Bacteria adopt a wide variety of sizes and shapes, with many species exhibiting stereotypical morphologies. How morphology changes, and over what timescales, is less clear. Previous work examining cell morphology in an experiment with Escherichia coli showed that populations evolved larger cells and, in some cases, cells that were less rod-like. That experiment has now run for over two more decades. Meanwhile, genome sequence data are available for these populations, and new computational methods enable high-throughput microscopic analyses. In this study, we measured stationary-phase cell volumes for the ancestor and 12 populations at 2,000, 10,000, and 50,000 generations, including measurements during exponential growth at the last time point. We measured the distribution of cell volumes for each sample using a Coulter counter and microscopy, the latter of which also provided data on cell shape. Our data confirm the trend toward larger cells while also revealing substantial variation in size and shape across replicate populations. Most populations first evolved wider cells but later reverted to the ancestral length-to-width ratio. All but one population evolved mutations in rod shape maintenance genes. We also observed many ghost-like cells in the only population that evolved the novel ability to grow on citrate, supporting the hypothesis that this lineage struggles with maintaining balanced growth. Lastly, we show that cell size and fitness remain correlated across 50,000 generations. Our results suggest that larger cells are beneficial in the experimental environment, while the reversion toward ancestral length-to-width ratios suggests partial compensation for the less favorable surface area-to-volume ratios of the evolved cells.

IMPORTANCE Bacteria exhibit great morphological diversity, yet we have only a limited understanding of how their cell sizes and shapes evolve and of how these features affect organismal fitness. This knowledge gap reflects, in part, the paucity of the fossil record for bacteria. In this study, we revived and analyzed samples extending over 50,000 generations from 12 populations of experimentally evolving Escherichia coli to investigate the relation between cell size, shape, and fitness. Using this “frozen fossil record,” we show that all 12 populations evolved larger cells concomitant with increased fitness, with substantial heterogeneity in cell size and shape across the replicate lines. Our work demonstrates that cell morphology can readily evolve and diversify, even among populations living in identical environments.