Genetic variation and evolutionary divergence within and among populations, species, and genera of the Cambarinae

Seven crayfish species from three genera of the subfamily Cambarinae were electrophoretically examined for genetic variation at a total of twenty-six loci. Polymorphism was detected primarily at three loci: Ao-2, Lap, and Pgi. The average heterozygosities over-all loci for each species were found to be very low when compared to most other invertebrate species that have been examined electrophoretically. With the exception of Cambarus bartoni, the interpopulation genetic identities are high within any given species. The average interspecific identities are somewhat lower and the average intergeneric identities are lower still. Populations, species and genera conform to the expected taxonomic progression. The two samples of ~ bartoni show high genetic similarity at only 50 percent of the loci compared. Locus by locus identity comparisons among species yield U-shaped distributions of genetic identities. Construction of a phylogenetic dendrogram using species mean genetic distances values shows that species grouping is in agreement with morphological taxonomy with the exception of the high similarity between Orconectespropinquus and Procambarus pictus. This high similarity suggests the possibility of a regulatory change between the two species. It appears that the low heterozygosities, high interpopulation genetic identities, and taxonomic mispositioning can all be explained on the basis of low mutation rates.

Alternative splicing of DNA polymerase beta in vertebrates

Alternative splicing (AS) is the predominant mechanism responsible for increasing eukaryotic transcriptome and proteome complexity. In this phenomenon, numerous mRNA transcripts are produced from a single pre-mRNA sequence. AS is reported to occur in 95% of human multi-exon genes; one specific gene that undergoes AS is DNA polymerase beta (POLB). POLB is the main DNA repair gene which performs short patch base excision repair (BER). In primate untransformed primary fibroblast cell lines, it was determined that the splice variant (SV) frequency of POLB correlates positively with species lifespan. To date, AS patterns of POLB have only been examined in mammals primarily through the use of cell lines. However, little attention has been devoted to investigating if such a relationship exists in non-mammals and whether cell lines reflect what is observed in vertebrate tissues. This idea was explored through cloning and characterization of 1,214 POLB transcripts from four non-mammalian species (Gallus gallus domesticus, Larus glaucescens, Xenopus laevis, and Pogona vitticeps) and two mammalian species (Sylvilagus floridanus and Homo sapiens) in two tissue types, liver and brain. POLB SV frequency occurred at low frequencies, < 3.2%, in non-mammalian tissues relative to mammalian (>20%). The highest POLB SV frequency was found in H. sapiens liver and brain tissues, occurring at 65.4% and 91.7%, respectively. Tissue specific AS of POLB was observed in L. glaucescens, P. vitticeps, and H. sapiens, but not G. gallus domesticus, X. laevis and S. floridanus.The AS patterns of a second gene, transient receptor potential cation channel subfamily V member 1 (TRPV1), were compared to those of POLB in liver and brain tissues of G. gallus domesticus, X. laevis and H. sapiens. This comparison was performed to investigate if any changes (either increase or decrease) observed in the AS of POLB were gene specific or if they were tissue specific, in which case similar changes in AS would be seen in POLB and TRPV1. Analysis did not reveal an increase or decrease in both the AS of POLB and TRPV1 in either the liver or brain tissues of G. gallus domesticus and H. sapiens. This result suggested that the AS patterns of POLB were not influenced by tissue specific rates of AS. Interestingly, an increase in the AS of both genes was only observed in X. laevis brain tissue. This result suggests that AS in general may be increased in the X. laevis brain as compared to liver tissue. No positive correlation between POLB SV frequency and species lifespan was found in non-mammalian tissues. The AS patterns of POLB in human primary untransformed fibroblast cell lines were representative of those seen in human liver tissue but not in brain tissue. Altogether, the AS patterns of POLB from vertebrate tissues and primate cell lines revealed a positive correlation between POLB SV frequency and lifespan in mammals, but not in non-mammals. It appears that this positive correlation does not exist in vertebrate species as a whole.

