Genome Based Analyses Reveals the Presence of Heterotypic Synonyms

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bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Genome based analyses reveals the presence of heterotypic

2 synonyms and subspecies in Bacteria and Archaea

3 Munusamy Madhaiyan1†, Venkatakrishnan Sivaraj Saravanan,2† & Wah-Seng See-Too3†

4 1Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore,

5 Singapore 117604

6 2Department of Microbiology, Indira Gandhi College of Arts and Science, Kathirkamam 605009,

7 Pondicherry, India

8 3Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of

9 Science, University of Malaya, Kuala Lumpur, Malaysia

10 †These authors have contributed equally to this work

11 Keywords: dDDH; AAI; genome-based taxonomy; 16S rRNA gene similarity; polyphasic taxonomy;

12 OGRI

13 Abbreviations: dDDH, digital DNA: DNA hybridization; ANI, average nucleotide identity; OGRI,

14 overall genome related indices

15 Abstract

16 Term heterotypic synonym refers to different names have been associated with different type

17 strains, however from the opinion of a bacteriologist, different names belongs to the same taxon

18 and term subspecies refers to strains and genetically close organisms that were diverging

19 phenotypically. In this study, sequenced and publicly available genomes in the Edgar 2.0 server

20 were carefully analysed and based on high (>98 %) amino acid identity value, synonyms were

21 putatively identified. The 16S rRNA gene sequence of those species were used for the

22 construction of maximum likelihood based phylogenetic trees to infer the genetic closeness or

23 distance by examining the tree topology and clustering of the organisms within clades. They

24 were further subjected to overall genome related indices like digital DNA-DNA hybridization,

25 average nucleotide identity to confirm the presence of synonyms or subspecies with phenotypic

26 data support. The outcome of this polyphasic taxonomic re-analysis was identification of 40 later

27 heterotypic synonyms and 13 subspecies spread over phylum Actinobacteria, Bacteroidetes,

28 Firmicutes, Nitrospirae, Proteobacteria and Thermotogae and in domain Archaea.

29 INTRODUCTION

30 A taxon refers to one or more elements, it can be of any taxonomic category from class to

31 subspecies that are designated as nomenclatural type. The term type refers to the nomenclatural

32 type strain designated as per the ICNP rule, it’s the element of the taxon with which the name is

33 permanently associated as correct name or heterotypic synonym [1]. In simple terms, synonym

34 refers to the same taxon under another scientific name, they usually come in pair or even swarms

35 [2]. In old literature, they were named as objective synonyms and subjective synonyms which are

36 referred in the recent times as homotypic and heterotypic synonyms [1]. In nutshell, homotypic

37 synonym referring to more than one name associated with same type strain whereas heterotypic

38 synonyms refer to different names have been associated with different type strains but based on

39 the opinion of the bacteriologist concerned both names belong to the same taxon.

40 Heterotypic synonyms were first used in conjunction with junior synonyms, in a study conducted

41 on Helicobacter type strains, where in Helicobacter nemestrinae [3] was identified as junior

42 heterotypic synonym of Helicobacter pylori [4], further, the presence of later heterotypic

43 synonyms were identified in different genera including Janibacter, Weissella and Streptomyces

44 [5, 6, 7]. Conventionally, heterotypic synonyms were proposed based on the 16S rRNA gene

45 analysis and DNA-DNA hybridization studies as in the case of Janibacter and Weissella [5, 6].

46 For instance, employing DNA-DNA hybridization technique on 13 Streptomyces species, two

47 subspecies and 8 later heterotypic synonyms were identified [7]. A study provided polyphasic

48 evidence for heterotypic synonym using fluorescent amplified fragment length polymorphism,

49 DNA-DNA hybridization and API 50 CHL analysis on Lactobacillus ferintoshensis and

50 Lactobacillus parabuchneri sharing similar genetic, biochemical and physiological features, this

51 led to proposal of L. ferintoshensis as the later heterotypic synonym of L. parabuchneri [8].

52 Another study focused on using of multilocus sequence typing analysis to identify heterotypic

53 synonyms, herein similarity of three housekeeping genes including pheS, rpoA and atpA were

54 used to propose Lactobacillus crypricasei as the later heterotypic synonym of Lactobacillus

55 acidipiscis [9]. In later studies, the heterotypic synonyms were identified when they attempted to

56 characterize certain isolated strains or serendipitously discovered when attempted to validate a

57 previously published strain [10, 11]. A new dimension in the study of heterotypic synonyms was

58 the suggestion of the use of average nucleotide identity (ANI), since the next generation

59 sequencing provided a rapid and cost effective way of obtaining the whole genome sequences, a

60 proposal for integrating the genomics as a polyphasic component of the Bacteria and Archaea

61 taxonomy and systematics was mooted out [12], ANI values and comparative genomic analysis

62 had become an inevitable part in the proposal of heterotypic synonyms in Neisseria,

63 Deinococcus and Bacillus [13, 14, 15].

64 The term subspecies denote the strains and genetically similar organism that are phenotypically

65 divergent [16, 17]. Previously, delineation of subspecies was based on qualitative measurement

66 of phenotypic characters rather than examining evolutionary distance or 16S rRNA gene

67 similarity. However, the current approach in the taxonomic assignment of subspecies was based

68 on the overall genome related indices such as digital DNA-DNA hybridization (dDDH) of 70-79

69 % [18].

70 The advent of genome sequencing technologies was reflected in bacterial systematics mainly in

71 reclassification of the taxa at different hierarchy level, such kind of proposals were attempted in

72 phylum Actinobacteria, Bacteroidetes, and Alphaproteobacteria [19, 20, 21]. As an exemplar,

73 within the taxa of Actinobacteria, by calculating intergenomic dDDH values, 29 new later

74 heterotypic synonyms, 8 new subspecies were proposed [19], in the phylum Bacteroidetes 6 later

75 heterotypic synonyms and 5 new subspecies were proposed [20]. The recent analysis of the

76 phylum Alphaproteobacteria revealed the presence of 33 later heterotypic synonyms, 12 new

77 subspecies and 2 new species [21]. The present study focusses on whole genome-based analyses

78 to identify heterotypic synonyms in the sequenced and publicly available bacterial genomes. The

79 idea behind this work originated when species of certain publicly available genomes in the Edgar

80 2.0 server [22] showed high amino acid similarity when their genomes were compared. Previous

81 studies show that genome analysis clearly envisage that strains from same species share an

82 amino acid identity (AAI) of > 95 % [23]. Based on this notion, on total 12479 genomes

83 comprising of 548 genera housed within 227 families were examined. Different species showing

84 > 98 % AAI similarity were assumed to contain heterotypic synonym or subspecies and they

85 were selected and used in the present study. Alternatively, genome results of certain recently

86 published type strains when critically examined, the possibility of heterotypic synonymy was

87 predicted. These putatively identified strains were also subjected to phylogenetic analysis,

88 overall genome related indices (OGRI) such as dDDH and average nucleotide identity (ANI)

89 analyses and their phenotypic traits were also considered to propose a holistic taxonomic

90 framework for the heterotypic synonyms and subspecies present in different genera.

91 METHODS

92 In this study, 95 type strains of different species of Bacteria and Archaea whose genome shared

93 > 98 % AAI were selected from the Edgar 2.0 server and certain recently published genome

94 contiaing putative heterotypic synonmy were downloaded from NCBI. Strains of these selected

95 genomes were distributed among phylum Actinobacteria, Bacteroidetes, Firmicutes,

96 Nitrospirae, Proteobacteria, Thermotogae and Domain Archaea, their genome details are

97 presented in Table S1.

98 For the selected strains, the pairwise 16S rRNA gene sequence similarity was worked out in a

99 phylum wise manner using the EzBioCloud database [24]. The Clustal W tool of MEGA 7 was

100 used for alignment of the sequences in an individual phylum level [25], distance between the

101 sequences was calculated using the Kimura correction in a pairwise deletion manner [26] and

102 maximum likelihood-based treeing program was used to reconstruct the type strains phylogeny

103 using the MEGA 7 software with 1000 non-parametric bootstrap replicates.

104 For OGRI analysis, 95 genome sequences of different type species were retrieved from

105 EzBioCloud database /GenBank. Using the retrieved genomes, OGRI analysis including Genome

106 distance in the form of digital DNA-DNA hybridization (dDDH) values calculated by the

107 Genome-to-Genome Distance Calculator (GGDC) version 2.1 (http://ggdc.dsmz.de) with

108 BLAST+ and with the recommended formula [27]. OrthoANI, that measures similarity between

109 two genome sequences was calculated using Oat 0.93.1 [27] with USEARCH

110 (https://www.ezbiocloud.net/tools/orthoaniu). Average amino acid identities (AAI) was

111 calculated using the EDGAR server 2.3 (https://edgar.computational.bio.uni-giessen.de). For

112 certain pairs retrived from database, the pairwise average AAI was calculated using the AAI

113 workflow with default settings in CompareM v0.0.23 (https://github.com/dparks1134/CompareM).

114 RESULTS AND DISCUSSION

115 When the genomes of the phyla were analysed, presence of synonyms or subspecies were

116 putatively identified by 16S rRNA gene sequence based phylogenetic tree topology, the gene

117 similarity values and clustering of the organismsm within the clades. The selected species pairs

118 or taxa were further subjected to dDDH [27], to underpin the subspecies or synonynm with a

119 threshold values of 70 - 79 % or > 80 % respectively [17, 18]. The complete list of subspecies

120 and heterotypic synonyms identified employing these criteria are listed in the Table 1.

121 Actinobacteria

122 Actinobacteria is one important phylum that was analysed for the presence of heterotypic

123 synonyms, the putative synonymous strains in different species were selected based on the high

124 AAI (>98 %) values of their genomes. The genetic closeness among the different species pairs

125 indicating the presence of heterotypic synonyms or subspecies was preliminarily identified based

126 on high similarity per centage of their 16S rRNA gene sequences (Table 1).

127 For instance, a genetically close pair consists of Actinokineospora spheciospongiae and

128 Actinokineospora mzabensis showing high similarity and bootstrap support in the 16S rRNA

129 gene analysis (Fig. 1). When it was subjected to the OGRI metrics (Table 1), this pair shared

130 dDDH of 89.9 %, their genome similarity was also reflected in the biochemical properties, as

131 only a few differences in mycelial pigments and carbon source utilization was observed between

132 the strains (Table S2). These data mooted the proposal of Actinokineospora spheciospongiae as

133 the later heterotypic synonym of Actinokineospora mzabensis. Further, when a Corynebacterium

134 clade was analysed, Corynebacterium ihumii recorded high dDDH value of 76.1 and 78 % with

135 Corynebacterium afermentans subsp. lipophilum HSID17239 and Corynebacterium afermentans

136 subsp. afermentans DSM 44280T respectively, phenotypically most of the traits are common

137 between these strains (Table S3). The dDDH value supported the reduction of taxon

138 Corynebacterium ihumii as C. afermentans subsp. ihumii. However, Corynebacterium ihumii

139 was not validly published and the subspecies was not proposed as per the taxonomic rules and

140 guidelines [29]. This clade also comprised of another pair of Corynebacterium pilbarense and C.

141 ureicelerivorans (Fig. 1) and its dDDH value was less than 70 % retaining their species status. A

142 recent study by Nouioui et al. [19] proposed Dietzia cinnamea as the later heterotypic synonym

143 of Dietzia maris, when the dDDH value was re-analysed it shared only 29 %, erroneous results

144 obtained may be attributed to the incorrect strain of D. maris DSM 43672 used in that study.

145 Thus D. cinnamea retaining its species status, but, when the genome of D. cinnamea was compared

146 with D. papillomatosis, dDDH was 81.55 %, phenotypically also both the strains were common in

147 morphological feaatures including their colony colour to substrate utilization profile (Table S4),

148 but D. maris strain was phenotypically different from D. cinnamea and D. papillomatosis. Thus,

149 the OGRI combined with phenotypic features supported the proposal of Dietzia papillomatosis

150 as the later heterotypic synonym of Dietzia cinnamea. A monophyletic clade of Nocardia

151 containing certain species sharing high 16S rRNA gene similarity (Table 1 & Fig. 1) was

152 noticed, all those three pairs were analysed for the dDDH to ascertain their taxonomic position

153 (Table 1), all strains recorded within a range of 73.7 - 75.2 %, which coincides with the value

154 (70-79 %) proposed as cutoff to delineate subspecies [18]. Further presence of similar

155 phenotypic properties is noticed within those pairs. For instance, the pair consist of N. exalbida

156 and N. gamkensis shared similarities in the degradation of substrates including arbutin, esculin

157 adenine, casein, hypoxanthine and xanthine (Table S5). The other pair comprising of Nocardia

158 ignorata and Nocardia coubleae when analysed, similarities in abiotic factors (temperature, pH

159 and NaCl concentration) affecting the growth was observed (Table S6). The third pair housing N.

160 elegans and N. nova were biochemically similar in utilization of carbon sources as sole carbon

161 source and energy (Table S7). The dDDH value combined with the phenotypic traits supported

162 the proposal of three different subspecies within Nocardia clade namely Nocardia exalbida

163 subsp. gamkensis subsp. nov., Nocardia ignorata subsp. coubleae subsp. nov. and Nocardia

164 nova subsp. elegans subsp. nov. In the Rhodococcus clade, genetic closeness of a pair was

165 identified through 16S rRNA based phylogenetic tree (Fig. 1), when it was subjected to dDDH

166 analysis, values supporting the claim of a heterotypic synonym was observed (Table 1). In fact,

167 phenotypically, most of the biochemical features are common between these strains (Table S8). This

168 led to the proposal of Rhodococcus imtechensis is later heterotypic synonym of Rhodococcus

169 opacus.

