conservation of endangered species, including elephants. We analyzed pathology data from 26 zoos and report that Asian elephants have increased neoplasia and malignancy prevalence compared with African bush elephants. This is consistent with observed higher susceptibility to tuberculosis and elephant endotheliotropic herpesvirus (EEHV) in Asian elephants. To investigate genetic mechanisms underlying disease resistance, including differential responses between species, among other elephant traits, we sequenced multiple elephant genomes. We report a draft assembly for an Asian elephant, and defined 862 and 1,017 conserved potential regulatory elements in Asian and African bush elephants, respectively. In the genomes of both elephant species, conserved elements were significantly enriched with genes differentially expressed between the species. In Asian elephants, these putative regulatory regions were involved in immunity pathways including tumor-necrosis factor, which plays an important role in EEHV response. Genomic sequences of African bush, forest, and Asian elephant genomes revealed extensive sequence conservation at TP53 retrogene loci across three species, which may be related to TP53 functionality in elephant cancer resistance. Positive selection scans revealed outlier genes related to additional elephant traits. Our study suggests that gene regulation plays an important role in the differential inflammatory response of Asian and African elephants, leading to increased infectious disease and cancer susceptibility in Asian elephants. These genomic discoveries can inform future functional and translational studies aimed at identifying effective treatment approaches for ill elephants, which may improve conservation.

The origins and evolution of chromosomes, dosage compensation, and mechanisms underlying venom regulation in snakes.

Here we use a chromosome-level genome assembly of a prairie rattlesnake (Crotalus viridis), together with Hi-C, RNA-seq, and whole-genome resequencing data, to study key features of genome biology and evolution in reptiles. We identify the rattlesnake Z Chromosome, including the recombining pseudoautosomal region, and find evidence for partial dosage compensation driven by an evolutionary accumulation of a female-biased up-regulation mechanism. Comparative analyses with other amniotes provide new insight into the origins, structure, and function of reptile microchromosomes, which we demonstrate have markedly different structure and function compared to macrochromosomes. Snake microchromosomes are also enriched for venom genes, which we show have evolved through multiple tandem duplication events in multiple gene families. By overlaying chromatin structure information and gene expression data, we find evidence for venom gene-specific chromatin contact domains and identify how chromatin structure guides precise expression of multiple venom gene families. Further, we find evidence for venom gland-specific transcription factor activity and characterize a complement of mechanisms underlying venom production and regulation. Our findings reveal novel and fundamental features of reptile genome biology, provide insight into the regulation of snake venom, and broadly highlight the biological insight enabled by chromosome-level genome assemblies.

The Evolution of Human Cancer Gene Duplications across Mammals.

Cancer is caused by genetic alterations that affect cellular fitness, and multicellular organisms have evolved mechanisms to suppress cancer such as cell cycle checkpoints and apoptosis. These pathways may be enhanced by the addition of tumor suppressor gene paralogs or deletion of oncogenes. To provide insights to the evolution of cancer suppression across the mammalian radiation, we estimated copy numbers for 548 human tumor suppressor gene and oncogene homologs in 63 mammalian genome assemblies. The naked mole rat contained the most cancer gene copies, consistent with the extremely low rates of cancer found in this species. We found a positive correlation between a species' cancer gene copy number and its longevity, but not body size, contrary to predictions from Peto's Paradox. Extremely long-lived mammals also contained more copies of caretaker genes in their genomes, suggesting that the maintenance of genome integrity is an essential form of cancer prevention in long-lived species. We found the strongest association between longevity and copy numbers of genes that are both germline and somatic tumor suppressor genes, suggesting that selection has acted to suppress both hereditary and sporadic cancers. We also found a strong relationship between the number of tumor suppressor genes and the number of oncogenes in mammalian genomes, suggesting that complex regulatory networks mediate the balance between cell proliferation and checks on tumor progression. This study is the first to investigate cancer gene expansions across the mammalian radiation and provides a springboard for potential human therapies based on evolutionary medicine.

Return to the Sea, Get Huge, Beat Cancer: An Analysis of Cetacean Genomes Including an Assembly for the Humpback Whale (Megaptera novaeangliae).

