Flatfishes (Pleuronectiformes), the commercially important soles, flounders, and halibuts, exhibit one of the most remarkable vertebrate metamorphoses, where one eye of a bilaterally (left-right) symmetrical larva migrates to the opposite side of the head, resulting in highly asymmetrical juvenile and adult forms. Despite the complexity and uniqueness of this transformation, virtually nothing is known about its evolutionary origin or the mechanism behind the change. Flatfishes are the only vertebrates to deviate so dramatically from a bilaterally symmetrical body plan, making them an ideal group to conduct the first comparative studies of regulatory genes and anatomical structures associated with this body asymmetry. Through analyses of DNA sequence data and MRI images we aim to resolve the evolutionary relationships of flatfishes, examine the evolution of developmental regulatory genes involved in left-right axis formation and assess the origins of structures in the head hypothesized to be unique to flatfishes and to have evolved as adaptations to a life on the sea floor. Examination of anatomical structures associated with vertebrate asymmetry and of the molecular mechanisms generating this bizarre anatomy will provide for a novel interpretation of the evolution of bilateral asymmetry.
Although adult flatfishes (order Pleuronectiformes) start out in life as bilaterally symmetrical (left and right sides are the same like humans) larvae, they undergo a remarkable metamorphosis, where one eye of the symmetrical larva migrates to the opposite side of the head, resulting in highly asymmetrical juvenile and adult forms. Because all flatfishes exhibit this bizarre morphology and variation, both the degree of asymmetry and handedness (direction of eye migration) exists within the order, this group provides multiple tests of hypotheses regarding the evolution of bilateral asymmetry and underlying mechanisms. Unfortunately, undertaking such studies has been elusive because of three major issues confounding flatfish evolutionary studies: 1) relationships of the major groups within the order remain mostly unresolved, 2) the closest living relative of flatfishes is unknown, and 3) monophyly (where there is one origin of the entire group) of the order is weakly supported. To resolve these issues in flatfish tree-of-life research, my dissertation research has focused on: 1) testing if there is one common ancestor, and origin, to the flatfishes and sister-group hypotheses and 2) resolving relationships within the order, re-examining characters of adult morphology and comparing them to often overlooked larval characters in light of new evolutionary trees. Once these evolutionary trees are generated it is possible to test hypotheses regarding the evolution of flatfish metamorphosis and the evolutionary shift from a left-right symmetrical body plan. Therefore, the final goal of this project was to test whether the developmental regulatory genes that determine normal left-right symmetry in all vertebrates are turned on during flatfish metamorphosis. In the first study new genes were identified and sequenced along with rho and rnf213 for 58 flatfishes and 90 putative close relatives to test monophyly, within-order relationships and sister-group hypotheses. Those sequences along with data from a previous study are analyzed to determine possible causes for phylogenetic error. I discovered that the new genes provide large amounts of much needed data and when combined with the others and analyzed simultaneously, provide overwhelming support for a single origin of the flatfishes. Additionally, I demonstrate that abundant missing data is likely the cause of the difficulty in determining the origin of flatfishes, validate the importance of investigating often overlooked causes of error and discuss the certain patterns of relationships as a cause of error at the base of the Carangimorpha (the larger group that the flatfishes are nested within) tree of life. In the next study, characters of adult anatomy were combined with new larval characters and used to determine if life history can predict the evolutionary tree for flatfishes. Further, I investigated the accuracy of statistical analyses to determine if these morphological characters provide additional support for hypotheses of relationships among major flatfish groups. My results suggest that larval characters should not be treated as a source of independent data, but do provide resolution and additional support for novel evolutionary relationships within flatfishes. Also shown was that because larval characters are similar among all flatfishes, and that larval anatomy is similar to that of the possible closest relative, these characters are a potential source of evidence needed to resolve the placement of this group within the larger evolutionary tree for spiny-rayed fishes. Finally, these evolutionary trees are being used to test whether the same genes that all vertebrates use to generate bilaterally symmetrical embryos are used to generate asymmetrical flatfishes. A profile of all the genes that are turned on, or expressed, in the heads of larval flatfishes undergoing metamorphosis were generated for six larvae from 3 families and one adult as a control. Currently, the results of the analysis of those data suggest that the same genes are re-expressed in flatfish larvae and are used to generate both a symmetrical larvae and asymmetrical adults.