170 Archaea

171 Presence of heterotypic synonyms were putatively selected in Domain Archaea through

172 preliminary examination of AAI value in genome belong to different species. Those showing

173 high AAI (>98 %) were taken up for further OGRI analysis. Presence of heterotypic synonym

174 was recognized within the clade of Haloferax (Fig. 2), high 16S rRNA gene similarity (Table 1)

175 was observed between three strains including Haloferax lucentense, Haloferax alexandrinus and

176 Haloferax volcanii, dDDH analysis revealed the genome similarity of those strains (Table 1).

177 Phenotypically, all the strains are oxidase positive and shared a similar concentration of NaCl

178 requirement (1-4.5 % w/v) pH and temperature optimum for growth. All strains produced H2S

179 when grown on thiosulfate medium and formed acid from arabinose and none of the strains

180 hydrolysed starch and casein, but variable results observed for gelatin hydrolysis (Table S9).

181 Based on the 16S rRNA gene sequence similarity, dDDH value and biochemical features,

182 Haloferax lucentense and Haloferax alexandrinus are proposed as the heterotypic synonyms of

183 Haloferax volcanii.

184 Another clade comprising of Methanobacterium contained two species that may be genetically

185 close based on 16S rRNA gene similairty (Fig. 2). Those species were subjected to dDDH

186 analysis (Table 1), high genetic similarity was noticed between Methanobacterium veterum and

187 Methanobacterium arcticum. Critical examination of their phenotypic traits revealed the fact that

188 both the strains share similar temperature, pH and NaCl concentration ranges supporting growth

189 (Table S10). The 16S rRNA gene sequence-based analysis (Fig. 2) revealed that other

190 Methanobacterium species present within the clade may also genetically similar, but currently

191 genome data was lacking for all those species except Methanobacterium bryantii, which showed

192 only 48 % dDDH value with M. veterum and M. arcticum. Considering together the OGRI and

193 biochemical properties, Methanobacterium arcticum is proposed as a later heterotypic synonym

194 of Methanobacterium veterum. Similarly, the heterotypic synonyms were identified in

195 Methanosarcina based on high 16S rRNA gene similarity between Methanosarcina soligelidi

196 and Methanosarcina mazei, it was also reflected in their genome based dDDH analysis (Fig. 2 &

197 Table 1), the observed results were supported by phenotypic properties including abiotic factors

198 (temperature, pH and NaCl concentration) and substrate utilization pattern, interestingly, both

199 shared similar archaeal lipids in their membrane (Table S11). These polyphasic data supported

200 the proposal of Methanosarcina soligelidi as the later heterotypic synonym of Methanosarcina

201 mazei.

202 Bacteroidetes

203 In Bacteroidetes, species pair consists of Rufibacter ruber and Rufibacter quisquiliarum were

204 similar in their genome which was evident from the values recorded in dDDH (Fig. 3 & Table 1).

205 Interestingly, in between these strains, abiotic factors such as temperature, pH and NaCl

206 concentrations favouring growth are common with few differences in carbon source assimilation

207 (Table S12). These polyphasic evidences support the proposal of R. quisquiliarum as the later

208 heterotypic synonym of R. ruber.

209 Firmicutes

210 A subspecies pair consists of Caldanaerobacter subterraneous subsp. yonseiensis and

211 Caldanaerobacter subterraneous subsp. tengcongensis showed high 16S rRNA gene similarity

212 (Fig. 4), they also shared dDDH and ortho ANI values of 81.7 and 98.0 % respectively. The

213 genome level similarity was also reflected in phenotypic properties with very few variable traits

214 (Table S13). Another organism that was phylogenetically close and found within this clade was

215 Thermoanaerobacter keratinophilus, which may be a taxon belonging to Caldanaerobacter, but

216 currently it was not validly published, due to the fact that only a single culture collection (T.

217 keratinophilus DSM14007T) deposition proof was available and the non-availability of the

218 genome details also hampered its re-classification. Based on the OGRI metrics analysed

219 Caldanaerobacter subterraneus subsp. yonseiensis was proposed as the heterotypic synonym of

220 C. subterraneus subsp tengcongensis.

221 Another pair consists of Carboxydothermus hydrogenoformans and Carboxydothermus

222 ferrireducens sharing 96.1 % 16S rRNA gene similarity (Fig. 4) and shared 79.8 and 97.8 % of

223 dDDH and ortho ANI respectively (Table 1). Biochemically there is not much difference existing

224 between these two species, except the utilization of ferric citrate as electron acceptor during

225 chemolithotrophic growth of cells with CO as electron donor (Table S14). The 16S rRNA

226 analysis also shows the presence of Carboxydothermus siderophilus within this clade (Fig. 4), its

227 taxonomic position was not analysed due to the non-availability of the genome data. Thus, the

228 dDDH recorded within the range of 70-79 % supported the reduction of taxon

229 Carboxydothermus ferrireducens to Carboxydothermans hydrogenoformans subsp. ferrireducens

230 subsp. nov.

231 The next pair analysed within the Firmicutes consists of Carnobacterium inhibens subsp.

232 inhibens and Carnobacterium inhibens subsp. gilichinskyi sharing 16S rRNA gene similarity of

233 99.4 % and their dDDH and ortho ANI recording 84.1 and 98.1 % respectively (Table 1). Both

234 strains also required a wider temperature (0-37 C) and pH (5.5-9.0) for growth, with a little

235 variability noticed in the utilization of substrates (Table S15). On careful observation of the 16S

236 rRNA gene based phylogenetic tree (Fig. 4), this clade has also nested Carnobacterium viridans,

237 C. pleistocenium and C. jeotgali. Especially C. pleistocenium, Carnobacterium inhibens subsp.

238 gilichinskyi and C. jeotgali shared a uniform bootstrap support, that lead to analysis of their

239 genomes to identify heterotypic synonyms or subspecies. However, all the three strains have

240 recorded <70 % dDDH retaining their species status. The OGRI including dDDH and ortho ANI

241 strongly supported proposing Carnobacterium inhibens subsp. inhibens as the later heterotypic

242 synonym of Carnobacterium inhibens subsp. gilichinskyi.

243 Another important genus analysed for the presence of heterotypic synonyms or subspecies in

244 Firmicutes was Geobacillus. On analyses of the twelve species of Geobacillus and four

245 Parageobacillus for OGRI metrics of dDDH and Ortho ANI. The two Geobacillus pairs consist

246 of G. kaustophilus; G. thermoleovorans and G. lituanicus; G. stearothermophilus shared a high

247 level of 16S rRNA gene similarity (Fig. 4), and the dDDH and ortho ANI ranged between (73.4-

248 88.0 %) and (96.9-98.5 %) respectively (Table 1). The careful examination of the phenotypic

249 traits showed only little variation within the taxa (Table S16) strongly supporting the claim that

250 Geobacillus kaustophilus is the later heterotypic synonym of Geobacillus thermoleovorans.

251 Another pair that consist of Geobacillus lituanicus and Geobacillus stearothermophilus has

252 recorded dDDH value of 73.4 %, Interestingly, both the species shared a common temperature

253 regime (Table S17), with optimum temperature requirement of 50 C for growth [30, 31, 32].

254 The dDDH recorded and similarity in phenotypic traits supported the subspecies delineation

255 [18], and proposal of different subspecies namely Geobacillus stearothermophilus subsp.

256 stearothermophilus subsp. nov. While these pairs were analysed, a pair of Geobacillus

257 comprising of Geobacillus yumthangensis and Geobacillus galactosidasius were found to be

258 clustered within the Parageobacillus clade (Fig. 4) in fact both the strains showed high genomic

259 similarity indices (Table 1) in the form of dDDH (82- 88 %) and ANI (97.9 - 98.5 %) with

260 Parageobacillus toebii, variability in the biochemical traits was observed between the three

261 strains are: production of acid from cellobiose, galactose, ribose, glycerol, lactose and rhamnose,

262 variation was recognized within the carbon source utilization also. But, the abiotic factors

263 (temperature, pH and NaCl concentration) required for these strains are similar (Table S18).

264 Thus the, 16S rRNA based topology with genome indices and biochemical data strongly

265 supported the proposal of Geobacillus yumthangensis and Geobacillus galactosidasius as later

266 heterotypic synonyms of Parageobacillus toebii, in fact Parageobacillus toebii was a homotypic

267 synonym of Geobacillus toebii [33].

268 Megamonas and Salimicrobium are the other two genera of Firmicutes that were analysed for the

269 OGRI metrics. Genome similarity of their strains can be understandable by the critical analysis

270 of the dDDH and ANI of the Megamonas pair (M. rupellensis and M. funiformis) and

271 Salimicrobium pair (S. salexigens and S. jeotgali) (Table 1). One more taxon (M. funiformis) was

272 associated with the Megamonas clade (Fig. 4) but, currently its genome sequence was

273 unavailable. Phenotypic similarity within these two pairs are also high, only a little difference in

274 the form of carbon substrate utilization and acid production from carbon sources are observed

275 (Table S19 - S20). Thus, the OGRI analyses supported the proposal of Megamonas rupellensis as

276 the later heterotypic synonym of Megamonas funiformis, previously also a synonym (M.

277 hypermegas) was identified in this genus [34]. In the Salimicrobium clade analysed, S. jeotgali

278 was reduced in taxon and proposed as Salimicrobium salexigens subsp. jeotgali subsp. nov. a

279 novel subspecies as per the previously suggested threshold value [18].

280 A paraphyletic clade comprised of eight Thermoanaerobacter species (Fig. 4) was subjected to

281 OGRI analyses (Table 1), subsequently, their biochemical characteristics was also compared, a

282 few differences in the form of motility and carbon substrate utilization was observed (Table S21)

283 The OGRI and phenotypic evidences supported the proposal of Thermoanaerobacter italicus and

284 Thermoanaerobacter mathranii subsp. mathranii as the later heterotypic synonyms of

285 Thermoanaerobacter thermocopriae. Thermoanaerobacter pseudethanolicus as the later

286 heterotypic synonym of Thermoanaerobacter brockii subsp. finnii. Finally, Thermoanaerobacter

287 wiegelii and Thermoanaerobacter siderophilus together constitute the later heterotypic

288 synonyms of Thermoanaerobacter ethanolicus.

289 Nitrospira

290 The phylum Nitrospira comprised of three valid genera namely Leptospirillum, Nitrospira and

291 Thermodesulfovibrio with Thermodesulfovibrio as the only genus containing five species. A

292 species pair of Thermodesulfovibrio was examined owing to high AAI value and the analysis of

293 its dDDH revealed the genome similarity between these two species (Table 1 & Fig. 5).

294 Phenotypically, these strains utilized pyruvate, hydrogen (plus acetate) and formate (plus acetate)

295 as both electron donor and carbon source in the presence of sulfate as terminal electron acceptor

296 and in addition they also utilized sulfate and thiosulfate as electron acceptors. However,

297 variability was noticed in utilization of sulfite and nitrate as terminal electron acceptors (Table 22).

298 Ecologically, both the strains were recovered from hot spring habitats [35, 36]. These polyphasic

299 evidences led to proposal of Thermodesulfovibrio islandicus as the later heterotypic synonym of

300 Thermodesulfovibrio yellowstonii.

301 Proteobacteria

302 Proteobacteria commonly refers to Gram negative bacteria, teems with versatile metabolic

303 capability and omnipresent in nature. The genetic closeness between several species those belong

304 to Proteobacteria were depicted clearly in the 16S rRNA gene sequence based phylogenetic tree

305 (Fig. 6).

306 All the Aeromonas taxa analysed in this study clustered together as single clade (Fig. 6) with

307 high (100 %) bootstrap support, this prompted to check the dDDH values for all the species

308 within that clade, however, genome data was available only for Aeromonas salmonicida subsp.

309 pectinolytica that recorded dDDH of < 70 %, retaining its subspecies status. Interestingly, based

310 on the 16S rRNA gene sequence similarity, a Haemophilus piscium was found within the

311 Aeromonas clade, and it was proposed as Aeromonas salmonicida subsp. piscium comb. nov. A

312 subspecies pair consist of Aeromonas salmonicida subsp. masoucida and Aeromonas

313 salmonicida subsp. salmonicida showed high AAI, so they were subjected for dDDH analysis

314 (Table 1), phenotypically these strains sharing similar phenotypic and biochemical traits (Table

315 S23). A. salmonicida subsp. masoucida was proposed as a later heterotypic synonym of

316 Aeromonas salmonicida subsp. salmonicida. Currently, Aeromonas salmonicida comprised of

317 five subspecies and the proposed one is the first heterotypic synonym of this genus.

318 A pair of Alteromonas species (A. addita and A. stellipolaris) was subjected for the dDDH

319 analysis (Table 1). Both these strains shared a similar abiotic factor (temperature and NaCl)

320 requirements for their growth and metabolism (Table S24). Ecologically, both strains share

321 similar environment as they were isolated from sea water samples [37, 38]. The genome metrics

322 and phenotypic evidences supported the proposal of Alteromonas addita as the later heterotypic

323 synonym of Alteromonas stellipolaris. A prevous phylogenetic analysis study of the Alteromonas

324 taxa, lead to transfer of 11 species to the newly proposed genus Pseudoalteromonas, leading to

325 large numbers of synonyms in this genus [39].

