Multiple rounds of whole genome duplication (WGD) have played a pivotal role in the expansion, elaboration, and evolutionary diversification of vertebrate genomes. In addition to sharing two rounds of whole genome duplication with all other vertebrates, a teleost-specific genome duplication (TGD) occurred in the stem of the teleost lineage ~350 million years ago (MYA) and is thus a genomic synapomorphy shared by all ~26,000 extant species. The TGD has variously been implicated in accelerated speciation, evolution of morphological complexity, increased rates of molecular evolution, and the evolution of novelty, and therefore is therefore of significant interest for its impact on teleost evolution and also as a model for understanding the evolutionary patterns and processes which accompany WGDs more generally. Investigation of the TGD has contributed extensively to the general understanding of WGDs however, until the present work, a relatively narrow taxonomic sampling of species within a single teleost subdivision, Clupeocephala, have been investigated. This taxonomic bias has left potentially relevant evolutionary changes to the teleost genome in the immediate wake of the TGD obscured. Due to their deeply branching ancestry, species belonging to the two other major teleost subdivisions, Osteoglossomorpha and Elopomorpha, are well positioned for deeper comparative genomic analyses of the TGD and the accompanying phenomenon of diploidization. The focus of the present work has been to develop the first genomic resources specifically for osteoglossomorphs and to investigate the evolutionary patterns and processes which accompanied diploidization prior the deep divergence of the three extant teleost subdivisions. To this end, I have generated de novo genome and transcriptome data from four osteoglossomorph taxa (Pantodon buchholzi, Osteoglossum bicirrhosum, Chitala ornata, and Gnathonemus petersii) and conducted comparative genomic and phylogenetic analysis with other teleosts and pre-TGD vertebrates including the gar Lepisosteus oculeatus. With a focus on Hox and other ANTP class homeobox-containing transcription factor families I provide evidence that speciation of the major teleost subdivisions occurred prior to the termination of the diploidization process following TGD and discuss the evolutionary implications of this model. Beginning with an analysis of the Hox clusters in P. buchholzi I show that divergent resolution of TGD-generated Hox duplicates occurred both at the individual gene level as well as at the level of whole cluster losses. Detailed phylogenetic analyses of the P. buchholzi Hox clusters further revealed that the transition from polyploid alleles to full paralogs during the diploidization process can occur independently in different lineages when speciation rapidly follows WGDs, causing duplicated genes to exhibit a special case of four-way gene homology which I have termed 'tetralogy'. A genome-wide survey of ANTP class homeobox genes in a de novo assembly of the P. buchholzi genome revealed that ancient TGD duplicates of at least 14 subfamilies were preserved uniquely in the P. buchholzi genome and lost from clupeocephalan teleosts. Finally, by comparing the Hox complements in gar and P. buchholzi with three additional osteoglossomorphs I show that the diversity in potential duplicate resolution patterns is also highly variable between osteoglossomorph families. Overall, this work highlights the importance of considering not only the relative timing of gene duplication and speciation in comparative genomic analyses but also their timing relative to diploidization. Going forward, the research community will need to carefully evaluate the effects differences in diploidization rate and pattern, both between lineages and across the genome, have had in influencing the fate of individual gene duplicates as well as upon the macroevolutionary phenomena frequently correlated with WGDs more generally.

The C₄ photosynthetic pathway acts to concentrate CO₂ around the enzyme Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco), ensuring that it catalyses a carboxylation rather than oxygenation reaction, which in turn suppresses photorespiration. In nearly all cases C₄ photosynthesis is underpinned by characteristic Kranz anatomy, with concentric wreaths of bundle sheath (BS) and mesophyll (M) cells surrounding closely spaced veins. The increased yields associated with the C₄ pathway have lead to the suggestion that C₃ crops such as rice should be engineered to undertake C₄ photosynthesis, however, this goal is currently held back by a lack of understanding about how the development of Kranz anatomy is regulated. Recently, a number of candidate Kranz regulators have been identified in an RNA-seq study that compared leaf development in maize foliar (Kranz) and husk (non-Kranz) leaves. However, this study did not consider the impact of a recent whole genome duplication in the maize lineage on the gene expression patterns analysed. Therefore, in this thesis maize homeolog gene-pair divergence during early leaf development was assessed. This revealed that expression divergence of homeolog gene-pairs is a significant evolutionary phenomenon. Functional validation of a subset of Kranz candidates revealed that a Zmscr1-1; Zmscr1h-1 double mutant exhibited defects in Kranz patterning, including increased formation of extra BS cells and veins with no separating M cells. Furthermore, Zmnkd1; Zmnkd2 double mutants exhibited a subtle increase in extra BS cell formation. Taken together, this indicates that both ZmSCR1/ZmSCR1h and ZmNKD1/ZmNKD2 function redundantly during Kranz development. No evidence was obtained that two additional genes, ZmSHR2 and ZmRVN1, play a role in Kranz development, and expression of candidate Kranz regulators in rice did not alter leaf anatomy. Together, this work has confirmed roles for a number of genes in Kranz regulation, and has provided insight into the complex regulation underpinning Kranz development in maize.