Cetaceans are a clade of highly specialized aquatic mammals that include the largest animals that have ever lived. The largest whales can have ∼1,000× more cells than a human, with long lifespans, leaving them theoretically susceptible to cancer. However, large-bodied and long-lived animals do not suffer higher risks of cancer mortality than humans-an observation known as Peto's Paradox. To investigate the genomic bases of gigantism and other cetacean adaptations, we generated a de novo genome assembly for the humpback whale (Megaptera novaeangliae) and incorporated the genomes of ten cetacean species in a comparative analysis. We found further evidence that rorquals (family Balaenopteridae) radiated during the Miocene or earlier, and inferred that perturbations in abundance and/or the interocean connectivity of North Atlantic humpback whale populations likely occurred throughout the Pleistocene. Our comparative genomic results suggest that the evolution of cetacean gigantism was accompanied by strong selection on pathways that are directly linked to cancer. Large segmental duplications in whale genomes contained genes controlling the apoptotic pathway, and genes inferred to be under accelerated evolution and positive selection in cetaceans were enriched for biological processes such as cell cycle checkpoint, cell signaling, and proliferation. We also inferred positive selection on genes controlling the mammalian appendicular and cranial skeletal elements in the cetacean lineage, which are relevant to extensive anatomical changes during cetacean evolution. Genomic analyses shed light on the molecular mechanisms underlying cetacean traits, including gigantism, and will contribute to the development of future targets for human cancer therapies.

Peto's Paradox: how has evolution solved the problem of cancer prevention?

The risk of developing cancer should theoretically increase with both the number of cells and the lifespan of an organism. However, gigantic animals do not get more cancer than humans, suggesting that super-human cancer suppression has evolved numerous times across the tree of life. This is the essence and promise of Peto's Paradox. We discuss what is known about Peto's Paradox and provide hints of what is yet to be discovered.

Hiding in the highlands: evolution of a frog species complex of the genus Ptychadena in the Ethiopian highlands.

The Ethiopian highlands are a biodiversity hotspot characterized by a high level of endemism, particularly in amphibians. Frogs of the genus Ptychadena have experienced an evolutionary radiation in these highlands. Thus, this group provides an excellent opportunity to study the process of speciation in this important biogeographic area. We sequenced two mitochondrial (16S and COI) and four nuclear (Rag-1, CXCR4, NCX1 and Tyr) genes in a sample of 236 frogs from 49 Ethiopian localities. Phylogenetic analyses of the mitochondrial genes revealed the presence of eight divergent mitochondrial lineages. We uncovered a near perfect concordance between these lineages and genetic clusters based on nuclear sequences. A Bayesian species delimitation analysis confirmed that these eight lineages correspond to eight genetically isolated populations which may represent eight species. Some of these species have already been recognized due to their distinct morphology (P. cooperi, P. nana and P. erlangeri) but we determined that the species P. neumanni is a complex of 5 cryptic species, thus increasing substantially the number of species in this genus and for this country. We resolved the phylogeny of Ethiopian highland Ptychadena using a species tree approach and determined that Ptychadena species group on the phylogeny according to their habitat preference. We propose that the diversity of Ethiopian Ptychadena results from an early phase of specialization to distinct elevations followed by a phase of ecological diversification within each elevational range. We estimated that the early phase of diversification of Ethiopian Ptychadena probably occurred in the late Miocene and the most recent speciation events in the late-Pliocene or Pleistocene.

Genetic variation in the green anole lizard (Anolis carolinensis) reveals island refugia and a fragmented Florida during the quaternary.

The green anole lizard (Anolis carolinensis) is a model organism for behavior and genomics that is native to the southeastern United States. It is currently thought that the ancestors of modern green anoles dispersed to peninsular Florida from Cuba. However, the climatic changes and geological features responsible for the early diversification of A. carolinensis in North America have remained largely unexplored. This is because previous studies (1) differ in their estimates of the divergence times of populations, (2) are based on a single genetic locus or (3) did not test specific hypotheses regarding the geologic and topographic history of Florida. Here we provide a multi-locus study of green anole genetic diversity and find that the Florida peninsula contains a larger number of genetically distinct populations that are more diverse than those on the continental mainland. As a test of the island refugia hypothesis in Pleistocene Florida, we use a coalescent approach to estimate the divergence times of modern green anole lineages. We find that all demographic events occurred during or after the Upper Pliocene and suggest that green anole diversification was driven by population divergence on interglacial island refugia in Florida during the Lower Pleistocene, while the region was often separated from continental North America. When Florida reconnected to the mainland, two separate dispersal events led to the expansion of green anole populations across the Atlantic Seaboard and Gulf Coastal Plain.

Neutral nuclear variation in Baboons (genus Papio) provides insights into their evolutionary and demographic histories.

