Rhodopsin, a visual pigment protein found in retinal photoreceptors, mediates vision at low-light levels. Recent studies focusing primarily in human and mouse have challenged the assumption of neutral evolution of synonymous substitutions in mammals. Using recently developed likelihood-based codon models accounting for mutational bias and selection, we find significant evidence for selective constraint on synonymous substitutions in mammalian rhodopsins, and a preference for cytosine at 3rd codon positions. A second project investigated adaptive evolution in rhodopsin, in view of theories of nocturnality in early mammals. We detected a significant acceleration of non-synonymous substitution rates at the origins of therian mammals, and a tendency of synonymous substitutions towards C-ending codons prior to that. These findings suggest an evolutionary scenario in which synonymous substitutions that increase mRNA stability and/or translation efficiency may have preceded adaptive non-synonymous evolution in early mammalian rhodopsins. These findings have important implications for theories of early mammalian nocturnality.

Auf der Basis von Fossilien wird angenommen, dass die ersten Säugetiere nachtaktiv waren. Diese Arbeit untersucht diese Hypothese mit bioinformatischen und molekularbiologischen Techniken. Der Fokus liegt auf dem Rhodopsin, ein Sehpigment im Wirbeltierauge, das für Sehen unter schlechten Lichtverhältnissen verantwortlich ist. Zunächst wurde das Rhodopsin der monotrematen Echidna, einem basalen Säugetier, sequenziert und mit zwei Mutanten mit Mutationen an Positionen 158 und 169 in vitro exprimiert. Die biochemische und funktionelle Charakterisierung ergab, dass das Echidna-Rhodopsin farbpigment-typische Charakteristika aufweist, was auf eine Expression auch in Zapfen hindeutet. Dies ist die erste Charakterisierung eines Rhodopsins eines nachtaktiven Tieres. Dann wurden anzestrale Rhodopsinsequenzen für die Knotenpunkte Amniota, Mammalia und Theria mithilfe der Maximum-Likelihood-Methode berechnet. Die in vitro Expression und biochemische und funktionelle Charakterisierung zeigt funktionale und rhodopsin-typische Sehpigmente. Das Mammalia- und Theria-Rhodopsin zeigen eine hohe Meta II Halbwertszeit. Dieses Ergebnis wird als eventuelle Anpassung an Sehen unter schlechten Lichtverhältnissen interpretiert, wobei, aufgrund von Unstimmigkeiten in der Literatur, Schlussfolgerungen auf ökologisch-bedingte Anpassungen basierend auf einzelnen Funktionstests problematisch sind, da die visuelle Signalkaskade ein sehr komplexes und durch viele Proteine vernetztes System darstellt. Zuletzt zeigen Selektionsanalysen, dass das Rhodopsin entlang der Theria-Linie positive Selektion auf nicht-synonyme Substitutionen erfahren hat, was zu Anpassungen in einem Protein führt. Der Fossilbericht belegt entlang dieser Linie mehrere Einnischungsevents in neue Lebensräume. Entlang der Mammalia-Linie wurde positive Selektion auf synonyme Substitutionen gemessen, was zu einer Zunahme an Rhodopsin-Molekülen führt und damit eine Anpassung an Sehen unter schlechten Lichtverhältnissen darstellt. / Based on information from the fossil record, the first mammals are thought to have been nocturnal. This thesis investigates this popular hypothesis using bioinformatic and molecular techniques, focusing on the rhodopsin, a visual pigment in the vertebrate eye that is responsible for vision at low-light levels. First, the rhodopsin gene of the monotreme echidna, a basal mammal, was sequenced and successfully expressed in vitro, together with two mutants with substitutions at sites 158 and 169. Biochemical and functional analyses revealed that the echidna rhodopsin displays cone-like characteristics, likely due to being also expressed in cones. With the echidna being nocturnal, this thesis comprises the first characterisation of a rhodopsin of a nocturnal animal. Second, ancestral rhodopsin sequences for the tetrapod nodes Amniota, Mammalia, and Theria were inferred using Maximum likelihood estimates. All expressed pigments were successfully expressed in vitro, functional and rod-like. Mammalia and Theria rhodopsins display a high meta II half life time, a pattern that is usually interpreted to facilitate better vision at low-light levels. However, due to inconsistency in the available data, the result also suggests that, with the visual signaling cascade being a complex and interconnected system, erecting ecological interpretations based on single biochemical and functional reactions is problematic. Third, selective constraint analyses performed on a set of tetrapod rhodopsin sequences indicate that positive selection on non-synonymous sites, was acting along the branch leading to Theria. This result reflects the rapid diversification into modern ecological habitats during the Triassic and Jurassic, as indicated by the fossil record. In addition, positive selection on synonymous sites, leading to an increase of rhodopsin molecules, was found along the branch leading to Mammalia and suggests adaptations to vision at low-light levels.

Rhodopsin, a visual pigment protein found in retinal photoreceptors, mediates vision at low-light levels. Recent studies focusing primarily in human and mouse have challenged the assumption of neutral evolution of synonymous substitutions in mammals. Using recently developed likelihood-based codon models accounting for mutational bias and selection, we find significant evidence for selective constraint on synonymous substitutions in mammalian rhodopsins, and a preference for cytosine at 3rd codon positions. A second project investigated adaptive evolution in rhodopsin, in view of theories of nocturnality in early mammals. We detected a significant acceleration of non-synonymous substitution rates at the origins of therian mammals, and a tendency of synonymous substitutions towards C-ending codons prior to that. These findings suggest an evolutionary scenario in which synonymous substitutions that increase mRNA stability and/or translation efficiency may have preceded adaptive non-synonymous evolution in early mammalian rhodopsins. These findings have important implications for theories of early mammalian nocturnality.