Evolutionary Dynamics of Asexual Hypermutators Adapting to a Novel Environment

GBE Evolutionary Dynamics of Asexual Hypermutators Adapting to a Novel Environment Wei-Chin Ho 1,*, Megan G. Behringer1,2,3, Samuel F. Miller1, Jadon Gonzales1, Amber Nguyen1, Meriem Allahwerdy1, Gwyneth F. Boyer1, and Michael Lynch1,* 1 Center for Mechanisms of Evolution, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA 2 Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, USA Department of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, Tennessee, USA *Corresponding authors: E-mails: ; . Accepted: 14 November 2021 Abstract How microbes adapt to a novel environment is a central question in evolutionary biology. Although adaptive evolution must be fueled by beneficial mutations, whether higher mutation rates facilitate the rate of adaptive evolution remains unclear. To address this question, we cultured Escherichia coli hypermutating populations, in which a defective methyl-directed mismatch repair pathway causes a 140-fold increase in single-nucleotide mutation rates. In parallel with wild-type E. coli, populations were cultured in tubes containing Luria-Bertani broth, a complex medium known to promote the evolution of subpopulation structure. After 900 days of evolution, in three transfer schemes with different population-size bottlenecks, hypermutators always exhibited similar levels of improved fitness as controls. Fluctuation tests revealed that the mutation rates of hypermutator lines converged evolutionarily on those of wild-type populations, which may have contributed to the absence of fitness differences. Further genome-sequence analysis revealed that, although hypermutator populations have higher rates of genomic evolution, this largely reflects strong genetic linkage. Despite these linkage effects, the evolved population exhibits parallelism in fixed mutations, including those potentially related to biofilm formation, transcription regulation, and mutation-rate evolution. Together, these results are generally inconsistent with a hypothesized positive relationship between the mutation rate and the adaptive speed of evolution, and provide insight into how clonal adaptation occurs in novel environments. Key words: adaptation, bottleneck effects, drift barrier, Escherichia coli, mutational load, mutation rate. Significance Although mutations are a critical source for the adaptation in a new environment, whether or not elevated mutation rates can lead to elevated adaptation rates remains unclear, especially when the environment is heterogeneous. To address this issue, we evolved E. coli populations with different starting mutation rates in a complex medium for 900 days and then examined their fitness and genome profiles. In the populations with higher starting mutation rates, despite faster rates of genome evolution, fitness improvement is not significantly elevated, and most of the accumulated mutations represent passive consequences of linkage effects. Introduction Beneficial mutations are the ultimate source of adaptive evolution. Therefore, it is of interest to study how changes to mutational processes can influence the pace of adaptive processes. In terms of mutation rates, a theoretically complicated relationship with the rate of adaptation in asexual populations was proposed in the early studies on the evolution of sex (Muller 1932; Crow and Kimura 1965). These studies posited that, when mutation rates are low, such that the waiting time ß The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact Genome Biol. Evol. 13(12) https://doi.org/10.1093/gbe/evab257 Advance Access publication 19 November 2021 1 3 GBE Ho et al. 2 (McDonald et al. 2012). Thus, it remains to be determined whether the relationship between rates of mutation and adaptation in richer and more complex environments follows the same patterns as observed in simpler settings. To study how the mutation rate affects adaptation in a more complex setting, we investigated the long-term evolutionary changes of Escherichia coli grown in culture tubes containing a complex medium, Luria-Bertani (LB) broth, comprised a nutritionally rich mixture of multiple amino-acid based carbon sources (Sezonov et al. 2007). In contrast to evolution in flasks containing glucose-limited media, such environments can facilitate the rapid emergence of stable subpopulations and clonal divergence based on spatial niche differentiation and amino-acid metabolism divergence (Behringer et al. 2018). To vary the mutation rate, we evolved both WT populations (methyl-directed mismatch repair [MMR]þ) and hypermutator populations with an impaired MMR pathway (MMR, obtained by mutL knockout), for which the single-nucleotide mutation rate is 140-fold higher than that for the WT genetic background (Lee et al. 2012). As different population-genetic environments can alter the fixation probability of mutations and the proportion of effectively beneficial or deleterious mutations (Wahl et al. 2002), and different demographic settings have been found to affect the results of experimental evolution (Vogwill et al. 2016; Wein and Dagan 2019), for both WT and MMR populations, we performed three different transfer sizes in daily transfers: 1/10 (large, L), 1/104 (medium, M), and 1/107 (small, S) to explore the generality of our experimental results. Here, we examine the differences in phenotypic and molecular evolution among these populations over the course of 900 days. Results Higher Initial Mutation Rates Do Not Lead to Faster Rates of Fitness Improvement When batch cultured, E. coli commonly adapt to their experimental environments and show fitness improvement compared with their ancestors (Van den Bergh et al. 2018; McDonald 2019). To study fitness improvement in populations originating from genetic backgrounds with different initial mutation rates (MMR and WT), we performed the experimental evolution in three different transfer dilution bottlenecks (L: 1/10, M: 1/104, and S: 1/107) for 900 days (supplementary fig. S1, Supplementary Material online). For each of six genetic background/transfer-size combinations (2  3), eight replicates were maintained. After 900 days of experimental evolution, we then performed head-to-head competition assays between evolved populations and their corresponding ancestors using four replicates. Across all 21 populations with data available (three were aborted; see Materials and Methods), mean fitness significantly increased relative to the time-zero ancestor, by a ratio of 1.14 (standard Genome Biol. Ev (...truncated)