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Accurate estimation of divergence times of soil bacteria that form nitrogen-fixing associations with most leguminous plants is challenging because of a limited fossil record and complexities associated with molecular clocks and phylogenetic diversity of root nodule bacteria, collectively called rhizobia. To overcome the lack of fossil record in bacteria, divergence times of host legumes were used to calibrate molecular clocks and perform phylogenetic analyses in rhizobia. The 16S rRNA gene and intergenic spacer region remain among the favored molecular markers to reconstruct the timescale of rhizobia. We evaluate the performance of the random local clock model and the classical uncorrelated lognormal relaxed clock model, in combination with four tree models (coalescent constant size, birth–death, birth–death incomplete sampling, and Yule processes) on rhizobial divergence time estimates. Bayes factor tests based on the marginal likelihoods estimated from the stepping-stone sampling analyses strongly favored the random local clock model in combination with Yule process. Our results on the divergence time estimation from 16S rRNA gene and intergenic spacer region sequences are compatible with age estimates based on the conserved core genes but significantly older than those obtained from symbiotic genes, such as nodIJ genes. This difference may be due to the accelerated evolutionary rates of symbiotic genes compared to those of other genomic regions not directly implicated in nodulation processes.
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