Duganella vulcania sp. nov., Rugamonas fusca sp. nov., Rugamonas brunnea sp. nov. and Rugamonas apoptosis sp. nov., isolated from subtropical streams, and phylogenomic analyses of the genera Janthinobacterium, Duganella, Rugamonas, Pseudoduganella and Massilia

Six Gram- stain-negative, catalase-and oxidase-positive, rod-shaped and motile strains (FT81W(T), FT82W, FT107W, FT3S(T), LX20W(T) and LX47W(T)) sharing high 16S rRNA gene sequence similarities with species of the genera Janthinobacterium (97.0- 98.3%), Duganella (96.3-98.8%), Rugamonas (97.8-98.6%), Pseudoduganella (96.8-97.5 %) and Massilia (94.5-98.6 %) were isolated from subtropical streams in PR China. The phylogenetic trees reconstructed using the 16S rRNA gene sequences indicated that the species of above five genera often mix together, indicating that the taxonomic statuses of some species were questionable. Phylogenomic reconstruction based on 369 single -copy orthologous clusters indicated that the species of the genus Janthinobacterium form a distinct cluster, strains FT81W(T), FT82W and FT107W form a tight cluster with the species of the genus Duganella, and strains FT3S(T), LX20W(T) and LX47WT form a tight cluster with the species of genus Rugamonas, and the species of genus Pseudoduganella form a tight cluster with Massilia guangdongensis, Massilia ginsengisoli, Massilia rivuli, Massi-lia namucuonensis, Massilia aquatica sensu Lu et al., Massilia buxea, Massilia armeniaca, Massilia plicata, Massilia flava, Massilia lurida, Massilia dura, Massilia lutea, Massilia umbonata, Massilia albidiflava and Massilia violacea. It should be noted that Massilia aquatica Lu et al. 2020 non Massilia aquatica Holochova et al. 2020 is a later homonym and an illegitimate name. The GTDB Release 202 also supported the proposal that M. guangdongensis, M. ginsengisoli, M. rivuli, M. namucuonensis, M. aquatica sensu Lu et al., M. buxea, M. armeniaca, M. plicata, M. flava, M. lurida, M. dura, M. lutea, M. umbonata, M. albidiflava and M. violacea should be transferred into the genus Pseudoduganella. The calculated pairwise orthologous average nucleotide identity by USEARCH (OrthoANIu) values were between 95.0 % and 95.6 % among strains FT81W(T), FT82W and FT107W, but were less than 91.5 % among strains FT3S(T), LX20W(T), LX47W(T)and other related strains. Combining the results of phylogenomic analyses, phenotypic, biochemical and genotypic characteristics, strains FT81W(T), FT82W and FT107W should represent a novel species of the genus Duganella, and strains FT3S(T), LX20W(T) and LX47W(T) should represent three novel species of the genus Rugamonas, for which the names Duganella vulcania sp. nov. (type strain FT81W(T)=GDMCC 1.1679T =KACC 21471(T)), Rugamonas fusca sp. nov. (type strain FT3S(T)=GDMCC 1.1907(T) =KACC 21952(T)), Rugamonas brunnea sp. nov. (type strain LX20W(T)=GDMCC 1.1911(T)=KACC 21956(T)) and Rugamonas apoptosis sp. nov. (type strain LX47W(T)=GDMCC 1.1914(T) =KACC 21959(T)) are proposed.