326 One of the important genus from the perspectives of clinical microbiology was Bordetella [40],

327 whose taxa were screened for heterotypic synonym owing to high 16S rRNA gene similarity

328 recorded within three species of Bordetella (Bordetella parapertussis, Bordetella bronchiseptica

329 and Bordetella pertussis) (Table 1), their genome were analysed for heterotypic synonyms or

330 subspecies (Fig. 6). Deduction of high dDDH values (Table 1) between these strains suggested

331 that at least two species among them may be heterotypic synonyms, further the analyses of

332 phenotypic properties also supported this claim as certain carbon utilization and enzyme activity

333 patterns are similar among all these species (Table S25). Genome based OGRI metrics combined

334 with biochemical features supported the proposal of Bordetella parapertussis and Bordetella

335 pertussis as the later heterotypic synonyms of Bordetella bronchiseptica.

336 Further, 16S rRNA gene similarity-based closeness was noticed in the following genera namely

337 Caldimonas, Cronobacter, Desulfotignum, Haemophilus, Methylomicrobium, Paraglaciecola,

338 Tepidiphilus and Thalassospira (Fig. 6). Their species pairs were analysed for OGRI metrics like

339 dDDH and ANI to identify heterotypic synonym or subspecies within their species clades (Table

340 1). In those eight pairs of different species analysed, dDDH values recorded with little variation

341 in the phenotypic properties that (Table S26-S33) supported the proposal of heterotypic

342 synonyms or subspecies.

343 Totally five heterotypic synonyms and two subspecies are proposed within those genera, they

344 are: Caldimonas taiwanensis as the later heterotypic synonym of Caldimonas manganoxidans;

345 Cronobacter dublinensis subsp. lactaridi as the later heterotypic synonym of Cronobacter

346 dublinensis subsp. lausannensis; Methylomicrobium agile as the later heterotypic synonym of

347 Methylomicrobium album, Paraglaciecola agarilytica as the later heterotypic synonym of

348 Paraglaciecola chathamensis and Tepidiphilus thermophilus as the later heterotypic synonym of

349 Tepidiphilus succinatimandens. The different subspecies proposed includes Desulfotignum

350 balticum subsp. phosphitoxidans subsp. nov. and Haemophilus influenzae subsp. aegyptius

351 subsp. nov. Analysis of the Neisseria clade showed the presence of two subspecies as the dDDH

352 value recorded (Table 1) was within the threshold (70-79 %) proposed for subspecies delineation

353 [18], for these two subspecies, similarity was also noticed in the substrate assimilation pattern

354 and enzyme activity (Table S34-35). The subspecies proposed are Neisseria sicca subsp.

355 macacae subsp. nov. and Neisseria mucosa subsp. cerebrosus subsp. nov.

356 Lastly, two important Proteobacteria clade comprised of Pseudoalteromonas and Shewanella

357 were anlaysed for heterotypic synonyms. The Pseudoalteromonas clade comprised of

358 Pseudoalteromonas agarivorans, Pseudoalteromonas donghaensis, Pseudoalteromonas

359 issachenkonii, Pseudoalteromonas atlantica, Pseudoalteromonas lipolytica and

360 Pseudoalteromonas tetraodonis (Fig. 6) all strains sharing high 16S rRNA gene simlairty (Fig. 6

361 & Table 1) When these species were subjected for dDDH analysis, results supported that among

362 each pair of different species identified in this clade, one may be a heterotypic synonym owing to

363 their strain’s genome similarity in the form of OGRI metrics (Table 1), they were also critically

364 examined for their phenotypic traits wherein, their similarity in phenotypic and biochemical

365 traits are noticed (Table S36-S38). When the 16S rRNA gene sequence-based phylogeny (Fig. 6)

366 was carefully examined, two other taxa of Pseudoalteromonas was also phylogenetically close.

367 When they were subjected to OGRI analysis, Pseudoalteromonas espejiana showed < 70 dDDH

368 with any other Pseudoalteromonas in the clade and for the other Pseudoalteromonas species,

369 currently genome data was unavailable. Based on these polyphasic evidences

370 Pseudoalteromonas agarivorans was proposed as later heterotypic synonym of

371 Pseudoalteromonas atlantica. Pseudoalteromonas donghaensis as the later heterotypic synonym of

372 Pseudoalteromonas lipolytica and Pseudoalteromonas issachenkonii as the later heterotypic

373 synonym of Pseudoalteromonas tetraodonis.

374 The clade comprised of Shewanella found to contain two pairs of species (Shewanella algae and

375 Shewanella upenei; Shewanella japonica and Shewanella pacifica). When their dDDH was

376 analysed, closeness in their 4 strains genome was observed (Table 1), phenotypic data also

377 supported the fact that between these two pairs most of the biochemical properties are shared

378 (Table S39-S40). These polyphasic evidences support the proposal of Shewanella upenei as later

379 heterotypic synonym of Shewanella algae and Shewanella pacifica as the later heterotypic

380 synonym of Shewanella japonica.

381 Thermotogae

382 Genetic closeness in the form of 16S rRNA gene similarity was identified in two genera that

383 belonged to the phylum Thermotogae (Fig. 7). A species pair of Pseudothermotoga (P. elfii and

384 P. lettingae) and Thermotoga pair (T. petrophila and T. naphthophila) were selected based 16S

385 rRNA gene similarity, they were subjected to dDDH analysis (Table 1). High similarity in their

386 phenotypic and biochemical traits are observed (Table S41 - S42). The 16S rRNA gene based

387 phylogenetic analysis combined with OGRI metrics and biochemical characters supported the

388 proposal of Pseudothermotoga lettingae as the later heterotypic synonym of Pseudothermotoga

389 elfii. Whereas in another pair, T. naphthophila was hierarchally reduced to subspecies

390 Thermotoga petrophila subsp. naphthophila subsp. nov.

391 Taxonomic consequences: New (combinations for) species

392 When the 16S rRNA gene similarity between the species of the different families or phylum

393 were examined, Firmicutes and Proteobacteria members namely Saccharococcus thermophilus

394 and Haemophilus piscium were taxonomically incorrect in their genus level placement.

395 Saccharococcus thermophilus was isolated from native sugar beet extraction plants in Sweden.

396 Based on cell morphology and its inability to form endospores, it could be differentiated from the

397 Geobacillus, and this trait was also considered as an important criterian in creation of this genus

398 [41]. In the present study, when the 16S rRNA gene phylogeny of this species was analysed, it

399 found to cluster within Parageobacillus, for instance, based on 16S rRNA gene similarity (98.9

400 %), it was phylogenetically similar to Parageobacillus caldoxylosilyticus, further the high ANI

401 value (90.3 %) recorded with P. caldoxylosilyticus also supported the claim that it belongs to

402 Parageobacillus species, since ANI more than 95 % was regarded a OGRI criteria supporting

403 species similarity [23]. Thus, based on the 16S rRNA gene similarity and the tree topology in

404 combination with the OGRI metrics strongly supported the proposal of Saccharococcus

405 thermophilus as Parageobacillus thermophilus as combination novel. Interestingly, the only

406 other member of Saccharococcus was S. caldoxylosilyticus, an obligately thermophilic, xylose

407 utilizing, endospore forming bacteria [42] which was subsequently reclassified to

408 Parageobacillus as a heterotypic synonym [43] and a recent phylogenomics based reassessment

409 study also insist the presence of such a heterotypic synonym in Geobacillus [33].

410 The Proteobacteria genus H. piscium was genetically distant from Haemophilus genus, its 16S

411 rRNA gene sequence similarity with the Haemophilus type species (H influenzae) was 85.6 %. In

412 the 16S rRNA gene sequence based analyis it was genetically close to Aeromonas salmonicida

413 subsp. salmonicida and Aeromonas salmonicida subsp. smithia with 99.9 and 99.6 % similarity.

414 However, the genome data was currently unavailable, hindering the OGRI analysis. Based on the

415 16S rRNA tree topology and on the similarity of the H. piscium 16S rRNA gene sequence with

416 the Aeromonas salmonicida subsp. salmonicida and its less similiar with the Haemophilus type

417 species, Haemophilus piscium was reclassified as Aeromonas salmonicida subsp. piscium.

418 TAXONOMIC CONSEQUENCES: EMENDATION OF SPECIES AND SUBSPECIES

419 ACTINOBACTERIA

420 EMENDED DESCRIPTION OF ACTINOKINEOSPORA MZABENSIS AOUICHE ET

421 AL. 2015

422 Heterotypic synonym: Actinokineospora spheciospongiae Kämpfer et al. 2015.

423 The species description is as given before [44, 45] with the following additions. The colonies

424 exhibited variable growth in terms of aerial (pinkish to white) and substrate mycelial (purple to

425 blackish and yellow to tan) pigments. Strains used cellulose, xylose, fructose, maltose and

426 mannitol as sole carbon source, they also reduced nitrate and liquified gelatin. Variability was

427 noticed in utilization of galactose, sucrose and rhamnose as sole carbon sources. The G+C

428 content of the strains are 72.8 % and the genome size is 7.55 Mbp. The type strain of

429 Actinokineospora mzabensis is PAL84T (=CECT 8578T = DSM 45961T) with GenBank

430 accession number for the whole-genome sequence is GCA_003182415.1.

431 EMENDED DESCRIPTION OF DIETZIA CINNAMEA YASSIN ET AL. 2006

432 Heterotypic synonym: Dietzia papillomatosis Jones et al. 2008.

433 The species description is as given before [46, 47] with the following additions. The colonies

434 produced orange colour, strains grew in 7 and 8 % of NaCl (w/v), and strains produced acid

435 using d-fructose as carbon source and utilized L-cysteine as sole carbon and nitrogen source.

436 Variability was noticed in degradation of chitin, L-tyrosine, acid production from d-glucose and

437 d-mannose and sucrose, variability was also noticed in utilization of L-arginine as sole carbon

438 and nitrogen source. The G+C content of the type strain genome is 70.8 % and the approximate

439 genome size is 3.60 Mbp. The GenBank accession number for the whole-genome sequence is

440 GCA_001571065.1. The type strain is IMMIB RIV-399T (=CCUG 50875T = DSM 44904T

441 = JCM 13663T = NBRC 102147T).

442 EMENDED DESCRIPTION OF RHODOCOCCUS OPACUS KLATTE ET AL. 1995

443 Heterotypic synonym: Rhodococcus imtechensis Ghosh et al. 2006.

444 The species description is as given before [48, 49] with the following additions. All strains

445 utilized maltose and lactose as sole carbon source and L-serine as sole nitrogen source.

446 Variability among the strains are observed for using of L-arabitol, D-melezitose, D-raffinose, L-

447 rhamnose, 2,4 dinitrophenol and p-nitrophenol as sole carbon source and energy and L-serine as

448 sole nitrogen source. Variability among the strains are also observed in Biolog GP2 plate assay

449 for utilization of acetic acid, 2,3 butanediol, -cyclodextrin, L-fucose, D-galacturonic acid, D-

450 lactic acid methyl ester, lactulose, methyl α-D-mannoside, propionic acid, putrescine, L-

451 pyroglutamic acid, D-trehalose and uridine 5-monophosphate as sole carbon source. The G+C

452 content of the type strain genome is 67.3 % and the approximate genome size is 8.53 Mbp. The

453 GenBank accession number for the whole-genome sequence is GCA_001646735.1. The type

454 strain is ATCC 51881T (=CIP 104549T = DSM 43205T = IFO 16217T = JCM 9703T = NBRC

455 100624T = NBRC 16217T).

456 ARCHAEA

457 EMENDED DESCRIPTION OF HALOFERAX VOLCANII (MULLAKHANBHAI AND

458 LARSEN 1975) TORREBLANCA ET AL. 1986

459 Heterotypic synonym: Haloferax lucentense corrig. Gutierrez et al. 2004; Haloferax alexandrinus

460 Asker and Ohta 2002.

461 The species description is as given before [50-53] with the following additions. Cell are Gram

462 negative, producing red to pinkish colonies, growth is observed under an optimum temperature

463 of 37 °C, pH of 7.0 and NaCl concentration of 1-4.5 % w/v. All strains are oxidase positive and

464 able to produce H2S from thiosulfate and produced acid from arabinose, and unable to hydrolyze

465 starch and casein but variability observed in gelatin hydrolysis. Other variable features observed

466 are cell motility, nitrate reduction, and usage of sucrose as sole carbon source. The G+C content

467 of the type strain genome is 65.5 % and the approximate genome size is 4.01 Mbp. The GenBank

468 accession number for the whole-genome sequence is GCA_000025685.1. The type strain is

469 DS2T (=ATCC 29605T = DSM 3757T = IFO 14742T = JCM 8879T = NBRC 14742T = NCCB

470 85050T = NCIMB 2012T = VKM B-1768T).

471 EMENDED DESCRIPTION OF METHANOBACTERIUM VETERUM KRIVUSHIN ET

472 AL. 2010

473 Heterotypic synonym: Methanobacterium arcticum Shcherbakova et al. 2011.

474 The species description is as given before [54, 55] with the following additions. Cells are Gram

475 negative, rod shaped with cyst like appearance, strains exhibit growth in temperature range of

476 15-45 C, pH of 5.5 – 8.5 and NaCl concentration of 0-3 % (w/v). Variability was observed in

477 utilization of substrates such as formate, methylamine/H2 and methanol/H2 and stimulation of

478 growth in presence of acetate. The G+C content of the type strain genome is 33.2 % and the

479 approximate genome size is 3.37 Mbp. The GenBank accession number for the whole-genome

480 sequence is GCA_000745485.1. The type strain is MK4T (=DSM 19849T= VKM B-2440T).

