*3.2. Polyphasic Taxonomic Characterization of NEAU-wh3-1*

Morphological observation of 2-week-old cultures of strain NEAU-wh3-1 grown on ISP 3 medium revealed that the strain has the typical characteristics of genus *Embleya* and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into spiral spore chains consisted of cylindrical spores (0.6–0.8μm × 0.9–1.3μm), the spores were rough-surfaced and non-motile (Figure 2). Strain NEAU-wh3-1 was found to grow at a temperature range of 4 to 37 ◦C (optimum temperature 28 ◦C), pH 5 to 12 (optimum pH 7), and NaCl tolerance of 0% to 3% (optimum NaCl of 1%). The physiological and biochemical properties of strain NEAU-wh3-1, *Embleya scabrispora* DSM 41855T, *Embleya hyalina* MB891-A1T, and *Streptomyces lasii* 5H-CA11<sup>T</sup> are given in Table 1**.**

**Figure 2.** Scanning electron micrograph of spore chains of strain NEAU-wh3-1 grown on ISP 3 agar for 2 weeks at 28 ◦C.



Strains: 1, NEAU-wh3-1; 2, *Embleya scabrispora* DSM 41855T; 3, *Embleya hyalina* MB891-A1T; 4, *Streptomyces lasii* 5H-CA11T. Abbreviation: +, positive; –, negative; ±, ambiguous; ND, not determined; w, weak; DPG, diphosphatidylglycerol; PME, phosphatidylmonomethylethanolamine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PIM, phosphatidylinositolmannoside; UL, unidentified lipid; GL, glucosamine-containing lipid; PGL, phospholipid containing glucosamine. All data are from this study except where marked. <sup>a</sup> Data from Ping et al. [43]; <sup>b</sup> Data from Komaki et al. [41]; <sup>c</sup> Data from Liu et al. [73].

Chemotaxonomic analyses revealed that strain NEAU-wh3-1 contained LL-diaminopimelic acid as cell wall diamino acid. The whole-cell sugar was found to contain arabinose, glucose, and ribose. The phospholipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and two unidentified lipids (ULs) (Supplementary Figure S1). The menaquinones detected were MK-9(H4) (46.5%), MK-9(H6) (45.8%), and MK-9(H8) (7.7%).

The almost complete 16S rRNA gene sequence of strain NEAU-wh3-1 (1487 bp) was determined and deposited with the accession number MN928616 in the GenBank/EMBL (European Molecular Biology Laboratory)/DDBJ (DNA Data Bank of Japan) databases. EzBioCloud analysis suggests that strain NEAU-wh3-1 shared the highest 16S rRNA gene sequence similarities with *Embleya scabrispora* DSM 41855<sup>T</sup> (99.65%), *Embleya hyalina* MB891-A1<sup>T</sup> (99.45%), and *Streptomyces lasii* 5H-CA11<sup>T</sup> (98.62%). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain formed a stable cluster with *E. scabrispora* DSM 41855T, *E. hyalina* MB891-A1T, and *S. lasii* 5H-CA11<sup>T</sup> based on neighbor-joining algorithm (Figure 3) and also supported by the maximum-likelihood algorithm (Supplementary Figure S2). To further clarify the affiliation of strain NEAU-wh3-1 to its closely related strains, partial sequences of housekeeping genes including *atp*D, *gyr*B, *rec*A, *rpo*B, and *trp*B were obtained. GenBank accession numbers of the sequences are displayed in Table S1. The phylogenetic tree based on the neighbor-joining tree constructed from the concatenated sequence alignment (1979 bp) of five housekeeping genes (Figure 4) suggested that the isolate clustered with *E. scabrispora* DSM 41855<sup>T</sup> and *E. hyalina* MB891-A1T, and also supported by the maximum-likelihood algorithm (*Streptomyces lasii* 5H-CA11<sup>T</sup> lacks housekeeping genes; Supplementary Figure S3). Moreover, pairwise distances calculated for NEAU-wh3-1 and the related species using concatenated sequences of *atp*D-*gyr*B-*rec*A-*rpo*B-*trp*B were well above 0.007 (Table S2) for the related species, which was considered to be the threshold for species determination [74].

**Figure 3.** Neighbor-joining tree showing the phylogenetic position of strain NEAU-wh3-1 (1487 bp) and related taxa based on 16S rRNA gene sequences. Bootstrap values > 50% (based on 1000 replications) are shown at branch points. *Arthrobacter globiformis* DSM 20124<sup>T</sup> (M23411) was used as an outgroup. *Asterisks* indicate branches also recovered in the maximum-likelihood tree; Bar, 0.01 substitutions per nucleotide position.

**Figure 4.** Neighbor-joining tree based on multilocus sequence analysis (MLSA) analysis of the concatenated partial sequences (1979 bp) from five housekeeping genes (*atp*D, *gyr*B, *rec*A, *rpo*B, and *trp*B) of strain NEAU-wh3-1 (in bold) with related taxa. Only bootstrap values above 50% (percentages of 1000 replications) are indicated. Asterisks indicate branches also recovered in the maximum-likelihood tree; Bar, 0.05 substitutions per nucleotide position.