With the proliferation of plant genomes being sequenced, assembled, and annotated, duplicate gene loss from whole genome duplication events, also known in plants as frac- tionation, has shown to have a different pattern from the classic gene duplication models described by Ohno in 1970. Models proposed more recently, the Gene Balance and Gene Dosage hypotheses, try to model this pattern. These models, however, disagree with each other on the relative importance of gene function and gene expression. In this thesis we explore the effects of gene function and gene expression on duplicate gene loss and retention. We use gene sequence similarity and gene order conservation to construct our gene fam- ilies. We applied multiple whole genome comparison methods across various plants in rosids, asterids, and Poaceae in looking for a general pattern. We found that there is great consistency across different plant lineages. Genes categorized as metabolic genes with low level of expression have relatively low fractionation resistance, losing duplicate genes readily, while genes categorized as regulation and response genes with high level of expression have relatively high fractionation resistance, retaining more duplicate gene pairs or triples. Though both gene function and gene expression have important effects on retention pattern, we found that gene function has a bigger effect than gene expression. Our results suggest that both the Gene Balance and Gene Dosage models account to some extent for fractionation resistance.

We propose a new, continuous model of the fractionation process (duplicate gene deletion after polyploidization) on the real line. The aim is to infer how much DNA is deleted at a time, based on segment lengths for alternating deleted (invisible) and undeleted (visible) regions. After deriving a number of analytical results for "one-sided" fractionation, we undertake a series of simulations that help us identify the distribution of segment lengths as a gamma with shape and rate parameters evolving over time. This leads to an inference procedure based on observed length distributions for visible and invisible segments. We suggest extensions of this mathematical and simulation work to biologically realistic discrete models, including two-sided fractionation.

For many years, researchers have studied the social lives of bacteria to understand intra- and inter-species interactions. Cell-cell communication, also known as quorum sensing (QS), is used by bacteria to coordinate their behaviour in response to environmental conditions. The QS system in Streptococcus species is well known to regulate competence. Studies show that Streptococcus pneumoniae has two homologous QS systems: 1) the competence (Com) system that regulates competence; and 2) a bacteriocin-like peptide (Blp) system that regulates the production of bacteriocins. Both functions are widespread in the genus. In S. pneumoniae, the Blp QS system shares a common ancestor and has similar features to the Com QS system. However, the evolutionary relationship between these QS systems remains obscure. SUCRE methodology was developed to identify the QS homologous genes in the streptococcal species. SUCRE uses four complementary approaches: homology search, putative gene finding, regulon construction, and evolutionary analysis. The performance of SUCRE was assessed in comparison with other orthology detection methods. SUCRE is precise in identifying the QS homologous genes and has similar performance to OrthoMCL. The QS system structures are found to be conserved across the streptococcal species. A streptococcal species phylogeny was constructed from the ribosomal and tRNA synthetase gene families. Using the QS genes identified from SUCRE and the streptococcal species phylogeny, the study infers the evolution of the QS systems in Streptococcus species. The study shows that the QS systems evolved as a regulon unit. The paralogous relationship between each of the QS systems suggests that duplication has a huge influence on functional divergence of the QS systems in the genus. Although, horizontal gene transfer (HGT) is commonly found in bacteria, little evidence is found to support that the effect of HGT on the functional divergence of the QS systems in this genus. However, the QS regulon genes of the same QS system are found to be non- vertically transferred across species that signifies that the HGT event promotes the sequence variation between these genes.

