1 Supplementary Information

2

3 Population Differentiation of Rhodobacteraceae Along Coral Compartments

4 Danli Luo, Xiaojun Wang, Xiaoyuan Feng, Mengdan Tian, Sishuo Wang, Sen-Lin Tang, Put

5 Ang Jr, Aixin Yan, Haiwei Luo

6

7

8

9

10

11 This PDF file includes:

12 Text 1. Supplementary methods

13 Text 2. Supplementary results

14 Figures S1 to S13

15 Supplementary references

16 17 Text 1. Supplementary methods

18 1.1 Coral sample collection and processing

19 1.2 Bacterial isolation

20 1.3 Genome sequencing, assembly and annotation

21 1.4 Ortholog prediction and phylogenomic tree construction

22 1.5 Analysis of population structure in core genomes

23 1.6 Inference of novel allelic replacement with external lineages in core genomes

24 1.7 Differentiation in the accessory genome and inference of evolutionary history

25 1.8 Identification of pseudogenes in the fla1 flagellar gene cluster

26 1.9 The physiological assays

27 1.10 Test of compartmentalization and dispersal limitation

28 1.11 Estimating the origin time for the Rhodobacteraceae and the Ruegeria populations

29 Text 2. Supplementary results

30 2.1 Population differentiation at the core genomes of the Ruegeria population

31 2.2 The Ruegeria population differentiation at the physiological level

32 2.3 Metabolic potential for utilizing other substrates by the mucus clade of the Ruegeria

33 population

34 2.4 Metabolic potential of the mucus clade in the Ruegeria population underlying

35 microbial interactions in the densely-populated mucus habitat

36 2.5 Adaptation of the skeleton clade in the Ruegeria population to the periodically

37 anoxic skeleton habitat

38

39 40 Text 1. Supplementary methods

41 1.1 Coral sample collection and processing

42 Coral samples of Platygyra acuta were collected by SCUBA diving in Hong Kong water

43 at Kiu Tsui Chau (N 22°22'04.4" E 114°17'42.0") on 24th April 2017, Wong Wan Chau (N

44 22°31'31.2" E 114°19'00.1") on 12th January 2018 and Ngo Mei Chau (N 22°31'47.2" E

45 114°19'02.9") and Chek Chau (N 22°30'03.3" E 114°21'22.7") on 25th February 2018 (Fig.

46 S1A). One coral rubble (2-8 cm in diameter) was sampled from each colony using a rock chisel,

47 separated in zip-lock bags with their ambient seawater, kept in a low-temperature oven, and

48 carefully transported to the laboratory. One sample of ambient seawater was collected by 50 mL

49 centrifuge tube at each site.

50 Separation of coral compartments followed an established procedure [1, 2]. In brief, coral

51 fragments were washed three times with filtered ambient seawater for 10 seconds with stirring to

52 disrupt the exogenous microbial contaminants from the ambient seawater or sediments. Mucus

53 samples were collected by exposing coral fragments to the air in the clean bench and waiting

54 until the mucus started to drip from the coral surface. A total of 150 μL dripping mucus was

55 collected using sterile syringes and transferred to 1.5 mL sterile centrifuge tubes. The collected

56 mucus was centrifuged at 2,000 rpm for five minutes. The cell debris on the bottom was

57 discarded and the transparent supernatant was kept.

58 Tissue samples were collected by spraying the coral surface using a Waterpik. Tissue

59 suspensions of 50 mL were collected with sterile zip-lock bags, and centrifuged at 12,000 rpm

60 for 15 min under 4 °C. The pellet was then suspended in 1 mL of autoclaved artificial seawater

61 (ASW). While procedures to collect clean mucus and skeleton were established [2], the accurate

62 method for collecting clean coral tissue remains unavailable due to the intersecting structure of 63 the coral compartments. For example, the mucocytes are part of the coral issue layer (Fig. S1B),

64 which keeps secreting mucus [3]. Besides, the tissue is embedded in the corallites (Fig. S1B),

65 which are part of the skeleton where the polyp sits and retracts, so the removal of tissue would

66 inevitably disturb the coral skeleton [4]. These anatomical features make the complete separation

67 of tissue from mucus and skeleton not possible by current methods, such as airbrush [5],

68 Waterpik [1] and centrifugation [6].

69 The core coral skeleton pieces of ~2 cm in diameter were carefully separated. To avoid

70 cross-contamination from the tissue, only the skeleton pieces located more than 2 cm apart from

71 the tissue layer were kept. Then the skeleton pieces were crushed into a slurry with sterilized

72 mortar and pestle with 1 mL ASW added. The slurry was filtered through a 100 μM mesh to

73 remove large fragments.

74

75 1.2 Bacterial isolation

76 The collected coral compartments were serially diluted and immediately transferred to

77 marine basal medium (MBM) agar plates. The MBM marine agar was prepared as the following

78 recipe (per liter): 8.47g of Tris-HCl, 0.37 g of NH4Cl, 0.0022 g of K2HPO4, 11.6 g of NaCl, 6 g

79 of MgSO4, 0.75 g of KCl, 1.47 g of CaCl2·2H2O, 2.5 mg of FeEDTA [pH 7.5], 1 mL of vitamins

80 [7], and 15g of agar. Taurine was added as the carbon source at the concentration of 0.5 mM.

81 The ambient seawater was treated in the same way as the samples of coral compartments, serially

82 diluted and spread over agar plates. Agar plates were incubated at 28 °C for at least 48 h.

83 Colonies were randomly selected and subject to streaking three times on 2216E marine agar [BD

84 Difco, USA] for purification. 85 The 16S rRNA gene was amplified using colony polymerase chain reaction (PCR) with

86 27F primer (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R primer (5'-

87 GGTTACCTTGTTACGACTT-3'). By following the protocol, Chelex 100 resin [Bio-rad, USA]

88 was used to prepare biomass samples, and the recipe of PCR was prepared using Premix Taq

89 [Takara Bio, USA]. The PCR was performed according to the following procedure: denaturing at

90 95℃ for 5 minutes, followed by 32 cycles (95℃ for 45 seconds, 55℃ for 45 seconds and 72℃

91 for 90 seconds) and a final extension at 72℃ for 10 minutes. The amplicons were sequenced

92 using 27F primer. The primers and the bases with low sequencing quality at the ends of the

93 amplicons were removed, and the remaining 600 bp were kept. The taxonomic information was

94 obtained by comparing the partial 16S rRNA gene sequences with those of all reported type

95 strains using EzBioCloud [8]. The partial 16S rRNA gene sequences were clustered to form

96 operational taxonomic units (OTUs) at the 98.7% identity level, which is used to delineate a

97 bacterial species [9]. Two Rhodobacteraceae OTUs each containing 12 (the Ruegeria

98 population) and 214 isolates (the Rhodobacteraceae population) covering two or more coral

99 compartments were chosen for population genomic analyses. For the Ruegeria population, an

100 additional closely related OTU with 8 strains was included as outgroup.

101

102 1.3 Genome sequencing, assembly and annotation

103 For each of the 234 isolates comprising the two populations, genomic DNA was extracted

104 using TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit [Takara Bio, USA]. The

105 quality of each extracted DNA sample was verified spectrophotometrically using NanoDropTM

106 2000 [Thermo Fisher, USA] (A260/A280 >1.8, A260/A230 > 2.0 and A260 >A270). Whole-genome

107 sequencing was performed using the BGISEQ-500 PE100 platform (Table S6) in Qingdao Huada 108 Gene Biotechnology Co., Ltd. The untrimmed adapters associated with raw reads were identified

109 with BBMerge implemented in BBmap v37 [10]. Next, adapters and low-quality reads were

110 trimmed using Trimmomatic v0.33 [11], reads each with less than 40 bp were discarded, and the

111 quality of the remaining reads was checked with FastQC v.0.11.4

112 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Contigs were assembled based on

113 the high-quality paired-end reads using SPAdes v3.9 [12] with default parameters. Only those

114 with a length of over 1,000 bp and with a k-mer coverage over five were kept for further

115 analyses. CheckM v0.9.7 [13] was used to assess the quality of assemblies, and statistics were

116 calculated with QUAST v4.5 [14]. The genome of HKCCD6109 in the Ruegeria population

117 showed a 50% heterogeneity (Table S6) by CheckM v0.9.7, suggesting potential DNA

118 contamination from very close relatives. To check the potential contamination, we re-purified

119 and re-sequenced the sample HKCCD6109 as described above. The new version of genome

120 assembly of HKCCD6109 was estimated to have completeness of 99.7% and heterogeneity of

121 50%. The old and new version of the assembled genome size is 4,472,332 bp and 4,522,316 bp,

122 respectively, and they differ at eight nucleotide sites across the aligned regions (3,595,109 bp).

123 Four of these sites are located together, and the other four are located randomly on the

124 chromosome. Apparently, differences at the former four sites cannot be ascribed to sequencing

125 error, and the possibility that the old HKCCD6109 culture contains very closely related

126 contamination cannot be ruled out. Note that our physiological assays (Supplemental Text 2.2)

127 involving HKCCD6109 used the old version.

128 Gene prediction was carried out using Prokka v1.14.6 [15]. The functions of the predicted

129 protein-coding genes were further annotated using NCBI Conserved Domain Database (CDD)

130 [16], RAST Annotation Server [17], and eggNOG [18]. 131 To obtain a closed genome as a reference for the Ruegeria population, PacBio Sequel

132 was used to sequence the strain HKCCD4315 isolated from coral mucus. Unicycler [19] was

133 used to assemble the complete genome of strain HKCCD4315 based on short reads from

134 BGISEQ-500 PE100 and long reads from PacBio Sequel. The gene prediction and functional

135 annotation were performed as described above.

136

137 1.4 Ortholog prediction and phylogenomic tree construction

138 Orthologous gene families were identified using OrthoFinder v2.2.1 [20] among the

139 strains in each population. Members of each single-copy gene family shared by all tested strains

140 were aligned at the amino acid sequence level using MAFFT v7.215 [21]. Gaps of alignments

141 were trimmed using trimAl v1.4.rev15 with parameters “-automated1 -resoverlap 0.55 -

142 seqoverlap 60” [22]. PartitionFinder2 v2.1.1 [23] was implemented to determine the best-fit

143 evolutionary model for each family. The maximum likelihood phylogeny was constructed based

144 on the concatenated sequences of trimmed alignments and model selection results using IQ-

145 TREE v1.6.5 [24] with 1,000 ultrafast bootstrap replicates.

146

147 1.5 Analysis of population structure in core genomes

148 For the Ruegeria population, ANI for each genome pair was calculated using FastANI

149 [25]. The whole-genome alignment of 12 strains comprising the clade-M and clade-S of was

150 produced using progressiveMauve v2.3.1 [26] with default settings. The core genomic regions

151 shared by the 12 strains were extracted. To measure the relative rate and effect of recombination

152 relative to point mutation among the population, ClonalFrameML v1.1 [27] was implemented 153 with the core genome alignments and the phylogenomic tree as inputs. The same analyses were

154 conducted for members of clade-M and those of clade-S separately.

155 To infer the population subdivision, we carried out the coancestry analysis. We generated

156 the haplotype data using SNPs from the core genomic alignments and the recombination map

157 files following the instructions of ChromoPainter [28]. Chromosome painting was implemented

158 to calculate the co-ancestry between pairwise strains using ChromoPainter. Next, the

159 fineSTRUCTURE [29] assigned strains to subpopulations based on the co-ancestry matrix using

160 a model-based clustering method. Assuming each individual as a recipient of DNA from the

161 remaining individuals (i.e., donors), the “chromosome painting” algorithm reconstructs the

162 genome of each recipient with chunks of DNA from other donors. Then the painting results were

163 summarized as a “co-ancestry matrix”, which represents ancestral relationships among

164 individuals. Based on the co-ancestry matrix, individuals were assigned to subpopulations by the

165 Markov chain Monte Carlo (MCMC) algorithm in the fineSTRUCTURE. Both the burn-in and

166 the MCMC step were run for 100,000 iterations to ensure convergence. The thin interval was

167 specified as 100. Two independent inferences were performed with the same parameters to

168 confirm the population assignment. The population structure was visualized with the R script

169 “fineRADstructure.R” [30].

170 To provide further information about the population differentiation between clade-M and

171 clade-S, we calculated the Fst values using Arlequin v3.5 [31] with 1,000 permutations. SNPs

172 within and between two clades were extracted and coordinated to the genome of strain

173 HKCCD4315 (a closed genome with one chromosome and three plasmids). The calculation of Fst

174 was performed with sliding windows of 10,000 bp moving in 5,000 bp steps across the core

175 genome alignment. 176

177 1.6 Inference of novel allelic replacement with external lineages in core genomes

178 To detect genes subject to homologous recombination from external lineages, we

179 employed a recently developed approach based on the synonymous substitution rate (dS) [32].

180 The synonymous mutations are largely neutral since they do not cause changes in amino acid

181 sequences. However, replacement with a divergent allele via recombination can import many

182 synonymous variants, which leads to an anonymously large dS value between recipient genomes

183 and the unaffected genome at this locus, compared to the remaining loci in other genomic

184 regions. Thus, if a gene family shows that pairwise dS values between two clades are enormously

185 large while pairwise dS values within each clade are small, it can be inferred that the allelic

186 replacement occurred at the last common ancestor (LCA) of either clade.

187 Other evolutionary mechanisms may also affect the synonymous substitution rate at

188 protein-coding genes, but they are expected to produce different dS patterns. In marine bacteria,

189 nitrogen (N) limitation and carbon (C) limitation act as selective pressures, driving genomic G+C

190 content to decrease and increase, respectively [33, 34]. As all genomic sites are subject to these

191 selective pressures, synonymous sites in all genes would be affected indiscriminately. Thus,

192 these selective pressures are not likely to affect a small proportion of gene families showing

193 unusually large dS values. Codon usage bias imposed by translational selection is another

194 potential force affecting the synonymous substitution rate. Different expression levels among

195 genes lead to a varied preference for alternative synonymous codons in fast-growing microbes

196 [35, 36]. For highly expressed genes, a stronger codon usage bias is expected to maximize the

197 translational speed or accuracy [37], which leads to a reduced synonymous substitution rate in

198 these genes. For genes at the regular or reduced expression level, the codon usage is a result of 199 stochastic mutation [38, 39]. In sum, codon usage bias is not likely to give rise to outlier gene

200 families with anonymously large dS values.

201 Based on these principles, we conclude that gene families with unusually large dS values

202 are most likely subject to recombination. In practice, pairwise dS values were calculated using the

203 YN00 program in PAML v4.9 [40] for each single-copy gene shared by the two clades. To

204 identify core gene families showing the above-described dS pattern, the K-means clustering

205 method was used to cluster gene families based on pairwise dS values. The number of optimal

206 clusters was determined using R package ‘NbClust’ [41], which provides a variety of indices for

207 cluster validity.

208 The above approach can identify which gene families were subjected to novel allelic

209 replacements and infer the candidate ancestral branches where allelic replacements occurred.

210 However, to determine the exact ancestral branch and the potential donor lineages, gene trees

211 were constructed for orthologs showing unusually large between-clade dS values and compared

212 to a phylogenomic tree. For the genome tree construction, a total of 202 published genomes are

213 phylogenetically related to the Ruegeria population according to a preliminary phylogenomic

214 tree of all publicly available Roseobacter genomes (data not shown) in NCBI Genbank before

215 October, 2019. Next, a maximum phylogenomic tree was constructed using IQ-TREE v1.6.5

216 [24] based on the concatenation of 120 conserved genes [42] from the 202 published

217 Roseobacter genomes, 20 newly sequenced genomes of the Ruegeria population and one

218 randomly chosen genome from the genetically uniform Rhodobacteraceae population consisting

219 of 214 strains. The phylogenomic tree was rooted using three Oceanicella genomes and one

220 Monaibacterium genome based on their phylogenetic position recorded in the Genome

221 Taxonomy Database (GTDB-release95) [42, 43]. For the gene tree construction, the putative 222 orthologs were identified from the above 202 genomes using the BLASTP v2.6.0 [44] program

223 with an E-value of 1e-5, and the best hit from each genome was kept. Next, MAFFT v7.215 [21]

224 was used to align the protein sequences, and TrimAl v1.4.rev15 [22] with parameters “-

225 automated1 -resoverlap 0.55 -seqoverlap 60” was used to trim poorly aligned sites. The

226 maximum likelihood gene trees were subsequently constructed for each gene family based on the

227 trimmed alignments using IQ-TREE v1.6.5 [24] with ModelFinder [45] assigning the best

228 substitution model and with 1,000 ultrafast bootstrap replicates. To root the gene trees, outgroup

229 lineages were chosen according to the phylogenetic placement of the 223 genomes (202

230 published Roseobacter group members, 20 strains of the Ruegeria population and one strain

231 from the Rhodobacteraceae population) in the above phylogenomic tree. After the genome tree

232 and the gene trees were constructed, the recombination history was manually checked by

233 comparing gene trees to the phylogenomic tree.

