Unlike most other vertebrates, many species in the genus Etheostoma do not utilize structural refraction to display blue or green color. Instead, blue and green mating coloration exhibited by male rainbow darters (E. caeruleum) and male greenside darters (E. blennioides) results from the presence of true chromoprotein pigments. This study was conducted in order to extract, purify, characterize, and compare these novel pigments. Pigments were extracted in aqueous buffer and partially purified by ammonium sulfate fractionation and gel filtration chromatography. Final purification consisted of preparative non-denaturing polyacrylamide gel electrophoresis for E. caeruleum and hydroxyapatite chromatography for E. blennioides. Isolation of the chromophore was accomplished using acetone precipitation. The chromophore is the same in both species and is believed to be biliverdin. The protein component differs between the species and appears to have a greater number of subunits in E. blennioides. Binding of the protein to the chromophore amplifies the absorbance in the visible region and causes spectral tuning of the absorbance profile of the chromophore, with slight differences between species. In E. caeruleum, the chromoprotein pigment has a lambda max of 683 nm and transmits light at slightly shorter wavelengths, causing it to appear blue. In E. blennioides, the chromoprotein pigment has a lambda max of 696 nm and transmits light at slightly longer wavelengths, causing it to appear green. This work has shown that the protein component, not the chromophore, is responsible for the difference in hue between these two pigments. Future work will involve obtaining amino acid sequences for the protein component of the pigments and ultimately sequencing the gene coding for these proteins in darters. / Bayer School of Natural and Environmental Sciences; / Environmental Science and Management (ESM) / MS; / Thesis;

The greenside darter, Etheostoma blennioides (Teleostomi: Percidae), is a wide-ranging polytypic taxon that occurs throughout eastern North America. A previous morphological study recognized four subspecies (blennioides, newmanii, gutselli, and pholidotum), several morphological races, and three zones of morphological intergradation. We generated complete cytochrome b (1140 bp) sequence data for 51 individuals from across the range of the greenside darter inclusive of all of the currently recognized taxa to assess genetic variation and taxonomic boundaries. Both maximum parsimony and mixed model Bayesian analyses resulted in two strongly supported deeply divergent clades including (1) a Tennessee River drainage clade, and (2) an Ohio River and Great Lakes basins, Interior Highlands, and Atlantic slope clade. Etheostoma blennius, a closely related congener, nested within the Tennessee River clade of E. blennioides, rendering the complex paraphyletic. Test of alternative topologies failed to support the current taxonomic designations. The inclusion of nuclear sequence data from intron 1 of the S7 ribosomal protein (523 bp) from a subset of the populations was included to independently test whether the currently recognized taxa conform to distinct evolutionary lineages and also to clarify potential issues associated with ancestral hybridization. Although the nuclear data was less variable than the mitochondrial data, the monophyly of several of the subspecies could not be rejected.