The Selection during Niche AdaPtation (SNAP) hypothesis aims to explain how the gene order in bacterial chromosomes can change as the result of bacteria adapting to a new environment. It starts with a duplication of a chromosomal segment that includes some genes providing a fitness advantage. The duplication of these genes is preserved by positive selection. However, the rest of the duplicated segment accumulates mutations, including deletions. This results in a rearranged gene order. In this work, we develop a method to identify SNAP in bacterial chromosomes. The method was tested in Salmonella and Bartonella genomes. First, each gene was assigned an orthologous group (OG). For each genus, single-copy panorthologs (SCPos), the OGs that were present in most of the genomes as one copy, were targeted. If these SCPos were present twice or more in a genome, they were used to build duplicated regions within said genome. The resulting regions were visualized and their possible compatibility with the SNAP hypothesis was discussed. Even though the method proved to be effective on Bartonella genomes, it was less efficient on Salmonella. In addition, no strong evidence of SNAP was detected in Salmonella genomes.

Gene prediction in newly sequenced genomes is a known challenging. Although sophisticated comparative pipelines are available, computationally derived gene models are often less than perfect. This is particularly true when multiple very similar paralogs are present. The issue is aggravated further when genomes are assembled only at a preliminary draft level to contigs or short scaffolds rather than to chromosomes. However, these genomes deliver valuable information for studying gene families. High accuracy models of protein-coding genes are needed in particular for phylogenetics and for the analysis of gene family histories. In this dissertation, I established a tool, the ExonMatchSolver-pipeline (EMS-pipeline), that can assist the assembly of genes distributed across multiple fragments (e.g. contigs). The tool in particular tackles the problem of identifying those coding exon groups that belong to the same paralogous genes in a fragmented genome assembly. The EMS-pipeline accommodates a homology search step with a protein input set consisting of several highly similar paralogs as query. The core of the pipeline uses an Integer Linear Programming Implementation to solve the paralog-to-contig assignment problem. An extension to the initial implementation estimates the number of paralogs encoded in the target genome and can handle several paralogs that are situated on the same genomic fragment. The EMS-pipeline was successfully applied to simulated data, several showcase examples and to deuterostome genomes in a large scale study on the evolution of the arrestin protein family. Especially at high genome fragmentation levels, the tool outperformed a naive assignment method. Arrestins are key signaling transducers that bind to activated and phosphorylated G protein-coupled receptors and can mediate their endocytosis into the cell. The refined annotations of arrestins resulting from the application of the EMS-pipeline are more complete and accurate in comparison to a conventional database search strategy. With the applied strategy it was possible to map the duplication- and deletion history of arrestin paralogs including tandem duplications, pseudogenizations and the formation of retrogenes in detail. My results support the emergence of the four arrestin paralogs from a visual and a non-visual proto-arrestin. Surprisingly, the visual ARR3 was lost in the mammalian clades afrotherians and xenarthrans. Segmental duplications in specific clades and the 3R-WGD in the teleost stem lineage, on the other hand, must have given rise to new paralogs that show signatures of diversification in functional elements important for receptor binding and phosphate sensing. The four vertebrate orthology groups show an interesting pattern of divergence of three endocytosis motifs: the minor and major clathrin binding site and the adapter protein-2 (AP-2) binding motif. Identification of such signatures, of residues that determine specificity between paralogs and are positively selected after duplication was made possible by high quality alignments obtained by genome inquiries, dense species sampling and consideration of fragmented loci from poorly assembled genomes in the framework of the EMS-pipeline, that was established in this dissertation.:1 Introduction 2 1.1 Basics and definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1.1 What is a gene? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.1.2 What is a tree in phylogenetics? . . . . . . . . . . . . . . . . . . 3 1.1.3 What are paralogs and orthologs? . . . . . . . . . . . . . . . . . 4 1.1.4 Central dogma in molecular biology: From DNA to protein . . 5 1.2 Gene duplications as evolutionary playground . . . . . . . . . . . . . . 12 1.2.1 Mechanisms of gene duplication . . . . . . . . . . . . . . . . . . 13 1.2.2 Evolutionary fate of duplicated genes . . . . . . . . . . . . . . . 14 1.3 Identification and annotation of protein homologs . . . . . . . . . . . . 15 1.3.1 Challenges of existing resources . . . . . . . . . . . . . . . . . . 