234

235 1.7 Differentiation in the accessory genome and inference of evolutionary history

236 The gene presence/absence matrix of the accessory gene families was summarized as

237 input. The Jaccard index is a measure of similarity between sample sets [46] and thus can be

238 applied to assess gene content similarity between two strains. It is defined as the size of the

239 intersection divided by the size of the union of the gene content in two strains, and is defined as

240 the size of the intersection divided by the size of the union of the gene content in two strains:

|푆1 ∩ 푆2| 241 퐽(푆1, 푆2) = |푆1| + |푆2| − |푆1 ∩ 푆2|

242 where 푆1 and 푆2 denote two strains. Thus, the Jaccard index between pairwise strains was

243 calculated to represent gene content similarity and visualized using R. 244 The presence/absence of genes alone cannot reveal the evolutionary history of accessory

245 genomes, which may provide insights into how different coral compartments drive the evolution

246 of the two clades (clade-M and clade-S). Among the accessory genomes, the population

247 differentiation may be largely driven by the clade-specific gene families, which are defined here

248 as genes that are present in at least two-thirds of the strains in one clade but are present in no

249 more than one-third of the strains in the other clade. The clade-specific genes could result from

250 either gene gain in one clade or gene loss in the other clade, depending on the presence/absence

251 of the gene family in the last common ancestor (LCA) shared by the two clades. As clade-M and

252 clade-S have a closely related lineage which serves as outgroup (eight genomes), inference of the

253 ancestral state of the LCA of the two clades could be assisted by the analysis of the phyletic

254 pattern of the gene family in the outgroup lineage. For example, a clade-M specific gene family

255 might be acquired at the LCA of clade-M or lost at that of clade-S. If this gene family is

256 prevalent among the outgroup members, it was likely present at the LCA shared by clade-M and

257 clade-S, and a reasonable inference is that this family was lost at the LCA of clade-S. Besides the

258 ancestral branch leading to the LCA of the two clades, the evolutionary gain or loss events could

259 also occur at the branches after the LCA. Apparently, we are more interested in the events

260 occurring at the branch leading to the LCA of clade-M or that of clade-S, as these events may

261 have been driving the speciation and ecological differentiation between the two clades.

262 In practice, the gene gain and loss history for clade-specific gene families was inferred

263 using BadiRate v1.35 [47]. The inference was based on the parsimony rule, and the number of

264 gene copies in each clade-specific gene family was summarized as the inputs with the parameters

265 “--ep CSP -rmodel BDI -bmodel FR”. Then the gene gain and loss history were inferred based

266 on the predicted copy number at the ancestral node of each clade. 267 The genomic islands (GIs) often contain many horizontally transferred genes [48, 49].

268 GIs were identified for each member of the Ruegeria population using IslandViewer 4 [50], with

269 the chromosome of the most closely related strain Ruegeria sp. AD91A (Accession number:

270 GCA_003443535.1) was chosen as a reference genome according to the genome tree shown in

271 Fig. S4. The clade-specific genes, the outlier genes showing unusually large dS values, and the

272 GIs were mapped to the pangenome of the Ruegeria population (Fig. 2), which was visualized

273 with Circos v0.64 [51].

274

275 1.8 Identification of pseudogenes in the fla1 flagellar gene cluster

276 The pseudogenes were identified using the program suite Psi-Phi [52] and following a

277 modified procedure described in a recent study [53]. We used the 20 genomes of the Ruegeria

278 population and another 22 closely related Ruegeria genomes sampled from other niches in Hong

279 Kong coastal ecosystems (Table S6) as the pool of protein for pseudogene identification. Using

280 the Psi-Phi program, the annotated proteins of each genome were searched against the complete

281 nucleotide sequence of every other genome using TBLASTN [54]. The pseudogenes were

282 recognized based on the reduced length of the protein (shorter than 80% of protein query), low

283 BLAST E-values (< 1e-15), and the occurrence of premature stop codons derived from

284 disruptive mutation.

285

286 1.9 The physiological assays

287 To compare the capability of clade-M and clade-S strains in utilizing relevant substrates,

288 three strains from each clade were chosen to grow on a defined minimal medium with added

289 substrates as a sole source of N and carbon C [55]. The minimum medium was modified from a 290 carbon-free marine ammonium mineral salts (MAMS) [56] by removing the original N source

291 (NH4Cl), and thus contained the following compounds (per liter): 20 g of NaCl, 1 g of

292 MgSO4·7H2O, 0.2 g of CaCl2·2H2O, 2 mg of FeSO4·7H2O, 20 mg of Na2MoO4·2H2O, 0.36 g of

293 KH2PO4, 2.34 g of K2HPO4, 1 mL of SL-10 trace metals solution [55] and 1 mL of vitamins [7].

294 The following organic substrates (5 mM) were added as a sole source of N and C: choline

295 (Fig. 4A-2), glycine betaine (GBT, Fig. 4A-3), dimethylglycine (DMG, Fig. 4A-4), sarcosine

296 (Fig. 4A-5), trimethylamine (TMA, Fig. 4A-6), trimethylamine-N-oxide (TMAO, Fig. 4A-7),

297 creatine (Fig. 4A-8), L-proline (Fig. 4A-9), taurine (Fig. 4A-10) and urea (Fig. 4A-11). The

298 minimum medium without any added N and C source was used as a negative control (Fig. 4A-1).

299 The positive control was set up with a rich medium to evaluate if the optimal growth was

300 consistent among the tested strains (Fig. 4A-1). The rich medium contained 5 g peptone and 1 g

301 yeast extract per liter as mixed C and N sources.

302 If a substrate can barely support the tested strains as a sole source of N and C, it was

303 further examined as a sole N source and a sole C source separately. Besides,

304 dimethylsulfoniopropionate (DMSP) was also tested as the sole C source. For the sole C source

305 assay, 10 mM NH4Cl was added as the N source (Fig. 4B-2, 4B-3, 4B-5, 4B-7, 4B-9 and 4B-11).

306 For the sole N source assay, 5 mM sodium pyruvate was added as the C source (Fig. 4B-4, 4B-6,

307 4B-8, 4B-10 and 4B-12). To control the potential growth bias introduced by the NH4Cl or

308 pyruvate, positive control was set up with 10 mM NH4Cl as a sole N source and 5 mM sodium

309 pyruvate as a sole C source (Fig. 4B-1), and negative control was set up with no added C and N

310 source (Fig. 4B-1).

311 Three replicates for each treatment were conducted in 50 mL tubes at 28°C, and growth

312 was examined by measuring the optical density at 600 nm (OD600). The growth rate was 313 calculated using the data points in the exponential phase. The relative growth yield was

314 represented by the OD600 of the final points in the exponential phase of the growth curve. Both

315 the growth rate and growth yield were compared between members from clade-M and those from

316 clade-S. The differences between clades were statistically evaluated with One-way Repeated

317 Measures ANOVA, with p < 0.05 indicating that the growth rates and the relative growth yield

318 between clades are significantly different [57].

319 The swimming, swarming, and twitching motility of the clade-M and clade-S members

320 were tested on agar plates with 2216E marine broth medium [BD Difco, USA]. Overnight

321 cultures of each strain were inoculated in 2216E marine broth medium with 1:200 dilution and

322 incubated at 28℃ under a shaking speed of 200 rpm until the OD600 of the culture reached 0.6-

323 0.8. For the swimming test, 0.3% (w/v) soft agar plates were point-inoculated with 3 μL of the

324 fresh cell suspension and incubated at 28℃ for eight days. The zone of swimming motility on

325 the surface of agar was measured. The swarming assay was conducted following the same

326 procedure but with the concentration of agar plates replaced by 0.6% (w/v). For the twitching

327 assay, cell cultures were stab-inoculated to the bottom of the Petri dish of 1.0 % (w/v) agar plate

328 with a sterile toothpick and incubated in the humidified box at 28°C for 10 days, and the zone of

329 twitching motility at the interface of agar and Petri dish was measured. All tests were done in

330 triplicates.

331

332 1.10 Test of compartmentalization and dispersal limitation

333 The genetically uniform Rhodobacteraceae population of 214 strains varying at only a

334 few dozen nucleotide sites across the whole genomes were partitioned into four subpopulations,

335 members of each exclusively isolated from distinct coral individuals. These four coral 336 individuals were collected from four different sampling locations (Fig. S1A). The two

337 subpopulations from Wong Wan Chau (WWC) and Ngo Mei Chau (NMC) each contain isolates

338 cultured from multiple compartments (Table S6), and thus they are amenable for calculating the

339 number of migrations between compartments.

340 The tree-based Slatkin-Maddison test [58] implemented in HyPhy v2.5 [59] was

341 employed to evaluate the compartmentalization of the two subpopulations. For each

342 subpopulation, the maximum likelihood phylogenetic tree was constructed using IQ-TREE

343 v1.6.5 [24] based on the core SNPs identified by kSNP v3.0, a software quickly and accurately

344 identifying SNPs among hundreds of genomes in an alignment-free approach [60]. Next, strains

345 were labeled according to their compartment of isolation (i.e., mucus, tissue and skeleton). The

346 strains isolated from ambient seawater of coral were not considered. Since the identical siblings

347 from the same compartment could amplify the signal of compartmentalization [61], only one of

348 the siblings with branch length zero from the same compartment was kept and the remaining

349 ones were pruned from the phylogenetic tree. To calculate the number of migrations observed in

350 each subpopulation, the pruned phylogenetic tree with compartment information was subject to

351 standard Slatkin-Maddison test, followed by 100,000 permutations of population structures to

352 generate a normal distribution representing the number of migrations expected by chance.

353 Whether isolates were significantly compartmentalized was determined by comparing the

354 observed and expected number of migrations

355

356 1.11 Estimating the origin time for the Rhodobacteraceae and the Ruegeria populations

357 To estimate the origin time of the two Rhodobacteraceae subpopulations (WWC and

358 NMC), we followed the formula: 359 푆 = 휇 ∗ 퐺 ∗ 퐿 ∗ 푇

360 where S is the number of point mutations that have occurred in a population, μ is the

361 base-substitution mutation rate (per nucleotide site per cell division), G is the growth rate in the

362 field (number of cell divisions per year), L is the number of nucleotide sites (i.e., average

363 genome size), T is the evolutionary time (years). Because both WWC and NMC subpopulations

364 showed genetic monomorphism which varies only at a few dozen SNP sites, the number of point

365 mutations can be approximated by the number of SNP sites (27 SNPs in WWC subpopulation

366 and 21 SNPs in NMC subpopulation). Since the mutation rate is not available for these

367 subpopulations, we turned to the model roseobacter Ruegeria pomeroyi DSS-3 [62], whose

368 unbiased spontaneous mutation rate (1.39 × 10-10 per site per generation) was determined using

369 the mutation accumulation experiment followed by whole genome sequencing of the mutant

370 lines [63]. Likewise, the growth rate is also not known for these subpopulations, so we used the

371 published data (averaging to one cell division per day) previously determined for pelagic

372 roseobacters in several coastal waters [64].

373 In the case of the Ruegeria population, which showed much greater diversity, purifying

374 selection at the protein sequence level may have acted to purge diversity at the nonsynonymous

375 (amino acid altering) nucleotide sites, leaving 342,831 SNPs at the synonymous (silent)

376 nucleotide sites useful for time estimation. In addition, a few core genes were subjected to allelic

377 replacement by homologous recombination with externally divergent species (Supplemental Text

378 1.6). As these genes showed unusually large synonymous substitution rates, they cannot be used

379 for time estimation. This led to the exclusion of 25,211 synonymous SNPs occurring in these

380 core genes. Next, 14,124 triallelic and 251 tetrallelic synonymous SNPs were identified, and for

381 a conservative estimate each of these SNPs was assumed to be introduced through a single event 382 of point mutation. Further, homologous recombination may also have occurred between

383 members within the population, which is best characterized by identifying homoplasious bi-

384 allelic SNPs [65], though a small proportion of homoplasious bi-allelic SNPs can be caused by

385 convergent mutations [66, 67]. Similarly, 34,567 homoplasious bi-allelic SNPs each were treated

386 as a single point mutation. The remaining biallelic 267,678 synonymous SNPs were either

387 autapomorphic or synapomorphic, each best explained by a single point mutation event.

388 Following this rationale, we estimated a total of 316,620 mutations at synonymous sites that have

389 occurred in the Ruegeria population, with the caveat that treating the triallelic, tetrallelic, and

390 homoplasious bi-allelic SNPs as single mutation events may underestimate the true number of

391 point mutation events. Next, the timescale was estimated following the procedure detailed for the

392 Rhodobacteraceae WWC and NMC subpopulations, with the L replaced by Lsyn (the number of

393 synonymous sites in all core genes of the Ruegeria population, Lsyn = 829,505).

394

395 Text 2. Supplementary results:

396 2.1 Population differentiation at the core genomes of the Ruegeria population

397 Speciation accompanies a decreased recombination frequency between differentiated

398 populations. The / ratio measures the relative rate of recombination to point mutation, and a

399 threshold of 0.25-0.5 delineates the clonality of a bacterial population [68]. The ClonalFrameML

400 v1.11 [27] analysis showed that this ratio between the two clades was only 0.05, whereas those

401 within clade-M and within clade-S were 0.51 and 0.34, respectively (Table S1). Likewise, the

402 r/m ratio assesses the relative effect of recombination to point mutation on genetic variation, and

403 the result of ClonalFrameML v1.11 showed this ratio between the two clades (0.67) to be much

404 lower than that within each clade (3.13 for clade-M and 4.63 for clade-S, Table S1). The 405 decreased / ratio and r/m ratio between the two clades compared to those within each clade

406 indicates that there is a strong barrier to gene flow between clade-M and clade-S.

407 Without a strong cohesive force by homologous recombination, the genetic

408 differentiation between the two clades is expected, which may have led to the fixation of

409 different alleles. In total, we identified 502,661 single nucleotide polymorphisms (SNPs) from 3.

410 47 Mbp core genomes shared by all members of clade-M and clade-S (Table 1). Among them,

411 302,836 (60.2%) were fixed differences between the two clades. Next, we measured the level of

412 differentiation by calculating Fst values along the core genome in a sliding window of 10 kbp

413 with 5 kbp steps (Fig. S2). Most of the genomic regions (96.8%) showed a high level of

414 differentiation (Fst ≥ 0.5), whereas only several patchy regions showed a lower level of

415 differentiation (Fst < 0.5) (Fig. S2). The permutation test showed that 924 out of the 934 genomic

416 regions were significantly differentiated (p < 0.05), suggesting that speciation between the two

417 clades may have already reached completion.

418

419 2.2 The Ruegeria population differentiation at the physiological level

420 As discussed above and in the main paper, the clade-specific accessory genes and core

421 genes that show unusually large dS values are involved in the utilization of several ecologically

422 relevant substrates, including choline, GBT, sarcosine, TMA, TMAO, creatine, L-proline, taurine

423 and urea. The utilization of these substrates each as a sole source of both C and N by these two

424 clades were tested and compared. As a control, all strains did not grow without C and N sources

425 (open circles in Fig. 4A-1), and grew equally well under optimal conditions with a replete supply

426 of C and N (open triangles in Fig. 4A-1). This indicates that any differences of growth traits in

427 the following assays supplemented with a specific substrate as a sole source of both C and N can 428 be ascribed to the distinct responses of the bacteria to the added substrate. Here, we provided

429 details of the assay results.

430 First, members of the two clades showed distinct responses to the addition of

431 methylamine-related coral osmolytes including choline, GBT, DMG, sarcosine, TMA, TMAO

432 and creatine. All six strains grew poorly when choline (Fig. 4A-2), GBT (Fig. 4A-3), TMA (Fig.

433 4A-6) and TMAO (Fig. 4A-7) each were used as a sole C and N source. However, for the

434 intermediates laying downstream of GBT (Fig. 3), such as DMG (Fig. 4A-4) and sarcosine (Fig.

435 4A-5), the assayed bacteria generally grew and the clade-M members showed significantly

436 higher growth rates and growth yields (p < 0.05, One-way Repeated-Measures ANOVA; the

437 same test used below unless stated otherwise) on sarcosine compared to the clade-S members.

