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.

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.

Occasional paternal inheritance of the germline-restricted chromosome in songbirds.

Songbirds have one special accessory chromosome, the so-called germline-restricted chromosome (GRC), which is only present in germline cells and absent from all somatic tissues. Earlier work on the zebra finch (Taeniopygia guttata castanotis) showed that the GRC is inherited only through the female line-like the mitochondria-and is eliminated from the sperm during spermatogenesis. Here, we show that the GRC has the potential to be paternally inherited. Confocal microscopy using GRC-specific fluorescent in situ hybridization probes indicated that a considerable fraction of sperm heads (1 to 19%) in zebra finch ejaculates still contained the GRC. In line with these cytogenetic data, sequencing of ejaculates revealed that individual males from two families differed strongly and consistently in the number of GRCs in their ejaculates. Examining a captive-bred male hybrid of the two zebra finch subspecies (T. g. guttata and T. g. castanotis) revealed that the mitochondria originated from a castanotis mother, whereas the GRC came from a guttata father. Moreover, analyzing GRC haplotypes across nine castanotis matrilines, estimated to have diverged for up to 250,000 y, showed surprisingly little variability among GRCs. This suggests that a single GRC haplotype has spread relatively recently across all examined matrilines. A few diagnostic GRC mutations that arose since this inferred spreading suggest that the GRC has continued to jump across matriline boundaries. Our findings raise the possibility that certain GRC haplotypes could selfishly spread through the population via occasional paternal transmission, thereby outcompeting other GRC haplotypes that were limited to strict maternal inheritance, even if this was partly detrimental to organismal fitness.

A Phylogenomic Assessment of Processes Underpinning Convergent Evolution in Open-Habitat Chats.

Insights into the processes underpinning convergent evolution advance our understanding of the contributions of ancestral, introgressed, and novel genetic variation to phenotypic evolution. Phylogenomic analyses characterizing genome-wide gene tree heterogeneity can provide first clues about the extent of ILS and of introgression and thereby into the potential of these processes or (in their absence) the need to invoke novel mutations to underpin convergent evolution. Here, we were interested in understanding the processes involved in convergent evolution in open-habitat chats (wheatears of the genus Oenanthe and their relatives). To this end, based on whole-genome resequencing data from 50 taxa of 44 species, we established the species tree, characterized gene tree heterogeneity, and investigated the footprints of ILS and introgression within the latter. The species tree corroborates the pattern of abundant convergent evolution, especially in wheatears. The high levels of gene tree heterogeneity in wheatears are explained by ILS alone only for 30% of internal branches. For multiple branches with high gene tree heterogeneity, D-statistics and phylogenetic networks identified footprints of introgression. Finally, long branches without extensive ILS between clades sporting similar phenotypes provide suggestive evidence for the role of novel mutations in the evolution of these phenotypes. Together, our results suggest that convergent evolution in open-habitat chats involved diverse processes and highlight that phenotypic diversification is often complex and best depicted as a network of interacting lineages.

Micro Germline-Restricted Chromosome in Blue Tits: Evidence for Meiotic Functions.

The germline-restricted chromosome (GRC) is likely present in all songbird species but differs widely in size and gene content. This extra chromosome has been described as either a microchromosome with only limited basic gene content or a macrochromosome with enriched gene functions related to female gonad and embryo development. Here, we assembled, annotated, and characterized the first micro-GRC in the blue tit (Cyanistes caeruleus) using high-fidelity long-read sequencing data. Although some genes on the blue tit GRC show signals of pseudogenization, others potentially have important functions, either currently or in the past. We highlight the GRC gene paralog BMP15, which is among the highest expressed GRC genes both in blue tits and in zebra finches (Taeniopygia guttata) and is known to play a role in oocyte and follicular maturation in other vertebrates. The GRC genes of the blue tit are further enriched for functions related to the synaptonemal complex. We found a similar functional enrichment when analyzing published data on GRC genes from two nightingale species (Luscinia spp.). We hypothesize that these genes play a role in maintaining standard maternal inheritance or in recombining maternal and paternal GRCs during potential episodes of biparental inheritance.

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.

The Enterprise, a massive transposon carrying Spok meiotic drive genes.