16 1.3.2 Similarity search approaches without consideration of the gene structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 1.3.3 Gene structure aware gene annotation approaches . . . . . . . . 19 1.3.4 Graph-based inference of orthology relationships . . . . . . . . 21 1.3.5 Chance and challenge of fragmented assemblies . . . . . . . . . 21 1.4 Applied phylogenetic methods . . . . . . . . . . . . . . . . . . . . . . . 22 1.4.1 Phylogenetic inference in a nutshell . . . . . . . . . . . . . . . . 23 1.4.2 Inference of natural selection in inter-species data sets . . . . . 29 1.4.3 Detection of specificity determining positions . . . . . . . . . . 32 1.5 Multi-talents in cell signaling: The cytosolic arrestin proteins . . . . . . 34 1.5.1 Functions of arrestins in cell signaling . . . . . . . . . . . . . . . 34 1.5.2 Arrestin activation by GPCR binding . . . . . . . . . . . . . . . 36 1.5.3 Functions of arrestins in cellular trafficking . . . . . . . . . . . . 37 1.5.4 Evolution of arrestins . . . . . . . . . . . . . . . . . . . . . . . . 39 2 The ExonMatchSolver-pipeline 42 2.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 2.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.1 Pipeline overview . . . . . . . . . . . . . . . . . . . . . . . . . . 43 2.2.2 Exon assembly as an assignment problem . . . . . . . . . . . . . 43 2.2.3 Solving the Paralog-to-Contig Assignment Problem . . . . . . . 46 2.2.4 Post-processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.2.5 Implementation and usage . . . . . . . . . . . . . . . . . . . . . 48 2.2.6 Performance assessment by simulations . . . . . . . . . . . . . . 50 2.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2.3.1 Performance on simulated data . . . . . . . . . . . . . . . . . . . 50 2.3.2 Performance on real data - Two Showcase Examples . . . . . . . 51 2.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 3 Evolution of the arrestin protein family in deuterostomes 61 3.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 3.2 Material and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.2.1 Database scan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3.2.2 Detailed gene annotation . . . . . . . . . . . . . . . . . . . . . . 63 3.2.3 Data resources used in the current study . . . . . . . . . . . . . 64 3.2.4 Alignment and building of phylogenetic trees . . . . . . . . . . 64 3.2.5 Identification of specificity determining positions . . . . . . . . 65 3.2.6 Testing for natural selection . . . . . . . . . . . . . . . . . . . . . 66 3.2.7 Assessement of conservation . . . . . . . . . . . . . . . . . . . . 66 3.2.8 Parsimonious reconstruction of exon gain and loss events . . . 67 3.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 3.3.1 Evolution of the arrestin fold family based on database inquiries 67 3.3.2 The refined arrestin annotations are more complete than database entries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 3.3.3 Arrestin paralog gain and loss patterns based on the refined annotations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73 3.3.4 Evolution of arrestin functional elements . . . . . . . . . . . . . 88 3.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 3.4.1 Limitation of arrestin database annotations . . . . . . . . . . . . 96 3.4.2 Arrestins in early vertebrate evolution . . . . . . . . . . . . . . . 98 3.4.3 Sub- and neofunctionalization as consequence of the 3R-WGD . 102 3.4.4 Independent arrestin duplications in deuterostomes . . . . . . . 104 3.4.5 Loss of arrestin paralogs in different vertebrate orders . . . . . 106 3.4.6 Previously unknown interaction partners and isoforms . . . . . 108 4 Improvements on the ExonMatchSolver-pipeline 110 4.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 4.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 4.2.1 Estimation of the paralog number . . . . . . . . . . . . . . . . . 111 4.2.2 Subdivision of gene loci on the same contig . . . . . . . . . . . . 113 4.2.3 Implementation details . . . . . . . . . . . . . . . . . . . . . . . 113 4.2.4 Assessment of the ExonMatchSolver-pipeline Version 2 . . . 115 4.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 4.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 5 Conclusion and Outlook 119 A Additional figures 123 B Additional tables 134 C CV 152 Bibliography 156

The ParaHox cluster contains three Hox‐related homeobox genes. The evolution of this sister of the Hox‐gene clusters has been studied extensively in metazoans with a focus on its early evolution. Its fate within the vertebrate lineage, and in particular following the teleost‐specific genome duplication, however, has not received much attention. Three of the four human ParaHox loci are linked with PDGFR family tyrosine kinases. We demonstrate that these loci arose as duplications in an ancestral vertebrate and trace the subsequent history of gene losses. Surprisingly, teleost fishes have not expanded their ParaHox repertoire following the teleost‐specific genome duplication, while duplicates of the associated tyrosine kinases have survived, supporting the hypothesis of a large‐scale duplication followed by extensive gene loss.