438 Next, we assayed the bacterial growth on other coral osmolytes including DMSP, L-

439 proline and taurine, as well as urea mainly from the excretions of animals in coral reef

440 ecosystems. When serving as a sole C source, DMSP supported a higher growth yield for the

441 clade-M members than the clade-S members (p < 0.05; Fig. 4B-2). When growing on L-proline

442 as a sole C and N source, the overall growth rates and growth yields of the clade-M members

443 were significantly higher than those of the clade-S members (p < 0.05; Fig. 4A-9). When taurine

444 was supplied as a sole C and N source, the clade-M members showed significantly higher growth

445 yields than the clade-S members (p < 0.05; Fig. 4A-10), but no significant growth rate difference

446 was observed. These results support the bioinformatics predictions on the additional copies

447 specific to clade-M (i.e., dddP, dmdABCD, tauABC and proVWX) and shared genes subjected to

448 novel allelic replacements (i.e., dddD and tauABC). When growing on urea, all tested strains

449 grew weakly and showed no significant difference in both the growth rates and yields (Fig. 4A-

450 11). 451 The physiological assays also showed that choline (Fig. 4B-2), GBT (Fig. 4B-3), TMA

452 (Fig. 4B-6), TMAO (Fig. 4B-7) and urea (Fig. 4B-11) cannot act as a sole C and N source. We

453 therefore further tested if they may serve as a sole C source or a sole N source separately. In the

454 control group, all strains did not grow without C and N sources (open circles in Fig. 4B-1) or

455 grew equally well when pyruvate and ammonium were used as C and N sources (open triangles

456 in Fig. 4B-1), respectively. These control experiments indicate that the addition of these common

457 C and N sources did not contribute to growth differences. In other words, when pyruvate or

458 ammonium was replaced by the tested substrates in the experimental group, the growth

459 differences, if any, can be ascribed to the differential responses of the bacteria to the tested

460 substrate. When choline (Fig. 4B-3), GBT (Fig. 4B-5), TMA (Fig. 4B-7), TMAO (Fig. 4B-9) and

461 urea (Fig. 4B-11) each was used as a sole C source, all bacteria grew poorly and did not show

462 significant between-clade differences. However, choline (Fig. 4B-4), GBT (Fig. 4B-6), TMA

463 (Fig. 4B-8) and TMAO (Fig. 4B-10) can serve as a sole N source to support both clade-M and

464 clade-S strains. Using choline or GBT as a sole N source respectively, the clade-M members

465 showed significantly higher growth yields than the clade-S members (p < 0.05), though the

466 growth rates between clades showed no significant difference. Moreover, there was no

467 significant difference in growth rates and yields between clade-M and clade-S when using TMA

468 or TMAO as a sole N source.

469

470 2.3 Metabolic potential for utilizing other substrates by the mucus clade of the Ruegeria

471 population

472 Carbohydrates also account for a large proportion of the osmolytes in corals [69]. L-

473 fucose is an osmolyte present in coral secretions and also part of oligosaccharides, mucins, and 474 other glycoconjugates in the surface mucus layer [2, 70]. A gene cluster involved in fucose

475 catabolism was found specific to the clade-M (HKCCD4315_03759-03763, Table S3), including

476 L-fucose mutarotase (fucU), L-fuconolactone hydrolase, L-fuconate dehydrogenase,

477 ketoglutarate semialdehyde dehydrogenase and 2-keto-3-deoxy-L-fuconate dehydrogenase. This

478 cluster was located on a plasmid, with three genes of this cluster inferred to be gained at the LCA

479 of clade-M, and two lost at the LCA of clade-S (Table S3). Through this pathway, fucose is

480 degraded to pyruvate and L-lactate via non-phosphorylated intermediates [71, 72]. Besides,

481 fucose in host mucus acts as important attractants for symbiotic microbiota, and the utilization of

482 fucose might also provide microbes with a competitive advantage in their niche colonization [72-

483 74]. Families are related to the utilization of other unknown monosaccharides (Table S3), and

484 these genes are located on a plasmid and were inferred to be gained at the LCA of clade-M,

485 suggesting that the carbohydrates in mucus are another important factor driving the

486 diversification of clade-M from clade-S.

487 Some of the aromatic compounds, such as polycyclic aromatic hydrocarbons (PAHs), are

488 ubiquitous pollutants in coral reefs, and are concentrated in coral mucus due to their high lipid-

489 solubility [75]. Hong Kong is one of the busiest seaports in the world, so oil spills occur

490 frequently in Hong Kong coastal waters [76, 77]. The coral mucus in this region is known to

491 contain aromatic pollutants such as PAHs [75]. Members of the Rhodobacteraceae play a major

492 role in degrading aromatics including PAHs in natural communities [78]. A clade-M specific

493 gene encoding the ring-cleaving enzyme which potentially acts on PAHs was likely acquired at

494 the LCA of clade-M (HKCCD4315_02148, Table S3). Besides, genes (pcaBDHG; Table S3) in

495 the protocatechuate pathway responsible for further degradation of the aromatic intermediates

496 were identified exclusively in the clade-M members. These results suggest that the clade-M 497 strains may be able to degrade aromatic pollutants like PAH and benefit the coral hosts.

498 However, the majority of aromatics degrading genes were likely acquired after the branching of

499 clade-M (Table S3), suggesting that these genes were a later innovation facilitating the mucus

500 niche adaptation.

501

502 2.4 Metabolic potential of the mucus clade in the Ruegeria population underlying microbial

503 interactions in the densely-populated mucus habitat

504 The coral mucus is a eutrophic niche enriched with native flora, and the associated

505 microbial community structure is shaped by microbial interactions [79]. Bacterial quorum

506 sensing (QS) is a widespread signaling mechanism acting at a high cell density [80]. Its

507 canonical signaling molecules, N-acyl-homoserine lactones (AHLs), have been detected in coral

508 mucus [81]. Bacteria respond to the AHLs signals and activate the QS circuits through the two-

509 component system, LuxIR, and degrade the signal molecular through N-acyl homoserine

510 lactonase [80]. The additional gene copies for the LuxIR system and N-acyl homoserine

511 lactonase were found located in the clade-M strains specific genome region (Table S3).

512 In the Rhodobacteraceae, biofilm formation is an effective strategy to compete with other

513 organisms for space and nutrients, which could be a response to QS signals [82]. Extracellular

514 polymeric substances (EPS) are important components of the biofilm matrix [83]. We found that

515 clade-M members possess several clade-specific genes involved in the synthesis of EPS such as

516 exopolysaccharides (HKCCD4315_04016, HKCCD4315_04166, and HKCCD4315_04162,

517 Table S3), and lipopolysaccharides (HKCCD4315_ 04159 and HKCCD4315_04161, Table S3).

518 These compounds are the structural components of the biofilm matrix [82]. Most of the

519 aforementioned genes (five out of eight) involved in the QS system and biofilm formation were 520 inferred to be acquired at the LCA of the clade-M, which may have facilitated the adaptation of

521 clade-M to the mucus niche of high cell density.

522

523 2.5 Adaptation of the skeleton clade in the Ruegeria population to the periodically anoxic

524 skeleton habitat

525 The skeleton is a diurnally anoxic environment [84]. The oxygen produced by the

526 photosynthesis of symbiotic and endolithic algae diffuses through the porous aragonite into the

527 skeleton core in the daytime, and is continuously consumed by the coral host and associated

528 community through respiration [84]. Thus, the skeleton undergoes a sharp decrease of dissolved

529 oxygen at night, and resulting in anoxic in the skeleton [85]. A gene cluster associated with

530 anaerobic respiration was identified exclusively in the clade-S members. This cluster encoded a

531 succinate dehydrogenase/fumarate reductase (sdh-frd, Table S4) and L(+)-tartrate dehydratase

532 (ttdAB, Table S4). The Sdh-Frd is bifunctional in some facultative anaerobes. It catalyzes

533 succinate oxidation to support the citric acid cycle under oxic conditions. Meanwhile, it does the

534 reverse reduction under anoxic condition, and employs fumarate as an electron acceptor to

535 maintain the anoxic respiration in the absence of oxygen [86]. The Ttd enzyme is responsible for

536 the fermentation of L-tartrate, which supports the anaerobic growth of bacteria as C and energy

537 source [87, 88]. As this gene cluster is located on a plasmid that is not linked to the chromosomal

538 genes encoding the canonical citric acid cycle-related enzymes, it is more likely related to

539 anaerobic respiration rather than the citric acid cycle. It was inferred to be acquired at the LCA

540 of clade-S, indicating an adaptation of clade-S strains in periodically anoxic skeleton niches.

541 Besides, a gene cluster encoding the dimethyl sulfoxide/trimethylamine oxide reductase

542 (dmsABC, Table S5) was identified as core genes with unusually large dS value. This gene is 543 involved in anaerobic respiration and enables the bacteria to use either dimethyl sulfoxide

544 (DMSO) or trimethylamine-N-oxide (TMAO) as a terminal electron acceptor anaerobically for

545 oxidative phosphorylation [89]. The gene trees showed that the three gene families subject to

546 distinct evolutionary history (Table S5). Together, the clade-S strains likely gained fitness

547 advantages in skeleton niches through enhanced anoxic tolerance. A B Bacteria Symbiodiniaceae Seawater Ngo Mei Chau Endolithic microalgae

N Wong Wan Chau Coral mucus Chek Chau Mucocyte

Coral tissue

Kiu Tsui Chau

Coral skeleton 4 km

Figure S1. Sampling information of coral Platygyra acuta. (A) The sampling sites in Hong Kong seawater: Wong Wan Chau (N 22°31‘31.2“ E 114°19’00.1”), Ngo Mei Chau (N 22°31‘47.2“ E 114°19’02.9”), Chek Chau (N 22°30‘03.3“ E 114°21’22.7”) and Kiu Tsui Chau (N 22°22‘04.4“ E 114°17’42.0”). (B) A cartoon shows the compartments of coral. The associated bacteria, endosymbiont Symbiodiniaceae and endolithic microalgae are showed in different compartments. Part of the cartoon is adapted from Bourne et al., 2016. Chromosome Plasmid 1 Plasmid 2 Plasmid 3

1.00 0.75

Fst

0.50

0.25

0 500000 1000000 1500000 2000000 2500000 3000000 3500000 4000000 4500000 Position on genome of HKCCD4315（bp）

Figure S2. Fst values between the clade-M and clade-S strains along the closed genome of strain HKCCD4315. The upper panel showed the genomic region of the chromosome and three plasmids of strain HKCCD4315. Each dot represents an Fst value calculated for a sliding window of 10 kb moving in 5 kb steps. A B Single-copy orthologous gene families 8

7 d s 2.0 6 ds within clade-M 5

4 ds within clade-S 0 3 d 2 s between clade-M and clade-S 1 0

# Indices supporting the number of clusters # Indices supporting the number 2 3 4 5 9 10 K-value Cluster I Cluster II

C D

HKCCD5851 HKCCD5851

HKCCD5849 HKCCD5849

HKCCD7303 HKCCD7303

HKCCD7319 HKCCD7319

HKCCD7296 HKCCD7296

HKCCD7318 HKCCD7318

or HKCCD4318 HKCCD4318

HKCCD4315 HKCCD4315

HKCCD4318-2 HKCCD4318-2

HKCCD4332 HKCCD4332

HKCCD4884 HKCCD4884

HKCCD6109 HKCCD6109

Figure S3. Illustration of allelic replacement inference using dS values. (A) The voting of the optimal number of cluster validity indices using R package “NbCluster”. The seven indices supporting K=2 as the optimal clustering number include ‘beale’, ‘duda’, ‘mcclain’, ‘pseudot2’, ‘ptbiserial’, ‘gap’ and ‘sdindex’. (B) The demo heatmap of the dS values were calculated for every possible pair of genomes across all single-copy orthologous gene families, with warmer colour indicating higher dS values. The gene families were grouped into two clusters using the K-means method. Cluster I shows enormously large dS values between clade-M and clade-S but small dS values within each clade, whereas all dS values are small in Cluster II. (C) The evolutionary history of an example gene from Cluster I was mapped to the genome tree. Due to the unusually large between-clade dS values and little diversity within each clade, the allelic replacement with distant lineages was inferred to have occurred at the LCA of clade-M or that of clade-S. (D) The evolutionary history of an example gene from Cluster II was mapped to the genome tree. Because of the small diversity both between- and within-clade, allelic replacement with distant lineages is less likely to occur. Clade-M: The Ruegeria population: Clade-S:

Mari {Outgroup: Roseovarius se

Roseobacterium elongatum DS

Thalassobacter Litor t

Rhodobacteraceae bacterium MA-7- i

The Rhodobacteraceae population: m Thalassobacter 3

Litor

2

i

Dinoroseobacter b D16 A eibacter a

M Albidovulum xiamenense CGMCC 1.10789 c C Tree scale: 0.1 t eibacter a

R e ensis

r

r

a Thalassobacter i diminilitoris DSM 29439

t t

a

i

a 1

l m

k

r Albidovulum xiamenense DSM 24422 janth e T stenotr Jannaschia sp. EhC01 a i M 26878

b

l r stenotr Nioella sediminis JS7-1 p Jannaschia sp. CCS1 ponti DSM 100977 i opicimonas sediminicola DSM 29339 a jejudon icola Maribius sali p Maribius pelagius DSM 26893 c m M 101533

h T3646 t e cticus 307

Rhodovulum inus DSM 26921 e t RhodovuluRhodovulum m i

l JL3646 r

shibae DFL u

a

ophicus CECT s s silacus DSM 16199

n

p

Rhodovulum ophicus DSM 16310 i p. JL Alg231-30 H

r sp. 16P . a DSM 28223

antar Nioella sp. Z7-4 HKCCD8482

a Confluent T U ascidiace temperatus SB 7735

C

B m temperatus temperatus DS

o

C A M 19469 ella sal visakhapatnam nus DSM 26892 t euryhalinum DSM 4868 ella sp. D2R18 ctica IMCC9565 steppense DSM 21 2 3 k 1

ALIMAR09 9 6

n

12 5 7 imicr a

l Actibacterium mu Rhodovulu marinumm DS 4 5

P Thalassobium sp. R2A62 oonia maricola DSM 29128 sp. CECT Rhodobacteraceae bacterium G7 Donghicola sp. 5294 Marivivens s 27 Loktan oonia maritima DS alea ar Y obium lipolyticum C Loktan Roseinatr Y Rhodovulum 1 Pseudooctadecabacter Rhodobacteraceae bacterium BH-S 7648 Octadecabacter Octadecabacter Octadecabacter cticum SM121 ense DSM 17937 Octadecabacter sedentarius DSM 104836 Rhodovulum Octadecabacter us DSM 26923 onobacter sp. ES.010 Planktot 1445 Rhodobacterales bacteriumOceanicola HTCC2255 s M 18063 Cognatishimia maritim cosum KCTC 23349 153 Phaeobacter alicic Shimia abyssi DSM 100673 Actibacterium pelagium sp. BSW8 Rhodobacteraceae bacterium Oceanicel thiooxidans DSM 13087 Shimia sagamensis DSM 29734 phth halenivorans DSM 19561 ECT Shimia sp. SK013 sp. P5 Shimia isoporae DSM 26433 halenivorans CECT Roseibaca ekhonensisp. MCTG156 Marinovum algicola napht Oceanicela actignisl CGMCC 1.10808 8621 Primorskyibacter Puniceibacterium sediminis naph DSMt 29052 Oceanicel heliothermus DSM 1 Puniceibacteriumropicibacter antar heliothermus SMS3 la actignis DSM 24423 T Monaibacterium marinum C Pelagimonasopicibacte variansr DSM 23678 r ctobacter la actignis DSM 22673 T ropicibacter T ctobacter ba KD53 Antar ba F15 Antar ba L6M1-5 Ruegeria sp. PBVC088 Mameliella al ba UMTAT08 1 Mameliella al ba CGMCC 1.7290 7 Mameliella al ba DSM 26384 Mameliella al ba JL351 8383 Mameliella al Mameliella al Leisingera sp. Mameliella al Rhodobacteraceae bacterium HIMB1 Leisingera sp. Thalassobius mediterraneus DSM 16398 4357 ANG-S5 Leisingera sp. Thalassobius mediterraneus CECT ANG-DT Thalassobius gelatinovorus DSM 5887 Leisingera sp. ANG-S Thalassobius gelatinovorus ensis DSM 28010 Leisingera sp.ANG1 JC1 Thalassobius p.gelatinovorus HL-35 CECT Leisingera sp. saemankum Leisingera daeponensis DSM 23529 Oceanicolaaribacte s r ANG-M7 Lutim Rhodobacterales bacterium Pseudooceanicola nanhaiensis DSM 18065 p. 22II-s10i educens CGMCC 1.7292 Leisingera caer Oceanicola s ulea DSM 24564Y4I Pseudooceanicola nitratireducens DSM 29619 Leisingera sp. Pseudooceanicola nitratir ANG-Vp salinarum Leisingera sp. NJS204 Roseovarius 7450 Leisingera methylohalidivorans DSM 14336 Roseovarius albus CECT Pelagicola litorisediminis CECT 8287 Leisingera aquimarina DSM 24565 Roseovarius lutimaris DSM 28463 e Roseobacter sp. MedPE-SWd Roseovarius gaetbuli CECT 8370 sp. MED193 Roseovarius sp. Roseobacter Roseovarius toleransA-2 DSM 1 arcticus DSM 23566 Roseovarius azor Pseudophaeobacter 1ANDIMAR09 1457 sp. 1 5382 Roseovarius sp. 217ensis DSM 100674 Phaeobacter sp. CECT Roseovarius mucosus DSM 17069 Phaeobacter sp. SK209-2-6 Roseovarius mucosus SMR3 Roseobacter sp. JL2872 Roseovarius sp. Phaeobacter obile 270-3 Roseovarius sp. Sulfitobact TM1035 Epibacterium mobile S2144 obile F2488 Sedimentitaleaer nanAK1035h Epibacterium m Sulfitobact sp. JL08 Ruegeria sp. P4 Sulfitobact Epibacterium m TrichCH4B er sp. Tateyamaria sp. sp. DFLaiensis- CGM obile G930 T er obile ateyamaria sp. pseudonitzschiae 23 SMR1 Silicibacter obile M41-2.2 Tateyamaria omph CC 1.10959 Roseobacter Epibacterium m TM1040 Alg231-49 Roseobacter ANG-S1 Sulfitobact EpibacteriumEpibacterium m m Sulfitobact denitrificansalii DOK1-4 OCh 1 Ruegeria sp. Roseobacter litoralis O Sulfitobact er omollicae DSM 25328 sp. SK012 inhibens P24/M2-4.4 Sulfitobact er inhibens P70 Sulfitobact donghicola JCMch 149 14565 inhibens P10/01/009 inhibens P88 Sulfitobact sp. GAI101 er 14 Sulfitobact sp. SK01 EpibacteriumPhaeobacte scott r inhibens piscinae S4Sm P71 Thalassococcus sp. S3er Phaeobacter Aestuariivita boseongensiser sp. BS-B2 DFL- Phaeobacter piscinae sp. P14LSS9 Phaeobacter Pseudodonghicola xiamenensiser mediterraneus DSM 18339 KCTC 32188 Rhodobacteraceae bacterium SM1703 1 er noctilucicola NB-77 Phaeobacter piscinae P13 7645 Jhaorihella thermophila DSM noctilucae 23413 NB-6814 porticola Thalassococcus sp. SH-1 Phaeobacter 1 Ruegeria pomer PhaeobactePhaeobacter r 14104 Cribrihabitans marinus DSM 29340 ensis DSM 26640 Ruegeria marisrubri ZGT1 Ruegeria ar W43 Ruegeria lacuscae Phaeobacter Ruegeria intermedia DSM 29341 53 Ruegeria pr gallaecPhaeobactei italicusr CECT Alg231-04 Ruegeria sp. ea R37 Ruegeria conchae ensis Ruegeria conchae DSM 29317 italicus DSM 26436 Rhodobacteraceae bacterium KLH1 Ruegeria sp. R1 Ruegeria sp. Ruegeria halocynthiae DSM 27839 Ruegeria faecimaris DSM 28009 oyi DSS-3 Ruegeria halocynthiae MOLA Ruegeria meonggei CECT enilitoris Ruegeria sp. Ruegeria sp. Ruegeria atlantica CECT Ruegeria sp. Ruegeria atlantica CECT Ruegeria sp. 6P Ruegeria sp. Phaeobacter gallaeci HKCCD6238 PhaeobacterNautella sp. ECSMB 8 4 19 ofund

