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1 Supporting Information Farrant et al /pnas Prochlorococcus % % 8% % % 5% 4% 3% 2% 1% % A Assignment level: Phylum Genus SubCluster Clade Synechococcus % % 8% % % 5% 4% 3% 2% 1% % Posi on of bp simulated reads along the petb sequence Assignment level: Phylum Genus SubCluster Clade SubClade B Fig. S1. Variation of the assignment ability of each individual -bp gene fragment along the sequence of petb gene using reference databases for Prochlorococcus (A)orSynechococcus (B). Simulated reads were generated by -bp sliding windows along the marker sequences, and the lowest taxonomic level at which they could be assigned is shown by a different blue tone (as indicated in the Inset;forProchlorococcus, the subcluster level actually corresponds to a LL or HL assignment, whereas the clade level corresponds to HLI IV and LLI IV, the lowest taxonomic level available for this genus). 1of1

2 A 15 HLI 3 2 HLII 3 2 HLIII 5 Prochlorococcus HLIV LLI LLII-III LLIV 2 5 Ia Ib Ic 8 5 IIa IIb IIc IId IIe IIf IIg IIh WPC2 2 IIIa IIIb IIIc Synechococcus IVa IVb V VIa VIb VIc VII VIII IX XVI XX CRD UC-A EnvA EnvB WPC Percent iden ty to best-hit in reference database 5 Fig. S2. (Continued) 2of1

3 B 3 2 HLI 3 2 HLII 4 2 HLIII Prochlorococcus HLIV 15 3 LLI LLII-III LLIV 2 5 Ia Ib Ic WPC2 IIh IIg IIf IIe IId IIc IIb IIa 2 IIIa IIIb IIIc IVa IVb V VIa VIb VIc 2 2 Synechococcus VII VIII IX XVI XX CRD UC-A EnvA EnvB WPC Percent iden ty to best-hit in reference database 5 Fig. S2. (A) Distribution of the percent identity of petb- mi tags recruited from the bacterial-size fraction of the Tara Oceans metagenomes with regard to their best hits in the reference database for each Prochlorococcus clade (top 7 graphs) and Synechococcus subclade (bottom 18 graphs) before addition of the 136 newly assembled environmental petb sequences. (B) Same as A but after addition of the 136 newly assembled environmental petb sequences. Note that clade XX was formerly called EnvC (12), but the name was changed here because there is at least one representative isolate (i.e., strain CC9616). 3of1

4 A SURFACE+DCM Prochlorococcus Synechococcus B SURFACE C DCM Prochlorococcus D E Synechococcus Fig. S3. Global recruitments of marine picocyanobacteria petb- mi tags in the bacterial size fraction of the Tara Oceans metagenomes. (A) All picocyanobacterial clades at both sampled depths. (B and C) Percentage of each Prochlorococcus clade in surface (B) and at the DCM (C). (D and E) Percentage of each Synechococcus clade in surface (D) and at the DCM (E). Note that clade XX was formerly called EnvC (12), but the name was changed here because there is now at least one representative isolate (i.e., strain CC9616). 4of1

5 Stations HLIA HLIB HLIIIA LLII-IIIA HLIC HLIVA LLIVA HLID Stations HLIIA HLIIB HLIIC Relative abundance Stations LLIA LLIB LLIC HLIID LLID Fig. S4. Prochlorococcus ESTUs based on the distribution patterns of within-clade 94% OTUs. At each station, the number of reads assigned to a given OTU is normalized by the total number of reads assigned to the clade in this station. Stations and OTUs are filtered based on the number of reads recruited. OTUs are hierarchically clustered (Bray Curtis distance) according to their distribution pattern. Stars indicate nodes supported by a P value <.5 as determined using similarity profile analysis (SIMPROF; test not applicable to pair comparisons). 5of1

6 A Temp ( C) B Genera Pro Syn ESTUs N A O MS RS IO SAO S O SPO NPO NAO Relative abundance Relative abundance 5.1 Normalized abundance C D reads 25 reads< N A O MS RS IO SAO S O SPO NPO NAO <25 reads Fig. S5. Marine picocyanobacteria community structure in Tara Oceans surface metagenomes based on petb- mi Tags recruitments. (A) Surface water temperature along the Tara Oceans transect. (B) Relative abundances of Prochlorococcus and Synechococcus normalized to the total number of reads at each station. (C and D) Relative abundances of Prochlorococcus and Synechococcus ESTUs, respectively. White, gray, and black dots indicate the number of reads used to build the profile, as detailed in the Inset. For readability, temperature for stations TARA_82 (7.3 C), TARA_84 (1.8 C), and TARA_85 (.7 C) are not shown on A. Abbreviations: IO, Indian Ocean; MS, Mediterranean Sea; NAO, North Atlantic Ocean; NPO, North Pacific Ocean; RS, Red Sea; SAO, South Atlantic Ocean; SO, Southern Ocean. 6of1

7 A Temp ( C) Depth (m) Genera Pro Syn ESTUs B N A O MS RS IO SAO S O SPO NPO NAO Relative abundance Relative abundance 5.1 Normalized abundance C D N A O MS RS IO SAO S O SPO NPO NAO reads 25 reads < < 25 reads Fig. S6. Same as Fig. S5 but at the DCM. A depth profile along the Tara Oceans transect was added. For readability, temperature for stations TARA_82 (7. C) and TARA_85 (.8 C) is not shown on A, and the temperature is missing for station TARA_7. 7of1

8 A B C Fig. S7. Distribution of minor Prochlorococcus ESTUs with regard to major ESTUs in the Tara Oceans metagenomes. Relative abundance normalized to the total number of reads per ESTU of (A) ESTUs HLIA and HLIC with regard to HLIIA in surface waters and (B and C) ESTUs LLIA C with regard to HLIIIA in surface waters and the DCM, respectively. For A, stations were sorted from the lowest to highest temperatures and for B by sampling date. 8of1

9 Spearman's rho Fig. S8. Correlation analysis between marine picocyanobacterial ESTUs and environmental parameters measured along the Tara Oceans transect for all sampled depths. (A) Prochlorococcus ESTUs. (B) Synechococcus ESTUs. The scale shows the degree of correlation (blue) or anticorrelation (red) between the two sets of data. Correlations with an adjusted P value >.5 are indicated by gray crosses. Abbreviations: DCM, deep chlorophyll maximum; fcdom, fluorescence, colored dissolved organic matter; MLD, mixed layer depth; Sal, salinity; Temp, temperature; Φsat, satellite-based NPQ-corrected quantum yield of fluorescence. Dataset S1. Summary data for picocyanobacterial petb reference sequences used in this study, including newly assembled sequences. The table includes subclade designation based on ref. 12 Dataset S1 Dataset S2. this study Summary data for petb reference sequences for photosynthetic organisms other than marine picocyanobacteria used in Dataset S2 Dataset S3. Tara Oceans sample description including the number of recruited petb reads per station. Iron and ammonium concentrations were simulated using the ECCO2-Darwin model, and an independent parameter to assess iron limitation (Φsat) was obtained using Behrenfeld et al. s (56) equation applied to monthly averaged satellite data (AMODIS chl_ocx, nflh and ipar) retrieved from the NASA website (oceandata.sci.gsfc.nasa.gov/) for each station and corresponding sampling date. Other environmental parameters measured during the Tara Oceans expedition and the methods used to acquire them are available at Dataset S3 9of1

10 Dataset S4. Sequence names of the members of each OTU defined for petb at 94% nucleotide sequence identity Dataset S4 1 of 1