481 EMENDED DESCRIPTION OF METHANOSARCINA MAZEI CORRIG. (BARKER

482 1936) MAH AND KUHN 1984

483 Heterotypic synonym: Methanosarcina soligelidi Wagner et al. 2013.

484 The species description is as given before [56, 57, 58] with the following additions. Cell are

485 Gram negative, irregular cocci, exhibiting wider temperature range of 0-50 C and narrow pH

486 6.1-8.0 for growth. Optimum NaCl concentration of 0.02 – 0.3 % (w/v) supported the growth of

487 the strains. Strains utilized H2/CO2, methanol, acetate. Strains cell membrane possessed archaeol

488 phosphatidylglycerol, hydroxyarchaeol phosphatidylglycerol, archaeol phosphatidylethanolamine

489 and hydroxyarchaeol phosphatidylethanolamine as predominant lipids. Variability among strains

490 was noticed in the utilization of dimethylamine. The G+C content of the type strain genome is

491 41.4 % and the approximate genome size is 4.14 Mbp. The GenBank accession number for the

492 whole-genome sequence is GCA_000970205.1. The type strain is OCM 26T (=DSM 2053T

493 = DSM 3318T = S-6T =VKM B-1636T).

494 BACTEROIDETES

495 EMENDED DESCRIPTION OF RUFIBACTER RUBER KÝROVÁ ET AL. 2016

496 Heterotypic synonym: Rufibacter quisquiliarum Felföldi et al. 2016.

497 The species description is as given before [59, 60] with the following additions. Cells are Gram

498 strain negative, producing pinkish to red coloured colonies on R2A medium, strains positive for

499 catalase, requires a temperature range of 20-37 °C, pH – 7-11 and NaCl concentration range of

500 0-1 % (w/v) for growth. Strains able to assimilate d-mannose, N-acetylglucosamine, aesculin,

501 glycogen, gentiobiose, α-D-glucose, maltose and D-trehalose. All strains produced esterase

502 lipase (C8), leucine arylamidase, valine arylamidase activity and α-Glucosidase enzyme.

503 Difference in assimilation of d-galactose, cellobiose, lactose, melibiose, sucrose and raffinose

504 was observed. The G+C content of the type strain genome is 51.5 % and the approximate

505 genome size is 5.5 Mbp. The GenBank accession number for the whole-genome sequence is

506 GCA_001647275.1. The type strain is CCM 8646T (=LMG 29438T).

507 FIRMICUTES

508 EMENDED DESCRIPTION OF CALDANAEROBACTER SUBTERRANEUS SUBSP.

509 TENGCONGENSIS (XUE ET AL. 2001) FARDEAU ET AL. 2004

510 Heterotypic synonym: Caldanaerobacter subterraneus subsp. yonseiensis (Kim et al. 2001)

511 Fardeau et al. 2004.

512 The species description is as given before [60, 61, 62, 63] with the following additions. Cells

513 grows in a temperature range of 50-80 C and pH range of 4.5 - 9 and NaCl concentration of 0-

514 2.5 % (w/v), strains assimilate CO, produced L-alanine, acetate, H2 and CO2 as diagnostic

515 fermentation products from glucose. Variability was observed in production of lactate as a

516 diagnostic fermentation product from glucose. The G+C content of the type strain genome is

517 37.6 % and the approximate genome size is 2.69 Mbp. The GenBank accession number for the

518 whole-genome sequence is GCA_000007085.1. The type strain is MB4T (=Chinese Collection of

519 Microorganisms AS 1.2430T = DSM 15242T = JCM 11007T = NBRC 100824T).

520 EMENDED DESCRIPTION OF CARNOBACTERIUM INHIBENS SUBSP. INHIBENS

521 (JÖBORN ET AL. 1999) NICHOLSON ET AL. 2015

522 Heterotypic synonym: Carnobacterium inhibens subsp. gilichinskyi Nicholson et al. 2015.

523 The species description is as given before [64, 65] with the following additions. Cells grows at a

524 wider temperature and pH range of 0-37 C and 5.5-9.0 respectively. Cells utilized maltose, D-

525 Mannose, sucrose and trehalose as substrate. Variability was observed in utilization of substrates

526 such as d-galactose, gentiobiose, d-glucose, pectin, potassium tellurite, starch and tetrazolium

527 blue. The G+C content of the type strain genome is 34.9 % and the approximate genome size is

528 2.75 Mbp. The GenBank accession number for the whole-genome sequence is

529 GCA_000746825.1. The type strain is K1T (=CCUG 31728T= CIP 106863T= DSM

530 13024T= JCM 16168T).

531 EMENDED DESCRIPTION OF GEOBACILLUS THERMOLEOVORANS (ZARILLA

532 AND PERRY 1988) NAZINA ET AL. 2001

533 Heterotypic synonym: Geobacillus kaustophilus (Priest et al. 1989) Nazina et al. 2001.

534 The species description is as given before [31, 66, 67] with the following additions. Strains are

535 positive for catalase activity and nitrate reduction, production of acids from glucose, fructose and

536 maltose and mannose, hydrolysis of gelatin and starch. However, variability was observed

537 among the strains in acid production from adonitol, cellobiose, glycerol, mannitol sucrose,

538 trehalose and D-xylose. Variability was also observed in aesculin and casein hydrolysis,

539 utilization of lactate and citrate as growth substrates. The G+C content of the type strain genome

540 is 52.3 % and the approximate genome size is 3.5 Mbp. The GenBank accession number for the

541 whole-genome sequence is GCA_001610955.1. The type strain is 2 LEH-1T (=ATCC 43513T =

542 BGSC 96A1T = DSM 5366T = LMG 9823T).

543 EMENDED DESCRIPTION OF PARAGEOBACILLUS TOEBII (SUNG ET AL. 2002)

544 ALIYU ET AL. 2019

545 Heterotypic synonym: Geobacillus galactosidasius Poli et al. 2012; Geobacillus yumthangensis

546 Najar et al. 2018.

547 The species description is as given before [33, 68, 69, 70] with the following additions. Abiotic

548 factors (temperature, pH and NaCl concentration) supporting the culture growth was similar for

549 all the strains, however, difference in the biochemical properties such as acid formation form

550 cellobiose, galactose, ribose, glycerol, lactose and rhamnose was observed, additionally variation

551 was observed in the carbon utilization profile. The G+C content of the type strain genome is 42.4

552 % and the approximate genome size is 3.32 Mbp. The GenBank accession number for the whole-

553 genome sequence is GCA_003688615.1. The type strain is BK-1T (=DSM 14590T = KCTC

554 0306BPT = LMG 23037T = NBRC 107807T = SK-1T).

555 EMENDED DESCRIPTION OF MEGAMONAS FUNIFORMIS SAKON ET AL. 2008

556 Heterotypic synonym: Megamonas rupellensis Chevrot et al. 2008.

557 The species description is as given before [71, 72] with the following additions. Strains produced

558 acids from glucose, maltose, lactose, mannose, arabinose, salicin, xylose and raffinose.

559 Variability was noticed in acid production from cellobiose and glycerol. The G+C content of the

560 type strain genome is 31.5 % and the approximate genome size is 2.57 Mbp. The GenBank

561 accession number for the whole-genome sequence is GCA_010669225.1. The type strain is YIT

562 11815T (=DSM 19343T = JCM 14723T).

563 EMENDED DESCRIPTION OF THERMOANAEROBACTER THERMOCOPRIAE (JIN

564 ET AL. 1988) COLLINS ET AL. 1994

565 Heterotypic synonyms: Thermoanaerobacter italicus Kozianowski et al. 1998;

566 Thermoanaerobacter mathranii subsp. mathranii (Larsen et al. 1998) Carlier et al. 2007.

567 The species description is as given before [73 -77] with the following additions. Cells exhibiting

568 Gram positive to variable reaction and non-spore forming, requires an optimum temperature of

569 60 C for growth. All strains degrade xylan, strains utilized glucose as growth substrate and

570 fermentation products are produced using acetate, lactate and ethanol. Variability was noticed in

571 motility and utiliztaio nof sucrose and xylose as growth substrates. The G+C content of the type

572 strain genome is 34.1 % and the approximate genome size is 2.46 Mbp. The GenBank accession

573 number for the whole-genome sequence is GCA_000518565.1. The type strain is JT3-3T

574 (=ATCC 51646T= IAM 13577T= JCM 7501T).

575 EMENDED DESCRIPTION OF THERMOANAEROBACTER BROCKII SUBSP. FINNII

576 (SCHMID ET AL. 1986) CAYOL ET AL. 1995

577 Heterotypic synonym: Thermoanaerobacter pseudethanolicus Onyenwoke et al. 2007.

578 The species description is as given before [78 -80] with the following additions. Cells exhibit

579 Gram variable reaction and produced spores and motile. Strains used glucose, sucrose, xylose as

580 growth substrates and fermentation products obtained from ethanol. The G+C content of the type

581 strain genome is 34.5 % and the approximate genome size is 2.34 Mbp. The GenBank accession

582 number for the whole-genome sequence is GCA_000175295.2. The type strain is Ako-1T

583 (=ATCC 43586T= DSM 3389T).

584 EMENDED DESCRIPTION OF THERMOANAEROBACTER ETHANOLICUS WIEGEL

585 AND LJUNGDAHL 1982

586 Heterotypic synonyms: Thermoanaerobacter wiegelii Cook et al. 1996; Thermoanaerobacter

587 siderophilus Slobodkin et al. 1999.

588 The species description is as given before [81- 83] with the following additions. Cells exhibit

589 Gram variable reaction with variability noticed in spore production. All strains are motile,

590 capable of starch hydrolysis, used glucose, sucrose, xylose as substrate for growth and

591 fermentation products are formed using acetate, lactase and ethanol as carbon sources.

592 Variability among the strains are noticed in utilization of ribose, mannitol, glycerol, pyruvate as

593 growth substrates. The G+C content of the type strain genome is 34.2 % and the approximate

594 genome size is 2.91 Mbp. The GenBank accession number for the whole-genome sequence is

595 GCA_003722315.1. The type strain is JW 200T (=ATCC 31550T= DSM 2246T).

596 NITROSPIRAE

597 EMENDED DESCRIPTION OF THERMODESULFOVIBRIO YELLOWSTONII HENRY

598 ET AL. 1994

599 Heterotypic synonym: Thermodesulfovibrio islandicus Sonne-Hansen and Ahring 2000.

600 The species description is as given before [35-36] with the following additions. Nutritionally

601 strains can utilize pyruvate, hydrogen (plus acetate) and formate (plus acetate) as both electron

602 donor and carbon source in the presence of sulfate as terminal electron acceptor and in addition

603 all strains utilized sulfate and thiosulfate as electron acceptors. However, variability was noticed

604 in utilization of sulfite and nitrate as terminal electron acceptors. Strains showed fermentative

605 growth with pyruvate. Strains preferred a thermophilic range of temperature (40 - 70 C) for

606 growth. The G+C content of the type strain genome is 34.1 % and the approximate genome size

607 is 2.0 Mbp. The GenBank accession number for the whole-genome sequence is

608 GCA_000020985.1. The type strain is YP87T (=ATCC 51303T= DSM 11347T).

609 PROTEOBACTERIA

610 EMENDED DESCRIPTION OF AEROMONAS SALMONICIDA (LEHMANN AND

611 NEUMANN 1896) GRIFFIN ET AL. 1953 (APPROVED LISTS 1980)

612 Heterotypic synonym: Aeromonas salmonicida subsp. masoucida Kimura 1969 (Approved Lists

613 1980).

614 The species description is as given before [85, 86, 87] with the following additions. Motile cells

615 utilized urocanic acid, sucrose and fermented mannitol. However, variability noticed in brown

616 diffusible pigment production, indole production and Voges-Proskauer test at 25 C,

617 fermentation of sucrose and gas production from glucose. The G+C content of the type strain

618 genome is 58.3 % and the approximate genome size is 4.94 Mbp. The GenBank accession

619 number for the whole-genome sequence is GCA_900445115.1. The type strain is ATCC 33658T

620 (=CAIM 346T = CIP 103209T = DSM 18220T = DSM 19634T = HAMBI 1984T = JCM 7874T

621 = LMG 3780T = NCIMB 1102T = NCTC 12959T).

622 EMENDED DESCRIPTION OF ALTEROMONAS STELLIPOLARIS VAN TRAPPEN ET

623 AL. 2004

624 Heterotypic synonym: Alteromonas addita Ivanova et al. 2005.

625 The species description is as given before [37, 38] with the following additions. All strains are

626 Gram-negative, motile, oxidase- and catalase-positive and negative for indole and H2S

627 production, grow at 3–6 % NaCl and produce lipase (Tween 80). Strains able to grow at 4 C but

628 growth was not observed at 40 C. Similarly, able to grow at 10 % but growth was not observed

629 at 15 % NaCl. Strains hydrolyzed starch. Variability observed in haemolysis and assimilation of

630 D-mannitol and L-lactate. The G+C content of the type strain genome is 43.5 % and the

631 approximate genome size is 4.90 Mbp. The GenBank accession number for the whole-genome

632 sequence is GCA_001562115.1. The type strain is ANT 69aT (=DSM 15691T = LMG 21861T).

633 EMENDED DESCRIPTION OF BORDETELLA BRONCHISEPTICA (FERRY 1912)

634 MORENO-LÓPEZ 1952 (APPROVED LISTS 1980)

635 Heterotypic synonym: Bordetella parapertussis (Eldering and Kendrick 1938) Moreno-López

636 1952 (Approved Lists 1980); Bordetella pertussis (Bergey et al. 1923) Moreno-López 1952

637 (Approved Lists 1980).

