2 *Tepidibacter hydrothermalis* sp. nov., a novel
3 anaerobic bacterium isolated from a deep-sea
4 hydrothermal vent

5 Jie Dai¹, Xue-Gong Li²,³,⁴,⁵*, Wei-Jia Zhang²,³,⁴,⁵, Long-Fei Wu¹,³*

6 ¹Aix Marseille Univ, CNRS, LCB, IMM, IM2B, Marseille, France

7 ²Laboratory of Deep-Sea Microbial Cell Biology, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, China

9 ³International Associated Laboratory of Evolution and Development of Magnetotactic Multicellular Organisms, CNRS-Marseille/CAS- Sanya

11 ⁴CAS Key Laboratory for Experimental Study under Deep-sea Extreme Conditions, Institute of Deep-sea Science and Engineering, Chinese Academy of Sciences, Sanya, China

13 ⁵Institution of Deep-sea Life Sciences, IDSSE-BGI, Hainan Deep-sea Technology Laboratory, Sanya, Hainan, China

15 Correspondence: Xue-Gong Li: xuegongli@idsse.ac.cn; Long-Fei Wu: wu@imm.cnrs.fr
<figure><img src="image_1.png" /></figure>

A

<figure><img src="image_2.png" /></figure>

B

Fig. S1. Phylogenetic tree based on 16S rRNA sequences of strain SWIR-1T and its relatives according to the minimum evolution (A) and maximum likelihood (B) method. Bootstrap values above 50 % based on 1,000 replicates are shown at nodes. GenBank accession numbers of 16S rRNA sequences are given in parentheses. Bar, 2 nucleotide substitutions per 1,000 nucleotide positions.

17

18
<figure><img src="image_3.png" /><figcaption>Fig. S2. Tree inferred with FastME 2.1.6.1 [1] from GBDP distances calculated from genome sequences. The branch lengths are scaled in terms of GBDP distance formula $d_5$. The numbers above branches are GBDP pseudo-bootstrap support values > 60 % from 100 replications, with an average branch support of 84.3 %. The tree was rooted at the midpoint [2].</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>Fig. S3. Polar lipids composition of strain SWIR-1T and other *Tepidibacter* species. (A) strain SWIR-1T; (B) *T. mesophilus* B1T; (C) *T. formicigenes* DV 1184T; (D) *T. thalassicus* SC 562T.
PL: Phospholipid, GL, glycolipid; L, unknown polar lipid; F-first dimension of TLC; S- second dimension of TLC.</figcaption></figure>
<table><caption>**Supplementary Table S1.** Whole genome-based comparisons between strain SWIR-1T and closely relatives.</caption><thead><tr><th>Strains</th><th>ANI (%)</th><th>DDH (%)</th><th>AAI (%)</th></tr></thead><tbody><tr><td><i>Tepidibacter mesophilus</i> B1<sup>T</sup></td><td>87.2</td><td>35.7</td><td>78.9</td></tr><tr><td><i>Tepidibacter formicigenes</i> DV1184<sup>T</sup></td><td>76.7</td><td>22.1</td><td>63.2</td></tr><tr><td><i>Tepidibacter thalassicus</i> SC 562<sup>T</sup></td><td>77.3</td><td>21.5</td><td>62.3</td></tr></tbody></table>

<table><caption>**Supplementary Table S2.** Fatty acid composition of strain SWIR-1T and its closely related strains. Strains: 1, Strain SWIR-1T; 2, *T. mesophilus* B1T; 3, *T. formicigenes* DV 1184T; 4, *T. thalassicus* SC 562T. Values are percentages of total fatty acids. Fatty acids that represented <1 % in all strains were omitted. TR, Traces (<1 %).</caption><thead><tr><th rowspan="2">Fatty acid*</th><th colspan="4">Composition (%) in:</th></tr><tr><th>1</th><th>2</th><th>3</th><th>4</th></tr></thead><tbody><tr><td>C<sub>10:0</sub></td><td>TR</td><td>TR</td><td>1.1</td><td>TR</td></tr><tr><td>C<sub>11:0</sub> iso</td><td>1.2</td><td>TR</td><td>TR</td><td>TR</td></tr><tr><td>C<sub>12:0</sub></td><td>3.5</td><td>5.3</td><td>TR</td><td>1.0</td></tr><tr><td>C<sub>13:0</sub> iso</td><td>3.7</td><td>TR</td><td>1.8</td><td>2.1</td></tr><tr><td>C<sub>14:1</sub>ω5c</td><td>1.5</td><td>TR</td><td>TR</td><td>TR</td></tr><tr><td>C<sub>14:0</sub></td><td>6.9</td><td>9.1</td><td>6.3</td><td>6.4</td></tr><tr><td>C<sub>13:0</sub> iso 3OH</td><td>1.7</td><td>TR</td><td>TR</td><td>TR</td></tr><tr><td>C<sub>15:1</sub> iso F</td><td>15.4</td><td>TR</td><td>10.1</td><td>5.2</td></tr><tr><td>C<sub>15:0</sub> iso</td><td>28.4</td><td>TR</td><td>57.5</td><td>59.5</td></tr><tr><td>C<sub>15:0</sub> anteiso</td><td>1.2</td><td>TR</td><td>2.2</td><td>1.4</td></tr><tr><td>C<sub>16:0</sub></td><td>9.8</td><td>23.8</td><td>4.8</td><td>5.5</td></tr><tr><td>C<sub>17:0</sub> iso</td><td>TR</td><td>TR</td><td>1.3</td><td>1.4</td></tr><tr><td>C<sub>17:0</sub> cyclo</td><td>TR</td><td>6.0</td><td>TR</td><td>TR</td></tr><tr><td>C<sub>18:1</sub>ω9c</td><td>6.0</td><td>1.8</td><td>2.8</td><td>4.4</td></tr><tr><td>C<sub>18:0</sub></td><td>6.7</td><td>1.1</td><td>2.6</td><td>3.0</td></tr><tr><td>Summed Feature 1</td><td>1.6</td><td>TR</td><td>TR</td><td>TR</td></tr><tr><td>Summed Feature 2</td><td>TR</td><td>7.8</td><td>TR</td><td>TR</td></tr><tr><td>Summed Feature 3</td><td>2.2</td><td>28.9</td><td>1.0</td><td>1.3</td></tr><tr><td>Summed Feature 8</td><td>2.23</td><td>13.27</td><td>TR</td><td>1.45</td></tr><tr><td>Summed Feature 9</td><td>1.5</td><td>TR</td><td>TR</td><td>TR</td></tr></tbody></table>

*Summed Features are fatty acids that cannot be resolved reliably from another fatty acid using the chromatographic conditions chosen. The MIDI system groups these fatty acids together as one feature with a single percentage of the total. Summed Feature 1 contained iso-C<sub>15:1</sub> iso H and/or C<sub>13:0</sub> 3-OH; Summed Feature 2 contained C<sub>12:0</sub> aldehyde; Summed Feature 3 contained C<sub>16:1</sub> ω7c and/or C<sub>16:1</sub> ω6c; Summed Feature 8 contained C<sub>18:1</sub> ω7c and/or C<sub>18:1</sub> ω6c; Summed Feature 9 contained C<sub>17:1</sub> iso ω9c.

1. Lefort V, Desper R, Gascuel O: **FastME 2.0: A Comprehensive, Accurate, and Fast Distance-Based Phylogeny Inference Program.** *Mol Biol Evol* 2015, **32**(10):2798-2800.
51 2. Farris JS: Estimating phylogenetic trees from distance matrices. *The American Naturalist*
52 1972, **106**(951):645-668.
53