HKCCD7296 2 0

Phaeobacter 6 Sedimentitalea sp. HKCCD7319 2 ANG-S4 rulensis ITI-1 Ruegeria litor HKCCD7318 6 6 HKCCD7303 HKCCD5849 D D i ZGT108 HKCCD5851 ANG-R C C 18 HKCCD4315 TW15

Ruegeria mediterranea M17 C C HKCCD4318-2 HKCCD4318 HKCCD4332 Alg231-54 HKCCD6109 A3M17 Phaeobacter HKCCD4884 K K HKCCD7559 AU67 HKCCD61

HKCCD6157 AD91A HKCCD6181 HKCCD6428

H H

Rhodobacteraceae bacterium ALISE 157

Rhodobacteraceae bacterium EL

P08 841

4292

4293

R1 1 1

Figure S4. The maximum likelihood phylogeny of the Roseobacter group, which was constructed using 223 publicly available Roseobacter genomes, the 20 strains of the Ruegeria population and one representative of the genetically uniform Rhodobacteraceae population. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 95%. The scale bar indicates number of substitutions per site. The members of clade-M, clade-S and outgroup clade were shadowed with blue, pink and gold, respectively. Figure S5. The maximum likelihood phylogeny of the core gene families ugpA , ugpB, ugpE, betA and dmgdh involved in methylamine utilization constructed by IQ-TREE v1.6.5. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 80%. The scale bar indicates number of substitutions per site. The members of clade-M, clade-S and outgroup clade were shadowed with blue, pink and gold, respectively. Rhodobacteraceae bacterium MA-7-27 Roseibacterium elongatum DSM 19469 Thalassococcus sp. S3 Cribrihabitans marinus DSM 29340 ugpA (HKCCD4315_00516) Phaeobacter sp. JL2872 Salinihabitans flavidus DSM 27842 Salinihabitans flavidus Ruegeria sp. 6PALISEP08 HKCCD4315 HKCCD4332 HKCCD4318-2 HKCCD6109 HKCCD4318 HKCCD4884 Rhodovulum sp. P5 Rhodovulum euryhalinum DSM 4868 Rhodovulum marinum DSM 18063 Tropicimonas sediminicola DSM 29339 Clade-M: Tropicimonas sp. IMCC34043 Tropicimonas sp. IMCC6043 Clade-S: Marinovum algicola Dinoroseobacter shibae DFL 12 Outgroup: Primorskyibacter sedentarius DSM 104836 Primorskyibacter marinus Pseudooceanicola flagellatus Tree scale: 0.1 Pseudooceanicola flagellatus CGMCC 1.12644 Pseudoruegeria sabulilitoris GJMS-35 Rhodobacteraceae bacterium CCMM004 Pelagibaca abyssi JLT2014 Pelagibaca abyssi Sagittula sp. P11 Sagittula stellata E-37 Sulfitobacter sp. SK012 Amylibacter cionae Neptunicoccus sediminis Oceaniglobus sp. YLY08 Sulfitobacter noctilucicola NB-77 Sulfitobacter sp. DFL-14 Sulfitobacter sp. EhC04 Oceaniglobus indicus 1-19b Oceaniglobus indicus Defluviimonas aquaemixtae Poseidonocella pacifica DSM 29316 Thalassococcus sp. SH-1 Sedimentitalea nanhaiensis Sedimentitalea nanhaiensis CGMCC 1.10959 Litoreibacter arenae DSM 19593 Litoreibacter meonggei DSM 29466 Litoreibacter halocynthiae DSM 29467 Litoreibacter janthinus DSM 26921 Oceaniovalibus guishaninsula JLT2003 Pseudoruegeria sp. SK021 Thioclava indica DT23-4 Thioclava indica Thioclava atlantica Thioclava atlantica 13D2W-2 Thioclava sp. L04-15 Thioclava marina Thioclava pacifica DSM 10166 Thioclava sp. JM3 Thioclava sp. F1Mire-8 Thioclava sp. IC9 Thioclava sp. F42-5 Thioclava sp. F36-6 Thioclava sp. DLFJ5-1 Thioclava sp. F28-4 Thioclava sp. F36-7 Thioclava sp. DLFJ4-1 Thioclava sediminum Thioclava electrotropha Thioclava electrotrophica elox9 Thioclava sp. ES.031 Ahrensia sp. R2A130 Aestuariivita boseongensis BS-B2 Pseudorhodobacter sp. E13 Kandeliimicrobium roseum Rhodobacteraceae bacterium BH-SD16 Planktomarina temperata RCA23 Octadecabacter antarcticus 307 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Roseovarius mucosus DSM 17069 Roseovarius mucosus Roseovarius mucosus SMR3 Roseovarius sp. 217 Roseovarius sp. TM1035 Roseovarius sp. AK1035 Roseovarius tolerans DSM 11457 Roseovarius sp. A-2 Roseovarius nitratireducens Roseovarius sp. A46 Roseovarius azorensis DSM 100674 Roseovarius lutimaris DSM 28463 Roseovarius gaetbuli CECT 8370 Roseovarius marisflavi DSM 29327 Aliiroseovarius sediminilitoris DSM 29439 Ruegeria halocynthiae DSM 27839 Sulfitobacter sp. SK011 Litoreibacter ponti DSM 100977 Roseovarius aestuarii Yoonia rosea DSM 29591 Loktanella sp. 1ANDIMAR09 Yoonia rosea Planktotalea frisia SH6-1 Planktotalea frisia DSM 23709 Thalassobium sp. R2A62 Sulfitobacter geojensis Roseobacter sp. GAI101 Pelagimonas varians DSM 23678 Pelagimonas varians Shimia abyssi DSM 100673 Ruegeria meonggei CECT 8411 Ruegeria conchae Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria profundi ZGT108 Ruegeria denitrificans Ruegeria denitrificans CECT 5091 Ruegeria halocynthiae MOLA R1 13b Ruegeria halocynthiae HKCCD7303 HKCCD5849 HKCCD7319 HKCCD5851 HKCCD7296 HKCCD7318 HKCCD6238 HKCCD6119 HKCCD7559 HKCCD6157 HKCCD6428 HKCCD6228 HKCCD6181 HKCCD6604 Ruegeria sp. AD91A Rhodobacteraceae bacterium KLH11 Ruegeria sp. A3M17 Ruegeria faecimaris DSM 28009 Ruegeria sp. Alg231-54 Ruegeria atlantica Ruegeria atlantica CECT 4292 Ruegeria atlantica CECT 4293 Ruegeria sp. AU67 ugpB (HKCCD4315_00515)

Clade-M: Clade-S: Thalassococcus sp. S3 Outgroup: Cribrihabitans marinus DSM 29340 Tree scale: 0.1 Phaeobacter sp. JL2872 Salinihabitans flavidus Salinihabitans flavidus DSM 27842 Ruegeria sp. 6PALISEP08 HKCCD4884 HKCCD6109 HKCCD4315 HKCCD4332 HKCCD4318 HKCCD4318-2 Octadecabacter antarcticus 307 Oceaniglobus indicus 1-19b Oceaniglobus indicus Litoreibacter arenae DSM 19593 Litoreibacter meonggei DSM 29466 Litoreibacter halocynthiae DSM 29467 Litoreibacter janthinus DSM 26921 Aliiroseovarius sediminilitoris DSM 29439 Ruegeria halocynthiae DSM 27839 Roseovarius aestuarii Litoreibacter ponti DSM 100977 Sulfitobacter sp. SK011 Roseobacter sp. GAI101 Sulfitobacter geojensis Thalassobium sp. R2A62 Planktomarina temperata RCA23 Pelagimonas varians Pelagimonas varians DSM 23678 Planktotalea frisia SH6-1 Planktotalea frisia DSM 23709 Yoonia rosea Loktanella sp. 1ANDIMAR09 Yoonia rosea DSM 29591 Shimia abyssi DSM 100673 Ruegeria faecimaris DSM 28009 Ruegeria sp. AD91A HKCCD6157 HKCCD6181 HKCCD6228 HKCCD6119 HKCCD6604 HKCCD7559 HKCCD6238 HKCCD6428 Ruegeria sp. A3M17 Ruegeria sp. AU67 Ruegeria sp. Alg231-54 Ruegeria atlantica CECT 4293 Ruegeria atlantica CECT 4292 Ruegeria atlantica HKCCD7303 HKCCD7319 HKCCD5851 HKCCD5849 HKCCD7296 HKCCD7318 Rhodobacteraceae bacterium KLH11 Ruegeria denitrificans CECT 5091 Ruegeria denitrificans Ruegeria conchae Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria profundi ZGT108 Ruegeria meonggei CECT 8411 Ruegeria halocynthiae Ruegeria halocynthiae MOLA R1 13b Phaeobacter sp. JL2872 Salinihabitans flavidus DSM 27842 Salinihabitans flavidus Cribrihabitans marinus DSM 29340 Ruegeria sp. 6PALISEP08 ugpE (HKCCD4315_00517) HKCCD4884 HKCCD6109 HKCCD4315 HKCCD4332 HKCCD4318 HKCCD4318-2 Sulfitobacter noctilucicola NB-77 Sulfitobacter sp. EhC04 Sulfitobacter sp. DFL-14 Oceaniglobus sp. YLY08 Sulfitobacter sp. SK012 Neptunicoccus sediminis Clade-M: Amylibacter cionae Defluviimonas aquaemixtae Clade-S: Poseidonocella pacifica DSM 29316 Rhodovulum euryhalinum DSM 4868 Outgroup: Rhodovulum marinum DSM 18063 Rhodovulum sp. P5 Rhodobacteraceae bacterium CCMM004 Tree scale: 0.01 Tropicimonas sediminicola DSM 29339 Marinovum algicola Dinoroseobacter shibae DFL 12 Primorskyibacter sedentarius DSM 104836 Primorskyibacter marinus Pseudooceanicola flagellatus Pseudooceanicola flagellatus CGMCC 1.12644 Oceaniglobus indicus 1-19b Oceaniglobus indicus Tropicimonas sp. IMCC34043 Tropicimonas sp. IMCC6043 Pseudoruegeria sabulilitoris GJMS-35 Sagittula sp. P11 Sagittula stellata E-37 Pelagibaca abyssi JLT2014 Pelagibaca abyssi Thalassococcus sp. SH-1 Sedimentitalea nanhaiensis CGMCC 1.10959 Sedimentitalea nanhaiensis Litoreibacter meonggei DSM 29466 Litoreibacter halocynthiae DSM 29467 Litoreibacter janthinus DSM 26921 Litoreibacter arenae DSM 19593 Oceaniovalibus guishaninsula JLT2003 Thioclava indica Thioclava indica DT23-4 Pseudorhodobacter sp. E13 Pseudoruegeria sp. SK021 Ahrensia sp. R2A130 Planktomarina temperata RCA23 Octadecabacter antarcticus 307 Rhodobacteraceae bacterium EL53 Phaeobacter gallaeciensis Kandeliimicrobium roseum Aestuariivita boseongensis BS-B2 Thioclava atlantica 13D2W-2 Thioclava pacifica DSM 10166 Thioclava marina Thioclava sp. L04-15 Thioclava sp. IC9 Thioclava sp. F36-7 Thioclava sp. F36-6 Thioclava sp. F28-4 Thioclava electrotrophica elox9 Thioclava sp. ES.031 Thioclava sediminum Thioclava sp. DLFJ4-1 Thioclava sp. JM3 Thioclava sp. DLFJ5-1 Thioclava electrotropha Thioclava sp. F42-5 Thioclava sp. F1Mire-8 Roseovarius azorensis DSM 100674 Roseovarius sp. A-2 Roseovarius tolerans DSM 11457 Roseovarius nitratireducens Roseovarius sp. A46 Roseovarius marisflavi DSM 29327 Roseovarius gaetbuli CECT 8370 Roseovarius lutimaris DSM 28463 Roseovarius mucosus DSM 17069 Roseovarius sp. 217 Roseovarius sp. AK1035 Roseovarius sp. TM1035 Roseovarius mucosus SMR3 Roseovarius mucosus Rhodobacteraceae bacterium BH-SD16 Ruegeria halocynthiae DSM 27839 Litoreibacter ponti DSM 100977 Roseovarius aestuarii Sulfitobacter sp. SK011 Sulfitobacter geojensis Roseobacter sp. GAI101 Thalassobium sp. R2A62 Pelagimonas varians DSM 23678 Pelagimonas varians Planktotalea frisia DSM 23709 Planktotalea frisia SH6-1 Yoonia rosea Yoonia rosea DSM 29591 Loktanella sp. 1ANDIMAR09 Aliiroseovarius sediminilitoris DSM 29439 Shimia abyssi DSM 100673 Ruegeria meonggei CECT 8411 Ruegeria halocynthiae MOLA R1 13b Ruegeria halocynthiae Ruegeria conchae TW15 Ruegeria conchae DSM 29317 Ruegeria conchae Ruegeria profundi ZGT108 HKCCD7303 HKCCD5849 HKCCD5851 HKCCD7318 HKCCD7296 HKCCD7319 Ruegeria sp. AD91A HKCCD6238 HKCCD6228 HKCCD6157 HKCCD6119 HKCCD6604 HKCCD6428 HKCCD6181 HKCCD7559 Ruegeria faecimaris DSM 28009 Rhodobacteraceae bacterium KLH11 Ruegeria denitrificans CECT 5091 Ruegeria denitrificans Ruegeria sp. A3M17 Ruegeria atlantica Ruegeria sp. AU67 Ruegeria atlantica CECT 4292 Ruegeria atlantica CECT 4293 Ruegeria sp. Alg231-54 Pseudooceanicola flagellatus CGMCC 1.12644 Pseudooceanicola flagellatus betA (HKCCD4315_00231) Primorskyibacter sedentarius DSM 104836 Roseovarius nanhaiticus Roseovarius nanhaiticus CGMCC 1.10961 Roseovarius nanhaiticus DSM 29590 Loktanella salsilacus DSM 16199 Clade-M: Sulfitobacter sp. 20 GPM-1509m Roseovarius sp. A-2 Clade-S: Loktanella sp. S4079 Outgroup: Thioclava dalianensis CGMCC 1.12325 Thioclava dalianensis Tree scale: 0.1 Thioclava sp. ES.031 Thioclava electrotrophica elox9 Thioclava electrotropha Sulfitobacter noctilucae NB-68 Actibacterium pelagium Ruegeria sp. AU671 HKCCD6119-1 HKCCD7559 Thalassobius mediterraneus DSM 16398 Thalassobius mediterraneus CECT 8383 Sulfitobacter marinus DSM 23422 Phaeobacter piscinae P71 Phaeobacter piscinae P13 Sulfitobacter geojensis GNM21425 Roseobacter sp. MedPE-SWde1 Sulfitobacter noctilucicola NB-77 Rhodobacteraceae bacterium Alg231-04 Phaeobacter sp. 11ANDIMAR09 Pelagimonas varians DSM 23678 Pelagimonas varians Roseobacter sp. MedPE-SWde Roseobacter sp. MED193 Leisingera sp. NJS204 Leisingera aquimarina DSM 24565 Leisingera caerulea DSM 24564 Leisingera sp. ANG-M7 Leisingera sp. JC1 Labrenzia sp. Alg231-36 Labrenzia alba CECT 5094 Labrenzia alba Labrenzia alba CECT 5096 Labrenzia alba CECT 5095 Thalassococcus sp. S3 Ahrensia marina Ahrensia marina LZD062 Sulfitobacter sp. JL08 Roseovarius nubinhibens ISM Rhodobacteraceae bacterium EL129 Ruegeria arenilitoris Ruegeria sp. ANG-S4 Ruegeria sp. ANG-R Rhodobacteraceae bacterium KLH11 Ruegeria sp. 6PALISEP08 Ruegeria halocynthiae DSM 27839 Ruegeria atlantica CECT 4293 Ruegeria sp. AU67 Ruegeria sp. A3M17 Ruegeria sp. Alg231-54 Ruegeria atlantica CECT 4292 Ruegeria atlantica Ruegeria meonggei CECT 8411 Ruegeria sp. AD91A HKCCD6238 HKCCD6157 HKCCD6604 HKCCD6119 HKCCD7559-1 HKCCD6228 HKCCD6181 HKCCD6428 Ruegeria faecimaris DSM 28009 Ruegeria denitrificans CECT 5091 Ruegeria denitrificans Ruegeria conchae TW15 Ruegeria conchae DSM 29317 Ruegeria conchae HKCCD4884 HKCCD6109 HKCCD4318-2 HKCCD4332 HKCCD4315 HKCCD4318 HKCCD5851 HKCCD7319 HKCCD7303 HKCCD7318 HKCCD5849 HKCCD7296 Roseovarius albus CECT 7450 Thalassobium sp. R2A62 dmgdh (HKCCD4315_03828) Planktotalea arctica IMCC9565 Litoreibacter meonggei DSM 29466 Tateyamaria sp. ANG-S1 Pelagicola litorisediminis CECT 8287 Sulfitobacter sp. JL08 Nioella sp. Z7-4 Nioella sediminis JS7-11 Rhodobacteraceae bacterium KLH11 Ruegeria sp. ANG-R Clade-M: Ruegeria halocynthiae DSM 27839 Ruegeria conchae TW15 Ruegeria conchae DSM 29317 Clade-S: Ruegeria conchae Labrenzia sp. PT13C1 Outgroup: Labrenzia alexandrii DFL-11 Labrenzia alexandrii CECT 5112 Labrenzia alexandrii Tree scale: 0.1 Ruegeria intermedia DSM 29341 Ruegeria arenilitoris Ruegeria lacuscaerulensis DSM 11314 Ruegeria lacuscaerulensis ITI-1157 Ruegeria faecimaris DSM 28009 Ruegeria meonggei CECT 8411 Ruegeria denitrificans CECT 5091 Ruegeria denitrificans Ruegeria halocynthiae Ruegeria halocynthiae MOLA R1 13b Ruegeria atlantica CECT 4293 Ruegeria sp. Alg231-54 Ruegeria sp. A3M17 Ruegeria sp. AU67 Ruegeria atlantica Ruegeria atlantica CECT 4292 Ruegeria sp. AD91A HKCCD6238 HKCCD6604 HKCCD6428 HKCCD6181 HKCCD6228 HKCCD6157 HKCCD6119 HKCCD7559 Ruegeria marisrubri ZGT118 Actibacterium pelagium Leisingera aquimarina DSM 24565 Leisingera sp. NJS204 Leisingera caerulea DSM 24564 Rhodobacterales bacterium Y4I Leisingera daeponensis DSM 23529 Leisingera sp. ANG-Vp Ruegeria pomeroyi DSS-3 Leisingera sp. ANG-M7 Leisingera sp. JC1 Leisingera sp. ANG-S5 Leisingera sp. ANG-DT Leisingera sp. ANG1 Leisingera sp. ANG-S Litoreibacter janthinus DSM 26921 Defluviimonas aquaemixtae Sulfitobacter sp. SK012 Shimia sp. SK013 Roseisalinus antarcticus Ahrensia sp. R2A130 Shimia abyssi DSM 100673 Roseovarius nubinhibens ISM Roseobacter sp. SK209-2-6 Phaeobacter sp. CECT 5382 Labrenzia sp. DG1229 Labrenzia sp. Alg231-36 Labrenzia alba CECT 5096 Labrenzia alba Labrenzia alba CECT 5095 Labrenzia alba CECT 5094 Loktanella sp. 1ANDIMAR09 Primorskyibacter sedentarius DSM 104836 Roseovarius sp. 217 Sulfitobacter sp. D7 Sulfitobacter indolifex HEL-45 Phaeobacter inhibens P10 1 9 21114 GNM8332 Leisingera methylohalidivorans MB2 Leisingera methylohalidivorans DSM 14336 Ruegeria sp. ANG-S4 Rhodobacteraceae bacterium Alg231-30 Aliiroseovarius pelagivivens Phaeobacter sp. 11ANDIMAR09 Rhodobacteraceae bacterium Alg231-04 Loktanella sp. D2R18 HKCCD4332 HKCCD6109 HKCCD4884 HKCCD4315 HKCCD4318 HKCCD4318-2 HKCCD7318 HKCCD7296 HKCCD7319 HKCCD5849 HKCCD7303 HKCCD5851 Roseovarius aestuarii Rhodobacteraceae bacterium HIMB11 Rhodobacteraceae sp. HIMB11 Marinovum algicola Pseudorhodobacter wandonensis KCTC 23672 Pseudorhodobacter wandonensis Litoreibacter albidus DSM 26922 Sulfitobacter noctilucae NB-68 Tropicimonas sediminicola DSM 29339 Roseovarius litoreus DSM 28249 Roseovarius sp. A-2 Sulfitobacter sp. SK011 Yoonia maritima DSM 101533 Sulfitobacter mediterraneus Yoonia litorea DSM 29433 Yoonia litorea Litoreibacter halocynthiae DSM 29467 Yoonia maricola Yoonia maricola DSM 29128 Cribrihabitans marinus DSM 29340 Thalassobacter sp. 16PALIMAR09 Thalassobacter stenotrophicus Thalassobacter stenotrophicus CECT 5294 Thalassobacter stenotrophicus DSM 16310 Roseovarius marisflavi DSM 29327 Roseovarius lutimaris DSM 28463 Sulfitobacter marinus DSM 23422 Roseovarius sp. YLY04 Jannaschia sp. EhC01 Planktotalea frisia SH6-1 Planktotalea frisia DSM 23709 Sulfitobacter mediterraneus DSM 12244 Sulfitobacter mediterraneus KCTC 32188 Roseobacter litoralis Och 149 Roseobacter denitrificans OCh 114 Roseobacter denitrificans DSM 7001 HKCCD4884 dddD (HKCCD4315_04324) HKCCD6109 HKCCD4332 Clade-M: HKCCD4315 HKCCD4318 Clade-S: HKCCD4318-2 Outgroup: Ruegeria sp. AD91A Tree scale: 0.01 HKCCD6428 HKCCD6119 HKCCD7559 HKCCD6181 HKCCD6228 HKCCD6604 HKCCD6238 HKCCD6157 HKCCD7296 HKCCD7319 HKCCD7318 HKCCD5849 HKCCD7303 HKCCD5851 Rhodobacteraceae bacterium KLH11 Roseovarius albus CECT 7450 Leisingera sp. ANG-Vp Leisingera methylohalidivorans DSM 14336 Leisingera aquimarina DSM 24565 Leisingera sp. NJS204

