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Genomic organisation of extinct lineages can be inferred from extant chromosome-level genome assemblies. Here, we apply bioinformatic and molecular cytogenetic approaches to determine the genomic structure of the diapsid common ancestor. We then infer the events that likely occurred along this lineage from theropod dinosaurs through to modern birds. Our results suggest that most elements of a typical ‘avian-like’ karyotype (40 chromosome pairs, including 30 microchromosomes) were in place before the divergence of turtles from birds ~255 mya. This genome organisation therefore predates the emergence of early dinosaurs and pterosaurs and the evolution of flight. Remaining largely unchanged interchromosomally through the dinosaur–theropod route that led to modern birds, intrachromosomal changes nonetheless reveal evolutionary breakpoint regions enriched for genes with ontology terms related to chromatin organisation and transcription. This genomic structure therefore appears highly stable yet contributes to a large degree of phenotypic diversity, as well as underpinning adaptive responses to major environmental disruptions via intrachromosomal repatterning., Ancient diapsids diverged into the lineages leading to turtles and birds over 250 million years ago. Here, the authors use genomic and molecular cytogenetic analyses of modern species to infer the genome structure of the diapsid common ancestor (DCA) and the changes occurring along the lineage to birds through theropod dinosaurs.

The ultimate aim of a genome assembly is to create a contiguous length of sequence from the p- to q- terminus of each chromosome. Most assemblies are however highly fragmented, limiting their use in studies of gene mapping, phylogenomics and genomic organisation. To overcome these limitations, we developed a novel scaffold-to-chromosome anchoring method combining reference-assisted chromosome assembly (RACA) and fluorescence in situ hybridisation (FISH) to position scaffolds from de novo genomes onto chromosomes. Using RACA, scaffolds were ordered and orientated into ‘predicted chromosome fragments’ (PCFs) against a reference and outgroup genome. PCFs were verified using PCR prior to FISH mapping. A universal set of FISH probes developed through the selection of conserved regions were then used to map PCFs of peregrine falcon (Falco peregrinus Tunstall, 1771), pigeon (Columba livia Gmelin, 1789), ostrich (Struthio camelus Linnaeus, 1758), saker falcon (Falco cherrug Gray, 1834) the budgerigar (Melopsittacus undulatus Shaw, 1805). Using this approach, we were able to improve the N50 of genomes seven-fold. Results revealed that Interchromosomal breakpoint regions are limited to regions with low sequence conservation, shedding light on why most avian species have very stable karyotypes.\ud \ud Our combined FISH and bioinformatics approach represents a step-change in the mapping of genome assemblies, allowing comparative genomic research at a higher resolution than was previously possible. The universal probe set facilitates research into avian karyotype evolution and the role of chromosome rearrangements in adaptation and phenotypic diversity in birds. Indeed, they have been used on over 20 avian species plus non-avian reptiles (including turtles), shedding light into the evolution of dinosaur species. Non-avian dinosaurs remain subjects of intense biological enquiry while pervading popular culture and the creative arts. While organismal studies focus primarily on their morphology, relationships, likely behaviour, and ecology there have been few academic studies that have made extensive extrapolations about the nature of non-avian dinosaur genome structure prior to the emergence of modern birds. We have used multiple avian whole genome sequences assembled at a chromosomal level, to reconstruct the most likely gross genome organization of the overall genome structure of the diapsid ancestor and reconstruct the sequence of inter and intrachromosomal events that most likely occurred along the Archosauromorpha-Archosauria-Avemetatarsalia-Dinosauria-Theropoda-Maniraptora-Avialae lineage from the lepidosauromorph-archosauromorph divergence ~275 million years ago through to extant neornithine birds.

