Final Report Summary - MICROSOCIOGENOMYX (Quantifying the roles that evolutionary forces play in shaping genomic and social divergence in natural populations of the cooperative bacterium Myxococcus xanthus.) The project proposed sought to quantify the roles that the key forces of evolution – de novo mutation, selection, genetic drift, hitchhiking, dispersal, and recombination – play in shaping genomic and social divergence in natural populations of the model cooperative myxobacterium Myxococcus xanthus. Moreover, the researcher planned to identify and quantify the forces that shape Myxococcus biogeography and social diversity across spatial scales ranging from micrometers (single fruiting bodies) up to 1000s of kilometers (continents). Based on these general goals the researcher has suggested three major objectives in his research and work plans, respectively.Objective 1: Phylogenomics and population genetics of natural M. xanthus isolatesObjective 2: Inferring mutation rates and signals of natural selection and genetic driftObjective 3: Linking phenotype to genotype in closely related natural isolatesPerformed work: Throughout the entire duration of the funded project, the objectives have been addressed by using a combination of bioinformatics and experimental approaches. In particular, the researcher performed the following tasks: (a) sequenced the whole genomes of both natural isolates (local and global scales) and evolved clones (from a mutation accumulation experiment); (b) inferred phylogenetic relationships after de novo assembly and whole genome alignment, respectively; (c) assessed evolutionary forces driving biogeographic patterns at different metric scales; (d) linking genotype and phenotype divergence data between closely related natural isolates from single fruiting bodies by utilizing molecular allele swapping techniques.Main results: (1) The researcher assessed and quantified the evolutionary forces shaping biogeography in the whole genomes of natural population isolates, completing aspects for local scales laid out in Objective 1 and parts of Objective 2. He concluded that local (cm-)scales barriers to recombination readily evolved and were strongest in most divergent clades, and lowest in socially compatible isolates. Moreover, he identified potential genomic regions in which sequence divergence aligns with the compatibility phenotype, one region of which involves CRISPR-Cas-locui implicated in multicellular development in M. xanthus. These data are summarized in a manuscript submitted to ISME Journal (Impact Factor: 9.1) and is currently under review.(2) Moreover, the researcher focused on the whole genome sequences of a wider set of isolates, sampled from a global scale, representing the M. xanthus species tree. These data comprised isolates from five different continents (Africa, Asia, Europe, and North and South America). This study was published in a co-authored paper in the Journal of Bacteriology, completing aspects laid out in Objective 1.(3) By combining the power of mutation accumulation assays and next generation sequencing the researcher inferred the most accurate mutation rate in M. xanthus to date, meeting criteria laid out in Objective 2, and thereby providing a gold standard for population genetics analyses with social myxobacteria. This rate is critical for inferring the extent to which natural selection vs. drift shape genomic biogeography in general and the evolution of genes responsible for social traits in particular.(4) The researcher deciphered the molecular mechanism underlying social diversity in a kin group from nature, completing aspects set out in Objective 3. For this completely closed genomes were inferred de novo, and used as new reference genome to assess genotypic diversity of multiple phenotypically diverse clones from the same social group. Finally, considering their phylogenetic relationships, candidate loci were inferred and the causal link between genotypic change and social phenotype switch was demonstrated by swapping the alleles in focal genes between socially proficient and deficient clones, respectively.Expected final results: During this project, we were able to sequence and assemble the genomes of many natural isolates. These data represent an invaluable resource, and we are currently trying to understand the genetic basis of different aspects of social interactions and their ecology and evolution, such as communal swarming, multicellular development, cheating and cheater control in natural myxobacterial isolates (derived from Objectives 1 and 2). Moreover, we demonstrated for the first time that molecular tools developed for laboratory strains of M. xanthus can be readily applied to manipulate the genomes of natural isolates in general (derived from Objective 3). This opens up the possibility to study genotype-phenotype relationships on a large scale. We have started to apply these techniques to more natural groups and seek to identify general (and potentially parallel) targets of molecular evolution.Socio-economic impacts: Few studies to date have analyzed large sets of whole genome sequences from geographically explicit environmental samples of free-living bacteria, the latter of which represent the vast majority of life forms found in nature, and thus likely contribute a majority of ecosystems functions. Here, we highlight key evolutionary forces that shape ecology and evolution of a fraction of those microbes that engage in social interactions. By increasing our conceptual understanding of microbiota in nature, we move closer toward being able to utilize their vast functional potential in nature, including nutrient cycling, soil remediation, and pest control.