Figure S6. The maximum likelihood phylogeny of the core gene families dddD involved in DMSP utilization constructed by IQ-TREE v1.6.5. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 80%. The scale bar indicates number of substitutions per site. The members of clade-M, clade-S and outgroup clade were shadowed with blue, pink and gold, respectively. Figure S7. The maximum likelihood phylogeny of the core gene families tauA, tauB and tauC involved in taurine utilization constructed by IQ-TREE v1.6.5. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 80%. The scale bar indicates number of substitutions per site. The members of clade-M, clade-S and outgroup clade were shadowed with blue, pink and gold, respectively. Ruegeria mediterranea M17 Ruegeria litorea R37 tauA (HKCCD4315_01945) Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Ruegeria profundi ZGT108 Ruegeria denitrificans CECT 5091 Ruegeria meonggei CECT 8411 Clade-M: Ruegeria sp. ANG-S4 Clade-S: Ruegeria sp. 6PALISEP08 HKCCD6109 Outgroup: HKCCD7296 HKCCD7319 Tree scale: 0.1 HKCCD7318 HKCCD5849 HKCCD7303 HKCCD5851 Ruegeria atlantica CECT 4293 Ruegeria sp. A3M17 Ruegeria faecimaris DSM 28009 Ruegeria halocynthiae MOLA R1 13b Ruegeria atlantica CECT 4292 Ruegeria sp. AU67 Ruegeria sp. Alg231-54 Ruegeria arenilitoris Ruegeria lacuscaerulensis ITI-1157 Ruegeria intermedia DSM 29341 Ruegeria sp. ANG-R Ruegeria halocynthiae DSM 27839 Rhodobacteraceae bacterium KLH11 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 HKCCD6604 HKCCD6119 Ruegeria sp. AD91A HKCCD6157 HKCCD6238 HKCCD7559 HKCCD6181 HKCCD6228 HKCCD6428 Rhodobacteraceae bacterium Alg231-04 Sedimentitalea nanhaiensis CGMCC 1.10959 Thalassococcus sp. S3 Ruegeria marina CGMCC 1.9108 Ruegeria pomeroyi DSS-3 Ruegeria marisrubri ZGT118 Rhodobacteraceae bacterium SM1703 Pseudodonghicola xiamenensis DSM 18339 Dinoroseobacter shibae DFL 12 Thalassococcus sp. SH-1 Phaeobacter sp. JL2872 Roseobacter sp. SK209-2-6 Phaeobacter sp. CECT 5382 Pseudophaeobacter arcticus DSM 23566 Phaeobacter sp. 11ANDIMAR09 Roseobacter sp. MED193 Roseobacter sp. MedPE-SWde Leisingera caerulea DSM 24564 Leisingera daeponensis DSM 23529 Rhodobacterales bacterium Y4I Leisingera sp. ANG-Vp Leisingera methylohalidivorans DSM 14336 Leisingera sp. NJS204 Leisingera aquimarina DSM 24565 Leisingera sp. ANG-S5 Leisingera sp. JC1 Leisingera sp. ANG-M7 Leisingera sp. ANG-DT Leisingera sp. ANG1 Leisingera sp. ANG-S Phaeobacter porticola Phaeobacter inhibens P88 Phaeobacter inhibens P70 Phaeobacter inhibens P24 M2-4.4 Phaeobacter inhibens S4Sm Phaeobacter inhibens P10 1 9 Phaeobacter piscinae P71 Phaeobacter piscinae P14 Phaeobacter gallaeciensis DSM 26640 Phaeobacter piscinae P13 Nautella sp. ECSMB14104 Phaeobacter italicus DSM 26436 Phaeobacter italicus CECT 7645 Ruegeria sp. R11 Ruegeria sp. TM1040 Epibacterium scottomollicae DSM 25328 Epibacterium mobile Epibacterium mobile S2144 Epibacterium mobile F2488 Epibacterium mobile 270-3 Ruegeria sp. P4 Epibacterium mobile G930 Epibacterium mobile M41-2.2 Silicibacter sp. TrichCH4B Sedimentitalea sp. W43 Roseibaca ekhonensis Tateyamaria sp. ANG-S1 Tateyamaria omphalii DOK1-4 Tropicimonas sediminicola DSM 29339 Jhaorihella thermophila DSM 23413 Aestuariivita boseongensis BS-B2 Cribrihabitans marinus DSM 29340 HKCCD4315 HKCCD4318-2 HKCCD4332 HKCCD4318 HKCCD4884 tauB (HKCCD4315_01947)

HKCCD4315 Clade-M: HKCCD6109 HKCCD4884 Clade-S: HKCCD4332 Outgroup: HKCCD4318 HKCCD4318-2 Tree scale: 0.01 Ruegeria sp. 6PALISEP08 HKCCD7296 HKCCD7319 HKCCD7318 HKCCD5849 HKCCD7303 HKCCD5851 Ruegeria faecimaris DSM 28009 Ruegeria sp. AD91A HKCCD6119 HKCCD7559 HKCCD6181 HKCCD6604 HKCCD6157 HKCCD6228 HKCCD6238 HKCCD6428 Ruegeria sp. ANG-S4 Rhodobacteraceae bacterium KLH11 Ruegeria profundi ZGT108 Ruegeria sp. ANG-R Ruegeria halocynthiae DSM 27839 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria denitrificans CECT 5091 Ruegeria atlantica CECT 4292 Ruegeria sp. AU67 Ruegeria sp. Alg231-54 Ruegeria sp. A3M17 Ruegeria atlantica CECT 4293 Ruegeria halocynthiae MOLA R1 13b Ruegeria meonggei CECT 8411 Ruegeria arenilitoris Ruegeria lacuscaerulensis ITI-1157 Ruegeria intermedia DSM 29341 Ruegeria marisrubri ZGT118 Thalassococcus sp. SH-1 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Ruegeria mediterranea M17 Ruegeria litorea R37 Rhodobacteraceae bacterium Alg231-04 Sedimentitalea sp. W43 Leisingera sp. ANG-Vp Rhodobacterales bacterium Y4I Phaeobacter italicus CECT 7645 Ruegeria sp. R11 Phaeobacter italicus DSM 26436 Nautella sp. ECSMB14104 Leisingera sp. JC1 Leisingera sp. ANG1 Leisingera sp. ANG-S Leisingera sp. ANG-S5 Leisingera sp. ANG-DT Leisingera sp. ANG-M7 Ruegeria pomeroyi DSS-3 Pseudodonghicola xiamenensis DSM 18339 Rhodobacteraceae bacterium SM1703 Cribrihabitans marinus DSM 29340 Rhodobacteraceae bacterium MA-7-27 Roseibacterium elongatum DSM 19469 Oceanicola sp. 22II-s10i Pseudooceanicola nitratireducens CGMCC 1.7292 Pseudooceanicola nitratireducens DSM 29619 Oceanicola sp. MCTG156 1a Actibacterium mucosum KCTC 23349 Nioella sp. Z7-4 Nioella sediminis JS7-11 Aestuariivita boseongensis BS-B2 Confluentimicrobium lipolyticum CECT 8621 Rhodobacteraceae bacterium BH-SD16 Thalassobium sp. R2A62 Octadecabacter ascidiaceicola tauC (HKCCD4315_01944) HKCCD6109 HKCCD7303 HKCCD5851 Clade-M: HKCCD5849 Clade-S: HKCCD7318 Outgroup: HKCCD7296 HKCCD7319 Tree scale: 0.1 Ruegeria sp. 6PALISEP08 Ruegeria sp. ANG-S4 Rhodobacteraceae bacterium KLH11 Ruegeria marisrubri ZGT118 Ruegeria lacuscaerulensis ITI-1157 Ruegeria intermedia DSM 29341 Ruegeria arenilitoris Ruegeria profundi ZGT108 Ruegeria meonggei CECT 8411 Ruegeria denitrificans CECT 5091 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria sp. ANG-R Ruegeria faecimaris DSM 28009 Ruegeria halocynthiae DSM 27839 Ruegeria sp. AD91A HKCCD6428 HKCCD6181 HKCCD6604 HKCCD6228 HKCCD6119 HKCCD6157 HKCCD6238 HKCCD7559 Ruegeria halocynthiae MOLA R1 13b Ruegeria sp. A3M17 Ruegeria atlantica CECT 4292 Ruegeria sp. AU67 Ruegeria atlantica CECT 4293 Ruegeria sp. Alg231-54 Aestuariivita boseongensis BS-B2 Cribrihabitans marinus DSM 29340 HKCCD4332 HKCCD4884 HKCCD4315 HKCCD4318 HKCCD4318-2 Figure S8. The maximum likelihood phylogeny of the core gene families coxG, coxF, coxE, coxD, coxL, coxS, coxM and coxC involved in CO oxidation constructed by IQ-TREE v1.6.5. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 80%. The scale bar indicates number substitutions per site. The members of clade-M, clade-S and outgroup clade were shadowed with blue, pink and gold, respectively. coxG (HKCCD4315_03676) HKCCD6238 HKCCD6157 HKCCD6228 HKCCD6428 HKCCD6181 HKCCD6604 Clade-M: HKCCD6109 Clade-S: HKCCD4884 HKCCD4315 Outgroup: HKCCD4332 HKCCD4318 Tree scale: 0.1 HKCCD4318-2 Shimia isoporae DSM 26433 Shimia sp. SK013 Actibacterium pelagium Litoreibacter ponti DSM 100977 Thalassobacter stenotrophicus CECT 5294 Thalassobacter stenotrophicus DSM 16310 Thalassobacter sp. 16PALIMAR09 Rhodobacteraceae bacterium Alg231-30 Thalassobius mediterraneus DSM 16398 Thalassobius mediterraneus CECT 8383 Thalassococcus sp. S3 Tateyamaria sp. ANG-S1 Ruegeria pomeroyi DSS-3 Rhodobacteraceae bacterium HIMB11 Tateyamaria sp. Alg231-49 Monaibacterium marinum C7 Tropicibacter phthalicicus DSM 26923 Loktanella sp. D2R18 Ruegeria halocynthiae MOLA R1 13b Octadecabacter temperatus DSM 26878 Octadecabacter temperatus SB1 Phaeobacter sp. CECT 7735 Ruegeria mediterranea M17 Loktanella salsilacus DSM 16199 Sulfitobacter sp. DFL-14 Litoreibacter janthinus DSM 26921 Roseobacter litoralis Och 149 Sulfitobacter sp. SK011 Sulfitobacter sp. SK012 Planktotalea arctica IMCC9565 Sulfitobacter sp. DFL-23 Sulfitobacter pseudonitzschiae SMR1 Jannaschia sp. EhC01 Ruegeria sp. AD91A Ruegeria litorea R37 Sulfitobacter sp. JL08 Ruegeria denitrificans CECT 5091 Jannaschia sp. CCS1 Yoonia maricola DSM 29128 Roseobacter sp. SK209-2-6 Planktomarina temperata RCA23 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 HKCCD7296 HKCCD7319 HKCCD7318 HKCCD5849 HKCCD7303 HKCCD5851 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Nioella sp. Z7-4 Nioella sediminis JS7-11 Ruegeria meonggei CECT 8411 Ruegeria faecimaris DSM 28009 Ruegeria profundi ZGT108 Ruegeria denitrificans CECT 50911 Ruegeria sp. A3M17 Ruegeria atlantica CECT 4293 Ruegeria sp. AU67 Ruegeria sp. Alg231-54 Ruegeria atlantica CECT 4292 Ruegeria sp. 6PALISEP08 Ruegeria sp. ANG-S4 Ruegeria arenilitoris Ruegeria sp. ANG-R Rhodobacteraceae bacterium KLH11 Shimia abyssi DSM 100673 Rhodobacteraceae bacterium G7 Marivivens sp. JLT3646 Donghicola sp. JL3646 Yoonia maritima DSM 101533 Leisingera sp. ANG-Vp Pelagimonas varians DSM 23678 Thalassobius gelatinovorus Thalassobius gelatinovorus CECT 4357 Thalassobius gelatinovorus DSM 5887 Confluentimicrobium lipolyticum CECT 8621 Phaeobacter sp. JL2872 Roseobacter sp. GAI101 Octadecabacter antarcticus 307 coxF (HKCCD4315_03678)