638 The species description is as given before [88-91] with the following additions. Minute

639 coccobacillus motile or non-motile cells, variability in oxidase activity and growth on the

640 MacConkey agar and Simmons citrate agar was noticed. Differences in urease activity, reduction

641 of nitrate to nitrite, brown pigment production on HI agar with tyrosine, litmus milk alkalization

642 was observed among the strains. None of the strains assimilated D-xylose and D-gluconate but

643 variability observed among assimilation of phenyl acetate. All strains are negative for lipase

644 (C14) and valine arylamidase, but variability observed on chymotrypsin and naphthol-AS-Bi-

645 phosphohydrolase activity. The G+C content of the type strain genome is 68.2 % and the

646 approximate genome size is 5.17 Mbp. The GenBank accession number for the whole-genome

647 sequence is GCA_900445725.1. The type strain is ATCC 19395T (=CCUG 219T = CIP 55.110T

648 = DSM 13414T = IFO 13691T = LMG 1232T = NBRC 13691T = NCTC 452T).

649 EMENDED DESCRIPTION OF CALDIMONAS MANGANOXIDANS TAKEDA ET

650 AL. 2002

651 Heterotypic synonym: Caldimonas taiwanensis Chen et al. 2005.

652 The species description is as given before [92, 93] with the following additions. Gram negative

653 rods, for growth, strains require an optimum temperature and pH of 50 C and 7.0 respectively.

654 Strains exhibit variable oxidase activity and variable utilization of substrates like galactose,

655 malate, malonate, sucrose, acetate, fructose and trehalose. The G+C content of the type strain

656 genome is 66 % and the approximate genome size is 3.53 Mbp. The GenBank accession number

657 for the whole-genome sequence is GCA_000381125.1. The type strain is HST (=ATCC BAA-

658 369T = IFO 16448T= JCM 10698T= NBRC 16448T).

659 EMENDED DESCRIPTION OF CRONOBACTER DUBLINENSIS SUBSP.

660 LAUSANNENSIS IVERSEN ET AL. 2008

661 Heterotypic synonym: Cronobacter dublinensis subsp. lactaridi Iversen et al. 2008.

662 The species description is as given before [95] with the following additions. Strains variable

663 Indole production, they utilized cis-Aconitate, trans-aconitate, palatinose, 1-O-methyl--

664 glucopyranoside and 4-aminobutyrate. But variability observed in the utilization of lactulose,

665 maltitol, putrescine, turanose, myo-inositol. However, strains area unable to utilize dulcitol,

666 malonate and Melezitose, The G+C content of the type strain genome is 57.9 % and the

667 approximate genome size is 4.61 Mbp. The GenBank accession number for the whole-genome

668 sequence is GCA_000409365.1. The type strain is E515T (=DSM 18706T= JCM 16469T = LMG

669 23824T).

670 EMENDED DESCRIPTION OF METHYLOMICROBIUM ALBUM (BOWMAN ET

671 AL. 1993) BOWMAN ET AL. 1995

672 Heterotypic synonym: Methylomicrobium agile (Bowman et al. 1993) Bowman et al. 1995.

673 The species description is as given before [95, 96] with the following additions. Cells are motile,

674 non-cyst forms able to grow at an optimum temperature of 25-30 C but unable to grow at 3 %

675 NaCl concentration and the optimum pH observed for growth is 7.0. Strains possessed

676 particulate methane mono oxygenase, but soluble methane mono oxygenase is absent. Strain

677 level variability was observed during growth at 37 C. The G+C content of the type strain

678 genome is 56.2 % and the approximate genome size is 4.49 Mbp. The GenBank accession

679 number for the whole-genome sequence is GCA_000214275.3. The type strain is BG8T (=ACM

680 3314T = ATCC 33003T = NCIMB 11123T = VKM-BG8T).

681 EMENDED DESCRIPTION OF PARAGLACIECOLA CHATHAMENSIS (MATSUYAMA

682 ET AL. 2006) SHIVAJI AND REDDY 2014

683 Heterotypic synonym: Paraglaciecola agarilytica (Yong et al. 2007) Shivaji and Reddy 2014.

684 The species description is as given before [97, 98, 99] with the following additions. Strains were

685 able to grow at temperature range of 7-30 C but growth was variable at 4 C, the NaCl range of

686 2-8 % supported the growth. Strains hydrolyzed aesculin, casein and starch; utilized D-galactose,

687 D-mannitol, D-mannose as carbon source. Strain variability was observed in usage of acetate,

688 cellobiose, maltose and sucrose as carbon substrates and hydrolysis of ONPG. The G+C content

689 of the type strain genome is 44.1 % and the approximate genome size is 5.27 Mbp. The GenBank

690 accession number for the whole-genome sequence is GCA_000314955.1. The type strain is

691 S18K6T (=JCM 13645T= NCIMB 14146T).

692 EMENDED DESCRIPTION OF PSEUDOALTEROMONAS ATLANTICA (AKAGAWA-

693 MATSUSHITA ET AL. 1992) GAUTHIER ET AL. 1995

694 Heterotypic synonym: Pseudoalteromonas agarivorans Romanenko et al. 2003.

695 The species description is as given before [39, 100, 101] with the following additions. Strains are

696 motile, positive for the sodium-ion requirement for growth, growth at 25–28 °C, produced acid

697 from mannitol and oxidized glycogen. Variability was noticed in hydrolysis of carrageenan, acid

698 production from glucose, D-mannitol, maltose, caprate, phenylacetate. Strain level variation was

699 also observed in oxidation of D-fructose, mannose, sucrose, glycerol, citrate, propionate

700 butyrate. The G+C content of the type strain genome is 40.8 % and the approximate genome size

701 is 4.47 Mbp. The GenBank accession number for the whole-genome sequence is

702 GCA_007988745.1. The type strain is NCIMB 301T (=ATCC 19262T = CIP 104721T = DSM

703 6840T = IAM 12927T = JCM 8845T = NBRC 103033T).

704 EMENDED DESCRIPTION OF PSEUDOALTEROMONAS LIPOLYTICA XU ET

705 AL. 2010

706 Heterotypic synonym: Pseudoalteromonas donghaensis Oh et al. 2011.

707 The species description is as given before [102, 103] with the following additions. Strains are

708 positive for catalase and oxidase activity, nitrate reduction, hydrolyzed gelatin and Tween 80,

709 they utilized N-acetyl-glucosamine, L-arabinose, maltose and mannose as sole carbon source.

710 Acid is produced from maltose and mannose as substrates. Variability among strains are

711 observed in acid production from glucose. The G+C content of the type strain genome is 41.4 %

712 and the approximate genome size is 4.54 Mbp. The GenBank accession number for the whole-

713 genome sequence is GCA_900116435.1. The type strain is LMEB 39T (=CGMCC

714 1.8499T= DSM 22356T = JCM 15903T).

715 EMENDED DESCRIPTION OF PSEUDOALTEROMONAS TETRAODONIS (SIMIDU ET

716 AL. 1990) IVANOVA ET AL. 2001

717 Heterotypic synonym: Pseudoalteromonas issachenkonii Ivanova et al. 2002.

718 The species description is as given before [104-106] with the following additions. Strains grow

719 at 4 C but strain variability noticed for growth at 37 C, strain growth is noticed upto 12 % NaCl

720 concentration. DNase activity observed in strains, but variability noticed in Chitinase activity but

721 none of the strains shows amylase, agarose and carrageenase activity. All strains used D-

722 galactose, sucrose, maltose, citrate, acetate, and pyruvate as substrates. But strain variability was

723 noticed in utilization of D-fructose, melibiose, lactose, succinate and mannitol as substrates. The

724 G+C content of the type strain genome is 40.3 % and the approximate genome size is 4.13 Mbp.

725 The GenBank accession number for the whole-genome sequence is GCA_002310835.1. The

726 type strain is GFCT (=ATCC 51193T = CIP 104758T = CIP 107120T = DSM 9166T = IAM

727 14160T = JCM 21038T = KMM 458T = NBRC 103034T).

728 EMENDED DESCRIPTION OF SHEWANELLA ALGAE CORRIG. SIMIDU ET AL. 1990

729 Heterotypic synonym: Shewanella upenei Kim et al. 2012.

730 The species description is as given before [106, 107] with the following additions. Strains

731 exhibited motility and positive for motility, catalase, oxidase; H2S production, nitrate reduction,

732 hydrolysis of casein, DNA, gelatin, tyrosine and Tweens 20, 40, 60, and 80; utilization of D-

733 glucose, acetate, L-malate, pyruvate and succinate; activity of alkaline phosphatase, esterase

734 (C4), esterase lipase (C8), leucine arylamidase (weak), Į-chymotrypsin. Variability among

735 strains are noticed for acid production from D-glucose and D-ribose, acid phosphatase and

736 naphthol-AS-BI-phosphohydrolase activity. The G+C content of the type strain genome is 53 %

737 and the approximate genome size is 4.87 Mbp. The GenBank accession number for the whole-

738 genome sequence is GCA_009183365.1. The type strain is OK-1T (=ATCC 51192T= CCUG

739 39064T = CIP 106454T = DSM 9167T = IAM 14159T = JCM 21037T = NBRC 103173T).

740 EMENDED DESCRIPTION OF SHEWANELLA JAPONICA IVANOVA ET AL. 2001

741 Heterotypic synonym: Shewanella pacifica Ivanova et al. 2004.

742 The species description is as given before [109, 110] with the following additions. Strains are

743 oxidase and catalase positive, reduce nitrate to nitrite, hemolytic and produced lipase, amylase,

744 gelatinase and Chitinase, strains can grow at 32 C but variability in growth is noticed at 4 C

745 and growth at 6 % NaCl. Strain level difference is also noticed in the utilization of d-galactose

746 and succinate as substrate. The G+C content of the type strain genome is 40.8 % and the

747 approximate genome size is 4.98 Mbp. The GenBank accession number for the whole-genome

748 sequence is GCA_002075795.1. The type strain is ATCC BAA-316T (=CIP 106860T = DSM

749 15915T = JCM 21433T = KMM 3299T = LMG 19691T = NBRC 103171T).

750 EMENDED DESCRIPTION OF TEPIDIPHILUS SUCCINATIMANDENS (BONILLA

751 SALINAS ET AL. 2004) PODDAR ET AL. 2014

752 Heterotypic synonym: Tepidiphilus thermophilus Poddar et al. 2014.

753 The species description is as given before [110, 111] with the following additions. All strains

754 used Tween 40, Tween 80, D-glucuronic acid and glucuronamide as substrates. Variability was

755 noticed in Urea hydrolysis and nitrite reduction, strain level difference is observed for valine

756 arylamidase, cysteine arylamidase, trypsin and α-glucosidase activity. Variability in oxidation of

757 carbon sources such as i-erythritol, xylitol, cis-aconitic acid, D-galacturonic acid, α-ketoglutaric

758 acid, α-ketovaleric acid, DL-lactic acid, D-alanine, L-alanine, L-asparagine, L- glutamic acid, L-

759 leucine, L-serine, glycerol and D-glucose 6-phosphate. The G+C content of the type strain

760 genome is 65.9 % and the approximate genome size is 2.37 Mbp. The GenBank accession

761 number for the whole-genome sequence is GCA_006503695.1. The type strain is 4BON T (= CIP

762 107790T = DSM 15512T).

763 THERMOTOGAE

764 EMENDED DESCRIPTION OF PSEUDOTHERMOTOGA ELFII (RAVOT ET AL. 1995)

765 BHANDARI AND GUPTA 2014

766 Heterotypic synonym: Pseudothermotoga lettingae (Balk et al. 2002) Bhandari and Gupta 2014.

767 The species description is as given before [112-114] with the following additions. All strains can

768 grow at a temperature range of 50-70 C, pH range of 5.5 – 8.5 and NaCl concentration of 1.2 %.

769 Strain utilized xylose a carbon source. However, strain level variability was observed in the

770 reduction of elemental sulfur and usage of acetate+thiosulfate as the substrate for growth. The

771 G+C content of the type strain genome is 38.7 % and the approximate genome size is 2.17 Mbp.

772 The GenBank accession number for the whole-genome sequence is GCA_000504085.1. The

773 type strain is SERBT (=ATCC 51869T= DSM 9442T = SEBR 6459T).

774 Taxonomic Consequences: New Subspecies

775 ACTINOBACTERIA

776 DESCRIPTION OF NOCARDIA EXALBIDA SUBSP. GAMKENSIS SUBSP. NOV.

777 Nocardia exalbida subsp. gamkensis (N.L. masc./fem. adj. gamkensis, pertaining to the river

778 Gamka in South Africa)

779 The description is as given for Nocardia gamkensis [115]. The G+C content of the type strain

780 genome is 68.4 % and the approximate genome size is 7.71 Mbp. The GenBank accession

781 number for the whole-genome sequence is GCA_001612985.1. The type strain is CZH20T

782 (=DSM 44956T= JCM 14299T = NRRL B-24450T).

783 DESCRIPTION OF NOCARDIA IGNORATA SUBSP. COUBLEAE SUBSP. NOV.

784 Nocardia ignorata subsp. coubleae (N.L. gen. fem. n. coubleae, of Couble, named after Andrée

785 Couble, in recognition of her contribution to the French Nocardiosis Observatory, Lyon, France).

786 The description is as given for Nocardia coubleae [116]. The G+C content of the type strain

787 genome is 67.9 % and the approximate genome size is 6.62 Mbp. The GenBank accession

788 number for the whole-genome sequence is GCA_001612805.1. The type strain is OFN N12T

789 (=CIP 108996T= DSM 44960T= JCM 15318T).

