Estimating the Divergence Times of Alphaproteobacteria Based on Mitochondrial Endosymbiosis and Eukaryotic Fossils.

Alphaproteobacteria is one of the most abundant bacterial lineages that successfully colonize diverse marine and terrestrial environments on Earth. In addition, many alphaproteobacterial lineages have established close association with eukaryotes. This makes Alphaproteobacteria a promising system to test the link between the emergence of ecologically important bacteria and related geological events and the co-evolution between symbiotic bacteria and their hosts. Understanding the timescale of evolution of Alphaproteobacteria is key to testing these hypotheses, which is limited by the scarcity of bacterial fossils, however. Based on the mitochondrial endosymbiosis which posits that the mitochondrion originated from an alphaproteobacterial lineage, we propose a new strategy to estimate the divergence times of lineages within the Alphaproteobacteria by leveraging the fossil records of eukaryotes. In this chapter, we describe the workflow of the mitochondria-based method to date Alphaproteobacteria evolution by detailing the software, methods, and commands used for each step. Visualization of data and results is also described. We also provide related notes with background information and alternative options. All codes used to build this protocol are made available to the public, and we strive to make this protocol user-friendly in particular to microbiologists with limited practical skills in bioinformatics.