Clade-M: HKCCD6238 Clade-S: HKCCD6181 Outgroup: HKCCD6228 HKCCD6604 Tree scale: 0.1 HKCCD6157 HKCCD6428 HKCCD6109 HKCCD4884 HKCCD4332 HKCCD4315 HKCCD4318 HKCCD4318-2 Shimia sp. SK013 Shimia isoporae DSM 26433 Nioella sp. Z7-4 Nioella sediminis JS7-11 Rhodobacteraceae bacterium KLH11 Ruegeria sp. ANG-S4 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 HKCCD7303 HKCCD5851 HKCCD7319 HKCCD5849 HKCCD7296 HKCCD7318

coxE (HKCCD4315_03679)

HKCCD6109 Clade-M: HKCCD4315 Clade-S: HKCCD4318-2 HKCCD4318 Outgroup: HKCCD4332 Tree scale: 0.1 HKCCD4884 HKCCD6604 HKCCD6228 HKCCD6428 HKCCD6181 HKCCD6238 HKCCD6157 Shimia isoporae DSM 26433 Shimia sp. SK013 Actibacterium pelagium Loktanella sp. D2R18 Nioella sp. Z7-4 Rhodobacteraceae bacterium KLH11 Ruegeria arenilitoris Ruegeria sp. ANG-R Ruegeria sp. ANG-S4 Ruegeria sp. 6PALISEP08 Ruegeria meonggei CECT 8411 Ruegeria profundi ZGT108 Ruegeria sp. AU67 Ruegeria sp. Alg231-54 Ruegeria sp. A3M17 Ruegeria atlantica CECT 4292 Ruegeria atlantica CECT 4293 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 HKCCD7296 HKCCD7319 HKCCD7318 HKCCD5849 HKCCD7303 HKCCD5851 coxD (HKCCD4315_03680) Rhodobacteraceae bacterium KLH11 Ruegeria profundi ZGT108 Ruegeria meonggei CECT 8411 Ruegeria sp. ANG-R Clade-M: Ruegeria sp. ANG-S4 Ruegeria sp. 6PALISEP08 Clade-S: Ruegeria arenilitoris Ruegeria atlantica CECT 4292 Outgroup: Ruegeria sp. Alg231-54 Ruegeria sp. AU67 Tree scale: 0.1 Ruegeria sp. A3M17 Ruegeria atlantica CECT 4293 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 HKCCD7303 HKCCD7318 HKCCD5849 HKCCD7319 HKCCD7296 HKCCD5851 Nioella sediminis JS7-11 Nioella sp. Z7-4 Jhaorihella thermophila DSM 23413 Albidovulum xiamenense CGMCC 1.10789 Albidovulum xiamenense DSM 24422 Oceanicella actignis DSM 22673 Oceanicella actignis DSM 24423 Oceanicella actignis CGMCC 1.10808 Roseovarius tolerans DSM 11457 Roseovarius sp. A-2 Antarctobacter heliothermus SMS3 Antarctobacter heliothermus DSM 11445 Confluentimicrobium lipolyticum CECT 8621 Octadecabacter antarcticus 307 Rhodobacteraceae bacterium MA-7-27 Ruegeria marina CGMCC 1.9108 Leisingera sp. ANG-Vp Pelagimonas varians DSM 23678 Yoonia maritima DSM 101533 Roseobacter sp. MED193 Roseobacter sp. MedPE-SWde Actibacterium pelagium Ruegeria mediterranea M17 Shimia isoporae DSM 26433 Shimia sp. SK013 HKCCD6228 HKCCD6157 HKCCD6428 HKCCD6238 HKCCD6181 HKCCD6604 HKCCD4884 HKCCD6109 HKCCD4332 HKCCD4315 HKCCD4318 HKCCD4318-2 Litoreibacter ponti DSM 100977 Monaibacterium marinum C7 Phaeobacter sp. CECT 7735 Thalassobacter sp. 16PALIMAR09 Thalassobacter stenotrophicus DSM 16310 Thalassobacter stenotrophicus CECT 5294 Tropicibacter phthalicicus DSM 26923 Rhodobacteraceae bacterium Alg231-30 Rhodobacteraceae bacterium HIMB11 Octadecabacter temperatus DSM 26878 Octadecabacter temperatus SB1 Tateyamaria sp. ANG-S1 Ruegeria pomeroyi DSS-3 Thalassococcus sp. S3 Thalassobius mediterraneus DSM 16398 Thalassobius mediterraneus CECT 8383 Loktanella sp. D2R18 Planktomarina temperata RCA23 Yoonia maricola DSM 29128 Ruegeria halocynthiae MOLA R1 13b Roseobacter sp. SK209-2-6 Sulfitobacter sp. JL08 Tateyamaria sp. Alg231-49 Jannaschia sp. EhC01 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Jannaschia sp. CCS1 Ruegeria denitrificans CECT 5091 Ruegeria litorea R37 Ruegeria sp. AD91A Puniceibacterium sediminis DSM 29052 Puniceibacterium antarcticum SM1211 Sulfitobacter donghicola JCM 14565 Sulfitobacter sp. DFL-23 Sulfitobacter pseudonitzschiae SMR1 Sulfitobacter sp. SK011 Planktotalea arctica IMCC9565 Sulfitobacter sp. SK012 Litoreibacter janthinus DSM 26921 Sulfitobacter sp. DFL-14 Roseobacter litoralis Och 149 Roseovarius azorensis DSM 100674 HKCCD6157 HKCCD6228 HKCCD6428 HKCCD6604 coxL (FormI, HKCCD4315_03681) HKCCD6181 HKCCD6238 HKCCD4884 HKCCD6109 HKCCD4315 Clade-M: HKCCD4332 HKCCD4318 Clade-S: HKCCD4318-2 Shimia isoporae DSM 26433 Outgroup: Shimia sp. SK013 Ruegeria mediterranea M17 Tree scale: 0.01 Actibacterium pelagium Sedimentitalea sp. W43 Phaeobacter sp. CECT 5382 Yoonia maritima DSM 101533 Roseobacter sp. MedPE-SWde Roseobacter sp. MED193 Pelagimonas varians DSM 23678 Thalassobius gelatinovorus Thalassobius gelatinovorus CECT 4357 Thalassobius gelatinovorus DSM 5887 Leisingera sp. ANG-Vp Rhodobacteraceae bacterium G7 Marivivens sp. JLT3646 Donghicola sp. JL3646 Rhodobacteraceae bacterium MA-7-27 Roseinatronobacter thiooxidans DSM 13087 Sulfitobacter sp. DFL-14 Litoreibacter janthinus DSM 26921 Roseobacter litoralis Och 149 Sulfitobacter sp. SK011 Sulfitobacter sp. SK012 Sulfitobacter donghicola JCM 14565 Planktotalea arctica IMCC9565 Thalassobacter stenotrophicus DSM 16310 Thalassobacter stenotrophicus CECT 5294 Thalassobacter sp. 16PALIMAR09 Thalassobius mediterraneus CECT 8383 Thalassobius mediterraneus DSM 16398 Thalassococcus sp. S3 Rhodobacteraceae bacterium HIMB11 Tateyamaria sp. ANG-S1 Ruegeria pomeroyi DSS-3 Tropicibacter phthalicicus DSM 26923 Litoreibacter ponti DSM 100977 Planktomarina temperata RCA23 Tateyamaria sp. Alg231-49 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Sulfitobacter sp. JL08 Ruegeria denitrificans CECT 5091 Ruegeria sp. AD91A Ruegeria litorea R37 Jannaschia sp. CCS1 Jannaschia sp. EhC01 Phaeobacter sp. CECT 7735 Roseobacter sp. SK209-2-6 Yoonia maricola DSM 29128 Ruegeria halocynthiae MOLA R1 13b Monaibacterium marinum C7 Rhodobacteraceae bacterium Alg231-30 Loktanella sp. D2R18 Octadecabacter temperatus SB1 Octadecabacter temperatus DSM 26878 Confluentimicrobium lipolyticum CECT 8621 Phaeobacter sp. JL2872 Dinoroseobacter shibae DFL 12 Roseovarius salinarum Rhodovulum sp. ES.010 Rhodovulum euryhalinum DSM 4868 Rhodovulum steppense DSM 21153 Roseovarius mucosus DSM 17069 Roseovarius sp. 217 Roseovarius mucosus SMR3 Roseovarius sp. AK1035 Roseovarius sp. TM1035 Ruegeria marina CGMCC 1.9108 Roseovarius tolerans DSM 11457 Roseovarius sp. A-2 Shimia abyssi DSM 100673 Antarctobacter heliothermus SMS3 Antarctobacter heliothermus DSM 11445 Nioella sediminis JS7-11 Nioella sp. Z7-4 Ruegeria profundi ZGT108 Rhodobacteraceae bacterium KLH11 Ruegeria sp. 6PALISEP08 HKCCD7318 HKCCD7296 HKCCD7319 HKCCD5849 HKCCD7303 HKCCD5851 Ruegeria sp. ANG-S4 Ruegeria arenilitoris Ruegeria meonggei CECT 8411 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria sp. ANG-R Ruegeria atlantica CECT 4292 Ruegeria atlantica CECT 4293 Ruegeria sp. AU67 Ruegeria sp. Alg231-54 Ruegeria sp. A3M17 Albidovulum xiamenense CGMCC 1.10789 Albidovulum xiamenense DSM 24422 Oceanicella actignis DSM 22673 Oceanicella actignis CGMCC 1.10808 Oceanicella actignis DSM 24423 HKCCD6228 HKCCD6238 coxS (HKCCD4315_03682) HKCCD6157 HKCCD6428 HKCCD4884 HKCCD6109 HKCCD4332 HKCCD4318-2 HKCCD4318 Clade-M: HKCCD6604 HKCCD6181 Clade-S: HKCCD4315 Actibacterium pelagium Outgroup: Shimia sp. SK013 Shimia isoporae DSM 26433 Tree scale: 0.1 Ruegeria mediterranea M17 Sulfitobacter sp. DFL-23 Sulfitobacter pseudonitzschiae SMR1 Planktotalea arctica IMCC9565 Sulfitobacter donghicola JCM 14565 Litoreibacter janthinus DSM 26921 Marivivens sp. JLT3646 Rhodobacteraceae bacterium G7 Ruegeria denitrificans CECT 5091 Thalassobacter sp. 16PALIMAR09 Thalassobacter stenotrophicus DSM 16310 Thalassobacter stenotrophicus CECT 5294 Tropicibacter naphthalenivorans DSM 19561 Tropicibacter naphthalenivorans CECT 7648 HKCCD5851 HKCCD7303 HKCCD5849 HKCCD7318 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 HKCCD7296 HKCCD7319 Ruegeria arenilitoris Ruegeria sp. 6PALISEP08 Ruegeria sp. ANG-S4 Ruegeria sp. ANG-R Ruegeria meonggei CECT 8411 Ruegeria atlantica CECT 4292 Ruegeria sp. AU67 Ruegeria sp. A3M17 Ruegeria atlantica CECT 4293 Ruegeria sp. Alg231-54 Rhodobacteraceae bacterium KLH11 Ruegeria profundi ZGT108 Shimia abyssi DSM 100673 Nioella sp. Z7-4 Nioella sediminis JS7-11 Roseovarius sp. A-2 Antarctobacter heliothermus SMS3 Antarctobacter heliothermus DSM 11445 Oceanicella actignis DSM 22673 Albidovulum xiamenense DSM 24422 Albidovulum xiamenense CGMCC 1.10789

coxC (HKCCD4315_03684)

Actibacterium pelagium Ruegeria mediterranea M17 Clade-M: Shimia sp. SK013 Shimia isoporae DSM 26433 Clade-S: HKCCD4315 Outgroup: HKCCD4318-2 HKCCD4332 Tree scale: 0.1 HKCCD4318 HKCCD4884 HKCCD6109 HKCCD6428 HKCCD6157 HKCCD6181 HKCCD6228 HKCCD6238 HKCCD6604 Nioella sp. Z7-4 Nioella sediminis JS7-11 Roseovarius tolerans DSM 11457 Ruegeria sp. ANG-S4 Ruegeria arenilitoris Ruegeria profundi ZGT108 Ruegeria meonggei CECT 8411 Ruegeria sp. Alg231-54 Ruegeria sp. A3M17 Ruegeria atlantica CECT 4293 Ruegeria sp. AU67 Ruegeria atlantica CECT 4292 Ruegeria sp. 6PALISEP08 Ruegeria sp. ANG-R Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Rhodobacteraceae bacterium KLH11 HKCCD7318 HKCCD7303 HKCCD5849 HKCCD5851 HKCCD7296 HKCCD7319 coxM (HKCCD4315_03683) HKCCD6428 HKCCD6157 HKCCD6604 HKCCD6181 HKCCD6238 Clade-M: HKCCD6228 HKCCD6109 Clade-S: HKCCD4884 Outgroup: HKCCD4315 HKCCD4318-2 Tree scale: 0.1 HKCCD4318 HKCCD4332 Actibacterium pelagium Ruegeria mediterranea M17 Shimia isoporae DSM 26433 Shimia sp. SK013 Rhodobacteraceae bacterium HIMB11 Thalassobius mediterraneus DSM 16398 Thalassobius mediterraneus CECT 8383 Tateyamaria sp. ANG-S1 Rhodobacteraceae bacterium Alg231-30 Thalassobacter sp. 16PALIMAR09 Thalassobacter stenotrophicus DSM 16310 Thalassobacter stenotrophicus CECT 5294 Octadecabacter temperatus DSM 26878 Octadecabacter temperatus SB1 Phaeobacter sp. CECT 7735 Monaibacterium marinum C7 Ruegeria pomeroyi DSS-3 Thalassococcus sp. S3 Ruegeria halocynthiae MOLA R1 13b Roseobacter sp. SK209-2-6 Loktanella sp. D2R18 Tateyamaria sp. Alg231-49 Jannaschia sp. EhC01 Litoreibacter ponti DSM 100977 Planktomarina temperata RCA23 Jannaschia sp. CCS1 Yoonia maricola DSM 29128 Sulfitobacter sp. JL08 Rhodobacteraceae bacterium EL53 Ruegeria denitrificans CECT 5091 Ruegeria sp. AD91A Ruegeria litorea R37 Rhodobacteraceae bacterium G7 Donghicola sp. JL3646 Marivivens sp. JLT3646 Planktotalea arctica IMCC9565 Sulfitobacter pseudonitzschiae SMR1 Sulfitobacter sp. DFL-23 Sulfitobacter sp. DFL-14 Loktanella salsilacus DSM 16199 Litoreibacter janthinus DSM 26921 Roseobacter litoralis Och 149 Ruegeria marina CGMCC 1.9108 Leisingera sp. ANG-Vp Confluentimicrobium lipolyticum CECT 8621 HKCCD7303 HKCCD5849 HKCCD5851 HKCCD7318 HKCCD7296 HKCCD7319 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria sp. ANG-S4 Ruegeria sp. ANG-R Ruegeria meonggei CECT 8411 Ruegeria arenilitoris Ruegeria profundi ZGT108 Ruegeria sp. A3M17 Ruegeria sp. Alg231-54 Ruegeria atlantica CECT 4292 Ruegeria atlantica CECT 4293 Ruegeria sp. AU67 Ruegeria sp. 6PALISEP08 Rhodobacteraceae bacterium KLH11 Nioella sp. Z7-4 Nioella sediminis JS7-11 Albidovulum xiamenense DSM 24422 Albidovulum xiamenense CGMCC 1.10789 Roseovarius sp. A-2 Roseovarius tolerans DSM 11457 Antarctobacter heliothermus DSM 11445 Antarctobacter heliothermus SMS3 Figure S9. The maximum likelihood phylogeny of the core gene families dmsA, dmsB and dmsC involved in anaerobic respiration constructed by IQ-TREE v1.6.5. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 80%. The scale bar indicates number of substitutions per site. The members of clade-M, clade-S and outgroup clade were shadowed with blue, pink and gold, respectively. dmsA (HKCCD4315_03090)