790 DESCRIPTION OF NOCARDIA NOVA SUBSP. ELEGANS SUBSP. NOV.

791 Nocardia nova subsp. elegans (e’le.gans L. adj. elegans, fastidious (with respect of utilization of

792 nutrients)

793 The description is as given for Nocardia elegans [117]. The G+C content of the type strain

794 genome is 67.9 % and the approximate genome size is 7.54 Mbp. The GenBank accession

795 number for the whole-genome sequence is GCA_001612845.1. The type strain is IMMIB N-

796 402T (=CCUG 50200T= CIP 108553T= DSM 44890T= JCM 13374T).

797 FIRMICUTES

798 DESCRIPTION OF CARBOXYDOTHERMUS HYDROGENOFORMANS SUBSP.

799 FERRIREDUCENS SUBSP. NOV.

800 Carboxydothermus hydrogenoformans subsp. ferrireducens [fer.ri.re.du’cens L. neut. n. ferrum,

801 iron; L. pres. part. reducens, converting to a different state; N.L. part. adj. ferrireducens,

802 reducing (ferric) iron]

803 The description is as given for Carboxydothermus ferrireducens [118-119]. The G+C content of

804 the type strain genome is 41.9 % and the approximate genome size is 2.44 Mbp. The GenBank

805 accession number for the whole-genome sequence is GCA_000427565.1. The type strain is

806 JW/AS-Y7T (=DSM 11255T= VKM B-2392T).

807 DESCRIPTION OF GEOBACILLUS STEAROTHERMOPHILUS SUBSP. LITUANICUS

808 SUBSP. NOV.

809 Geobacillus stearothermophilus subsp. lituanicus (li.tu.a’ni.cus M.L. adj. lituanicus, of

810 Lithuania, referring to the Lithuanian oilfield from where the type strain was isolated)

811 The description is as given for Geobacillus lituanicus [32]. The G+C content of the type strain

812 genome is 52.1 % and the approximate genome size is 3.5 Mbp. The GenBank accession number

813 for the whole-genome sequence is GCA_002243605.1. The type strain is N-3T (=DSM 15325T

814 = VKM B-2294T).

815 DESCRIPTION OF SALIMICROBIUM SALEXIGENS SUBSP. JEOTGALI SUBSP. NOV.

816 Salimicrobium salexigens subsp. jeotgali (je.ot.ga’li N.L. gen. n. jeotgali, of jeotgal, from which

817 the organism was first isolated).

818 The description is as given for Salimicrobium jeotgali [120]. The G+C content of the type strain

819 genome is 46.3 % and the approximate genome size is 2.78 Mbp. The GenBank accession

820 number for the whole-genome sequence is GCA_001685435.3. The type strain is MJ3T (=JCM

821 19758T= KACC 16972T).

822 PROTEOBACTERIA

823 DESCRIPTION OF DESULFOTIGNUM BALTICUM SUBSP. PHOSPHITOXIDANS

824 SUBSP. NOV.

825 Desulfotignum balticum subsp. phosphitoxidans (phos.phit.o’xi.dans N.L. part.

826 adj. phosphitoxidans, oxidizing phosphite, referring to its utilization of this unusual electron

827 source).

828 The description is as given for Desulfotignum phosphitoxidans [121]. The G+C content of the

829 type strain genome is 51.3 % and the approximate genome size is 4.50 Mbp. The GenBank

830 accession number for the whole-genome sequence is GCA_000350545.1. The type strain is

831 FiPS-3T (=DSM 13687T= OCM 818T).

832 DESCRIPTION OF HAEMOPHILUS INFLUENZAE SUBSP. AEGYPTIUS SUBSP. NOV.

833 Haemophilus influenzae subsp. aegyptius (L. masc. adj. aegyptius, Aegyptian).

834 The description is as given for Haemophilus aegyptius [122, 123]. The G+C content of the type

835 strain genome is 37.1 % and the approximate genome size is 1.96 Mbp. The GenBank accession

836 number for the whole-genome sequence is GCA_000195005.1. The type strain is ATCC 11116T

837 (=CCUG 25716T = CIP 52.129T = DSM 21187T = NCTC 8502T).

838 DESCRIPTION OF NEISSERIA SICCA SUBSP. MACACAE SUBSP. NOV.

839 Neisseria sicca subsp. macacae (ma.ca’cae N.L. gen. n. macacae, of a monkey, referring to the

840 source of the isolate (isolated from the oropharynges of rhesus monkeys); from Portuguese

841 n. macaco, female monkey; from N.L. n. Macaca, the generic name of macaques).

842 The description is as given for Neisseria macacae [124]. The G+C content of the type strain

843 genome is 50 % and the approximate genome size is 2.75 Mbp. The GenBank accession number

844 for the whole-genome sequence is GCA_000220865.1. The type strain is M-740T (=ATCC

845 33926T = CIP 103346T = DSM 19175T).

846 DESCRIPTION OF NEISSERIA MUCOSA SUBSP. CEREBROSUS SUBSP. NOV.

847 Neisseria mucosa subsp. cerebrosus (L. masc. adj. cerebrosus, having a madness of the brain,

848 hare-brained, hotbrained, passionate, intended to mean pertaining to the brain, the original source

849 of isolation of this organism)

850 The description is as given for Morococcus cerebrosus [125]. The G+C content of the type strain

851 genome is 51.4 % and the approximate genome size is 2.45 Mbp. The GenBank accession

852 number for the whole-genome sequence is GCA_000813705.1. The type strain is UQM 858T

853 (=ATCC 33486T= CIP 81.93T = DSM 24335T= NCTC 11393T).

854 DESCRIPTION OF THERMOTOGA PETROPHILA SUBSP. NAPHTHOPHILA SUBSP.

855 NOV.

856 Thermotoga petrophila subsp. naphthophila (Gr. n. naphtha, naphta, bitumen; N.L. fem.

857 adj. phila, friend, loving; from Gr. fem. adj. philê, loving; N.L. fem. adj. naphthophila, bitumen-

858 loving).

859 The description is as given for Thermotoga naphthophila [126]. The G+C content of the type

860 strain genome is 46.1 % and the approximate genome size is 1.81 Mbp. The GenBank accession

861 number for the whole-genome sequence is GCA_000025105.1. The type strain is RKU-10T

862 (=DSM 13996T= JCM 10882T).

863 Taxonomic Consequences: New (Combinations for) Species

864 DESCRIPTION OF AEROMONAS SALMONICIDA SUBSP. PISCIUM COMB. NOV.

865 Aeromonas salmonicida subsp. piscium (pis’ci.um L. gen. pl. n. piscium, of fishes).

866 Basonym: Haemophilus piscium Snieszko et al. 1950 (Approved Lists 1980).

867 The description is same as given before [127-133] for Haemophilus piscium with the following

868 additions. 16S rRNA phylogeny analyses provided strong evidence for assignment of this species

869 to the genus Aeromonas. The type strain is ATCC 10801T (=CCUG 15943= CIP 106116= JCM

870 7872T).

871 DESCRIPTION OF PARAGEOBACILLUS THERMOPHILUS COMB. NOV.

872 Parageobacillus thermophilus (ther.mo’phi.lus Gr. fem. n. thermê, heat; N.L. adj. philus -a -um,

873 friend, loving; from Gr. adj. philos -ê -on, loving; N.L. masc. adj. thermophilus, heat-loving)

874 Basonym: Saccharococcus thermophilus Nystrand 1984.

875 The description is same as given before [41] for Saccharococcus thermophilus with the

876 following additions. Phylogenomic analyses and 16S rRNA phylogeny provided strong evidence

877 for assignment of this species to the genus Parageobacillus. The G+C content of the type-strain

878 genome is 44.88 %, its approximate size 3.15 Mbp, its NCBI GenBank assembly accession

879 GCA_011761475.1. The type strain is 657T (=ATCC 43125T= CCM 3586T= DSM 4749T).

880 Funding information

881 The authors received no specific grant from any funding agency.

882 Acknowledgements

883 Conflicts of interest

884 The author(s) declare that there are no conflicts of interest.

885 References

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1293 1999; 35:155-158.

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Table 1. 16S rRNA gene sequence percent identity and overall genome-relatedness indices for specific clades in the phylum Actinobacteria, Bacteroidetes, Firmicutes, Nitrospirae, Proteobacteria, Thermotogae and in domain Archaea. 16S Strain 1 Strain 2 dDDH* OrthoANI$ AAI† Taxonomic Consequences rRNA Actinobacteria Actinokineospora spheciospongiae EG49T Actinokineospora mzabensis CECT 8578T 99.79 89.9 98.84 98.72 A. spheciospongiae is later heterotypic synonym “Corynebacterium ihumii” GD7T C. afermentans subsp. afermentans DSM 44280T 99.93 78.0 97.47 99.23 New subspecies of C. afermentans from C. ihumii Dietzia papillomatosis NBRC 105045T Dietzia cinnamea NBRC 102147T 99.58 81.5 97.97 98.73 D. papillomatosis is later heterotypic synonym Dietzia cinnamea NBRC 102147T Dietzia maris 97T 98.11 29.0 85.45 86.6 Not heterotypic synonyms (see discussion) Nocardia coubleae NBRC 108252T Nocardia ignorata DSM 44496T 99.78 74.8 96.59 99.48 New subspecies of N. ignorata from N. coubleae Nocardia gamkensis NBRC 108242T Nocardia exalbida NBRC 100660T 99.78 73.7 96.24 99.06 New subspecies of N. exalbida from N. gamkensis Nocardia nova NBRC 15556T Nocardia elegans NBRC 108235T 98.11 75.1 96.61 98.96 New subspecies of N. nova from N. elegans Rhodococcus imtechensis RKJ300T Rhodococcus opacus DSM 43205T 98.89 81.2 97.89 99.06 R. imtechensis is later heterotypic synonym Archaea Haloferax alexandrinus JCM 10717T Haloferax volcanii DS2T 99.72 80.4 97.84 97.82 H. alexandrinus is later heterotypic synonym Haloferax lucentense DSM 14919T Haloferax alexandrinus JCM 10717T 99.86 94.0 99.26 99.69 H. lucentense is later heterotypic synonym Haloferax lucentense DSM 14919T Haloferax volcanii DS2T 99.72 79.9 97.88 97.85 H. lucentens is later heterotypic synonym Methanobacterium veterum MK4T Methanobacterium arcticum M2T 99.85 99.0 99.99 99.92 M. arcticum is later heterotypic synonym Methanosarcina mazei S-6T Methanosarcina soligelidi SMA-21T 99.80 81.5 97.89 99.17 M. soligelidi is later heterotypic synonym Bacteroidetes Rufibacter ruber CCM 8646T Rufibacter quisquiliarum DSM 29854T 99.93 83.0 98.04 nd R. quisquiliarum is later heterotypic synonym Firmicutes Caldanaerobacter subterraneus subsp. yonseiensis KB-1T C. subterraneus subsp. tengcongensis MB4T 98.35 81.7 97.95 99.14 C. subterraneus subsp. yonseiensis is later heterotypic synonym Carboxydothermus ferrireducens DSM 11255T Carboxydothermus hydrogenoformans Z-2901T 96.11 79.8 97.75 98.27 New subspecies of C. hydrogenoformans from C. ferrireducens Carnobacterium inhibens subsp. inhibens DSM 13024T C. inhibens subsp. gilichinskyi WN1359T 99.53 84.1 98.06 99.55 C. inhibens subsp. gilichinskyi is later heterotypic synonym Geobacillus galactosidasius DSM 18751T Geobacillus yumthangensis AYN2T 99.76 81.2 97.84 nd G. yumthangensis is later heterotypic synonym Geobacillus kaustophilus NBRC 102445T Geobacillus thermoleovorans KCTC 3570T 99.86 84.0 98.18 99.76 G. kaustophilus is later heterotypic synonym Geobacillus lituanicus N-3T Geobacillus stearothermophilus ATCC 12980T 99.05 73.4 96.93 nd New subspecies of G. stearothermophilus from G. lituanicus Geobacillus yumthangensis AYN2T Parageobacillus toebii DSM 14590T 99.92 82.0 97.9 nd G. yumthangensis is later heterotypic synonym Geobacillus galactosidasius DSM 18751T Parageobacillus toebii DSM 14590T 99.50 88.0 98.53 98.36 G. galactosidasius is later heterotypic synonym Megamonas rupellensis DSM 19944T Megamonas funiformis YIT 11815T 97.18 83.9 98.18 99.43 M. rupellensis is later heterotypic synonym Parageobacillus caldoxylosilyticus NBRC 107762T Saccharococcus thermophilus DSM 4749T 98.92 40.5 90.25 92.55 Transfer of S. thermophilus to the genus Parageobacillus Salimicrobium salexigens DSM 22782T Salimicrobium jeotgali MJ3T 99.93 79.1 97.57 99.4 New subspecies of S. salexigens from S. jeotgali Thermoanaerobacter ethanolicus JW 200T Thermoanaerobacter wiegelii Rt8.B1T 98.3 76.2 97.41 99.08 T. wiegelii is later heterotypic synonym Thermoanaerobacter ethanolicus JW 200T Thermoanaerobacter siderophilus SR4T 97.7 86.6 98.47 99.38 T. siderophilus is later heterotypic synonym Thermoanaerobacter italicus Ab9T T. mathranii subsp. mathranii A3T 98.35 82.8 98.0 99.11 T. italicus is later heterotypic synonym Thermoanaerobacter italicus Ab9T Thermoanaerobacter thermocopriae JCM 7501T 98.15 84.6 98.15 99.03 T. italicus is later heterotypic synonym Thermoanaerobacter mathranii subsp. mathranii A3T Thermoanaerobacter thermocopriae JCM 7501T 99.66 89.3 98.77 99.35 T. mathranii subsp. mathranii is later heterotypic synonym Thermoanaerobacter pseudethanolicus ATCC 33223T Thermoanaerobacter brockii subsp. finnii Ako-1T 100 97.6 99.95 99.98 T. pseudethanolicus is later heterotypic synonym Thermoanaerobacter siderophilus SR4T Thermoanaerobacter wiegelii Rt8.B1T 98.23 77.7 97.46 98.94 T. siderophilus is later heterotypic synonym

