The phylogenetic position of Xenoturbella bocki has been a matter of controversy since its description in 1949. We sequenced a second complete mitochondrial genome of this species and performed phylogenetic analyses based on the amino acid sequences of all 13 mitochondrial protein-coding genes and on its gene order. Our results confirm the deuterostome relationship of Xenoturbella. However, in contrast to a recently published study (Bourlat et al. in Nature 444:85–88, 2006), our data analysis suggests a more basal branching of Xenoturbella within the deuterostomes, rather than a sister-group relationship to the Ambulacraria (Hemichordata and Echinodermata).

The thermodynamics of RNA-RNA interaction consists of two components: the energy necessary to make a potential binding region accessible, i.e., unpaired, and the energy gained from the base pairing of the two interaction partners. We show here that both components can be efficiently computed using an improved variant of RNAup. The method is then applied to a set of bacterial small RNAs involved in translational control. In all cases of biologically active sRNA target interactions, the target sites predicted by RNAup is in perfect agreement with literature. In addition to prediction of target site location, RNAup can be also be used to determine the mode of sRNA action. Using information about target site location and the accessibility change resulting form sRNA binding we can discriminate between positive and negative regulators of translation.

Toll-like receptors (TLRs) play important role in the innate immune system. TLR15 is reported to have a unique role in defense against pathogens, but its structural and evolution characterizations are still poorly understood. In this study, we identified 57 completed TLR15 genes from avian and reptilian genomes. TLR15 clustered into an individual clade and was closely related to family 1 on the phylogenetic tree. Unlike the TLRs in family 1 with the broken asparagine ladders in the middle, TLR15 ectodomain had an intact asparagine ladder that is critical to maintain the overall shape of ectodomain. The conservation analysis found that TLR15 ectodomain had a highly evolutionarily conserved region on the convex surface of LRR11 module, which is probably involved in TLR15 activation process. Furthermore, the protein-protein docking analysis indicated that TLR15 TIR domains have the potential to form homodimers, the predicted interaction interface of TIR dimer was formed mainly by residues from the BB-loops and αC-helixes. Although TLR15 mainly underwent purifying selection, we detected 27 sites under positive selection for TLR15, 24 of which are located on its ectodomain. Our observations suggest the structural features of TLR15 which may be relevant to its function, but which requires further experimental validation.

Toll-like receptors (TLRs) are important pattern recognition receptors in the innate immune system of fish. Although ten years have passed since the first identification, the systematic knowledge about fish-specific TLR19 is still far insufficient. In present study, a phylogenetic analysis showed that TLR19 belonged to family 11, and clustered with TLR20 and TLR11/12 on the evolutionary tree. TLR20 is the closest paralogue of TLR19. The ectodomain of TLR19 contains 24 leucine-rich repeat (LRR) modules. The electrostatic surface potential analysis indicated that the modeled structure of TLR19 ectodomain showed much stronger polarity on the ascending lateral surface than on the descending lateral surface. The ascending lateral surface with strong electrostatic surface potential possibly mainly participates in the ligand binding of TLR19 ectodomain. The quite small dN/dS value at the TLR19 locus showed that TLR19 was very conserved. Approximately one third codons in the coding sequence of TLR19 were subjected to significantly negative selection, whereas only 5 codons underwent significantly positive selection. Overall, these findings possibly help in deepening the understanding to fish-specific TLR19.

Toll-like receptors (TLRs) are critical components of the innate immune response of fish. In a phylogenetic analysis, TLR27 from three fish species, which belongs to TLR family 1, clustered with TLR14/18 and TLR25 on the evolutionary tree. The ectodomain of TLR27 is predicted to include 19 leucine-rich repeat (LRR) modules. Structural modeling showed that the TLR27 ectodomain can be divided into three distinctive sections. The lack of conserved asparagines on the concave surface of the central subdomain causes a structural transition in the middle of the ectodomain, forming a distinct hydrophobic pocket at the border between the central subdomain and the C-terminal subdomain. We infer that, like other functionally characterized TLRs in family 1, the hydrophobic pocket located between LRR11 and LRR12 participates in ligand recognition by TLR27. An evolutionary analysis showed that the dN/dS value at the TLR27 locus was very low. Approximately one quarter of the total number of TLR27 sites are under significant negatively selection pressure, whereas only two sites are under positive selection. Consequently, TLR27 is highly evolutionarily conserved and probably plays an extremely important role in the innate immune systems of fishes.

Composed of a chromophore bound to an integral membrane protein (opsin), visual pigments are phenotypically characterized by the wavelength of maximal absorption (λmax). The underlying molecular mechanism controlling λmax is the interaction between the opsin amino acid sequence and the chromophore. While a plethora of studies have looked at structure/function relationships in vertebrate opsins, fewer studies have investigated similar issues in invertebrates. Furthermore, those few studies undertaken in invertebrate systems suggest different mechanisms of spectral tuning and photoactivation compared to vertebrate systems. This dissertation research is focused on expanding our knowledge of opsin evolution in invertebrate systems, particularly from non-insect taxa. First, issues related to opsin evolution and the maintenance of supposedly ‘non-functional’ genes were explored in a review of regressive and reverse evolution. Second, in order to place studies of crustacean opsin evolution in context, phylogenetic studies of two crustacean groups (Mysidae and Decapoda) were completed. Studies of Mysidae utilized 16S mtDNA, and 18S and 28S rDNA to reconstruct phylogenetic relationships and assess newly developed Bayesian methods of assessing pattern heterogeneity. Using this suite of genetic markers, there are incongruencies between current taxonomy and inferred phylogenetic relationships. Studies of Decapoda assessed phylogenetic relationships and estimated divergence times using 16S mtDNA, H3 nDNA, and 18S and 28S rDNA sequence data in conjunction with a set of eight fossil calibrations. Reconstructed phylogenies show support for two well supported nodes corresponding to the Pleocyemata and the informal ‘Reptantia’ and place the emergence of the Decapod lineage in the early Devonian (407 MYA. Finally, opsin sequences and spectral sensitivity data from species within the Mysidae and Decapoda were combined with previously characterized invertebrate sequences to investigate opsin evolution. Standard dN/dS methods did not detect any evidence of selection. Methods investigating selection on amino acid properties, however, identified four properties (coil tendencies, compressibility, power to be at the middle of the alpha helix, and refractive index) to be under positive destabilizing selection. These properties occurred mostly at sites in transmembrane helices and included residues previously identified to affect spectral tuning as well as identifying novel sites.