BACKGROUND: Thamnophilidae birds are the result of a monophyletic radiation of insectivorous Passeriformes. They are a diverse group of 225 species and 45 genera and occur in lowlands and lower montane forests of Neotropics. Despite the large degree of diversity seen in this family, just four species of Thamnophilidae have been karyotyped with a diploid number ranging from 76 to 82 chromosomes. The karyotypic relationships within and between Thamnophilidae and another Passeriformes therefore remain poorly understood. Recent studies have identified the occurrence of intrachromosomal rearrangements in Passeriformes using in silico data and molecular cytogenetic tools. These results demonstrate that intrachromosomal rearrangements are more common in birds than previously thought and are likely to contribute to speciation events. With this in mind, we investigate the apparently conserved karyotype of Willisornis vidua, the Xingu Scale-backed Antbird, using a combination of molecular cytogenetic techniques including chromosome painting with probes derived from Gallus gallus (chicken) and Burhinus oedicnemus (stone curlew), combined with Bacterial Artificial Chromosome (BAC) probes derived from the same species. The goal was to investigate the occurrence of rearrangements in an apparently conserved karyotype in order to understand the evolutionary history and taxonomy of this species. In total, 78 BAC probes from the Gallus gallus and Taeniopygia guttata (the Zebra Finch) BAC libraries were tested, of which 40 were derived from Gallus gallus macrochromosomes 1-8, and 38 from microchromosomes 9-28. RESULTS: The karyotype is similar to typical Passeriformes karyotypes, with a diploid number of 2n = 80. Our chromosome painting results show that most of the Gallus gallus chromosomes are conserved, except GGA-1, 2 and 4, with some rearrangements identified among macro- and microchromosomes. BAC mapping revealed many intrachromosomal rearrangements, mainly inversions, when comparing Willisornis vidua karyotype with Gallus gallus, and corroborates the fissions revealed by chromosome painting. CONCLUSIONS: Willisornis vidua presents multiple chromosomal rearrangements despite having a supposed conservative karyotype, demonstrating that our approach using a combination of FISH tools provides a higher resolution than previously obtained by chromosome painting alone. We also show that populations of Willisornis vidua appear conserved from a cytogenetic perspective, despite significant phylogeographic structure.

Background: Thamnophilidae birds are the result of a monophyletic radiation of insectivorous Passeriformes. They are a diverse group of 225 species and 45 genera and occur in lowlands and lower montane forests of Neotropics. Despite the large degree of diversity seen in this family, just four species of Thamnophilidae have been karyotyped with a diploid number ranging from 76 to 82 chromosomes. The karyotypic relationships within and between Thamnophilidae and another Passeriformes therefore remain poorly understood. Recent studies have identified the occurrence of intrachromosomal rearrangements in Passeriformes using in silico data and molecular cytogenetic tools. These results demonstrate that intrachromosomal rearrangements are more common in birds than previously thought and are likely to contribute to speciation events. With this in mind, we investigate the apparently conserved karyotype of Willisornis vidua, the Xingu Scale-backed Antbird, using a combination of molecular cytogenetic techniques including chromosome painting with probes derived from Gallus gallus (chicken) and Burhinus oedicnemus (stone curlew), combined with Bacterial Artificial Chromosome (BAC) probes derived from the same species. The goal was to investigate the occurrence of rearrangements in an apparently conserved karyotype in order to understand the evolutionary history and taxonomy of this species. In total, 78 BAC probes from the Gallus gallus and Taeniopygia guttata (the Zebra Finch) BAC libraries were tested, of which 40 were derived from Gallus gallus macrochromosomes 1–8, and 38 from microchromosomes 9–28. Results: The karyotype is similar to typical Passeriformes karyotypes, with a diploid number of 2n = 80. Our chromosome painting results show that most of the Gallus gallus chromosomes are conserved, except GGA-1, 2 and 4, with some rearrangements identified among macro- and microchromosomes. BAC mapping revealed many intrachromosomal rearrangements, mainly inversions, when comparing Willisornis vidua karyotype with Gallus gallus, and corroborates the fissions revealed by chromosome painting. Conclusions: Willisornis vidua presents multiple chromosomal rearrangements despite having a supposed conservative karyotype, demonstrating that our approach using a combination of FISH tools provides a higher resolution than previously obtained by chromosome painting alone. We also show that populations of Willisornis vidua appear conserved from a cytogenetic perspective, despite significant phylogeographic structure.