The genomes of eukaryotes are full of parasitic sequences known as transposable elements (TEs). Here, we report the discovery of a putative giant tyrosine-recombinase-mobilized DNA transposon, Enterprise, from the model fungus Podospora anserina Previously, we described a large genomic feature called the Spok block which is notable due to the presence of meiotic drive genes of the Spok gene family. The Spok block ranges from 110 kb to 247 kb and can be present in at least four different genomic locations within P. anserina, despite what is an otherwise highly conserved genome structure. We propose that the reason for its varying positions is that the Spok block is not only capable of meiotic drive but is also capable of transposition. More precisely, the Spok block represents a unique case where the Enterprise has captured the Spoks, thereby parasitizing a resident genomic parasite to become a genomic hyperparasite. Furthermore, we demonstrate that Enterprise (without the Spoks) is found in other fungal lineages, where it can be as large as 70 kb. Lastly, we provide experimental evidence that the Spok block is deleterious, with detrimental effects on spore production in strains which carry it. This union of meiotic drivers and a transposon has created a selfish element of impressive size in Podospora, challenging our perception of how TEs influence genome evolution and broadening the horizons in terms of what the upper limit of transposition may be.

Genomic landscapes of divergence among island bird populations: Evidence of parallel adaptation but at different loci?

When populations colonise new environments, they may be exposed to novel selection pressures but also suffer from extensive genetic drift due to founder effects, small population sizes and limited interpopulation gene flow. Genomic approaches enable us to study how these factors drive divergence, and disentangle neutral effects from differentiation at specific loci due to selection. Here, we investigate patterns of genetic diversity and divergence using whole-genome resequencing (>22× coverage) in Berthelot's pipit (Anthus berthelotii), a passerine endemic to the islands of three north Atlantic archipelagos. Strong environmental gradients, including in pathogen pressure, across populations in the species range, make it an excellent system in which to explore traits important in adaptation and/or incipient speciation. First, we quantify how genomic divergence accumulates across the speciation continuum, that is, among Berthelot's pipit populations, between sub species across archipelagos, and between Berthelot's pipit and its mainland ancestor, the tawny pipit (Anthus campestris). Across these colonisation timeframes (2.1 million-ca. 8000 years ago), we identify highly differentiated loci within genomic islands of divergence and conclude that the observed distributions align with expectations for non-neutral divergence. Characteristic signatures of selection are identified in loci associated with craniofacial/bone and eye development, metabolism and immune response between population comparisons. Interestingly, we find limited evidence for repeated divergence of the same loci across the colonisation range but do identify different loci putatively associated with the same biological traits in different populations, likely due to parallel adaptation. Incipient speciation across these island populations, in which founder effects and selective pressures are strong, may therefore be repeatedly associated with morphology, metabolism and immune defence.

Genotype-environment associations reveal genes potentially linked to avian malaria infection in populations of an endemic island bird.

Patterns of pathogen prevalence are, at least partially, the result of coevolutionary host-pathogen interactions. Thus, exploring the distribution of host genetic variation in relation to infection by a pathogen within and across populations can provide important insights into mechanisms of host defence and adaptation. Here, we use a landscape genomics approach (Bayenv) in conjunction with genome-wide data (ddRADseq) to test for associations between avian malaria (Plasmodium) prevalence and host genetic variation across 13 populations of the island endemic Berthelot's pipit (Anthus berthelotii). Considerable and consistent spatial heterogeneity in malaria prevalence was observed among populations over a period of 15 years. The prevalence of malaria infection was also strongly positively correlated with pox (Avipoxvirus) prevalence. Multiple host loci showed significant associations with malaria prevalence after controlling for genome-wide neutral genetic structure. These sites were located near to or within genes linked to metabolism, stress response, transcriptional regulation, complement activity and the inflammatory response, many previously implicated in vertebrate responses to malarial infection. Our findings identify diverse genes - not just limited to the immune system - that may be involved in host protection against malaria and suggest that spatially variable pathogen pressure may be an important evolutionary driver of genetic divergence among wild animal populations, such as Berthelot's pipit. Furthermore, our data indicate that spatio-temporal variation in multiple different pathogens (e.g. malaria and pox in this case) may have to be studied together to develop a more holistic understanding of host pathogen-mediated evolution.

Satellite DNA evolution in Corvoidea inferred from short and long reads.