The application of Y-chromosome analysis is expanding in fields such as forensic science and genealogy. By researching the potential polymorphisms this chromosome can present, we can further our ability to assess DNA profiles for these disciplines to avoid erroneous exclusions of paternal linkage, wrongful convictions based on forensic evidence, and other misinformed genetic conclusions. The conservation of Y-haplotypes during transmission occurs due to a relative lack of genetic recombination events in the inheritance of the Y-chromosome [1]. However, random mutation events can occur in a paternal line resulting in haplotype changes. These changes can include allele duplications and deletions that occur at the STR and SNP loci used in forensic DNA analysis. This can become important in cases of sexual assault where male-female mixture samples have low amounts of male DNA such that the male signal is not amplified in currently used STR multiplexes [7]. In this study, we analyzed a father and his eleven sons using two different methodologies for genetic analysis; next generation sequencing and capillary electrophoresis. The samples were obtained from the Coriell Institute for Medical Research located in Hamden, NJ, in the form of frozen DNA extracts isolated from a blood-sourced lymphocyte cell culture [22]. DNA from these samples was tested with the ForenSeqTM DNA Signature Prep Kit [14] (Verogen, San Diego, CA) primer set A and the YFilerTM Plus PCR Amplification Kit [24] (Thermo Fisher Scientific, Waltham, MA). Using these two platforms, three Y-STR loci were identified as discordant between the father and all of his eleven sons. In all three instances, the father possessed the same allele as the sons as well as one additional allele. At two of these loci (DYS449 and DYS635), the additional allele was one repeat (4bp) longer than that of the shared allele. At the other locus (DYS458), the additional allele was three repeats (12bp) longer than that of the shared allele. Following read count and peak height analysis, it was concluded that these double allele loci are not the product of stutter and are potentially the product of a non-inheritable mutation. With the knowledge that the DNA was extracted from a blood lymphocyte cell culture, it is believed that a somatic mutation may be present in the cell line. We are not able to determine whether the mutations exist in the blood of the father (true somatic mutations) or occurred as a result of the cell culture process. Throughout the study, details concerning the position of these loci on the Y-chromosome, the repeat motifs of the alleles, and the potential for duplication and/or stutter as the originating event are discussed in an effort to further understand this phenomenon. Potential locus duplications were compared to those reported on the National Institute of Standards and Technology STRBase [21] list of allele variations and also to information found in literature. The observed DYS635 locus had an allele designation of 21,22 which is reported on STRBase. The DYS449 and DYS458 loci showed potential allele-specific locus duplications that were not found on STRBase. The implications of potentially undocumented non-inheritable allele patterns in the Y-chromosome, such as this, are significant when considering comparisons between DNA obtained from germline cells (sperm) versus a known casework sample which is usually obtained from blood or saliva [7].

Partial circuit replication is a soft error mitigation technique that uses redundant copies of a circuit to mask or detect the effects of soft errors. By masking or detecting the effect of soft errors on SRAM-based FPGAs, implemented circuits can be made more reliable. The technique is applied selectively, to only a portion of the components within a circuit. Partial application lowers the cost of implementation. The objective of partial circuit replication is to provide maximal benefit at limited or minimized cost. The greatest challenge of partial circuit replication is selecting which components within a circuit to replicate. This dissertation advances the state of the art in the effective use of partial circuit replication for masking and detecting soft errors in SRAM-based FPGAs. It provides a theoretical foundation in which the expected benefits and challenges of partial circuit replication can be understood. It proposes several new selection approaches for identifying the most beneficial areas of a circuit to replicate. These approaches are applied to two complex FPGA-based computer networking systems and another FPGA design. The effectiveness of the selection approaches are evaluated through fault injection and accelerated radiation testing. More benefit than expected is obtained through partial circuit replication when applied to critical components and sub-regions of the designs. In one example, in an open-source computer networking design, partial circuit replication masks and detects approximately 70% of failures while replicating only 5% of circuit components, a benefit-cost ratio of 14.0.