63 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Nitrospirae Thermodesulfovibrio islandicus DSM 12570T Thermodesulfovibrio yellowstonii DSM 11347T 99.86 92.4 99.07 99.37 T. islandicus is later heterotypic synonym Proteobacteria Aeromonas salmonicida subsp. masoucida NBRC 13784T A. salmonicida subsp. salmonicida NCTC 12959T 99.86 97.7 99.74 99.86 A. salmonicida subsp. masoucida is later heterotypic synonym Alteromonas addita R10SW13T Alteromonas stellipolaris LMG 21861T 99.73 90.1 98.86 99.76 A. addita is later heterotypic synonym Bordetella parapertussis FDAARGOS 177T Bordetella bronchiseptica NCTC 452T 100 91.0 98.95 99.64 B. parapertussis is later heterotypic synonym Bordetella parapertussis FDAARGOS 177T Bordetella pertussis 18323T 99.79 88.7 98.7 99.4 B. parapertussis is later heterotypic synonym Bordetella pertussis 18323T Bordetella bronchiseptica NCTC 452T 99.79 86.5 98.45 99.39 B. pertussis is later heterotypic synonym Caldimonas taiwanensis NBRC 104434T Caldimonas manganoxidans ATCC BAA-369T 100 87.6 98.58 99.63 C. taiwanensis is later heterotypic synonym Cronobacter dublinensis subsp. lausannensis LMG 23824T C. dublinensis subsp. lactaridi LMG 23825T 99.11 85.8 98.43 99.2 C. dublinensis subsp. lactaridi is later heterotypic synonym Desulfotignum balticum DSM 7044T Desulfotignum phosphitoxidans DSM 13687T 98.92 75.7 97.03 98.71 New subspecies of D. balticum from D. phosphitoxidans Haemophilus influenzae NCTC 8143T Haemophilus aegyptius ATCC 11116T 99.32 72.9 96.87 98.44 New subspecies of H. influenzae from H. aegyptius Methylomicrobium album BG8T Methylomicrobium agile ATCC 35068T 100 90.0 98.93 99.64 M. agile is later heterotypic synonym Neisseria macacae ATCC 33926T Neisseria sicca ATCC 29256T 99.86 70.7 96.34 99.09 New subspecies of N. sicca from N. macacae Neisseria mucosa ATCC 19696T Morococcus cerebrosus CIP 81.93T 99.93 72.6 96.91 nd New subspecies of N. mucosa from M. cerebrosus Paraglaciecola chathamensis S18K6T Paraglaciecola agarilytica NO2T 99.86 86.3 98.39 99.6 Paraglaciecola agarilytica is later heterotypic synonym Pseudoalteromonas agarivorans DSM 14585T Pseudoalteromonas atlantica NBRC 103033T 99.79 83.2 98.14 98.61 Pseudoalteromonas agarivorans is later heterotypic synonym Pseudoalteromonas donghaensis HJ51T Pseudoalteromonas lipolytica CGMCC 1.8499T 99.45 92.6 99.02 99.14 Pseudoalteromonas donghaensis is later heterotypic synonym Pseudoalteromonas issachenkonii KCTC 12958T Pseudoalteromonas tetraodonis GFCT 99.86 83.8 98.11 98.65 Pseudoalteromonas issachenkonii is later heterotypic synonym Shewanella algae CECT 5071T Shewanella upenei 20-23RT 99.93 83.8 98.2 98.64 Shewanella upenei is later heterotypic synonym Shewanella japonica KCTC 22435T Shewanella pacifica KCTC 12235T 99.31 88.9 98.79 99.01 Shewanella pacifica is later heterotypic synonym Tepidiphilus thermophilus JCM 19170T Tepidiphilus succinatimandens DSM 15512T 99.71 88.4 98.62 98.69 Tepidiphilus thermophilus is later heterotypic synonym “Thalassospira permensis” NBRC 106175T Thalassospira xiamenensis M-5T 99.93 79.6 97.78 99.53 New subspecies of T. xiamenensis from T. permensis Thermotogae Pseudothermotoga lettingae TMOT Pseudothermotoga elfii NBRC 107921T 99.93 93.7 99.33 99.59 Pseudothermotoga lettingae is later heterotypic synonym Thermotoga petrophila RKU-1T Thermotoga naphthophila RKU-10T 99.53 71.2 96.8 98.11 New subspecies of T. petrophila from T. naphthophila

*dDDH similarities were calculated using Genome-to-Genome Distance Calculator (formula 2) web server 2.1 (http://ggdc.dmz.de).

$OrthoANI values were calculated by using the standalone Orthologous Average Nucleotide Identity Tool (Lee et al. 2016) [27].

†Average amino acid identities (AAI) were calculated using the EDGAR server 2.3 (https://edgar.computational.bio.uni-giessen.de).

nd, not detected. Only results that yield taxonomic consequences are shown.

64 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Fig. 1. Phylogenetic analysis based on the 16S rRNA gene sequences comparison highlighting

the position of 8 heterotypic synonyms from 4 different genera and other closely related strains

belongs to the phylum Actinobacteria. The numbers at the nodes are given as percent and

represent the levels of bootstrap support (>70%) based on maximum likelihood analyses of 1000

re-sampled data sets. GenBank accession numbers are indicated in parentheses. Heterotypic

synonyms of the type species are marked in bold. Sequences of the strains were retrieved from

EzBioCloud. Scale bar, 0.01 nt substitutions per position.

Fig. 2. Maximum- likelihood phylogenetic tree based on the 16S rRNA gene sequences of taxa

belonging to domain Archaea, the tree depicts the phylogenetic position of 5 heterotypic

synonyms identified in 3 different genera and their closely related members. Bootstrap values are

expressed as percentages of 1000 replications and are shown at branch nodes (>70 %). GenBank

accession numbers are indicated in parentheses. Heterotypic synonyms of the type species are

marked in bold. Sequences of the strains were retrieved from EzBioCloud. Scale bar, 0.05 nt

substitutions per position.

Fig. 3. Bacteroidetes members 16S rRNA gene phylogeny reconstructed using Maximum-

likelihood method, it shows the heterotypic synonomous strains Rufibacter ruber CCM 8646T

and Rufibacter quisquiliarum CAI-18bT and its top 30 closest relatives with validly published

name. The numbers at the nodes are given as percent and represent the levels of bootstrap

support (>70%) based on maximum likelihood analyses of 1000 re-sampled data sets. GenBank

accession numbers are indicated in parentheses. Scale bar, 0.01 nt substitutions per position.

Fig. 4. Maximum-likelihood tree based on 16S rRNA gene sequences of 18 heterotypic

synonyms belongs to 8 different genera within the phylum Firmicutes. The numbers at the nodes

65 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

are given as percent and represent the levels of bootstrap support (>70%) based on maximum

likelihood analyses of 1000 re-sampled data sets. GenBank accession numbers are indicated in

parentheses. Heterotypic synonyms of the type species are marked in bold. Scale bar, 0.01 nt

substitutions per position.

Fig. 5. Maximum likelihood tree based on 16S rRNA gene sequences showing the phylogenetic

relationship of strains Thermodesulfovibrio yellowstonii DSM 11347T and Thermodesulfovibrio

islandicus DSM 12570T and other closely related strains belonged to the phylum Nitrospirae.

The numbers at the nodes are given as percent and represent the levels of bootstrap support

(>70%) based on maximum likelihood analyses of 1000 re-sampled data sets. GenBank

accession numbers are indicated in parentheses. Heterotypic synonyms of the type species are

marked in bold. Scale bar, 0.02 nt substitutions per position.

Fig. 6. Maximum-likelihood tree based on 16S rRNA gene sequences of 27 heterotypic

synonyms that belongs to 9 different genera within the phylum Proteobacteria. The numbers at

the nodes are given as percent and represent the levels of bootstrap support (>70%) based on

maximum likelihood analyses of 1000 re-sampled data sets. GenBank accession numbers are

indicated in parentheses. Heterotypic synonyms of the type species are marked in bold. Scale

bar, 0.02 nt substitutions per position.

Fig. 7. 16S rRNA gene sequence-based Maximum likelihood phylogenetic tree showing four

heterotypic synonyms from two different genera (in bold) and the closest relatives with validly

published names that belonged to phylum Thermotogae. Bootstrap values are expressed as

percentages of 1000 replications and are shown at branch nodes (>70 %). GenBank accession

66 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

numbers are indicated in parentheses. Heterotypic synonyms of the type species are marked in

bold. Scale bar, 0.02 nt substitutions per position.

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100 Nocardia ignorata DSM 44960T (GCA_004362495.1) Nocardia coubleae NBRC 108252T (GCA_001612805.1) 78 Nocardia nova NBRC 15556T (BDBN01000167)

T 99 Nocardia elegans NBRC 108235 (GCA_001612845.1) Nocardia Nocardia arthritidis NBRC 100137T (BDBB01000084) Nocardia abscessus IMMIB D-1592T (AF218292) 79 Nocardia exalbida NBRC 100660T (BAFZ01000028) 81 Nocardia gamkensis NBRC 108242T (GCA_001612985.1) Rhodococcus opacus DSM 43205T (X80630) Rhodococcus imtechensis RKJ300T (AY525785) 99 Rhodococcus Rhodococcus percolatus MBS1T (X92114) 100 Rhodococcus jostii DSM 44719T (FNTL01000001)

95 Dietzia papillomatosis NBRC 105045T (BCSL01000097) 100 Dietzia cinnamea IMMIB RIV-399T (AJ920289) Dietzia Dietzia lutea YIM 80766T (EU821598) 75 Dietzia maris DSM 43672T (X79290) 97 Corynebacterium pilbarense IMMIB WACC 658T (FN295567) Corynebacterium ureicelerivorans IMMIB RIV-2301T (CP009215) 100 Corynebacterium afermentans subsp. lipophilum CIP 103500T (X82055) Corynebacterium 74 T 99 Corynebacterium ihumii GD7 (HG001324) 96 C. afermentans subsp. afermentans DSM 44280T (GCA_900156035.1)

86 Streptomyces reticuliscabiei NRRL B-24446T (MUNI01000325)

100 Streptomyces turgidiscabies ATCC 700248T (AB026221) Streptomyces Streptomyces lacrimifluminis Z1027T (KJ829342) Streptomyces graminilatus NRRL B-59124T (LIQQ01000213) Actinokineospora cibodasensis ID03-0748T (AB447489)

100 Actinokineospora spheciospongiae EG49T (AYXG01000061) 86 Actinokineospora mzabensis PAL84T (KJ504177) Actinokineospora 99 Actinokineospora baliensis ID03-0561T (AB447488) 92 Actinokineospora cianjurensis DSM 45657T (RCDD01000016)

0.01 99 Methanobacterium aggregans E09F.3T (KP006499) Methanobacterium congolense CT (AF233586) Methanobacterium paludis SWAN1T (CP002772) Methanobacterium movilense MC-20T (JF812256) bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756Methanobacterium; this version postedlacus December17A1T (HQ110085) 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Methanobacterium beijingense 8-2T (AY350742) Methanobacterium alcaliphilum DSM 3387T (DQ649335)

71 Methanobacterium subterraneum DSM 11074T (X99044) 93 Methanobacterium ferruginis Mic6c05T (AB542743) Methanobacterium 77 Methanobacterium formicicum DSM 1535T (LN515531) Methanobacterium palustre DSM 3108T (AF093061) Methanobacterium bryantii M.o.HT (LMVM01000014) Methanobacterium uliginosum Kf1-F1T (AF095265) 100 99 Methanobacterium ivanovii OCM140T (AF095261) 73 Methanobacterium veterum MK4T (EF016285) 82 Methanobacterium espanolae GP9T (AF095260) Methanobacterium arcticum M2T (DQ517520) Methanobacterium oryzae FPiT (AF028690)

81 Methanosarcina semesiae DSM 12914T (AJ012742) Methanosarcina baltica GS1-AT (AJ238648) Methanosarcina lacustris ZST (AF432127) 100 Methanosarcina subterranea HC-2T (AB288264)

82 Methanosarcina mazei S-6T (CP009512) Methanosarcina soligelidi SMA-21T (JQLR01000001)

T Methanosarcina horonobensis HB-1 (CP009516) Methanosarcina 72 Methanosarcina spelaei MC-15T (LMVP01000249)

T 65 Methanosarcina vacuolata Z-761 (CP009520) Methanosarcina barkeri MST (CP009528) Methanosarcina acetivorans C2AT (AE010299) Methanosarcina siciliae T4/MT (CP009506) Methanosarcina flavescens E03.2T (LKAZ01000047) 77 Methanosarcina thermophila DSM 1825T (M59140) Salinigranum salinum YJ-50-S2T (KC918822)

