Exceptions to paradigms are important in at least two ways, including highlighting limits of existing paradigms and in providing novel opportunities to test general hypotheses. In the context of genetic processes and systems, non-paradigmatic processes such as complex patterns of inheritance can reveal what may be overlooked in conventional systems and even inform us about established general ideas in evolutionary and population genetics. This thesis explores the potential of non-paradigmatic genetic systems as valuable tools for understanding evolutionary processes. In Chapters 1 and 2, I focus on an atypical genetic system in arthropods with genetic peculiarities that allow for less biased natural experiments for testing hypotheses regarding several fundamental questions regarding selection, mutation, and drift, and about sex determination system turnover. To highlight this system’s use for several fundamental evolutionary questions, chapter 1 presents the power of X chromosomes for evolutionary analysis, the limitations of this approach, and how the atypical system resolves these limitations. I first present both the value in comparing sex chromosomes to autosomes, and the challenges in interpreting data from these studies due to the co-occurrence of confounding differences between X chromosomes and autosomes— primarily the hemizygosity of the X chromosome in XY males, and the unequal transmission of X chromosomes through the sexes. I describe ways in which this confounded problem has been previously addressed, then introduce an atypical arthropod genetic system and explain how the system allows for the isolation of hemizygosity as a driver of X chromosome and autosomal difference. Finally, I discuss the implications of potential findings in evolutionary rate, sequence diversity, and codon bias. In Chapter 2, I employ the same exceptional genetic system in arthropods in comparison to relatives with typical genetic systems to test a very different question, namely the origins of haplodiploidy. In this chapter, I specifically investigate the origins of male-haploid systems and the relationship of gene-rich X chromosomes with sex-determination system turnover and find evidence to support intragenomic conflict as a driver of turnover. Intragenomic conflict is generally understudied and its effects on evolutionary processes, potentially substantial, remain largely untested. Thus, I offer some of the first empirical testing of its contribution to major genetic and evolutionary shifts. In Chapter 3, I study the history of a gene fusion of two enzymes involved in subsequent steps of fungal leucine biosynthesis, an essential metabolic process in fungi, through phylogenetic reconstruction on the fusion domains. I find a very complex history, featuring ancestral fusion, multiple instances of gene fission and loss, and abundant gain of the fused construct through horizontal gene transfer. This result is somewhat surprising given the core nature of these enzymes and longstanding paradigms of eukaryotic metabolic and genic evolution. Altogether, this doctoral body of work aims to demonstrate the value in studying exceptional genetic systems and evolutionary processes and how they are revealing for a broad set of important ideas in molecular evolution and genetics.

Nucleosomes in eukaryotes achieve the tradeoff between compact packaging for genome stability and inheritance, and accessibility for gene expression, by deploying histone variants with specialized chromatin functions. While some histone variants are highly conserved across eukaryotes, others have evolved to play crucial roles and carry out lineage-specific functions. The origins and functional roles of many histone variants remain largely unknown, and investigations into the evolution and functional roles of histone variants can provide a foundation for understanding the participation of chromatin in epigenetic memory and in critical cellular processes. This doctoral study furthers the understanding of the regulatory roles and evolutionary history of the mysterious histone H2A variants of C. elegans, namely, the ancient H2A.ZHTZ-1, sperm-specific HTAS-1, and HIS-35. This study describes the mechanism of the incorporation and dynamics of the H2A variants, HTAS-1 and HTZ-1, during spermatogenesis. It elucidates that the variant HTZ-1 is expressed throughout the male germline, whereas HTAS-1 is expressed in the proximal germline during later stages of sperm formation. HTZ-1 regulates transcription around the promoters during sperm formation, thus turning the gene On/OFF, whereas HTAS-1 may replace HTZ-1 during transcription and can provide sperm-specific re-packaging. Therefore, HTAS-1 may act for temporal gene regulation, as a replacement histone, for specific packaging of paternal info. The findings of the study suggest that in hermaphrodites, HTZ-1 activates germline genes during oocyte formation, whereas it represses sperm-specific genes during oocyte formation. This investigation also restructures the story of ‘how’ and ‘when’ the three H2A variants of C. elegans got evolved from their canonical counterparts. It is depicted that HTZ-1 is an ancestral histone which appeared long before the nematodes. HTAS-1, on the other hand, evolved fairly recently, and it was incorporated in the ancestor of Caenorhabditis, whereas HIS-35 evolved somewhere in the ancestor of Caenorhabditis and Diploscapter.

Spliceosomal introns, sequences interspersed throughout genes that are removed during mRNA production by machinery called the spliceosome, are a hallmark feature of eukaryotic genomes and gene structure. They are ancient genomic elements which can vary widely in number and size between species, and have remained a puzzling aspect of genome evolution in the decades since they were first discovered. To make the situation more complicated, there are in fact two separate spliceosomal systems, termed major (or U2) and minor (U12), which are responsible for the removal of ≥ 99.5% and less than half a percent of introns in most genomes, respectively. Minor introns in particular display a puzzling evolutionary pattern: in many cases, they are maintained with high degrees of conservation between deeply-diverged species, yet in others are either mostly or entirely missing. A large amount of the foundational work on minor introns was completed before the advent of next-generation sequencing, and almost all of the existing comparative genomics work in the field involves fewer than a dozen or so model organisms. In addition, the primary resources used by the field to identify minor introns are static databases containing information on a limited number of species. This dissertation contributes to our understanding of minor introns over the course of four distinct but related projects: First, it describes an effective and accessible method for identifying minor introns in intron sequence data, as well as the creation of a database containing the largest collection of minor intron orthology data available. Second, this dissertation highlights an extraordinary case of minor intron enrichment in a slime mold genome, reshaping our understanding of the limits of minor intron evolution. Third, it contains a sweeping analysis of minor intron diversity across more than 3000 eukaryotic genomes, uncovering a number of novel aspects of minor intron evolution and providing a rich substrate for future work. Finally, this dissertation describes a novel finding of a fungal genome with significant numbers of minor introns, and uses it to attempt to clarify a set of longstanding theories about the role of minor introns in eukaryotic evolution.

Background: Eukaryotic protein-coding genes are interrupted by spliceosomal introns, which are removed from transcripts before protein translation. Many facets of spliceosomal intron evolution, including age, mechanisms of origins, the role of natural selection, and the causes of the vast differences in intron number between eukaryotic species, remain debated. Genome sequencing and comparative analysis has made possible whole genome analysis of intron evolution to address these questions.

Results: We analyzed intron positions in 1,161 sets of orthologous genes across 25 eukaryotic species. We find strong support for an intron-rich fungus-animal ancestor, with more than four introns per kilobase, comparable to the highest known modern intron densities. Indeed, the fungus-animal ancestor is estimated to have had more introns than any of the extant fungi in this study. Thus, subsequent fungal evolution has been characterized by widespread and recurrent intron loss occurring in all fungal clades. These results reconcile three previously proposed methods for estimation of ancestral intron number, which previously gave very different estimates of ancestral intron number for eight eukaryotic species, as well as a fourth more recent method. We do not find a clear inverse correspondence between rates of intron loss and gain, contrary to the predictions of selection- based proposals for interspecific differences in intron number.

Conclusion: Our results underscore the high intron density of eukaryotic ancestors and the widespread importance of intron loss through eukaryotic evolution.

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