Leisingera sp. ANG-DT Leisingera sp. ANG-S5 Clade-M: Leisingera sp. ANG1 Leisingera sp. ANG-S Clade-S: Leisingera sp. ANG-Vp Outgroup: Leisingera sp. ANG-M7 Leisingera sp. JC1 Tree scale: 0.1 Leisingera methylohalidivorans MB2 Leisingera methylohalidivorans DSM 14336 Leisingera aquimarina DSM 24565 Leisingera sp. NJS204 Ruegeria pomeroyi DSS-3 Roseobacter sp. SK209-2-6 Phaeobacter sp. CECT 5382 Roseobacter sp. MedPE-SWde Phaeobacter sp. 11ANDIMAR09 Phaeobacter inhibens P24/M2-4.4 Phaeobacter inhibens P70 Phaeobacter inhibens P88 Phaeobacter inhibens S4Sm Phaeobacter porticola Phaeobacter inhibens P10/01/009 Phaeobacter piscinae P71 Phaeobacter piscinae P14 Phaeobacter piscinae P13 Ruegeria litorea R37 Ruegeria mediterranea M17 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Ruegeria arenilitoris Ruegeria lacuscaerulensis DSM 11314 Ruegeria lacuscaerulensis ITI-1157 Ruegeria sp. ANG-R Ruegeria halocynthiae DSM 27839 Ruegeria conchae Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria atlantica CECT 4293 Ruegeria atlantica CECT 4292 Ruegeria atlantica Ruegeria sp. Alg231-54 Ruegeria sp. A3M17 Ruegeria sp. AU67 Ruegeria sp. 6PALISEP08 HKCCD7296 HKCCD7319 HKCCD7303 HKCCD5849 HKCCD7318 HKCCD5851 Phaeobacter sp. JL2872 Sedimentitalea sp. W43 Leisingera caerulea DSM 24564 Leisingera daeponensis DSM 23529 Rhodobacterales bacterium Y4I Ruegeria marisrubri ZGT118 Cribrihabitans marinus DSM 29340 Ruegeria intermedia DSM 29341 Ruegeria profundi ZGT108 Ruegeria sp. ANG-S4 Ruegeria denitrificans CECT 5091 Ruegeria denitrificans HKCCD6157 HKCCD6119 Ruegeria sp. AD91A HKCCD7559 HKCCD6238 HKCCD6228 HKCCD6181 HKCCD6604 HKCCD6428 Rhodobacteraceae bacterium KLH11 Ruegeria faecimaris DSM 28009 Ruegeria meonggei CECT 8411 Ruegeria halocynthiae Ruegeria halocynthiae MOLA R1 13b HKCCD6109 HKCCD4884 HKCCD4332 HKCCD4315 HKCCD4318 HKCCD4318-2 dmsB (HKCCD4315_03091)

Leisingera caerulea DSM 24564 Leisingera daeponensis DSM 23529 Clade-M: Rhodobacterales bacterium Y4I Leisingera sp. JC1 Clade-S: Leisingera sp. ANG-S5 Outgroup: Leisingera sp. ANG-DT Leisingera sp. ANG1 Tree scale: 0.1 Leisingera sp. ANG-S Leisingera sp. ANG-Vp Leisingera sp. ANG-M7 Leisingera aquimarina DSM 24565 Leisingera sp. NJS204 Leisingera methylohalidivorans DSM 14336 HKCCD6109 HKCCD4884 HKCCD4315 HKCCD4332 HKCCD4318 HKCCD4318-2 Ruegeria sp. ANG-S4 Phaeobacter sp. JL2872 Phaeobacter sp. CECT 5382 Roseobacter sp. SK209-2-6 Pseudophaeobacter arcticus DSM 23566 Phaeobacter sp. 11ANDIMAR09 Roseobacter sp. MED193 Roseobacter sp. MedPE-SWde Phaeobacter porticola Phaeobacter inhibens P10 1 9 Phaeobacter piscinae P13 Phaeobacter piscinae P71 Phaeobacter piscinae P14 Nautella sp. ECSMB14104 Phaeobacter italicus CECT 7645 Ruegeria sp. R11 Phaeobacter italicus DSM 26436 Phaeobacter gallaeciensis DSM 26640 Phaeobacter inhibens S4Sm Phaeobacter inhibens P88 Phaeobacter inhibens P24 M2-4.4 Phaeobacter inhibens P70 Sedimentitalea sp. W43 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Rhodobacteraceae bacterium Alg231-04 Ruegeria marina CGMCC 1.9108 Ruegeria pomeroyi DSS-3 Ruegeria sp. 6PALISEP08 HKCCD7318 HKCCD7296 HKCCD7319 HKCCD5849 HKCCD7303 HKCCD5851 Ruegeria sp. AD91A HKCCD6228 HKCCD6238 HKCCD6604 HKCCD6157 HKCCD6428 HKCCD6181 HKCCD6119 HKCCD7559 Ruegeria arenilitoris Ruegeria lacuscaerulensis ITI-1157 Ruegeria intermedia DSM 29341 Ruegeria atlantica CECT 4293 Ruegeria sp. A3M17 Ruegeria atlantica CECT 4292 Ruegeria sp. AU67 Ruegeria sp. Alg231-54 Rhodobacteraceae bacterium KLH11 Ruegeria halocynthiae DSM 27839 Ruegeria sp. ANG-R Ruegeria denitrificans CECT 5091 Ruegeria profundi ZGT108 Ruegeria halocynthiae MOLA R1 13b Ruegeria faecimaris DSM 28009 Ruegeria meonggei CECT 8411 Ruegeria mediterranea M17 Ruegeria litorea R37 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Ruegeria marisrubri ZGT118 Roseovarius albus CECT 7450 Sedimentitalea nanhaiensis CGMCC 1.10959 Roseobacter litoralis Och 149 Roseobacter denitrificans OCh 114 Roseobacter sp. SK209-2-6 Leisingera aquimarina DSM 24565 Rhodobacteraceae bacterium Alg231-04 Leisingera sp. NJS204 dmsC (HKCCD4315_03092) Leisingera methylohalidivorans MB2 Leisingera methylohalidivorans DSM 14336 Leisingera caerulea DSM 24564 Leisingera daeponensis DSM 23529 Rhodobacterales bacterium Y4I Leisingera sp. JC1 Leisingera sp. ANG-Vp Clade-M: Leisingera sp. ANG-M7 Leisingera sp. ANG-DT Clade-S: Leisingera sp. ANG-S5 Outgroup: Leisingera sp. ANG1 Leisingera sp. ANG-S Phaeobacter italicus CECT 7645 Tree scale: 0.1 Phaeobacter italicus DSM 26436 Nautella sp. ECSMB14104 Phaeobacter inhibens S4Sm Phaeobacter piscinae P13 Phaeobacter sp. JL2872 Sedimentitalea sp. W43 Phaeobacter sp. CECT 5382 Pseudophaeobacter arcticus DSM 23566 Phaeobacter sp. 11ANDIMAR09 Roseobacter sp. MED193 Roseobacter sp. MedPE-SWde Phaeobacter gallaeciensis DSM 26640 Phaeobacter piscinae P71 Phaeobacter piscinae P14 Phaeobacter inhibens P10/01/009 Phaeobacter porticola Phaeobacter inhibens P70 Phaeobacter inhibens P88 Phaeobacter inhibens P24/M2-4.4 Ruegeria marisrubri ZGT118 Ruegeria litorea R37 Ruegeria mediterranea M17 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 HKCCD7303 HKCCD5849 HKCCD7319 HKCCD5851 HKCCD7296 HKCCD7318 Ruegeria sp. 6PALISEP08 HKCCD4884 HKCCD4315 HKCCD4332 HKCCD6109 HKCCD4318 HKCCD4318-2 Ruegeria sp. AD91A HKCCD6228 HKCCD6604 HKCCD6238 HKCCD6428 HKCCD6119 HKCCD7559 HKCCD6181 HKCCD6157 Ruegeria atlantica CECT 4292 Ruegeria atlantica Ruegeria sp. AU67 Ruegeria atlantica CECT 4293 Ruegeria sp. Alg231-54 Ruegeria sp. A3M17 Rhodobacteraceae bacterium KLH11 Ruegeria conchae TW15 Ruegeria conchae DSM 29317 Ruegeria conchae Ruegeria halocynthiae DSM 27839 Ruegeria halocynthiae Ruegeria halocynthiae MOLA R1 13b Ruegeria sp. ANG-R Ruegeria meonggei CECT 8411 Ruegeria denitrificans CECT 5091 Ruegeria denitrificans Ruegeria faecimaris DSM 28009 Ruegeria sp. ANG-S4 Ruegeria profundi ZGT108 Ruegeria marina CGMCC 1.9108 Ruegeria pomeroyi DSS-3 Ruegeria arenilitoris Ruegeria intermedia DSM 29341 Ruegeria lacuscaerulensis DSM 11314 Ruegeria lacuscaerulensis ITI-1157 Cribrihabitans marinus DSM 29340 Roseovarius litoreus DSM 28249 Roseovarius pacificus DSM 29589 Roseovarius halotolerans DSM 29507 Roseovarius halotolerans Rhodobacteraceae bacterium EL129 Pelagicola litorisediminis CECT 8287 Roseovarius atlanticus R12b Roseovarius nitratireducens Roseovarius sp. A46 Antarctobacter heliothermus DSM 11445 Antarctobacter heliothermus SMS3 Thalassococcus sp. S3 Sulfitobacter sp. JL08 Litoreibacter arenae DSM 19593 Litoreibacter meonggei DSM 29466 Litoreibacter halocynthiae DSM 29467 Sulfitobacter sp. SK012 Sulfitobacter donghicola KCTC 12864 Sulfitobacter donghicola JCM 14565 Sulfitobacter donghicola DSW-25 Sulfitobacter sp. DFL-14 Sulfitobacter sp. EhC04 Sulfitobacter mediterraneus Sulfitobacter mediterraneus KCTC 32188 Sulfitobacter mediterraneus DSM 12244 Sulfitobacter sp. SK011 Sulfitobacter noctilucicola NB-77 Sulfitobacter noctilucae NB-68 Tateyamaria omphalii DOK1-4 Nioella sp. Z7-4 Nioella sediminis JS7-11 Figure S10. The maximum likelihood phylogeny of the core gene families ureA, ureB, ureC, ureD, ureE and ureF involved in urea utilization constructed by IQ-TREE v1.6.5. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 80%. The scale bar indicates number of substitutions per site. The members of clade-M, clade-S and outgroup clade were shadowed with blue, pink and gold, respectively. ureA (HKCCD4315_03756)

HKCCD4315 HKCCD4318 Clade-M: HKCCD4318-2 Clade-S: Ruegeria sp. 6PALISEP08 Ruegeria sp. Alg231-54 Outgroup: Ruegeria sp. AU67 Ruegeria atlantica CECT 4292 Tree scale: 0.01 HKCCD6604 Ruegeria atlantica CECT 4293 Cognatishimia maritima DSM 28223 Ruegeria sp. AD91A HKCCD7296 HKCCD7319 HKCCD6238 HKCCD6157 HKCCD7318 HKCCD6228 HKCCD7303 HKCCD5851 HKCCD6428 HKCCD6181 HKCCD5849 HKCCD4884 HKCCD6109 Ruegeria conchae TW15 Ruegeria conchae DSM 29317 Ruegeria halocynthiae MOLA R1 13b Maribius pelagius DSM 26893 Maribius salinus DSM 26892 Ruegeria meonggei CECT 8411 Ruegeria profundi ZGT108 Ruegeria sp. ANG-S4 Aestuariivita boseongensis BS-B2 Maritimibacter sp. UBA3975 Maritimibacter alkaliphilus HTCC2654 Ruegeria sp. TM1040 Epibacterium scottomollicae DSM 25328 Epibacterium mobile M41-2.2 Ruegeria sp. P4 Silicibacter sp. TrichCH4B Epibacterium mobile S2144 Epibacterium mobile F2488 Epibacterium mobile G930 Epibacterium mobile 270-3 Epibacterium mobile Oceanicola sp. HL-35 HKCCD4332 Rhodovulum sp. P5 Mameliella alba UMTAT08 Mameliella alba JL351 Mameliella alba L6M1-5 Ruegeria sp. PBVC088 Mameliella alba F15 Mameliella alba KD53 Mameliella alba DSM 26384 Mameliella alba CGMCC 1.729 Shimia sp. SK013 Actibacterium pelagium Pseudooctadecabacter jejudonensis Tateyamaria omphalii DOK1-4 Tateyamaria sp. ANG-S1 Roseobacter denitrificans OCh 114 Pelagicola litorisediminis CECT 8287 Roseovarius gaetbuli CECT 8370 Rhodovulum marinum DSM 18063 Marinovum algicola Sulfitobacter noctilucae NB-68 Nioella sediminis JS7-11 Nioella sp. Z7-4 Lutimaribacter saemankumensis DSM 28010 Rhodovulum steppense DSM 21153 Rhodovulum visakhapatnamense DSM 17937 Rhodovulum sp. BSW8 ureB (HKCCD4315_03755)

Clade-M: HKCCD4332 Clade-S: HKCCD4315 Outgroup: HKCCD6109 HKCCD4884 Tree scale: 0.1 HKCCD4318 HKCCD4318-2 HKCCD7303 HKCCD7318 HKCCD7319 HKCCD5851 HKCCD7296 HKCCD5849 Ruegeria halocynthiae MOLA R1 13b Ruegeria sp. ANG-S4 Ruegeria meonggei CECT 8411 Ruegeria profundi ZGT108 HKCCD6157 HKCCD6228 HKCCD6238 HKCCD6604 HKCCD6181 HKCCD6428 Ruegeria sp. AU67 Ruegeria atlantica CECT 4292 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria sp. Alg231-54 Ruegeria atlantica CECT 4293 Ruegeria sp. 6PALISEP08

ureC (HKCCD4315_03752) HKCCD7296 HKCCD7319 HKCCD7318 Clade-M: HKCCD5849 HKCCD7303 Clade-S: HKCCD5851 HKCCD4884 Outgroup: HKCCD6109 HKCCD4332 Tree scale: 0.01 HKCCD4315 HKCCD4318 HKCCD4318-2 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria atlantica CECT 4293 Ruegeria sp. AD91A Ruegeria sp. 6PALISEP08 Ruegeria sp. AU67 Ruegeria atlantica CECT 4292 Ruegeria sp. Alg231-54 HKCCD6181 HKCCD6157 HKCCD6238 HKCCD6428 HKCCD6604 HKCCD6228 Ruegeria halocynthiae MOLA R1 13b Ruegeria sp. ANG-S4 Ruegeria profundi ZGT108 Ruegeria meonggei CECT 8411 Roseovarius lutimaris DSM 28463 Sulfitobacter noctilucae NB-68 Sulfitobacter noctilucicola NB-77 Shimia sagamensis DSM 29734 Ruegeria mediterranea M17 Phaeobacter sp. CECT 7735 Tropicibacter phthalicicus DSM 26923 Sulfitobacter donghicola JCM 14565 ureD (HKCCD4315_03757)

HKCCD7296 Clade-M: HKCCD7319 HKCCD7318 Clade-S: HKCCD5849 Outgroup: HKCCD7303 Tree scale: 0.01 HKCCD5851 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 Ruegeria sp. Alg231-54 Ruegeria sp. AD91A Ruegeria sp. 6PALISEP08 Ruegeria sp. AU67 Ruegeria atlantica CECT 4293 Ruegeria atlantica CECT 4292 HKCCD6604 HKCCD6428 HKCCD6228 HKCCD6157 HKCCD6181 HKCCD6238 Ruegeria meonggei CECT 8411 Ruegeria halocynthiae MOLA R1 13b Ruegeria profundi ZGT108 Ruegeria sp. ANG-S4 HKCCD4332 HKCCD6109 HKCCD4884 HKCCD4315 HKCCD4318 HKCCD4318-2 Roseovarius gaetbuli CECT 8370 Primorskyibacter sedentarius DSM 104836 Shimia sagamensis DSM 29734 Sulfitobacter mediterraneus KCTC 32188

ureE (HKCCD4315_03750)

