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Fig. S1. Differential representation of gene families in free-living oceanic compared with cultured roseobacters (M versus A plot). For the latter, the gene families were directly sampled from all annotated genes in the 39 Roseobacter genomes, rather than only from those linked to a Roseobacter core gene. Families plotting above the line are enriched and those plotting below the line are depleted in the oceanic roseobacters. The colour scheme is the same as for Fig. 1.

Fig. S2. Maximum likelihood phylogeny of the tRNA-dihydrouridine synthase encoded in the anchor end of JCVI_READ_1172364. The tree was constructed using RAxML 7.0.4 software with the ‘PROTGAMMAWAGF’ model. Values at the nodes show the number of times the clade defined by that node appeared in the 100 bootstrapped data sets.

Fig. S3. Maximum likelihood phylogeny of the Slt transglycosylase encoded in the anchor end of JCVI_READ_926014. The tree was constructed using the method described in Fig. S2.

Fig. S4. Maximum likelihood phylogeny of the pyrophosphatase encoded in the mate read of JCVI_READ_1172365. The tree was constructed using the method described in Fig. S2.

Fig. S5. Maximum likelihood phylogeny of the acyltransferase encoded in the mate read of JCVI_READ_1172365. The tree was constructed using the method described in Fig. S2.

Table S1. The 348 orthologous genes shared by sequenced 45 marine Alphaproteobacteria. The locus tags in the 45 genomes are given, arranged by gene (row) and organism (column).

Table S2. The 801 orthologous genes shared by sequenced marine Roseobacters. The locus tags in the 39 Roseobacter genomes are given, arranged by gene (row) and organism (column).

Table S3. GOS reads identified by dN analysis to be of Roseobacter origin.

Table S4. Phylogenetic trees for individual gene families, each of which includes GOS sequences identified by the dN-based pipeline and reference genes from the 45 Alphaproteobacteria genomes. Escherichia coli str. K-12 substr. W3110 was used as an outgroup. Trees were constructed using RAxML.

Table S5. Differential representation of gene families in free-living oceanic compared with cultured roseobacters. Three simulated metagenomes were constructed with random 2 kb fragments from cultured roseobacters, and each was compared with the oceanic Roseobacter metagenome according to the dN-based pipeline. A ‘1’ indicates that the gene family was significantly different between the oceanic and cultured metagenome.

Table S6. GOS sequences identified as Roseobacter by dN analysis but not by blast best hit analysis.

Table S7. Differential representation of gene families in Roseobacter genomes obtained as HTCC isolates (7 genomes) compared with those obtained by standard culturing methods (32 genomes). Three simulated metagenomes were constructed as random 2 kb fragments from each set of cultured roseobacter genomes and each was compared with the free-living oceanic Roseobacter metagenome according to the dN pipeline. A ‘1’ indicates that the gene family was significantly different between the oceanic and cultured metagenome. Values in the last column (‘Total’) range from 1 (significant in only one replicate simulated metagenome for one genome set) to 6 (significant for all replicate simulated metagenomes for both genome sets).

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