CHROMOSOME-LEVEL DE NOVO GENOME ASSEMBLIES OF AVIAN SPECIES AND THEIR RELEVANCE FOR COMPARATIVE GENOMICS, PANGENOMICS, POPULATION GENOMICS AND SPECIES CONSERVATION.

Life on Earth is currently experiencing the sixth mass extinction. A major loss of species is being caused by anthropogenic habitat destruction, illegal wildlife trade, overfishing, massive fossil fuel consumption and the consequent climate changes, leading to a progressive collapse of biodiversity. The establishment of conservation initiatives is now more crucial than ever and the genetic management of threatened species is currently gaining critical significance. Previous genetic studies have been limited by the lack of complete reference genomes, thus having to focus on a limited number of genes or incomplete genomic sequencing data. In several cases, this led to wrong or incomplete assumptions and to conservation decisions which did not carry the desired effects due to incorrect evaluations. The availability of high-throughput sequencing technologies and the development of advanced computational methods have recently allowed the generation of cost-effective chromosome-level reference genomes. In the last decades, tremendous efforts were made to generate the most complete human genome. However, additional reference genomes spanning the entire tree of life are a necessary foundation for the study of biology in the 21st century, which was made possible with the establishment of large sequencing consortia. This thesis work represents a contribution to the collective effort of describing and preserving our planet's genetic diversity, being part of international consortia such as the Vertebrate Genomes Project (VGP), Darwin Tree of Life (DToL) and European Reference Genome Atlas (ERGA). Here I report new methods to assemble highly contiguous reference genomes for vertebrate species in the context of the VGP and their relevance in deciphering the biology of a species, its evolution, its intrinsic variability and in planning conservation actions, providing a comprehensive collection of genomic markers. Using the VGP pipelines, I have assembled 6 chromosome-level bird species: barn swallow (Hirundo rustica), lesser kestrel (Falco naumanni), American flamingo (Phoenicopterus ruber), common yellowthroat (Geothlypis trichas), rifleman (Acanthisitta chloris) and red-crested turaco (Tauraco erythrolophus), which fulfilled and exceeded the standard VGP metrics for genome assembly. Moreover, I contributed to the generation of the European nightjar (Caprimulgus europaeus) reference genome for the DToL. The genomes I generated will be exploited in the future to get insights into the biology of these species, but also for comparative genomics analyses using all available VGP-quality species. In particular, the lesser kestrel reference genome will be a fundamental resource for a future study that involves the assessment of how this species coped with climatic fluctuations in the past and how it is expected to cope with them in the future under the current scenario of climate changes. Moreover, the European nightjar genome will help to deepen the knowledge on the biology of this cryptic and 2 elusive bird, also boosting the sequencing of other members of the Caprimulgidae family to reconstruct a comprehensive phylogeny. During my PhD, I particularly focused on barn swallow (Hirundo rustica) genomics, an iconic migratory passerine bird with a long-standing association with humans. Using the VGP-quality assembly, I performed comparative genomics, population genomics and pangenomics analyses. Comparative genomics work was carried out with the reference-free aligner Cactus, with which I have aligned the barn swallow reference with other chromosome-level bird species. Using the alignment, I performed a positive and negative selection analysis across the genome to find candidate genes under selection important for barn swallow biology. For population genomics analyses, we aligned all publicly available data for the barn swallow subspecies and populations to the reference genome and performed a Linkage Disequilibrium scan. Comparative and population genomics approaches both pointed at candidate genes under selective constraint which may have a role in the onset of the synanthropic behavior of the species. Finally, with the reference genome and other HiFi-based barn swallow assemblies, I constructed the first pangenome graph for the species and preliminarily evaluated the extent of core and accessory genes among individuals. We also complemented the nuclear genome-based study with the generation of the complete mitochondrial genome for the species, which allowed us to dissect the phylogenetic relationships between barn swallow subspecies and to clarify the species’ phylogeographic history. In conclusion, this thesis work represents a valuable step forward and a contribution to international genome sequencing efforts. I outlined how complete genomic resources can revolutionize studies on the biology and evolution of a species, but also how they represent a pivotal resource to correctly plan threatened species conservation actions during the sixth mass extinction.