Rhinichthys osculus, the Speckled Dace, is one of the most ubiquitous fish in western North America. Within the Southern California region, the local taxon is known as the Santa Ana Speckled Dace. The purpose of this study was to characterize and identify polymorphic microsatellite markers for R. osculus in which twenty-three were identified through Illumina pair-end sequencing. Seven of these loci were then used to examine the patterns of genetic variation and population structure that occurred within and among the watersheds in the Southern California. The study also examined the regional relationships among Southern California, Central California and Owen’s River Valley. Analysis of the microsatellite data revealed highly significant moderate levels of population structure exist within the Southern California region (RST=0.160, p=0.001). This structure is best explained by watershed as well as isolation by distance (R2=.2286, p=0.010). Highly significant geographic structure also exists among the geographic regions of Southern California, Central Coast, and Owen’s River Valley regions (RST= 0.600, p-value=0.001) that are congruent with the regional differentiation elucidated by mtDNA sequence data. In both cases, the degree of population differentiation was correlated with isolation by distance. Utilizing this information we were able gain a better understanding of the evolutionary relationships among the Southern California populations of Santa Ana Speckled Dace. Within the Santa Ana Speckled Dace populations we examined four models to explain the geographic structure: watershed, mountain range, tributary, and isolation by distance. While all were significant, the tributary model exhibited the higher level of population structure (RST= 0.160, p-value=0.001) and a significant correlation was exhibited between geographic distance and population structure, suggesting isolation by distance may be playing a role. The results of the microsatellite analysis are congruent with an earlier broad scale analysis of mtDNA sequence data that suggests the Central California and the Owens Valley populations diverged from each other prior to the divergence of the Santa Ana Speckled Dace populations from the Colorado Basin populations, and that the Central Coast populations were not established as a result of a migration event from the Southern California populations, as was previously hypothesized. Primarily due to human activity, Santa Ana Speckled Dace habitat has become highly fragmented resulting in some populations becoming extirpated. We hope this study will guide the strategies for the conservation of the remaining populations of Santa Ana Speckled Dace and watershed management in Southern California.

In this study, I sequenced the mitochondrial cytochrome b gene to elucidate the extent and pattern of genetic variations among and within populations of Rhinichthys osculus (Santa Ana Speckled Dace) found in the different watersheds in Southern California, Central California Coast and Eastern California Desert. I described and analyzed the structural characteristics and pattern of base sequence substitutions in the cytochrome b gene to understand the molecular evolution of the gene. The Phylogenetic analyses showed that the Southern California Santa Ana Speckled dace is a distinct population from the Central California Coast dace population and Eastern California Desert dace population, and is more closely related to the Colorado River speckled dace population. There is a high degree of genetic variation among all populations including a significant genetic structure associated with watersheds, mountain ranges, and geographic grouping based on locations. The SWISS-Model automated protein structure homology elucidated the conserved and invariant residues within the cytochrome b gene where the amino acid substitutions are located in the trans-membrane of the protein sequence. The implication for conservation and management of the Southern California Santa Ana Speckled dace is high because of habitat lost for this distinct dace population. The data from this study will contribute to preserving the genetic variability of the Santa Ana Speckled Dace as a separate taxa and species, as well as to help maintain intact the population in the different Southern California creeks.