Abstract
All individuals in an evolving population compete for resources, and their performance is measured by a fitness metric. The performance of the individuals is relative to their abilities and to the biotic surroundings--the conditions under which they are competing--and involves many components. Molecules evolving in a test tube can also face complex environments and dynamics, and their fitness measurements should reflect the complexity of various contributing factors as well. Here, the fitnesses of a set of ligase ribozymes evolved by the continuous in vitro evolution system were measured. During these evolution cycles there are three different catalytic steps, ligation, reverse transcription, and forward transcription, each with a potential differential influence on the total fitness of each ligase. For six distinct ligase ribozyme genotypes that resulted from continuous evolution experiments, the rates of reaction were measured for each catalytic step by tracking the kinetics of enzymes reacting with their substrates. The reaction products were analyzed for the amount of product formed per time. Each catalytic step of the evolution cycle was found to have a differential incidence in the total fitness of the ligases, and therefore the total fitness of any ligase cannot be inferred from only one catalytic step of the evolution cycle. Generally, the ribozyme-directed ligation step tends to impart the largest effect on overall fitness. Yet it was found that the ligase genotypes have different absolute fitness values, and that they exploit different stages of the overall cycle to gain a net advantage. This is a new example of molecular niche partitioning that may allow for coexistence of more than one species in a population. The dissection of molecular events into multiple components of fitness provides new insights into molecular evolutionary studies in the laboratory, and has the potential to explain heretofore counterintuitive findings.