The seed storage globulins found among virtually all spermatophytes comprise a multi-gene family of proteins with ancient evolutionary origins. The two main groups of storage globulins include the legumins (11S) and vicilins (7S), both of which play a main role in protein deposition and storage in the seed endosperm. Composed of two cupin domains (bicupin), these proteins have been recently noted not only for their close structural relationships among the two subfamilies (7S and 11S) but also for their similarity to other proteins such as germin-like proteins (GLP's), bacterial oxalate decarboxylases, and other cupin containing proteins. Previous studies have investigated the evolutionary relationships among the legumin and vicilin groups, as well as their presumed evolutionary link to other cupin containing proteins; however these have each come short of any comprehensive resolved evolutionary history of the globulin family. This study focuses first on resolving the relationships among the cupin super-family in relation to the storage globulins, as well as the GLP's, which have been postulated to be the single domain ancestors of the bicupin storage globulins. Nucleotide coding sequences for both N-terminus and C-terminus cupin domains of the storage globulins, including conserved non-cupin domain helical repeats and inter-domain spacers were aligned to a comparably sized set of single cupin coding sequences (CDS). The phylogenetic relationships among the two globulin domains as well as the single cupin genes were elucidated using Bayesian inference of tree likelihoods. Further phylogenetic analysis was performed on the complete CDS's for all storage globulin sequences in the study, using an appropriate out-group of similar overall domain architecture determined by the overall topology of the cupin super-family. This globulin muti-gene tree was used, along with an alignment corresponding to structurally resolved portions of the mature globulin peptides, to perform an analysis of patterns of selection among the various lineages of cupin-containing globulins. The results of these analyses provide evidence for a common origin of all cupin containing genes. The GLP and storage globulin domains do not appear to be immediate ancestors of one another, but are grouped with the fungal spherulins as well, suggesting that the single cupin genes which gave rise to these groups had already diverged prior to the rise of land plants. The storage globulin gene tree provides evidence supporting the notion that true legumins and vicilins were recruited as seed storage proteins independent of one another, after their divergence. This is evidenced by the fact that they comprise two separate groups each with basal non-storage 11S/7S-like proteins. Additional insight into the differentiating selection pressures provides a clearer picture of how similar suites of physicochemical properties came under selection after the recruitment of the 11S and 7S families as seed specific proteins. Regions under strong destabilizing selection correspond to regions known to be of importance in the overall structure of storage globulins. Strong destabilizing selection at the pore of the globulin subunit suggests that this region may have undergone more functional diversification than previously thought to have occurred among the legumins and vicilins.

Picornaviruses are responsible for some of the most common and debilitating diseases affecting humans and animals worldwide. The objectives of this dissertation research were (1) estimate phylogenetic relationships among 11 picornavirus genera and within three species: foot-and-mouth disease virus (FMDV: Aphthovirus) which afflicts cloven-hoofed animals and human rhinovirus A and B (HRV: Enterovirus) which cause the common cold; (2) better understand the impact recombination has on genomic organization and evolution; (3) characterize where positive and purifying selection has occurred in proteins and how selection has influenced phenotype. The dissertation includes four studies. The first chapter provides an overview of the evolutionary significance of recombination, its detection and estimation, and its effect on phylogenetic analysis in four biological systems: bacteria, viruses, mitochondria, and the human genome. Chapter two investigates the inter- and intra-serotypic relationships within FMDV by examining 12 genes. Gene sequences were analyzed to assess recombination breakpoint locations, genetic diversity, and natural selection in FMDV. Recombination breakpoints were located throughout the genome. Paraphyletic relationships among serotypes were not as prevalent as previously reported, suggesting that convergent evolution was prevalent. Purifying selection was the dominant evolutionary force influencing both genotype and phenotype. Chapter three examines inter- and intra-specific relationships of HRV using 11 genes. Similar hypotheses were tested as in chapter two. No recombination was detected and phylogenetic relationships among enteroviruses, HRV-A, and HRV-B remain unresolved. The evolution of HRV-A major serotypes appeared to be under extensive purifying selection, HRV-A minor serotypes under predominantly positive selection, and a nearly equal influence from both kinds of selection was evident for HRV-B serotypes. Chapter four examines phylogenetic relationships among genera using three conserved genes. The hypothesis of cospeciation between picornaviruses and their hosts was also tested. The deepest split in the family separated Hepatovirus, ‘Tremovirus’, Parechovirus, and seal picornavirus type 1 from the remainder of the family. Enterovirus and ‘Sapelovirus’ were sister taxa. Cardiovirus, ‘Senecavirus’, Aphthovirus, Erbovirus, Teschovirus, and Kobuvirus were derived from a common ancestor with Kobuvirus occupying a basal position relative to the other genera in this clade. My analyses suggest that picornaviruses have not cospeciated with their known hosts.

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