The diversity of multicellular organisms is, in large part, due to the fact that multicellularity has independently evolved many times. Nonetheless, multicellular organisms all share a universal biophysical trait: cells are attached to each other. All mechanisms of cellular attachment belong to one of two broad classes; intercellular bonds are either reformable or they are not. Both classes of multicellular assembly are common in nature, having independently evolved dozens of times. In this review, we detail these varied mechanisms as they exist in multicellular organisms. We also discuss the evolutionary implications of different intercellular attachment mechanisms on nascent multicellular organisms. The type of intercellular bond present during early steps in the transition to multicellularity constrains future evolutionary and biophysical dynamics for the lineage, affecting the origin of multicellular life cycles, cell–cell communication, cellular differentiation, and multicellular morphogenesis. The types of intercellular bonds used by multicellular organisms may thus result in some of the most impactful historical constraints on the evolution of multicellularity.

During the biofilm life cycle, bacteria attach to a surface and then reproduce, forming crowded, growing communities. Many theoretical models of biofilm growth dynamics have been proposed; however, difficulties in accurately measuring biofilm height across relevant time and length scales have prevented testing these models, or their biophysical underpinnings, empirically. Using white light interferometry, we measure the heights of microbial colonies with nanometer precision from inoculation to their final equilibrium height, producing a detailed empirical characterization of vertical growth dynamics. We propose a heuristic model for vertical growth dynamics based on basic biophysical processes inside a biofilm: diffusion and consumption of nutrients and growth and decay of the colony. This model captures the vertical growth dynamics from short to long time scales (10 min to 14 d) of diverse microorganisms, including bacteria and fungi.

During the evolution of multicellularity, cells undergo an evolutionary transition in individuality, such that groups become the subject of Darwinian evolution. Comparative work, supported by theory, suggests that a life cycle in which cells 'stay together' following cellular division (termed clonal development) should facilitate this transition. While central to our understanding of multicellular evolution, this hypothesis has never been directly tested in a single experimental system. We circumvent this limitation by creating an isogenic yeast system capable of either clonal or aggregative development. We evolved 20 populations of either clonally-reproducing 'snowflake' yeast or aggregative 'floc' yeast with daily selection for rapid growth followed by sedimentation, an environment where multicellularity is adaptive. While both genotypes adapted to this regime, growing faster and having higher survival during the group-selection phase, there was a stark difference in evolutionary dynamics. Competitions reveal that evolved floc obtained nearly all of their increased fitness from faster growth, not improved group survival, while snowflake yeast mainly benefited from higher group-dependent fitness. Through a combination of genome sequencing and mathematical modeling, we identify a trade-off: clonal development can allow selection to act more efficiently on group-beneficial traits, but dramatically increases the overall rate of genetic drift due to mutational bottlenecking. Our results demonstrate how simple differences in the mode of group formation can have profound impacts on the transition to multicellularity: clonal development, but not aggregation, precipitated a transition from cells to groups as the primary level of Darwinian individuality.

During the biofilm life cycle, bacteria attach to a surface then reproduce, forming crowded, growing communities. As these colonies develop, they expand horizontally and vertically. This horizontal expansion, known as the range expansion, occurs at a constant rate, which is often used as a proxy for bacterial fitness. Conversely, the vertical growth of biofilms is much less studied, despite representing a fundamental aspect of bacterial physiology. Many theoretical models of vertical growth dynamics have been proposed; however, difficulties in measuring biofilm height accurately across relevant time and length scales have prevented testing these models or their biophysical underpinnings empirically. Using white light interferometry, we measure the heights of microbial colonies with nanometer precision from inoculation (sub-micron) to their final equilibrium height (hundreds of microns), producing a novel and detailed empirical characterization of vertical growth dynamics. We show that models relying on logistic growth or nutrient depletion fail to capture biofilm height dynamics on short and long time scales. Our empirical results support a simple model inspired by the fact that biofilms only interact with the environment through their interfaces. This interface model captures the growth dynamics from short to long time scales (10 minutes to 14 days) of diverse microorganisms, including prokaryotes like gram-negative and gram-positive bacteria and eukaryotes like aerobic and anaerobic yeast. This model provides heuristic value, highlighting the biophysical constraints that limit vertical growth as well as establishing a quantitative model for biofilm development.

Many microbial communities are characterized by intense competition for nutrients and space. One way for an organism to gain control of these resources is by eliminating nearby competitors. The Type VI Secretion System (T6) is a nano-harpoon used by many bacteria to inject toxins into neighboring cells. While much is understood about mechanisms of T6-mediated toxicity, little is known about the ways that competitors can defend themselves against this attack, especially in the absence of their own T6. Here we use directed evolution to examine the evolution of T6 resistance, subjecting eight replicate populations of Escherichia coli to T6 attack by Vibrio cholerae. Over ~500 generations of competition, the E. coli evolved to survive T6 attack an average of 27-fold better than their ancestor. Whole genome sequencing reveals extensive parallel evolution. In fact, we found only two pathways to increased T6 survival: apaH was mutated in six of the eight replicate populations, while the other two populations each had mutations in both yejM and yjeP. Synthetic reconstruction of individual and combined mutations demonstrate that yejM and yjeP are synergistic, with yejM requiring the mutation in yejP to provide a benefit. However, the mutations we identified are pleiotropic, reducing cellular growth rates, and increasing susceptibility to antibiotics and elevated pH. These trade-offs underlie the effectiveness of T6 as a bacterial weapon, and help us understand how the T6 shapes the evolution of bacterial interactions.