Satellite DNA (satDNA) is a fast-evolving portion of eukaryotic genomes. The homogeneous and repetitive nature of such satDNA causes problems during the assembly of genomes, and therefore it is still difficult to study it in detail in nonmodel organisms as well as across broad evolutionary timescales. Here, we combined the use of short- and long-read data to explore the diversity and evolution of satDNA between individuals of the same species and between genera of birds spanning ~40 millions of years of bird evolution using birds-of-paradise (Paradisaeidae) and crow (Corvus) species. These avian species highlighted the presence of a GC-rich Corvoidea satellitome composed of 61 satellite families and provided a set of candidate satDNA monomers for being centromeric on the basis of length, abundance, homogeneity and transcription. Surprisingly, we found that the satDNA of crow species rapidly diverged between closely related species while the satDNA appeared more similar between birds-of-paradise species belonging to different genera.

Runs of homozygosity reveal past bottlenecks and contemporary inbreeding across diverging populations of an island-colonizing bird.

Genomes retain evidence of the demographic history and evolutionary forces that have shaped populations and drive speciation. Across island systems, contemporary patterns of genetic diversity reflect population demography, including colonization events, bottlenecks, gene flow and genetic drift. Here, we investigate genome-wide diversity and the distribution of runs of homozygosity (ROH) using whole-genome resequencing of individuals (>22× coverage) from six populations across three archipelagos of Berthelot's pipit (Anthus berthelotii)-a passerine that has recently undergone island speciation. We show the most dramatic reduction in diversity occurs between the mainland sister species (the tawny pipit) and Berthelot's pipit and is lowest in the populations that have experienced sequential bottlenecks (i.e., the Madeiran and Selvagens populations). Pairwise sequential Markovian coalescent (PSMC) analyses estimated that Berthelot's pipit diverged from its sister species ~2 million years ago, with the Madeiran archipelago founded 50,000 years ago, and the Selvagens colonized 8000 years ago. We identify many long ROH (>1 Mb) in these most recently colonized populations. Population expansion within the last 100 years may have eroded long ROH in the Madeiran archipelago, resulting in a prevalence of short ROH (<1 Mb). However, the extensive long and short ROH detected in the Selvagens suggest strong recent inbreeding and bottleneck effects, with as much as 38% of the autosomes consisting of ROH >250 kb. These findings highlight the importance of demographic history, as well as selection and genetic drift, in shaping contemporary patterns of genomic diversity across diverging populations.

Mendelian nightmares: the germline-restricted chromosome of songbirds.

Germline-restricted chromosomes (GRCs) are accessory chromosomes that occur only in germ cells. They are eliminated from somatic cells through programmed DNA elimination during embryo development. GRCs have been observed in several unrelated animal taxa and show peculiar modes of non-Mendelian inheritance and within-individual elimination. Recent cytogenetic and phylogenomic evidence suggests that a GRC is present across the species-rich songbirds, but absent in non-passerine birds, implying that over half of all 10,500 bird species have extensive germline/soma genome differences. Here, we review recent insights gained from genomic, transcriptomic, and cytogenetic approaches with regard to the genetic content, phylogenetic distribution, and inheritance of the songbird GRC. While many questions remain unsolved in terms of GRC inheritance, elimination, and function, we discuss plausible scenarios and future directions for understanding this widespread form of programmed DNA elimination.

Genetic Barriers to Historical Gene Flow between Cryptic Species of Alpine Bumblebees Revealed by Comparative Population Genomics.

Evidence is accumulating that gene flow commonly occurs between recently diverged species, despite the existence of barriers to gene flow in their genomes. However, we still know little about what regions of the genome become barriers to gene flow and how such barriers form. Here, we compare genetic differentiation across the genomes of bumblebee species living in sympatry and allopatry to reveal the potential impact of gene flow during species divergence and uncover genetic barrier loci. We first compared the genomes of the alpine bumblebee Bombus sylvicola and a previously unidentified sister species living in sympatry in the Rocky Mountains, revealing prominent islands of elevated genetic divergence in the genome that colocalize with centromeres and regions of low recombination. This same pattern is observed between the genomes of another pair of closely related species living in allopatry (B. bifarius and B. vancouverensis). Strikingly however, the genomic islands exhibit significantly elevated absolute divergence (dXY) in the sympatric, but not the allopatric, comparison indicating that they contain loci that have acted as barriers to historical gene flow in sympatry. Our results suggest that intrinsic barriers to gene flow between species may often accumulate in regions of low recombination and near centromeres through processes such as genetic hitchhiking, and that divergence in these regions is accentuated in the presence of gene flow.

