Supporting material for: Evidence for shared ancestry between Actinobacteria and Firmicutes bacteriophages Matthew Koert1, Júlia López-Pérez2, Courtney Mattson1, Steven Caruso1 & Ivan Erill1,2 1 Department of Biological Sciences, University of Maryland Baltimore County (UMBC), Baltimore, MD (USA) 2 Departament de Genètica i Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, Spain Supporting Information Captions Figure S1 - Section of the multiple sequence alignment of the FP_oengus (AUV56548.1) gene product annotated as “putative RNA polymerase. Signature motifs shared by RDRPs and DNA-dependent RNA polymerases are highlighted in red. The signature metal-binding DxDxD motif is thought to be part of the primary catalytic loop for these RNA polymerases. Figure S2 - Consensus network inferred on SplitTree with the NeighborNet algorithm using a gene content distance. The branches corresponding to the actinobacteriophage supercluster and the Lactococcus and Faecalibacterium phage group are highlighted in red. Figure S3 - Phylogenetic tree from Neighbor Joining inference on the multiple sequence alignment of TerL sequences (Data S2) Figure S4 - Phylogenetic tree from minimal evolution inference on BLAST-derived inter-genomic distances. The branches corresponding to the actinobacteriophage supercluster and the Lactococcus and Faecalibacterium phage group are highlighted in red. Figure S5 - Detail of the reference viral proteomic tree generated by VipTree, highlighting the clustering of actinobacteriophage supercluster and the Lactococcus phages. Figure S6 - Average %GC and CAI of phage cluster genomes and of complete genomes from each cluster host genus. Cluster designations for actinobacteriophages are as assigned by PhagesDB. Host data is shown using triangles and phage data with circles. Data for hosts, for actinobacteriophage supercluster clusters (BI, AM, AU, AW, DJ, CC and EL) and for the Faecalibacterium and Lactococcus clusters studied here (Faec* and Lacto*) are highlighted. Computations for Faecalibacterium hosts used available whole genome shotgun assemblies. All phage and host information is available in Table S4. Table S1 - Groups of orthologous proteins (phams) in the set of analyzed phage genomes. Table S2 - List of phage genomes analyzed in phylogenetic analyses. Table S3 - Average fraction of protein clusters (PCs) shared between members of the reported supercluster and versus other phages showing a significant fraction of shared protein clusters with them, as reported by Jang H, Bolduc B, Zablocki O, Kuhn JH, Roux S, Adriaenssens EM, et al. Nat Biotechnol. 2019;37: 632–639. doi:10.1038/s41587-019-0100-8. Table S4 - %GC content, nRCA and CAI values of phages and their hosts. Data S1 - FASTA-formatted files for host nRCA reference sets inferred with scnRCA. Data S2 - FASTA-formatted file with TermL sequences for the terminase tree. Data S3 - Nexus-formatted SplitTree file for the pham-based tree. Data S4 - Newick-formatted file for the TerL NJ tree.