*2.2. Phylogenomic Analysis of Nine Prioritized Indonesian Streptomyces Strains*

To obtain a better understanding of the phylogenetic relationship about the prioritized nine *Streptomyces* strains, a phylogenetic analysis based on their full-length genomes sequences was performed. For this purpose and the genome mining studies mentioned below, the genomic DNA was isolated from each sample and sequenced by using the Pacific Biosciences RS II (PacBioRSII) platform [57–59]. The resultant genome sequences ranged in sizes between 7.05 Mbp (*Streptomyces* sp. I6) and 8.36 Mbp (DHE 17-7) and GC contents between 72.08% (DHE 7-1) and 72.47% (*Streptomyces* sp. I6) (Table S1), which share comparable values reported for *Streptomyces* species (genome sizes of 6-12 Mb [60] and GC contents of 72–73% [61,62]).

In order to run a whole-genome phylogenetic analysis, the genome sequences were submitted to the Type (Strain) Genome Server (TYGS) (https://tygs.dsmz.de, accessed on 13 December 2019) [63], which allows a phylogenetic analysis based on full-length genome sequences and compares genomic data with the database genomes. The resulting phylogenetic information is more authentic than those obtained from 16S rDNA- or multi-locus sequence analysis (MLSA)-based classifications, which only use small sequence fragments as a basis for sequence comparisons [63]. The TYGS analysis provides information on the similarity of a strain to its nearest related type strain, derived from the digital DNA-DNA hybridization (dDDH) values calculated by the genome-to-genome distance calculator (GGDC) 2.1 (http://ggdc.dsmz.de, accessed on 13 December 2019) [64]. TYGS phylogenomic analysis revealed that all nine isolates belong to the genus *Streptomyces*. The dDDH values between the nine Indonesian strains and their closest relatives ranged between 31.4% (*Streptomyces* sp. I4) and 51.5% (*Streptomyces* sp. I6) (using GGDC distance formula *d4*) (Table 2), which is below the threshold of 70% used for species delineation [65,66], proposing a novel collection of *Streptomyces* species.



According to the TYGS phylogenomic tree, the terrestrial Enggano Island strains SHP 22-7 and DHE 17-7 belong to the same clade (clade A) (Figure 3) and most likely resemble the same type of species with a dDDH value of 86.7% (Table 2). Both bacteria are found to be closely related to *S. luteus* TRM 45540, isolated from a soil sample from China [67]. All mangrove isolates originating from sediments of Lombok Island (*Streptomyces* sp. I3, I4, and I5) and Bali Island (BSE 7F, and BSE 7-9) were allied in clade B, suggesting a correlative connection (Figure 3). Additionally, the dDDH analysis showed that BSE 7F is closely related to BSE 7-9 with a value of 95.7% and thus most likely represent the same subspecies (Table 2), while *Streptomyces* sp. I3 and I4 probably represent the same species having a dDDH score of almost 100% (Table 2). The nearest related type strain of all five mangrove strains is *S. capillispiralis* DSM 41695 isolated from a Sweden soil sample [68].

**Figure 3.** Whole-genome sequence tree generated with the TYGS web server for nine Indonesian *Streptomyces* isolates (highlighted by red boxes) and closely related type strains. Tree inferred with FastME from GBDP distances was determined from genome sequences. The branch lengths are scaled in terms of GBDP distance formula *d*5. The numbers above branches indicate GBDP pseudobootstrap support values > 60% from 100 replications, with an average branch support of 84.4%. The tree was rooted at the midpoint.

By contrast, the soil sample DHE 7-1 and mangrove *Streptomyces* sp. I6 were found to group separately in distinct clades (clade C and D, respectively) (Figure 3). The soil *S. bungoensis* DSM 41781 collected in Japan [69] shares a dDDH value of 32.3% as the closest related strain to DHE 7-1 (Table 2), while the nearest related neighbor of *Streptomyces* sp. I6 is *S. spongiicola* HNM0071, isolated from a marine sponge collected from China [70] with a dDDH value of 51.5% (Table 2). Additional information on the specific polyphasic characteristics of the representative type strains from each clade can be found in the Supplementary Material. Altogether, 16S rRNA gene-based phylogenetic and phylogenomic studies revealed that all nine prioritized isolates belong to the genus *Streptomyces* and, based on dDDH analysis, represent novel species (Figure 3).