Seasonal migration patterns and the maintenance of evolutionary diversity in a cryptic bird radiation.

Morphological differentiation associated with evolutionary diversification is often explained with adaptive benefits but the processes and mechanisms maintaining cryptic diversity are still poorly understood. Using genome-wide data, we show here that the pale sand martin Riparia diluta in Central and East Asia consists of three genetically deeply differentiated lineages which vary only gradually in morphology but broadly reflect traditional taxonomy. We detected no signs of gene flow along the eastern edge of the Qinghai-Tibetan plateau between lowland south-eastern Chinese R. d. fohkienensis and high-altitude R. d. tibetana. Largely different breeding and migration timing between these low and high altitude populations as indicated by phenology data suggests that allochrony might act as prezygotic isolation mechanism in the area where their ranges abut. Mongolian populations of R. d. tibetana, however, displayed signs of limited mixed ancestries with Central Asian R. d. diluta. Their ranges meet in the area of a well-known avian migratory divide, where western lineages take a western migration route around the Qinghai-Tibetan plateau to winter quarters in South Asia, and eastern lineages take an eastern route to Southeast Asia. This might also be the case between western R. d. diluta and eastern R. d. tibetana as indicated by differing wintering grounds. We hypothesize that hybrids might have nonoptimal intermediate migration routes and selection against them might restrict gene flow. Although further potential isolation mechanisms might exist in the pale sand martin, our study points towards contrasting migration behaviour as an important factor in maintaining evolutionary diversity under morphological stasis.

Genomic associations with poxvirus across divergent island populations in Berthelot's pipit.

Understanding the mechanisms and genes that enable animal populations to adapt to pathogens is important from an evolutionary, health and conservation perspective. Berthelot's pipit (Anthus berthelotii) experiences extensive and consistent spatial heterogeneity in avian pox infection pressure across its range of island populations, thus providing an excellent system with which to examine how pathogen-mediated selection drives spatial variation in immunogenetic diversity. Here, we test for evidence of genetic variation associated with avian pox at both an individual and population-level. At the individual level, we find no evidence that variation in MHC class I and TLR4 (both known to be important in recognising viral infection) was associated with pox infection within two separate populations. However, using genotype-environment association (Bayenv) in conjunction with genome-wide (ddRAD-seq) data, we detected strong associations between population-level avian pox prevalence and allele frequencies of single nucleotide polymorphisms (SNPs) at a number of sites across the genome. These sites were located within genes involved in cellular stress signalling and immune responses, many of which have previously been associated with responses to viral infection in humans and other animals. Consequently, our analyses indicate that pathogen-mediated selection may play a role in shaping genomic variation among relatively recently colonised island bird populations and highlight the utility of genotype-environment associations for identifying candidate genes potentially involved in host-pathogen interactions.

Long-term persistence of supernumerary B chromosomes in multiple species of Astyanax fish.

BACKGROUND: Eukaryote genomes frequently harbor supernumerary B chromosomes in addition to the "standard" A chromosome set. B chromosomes are thought to arise as byproducts of genome rearrangements and have mostly been considered intraspecific oddities. However, their evolutionary transcendence beyond species level has remained untested. RESULTS: Here we reveal that the large metacentric B chromosomes reported in several fish species of the genus Astyanax arose in a common ancestor at least 4 million years ago. We generated transcriptomes of A. scabripinnis and A. paranae 0B and 1B individuals and used these assemblies as a reference for mapping all gDNA and RNA libraries to quantify coverage differences between B-lacking and B-carrying genomes. We show that the B chromosomes of A. scabripinnis and A. paranae share 19 protein-coding genes, of which 14 and 11 were also present in the B chromosomes of A. bockmanni and A. fasciatus, respectively. Our search for B-specific single-nucleotide polymorphisms (SNPs) identified the presence of B-derived transcripts in B-carrying ovaries, 80% of which belonged to nobox, a gene involved in oogenesis regulation. Importantly, the B chromosome nobox paralog is expressed > 30× more than the A chromosome paralog. This indicates that the normal regulation of this gene is altered in B-carrying females, which could potentially facilitate B inheritance at higher rates than Mendelian law prediction. CONCLUSIONS: Taken together, our results demonstrate the long-term survival of B chromosomes despite their lack of regular pairing and segregation during meiosis and that they can endure episodes of population divergence leading to species formation.