HKCCD7303 Clade-M: HKCCD7318 Clade-S: HKCCD7319 HKCCD5849 Outgroup: HKCCD7296 HKCCD5851 Tree scale: 0.01 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 HKCCD4315 HKCCD4318 HKCCD4318-2 HKCCD4884 HKCCD4332 HKCCD6109 HKCCD6181 HKCCD6604 HKCCD6238 HKCCD6157 HKCCD6228 HKCCD6428 Ruegeria sp. 6PALISEP08 Ruegeria sp. AD91A Ruegeria sp. Alg231-54 Ruegeria atlantica CECT 4293 Ruegeria atlantica CECT 4292 Ruegeria sp. AU67 Ruegeria meonggei CECT 8411 Ruegeria sp. ANG-S4 ureF (HKCCD4315_03749)

Clade-M: HKCCD4884 Clade-S: HKCCD4332 Outgroup: HKCCD6109 HKCCD4315 Tree scale: 0.01 HKCCD4318 HKCCD4318-2 Ruegeria conchae DSM 29317 Ruegeria conchae TW15 HKCCD6428 HKCCD6238 HKCCD6157 HKCCD6181 HKCCD6228 Ruegeria sp. Alg231-54 Ruegeria sp. AD91A Ruegeria sp. 6PALISEP08 Ruegeria sp. AU67 Ruegeria atlantica CECT 4293 Ruegeria atlantica CECT 4292 HKCCD6604 HKCCD7296 HKCCD7319 HKCCD7318 HKCCD5851 HKCCD7303 HKCCD5849 Ruegeria sp. ANG-S4 Ruegeria profundi ZGT108 Ruegeria halocynthiae MOLA R1 13b Ruegeria meonggei CECT 8411 Ruegeria pomeroyi DSS-3 Primorskyibacter sedentarius DSM 104836 Phaeobacter gallaeciensis Rhodobacteraceae bacterium EL53 Shimia sagamensis DSM 29734 Ruegeria mediterranea M17 Ruegeria litorea R37 Vertical transmission: Zygotes LCA of clade-M and clade-S Clade-S strains Planulea Clade-M strains Eggs Horizontal transmission: Sperms Clade-S strains Clade-M strains Settlement Seawater Adults

Mucus

Tissue

Skeleton Metamorphism (Single polyp)

Calcification (Colony) Figure S11. The life cycle of Platygyra acuta showing the origin of mucus and skeleton associated bacteria. The grey arrows indicate the vertical transmission of bacteria. The cyan arrows indicate the horizontal transmission of bacteria. Coral, mucus: Coral, skeleton: Coral, tissue:

H Seawater: HKCCA5983 H HKCCA5693 HKCCC1522 HKCCC1591 HKCCA551

HKCCA5505 6 HKCCD8504

K HKCCD7298 K

HKCCA5270 1 HKCCD8795 HKCCD7251

7

C HKCCD7239 C HKCCA5491 Mangrove, rhizosphere: 4 HKCCA4707

C HKCCD7221 C

HKCCA5426 A

A HKCCD6964 A

HKCCA6666 C

5 Macroalgae, tissue: HKCCA6670 5 C

4 HKCCC2121 4 HKCCA6631

5 8

HKCCA6521 K

4 HKCCD5879 9 1

H HKCCD5912 HKCCA5751 HKCCA6346 HKCCA5777 HKCCC2149 HKCCA6172 HKCCD9147 Sediments: HKCCA5471 HKCCD6438 HKCCA5471-2 HKCCD7225 HKCCA0712 HKCCC1264 HKCCA5588 HKCCD7206-1 18 HKCCC1251 HKCCD7206-2 157 HKCCA5690 HKCCD7232 HKCCD7218 HKCCA4830 HKCCD7223 HKCCD8719 oyi DSS-3 Tree scale: 0.1 HKCCD6438-2HKCCD7266 HKCCD7583 Clade-2 HKCCD9208 HKCCD9151 Ruegeria pr HKCCD9148 rulensis ITI-1 HKCCD6430 Clade-1 HKCCD6128 HKCCD6218 Ruegeria pomer HKCCA5280 Ruegeria marina ofunHKCCD9103d Cribrihabitans marinus HKCCD8929 Ruegeria marisrubri ZGT1 HKCCD9i ZGT1081 Ruegeria intermedia HKCCD9150 Ruegeria lacuscae HKCCA5460 HKCCA4775 HKCCD9162 HKCCA5725 HKCCD8343 HKCCD8206 1 HKCCA5403 enilitoris HKCCA0214 1 HKCCA4685 HKCCD9204 HKCCA5708 Ruegeria denitrificans CECTHKCCA0238 HKCCD9190 HKCCA0501 HKCCD8755 Ruegeria ar HKCCD6168 HKCCD6232 HKCCA4692 HKCCD6120 Ruegeria halocynthiae MOLA R1/13bHKCCD7052 HKCCA5932 Ruegeria meonggei HKCCA5915 Ruegeria sp. EL01 HKCCA6437 Ruegeria faecimaris HKCCA6764 5091 HKCCD8500 HKCCA0519 Ruegeria atlantica CECT HKCCD6425 HKCCD5857 HKCCD6598 HKCCD5876 HKCCD6598-2 Ruegeria sp. HKCCA5930 Ruegeria sp. HKCCA0037 Ruegeria sp. 4293 HKCCA0154 AU67 HKCCA0510 A3M17 Alg231-54 HKCCA0515 HKCCD6212 HKCCA5546 HKCCA5752 HKCCA5369 HKCCA4777 HKCCA5275 HKCCA0240 HKCCA6269 HKCCA5923 HKCCE4148 HKCCD9083165 HKCCE4150 HKCCC1 HKCCA4633 HKCCD7731182 HKCCD7318 HKCCC1 HKCCD5849 HKCCC1588 HKCCD5851 HKCCA4909 HKCCD7303 HKCCA5416 HKCCC1266 HKCCD7319 HKCCC1208 HKCCD7296 HKCCA4852 HKCCA5763 HKCCD7214 HKCCA6837 HKCCA6043 HKCCD4318 HKCCA5649 HKCCD4318-2 HKCCA4756 HKCCD4315 HKCCD6567 HKCCD4332 HKCCC1212 17 HKCCA5019 HKCCC21 HKCCA6041 HKCCD4884 HKCCA6064 HKCCD610911 HKCCD7021 HKCCC2112 HKCCD7021-2 HKCCC21 HKCCC1285 HKCCD9179 HKCCC1 HKCCA5929P08 HKCCA5755157 ALISE HKCCD7041 HKCCA5663 Ruegeria sp. 6HKCCA5463P HKCCA4796 HKCCD6228 HKCCD7235 HKCCA0235A HKCCD7099 HKCCD6181 HKCCA5738 HKCCD6238 HKCCA6553 HKCCD6428 HKCCD9169 HKCCD4060 HKCCD6604 HKCCD4065 HKCCA0370 HKCCD4061 HKCCA4008 HKCCD8929-2 HKCCA4812 HKCCD6397 HKCCD6157 HKCCA5371 HKCCSP346 HKCCA5913 19 HKCCA4698 HKCCE3926 HKCCD7176 HKCCD7559 HKCCA5443 HKCCD61 HKCCA4734 HKCCD9122AD91A HKCCD6457 HKCCD8344 HKCCC1088 HKCCD7299 HKCCC2136 HKCCSA071 HKCCA0806 Ruegeria sp.HKCCSP335 Ruegeria sp. HKCCSP351 HKCCC1 HKCCA5689 HKCCA5739 HKCCA5681 HKCCC1288 10 HKCCA6308 HKCCA4945 HKCCD9203-2HKCCA0310 HKCCC1250 153 ANG-S4 HKCCD9198 HKCCA6259 HKCCA5687 HKCCA6158 HKCCA6948 HKCCD91 HKCCA5839 HKCCA5916 HKCCD7255 HKCCA5014 HKCCA4748 HKCCC1038 HKCCA5917 HKCCA6707 HKCCA4742 HKCCD7171 Rhodobacteraceae bacterium KLH1 HKCCD6401 Ruegeria halocynthiae HKCCD7314 Ruegeria sp. HKCCA0302-2 HKCCD9079 HKCCA0302 HKCCA4717 HKCCA0466 HKCCA0468 HKCCA4709 Ruegeria conchae DSM 29317 HKCCD7301 HKCCA4710 HKCCD7217 HKCCD8715 HKCCA5794 HKCCA6783 HKCCA4785 HKCCC1043 HKCCC2131 HKCCA0503 1 HKCCC2122 HKCCD9012 HKCCD6461 HKCCD8727 HKCCD9105 HKCCD4580 ANG-R HKCCC1047 HKCCD4579 HKCCA5680 HKCCA4776 HKCCA5816 HKCCD7109 HKCCD7172 HKCCD6234 HKCCA1383-2 HKCCD61 HKCCD61 HKCCD6210 HKCCA4749 H

HKCCA0208 8 HKCCD9203 K HKCCA0065 5

1 HKCCA6604 C

HKCCA6601 0 HKCCD6421 C

HKCCD4871 A

HKCCD481 A HKCCA6196 HKCCA0209 C HKCCA6533 0

HKCCD7291 C HKCCD6459 0

15 HKCCA0219 16 HKCCD6291 4

HKCCA5940 K HKCCD4449 HKCCD6593 4 HKCCA4608 HKCCA4609 HKCCC1226 HKCCA4824 HKCCD7560 HKCCD6251 H 1

Clade-1: Tree scale 0.05: Clade-2：Tree scale 0.05:

Parasedimentitalea marina W43 Rhodobacterales bacterium 56 14 T64 Ruegeria sp. 318-1 Rhodobacteraceae bacterium Alg231-04 Sulfitobacter sp. S4J41 Phaeobacter gallaeciensis C3M10 Pseudodonghicola xiamenensis DSM 18339 Rhodobacteraceae bacterium EL53 Thalassococcus sp. SH-1 Ruegeria mediterranea M17 Jhaorihella thermophila Ruegeria litorea R37 Rhodobacteraceae bacterium CPC19 Pseudophaeobacter arcticus DSM 23566 Marinibacterium profundimaris 22II1-22F33 Pseudophaeobacter sp. EL27 Chachezhania antarctica SM1703 Phaeobacter sp. 11ANDIMAR09 Rhodobacteraceae bacterium Glo 4 Roseobacter sp. MedPE-SWde Sedimentitalea nanhaiensis DSM 24252 Roseobacter sp. MED193 Phaeobacter marinintestinus Pseudophaeobacter leonis 306 Pseudooceanicola lipolyticus 157 Phaeobacter sp. CECT 5382 Phaeobacter sp. 22II1-1F12B Roseobacter sp. SK209-2-6 Ruegeria kandeliae J95 Phaeobacter sp. JL2872 Pseudooceanicola sp. SAT54 Phaeobacter inhibens DSM 16374 Phaeobacter gallaeciensis DSM 26640 Phaeobacter sp. S60 Phaeobacter piscinae P42 Phaeobacter sp. LSS9 Ruegeria sp. R11 Phaeobacter italicus CECT 7645 Nautella sp. ECSMB14104 Leisingera sp. JC1 Epibacterium ulvae U95 Epibacterium multivorans CECT 7557 Tritonibacter horizontis O3.65 Phaeobacter sp. O365 Epibacterium scottomollicae DSM 25328 Ruegeria sp. TM1040 Ruegeria sp. P4

Figure S12. The maximum likelihood phylogeny of 454 Ruegeria and related strains. Solid circles at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 95%. The scale bar indicates number of substitutions per site. The habitats of the expanded Ruegeria population were shadowed with different colours. H

HKCCD HKCCD HKCCD

3

HKCCD K HKCCD

9 HKCCD C

HKCCD 1

C

HKCCD 8 8431 8914 8291 HKCCD 8831

D

D HKCCD 8264 8284

5 HKCCD C 7121 7924 9

5916 HKCCD 7886 C 8659 8309

1 8269 HKCCD 91 K 8198 6 8414 8515

7327 H HKCCD - 17-2 8484 2 6301

HKCCD HKCCD 9021 HKCCD HKCCD HKCCD 7389 Seawater 6543 HKCCD HKCCD HKCCD 7357 9042 HKCCD HKCCD 7906 HKCCD 9066 HKCCD 9028 8435 HKCCD HKCCD 5930 HKCCD HKCCD 9026 HKCCD 8417 Mucus HKCCD 8773 HKCCD 8654 HKCCD 5856 HKCCD 8721 HKCCD 8629 HKCCD 8469 HKCCD 7415 HKCCD 8088 Tissues HKCCD HKCCD 6502 9024 HKCCD HKCCD 8752 1 HKCCD 7086 9030 HKCCD HKCCD 5934 7120- Skeleton HKCCD HKCCD 8268 8272 HKCCD HKCCD 7103 8504- 8214 HKCCD HKCCD 9054 HKCCD 2 9020 HKCCD 6571 HKCCD Wong Wan Chau 9065 HKCCD 9152 HKCCD 8190 HKCCD 7959 HKCCD 8304 HKCCD 8926 HKCCD Ngo Mei Chau HKCCD 7100 HKCCD 7580 8134 HKCCD 9052 HKCCD HKCCD Chek Chau 6135 6497 HKCCD HKCCD 8877 8432 HKCCD HKCCD 7577 4356 HKCCD Kiu Tsui Chau 8628 7387 HKCCD 1 841 HKCCD Tree scale: 0.001 HKCCD 8507 HKCCD 9072 HKCCD 9199 HKCCD 7397 HKCCD HKCCD 8979 HKCCD HKCCD 8199 9055 HKCCD HKCCD 7538 9043 8882 HKCCD HKCCD 8381 HKCCD 8861 9056 HKCCD HKCCD 8267 8769 HKCCD HKCCD 7635 7390 HKCCD HKCCD 8749 9027 HKCCD HKCCD9180 HKCCD9063 HKCCD9159 HKCCD7378 HKCCD8733 HKCCD8499 HKCCD8626 HKCCD6540 HKCCD8710 HKCCD8809 HKCCD8657 HKCCD8482 HKCCD8430 HKCCD5941 HKCCD9144 HKCCD8812 HKCCD8195 HKCCD 5941-2 9050 HKCCD HKCCD 8495 8700 HKCCD HKCCD 7543 6578 HKCCD HKCCD 8801 6491 HKCCD HKCCD 8473 9036 HKCCD HKCCD 2 6576- HKCCD 8415 HKCCD 9051 HKCCD 6795 HKCCD 6490 HKCCD 7022 HKCCD 6508 HKCCD 6507 2 HKCCD 7120- HKCCD 9025 HKCCD 8413 HKCCD 9041 HKCCD 6510 HKCCD 6505 HKCCD HKCCD 9073 8487 HKCCD HKCCD 5919 9057 HKCCD 5938 7391 HKCCD HKCCD 8481 6503 HKCCD HKCCD 9019 HKCCD HKCCD 6541 8485 HKCCD 6925 HKCCD 8822 HKCCD 7371 HKCCD 6132 HKCCD 8429 HKCCD 2 7106 HKCCD 8421 HKCCD 5934- HKCCD 8483 7634 HKCCD HKCCD 7328 HKCCD 7542 HKCCD 9039 9005 HKCCD HKCCD 8767 6544 HKCCD HKCCD 8420 HKCCD 7386 HKCCD 8419 HKCCD 5928 HKCCD HKCCD 8410 9061 HKCCD HKCCD 8806 HKCCD 9037 HKCCD 8768 HKCCD HKCCD 6547 9022 HKCCD 7372 HKCCD 5927 HKCCD 5862 HKCCD 8503 HKCCD 8418 7632 HKCCD HKCCD 7100- 8474 7120 HKCCD HKCCD 8192 2 HKCCD 7413- HKCCD 7373 HKCCD HKCCD 8191 HKCCD 9054- HKCCD HKCCD 8832 2 7561 HKCCD HKCCD HKCCD 8430- HKCCD HKCCD 1 HKCCD 8472 H 8434 HKCCD 8505 0 5935 7422 K 2

HKCCD 8765 1 8486

5929 C 7581

HKCCD 0 6576 6575 7382

8810

C 8703 HKCCD 9 9032 5914 HKCCD D D 7124 6565

HKCCD 6265 5 5932 HKCCD C 7780

HKCCD 9 HKCCD C

2 HKCCD

HKCCD K 2 HKCCD HKCCD HKCCD

HKCCD

H Figure S13. The maximum-likelihood phylogenomic tree of the genetically uniform Rhodobacteraceae population consisting of 214 strains isolated from four coral individuals of the same coral species Platygyra acuta, each collected from a distinct location in Hong Kong. Solid black dots at the nodes indicate that the ultrafast bootstrap support value of the branch is ≥ 80%. The scale bar indicates number of substitutions per site. Strain names are coloured with coral compartment information. The coloured dots at tree tips represent the sampling sites of associated coral individuals. References:

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