HUMAN GENOME VARIATIONS AND EVOLUTION WITH A FOCUS ON THE ANALYSIS OF TRANSPOSABLE ELEMENTS

Genome sequence varies in numerous ways among individuals although the gross architecture is fixed for all humans. Retrotransposons create one of the most abundant structural variants in the human genome and are divided in many families, with certain members in some families, e.g., L1, Alu, SVA, and HERV-K, remaining active for transposition. Along with other types of genomic variants, retrotransponson-derived variants contribute to the whole spectrum of genome variants in humans. With the advancement of sequencing techniques, many human genomes are being sequenced at the individual level, fueling the comparative research on these variants among individuals. In this thesis, the evolution and functional impact of structural variations is examined primarily focusing on retrotransposons in the context of human evolution. The thesis comprises of three different studies on the topics that are presented in three data chapters. First, the recent evolution of all human specific AluYb members, representing the second most active subfamily of Alus, was tracked to identify their source/master copy using a novel approach. All human-specific AluYb elements from the reference genome were extracted, aligned with one another to construct clusters of similar copies and each cluster was analyzed to generate the evolutionary relationship between the members of the cluster. The approach resulted in identification of one major driver copy of all human specific Yb8 and the source copy of the Yb9 lineage. Three new subfamilies within the AluYb family – Yb8a1, Yb10 and Yb11 were also identified, with Yb11 being the youngest and most polymorphic. Second, an attempt to construct a relation between transposable elements (TEs) and tandem repeats (TRs) was made at a genome-wide scale for the first time. Upon sequence comparison, positional cross-checking and other relevant analyses, it was observed that over 20% of all TRs are derived from TEs. This result established the first connection between these two types of repetitive elements, and extends our appreciation for the impact of TEs on genomes. Furthermore, only 6% of these TE-derived TRs follow the already postulated initiation and expansion mechanisms, suggesting that the others are likely to follow a yet-unidentified mechanism. Third, by taking a combination of multiple computational approaches involving all types of genetic variations published so far including transposable elements, the first whole genome sequence of the most recent common ancestor of all modern human populations that diverged into different populations around 125,000-100,000 years ago was constructed. The study shows that the current reference genome sequence is 8.89 million base pairs larger than our common ancestor’s genome, contributed by a whole spectrum of genetic mechanisms. The use of this ancestral reference genome to facilitate the analysis of personal genomes was demonstrated using an example genome and more insightful recent evolutionary analyses involving the Neanderthal genome. The three data chapters presented in this thesis conclude that the tandem repeats and transposable elements are not two entirely distinctly isolated elements as over 20% TRs are actually derived from TEs. Certain subfamilies of TEs themselves are still evolving with the generation of newer subfamilies. The evolutionary analyses of all TEs along with other genomic variants helped to construct the genome sequence of the most recent common ancestor to all modern human populations which provides a better alternative to human reference genome and can be a useful resource for the study of personal genomics, population genetics, human and primate evolution.

The Cenozoic Gonyaulacacean Dinoflagellate Genera Operculodinium Wall, 1967 and Protoceratium Bergh, 1881 and Their Phylogenetic Relationships

The palynology of Ocean Drilling Program Site 1007, leeward of the present Bahamas Bank, provides insights into upper Oligocene–lower Pleistocene dinoflagellate cyst associations in the tropical Americas. These associations are reviewed along with the sedimentary paleoenvironment to provide context for a morphological study of the cystdefined dinoflagellate Operculodinium bahamense and its comparison with the thecadefined dinoflagellate Protoceratium reticulatum which produces a cyst assignable to the cyst-defined genus Operculodinium. Detailed reconstructions of the tabulation in both species reveal strong similarities, having a sexiform hyposomal tabulation and L-type or modified L-type ventral organization. Protoceratium reticulatum has dextral torsion of the hypotheca, requiring assignation of the genus to the subfamily Cribroperidinioideae, whereas Operculodinium bahamense has neutral torsion requiring assignation to the subfamily Leptodinioideae. Results either imply polyphyletic origins for the genus Operculodinium or that combinations of ventral organization and torsion cannot always be applied rigidly to subdivide the family Gonyaulacaceae.