Unboxing mutations: Connecting mutation types with evolutionary consequences.

A key step in understanding the genetic basis of different evolutionary outcomes (e.g., adaptation) is to determine the roles played by different mutation types (e.g., SNPs, translocations and inversions). To do this we must simultaneously consider different mutation types in an evolutionary framework. Here, we propose a research framework that directly utilizes the most important characteristics of mutations, their population genetic effects, to determine their relative evolutionary significance in a given scenario. We review known population genetic effects of different mutation types and show how these may be connected to different evolutionary outcomes. We provide examples of how to implement this framework and pinpoint areas where more data, theory and synthesis are needed. Linking experimental and theoretical approaches to examine different mutation types simultaneously is a critical step towards understanding their evolutionary significance.

Horizontal transfer and subsequent explosive expansion of a DNA transposon in sea kraits (Laticauda).

Transposable elements (TEs) are self-replicating genetic sequences and are often described as important 'drivers of evolution'. This driving force is because TEs promote genomic novelty by enabling rearrangement, and through exaptation as coding and regulatory elements. However, most TE insertions potentially lead to neutral or harmful outcomes, therefore host genomes have evolved machinery to suppress TE expansion. Through horizontal transposon transfer (HTT) TEs can colonize new genomes, and since new hosts may not be able to regulate subsequent replication, these TEs may proliferate rapidly. Here, we describe HTT of the Harbinger-Snek DNA transposon into sea kraits (Laticauda), and its subsequent explosive expansion within Laticauda genomes. This HTT occurred following the divergence of Laticauda from terrestrial Australian elapids approximately 15-25 Mya. This has resulted in numerous insertions into introns and regulatory regions, with some insertions into exons which appear to have altered UTRs or added sequence to coding exons. Harbinger-Snek has rapidly expanded to make up 8-12% of Laticauda spp. genomes; this is the fastest known expansion of TEs in amniotes following HTT. Genomic changes caused by this rapid expansion may have contributed to adaptation to the amphibious-marine habitat.

Comparative analysis of morabine grasshopper genomes reveals highly abundant transposable elements and rapidly proliferating satellite DNA repeats.

BACKGROUND: Repetitive DNA sequences, including transposable elements (TEs) and tandemly repeated satellite DNA (satDNAs), collectively called the "repeatome", are found in high proportion in organisms across the Tree of Life. Grasshoppers have large genomes, averaging 9 Gb, that contain a high proportion of repetitive DNA, which has hampered progress in assembling reference genomes. Here we combined linked-read genomics with transcriptomics to assemble, characterize, and compare the structure of repetitive DNA sequences in four chromosomal races of the morabine grasshopper Vandiemenella viatica species complex and determine their contribution to genome evolution. RESULTS: We obtained linked-read genome assemblies of 2.73-3.27 Gb from estimated genome sizes of 4.26-5.07 Gb DNA per haploid genome of the four chromosomal races of V. viatica. These constitute the third largest insect genomes assembled so far. Combining complementary annotation tools and manual curation, we found a large diversity of TEs and satDNAs, constituting 66 to 75% per genome assembly. A comparison of sequence divergence within the TE classes revealed massive accumulation of recent TEs in all four races (314-463 Mb per assembly), indicating that their large genome sizes are likely due to similar rates of TE accumulation. Transcriptome sequencing showed more biased TE expression in reproductive tissues than somatic tissues, implying permissive transcription in gametogenesis. Out of 129 satDNA families, 102 satDNA families were shared among the four chromosomal races, which likely represent a diversity of satDNA families in the ancestor of the V. viatica chromosomal races. Notably, 50 of these shared satDNA families underwent differential proliferation since the recent diversification of the V. viatica species complex. CONCLUSION: This in-depth annotation of the repeatome in morabine grasshoppers provided new insights into the genome evolution of Orthoptera. Our TEs analysis revealed a massive recent accumulation of TEs equivalent to the size of entire Drosophila genomes, which likely explains the large genome sizes in grasshoppers. Despite an overall high similarity of the TE and satDNA diversity between races, the patterns of TE expression and satDNA proliferation suggest rapid evolution of grasshopper genomes on recent timescales.