*3.9. Phylogenomics*

We compared the whole chromosome structure of *Rhodovulum* sp. MB263 and the two authentic *Rdv. sulfidophilum* strains by dot plotting. The arrangemen<sup>t</sup> of chromosomes is less similar between strains MB263 and DSM 1374<sup>T</sup> than between strains DSM 1374<sup>T</sup> and DSM 2351 (Figure S7). In agreemen<sup>t</sup> with this, the pair-wise ANI scores for whole genomes calculated were 91.21% between strains MB263 and DSM 1374<sup>T</sup> and 97.35% between strains DSM 1374<sup>T</sup> and DSM 2351. *Rhodovulum* sp. MB263 had lower ANI values to the type strains of other *Rhodovulum* species (Table S2). Since the ANI value between strain MB263 and its closest relative *Rdv. sulfidophilum* DSM 1374<sup>T</sup> is lower than the threshold

value (95–96%) recommended for the boundary of prokaryotic species delineation [52,68], the former strain may represent a novel genospecies of the genus *Rhodovulum*.

A phylogenomic analysis of 25 strains of *Rhodovulum* species on which whole-genome information is available (Table S3) [63,69–71] was performed on the basis of 92 protein-coding gene sequences. The reconstructed phylogenomic tree revealed that *Rhodovulum* sp. MB263 is closely related to *Rdv. sulfidophilum* but clearly branched off the clade of the latter species. This suggests that strain MB263 has a distinct phylogenetic position at the species level within the genus *Rhodovulum* (Figure 7).

**Figure 7.** Maximum likelihood phylogenomic tree based on UBCGs (concatenated alignment of 92 core gene sequences) showing genealogical relationships between *Rhodovulum* sp. MB263 and *Rdv. sulfidophilum* DSM 1374<sup>T</sup> (shown by red letters) or other members of the genus *Rhodovulum*. The database accession numbers for the sequences incorporated are shown in Table S2. The genome sequence of *Rhodospirillum rubrum* ATCC 11170<sup>T</sup> was used as an outgroup to root the tree. Gene support indices and percentage bootstrap values are given at the nodes of the tree. Scale bar = 0.2 substitution per position.
