Illuminating the laws that produce Darwin's 'tangled bank' remains one of the great challenges of biology, one that requires understanding how differences among forms are selected for, and how interdependence among forms is generated. Amongst microbial communities, be they pathogenic or natural, interdependence is surprisingly common, but evidence that it allows biocomplexity to arise and be sustained is so far lacking. Our vision is to use a systems biology approach to dramatically extend our understanding of the genetic and environmental factors that drive biological innovation, community structuring and interdependence within microbial communities. Addressing these issues in natural environments is complicated by the fact that selection pressures often vary widely over space and time. Laboratory evolution experiments offer an alternative by which to study, under controlled conditions, both the interplay between genotype and phenotype as well as the interactions among phenotypes in simple communities. Our experiments combine the power of next-generation sequencing with the precision of resource-limited chemostats, using the model organism Escherichia coli. Here we seek a Planetary Major Equipment award to augment this experimental system with fluorescence cytometry and cell sorting capability. This stand-alone Planetary Major Equipment grant will further empower our parent Exobiology grant, NNX12AD87G (PROJECT 1), and enable us to accelerate towards our goal of answering five fundamental questions: What causes mutualism to evolve? How clonal is a 'clonal population?' Are cooperative interactions driven more by structural than by regulatory mutations? Does cooperation create communities that are more energetically efficient and/or more ecologically stable than those driven by competition? Are mutualisms that have a long co-evolutionary history more stable than those having a short history? Equipped with fluorescence cytometry and cell sorting capability, Montana researchers will be able to more rapidly advance towards the goals of other NASA-supported investigations aimed at understanding how biocomplexity emerges in the forms of new species (PROJECT 2), multicellularity (PROJECT 3) and mitochondria (PROJECT 4).The instrumentation we seek in this PME is the Partech CyFlow Space Cytometer/Sorter and CyPad high speed sampler. The configuration we have chosen (3 lasers+365 nm UV-LED) will enable us to detect and sort diverse fluorescent protein (FP) tags, to perform live-dead analysis, and to carry out a variety of DNA analyses by which we can ascertain cell cycle status, apoptosis, genome size and ploidy. The size range of objects that can be counted and sorted using the CyFlow greatly exceeds, for example, Coulter counters, inasmuch as it can size and separate objects as small as viruses and as large as clusters of eukaryotic cells. Unlike conventional sorters, which produce aerosols and require biosafety cabinets, the CyFlow is a totally closed fluidic system that can be operated on the benchtop. This instrument package includes an autoloader that can accommodate 96-well plates as well as sample tube racks. Because FP signals in fixed cells are stable over 24 h, we can accumulate multiple samples per day from multiple experiments, count these overnight, and thereby monitor experiments' progress in real time. Anticipated uses of this instrument include real-time FP-based monitoring of genotypes in evolving cell cultures, FP-based live sorting of individual clones in mixed continuous culture (PROJECT 1), analysis of ploidy, viability and individual chromosome copy number (PROJECT 2), live sorting of morphological variants (PROJECT 3), and live sorting of symbionts that exhibit co-localization of different FP signals. The footprint of this instrument is small and can easily be accommodated on the benchtop in the PI's lab.