87 Halogeometricum limi CGMCC 1.8711T (jgi.1058052) Halobellus clavatus TNN18T (GQ282620) 100 Haloferax denitrificans ATCC 35960T (AOLP01000013)

T 83 Haloferax chudinovii RS75 (JX669135) Haloferax alexandrinusT TM (AB037474) Haloferax volcanii DS2T (CP001956) 99 Haloferax gibbonsii ATCC 33959T (AOLJ01000012) Haloferax prahovense TL6T (AB258305) Haloferax Haloferax lucentense DSM 14919T (AOLH01000027) Haloferax mucosum ATCC BAA-1512T (AOLN01000016) Haloferax sulfurifontis ATCC BAA-897T (AOLM01000015) Haloferax namakaokahaiae Mke2.3T (KT970727) Haloferax larsenii JCM 13917T (AOLI01000021) 100 Haloferax elongans SA5T (DQ860977)

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Pontibacter litorisediminis YKTF-7T (KX198137) Pontibacter odishensis JC130T (HE681883) Pontibacter korlensis X14-1T (DQ888330) Pontibacter oryzae KIRANT (MH165268) 86 Pontibacter actiniarum DSM 19842T (CP021235) Pontibacter roseus DSM 17521T (ARDO01000013) Pontibacter amylolyticus 9-2T (KF660586) 82 Pontibacter virosus W14T (KT719408)

99 Pontibacter akesuensis DSM 18820T (jgi.1058912) 94 Pontibacter rugosus KYW1030T (KR674127) Pontibacter silvestris XAAS-R86T (MG547902) Pontibacter terrae 17SD1-15T (KY924851) Pontibacter yuliensis H9XT (KF146891) 79 Pontibacter diazotrophicus H4XT (KF146887) 100 Pontibacter humi SWU8T (KF975403) Pontibacter brevis XAAS-2T (MF410355) Pontibacter xinjiangensis 311-10T (FJ004994) Pontibacter ummariensis NKM1T (jgi.1118304) Pontibacter aydingkolensis XAAS-1T (KX530203) Pontibacter locisalis Sy30T (KR080555)

100 Adhaeribacter terreus DNG6T (EU682684) Adhaeribacter terrae HY02T (LC177335) Nibribacter koreensis GSR3061T (JN607156) Rufibacter glacialis MDT1-10-3T (JX949546) 81 100 Rufibacter ruber CCM 8646T (LRMM01000126)

86 T 95 Rufibacter quisquiliarum CAI-18b (KM083132) Rufibacter roseus CCM 8621T (LRML01000033) Rufibacter tibetensis 1351T (CP012643) Rufibacter sediminis H-1T (KY660624) 69 Rufibacter immobilis MCC P1T (RJJE01000005)

0.01 Thermoanaerobacter sulfurophilus L-64T (Y16940) Thermoanaerobacter wiegelii Rt8.B1T (CP002991) 73 Thermoanaerobacter thermohydrosulfuricus E100-69T (L09161)

84 Thermoanaerobacter ethanolicus JW 200T (AEYS01000048) Thermoanaerobacter siderophilus SR4T (CM001486) Thermoanaerobacter acetoethylicus ATCC 33265T (X69336) Thermoanaerobacter brockii subsp. brockii DSM 1457T (L09165) Thermoanaerobacter brockii subsp. lactiethylicus DSM 9801T (U14330) 90 97 T Thermoanaerobacter brockii subsp. finnii Ako-1 (CP002466) Thermoanaerobacter Thermoanaerobacter pseudethanolicus ATCC 33223T (CP000924) 92 Thermoanaerobacter kivui DSM 2030T (CP009170) Thermoanaerobacter italicus Ab9T (CP001936) bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756; this version posted December 13, 2020. The copyright holder for this preprint Thermoanaerobacter pentosaceus DTU01T (GU176611) (which was not certified by peer review) is the99 author/funder. All rights reserved. No reuse allowed without permission. 78 Thermoanaerobacter thermocopriae IAM 13577T (L09167) Thermoanaerobacter mathranii subsp. mathranii A3T (CP002032) Thermoanaerobacter mathranii subsp. alimentarius AIP 505.99T (AY701758) 99 Thermoanaerobacter sulfurigignens JW/SL-NZ826T (AF234164) Thermoanaerobacter uzonensis JW/IW010T (EF530067) Caldanaerobacter subterraneus subsp. subterraneus SEBR 7858T (AF195797) Caldanaerobacter subterraneus subsp. yonseiensis KB-1T (AXDC01000042) 94 99 “Thermoanaerobacter keratinophilus” 2KXIT (AY278483)† Caldanaerobacter 84 Caldanaerobacter subterraneus subsp. tengcongensis MB4T (AE008691) 95 Caldanaerobacter subterraneus subsp. pacificus DSM 12653T (ABXP02000106) Carboxydothermus pertinax Ug1T (BDJK01000046) Carboxydothermus ferrireducens JW/AS-Y7T (U76363) 100 Carboxydothermus islandicus SET IS-9T (GQ324698) Carboxydothermus 76 Carboxydothermus hydrogenoformans Z-2901T (CP000141) 91 Carboxydothermus siderophilus 1315T (EF542810) Megamonas hypermegale NCTC 10570T (LT906446)

T 100 Megamonas rupellensis FM1025 (EU346729) Megamonas 96 Megamonas funiformis YIT 11815T (AB300988) Geobacillus vulcani 3S-1T (AJ293805) Geobacillus proteiniphilus 1017T (GU459251) Geobacillus kaustophilus NBRC 102445T (BBJV01000091) Geobacillus subterraneus subsp. aromaticivorans Ge1T (HE613733) Geobacillus stearothermophilus NBRC 12550T (AB271757) 74 Geobacillus 89 Geobacillus lituanicus N-3T (CP017692) Geobacillus thermoleovorans KCTC 3570T (CP014335) Geobacillus icigianus G1w1T (KF631430) Geobacillus gargensis Ga (AY193888) 82 Geobacillus thermocatenulatus KCTC 3921T (CP018058) 99 Saccharococcus thermophilus DSM 4749T (GCA_011761475.1)

72 Parageobacillus thermoglucosidasius NBRC 107763T (BAWP01000055) 66 Parageobacillus thermantarcticus DSM 9572T (FR749957) Geobacillus galactosidasius CF1BT (GCA_002217735.1) 87 Parageobacillus Geobacillus yumthangensis AYN2T (GCA_002494375.1) 77 Parageobacillus toebii NBRC 107807T (BDAQ01000034) Parageobacillus caldoxylosilyticus NBRC 107762T (BAWO01000028)

96 Carnobacterium inhibens subsp. inhibens DSM 13024T (JQIV01000006) Carnobacterium viridans MPL-11T (FNJW01000008)

T 100 Carnobacterium pleistocenium FTR1 (JQLQ01000002) Carnobacterium Carnobacterium jeotgali MS3T (JEMH01000060) Carnobacterium inhibens subsp. gilichinskyi WN1359T (CP006812) 99 Salimicrobium album DSM 20748T (X90834) Salimicrobium jeotgali MJ3T (AMPQ01000045) 100 Salimicrobium salexigens DSM 22782T (GCA_900156705.1) Salimicrobium 85 Salimicrobium flavidum DSM 23127T (GCA_900156645.1) Salimicrobium luteum BY-5T (DQ227305) 92 Salimicrobium halophilum DSM 4771T (AJ243920)

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Thermodesulfovibrio islandicus DSM 12570T (AXWU01000024) 100

89 Thermodesulfovibrio yellowstonii DSM 11347T (CP001147)

Thermodesulfovibrio thiophilus DSM 17215T (AUIU01000004)

100 Thermodesulfovibrio aggregans TGE-P1T (BCNO01000001)

Thermodesulfovibrio hydrogeniphilus Hbr5T (EF081294)

Acidothermus cellulolyticus ATCC 43068T (CP000481)

Dissulfuribacter thermophilus S69T (MAGO01000004)

Thermodesulfitimonas autotrophica SF97T (KX450231)

98 Thermanaeromonas toyohensis ToBET (LT838272)

Thermoanaerobacter mathranii subsp. mathranii A3T (CP002032)

0.02 Paraglaciecola chathamensis S18K6T (BAEM01000005) Paraglaciecola agarilytica NO2T (BAEK01000058) 99 Paraglaciecola oceanifecundans 162Z-12T (JX310203) Paraglaciecola Paraglaciecola mesophila KMM 241T (BAEP01000046) 100 88 Paraglaciecola polaris LMG 21857T (BAER01000081) Alteromonas gracilis 9a2T (AB920393)

T 100 Alteromonas addita R10SW13 (CP014322) 73 Alteromonas 99 Alteromonas stellipolaris LMG 21861T (CP013926) Pseudoalteromonas donghaensis HJ51T (CP032090) Pseudoalteromonas lipolytica CGMCC 1.8499T (jgi.1058048) Pseudoalteromonas fuliginea CIP 105339T (AF529062) 100 97 Pseudoalteromonas tetraodonis GFCT (CP011041) 95 Pseudoalteromonas issachenkonii KCTC 12958T (CP013350) Pseudoalteromonas

90 Pseudoalteromonas agarivorans DSM 14585T (CP011011)

T 89 Pseudoalteromonas atlantica NBRC 103033 (BJUT01000111) bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.41875689 Pseudoalteromonas; thisespejiana versionATCC posted 29659 DecemberT (CP011028) 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 100 Shewanella algae JCM 21037T (BALO01000089) 97 Shewanella upenei 20-23RT (GQ260190) Shewanella submarina NIO-S14T (KX645967) Shewanella 98 Shewanella japonica KCTC 22435T (CP020472)

99 T 100 Shewanella pacifica KMM 3597 (AF500075) 82 Shewanella electrodiphila MAR441T (FR744784) Aeromonas salmonicida subsp. pectinolytica 34melT (ARYZ01000167) Aeromonas salmonicida subsp. smithia CCM 4103T (AJ009859) 100 Aeromonas salmonicida subsp. masoucida NBRC 13784T (BAWQ01000150) Aeromonas Aeromonas salmonicida subsp. achromogenes NCIMB 1110T (X60407) Haemophilus piscium CIP 106116T (JN175340) Aeromonas salmonicida subsp. salmonicida ATCC 33658 (LSGW01000109)

85 100 Haemophilus influenzae NCTC 8143T (LN831035) 100 Haemophilus aegyptius ATCC 11116T (GL878535) Haemophilus Haemophilus seminalis SZY H1T (MK796039)

75 Cronobacter turicensis z3032T (FN543093) Cronobacter dublinensis subsp. lactaridi LMG 23825T (AJKX01000065) 100 Cronobacter Cronobacter dublinensis subsp. lausannensis LMG 23824T (AJKY01000076) Cronobacter dublinensis subsp. dublinensis LMG 23823T (CP012266) Methylomicrobium lacus LW14T (AZUN01000001)

T 100 Methylomicrobium agile ATCC 35068 (JPOJ01000001) Methylomicrobium 100 Methylomicrobium album BG8T (CM001475) Thalassospira xianhensis P-4T (EU017546) Thalassospira profundimaris WP0211T (AY186195) 100 Thalassospira permensis SMB34T (FJ860275.1) Thalassospira

T 90 Thalassospira xiamenensis M-5 (CP004388) Desulfotignum toluenicum H3/clone1T (EF207157) Desulfotignum phosphitoxidans DSM 13687T (APJX01000002) Desulfotignum 100 Desulfotignum balticum DSM 7044T (ATWO01000001)

99 Tepidiphilus thermophilus JHK30T (HM543264) 80 100 Tepidiphilus succinatimandens 4BONT (AY219713) Tepidiphilus Tepidiphilus margaritifer DSM 15129T (AUDR01000008) Neisseria flava U40T (AJ239301) 87 Neisseria sicca ATCC 29256T (ACKO02000016) Neisseria macacae ATCC 33926T (AFQE01000146) 100 100 Neisseria Neisseria mucosa JCM 12992T (AB910739) Morococcus cerebrosus CIP 81.93T (JUFZ01000072)

98 Caldimonas manganoxidans JCM 10698T (AB008801) 100 Caldimonas taiwanensis NBRC 104434T (BCWK01000040) Caldimonas Caldimonas meghalayensis AK31T (HF562216)

80 Bordetella flabilis AU10664T (CP016172) Bordetella petrii DSM 12804T (AM902716) 100 Bordetella pertussis Tohama IT (BX470248) Bordetella Bordetella parapertussis NCTC 5952T (LRII01000001) 87 Bordetella bronchiseptica ATCC 19395T U04948

0.02 bioRxiv preprint doi: https://doi.org/10.1101/2020.12.13.418756; this version posted December 13, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Pseudothermotoga lettingae TMOT (CP000812) 100 Pseudothermotoga subterranea DSM 9912T (U22664) 99 Pseudothermotoga elfii NBRC 107921T (AP014507)

86 Thermotoga profunda AZM34c06T (AP014510)

Thermotoga caldifontis AZM44c09T (AP014509) 94 90 Pseudothermotoga hypogea NBRC 106472T (AP014508)

Pseudothermotoga thermarum DSM 5069T (CP002351)

Thermotoga naphthophila RKU-10T (ACXW01000001)

Thermotoga petrophila RKU-1T (CP000702) 100 Thermotoga maritima MSB8T (AE000512)

94 Thermotoga neapolitana DSM 4359T (CP000916)

Thermosipho activus Rift-s3T (KF931642)

Thermoflexus hugenholtzii JAD2T (FYEK01000008)

0.02