Cyprinodon variegatus is a pupfish that inhabits the Atlantic coast of North America, nearly continuously, from Massachusetts to Belize. This research attempts to resolve the phlylogeography of C. variegatus by investigating the genetic sequence structure of the mitochondrial control region (D-loop, non-coding origin of replication) and cytochrome-b gene for evidence of northern and southern subspecies within the Atlantic Coast of the eastern United States. Additionally, it will be may be possible to determine if secondary hybrid zones developed as a result of the last retreat of ice from North America during the Pleistocene, about 17,000 years ago. A definitive monophyletic northern clade was found using parsimony, distance, and maximum likelihood phylogenetic methods to analyze the control region data. The cytochrome-b sequence data supported this monophyletic northern clade, although their utility for this analysis is marginal. Little evidence was found for the existence of a hybrid zone. More thorough sampling will be needed to make more accurate determinations regarding the existence of such a zone. / Master of Science

The study of the process of speciation is instrumental to understanding the species diversity observed today. Diverging populations are intriguing, because speciation has not reached an endpoint, yet the process that may eventually lead to distinct species can be studied. Systems that contain many putative species and/or parallel divergences, such as many species flocks and species pairs, are extraordinary examples of divergence and therefore are critical to the understanding of the speciation process. A "miniature" species flock of pupfish (Cyprinodon variegatus) discovered in lakes on San Salvador Island, Bahamas has evolved in less than 6 000 years, and is, therefore, important to the study of the pace of evolutionary processes. The San Salvador Island pupfish flock is composed of a normal form, which resembles coastal C. variegatus, and bulldog and bozo morphs, which diverge ecologically and morphologically from the normal morph. In Chapter 1, I sequenced the mtDNA control region and used haplotype frequency analyses to assess the level of differentiation between sympatric normals and bulldogs sampled from Osprey Lake and Little Lake on San Salvador Island. The bozo morph was too rare to include in the study. I also included samples of normals that occur in lakes without bulldog and bozo morphs to assess any differences between lakes on the island. All haplotype frequency comparisons for sympatric normals and bulldogs were highly significant, which suggests these morphs are distinct populations in sympatry and, therefore, have characteristics of biological species. Further, an estimation of Time for Speciation supports geological data that suggest this fauna is very young (6 000 years). The San Salvador Island pupfish species flock is, therefore, the youngest known species flock and presents an important model system for the study of how morphological and ecological divergence can promote speciation in Cyprinodon. In Chapter 2, I first compared the San Salvador Island pupfishes to other Bahamian C. variegatus populations to assess the level of inter- and intra-island pupfish population differentiation in the Bahamas. The mtDNA control region was sequenced for bulldogs and normals from San Salvador Island and normals sampled from New Providence and Exuma Islands. San Salvador Island bulldogs were found to be distinct from all normal populations sampled, and comparisons of shared haplotypes suggest they originated on San Salvador Island rather than any of the other islands sampled. This was intriguing, because a "bulldog-like" morph has recently been observed in a lake on New Providence Island, which suggests parallel divergences may be occurring throughout the Bahamas. I also sequenced the mtDNA cytochrome b gene to assess the phylogeography of C. variegatus. Populations were sampled from the Bahamas and the east coast of North America, and the results suggest the Bahamas were only recently colonized by the Southern coastal lineage of C. variegatus. A distinct Northern lineage of C. variegatus, which may warrant species designation, was also supported by the cytochrome b data. Overall, the results supported a San Salvador Island origin for the Little Lake and Osprey Lake bulldog morphs, and also suggest the Bahamian C. variegatus populations are very young. / Master of Science

The microbiome plays a key symbiotic role in maintaining host health and aids in acquiring nutrients, supporting development and immune function, and modulating behavior. However, more research is needed to elucidate the potential impact of environmental pollutants on host microbial communities and how microbiomes can modulate the toxicity of contaminants to the host. Through a literature review of 18 studies that assessed the impacts of various anthropogenic chemicals on fish-associated microbiomes, we found that toxicants generally decrease microbial diversity, which could lead to long-term health impacts if chronically stressed, and can increase the host’s susceptibility to disease as well as the chemical resistance of certain microbes. These findings led us to explore the impacts of one of the reviewed contaminants, polycyclic aromatic hydrocarbons (PAHs), typically found in oil. The Deepwater Horizon disaster of April 2010 was the largest oil spill in U.S. history and had catastrophic effects on several ecologically important fish species in the Gulf of Mexico (GoM). This study tested the hypotheses that exposure to weathered oil would cause significant shifts in fish gut-associated microbial communities, with taxa known for hydrocarbon degradation increasing in abundance and that foraging behavior would decrease, potentially due to microbial dysbiosis via the gut-brain axis. We characterized the gut microbiome (with 16S rRNA gene sequencing) of a native GoM estuarine species, the sheepshead minnow (Cyprinodon variegatus). Fish were exposed to High Energy Water Accommodated Fractions (HEWAF; tPAH = 80.99 ± 12.5 μg/L) of oil over a 7-day period and whole gastrointestinal tracts were sampled for microbiome analyses. A foraging behavioral assay was used to determine feeding efficiency before and after oil exposure. The fish gut microbiome did not experience any significant changes in alpha or beta diversity but known hydrocarbon degrading taxa were noticeably present in oil-exposed communities and were absent in controls. We found the order Pseudomonadales, the family Paenibacillaceae, and Pseudomonas pachastrellae to be among these, with Pseudomonadales increasing in abundance. Foraging behavior was not significantly affected by oil exposure. This work highlights the need for further research to elucidate the functional metagenomic responses of the fish gut-microbiome under oil spill conditions.

<div>Following the 2010 Deepwater Horizon oil spill, extensive research has been conducted on the toxicity of oil and polycyclic aromatic hydrocarbons (PAHs) in the aquatic environment. The location and timing of the Deepwater Horizon surface slick coincided with the spawning seasons of many important pelagic and estuarine fish species. As such, there has been particular emphasis placed on the effects of PAHs on sensitive life history stages in fish, such as the embryonic and larval periods. Additionally, the spill occurred throughout the spring and summer months which, in estuaries, are marked by regular fluctuations in abiotic environmental factors such as dissolved oxygen, salinity, and temperature. Until recently, there has been little work done to elucidate the combined effects that PAHs from oil spills and adverse environmental conditions (hypoxia, increased salinity, and elevated temperatures).</div><div>Work presented in this dissertation uses next generation sequencing technology (RNA Seq) to determine differential gene expression in larval estuarine teleosts following exposure to adverse environmental conditions and PAHs. Downstream canonical pathway and toxicological function analysis were then applied to the identified differentially expressed genes (DEGs) to predict cardiotoxic responses at the organismal level. To verify the predicted responses, a phenotypic anchoring study was conducted and identified a cardiotoxic phenotype (pericardial edema) and reduced cardiac output in embryos exposed to oil. Finally, the mechano-genetic interplay governing the morphological development of the teleost heart was investigated and correlations between developmental gene expression and blood flow forces within the cardiovascular system were identified.</div>