Baboons (genus Papio) are distributed over most of sub-Saharan Africa and in the southern portion of the Arabian Peninsula. Six distinct morphotypes, with clearly defined geographic distributions, are recognized (the olive, chacma, yellow, Guinea, Kinda, and hamadryas baboons). The evolutionary relationships among baboon forms have long been a controversial issue. Phylogenetic analyses based on mitochondrial DNA sequences revealed that the modern baboon morphotypes are mitochondrially paraphyletic or polyphyletic. The discordance between mitochondrial lineages and morphology is indicative of extensive introgressive hybridization between ancestral baboon populations. To gain insights into the evolutionary relationships among morphotypes and their demographic history, we performed an analysis of nuclear variation in baboons. We sequenced 13 noncoding, putatively neutral, nuclear regions, and scored the presence/absence of 18 polymorphic transposable elements in a sample of 45 baboons belonging to five of the six recognized baboon forms. We found that the chacma baboon is the sister-taxon to all other baboons and the yellow baboon is the sister-taxon to an unresolved northern clade containing the olive, Guinea, and hamadryas baboons. We estimated that the diversification of baboons occurred entirely in the Pleistocene, the earliest split dating ∼1.5 million years ago, and that baboons have experienced relatively large and constant effective population sizes for most of their evolutionary history (∼30,000 to 95,000 individuals).

Differential Conservation and Loss of CR1 Retrotransposons in Squamates Reveals Lineage-Specific Genome Dynamics across Reptiles.

Transposable elements (TEs) are repetitive DNA sequences which create mutations and generate genetic diversity across the tree of life. In amniotic vertebrates, TEs have been mainly studied in mammals and birds, whose genomes generally display low TE diversity. Squamates (Order Squamata; ~11,000 extant species of lizards and snakes) show as much variation in TE abundance and activity as they do in species and phenotypes. Despite this high TE activity, squamate genomes are remarkably uniform in size. We hypothesize that novel, lineage-specific dynamics have evolved over the course of squamate evolution to constrain genome size across the order. Thus, squamates may represent a prime model for investigations into TE diversity and evolution. To understand the interplay between TEs and host genomes, we analyzed the evolutionary history of the CR1 retrotransposon, a TE family found in most tetrapod genomes. We compared 113 squamate genomes to the genomes of turtles, crocodilians, and birds, and used ancestral state reconstruction to identify shifts in the rate of CR1 copy number evolution across reptiles. We analyzed the repeat landscapes of CR1 in squamate genomes and determined that shifts in the rate of CR1 copy number evolution are associated with lineage-specific variation in CR1 activity. We then used phylogenetic reconstruction of CR1 subfamilies across amniotes to reveal both recent and ancient CR1 subclades across the squamate tree of life. The patterns of CR1 evolution in squamates contrast other amniotes, suggesting key differences in how TEs interact with different host genomes and at different points across evolutionary history.

The State of Squamate Genomics: Past, Present, and Future of Genome Research in the Most Speciose Terrestrial Vertebrate Order.

Squamates include more than 11,000 extant species of lizards, snakes, and amphisbaenians, and display a dazzling diversity of phenotypes across their over 200-million-year evolutionary history on Earth. Here, we introduce and define squamates (Order Squamata) and review the history and promise of genomic investigations into the patterns and processes governing squamate evolution, given recent technological advances in DNA sequencing, genome assembly, and evolutionary analysis. We survey the most recently available whole genome assemblies for squamates, including the taxonomic distribution of available squamate genomes, and assess their quality metrics and usefulness for research. We then focus on disagreements in squamate phylogenetic inference, how methods of high-throughput phylogenomics affect these inferences, and demonstrate the promise of whole genomes to settle or sustain persistent phylogenetic arguments for squamates. We review the role transposable elements play in vertebrate evolution, methods of transposable element annotation and analysis, and further demonstrate that through the understanding of the diversity, abundance, and activity of transposable elements in squamate genomes, squamates can be an ideal model for the evolution of genome size and structure in vertebrates. We discuss how squamate genomes can contribute to other areas of biological research such as venom systems, studies of phenotypic evolution, and sex determination. Because they represent more than 30% of the living species of amniote, squamates deserve a genome consortium on par with recent efforts for other amniotes (i.e., mammals and birds) that aim to sequence most of the extant families in a clade.

Life history and cancer in birds: clutch size predicts cancer.

