Molecular Systematics and Traits Evolution in Phasmatodea Leach, 1815 (Hexapoda; Insecta)

Phasmatodea Leach, 1815 (Hexapoda; Insecta) is a polyneopteran order which counts approximately 3000 described species, often known for their remarkable forms of mimicry. In this thesis, I provide a comprehensive systematic framework which includes over 180 species never considered in a phylogenetic framework: the latter can facilitate a better understanding of the processes underlying phasmids evolutionary history. The clade represents in fact an incredible testing ground to study trait evolution and its striking disparity of reproductive strategies and wing morphologies have been of great interest to the evolutionary biology community. Phasmids wings represent one of the first and most notable rejection of Dollo’s law and they played a central role in initiating a long- standing debate on the irreversibility of complex traits loss. Macroevolutionary analyses presented here confirm that wings evolution in phasmids is a reversible process even when possible biases - such as systematic uncertainty and trait-dependent diversification rates - are considered. These findings remark how complex traits can evolve in a dynamic, reversible manner and imply that their molecular groundplan can be preserved despite its phenotypical absence. This concept has been further tested with phylogenetic and transcriptomic approaches in two phasmids parthenogenetic lineages and a bisexual congeneric of the European Bacillus species complex. Leveraging a gene co-expression network approach, male gonad associated genes were retrieved in the bisexual species and then their modifications in the parthenogens were charachterized. Pleiotropy appears to constrain gene modifications associated to male reproductive structures after their loss in parthenogens, so that the lost trait molecular groundplan can be largely preserved in both transcription patterns and sequence evolution. Overall, the results presented in this thesis contribute to shape our understanding of the interplay between the phenotypic and molecular levels in trait evolution.

Mitochondrial Inheritance, Mito-nuclear Coevolution, and Sex-associated Genes in Bivalve Molluscs: Transcriptomics and Molecular Evolution

Bivalvia represents an ancient taxon including around 25,000 living species that have adapted to a wide range of environmental conditions, and show a great diversity in body size, shell shapes, and anatomic structure. Bivalves are characterized by highly variable genome sizes and extremely high levels of heterozygosity, which obstacle complete and accurate genome assemblies and hinder further genomic studies. Moreover, some bivalve species presented a stable evolutionary exception to the strictly maternal inheritance of mitochondria, namely doubly uniparental inheritance (DUI), making these species a precious model to study mitochondrial biology. During my PhD, I focused on a DUI species, the Manila clam Ruditapes philippinarum, and my work was two-folded. First, taking advantage of a newly assembled draft genome and a large RNA-seq dataset from different tissues of both sexes, I investigated 1) the role of gene expression and alternative splicing in tissue differentiation; 2) the relationship across tissue specificity, regulatory network connectivity, and sequence evolution; 3) sexual contrasting genetic markers potentially associated with sexual differentiation. The detailed information for this part is in Chapter 2. Second, using the same RNA-seq data, I investigated how nuclear oxidative phosphorylation (OXPHOS) genes coordinate with two divergent mitochondrial genomes in DUI species (mito-nuclear coordination and coevolution). To address this question, I compared transcription, polymorphism, and synonymous codon usage in the mitochondrial and nuclear OXPHOS genes of R. philippinarum in Chapter 3. To my knowledge, this thesis represents the first study exploring the role of alternative splicing in tissue differentiation, and the first study analyzing both transcriptional regulation and sequence evolution to investigate the coordination of OXPHOS genes in bivalves.

Genes involved in germline regulative pathways in metazoa: an integrative approach to investigate a key feature of animal biology

In Metazoa, the germline represents the cell lineage devoted to transmission of genetic heredity across generations. Its functions intuitively evoke the crucial roles that it plays in the development of a new organism and in the evolution of the species. Germline establishment is tightly tied to animal multicellularity itself, in which the complex differentiation of cell lineages is favoured by the confinement of totipotency in specific cell populations. In the present thesis, I addressed the subject of germline characterization in animals through different approaches, in an attempt to cover different sides and scales. First, I investigated the extent and nature of shared differentially transcribed molecular factors in 10 different species germline-related lineages. I observed that newly evolved genes are less likely to be involved in germline-related mechanisms and that the mostly shared transcriptional signal across the species considered was the upregulation of genes associated to proper DNA replication, instead of the expected transcriptional and post-transcriptional regulation, that apparently have a higher level of lineage-specificity. I then focused on the evolutionary history of Tudor domain containing proteins, a gene family that underwent germline-associated expansions in animals. Using data from 24 holozoan phyla, I could confirm the previously proposed evolution of the Tudor domain secondary structure. Also, I associated lineage-specific family reductions and expansions to peculiar genomic dynamics and to the evolution of germline-associated piRNA pathway of retrotransposon silencing. Lastly, I characterized and investigated the expression of the Tudor protein TDRD7 in the clam Ruditapes philippinarum. Through immunolocalization, I could compare its expression profiles in gametogenic specimens to the previously characterized germline marker vasa. Combining results with literature, I proposed that, in this species, TDRD7 is involved in the assembly of germ granules, i.e. cytoplasmic structures associated to germline differentiation in virtually all animals, but whose assemblers can be taxon specific.