Bacteriophages are highly understudied considering their abundance but have significant impacts on ecosystems like the soil microbiome, which is becoming increasingly important in efforts to improve agricultural techniques. In this study, the effort to characterize more soil-dwelling bacteriophages is advanced through investigation of the Arthrobacter phage Lego. Exploration of Lego’s genome included the interrogation of evolutionary relationships among members of the AZ cluster, or the group of phages with at least 50% nucleotide similarity to Lego, in order to better understand the process of phage evolution and to characterize diversity among phages. These analyses contribute to a better understanding of the soil microbiome. In order to better understand the evolutionary relationships, bioinformatic analyses including dotplots, ANI, and protein family analyses were performed. Genome-wide analyses illuminated patterns that suggested three sub-groups within Lego’s cluster, AZ. In addition, single-gene analyses of conserved genes in the cluster can be used to support these groupings, as shown through homology and phylogenetic analysis. Horizontal gene transfer is likely responsible for some of the differentiation events that led to separate lineages in the cluster. These groupings help show how soil phages interact and change in the environment and contribute information about phage evolution and specifically the AZ cluster. The greater impact of this work is that it will inform future studies into soil bacteriophages by laying the groundwork of phage analysis in this cluster of temperate Arthrobacter phages. In addition, it provides a model for analyzing evolutionary relationships within phage clusters using several bioinformatic techniques
