*4.2. Genomic DNA Extraction*

Strains were grown on ISP2 media for 24–48 h, and cells were collected by centrifugation (13,000× *g*, 2 min), resuspended in 50 mM EDTA, and then mechanically separated with a shank. For cell rupture, disaggregated cells were treated with Lysozyme 10 mg/mL, Lysostaphin 10 mg/mL, and Proteinase K 1 mg/mL for 1 h at 37 ◦C. Then, for DNA purification, the Promega Wizard DNA extraction kit was used, according to the manufacturer's description. Finally, an alcohol treatment was used for DNA precipitation and cleaning, with 100% isopropanol for DNA precipitation, followed by 70% ethanol to wash the DNA

pellet. The sample was rehydrated and stored according to the Promega Wizard DNA Extraction Kit instructions.

#### *4.3. Genome Sequencing, Assembly, and Annotation*

Sequencing was performed using the Illumina NextSeq platform with a 150 bp × 2 configuration. Raw reads were examined for coverage and trimmed for a minimum quality of 20 using Sickle v.1.33 [69]. Genome assembly was performed using SPAdes v.3.13.1 with default parameters [70,71] and Unicycler in normal mode [72] (internally using SPAdes v.3.11.1. The SPAdes assembly pipeline contains a final step of scaffolding using SSPACE [73]. Assembly quality was measured using QUAST v.5.0.2 [74], and genome completeness and contamination were evaluated using CheckM v1.0.18 [75]. A unique working assembly for each strain was selected based on the one with a smaller number of contigs, largest contigs (N50, L50 evaluation), and the lowest contamination metrics. Assembled genomes were annotated using Prokka v.1.13.4 [76], PANNZER2 [77], and eggNOG 5.0 [78] web servers.

## *4.4. Phylogenomic Tree Inference*

*Streptomyces* genomes were selected from the tree described in Nouioui et al. [79], spanning over subclades. The selection criteria used was genome quality (fewer contigs and higher completeness based on CheckM), and type strains were preferred for the analysis. Assembly metrics were obtained using QUAST v.5.0.2 and CheckM v.1.0.18. The genomes of strains that closely resembled our strains by 16S rRNA identity [37] were also added: *Streptomyces albidoflavus* NBRC 13010 (=NRRL B-1271) and *Streptomyces aurantiogriseus* NRRL B-5416. *S. albogriseoleus* genome was not included, since assemblies presented anomalies, according to NCBI Genbank. The genomes of a *Catenulispora* strain (DSM 44928) and a *Kitasatospora* strain (KM-6054) were used as outgroups. Selected genomes and the outgroup genomes were retrieved from the Pathosystems Resource Integration Center (PATRIC) database and are described in Table S2. Phylogenomic reconstruction was performed using Orthofinder v.2.3.11 [42], with FastTree v.2.1.10 [80], in multiple sequence alignment mode (-M msa option), employing the default parameters for the orthogroup definition. Complementary to the phylogenomic analysis, average nucleotide identity calculation was performed using all of the selected genomes (Table S3), with the Python PyANI package [81] and depicted using the seaborn package in Python.
