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Owing to the highly similar phenotypic profiles, protein spectra and 16S rRNA gene sequences observed between three pairs of  Tsukamurella  species ( Tsukamurella pulmonis / Tsukamurella spongiae ,  Tsukamurella tyrosinosolvens / Tsukamurella carboxy-divorans , and  Tsukamurella pseudospumae / Tsukamurella sunchonensis ), we hypothesize that and the six  Tsukamurella  species may have been misclassified and that there may only be three  Tsukamurella  species. In this study, we characterized the type strains of these six  Tsukamurella  species by tradition DNA–DNA hybridization (DDH) and “digital DDH” after genome sequencing to determine their exact taxonomic positions. Traditional DDH showed 81.2 ± 0.6% to 99.7 ± 1.0% DNA–DNA relatedness between the two  Tsukamurella  species in each of the three pairs, which was above the threshold for same species designation. “Digital DDH” based on Genome-To-Genome Distance Calculator and Average Nucleotide Identity for the three pairs also showed similarity results in the range of 82.3–92.9 and 98.1–99.1%, respectively, in line with results of traditional DDH. Based on these evidence and according to Rules 23a and 42 of the  Bacteriological Code , we propose that  T. spongiae  Olson et al. 2007, should be reclassified as a later heterotypic synonym of  T. pulmonis  Yassin et al. 1996,  T. carboxydivorans  Park et al. 2009, as a later heterotypic synonym of  T. tyrosinosolvens  Yassin et al. 1997, and  T. sunchonensis  Seong et al. 2008 as a later heterotypic synonym of  T. pseudospumae  Nam et al. 2004. With the advancement of genome sequencing technologies, classification of bacterial species can be readily achieved by “digital DDH” than traditional DDH.

Phylogenomic Analyses and Reclassification of Species within the Genus Tsukamurella: Insights to Species Definition in the Post-genomic Era