Genome-scaled molecular clock studies of invasive mosquitoes and other organisms of societal relevance

Molecular dating (or molecular clock) is a powerful technique that uses the mutation rate of biomolecules to estimate divergence times among organisms. In the last two decades, the theory behind the molecular clock has been intensively developed, and it is now possible to employ sophisticated evolutionary models on genome-scaled datasets in a Bayesian framework. The molecular clock has been successfully applied to virtually all types of organisms and molecules to estimate timing of speciation, timing of gene duplications, and generation times: this knowledge allows contextualizing past and present events in the light of (paleo)ecological scenarios. Molecular clock studies are routinely used in evolutionary and ecological studies, but their use in applied fields such as agricultural and medical entomology is still scarce in particular because of a paucity of genome data. Genome-scaled clocks have been successfully applied, for example, to various model organisms such as Anopheles and Drosophila, as well as to invasive mosquitoes Aedes aegypti and Aedes albopictus. Many other invasive pests are emerging worldwide aided by global trade, increased connectivity among countries, lack of prevention, and flawed invasive species management. Among them, there is Aedes koreicus and Aedes japonicus, two invasive mosquito species which are monitored for public health concerns because of their harboured human pathogenic viruses. For these, as well as for other insects of societal relevance, such as the parasitoid Trissolcus japonicus, there is a paucity of gene markers and no genome data for large scale molecular clock studies. Invasive pests are typically studied using microevolutionary approaches that tackle events at an intraspecific level: these approaches provide important information for the pest management, for example, by revealing invasion routes and insecticide resistances. Approaches that tackle the deep-time evolution of the pest, such as the molecular clock, are instead less used in pest science. Many important traits associated with invasiveness have evolved by speciation over a long time frame: the molecular clock can reveal the paleo-ecological conditions that favoured these traits helping a better understanding of pest biology. Molecular clock, when coupled with phylogenomics, can further identify genes and patterns that characterize the pest: this knowledge can be used to enhance management practices. Although this is a data-driven thesis, its major aim is to provide new results to demonstrate the utility of the molecular clock in pest science. This has been done by systematically apply the molecular clock to various neglected organisms of medical and agricultural relevance. To this aim, I generated new genome data and/or assembled the largest genome-scaled data to date. I studied the molecular clock in mosquitoes, focusing on the Aedini radiation (Chapter 2) and identified a strong incongruence between the mitochondrial and nuclear phylogeny for what concerns their molecular clock. This result highlighted the importance of employing genome scaled data for these species to exclude stochastic effects due to poor/inaccurate sampling in clock studies. To tackle the absence of data, I further assembled the whole mitogenome of emerging invasive species Aedes koreicus and Aedes japonicus with the aim of producing useful data for molecular typing and of inferring divergence estimates using whole mitogenomes (Chapter 3). Dated phylogenies point toward more recent diversification of Aedini and Culicini compared to estimates from previous works, addressing the issue of taxon sampling sensitivity in dated phylogeny. Although it is possible to perform molecular clock studies on single/few gene markers, the current trend is to couple this methodology with genome-scaled datasets to reduce the stochastic effect of using few genes. For this reason, I sequenced the draft genome of A. koreicus and A. japonicus (Chapter 4). The assemblies were extremely fragmented, highlighting the problem of sequencing large genomes using short reads. The assemblies provided, however enough information for genome skimming allowing extraction of BUSCO genes for downstream analyses, whole mitogenome assemblies (used in Chapter 3), and characterisation of the associated metagenome. These data need to be integrated by long reads; it provides, however a first framework to investigate the genome evolution of these species. I further sequenced and assembled the genome of Trissolcus japonicus, the parasitoid wasp of the invasive pest Halyomorpha halys. To elucidate its divergence, estimate and define an intraspecific typing system to differentiate strains for biocontrol strategies, I reconstructed the mitochondrial genomes of two populations: the mitogenomes were surprisingly identical, suggesting that they belong to the same de facto population. I further provide a detailed clock investigation of Zika, a virus harboured and transmitted by some Aedes species (Chapter 5). Using the largest set of genomes to date, I could set the origin of ZIKV in the middle age and its first diversification in the mid-19th century. From a methodological point of view, the clocking of this virus highlighted the importance of checking for recombination and for cell-passages to obtain correct divergence estimates. I finally show my contributions to molecular clock studies of three other invasive species (Chapter 6): I helped disentangle the divergence times of Bactrocera, a genus of invasive fruit files pest of agriculture; I contributed in performing a phylogenomics study of opsin genes in Diptera; I used chloroplast and nuclear genome data to reconstruct the divergences of the invasive reed Arundo. In the various Chapters of my thesis, I highlighted the limits and the problems of current molecular clock methodologies and identified the best practices for different types of organisms in order to develop a cross-discipline understanding of the molecular clock techniques. The various results presented in this thesis further demonstrate the utility of the molecular clock approach in pest studies.