Cancer is a disease that affects nearly all multicellular life, including birds. However, little is known about what factors explain the variance in cancer prevalence among species. Litter size is positively correlated with cancer prevalence in managed species of mammals, and larger body size, but not incubation or nestling period, is linked to tumor prevalence in wild birds. Also, birds that produce more elaborate sexual traits are expected to have fewer resources for cancer defenses and thus higher cancer prevalence. In this study, we examined whether cancer prevalence is associated with a wide variety of life history traits (clutch size, incubation length, body mass, lifespan, and the extent of sexual dimorphism) across 108 species of managed birds in 25 different zoological facilities, sanctuaries, and veterinary clinics. We found that clutch size was positively correlated with cancer and neoplasia (both benign and malignant) prevalence, even after controlling for body mass. Cancer prevalence was not associated with incubation length, body mass, lifespan, or sexual dimorphism. The positive correlations of clutch size with cancer prevalence and neoplasia prevalence suggest that there may be life-history trade-offs between reproductive investment and somatic maintenance (in the form of cancer prevention mechanisms) in managed birds.

Genome Report: chromosome-scale genome assembly of the African spiny mouse (Acomys cahirinus).

There is increasing interest in the African spiny mouse (Acomys cahirinus) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from 4 different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

GENOME REPORT: Chromosome-scale genome assembly of the African spiny mouse ( Acomys cahirinus ).

There is increasing interest in the African spiny mouse ( Acomys cahirinus ) as a model organism because of its ability for regeneration of tissue after injury in skin, muscle, and internal organs such as the kidneys. A high-quality reference genome is needed to better understand these regenerative properties at the molecular level. Here, we present an improved reference genome for A. cahirinus generated from long Nanopore sequencing reads. We confirm the quality of our annotations using RNA sequencing data from four different tissues. Our genome is of higher contiguity and quality than previously reported genomes from this species and will facilitate ongoing efforts to better understand the regenerative properties of this organism.

Cancer Prevalence Across Vertebrates.

Cancer is pervasive across multicellular species. Are there any patterns that can explain differences in cancer prevalence across species? Using 16,049 necropsy records for 292 species spanning three clades (amphibians, sauropsids and mammals) we found that neoplasia and malignancy prevalence increases with adult weight and decreases with gestation time, contrary to Peto’s Paradox. Evolution of cancer susceptibility appears to have undergone sudden shifts followed by stabilizing selection. Outliers for neoplasia prevalence include the common porpoise (<1.3%), the Rodrigues fruit bat (<1.6%) the black-footed penguin (<0.4%), ferrets (63%) and opossums (35%). Discovering why some species have particularly high or low levels of cancer may lead to a better understanding of cancer syndromes and novel strategies for the management and prevention of cancer.

Cancer Prevalence Across Vertebrates.

Cancer is pervasive across multicellular species, but what explains differences in cancer prevalence across species? Using 16,049 necropsy records for 292 species spanning three clades (amphibians, sauropsids and mammals) we found that neoplasia and malignancy prevalence increases with adult weight (contrary to Peto's Paradox) and somatic mutation rate, but decreases with gestation time. Evolution of cancer susceptibility appears to have undergone sudden shifts followed by stabilizing selection. Outliers for neoplasia prevalence include the common porpoise (<1.3%), the Rodrigues fruit bat (<1.6%) the black-footed penguin (<0.4%), ferrets (63%) and opossums (35%). Discovering why some species have particularly high or low levels of cancer may lead to a better understanding of cancer syndromes and novel strategies for the management and prevention of cancer.

Chromosome-scale genome assembly of the brown anole (Anolis sagrei), an emerging model species.

Rapid technological improvements are democratizing access to high quality, chromosome-scale genome assemblies. No longer the domain of only the most highly studied model organisms, now non-traditional and emerging model species can be genome-enabled using a combination of sequencing technologies and assembly software. Consequently, old ideas built on sparse sampling across the tree of life have recently been amended in the face of genomic data drawn from a growing number of high-quality reference genomes. Arguably the most valuable are those long-studied species for which much is already known about their biology; what many term emerging model species. Here, we report a highly complete chromosome-scale genome assembly for the brown anole, Anolis sagrei - a lizard species widely studied across a variety of disciplines and for which a high-quality reference genome was long overdue. This assembly exceeds the vast majority of existing reptile and snake genomes in contiguity (N50 = 253.6 Mb) and annotation completeness. Through the analysis of this genome and population resequence data, we examine the history of repetitive element accumulation, identify the X chromosome, and propose a hypothesis for the evolutionary history of fusions between autosomes and the X that led to the sex chromosomes of A. sagrei.